Molecular Aspects of Medicine 62 (2018) 1e11

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Molecular Aspects of Medicine

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PDGF-C and PDGF-D signaling in vascular diseases and animal models

* Erika Folestad a, Anne Kunath b, Dick Wågsater€ b, a Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden b Division of Drug Research, Department of Medical and Health Sciences, Linkoping€ University, Linkoping,€ Sweden article info abstract

Article history: Members of the platelet-derived (PDGF) family are well known to be involved in different Received 31 August 2017 pathological conditions. The cellular and molecular mechanisms induced by the PDGF signaling have Received in revised form been well studied. Nevertheless, there is much more to discover about their functions and some 14 November 2017 important questions to be answered. This review summarizes the known roles of two of the PDGFs, Accepted 22 January 2018 PDGF-C and PDGF-D, in vascular diseases. There are clear implications for these growth factors in several Available online 14 February 2018 vascular diseases, such as atherosclerosis and stroke. The PDGF receptors are broadly expressed in the cardiovascular system in cells such as fibroblasts, smooth muscle cells and pericytes. Altered expression Keywords: Aneurysm of the receptors and the ligands have been found in various cardiovascular diseases and current studies fi Atherosclerosis have shown important implications of PDGF-C and PDGF-D signaling in brosis, neovascularization, Growth factor atherosclerosis and restenosis. Myocardial infarction © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND Smooth muscle cells license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction LaRochelle et al., 2001). This review will focus on and summarize the literature of PDGF-C and PDGF-D, and their receptors PDGFR-a Cardiovascular disease accounts for a large proportion of deaths, and PDGFR-b, in the field of vascular physiology and pathology, and the disease's development depends on a pathological change in including angiogenesis and vascular disease. a large number of factors. Infiltrated leukocytes and activated vascular cells such as endothelial cells, smooth muscle cells (SMC) 2. PDGF structure and signaling and fibroblasts secrete different growth factors, cytokines and other inflammatory factors that facilitates a local low grade inflammation The structure and signaling of the PDGF family has been resulting in tissue remodeling. Among these factors, the platelet- described and reviewed elsewhere in detail, therefore it is only derived growth factors (PDGF) play an important role involving summarized in brief in this review. Members of the PDGF family multiple mechanisms responsible for vascular pathologies such as binds to and signals through the PDGF receptors (Fig. 1). They are atherosclerosis, restenosis, aortic aneurysm and pulmonary arterial receptors, which are expressed in two different hypertension, resulting in ischemia, myocardial infarction and forms, PDGFR-a and PDGFR-b, that encode a transmembrane pro- stroke as example. tein with an extracellular ligand-binding domain and an intracel- The PDGF family is probably one of the best studied growth lular tyrosine kinase domain (Heldin and Westermark, 1999; factor systems. The PDGF family consists of four members; the Kazlauskas, 2017; Westermark et al., 1989, 1990). These two re- classical PDGF-A and PDGF-B, but also the novel PDGF-C and PDGF- ceptor isoforms dimerize upon ligand binding, which leads to one D. The classical PDGFs are well studied, however, even though it of the three possible receptor combination -aa,-ab and -bb. The was more than 15 years ago, since the novel PDGF-C and PDGF-D dimerization results in receptor autophosphorylation on tyrosine were discovered, they have not yet been studied in detail in the residues in the intracellular domain (Heldin and Westermark, 1999; context of vascular and cardiovascular disease. The two were Kelly et al., 1991; Westermark et al., 1989, 1990). Autophosphor- discovered in 2000 (Li et al., 2000) and 2001 (Bergsten et al., 2001; ylation further activates the receptor kinase and docking sites for downstream signaling molecules and modulation of different pathways (Kazlauskas and Cooper, 1989; Reigstad et al., 2005; * € € Westermark et al., 1989, 1990). Corresponding author. Linkoping University, entrence 68, 581 85, Linkoping, aa Sweden. PDGF-AA can bind the PDGFR- and PDGF-BB binds all forms, E-mail address: [email protected] (D. Wågsater).€ (i.e., PDGFR-aa, PDGFR-ab and PDGFR-bb)(Andrae et al., 2008; https://doi.org/10.1016/j.mam.2018.01.005 0098-2997/© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 E. Folestad et al. / Molecular Aspects of Medicine 62 (2018) 1e11

Fig. 1. Simplified schematic overview demonstrating PDGF processing, secretion and receptor binding from both in vitro and in vivo studies. Furin is mainly responsible for the intracellular cleavage of propeptides in PDGF-A and PDGF-B that can bind to the extracellular matrix or diffuse if the c-terminal retention motif is cleaved. PDGF-C and PDGF-D are secreted in inactive forms and are activated by tPA and uPA or matriptase, respectively.

