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List of Papers
This thesis is based on the following papers, which are referred to in the text by their Roman numerals.
I Rodriguez, A., Karén, J., Gardner, H., Gerdin B., Rubin K., Sundberg C (2008) Integrin 1 1 is involved in the differentiation into myofibroblasts in adult reactive tissues in vivo. Journal of Cellular and Molecular Medicine, 13(9b): 3449–3462 II Fuchs D*., Rodriguez A*., Eriksson S., Christofferson R., Sundberg C., Azarbayjani F (2010) Metronomic administration of the drug GMX1777, a cellular NAD synthesis inhibitor, results in neuroblastoma regression and vessel maturation without inducing drug resistance. International Journal of cancer (Published online January 2010) * Shared first authorship III Rodriguez A., Friman T., Gustafsson R., Kowanetz M., van Wieringen T., Sundberg C (2010) Phenotypical differences in connective tissue cells emerging from microvascular pericytes in response to over-expression of PDGF-B and TGF- 1 in normal skin in vivo. Manuscript
Reprints were made with permission from the respective publishers.
Contents
Introduction...... 9 PDGF...... 10 TGF- ...... 12 Integrins...... 13 Fibroblasts and myofibroblasts ...... 15 Pericytes ...... 16 Angiogenesis ...... 18 Fibrosis...... 19 Tumor stroma formation ...... 20 Targeting the vasculature ...... 22 Present investigations...... 24 Aims ...... 24 Results and discussion Paper I: Integrin 1 1 is involved in the differentiation into myofibroblasts in adult reactive tissues in vivo ....24 Results and discussion paper II: Metronomic administration of the drug GMX1777, a cellular NAD synthesis inhibitor, results in neuroblastoma regression and vessel maturation without inducing drug resistance ...... 25 Results and dicussion paper III: Phenotypical differences in connective tissue cells emerging from the microvascular pericyte in response to over-expression of PDGF-B and TGF- 1 in normal skin in vivo...... 26 Future perspectives ...... 27 Populärvetenskaplig sammanfattning ...... 28 Acknowledgements...... 30 References...... 32
Abbreviations
ECM Extra cellular matrix -SMA - smooth muscle actin PDGF Platelet derived growth factor PDGFR Platelet derived growth factor receptor HPSG Heparan sulfate proteoglycan VEGF Vascular endothelial growth factor CAF Carcinoma associated fibroblast TGF- Transforming growth factor- ALK Activin like kinase MMP Matrix metalloproteinase EMT Epithelial mesenchymal transition GFP Green fluorescence protein PlGF Placental growth factor EC Endothelial cell SLRP Small leucin rich proteoglycan TAM Tumor associated macrophage
Introduction
Tissue architecture and function is established during fetal development and postnatally. This is achieved through the concerted action and interaction of signaling molecules, generation of protein gradients, and cellular migration, proliferation and differentiation. As a consequence an extra cellular matrix (ECM) and a hierarchical defined vasculature are formed that resist physical tensional stress and fluid pressure in the newly formed tissues. The stroma of a tissue is defined as the supportive connective tissue where cellular waste products are cleared but also where gas exchange and delivery of nutrients occur. In addition it is also the site for inflammatory reactions. The stromal compartment consists of several components such as ECM proteins, blood vessels, and loose connective tissue cells. The parenchymal compartment is the functional unit of the tissue, for instance the epithelium of the intestine. In pathological conditions characterized by chronic inflammation, tissue architecture and function will be altered. In some cases an excessive production and deposition of ECM proteins will result in a pathological condition termed fibrosis that is reversible in some cases, but in general the fibrotic lesion persists. The reasons for this are largely unknown. The deposition and remodeling of the ECM is mainly mediated by activated fibroblasts and macrophages. Myofibroblasts are mesenchymal cells with a phenotype resembling that of fibroblasts, smooth muscle cells and pericytes. In addition to their role in ECM deposition and remodeling they also exhibit a contractile phenotype, for instance in wound healing, a characteristic highlighted by their expression of -smooth muscle actin ( -SMA) 1. In non- pathological conditions the formation of a functional vasculature is ensured by a tight regulation of signaling events in the tissue. In conditions characterized by chronic inflammation however, the cell signaling is less regulated and leads to the activation and formation of a defective vasculature, and consequently a sub-optimal oxygenation and clearing of waste products from the tissue. The pathological deposition of ECM and formation of blood vessels takes place in the stroma of a tissue and is regulated by paracrine and autocrine signaling. The aim of the studies that are discussed in this thesis was to elucidate processes that take place in the activated stroma during pathological conditions and the underlying molecular mechanisms. The studies have focused on the tumor stroma but also other pathologies and in vivo models where stroma formation and tissue activation is a pathological hallmark.
