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dissertationes MARKUS RÄSÄNEN MARKUS AND FACTOR-B GROWTH ENDOTHELIAL VASCULAR HYPERTROPHY CARDIAC IN KINASE THE BMX TYROSINE AND REVASCULARIZATION WIHURI RESEARCH INSTITUTE CANCER MEDICINE RESEARCH PROGRAM TRANSLATIONAL OF MEDICINE FACULTY PROGRAMME IN BIOMEDICINE DOCTORAL OF HELSINKI UNIVERSITY
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Markus Räsänen
Wihuri Research Institute,
Translational Cancer Medicine Research Program
Doctoral Programme in Biomedicine (DPBM)
Faculty of Medicine, University of Helsinki, Finland
ACADEMIC DISSERTATION
Doctoral dissertation, to be presented for public discussion with the permission of the Faculty of Medicine of the University of Helsinki, in Lecture Hall 2, Biomedicum Helsinki, on the 13th of September, 2019 at 12 o’clock
Helsinki 2019
1 1
ISBN 978-951-51-5404-0 (paperback) ISBN 978-951-51-5405-7 (PDF)
ISSN 2342-3161 (print) ISSN 2342-317X (online)
Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis No. 58/2019 Cover layout by Anita Tienhaara
Hansaprint Helsinki 2019
2 2 Supervisors:
Kari Alitalo, M.D., Dr. Med. Sci. Research Professor of the Finnish Academy of Sciences Wihuri Research Institute and Translational Cancer Medicine Research Program University of Helsinki Finland
Riikka Kivelä, PhD Adjunct Professor, Principal Investigator, Research Fellow of the Finnish Academy of Sciences Wihuri Research Institute and Research Programs Unit University of Helsinki Finland
Thesis committee:
Ilkka Tikkanen, MD, Dr. Med. Sci Professor Minerva Foundation Institute for Medical Research University of Helsinki Finland
Jyri Lommi, MD, Dr. Med. Sci Associate Professor, Chief physician of cardiology Heart and Lung Center Helsinki University Central Hospital Finland
Reviewers appointed by the Faculty:
Heikki Ruskoaho, MD, Dr. Med. Sci Professor Division of Pharmacology and Pharmacotherapy University of Helsinki Finland
Christian Kupatt, Dr. med. Professor 1. Medizinische Klinik, Klinikum rechts der Isar Technical University of Munich Germany
Opponent appointed by the Faculty:
Kristy Red-Horse, PhD Associate Professor Department of Biology Stanford University
3 3
To my family
4 4
Above all else, guard your heart,
for everything you do flows from it.
Proverbs 4:23
‘
5 5 TABLE OF CONTENTS
LIST OF ORIGINAL PUBLICATIONS...... 9
ABSTRACT...... 10
REVIEW OF THE LITERATURE ...... 12
Introduction...... 12
1. The cardiovascular system and the heart...... 12
2. Angiogenesis, VEGF-family and their receptors...... 14
3. Cardiac development...... 24
4. Cardiac remodeling in the adult heart...... 29
5. Translational insights of an angiogenic approach to cardiovascular diseases...... 32
AIMS OF THE STUDY ...... 34
MATERIALS AND METHODS ...... 35
RESULTS AND DISCUSSION ...... 44
Study I VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart...... 44
Study II Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling...... 46
Study III. VEGF-B gene therapy inhibits doxorubicin-induced cardiotoxicity by endothelial protection...... 50
Study IV VEGF-B promotes endocardium-derived coronary vessel development and regeneration...... 53
CONCLUDING REMARKS ...... 57
ACKNOWLEDGEMENTS ...... 61
REFERENCES ...... 63
6 6 ABBREVIATIONS
AAV (adeno-associated viral vector) αMHC (α-myosin heavy chain) AngII (angiotensin II) Ad (adenoviral) Apln (apelin) CMC/CM (cardiomyocyte) DOX (doxorubicin) EC (endothelial cell) ECG (electrocardiogram) 5-ethynyl-2’-deoxyuridine (EdU) EndMT (endothelial/endocardial-to-mesenchymal transformation) eNOS (endothelial nitric oxide synthase) ERK (Extracellular Signal-regulated Kinase) FDA (The United Stated Food and Drug Administration) HCMEC (human cardiac microvascular endothelial cells) HCAEC (human cardiac arterial endothelial cells) HGF (hepatocyte growth factor) HIF (hypoxia-inducible factor) HUVECs (human umbilical venous endothelial cells) IL (interleukin) KO (knock-out) LEC (lymphatic endothelial cell) LLC (Lewis’ lung carcinoma) MI (myocardial infarction) Nkx2-4 (NK2 homeobox 4) NO (nitric oxide) NRP (neuropilin) PlGF (placental growth factor) PKC (protein kinase C) ROS (reactive oxygen species)
7 7 SDF-1α (stromal cell-derived factor 1 alpha) Sema (semaphorin) sFlt1 (soluble VEGFR-1) Scx (scleraxis) SV (sinus venosus) TAC (transverse aortic constriction) TEC (tyrosine-protein kinase) TG (transgene) TNF (tumor necrosis factor) VEGF (vascular endothelial growth factor A) VEGF-B (vascular endothelial growth factor B)
8 8 LIST OF ORIGINAL PUBLICATIONS
The thesis is based on the following original publications. The original publications have been reproduced at the end of the thesis with the permission of the copyright holders.
I Kivelä R, Bry M, Robciuc MR, Räsänen M, Taavitsainen M, Silvola JM, Saraste A, Hulmi JJ, Anisimov A, Mäyränpää MI, Lindeman JH, Eklund L, Hellberg S, Hlushchuk R, Zhuang ZW, Simons M, Djonov V, Knuuti J, Mervaala E, Alitalo K.VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart. EMBO Mol Med. 2014 Mar;6(3):307-21. doi: 10.1002/emmm.201303147. Epub 2014 Jan 21.
II Holopainen T, Räsänen M, Anisimov A, Tuomainen T, Zheng W, Tvorogov D, Hulmi JJ, Andersson LC, Cenni B, Tavi P, Mervaala E, Kivelä R, Alitalo K. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling. Proc Natl Acad Sci U S A. 2015 Oct 20;112(42):13063-8. doi: 10.1073/pnas.1517810112. Epub 2015 Oct 1.
III Räsänen M, Degerman D, Nissinen T A, Miinalainen I, Kerkelä R, Siltanen A, Backman JT, Mervaala E, Hulmi JJ, Kivelä R and Alitalo K VEGF-B gene therapy inhibits doxorubicin-induced cardiotoxicity by endothelial protection Proc Natl Acad Sci U S A. 2016, Nov 15;113(46):13144-13149. Epub 2016 Oct 31.
IV Markus Räsänen, Jennifer Paech*, Karthik Hemanthakumar Amudhala*, Ibrahim Sultan*, Wei Yu, Juan Tang, Liqun He, Ying Sun, Ruslan Hlushchuk, Oleksiy-Zakhar Khoma, Eero Mervaala, Valentin Djonov, Christer Betsholtz#, Riikka Kivelä1#, Bin Zhou# and Kari Alitalo VEGF-B promotes endocardium-derived coronary vessel development and regeneration Submitted *,# these authors contributed equally to this work
Publication I was included in the doctoral thesis of Maija Bry (University of Helsinki).
9 9 ABSTRACT
Cardiovascular diseases are the leading cause of mortality worldwide, and they have become the most important and most significant cause of non-communicable diseases. In the Western world, they also rank first when listed by disability-adjusted life years (DALY). In particular, ischemic heart disease is still a major burden to the society, causing up to one third of the deaths of persons over 35 years.
My aim in this thesis was to investigate the translational potential of vascular endothelial growth factor B (VEGF-B) and the downstream Bone marrow kinase X (Bmx) tyrosine kinase in the treatment of pathological cardiac conditions, such as myocardial ischemia, pathological cardiac hypertrophy and cardiotoxicity. Based on earlier studies, VEGF-B seemed like an interesting angiogenic growth factor because of its relative specificity to the heart and adipose tissues. Furthermore, I was interested in analyzing the Bmx tyrosine kinase, which has been shown to receive signals from the VEGF-B receptor, and to be required for cardiac hypertrophy, but not for normal physiological functions in mice (Rajantie et al. 2001, Mitchell-Jordan et al. 2008).
In the first study of my thesis, we showed that expression of a VEGF-B transgene (TG) in the cardiomyocytes leads to an expansion of the coronary arterial tree. The VEGF-B transgenic hearts were also protected against myocardial ischemia. This opened further possibilities to study VEGF-B and its signaling in different pathological conditions in the heart.
In the second publication of the thesis, we showed that angiotensin II (Ang II)-induced cardiac hypertrophy was significantly reduced in mice, which were either deleted for the Bmx gene or the tyrosine kinase activity of Bmx was inactivated. This suggests that a selective Bmx tyrosine kinase inhibitor could be used in the treatment of pathological cardiac hypertrophy.
Cardiotoxicity and congestive heart failure are growing problems in cardio-oncology. This is mainly due to decreased cancer mortality and associated long-term use of cytotoxic drugs by an increasing number of cancer patients. Doxorubicin (DOX) is an antibiotic of the anthracycline class, which is associated with cardiotoxicity that may further develop into congestive heart failure. In the third paper of my thesis we showed that, in mice, VEGF-B gene therapy can be used to protect the endothelium and further to inhibit the cardiac atrophy and capillary rarefaction induced by DOX.
10 10 In the fourth study of this thesis we found that VEGF-B gene therapy can induce endocardium to venous endothelial cell (EC) transition, opening the possibility of a new pathway for coronary reperfusion. Currently, coronary occlusion is mainly treated by catheter-assisted reperfusion and thrombolysis of the coronary vessels. However, regardless of the reperfusion, insufficient subendocardial vessel perfusion (“no-flow reperfusion”) occurs frequently. Recent discoveries have indicated that the sinus venosus (SV) and the endocardium are the major sources of the coronary vessels that support the expanding myocardium in the developing heart, but the adult heart can no longer generate new blood vessels from the endocardium, because the endocardium-to-coronary vessel transition is inhibited after the neonatal stage.
We, however, observed that cardiomyocyte overexpression of VEGF-B in mice and rats promotes the growth of vessels derived from the endocardium that then perfuse the subendocardial myocardium. The findings demonstrate that VEGF-B promotes endocardial to vascular EC reprogramming and endocardium-derived vessel invasion into the myocardium during development. Importantly, subendocardial EC activation and proliferation was obtained also in adult mice by VEGF-B gene delivery to the cardiomyocytes. Furthermore, we were able to induce the endocardial to endothelial cell transdifferentiation by VEGF-B gene delivery during tissue remodeling after myocardial infarction (MI). Thus, re-activation and reprogramming of the adult endocardium by growth factors could be a new therapeutic strategy for cardiac neovascularization after myocardial ischemic injury.
Overall, these findings open translational therapeutic possibilities for the treatment of coronary artery disease, cardiac hypertrophy and anthracycline-induced cardiotoxicity.
11 11 REVIEW OF THE LITERATURE
Introduction
The importance of the heart is reflected by the finding that already 30 000 years ago, prehistoric drawings sketched a mammoth with a heart-shaped organ in the middle of it, highlighting its vital importance to the hunters. Aristotle instead believed the heart to be the organ containing one’s soul and the power of thought. Furthermore, in the 14th century, Vesalius found the heart to be the central organ of the vascular system, which opened possibilities for further advances by Harvey and others (Savona and Grech, 1999).
At the current state, our understanding is developed to the extent that we are capable of efficiently treating cardiac disease, spanning from acute myocardial infarctions to various arrhythmias. However, many questions remain unanswered, and many of the therapeutic strategies remain rather symptomatic than de facto causal. The understanding is still limited and many actual molecular pathways are still to be characterized. However, the possibilities of using biological therapeutics, such as tissue engineering, angiogenic growth factors and genome editing, promise a brighter future.
1. The cardiovascular system and the heart
Heart – the composition of cells and their architecture
The heart is a complex organ, consisting of several cell types, the three main ones being cardiomyocytes (CMCs), cardiac ECs and fibroblasts. Based on today’s knowledge, cardiomyocytes, endothelial cells and their interaction play an essential role regulating heart function and remodeling (Talman and Kivela, 2018, Hsieh et al., 2006). Pericytes and vascular smooth muscle cells are an important part of the cardiac vasculature. Pericytes have been suggested to provide a stem cell-like pool of cardiac cells and have been suggested to have a role in the regenerative and repair processes of the heart (Chen et al., 2013a). They have been recently shown to be capable of further differentiation into vascular smooth muscle cells (VSMC) (Volz et al., 2015). VSMC proliferation, and perhaps their transdifferentiation have important roles in the development of atherosclerotic plaques and coronary restenosis after percutaneous coronary intervention (Bennett et al., 2016, Libby et al., 2011).
12 12 CMCs, which are responsible for cardiac muscle contractions, can be divided into pacemaker cells and the actual force-giving CMCs. The ECs are important in regulating several physiological functions in the heart, including cardiac oxygen and nutrient supply, non-thrombogenic vessel surface and the VSMC tone. Single-cell RNA sequencing shows that ECs are the major non-myocyte population in the heart (Pinto et al., 2016). The heart contains also lymphatic ECs that are believed to participate for example in the recovery from MI (Henri et al., 2016, Klotz et al., 2015). The focus of my thesis, however, is on the blood vascular ECs in the heart and their interaction with the CMCs.
The cardiac ventricular wall consists of epicardium (ECs), myocardium (CMCs) and vessels (ECs, PCs, cardiac fibroblasts) and innermost the single layer of endocardial cells lining the ventricle (Brutsaert, 2003). The coronary ECs have been demonstrated to develop from three main sources: the epicardium, the SV (Red-Horse et al., 2010) and the endocardium (Wu et al., 2012, Tian et al., 2014, Tian et al., 2015).
Paracrine signaling in the heart
All cardiac cell types produce and secrete proteins and peptides for interaction and communication with other cardiac cells. The CMC-secreted factors are called cardiokines, among them the vascular endothelial growth factors (VEGFs), which bind to their receptors on the ECs and stimulate angiogenesis. Upon suitable stimuli, ECs also secrete proteins and peptides, called angiocrines, which then on their part regulate the growth and function of their neighboring parenchymal cells. Even though the angiocrines have been widely studied in other organs such as liver and lung (Ding et al., 2011, Ding et al., 2014), their role in the cardiac homeostasis regulation has remained poorly known (Brutsaert, 2003, Kamo et al., 2015). Additionally, the cardiac cells establish cell contacts through gap junction proteins, such as connexins (Cx43, Cx40, and Cx37) that allow conduction of electrical stimuli throughout the myocardium (Sohl and Willecke, 2004).
Angiocrine signaling in the heart
Endothelial cells in tissue specific vascular niches secrete angiocrine molecules, which can stimulate organ repair in damaged or diseased organs, ultimately preventing scar formation (Rafii et al., 2016). Angiocrine signaling has been shown to play an important role in lung alveogenesis after injury (Ding et al., 2011) and in liver regeneration after partial hepatectomy (Hu et al., 2014, Ding et al., 2014). In addition, EC Notch-signaling and angiocrine secretion have been shown to be crucial in promoting
13 13 angiogenesis and osteogenesis in the bone (Ramasamy et al., 2014). In the heart, nitric oxide (NO), endothelin-1 (ET-1), neuregulin-1 (NRG1), and apelin are secreted by cardiac ECs and they have been shown to regulate cardiomyocyte hypertrophy (Oka et al., 2014, Kamo et al., 2015). Recent work showed that EC stimulation by VEGF-B or placental growth factor (PlGF) leads to secretion of angiocrines, which promote cardiac hypertrophy via NO and ErbB signaling (Jaba et al., 2013, Kivela et al., 2019b).
2. Angiogenesis, VEGF-family and their receptors
In order for tissues to grow and to survive, nutrients and oxygen must be transported to tissues by blood vessel capillaries (Adams and Alitalo, 2007). Vessel growth is mainly regulated by vascular endothelial growth factors (VEGFs) and their receptors, which are expressed on ECs. However, also numerous other molecules have been shown to take part in the regulation of vascular growth. It was recently been shown that VEGF receptor 1 (VEGFR-1) and VEGFR-2 are expressed exclusively in ECs in the heart (Kurotsu et al., 2017).
The VEGF family in mammals consists of five secreted growth factors, VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D and PlGF. All of these factors have specific patterns of binding to the VEGFR-1, VEGFR-2 and VEGFR-3 on ECs, and they specifically regulate both blood and lymphatic vessel development and growth (Ferrara et al., 2003, Karaman et al., 2018). The VEGFs occur in several isoforms, for example, VEGF has four and VEGF-B has two isoforms, which have been shown to have different effects in various studies. The VEGF/VEGFR-2 pathway is the most crucial one for blood vascular development and angiogenesis (Shibuya, 2011).
VEGF and its receptors
VEGF is the key player in angiogenesis, EC migration and proliferation (Shibuya, 2011). VEGFA165 is biologically the most important isoform (Robinson and Stringer, 2001). Overexpression of VEGF boosts angiogenesis robustly in various tissues (Detmar et al., 1998, Isner et al., 1996, Leung et al., 1989). VEGF also has an important function in regulating endothelial nitric oxide synthase (eNOS), and therefore influencing vasodilatation (Fukumura et al., 2001). The vascular permeability promoting function of VEGF has hindered its therapeutic use in proangiogenic treatment of ischemic tissues. The described functions are mediated through the VEGF signals, mainly acting through VEGFR-2. VEGFR-1 mainly functions as an anti-angiogenic decoy receptor (Boucher et al., 2017).
14 14 VEGFR-1 is required for proper embryonic vasculature development (Shibuya, 2006, Olsson et al., 2006, Nesmith et al., 2017). In the heart, VEGFR-1 is not expressed in the CMCs (Muhl et al., 2016, Kivela et al., 2019a, Kurotsu et al., 2017). Deletion of the VEGFR-1 in adult mice leads to tissue hypervascularization (Kivelä, 2019, Ho et al., 2012). Furthermore, VEGF also binds to Neuropilin- 1 (NRP-1) and Neuropilin-2 (NRP-2) (Soker et al., 1998, Gluzman-Poltorak et al., 2000), which are discussed in the following chapter.
VEGF has an essential developmental role for the vasculature, and mice lacking even a single allele die during embryonic development due to impaired angiogenesis and blood island formation (Carmeliet et al., 1996, Ferrara et al., 1996). Hypoxia is known to regulate VEGF and angiogenesis by stabilizing the hypoxia-inducible factor (HIF), via its HIF-1α subunit (Pugh and Ratcliffe, 2003, Forsythe et al., 1996, Shweiki et al., 1992). However, also growth factors, inflammatory cytokines and hormones have been shown to stimulate VEGF (Ferrara et al., 2003).
In translational proof-of-principle studies, adenoviral gene delivery of VEGF165 (AdVEGF165) was shown to lead to EC proliferation and dilation of myocardial capillaries and therefore to an improved cardiac ejection fraction in pigs (Lahteenvuo et al., 2009). In mice, AdVEGF165 also promoted the expression of VEGR-1 and VEGFR-2 (Huusko et al., 2010). These phenotypic findings are in line with previous studies showing that VEGFR-2 and its downstream signaling activates angiogenesis by inducing EC proliferation, migration and sprouting (Shibuya, 2011). However, it has proven difficult to balance the various functions of VEGF for a controlled angiogenic response that supports oxygen and nutrient delivery adequately, without simultaneously inducing excessive vascular leakage. Titration of an optimal dosage of VEGF and combinations with other growth factors such as platelet derived growth factor-B (PDGF-B) may ultimately improve these possibilities (Banfi et al., 2012, Kupatt et al., 2010, Gianni-Barrera et al., 2018).
VEGF-B
VEGF-B was discovered in 1996 (Olofsson et al., 1996, Grimmond et al., 1996). Structurally VEGF- B is rather similar to VEGF, as mouse VEGF-B is up to 43% identical in its amino acid sequence with mouse VEGF164 (Olofsson et al., 1996). VEGF-B is endogenously highly expressed in mitochondria-rich tissues such as the heart, aerobic skeletal muscle and brown adipose tissue. These tissues are metabolically active and use lipid oxidation as a major source of energy (Hagberg et al., 2010, Bry et al., 2014). The VEGF-B gene has seven exons and due to alternative splicing acceptor sites in exon 6, it generates two isoforms (Olofsson et al., 1996, Grimmond et al., 1996). VEGF-B167,
15 15 the more common isoform in vivo, has a heparin-binding carboxy-terminus. The other isoform,
VEGF-B186, contains a hydrophobic carboxy-terminus that is modified by O-glycosylation and proteolytic processing (Olofsson et al., 1996), making it freely diffusible and offering a wider use. Interestingly, both VEGF-B isoforms bind to the VEGFR-1 and NRP-1 but not to VEGFR-2 or VEGFR-3 (Olofsson et al., 1998, Makinen et al., 1999). Nevertheless, VEGF-B and VEGF have similar affinities to the VEGFR-1 high-affinity receptor (Koch and Claesson-Welsh, 2012). However, VEGF-B induces only very little phosphorylation or activation of VEGFR-1 (Anisimov et al., 2013). High tissue concentrations of VEGF-B can displace VEGF from VEGFR-1, which acts as a decoy receptor, increasing endogenous VEGF binding to the signal transducing VEGFR-2 (Kivela et al., 2014, Kivelä, 2019, Boucher et al., 2017, Robciuc et al., 2016) (Schematic Figure 1, below).
Schematic figure 1: VEGF-B functions via activation of the VEGF/VEGFR-2 pathway. A. Excessive VEGF-B (transgenic or gene transfer) displaces VEGF from VEGFR-1, which can then activate VEGFR-2 signaling, leading to enhanced angiogenesis. B. Similar effects are also obtained through a genetic deletion of VEGFR-1. References Kivelä et al. 2014 and 2019.
16 16 Another interesting aspect is that, unlike VEGF, VEGF-B does not seem to be hypoxia-regulated (Enholm et al., 1997). Only one study reported hypoxia induction of VEGF-B in mouse retina (Singh et al., 2013), presumably via an indirect mechanism.
VEGF-B has only minimal angiogenic effects in other tissues than the heart or the adipose tissue. Transgenic overexpression in the skin increased the capillary diameter, but only minimally blood vessel density (Karpanen et al., 2008). Vector-mediated delivery into skeletal muscle or periadventitial tissue did not induce angiogenesis (Bhardwaj et al., 2003, Rissanen et al., 2003), (Bry et al., 2010). In hind limb ischemia, VEGF-B failed to provide vascular growth (Lahteenvuo et al., 2009, Li et al., 2008a), but rather potentiated the already existing angiogenic response (Mould et al., 2005). In contrast to VEGF or other VEGF-family members, it did not increase vascular permeability either (Brkovic and Sirois, 2007, Abraham et al., 2002). One study has suggested that VEGF-B is a survival factor for ECs via VEGFR-1 and NRP-1 (Zhang et al., 2009). Recent studies showed that VEGF-B is a potent anti-oxidant (Arjunan et al., 2018), and suggested a possible role for VEGF-B in oxidative stress-related diseases such as age-related macular degeneration, Alzheimer’s disease and Parkinson’s disease (Chen et al., 2019). It was also suggested that VEGF-B plays a role in tumor development, as elevated levels of VEGF-B were observed in various types of cancer (Gunningham et al., 2001). However, in a genetic VEGF-B model, ectopic expression of VEGF-B actually reduced tumor growth, whereas in mice lacking VEGF-B, the tumors were larger (Albrecht et al., 2010, Hanrahan et al., 2003).
VEGF-B in the heart
In the healthy heart, VEGF-B is abundantly expressed in the cardiomyocytes, whereas PlGF, which binds to the same receptors, shows low levels of expression (Bry et al., 2014). Mice lacking the endogenous VEGF-B are viable and fertile. However, one study has reported that in a certain strain, mice lacking VEGF-B have smaller hearts, their coronary vasculature is not fully functional and their recovery from cardiac ischemia was impaired (Bellomo et al., 2000). These mice were also more resistant against development of pulmonary hypertension by contributing to the vascular remodeling during chronic hypoxia (Wanstall et al., 2002). Such phenotype could not be reproduced using an independent VEGF-B targeted allele in the C57Bl6 genetic background (Aase et al., 2001). Instead a delay in the atrioventricular conduction (prolonged PQ-time) was reported in this strain of VEGF-B gene deleted mice (Naumenko et al., 2017, Aase et al., 2001). No obvious changes in the vasculature or cardiac development were observed in VEGF-B deficient rats. After an MI their myocardial scar
17 17 tissue was slightly bigger, but their cardiac function was unaltered (Kivela et al., 2014). Two studies have reported VEGF-B involvement in fatty acid transport across the endothelium in highly metabolically active tissues, such as the heart (Hagberg et al., 2010, Hagberg et al., 2012), but subsequent studies failed to reproduce the key findings in these papers (Kivela et al., 2014, Dijkstra et al., 2014).
