The Roles of Growth Factor Interactions and Mechanical

          

             

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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 Rennel, E., Mellberg, S., Dimberg, A., Petersson, L., Botling, J., Ameur, A., Orzechowski Westholm, J., Komorowski, J., Lassalle, P., Cross, M.J., Gerwins, P. (2007) Endocan is a VEGF-A and PI3K regulated gene with increased expression in human renal cancer Experimental cell research, 313(7):1285– 94 II Kilarski, W.W., Samolov, B.,§, Petersson, L.,§, Kvanta A., Gerwins, P. (2009) Biomechanical regulation of blood vessel growth during tissue vascularization. Nature Medicine 15 (6) 657-64 §equal contribution III Kilaski, W.W., Petersson, L., Gerwins, P. A quantitative neo- vascularization assay for screening pro- and anti-angiogenic factors. Manuscript IV Petersson, L., Fredlund-Fuchs, P., Kilarski W.W., Gerwins, P. The role of growth factor receptor signaling for angiogenesis and vessel stability in an experimental wound healing model. Manuscript

Reprints were made with permission from the respective publishers.

Contents

Introduction...... 11 The vascular system ...... 12 Molecular markers of the vascular system ...... 13 Angiogenesis ...... 14 Sprouting angiogenesis...... 14 Intussusceptive angiogenesis...... 16 Non-angiogenic rearrangements of vessels ...... 17 Angiogenesis in physiological and pathological conditions...... 18 Lymphangiogensis...... 19 Myofibroblasts ...... 19 Wound healing ...... 22 Growth factors and cell signaling...... 24 FGF...... 25 VEGF...... 27 PDGF...... 29 TGF- ...... 31 Integrins...... 33 Mechanical regulation of cell signaling and response ...... 34 Methods ...... 36 Microarray...... 36 CAM angiogenesis model ...... 37 Cornea angiogenesis model...... 38 Cell sprouting assays...... 38 Present investigations...... 40 Results and discussion Paper I: Endocan is a VEGF-A and PI3K regulated gene with increased expression in human renal cancer...... 40 Result and discussion Paper II: Biomechanical regulation of blood vessel growth during tissue vascularization...... 41 Result and discussion: Paper III: A quantitative neo-vascularization assay for screening pro- and anti-angiogenic factors...... 42 Result and discussion Paper IV: The role of growth factor receptor signaling for angiogenesis and vessel stability in an experimental wound healing model...... 43 Future perspectives ...... 45

Populärvetenskaplig sammanfattning ...... 47 Acknowledgements...... 49 References...... 51

Abbreviations

ALK Activin receptor-like kinase SMA Alpha smooth muscle actin BMP Bone morphogenic protein CAM Chorioallantoic membrane ECM Extracellular matrix EGF Epidermal growth factor EMT Epithelial to mesenchymal transition eNOS Endothelial nitric oxide synthetase ERK Extracellular signal regulated kinase FAK Focal adhesion kinase FGF Fibroblast growth factor FGFR Fibroblast growth factor receptor HGF Hepatocyte growth factor HIF Hypoxia inducible factor LYVE Lymphatic vessel endothelial hyaluronan receptor MEK MAP (Mitogen activated protein kinase) kinase / ERK kinase MMP Matrix metalloproteinase NG2 Neuron glial antigen 2 N-cadherin Neural calcium dependent adhesion molecule OB-cadherin Osteoblast calcium dependent adhesion molecule PCR Polymerase chain reaction PDGF Platelet derived growth factor PDGFR Platelet derived growth factor receptor PECAM Platelet endothelial cell adhesion molecule PI3K Phosphatidylinositol 3 kinase PKC Protein kinase C PLC Phosholipase C PlGF Placental growth factor SMAD Sma/Mad (Small / Mothers against decapentaplegic) related TGF- Transforming growth factor beta TIMP Tissue inhibitor of metalloproteinase VE-cadherin Vascular endothelial calcium dependent adhesion molecule VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor VPF Vascular permeability factor

Introduction

The cells in our bodies, like in other multicellular organisms, need nutrients and oxygen to function which are delivered through blood vessels. If new blood vessels are needed they can form by a process called angiogenesis. This formation of blood vessels is an important component in many pathological conditions and it is therefore of great interest to understand how this process is regulated. In this thesis a number of results are presented that can hopefully increase our understanding in this area.

Figure 1. The blood vascular system as it was magnificently presented by Vesalius in the year 1543 [1] slightly cropped via http://commons.wikimedia.org.

11 The vascular system The blood vascular system in vertebrates and many other animals serve the function of transporting oxygen and nutrients to the cells throughout the body and to eliminate carbon dioxide and other waste products. It is also important for transport of signal molecules, like hormones, to their targets. The function of the vascular system was described by William Harvey in 1623 [2]. It is composed by two serially connected circulatory networks, the systemic and pulmonary circulations. The heart pumps blood through these networks: from the right ventricle into the pulmonary circulation and from the left ventricle into the systemic circulation. In the pulmonary circulation blood is oxygenated and cleared from carbon dioxide passing alveoli in the lungs. Oxygenated blood goes via the left heart into the systemic circulation to oxygenate the whole body before returning to the right atrium. Blood vessels are morphologically and functionally divided into arteries, veins and capillaries. Arteries are the vessels transporting blood from the heart and have a thick smooth muscle wall to withstand the high pressure and regulate blood flow by contraction. The inside of the arteries are lined with a continuous layer of endothelial cells that rest on a basement membrane that separates the endothelium from the smooth muscle layer. Arteries branch into smaller and smaller vessels and the smallest, called arteriole, feed blood into the capillaries. Capillaries are the smallest possible functional vessels, with only one endothelial cell forming the lumen and a diameter that only allows one blood cell to pass through at a time. The capillary endothelial cells also rest on a basement membrane and on the outside sit specialized mural cells called pericytes. These, like mural cells in larger vessels, can regulate flow by contracting the vessel. Pericytes differ from other mural cells both in their morphology and in their expression of markers. It is through the capillary wall that oxygen and nutrients diffuse to the neighboring cells. Capillaries drain into vessels called venulae that are the smallest veins and which merge into subsequently larger and larger veins until they feed into vena cava back to the heart. In veins the smooth muscle layer is much thinner than in arteries and large veins have valves that extend from the endothelium to prevent blood backflow.

12 Adventitia

Media - Smooth Muscle Layer

PericytePericyte Elastica interna - Basement membrane

Intima

Endothelial layer ARTERY VEIN EndothelialEndothelial CCellell

Endothelial Cells Basement Membrane

Vascular Smooth BasementBasement MMembraneembrane Muscle Cells CAPILLARY Figure 2. The structure of the different blood vessel types in the circulation.

In addition to the blood vessel network most vertebrates have a second vascular network, the lymphatic vessels. The lymphatic vascular system in humans was described in parallel by Olaus Rudbeck and Thomas Bartholin in the middle of the 17th century [3, 4]. Lymph vessels consist of blind-ended lymph capillaries merging into subsequently larger vessels. The largest lymphatic vessels empty into the venous circulation. The fluid in the lymph capillaries comes from the surrounding tissue by tissue pressure, and in this way excessive fluid is removed from tissues. Lymphatic fluid passes lymph nodes where immune cells can identify pathogens and atypical cells taken up. In addition lymph vessels take part in fat transport from the intestine. Lymph endothelial cells are similar to blood endothelial cells, but are a defined specialized cell type. Backflow in lymph vessels is prevented by valves similar to veins.

Molecular markers of the vascular system When identifying the vascular cells a number of proteins are used as markers. Endothelial cells express Platelet Endothelial Cell Adhesion Molecule, PECAM [5-7], also known as CD31 that is also expressed on lymphocytes and facilitate contact between these cell types. Both blood and lymph endothelium can express this receptor [8]. Another common endothelial cell marker is von Willebrand factor (vWF) that is also expressed in platelets and is involved in coagulation [9]. Smooth muscle cells in the vessel wall express smooth muscle actins (SMAs), including SMA [10]. Pericytes do sometimes contain SMA, but also express the chondrotin proteoglycan neuron glial antigen 2 (NG2) on their surface [11]. Lymph endothelium express the surface protein lymphatic vessel endothelial hyaluronan receptor one; LYVE-1 [12]. In addition the vascular cells

13 express, with some specificity, a number of receptors on their surface that will be described in more detail in the following chapters.

Angiogenesis Angiogenesis is defined as the process of formation of new blood vessels from pre-existing ones. This separates angiogenesis from vasculogenesis, where blood vessels form de novo. A new vessel is defined by the formation of a new branch point in the vascular tree. Thus other modifications of the blood vessels, such as enlargement, elongation or relocation strictly speaking are not angiogenesis. There are many ways described for formation of new branch points that can contribute to angiogenesis in vivo. Two major modes have been described with distinct differences, sprouting angiogenesis [13, 14] and intussusceptive (or splitting) angiogenesis [15-18].