9 PDGF Platelet derived growth factor (PDGF) is a group of glycosylated growth factors that consists of four different polypeptide chain monomers designated PDGF-A, -B, -C and D, that in order to bind to their receptors and exert their biological functions have to form cystein linked homodimers or heterodimers 2. The homodimers are AA, BB, CC and DD and the heterodimer AB. The PDGFs are in addition divided into two groups depending on the presence of a CUB domain which is present on the PDGF- C and PDGF-D isoforms but are absent on PDGF-A and-B. These different dimers have different affinities to their cognate receptors, the platelet derived growth factor receptors PDGFR- and . These receptors belong to the family of receptor tyrosine kinases (RTK) 3, and as with the ligands they form homodimers or heterodimers with each other. The two different receptor chains are able to dimerize as / , / and / receptors. The interaction between ligands and receptors are shown in figure 1. PDGF-AA and PDGF-BB are secreted as cystein linked dimers. PDGF- AA exerts its biological functions through binding to the PDGFR / . PDGF- AA have been shown to have a mitogenic effect on cells of mesenchymal origin as well as being an important factor in development of tumors and as an angiogenic factor 4-6. In vitro studies have shed evidence that PDGF-BB is a potent mitogen and chemoattractor for cells of mesenchymal origin7. In tumors it can regulate growth of tumor cells both in a paracrine and autocrine fashion 8. During development it has been shown that it plays a crucial role in organ formation and in pericyte recruitment to blood vessels. Deletion studies of the PDGF-B gene in mice leads to lethality at birth or just before birth, predominantly around embryonic day 18,5, and is associated with defects in renal and cardiovascular development but also with hematological abnormalities 9. Furthermore and of major importance is the finding that pericyte recruitment is compromised in PDGF-B null animals. Loss of the PDGF-B genes results in aneurysm formation in the brain microvasculature due to failure to attract pericytes to endothelial cells 10. The same observation was also made for mice lacking the PDGFR- 11. The structure of blood vessels in PDGF-B animals is also severely compromised in that they exhibit vessel dilatation and endothelial cell hyperplasia 12. The role of PDGF-B has also been examined in animal models where the heparan sulfate proteoclycan (HPSG) binding part of PDGF-B has been deleted 13. This modification leads to poor pericyte coverage of blood vessels compared to wild type animals. The requirement of PDGF-BB might differ in development and in pathological conditions as has been suggested in studies using PDGFR- chimeric animals that, in contrast to PDGF-B and PDGFR- knockout animals, are viable 14, 15. The approach of these studies was to generate chimeric mice by preparing blastocysts from cells that were PDGFR- +/+, +/- and -/-. Cells that are
10 dependent on PDGFR- signaling, that is PDGFR- +/+, will outcompete PDGFR- -/- cells. The findings of these studies were that PDGFR- expression on fibroblasts is important in reparative processes in the adult but not during proliferation and migration in development. The role of PDGF-B in pericyte recruitment to blood vessels in tumors and other pathological conditions have been the subject of extensive studies. B16 melanoma tumor cells engineered to over-express PDGF-B and subsequently injected into mice led to an increased coverage of pericytes in the vessels of the formed tumors 16. Furthermore, it also led to an increase in tumor size but without increasing the vascular density of the tumors. In another study, glioma cells were engineered to over-express PDGF-B. Here, the over-expression also led to an increased pericyte coverage of vessels, but it did also increase the vascular density, in part by increasing the expression of vascular endothelial growth factor (VEGF) in tumor endothelial cells 17. In contrast, tumor cell over-expression of PDGF-B in a pancreatic tumor model resulted in increased pericyte coverage of tumor blood vessels and a decreased tumor growth due to a decreased microvessel density 18. PDGF-C and PDGF-D are secreted in a latent form that has to be cleaved by limited proteolysis to remove the CUB domains, which inhibits the function of the factors. PDGF-C can be activated by tissue plasminogen activator (tPA) whereas PDGF-D is activated by urokinase plasminogen activator (uPA) 19, 20. Both forms have been reported to play important roles in tumor progression 21. PDGF-C has been shown to promote tumor growth by recruitment of carcinoma associated fibroblasts (CAFs) that drives tumor growth through the expression of osteopontin, in a model for malignant melanoma 22. PDGF-D has also been shown to affect tumor progression depending on if it is secreted as a latent peptide or if it is subjected to proteolytic activation 23.
11
Figure 1. PDGF-PDGFR interactions. Dashed arrows indicate weak interactions.
TGF- Transforming growth factor- 1 (TGF- 1) is a multifunctional cytokine belonging to the TGF superfamily that consists of more than 40 members. It regulates different cell functions such as proliferation, apoptosis and migration. TGF- 1 is released as an 390 amino acid long precursor that needs to be cleaved to become biologically active and bind to its receptor 24. Binding of active TGF- is initiated by binding to the intrinsically active TGF- receptor type II. This complex will subsequently recruit the TGF- receptor type I 25. The TGF- receptor I belongs to a family of receptors consisting of at least 7 members known as activin like kinases (ALK). The most important members are ALK1 and 5, and they are serine/threonine kinases. Downstream signaling is mediated by the Smad proteins that are phosphorylated by the type I receptor. TGF- 1 promote signaling pathways that lead to fibrosis such as myofibroblast differentiation, stimulation of ECM protein synthesis and the induction of epithelial to mesenchymal transitions EMT 26. It is well established that TGF- 1 is required for myofibroblast differentiation both in vivo and in vitro by inducing expression of -SMA 27. TGF- 2 does also have the ability to promote myofibroblast differentiation, whereas TGF- 3 has the opposite effect 28. Tensional environmental forces
12 in combination with TGF- induction do also modulate myofibroblast differentiation. Fibroblasts cultivated in collagen gels with varying degrees of compliance indicated that incorporation of -SMA into the actin cytoskeleton was decreased in free floating gels compared with anchored gels when stimulated with TGF- 1 29. Another study utilizing an in vitro model for interstitial flow showed that antibody mediated blocking of the 1 1 integrin inhibited myofibroblast differentiation 30. Other approaches to study the role of TGF- induction and myofibroblasts in fibrosis include adenoviral overexpression of TGF- in models for lung fibrosis 31 and the use of transgenic animals 32.