From the developmental point of view, it is interesting that VEGF-B expression correlates well with the spatiotemporal progression of the coronary vessel development. During embryogenesis, VEGF- B expression begins in the basal parts of the heart and expands then further on along the interventricular septum to the apical part or the heart, mimicking the pathway of coronary vessel development (Bellomo et al., 2000, Sharma et al., 2017a). In quail embryos, coronary artery development was inhibited by (poorly characterized) antibodies against VEGF-B (Tomanek et al., 2006, Tomanek et al., 2002). However, loss of endogenous VEGF-B by gene deletion, doesn’t seem to lead to any developmental defects in mouse embryos (Bellomo et al., 2000).
By now, VEGF-B overexpression has been reported to be cardioprotective in several studies. This is well in line with the findings that myocardial VEGF-B levels are decreased in human heart disease (Kivela et al., 2014) and in rats after an MI or transverse aortic constriction (TAC) (Zhao et al., 2013, Huusko et al., 2012). Several studies have suggested usage of VEGF-B as a biomarker in cardiac diseases as low plasma levels correlate well with cardiac remodeling changes and left ventricular cardiac dysfunction after an MI (Zhu et al., 2019, Devaux et al., 2010, Devaux et al., 2012).
Both VEGF-B isoforms, VEGF-B167 and VEGF-B186, have been shown to induce coronary vessel enlargement and coronary arteriogenesis (Bry et al., 2010, Karpanen et al., 2008, Lahteenvuo et al., 2009). It has also been suggested that VEGF-B binds CMC VEGFR-1 and thereby directly improves myocardial contractility and CMC survival (Zentilin et al., 2009). Recent studies, however, have shown that the expression of VEGFR-1 and VEGFR-2 are restricted to ECs in the heart, whereas there is very low, if any expression, in CMCs (Kurotsu et al., 2017, Kivela et al., 2014, Kivela et al., 2019b). VEGF-B transgene expression in CMCs induced an expansion of the coronary vasculature and promoted coronary arteriogenesis, especially in the epicardial and subendocardial regions, whereas capillary sprouting angiogenesis was not detected. The expansion of the coronary vasculature led to development of mild cardiac hypertrophy without compromising heart function (Bry et al., 2010). Unlike VEGF, VEGF-B did not induce inflammation or vascular leakage in the heart or in skeletal muscle (Bry et al., 2010).
18 18 Controversial results have been reported on VEGF-B function in the diabetic setting. One study showed that CMC surface pool of VEGF-B promotes CMC and EC survival. However, ECs secreting heparanase in hyperglycemic setting can replace the VEGF-B bound to the heparan sulfate proteoglycans on the CMC surface. The authors also showed that VEGF-B promotes cell survival: loss of VEGF-B and downstream Extracellular signal-regulated kinase (ERK)-signaling leads to hyperglycemia, concluding that loss of VEGF-B could have a role in the development of diabetic cardiomyopathy (Lal et al., 2017). This idea was supported by a review concluding that VEGF- B:VEGFR-1 signaling seems cause dysfunction in the diabetic heart and that VEGF-B could be a potential therapeutic target (Rowe and Young, 2017). On the contrary, studies by Hagberg et al. claimed the VEGF-B has a role in controlling the endothelial fatty acid transport. They claimed, that VEGF-B loss of function in mice led to decreased uptake and accumulation of lipids in the heart (Hagberg et al., 2010) and attenuated insulin resistance (Hagberg et al., 2012).
Validating VEGF-B’s translational potential, systemic delivery of AAV-mVEGF-B186 mimicked the angiogenic response and the physiological cardiac hypertrophy seen in VEGF-B transgenic mice and rats (Kivela et al., 2014). Adenoviral vector-mediated VEGF-B gene delivery has been also studied in pigs, in which it was shown to be safe, and produced efficient myocardial angiogenesis as evidenced by an increase in capillary diameter and density. This led to increased perfusion of the myocardium as measured by position emission tomography (PET), without inducing a coronary steal syndrome (Nurro et al., 2016). Furthermore, adenoviral VEGF-B delivery did not induce alterations in ECG (Huusko et al., 2010). Interestingly, also intracoronary AAV-VEGF-B167 in canine dilated cardiomyopathy (DCM) appeared to be safe and feasible (Paradies et al., 2019). In this study, VEGF- B gene therapy halted the progression of DCM without inducing arrhythmias. Other studies have reported that a CMC specific human VEGF-B167-transgene in mice leads to increased ceramide levels in the heart (Karpanen et al., 2008) and later, heart failure (Lottonen-Raikaslehto et al., 2017). However, also in healthy dogs that were subjected to tachypacing to induce DCM, development of the disease could be prevented by AAV-VEGF-B167 treatment. AAV-VEGF-B167 treatment prevented the left ventricle thinning and inhibited apoptosis, oxidative stress and cardiac changes caused by tachypacing (Pepe et al., 2010). The anti-apoptotic properties of VEGF-B were also confirmed in studies showing that VEGF-B167 suppressed the expression of numerous apoptosis-associated genes and reduced the expression levels of caspase-2, -8, -9 and -12 as well as p53 in various cell types (Li et al., 2008c). Nevertheless, in a cardiac transplant-associated ischemia-reperfusion study, VEGF-B overexpression or gene delivery did not protect the hearts from injury (Raissadati et al., 2017). This
19 19 might be due to the already hypertrophic and more oxygen-consuming heart that is more sensitive to the transplantation injury.
A recent study has shed light on the EC-CMC crosstalk in cardiac hypertrophy development. It was demonstrated that AAV-VEGF-B -induced mild cardiac hypertrophy could be inhibited and also reversed by VEGFR-2 blocking antibody or deleting VEGFR2 from the ECs. Reversibility is one of the important hallmarks of physiological cardiac hypertrophy, thus in the light of these findings it can be speculated that angiogenesis is an important trigger of physiological hypertrophy and it is regulated by VEGF-VEGFR2 signaling (Kivela et al., 2019).
While most previous studies have concentrated on the protective effects of VEGF-B on the ECs, a recent study suggested that its beneficial cardiac effects during ischemia-reperfusion depend on its direct signaling activity in CMCs (Li et al., 2016). In this study, the authors also claimed that VEGF- B protein treatment induced the expression of stromal cell-derived factor 1 alpha (SDF-1α) and hepatocyte growth factor (HGF), which enhanced the homing of c-Kit positive CMCs . However, the role of c-Kit positive cells in cardiac repair and regeneration has been recently challenged by several investigators (Zhou and Wu, 2018, Marino et al., 2019). Another study suggested that stimulation of the vagus nerve induces an angiogenic response in a post-infarction heart by activating the VEGF and VEGF-B in the ECs and vascular smooth muscle cells (Lv et al., 2018).
PlGF
PlGF, placental growth factor, was found first in the human placenta shortly after VEGF (Maglione et al., 1991). In humans, the PlGF gene encodes four isoforms but in mice only one, PlGF-2 (Maglione et al., 1993, Cao et al., 1997, Yang et al., 2003). Besides the placenta, PlGF is also expressed in skeletal muscle, lungs and in the heart (De Falco, 2012). VEGF-B and PlGF are very similar structurally and also bind to the same receptors, VEGFR-1 and Nrp1 (Park et al., 1994, Migdal et al., 1998). However, they differ in their interaction with VEGFR-1 (Anisimov et al., 2013).
Loss of mouse PlGF was claimed to impair angiogenesis and collateral growth during ischemia, inflammation, wound healing and cancer (Carmeliet et al., 2001). On the other hand, PlGF treatment stimulated angiogenesis and collateral growth in ischemic heart and limb with an efficacy comparable to VEGF (Luttun et al., 2002). However, similarly to VEGF, also PlGF induced vasculature permeability (Odorisio et al., 2002, Bry et al., 2010). PlGF may function by inducing crosstalk between VEGFR-1 and VEGFR-2 and perhaps enhance VEGF signals via VEGF/PlGF heterodimer
20 20 formation (Park et al., 1994, Cao et al., 1996, Carmeliet et al., 2001). However, further studies using anti-PlGF antibodies could not reproduce the results in inhibiting tumor angiogenesis (Bais et al., 2010).
An interesting aspect regarding VEGFR-1 and PlGF is pregnancy induced cardiac hypertrophy. The female heart is known to undergo significant physiological hypertrophy during pregnancy, when the placenta produces high amounts of PlGF, plus a substantial amount of soluble VEGFR-1 (sFlt1), which acts as an inhibitor of VEGF, VEGF-B and PlGF function (Chau et al., 2017). An increased ratio between sFlt1 and PlGF has been linked to peripartum cardiomyopathy (Patten et al., 2012). A preclinical mouse study concluded that high concentration of PlGF in plasma during pregnancy is associated with protective functions on the maternal cardiovascular system across midgestation. Near the end of gestation, PlGF concentrations decline (Aasa et al., 2015). Pre-eclampsia is associated to the excess of circulating soluble vascular endothelial growth factor receptor 1 (sFlt1) from the placenta (Maynard et al., 2003, Young et al., 2010). A recent paper showed that by using short interfering RNAs (siRNAs) to silence the main three sFlt1 mRNA isoforms led to reduced circulating sFlt1, and was effective in the treatment of the common clinical signs of pre-eclampsia, for example hypertension, in baboons (Turanov et al., 2018).
Neuropilins
Neuropilins 1 and 2 are receptors lacking cytosolic enzyme activity. They function as co-receptors for several transmembrane receptors, including VEGFR-1, VEGFR-2 and VEGFR-3 (Pellet-Many et al., 2008). Neuropilins were found initially as receptors for semaphorins responsible for neuronal axon guidance (Chen et al., 1997, Kolodkin et al., 1997). VEGF-B and PlGF-2 can bind only NRP-1
(Migdal et al., 1998, Makinen et al., 1999). VEGF binding is very isoform-specific: VEGF165 and
VEGF121 interact also with NRP-1 (Soker et al., 1998), whereas VEGF165 and VEGF145 interact with NRP-2 (Gluzman-Poltorak et al., 2000). NRP-1 and NRP-2 can both form complexes with VEGFR- 1 (Gluzman-Poltorak et al., 2001, Fuh et al., 2000). In the cardiac tissue, NRP-1 is mostly colocalized with VEGFR-1 in the cardiac coronaries and capillaries, but also in the epicardial vessels (Partanen et al., 1999). During development, this colocalization is also found in the endocardium and myocardium (Makinen et al., 1999).
Neuropilin-1 is involved in the formation of the vasculature, and mice lacking NRP-1 die during embryonic development at E13.5 due to vascular defects (Kawasaki et al., 1999). Mice lacking neuropilin-2 are instead viable, but show defective neuronal, lymphatic and small capillary
21 21 development (Yuan et al., 2002). However, after a simultaneous deletion of Neuropilin-1 and Neuropilin-2, mouse embryos are no longer viable at E8.5, showing severe yolk sac and angiogenic abnormalities (Takashima et al., 2002).
Other VEGFs and their receptors
VEGF-C and VEGF-D bind to VEGFR-3 and Neuropilin-2, and act as lympangiogenic factors and therefore control and regulate the growth of lymphatic vessels (Tammela and Alitalo, 2010). The proteolytically processed forms of human VEGF-C and VEGF-D have been shown to bind also to VEGFR-2. Full-length mouse VEGF-D, however, only binds to VEGFR-3. Overexpression of VEGF-C or VEGF-D promotes lymphangiogenesis in adult skin (Jeltsch et al., 1997, Veikkola et al., 2001, Joukov et al., 1997), but, as mentioned, the processed human forms also have angiogenic properties, presumably through VEGFR-2 signaling (Saaristo et al., 2002, Rissanen et al., 2003). VEGF-C and VEGF-D are the main drivers of lymphatic endothelial cell (LEC) migration, proliferation, and differentiation in the heart (Brakenhielm and Alitalo, 2019). These transgenic mice expressing soluble VEGFR-3 as a trap for VEGF-C and VEGF-D developed lymphatic hypoplasia or aplasia in the heart as well as in many other organs leading to severe pre- and postnatal oedema and pericardial fluid accumulation (Makinen et al., 2001). However, the lymphatic vasculature eventually forms postnatally in these mice, indicating that there are also other lymphangiogenic growth factors that can compensate (Vaahtomeri et al., 2017).
Apart from its role in forming and maintaining the cardiac lymphatics, VEGF-C’s connection to the coronary vascular development has also been studied. The coronary artery stem development has been shown to require VEGF-C to stimulate vessel growth around the outflow tract and interactions between the vessels and CMC facilitate further coronary development (Chen et al., 2014a). VEGF-D has recently been tested in a clinical trial to induce coronary angiogenesis. In this study, refractory angina patients received AdVEGF-DΔNΔC gene therapy (the processed form of VEGF-D). A modest improvement of myocardial perfusion was obtained after the treatment (Hartikainen et al., 2017).
22 22 Bone marrow kinase X (Bmx)
The Bmx (Etk) gene is located on chromosome X, region XP22.2 and encodes a cytosolic tyrosine kinase, which belongs to the TEC family (Tamagnone et al., 1994). Bmx is expressed in myeloid hematopoietic cells, in certain epithelial cells and in the endocardium of the heart as well as in the arterial endothelium. Previous studies have highlighted its role in regulating the immune response (Guryanova et al., 2011, Gottar-Guillier et al., 2011) and it has been shown to be important in a variety of pathological states, including tumor growth (Holopainen et al., 2012). It has also been shown to be involved in the transactivation of epidermal growth factor receptor (EGFR1) via an angiotensin receptor 1 activated pathway. Bmx was shown to attenuate the downstream signal activation of ERK serine kinase activity (Rajantie et al., 2001).
Schematic figure 2. Schematic illustration of VEGFR-1, VEGFR-2, Bmx signaling pathways in the coronary arterial endothelium. Bmx interacts with VEGFR signaling pathways which can modulate signals regulating vascular leakage, inflammation and vessel stabilization. References Karaman et al., 2018, Rajantie et al., 2001, He et al., 2006.
23 23 Recent signal transduction studies have shown that Bmx phosphorylates proteins at a dual tyrosine (pYpY) peptide (Chen et al., 2013b). Its overexpression promotes ischemia-induced arteriogenesis and inflammatory angiogenesis (Paavonen et al., 2004). In 2004, a study linked Bmx as a participant in the protein kinase C (PKC)-ε signaling system in cardioprotection (Zhang et al., 2004). Thereafter, Bmx was suggested to participate in the vascular growth signals that are triggered by ischemic preconditioning of the heart (Mathur et al., 2005). It was then shown that Bmx deficient mice tolerated well cardiac pressure overload induced by TAC without developing a pathological cardiac hypertrophy, as opposed to their wildtype littermates. In these experiments, Bmx deletion also preserved the cardiac function and reduced the mortality (Mitchell-Jordan et al., 2008). Furthermore, a role for Bmx in nitric oxide (NO)-mediated cardioprotection has also been suggested (Zhang et al., 2004).
Previous data indicate that Bmx, ectopically expressed in 293T cells, is activated by VEGFR-1 transfected to the same cells (Rajantie et al., 2001), suggesting that it is involved in the signaling pathway employed by the VEGFR-1 ligands, such as VEGF-B and placenta growth factor (PlGF) in the arterial endothelium. Therefore, Bmx may link to VEGF-B, especially in the heart. Activation of VEGFR-2 upon TNF stimulation has been also shown to be Bmx dependent suggesting that Bmx is not only a downstream but also a feedback activator VEGFR-2 ((He et al., 2006), schematic Figure 2, above).
3. Cardiac development
Cardiac development and remodeling
Cardiac muscle and vessel development, has been extensively studied in the past decades, because the involved developmental mechanistic pathways might teach us on how to redeploy these developmental pathways also in pathological conditions in the adult heart (Smart, 2017).
The heart develops from the anterior splanchnic mesoderm (Gittenberger-de Groot et al., 2005) and later on the cardiac tube is then formed of two cardiac plates. The cardiac tube has gene transcripts regulating the anterior (ventricular) and posterior (atrial) specification, but the definitive differentiation takes place only after cardiac looping (Olson and Srivastava, 1996). After the primary heart tube is formed, cardiac development continues to secondary looping, which is followed by the formation of the outflow tract of the primary heart tube.
24 24 After septation and valve formation, the resulting two mature atria and ventricles still contain elements of the primitive chambers as well as transitional zones. Extracardiac cell populations, such as neural crest cells and epicardium-derived cells, participate in the developmental process. Neural crest cells are needed for outflow tract septation and the epicardium-derived cells for maturation of the coronary ECs and the myocardium. Extracardiac cell signaling is also considered to be important for the differentiation of the conduction system (Gittenberger-de Groot et al., 2005). Compaction, meaning the development of ventricular trabecular myocardium from a sponge-like CMC mass to a solid myocardium, is a crucial step of cardiac development and during this process endocardium contributes to the coronary ECs (Moorman and Christoffels, 2003). For this, communication between the endocardium and myocardium is crucial and previous studies have shown that Notch (Grego- Bessa et al., 2007, D'Amato et al., 2016) and neuregulin-ErbB (Lai et al., 2010) pathways are important in these.
Development of the heart is guided by secreted morphogens, including members of the transforming growth factor (TGF)–β superfamily (Noseda et al., 2011). In addition to their regulatory function in organogenesis, the TGF-β family of growth factors, including activins, bone morphogenetic proteins (BMP) as well as growth and differentiation factors (GDF), regulates cardiac physiology and pathophysiology in the adult heart. These factors signal through their type I and type II receptors, both of which are transmembrane serine/threonine kinases. Recent studies have also shown that the endocardium has an evident role in the development of the heart. The endocardium is very plastic during development and able to adopt different cell fates: mesenchymal cushion cells, hematopoietic cells, coronary ECs and, arguably, even CMCs (Zhang et al., 2018).
Coronary development
Coronary vessels have a crucial role in cardiac development and the physiological function of the heart, as they are responsible for supplying oxygen and nutrients for the cardiac muscle. In the early stages of development, the thin layer of CMCs acquires its oxygen through diffusion. As the hearts grows in size, an immature vascular plexus is formed, that then infiltrates in to the myocardium (Red- Horse et al., 2010). The plexus anastomoses with the aorta and after the establishment of blood flow, starts to mature to arteries, veins and capillaries (Waldo et al., 1990, Vrancken Peeters et al., 1997).
The cellular origin and progenitor sources of the coronary ECs have been studied extensively for over a century (Sharma et al., 2017a), as better understanding of coronary vessel development might offer new therapeutic possibilities. Cumulative data and observations have suggested that coronary ECs
25 25 develop from three different sources. A fundamental study in the field showed that both the coronary veins and arteries as well as capillaries arise from progenitor cells in the SV and endocardium (Red- Horse et al., 2010). Furthermore, epicardial Scx+ and Sema3D+ cells have been shown to contribute to coronary vasculature formation during development (Katz et al., 2012). Previous studies using other epicardial markers in lineage tracing experiments concluded that only a few or none of the coronary ECs arise from the epicardial cells (Zhou et al., 2008, Cai et al., 2008, Merki et al., 2005). In these studies epicardial heterogeneity poses a challenge for the finding of reliable lineage tracers. This could explain the different extents of epicardial contribution to coronary vasculature, from minimal (Zhou et al., 2008, Cai et al., 2008) to up to 20% (Katz et al., 2012, Cano et al., 2016). For example, a recent study suggested that of all epicardial cells, only the Sema3D+ cells express the Yap/Taz transcription factors that are needed for the endothelial-to-mesenchymal transformation (EndMT) and differentiation into coronary ECs (Singh et al., 2016).
A study using clonal tracing analysis has shown that coronary vessels that arise from the SV, migrate along the subepicardium to cover the heart surface, and part of them grow and differentiate to coronary arteries in the myocardium; the other part differentiates to coronary veins (Red-Horse et al., 2010). The SV contribution of coronary vessels is regulated by VEGF-C and Apelin signaling pathways (Sharma et al., 2017b, Chen et al., 2014b). Recent evidence indicates that the endocardium grows in the developing ventricles, forming circular structures lined by ECs and filled with blood cells, which then later on contribute mostly to septal vessels (Red-Horse et al., 2010, Zhang and Zhou, 2013). The endocardium is also believed to be hemogenic and to give rise to blood cells (Nakano et al., 2013). Another study challenged this view, claiming that ventricular endocardial cells directly migrate into the compact myocardium and form the majority of intramyocardial coronary arterial ECs and capillaries, but only a few veins (Wu et al., 2012). It was shown that this was driven by myocardial VEGF binding on endocardial VEGFR-2, since deletion either of these resulted in a very sparse coronary vessel network. Interestingly, deletion of soluble VEGFR-1 (sFlt1) from the endocardium induced a robust coronary budding, underlining the fact that the endocardium can give rise to hypoxia-driven angiogenesis, unlike the SV (Zhang and Zhou, 2013, Sharma et al., 2017b). A recent study using a novel endocardial specific lineage tracer (Npr3-CreER) showed that the endocardial contribution is not as extensive as previously thought (Zhang et al., 2016, Tang et al., 2018). Instead, the subepicardial vessels form the majority of the coronary vessels in the ventricular free walls, as shown with both the Apln-CreER and Nkx2-4-Cre lineage tracing lines (Tian et al., 2013, Chen et al., 2014b). Thus, current knowledge suggests that the majority of coronary vessels in the free walls of the cardiac ventricle originate from the SV, whereas septal vessels develop mainly from the
26 26 endocardial side (Zhang et al., 2018). In the neonatal heart, there is an expansion of the existing coronary EC pool and a phenotypic transition of endocardial cells to coronary ECs (Tian et al., 2014). Recent studies have addressed how the SV-derived venous ECs then undergo an early cell fate switch to create pre-artery ECs and further on, the actual coronary artery ECs (Su et al., 2018).
VEGFs have been shown to have important functions in coronary development. VEGF has been shown to act as a negative regulator of endocardial-to-mesenchymal transformation (EndMT) and therefore also in the formation of endocardial cushions (Dor et al., 2001). On the other hand, VEGF- C has been previously shown to play an important role in guiding the coronary artery stem development (Chen et al., 2014a). VEGF-B’s role in cardiac vasculature development has remained mostly unknown, though it has been suggested to be linked to avian stem development (Tomanek et al., 2006). However, as mentioned above, the knocking out soluble VEGFR-1 in the endocardial cells led to accelerated and excessive coronary plexus formation, underlining that endocardial produced VEGFR-1 has a crucial functions in the endocardial-to-coronary EC transition (Zhang and Zhou, 2013).
The endocardium also functions in other processes of cardiac development besides the coronary EC development. In fact, the heart valves are formed by extracellular matrix, so called cardiac jelly consisting mainly of endocardial cells that undergo EndMT and adopt a fibroblast cell fate (Garside et al., 2013). The EndMT process is mostly coordinated by TGF-β superfamily signaling, initiated by SMAD2/3/4 and further controlled by myocardial BMP signaling (Somi et al., 2004, Nakajima et al., 2000). Also Notch1 signaling (Timmerman et al., 2004) and canonical Wnt/β-catenin signaling (Liebner et al., 2004) are essential for EndMT and inhibiting these blocks the process.
In conclusion, coronaries develop in three waves: first wave from the SV, the second wave from the ventricular endocardium into the interventricular septum, and the third wave neonatally again from then ventricular endocardium into the inner myocardial layer.
In the adult murine heart, the extent of the endocardial contribution to the formation of coronary ECs after injury, such as MI, is minimal but may depend on the extent of the damage (Tang et al., 2018, Dube et al., 2017). Another study reported that myocardial ischemia activates endogenous arterialization in endocardial cells, as arterial markers were seen in the endocardium (Miquerol et al., 2015). Furthermore, during pathological and regenerative processes, the endocardium can assume
27 27 multiple cell fates (Zhang et al., 2018) and one could speculate that these fates could be influenced by certain external stimuli.
Cardiac regeneration
Whereas adult mammals lack the ability for cardiac regeneration, some vertebrates and reptiles can regenerate their myocardium and are capable of CMC proliferation (Uygur and Lee, 2016). Harnessing the regenerative potential of the heart for therapeutic purposes has been an intriguing possibility. However, despite the positive results in preclinical models, human clinical regenerative trials have failed (Bertero and Murry, 2018). The problem in CMC proliferation is that their potential undergo cell cycling in mammals drastically decreases right after birth (Laflamme and Murry, 2011). Although some CMCs in the infarct border zone enter the cell cycle after MI (Soonpaa and Field, 1997), this is far from being sufficient for full regeneration of the damaged tissue.
Different approaches have been taken to regenerate myocardial tissue after MI, including induction of CMC proliferation (Leach and Martin, 2018, Gabisonia et al., 2019). On the other hand, cardiac stromal cells, such as cardiac fibroblasts, could be reprogrammed into CMCs (Srivastava and DeWitt, 2016). Another strategy was to use cardiac progenitor or stem-like cells that have cardiogenic potential (Liu et al., 2018). Cardiac stem cells have been extensively studied and multiple other (both endogenous and exogenous) cell sources have been suggested for regenerative purposes. c-KIT+ or SCA-1+ cells are developmental progenitors of cardiac ECs and CMCs; studies have been performed to implant stem cells to induce regeneration and formation of new myocardium after MI (Tang et al., 2010, Bearzi et al., 2007). Also bone-marrow derived cells have been tried, but their cardiogenic potential has remained very modest (Bartunek et al., 2013). Recently, attemps have been made to implant human pluripotent stem cells (hPSCs) (Shiba et al., 2016, Stevens and Murry, 2018).
