Ординатура / Офтальмология / Учебные материалы / Retinal Vascular Disease Joussen Springer

3.1 General Concepts of Angiogenesis and Vasculogenesis

Table 3.1.1. Diseases characterized or caused by abnormal or excessive angiogenesis

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gressive loss of the microvasculature has been implicated in nephropathy [157], bone loss [210], and impaired reendothelialization after arterial injury [101]. Diabetes, atherosclerosis and hyperlipidemia also impair vessel growth [319, 328, 343], whereas hypertension causes microvascular rarefaction [29]. Reduced angiogenic signaling also causes pulmonary fibrosis [171] and emphysema [159]. Insufficient vascular growth or function may also significantly impact on the nervous system, as the extent of angiogenesis correlated with survival in stroke patients [172] and insufficient VEGF levels causes motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis [239]. Amyloid-, a molecule believed to play a central role in the pathogenesis of Alzheimer’s disease and plaque formation, has negative effects on the cerebral vasculature [67].

Dysregulation of lymphatic vessels is also known to give rise to severe diseases, of which lymphedema and lymphatic metastasis are the most common. In lymphedema, the transport ability of lymphatic vessels is impaired and this leads to fluid accumulation in the tissue. The effects are manifested as chronic and disabling swelling, tissue fibrosis, adipose degeneration, poor immune function, susceptibility to infections and impaired wound healing [5, 238, 270].

Primary lymphedemas are rare genetic disorders while secondary lymphedema results from various types of damage to the lymphatic vessels caused by for example radiation therapy, surgery or parasite infections. At present, there is no cure to this disease and only symptomatic use of supportive stockings to reduce the swellings can be offered. Cancer metastasis today is one of the leading causes of mortality in our society. Excessive proliferation of lymphatic vessels is mainly linked to cancer progression and tumor cell metastasis to lymph nodes and represents a major criterion for evaluating the prognosis of patients.

Thus, as an excess or impairment of blood and lymphatic vessel growth contributes to the pathogenesis of numerous disorders, it is important to define the molecular basis of these defects and how vessels grow, fail to grow or grow excessively. The development of compounds or strategies, stimulating or inhibiting the growth of blood vessels (a process called “angiogenesis”) and lymphatic vessels (a process termed “lymphangiogenesis”) offers unprecedented opportunities for treating lymphedema and cancer among many other diseases. We will therefore discuss some key principles of the various steps of vessel growth in the embryo and adult.

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Fig. 3.1.3. Vasculogenesis in the adult. Bone marrow progenitor cells (EPCs and myeloid cells) are recruited from the bone marrow and incorporated into nascent vessels or stimulate new vessel growth by releasing proangiogenic factors and inducing the proliferation of resident endothelial cells

might be alternative sources of endothelial progenitor cells. Indeed, multipotent adult progenitor cells (MAPCs) with angioblastic potency have been identified in the bone marrow [261], while tissue specific stem cells might also exist. In skeletal muscle, myoendothelial progenitors might differentiate locally into muscle or ECs [315]. Unlike mature ECs, EPCs are characterized by a greater capacity to proliferate [195] and by the fact that they have not yet acquired mature endothelial markers. EPCs should be distinguished from circulating ECs (CECs), which are sloughed off due to shedding from the existing vasculature and enter the circulation as a result of traumatic and infectious vascular injury or tumor growth. EPCs express EC surface antigens such as VEGFR-2, CD34, vascular endothelial cadherin (VE-cadherin) and AC133 among other markers [257]. AC133 is lost upon differentiation of the EPCs into more mature ECs [146]. Since mature myelomonocytic cells have lost surface expression of AC133, this marker also provides an effective means to distinguish EPCs from mature cells of myelomonocytic origin [259].