Integrins The integrin family of adhesion receptors plays an important role in mediating the contact between cells and ECM and in cell-cell interactions. In humans, there are 18 subunits and 8 subunits that are able to form 24 different heterodimers 33(Figure 2). Signaling mediated by the various heterodimers regulate various cellular responses such as cellular migration, survival, proliferation and differentiation. A majority of the integrins bind ECM proteins. Integrins are also involved in cell-cell contacts by binding to non integrin receptors. The binding of integrin 9 1 to vascular adhesion molecule-1 (VCAM-1) permits leukocyte extravasation for example 34. Integrins when bound to the cell surface can exist in an activated form (stretched) or inactive form (bent). Different mechanism for how integrins bind their ligands have been proposed. One of them suggests that for an integrin to bind its ligand and be able to signal it has to adapt an activated form, which means that they have to change their structural conformation 35 and consequentially they increase the affinity for the ligand. Another mechanism by which integrins increase their ligand binding is by integrin clustering by lateral diffusion 36 37, a process referred to avidity modulation. Both these events will lead to assembly of actin filaments leading to a formation of a functional complex containing integrins, other components of the cytoskeleton and signaling molecules. The resulting complex is termed focal adhesion (FA) 38 and its main role is to enable signaling as well as anchoring the cytoskeleton with the ECM using integrins as a conduit. The largest family of integrins includes the 1 subunit. To date 12 different heterodimers containing the 1 subunit have been identified and their common theme is their binding to ECM proteins. Signaling of 1 integrins have been shown to play a critical role in both development and pathological conditions. When this subunit is knocked down in mice, embryonic lethality is observed at E5.5 for null homozygous mice whereas heterozygous mice developed normal 39. Integrins 1 1, 2 1, 10 1 and
13 11 1 bind native triple-helical collagens and in the case of 1 1 and 2 1 also laminin. Knockout animals have been generated for all these subunits. Integrin 1 knockout animals: Knockout animals are viable and fertile without showing gross abnormalities. They exhibit a hypocellular dermis compared to wild type animals. Embryonic 1 1 deficient fibroblasts proliferate at a slower rate in vitro 40. Furthermore, deficiency of 1 subunit shows that it is involved in the regulation of collagen41, suggesting that the presence of this integrin subunit act as a negative regulator of collagen synthesis. In tumor models, knockout mice show a reduced angiogenic response compared to wild type animals 42. This mechanism is mediated by increased protein levels of matrix metalloproteinase 9 (MMP9) and MMP7 in knockout mice, that in turn increase levels of the anti-angiogenic molecule angiostatin. Integrin 2 knockout animals: the integrin 2 1, binding collagen and laminin, is expressed in a variety of cells including mesenchymal cells, epithelial cells and platelets. Deletion of the 2 subunit in mice does not result in lethality. The observed phenotypes in knockout mice are reduced morphogenesis of mammary glands 43 and a defective ability of platelets to interact with collagen 44. The process of wound healing in these animals does not seem to be affected compared with wild type animals 43 although differences in neovascularization in wounds have been observed indicating that knockout animals have an increased ability to induce angiogenesis compared to wild type animals 45. In a tumor model utilizing 2 null mice, angiogenesis was increased in null animals compared to wild type animals in a VEGFR1 dependent fashion 46. Integrin 10 knockout animals: the 10 1 integrin is a receptor for collagen. When the 10 gene is inactivated, null animals exhibit a mild form of chondrodysplasia 47 and a reduced proliferative capacity of chondrocytes, but otherwise the animals are viable. Integrin 11 knockout animals: These animals are viable and fertile but exhibit defective incisors that lead to dwarfism and early postnatal lethality 48. When acute inflammation is induced in knockout animals they fail to reduce the interstitial fluid pressure compared to wild type animals49. Integrins play an important role in pathological conditions such as tumor development making it possible for malignant cells to migrate, and thereby facilitating metastasis 50. Integrins have also been shown to be involved in angiogenesis in reactive tissues which is the case for v 3 and v 5 integrins which are expressed on endothelial cells 51 and 1 1 and 2 1 integrins 52. Blocking of the v 3 integrin in tumors and cutaneous wound healing, leads to a down regulation of angiogenesis and blocking of tumor growth 53. In contrast to these findings however, is the angiogenic response encountered in v 3 knockout mice 54. When tumors are implanted in knockout animals they grow much faster and display an increased
14 angiogenic response compared to tumors injected in wild type animals, indicating that this integrin is not required for tumor angiogenesis.
Figure 2: The integrin family of receptors. Heterodimers of the and subunits are connected with lines.
Fibroblasts and myofibroblasts Fibroblasts are the main connective tissue cell type populating the loose connective tissues in the body. Fibroblasts have been shown to be a more heterogeneous cell type than previously believed, exhibiting differences both within and between tissues, as revealed by microarray experiments 55, 56. In resting tissues they exhibit a low production of ECM proteins. Upon tissue injury fibroblasts differentiate to myofibroblasts, a cell type that expresses - smooth muscle actin ( -SMA) and plays an important role in secretion and organization of ECM proteins. The differentiation process is preceded by an intermediate cell phenotype called proto-myofibroblast, observed at early phases of forming of granulation tissue for example. It is negative for - SMA but exhibits stress fibers. They also secrete a splice variant of fibronectin called EDA-fibronectin and TGF- , two factors important for myofibroblast differentiation 57. The presence of -SMA in myofibroblasts is associated with their contractile abilities 1 and is a widely used marker for identifying them. In addition other molecular markers have also been found
15 to be expressed such as vimentin, desmin, prolyl-4-hydroxylase and endosialin. At the ultrastructural level structures known as fibronexus, which are a specialized form of focal adhesions, are present and enables anchoring of -SMA with EDA fibronectin58, These structures are important for transmitting the forces that are generated by the stress fibers in the cytoskeleton to the ECM . Fibronexus are absent in fibroblasts and smooth muscle cells in vivo 59. In vivo and in vitro studies suggests that myofibroblasts are derived from residing fibroblasts 60 but other sources have also been suggested such as bone marrow derived mesenchymal cells 61, epithelial cells through the process of epithelial mesenchymal transition (EMT)62 and pericytes (discussed below). EMT is a term that refers to the process whereby epithelial cells change their phenotype towards a mesenchymal phenotype. This includes the down-regulation of adhesion proteins, particularly E- cadherin, involved in cell-cell contacts 63. EMT is a process that plays an important role during development. EMT has been implicated in a number of pathological conditions in the adult such as various diseases characterized by fibrosis e.g. kidney fibrosis 64, liver fibrosis 65 and lung fibrosis 66. In these conditions it is believed that the differentiation of epithelial cells to mesenchymal cells leads to the generation of myofibroblasts that contribute to the tissue fibrosis through their synthesis and deposition of ECM components. In solid tumors there is also evidence for this process as a source of myofibroblasts 67. In addition to epithelial cells it has also been shown that endothelial cells in tumors can undergo the process of EMT. In this case however the process is called endothelial to mesenchymal transition (EndMT). In one study this was shown in two in vivo tumor models employing the B16F10 melanoma model and the Rip-Tag2 model 68. EndEMT has also been shown to take place in cardiac fibrosis and kidney fibrosis 69, 70. Fibrocytes is the term used for bone marrow derived (BM) stem/progenitor cells that have the ability to home in and differentiate into myofibroblasts at injury sites and thus contribute to tissue fibrosis. Circulating fibrocytes can be identified by the expression of markers such as CD34, CD45 and collagen type I 71. Studies on human tissues and animal models have revealed a bone marrow origin for myofibroblasts in activated tissues. 72, 73