Sprouting angiogenesis The naming sprouting angiogenesis comes from the resemblance to how a new branch sprouts from a tree. An elongating blind-ended endothelial cell row or tube, the sprout, will grow out from a pre existing vessel and connect with another vessel or sprout to form a new functional blood vessel. The origin vessel is usually a capillary or small venulae [13]. This process can be broken down in a series of defined steps: First, the basement membrane and the extracellular matrix (ECM) at the point of future sprout formation must be degraded to permit the endothelial cells to migrate out. Matrix degrading enzymes called matrix metalloproteinases, MMP, are secreted by endothelial cells at the point along the vessel where the sprout will form. The enzyme activity is regulated by specific endogenous MMP inhibitors, TIMPs. The most potent factor described stimulating this initial process is vascular endothelial growth factor (VEGF) [19-21]. To avoid excessive sprouting, where all endothelial cells along the origin vessel simultaneously degrade the basement membrane and start to sprout, there is a mechanism for lateral inhibition so that the endothelial cell first being activated will suppress this process in the adjacent cells [22-24]. With the basement membrane locally degraded the endothelial cell change shape and send a protrusion into the surrounding matrix. This cell that leads the sprout is called the tip cell. As it exits into the matrix the adjacent cells in the vessel will follow and are now called stalk cells. Sprout growth requires endothelial cell proliferation which takes place in the stalk, while the tip cell does not [25]. The tip cell, while not proliferating, migrates continuously through the ECM by degrading it guided by environmental cues such as concentration

14 gradients of growth factors such as VEGF [25]. It has also been shown that repulsive factors may influence the direction of tip cell migration [26, 27]. The stalk cells keep physical contact with each other and the first stalk cell also with the tip cell, forming a continuous solid cell row. The growing endothelial sprout needs to form a new lumen. The exact timing and mechanism of lumen formation varies slightly between different reports. One mechanism is through vacuole formation inside stalk cells [28, 29]. Theses vacuoles will fuse with each other and the plasma membrane in the ends of the cell forming a tube shaped cell. Another mechanism is by reorganization of the stalk cells that are laying parallel and overlapping to form a tube resembling a normal capillary [30]. Vascular smooth muscle cells are recruited to the sprout, either from the origin vessel, migrating along the new basement membrane laid out by stalk cells [13], or by differentiation of stromal cells [31]. Endothelial cells secrete growth factors such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-) to stimulate this process. Mural cells will wrap around the vessel and secrete factors that promote survival of the endothelium and make the vessel less sensitive to VEGF withdrawal [32- 36].

Figure 3. The steps in classic sprouting angiogenesis. First, endothelial activation and degradation of basement membrane allow tip cell to migrate out. The stalk of proliferating endothelial cells follows and forms a new vessel that can connect to another vessel. Mural cells (in light grey) wrap around the new vessel.

The last step in the formation of a new vessel through sprouting is connection with another vessel or sprout. If the sprout fails to connect and form a new vessel with blood flow, it will later regress by cell apoptosis since a blind ended blood vessel is not a functional part of the circulation. If the connected sprout is still not covered by mural cells it continues to be sensitive to growth factor withdrawal [32, 33, 37]. Some of the endothelial cells in the new vessels may have a different origin than the “old” vessel. I has been proposed that cells circulating in the bloodstream called endothelial circulating progenitor cells, of bone marrow origin, may integrate into the forming vessel and differentiate to endothelial

15 cells [38-40]. This is also a suggested mechanism for replacement of dead or injured endothelial cells in mature vessels. However the practical importance of this mechanism and to which degree it could provide support for angiogenesis is not established. In a similar fashion mural cell may originate from circulating progenitors [41, 42].

Intussusceptive angiogenesis Intussusceptive angiogenesis is also referred to as splitting angiogenesis. Similar to sprouting the process is described to take place in capillaries and venules. In contrast to sprouting it is not the endothelial cells that are initiating the process. A mural cell or adjacent stroma cell starts to push into the vessel, forming an invagination. The vessel wall with endothelium and basement membrane is intact during the process. When the invagination meets the contra lateral wall or a responding invagination from it, the endothelial cells on the opposing sides will make contact and rearrange to form two new vessel lumens [15, 16, 18, 43]. This division creates a new branch point that can be enlarged by elongation along the length of the origin vessel. The process can be likened to opening a zipper. The new vessels will lie parallel and close to each other. Proliferation of cells lying between the vessels and production of ECM may further push the vessels apart. This is a difference between intussusceptive angiogenesis and the more invasive sprouting. Another difference is that the vessels maintain functional blood flow during the process of intussusception, while a growing sprout is a blind ended non functional vessel until it fuses with another vessel [14, 17]. A variant to classic splitting angiogenesis has been described where endothelial cells form the branch point by sprouting into the vessel forming an endothelial bridge through the vessel. This process is referred to as bridging angiogenesis [44-46].

16 A

B

Figure 4. In the classic model for intussusceptive angiogenesis mural cells rearrange to form an invagination in the vessel, and finally splitting it in half (A). In the (B) picture is shown how the newly formed branchpoint can grow along the parent vessel to form two parallel new vessels.

Non-angiogenic rearrangements of vessels Enlargement of vessel diameter leads to that an increased blood volume can pass through the vessel. Fast regulation of diameter is provided by relaxation or contraction of the smooth muscle cells, and only persistent changes in the need of blood flow gives proliferative changes. Capillaries that enlarge can become either venulae or arteriole while the pericytes reform into specific smooth muscle cells [47-49]. Artierogenesis is a more specific process whereby smaller vessels (usually arteriole or small arteries) differentiate into larger arteries [50-53]. This involves changes to adopt the vessel to higher pressure and increased blood flow. The most prominent change is vessel wall thickening by proliferation of smooth muscle cells that also undergo changes in their surface receptor expression pattern and intracellular skeleton arrangement. In addition the endothelial cells proliferate and rearrange the basement membrane to generate an enlarged lumen. Since both sprouting and splitting angiogenesis has been shown to take place on the venous side of the vessel network this provides a secondary wave of adaption, when parts of the enriched venular/capillary network closest to the arterial side first differentiate into arteriole and through arteriogenesis grow to become arteries [50]. Vessel elongation usually takes place in parallel to changes in the surrounding tissue structure that can push or stretch the vessel[54]. To a

17 degree the vessel can elongate by only cell elongation and enlargement without cell proliferation [55].

Figure 5. Vessel elongation can initially occur without proliferation. In the picture the vessel is also relocated. The elongation can be initiated by surrounding tissue and cells by pushing or stretching.

Angiogenesis in physiological and pathological conditions Angiogenesis is involved in many physiological processes where expansion of the vascular network is needed. During the time an organism grows, angiogenesis takes place to support the demand of oxygen and nutrient transportation. In the full grown body angiogenesis takes place mainly in muscle growth, endometrial growth and the ovary [53, 56, 57]. Sprouting, intussusceptive and bridging angiogenesis as well as vessel elongation have been described in physiological angiogenesis. The role of angiogenesis in wound healing is discussed in a separate chapter. In many pathological processes excessive or insufficient angiogenesis plays a role. I malignancies vessel growth supports the growing tumor with oxygen and nutrients [58, 59]. The tumor environment provides a constant angiogenic stimulus by overproduction of factors such as fibroblast growth factors (FGFs), VEGFs and PDGFs [59]. In addition to formation of new blood vessels, VEGF-A also increase vascular permeability giving the tumor a high tissue pressure that lessens the availability of anti cancer treatment drugs to the tumor cells [60-65]. This high intratumoral pressure also leads to inefficient circulation and oxygenization driving further hypoxic responses.

18 Other pathological conditions where undesired angiogenesis is involved include diabetes mellitus, rheumatoid arthritis, psoriasis, retinopathies, asthma, and endometriosis [66-72]. If the angiogenic process is insufficient pathological conditions with ischemia can occur such as heart infarction, diabetic vascular insufficiency or peripheral arterial disease [72-74]. Ischemia due to arteriosclerosis or thrombosis triggers a pro-angiogenic reaction by VEGF release. The local effect of occlusion of smaller vessels could be reverted by forming new vessels through the hypoxic area, and therapies to promote this is being developed [73, 74]. However clinical conditions where vessel occlusions occur usually also involve a general negative effect on endothelial cell function and their angiogenic ability [72, 73, 75]. If the avascular area is too large angiogenesis will be insufficient. The effects of occlusion of larger vessels cannot be reverted directly by angiogenesis although arteriogenesis can improve the condition by transforming small collateral arteriole into larger arteries to replace the function of the occluded vessels [51].

Lymphangiogensis Lymphatic angiogenesis occur similarly to sprouting angiogenesis [76-78]. Two members of the VEGF family of ligands, VEGF-C and VEGF-D are the main known inducers of lymphangiogenesis. The lymph endothelial cells proliferate to form the stalk of the new capillary. Lymph capillaries are not covered by mural cells so no mural cells recruitment is needed. The sprout does not connect with another vessel since functional lymph capillaries are blind ended, but preexisting lymphatic capillaries can restart their sprouting to elongate. Intussusceptive mechanisms for lymphangiogenesis have not been described. Insufficient lymphangiogenesis might cause reduced fluid drainage and edema will form. Lymphatic capillary remodeling to form enlarged vessels include recruitment of the mural cells that normally cover larger lymphatic vessels.

Myofibroblasts In the 1970-ties a type of stromal cell was discovered in wound tissue that had a smooth muscle like phenotype [79], with a characteristic intracellular microfilamentous apparatus later identified containing smooth muscle actins [10, 80]. The cell type was based on this findings first proposed to participate in wound contraction. Since this stromal cell share some characteristics with fibroblasts, and it was later shown that normal tissue fibroblast could differentiate into these cells, they were named myofibroblasts. Sometimes the term activated fibroblast is used to include both myofibroblasts and intermediate cell types.