EPCs in the bone marrow likely reside in close association with hematopoietic stem cells and stromal cells in bone marrow niches. Though still largely undefined, these cells are possibly involved in promoting local EPC proliferation and transmigration across the bone marrow/blood barrier via secretion of VEGF, PlGF (placental growth factor, a homologue of VEGF) and other angiogenic factors [324]. Mobilization of EPCs from the bone marrow, as well as their recruitment to sites of adult vasculogenesis, involves a number of similar cues that regulate EC sprouting (angiogenesis), such as VEGF [11], fibroblast growth factor 2 (FGF-2), PlGF [49, 257], the recently discovered platelet-derived growth factor family member PDGF-CC [192], and angiopoietin-1 (Ang-1) [124]. In addition to stimulating proliferation of ECs, the chemokine stromal cell-derived factor (SDF-1), a chemoattractant for hematopoietic progenitor cells (HPCs), also induces migration of EPCs, which

express CXCR4, a receptor for SDF-1 [229, 250, 353], while inhibition of SDF-1 blocks EPC recruitment to sites of neovascularization [117].

It remains an outstanding question to what extent and how precisely EPCs contribute to vascular growth. Apart from differentiating to mature ECs that become incorporated as building blocks in the endothelial layer of nascent vessels [250], mononuclear cells might, together with accessory cells derived from the bone marrow, create a proangiogenic microenvironment to facilitate neovascularization. CD34-expressing cells mobilized from the bone marrow stimulate vascularization in myocardial infarcts both via vasculogenic in situ vessel formation and via stimulation of angiogenic sprouting from resident endothelium by secretion of angiogenic growth factors [168]. Recently, a new role has been described for SDF-1 as a retention factor for angiocompetent cells recruited from the bone marrow to the site of neovascularization [115].

The relative numeric contribution of bone mar- row-derived EPCs to adult organ and tumor neovascularization is highly variable. In different experimental settings of pathological angiogenesis, incorporation of EPCs into the growing vasculature has been reported to be remarkably high [98] or negligibly low [258, 365]. Apart from differences in the genetic background of mouse strains used for these studies, the variability might also reflect remarkable differences of spontaneous and xenografted tumors in their dependence on bone marrow derived endothelial precursors [277]. Mathematical models have been suggested to calculate – and possibly predict – the contribution of EPCs to tumor neovascularization [306].

Despite these unresolved questions, the concept of postnatal vasculogenesis offers challenging clinical perspectives for the treatment of cardiovascular disorders and cancer. Mobilization of endothelial progenitor cells from the bone marrow is enhanced in patients with ischemic conditions [294], and levels of circulating endothelial progenitor cells have been

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introduced as a valuable clinical parameter for cardiovascular risk assessment [131]. In tumor bearing mice, EPC levels in peripheral blood correlate with the anti-angiogenic effect of angiogenesis inhibitors on tumor angiogenesis and growth [131], suggesting that EPCs (and CECs) may be useful biomarkers for dose finding and monitoring the effect of anti-angio- genic treatment in cancer. Intriguingly, a recent study has shown that in multiple myeloma (MM) patients with 13q14 deletion, CECs carried the same chromosome aberration as the neoplastic plasma cells, suggesting that not only EPCs or host-derived ECs, but also MM-derived CECs, may contribute to tumor vasculogenesis [265].

3.1.4.3The Endothelial/Hematopoietic Connection – An Emerging Theme

In the embryo, hematopoietic stem cells (HSCs) migrate into avascular areas and attract sprouting ECs by releasing angiogenic factors, such as angio- poietin-1 [314]. In the adult, bone marrow-derived hematopoietic cells, expressing markers such as Sca- 1, c-Kit, CXCR4 and/or VEGFR1, become recruited, often together with EPCs, to tumors or ischemic tissues in response to VEGF and PlGF [115, 240, 257]. These angiocompetent cells extravasate around nascent vessels, where they are retained by SDF-1, and stimulate growth of resident vessels by releasing angiogenic factors such as VEGF, PlGF and angiopo- ietin-2 (Ang-2) [15, 34, 237]. In other cases, these cells function as hemangioblasts, producing both hematopoietic and endothelial progenitors which give rise to new blood vessels [257]. Furthermore, in response to PlGF released by tumor cells, VEGFR-1 expressing hematopoietic bone marrow progenitors home in to tumor-specific premetastatic sites, where they recruit tumor cells and EPCs; anti-VEGFR1 antibodies prevent the formation of such premetastatic niches [263].