Ultimately, cardiac regeneration aiming at reconstitution of functional myocardium has to be able to offer remuscularization with CMCs that are not only contracting but also electromechanically stable in order not to induce arrhythmias. Additionally, sufficient angiogenesis and vessel arterialization has to be reassured. In the post-ischemic remodeling, initial inflammation and the resulting fibrosis protects the ventricle from rupturing under pressure, but later, the infarcted part of the heart is electrically inert and has low blood flow. Postischemic inflammation and fibrosis are therefore issues that need to be addressed to ensure proper regeneration of cardiac tissue (Bertero and Murry, 2018).
28 28 4. Cardiac remodeling in the adult heart
Cardiac hypertrophy
Due to various reasons, preload or afterload in the heart can increase and as CMCs are terminally differentiated in the adult heart, the way to adapt to external stimuli is through an increase in the size of the CMCs instead of proliferation. This process is called cardiac hypertrophy and is defined as growth of the cardiac muscle and CMCs in response to a physiological or pathological stimulus (Nakamura and Sadoshima, 2018). Pathological hypertrophy and cardiac remodeling take place usually due to a lasting and intrinsic stimulus, for example, hypertension, aortic stenosis or inflammation, post MI or due to a genetic mutation. It is often accompanied by fibrosis (van Berlo et al., 2013). Reactive or physiological hypertrophy develops as a response to an extrinsic stimulus or increase in cardiac work. This can be, for example, exercise-induced or it can happen during pregnancy, where the heart has to adapt up to a 40% increase in blood volume and a 45% increase in cardiac output (Hunter and Robson, 1992). It is reversible, unlike pathological remodeling, which in many cases eventually leads to heart failure (Mann and Rosenzweig, 2012, Vega et al., 2017). A detailed understanding of the molecular basis and differences in reversible physiological and pathological hypertrophy would serve in developing and targeting the treatment of pathological cardiac hypertrophy (Mann and Rosenzweig, 2012).
Some of the intracellular signaling pathways essential for cardiac hypertrophy are mitogen-activated protein kinase (MAPK) signaling (Bueno et al., 2000), the phosphoinositide 3-kinase (PI3K)–Akt and mammalian target of rapamycin (mTOR) pathways (Condorelli et al., 2002, Shioi et al., 2003, Shioi et al., 2000, Patrucco et al., 2004, Sciarretta et al., 2018). Also, calcineurin–NFAT signaling pathways (Molkentin et al., 1998, Wilkins and Molkentin, 2004), and other calmodulin-dependent protein kinases (Passier et al., 2000) have been shown to contribute. The role of glycogen synthase kinase-3β (GSK-3β) is also interesting, as it can inhibit both physiological and pathological cardiac growth (Kerkela et al., 2007, Antos et al., 2002).
Of the different cardiac cells, the CMC role in biomechanical responses to external stimuli has understandably been in focus (Hunter and Chien, 1999, Sadoshima et al., 1993). Therefore, most of the therapeutic approaches have concentrated on modulating the CMC function (Esposito et al., 2002, Hill et al., 2002). However, angiogenesis plays an important role in the development and type of cardiac hypertrophy. In exercise-induced physiological hypertrophy, oxygen supply is matched to the
29 29 proportional increases in cardiac myocyte size and muscle mass by increasing the size and extent of coronary microvasculature (Hudlicka et al., 1992, Shimizu and Minamino, 2016). Pathological hypertrophy is associated with a mismatch between oxygen supply and demand, as the increase in CMC size is not matched by a sufficient increase in the vasculature (Hudlicka et al., 1992, Shimizu and Minamino, 2016). Studies have also shown that angiogenesis alone can induce cardiac hypertrophy in mice (Tirziu et al., 2007, Jaba et al., 2013). Angiogenic VEGF-signaling has been linked to both pathological and physiological hypertrophic processes. ECs are the most abundant cell type in the heart in absolute numbers (Pinto et al., 2016), and their role in regulating the angiogenic hypertrophy responses have been somewhat underappreciated. Several studies have linked VEGF-B and its cardiac overexpression to physiological and beneficial hypertrophy (Kivela et al., 2014, Bry et al., 2010, Lahteenvuo et al., 2009).
Myocardial ischemia
Myocardial ischemia occurs due to an imbalance between myocardial oxygen supply and demand (Hoffman, 1987). The severity of the ischemia is determined by the size of the affected region, the extent of the decrease in the blood supply and possible protecting factors, such as collateral vessels, well-functioning endothelium and vasodilators (Crossman, 2004). Mechanistically, obstruction in the coronary flow can be due to many reasons: atherosclerotic plaques, thrombosis, stenosis or spasms in the epicardial coronaries or in the microvasculature. The coronary flow is regulated by microvascular tone, and ECs work by reducing or increasing resistance in the coronary vessels. Release of NO or prostaglandins for example reduces the vascular smooth muscle cell tone (Shimokawa, 1999, Ross, 1999). Dysfunction in this EC-driven process can lead to atherosclerosis and ultimately myocardial ischemia.
During the last years, the focus in the treatment of ischemic heart disease has been drawn to the microvascular coronary dysfunction (Camici and Crea, 2007): for patients undergoing a coronary artery reperfusion, dysfunction of the microvasculature might cause the “no-reflow”-phenomenon, where a sufficient perfusion to the myocardium cannot be sustained, even as the coronary artery occlusion has been treated (Shimokawa and Yasuda, 2008) and resulting in a worse outcome (Wu et al., 1998).
30 30 Cardiotoxicity
In recent years, as oncological therapies and their efficacy have advanced and cancer mortality has decreased, the amount of patients suffering from the adverse effects of cardiotoxic antineoplastic drugs has drastically increased (Levis et al., 2017). This development has led to a new field in clinical medicine called cardio-oncology, which aims in significantly reducing cardiovascular morbidity and mortality. Indeed, there is a clear increase in the interest to study the mechanism and possible therapeutic approaches to treat and prevent cardiotoxicity (Koutsoukis et al., 2018).
Cardiotoxic drugs side-effects are divided into reversible and irreversible cardiac changes. Reversible changes occur for example during monoclonal antibody therapy with trastuzumab. In contrast, anthracyclines are associated with irreversible and cumulative toxicity leading to cardiac failure (Suter and Ewer, 2013, Yu et al., 2014).
Doxorubicin (DOX) is an effective anthracyclic cytostatic agent, which has been widely used to treat various cancers already for decades, including different hematological cancers (for example leukemias, lymphomas, sarcomas, multiple myeloma), breast cancers and lung cancers (Koutsoukis et al., 2018, Volkova and Russell, 2011, Segredo et al., 2014) . It was originally obtained in the 1960s from mutagen treated bacteria (Arcamone et al., 1969). DOX acts mainly by inhibiting topoisomerase IIb, inducing DNA double-strand breaks that lead to increased apoptosis as reviewed in (Tacar et al., 2013). Mitochondrial iron accumulation and associated redox reactions (Ichikawa et al., 2014, Doroshow, 1983), abnormal protein processing, activation of innate immune responses, DNA damage, failure of cardiac repair and decreased vasculature have all been suggested to contribute to the cardiotoxicity of DOX (Zhang et al., 2012).
Numerous ways to protect the heart against the DOX-induced toxic effects have been suggested, most of them aim to protect the CMCs. In current clinical practice, DOX is administered in combination with other antineoplastic drugs to maximize the effects on the malignant tissue and to minimize the cumulative dose of DOX (Price et al., 1993, Singal and Iliskovic, 1998). DOX administration using PEGylated liposomal packaging has been also suggested to alleviate the cardiotoxicity (Franco et al., 2018). This form of encapsulation in a phospholipid bilayer protects it from being recognized by the mononuclear phagocytes allowing a longer circulation time and simultaneously reducing the exposure of free DOX in the plasma (Franco et al., 2018). Since mitochondrial iron accumulation has been linked to the DOX-related cardiotoxicity, dexrazoxane as an iron chelator has been tested to alleviate
31 31 DOX-induced toxicity with relatively good results (Simunek et al., 2009). This drug has been approbed by the United Stated Food and Drug Administration (FDA) approved for the treatment of the DOX-induced cardiotoxicity (Ichikawa et al., 2014). Nevertheless, not all iron chelators have proved such positive outcomes, raising questions on the role of iron accumulation in DOX-induced cardiotoxicity (Simunek et al., 2009). Dexrazone has also been linked with induction of myelosuppression (Seymour et al., 1999).
As oxidative stress has been linked to DOX toxicity several antioxidative compounds have also been (Hideg and Kalai, 2007). For example, N-acetylcysteine, coenzyme Q10, phenethylamines and beta- blockers such as carvedilol and statins have been used in randomized controlled trials, but in a meta- analysis their efficacy could not be confirmed (van Dalen et al., 2011). An evident need for an efficient and causal therapy of the DOX-induced cardiac toxicity therefore clearly exists.
5. Translational insights of an angiogenic approach to cardiovascular diseases
Translational insights are in urgent need for novel cardiac therapies. During the last years, the first approved gene therapy in humans has been for the treatment of lipoprotein lipase deficiency, that causes high plasma triglyceride levels (Yla-Herttuala, 2012). Considering the ability of vascular growth factor pathways to modulate cardiac remodeling, cardiac gene therapy offers an interesting solution for coronary artery disease, cardiotoxicity and cardiac failure. However, clinical randomized controlled VEGF trials have provided only modest results (Yla-Herttuala and Baker, 2017, Lahteenvuo and Yla-Herttuala, 2017). A recent review compared 14 clinical proangiogenic approaches using different VEGF and VEGF-D forms. The VEGF therapies were able to slightly increase the left ventricular ejection fraction (LVEF), and improve symptoms measured with Canadian Cardiovascular Society angina class. However, mortality and angina frequency scores were unimproved over the existing treatments (Yuan et al., 2018). Many reasons can explain the rather modest results. A proper and efficient gene transfer to the myocardium can be challenging. In clinical studies, main issues have concerned inadequate dosing of the vectors, insufficient duration of exposure, compromised delivery and poor vector transduction efficacy (Dragneva et al., 2013).
Gene therapy delivery can be divided into two major categories; nonviral and recombinant viral. The nonviral methods include plasmids, liposomal DNA complexes, oligonucleotids, polymer-carried DNA and the viral vectors include lenti-, adeno- (Ad) and adeno-associated (AAV) viruses (Hajjar, 2013). The immune response, the expression peak and the length and size of the vector vary when
32 32 using the various vectors. AAV-mediated gene therapy has advantages, when compared to the rather robust but transient overexpression via adenoviral delivery. In a study using AdVEGF165, the expression levels faded out in already 2 weeks (Huusko et al., 2010). With AAV-mediated gene delivery, clear effects with minimal immune response and long lasting steady expression can be achieved (Lopez-Gordo et al., 2019). However, AAVs are relatively small (20 nm) and thus limited in their cargo capacity of 4.7 kb. Of the 13 reported serotypes with varying degrees of tissue tropism AAV1, -6, -8, and -9 have been identified as being the most cardiotropic (Asokan et al., 2012), but transduce also the liver, skeletal muscle and lung (Hajjar, 2013). AAVs have already been successfully used in humans to treat congenital muscle dystrophy (Mendell et al., 2017). Additionally, the results from large animal AAV-based cardiac studies are promising so the use of AAV-based cardiac therapies also in humans looks possible (Ishikawa and Hajjar, 2015).
AAV-gene therapy using heme-oxygenase 1 (HO-1) seems to protect the rat heart against MI (Hinkel et al., 2015). A previous report studied HO-1 effects through carbon monoxide (CO) showing that it induced c-kit+ stem/progenitor cells in the infarct area and leads to formation of new coronary arteries. Interestingly, in the infarct region, increased expression of VEGF-B and HIF-1α and SDF- 1α was also observed (Lakkisto et al., 2010).
Identifying the optimal vectors is key to avoid adverse or unwanted systemic effects and to target the treatment to the ischemic region of the heart (Yla-Herttuala et al., 2007, Yla-Herttuala et al., 2017). For example, prolonged overexpression of VEGF is associated with increased permeability and leakiness of the vessels (Hulot et al., 2016). Recent study using VEGF-D reasoned that by utilizing both the angiogenic and lympangiogenic response, the oedematic side effects of proangiogenic therapy could be avoided. In the study the adenoviral vector was applied using intramyocardially via a catheter and the safety of this approach was confirmed, although an increase in anti-adenoviral titers was noted in the treated patients (Hartikainen et al., 2017, Brakenhielm and Alitalo, 2019).
33 33 AIMS OF THE STUDY
My thesis studies were undertaken to investigate the therapeutic potential of vascular growth factor B (VEGF-B) and its downstream Bmx tyrosine kinase in cardiac vasculature in some heart disease models.
Studies I and IV were conducted to explore the therapeutic potential of VEGF-B for vessel growth and cardiac remodeling in ischemic heart and to examine the mechanisms involved.
Study II was carried out to analyze the possibility that Bmx tyrosine kinase activity is involved in the development of pathological cardiac hypertrophy, because specific Bmx tyrosine kinase inhibitors would then provide a possibility for clinical development.
Study III was carried out to examine if VEGF-B gene delivery has a protective effect in anthracycline- induced cardiotoxicity.
Study IV was carried out to determine if VEGF-B could induce growth of de novo vessels from the endocardium in the adult heart after MI.
34 34 MATERIALS AND METHODS
MATERIALS
I have used various genetic mouse models in my thesis studies. To study Bmx deletion, we used both constitutive Bmx knockout (KO) and tyrosine kinase inactivated knockout mouse. For the VEGF-B studies, I have used transgenic overexpression models specifically in the cardiomyocytes (using α- myosin heavy chain (αMHC promoter). I have also used AAV-constructs to induce VEGF-B overexpression in the heart and Npr3-CreERT2 to lineage trace the endocardium and Apelin- CreERT2 in order to study the angiogenic sprouting in the heart (Table 1).
Table 1. Mouse and rat lines
Strain name Desription of Used Source/Reference the strain in αMHC-VEGF-B Overexpression I, IV Bry et al. Circ 2010 mouse of both isoforms of human VEGF- B in cardiomyocytes
αMHC-VEGF-B Overexpression I, IV Bry et al. Circ 2010 rat of both isoforms of human VEGF- B in cardiomyocytes
Bmx KO in C57Bl Bmx gene II Rajantie et al. Moll Cell Biol mouse deletion 2001
Bmx KO in Balb/C Bmx gene II Rajantie et al. Moll Cell Biol mouse deletion 2001
Bmx TK -/- mouse Bmx gene point II Holopainen et al. mutation in the PNAS 2016 tyrosine kinase domain Apln-CreERT2 Inducible Cre- IV Tian et al. Cell Res 2013 mouse allele under the Apelin promoter
35 35 Npr3-CreERT2 Inducible Cre- IV Tang et al. Circ Res 2018 mouse allele under the Npr3-promoter
Rosa26- Reporter line IV Wu et al. Cell 2012 TdTomatolox/STOP/lox
Table 2. Cell lines, recombinant viral vectors
Cell lines Host Used in Source HCAEC Human II, III Promo Cell HUVEC Human II Sigma HMEC Human II Promo Cell HCM Human II Promo Cell 293T Human II, III Promo Cell HCF Human II Promo Cell LLC Mouse III ATCC RECOMBINANT VIRAL Description Used in Source/Reference VECTORS AAV-VEGF-B-M/H186 Adeno-associated I, III, IV II, III, IV virus encoding mouse and human VEGF- B186 AAV-S2/CTRL Scrambled adeno- I, III, IV II, III, IV associated virus AAV-HSA Adeno-associated I Bry et al. Circ 2010 virus encoding human serum albumin
AAV-VEGF-B-M167 Adeno-associated I Bry et al. Circ 2010 virus encoding mouse VEGF-B167 AAV-ANG1 Adeno-associated IV Nykänen et al. Circ Res. 2006 virus encoding mouse Angiopoietin 1
36 36 Table 3. Antibodies
Antigen Antibody Staining Manufacturer Catalogue host and number dilution DYSTROPHIN-2 Mouse IF Novocastra #NCL-DYS2 (1:500) CD-31 Rat IF BD #553370 Arm. (1:500) Pharmingen #MAB1398Z hamster IF EMD Millipore (1:600) Corp. FITC-CD31 Mouse IF Invirtrogen RM5201 (1:1000) PACIFICBLUE-CD45 Mouse FACS Biolegend # 103125 (1:100) PACIFICBLUE-TER119 Mouse FACS Biolegend #116231 (1:100) PE-CYANINE7-CD140A Mouse FACS eBioscience #251401 (1:100) DAPI FACS Sigma-Aldrich #D9542 (1:100) VEGF-B Goat IF R&D systems #AF590 (1:500) FABP-4 Rabbit IF Abcam #ab13979 (1:500) ENDOMUCIN Rat IF Santa Cruz #sc-65495 (1:500) TROPONIN Mouse IF CV-2 (1:100) CASPASE 3 Rabbit IF R&D Systems AF835 (1:300) VEGFR-2 Goat IF R&D Systems AF466 (1:500) KI67 Rabbit IF Leica #KI67P-CE (1:500) SMA Mouse IF Sigma Cy3 monoclonal- (1:500) Cy3
RECA-1 Mouse IF Serotec MCA970 (1:500)
CD45 Mouse IF BD Biosciences 550566 (1:500)
37 37 ED1 Mouse IF Serotec MCA341R (1:500)
VEGFR-1 Rat IF ImClone 5B12 (1:500) gamma H2AX Mouse IF Abcam ab22551 (1:250) LAMININ Rabbit IF Thermo TB-082 (1:250) scientific PODOCALYXIN Goat IF R&D AF1556 (1:250) phospho-ERK1/2 WB (1:500) phosphor-rpS6 rabbit WB Cell Signaling (1:500) rpS6 mouse WB Cell Signaling 54D02 (1:500) Technology FITC-conjugated lectin mouse IF Vector FL1171 (1:250) Laboratories RECA-1 mouse IF Serotec MCA970 (1:500) Cyclin D1 rabbit IF Thermo RM-9104-5 (1:500) Scientific PERILIPIN5 goat IF Proge GP31S (1:500) PDK4 rabbit IF Novus 07047 (1:500) Biologicals Bmx human IF BD 610792 (1:500) Transduction Laboratories phospho-Bmx Y566 rabbit WB Abcam 59409 (1:500) pSTAT3 mouse WB Cell signaling 9131 (1:500) STAT3 mouse WB Cell signaling 9132 (1:500) mouse IF (1:500)
38 38 METHODS
Table 4. Summary of methods
Method Study AAV transduction of mice and rats I,III, IV Cell culture II, III Cell transfections and transductions II, III Characterization of a new transgenic mouse strain I Clinical chemistry III FACS analysis III Functional analysis of endothelial cells II Histological analysis of mouse aortas II Immunofluoresence/immunohistochemistry I - IV Immunoprecipitation and immunoblotting II Microscopy I - IV Polymerase chain reaction I - IV Real-time quantitative PCR I - IV Tumor experiments III X-gal staining of the tissues I, IV Computer tomography IV Echocardiography I, III, IV Transmission electron microscopy III, AngII-minipump II Blood pressure measurement II Microarray and GO-analysis II, III Mitochondrial function analysis III DXA III Treadmill running protocol III EC tube formation III ELISA III, IV Bulk RNA sequencing IV Single cell RNA sequencing IV Lineage tracing IV LAD ligation I, IV Coronary ligation and DiI perfusion staining IV
Functional analysis of endothelial cells
To study endothelial function, we isolated the descending aorta and placed it carefully between stainless steel hooks. The aorta was then placed in an organ bath chamber containing physiological salt solution (pH 7.4). The salt solution consisted of: NaCl 11.9%, NaHCO3 25.0, glucose 11.1, CaCl2
39 39 1.6, KCl 4.7, KH2PO4 1.2, and MgSO4 1.2 aerated with 95% O2 and 5% CO2 (mmol/L). These aortic rings were hung with a resting tension of 1.0 g for an hour at 37 degrees to create a baseline. To measure the force of contraction we used an isometric force-displacement transducer and recorded with IOX Software 1.569 and Data-analyst V1.68 (EMKA Technologies). After this we precontracted the aortic artery rings with 1 μmol/L phenylephrine for 6 min. Relaxation with acetylcholine (ACh) was used to confirm that the endothelium in the vascular preparations was intact and not damaged during the preparation. This also then served as a parameter for the endothelial function. To rule out that the relaxation effect would come from other tissues than the endothelium, endothelium- dependent relaxation was blocked with L-NAME 0.1 mmol/L.
Immunofluorescence/Immunohistochemistry. Tissue sections ranging between 8 and 100 μm in thickness were fixed overnight with 4% paraformaldehyde (PFA) or with cold acetone immediately before the staining, washed with PBS and blocked with TNB (PerkinElmer). Sections were washed with TNT buffer and the primary antibodies were detected with the appropriate Alexa 488, 594 or 647 secondary antibody conjugates (Molecular Probes/Invitrogen).
For the whole mount tissues, the samples were fixed overnight in 4 % PFA, followed by washing in PBS and staining with antibodies in donkey immune mix. All samples were mounted with Vectashield containing 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories).
In vivo studies. Cre-recombinase activation. For induction of CreERT2 mediated recombination in embryos, the mother was injected at the indicated days with two consecutive intragastric doses of 4- OH tamoxifen (4-OHT) (Sigma) (25 mg/ml dissolved in 100 μl of ethanol/olive oil). In the neonatal mice, the Cre-mediated recombination was induced between P1 and P5 by daily intragastric administration of 4-OHT (2 μl at 25 mg/ml dissolved in ethanol). Recombination in adult mice (7–8 weeks old) was done by intragastric tamoxifen (Sigma, dissolved in 100 μl of corn oil at 2 mg/ml) administration during five consecutive days.
Microscopy. Immunofluorescence images were taken with a compound fluorescent microscope (Zeiss Axioplan 2, Zeiss Axioimager) or a confocal microscope (Zeiss LSM 510Meta, Leica SP8x). All confocal images were recorded sequentially and averaged at least twice. Three-dimensional projections were digitally reconstructed from confocal z-stacks. Bright-field sections were viewed with a Leica DM LB microscope (Leica Microsystems), and images were captured with an Olympus DP50 color camera (Olympus Soft Imaging Solutions GMBH). MicroCT imaging was performed using Skyscan 1272 (Bruker microCT, Belgium). NRecon 1.7.1.0 software was used to reconstruct
40 40 the scanned hearts. Acquired projection images were used for reconstruction using NRecon or CTvox Software of the microCT scanner manufacturer (Bruker microCT, Belgium).
MicroCT-imaging. For the MicroCT-imaging with the Angiofil, the aorta of the mice was retrogradely cannulated and perfused with microAngiofil followed by polymerization at room temperature (RT) for at least 30 minutes. Next, the hearts were fixed in 4% PFA for 4h at RT and dehydrated in an alcohol array. They were then stained with 0.3% phosphotungstic acid in 70% ethanol for 48 h, and embedded in 4% low melting point agarose.
Real-time quantitative PCR. Total RNA from tissues or cultured cells was isolated using the RNeasy Mini Kit (Qiagen) or NucleoSpin RNA II Kit (Macherey-Nagel). Homogenization was carried out using rotor-stator homogenization, followed by on-column DNase digestion (RNase-Free DNase Set, 79254). Quality control of samples was carried out using a Nanodrop ND-1000 spectrophotometer. RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Three qRT-PCR reactions were carried out in triplicates using TaqMan Gene Expression Assays (Applied Biosystems) and the DyNAmo Probe qPCR Kit (F-450S, Finnzymes) or the iQ Supermix Kit. qRT-PCR was carried out using a BIO-RAD C1000 Thermal cycler according to a standardized protocol.
Adeno-associated viral vectors. The recombinant AAVs encoding mVEGFB186 (AAV9-VEGF-B), hVEGFB186 (AAV9-hVEGF-B), mANGPT1 (AAV-Ang1) or scrambled control (AAV-Ctrl) vector were constructed and generated as described in a previous publication (Anisimov et al., 2009). All vectors contained the CAG-promoter. 2 x 1011 AAV9 particles were injected to mice intraperitoneally (i.p.).
Isolation of cardiac ECs. The hearts were digested using 1mg/ml collagenases of type I, II and IV (Gibco) at 37°C for 25 min, then filtered through a 70 μm nylon cell strainer (Corning, #352350), rinsed with Dulbecco’s modified eagle medium (DMEM) with 10% heat-inactivated serum and suspended in staining buffer (Dulbecco’s phosphate-buffered saline containing 2% heat-inactivated fetal calf serum (FCS) plus 1mM EDTA). The formed suspension was centrifuged for 5 min at 300 g at 4°C. Cardiac ECs (CD31+ CD45- Ter119- CD140a- DAPI-) were isolated using FACS Aria II (BD Biosciences), and the data was analyzed using FlowJo v10.1 (FlowJo, LLC).