19 The process of differentiation from fibroblast to myofibroblast in tissues can be described in a series of steps. Normally the intact stroma protects the cells within it from any mechanical stress enforced upon the tissue. Damaged tissue, such as a wound, however loses this stress shielding ability. Fibroblasts react to mechanical stress first by changing their intracellular filament structure and contacts with the ECM [81]. This is triggered by different mechanosensing systems; both surface receptors and the cell skeleton itself. The intracellular fibrils formed during tensional stress are called stress fibers and are composed of cytoplasmic actins [81-85]. These connect to focal adhesion junctions at the cell membrane containing integrin v3 and paxillin that in turn connect to the ECM [83, 85] Similar connections to other cells are provided through N-cadherin containing cell- cell junctions [84]. These changes make the cell contractile to counteract mechanical stress. This cell is called a protomyofibroblast. Protomyofibroblasts can differentiate into mature myofibroblasts by specific chemical stimuli, but continuous mechanical stress is considered a prerequisite for full differentiation. The most important cytokine driving the differentiation is TGF- [86, 87]. In addition exposure to a splice variant of fibronectin, ED-A fibronectin expressed and secreted by the protomyofibroblasts themselves promote differentiation [88]. Even though TGF- is considered necessary for myofibroblast differentiation some TGF- independent stimuli have been described such as thrombin and endothelin [89-91]. The mature myofibroblast have a stress fiber network containing -SMA [10, 79, 80, 92]. The focal adhesions are enlarged and containing integrin 51 and tensin leading to stronger attachments to the ECM [83, 85]. In addition specialized cell-cell connections containing OB cadherin ensures a strong contractile cell network [84].

20 Mechanical Stress Fibroblast

Protomyofibroblast

Cytoplasmic actin TGF-β

alpha smotth muscle actin

Focal adhesions

Adherens junction (N-cadherin)

Supermature focal adhesion

Adherens junction (N & OB-cadherin)

Myofibroblast

Figure 6. The steps in the fibroblast to myofibroblast differentiation include expression of specific cell adhesion cytoskeleton proteins.

While tissue fibroblasts are thought to be the main source of myofibroblast, other cell types can also differentiate into myofibroblasts. Vascular smooth muscle cells can after migration from vessel wall into the stroma change marker expression to become myofibroblasts [93, 94]. Circulating bone marrow derived cells such as fibrocytes, a fibroblast precursor cell with stem cell characteristics [95], and monocytes [96] have also been shown to acquire a myofibroblast phenotype after leaving the blood stream. Epithelial cells can differentiate into myofibroblasts through a process called epithelial mesenchymal transition, (EMT) [97-99] which is driven by TGF- [100]. Endothelial cells have been shown to be able to transdifferentiate into myofibroblasts [101], in a similar manner as epithelial cells.

21 Pos sible de -differe ntiati on, FG F? O the r Fa cto rs?

Mechanical Stress Fibroblast

Differentiation

Protomyofibroblast TGF- β

Fibrocyte

β TGF- Vascular Smooth Muscle Cell GF PD β? TGF-

Pericyte Myofibroblast Epithelial Cell

Endothelial Cell Figure 7. Many cells in addition to fibroblasts have a capacity to become functional myofibroblasts.

Wound healing Repair of damaged tissue through wound healing serves to restore tissue function fast and efficient [102, 103]. While speed of recovery is achieved the healing process may not be able to fully replicating the previous tissue organization, resulting in a scar. The remaining scar tissue in skin after the wound healing process is finished can vary from minutiae to functionally impairing depending on the damage type. In more complex organs, however functional impairment by remaining scar tissue can give more serious long term problems. It is notable how the need for fast healing has evolutionary influenced the process. The initial stages with formation of disorganized wound tissue is quick during a time span of days, while the later stages where the provisional wound tissue is replaced by normal tissue can take from weeks to years in time. An overview of the different steps in skin wound healing serves to illustrate the wound healing process. A physical trauma damages the epidermis and dermis causing vessels damage and bleeding which triggers the coagulation cascade and formation of a blood clot containing fibrin, activated platelets and trapped blood cells, that forms the first physical protection of the wounded area. The fibrin network form by enzymatic cleavage of the soluble protein fibrinogen. Platelets, in addition to participate

22 in coagulation, also store cytokines in their intracellular granulae. These are released when the platelet is activated [103-105]. Together with other factors released by trapped and damaged cells and peptides released during the clotting [106, 107], these cytokines will act as chemoattractants for many cell types. The first cells to enter the wound are polymorphonuclear inflammatory cells from the circulation [103], that eradicate invading pathogens. Slightly later monocytes leave the blood vessels, migrate into the wound and differentiate into mature macrophages. These are versatile cells that are involved in almost all the processes in wound healing [103, 108]. They remove pathogens by phagocytosis, secrete cytokines and chemokines to promote cell differentiation and migration and have the capability to differentiate into other cell types such as myofibroblasts. Tissue fibroblasts resident in the wounded area or attracted to migrate into the wound from adjacent tissue by secreted growth factors are exposed to mechanical stress in the provisional unshielded wound tissue. The cells differentiate to protomyofibroblasts [87, 103]. Endothelial cells and vessel mural cells are affected by secreted cytokines initiating vessel growth into the wound by angiogenesis, possibly including both intussusception and sprouting. A vessel network is hereby formed in the wounded area that is denser than the one in the tissue before wounding. The vessels are also enlarged by proliferation and dilation and the dilated vessels increase the possible blood flow volume to the wound. The rich vasculature gives the wound its reddish color and is called granulation tissue [103]. During the granulation phase the protomyofibroblasts continue their full differentiation into myofibroblasts and their contractile network continue to contract the wound [87, 103]. They also begin to produce a new ECM network that restores protection against mechanical stress [79, 109, 110]. The epidermis is concurrently restored to cover the wounded area by epithelial proliferation and migration. The initial blood clot is thus replaced by granulation tissue and new epithelium. After this the wound resolution phase begins where granulation tissue is slowly resolved into normal dermis by the production of new ECM, blood vessel regression and disappearance of inflammatory cells and myofibroblasts [87, 103, 108]. It is still unclear if the myofibroblasts dedifferentiate into fibroblasts or undergo apoptosis. In the remaining fibrotic scar tissue the ECM will not fully resolve and stiffness of the matrix remains. In this milieu myofibroblasts may remain for a long time. In pathological wound healing these overactive remaining myofibroblasts can cause excessive scar tissue or contractures [87].

23 CLOT FORMATION INFLAMMATORY RESPONSE

Blood vessel

Neoutrophil

Macrophage Before injury Minutes 24 hours Fibroblast GRANULATION TISSUE WITH NEW TISSUE FORMED Myofibroblast INITIATION OF HEALING MASSIVE ANGIOGENSIS REMODELING OF VESSELS CELL MIGRATION AND VESSEL REPAIR MYOFIBROBLAST DIFFERNTIATION MYOFIBROBLAST REDUCTION

3 days 7 days 3 Weeks Figure 8. Overview of the steps in dermal wound healing. While the initial steps follow a specific pattern, the final resolving from wound tissue to normal tissue can take longer time.

Growth factors and cell signaling All cells in the organism need to interact and respond to changes in a controlled manner to serve their function. Any cell in an organism is exposed to a number of stimuli, both chemical and mechanical. Chemical stimuli include small organic molecules, ions and peptides/proteins that bind to specific receptors on the cell surface, thereby triggering a conformational change in the receptor that in turn can give an intracellular signal. In this thesis two groups of receptors are described that regulate angiogenesis and myofibroblast differentiation; tyrosine kinase receptors (receptors for FGFs, VEGFs and PDGFs) and the serine/threonine kinase receptors (receptors for TGF-). These receptors are transmembrane proteins that form receptor complexes after extracellular ligand binding, which induce kinase activity in their intracellular part. This kinase activity leads to phosphorylation of specific amino acids (tyrosine or serine and threonine respectively) in the receptor itself and/or in other intracellular proteins. Other growth factors involved in angiogenesis that signal through receptor tyrosine kinases are Hepatocyte Growth factor (HGF), Epidermal Growth Factor (EGF), Insulin-like Growth Factor (IGF) and the Angiopoietins (Ang1-4) [111-114].

24 A different group of cell surface receptors are the integrins. These are dimers consisting of one and one subunit that bind different ECM and basement membrane proteins via their extracellular part. They are known to interact with other receptors affecting their function. Co-receptors are the designation for additional cell surface structures facilitating and modifying binding between ligand and receptor. One important group of co-receptors are the proteoglycans, that consist of a protein core to which long glycosaminoglycan chains are covalently attached. The side chains can be of various compositions such as heparan sulfate, chondroitin sulfate or dermatan sulfate [115, 116]. Important membrane bound proteoglycans are syndecans and glypicans. There are also proteoglycans not attached to the cell membrane residing in the ECM or circulation. Endocan is an example of a proteoglycan with dermatan sulphate side chains that is produced by endothelial cells and released as a soluble molecule [117, 118].