Pericytes Microvascular pericytes are defined as abluminal cells juxtapositioned to the endothelial cells of capillaries, venules and small arterioles74. They are embedded in a shared basement membrane with cellular protrusions that surrounds the endothelial tube. The basement membrane is a specialized
16 form of the ECM and consists mainly of collagen type IV, heparan sulfate proteoglycans (HPSGs) and laminin75. It has been suggested that pericytes have a role in mediating contraction of blood vessels 76 as well as to stabilize the vasculature by e.g. contributing to the formation of the endothelial basement membrane 77. Pericytes are a heterogeneous cell type differing in morphology, marker expression and function depending on the organ in which they reside. Pericyte coverage of vessels may vary in different organs reflected by different ratios of endothelial cells vs. pericytes 78. In the liver pericytes are called stellate cells and in the kidney mesangial cells. In these cases they are associated with epithelial cells of ducts and glomeruli respectively. In the microvasculature of the brain they play an important role in maintaining the integrity of the blood brain barrier 79, 80. There are no specific molecular markers of pericytes in tissues in vivo. Combination of markers has to be employed as well as spatial distribution i.e. their juxtaposition to the endothelial cells in the microvasculature. The heterogeneity depends on the organ, but also if the tissues are activated or not. Common markers for pericytes are -SMA, PDGFR- , Rgs-5, Ang-1, desmin and NG2. In activated tissues, NG2 is most commonly used 81. PDGFR- have also been suggested as a marker for pericytes in activated tissues 82, 83. Numerous studies indicate that pericytes behave like a multipotent stem cell/progenitor cell. Evidence for differentiation toward a number of lineages has been reported 84-86. Pericyte differentiation to fibroblasts and myofibroblasts suggests that they play an important role in wound healing, but also in conditions characterized by chronic inflammation such as rheumatoid arthritis and cancer. It has been shown that upon tissue activation, pericytes undergo a proliferative burst 87 followed by a migration towards the surrounding connective tissue, changing their marker profile into that of a collagen type I producing fibroblast 82, 88. In human pathologies a pericyte origin for activated fibroblasts and myofibroblasts have been suggested such as in systemic sclerosis 89, in tumors such as breast cancer 90 and in kidney fibrosis 91. Most of the studies showing a link between the differentiation of pericytes to myofibroblasts and activated fibroblasts in vivo are based on immnunohistochemical techniques that gives indirect evidence for the pericyte origin of myofibroblasts. However, the use of transgenic animals has enabled more direct investigation of the origin of myofibroblasts. One study, employing a transgenic mouse model that expressed the reporter gene green fluorescent protein (GFP) under the promoter and enhancer of the pro- (I) collagen gene (col1a1), revealed that the major source for Col1 1 expressing myofibroblasts in inflammatory injury in kidney were derived from pericytes 92. Moreover, the study showed that blood borne precursors, termed fibrocytes (see above), contributed to a minor extent to the pool of myofibroblasts. In a subsequent study another
17 transgenic animal model was used, this time to evaluate to what extent kidney epithelial cells contributed to myofibroblasts through EMT. By using cre/lox techniques, epithelial cells were genetically labeled making fate tracing of cells possible. This study revealed that epithelial cells do not contribute to myofibroblasts in inflammatory injury, but instead it is the pericyte that is the main source 93. Taken together with other differentiation fates, pericytes may be an important source of various cell lineages in conditions requiring reparative processes as well in fibrosis, making the pericyte an attractive candidate for the purpose of regenerative medicine.
Angiogenesis Vasculogenesis and angiogenesis are two principally different mechanisms by which blood vessels are formed. Vasculogenesis takes place primarily during embryonic development and is characterized by the differentiation of a vascular precursor cell, termed angioblast, to endothelial cells that subsequently forms a primitive vascular network. Arterial and venous identity is established at this stage 94. Angiogenesis is on the other hand the process whereby new blood vessels are formed from already existing ones. Angiogenesis plays an important role in physiological processes in the adult, such as in the growth of the endometrium during the ovarian cycle and placenta formation during pregnancy. During physiological conditions, angiogenesis is tightly regulated by a number of growth factors. In pathological conditions however, such as in cancer and rheumatoid arthritis, this process is deregulated, leading to abnormal blood vessels as well as an abnormal ECM with regards to structure, composition and function. In tumors, hypoxic regions develop as a consequence to deficient oxygenation due to aberrant blood vessel formation and function. The angiogenic switch is initiated by the tipping of a balance, consisting of pro-angiogenic factors on the one hand and anti-angiogenic factors on the other, in favor of the pro-angiogenic factors 95. This leads to activation of blood vessels resulting in dissociation of the basement membrane and the subsequent detachment of pericytes. This is followed by proliferation and migration of endothelial cells. Eventually the formation of endothelial sprouts will take place, a process termed sprouting angiogenesis that ensures migration of blood vessels into areas with high production of pro-angiogenic factors. In addition to sprouting angiogenesis, other modes of angiogenesis have been described such as intussuseptive angiogenesis and glomeruloid body formation. In intussuseptive angiogenesis connective tissue pillars forms into the lumen of vessels, there by splitting them into more vessels 96, 97. This process is not dependent on endothelial cell (EC) proliferation. Glomeruloid body formation have been observed in tumors
18 such as gliomas but also in experimental tumor models 98, 99. The name is derived from the structure that rapidly proliferating endothelial cells give rise to, resembling a kidney glomeruli. Initially this structure consists of endothelial cells but pericytes and macrophages are also incorporated at later stages. These structures can also be generated by over-expressing vascular endothelial growth factor (VEGF) in normal tissues 100. VEGF is the most studied pro angiogenic factor. Since its discovery, different isoforms have been discovered as well as their cognate receptors. To date, five different mammalian isoforms have been identified; VEGF-A, - B, -C, -D and placental growth factor (PlGF). In addition a viral encoded variant showing resemblance to the above mentioned isoforms have been identified and named VEGF-E. The receptors for the ligands are receptors VEGFR1, -2 and -3. The VEGFs bind to their cognate receptors as either homo or heterodimers101.