Single-cell RNA sequencing of cardiac ECs. Cardiac ECs were sorted in PBS containing 0.05% sterile bovine serum albumin (BSA). Cell viability and clusters of FACS sorted ECs were evaluated
41 41 using an automated cell counter (Luna, Logos Biosystems). Single cells were encapsulated with emulsion beads by using the 10X GEMCode technology (10X Genomics). The Single-Cell 3’ Reagent kit v2 (10X Genomics) was used on all cells and individual transcripts with unique molecular identifier (UMI) and cDNA libraries were generated. The libraries were sequenced in an S1 flow cell using the NovaSeq 6000 system (Illumina) in paired-end with the following read length values: 26bp (Read 1), 8bp (i7 Index), 0 bp (i5 Index) and 91 bp (Read 2).
Single-cell RNA data analysis. The pipelines mkfastq and count were used in the Cell Ranger software (v.2.1.1) to demultiplex the data, to convert chromium single-cell 3’ RNA sequencing barcodes and to generate FASTQ files and a gene-cell matrix. The reads were aligned to the mouse reference genome mm10 using STAR aligner. The R (version 3.5.1) package Seurat (v2.3.4) was used for quality control, filtering, normalization and downstream analysis of the data (Butler et al., 2018). Cells containing less than 200 genes or mitochondrial genes greater than 10% were filtered out, a scale factor of 10,000 was used to normalize the qualified cells. The t-distributed stochastic neighbor embedding (tSNE) was created using the Pagoda2 package (version 0.0.0.9003). The cell clustering was created using the spliced RNA matrix. The velocyto.R package (La Manno et al., 2018) (version 0.6) was applied to analyze the expression dynamics of the cells. Pseudotime analysis of the endocardial and putative venous EC was performed using the monocle package (Trapnell et al., 2014) (version: 2.8.0) with default parameters. As to compare the cell-wide differences between the control vs experimental groups, canonical correlation analysis (CCA) was performed and, successively, the common marker genes for each cluster and differentially expressed genes within clusters were identified using the FindConservedMarkers function and the FindMarkers function in the Seurat package, respectively.
Endocardial lineage tracing. Npr3-CreERT2;R26-Td-Tomato mice were treated with 3 daily doses of tamoxifen through gavage (200 mg/g weight). AAV-VEGFB or AAV-Ctrl virus particles were injected to mice intraperitoneally (i.p.) one week after the last tamoxifen treatment, and the week thereafter, the LAD was ligated to generate an MI. 4 weeks after this, the cardiac samples were collected and analyzed for Npr3-CreER labeled cells (TdTomato+).
Analysis of sprouting angiogenesis and EC proliferation in the heart. To detect ECs arising from AAV-VEGF-B induced vessel sprouts in the heart, the Apln-CreERT2 mice were mated with the Rosa26-TdTomatolox/STOP/lox reporter line. The mice were then first injected with the viral vectors (AAV-Ctrl/VEGF-B/Ang1) and after three days, a two-day tamoxifen treatment (2mg/day) by oral gavage was started. To study the EC proliferation and VEGF-B’s effect on it, we injected the mice
42 42 intraperitoneally (i.p.), first with AAV-Ctrl or AAV-VEGF-B and then 10 days later, with 50 μg/g of 5-Ethynyl-2'-deoxyuridine (EdU) twice a day for three consecutive days to label the proliferating cells. At two weeks, the mice were euthanized and the TdTomato+ ECs and EdU stained coronary vasculature were analyzed.
Experimental myocardial infarction, coronary ligation and DiI-perfusion staining. Myocardial infarction was induced in rats and in mice by ligation of the left anterior descending artery (LAD). For these studies, the hearts were first perfused with PBS through the abdominal aorta retrogradely, followed by ligation of coronaries at the aortic root both in mice and in rats in order to close all coronary circulation. Successively, the aorta was perfused again retrogradely with 1,1-dioctadecyl- 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) and 1% PFA, and immersion fixed in 4% PFA overnight at RT.
Statistical analysis. Quantitative data was compared between groups using a two-tailed unpaired t- test or a one-way ANOVA followed by post hoc tests for multiple comparisons. Values are expressed as mean +/- SEM or +/- SD. A p-value < 0.05 was considered significant.
A detailed description of the methods can be found in the original publications.
43 43 RESULTS AND DISCUSSION
Study I: VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart
VEGF-B’s role in inducing cardiac hypertrophy was evident from previous studies (Karpanen et al., 2008, Bry et al., 2010, Lahteenvuo et al., 2009). In this project, I wanted to study the phenotype in a more detailed approach and address how increased dosage vs. loss of VEGF-B alters the cardiac metabolism, as previous studies had linked it to endothelial fatty acid uptake (Hagberg et al., 2010, Hagberg et al., 2012). We performed high-resolution microCT imaging of WT and VEGF-B transgene expressing hearts using a contrast agent and observed a very significant expansion in the coronary tree (Figure 1, below). Cardiomyocyte overexpression of VEGF-B led to an increased number of coronary vessels and to larger capillaries observed in electron microscopy (EM). Importantly, we observed that a similar phenotype developed in adult mice transduced with AAV- VEGF-B, which led to long-lasting expression of VEGF-B without unwanted immune response.
Figure 1: Coronary arterial vasculature in αMHC-VEGF-B WT and TG rats. Representative microCT images of WT and TG hearts at three months of age.
44 44 In order to study the effects of the loss of VEGF-B in the heart, we generated a rat model of VEGF- B gene deletion. VEGF-B gene deletion in rats did not have obvious effects on blood vessel development or cardiac function after MI when compared with littermate control rats. Histologically quantified infarct areas were, however, larger in the VEGF-B deficient hearts. In the VEGF-B overexpressing rats, the resulting infarcts were significantly smaller than in control rats, apparently because of the protective effect of the increased cardiac vasculature. The VEGF-B TG hearts also showed improved blood perfusion and cardiac function after MI when compared to WT hearts. To mimic the insufficient reperfusion after coronary occlusion, we also performed ischemia/reperfusion (I/R) experiments by ligating the left anterior descending (LAD) coronary artery for 30 min, followed by reperfusion for 2 h. Interestingly, in analysis of mitochondrial function in these hearts, respiratory chain complex I activity and function was significantly better preserved in the TG hearts than in the WT hearts, when using both carbohydrates and fatty acids as substrates, suggesting that VEGF-B protected the CMCs in the I/R injury setting.
Mechanistically, we found that VEGFR-2 and its downstream signaling are responsible for the VEGF-B induced growth of the cardiac muscle and vessel expansion. We could also show that VEGFB induces activation of ERK1/2, Akt, and mTORC1 pathways in the heart. Because previous publications had suggested that VEGF-B regulates endothelial cell fatty acid uptake (Hagberg et al., 2010), we studied the cardiac uptake of fatty acids in TG and KO rat models, but did not observe any differences between the VEGF-B WT, TG or KO rats. VEGF-B overexpression, however, led to a shift from fatty acid to glucose usage in the heart. Such shift often occurs in pathological hypertrophy (Kolwicz and Tian, 2011), but in our model we did not observe any increase in transcripts associated with pathological hypertrophy. It should be noted that efficient cardiac glucose utilization has been suggested to slow down the progression of cardiac failure (Taegtmeyer, 2002).
In conclusion, this study indicated that cardiomyocyte overexpression of VEGF-B leads to the expansion of the coronary vasculature and the reprogramming of cardiac metabolism to favor glucose utilization, resulting in cardioprotection in myocardial ischemia. Furthermore, AAV-VEGF-B phenotype mimicked the cardiac phenotype of the TG rats, which suggests that VEGFB can be used therapeutically to improve cardiac function in ischemic heart disease.
45 45 Study II: Endothelial Bmx tyrosine kinase activity regulates pathological myocardial hypertrophy and remodeling
Bmx is a tyrosine kinase expressed in the arterial endothelium, in the endocardium and in myeloid cells. A recent collaborative study using single-cell RNA sequencing (scRNA seq) defines Bmx as a very suitable arterial marker in the murine brain (Vanlandewijck et al., 2018), and my scRNA seq data of the cardiac endothelium shows similar results in the cardiac ECs (Figure 2, below). Bmx has been linked to the downstream signaling pathway of VEGFR-1 (Ekman et al., 1997). For these reasons, Bmx is interesting for cardiovascular studies in which the arterial endothelium and the endocardium serve as potential druggable targets. Pharmacological inhibitors of the Bmx kinase activity could offer a possibility for therapeutic studies. However, up to date, the degree of specificity
Figure 2: Bmx expression in the heart. A. Single cell RNA sequencing of the cardiac endothelium, demonstrating Bmx expression in the endocardial and arterial EC clusters. B. Macroscopic images of Bmx LacZ/LacZ heart after betagal- staining. Note the expression in auricles, endocardium and arteries.
46 46 and safety of Bmx tyrosine kinase inhibitors are still under investigation (Chen et al., 2018, Liu et al., 2013). Prior to my studies, one report showed that cardiac hypertrophy in response to TAC was significantly attenuated in mice lacking the Bmx gene (Mitchell-Jordan et al., 2008). Another study showed that VEGF-B prevented AngII-induced CMC hypertrophy. The authors of these papers suggested that this is mediated by VEGF-B:VEGFR-1 signaling, as knockdown of VEGFR-1 canceled the effect of VEGF-B (Shen et al., 2018).
In the constitutive Bmx knockout (KO) mice, the AngII induced cardiac hypertrophy at two and at six weeks was inhibited by Bmx deletion, based on the heart weight/bodyweight-ratio (HW/BW) and CMC size. Next, we used the Bmx TK-/- mouse line, where Bmx tyrosine kinase activity has been inactivated by a K421R mutation (equivalent to human K445R) (Ekman et al., 2000, Gottar-Guillier et al., 2011). Also, these mice failed to develop AngII-induced pathological hypertrophy. Skeletal α- actin, used as a marker of pathological hypertrophy or markers of cardiac failure (ANP, BNP) were not increased after the AngII-treatment in the Bmx KO or TK-/- mice, but were clearly upregulated in the WT mice. Interestingly, blood pressure was increased by AngII similarly in the Bmx KO or TK-/- or WT mice.
We observed high expression levels of Bmx RNA in human microvascular and arterial endothelial cells, but little or no expression in CMCs or in cardiac fibroblasts, or even after AngII stimulation of CMC in vitro. The AngII-induced cardiac hypertrophy thus has to be mediated by EC-CMC crosstalk, where Bmx plays an important role.
The supply of sufficient amounts of oxygen and nutrients to the cardiac tissue is critical for the physiological function of the heart, and capillary rarefaction is one of the hallmarks of pathological cardiac hypertrophy (Mohammed et al., 2015). I observed a decrease in cardiac vascular density after AngII treatment, but Bmx gene deletion or Bmx tyrosine kinase inactivation inhibited the capillary rarefaction. Increased Collagen I and III mRNA-levels in the WT hearts after AngII treatment were observed, indicating increased fibrotic changes in the heart (Mohammed et al., 2015, Kong et al., 2014), and these were attenuated in the AngII-treated Bmx KO and TK-/- mice.
Whole-genome wide gene expression analysis revealed that in homeostatic conditions, only a few cardiac transcripts differed between the Bmx KO and WT mice. However, upon the AngII treatment, the WT heart showed numerous altered transcripts that have been linked to signaling in cardiac hypertrophy, for example increased expression of genes encoding extracellular matrix proteins. Again, Bmx KO and TK-/- mice expressed significantly less of such transcripts. The gene expression
47 47 analysis also demonstrated that AngII induces less changes in gene expression in the Bmx KO and TK hearts than in WT hearts. Furthermore, genes encoding components of the inner mitochondrial membrane and electron transport chain were downregulated by AngII only in the WT hearts. These results, together with the well preserved mitochondrial function and expression of PCG-1-α, the major regulator of mitochondrial biosynthesis (Rosca et al., 2013), suggest that loss of BMX function protects the function of cardiac mitochondria under pathological conditions.
As increased inflammation has been linked to cardiac hypertrophy, we studied the inflammatory cytokine response upon the AngII treatment. Previous publications have linked Bmx to inflammatory processes and their regulation (Gottar-Guillier et al., 2011, Paavonen et al., 2004). We observed an AngII-induced increase in transcripts encoding inflammatory mediators, for example interleukins IL- 6, IL-8, Tnfr2. Bmx KO and TK-/- mice showed a blunted inflammatory response after AngII treatment. Also expression levels of tissue inhibitor of metalloproteinases 1 (Timp1) and heat shock protein 70 (Hsp70) that have been linked to hypertrophic cardiomyopathy, were decreased in KO and TK-/- hearts when compared to WT hearts (Octavia et al., 2012).
Finally, we studied the effects of Bmx deficiency on the mTORC1 pathway, which is a major regulator of protein synthesis during cell growth (Ma and Blenis, 2009). Phosphorylation of ribosomal protein S6 (rpS6), a downstream target of the mTORC1 pathway, was less in the Bmx KO and TK-/- hearts than in the WT hearts, consistent to the smaller CMC size.
To study the changes in protein phosphorylation, we conducted a study where Angiotensin II was injected to the tail vein of the mice and the cardiac lysates of these mice were analyzed 10 minutes after the injection. AngII treatment increased phosphorylation of mTORC1, Akt and STAT3 in WT hearts, but not in TK-/- hearts. Microarray analysis showed increased Stat3 gene expression in AngII treated WT hearts, which was inhibited in the KO mice. Silencing of Bmx using three different shRNA constructs indicated that Bmx is needed for AngII induced STAT3 activation also in human umbilical venous endothelial cells (HUVECs). This is well in line with previous findings showing that Bmx mediates STAT3 activation in a pressure overload-induced cardiac hypertrophy model (Willey et al., 2008).
In conclusion, Bmx tyrosine kinase provides a potential target for the treatment of pathological cardiac hypertrophy. However, some important questions remain unanswered. So far, studies concentrated on constitutive mouse lines. Future studies need to address the question using inducible deletion of Bmx in adult mice. Also, a selective Bmx tyrosine kinase inhibitor would be needed for
48 48 further translational studies on Bmx. Recently, a group studying prostate cancer showed that inhibition of Bmx with ibrutinib (originally an inhibitor of the related Tec kinase BTK) and with another Bmx kinase inhibitor Bmx-IN-1 efficiently inhibited Bmx induced STAT3 activation (Chen et al., 2018). The functional benefit of Bmx inhibition needs to be addressed in future studies.
49 49 Study III: VEGF-B gene therapy in the treatment of anthracycline -induced cardiotoxicity
In this study, I aimed to determine if VEGF-B gene therapy can alleviate doxorubicin-induced cardiotoxicity. I injected AAV9-mVEGF-B186 into mice and four days later, started DOX treatment (6 mg/kg every third day for 12 days). This led into cardiac atrophy as evidenced by decreased heart weight to tibial length-ratio (HW/TL), left ventricle (LV) posterior wall and septum thickness as well as smaller cardiomyocytes at termination of the mice two and four weeks after the start of treatment. Comparison with mice that only received DOX showed that the VEGF-B treatment inhibits the cardiac atrophy in both two- and four-week experiments.
DOX treatment also led to whole-body cachexia, and body composition analysis revealed this was mainly due to a decrease in fat and muscle mass. Surprisingly, the AAV-VEGF-B pre-treatment inhibited also the loss of body weight in both two- and four-week experiments. Our previous studies indicate, that in homeostatic physiological conditions, VEGF-B has no effect on body weight (Bry et al., 2010).
Most of the cardiotoxicity studies so far have concentrated on the cardiomyocytes. However, in this study I focused on the protective effects of VEGF-B and toxic effects of DOX on the cardiac ECs. DOX caused myocardial capillary rarefaction in the two- and four-week experiments in vivo, which was fully prevented by VEGF-B treatment. In order to evaluate their effects on endothelial function more closely, I measured endothelial dependent aortic relaxation to acetylcholine (ACh) after phenylephrine-induced contraction. DOX treatment impaired aortic relaxation, which could be in part prevented by AAV-VEGF-B pre-treatment. Inhibition of the endothelium-dependent vascular relaxation by N[omega]-nitro-L-arginine methyl ester (L-NAME) abolished the differences suggesting that the effect is mediated by NO produced by the ECs. In accordance, the in vitro tube formation by human cardiac arterial endothelial cells (HCAECs) was inhibited by DOX treatment and rescued by AAV-VEGF-B. DOX-induced EC and mitochondrial damage in AAV-Ctrl but not in AAV-VEGF-B treated hearts was also observed in electron microscopy (Figure 3, below). Mechanistic studies by western blotting of heart tissue indicated that the activation of ERK, an important regulator of angiogenesis and arteriogenesis downstream of VEGFR-2 signaling (Simons and Eichmann, 2015), was also decreased by DOX and this decrease was prevented by VEGF-B pre- treatment.
50 50
Figure 3. DOX-induced EC damage. Electron microscopic images of mouse hearts treated with AAV-Ctrl+DOX and AAV-VEGF-B+DOX. Magnification 4800x.
As DOX is known to induce DNA damage (Zhang et al., 2012), I studied if VEGF-B has an effect on this in an acute setting. DOX was given intraperitoneally at 15 mg/kg and the hearts were collected 20 h after the injection. VEGF-B pre-treatment prevented the DOX-induced DNA damages shown by staining for phosphorylated histone H2Ax (γH2Ax). The mRNA encoding the DNA damage- inducible transcript 4 (Ddit4/REDD1), a mediator of cardiomyocyte autophagy and apoptosis through the mTOR pathway, was also elevated in the DOX treated Ctrl, but not VEGF-B treated hearts. Additionally, many markers of pathological cardiac remodeling, including BNP, ANP and β-MHC were increased by DOX and inhibited by the VEGF-B gene therapy.
Considering the translational potential of VEGF-B in cancer patients, it was important to exclude the possibility that VEGF-B increases tumor growth as an angiogenic factor. I implanted Lewis lung carcinoma (LLC) cells subcutaneosly into isogenic C57Bl6 mice. AAV–VEGF-B or AAV–Ctrl was injected three days and DOX six and 11 days after tumor implantation. Importantly, I found that VEGF-B administration did not affect the growth of the LLC xenotransplants. Interestingly, a previous study reported that VEGF-B167 even inhibits the growth of a transgenic pancreatic endocrine adenocarcinoma (Albrecht et al., 2010).
In conclusion, this study shows that VEGF-B protects the heart by maintaining the vascular endothelial cell viability and function. VEGF-B gene transfer therefore offers a therapeutic strategy to alleviate anthracycline therapy-related cardiotoxic effects. As the sensitivity to anthracycline- induced cardiotoxicity varies among different individuals, pre-therapeutic screening using human
51 51 induced pluripotent stem cell (hiPSC) -CMCs has been suggested for the identification of its genetic basis and molecular mechanisms (Burridge et al., 2016). Recent studies on DOX cardiotoxicity have identified resveratrol as a possible alleviating agent. Interestingly, it was suggested that the resveratrol-induced cardioprotection in MI is achieved through the VEGF-B/AMPK/eNOS/NO signaling pathway (Feng et al., 2019). Also, a recent study reported that VEGF-B is a potent antioxidant and speculated on its potential in the treatment of oxidative-stress related diseases (Chen et al., 2019). This supports my findings on the cardioprotective effect of VEGF-B, especially because previous publications have highlighted the role of reactive oxygen species in the cardiotoxic effects and have suggested that antioxidants could provide a therapeutic option (Songbo et al., 2019).
Most importantly, my results show that by protecting the endothelium and the endothelial function of the heart, one can achieve substantial cardioprotection. During the past years, the endothelium has been out of focus in this context, but emerging evidence shows that especially in DOX-induced toxicity, EC damage leads to release of angiocrines that can boost cardiomyopathy (Luu et al., 2018). DOX increases endothelial permeability, and therefore exposes CMCs to direct damage. It also induces, via reactive oxygen species (ROS), the secretion of NO (Octavia et al., 2017), neuregulin-1 (NRG-1) (Geisberg et al., 2013) and prostaglandin I2 (PGI2) (Neilan et al., 2006), which boost cardiomyopathy. It seems that by protecting the endothelium, we can efficiently protect the heart from cytotoxic damage.
52 52 Study IV: VEGF-B promotes endocardium-derived coronary vessel development and regeneration
The endocardium has an important role in coronary development and endocardial cells can differentiate into several other cell types (Zhang et al., 2018, Sharma et al., 2017a). Previous studies with VEGF-B demonstrated that VEGF-B transgene overexpression in cardiomyocytes leads to an expansion of the coronary tree and increased vasculature density, which seemed to protect the heart against myocardial ischemia. Prophylactic AAV-VEGF-B expression in the heart protected the heart from ischemia and cardiomyopathy. I therefore wanted to study in more detail how VEGF-B affects coronary development and expansion of the coronary vasculature.
Increased coronary vasculature and about a 20-30 % increase in heart weight/body weight ratio and cardiomyocyte size was observed in adult αMHC-VEGF-B TG mice compared to WT mice (Bry et al., 2010). Importantly however, no increase in pathological cardiac markers was observed. In this study, I analyzed the TG vs WT ratio in newborn pups born from crosses of heterozygous transgenic and WT mice. We observed that the αMHC-VEGF-B embryos develop normally and are born in normal Mendelian ratios. A significant increase in both the coronary vessel area and heart weight/body weight ratio was observed first at postnatal day 7 (P7).
Bioinformatic analysis of data from single-cell RNA sequencing of the FACS-isolated cardiac CD31- positive ECs at P7 revealed that there was a very significant shift in one of the clusters of cells, expressing endocardial markers such as Npr3, Cdh11 and Bmx. This data indicated that VEGF-B overexpression leads to a reduced number of endocardial cells at P7, suggesting that part of the endocardial cells have altered their cell fate.
To study this phenotype more closely, I used high-resolution microCT imaging with µAngiofil to visualize three-dimensionally the architecture of the coronary vasculature in the αMHC-VEGF-B TG hearts. Strikingly, numerous myocardial vessels were seen originating from the endocardium into the myocardium in the TG hearts. To test if these vessels actually connect to the ventricles, I ligated the coronaries at the aortic root in the TG and WT mice and rats and then perfused the ventricles retrogradely via the aorta with the lipophilic DiI carbocyanine dye that incorporates into endothelial cell membranes (Li et al., 2008b). Analysis of the sections showed DiI-staining within subendocardial vessels in the TG hearts, but not in the WT hearts. Staining with DyLight488-labeled tomato-lectin as an endothelial marker in the thick cardiac sections confirmed that these DiI-positive subendocardial vessels are connected to the rest of the coronary vasculature in the TG mice. The
53 53 VEGF-B TG hearts had also more SMA staining per field than the WT hearts, consistent with our previous findings (Bry et al., 2010). In single-chambered hearts of fishes and reptiles, where the coronaries are absent or very small, such connections are known to be present. Additionally, also some reports of humans that have coronary abnormalities are described to have coronary blood supply via myocardial sinusoids (Tsang and Chiu, 1995). Transmyocardial revascularization (TMR) has been approved to treat patients with severe angina pectoris (Horvath, 2008). In TMR, conducted either percutaneously or through thoracotomy, the YAG-laser beam creates small channels into the LV myocardium, allowing perfusion of oxygen rich blood into the myocardium. Interestingly, TMR is believed to activate angiogenesis in the treated areas and its use with VEGF-B might provide benefits in the treatment of angina pectoris (Hughes et al., 1998).
We treated adult mice with AAV-VEGF-B to study how VEGF-B transduction modulates the endothelial transcriptome of the adult heart. We performed scRNA sequencing of the cardiac ECs two weeks after AAV-VEGF-B-transduction. The results indicated an increase of cells in a cluster presumably representing highly proliferating ECs, as indicated by expression of, for example, survivin/Birc5 and Top2a (Altieri and Marchisio, 1999, Neubauer et al., 2016). Increased EC proliferation in the AAV-VEGF-B-transduced hearts was confirmed by an in vivo experiment, where 5-ethynyl-2’-deoxyuridine (EdU) was injected to the mice twice a day for 3 days. EdU incorporation into DNA in proliferating cells was then visualized together with CD31 and troponin in cardiac sections. AAV-VEGF-B-transduced hearts had three times more EdU-positive EC nuclei in the subendocardium, whereas the control AAV-transduced myocardium showed essentially no EdU- labeling. Staining of the myocardium with antibodies binding to the Ki67 cell cycle s-phase marker confirmed AAV-VEGF-B stimulated cell proliferation, supporting the EdU results. We then used the AplnCreERT2;Tdtomato reporter mouse line to detect and quantify angiogenic activation of ECs in the AAV-VEGF-B overexpressing hearts (Liu et al., 2015, Sharma et al., 2017b). Mice were first injected with AAV-Ctrl (scrambled vector), AAV-VEGF-B, or AAV-Angiopoietin 1 (Ang1, as a negative control), and tamoxifen was administered four and five days later to induce the Td-tomato reporter, which marks the Apelin+ ECs and cells derived from them. VEGF-B induced about a two- to threefold increase of Apelin+ ECs. These results indicated that VEGF-B increases EC activation throughout the myocardium, and proliferation especially in the subendocardial myocardium.