FGF The first member of the fibroblast growth factor family was identified in 1974. Since then 22 members have been discovered [119]. FGFs form dimers and are produced and secreted from a variety of cell types. Several of the FGFs have a well defined restricted expression pattern while some, most notably FGF-1 (acidic FGF) and FGF-2 (basic FGF), are expressed generally in both physiological and pathological situations. The FGF receptors consists of four members FGFR 1-4, where 1-3 each have two splice variants designated b and c[119]. FGF receptors are present on the surface of a wide variety of cell types and mediate a number of cellular responses. Receptor activation by FGFs requires interactions with heparan sulphate proteoglycan, usually syndecan or glypican. These are therefore referred to as co-receptors [120-122]. The affinity for FGF to bind to heparin was important for their initial discovery [123, 124].

25 FGF-1 FGF-2 FGF-1 FGF-4 FGF-1 FGF-2 FGF-6 FGF-2 FGF-4 FGF-8 FGF-1 FGF-1 FGF-4 FGF-8 FGF-9 FGF-1 FGF-2 FGF-3 FGF-6 FGF-9 FGF-16 FGF-2 FGF-4 FGF-7 FGF-9 FGF-17 FGF-17 FGF-3 FGF-5 FGF-10 FGF-17 FGF-1 FGF-18 FGF-18 FGF-10 FGF-6 FGF-22 FGF-18 FGF-9 FGF-23 FGF-19

FGFR-1b FGFR-1c FGFR-2b FGFR-2c FGFR-3b FGFR-3c FGFR-4 Figure 9. The redundancy between the members of the large FGF family visualized. The different FGFs have specific expression patterns both temporally and spatially and loss of one specific ligand usually gives a limited phenotype. The most abundantly expressed FGF 1 and 2 can be functionally replaced by other family members See reference[119]for details.

FGF-1 and 2 where the first identified growth factors with pro-angiogenic activity [123, 124]. Gene targeting studies in mice showed that Fgfr1 and Fgfr2 null mice die early in development[125-127] while neither Fgfr3 nor Fgfr4 knockouts have vascular defects[128-130]. Regarding ligands, mice with targeted deletion of Fgf1 and Fgf2 have normal developmental angiogenesis, showing that other ligands can compensate them in vivo [131- 133]. The double knockout of Fgf1 and Fgf2 also has normal vascular development [133]. However wound healing is delayed and vascular tone is decreased in Fgf2 knockouts [131, 132]. Targeted deletion of Fgf4, Fgf8, Fgf9, Fgf10 and Fgf18 are lethal, but not directly due to vascular development defects (a complete review is found in reference [119]). In endothelial cells FGF-2 stimulate proliferation and secretion of proteolytic enzymes that can degrade basement membrane which are important functions during sprouting angiogenesis. FGF-2 can also increase the production of VEGF-A and expression of VEGF receptors and integrins. In vascular mural cells maturation and stabilization is promoted by the synergistic effects of FGF and PDGF receptor activation [134, 135]. On fibroblasts FGFR activation stimulates both proliferation and migration. FGF can oppose the effect of TGF- by inhibiting the differentiation of protomyofibroblasts into myofibroblasts [136-138]. The downstream targets of FGF receptors vary between receptor type and cell type. Important pathways are the Ras/Raf/MEK/ERK pathway, PLC-,

26 PKC- and the PI3 kinase [139, 140]. ERK is known to regulate both migration and proliferation [141-143], while PI3K is thought to support cell survival [144].

VEGF Since their discovery the Vascular Endothelial Growth Factors, VEGFs, have been studied mainly for their pivotal role in endothelial cell biology. The VEGF family members are VEGF-A, B, C, D and Placental Growth Factor (PlGF) [145-149]. The three known VEGF receptors are VEGFR 1, 2 and 3 [147, 150-152], and can form both homodimers and the hetrodimers 1/2 and 2/3 [153]. Important co-receptors for VEGF are Neuropilin and various heparan sulphate proteoglycans [152, 154-157]. VEGF-A is the most important ligand for VEGFR-1 and 2 while VEGF-C and VEGF-D are ligands for VEGFR-3 [152]. Both VEGF-C and VEGF-D can after proteolytic processing bind to VEGFR-2 [158, 159].

PlGF VEGF-B VEGF-A VEGF-C VEGF-D

VEGFR-1 VEGFR-2 VEGFR-3 Figure 10. The VEGF family of ligands have a specific affinity for the VEGF receptors. The dotted line indicate that the ligand needs to be proteolytically processed before binding.

Various splice variants of VEGF-A have been defined, categorized by number of amino acids. They differ in their ability to bind co-receptors and ECM. VEGF-A 121 binds neither ECM heparan sulphate, membrane bound proteoglycans or neuropilin while VEGF-A 145, 165 and 189 to varying

27 degree do, where VEGF-A 189 bind strongest to the ECM heparan sulphate [154-157]. Blood vessel endothelial cells express mostly VEGFR-2, while VEGFR-3 is the dominant receptor on lymphatic endothelial cells [160-162]. The specificity, however, is not absolute and both receptors can be found on both cell types. Other cell types that express VEGFRs are monocytes/macrophages and hematopoietic cells [152]. Mouse gene inactivation studies revealed that VEGF-A is necessary for development, since knockouts die at embryonic day 9.5-10.5 presumably due to vascular defects [163]. The heterozyogotes die slightly later at embryonic day 11-12 [163, 164], suggesting that the level of VEGF-A expression is important for vascular development. VEGF-C is indispensible for lymph vessel development since the knockouts die prenatally lacking lymph vessels and the heterozygotes, while viable, have lymph vessel defects [165]. VEGF receptor knockout mice have phenotypes that underline their importance, Vegfr2 and Vegfr1 knockouts die even earlier than Vegfa knockouts [166-168], and Vegfr3 knockouts die before forming of the lymphatics due to vascular defects [169]. This shows the importance of VEGFR-3 not only for lymph vessels but also blood vessels. VEGF-A is produced and secreted by most cell types during hypoxic conditions. The transcription factor hypoxia inducible factor (HIF) is regulated by oxygen levels and initiate expression of a number of genes including VEGF-A in hypoxic cells [170]. Thus if tissues experience low oxygen levels due to insufficient vascularization, VEGF-A production can stimulate growth of new blood vessels. Several downstream signaling targets of VEGFR-2 have been identified [152], and many of them are shared with other receptors such as FGFRs. The Ras/Raf/MEK/ERK pathway mediates proliferation [171, 172], while the PLC-/PKC pathway regulates both cell proliferation and vascular permeability [173-175]. The induction of permeability is a unique feature of VEGFRs compared to its relatives the FGFRs and PDGFRs and has given VEGF-A its alternative name Vascular permeability factor (VPF). The PI3K/PKB pathway can mediate survival through anti-apoptosis, increase permeability through eNOS and stimulate migration [176-178]. VEGFR-2 also interacts with Src and focal adhesion kinase, FAK, and this pathway mediates the VEGF-A induced directed cell migration [179]. In addition Src activates PI3K and through this pathway increase permeability [180]. VEGF-A and FGFs commonly act together to stimulate angiogenesis, and although they induce similar cellular responses they each have unique effects. Both in vivo and in vitro, they stimulate the production of each other and are often produced simultaneously making it difficult to distinguish specific responses [139, 181, 182].

28 VEGF-A

VEGFR-2

GRB2 PLCy Ras SHC SoS PI3K PKC Src Raf

PDK

FAK MEK

eNOS AKT/PKB ERK

Permeability Survival Migration Proliferation Figure 11. Summary of the important downstream pathways and cellular responses from the VEGF receptor. Many of the pathways are shared with PDGFRs and FGFRs, but vessel permeability is considered exclusive for VEGFR signaling.

Anti-VEGF or anti-VEGFR therapy is used in antitumor therapy to reduce angiogenesis and decrease vessel permeability [183, 184]. Decreased vessel permeability will reduce tumor pressure, making the tumor more accessible to chemotherapy. Many tumors contain immature vessels that are sensitive to VEGF-A withdrawal compared to other blood vessels.

PDGF The members of the platelet derived growth factor family are PDGF-A, B, C and D. These monomers pair together into dimers in combinations AA, AB, BB, CC and DD. Their receptors, PDGF receptor and are tyrosine kinase receptors of the same receptor family as VEGFRs [185-187].

29 PDGF-AA PDGF-AB PDGF-BB PDGF-CC PDGF-DD

PDGFR αα PDGFR αβ PDGFR ββ

Figure 12. The PDGF family of growth factors bind with a specific pattern to the PDGF receptors. Note that PDGF-BB can bind to all three receptor dimer forms.