Heldin and Westermark, 1999; Raines, 2004; Seifert et al., 1989). factor domain active and induce receptor signaling (Bergsten et al., The heterodimer PDGF-AB can bind PDGFR-aa and PDGFR-ab. 2001; LaRochelle et al., 2001; Li et al., 2000). The hinge regions of As for PDGF-A and PDGF-B, PDGF-C and PDGF-D also exert their PDGF-C and PDGF-D are between the CUB domain and the growth biological functions by binding to these receptors (Andrae et al., factor domain and contain cleavage sites for proteolytic removal of 2008; Fredriksson et al., 2004; Heldin and Westermark, 1999). the CUB domain before receptor binding. PDGF-C and PDGF-D PDGF-CC preferentially binds to and signals through PDGFR-a occur as homodimers (PDGF-CC and PDGF-DD), which are homodimers (Li and Eriksson, 2003; Li et al., 2000), while PDGF-DD secreted in their full-length form before proteolytic activation. The mainly binds to PDGFR-b homodimers (Bergsten et al., 2001; extracellular cleavage is performed by serine proteases. Plasmin LaRochelle et al., 2001). The signaling effects of PDGF-A and and tissue plasminogen activator (tPA) cleave PDGF-C (Fredriksson PDGF-B have been extensively investigated, and since PDGF-C and et al., 2004, 2005; Gilbertson et al., 2001; Li et al., 2000), while D also bind these receptors, one could predict similar effects. plasmin, urokinase plasminogen activator (uPA) and matriptase Due to their highly conserved cysteine knot motif, PDGF-C and were found to cleave PDGF-D (Ehnman et al., 2009; Reigstad et al., PDGF-D belong to the PDGF/vascular endothelial growth factor 2005; Ustach et al., 2010; Ustach and Kim, 2005). (PDGF/VEGF) family and reside in humans on 4 and Riehle and colleagues used tPA knockout mice crossed with 11, respectively (Uutela et al., 2001). In addition to having the transgenic mice overexpressing PDGF-C and showed that cleaved common structure of classic PDGFs (i.e. PDGF-A and PDGF-B), PDGF-C levels remained high (Riehle et al., 2014), indicating that PDGF-C and PDGF-D both have an additional unique N-terminal other proteases might also be involved in cleavage of PDGF-C. A CUB domain (Complement subcomponents c1r/c1s, Urchin study also showed that using matriptase to remove the CUB domain (EGF)-like and Bone morphogenic increases the binding of PDGF-DD to extracellular matrix, whereas protein 1), which binds to the extracellular matrix to prevent the cleavage of the growth factor domain reduces the association of diffusion (Andrae et al., 2008; Bergsten et al., 2001; Bork and PDGF-D and extracellular matrix and can act as dominant-negative Beckmann, 1993; LaRochelle et al., 2001; Li et al., 2000). The CUB ligand that prevents PDGF-B mediated PDGFR-b activation (Huang domain blocks the receptor binding of the C-terminal growth factor and Kim, 2015). Interestingly, recent findings show that PDGFR domain and needs to be cleaved extracellularly to make the growth signaling can be modified by neuropilin-1, which binds PDGF-D and E. Folestad et al. / Molecular Aspects of Medicine 62 (2018) 1e11 3 functions as a co-receptor in PDGF-D/PDGFR-b signaling (Muhl in infarcted heart reduces the interstitial fibrosis (Liu et al., 2014). et al., 2017). PDGF ligand signaling is involved in several pathophysiological 3.1. Atherosclerosis conditions. Detailed expression patterns of the individual PDGF li- gands and receptors are complex and the in vitro and in vivo A major risk factor for developing vascular and cardiovascular expression have been found to differ. PDGFs are stored and released disease is atherosclerosis, a process in which lipid-containing pla- by a-granules of activated platelets, but can be further secreted by ques are slowly formed in the vascular wall followed by inflam- macrophages, fibroblasts, vascular SMCs, endothelial cells, epithe- mation that stimulates SMCs to proliferate and migrate. The cellular lial cells and glial cells (Andrae et al., 2008; Demoulin and Essaghir, changes in the vascular wall eventually lead to vessel occlusion 2014; Huang and Kim, 2015). PDGFs act on neighboring cells pri- (Raines, 2004). In atherosclerosis expression of both PDGFR-a and marily in a paracrine manner (Andrae et al., 2008). PDGFR-b are widely upregulated in the vessel wall, and they are PDGF-C promotes migration and proliferation of macrophages expressed by vascular SMCs and macrophages. PDGFR-b is more (Boor et al., 2010; Wågsater€ et al., 2009), endothelial cells (Li et al., abundantly expressed than PDGFR-a, and PDGFR-b signaling also 2005) and vascular SMCs (Crawford et al., 2009). Through its effects appears to be the driving force in the atherogenic process where on multiple cell types, PDGF-C has been shown to have a critical the receptor activation induces leukocyte migration (Andrae et al., role in embryonic development (Ding et al., 2000), fibrosis (Ponten 2008; Karvinen et al., 2009; Raines, 2004; Wågsater€ et al., 2009). et al., 2003), wound healing (Grazul-Bilska et al., 2003) and Furthermore, PDGFR-b expression is also induced during angiogenesis (Cao et al., 2002; Crawford et al., 2009; Li et al., 2005). monocyte-to-macrophage differentiation, and strongly expressed PDGF-D is mainly expressed in endothelial cells and SMCs in fatty streaks. Active vascular SMCs play a central role because (Gladh et al., 2016; Karvinen et al., 2009; Ponten et al., 2005; they proliferate and contribute to the thickening of the vascular Wågsater€ et al., 2009). It also plays a role in wound healing wall. Activation of vascular SMCs from a contractile to synthetic (Uutela et al., 2004), fibrotic processes, various cancers (Andrae phenotype of vascular SMCs is also associated with increased et al., 2008; Cortez et al., 2016; Ponten et al., 2005; Wang et al., PDGFR-b expression and signaling (Hansson, 2005; He et al., 2015). 2010) and cardiovascular diseases such as arteriosclerosis (Hsu Furthermore, PDGFR-b antibodies (Sano et al., 2001) or PDGFR and Smith, 2012; Karvinen et al., 2009). blocking kinase inhibitor (Kozaki et al., 2002) reduced the athero- sclerotic progression in an atherosclerotic mouse model. 