3.1.4.4Arterial, Venous and Lymphatic Cell Fate Specification

Arteries and veins have evolved as anatomically distinct but closely interconnected blood vessel types. The structural differences between arteries and veins were attributed to different flow dynamics and distinct physiological requirements. But, evidence has recently emerged that molecular differences between arterial and venous ECs already exist, even before blood vessels are formed, and that complex genetic pathways are responsible for this arterial versus venous specification. The expression of the ligand ephrinB2 in arteries and of the Eph receptor tyrosine kinase EphB4 in veins occurs before the

onset of circulation [103, 104]. This indicates that while ephrins are essential for proper distinction between arterial and venous cells, they are not required for the initial fate decision that distinguishes arterial and venous endothelial progenitors.

Lineage tracking in zebrafish embryos indicates that angioblasts in the lateral posterior mesoderm receive signals from the notochord and the ventral endoderm, and become restricted to the aorta or trunk vein [363]. Studies in zebrafish and Xenopus indicate that sonic hedgehog (Shh), produced by the notochord, specifies arterial EC fate [181]. Indeed, formation of the aorta is impaired in zebrafish embryos mutant for sonic you (syu), the zebrafish homolog of Shh [31, 56] or after morpholino knockdown of Shh [181]. Shh induces the expression in the adjacent somites of VEGF, which, in turn, drives arterial differentiation of angioblasts. In the chick, the early extraembryonic blood islands contain a mixture of subpopulations of cells expressing neuropilins 1 and 2 (Nrp-1 and Nrp-2), which subsequently become lineage markers of arteries and veins respectively [128]. This suggests that even early angioblasts may already be committed to either the arterial or venous lineage. Further evidence that VEGF has a role stems from findings that, when released from Schwann cells, it induces arterial specification of vessels, tracking alongside these nerves [224] and that Nrp-1, a receptor selective for the VEGF165 isoform, is expressed in arterial beds [222, 223, 304]. VEGF also determines arterial EC specification after birth in the heart and retina, where the matrix-bind- ing VEGF188 isoform is critical for arterial development [304, 335].

The Notch pathway acts downstream of VEGF in arterial EC specification [181]. Notch signaling is initiated when the Notch receptors (Notch-1 – 4) are activated by their ligands Jagged-1, Jagged-2 and Delta-like-1, -3 and -4 [7]. During vascular development, defects in Notch signaling disrupt normal arterial-venous differentiation, resulting in loss of artery-specific markers (e.g., ephrin-B2) and ectopic expression of venous markers (e.g., VEGFR-3/Flt-4) in the aorta [180]. Conversely, overactivation of Notch induces ectopic arterial markers in veins, thereby suppressing vein differentiation. Mutation in Delta-4 (dll4), which is specifically expressed in developing arterial endothelium, leads to defective development of the dorsal aorta, with development of arteriovenous shunts [77, 95]. This is associated with downregulation of arterial markers and upregulation of venous markers in the dorsal aorta. Furthermore, Hey2, a transcription factor induced by Notch signaling, confers features of arterial EC gene expression to venous ECs, upregulating arterial-spe- cific genes (ADHA1, EVA1, and keratin 7), while

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tinuous and fenestrated, allowing high volume molecular and ion transport as well as hormone trafficking. Overall, the factors that regulate acquisition of specific endothelial properties are largely unknown. However, it appears that the interaction with the host environmental extracellular matrix, in concert with VEGF, plays a major role [267, 268]. Besides vascular cell heterogeneity in distinct organs, ECs within the same organ can even be heterogeneous. In the heart, for instance, ECs in distinct locations of the coronary vascular tree differ in their expression of the endothelial constitutive nitric oxide synthase isoform [9], brain-derived neurotrophic factor [76] or adhesion molecules [69, 70]. Even within a single vessel, ECs may have distinct cell fates. For example, three types of ECs, each with a distinct cell fate, build the intersegmental vessels in the zebrafish embryo [58].