PDGFRs are expressed on the surface on a variety of cell types, including vascular smooth muscle cells and fibroblasts. Endothelial cells have been shown to express PDGFRs in some cases, but generally they are absent [36]. Endothelial cells however, are an important source for PDGF production during many biological processes both physiological and pathological. Similar to FGFR and VEGFR autophoshorylation of the PDGFR is caused by ligand binding and dimerization. Downstream signaling by binding of docking proteins to the phosphorylated tyrosines or phoshorylation of binding proteins is thereby initiated. The downstream targets are overlapping with the ones for FGF and VEGF. PDGFR- signaling has been shown to work through Ras/Raf, PLC- and PI3 kinase [188-193]. During angiogenesis and vascular remodeling endothelial cells that are not yet covered by mural cells stimulate mural cell migration and proliferation by secreting PDGF-BB [194, 195]. As the name indicates PDGFs are also released by activated platelets which in the initial phase of wound healing attract fibroblasts and stimulate their proliferation [87]. PDGFs can also act together with TGF- in myofibroblast maturation [195]. Overexpression of PDGFs by stromal or tumor cells can seemingly contradictory reduce vessel maturation since PDGF-BB gradients mediating recruitment to the naked vessels are disrupted [196, 197]. PDGF-BB secreted by endothelium is important for maturation of the mural cell after it

30 is recruited. FGF-2 also plays a role and although the two factors have overlapping downstream signaling effects FGF-2 and PDGF-BB have been shown to work synergistically in vessel maturation [134, 135]. Interestingly FGF-2 has been shown to induce PDGFR expression on endothelial cells and this may influence the maturation process that is dependent on a tight interaction between mural and endothelial cell [36].

TGF- Transforming Growth Factor beta (TGF-) affects a number of cell types and has three subtypes designated TGF- a, b and c. The transforming growth factor family also includes Bone Morphogenic Proteins (BMPs) and activins [198]. As the name implies, TGF- is important for cell transformation like EMT [100]. It is produced as large precursor proteins that form a dimer through covalent disulfide bindings. To be active it needs to be specifically cleaved before release from the plasma membrane [198, 199]. TGF- receptors have serine/threonine kinase activity. In contrast to the tyrosine kinase receptors they are heterotetramers consisting of one TGF- receptor type I dimer and one TGF- receptor type II dimer. The type II receptor involved in TGF- signaling is TGFRII and the type I receptors are called Activin receptor Like Kinase, ALK [198, 200]. In addition a third type of receptor, the type III receptor can associate with the receptor ligand complex and influence its abilities as a co-receptor [198, 200]. The most commonly expressed type I receptor for TGF- ALK5, is expressed on a wide variety of cell types including endothelial cells, mural cells and fibroblasts. Endothelial cells in addition express ALK1 that can mediate TGF- signaling [200, 201]. TGF- stimulation can be either pro or anti angiogenic depending on the relative levels of the different receptor type I subtypes that are expressed in the endothelium [200, 202, 203]. The ALK1 receptor activates signaling pathways that mediate proliferation and migration while ALK5 activated signaling suppresses these responses. TGF- is not the only factor activating ALK1, since BMPs bind and activate ALK1/BMPRII receptor complexes. The role of BMPs relative to TGF- in angiogenesis is not fully known [204]. Recruitment of mural cells to an immature vessel is in addition to PDGFs and FGFs, regulated by TGF- through ALK5 receptors [203]. TGF- is a strong inducer of the differentiation of protomyofibroblasts into myofibroblasts through ALK5 activation. Although TGF- works synergistically with PDGF-BB and to some extent FGF-2 during mural cell recruitment, there are situations when the growth factors have opposing effects. While TGF- induce differentiation of both mural cells and myofibroblasts from precursors such as fibroblasts [86, 87], FGF-2 opposes this differentiation [136, 138]. On the other hand TGF- stimulation can reduce FGF-2 induced proliferation [136].

31 The most important second messenger molecules belong to a family of intracellular proteins called Smads with seven members Smad 1-7. ALK1 mainly phosphorylates Smad1 and Smad5 [202, 205], while ALK5 phosphorylates Smad2 and Smad3 [206-209]. Phosphorylated Smad monomers then form dimers with another Smad, called Smad4 or Co-Smad [209, 210]. This heterodimer is translocated into the cell nucleus where it acts as a transcription factor. Depending on what Smad the dimer contains transcription of different genes is affected. Opposing effects of different Smads on the endothelial cell function means that the relative levels of ALKs and Smads are important to determine the response. TGF-β

TGFßRII TGFßRII

ALK-1 ALK-5 P P SMAD2/3SMAD2/3 SMAD1/5SMAD1/5

P SMAD4SMAD4 P

Endothelial cell: Endothelial cell: Inhibition of proliferation and migration Stimulates proliferation and migration Mural cell: Differentiation and proliferation Fibroblast and protomyofibroblast: Stimulates maturation into myofibroblast

Figure 13. TGF- receptor signaling through different pathways with partially opposite effects in endothelial cells. In many other cell types TGF- mainly acts through ALK-5 activation.

32 In addition to signaling through SMADs, TGF- receptors can initiate activation of other signaling pathways in some cells. It has been shown that by binding of adaptor proteins to the receptor, activation of signaling pathways towards ERK, AKT, p38, JNK and Rho are initiated [211-218]. This plays a role in stimulating EMT [100]. In some cells TGF- can be pro- proliferative or pro-apoptotic and these responses are partially attributed to be dependent on non-SMAD signaling.

Integrins Integrins are cell surface receptors composed of heterodimers consisting of one and one subunit. In contrast to growth factor receptors, whose ligands are diffusible proteins, integrins have affinity for ECM and basement membrane components. Another difference is that integrins do not have intrinsic kinase activity. In mammals the integrin family consists of 18 and 8 units [219, 220]. There are 24 known integrin / dimers, so not all can dimerize with all units and vice versa. Several dimer combinations can bind the same ECM ligand, and usually a specific integrin dimer can bind different matrix ligands [219, 220]. Integins participate in both outside-in and inside-out signal transduction [219, 221, 222]. Outside-in signaling refers to that ligand binding to the extracellular part change the conformation of the intracellular part so that other proteins may bind or detach initiating an intracellullar signal cascade similar to other receptors. Inside-out signaling denotes that binding of proteins to the intracellular part of the integrin can change the affinity for the extracellular part to bind its ligands. During angiogenesis endothelial cells express v3, v1, v5 and 51 integrins [219]. These bind to extracellular ligands not normally present in endothelial basal lamina but present during angiogenesis most notably fibronectin and vitronectin [223-226]. Integin v3 is notable in that it binds to a large number of extracellular molecules, in addition to the above mentioned such as fibrinogen and denatured collagen [219, 222]. Blocking the function of v3 affects tumor angiogenesis [227] but contrasting in knockouts developmental is normal and pathological angiogenesis is enhanced [228]. On the other hand 51 integrin is essential for the formation of a functional vascular network during development [229]. In wound healing 12 integrin by its collagen binding ability is important for vessel growth[230]. As noted above, myofibroblasts have a similar integrin expression pattern, with v3 and 51 in their focal adhesion junctions [83, 85]. Integrin 12 is however not necessary for the myofibroblast function[230]. This shows how the same integrins have can have important functions in different processes.

33 Mechanical regulation of cell signaling and response In addition to chemical outside stimuli, the living cells are also able to register and respond to mechanical forces [231-233]. Similar to chemical stimuli the cell registers these stimuli through receptors systems. As described, mechanical force sensing is important for the fibroblast to myofibroblast differentiation. Another example is how endothelial cells sense mechanical shear stress from variations in intravascular flow. The surrounding smooth muscle cells sense the stretching when the vessels dilate. Shear stress caused by increased blood flow can stimulate intussusceptive angiogenesis [53]. Interestingly, VEGFR-2 on the vessel lumen side can be activated by shear stress without binding of VEGF-A possibly by interaction between receptor and integrin v3[234]. Another example of growth factor independent activation of their receptors is the interaction between VEGFR-2, PECAM and VE-cadherin at endothelial cell junctions by shear stress on the vessel [235-237]. Stretching has been proposed to induce sprouting angiogenesis [46, 53, 238, 239], while the precise molecular mechanism for this mechanosensing is not known.

Outside tension

ECM

Integrin

Cytoskeleton

Cellular response Figure 14. The integrins can act as the mechanical connector between the outside and intracellular protein network making it important for both sensing and reaction to mechanical impulses.

Integrins can participate in mechanosensing and apart from inducing interactions with other receptors, mechanical stretching may also expose cryptic sites in extracellular proteins by conformational changes modifying integrin/ECM affinity [232, 240]. Since the integrins act as links between the intra cellular cytoskeleton and ECM they transmit both extracellular stress and cytoskeletal changes in response to such stress. In many situations

34 cellular response to mechanical stress can be described as a way for the cell to try to counter an outside force by manipulation of the cytoskeleton and cell to outside contacts. As seen in the case of fibroblast differentiation in wound healing it is more complex, and involves both short term cytoskeletal contraction response and long term differentiation and expression of all involved components, the ECM, the integrin and cadherin contacts and the cytoskeleton [85, 87, 233, 241]. In fibroblast and myofibroblasts the focal adhesions and cell-cell contacts contain the mechanosensing and mechotransductive apparatus, where the mechanical stress will be highest, since it is the contact between extra- and intracellular forces. The development of mature contacts in myofibroblasts is thus an adaption to improve both mechanosensing and mechanoresponse In addition to integrins, ion channel linked stress receptors are activated by mechanical stress [242, 243]. The opening of a channel can be initiated by stretching of the membrane or by movement of intra and extracellular molecules bound to the channel [232, 242, 243]. This initiates fast responses, since inflow of ions such as Ca2+ leads to immediate activation of different pathways. Opening of other ion channels like K+ channels change the membrane potential, triggering other responses [242, 243]. Other surface molecules, such as cadherins, cell adhesion molecules and G-protein coupled receptors all participate in mechanotransduction due to conformal changes in the molecules themselves or their ligands, both extracellular and intracellular[231-233].