I then wanted to confirm that VEGF-B can reprogram adult endocardial cells into the endothelium of coronary vessels by using endocardial specific lineage tracing. The Npr3-CreER mouse line has been previously shown to label adult endocardium (Tang et al. Circ Res 2018). After tamoxifen induction
54 54 of the Npr3-CreER;R26-tdTomato mice, we ligated the LAD coronary artery to cause MI. When analyzed four weeks after the MI or sham operation, we found that almost all endocardial cells remained in the innermost layer of myocardial wall in the AAV-control group, but strikingly a subset of the endocardium-derived (tdTomato+) cells contributed to FABP4+ vascular endothelial cells in the myocardium. Quantification of this difference showed a significant increase in myocardial Npr+ vascular ECs in the AAV-VEGF-B group. Lectin staining together with the DiI-perfusion staining of the myocardium confirmed that these vessels are connected to the coronary vessels. This shows that VEGF-B can also de facto reprogram adult endocardial cells into de novo coronary ECs in the adult heart.
A previous study has shown that although endocardial cells generate coronary vessels in the developing neonatal mouse heart (Tian et al., 2014), they no longer contribute to new coronary vessels in the adult myocardium even after injury (Tang et al., 2018, Chen and Simons, 2018). During mouse development such transdifferentiation is known to occur during trabecular compaction morphogenesis, which happens at E9.0 (Del Monte-Nieto et al., 2018). Cardiac compaction is a part of normal cardiac development. It starts by endocardial cell trapping between the compacting myocardial trabeculae after which the endocardial cells change their fate to vascular ECs, fibroblasts and vascular smooth muscle cells, contributing to the coronary vessels (Zhang et al., 2018, Del Monte-Nieto et al., 2018, He and Zhou, 2018). Recent reports have suggested that the plasticity of the endocardium is increased after MI or other pathological challenges, and that subendocardial remodeling associated with MI reverts the subendocardial myocardium to the developmental-like hypertrabeculated state, which could enable endocardium-derived neovascularization (Miquerol et al., 2015, Dube et al., 2017). This concept and our results of VEGF-B induced endocardial neovascularization open a new direction for cardiac revascularization research.
In summary, in this study I showed that VEGF-B can be utilized for activating and redeploying endogenous developmental mechanisms of coronary vasculature development from the endocardium (Figure 4, below). To our knowledge, this organ-specific developmental transdifferentiation program is distinct from angiogenic sprouting in other developing vascular beds and organs. In possible clinical applications, VEGF-B gene therapy could be administered upon first evidence of MI, or for example to patients having an unstable coronary artery disease. The aim would be to induce de novo endocardial derived vessels for attenuation of myocardial hypoxia and prevention of any additional CMC death. This clinically relevant area for further research may provide a solution for better myocardial perfusion to injured heart directly from the cardiac ventricles. Our finding provides new
55 55 insights into cardiovascular regenerative strategies in the ischemic heart. To conclude, the study presents a novel function of VEGF-B in reprogramming of the endocardial cell fate, even in adult heart after a myocardial injury, perhaps offering a future tool for redeploying developmental mechanisms in various cardiac pathologies.
Figure 4: VEGF-B stimulates the endocardial-to-coronary EC transdifferentiation. On the left, effects of VEGF-B on the heart. On the right, microscopic demonstration of the endocardial trabeculation.
56 56 CONCLUDING REMARKS
Angiogenesis is essential for cardiac development and studies have shown its importance also in physiological cardiac hypertrophy. Nevertheless, proangiogenic therapies in the heart have not yet been able to provide significant improvement in the treatment of cardiovascular disease. Success has been limited mainly because of insufficient efficacy and inadequate vectors in clinical trials (Yla- Herttuala et al., 2017, Yla-Herttuala et al., 2007). Increased permeability and inflammation caused by VEGF-based viral vectors have posed additional problems (Lee et al., 2000). This is likely to change as we gain a better understanding of the underlying biology of angiogenic growth factors as well as of their regulation and functions, allowing us to develop more targeted and steerable vectors.
The functions of VEGF-B in the heart have been shown as cardioprotective by multiple international research groups in the last years. In accordance with these, the first study of this thesis showed that the myocardial overexpression of VEGF-B induces a significant coronary expansion and induces mild physiological cardiac hypertrophy. VEGF-B protected the hearts against myocardial ischemia and the following reperfusion injury. Importantly, AAV-VEGF-B reproduced a similar phenotype, confirming its translational potential.
The second study of my thesis concentrated on bone marrow kinase X, a tyrosine kinase that is linked to the VEGF family, as it is a downstream signaling molecule of VEGFRs. We showed, that the deletion of Bmx can attenuate the development of pathological cardiac hypertrophy. We were also able to confirm that the tyrosine kinase activity of the Bmx is needed for the development of pathological cardiac hypertrophy, and that the effects were mediated from endothelial cells expressing Bmx to cardiomyocytes. This opens a possibility for the treatment of pathological cardiac hypertrophy using Bmx tyrosine kinase inhibitors, which should be further studied in pre-clinical and clinical settings.
In the third study, I studied the cardiac effects of a known cardiotoxic drug, doxorubicin. I demonstrated that DOX induced cardiac atrophy, DNA damage and mitochondrial damage. Promisingly, a pre-treatment using the AAV-VEGF-B inhibited the atrophy and damage, confirming VEGF-B’s cardioprotective qualities against DOX-induced cardiotoxicity. The cardiotoxic changes in the heart occur rather slowly so a gene treatment prior to the elective cytotoxic treatment could be well planned. This study is also the first to show that by protecting the cardiac endothelium and its
57 57 function, the DOX-induced cardiotoxicity can be efficiently treated. Subsequent studies support the importance of protecting the endothelium in cardiotoxic damage (Luu et al., 2018).
In the last study, I observed a striking novel function for VEGF-B, as it induced the growth of myocardial vessels from the endocardium and the cardiac ventricles. Translationally interestingly, I also showed that in MI, VEGF-B gene therapy can reactivate the same endogenous developmental process and reprogram endocardial cells to convert into coronary ECs, and thus provide reperfusion of the ischemic myocardium directly from the cardiac ventricles.
Overall our studies show that an excess of VEGF-B produced by a transgene leads to coronary expansion, non-pathological hypertrophy, metabolic reprogramming and ischemia resistance in the heart. I discovered that the VEGF-B transgene greatly enhances the developmental contribution of endocardium to coronary vascularization. I showed that this could be mimicked by AAV-mediated VEGF-B gene expression upon the tissue remodeling that is associated with the subendocardium during myocardial infarction. VEGF-B gene delivery to adult heart protected the myocardium also from the cumulative toxic effects of anthracycline cytostatics. I furthermore demonstrated that, downstream of the VEGF-B receptor VEGFR-1, tyrosine kinase activity of the Bmx signal transducer is needed for pathological cardiac hypertrophy.
VEGF-B fulfills many of the desired features for a vascular perfusion-enhancing agent (Figure 5). It has been shown to protect from microvascular defects generated during cardiac stress (Woitek et al., 2015, Kivela et al., 2014), which is an important aspect for avoidance of the no-flow phenomenon and successful cardiac reperfusion (Smart, 2017). Long-term VEGF-B expression also induces vessel arterialization (Kivela et al., 2014, Bry et al., 2010), which is crucial for pressurized blood delivery into the cardiac muscle. Furthermore, a clear advantage of VEGF-B in comparison to other angiogenic factors is that it cannot be overdosed, as an excess of VEGF-B merely works by binding to the VEGFR-1 decoy receptor, thereby displacing the endogenous VEGF to induce angiogenesis through the VEGFR-2-signaling pathway (Robciuc et al., 2016, Kivela et al., 2019a).
58 58 "#% (! & ! "
% ! % ! ! !! ! ! % %* ' ! $ ' +&+" " ' ("$( & ! !! ) + ' " ' %# $ +&+" " ' ("$( & #% +%$+" " ' ! ! !!% & '# ! %* ' " ## ! %$ ' ' %$" " ' "' " ' !! ' ")(# ' ) + '
Figure 5: Effects of VEGF-B overexpression in cardiac cells. Schematic view of the subendocardial myocardium of the heart. FB=fibroblast/connective tissue layer under the endocardium, PC=pericyte, CMC=cardiomyocyte, EC=endothelial cell.
All in all, VEGF-B has an unredeemed potential as a future gene therapeutic use to treat cardiac ischemia, cardiotoxicity and congestive heart failure. Data from our group suggest that circulating levels of VEGF-B are not sufficient to induce and maintain the cardiac hypertrophy, but it needs to be expressed by CMCs. In addition, previous experiments using VEGF-B protein instead of a viral- vector mediated expression have not turned out particularly successful, except a study showing enhanced myocardial revascularization using VEGF-B167 protein from osmotic minipumps (Li et al., 2008a). Therefore, the actual therapeutic approach for VEGF-B has to be carefully considered and a detailed understanding of its molecular way of action and downstream signaling molecules, like Bmx, enable even more targeted vectors for therapeutic use. A systemic untargeted genetic treatment may also induce unwanted effects elsewhere in the body, so future studies need to aim at a more targeted
59 59 approach. This could include an inducible switch-on/off mechanism for the vector, for example by a drug ligand or by using an optogenetic approach.
60 60 ACKNOWLEDGEMENTS
The studies were carried out during the years 2013-2019 in the Wihuri Research Institute and Translational Cancer Medicine Laboratory, both located within the excellent research facilities of the Research Programs Unit, Biomedicum Helsinki.
I would like to convey my deepest gratitude to my supervisor and mentor Kari Alitalo. Firstly, for the opportunity to work in his world-class research group of outstanding scientists and high-quality laboratory facilities. Kari is the first person to teach me what true commitment to passion is, and how hard I must work if I want to aim high and contribute to human understanding of biology. Secondly, I want to thank Kari for his guidance and teaching on how to plan, execute and report in a professional, scientific manner.
Riikka Kivelä, my second supervisor, has been invaluable to me throughout my PhD studies. In the beginning she was a hands-on teacher; as my confidence built in the techniques, she has been a constant guiding force. What I value most is that Riikka never stops encouraging young scientists, and there is nothing too stupid or too unimportant that I could have asked her. I never felt stranded, knowing she was there to help.
I am indebted to Heikki Ruskoaho and Christian Kupatt for their thorough and professional review of my thesis, and I am grateful for the subsequent improvement of my thesis following their valuable comments. I wish to express my gratitude to Associate professor Kristy Red-Horse from Stanford University for agreeing to act as my opponent and Professor Juha Sinisalo for promising to be the custos at my public examination. I am also grateful to the Helsinki Biomedical Graduate Program and its faculty for the funding and opportunities provided by the graduate school. I want to thank the many wonderful collaborators I have had the chance to work with over the years, especially Eero Mervaala, Juha Hulmi, Christer Betsholtz, Bin Zhou and Seppo Ylä-Herttuala.
I would like to acknowledge the support and companionship of all present and past members of the Alitalo lab and neighboring labs. Maija B., Joni, Tanja H., Andrey, Ibrahim, Jennifer, Karthik and Emma are acknowledged for their important contributions to various aspects of the VEGF-B project. Special thanks go to Ibrahim and Pikku-Markus for their wonderful friendship. Additionally, I want to give warm thanks to Olli Ritvos for being my mentor in the field of science; advising and encouraging me through multiple challenges.
61 61 Thanks to Tapio for being the one who can make the lab run so smooth and for always being available to help. Maria has been my “lab-mom” throughout the years, thank you for that. I am also grateful to Katja S., Kirsi, Tanja L., Mari, Maija, Laura, and Päivi for their professional assistance, as well as to the expert staff at the Biomedicum Imaging Unit and Meilahti and Viikki animal facilities. Thanks to Kaisa S., Elina P. and Saija P. for their administrative help.
My friends and colleagues in Finland, Germany and in Switzerland, Marie Curie fellows, friends from the Finnish cardiac society, high school friends, EpioMed guys, you all have been valuable during this journey. My aunt Paula, uncles Erkki and Pentti and my dear godchild Lauri and his family are also acknowledged. To Teemu, I profess my gratitude, for being the closest thing to a brother and for his constant support. I would like to express my appreciation and gratitude for Anna for her support and presence during a major part of this journey. Special thanks goes to Amanda for sharing a short yet crucial part of this journey. Thank you for caring and helping in innumerable ways.
I have been financially supported by grants from the Biomedicum Helsinki Foundation, Finnish Foundation for Cardiovascular Research, Finnish Medical Foundation, Emil Aaltonen Foundation, Finnish Cardiac Society, Paolo Foundation, Ida Montin Foundation, Aarne Koskelo Foundation, Paavo Nurmi Foundation, and the Aarne and Aili Turunen Foundation, which are sincerely acknowledged.
I dedicate this thesis to my parents, whom I cannot thank enough for their support. When I was a young boy, my father, following the defence of his own thesis, gave me a scroll. The scroll was addressed to “doctor in spe”. Now, many years later, the dreams of a young boy inspired by the words of his father seem to be coming true. The lifelong support of both of my parents means the world to me.
Helsinki, September 2019
62 62 REFERENCES
AASA, K. L., ZAVAN, B., LUNA, R. L., WONG, P. G., VENTURA, N. M., TSE, M. Y., CARMELIET, P., ADAMS, M. A., PANG, S. C. & CROY, B. A. 2015. Placental growth factor influences maternal cardiovascular adaptation to pregnancy in mice. Biol Reprod, 92, 44. AASE, K., VON EULER, G., LI, X., PONTEN, A., THOREN, P., CAO, R., CAO, Y., OLOFSSON, B., GEBRE-MEDHIN, S., PEKNY, M., ALITALO, K., BETSHOLTZ, C. & ERIKSSON, U. 2001. Vascular endothelial growth factor-B-deficient mice display an atrial conduction defect. Circulation, 104, 358-64. ABRAHAM, D., TAGHAVI, S., RIML, P., PAULUS, P., HOFMANN, M., BAUMANN, C., KOCHER, A., KLEPETKO, W. & AHARINEJAD, S. 2002. VEGF-A and -C but not -B mediate increased vascular permeability in preserved lung grafts. Transplantation, 73, 1703- 6. ADAMS, R. H. & ALITALO, K. 2007. Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol, 8, 464-78. ALBRECHT, I., KOPFSTEIN, L., STRITTMATTER, K., SCHOMBER, T., FALKEVALL, A., HAGBERG, C. E., LORENTZ, P., JELTSCH, M., ALITALO, K., ERIKSSON, U., CHRISTOFORI, G. & PIETRAS, K. 2010. Suppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis. PLoS One, 5, e14109. ALTIERI, D. C. & MARCHISIO, P. C. 1999. Survivin apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest, 79, 1327-33. ANISIMOV, A., ALITALO, A., KORPISALO, P., SORONEN, J., KAIJALAINEN, S., LEPPANEN, V. M., JELTSCH, M., YLA-HERTTUALA, S. & ALITALO, K. 2009. Activated forms of VEGF-C and VEGF-D provide improved vascular function in skeletal muscle. Circ Res, 104, 1302-12. ANISIMOV, A., LEPPANEN, V. M., TVOROGOV, D., ZARKADA, G., JELTSCH, M., HOLOPAINEN, T., KAIJALAINEN, S. & ALITALO, K. 2013. The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands. Sci Signal, 6, ra52. ANTOS, C. L., MCKINSEY, T. A., FREY, N., KUTSCHKE, W., MCANALLY, J., SHELTON, J. M., RICHARDSON, J. A., HILL, J. A. & OLSON, E. N. 2002. Activated glycogen synthase-3 beta suppresses cardiac hypertrophy in vivo. Proc Natl Acad Sci U S A, 99, 907- 12. ARCAMONE, F., CASSINELLI, G., FANTINI, G., GREIN, A., OREZZI, P., POL, C. & SPALLA, C. 1969. Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius. Biotechnol Bioeng, 11, 1101-10. ARJUNAN, P., LIN, X., TANG, Z., DU, Y., KUMAR, A., LIU, L., YIN, X., HUANG, L., CHEN, W., CHEN, Q., YE, Z., WANG, S., KUANG, H., ZHOU, L., XU, K., CHEN, X., ZENG, H., LU, W., CAO, Y., LIU, Y., ZHAO, C. & LI, X. 2018. VEGF-B is a potent antioxidant. Proc Natl Acad Sci U S A, 115, 10351-10356. ASOKAN, A., SCHAFFER, D. V. & SAMULSKI, R. J. 2012. The AAV vector toolkit: poised at the clinical crossroads. Mol Ther, 20, 699-708. BAIS, C., WU, X., YAO, J., YANG, S., CRAWFORD, Y., MCCUTCHEON, K., TAN, C., KOLUMAM, G., VERNES, J. M., EASTHAM-ANDERSON, J., HAUGHNEY, P., KOWANETZ, M., HAGENBEEK, T., KASMAN, I., RESLAN, H. B., ROSS, J., VAN BRUGGEN, N., CARANO, R. A., MENG, Y. J., HONGO, J. A., STEPHAN, J. P., SHIBUYA, M. & FERRARA, N. 2010. PlGF blockade does not inhibit angiogenesis during primary tumor growth. Cell, 141, 166-77.
63 63 BANFI, A., VON DEGENFELD, G., GIANNI-BARRERA, R., REGINATO, S., MERCHANT, M. J., MCDONALD, D. M. & BLAU, H. M. 2012. Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB. FASEB J, 26, 2486-97. BARTUNEK, J., BEHFAR, A., DOLATABADI, D., VANDERHEYDEN, M., OSTOJIC, M., DENS, J., EL NAKADI, B., BANOVIC, M., BELESLIN, B., VROLIX, M., LEGRAND, V., VRINTS, C., VANOVERSCHELDE, J. L., CRESPO-DIAZ, R., HOMSY, C., TENDERA, M., WALDMAN, S., WIJNS, W. & TERZIC, A. 2013. Cardiopoietic stem cell therapy in heart failure: the C-CURE (Cardiopoietic stem Cell therapy in heart failURE) multicenter randomized trial with lineage-specified biologics. J Am Coll Cardiol, 61, 2329- 38. BEARZI, C., ROTA, M., HOSODA, T., TILLMANNS, J., NASCIMBENE, A., DE ANGELIS, A., YASUZAWA-AMANO, S., TROFIMOVA, I., SIGGINS, R. W., LECAPITAINE, N., CASCAPERA, S., BELTRAMI, A. P., D'ALESSANDRO, D. A., ZIAS, E., QUAINI, F., URBANEK, K., MICHLER, R. E., BOLLI, R., KAJSTURA, J., LERI, A. & ANVERSA, P. 2007. Human cardiac stem cells. Proc Natl Acad Sci U S A, 104, 14068-73. BELLOMO, D., HEADRICK, J. P., SILINS, G. U., PATERSON, C. A., THOMAS, P. S., GARTSIDE, M., MOULD, A., CAHILL, M. M., TONKS, I. D., GRIMMOND, S. M., TOWNSON, S., WELLS, C., LITTLE, M., CUMMINGS, M. C., HAYWARD, N. K. & KAY, G. F. 2000. Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia. Circ Res, 86, E29-E35. BENNETT, M. R., SINHA, S. & OWENS, G. K. 2016. Vascular Smooth Muscle Cells in Atherosclerosis. Circ Res, 118, 692-702. BERTERO, A. & MURRY, C. E. 2018. Hallmarks of cardiac regeneration. Nat Rev Cardiol, 15, 579-580. BHARDWAJ, S., ROY, H., GRUCHALA, M., VIITA, H., KHOLOVA, I., KOKINA, I., ACHEN, M. G., STACKER, S. A., HEDMAN, M., ALITALO, K. & YLA-HERTTUALA, S. 2003. Angiogenic responses of vascular endothelial growth factors in periadventitial tissue. Hum Gene Ther, 14, 1451-62. BOUCHER, J. M., CLARK, R. P., CHONG, D. C., CITRIN, K. M., WYLIE, L. A. & BAUTCH, V. L. 2017. Dynamic alterations in decoy VEGF receptor-1 stability regulate angiogenesis. Nat Commun, 8, 15699. BRAKENHIELM, E. & ALITALO, K. 2019. Cardiac lymphatics in health and disease. Nat Rev Cardiol, 16, 56-68. BRKOVIC, A. & SIROIS, M. G. 2007. Vascular permeability induced by VEGF family members in vivo: role of endogenous PAF and NO synthesis. J Cell Biochem, 100, 727-37. BRUTSAERT, D. L. 2003. Cardiac endothelial-myocardial signaling: its role in cardiac growth, contractile performance, and rhythmicity. Physiol Rev, 83, 59-115. BRY, M., KIVELA, R., HOLOPAINEN, T., ANISIMOV, A., TAMMELA, T., SORONEN, J., SILVOLA, J., SARASTE, A., JELTSCH, M., KORPISALO, P., CARMELIET, P., LEMSTROM, K. B., SHIBUYA, M., YLA-HERTTUALA, S., ALHONEN, L., MERVAALA, E., ANDERSSON, L. C., KNUUTI, J. & ALITALO, K. 2010. Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation. Circulation, 122, 1725-33. BRY, M., KIVELA, R., LEPPANEN, V. M. & ALITALO, K. 2014. Vascular endothelial growth factor-B in physiology and disease. Physiol Rev, 94, 779-94. BUENO, O. F., DE WINDT, L. J., TYMITZ, K. M., WITT, S. A., KIMBALL, T. R., KLEVITSKY, R., HEWETT, T. E., JONES, S. P., LEFER, D. J., PENG, C. F., KITSIS, R. N. & MOLKENTIN, J. D. 2000. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J, 19, 6341-50.