3. PDGF receptors in vascular disease Low-density lipoprotein receptor-related protein 1 (LRP-1) forms a complex with PDGFR-b, which gives it an important role in The PDGFRs are broadly expressed in the cardiovascular system controlling this activation. Boucher and colleagues demonstrated in cells of mesenchymal origin, such as fibroblasts, SMCs and per- that treatment with the tyrosine kinase inhibitor blocked icytes. Consequently, PDGF receptor signaling is important for the activation of LRP-1-induced phosphorylation of PDGFR-b and proper development and maintenance of the vascular and cardio- reduced the size and area of the atherosclerotic lesions in mice vascular system. This was shown in knockout mouse studies, as (Boucher et al., 2003). both PDGFR null mice phenotypes are embryonically lethal (Andrae et al., 2008). PDGFR-a signaling is indispensable in organogenesis 3.2. Restenosis (Soriano, 1997), and PDGFR-b signaling is crucial for proper devel- opment of the vascular system. PDGFR-b is mainly expressed by Restenosis and chronic rejection are vascular diseases that give pericytes and its signaling is necessary for proper recruitment of rise to conditions similar to atherosclerosis. Following blood vessel vascular SMC and pericytes (Leveen et al., 1994). PDGF receptor injury and angioplasty, medial vascular SMCs migrate to the intima, activation has been particularly linked to angiogenesis where where they proliferate and switch to a synthetic phenotype in a PDGFR-b expression especially correlates with increased vascu- PDGFR-b dependent manner, resulting in neointimal hyperplasia larity and maturation of the vascular wall (Cao et al., 2002; Laschke (Raines, 2004). PDGFR-a immunoreactivity is increased in the et al., 2006; Raica and Cimpean, 2010). media of arteries in acute rejection (Tuuminen et al., 2009). Many signaling pathways with essential functions in normal PDGF inhibitors have inhibitory effects physiology are also often key players in the development and on restenosis after balloon angioplasty (Bilder et al., 1999; Sihvola progression of diseases. Dysregulation of PDGF receptor activity is et al., 1999). Micke and colleagues showed that protein tyrosine seen in a number of common pathological conditions such as phosphatases are important in PDGF signaling in vascular remod- atherosclerosis, which is the main underlying cause of cardiovas- eling (Micke et al., 2009). The adventitial layers of vessels were cular diseases, including myocardial infarction and stroke (Hu and studied in a rat carotid artery model. PDGFR-b were upregulated Huang, 2015; Raines, 2004). Altered expression of PDGFR-a and together with the receptor antagonists density-enhanced phos- PDGFR-b have been found in cardiovascular diseases such as phatase-1 (DEP-1) and T-cell protein tyrosine phosphatase (TC- myocardial infarction and stroke and in vascular diseases such as PTP). The mRNA expression of PDGF ligands, PDGFR-b and protein aortic aneurysm and pulmonary arterial hypertension (Hu and tyrosine phosphatases DEP-1, TC-PTP, SHP-2 and PTP1B correlated Huang, 2015). In general, two types of cells appear to respond in with vascular morphometrics, proliferation and PDGFR-b activity. a pathological fashion to increased PDGF receptor activity, namely SMCs and fibroblasts. As a response to the enhanced receptor 3.3. Stroke expression and signaling, these cell types increase in proliferation, differentiation, apoptosis, migration and invasion, which promotes PDGF signaling is also important in stroke where activation of vessel wall pathologies and fibrotic tissue scarring. PDGFR-b seems PDGFR-a on perivascular astrocytes is involved in the acute opening to be the PDGF receptor principally involved in vascular pathology. of the blood-brain barrier (BBB). In a study it was shown that The role for PDGFR-a signaling is to stimulate various types of treatment of mice with the PDGFR antagonist imatinib after an mesenchymal cell/fibroblast-driven pathological processes, ischemic stroke reduced brain damage by decreasing the cerebro- including fibrotic responses. In infarcted heart, expression of the vascular permeability and hemorrhagic complications (Su et al., PDGF receptors are augmented in the myocardium in both early 2008, 2009). These data suggested potential new strategies for and late stages (Zhao et al., 2011). Blocking of PDGF receptor activity stroke treatment, and a phase II study showed that imatinib is safe 4 E. Folestad et al. / Molecular Aspects of Medicine 62 (2018) 1e11 and reduces neurological disability in patients treated with intra- 4.1. Atherosclerosis venous thrombolysis after an ischemic stroke (Wahlgren et al., 2017). PDGFR-b also play important roles in stroke. In adult In the atherosclerotic vessel wall, both PDGF-C and PDGF-D are normal matured brain PDGFR-b expression is low. It is, however, increased and mainly secreted by endothelial cells, vascular SMCs gradually induced predominantly in pericytes and fibroblast-like and macrophages (Karvinen et al., 2009; Raines, 2004; Wågsater€ cells in stroke areas after the induction of brain ischemia. After et al., 2009). PDGF-D is upregulated in endothelial cells exposed brain ischemia PDGFR-b mediates various function such as regu- to atheroprone blood flow and is induced during macrophage dif- lation of the BBB and tissue repair of infarcted areas (Cai et al., 2017; ferentiation. PDGF-D is strongly expressed in fatty streaks and its Nakamura et al., 2016). increased expression promotes vascular SMC phenotypic switch by inhibiting expression of multiple SMC , including smooth muscle a-actin (a-SMA) and smooth muscle myosin heavy chain 3.4. Pulmonary arterial hypertension (Raines, 2004; Thomas et al., 2009). Furthermore, PDGF-D is upregulated in the intima following angioplasty and has been Pulmonary arterial hypertension is a vascular