ECM

Ion channel Integrin VEGFR Integrin VEGFR VE-cadherin PECAM Actin fiber

Figure 15. Mechanical regulation of cell surface receptors. Integrins act as links between the ECM and cytoskeleton and thus are important in both transmission of and response to mechanical tension. Ion channels can open by mechanical stress either through rearrangements of the cell membrane or through linked structural proteins. Growth factor receptors can be activated by interaction with other surface proteins, such as adhesion molecules or integrins.

35 Methods

Microarray Microarray is a powerful method for screening gene expression for a large number of genes at the same time. Ideally, if all genes in an organism are known their expression levels can be compared between cells or tissues under different conditions in one experiment. Available arrays cover a majority of known and predicted genes for a number of organisms. For a review of the method see references [244, 245]. Microarray glass slides have a number of spots with DNA fragments that are either PCR products or synthetic oligonucleotides printed on them. Each spot contains several copies of the same fragment and the spots are arranged in a regular grid. Usually the same fragment is spotted multiple times on the glass in different positions to serve as a internal control. The arrays used in our studies contained 30 000 different fragments.

ARRAY

RNA

cDNA Gene A 2.15 DATA Gene B 1.10 Gene C 1.55 Gene D 1.40 Gene E 4.05 Gene F 1.00 ...... ARRAY GLASS Gene XYZ -1.15

Figure 16. Microarray. RNA is isolated from biological material and marked cDNA is made by rt PCR. The cDNA is bound to the array spots and the fluorescence analyzed. Raw data of gene regulation is collected by comparing intensities from different conditions.

Expression microarray is often used to evaluate gene expression profiles between stimulated and unstimulated cells. mRNA from the cells is purified and cDNA fragments are made by reverse transcription. These

36 cDNA fragments are marked with a fluorophore, in this case Cy3 or Cy5. Fragments from two experimental conditions are each marked with a different fluorophore for comparison on a single array glass. These solutions are mixed and applied on the array glass, to hybridize cDNA to the immobilized spotted DNA fragments on the array. After stringent washing the amount of bound cDNA from the samples is measured through reading of the fluorophore intensity in a flourescent scanner. To gain reliable results multiple array glasses with the same samples hybridized are used. Dye switching, that each sample pair are used with both combinations of fluorophore, increase the reliability of data. By data processing genes that are up- or down-regulated are picked out. Genes previously known to be up or down regulated during the tested conditions provide an extra control. The differential expressions of a selected number of genes are further verified by real time PCR which is a more sensitive method to determine gene expression.

CAM angiogenesis model The chicken chorioallantoic membrane (CAM) model is a model for extra- embryonic angiogenesis. The model takes advantage of the easiness of studying the development of the extra embryonic vascular tree of the avian embryo, since the vessels are located just under the egg shell in the thin but highly vascularized CAM. The assay used in this thesis is a development of the model described by Nguyen et al [246], where a gel is placed on the CAM with addition of pro- or anti-angiogenic substances. A gel composed of collagen I and fibrin is formed in a plastic chamber glued on a nylon grid [247, 248]. The grid separates ingrowing vessels from the underlying native CAM vasculature and gives the possibility to distinguish new from pre- existing vessels. To initiate vessel ingrowth a stimulatory factor like FGF-2 is added to the gel. The gel-chamber is placed on outgrown CAM on day of 12 embryo development and the egg is further incubated for 3-6 days. The tissue is then fixed in a zink fixative, followed by incubation with diaminobenzidine that stains erythrocytes with a strong red-brown color. Thereafter samples are dehydrated to a mixture of benzylbenzoate and benzylalcohol to make the remaining tissue transparent, while the diaminobenzidine staining remains, allowing visualization of the entire vasculature in the ingrown tissue. The samples are scored as positive or negative for ingrowth and to achieve a more fine-tuned measurement quantification of vessels area and length can be done after image processing.

37 Cornea angiogenesis model The cornea model reflects angiogenisis in an adult animal. The cornea is normally avascular in most animals, including mice and humans. Wounding of the cornea gives an inflammatory response similar to dermal wound healing, with invasion of inflammatory cells, transformation of stromal cells into myofibroblasts and ingrowth of blood and lymph vessels. Vessels originate from the limbal vasculature that surrounds the cornea. In the model used in this thesis, the inflammatory response is triggered by a small silk thread sutured through the mouse cornea in its center[249]. This causes a vessel ingrowth from the whole circumference of the cornea. After 1 to 6 days the animal is sacrificed and before the eye is taken out the circulation perfused with a vascular casting or a flouruscent marker that stain endothelium to mark perfused vessels. Staining of the corneas for microscopy is done either on flatmounts of the cornea or paraffin sections of the whole eye. The degree of vascularization and the vessel morphology can be evaluated. Immunostaining of specific cell type markers can be done to distinguish between lymph and blood vessels and identifying vascular mural cells, inflammatory cells and myofibroblasts. Proliferation markers, such as BrdU or PCNA, are used to evaluate the degree of proliferation

Cell sprouting assays Cell sprouting ability can be measured in vitro with embedded cell spheroids. This has been used to study both endothelial cell, fibroblast and myofibroblast sprouting. Cells in suspension are let to aggregate in drops of medium hanging from the inside of a lid of a Petri dish. After a few days, exact time dependent on cell line and cell number, cell spheroids have formed in the hanging drops [248, 250]. These are diluted in a non- polymerized matrix solution, such as fibrinogen and/or neutralized collagen. The suspension is pipetted into wells in a cell plate and the gel is polymerized, if fibrinogen is in the mix by addition of thrombin[248, 250]. In the papers in thesis fibrin gels were used. After gel polymerization, cell medium with addition of growth factors and/or inhibitors is added on top of the gel, and the gels are further incubated. Cells from the spheroid will begin to either sprout or migrate away into the surrounding gel. The progress of sprouting can be checked daily by light microscope and pictures taken [248, 250]. Different cell types differ in how fast they sprout into the gel, so incubation time vary. In this thesis the model was used to evaluate the effect of inhibition of specific receptor kinases on cell sprouting. Staining of the cells for cell markers with immunofluorecense after fixation of the gels is used for quantification of sprouts number and length.

38 An alternative to making cell spheroids cells were used in paper I in this thesis where endothelial cell instead were seeded on top of a fibrin gel, and the sprouting is going down into the gels [251, 252].

39 Present investigations

Results and discussion Paper I: Endocan is a VEGF-A and PI3K regulated gene with increased expression in human renal cancer VEGF-A is an important initiator of angiogenesis and vessel growth and we therefore used microarray analysis in order to find differentially expressed genes in VEGF-A treated cells. This gave us material to identify genes with different expression patterns after stimulation. We identified a number of genes previously known to regulate angiogenesis but also genes previously not firmly associated with angiogenesis such as endocan, pinin, plakophilin, phosphodiesterase 4B and gelsoline. Endocan, also known as Endothelial Specific Molecule 1, ESM-1, is a proteoglycan known to be expressed by endothelial cells and released as a soluable molecule. Endocan expression was upregulated in all time points after VEGF-A stimulation from 3 to 72 hours confirmed by real time PCR and measurement of protein levels in cells and conditioned media. No increase in expression was seen after stimulation with FGF-2, PDGF-BB, EGF or HGF. Evaluation of downstream signaling pathways known to be activated by VEGFR-2 showed that inhibition of neither Src nor MEK/ERK affected VEGF-A induced endocan expression. Surprisingly we found that PI3K had a repressive effect since inhibition of PI3K increased the VEGF-A induced Endocan expression. This indicates a negative feedback loop involved in in regulation of endocan expression. Endocan did not induce angiogenesis in the CAM model, and did not modulate VEGF-A induced endothelial cell invasion into fibrin gels. Inhibition of endogenous endocan with a blocking antibody did not affect VEGF-A induced endothelial cell sprouting. Staining of human renal cancer tissue revealed that endocan was expressed in tumor cells as well as in the vasculature. In normal kidney tissue endocan was expressed in low levels in renal epithelium. The function of endocan in angiogenesis regulation thus remains unclear. While being secreted by endothelial cells after VEGF-A stimulation it does not influence the endothelial cells directly. It is possible that endothelial produced endocan act on tumor cells in a paracrine manner. At least in renal tumors it may be used as a tumor marker, indicating active VEGF-A driven angiogenesis.