Fibrosis Fibrosis is a condition characterized by an excessive deposition and stiffening of ECM proteins, mainly collagen that ultimately leads to organ dysfunction. Tissue fibrosis is the deleterious sequel in adult tissues of a suboptimal reparative process that is preceded by chronic inflammation. The main cell implicated in fibrosis is the myofibroblast. Fibrotic tissue is characterized by an accumulation of ECM proteins, with fibrillar collagen type I as the dominant component. The deposition of collagen is a process regulated by degradation and production of collagen as well as assembly of collagen into fibers. Cross linking of collagen molecules is also important for the formation of a fibrotic collagen network 102. For proper wound healing a provisional matrix has to be formed followed by wound closure. In cutaneous wound healing this is a regulated process that can be divided into different stages based on the morphological features of the tissue and presence of inflammatory cells103. Initially, the formed clot serves as a provisional matrix that facilitates the migration of inflammatory cells into the wound area. A wounded area will exhibit a disrupted ECM, and as a consequence cryptic sites of ECM proteins, such as collagens, will be exposed enabling integrin-mediated migration of various cell types. Invading macrophages will start to synthesize a broad range of cytokines and growth factors that will promote further infiltration of cells 104. Subsequently, epithelization takes place, covering the newly formed connective tissue, termed granulation tissue which contains a high density of newly formed capillaries. The growth factors secreted by infiltrating macrophages, PDGF- BB and TGF- in particular, play important roles. PDGF-BB stimulates fibroblasts migration and TGF- drives the differentiation of fibroblasts
19 toward a myofibroblast phenotype. In addition, ECM protein expression is also up-regulated, mainly in the form of collagen type I. In adults the reparative processes are not optimal and may result in fibrosis. Aberrant cutaneous wound healing in adults can result in keloids or hypertrophic scars. In both cases there is an excessive deposition of collagen and glycoproteins, however, whereas hypertrophic scars have the ability to regress keloids seldom do. With regard to structure, keloids exhibit thick collagen fibers whereas hypertrocphic scars exhibit nodular structures containing -SMA containg cells and blood vessels and in addition fine fibrillar collagen fibers 105. In addition to the deposition of collagen the expression of collagen organizing proteins play an important role in the stiffening of the fibrotic stroma. Lysyl oxidase is an enzyme that have an important role in collagen cross linking 106. In a model for liver fibrosis an inhibitor of lysyl oxidase has been shown to reduce stiffness in the liver in a tetra chloride induced liver fibrosis 107. The activity of Lysol oxidase has also been shown to increase tumor progression in a breast tumor model 108. Proteins belonging to the family of Small leucin rich proteoglycans (SLRP), such as the collagen binding biglycan, fibromodulin and the fibronectin/collagen binding decorin have also been implicated in fibrosis 109. Furthermore, there also exist differences in scar formation in fetuses and adults. Fetal dermal wound healing proceeds virtually without scarring in a regenerative manner with restoration of tissue architecture and function 110. The mechanism behind this important difference remains to this date unknown but various suggestions have been proposed. One mechanism may be differences in the activation of apoptotic programs at different gestational phases 111 Other suggestions are different responses of fetal and adult fibroblasts to the pro-fibrotic growth factor TGF- 112, expression of matrix metalloproteinases (MMPs) 113 and collagen cross-linking enzymes such as lysyl oxidase 114.
Tumor stroma formation Most of the processes involved in stroma formation have their counterpart in wound healing. The important difference lies in the sequence and duration of events such as fibrin clot formation, migration of cells and blood vessels and formation of granulation tissue. In cutaneuos wound healing for example the different stages of the wound healing process are tightly regulated and the end product is the formation of a scar. In tumors, angiogenesis and collagen deposition is constantly progressing through paracrine and autocrine signalling. Therefore it has been proposed that tumors are wounds that do not heal 115.
20 In the initial steps of tumor stroma formation, deposition of a provisional matrix takes place and is sustained throughout tumor progression. Formation of this provisional matrix is preceded by extravasation of plasma proteins across permeable venules, and consists of cross linked fibrin 115 which facilitates the invasion of mesenchymal cells and blood vessels 116, 117. Formation of this fibrin network is enabled by the extravasation of plasma fibrinogen and the enzymatic activity of thrombin and factor XIII 118. The observed permeability of blood vessels is chiefly mediated by the cytokine vascular endothelial growth factor-A (VEGF-A). Initially it was given the name vascular permeability factor (VPF) due to its ability to render the microvasculature permeable to plasma proteins 119. The discovery of VEGF/VPF was made in tumors and subsequent studies revealed that a wide range of human and murine tumor cell lines secreted this protein 120. The provisional stroma is gradually replaced by inflammatory cells, fibroblasts, blood vessels and an extra cellular matrix (ECM) composed of mainly collagen. Infiltrating cells, such as myofibroblasts degrade the cross- linked fibrin through the enzymatic activities of secreted proteins and in turn deposit collagen. These myofibroblasts play a crucial role in the formation and function of the tumor stroma 121. The presence and action of myofibroblasts will eventually lead to a desmoplastic tumor stroma, defined as a fibrotic scar like formation122-124. Fibroblast activation is driven by various growth factors secreted by tumor cells and inflammatory cells. Important among these factors is platelet derived growth PDGF-B125. The fibrotic reaction in tumors play a critical role in tumor progression 126. The fibrotic stroma in cancer can be classified depending on its cellularity and structure of the deposited ECM 127. The role of collagen deposition in tumors has been suggested to enhanced tumor progression. High levels of collagen I have been shown to increase metastasis 128, 129 The reactive stroma formed in tumor development exhibits structural and functional abnormalities compared to healthy tissues. In the tumor stroma, oxygen supply is hampered due to uncontrolled growth and development of defective blood vessels, creating an hypoxic environment that in turn have a promoting effect on tumor progression and impairs treatment efficacy130. Hypoxia has many consequences. For example, it has been shown that it promotes further DNA damage in tumor cells 131-133 and to increase the metastatic potential of the tumor either by directly affecting the tumor cells or by altering the behavior of stromal cells. The tumor stroma microenvironment contains a wide variety of cells that can be divided into the following groups: inflammatory cells, mesenchymal cells and endothelial vascular cells. The stroma microenvironment has emerged as a potential target for therapeutic intervention in the treatment of cancer. Paracrine signaling takes place between the tumor cells and stromal cells but signaling also takes place between the different kinds of stromal cells. In addition to cellular communication, there is also an interaction