disease involving suggested to be involved in the formation of neointimal hyperplasia endothelial dysfunction and proliferation and migration of vascular induced at vascular injury, and might serve as a target in preventing SMCs causing increased vascular resistance, which eventually leads vascular restenosis (Chen et al., 2005; Raines, 2004). PDGF-C and to right ventricular failure and death. Increased expression and PDGF-D were upregulated in adventitial layers of vessels in a rat phosphorylation of PDGF receptor has been correlated with pul- carotid artery model (Micke et al., 2009). As a result of macrophage monary arterial hypertension in various experimental animal accumulation, PDGF-C levels increase in the arterial wall, resulting models (Guignabert et al., 2009; Hu and Huang, 2015) and in in SMC proliferation (Chen et al., 2014). It was shown that PDGF-C humans (Schermuly et al., 2005). The tyrosine kinase inhibitor and PDGF-D might play important roles in atherosclerosis by imatinib was shown to reverse experimental hypertension in ani- stimulating the activity of matrix metalloproteinase (MMP)-9 and mal models of pulmonary arterial hypertension (Schermuly et al., by influencing monocyte migration (Wågsater€ et al., 2009). 2005). In a human case of pulmonary arterial hypertension, ima- tinib showed promising preliminary results, but unfortunately 4.2. Fibrosis some serious adverse events, including subdural hematomas, also occurred (Hoeper et al., 2013; Shah et al., 2015). PDGF/PDGFR signaling plays essential roles during embryonic development, but its function in adulthood is often unclear and seems to be more detrimental than constructive in human physi- 3.5. Abdominal aortic aneurysm ology, except in wound healing (Chen et al., 2013). PDGF signaling plays a critical role during the proliferation and migration stage of An abdominal aortic aneurysm (AAA) is a disease characterized wound healing (Grazul-Bilska et al., 2003). Fibrosis results from the by vascular remodeling, infiltration of inflammatory cells and deposition of extracellular matrix primarily by myofibroblasts. modulation of vascular SMCs. Expression of PDGFR-a and PDGFR-b PDGFs, in particular PDGF-A and PDGF-B, play an important role in has been found in both human and mouse AAAs, and PDGFR-b is the phenotypic change of fibroblasts into myofibroblasts by stim- strongly expressed by vascular SMCs (Kanazawa et al., 2005; ulating proliferation and migration (Kinnman et al., 2003; Vorkapic et al., 2016). Using imatinib to inhibit PDGF receptor Tangkijvanich et al., 2002). activation in an animal model of AAA reduced the expression of The link between PDGF and transforming growth factor (TGF)-b phosphorylated PDGFR-b in aortas and hindered aneurysm for- signaling in the development of fibrosis is well described. TGF-b is mation, suggesting that it is beneficial to reduce PDGFR activation overexpressed and regulates fibroblast differentiation into myofi- in pathological vascular inflammation such as AAA (Vorkapic et al., broblasts. In addition, the connection of PDGF-C and PDGF-D and 2016). TGF-b has been studied. Chaabane and colleagues demonstrated the first link between TGF-b and PDGF-C and PDGF-D ligands. TGF- b 3.6. Adipocyte metabolism and angiogenesis reduced expression of PDGF-D and induced expression of PDGF-C. PDGF-D was downregulated in a model of lung fibrosis and this b fi Impaired regulation of the PDGF receptors is also linked to some correlated with TGF- and other brotic growth factors (Chaabane of the underlying causes of cardiovascular disease such as adult et al., 2014). fi obesity and type II diabetes. In patients with diabetes, increased In another brosis model, which used bile duct ligation or fi PDGFR-b signaling was found to accelerate the growth of aortic induced hepatic brosis model, PDGF-D was upregulated and vascular SMCs (Hu and Huang, 2015). Furthermore, PDGFR-b acti- adenoviral PDGF-D transfer induced hepatic stellate cell fi vation plays a significant role in the development of adipose tissue proliferation and liver brosis (Borkham-Kamphorst et al., 2015). neovascularization, and targeting PDGFR-b could be a strategy for In vitro stimulation of hepatic stellate cells upregulated expression preventing obesity and type II diabetes (Onogi et al., 2017). of TIMP-1, which attenuated MMP-2 and MMP-9 activity whereas levels of collagen I and a-SMA were unchanged. Macrophages are also known to play an important role in wound 4. PDGF-C and PDGF-D in vascular disease healing (Gilbertson et al., 2001; Glim et al., 2013). PDGF-D affects different stages of wound healing by enhancing recruitment of In healthy vasculature, the expression of PDGF ligands is low or macrophages in the skin and increasing macrophage accumulation undetectable. Yet, increased expression of all PDGF ligands, in skeletal muscle, which leads to acceleration of wound healing including PDGF-C and PDGF-D, are found in many vascular and (Gilbertson et al., 2001; Pierce et al., 1991; Uutela et al., 2004). Anti- cardiovascular diseases. Dysregulation of PDGF-D appears to be inflammatory M2 macrophages have increased mRNA and protein more prevalent than PDGF-C in classical cardiovascular diseases levels of PDGF-C, which is able to induce a-SMA expression in fi- involving proliferation and differentiation of vascular SMC. Fig. 2 broblasts (Glim et al., 2013). In contrast, lack of PDGF-C in M2 briefly summarizes expression and role of PDGF-C and PDGF-D in macrophages leads to decreased a-SMA expression in fibroblasts vascular wall. (Glim et al., 2013). Overexpression of PDGF-C could contribute to E. Folestad et al. / Molecular Aspects of Medicine 62 (2018) 1e11 5

Fig. 2. Schematic figure describing expression and role of PDGF-C and PDGF-D in vascular wall in vivo. PDGF-C and PDGF-D are produced by endothelial cells and SMCs and promotes migration and proliferation primarily of SMC in vivo and activation of MMPs as well as activation of fibroblasts.

