Differential Angiogenic Pathways

Complex angiogenic pathways involve the discrete regulation of each stage in this multistep process. Tissue- and disease-specific expression of angiogenic factors and inhibitors may vary through the different phases of a pathological process. For example, an angiogenic switch may turn slow growing neoplasms into aggressive, metastasizing tumors [44]. Sequential activation of various angiogenic and inhibitory pathways may coordinate the beneficial progression from hemostasis through successful repair during wound healing, or the arguably less desirable transition from acute to chronic inflammation.

5.1. Angiogenic Factor Expression in Malignant Tumors

The expression of angiogenic factors by tumor cells varies between different tumor cell types and with time in any particular tumor or its subclones. In cross-sectional studies, malignant tumors of increasingly advanced stage display higher expression of angiogenic factors. For example, VEGF expression increased from metaplasia through advanced esophageal adenocarcinoma, and similarly varied between adenomas through various grades of colorectal carcinoma [45, 46].

There is no single angiogenic factor that is expressed by all tumor cells. For example, platelet-derived endothelial cell growth factor (PD-ECGF, thymidine phosphorylase) was found in one study to be expressed by more than 50% of tumor cells in only 32% of squamous cell carcinomas and 42% of adenocarcinomas of the lung, and in none of 22 small cell carcinomas [47]. Individual tumor types may variously express different angiogenic factors, any one or combination of which may be responsible for vascular growth in that tumor. For example, VEGF and PD-ECGF are each independently associated with high microvascular density within pancreatic carcinomas; although, in one study, only 56% of pancreatic carcinomas were found to be positive for VEGF and 32% for PD-ECGF [48]. A variety of factors including VEGF, PD-ECGF, and the chemokine CXCL8 may correlate with vascular densities within some tumors, whereas in other tumor types the extent of expression of, for example, VEGF may not show any clear relationship with tumor vascularization [48-51].

As well as differing between tumors of different types, angiogenic factor expression may vary between individual tumor cells within a tumor. This heterogeneous production of factors between subclones of tumor cells within a primary tumor is reflected by variations in factor production between primary tumors and their metastases [52]. Indeed, inconsistent relationships between matched individual tumors and their metastatic deposits in their levels of vascularity and endothelial cell proliferation may indicate that angiogenic factor production by the metastasizing clone differs from that in the primary tumor [53].

5.2. Consequences of Differential Angiogenesis in Tumors

The hypothesis that an "angiogenic switch'' can explain progression of malignant tumors was eloquently put forward by Folkman over a decade ago [44]. As originally described, tumor growth may be restricted by the inadequacy of its blood supply until it "switches on'' a production of angiogenic factors that in turn stimulates its neovascularization and facilitates tumor growth and subsequent metastasis. The molecular entities that may be expressed by tumor cells to induce this angiogenic switch have been more difficult to pin down, and, indeed, the expression of several or any of a variety of angiogenic factors may be responsible for such a switch.

It is difficult to determine from cross-sectional studies whether new induction of angiogenic factors leads sequentially to tumor progression, as implied by the angiogenic switch hypothesis, or whether angiogenic factor expression identifies a subgroup of tumors that were likely to progress from the start. The potential for tumor cells themselves to drive an angiogenic switch has been complemented by the recognition of host cells within the tumor environment such as fibroblasts and macrophages, which may express a variety of angiogenic factors and thereby induce angiogenesis, as it were, from the outside.

Differences in angiogenic factor expression explain some of the differences in appearance and behavior between different tumor types. For example, higher VEGF expression by breast carcinoma cells to some extent distinguishes ductal tumors from those of a lobular type [54]. Basal cell carcinomas of the skin are locally invasive but have very low metastatic potential compared with squamous cell carninomas. VEGF expression is localized to the invasive edge of basal cell carcinomas, whereas it is expressed throughout the tumor in squamous cell carcinomas [55]. Similarly, CXCL8 is more strongly expressed and associated with high vascularity in diffuse rather than in intestinal-type gastric carcinomas [50].

Variations in the expression of angiogenic factors may modulate tumor vascularization and therefore oxygenation. This, in turn, may contribute to differing responsiveness to treatments between different tumor cell types. For example, increased radioresistance in pancreatic carcinoma cell lines was associated with an increased expression of angiopoietin (Ang)-2, which plays an important role in vascular maturation [56].