40 Result and discussion Paper II: Biomechanical regulation of blood vessel growth during tissue vascularization We observed that neovessels in the gel chamber in the CAM model grew as vascular loops which suggested that vessels grew through elongation of preexisting ones. Microscopic analysis of sections of the gel and staining with endothelial and mural cell markers revealed no clear sprouts. This indicated that, at least initially, vessels grew not mainly by classic angiogenesis but by elongation. Vessels did not enter into the premade collagen/fibrin matrix, but where contained within the expanding CAM tissue. Initially, the outer CAM epithelium remained largely intact. The gel itself was early populated with invading inflammatory cells and a (proto)myofibroblast population. These cells formed a contractile cell network and the gel was contracted. The vascular tissue seemed to be pulled into the chamber since a clear border between the preformed gel and the ingrowing tissue was seen with CAM epithelial cells marking the border during the first days. Vascularized tissue first entered in the periphery, where tensional forces expected to be the highest. Vessel loops that went through the grid often had a sunflower shape in accordance with the tissue being dragged towards the centre of the chamber as the gel contracted. The contractile force thus seemed to guide the direction of vessel elongation. We tested our hypothesis that contraction was necessary for vascularization by inhibition gel contraction. First, rat tail collagen purified by acid solubilization was replaced with pepsinized collagen, that even if it forms a gel lacks telopetides that myofibroblasts need for contraction which was confirmed with in vitro collagen contraction assay. Use of this non- contractile collagen inhibited vascularization and ingrowing cells expressing SMA failed to form a contractile network. Treatment of the gels with a chemical crosslinking reagent that made it more rigid and less contractible gave similar results. Both use of non contractile collagen and crosslinking might result in chemical changes to the gel, which could affect the ability of cells to grow into the gel and differentiate as well as affecting their secretion of cytokines. Gels with rat tail collagen and armored with glass fibers were resistant to contraction, but the gel itself was biochemically unchanged and should not affect cell ingrowth and differentiation. Glass fiber armored gels blocked vessel ingrowth. Taken together these results supported the hypothesis that tensile forces induce and mediate vessel ingrowth. The proposed model was further validated using the cornea model of neovascularization in mice. A similar pattern of ingrowing vascular loops was observed both three and six days after suture of the cornea. The cornea itself was deformed from a spherical to a more conical shape indicative of tension development. Immunuhistochemistry of the corneal tissue revealed a

41 network of SMA positive cells, myofibroblasts, populating the central cornea. Vessels where covered by mural cells positive for SMA and/or NG2. Slightly larger blind ended vessel sprouts were positive for LYVE-1 and identified as lymphatic vessels. Corneal flatmounts with fluorescent staining demonstrated that the invading vascular loops in addition had short endothelial sprouts, usually one cell long. Inhibition of VEGF induced signaling was performed by treatment with a VEGFR-2 blocking antibody. This treatment reduced the number of vascular sprouts, increased mural cell coverage and reduced endothelial cell proliferation. Blocking of VEGFR-2 did not reduce vascularization during the initial 3 days and caused only partial inhibition at later time points. This model of looping vascularization where pre-existing vessels with maintained circulation are pulled by tensile forces is sufficient to mediate and direct vascularization independent of endothelial sprouting and proliferation. New branch points might be formed through intussusception or fusion of short sprouts.

Result and discussion: Paper III: A quantitative neo- vascularization assay for screening pro- and anti- angiogenic factors. The CAM angiogenesis assay is used to evaluate the effect of angiogenesis stimulators and inhibitors. The CAM assay used in paper I and II has an advantage in its easy distinction between newly formed and pre-existing vessels. In addition, the findings in paper II indicate that it can be used to evaluate effects on non-sprouting angiogenesis. Comparing the ability for growth factors to induce neovascularization we found that PDGF-BB induced vessel growth similar to FGF-2 while EGF, HGF, VEGF-A and TGF- had no effect in a similar dose range. Previously described angiogenesis inhibitors thalidomide, fumagillin, wortmannin, cortisol and U0126 were tested against FGF-2. Cortisone affected the vasculature, reducing ingrowth and giving thinner vessels in accordance with its general catabolic nature. Fumigallin and U0126 blocked neovessel growth, while wortmannin caused a retraction of the pre-existing vasculature in addition to blockade of neovessels. By image processing the vessels that grew from the CAM into the gel chamber could be quantified. By applying Photoshop filters and measuring tools the total length of the ingrown vasculature was measured. Subtler effects of inhibitors were thus seen. Using this setup we found that thalidomide reduced the degree of vascularization inside the construct, while not significantly reducing the fraction of constructs positive for vascularization.

42 Result and discussion Paper IV: The role of growth factor receptor signaling for angiogenesis and vessel stability in an experimental wound healing model. Since we found in paper II that vascularization in the CAM assay for vessel growth was guided by mechanical tension and that vessel elongation was part of the process we wanted to evaluate the importance of various growth factors on this mode of vascularization. While both FGF-2 and PDGF-BB induced vessel ingrowth at comparable levels, TGF- did not induce vessel ingrowth but instead had an inhibitory effect on FGF-2 induced vascularization. Since both FGF-2 and PDGF-BB can stimulate the production of other growth factors we evaluated the necessity for this endogenous growth factor production. Inhibition of either VEGFR or PDGFR signaling with kinase inhibitors blocked the vessel ingrowth induced by FGF-2 or PDGF-BB. The preexisting vasculature was sensitive to these inhibitors, but at a higher dose. There was a difference in how the inhibitors affected the pre existing vasculature at high doses. Inhibition of PDGFR via Imatinib caused total retraction of the vessel underneath the gel chamber, whereas inhibition of VEGFR via PTK787 only affected the smaller vessels. Inhibition of FGFR kinase with PD173074 did not result in specific inhibition of PDGF-BB induced ingrowth, but retraction of underlying vasculature at higher inhibitor doses was similar to PDGFR inhibition. Interestingly inhibition of TGF- signaling by the ALK inhibitor SB431542 was insufficient to block vessel ingrowth. None of the inhibitors could block myofibroblast sprouting in fibrin gels apart from Imatinib in high doses. These results suggest that although biomechanical factors are necessary for vascularization, there is still a need of functional growth factor receptors. This is not surprising since the continued elongation of the vessels necessary for extending and forming the vessel network in the chamber must be supported by proliferation, induced by growth factor stimulation, in both endothelial cells and mural smooth muscle cells. In summary functional VEGFR and PDGFR are necessary for FGF-2 initiated vessel growth. Pervious studies have shown that VEGF and PDGF are important for these cells to maintain neovessels. The observed effect might be due to inhibition of mitosis and/or apoptosis if the contact between endothelial and mural cell is disrupted. That FGFR activity was not essential in PDGF-BB induced vascularization, on the other hand, suggests that other receptor systems can replace FGFR function in vessel elongation and myofibroblast recruitment. The effects of TGF- stimulation and inhibition can be explained by a combination of factors. An early stimulation with TGF- may give premature differentiation into myofibroblasts before they have invaded far into the gel. The stimulatory effect of FGF-2 on fibroblast migration and

43 proliferation has previously been shown to be opposed by TGF-. On endothelial cells the dual opposing TGF- induced pathways might lead to that an exogenous stimulation gives dysregulated response or a concentration favoring the antiangiogenic ALK5 pathway. That inhibition of ALK5 with SB431542 did not block vessel ingrowth even though it supposedly is important for both modulation of endothelial cell activity in angiogenesis and fibroblast to myofibroblast differentiation was notable. TGF- may thus not be necessary for vessel ingrowth via elongation, in contrast to its role in classic angiogenesis. Myofibroblast differentiation has previously been shown to occur independent of TGF- in cells exposed to mechanical tension and non SMA expressing cells, protomyofibroblasts, which do not need TGF- to form, are efficient contractors in themselves.

44 Future perspectives

Our findings in paper II show that the contractibility of the matrix is important for vascularization. Lymphangiogenesis is an important part of both wound healing and many pathological conditions. We earlier tried to use the CAM model as a model for study of lymphangiogenesis. However, we found very little to none expression of lymph vasculature markers using in situ hybridization on sections from the ingrowing tissue compared to the underlying CAM. In contrast the cornea angiogenesis model proved to be very useful for lymph vessel studies. Interestingly, a method used in the clinic involves crosslinking of cornea matrix with UV light. In the mouse cornea model the cornea matrix could thus be crosslinked before the suture is placed. The effect on both blood and lymphatic vessels of mechanical tension reduction could thus be investigated. This can be complemented with treatment with inhibitors for FGF, PDGF and TGF- systems to evaluate for effect on vessel formation and myofibroblast function. In addition it would be of interest to specifically inhibit lymph or blood vessel formation. Receptor inhibitors are available with specificity for either VEGFR2 or 3 in addition to soluble ligand to receptor binding competing fusion proteins and inhibitory antibodies. In situ hybridization of whole mount corneas can be used in addition to immunostaining for lymph and blood vessel marking. Gene arrays and qPCR from cornea material can be used to deduce differences between blood and lymph vessel regarding gene expression to find suitable new markers. Optimally arrays from material consisting of cornea invaded by only blood or lymph vessels can be used. Novel gene findings can be further studied in isolated human endothelial cell lines available. By inhibition of gene expression with RNAi in the cell lines we can investigate the effect on migration, proliferation and sprouting. The array findings in paper I include data regarding many genes of interest that we yet have not looked into in detail. A comparison with findings from cornea studies can give valuable information. The findings in paper II imply interesting consequences for the efficiency of antiangiogenic treatments. It would be of interest to gain information regarding the importance of mechanical guidance in vascularization and angiogenesis in clinical conditions. Studies of clinical tissue material for clues to if these processes occur in tumor tissues could be a future way. Wound tissues samples from the clinic could also be analyzed for signs of mechanically regulated vessel growth. Differences between normal wounds

45 and tumor tissue regarding the amount of non sprouting vessel growth could give information for making specific therapies against pathological but not physiological vascularization. Specific inhibition of myofibroblast function has been tried by targeting the expression of SMA. However the effect has been limited, with only a small reduction in contraction. In our CAM model in paper II we saw no sufficient effect of targeting SMA. This is explained by the notion that even the immature protomyfibroblasts without SMA can contract the matrix. As mentioned earlier there are factors such as TGF- and ED-A fibronectin that support myofibroblast differentiation. Even if it would be possible to block the effect of those, disregarding multiple possible side effects of blocking such a multifunctional factor as TGF-, it may be insufficient since it targets the later steps of differentiation. This skepticism is supported by the findings in paper IV considering TGF- receptor inhibition. Thus a factor inhibiting the contraction of also protomyofibroblasts would be desired, but no appropriate candidates are known. On the other hand, support of physical vascularization is desired in other diseases where it is hampered, often with consequences as tissue hypoxia and ultimately necrosis. If sprouting angiogenesis is insufficient to give enough functional new vessels in these conditions therapy to increase other vascularization modes would be a more effective therapy. Stimulation of myofibroblast differentiation is the obvious therapy consideration, but another alternative may already be in use in clinical wound care. This is vacuum assisted wound healing, which could assist by applying indirect contractile forces upon the vascular bed. Tissue samples from such wounds cold give information if increased vascularization by looping is present.