fibrosis formation, whereas low expression of this growth factor rejection of cardiac allografts. could be involved in delayed wound healing (Eitner et al., 2008; Fan and colleagues investigated regulation of PDGF signaling Glim et al., 2013). In line with these results, increased wound pathway in myocardial fibrosis using desoxycorticosterone- healing was observed in diabetic mice when PDGF-CC was applied induced hypertensive, salt-sensitive rats (Fan et al., 2013). Expres- (Gilbertson et al., 2001). Diabetes mellitus is one of the leading sion of all members of the PDGF family increased, with the causes of impaired wound healing. A study on diabetic rats dem- exception of PDGF-D, which remained unchanged. Use of the anti- onstrates that a certain expression level of PDGF is necessary for inflammatory drug fasudil inhibited myocardial inflammation and normal tissue repair (Li et al., 2008). expression of PDGF-A and PDGF-B. PDGF-C and PDGFR-a were reduced whereas PDGFR-b was unchanged.

4.3. Cardiac remodeling 4.4. Adipocyte metabolism and angiogenesis PDGF-D is also suggested to be involved in cardiac remodeling and cardiac repair in infarcted hearts of rats. All PDGF ligands and Interestingly, results by Seki and colleagues suggest that receptors were expressed in normal myocardium. After cardiac angiogenic endothelial cells may modulate adipocyte metabolism infarction, increased expression of PDGF-D had been significantly and may provide a new target for treatment of obesity and meta- induced in the infarcted myocardium and was primarily expressed bolic disease (Seki et al., 2016). Endothelial production of PDGF-C by endothelial cells, macrophages and myofibroblasts, whereas during white adipose tissue angiogenesis regulated browning of expression of PDGF-B and PDGF-C were reduced (Zhao et al., 2011). the fat. Further, deletion of endothelial knockout of the Pdgf-c gene a In another study, PDGF-D induced cardiac remodeling in a or usage of PDGFR- neutralizing antibodies impaired conversion of mouse model where PDGF-D was overexpressed, specifically in the white fat to beige fat. heart. Overexpression of PDGF-D caused cardiac fibrosis followed by dilated cardiomyopathy, proliferation of interstitial fibroblasts, 4.5. Neovascularization in ischemia extensive deposition of collagen, vessel dilation, locally decreased capillary density, and an increased number of SMC-coated vessels The formation of collaterals and neovascularization to maintain (Ponten et al., 2005). Overexpression of PDGF-C in heart-specific blood circulation in ischemic vessels is important for maintaining transgenic mice also induced a less developed cardiac fibrosis normal function but may also be a disadvantage in cases of with hyperplasia of fibroblast-like cells and dilated cardiomyopathy tumorigenesis. (Ponten et al., 2003). The influence of PDGFs in cardiac allograft In cell culture, several types of vascular endothelial cells are able rejection was shown in studies by Tuuminen and colleagues to express PDGFR-a and PDGFR-b (Lee et al., 2013; Marx et al., 1994; showing that they all, with the exception of PDGF-B, increased TGF- Plate et al., 1992; Smits et al., 1989). The in vitro finding of functional b1 chronic rejection in rat cardiac allografts (Tuuminen et al., 2006, PDGFR on human microvascular endothelial cells suggests a direct 2009). Intracoronary adeno-associated virus-mediated PDGF-C and role of PDGFs in endothelial cells and SMCs and the importance of PDGF-D transfer augmented cardiac allograft inflammation, fibrosis PDGF in neovascularization (Beitz et al., 1992; Marx et al., 1994; and chronic rejection. Based on these results, there is a potential Risau et al., 1992; Smits et al., 1989). However, whether PDGF that inhibition of PDGF-A, -C and -D signaling may inhibit chronic signaling has a direct effect on endothelial cells in vivo is still 6 E. Folestad et al. / Molecular Aspects of Medicine 62 (2018) 1e11 unclear. Further, PDGFR-a and PDGFR-b were found to be expressed normalizing blood vessels in tumors and thereby improves the on newly formed blood vessels (Cao et al., 2002). efficacy of chemotherapy (Liu et al., 2011). Li and others have reported direct effects of PDGF-CC on endo- thelial cells or their progenitor cells in in vitro studies (Gilbertson 4.7. Single nucleotide polymorphisms et al., 2001; Li et al., 2005). Endothelial progenitor cells partici- pate in the neovascularization and re-endothelialization process PDGF-C and PDGF-D have several potential synonymous and that follows vascular injury. Endothelial progenitor cells’ role in non-synonymous single nucleotide polymorphisms (SNPs). In an vascular injury repair involves an interaction with platelets, which early attempt to identify novel SNPs in cardiovascular disease, are involved in the recruitment of endothelial progenitor cells to Peden et al. found several new loci (Coronary Artery Disease sites of vascular injury. PDGF-C is able to stimulate the endothelial Genetics, 2011). One of the hits belonged to rs974849 and was progenitor cells recruitment from bone marrow and promote their thought to belong to the PDGF-D gene. Further, Zhou and col- differentiation (Dimmeler, 2005; Li et al., 2005), suggesting that leagues evaluated the results from Peden et al. in