21 between cells and the deposited ECM either through direct interactions by cell surface receptors of the integrin family or by release of growth factors that bind the ECM proteins, such as PDGF-B and TGF- . Remodeling by MMPs and other matrix degrading enzymes play an important role in this context. Inflammatory cells have for a long time been recognized as an important component of solid tumors, contributing in both tumor promotion and inhibiting tumor growth. An inflammatory environment has been proposed as a tumor initating event, especially in the case of carcinomas. This hypothesis was suggested over 150 years ago by Rudolf Virchow 134. Inflammation provokes an environment that promotes cell proliferation and the release of free radicals that induce DNA damage. Causative agents of the inflammatory state can be viral or bacterial infections or exposure to carcinogens. Inflammatory cells belonging to both the adaptive and innate immunity in tumors have various effects on the microenvironment. Recruitment of macrophages correlates with increased tumor growth in both experimental tumor models and human cancers. Recruitment of Tumor Associated Macrophages (TAM) by colony stimulating factor-1 (CSF-1) has been shown to promote tumor invasion 135. There are various mechanisms that have been suggested by which macrophages promote tumor growth. It can for example be either by inducing invasiveness of tumor cells 136, 137 and by promoting angiogenesis 138 through the release of pro-angiogenic factors. Infiltration of cells belonging to the adaptive immunity, on the other hand, is associated with a favorable prognosis. The proposed immunosurveillance theory is based on the observation that these cells limit tumor growth and tumor cell dissemination 139, 140. Carcinoma Associated Fibroblasts (CAF) is a term used for the heterogenic population of stromal fibroblasts. Myofibroblasts are defined as a subpopulation of CAFs. It is well known that CAFs provide a pro- angiogenic environment by secretion of various cytokines. One study utilized a co-injection approach where stromal fibroblasts isolated from invasive mammary ductal carcinoma together with MCF-7 human breast cancer cells were injected into mice 141. This mix caused tumors to grow faster compared with tumor cells injected with healthy fibroblasts. This effect depended on the secretion of the chemokine SDF-1 that recruited endothelial cell progenitors to the tumor and thus enhanced angiogenesis.
Targeting the vasculature In tumors, pericyte coverage of blood vessels is heterogeneous. A more abundant coverage of a subset of vessels might confer increased resistance for antiangiogenic therapies 142, probably by rendering pericyte covered ECs resistance to apoptosis. Therefore combinatorial therapies that target both
22 endothelial cells and pericytes as suggested by administration of both VEGFR and PDGFR inhibitors might hold a greater promise for optimal antiangiogenic treatment 143. PDGFR- inhibitors in tumors have also been shown to increase drug uptake due to lowering of interstitial fluid pressure144, 145. Thus by disrupting the contacts between ECs and pericytes, ECs will be more sensitive for antiangiogenic and chemotherapy treatment. Another strategy for more efficient treatment of tumors is to normalize the tumor vasculature i.e. increasing the fraction of pericyte covered vessels. Tumor vessels are leaky torturous and dilated. They do also show abnormalities in their basement membranes and pericytes are in loose association to the endothelium or completely absent 146, 147. A therapeutic consequence of this is that efficient delivery of chemotherapeutic drugs will be impaired. By inhibiting VEGFR signaling in both experimental models and in the clinic, it has been shown that drug delivery is facilitated by inducing normalization of the tumor vasculature 148, 149. Targeting the endothelial compartment of tumor vessels have been shown to selectively decrease the amount of defective vessels and thus leaving vessels with a more normal morphology. This phenomenon is termed vascular normalization or maturation 150. Targeting the signaling of VEGFR and epidermal growth factor receptor (EGFR) has been shown effective in normalizing tumor vessels and at the same time increasing radiation efficacy 151, 152 possibly by relieving tumor hypoxia.
23 Present investigations
Aims The aims of the studies were to 1. Investigate the role of integrins in myofibroblast differentiation. 2. Investigate the role of interference in cellular energy turnover in tumor. growth, resistance, stroma composition, vessel maturation. 3. Develop models for tissue fibrosis. Elucidate the roles of TGF- and PDGF-B in connective tissue cell phenotype.
Results and discussion Paper I: Integrin 1 1 is involved in the differentiation into myofibroblasts in adult reactive tissues in vivo The nature of the stroma, with regards to cell types and extracellular matrix, that forms in diseases characterized by chronic inflammation play an important role in disease progression. Common denominators in these conditions are the infiltration of inflammatory cells, deposition of ECM proteins and angiogenesis. Tissue activation leads to a disturbed balance between processes that regulate matrix turnover and angiogenesis. Myofibroblasts have been implicated as key players in matrix production. Deposition of ECM in activated tissues will alter the environment for a wide variety of resident tissue cells. Notably, cell surface receptors that mediate interaction between the cells and ECM will be upregulated or downregulated depending on the kind of ECM proteins that is being deposited. In this study we set out to study integrin expression and other activation markers on connective tissue cells in three different kinds of human activated tissues: wound healing, pannus formation in rheumatoid arthritis and colorectal adenocarcinoma. As control tissues, biopsies from normal skin, normal synovia and normal colon were used. Initially, immunohistochemistry was performed on tissues to identify where in the tissue the markers localized i.e. in the interstitium or in the vasculature. Next double immunofluorescence was performed with the same markers to identify the degree of overlap between the markers using computer aided image analysis 83. Differences in expression patterns emerged in the different conditions, most notably the overlapping pattern between the 1 1 integrin and -SMA on interstitial fibroblasts. This co-distribution led us to hypothesize that 1 1 integrin is important for the acquisition of the myofibroblast phenotype. To test this
24 hypothesis we employed one wound healing model and one tumor model in 1 integrin knockout animals. The wound healing model consisted in injecting Matrigel subcutaneously in wild type and knockout mice. The Matrigel data indicated that fibroblasts are able to migrate into the Matrigel implanted in knockout mice as detected by staining with the fibroblast antibody Reticular Fibroblast Marker (RFM). These cells however lacked expression of -SMA. Furthermore infiltration of CD31 positive blood vessels was decreased in knockout animals compared to controls. In inoculated tumors, the -SMA expressing cells were mainly confined to the vascular compartment, while interstitial cells were positive for RFM but negative for -SMA in knockout animals. A lower vascularity was also observed in knockout animals, in agreement with other studies on 1 deficient animals 42. However, in contrast to the Matrigel model the expression of RFM did not show any significant difference between knockout and wild type animals. These results indicate that 1 1 integrin plays a positive role regarding the acquisition of myofibroblasts. It is also a regulator of angiogenesis. The differences in RFM expression in both models could be explained by different mechanisms of recruitment of fibroblasts in tumors.