64 64 BURRIDGE, P. W., LI, Y. F., MATSA, E., WU, H., ONG, S. G., SHARMA, A., HOLMSTROM, A., CHANG, A. C., CORONADO, M. J., EBERT, A. D., KNOWLES, J. W., TELLI, M. L., WITTELES, R. M., BLAU, H. M., BERNSTEIN, D., ALTMAN, R. B. & WU, J. C. 2016. Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity. Nat Med, 22, 547-56. BUTLER, A., HOFFMAN, P., SMIBERT, P., PAPALEXI, E. & SATIJA, R. 2018. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol, 36, 411-420. CAI, C. L., MARTIN, J. C., SUN, Y., CUI, L., WANG, L., OUYANG, K., YANG, L., BU, L., LIANG, X., ZHANG, X., STALLCUP, W. B., DENTON, C. P., MCCULLOCH, A., CHEN, J. & EVANS, S. M. 2008. A myocardial lineage derives from Tbx18 epicardial cells. Nature, 454, 104-8. CAMICI, P. G. & CREA, F. 2007. Coronary microvascular dysfunction. N Engl J Med, 356, 830- 40. CANO, E., CARMONA, R., RUIZ-VILLALBA, A., ROJAS, A., CHAU, Y. Y., WAGNER, K. D., WAGNER, N., HASTIE, N. D., MUNOZ-CHAPULI, R. & PEREZ-POMARES, J. M. 2016. Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio-venous connections. Proc Natl Acad Sci U S A, 113, 656-61. CAO, Y., JI, W. R., QI, P., ROSIN, A. & CAO, Y. 1997. Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing. Biochem Biophys Res Commun, 235, 493-8. CAO, Y., LINDEN, P., SHIMA, D., BROWNE, F. & FOLKMAN, J. 1996. In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor. J Clin Invest, 98, 2507-11. CARMELIET, P., FERREIRA, V., BREIER, G., POLLEFEYT, S., KIECKENS, L., GERTSENSTEIN, M., FAHRIG, M., VANDENHOECK, A., HARPAL, K., EBERHARDT, C., DECLERCQ, C., PAWLING, J., MOONS, L., COLLEN, D., RISAU, W. & NAGY, A. 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature, 380, 435-9. CARMELIET, P., MOONS, L., LUTTUN, A., VINCENTI, V., COMPERNOLLE, V., DE MOL, M., WU, Y., BONO, F., DEVY, L., BECK, H., SCHOLZ, D., ACKER, T., DIPALMA, T., DEWERCHIN, M., NOEL, A., STALMANS, I., BARRA, A., BLACHER, S., VANDENDRIESSCHE, T., PONTEN, A., ERIKSSON, U., PLATE, K. H., FOIDART, J. M., SCHAPER, W., CHARNOCK-JONES, D. S., HICKLIN, D. J., HERBERT, J. M., COLLEN, D. & PERSICO, M. G. 2001. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med, 7, 575-83. CHAU, K., HENNESSY, A. & MAKRIS, A. 2017. Placental growth factor and pre-eclampsia. J Hum Hypertens, 31, 782-786. CHEN, C. W., OKADA, M., PROTO, J. D., GAO, X., SEKIYA, N., BECKMAN, S. A., CORSELLI, M., CRISAN, M., SAPAROV, A., TOBITA, K., PEAULT, B. & HUARD, J. 2013a. Human pericytes for ischemic heart repair. Stem Cells, 31, 305-16. CHEN, D. & SIMONS, M. 2018. Reprogramming the Endocardium: Trials and Tribulations. Circ Res, 122, 913-915. CHEN, H., CHEDOTAL, A., HE, Z., GOODMAN, C. S. & TESSIER-LAVIGNE, M. 1997. Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron, 19, 547-59. CHEN, H. I., PODURI, A., NUMI, H., KIVELA, R., SAHARINEN, P., MCKAY, A. S., RAFTREY, B., CHURKO, J., TIAN, X., ZHOU, B., WU, J. C., ALITALO, K. & RED-
65 65 HORSE, K. 2014a. VEGF-C and aortic cardiomyocytes guide coronary artery stem development. J Clin Invest, 124, 4899-914. CHEN, H. I., SHARMA, B., AKERBERG, B. N., NUMI, H. J., KIVELA, R., SAHARINEN, P., AGHAJANIAN, H., MCKAY, A. S., BOGARD, P. E., CHANG, A. H., JACOBS, A. H., EPSTEIN, J. A., STANKUNAS, K., ALITALO, K. & RED-HORSE, K. 2014b. The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis. Development, 141, 4500-12. CHEN, R., LEE, C., LIN, X., ZHAO, C. & LI, X. 2019. Novel function of VEGF-B as an antioxidant and therapeutic implications. Pharmacol Res, 143, 33-39. CHEN, S., CAI, C., SOWALSKY, A. G., YE, H., MA, F., YUAN, X., SIMON, N. I., GRAY, N. S. & BALK, S. P. 2018. BMX-Mediated Regulation of Multiple Tyrosine Kinases Contributes to Castration Resistance in Prostate Cancer. Cancer Res, 78, 5203-5215. CHEN, S., JIANG, X., GEWINNER, C. A., ASARA, J. M., SIMON, N. I., CAI, C., CANTLEY, L. C. & BALK, S. P. 2013b. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases. Sci Signal, 6, ra40. CONDORELLI, G., DRUSCO, A., STASSI, G., BELLACOSA, A., RONCARATI, R., IACCARINO, G., RUSSO, M. A., GU, Y., DALTON, N., CHUNG, C., LATRONICO, M. V., NAPOLI, C., SADOSHIMA, J., CROCE, C. M. & ROSS, J., JR. 2002. Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice. Proc Natl Acad Sci U S A, 99, 12333-8. CROSSMAN, D. C. 2004. The pathophysiology of myocardial ischaemia. Heart, 90, 576-80. D'AMATO, G., LUXAN, G., DEL MONTE-NIETO, G., MARTINEZ-POVEDA, B., TORROJA, C., WALTER, W., BOCHTER, M. S., BENEDITO, R., COLE, S., MARTINEZ, F., HADJANTONAKIS, A. K., UEMURA, A., JIMENEZ-BORREGUERO, L. J. & DE LA POMPA, J. L. 2016. Sequential Notch activation regulates ventricular chamber development. Nat Cell Biol, 18, 7-20. DE FALCO, S. 2012. The discovery of placenta growth factor and its biological activity. Exp Mol Med, 44, 1-9. DEL MONTE-NIETO, G., RAMIALISON, M., ADAM, A. A. S., WU, B., AHARONOV, A., D'UVA, G., BOURKE, L. M., PITULESCU, M. E., CHEN, H., DE LA POMPA, J. L., SHOU, W., ADAMS, R. H., HARTEN, S. K., TZAHOR, E., ZHOU, B. & HARVEY, R. P. 2018. Control of cardiac jelly dynamics by NOTCH1 and NRG1 defines the building plan for trabeculation. Nature, 557, 439-445. DETMAR, M., BROWN, L. F., SCHON, M. P., ELICKER, B. M., VELASCO, P., RICHARD, L., FUKUMURA, D., MONSKY, W., CLAFFEY, K. P. & JAIN, R. K. 1998. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol, 111, 1-6. DEVAUX, Y., AZUAJE, F., VAUSORT, M., YVORRA, C. & WAGNER, D. R. 2010. Integrated protein network and microarray analysis to identify potential biomarkers after myocardial infarction. Funct Integr Genomics, 10, 329-37. DEVAUX, Y., VAUSORT, M., AZUAJE, F., VAILLANT, M., LAIR, M. L., GAYAT, E., LASSUS, J., NG, L. L., KELLY, D., WAGNER, D. R. & SQUIRE, I. B. 2012. Low levels of vascular endothelial growth factor B predict left ventricular remodeling after acute myocardial infarction. J Card Fail, 18, 330-7. DIJKSTRA, M. H., PIRINEN, E., HUUSKO, J., KIVELA, R., SCHENKWEIN, D., ALITALO, K. & YLA-HERTTUALA, S. 2014. Lack of cardiac and high-fat diet induced metabolic phenotypes in two independent strains of Vegf-b knockout mice. Sci Rep, 4, 6238. DING, B. S., CAO, Z., LIS, R., NOLAN, D. J., GUO, P., SIMONS, M., PENFOLD, M. E., SHIDO, K., RABBANY, S. Y. & RAFII, S. 2014. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis. Nature, 505, 97-102.
66 66 DING, B. S., NOLAN, D. J., GUO, P., BABAZADEH, A. O., CAO, Z., ROSENWAKS, Z., CRYSTAL, R. G., SIMONS, M., SATO, T. N., WORGALL, S., SHIDO, K., RABBANY, S. Y. & RAFII, S. 2011. Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell, 147, 539-53. DOR, Y., CAMENISCH, T. D., ITIN, A., FISHMAN, G. I., MCDONALD, J. A., CARMELIET, P. & KESHET, E. 2001. A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects. Development, 128, 1531-8. DOROSHOW, J. H. 1983. Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res, 43, 460-72. DRAGNEVA, G., KORPISALO, P. & YLA-HERTTUALA, S. 2013. Promoting blood vessel growth in ischemic diseases: challenges in translating preclinical potential into clinical success. Dis Model Mech, 6, 312-22. DUBE, K. N., THOMAS, T. M., MUNSHAW, S., ROHLING, M., RILEY, P. R. & SMART, N. 2017. Recapitulation of developmental mechanisms to revascularize the ischemic heart. JCI Insight, 2. EKMAN, N., ARIGHI, E., RAJANTIE, I., SAHARINEN, P., RISTIMAKI, A., SILVENNOINEN, O. & ALITALO, K. 2000. The Bmx tyrosine kinase is activated by IL-3 and G-CSF in a PI- 3K dependent manner. Oncogene, 19, 4151-8. EKMAN, N., LYMBOUSSAKI, A., VASTRIK, I., SARVAS, K., KAIPAINEN, A. & ALITALO, K. 1997. Bmx tyrosine kinase is specifically expressed in the endocardium and the endothelium of large arteries. Circulation, 96, 1729-32. ENHOLM, B., PAAVONEN, K., RISTIMAKI, A., KUMAR, V., GUNJI, Y., KLEFSTROM, J., KIVINEN, L., LAIHO, M., OLOFSSON, B., JOUKOV, V., ERIKSSON, U. & ALITALO, K. 1997. Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors, oncoproteins and hypoxia. Oncogene, 14, 2475-83. ESPOSITO, G., RAPACCIUOLO, A., NAGA PRASAD, S. V., TAKAOKA, H., THOMAS, S. A., KOCH, W. J. & ROCKMAN, H. A. 2002. Genetic alterations that inhibit in vivo pressure- overload hypertrophy prevent cardiac dysfunction despite increased wall stress. Circulation, 105, 85-92. FENG, L., REN, J., LI, Y., YANG, G., KANG, L., ZHANG, S., MA, C., LI, J., LIU, J., YANG, L. & QI, Z. 2019. Resveratrol protects against isoproterenol induced myocardial infarction in rats through VEGF-B/AMPK/eNOS/NO signalling pathway. Free Radic Res, 53, 82-93. FERRARA, N., CARVER-MOORE, K., CHEN, H., DOWD, M., LU, L., O'SHEA, K. S., POWELL-BRAXTON, L., HILLAN, K. J. & MOORE, M. W. 1996. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature, 380, 439- 42. FERRARA, N., GERBER, H. P. & LECOUTER, J. 2003. The biology of VEGF and its receptors. Nat Med, 9, 669-76. FORSYTHE, J. A., JIANG, B. H., IYER, N. V., AGANI, F., LEUNG, S. W., KOOS, R. D. & SEMENZA, G. L. 1996. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol, 16, 4604-13. FRANCO, Y. L., VAIDYA, T. R. & AIT-OUDHIA, S. 2018. Anticancer and cardio-protective effects of liposomal doxorubicin in the treatment of breast cancer. Breast Cancer (Dove Med Press), 10, 131-141. FUH, G., GARCIA, K. C. & DE VOS, A. M. 2000. The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1. J Biol Chem, 275, 26690-5. FUKUMURA, D., GOHONGI, T., KADAMBI, A., IZUMI, Y., ANG, J., YUN, C. O., BUERK, D. G., HUANG, P. L. & JAIN, R. K. 2001. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc Natl Acad Sci U S A, 98, 2604-9.
67 67 GABISONIA, K., PROSDOCIMO, G., AQUARO, G. D., CARLUCCI, L., ZENTILIN, L., SECCO, I., ALI, H., BRAGA, L., GORGODZE, N., BERNINI, F., BURCHIELLI, S., COLLESI, C., ZANDONA, L., SINAGRA, G., PIACENTI, M., ZACCHIGNA, S., BUSSANI, R., RECCHIA, F. A. & GIACCA, M. 2019. MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature, 569, 418-422. GARSIDE, V. C., CHANG, A. C., KARSAN, A. & HOODLESS, P. A. 2013. Co-ordinating Notch, BMP, and TGF-beta signaling during heart valve development. Cell Mol Life Sci, 70, 2899- 917. GEISBERG, C. A., ABDALLAH, W. M., DA SILVA, M., SILVERSTEIN, C., SMITH, H. M., ABRAMSON, V., MAYER, I., MEANS-POWELL, J., FREEHARDT, D., WHITE, B., LENIHAN, D. & SAWYER, D. B. 2013. Circulating neuregulin during the transition from stage A to stage B/C heart failure in a breast cancer cohort. J Card Fail, 19, 10-5. GIANNI-BARRERA, R., BUTSCHKAU, A., UCCELLI, A., CERTELLI, A., VALENTE, P., BARTOLOMEO, M., GROPPA, E., BURGER, M. G., HLUSHCHUK, R., HEBERER, M., SCHAEFER, D. J., GURKE, L., DJONOV, V., VOLLMAR, B. & BANFI, A. 2018. PDGF- BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation. Angiogenesis, 21, 883-900. GITTENBERGER-DE GROOT, A. C., BARTELINGS, M. M., DERUITER, M. C. & POELMANN, R. E. 2005. Basics of cardiac development for the understanding of congenital heart malformations. Pediatr Res, 57, 169-76. GLUZMAN-POLTORAK, Z., COHEN, T., HERZOG, Y. & NEUFELD, G. 2000. Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF- 165 [corrected]. J Biol Chem, 275, 18040-5. GLUZMAN-POLTORAK, Z., COHEN, T., SHIBUYA, M. & NEUFELD, G. 2001. Vascular endothelial growth factor receptor-1 and neuropilin-2 form complexes. J Biol Chem, 276, 18688-94. GOTTAR-GUILLIER, M., DODELLER, F., HUESKEN, D., IOURGENKO, V., MICKANIN, C., LABOW, M., GAVERIAUX, S., KINZEL, B., MUELLER, M., ALITALO, K., LITTLEWOOD-EVANS, A. & CENNI, B. 2011. The tyrosine kinase BMX is an essential mediator of inflammatory arthritis in a kinase-independent manner. J Immunol, 186, 6014- 23. GREGO-BESSA, J., LUNA-ZURITA, L., DEL MONTE, G., BOLOS, V., MELGAR, P., ARANDILLA, A., GARRATT, A. N., ZANG, H., MUKOUYAMA, Y. S., CHEN, H., SHOU, W., BALLESTAR, E., ESTELLER, M., ROJAS, A., PEREZ-POMARES, J. M. & DE LA POMPA, J. L. 2007. Notch signaling is essential for ventricular chamber development. Dev Cell, 12, 415-29. GRIMMOND, S., LAGERCRANTZ, J., DRINKWATER, C., SILINS, G., TOWNSON, S., POLLOCK, P., GOTLEY, D., CARSON, E., RAKAR, S., NORDENSKJOLD, M., WARD, L., HAYWARD, N. & WEBER, G. 1996. Cloning and characterization of a novel human gene related to vascular endothelial growth factor. Genome Res, 6, 124-31. GUNNINGHAM, S. P., CURRIE, M. J., HAN, C., TURNER, K., SCOTT, P. A., ROBINSON, B. A., HARRIS, A. L. & FOX, S. B. 2001. Vascular endothelial growth factor-B and vascular endothelial growth factor-C expression in renal cell carcinomas: regulation by the von Hippel-Lindau gene and hypoxia. Cancer Res, 61, 3206-11. GURYANOVA, O. A., WU, Q., CHENG, L., LATHIA, J. D., HUANG, Z., YANG, J., MACSWORDS, J., EYLER, C. E., MCLENDON, R. E., HEDDLESTON, J. M., SHOU, W., HAMBARDZUMYAN, D., LEE, J., HJELMELAND, A. B., SLOAN, A. E., BREDEL, M., STARK, G. R., RICH, J. N. & BAO, S. 2011. Nonreceptor tyrosine kinase BMX maintains self-renewal and tumorigenic potential of glioblastoma stem cells by activating STAT3. Cancer Cell, 19, 498-511.
68 68 HAGBERG, C. E., FALKEVALL, A., WANG, X., LARSSON, E., HUUSKO, J., NILSSON, I., VAN MEETEREN, L. A., SAMEN, E., LU, L., VANWILDEMEERSCH, M., KLAR, J., GENOVE, G., PIETRAS, K., STONE-ELANDER, S., CLAESSON-WELSH, L., YLA- HERTTUALA, S., LINDAHL, P. & ERIKSSON, U. 2010. Vascular endothelial growth factor B controls endothelial fatty acid uptake. Nature, 464, 917-21. HAGBERG, C. E., MEHLEM, A., FALKEVALL, A., MUHL, L., FAM, B. C., ORTSATER, H., SCOTNEY, P., NYQVIST, D., SAMEN, E., LU, L., STONE-ELANDER, S., PROIETTO, J., ANDRIKOPOULOS, S., SJOHOLM, A., NASH, A. & ERIKSSON, U. 2012. Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes. Nature, 490, 426- 30. HAJJAR, R. J. 2013. Potential of gene therapy as a treatment for heart failure. J Clin Invest, 123, 53-61. HANRAHAN, V., CURRIE, M. J., GUNNINGHAM, S. P., MORRIN, H. R., SCOTT, P. A., ROBINSON, B. A. & FOX, S. B. 2003. The angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma-carcinoma sequence during colorectal cancer progression. J Pathol, 200, 183-94. HARTIKAINEN, J., HASSINEN, I., HEDMAN, A., KIVELA, A., SARASTE, A., KNUUTI, J., HUSSO, M., MUSSALO, H., HEDMAN, M., RISSANEN, T. T., TOIVANEN, P., HEIKURA, T., WITZTUM, J. L., TSIMIKAS, S. & YLA-HERTTUALA, S. 2017. Adenoviral intramyocardial VEGF-DDeltaNDeltaC gene transfer increases myocardial perfusion reserve in refractory angina patients: a phase I/IIa study with 1-year follow-up. Eur Heart J, 38, 2547-2555. HE, L. & ZHOU, B. 2018. The Development and Regeneration of Coronary Arteries. Curr Cardiol Rep, 20, 54. HE, Y., LUO, Y., TANG, S., RAJANTIE, I., SALVEN, P., HEIL, M., ZHANG, R., LUO, D., LI, X., CHI, H., YU, J., CARMELIET, P., SCHAPER, W., SINUSAS, A. J., SESSA, W. C., ALITALO, K. & MIN, W. 2006. Critical function of Bmx/Etk in ischemia-mediated arteriogenesis and angiogenesis. J Clin Invest, 116, 2344-55. HENRI, O., POUEHE, C., HOUSSARI, M., GALAS, L., NICOL, L., EDWARDS-LEVY, F., HENRY, J. P., DUMESNIL, A., BOUKHALFA, I., BANQUET, S., SCHAPMAN, D., THUILLEZ, C., RICHARD, V., MULDER, P. & BRAKENHIELM, E. 2016. Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction. Circulation, 133, 1484-97; discussion 1497. HIDEG, K. & KALAI, T. 2007. Novel antioxidants in anthracycline cardiotoxicity. Cardiovasc Toxicol, 7, 160-4. HILL, J. A., ROTHERMEL, B., YOO, K. D., CABUAY, B., DEMETROULIS, E., WEISS, R. M., KUTSCHKE, W., BASSEL-DUBY, R. & WILLIAMS, R. S. 2002. Targeted inhibition of calcineurin in pressure-overload cardiac hypertrophy. Preservation of systolic function. J Biol Chem, 277, 10251-5. HINKEL, R., LANGE, P., PETERSEN, B., GOTTLIEB, E., NG, J. K., FINGER, S., HORSTKOTTE, J., LEE, S., THORMANN, M., KNORR, M., EL-AOUNI, C., BOEKSTEGERS, P., REICHART, B., WENZEL, P., NIEMANN, H. & KUPATT, C. 2015. Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model. J Am Coll Cardiol, 66, 154-65. HO, V. C., DUAN, L. J., CRONIN, C., LIANG, B. T. & FONG, G. H. 2012. Elevated vascular endothelial growth factor receptor-2 abundance contributes to increased angiogenesis in vascular endothelial growth factor receptor-1-deficient mice. Circulation, 126, 741-52. HOFFMAN, J. I. 1987. Transmural myocardial perfusion. Prog Cardiovasc Dis, 29, 429-64.
69 69 HOLOPAINEN, T., LOPEZ-ALPUCHE, V., ZHENG, W., HELJASVAARA, R., JONES, D., HE, Y., TVOROGOV, D., D'AMICO, G., WIENER, Z., ANDERSSON, L. C., PIHLAJANIEMI, T., MIN, W. & ALITALO, K. 2012. Deletion of the endothelial Bmx tyrosine kinase decreases tumor angiogenesis and growth. Cancer Res, 72, 3512-21. HORVATH, K. A. 2008. Transmyocardial laser revascularization. J Card Surg, 23, 266-76. HSIEH, P. C., DAVIS, M. E., LISOWSKI, L. K. & LEE, R. T. 2006. Endothelial-cardiomyocyte interactions in cardiac development and repair. Annu Rev Physiol, 68, 51-66. HU, J., SRIVASTAVA, K., WIELAND, M., RUNGE, A., MOGLER, C., BESEMFELDER, E., TERHARDT, D., VOGEL, M. J., CAO, L., KORN, C., BARTELS, S., THOMAS, M. & AUGUSTIN, H. G. 2014. Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat. Science, 343, 416-9. HUDLICKA, O., BROWN, M. & EGGINTON, S. 1992. Angiogenesis in skeletal and cardiac muscle. Physiol Rev, 72, 369-417. HUGHES, G. C., LOWE, J. E., KYPSON, A. P., ST LOUIS, J. D., PIPPEN, A. M., PETERS, K. G., COLEMAN, R. E., DEGRADO, T. R., DONOVAN, C. L., ANNEX, B. H. & LANDOLFO, K. P. 1998. Neovascularization after transmyocardial laser revascularization in a model of chronic ischemia. Ann Thorac Surg, 66, 2029-36. HULOT, J. S., ISHIKAWA, K. & HAJJAR, R. J. 2016. Gene therapy for the treatment of heart failure: promise postponed. Eur Heart J, 37, 1651-8. HUNTER, J. J. & CHIEN, K. R. 1999. Signaling pathways for cardiac hypertrophy and failure. N Engl J Med, 341, 1276-83. HUNTER, S. & ROBSON, S. C. 1992. Adaptation of the maternal heart in pregnancy. Br Heart J, 68, 540-3. HUUSKO, J., LOTTONEN, L., MERENTIE, M., GURZELER, E., ANISIMOV, A., MIYANOHARA, A., ALITALO, K., TAVI, P. & YLA-HERTTUALA, S. 2012. AAV9- mediated VEGF-B gene transfer improves systolic function in progressive left ventricular hypertrophy. Mol Ther, 20, 2212-21. HUUSKO, J., MERENTIE, M., DIJKSTRA, M. H., RYHANEN, M. M., KARVINEN, H., RISSANEN, T. T., VANWILDEMEERSCH, M., HEDMAN, M., LIPPONEN, J., HEINONEN, S. E., ERIKSSON, U., SHIBUYA, M. & YLA-HERTTUALA, S. 2010. The effects of VEGF-R1 and VEGF-R2 ligands on angiogenic responses and left ventricular function in mice. Cardiovasc Res, 86, 122-30. ICHIKAWA, Y., GHANEFAR, M., BAYEVA, M., WU, R., KHECHADURI, A., NAGA PRASAD, S. V., MUTHARASAN, R. K., NAIK, T. J. & ARDEHALI, H. 2014. Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. J Clin Invest, 124, 617-30. ISHIKAWA, K. & HAJJAR, R. J. 2015. Revisiting Old Players in the Revitalized Field of Cardiovascular Gene Therapy. J Am Coll Cardiol, 66, 166-8. ISNER, J. M., PIECZEK, A., SCHAINFELD, R., BLAIR, R., HALEY, L., ASAHARA, T., ROSENFIELD, K., RAZVI, S., WALSH, K. & SYMES, J. F. 1996. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet, 348, 370-4. JABA, I. M., ZHUANG, Z. W., LI, N., JIANG, Y., MARTIN, K. A., SINUSAS, A. J., PAPADEMETRIS, X., SIMONS, M., SESSA, W. C., YOUNG, L. H. & TIRZIU, D. 2013. NO triggers RGS4 degradation to coordinate angiogenesis and cardiomyocyte growth. J Clin Invest, 123, 1718-31. JELTSCH, M., KAIPAINEN, A., JOUKOV, V., MENG, X., LAKSO, M., RAUVALA, H., SWARTZ, M., FUKUMURA, D., JAIN, R. K. & ALITALO, K. 1997. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science, 276, 1423-5.
70 70 JOUKOV, V., SORSA, T., KUMAR, V., JELTSCH, M., CLAESSON-WELSH, L., CAO, Y., SAKSELA, O., KALKKINEN, N. & ALITALO, K. 1997. Proteolytic processing regulates receptor specificity and activity of VEGF-C. EMBO J, 16, 3898-911. KAMO, T., AKAZAWA, H. & KOMURO, I. 2015. Cardiac nonmyocytes in the hub of cardiac hypertrophy. Circ Res, 117, 89-98. KARAMAN, S., LEPPANEN, V. M. & ALITALO, K. 2018. Vascular endothelial growth factor signaling in development and disease. Development, 145. KARPANEN, T., BRY, M., OLLILA, H. M., SEPPANEN-LAAKSO, T., LIIMATTA, E., LESKINEN, H., KIVELA, R., HELKAMAA, T., MERENTIE, M., JELTSCH, M., PAAVONEN, K., ANDERSSON, L. C., MERVAALA, E., HASSINEN, I. E., YLA- HERTTUALA, S., ORESIC, M. & ALITALO, K. 2008. Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy. Circ Res, 103, 1018-26. KATZ, T. C., SINGH, M. K., DEGENHARDT, K., RIVERA-FELICIANO, J., JOHNSON, R. L., EPSTEIN, J. A. & TABIN, C. J. 2012. Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells. Dev Cell, 22, 639-50. KAWASAKI, T., KITSUKAWA, T., BEKKU, Y., MATSUDA, Y., SANBO, M., YAGI, T. & FUJISAWA, H. 1999. A requirement for neuropilin-1 in embryonic vessel formation. Development, 126, 4895-902. KERKELA, R., WOULFE, K. & FORCE, T. 2007. Glycogen synthase kinase-3beta -- actively inhibiting hypertrophy. Trends Cardiovasc Med, 17, 91-6. KIVELÄ, R. 2019. Endothelial cells regulate physiological cardiomyocyte growth via VEGFR2 - mediated paracrine signaling. Circulation, In Press. KIVELA, R., BRY, M., ROBCIUC, M. R., RASANEN, M., TAAVITSAINEN, M., SILVOLA, J. M., SARASTE, A., HULMI, J. J., ANISIMOV, A., MAYRANPAA, M. I., LINDEMAN, J. H., EKLUND, L., HELLBERG, S., HLUSHCHUK, R., ZHUANG, Z. W., SIMONS, M., DJONOV, V., KNUUTI, J., MERVAALA, E. & ALITALO, K. 2014. VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart. EMBO Mol Med, 6, 307-21. KIVELA, R., HEMANTHAKUMAR, K., VAPARANTA, K., ROBCIUC, M. R., IZUMIYA, Y., KIDOYA, H., TAKAKURA, N., PENG, X., SAWYER, D., ELENIUS, K., WALSH, K. & ALITALO, K. 2019a. Endothelial cells regulate physiological cardiomyocyte growth via VEGFR2 -mediated paracrine signaling. Circulation, In Press. KIVELA, R., HEMANTHAKUMAR, K. A., VAPARANTA, K., ROBCIUC, M., IZUMIYA, Y., KIDOYA, H., TAKAKURA, N., PENG, X., SAWYER, D. B., ELENIUS, K., WALSH, K. & ALITALO, K. 2019b. Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2 -Mediated Paracrine Signaling. Circulation. KLOTZ, L., NORMAN, S., VIEIRA, J. M., MASTERS, M., ROHLING, M., DUBE, K. N., BOLLINI, S., MATSUZAKI, F., CARR, C. A. & RILEY, P. R. 2015. Cardiac lymphatics are heterogeneous in origin and respond to injury. Nature, 522, 62-7. KOCH, S. & CLAESSON-WELSH, L. 2012. Signal transduction by vascular endothelial growth factor receptors. Cold Spring Harb Perspect Med, 2, a006502. KOLODKIN, A. L., LEVENGOOD, D. V., ROWE, E. G., TAI, Y. T., GIGER, R. J. & GINTY, D. D. 1997. Neuropilin is a semaphorin III receptor. Cell, 90, 753-62. KOLWICZ, S. C., JR. & TIAN, R. 2011. Glucose metabolism and cardiac hypertrophy. Cardiovasc Res, 90, 194-201. KONG, P., CHRISTIA, P. & FRANGOGIANNIS, N. G. 2014. The pathogenesis of cardiac fibrosis. Cell Mol Life Sci, 71, 549-74.