Europeans and in PDGF-C stimulates angiogenesis by affecting endothelial cells. South Asians in the Han Chinese population (Zhou et al., 2012). There is further evidence that PDGF-C and PDGF-D contribute to Although they included only 161 patients with coronary heart angiogenesis (Li and Eriksson, 2003; Li et al., 2005; Ustach et al., disease and 112 controls, they could determine that SNP rs974849 2004). PDGF-C also induced angiogenesis in chicken embryos and was associated with an increased risk of coronary heart disease. mouse corneas (Cao et al., 2002). In addition, administration of This SNP has also been associated with increased cardiovascular PDGF-C has been found to promote post-ischemic revascularization mortality in elderly males (Alehagen et al., 2016). However, recent of mouse hearts and hind limbs (Li et al., 2005). Recently it was data reveals that this SNP is located about 120 Kb away from the reported that dysregulation of PDGF-C expression in limb tissues of PDGF-D gene and is actually much closer to a microRNA in an area diabetic mice contributed to impaired angiogenesis and that PDGF- of long intergenic noncoding RNAs. C might be a novel target for the treatment of ischemic cardiovas- Another SNP more close to the PDGF-D gene, 858A/C, cular diseases in diabetes (Moriya et al., 2014). rs3809021, was studied in patients with stroke, and the AA geno- PDGF-C acts on the PDGFR-a, which is upregulated in vessels in type was associated with increased risk for non-hypertensive ischemic hearts and it thus stimulates the migration of endothelial intracerebral hemorrhage (Bai et al., 2012). Not far from this SNP, cells and SMCs (Dimmeler, 2005). Indirect evidence that PDGF-C in the first intron at position þ3166, rs7950273 was studied in contributes to angiogenesis was shown by high levels of PDGF-C ischemic stroke but no significant differences were found (Han in angiogenic tissues, such as tumors, placenta, ovary and embry- et al., 2016). However, the study found an association between onic tissues, suggesting that PDGF-C induces growth of new blood the SNP and ischemic stroke in subjects without a history or family vessels (Cao et al., 2002; Clara et al., 2014). Furthermore, PDGF-C history of hypertension or diabetes in a Chinese population. was demonstrated to be a novel potent pro-angiogenic factor that Current studies indicate that abnormal PDGF-D activity could be induces endothelial cells proliferation in the kidney (Boor et al., causative in cardiovascular disease, emphasizing the importance of 2010). PDGF-D in these conditions. However, the review of the literature In 2007 Wang and colleagues showed that downregulation of revealed that not many studies have been done on genetic varia- PDGF-D led to the inactivation of Notch-1 and NF-kappaB DNA- tions of PDGF-C and PDGF-D in vascular disease. binding activity, which resulted in downregulation of target genes such as VEGF and the activity of MMP-9 (Wang et al., 2007). Their 5. Animal models to study the role for PDGF-C and PDGF-D in results also showed that the medium from downregulated PDGF-D vascular diseases inhibited tube formation of human umbilical vascular endothelial cells. This is in agreement with results from Zhao et al. that showed Diseases involving cardiac and vascular complications are often that silencing of PDGF-D also resulted in the reduction of VEGF in complex and studying the molecular mechanisms controlling these gastric cancer cells (Zhao et al., 2010). diseases is a challenge. However, the development of different animal models of vascular and cardiovascular diseases has provided 4.6. Neovascularization in tumors us with important tools for investigating the pathophysiology of different diseases and evaluating new therapeutic strategies. Uncontrolled blood vessel growth from existing blood vessel Further, the ability to generate knockout mice has made it possible tissue, which is one of the important mechanism activated in to understand the in vivo function(s) of different genes and spe- cancer, involves a large spectrum of signaling molecules as well as cifically studying the lack of gene of interested in disease situations. vascular cells, stromal fibroblasts and inflammatory cells (Cao, In addition, the use of mouse models, where genetic gain-of- 2013; Manzat Saplacan et al., 2017). These cells are constantly function and loss-of-function mutations are introduced, are also exposed to growth factors and cytokines, which modulate tumor well established approaches to test the role of certain genes in growth, invasiveness and metastasis (Hanahan and Weinberg, different pathological conditions such as different vascular dis- 2011). PDGFs and their receptors play a critical role for angiogen- eases. PDGF-C and PDGF-D have been studied in relation to vascular esis because they are often expressed in diverse tumors, which and cardiovascular disease by using several different mouse correlates with tumor growth and invasion (Cao, 2013). The critical models. role for PDGF-C is its expression and upregulation in tumor endo- thelial cells (Clara et al., 2014; Crawford et al., 2009), where it most 5.1. Knockout mice likely promote angiogenesis by activation of PDGFR-aa and PDGFR- ab in endothelial cells (Cao et al., 2002). Conversely, reduction