Results and discussion paper II: Metronomic administration of the drug GMX1777, a cellular NAD synthesis inhibitor, results in neuroblastoma regression and vessel maturation without inducing drug resistance Neuroblastoma is a common solid pediatric tumor of neuroendocrine origin with a low survival rate. It develops extracranially and it often originates in the adrenal glands. It is a highly vascularized tumor with a low abundance of stroma. In this study we administered the drug GMX1777 with the unique mechanism of inhibiting the enzyme nicotinamide phosphoribosyl transferase (NAMPRT) that plays an important role in the re-synthesis of NAD, which has an important role in cell metabolism. The neuroblastoma cell line IMR-32 was used. Cells were injected in the right hind leg and when tumors reached 0,5 ml the animals were randomized. This drug was administered subcutaneously by metronomic dosing i.e. repetitive at low doses in order to reduce toxicity. In order to identify the optimal dose, primary tumors were treated with 7,5 and 15 mg/kg/day. Treatment with 15 mg/kg/day gave the highest tumor size reducing response without inducing drug toxicity. In a third treatment group tumors were allowed to relapse 3 times before sacrificing the animal. In addition, 2 control groups were included; one to match the relapse group and one to match the time frame for the high dose treatment of primary tumors. Differences in tumor stroma formation, vessel maturation, apoptosis and proliferation were observed in
25 the different treatment modalities. Primary tumors treated with 15 mg/kg/day showed to our surprise an increased microvascular density compared to control tumors. When analyzed with immunofluorescence these vessels had an increased coverage of pericytes compared to controls, indicating maturation of blood vessels. Furthermore, stromal expression of PDGF-B, VEGF and GLUT-1, a marker for hypoxia were also reduced. The reduction of growth factor expression suggested a silencing of the stroma and in addition a reduction of tumor hypoxia. A decrease of tumor cell proliferation was observed. These data suggests that metronomic administration of the drug GMX1777 affects both the tumor cell compartment and stromal compartment.
Results and discussion paper III: Phenotypical differences in connective tissue cells emerging from the microvascular pericyte in response to over-expression of PDGF-B and TGF- 1 in normal skin in vivo Fibrosis is regulated by a wide variety of growth factors. In order elucidate the role of PDGF-B and TGF- over-expression in resting tissues, we developed a model based on adenoviral over-expression by these two factors. In addition we used an adenovirus expressing the reporter gene green fluorescent protein (GFP) as a control virus. By injecting these recombinant adenoviruses and the harvesting the tissues at different time points (3, 7 and 14 days) we could perform a temporal study of the transient over-expression of the growth factors. Tissues were harvested and analyzed by regular immunohistochemistry and immunofluorescens in order to follow cellular events in the vasculature and in the interstitium. Over-expression of both growth factors lead to activation and detachment of pericytes from the endothelium and a concomitant decrease in vascularity. This decline was visible after three days. In both condition an influx of macrophages was also observed. The differences were formation of glomeruloid bodies in PDGF treated ears but not in TGF- treated ears. Furthermore, activated fibroblasts in both conditions originated from microvascular pericytes but their phenotypes differed in the different treatment modalities. TGF- over- expression led to the generation of -SMA expressing fibroblasts, while in PDGF-B over-expressing tissues the fibroblasts were negative for -SMA but positive for the fibroblast marker RFM and for the pericyte marker NG2. These results confirm the role of TGF- in myofibroblast differentiation whereas PDGF-B does not seem to be involved in this process.
26 Future perspectives
Understanding the origin and differentiation of activated fibroblasts is an important foundation for the treatment of fibrosis. Various kinds of progenitor cells for activated fibroblasts have been suggested such as bone marrow derived precursors, epithelial cells through EMT and pericytes. The use of knockout technology in mice has shown a great potential in elucidating how different proteins are involved in physiological and pathological processes. The use of integrin 1 1 in the present study elucidated an important role in myofibroblast differentiation. One attractive approach that could elucidate the underlying mechanism more in detail would be to use microarray analysis of the tissues that were examined in the study. Hopefully new signaling pathways could be discovered in this way. This information could be used for designing new drugs that target candidate molecules. A proteomics based approach would of course be equally valuable. The approach mentioned above could also be used in a continuation of the neuroblastoma study. If coupled to laser capture microdissection, and combined with cDNA microarray techniques, a high throughput screening could be done for the stromal compartment and the tumor compartment and thus give a more detailed information of the regulation of the angiogenic switch in treated and non-treated tumors and of course what drives the myofibroblast differentiation in this model. The observed down-regulation of hypoxia, as indicated by GLUT-1 expression, and the normalization of blood vessels open up for other interesting treatment approaches. It would for example be interesting to use GMX1777 in combination with radiation therapy, or in combination with other drugs. The great advantage with the adenovirus study is that it allows examining one growth factor at the time. All the effects observed in the tissue are initially triggered by the over-expression of just one gene. Since the expression is transient, a long term study would be valuable in evaluating persistence of tissue fibrosis long after expression have ceased. Once the system is fully characterized, adenoviral over-expression of TGF- and PDGF-B could be used in combinations with for example tyrosine kinase inhibitors or inhibitors of other signaling pathways in order to link morphological and cellular processes with signaling pathways.