71 71 KOUTSOUKIS, A., NTALIANIS, A., REPASOS, E., KASTRITIS, E., DIMOPOULOS, M. A. & PARASKEVAIDIS, I. 2018. Cardio-oncology: A Focus on Cardiotoxicity. Eur Cardiol, 13, 64-69. KUPATT, C., HINKEL, R., PFOSSER, A., EL-AOUNI, C., WUCHRER, A., FRITZ, A., GLOBISCH, F., THORMANN, M., HORSTKOTTE, J., LEBHERZ, C., THEIN, E., BANFI, A. & BOEKSTEGERS, P. 2010. Cotransfection of vascular endothelial growth factor-A and platelet-derived growth factor-B via recombinant adeno-associated virus resolves chronic ischemic malperfusion role of vessel maturation. J Am Coll Cardiol, 56, 414-22. KUROTSU, S., OSAKABE, R., ISOMI, M., TAMURA, F., SADAHIRO, T., MURAOKA, N., KOJIMA, H., HAGINIWA, S., TANI, H., NARA, K., KUBOTA, Y., EMA, M., FUKUDA, K., SUZUKI, T. & IEDA, M. 2017. Distinct expression patterns of Flk1 and Flt1 in the coronary vascular system during development and after myocardial infarction. Biochem Biophys Res Commun. LA MANNO, G., SOLDATOV, R., ZEISEL, A., BRAUN, E., HOCHGERNER, H., PETUKHOV, V., LIDSCHREIBER, K., KASTRITI, M. E., LONNERBERG, P., FURLAN, A., FAN, J., BORM, L. E., LIU, Z., VAN BRUGGEN, D., GUO, J., HE, X., BARKER, R., SUNDSTROM, E., CASTELO-BRANCO, G., CRAMER, P., ADAMEYKO, I., LINNARSSON, S. & KHARCHENKO, P. V. 2018. RNA velocity of single cells. Nature, 560, 494-498. LAFLAMME, M. A. & MURRY, C. E. 2011. Heart regeneration. Nature, 473, 326-35. LAHTEENVUO, J. & YLA-HERTTUALA, S. 2017. Advances and Challenges in Cardiovascular Gene Therapy. Hum Gene Ther, 28, 1024-1032. LAHTEENVUO, J. E., LAHTEENVUO, M. T., KIVELA, A., ROSENLEW, C., FALKEVALL, A., KLAR, J., HEIKURA, T., RISSANEN, T. T., VAHAKANGAS, E., KORPISALO, P., ENHOLM, B., CARMELIET, P., ALITALO, K., ERIKSSON, U. & YLA-HERTTUALA, S. 2009. Vascular endothelial growth factor-B induces myocardium-specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor-1- and neuropilin receptor-1-dependent mechanisms. Circulation, 119, 845-56. LAI, D., LIU, X., FORRAI, A., WOLSTEIN, O., MICHALICEK, J., AHMED, I., GARRATT, A. N., BIRCHMEIER, C., ZHOU, M., HARTLEY, L., ROBB, L., FENELEY, M. P., FATKIN, D. & HARVEY, R. P. 2010. Neuregulin 1 sustains the gene regulatory network in both trabecular and nontrabecular myocardium. Circ Res, 107, 715-27. LAKKISTO, P., KYTO, V., FORSTEN, H., SIREN, J. M., SEGERSVARD, H., VOIPIO-PULKKI, L. M., LAINE, M., PULKKI, K. & TIKKANEN, I. 2010. Heme oxygenase-1 and carbon monoxide promote neovascularization after myocardial infarction by modulating the expression of HIF-1alpha, SDF-1alpha and VEGF-B. Eur J Pharmacol, 635, 156-64. LAL, N., CHIU, A. P., WANG, F., ZHANG, D., JIA, J., WAN, A., VLODAVSKY, I., HUSSEIN, B. & RODRIGUES, B. 2017. Loss of VEGFB and its signaling in the diabetic heart is associated with increased cell death signaling. Am J Physiol Heart Circ Physiol, 312, H1163-H1175. LEACH, J. P. & MARTIN, J. F. 2018. Cardiomyocyte Proliferation for Therapeutic Regeneration. Curr Cardiol Rep, 20, 63. LEE, R. J., SPRINGER, M. L., BLANCO-BOSE, W. E., SHAW, R., URSELL, P. C. & BLAU, H. M. 2000. VEGF gene delivery to myocardium: deleterious effects of unregulated expression. Circulation, 102, 898-901. LEUNG, D. W., CACHIANES, G., KUANG, W. J., GOEDDEL, D. V. & FERRARA, N. 1989. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science, 246, 1306-9.
72 72 LEVIS, B. E., BINKLEY, P. F. & SHAPIRO, C. L. 2017. Cardiotoxic effects of anthracycline- based therapy: what is the evidence and what are the potential harms? Lancet Oncol, 18, e445-e456. LI, X., TJWA, M., VAN HOVE, I., ENHOLM, B., NEVEN, E., PAAVONEN, K., JELTSCH, M., JUAN, T. D., SIEVERS, R. E., CHORIANOPOULOS, E., WADA, H., VANWILDEMEERSCH, M., NOEL, A., FOIDART, J. M., SPRINGER, M. L., VON DEGENFELD, G., DEWERCHIN, M., BLAU, H. M., ALITALO, K., ERIKSSON, U., CARMELIET, P. & MOONS, L. 2008a. Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium. Arterioscler Thromb Vasc Biol, 28, 1614-20. LI, Y., SONG, Y., ZHAO, L., GAIDOSH, G., LATIES, A. M. & WEN, R. 2008b. Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI. Nat Protoc, 3, 1703-8. LI, Y., ZHANG, F., NAGAI, N., TANG, Z., ZHANG, S., SCOTNEY, P., LENNARTSSON, J., ZHU, C., QU, Y., FANG, C., HUA, J., MATSUO, O., FONG, G. H., DING, H., CAO, Y., BECKER, K. G., NASH, A., HELDIN, C. H. & LI, X. 2008c. VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats. J Clin Invest, 118, 913-23. LIBBY, P., RIDKER, P. M. & HANSSON, G. K. 2011. Progress and challenges in translating the biology of atherosclerosis. Nature, 473, 317-25. LIEBNER, S., CATTELINO, A., GALLINI, R., RUDINI, N., IURLARO, M., PICCOLO, S. & DEJANA, E. 2004. Beta-catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse. J Cell Biol, 166, 359-67. LIU, F., ZHANG, X., WEISBERG, E., CHEN, S., HUR, W., WU, H., ZHAO, Z., WANG, W., MAO, M., CAI, C., SIMON, N. I., SANDA, T., WANG, J., LOOK, A. T., GRIFFIN, J. D., BALK, S. P., LIU, Q. & GRAY, N. S. 2013. Discovery of a selective irreversible BMX inhibitor for prostate cancer. ACS Chem Biol, 8, 1423-8. LIU, Q., HU, T., HE, L., HUANG, X., TIAN, X., ZHANG, H., HE, L., PU, W., ZHANG, L., SUN, H., FANG, J., YU, Y., DUAN, S., HU, C., HUI, L., ZHANG, H., QUERTERMOUS, T., XU, Q., RED-HORSE, K., WYTHE, J. D. & ZHOU, B. 2015. Genetic targeting of sprouting angiogenesis using Apln-CreER. Nat Commun, 6, 6020. LOPEZ-GORDO, E., KOHLBRENNER, E., KATZ, M. G. & WEBER, T. 2019. AAV Vectors for Efficient Gene Delivery to Rodent Hearts. Methods Mol Biol, 1950, 311-332. LOTTONEN-RAIKASLEHTO, L., RISSANEN, R., GURZELER, E., MERENTIE, M., HUUSKO, J., SCHNEIDER, J. E., LIIMATAINEN, T. & YLA-HERTTUALA, S. 2017. Left ventricular remodeling leads to heart failure in mice with cardiac-specific overexpression of VEGF-B167: echocardiography and magnetic resonance imaging study. Physiol Rep, 5. LUTTUN, A., TJWA, M., MOONS, L., WU, Y., ANGELILLO-SCHERRER, A., LIAO, F., NAGY, J. A., HOOPER, A., PRILLER, J., DE KLERCK, B., COMPERNOLLE, V., DACI, E., BOHLEN, P., DEWERCHIN, M., HERBERT, J. M., FAVA, R., MATTHYS, P., CARMELIET, G., COLLEN, D., DVORAK, H. F., HICKLIN, D. J. & CARMELIET, P. 2002. Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med, 8, 831-40. LUU, A. Z., CHOWDHURY, B., AL-OMRAN, M., TEOH, H., HESS, D. A. & VERMA, S. 2018. Role of Endothelium in Doxorubicin-Induced Cardiomyopathy. JACC Basic Transl Sci, 3, 861-870. LV, Y. X., ZHONG, S., TANG, H., LUO, B., CHEN, S. J., CHEN, L., ZHENG, F., ZHANG, L., WANG, L., LI, X. Y., YAN, Y. W., PAN, Y. M., JIANG, M., ZHANG, Y. E., WANG, L., YANG, J. Y., GUO, L. Y., CHEN, S. Y., WANG, J. N. & TANG, J. M. 2018. VEGF-A and
73 73 VEGF-B Coordinate the Arteriogenesis to Repair the Infarcted Heart with Vagus Nerve Stimulation. Cell Physiol Biochem, 48, 433-449. MA, X. M. & BLENIS, J. 2009. Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol, 10, 307-18. MAGLIONE, D., GUERRIERO, V., VIGLIETTO, G., DELLI-BOVI, P. & PERSICO, M. G. 1991. Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc Natl Acad Sci U S A, 88, 9267-71. MAGLIONE, D., GUERRIERO, V., VIGLIETTO, G., FERRARO, M. G., APRELIKOVA, O., ALITALO, K., DEL VECCHIO, S., LEI, K. J., CHOU, J. Y. & PERSICO, M. G. 1993. Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14. Oncogene, 8, 925-31. MAKINEN, T., JUSSILA, L., VEIKKOLA, T., KARPANEN, T., KETTUNEN, M. I., PULKKANEN, K. J., KAUPPINEN, R., JACKSON, D. G., KUBO, H., NISHIKAWA, S., YLA-HERTTUALA, S. & ALITALO, K. 2001. Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat Med, 7, 199-205. MAKINEN, T., OLOFSSON, B., KARPANEN, T., HELLMAN, U., SOKER, S., KLAGSBRUN, M., ERIKSSON, U. & ALITALO, K. 1999. Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1. J Biol Chem, 274, 21217- 22. MANN, N. & ROSENZWEIG, A. 2012. Can exercise teach us how to treat heart disease? Circulation, 126, 2625-35. MARINO, F., SCALISE, M., CIANFLONE, E., MANCUSO, T., AQUILA, I., AGOSTI, V., TORELLA, M., PAOLINO, D., MOLLACE, V., NADAL-GINARD, B. & TORELLA, D. 2019. Role of c-Kit in Myocardial Regeneration and Aging. Front Endocrinol (Lausanne), 10, 371. MATHUR, P., KAGA, S., ZHAN, L., DAS, D. K. & MAULIK, N. 2005. Potential candidates for ischemic preconditioning-associated vascular growth pathways revealed by antibody array. Am J Physiol Heart Circ Physiol, 288, H3006-10. MAYNARD, S. E., MIN, J. Y., MERCHAN, J., LIM, K. H., LI, J., MONDAL, S., LIBERMANN, T. A., MORGAN, J. P., SELLKE, F. W., STILLMAN, I. E., EPSTEIN, F. H., SUKHATME, V. P. & KARUMANCHI, S. A. 2003. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest, 111, 649-58. MENDELL, J. R., AL-ZAIDY, S., SHELL, R., ARNOLD, W. D., RODINO-KLAPAC, L. R., PRIOR, T. W., LOWES, L., ALFANO, L., BERRY, K., CHURCH, K., KISSEL, J. T., NAGENDRAN, S., L'ITALIEN, J., SPROULE, D. M., WELLS, C., CARDENAS, J. A., HEITZER, M. D., KASPAR, A., CORCORAN, S., BRAUN, L., LIKHITE, S., MIRANDA, C., MEYER, K., FOUST, K. D., BURGHES, A. H. M. & KASPAR, B. K. 2017. Single- Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. N Engl J Med, 377, 1713- 1722. MERKI, E., ZAMORA, M., RAYA, A., KAWAKAMI, Y., WANG, J., ZHANG, X., BURCH, J., KUBALAK, S. W., KALIMAN, P., IZPISUA BELMONTE, J. C., CHIEN, K. R. & RUIZ- LOZANO, P. 2005. Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation. Proc Natl Acad Sci U S A, 102, 18455-60. MIGDAL, M., HUPPERTZ, B., TESSLER, S., COMFORTI, A., SHIBUYA, M., REICH, R., BAUMANN, H. & NEUFELD, G. 1998. Neuropilin-1 is a placenta growth factor-2 receptor. J Biol Chem, 273, 22272-8.
74 74 MIQUEROL, L., THIREAU, J., BIDEAUX, P., STURNY, R., RICHARD, S. & KELLY, R. G. 2015. Endothelial plasticity drives arterial remodeling within the endocardium after myocardial infarction. Circ Res, 116, 1765-71. MITCHELL-JORDAN, S. A., HOLOPAINEN, T., REN, S., WANG, S., WARBURTON, S., ZHANG, M. J., ALITALO, K., WANG, Y. & VONDRISKA, T. M. 2008. Loss of Bmx nonreceptor tyrosine kinase prevents pressure overload-induced cardiac hypertrophy. Circ Res, 103, 1359-62. MOHAMMED, S. F., HUSSAIN, S., MIRZOYEV, S. A., EDWARDS, W. D., MALESZEWSKI, J. J. & REDFIELD, M. M. 2015. Coronary microvascular rarefaction and myocardial fibrosis in heart failure with preserved ejection fraction. Circulation, 131, 550-9. MOLKENTIN, J. D., LU, J. R., ANTOS, C. L., MARKHAM, B., RICHARDSON, J., ROBBINS, J., GRANT, S. R. & OLSON, E. N. 1998. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell, 93, 215-28. MOORMAN, A. F. & CHRISTOFFELS, V. M. 2003. Cardiac chamber formation: development, genes, and evolution. Physiol Rev, 83, 1223-67. MOULD, A. W., GRECO, S. A., CAHILL, M. M., TONKS, I. D., BELLOMO, D., PATTERSON, C., ZOURNAZI, A., NASH, A., SCOTNEY, P., HAYWARD, N. K. & KAY, G. F. 2005. Transgenic overexpression of vascular endothelial growth factor-B isoforms by endothelial cells potentiates postnatal vessel growth in vivo and in vitro. Circ Res, 97, e60-70. MUHL, L., MOESSINGER, C., ADZEMOVIC, M. Z., DIJKSTRA, M. H., NILSSON, I., ZEITELHOFER, M., HAGBERG, C. E., HUUSKO, J., FALKEVALL, A., YLA- HERTTUALA, S. & ERIKSSON, U. 2016. Expression of vascular endothelial growth factor (VEGF)-B and its receptor (VEGFR1) in murine heart, lung and kidney. Cell Tissue Res, 365, 51-63. NAKAJIMA, Y., YAMAGISHI, T., HOKARI, S. & NAKAMURA, H. 2000. Mechanisms involved in valvuloseptal endocardial cushion formation in early cardiogenesis: roles of transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP). Anat Rec, 258, 119-27. NAKAMURA, M. & SADOSHIMA, J. 2018. Mechanisms of physiological and pathological cardiac hypertrophy. Nat Rev Cardiol, 15, 387-407. NAKANO, H., LIU, X., ARSHI, A., NAKASHIMA, Y., VAN HANDEL, B., SASIDHARAN, R., HARMON, A. W., SHIN, J. H., SCHWARTZ, R. J., CONWAY, S. J., HARVEY, R. P., PASHMFOROUSH, M., MIKKOLA, H. K. & NAKANO, A. 2013. Haemogenic endocardium contributes to transient definitive haematopoiesis. Nat Commun, 4, 1564. NAUMENKO, N., HUUSKO, J., TUOMAINEN, T., KOIVUMAKI, J. T., MERENTIE, M., GURZELER, E., ALITALO, K., KIVELA, R., YLA-HERTTUALA, S. & TAVI, P. 2017. Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart. Front Physiol, 8, 373. NEILAN, T. G., JASSAL, D. S., SCULLY, M. F., CHEN, G., DEFLANDRE, C., MCALLISTER, H., KAY, E., AUSTIN, S. C., HALPERN, E. F., HARMEY, J. H. & FITZGERALD, D. J. 2006. Iloprost attenuates doxorubicin-induced cardiac injury in a murine model without compromising tumour suppression. Eur Heart J, 27, 1251-6. NESMITH, J. E., CHAPPELL, J. C., CLUCERU, J. G. & BAUTCH, V. L. 2017. Blood vessel anastomosis is spatially regulated by Flt1 during angiogenesis. Development, 144, 889-896. NEUBAUER, E., WIRTZ, R. M., KAEMMERER, D., ATHELOGOU, M., SCHMIDT, L., SANGER, J. & LUPP, A. 2016. Comparative evaluation of three proliferation markers, Ki- 67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects. Oncotarget, 7, 41959-41973.
75 75 NOSEDA, M., PETERKIN, T., SIMOES, F. C., PATIENT, R. & SCHNEIDER, M. D. 2011. Cardiopoietic factors: extracellular signals for cardiac lineage commitment. Circ. Res, 108, 129-152. NURRO, J., HALONEN, P. J., KUIVANEN, A., TARKIA, M., SARASTE, A., HONKONEN, K., LAHTEENVUO, J., RISSANEN, T. T., KNUUTI, J. & YLA-HERTTUALA, S. 2016. AdVEGF-B186 and AdVEGF-DDeltaNDeltaC induce angiogenesis and increase perfusion in porcine myocardium. Heart, 102, 1716-1720. OCTAVIA, Y., KARARIGAS, G., DE BOER, M., CHRIFI, I., KIETADISORN, R., SWINNEN, M., DUIMEL, H., VERHEYEN, F. K., BRANDT, M. M., FLIEGNER, D., CHENG, C., JANSSENS, S., DUNCKER, D. J. & MOENS, A. L. 2017. Folic acid reduces doxorubicin- induced cardiomyopathy by modulating endothelial nitric oxide synthase. J Cell Mol Med, 21, 3277-3287. OCTAVIA, Y., TOCCHETTI, C. G., GABRIELSON, K. L., JANSSENS, S., CRIJNS, H. J. & MOENS, A. L. 2012. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol, 52, 1213-25. ODORISIO, T., SCHIETROMA, C., ZACCARIA, M. L., CIANFARANI, F., TIVERON, C., TATANGELO, L., FAILLA, C. M. & ZAMBRUNO, G. 2002. Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. J Cell Sci, 115, 2559-67. OKA, T., AKAZAWA, H., NAITO, A. T. & KOMURO, I. 2014. Angiogenesis and cardiac hypertrophy: maintenance of cardiac function and causative roles in heart failure. Circ Res, 114, 565-71. OLOFSSON, B., KORPELAINEN, E., PEPPER, M. S., MANDRIOTA, S. J., AASE, K., KUMAR, V., GUNJI, Y., JELTSCH, M. M., SHIBUYA, M., ALITALO, K. & ERIKSSON, U. 1998. Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. Proc Natl Acad Sci U S A, 95, 11709-14. OLOFSSON, B., PAJUSOLA, K., KAIPAINEN, A., VON EULER, G., JOUKOV, V., SAKSELA, O., ORPANA, A., PETTERSSON, R. F., ALITALO, K. & ERIKSSON, U. 1996. Vascular endothelial growth factor B, a novel growth factor for endothelial cells. Proc. Natl Acad. Sci. USA, 93, 2576-2581. OLSON, E. N. & SRIVASTAVA, D. 1996. Molecular pathways controlling heart development. Science, 272, 671-6. OLSSON, A. K., DIMBERG, A., KREUGER, J. & CLAESSON-WELSH, L. 2006. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol, 7, 359-71. PAAVONEN, K., EKMAN, N., WIRZENIUS, M., RAJANTIE, I., POUTANEN, M. & ALITALO, K. 2004. Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing. Mol Biol Cell, 15, 4226-33. PARADIES, P., CARLUCCI, L., WOITEK, F., STAFFIERI, F., LACITIGNOLA, L., CECI, L., ROMANO, D., SASANELLI, M., ZENTILIN, L., GIACCA, M., SALVADORI, S., CROVACE, A. & RECCHIA, F. A. 2019. Intracoronary Gene Delivery of the Cytoprotective Factor Vascular Endothelial Growth Factor-B167 in Canine Patients with Dilated Cardiomyopathy: A Short-Term Feasibility Study. Vet Sci, 6. PARK, J. E., CHEN, H. H., WINER, J., HOUCK, K. A. & FERRARA, N. 1994. Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem, 269, 25646-54. PARTANEN, T. A., MAKINEN, T., AROLA, J., SUDA, T., WEICH, H. A. & ALITALO, K. 1999. Endothelial growth factor receptors in human fetal heart. Circulation, 100, 583-6. PASSIER, R., ZENG, H., FREY, N., NAYA, F. J., NICOL, R. L., MCKINSEY, T. A., OVERBEEK, P., RICHARDSON, J. A., GRANT, S. R. & OLSON, E. N. 2000. CaM
76 76 kinase signaling induces cardiac hypertrophy and activates the MEF2 transcription factor in vivo. J Clin Invest, 105, 1395-406. PATRUCCO, E., NOTTE, A., BARBERIS, L., SELVETELLA, G., MAFFEI, A., BRANCACCIO, M., MARENGO, S., RUSSO, G., AZZOLINO, O., RYBALKIN, S. D., SILENGO, L., ALTRUDA, F., WETZKER, R., WYMANN, M. P., LEMBO, G. & HIRSCH, E. 2004. PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase- dependent and -independent effects. Cell, 118, 375-87. PATTEN, I. S., RANA, S., SHAHUL, S., ROWE, G. C., JANG, C., LIU, L., HACKER, M. R., RHEE, J. S., MITCHELL, J., MAHMOOD, F., HESS, P., FARRELL, C., KOULISIS, N., KHANKIN, E. V., BURKE, S. D., TUDORACHE, I., BAUERSACHS, J., DEL MONTE, F., HILFIKER-KLEINER, D., KARUMANCHI, S. A. & ARANY, Z. 2012. Cardiac angiogenic imbalance leads to peripartum cardiomyopathy. Nature, 485, 333-8. PELLET-MANY, C., FRANKEL, P., JIA, H. & ZACHARY, I. 2008. Neuropilins: structure, function and role in disease. Biochem J, 411, 211-26. PEPE, M., MAMDANI, M., ZENTILIN, L., CSISZAR, A., QANUD, K., ZACCHIGNA, S., UNGVARI, Z., PULIGADDA, U., MOIMAS, S., XU, X., EDWARDS, J. G., HINTZE, T. H., GIACCA, M. & RECCHIA, F. A. 2010. Intramyocardial VEGF-B167 gene delivery delays the progression towards congestive failure in dogs with pacing-induced dilated cardiomyopathy. Circ Res, 106, 1893-903. PINTO, A. R., ILINYKH, A., IVEY, M. J., KUWABARA, J. T., D'ANTONI, M. L., DEBUQUE, R., CHANDRAN, A., WANG, L., ARORA, K., ROSENTHAL, N. A. & TALLQUIST, M. D. 2016. Revisiting Cardiac Cellular Composition. Circ Res, 118, 400-9. PRICE, F. V., CHAMBERS, S. K., CARCANGIU, M. L., KOHORN, E. I., SCHWARTZ, P. E. & CHAMBERS, J. T. 1993. Intravenous cisplatin, doxorubicin, and cyclophosphamide in the treatment of uterine papillary serous carcinoma (UPSC). Gynecol Oncol, 51, 383-9. PUGH, C. W. & RATCLIFFE, P. J. 2003. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med, 9, 677-84. RAFII, S., BUTLER, J. M. & DING, B. S. 2016. Angiocrine functions of organ-specific endothelial cells. Nature, 529, 316-25. RAISSADATI, A., TUUMINEN, R., DASHKEVICH, A., BRY, M., KIVELA, R., ANISIMOV, A., SYRJALA, S., ARNAUDOVA, R., ROUVINEN, E., KERANEN, M. A., KREBS, R., NYKANEN, A. I. & LEMSTROM, K. B. 2017. Vascular Endothelial Growth Factor-B Overexpressing Hearts Are Not Protected From Transplant-Associated Ischemia- Reperfusion Injury. Exp Clin Transplant, 15, 203-212. RAJANTIE, I., EKMAN, N., ILJIN, K., ARIGHI, E., GUNJI, Y., KAUKONEN, J., PALOTIE, A., DEWERCHIN, M., CARMELIET, P. & ALITALO, K. 2001. Bmx tyrosine kinase has a redundant function downstream of angiopoietin and vascular endothelial growth factor receptors in arterial endothelium. Mol Cell Biol, 21, 4647-55. RAMASAMY, S. K., KUSUMBE, A. P., WANG, L. & ADAMS, R. H. 2014. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature, 507, 376-380. RED-HORSE, K., UENO, H., WEISSMAN, I. L. & KRASNOW, M. A. 2010. Coronary arteries form by developmental reprogramming of venous cells. Nature, 464, 549-53. RISSANEN, T. T., MARKKANEN, J. E., GRUCHALA, M., HEIKURA, T., PURANEN, A., KETTUNEN, M. I., KHOLOVA, I., KAUPPINEN, R. A., ACHEN, M. G., STACKER, S. A., ALITALO, K. & YLA-HERTTUALA, S. 2003. VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ Res, 92, 1098-106. ROBCIUC, M. R., KIVELA, R., WILLIAMS, I. M., DE BOER, J. F., VAN DIJK, T. H., ELAMAA, H., TIGISTU-SAHLE, F., MOLOTKOV, D., LEPPANEN, V. M., KAKELA, R., EKLUND, L., WASSERMAN, D. H., GROEN, A. K. & ALITALO, K. 2016.