or Genetic deletions of PDGF-C and PDGF-D have been reported. blocking of tumor angiogenesis and growth was found by inhibiting However, the phenotype of PDGF-C is complicated and background PDGF-C (Crawford et al., 2009; Hou et al., 2010). In a melanoma dependent; Pdgfc / mice on a 129S1/Sv genetic background die model, tumor-derived PDGF-C recruited fibroblasts and promoted perinatally from feeding and respiratory problems due to cleft tumor growth and angiogenesis (Anderberg et al., 2009). An palates. However, Pdgfc / mice on a C57BL/6 genetic background interesting finding is that overexpression of PDGF-D increases drug survive, but display cerebral ventricular malformations, abnormal delivery of the chemotherapy drug doxorubicin in mice by vascularization and skeletal deformations (Ding et al., 2004; E. Folestad et al. / Molecular Aspects of Medicine 62 (2018) 1e11 7

Fredriksson et al., 2012). The first article describing a PDGF-D complications such as vascular remodeling, including dilation of knockout mouse was recently published and, it showed that in vessels, increased density of SMC-coated vessels, and proliferation contrast to the other PDGF knockouts, the Pdgfd / is viable, fertile of vascular SMCs, leading to a thickening of tunica media was seen and show no gross abnormalities (Gladh et al., 2016). The pheno- when PDGF-D was overexpressed. Similar vascular abnormalities type of the PDGF-C deficient mouse makes it less suitable for use in were seen overexpressing PDGF-C but the thickening of arterial vascular disease models while the PDGF-D deficient mouse is much walls was a unique feature induced by PDGF-D overexpression. more useful in different pathological models. In another transgenic mouse model where PDGF-D was over- Although a severe phenotype was reported in the PDGF-C expressed in the basal epidermal keratinocytes using the K14 knockout mice it have been used to evaluate the effect of PDGF-C promoter, PDGF-D transgenic mice displayed vascular impact such activity in a neurodegenerative vascular disease such as amyo- as increased numbers of macrophages and elevated interstitial fluid trophic lateral sclerosis where the SOD1G93A strain was crossed pressure in the dermis (Uutela et al., 2004). with Pdgfc / mice (SOD1G93APdgfc / ). Decrease of Pdgfc expression restored vascular barrier properties, reduced motor 5.3. Other animal models neuron loss and delayed symptom onset (Lewandowski et al., 2016). Several animal models of AAA are available and they have pro- In addition, Pdgfc / mice has also been used in several other vided insights into the mechanisms of human AAAs. One pathological mouse models to assess role of PDGF-C. Since PDGF-C commonly used model is infusion of angiotensin II into a hyper- has been suggested to have an important role in kidney fibrosis, lipidemic mouse such as apoE / mice, where angiotensin II pro- PDGF-C deficiency has been investigated in several different mouse duces large suprarenal AAAs (Zaragoza et al., 2011). Dysregulation models (unilateral ureteral obstruction (UUO), unilateral ischemia- of PDGF-D in the form of decreased PDGF-D expression is found in reperfusion (I/R) and Col4a3-deficient (Alport) mice) of renal this model (Vorkapic et al., 2016) fibrosis. In the UUO model it was demonstrated that lack of PDGF-C Mice are the most commonly used animal model to study reduced the development of renal fibrosis as well as decreased the different pathological conditions but due to their small size some leukocyte infiltration. Yet, lack of PDGF-C did not have any effect on procedures may be difficult to perform. Rat models are often used microvascular function and tissue perfusion when assessed as to study heart failure because they are larger than mice, which capillary rarefaction it the three different kidney fibrosis models greatly facilitate surgical and postsurgical procedures. Myocardial mentioned above (Boor et al., 2015; Eitner et al., 2008). damage in rat hearts can be induced by several different methods Lately, the role of PDGF-C in adipocyte metabolism was inves- but the most common consists of ligation of the left coronary artery tigated in a mouse model where PDGF-C deficient mice was treated (Zaragoza et al., 2011). Through the use of this method, decreased with a b3-adregenic agonist to induce adipose angiogenesis and expression of PDGF-C and increased expression of PDGF-D was browning of subcutaneous white adipose tissue. In this study it is found in the infarcted myocardium (Zhao et al., 2011). suggested that lack of PDGF-C impairs browning of white adipose tissue (Seki et al., 2016). 