27 Populärvetenskaplig sammanfattning
Vid vävnadsskador sätter kroppen igång en läkningsprocess för att reparera skadan. Den skadade vävnaden börjar producera proteiner som t.ex. PDGF- B och TGF- vars uppgift är rekryteringen av omgivande celler för att läka skadan, samt att stimulera produktionen av bindväv. Huvudmålet är att återställa den skadade vävnadens struktur och framför allt dess funktion. Detta är en komplicerad process som omfattar flera steg, bland annat den nyss nämnda rekryteringen av celler, såsom bindvävsproducerande celler som kallas fibroblaster men även blodkärl och immunsystemets celler. Fibroblaster är vanligtvis vilande celler i frisk, vilket bl.a. betyder att deras produktion av bindväv är lågt. Fibroblasterna som attraheras till vävnadsskadan däremot, börjar producera bindväv och ger upphov till ärrvävnad. Produktionen av ärrvävnad leder till en nedsatt funktion av den skadade vävnaden. I vissa fall kan bildningen av ärrvävnad bli så omfattande att den skadade delen förlorar sin funktion helt. Att försöka förstå mekanismerna bakom fibroblast aktivering skulle underlätta att hitta behandlingar för sjukdomar som kännetecknas av ärrbildning. I den första studien studerades human friska och sjuka vävnader. De sjuka vävnaderna kännetecknas av ärrbildning och dessa var reumatisk artrit, sårläkning och coloncancer. Med hjälp av biokemiska tekniker identifierades cellassocierade proteiner i dessa vävnader. Aktiverade fibroblaster har på sin yta ett protein som heter -SMA. Ett annat protein, integrin 1 1 överlappade med -SMA, d.v.s. på aktiverade fibroblaster. Utifrån detta lade vi fram hypotesen att 1 1 på något sätt är inblandad i omvandlingen från vilande fibroblast till aktiverad fibroblast. Vi gick därför vidare att jobba med möss som saknar denna integrin. Genom att studera sårläkning och tumörbildning fann vi att aktiverade fibroblaster fanns i mindre utsträckning i dessa möss jämfört med normala möss. I den andra studien studerades effekterna av ett cellgift, GMX1777, på tumörer som tilläts växa i möss. Effekterna av cellgiftet var att tumörerna krympte men att det bildades fler blodkärl i dom jämfört med icke behandlade tumörer. Samtidigt kunde man i behandlade tumörer se att ärrbildningsvävnaden I tumören inte var aktiverad. Cellgiftet bidrog till att tumörcellerna dog och att den resterande vävnaden återgick till det normala. I den tredje studien undersöktes vad som händer om man på konstgjord väg producerar höga nivåer av PDGF-B och TGF- i normal hudvävnad på möss. Genförändrade virus som bar på den genetiska informationen för
28 proteinerna PDGF-B och TGF- , injicerade in i huden på möss i 3 olika tidpunkter (3 dagar, 7 dagar och 14 dagar) för att kunna följa temporala förändringar i huden som uppstod till följd av överproduktionen av proteinerna. I båda fallen bildades ärrvävnad samtidigt som tätheten av blodkärl försvann jämfört med möss som blivit injicerade med ett kontroll virus utan den genetiska informationen för ett aktivt protein. I båda fallen uppstod det aktiverade fibroblaster i vävnaden men dessa skilde sig åt i de två olika behandlingarna med avseende på vilka proteiner som de uttryckte på sin yta.
29 Acknowledgements
The work presented in this thesis was carried out at the department of medical biochemistry and microbiology at Uppsala University, Sweden. It was financed by Children’s Cancer Foundation in Sweden, Swedish Cancer Foundation, Swedish Medical Research Council and Lions Cancer Foundation.
I would like to express my gratitude to all people that have supported my all these years. I would especially like to thank the following people:
Christian Sundberg, for all interesting discussions and support all these years. I have learned more from you than you can imagine.
My examiner Kristofer Rubin for valuable help and interesting as well as helpful discussions
My co-supervisor Catharina Svensson for sharing your enthusiasm and encouraging words.
Anders Ahlander för ovärderlig hjälp med vävnadssnittning, men också för trevliga och givande samtal
My co-authors. In particular Dieter and Faranak for a fantastic collaboration. With all tumor material we have left in the freezer I am sure it’s enough for one more article .
The Sundberg Team. Tomas och Jakob för några fantastiska år med intressanta diskussioner om allt mellan himmel och jord, men även ett bra sammarbete. Och en hel del öl också. To Anqi for conversations about science and pericytes and movies.
Tijs and Vahid. Thank you for valuable discussions over the lab bench and elsewhere.
To all IMBIM people, past and present
30 Ett speciellt tack till Birger, för alla trevliga år vi känt varandra och för alla roliga aktiviteter. Allt från samtal, bio, pubkvällar på GH eller O’Connors, forskningssamarbete, jazzfestival och födelsedagar
Axel, Martin Kellner, Martin Sjödin, Sebastian, Martijn, Pontus, for being such wonderful friends. I promise I will keep in touch a little bit more frequent than the last years.
Eva och Hans-Erik. Dom bästa svärföräldrarna man kan ha.
Maria Nordin och Mattias Nordin. För alla trevliga stunder. Dom kanske är få men alltid väldigt trevliga.
Barbro Sandin. För trevliga samtal och för din stora värme
Pauli, Jens, Aron, Herman, och Samuel. Ert sällskap och ert stöd är ovärderligt. Med er är det bara glädje.
Mina underbara föräldrar. För all uppmuntran och allt stöd som ni har gett mig. Ganska säker på att David Attenborough och “Liv på jorden” har spelat in i mina studieval. Tack för att ni alltid finns där.
Hanna. Min underbara älskling. För ditt obeskrivliga tålamod. För all kärlek och förståelse du visat ända sen den dagen vi först träffades och jag berättade att doktorerandet var på sluttampen. Nu blir det lugnare framöver och mer tid för oss tre. Jag älskar dig!
31 References
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