Recently, genetic studies in mice, zebrafish and Xenopus are starting to unravel the transcriptional code that determines EC fate [31, 193]. This code involves bHLH transcriptional activators (hypoxiainducible factor-2, stem cell leukemia factor, Tfeb) [39] as well as Id repressors as demonstrated by the perturbation of developmental and tumor associated angiogenesis in mice lacking Id-1/3. Several members of the homeobox transcription factors (HOX) family are expressed in the vascular system during development and have also been implicated in EC fate determination [110]. Within the lymphatic system, LYVE-1 is predominantly expressed in the lymphatic capillary endothelium, while podoplanin is expressed both in the capillaries and valved collecting lymphatic vessels [209]. However, there are other mechanisms determining endothelial heterogeneity and organ-specific angiogenesis. For instance, the activity of VEGF and Ang-1 varies in different tissues. Low permeability tumors overexpress Ang-1 and/or underexpress VEGF or PlGF, whereas tumors with high permeability lack Ang-1, but overexpress Ang-2 [149]. Another example is the effect of Ang-1, which stimulates angiogenesis in the skin but suppresses vascular growth in the heart [312, 335].

An exciting new development is the discovery of tissue-specific vascular endothelial growth factors. A striking example of this novel class of cues is the endocrine gland-derived VEGF (EG-VEGF), which selectively affects EC growth and differentiation (fenestration) in endocrine glands [185, 186]. Other organ-specific angiogenic factors include prokineti- cin-2 and Bv8 in endocrine glands [184], Bves and fibulin-2 in the heart [342], and glial derived neurotrophic factor in the brain. That ECs in different tissues are distinct is further suggested by their considerably different response to anti-angiogenic factors. Indeed, ECs in endocrine glands rapidly lose their

fenestrations and even become apoptotic in response to VEGF inhibitors, resulting in a 70 % loss of the microvasculature in these organs [156]. By contrast, the microvasculature in other organs is much more resistant to such pruning in response to anti-VEGF therapy [156, 316]. Malignant cells also induce ECs in tumor vessels to acquire a distinct fate and express unique markers (“vascular zip codes”) that are absent or barely detectable in quiescent blood vessels of normal tissue [273, 274]. Tumor cells also change the responsiveness of ECs to cues – for instance, epidermal growth factor (EGF) upregulates its receptors in tumor-associated vessels and thereby makes these ECs responsive to the mitogenic activity of EGF [20]

– a finding of significant therapeutic relevance.

3.1.5 Angiogenesis

After vasculogenesis, the nascent primitive vascular labyrinth expands and becomes remodeled into a more complex, hierarchically and stereotypically organized network of larger vessels, ramifying into smaller vessels. This process involves sprouting, bridging and intussusceptive growth from preexisting vessels, navigation and guidance, remodeling and pruning. Many experimental models have been developed to study angiogenesis. Among them we have highlighted the most commonly used to study angiogenesis in the eye in Box 2. We will now discuss this process as the following orderly series of events (Figs. 3.1.1, 3.1.4). The avascular areas in the embryo release angiogenic cues that diffuse into the nearby tissues and activate ECs to induce extracellular matrix (ECM) degradation. ECs then proliferate and navigate toward these cues forming a sprouting. Finally, arteriogenesis takes place, whereby the newly formed blood vessel are stabilized by smooth muscle cells and pericytes.