6. Proteolytic activation of PDGF-C and PDGF-D Although the PDGF-D knockout mouse was recently reported, genetic deletion of have been described in two distinct Proteolytic activation is an important regulatory step for many models of renal fibrosis UUO and I/R. Pdgfd / mice showed . Proteases often play key roles in many signal transduction significantly reduced renal interstitial fibrosis in both models of pathways by regulating and controlling the levels of critical com- renal scarring (Buhl et al., 2016). Furthermore, disruption of PDGF- ponents that signals through the pathway. Therefore the amount of D signaling in the RIP1-TAg2 mouse model of pancreatic neuro- protease available can be an important aspect in many pathological endocrine tumors delayed tumor growth and prolonged survival conditions. The PDGFs are all proteolytically processed to become without any effects on the vasculature (Cortez et al., 2016). mature and active. The classical PDGF-A and PDGF-B chains un- Atherosclerotic mouse models such as low-density lipoprotein dergo intracellular proteolytic activation, while PDGF-C and PDGF- receptor-deficient mice (LDLR / mice) and apolipoprotein E-defi- D chains undergo extracellular activation to generate active pro- cient mice (apoE / mice) are widely used in studying the devel- teins. It has been reported that plasmin is able to activate both opment and progression of atherosclerotic lesions. These models PDGF-C and PDGF-D. However, due to the wide substrate specificity are useful tools in determine the role of different genes in athero- of plasmin it is probably not the physiologically relevant protease sclerotic development as well as they are also versatile for evalu- (Fredriksson et al., 2004, 2005; Reigstad et al., 2005). More specific ating the effectiveness of drugs for treating atherosclerosis extracellular cleavage of PDGF-C and PDGF-D is performed by (Whitman, 2004; Zadelaar et al., 2007; Zaragoza et al., 2011). Still, serine proteases, tPA activates PDGF-C, while PDGF-D is cleaved by descriptions of PDGF-C or PDGF-D deficient mice crossed with any uPA and matriptase (Ehnman et al., 2009; Fredriksson et al., 2004; of these mice models are lacking. However, upregulation of PDGF-D Reigstad et al., 2005; Ustach et al., 2010; Ustach and Kim, 2005). expression in apoE deficient mice have been described (Ponten However, there might be other proteases capable of cleaving PDGF- et al., 2005). C and PDGF-D that are still unknown. The fact that PDGF-C and PDGF-D are secreted as inactive pro- 5.2. Transgenic mice teins that dependend on the availability of the correct proteases complicates the biology and the investigations of their signaling Transgenic overexpression of PDGF-C and PDGF-D in mice has outcomes. been described in several different models. Generally, both PDGF-C and PDGF-D induces fibrosis when overexpressed. 7. Future perspectives Focusing in particular on mouse models of vascular and car- diovascular diseases, PDGF-C and PDGF-D has been transgenic 7.1. Pathological conditions overexpressed, specifically in the heart using the a-myosin heavy chain promoter (Ponten et al., 2003, 2005). Both growth factors Several of the suggested functions of the novel PDGFs in induced cardiac fibrosis, which is commonly seen in several car- different pathological vascular conditions come from studies diovascular complications. Furthermore, several vascular reporting dysregulation of PDGF-C and PDGF-D where they are 8 E. Folestad et al. / Molecular Aspects of Medicine 62 (2018) 1e11 either up or down regulated. It might be that PDGF-C and PDGF-D Another important aspect of PDGF-C and PDGF-D signaling is in do not need to be overexpressed to cause undesired cellular acti- the role of adipogenesis and thermogenesis, and especially in vation and pathological conditions. If the protease that can activate regulation of or by perivascular adipose tissue, which has received PDGF-C or PDGF-D is presented in an excess amount, it can cause more attention recently. Here, the involvement of the anti- increased activation of PDGF-C or PDGF-D, without any need for inflammatory pathway of peroxisome proliferator activator re- increased expression of the ligand itself. In order to accurately ceptors is of interest (Gabrielson et al., 2016). understand the role novel PDGFs play in different vascular patho- Finally, genetic variations within the PDGF-C and PDGF-D genes logical conditions, it is important to further study the endogenous and their implications for vascular disease as well as their regula- factors involved in their regulation. The following description of the tion by microRNAs is still relatively unstudied and so needs further suggested role of PDGF-C in stroke illustrates why it is so important investigation. to understand the exact role of all the players involved in the biology of the novel PDGFs. The serine protease tPA, which is Acknowledgements known to dissolve blood clots and to be involved in the activation of PDGF-C, is used to treat ischemic stroke patients. Thus, while tPA This research did not receive any specific grant from funding helps to dissolve the clot quickly and restore blood flow to the brain agencies in the public, commercial, or not-for-profit sectors. tissue it can also activate PDGF-C that is expressed in the brain, which enhances the opening of the blood-brain barrier. This allows Appendix A. Supplementary data an influx of inflammatory cells into the brain, contributing to edema, hemorrhagic transformation, and increased mortality. Supplementary data related to this article can be found at Treatment with tPA is the only FDA-approved treatment for stroke, https://doi.org/10.1016/j.mam.2018.01.005. but because of the effects caused by the opening of the blood-brain barrier, it must be started within 4.5 h of symptom onset. This limits References the number of patients that can be treated with tPA. 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