46 Populärvetenskaplig sammanfattning

Cellerna i våra kroppar behöver både syre och näringsämnen för att överleva. Dessa transporteras i blodkärl, som på insidan är täckta av så kallade endotelceller och på utsidan är omlindade av glatta muskelceller. Genom de minsta kärlen, kapillärerna, sker utbytet av näringen och syret till cellerna. När ett sår läker behövs nya blodkärl för att understödja uppbyggnaden av den läkande vävnaden. Nybildning av blodkärl kallas angiogenes, och kan beskrivas som att nya grenar i kärlträdet skapas. Den vanligaste modellen för hur detta sker är att endotelceller växer ut i rad, som en gren, från ett befintligt kärl. Det finns många specialiserade proteiner som stimulerar nybildning av blodkärl. Dessa är så kallade tillväxtfaktorer och frisätts till exempel av celler som behöver nya blodkärl för att få tillräckligt med syre. En viktig faktor som utsöndras av celler med syrebrist är VEGF, men blodkärlsnybildning kan också stimuleras av andra faktorer såsom FGF och PDGF. Dessa påverkar cellerna efter att ha bundit till specifika proteiner, receptorer, på cellytan. Endotelcellerna är känsligast för VEGF och FGF medan kärlens glatta muskelceller är känsliga för FGF och PDGF. All angiogenes är inte en fördel, en cancertumör behöver till exempel nya blodkärl när den växer och utnyttjar samma faktorer som stimulerar vanlig angiogenes för att rekrytera dessa. I det första arbetet i denna avhandling studerades hur VEGF påverkade vilka proteiner endotelceller tillverkar. Ett protein som endotelcellerna börjar tillverka var endocan, som är ett protein på vilket långa sockerkedjor kopplats. Endocanproduktionen ökade bara om cellerna fick VEGF men inte efter att de fått FGF, EGF och HGF. Endocan påverkade dock inte kärlnybildning eller endotelcellerna själva. Däremot fanns i tumörvävnad anrikningar av endocan i blodkärlen och i tumörcellerna själva. I arbete II studerades mekanismen för hur blodkärl växer. Vi använde två sårläkningsmodeller, dels en baserad på studier av blodkärlen som syresätter ett kycklingembryo i ägget och dels en där sårläkning i ögat på en mus studeras. Vi såg att nya kärl växte som loopar, inte som de små grenar som beskrivs i den vanliga angiogenesmodellen. Dessa kärlloopar var sammankopplade med blodcirkulationen och fungerade som vanliga kärl under det att de växte. I den kärlfria sårvävnaden mot vilken kärllooparna sträckte sig fanns en speciell celltyp kallad myofibroblast. Dessa härstammar från vanliga vävnadsceller, fibroblaster, som fått förmågan att dra samman den omgivande vävnaden. De liknar på många sätt de glattmuskelceller som

47 omger våra blodkärl. Om den kärlfria vävnaden där myofibroblasterna fanns gjordes mindre kontrakterbar växte kärlen inte in. De inväxande kärlloparna vägleddes således av dragkraften från den sammandragande vävnaden, och inte efter de gradienter av tillväxtfaktor som antagits vara en förutsättning för kärlnybildning. I det tredje och fjärde arbetet visade vi hur olika tillväxtfaktorer kunde påverka blodkärlsväxten i embryomodellen från arbete II. Vi fann att det behövdes fungerande receptorer för både PDGF och VEGF för att de nya kärlen skulle växa. Däremot var inte FGF receptorerna nödvändiga. FGF och PDGF kunde stimulera nya kärl att växa, men andra tillväxtfaktorer som tidigare påvisats kunna påverka blodkärl som TGF-, EGF och HGF inte stimulerade kärlnybildning. När man vill undertrycka nybildning av blodkärl i cancerbehandling söker man efter en substans som ska påverka tumörens kärl att inte växa medan de vanliga friska kärlen inte påverkas. Därför är det intressant om vissa proteiner som endocan finns mer i tumörer än i frisk vävnad. Den modell för kärlnybildning vi beskriver i arbete II ger ett intressant perspektiv på möjliga framtida terapier. För sjukdomar när den vanliga sårläkningen inte fungerar tillfredställande kan en behandling som hjälper både myofibroblaster och endotelceller kanske att fungera mer effektivt än om bara endotelcellerna behandlas. I vissa tumörer är myofibroblaster vanligt förekommande. Om dessa liksom i våra sårläkningsmodeller stödjer kärltillväxt kan en behandling som undertrycker deras funktion förhoppningsvis minska tumörens förmåga att få nya blodkärl, och minska tumörens tillväxt. Hur viktigt mekaniska stimulanser är för celler är fortfarande inte helt känt. Mycket av fokus i angiogenesforskning ligger på tillväxtfaktorer och andra signalmolekyler. Det är dock även sedan tidigare känt att endotelceller, liksom många andra celltyper, reagerar på mekaniska faktorer som sträckning eller tryck. Att vi nu visar hur mekaniska stimuli kan dirigera även kärltillväxt tyder på att det i praktiken kan vara väl så viktigt som tillväxtfaktorerna.

48 Acknowledgements

This work has been carried out partly at the Department of Medical Biochemistry and Microbiology and partly at the Department of Genetics and Pathology at Uppsala University. Financial external support was provided by the Swedish Research Council, the Children's Cancer Foundation, the Lions Cancer Research Foundation in Uppsala, Göran Gustafsson Foundation and the Swedish Cancer Foundation.

I would like to express my gratitude to all those contributed to this work and that gave support. Thank you all! I would especially like to thank the following people.

My supervisor Pär Gerwins, for all support, great discussions and an invaluable enthusiasm. It is a great privilege to work with someone with such a broad interest and knowledge from basic science to practical medical reality.

Witold "Witek" Kilarski for practical supervision during my first years, and theoretical teachings in the ever changing field of angiogenesis research. It was fun to discuss science with a born skeptic and philosopher, that always is ready to go your way.

My co-supervisor Carl-Henrik Heldin.

All co-authors in the publications for all the work done and the privilege to have such great collaborators, with many extra thanks to Branka Samolov for all work during the revisions for paper II.

The vascular biology group in D9 at BMC, Johan K, Sebastien, Irmeli, Peder, Johan H, Zsolt, Judith and Katarina. Great colleagues, you are all always very helpful. Also thanks for all non-scientific discussion and great humor that makes the days more fun between all work.

The colleagues in corridor D94, past and present: Christoffer T, Anna E, Jimmy, Tobias, Ulrika, Hanna, Fredrik, Maud, PH, Anders, Bo, Suomi, John, Inger, Cecilia, Lars L, Karin H, Pernilla, Elina, Rashimi, Dorothe, Ulf L, Jin-Ping, Juan, Eva G, Eva H, Nadja, Karin F-N, Lena K and everyone

49 else. For interesting insights into other fields of research, professional help and support, but above all a great social interaction.

All past members of the vascular biology group, both at BMC and all former colleagues at Rudbeck. Dan, Lars J, Sofie, Peetra, Maria, Ingrid, Chunsik, Lars L, Anna-Karin, Lena C-W, Emma, Johan D, Anna, Irja, Charlotte, Stefan, Mike, Svante, Ed, Åsa, Harukiyo, Svetlana, Fuad, Pacho, Petter, Xiujuan, Zuyue, Lothar and everyone that I forgot in this list. The best thing about these years was always the friendliness, support and all other great memories.

The administrative staff at IMBIM: Barbro L, Marianne W, Rehne Å, Kerstin L, Olav N, for helping out (usually when I being somewhat panicked)

All old friends (yup, all of you cannot list everyone here). Pity that us all having responsible adult lives usually leads to that we see each other too rarely, but it is always great to have you.

All new friends I have gotten the chance to learn to meet during these years for that matter.

Most of all I must thank my family, for all love and support. Pappa Lasse and Bodil, it is many times great to have the parental home nearby, to go somewhere where to just relax and being oneself. My wonderful younger sisters Linnea, Maja and Agnes for being fun, smart, honest and helpful. My younger brother Albin for simply being the best in the world. Andreas and Sofie with little Erik and Ella that always makes the day more interesting when I get the chance to see you.

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