5.3. Host Tissue Angiogenic Responses

Cells in the environment of tumors also express angiogenic factors and may be no less important than the tumor cells themselves in influencing tumor vascularization. Whereas the tumor cells appear to be the main source of VEGF in supraglottic squamous cell carcinomas of the head and neck, the majority of VEGF positive cells in non-small cell lung carcinomas and invasive ductal breast carcinomas may be macrophages and fibroblasts [57]. Indeed, some angiogenic factors such as FGF-2 and transforming growth factor (31 may be predominantly expressed by macrophages and fibroblasts in most tumor types.

The importance both of the tumor cells and their environment as sources of angiogenic factors is supported by their independent associations with tumoral vascular density in human tissues [58]. This principle has been elegantly demonstrated using species-specific reagents in mice bearing human xeno-grafts. Antibodies that block both human and mouse VEGF inhibited equally the growth of three tumor cell types, HM-7, A673, and HPAC [59]. Avastin is a monoclonal blocking antibody that binds and inhibits human but not mouse pathophysiological mechanisms of angiogenesis 201

  1. It was found to be almost completely effective in inhibiting HM-7 and A673 tumor cell growth, but less than 50% effective in inhibiting HPAC growth. In the context of HPAC tumor growth, it appears that VEGF produced by the mouse is sufficient to support the growth of the human tumor cells.
  2. 4. Vascular Phenotype

Different patterns of vascularization may also suggest differential regulation of angiogenesis in different tissues. Corrosion casts of the vasculatures of bladder carcinomas revealed two types of capillary systems, dense flat networks, and tightly packed tortuous loops [60]. The distinction between flat and tortuous microvascular systems has also been observed directly by arthros-copy in patients with inflammatory arthritis [61]. Patients with seronegative arthritis associated with psoriasis typically display more tortuous vascular patterns than do patients with the more common rheumatoid arthritis. The fact that the creation of different vascular patterns may reflect regulation by different angiogenic factors has been suggested also in carcinoma in situ of the breast [62]. Two vascular patterns have been described in this condition: first, a diffuse increase in stromal vascularity and second, a dense rim of microvessels close to the basement membrane of involved ducts. PD-ECGF expression correlates with the presence of a dense vascular rim but not with increased stromal vascularity.

In addition to differences between tumors and their environment, the neovascular phenotype itself may differ between tissues. A variety of vascular morphologies has been discussed above, and specific molecules expressed by the neovasculature may also vary. For example, binding of the antiangiogenic factor endostatin was found to almost all bladder tumor vessels, three quarters of the vessels in prostatic carcinomas, and only 11% of renal tumor vessels [63]. VEGFR3, which, in most tissues, is restricted to lymphatics, has been identified in the new blood vessels of inflamed synovium [26]. These and other characteristics of different neovascular beds may contribute to heterogeneous responses to therapies that target the vasculature.

5.5. Plasticity of Angiogenic Pathways

Angiogenesis requires that endogenous agonists and receptors are sequentially expressed from the initiation of vascular growth through the assembly of a mature and functional blood vessel. This is illustrated by the VEGF and angiopoietin systems. VEGF stimulates endothelial cell migration, proliferation, and capillary tube formation. VEGFRs are upregulated by the new endothelium, and VEGF facilitates the survival of these newly formed blood vessels [64]. Recruitment of pericytes and other ancillary cells results in the local expression of Ang-1 that can interact with its Tie-2 receptor on endothelial cells [8, 65]. These and other changes shift the new vessel toward a maturing phenotype. Expression of Ang-2, a naturally occurring antagonist of Tie-2, further buffers the angiogenic response, thereby stabilizing the neovasculature [66, 67].

This normal, sequential, coordinated expression of complementary factors which leads from blood vessel growth to establish a mature, resting vascula-ture, is observed during embryonic development and during physiological angiogenesis in the adult. It is distinct from the sequential expression of angiogenic factors that can lead from the initiation of angiogenesis to sustained vessel growth over months or years, for example, during chronic inflammation. Persistent angiogenesis, far from resulting in a functional, normally regulated vascular bed appropriate to the tissue's metabolic demands, instead leads to a disorganized, dysregulated, leaky, and fragile network of blood vessels [68].