3.1.5.1Vascular Permeability and Extracellular Matrix Degradation

Water and nutrients move from blood to tissues across the walls of capillaries and venules. The wall of blood vessels is composed of ECs and mural cells, namely pericytes and smooth muscle cells, which are embedded in an ECM. The expression of cell adhesion molecules such as VE-cadherin in adherent junctions and claudins, as well as occludins and JAM-1 in tight junctions of quiescent vessels, provides mechanical strength and tightness to the vessel wall and establishes a permeability barrier. Between vascular cells, an interstitial matrix of collagen-I and elastin provides both viscoelasticity and strength to the vessel wall. The ECM is responsible for the contacts between ECs and the surrounding tissue, and

thus prevents vessels from collapsing. Before new vessels can sprout, this stable vessel must first be destabilized.

Hypoxia is an important stimulus of angiogenesis. In response to oxygen deprivation, a number of genes including nitric oxide synthase, VEGF, and angiopoietin (Ang)-2 [165] are rapidly upregulated. One of the early changes during angiogenesis involves an increase in vascular permeability and extravasation of plasma proteins, which serve as a provisional matrix stimulating EC proliferation and migration. VEGF, which was originally discovered as a vascular permeability factor (VPF) [87, 284], induces EC fenestration and a redistribution of intercellular adhesion molecules, including PECAM-1 and VE-cadherin, as well as alterations in cell membrane structure involving Src kinase [78, 96]. However, excessive vascular leakage may result in pathological outcomes, such as circulatory collapse, intracranial hypertension, blindness, psoriasis, myocardial or brain infarction and psoriasis [329, 347]. It also leads to increased interstitial fluid pressure in tumors which, along with the abnormal properties of tumor-associated vessels, compromises drug delivery to the tumor [323]. Thus, blocking the action of VEGF is a promising anti-angiogenic approach with which to treat solid tumors [148]. Angiopoietin-1 is a natural inhibitor of vascular permeability, which protects against excessive plasma leakage by tightening preexisting vessels via effects on endothelial junctions and pericyte-EC interactions [320].

To emigrate from their resident site, ECs need to loosen interendothelial cell contacts, to relieve periendothelial cell support and break down the surrounding ECM (Fig. 3.1.4). Ang-2, an inhibitor of

Tie-2 signaling and antagonist of Ang-1, is involved in the destabilization of mature vessels by detaching smooth muscle cells and loosening the underlying matrix [96, 208]. Breakdown of the ECM is mediated by several proteinase families, including plasminogen activators [such as urokinase plasminogen activator (uPA) and its inhibitor, PAI-1], matrix metalloproteinases [MMPs and tissue inhibitors of metalloproteinases (TIMPs)], chymases, heparanases, tryptases, cathepsins, and kallikreins (and their inhibitor kallistatin) [25, 147, 170, 201, 220, 362]. Proteolytic degradation of ECM molecules is an integral part of angiogenesis, as it not only provides scaffold support to migrating ECs but also results in liberating matrix-bound angiogenic growth factors, including FGF-2, VEGF, insulin-like growth factor (IGF)-1, transforming growth factor (TGF)- and tumor necrosis factor (TNF)- , and proteolytically activating angiogenic chemokines such as interleukin (IL)-1 [26, 62]. In addition, proteinases cleave VEGF into shorter isoforms with different solubility, receptor binding and biological activities [246]. Proteinases also have a role in the resolution of angiogenesis, as they liberate matrix-bound inhibitors [thrombospondin (TSP)-1, arresten, canstatin, tumstatin, angiostatin, endostatin, cleaved anti-throm- bin III, platelet factor 4] [232] and inactivate angiogenic cytokines, such as SDF-1 [240]. These pleiotropic activities may explain why proteinases and their receptors and inhibitors often have activities that are context and concentration dependent.

Proteolytic remodeling of the ECM must occur in a balanced manner. Excessive breakdown removes critical support and guidance cues from migration EC, thereby impairing angiogenesis. On the other

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