77 77 VEGFB/VEGFR1-Induced Expansion of Adipose Vasculature Counteracts Obesity and Related Metabolic Complications. Cell Metab, 23, 712-24. ROBINSON, C. J. & STRINGER, S. E. 2001. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci, 114, 853-65. ROSCA, M. G., TANDLER, B. & HOPPEL, C. L. 2013. Mitochondria in cardiac hypertrophy and heart failure. J Mol Cell Cardiol, 55, 31-41. ROSS, R. 1999. Atherosclerosis--an inflammatory disease. N Engl J Med, 340, 115-26. ROWE, G. C. & YOUNG, M. E. 2017. VEGF-B: friend or foe to the heart in times of nutrient excess? Am J Physiol Heart Circ Physiol, 313, H244-H247. SAARISTO, A., VEIKKOLA, T., ENHOLM, B., HYTONEN, M., AROLA, J., PAJUSOLA, K., TURUNEN, P., JELTSCH, M., KARKKAINEN, M. J., KERJASCHKI, D., BUELER, H., YLA-HERTTUALA, S. & ALITALO, K. 2002. Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J, 16, 1041-9. SADOSHIMA, J., XU, Y., SLAYTER, H. S. & IZUMO, S. 1993. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell, 75, 977-84. SAVONA, V. C. & GRECH, V. 1999. Concepts in cardiology - a historical perspective. Images Paediatr Cardiol, 1, 22-31. SCIARRETTA, S., FORTE, M., FRATI, G. & SADOSHIMA, J. 2018. New Insights Into the Role of mTOR Signaling in the Cardiovascular System. Circ Res, 122, 489-505. SEGREDO, M. P., SALVADORI, D. M., ROCHA, N. S., MORETTO, F. C., CORREA, C. R., CAMARGO, E. A., DE ALMEIDA, D. C., REIS, R. A., FREIRE, C. M., BRAZ, M. G., TANG, G., MATSUBARA, L. S., MATSUBARA, B. B., YEUM, K. J. & FERREIRA, A. L. 2014. Oxidative stress on cardiotoxicity after treatment with single and multiple doses of doxorubicin. Hum Exp Toxicol, 33, 748-60. SEYMOUR, L., BRAMWELL, V. & MORAN, L. A. 1999. Use of dexrazoxane as a cardioprotectant in patients receiving doxorubicin or epirubicin chemotherapy for the treatment of cancer. The Provincial Systemic Treatment Disease Site Group. Cancer Prev Control, 3, 145-59. SHARMA, B., CHANG, A. & RED-HORSE, K. 2017a. Coronary Artery Development: Progenitor Cells and Differentiation Pathways. Annu Rev Physiol, 79, 1-19. SHARMA, B., HO, L., FORD, G. H., CHEN, H. I., GOLDSTONE, A. B., WOO, Y. J., QUERTERMOUS, T., REVERSADE, B. & RED-HORSE, K. 2017b. Alternative Progenitor Cells Compensate to Rebuild the Coronary Vasculature in Elabela- and Apj- Deficient Hearts. Dev Cell, 42, 655-666 e3. SHEN, Z., ZHANG, Z., WANG, X. & YANG, K. 2018. VEGFB-VEGFR1 ameliorates Ang II- induced cardiomyocyte hypertrophy through Ca(2+) -mediated PKG I pathway. J Cell Biochem, 119, 1511-1520. SHIBUYA, M. 2006. Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1): a dual regulator for angiogenesis. Angiogenesis, 9, 225-30; discussion 231. SHIBUYA, M. 2011. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes Cancer, 2, 1097-105. SHIMIZU, I. & MINAMINO, T. 2016. Physiological and pathological cardiac hypertrophy. J Mol Cell Cardiol, 97, 245-62. SHIMOKAWA, H. 1999. Primary endothelial dysfunction: atherosclerosis. J Mol Cell Cardiol, 31, 23-37. SHIMOKAWA, H. & YASUDA, S. 2008. Myocardial ischemia: current concepts and future perspectives. J Cardiol, 52, 67-78.
78 78 SHIOI, T., KANG, P. M., DOUGLAS, P. S., HAMPE, J., YBALLE, C. M., LAWITTS, J., CANTLEY, L. C. & IZUMO, S. 2000. The conserved phosphoinositide 3-kinase pathway determines heart size in mice. EMBO J, 19, 2537-48. SHIOI, T., MCMULLEN, J. R., TARNAVSKI, O., CONVERSO, K., SHERWOOD, M. C., MANNING, W. J. & IZUMO, S. 2003. Rapamycin attenuates load-induced cardiac hypertrophy in mice. Circulation, 107, 1664-70. SHWEIKI, D., ITIN, A., SOFFER, D. & KESHET, E. 1992. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature, 359, 843-5. SIMONS, M. & EICHMANN, A. 2015. Molecular controls of arterial morphogenesis. Circ Res, 116, 1712-24. SIMUNEK, T., STERBA, M., POPELOVA, O., ADAMCOVA, M., HRDINA, R. & GERSL, V. 2009. Anthracycline-induced cardiotoxicity: overview of studies examining the roles of oxidative stress and free cellular iron. Pharmacol Rep, 61, 154-71. SINGAL, P. K. & ILISKOVIC, N. 1998. Doxorubicin-induced cardiomyopathy. N Engl J Med, 339, 900-5. SINGH, A., RAMESH, S., CIBI, D. M., YUN, L. S., LI, J., LI, L., MANDERFIELD, L. J., OLSON, E. N., EPSTEIN, J. A. & SINGH, M. K. 2016. Hippo Signaling Mediators Yap and Taz Are Required in the Epicardium for Coronary Vasculature Development. Cell Rep, 15, 1384-1393. SINGH, N. K., HANSEN, D. E., 3RD, KUNDUMANI-SRIDHARAN, V. & RAO, G. N. 2013. Both Kdr and Flt1 play a vital role in hypoxia-induced Src-PLD1-PKCgamma-cPLA(2) activation and retinal neovascularization. Blood, 121, 1911-23. SMART, N. 2017. Prospects for improving neovascularization of the ischemic heart: Lessons from development. Microcirculation, 24. SOHL, G. & WILLECKE, K. 2004. Gap junctions and the connexin protein family. Cardiovasc Res, 62, 228-32. SOKER, S., TAKASHIMA, S., MIAO, H. Q., NEUFELD, G. & KLAGSBRUN, M. 1998. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell, 92, 735-45. SOMI, S., BUFFING, A. A., MOORMAN, A. F. & VAN DEN HOFF, M. J. 2004. Dynamic patterns of expression of BMP isoforms 2, 4, 5, 6, and 7 during chicken heart development. Anat Rec A Discov Mol Cell Evol Biol, 279, 636-51. SONGBO, M., LANG, H., XINYONG, C., BIN, X., PING, Z. & LIANG, S. 2019. Oxidative stress injury in doxorubicin-induced cardiotoxicity. Toxicol Lett, 307, 41-48. SOONPAA, M. H. & FIELD, L. J. 1997. Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts. Am J Physiol, 272, H220-6. SRIVASTAVA, D. & DEWITT, N. 2016. In Vivo Cellular Reprogramming: The Next Generation. Cell, 166, 1386-1396. STEVENS, K. R. & MURRY, C. E. 2018. Human Pluripotent Stem Cell-Derived Engineered Tissues: Clinical Considerations. Cell Stem Cell, 22, 294-297. SU, T., STANLEY, G., SINHA, R., D'AMATO, G., DAS, S., RHEE, S., CHANG, A. H., PODURI, A., RAFTREY, B., DINH, T. T., ROPER, W. A., LI, G., QUINN, K. E., CARON, K. M., WU, S., MIQUEROL, L., BUTCHER, E. C., WEISSMAN, I., QUAKE, S. & RED- HORSE, K. 2018. Single-cell analysis of early progenitor cells that build coronary arteries. Nature, 559, 356-362. SUTER, T. M. & EWER, M. S. 2013. Cancer drugs and the heart: importance and management. Eur Heart J, 34, 1102-11. TACAR, O., SRIAMORNSAK, P. & DASS, C. R. 2013. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol, 65, 157-70.
79 79 TAEGTMEYER, H. 2002. Switching metabolic genes to build a better heart. Circulation, 106, 2043-5. TAKASHIMA, S., KITAKAZE, M., ASAKURA, M., ASANUMA, H., SANADA, S., TASHIRO, F., NIWA, H., MIYAZAKI JI, J., HIROTA, S., KITAMURA, Y., KITSUKAWA, T., FUJISAWA, H., KLAGSBRUN, M. & HORI, M. 2002. Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis. Proc Natl Acad Sci U S A, 99, 3657-62. TALMAN, V. & KIVELA, R. 2018. Cardiomyocyte-Endothelial Cell Interactions in Cardiac Remodeling and Regeneration. Front Cardiovasc Med, 5, 101. TAMAGNONE, L., LAHTINEN, I., MUSTONEN, T., VIRTANEVA, K., FRANCIS, F., MUSCATELLI, F., ALITALO, R., SMITH, C. I., LARSSON, C. & ALITALO, K. 1994. BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2. Oncogene, 9, 3683-8. TAMMELA, T. & ALITALO, K. 2010. Lymphangiogenesis: Molecular mechanisms and future promise. Cell, 140, 460-76. TANG, J., ZHANG, H., HE, L., HUANG, X., LI, Y., PU, W., YU, W., ZHANG, L., CAI, D., LUI, K. O. & ZHOU, B. 2018. Genetic Fate Mapping Defines the Vascular Potential of Endocardial Cells in the Adult Heart. Circ Res, 122, 984-993. TANG, X. L., ROKOSH, G., SANGANALMATH, S. K., YUAN, F., SATO, H., MU, J., DAI, S., LI, C., CHEN, N., PENG, Y., DAWN, B., HUNT, G., LERI, A., KAJSTURA, J., TIWARI, S., SHIRK, G., ANVERSA, P. & BOLLI, R. 2010. Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction. Circulation, 121, 293-305. TIAN, X., HU, T., ZHANG, H., HE, L., HUANG, X., LIU, Q., YU, W., HE, L., YANG, Z., YAN, Y., YANG, X., ZHONG, T. P., PU, W. T. & ZHOU, B. 2014. Vessel formation. De novo formation of a distinct coronary vascular population in neonatal heart. Science, 345, 90-4. TIAN, X., HU, T., ZHANG, H., HE, L., HUANG, X., LIU, Q., YU, W., HE, L., YANG, Z., ZHANG, Z., ZHONG, T. P., YANG, X., YANG, Z., YAN, Y., BALDINI, A., SUN, Y., LU, J., SCHWARTZ, R. J., EVANS, S. M., GITTENBERGER-DE GROOT, A. C., RED- HORSE, K. & ZHOU, B. 2013. Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries. Cell Res, 23, 1075-90. TIAN, X., PU, W. T. & ZHOU, B. 2015. Cellular origin and developmental program of coronary angiogenesis. Circ Res, 116, 515-30. TIMMERMAN, L. A., GREGO-BESSA, J., RAYA, A., BERTRAN, E., PEREZ-POMARES, J. M., DIEZ, J., ARANDA, S., PALOMO, S., MCCORMICK, F., IZPISUA-BELMONTE, J. C. & DE LA POMPA, J. L. 2004. Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev, 18, 99-115. TIRZIU, D., CHORIANOPOULOS, E., MOODIE, K. L., PALAC, R. T., ZHUANG, Z. W., TJWA, M., RONCAL, C., ERIKSSON, U., FU, Q., ELFENBEIN, A., HALL, A. E., CARMELIET, P., MOONS, L. & SIMONS, M. 2007. Myocardial hypertrophy in the absence of external stimuli is induced by angiogenesis in mice. J Clin Invest, 117, 3188-97. TOMANEK, R. J., HOLIFIELD, J. S., REITER, R. S., SANDRA, A. & LIN, J. J. 2002. Role of VEGF family members and receptors in coronary vessel formation. Dev Dyn, 225, 233-40. TOMANEK, R. J., ISHII, Y., HOLIFIELD, J. S., SJOGREN, C. L., HANSEN, H. K. & MIKAWA, T. 2006. VEGF family members regulate myocardial tubulogenesis and coronary artery formation in the embryo. Circ Res, 98, 947-53. TRAPNELL, C., CACCHIARELLI, D., GRIMSBY, J., POKHAREL, P., LI, S., MORSE, M., LENNON, N. J., LIVAK, K. J., MIKKELSEN, T. S. & RINN, J. L. 2014. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol, 32, 381-386.
80 80 TSANG, J. C. & CHIU, R. C. 1995. The phantom of "myocardial sinusoids": a historical reappraisal. Ann Thorac Surg, 60, 1831-5. TURANOV, A. A., LO, A., HASSLER, M. R., MAKRIS, A., ASHAR-PATEL, A., ALTERMAN, J. F., COLES, A. H., HARASZTI, R. A., ROUX, L., GODINHO, B., ECHEVERRIA, D., PEARS, S., ILIOPOULOS, J., SHANMUGALINGAM, R., OGLE, R., ZSENGELLER, Z. K., HENNESSY, A., KARUMANCHI, S. A., MOORE, M. J. & KHVOROVA, A. 2018. RNAi modulation of placental sFLT1 for the treatment of preeclampsia. Nat Biotechnol. UYGUR, A. & LEE, R. T. 2016. Mechanisms of Cardiac Regeneration. Dev Cell, 36, 362-74. VAAHTOMERI, K., KARAMAN, S., MAKINEN, T. & ALITALO, K. 2017. Lymphangiogenesis guidance by paracrine and pericellular factors. Genes Dev, 31, 1615-1634. VAN BERLO, J. H., MAILLET, M. & MOLKENTIN, J. D. 2013. Signaling effectors underlying pathologic growth and remodeling of the heart. J Clin Invest, 123, 37-45. VAN DALEN, E. C., CARON, H. N., DICKINSON, H. O. & KREMER, L. C. 2011. Cardioprotective interventions for cancer patients receiving anthracyclines. Cochrane Database Syst Rev, CD003917. VANLANDEWIJCK, M., HE, L., MAE, M. A., ANDRAE, J., ANDO, K., DEL GAUDIO, F., NAHAR, K., LEBOUVIER, T., LAVINA, B., GOUVEIA, L., SUN, Y., RASCHPERGER, E., RASANEN, M., ZARB, Y., MOCHIZUKI, N., KELLER, A., LENDAHL, U. & BETSHOLTZ, C. 2018. A molecular atlas of cell types and zonation in the brain vasculature. Nature, 554, 475-480. VEGA, R. B., KONHILAS, J. P., KELLY, D. P. & LEINWAND, L. A. 2017. Molecular Mechanisms Underlying Cardiac Adaptation to Exercise. Cell Metab, 25, 1012-1026. VEIKKOLA, T., JUSSILA, L., MAKINEN, T., KARPANEN, T., JELTSCH, M., PETROVA, T. V., KUBO, H., THURSTON, G., MCDONALD, D. M., ACHEN, M. G., STACKER, S. A. & ALITALO, K. 2001. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J, 20, 1223-31. VOLKOVA, M. & RUSSELL, R., 3RD 2011. Anthracycline cardiotoxicity: prevalence, pathogenesis and treatment. Curr Cardiol Rev, 7, 214-20. VOLZ, K. S., JACOBS, A. H., CHEN, H. I., PODURI, A., MCKAY, A. S., RIORDAN, D. P., KOFLER, N., KITAJEWSKI, J., WEISSMAN, I. & RED-HORSE, K. 2015. Pericytes are progenitors for coronary artery smooth muscle. Elife, 4. VRANCKEN PEETERS, M. P., GITTENBERGER-DE GROOT, A. C., MENTINK, M. M., HUNGERFORD, J. E., LITTLE, C. D. & POELMANN, R. E. 1997. The development of the coronary vessels and their differentiation into arteries and veins in the embryonic quail heart. Dev Dyn, 208, 338-48. WALDO, K. L., WILLNER, W. & KIRBY, M. L. 1990. Origin of the proximal coronary artery stems and a review of ventricular vascularization in the chick embryo. Am J Anat, 188, 109- 20. WANSTALL, J. C., GAMBINO, A., JEFFERY, T. K., CAHILL, M. M., BELLOMO, D., HAYWARD, N. K. & KAY, G. F. 2002. Vascular endothelial growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension. Cardiovasc Res, 55, 361-8. WILKINS, B. J. & MOLKENTIN, J. D. 2004. Calcium-calcineurin signaling in the regulation of cardiac hypertrophy. Biochem Biophys Res Commun, 322, 1178-91. WILLEY, C. D., PALANISAMY, A. P., JOHNSTON, R. K., MANI, S. K., SHIRAISHI, H., TUXWORTH, W. J., ZILE, M. R., BALASUBRAMANIAN, S. & KUPPUSWAMY, D. 2008. STAT3 activation in pressure-overloaded feline myocardium: role for integrins and the tyrosine kinase BMX. Int J Biol Sci, 4, 184-99. WOITEK, F., ZENTILIN, L., HOFFMAN, N. E., POWERS, J. C., OTTIGER, I., PARIKH, S., KULCZYCKI, A. M., HURST, M., RING, N., WANG, T., SHAIKH, F., GROSS, P.,
81 81 SINGH, H., KOLPAKOV, M. A., LINKE, A., HOUSER, S. R., RIZZO, V., SABRI, A., MADESH, M., GIACCA, M. & RECCHIA, F. A. 2015. Intracoronary Cytoprotective Gene Therapy: A Study of VEGF-B167 in a Pre-Clinical Animal Model of Dilated Cardiomyopathy. J Am Coll Cardiol, 66, 139-53. WU, B., ZHANG, Z., LUI, W., CHEN, X., WANG, Y., CHAMBERLAIN, A. A., MORENO- RODRIGUEZ, R. A., MARKWALD, R. R., O'ROURKE, B. P., SHARP, D. J., ZHENG, D., LENZ, J., BALDWIN, H. S., CHANG, C. P. & ZHOU, B. 2012. Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling. Cell, 151, 1083-96. WU, K. C., ZERHOUNI, E. A., JUDD, R. M., LUGO-OLIVIERI, C. H., BAROUCH, L. A., SCHULMAN, S. P., BLUMENTHAL, R. S. & LIMA, J. A. 1998. Prognostic significance of microvascular obstruction by magnetic resonance imaging in patients with acute myocardial infarction. Circulation, 97, 765-72. YANG, W., AHN, H., HINRICHS, M., TORRY, R. J. & TORRY, D. S. 2003. Evidence of a novel isoform of placenta growth factor (PlGF-4) expressed in human trophoblast and endothelial cells. J Reprod Immunol, 60, 53-60. YLA-HERTTUALA, S. 2012. Endgame: glybera finally recommended for approval as the first gene therapy drug in the European union. Mol Ther, 20, 1831-2. YLA-HERTTUALA, S. & BAKER, A. H. 2017. Cardiovascular Gene Therapy: Past, Present, and Future. Mol Ther, 25, 1095-1106. YLA-HERTTUALA, S., BRIDGES, C., KATZ, M. G. & KORPISALO, P. 2017. Angiogenic gene therapy in cardiovascular diseases: dream or vision? Eur Heart J, 38, 1365-1371. YLA-HERTTUALA, S., RISSANEN, T. T., VAJANTO, I. & HARTIKAINEN, J. 2007. Vascular endothelial growth factors: biology and current status of clinical applications in cardiovascular medicine. J Am Coll Cardiol, 49, 1015-26. YOUNG, B. C., LEVINE, R. J. & KARUMANCHI, S. A. 2010. Pathogenesis of preeclampsia. Annu Rev Pathol, 5, 173-92. YU, A. F., STEINGART, R. M. & FUSTER, V. 2014. Cardiomyopathy associated with cancer therapy. J Card Fail, 20, 841-52. YUAN, L., MOYON, D., PARDANAUD, L., BREANT, C., KARKKAINEN, M. J., ALITALO, K. & EICHMANN, A. 2002. Abnormal lymphatic vessel development in neuropilin 2 mutant mice. Development, 129, 4797-806. YUAN, R., XIN, Q., SHI, W., LIU, W., LEE, S. M., HOI, P., LI, L., ZHAO, J., CONG, W. & CHEN, K. 2018. Vascular endothelial growth factor gene transfer therapy for coronary artery disease: A systematic review and meta-analysis. Cardiovasc Ther, 36, e12461. ZENTILIN, L., PULIGADDA, U., LIONETTI, V., ZACCHIGNA, S., COLLESI, C., PATTARINI, L., RUOZI, G., CAMPORESI, S., SINAGRA, G., PEPE, M., RECCHIA, F. A. & GIACCA, M. 2009. Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction. Faseb J. ZHANG, F., TANG, Z., HOU, X., LENNARTSSON, J., LI, Y., KOCH, A. W., SCOTNEY, P., LEE, C., ARJUNAN, P., DONG, L., KUMAR, A., RISSANEN, T. T., WANG, B., NAGAI, N., FONS, P., FARISS, R., ZHANG, Y., WAWROUSEK, E., TANSEY, G., RABER, J., FONG, G. H., DING, H., GREENBERG, D. A., BECKER, K. G., HERBERT, J. M., NASH, A., YLA-HERTTUALA, S., CAO, Y., WATTS, R. J. & LI, X. 2009. VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis. Proc Natl Acad Sci U S A, 106, 6152-7. ZHANG, H., LUI, K. O. & ZHOU, B. 2018. Endocardial Cell Plasticity in Cardiac Development, Diseases and Regeneration. Circ Res, 122, 774-789.
82 82 ZHANG, H., PU, W., LI, G., HUANG, X., HE, L., TIAN, X., LIU, Q., ZHANG, L., WU, S. M., SUCOV, H. M. & ZHOU, B. 2016. Endocardium Minimally Contributes to Coronary Endothelium in the Embryonic Ventricular Free Walls. Circ Res, 118, 1880-93. ZHANG, J., PING, P., WANG, G. W., LU, M., PANTALEON, D., TANG, X. L., BOLLI, R. & VONDRISKA, T. M. 2004. Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection. Am J Physiol Heart Circ Physiol, 287, H2364-6. ZHANG, S., LIU, X., BAWA-KHALFE, T., LU, L. S., LYU, Y. L., LIU, L. F. & YEH, E. T. 2012. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med, 18, 1639-42. ZHANG, Z. & ZHOU, B. 2013. Accelerated coronary angiogenesis by vegfr1-knockout endocardial cells. PLoS One, 8, e70570. ZHAO, T., ZHAO, W., CHEN, Y., LIU, L., AHOKAS, R. A. & SUN, Y. 2013. Differential expression of vascular endothelial growth factor isoforms and receptor subtypes in the infarcted heart. Int J Cardiol, 167, 2638-45. ZHOU, B., MA, Q., RAJAGOPAL, S., WU, S. M., DOMIAN, I., RIVERA-FELICIANO, J., JIANG, D., VON GISE, A., IKEDA, S., CHIEN, K. R. & PU, W. T. 2008. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature, 454, 109-13. ZHOU, B. & WU, S. M. 2018. Reassessment of c-Kit in Cardiac Cells: A Complex Interplay Between Expression, Fate, and Function. Circ Res, 123, 9-11. ZHU, L., LI, C., LIU, Q., XU, W. & ZHOU, X. 2019. Molecular biomarkers in cardiac hypertrophy. J Cell Mol Med, 23, 1671-1677.
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dissertationes MARKUS RÄSÄNEN MARKUS AND FACTOR-B GROWTH ENDOTHELIAL VASCULAR HYPERTROPHY CARDIAC IN KINASE THE BMX TYROSINE AND REVASCULARIZATION WIHURI RESEARCH INSTITUTE CANCER MEDICINE RESEARCH PROGRAM TRANSLATIONAL OF MEDICINE FACULTY PROGRAMME IN BIOMEDICINE DOCTORAL OF HELSINKI UNIVERSITY
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