Several vasoactive peptides provide examples where one angiogenic pathway gives way to another, thereby maintaining angiogenesis. This plasticity of angiogenic pathways emphasizes the need to extend studies in normal tissues by undertaking research on pathological samples, since factors that are important in one may be redundant in the other.

5.5.1. Neuropeptides

Substance P is present in fine, unmyelinated nerves around resting blood vessels, whose endothelia express the NK1 tachykinin receptor [69]. The release of preformed substance P therefore is well placed to initiate angiogenesis immediately following tissue injury [70]. Sensory nerves are, however, depleted during chronic inflammation [71]. Nerve growth progresses much more slowly than does that of blood vessels, and the neovascu-latures of, for example, tumors, skin grafts, and arthritic joints are often relatively noninnervated, despite expressing NK1 receptors [17, 72-74].

The recent discovery of endokinins and hemokinins provides an alternative pathway that may take over from neuronally derived substance P as an increasing proportion of vessels are noninnervated. These novel tachykinins are expressed by bone marrow-derived inflammatory cells and endothelial cells themselves [75]. Both hemokinin-1 and endokinins A and B are selective and full agonists at NK1 receptors in man, rat, and mouse, inducing acute plasma extravasation and vasodilation [76]. It is likely that these novel tachykinins will also, like substance P, induce angiogenesis.

Such a "handover" of angiogenesis from nerves to inflammatory cells, using analogous signaling mechanisms, may also occur with calcitonin gene-related peptide (CGRP) and adrenomedullin. CGRP is a cotransmitter of substance P that also may induce angiogenesis [77]. Adrenomedullin is a hypoxia-inducible angiogenic factor that is produced by, among other nonneuronal cells, macrophages [78-80]. Adrenomedullin stimulates endothelial cell proliferation, capillary tube formation, and vascular survival in vitro [81, 82] and enhances vascular growth in vivo [83, 84].

Biological effects of CGRP and adrenomedullin are mediated through cell surface, G-protein-coupled receptors, the CGRP receptor-1, and adrenomedullin receptors-1 and -2. The pharmacological selectivities of CGRP and adrenomedullin receptors depend on the heterodimeric association of a single calcitonin receptor-like receptor (CRLR) with one of three receptor activity-modifying proteins (RAMPs) [85]. CRLR is a seven-transmembrane G-protein-coupled receptor. CRLR association with RAMP1 confers selectivity for CGRP, whereas association of CRLR with RAMP2 or RAMP3 confers selectivity for adrenomedullin.

5.5.2. Other Vasoactive Peptides

Kinins are produced by enzymatic cleavage of kininogen during acute inflammation [86]. Normal tissues express B2 receptors on their blood vas-culature, which have relative selectivity for the intact bradykinin molecule. During inflammation, B1 receptors are induced, and carboxypeptidase A cleaves kinins to produce their des-Arg fragments which have a relative selectivity for this receptor. B2 and B1 receptors each can mediate kinin-enhanced angiogenesis [87, 88]. Experiments using selective receptor antagonists have indicated a shift from B2 receptor- to B1 receptor signaling as inflammation evolves [89].

Angiotensin can also stimulate angiogenesis, despite being a vasoconstrictor peptide [90, 91]. Angiotensin displays equal affinity for each of two receptors, AT1 and AT2. Angiotensin-enhanced angiogenesis is mediated by AT1 receptors, whereas upregulation of AT2 in the granulation tissue damps down this angiogenic activity, providing a negative feedback loop [91]. Angiotensin is formed by enzymatic cleavage of angiotensinogen, initially involving the enzyme renin. The component of angiotensinogen that is cleaved from angio-tensin I [des(angiotensin I)angiotensinogen] is a member of the serpin family of folded proteins, with structural similarity to PEDF. This fragment also displays antiangiogenic activity, suggesting an additional possible level of angiogenesis regulation by the rennin-angiotensin system [92].

In summary, factors that initiate angiogenesis may well differ from those that sustain it in chronic diseases. Some angiogenic factors and their receptors are present in normal resting tissues, or may be present in inactive forms rapidly released during tissue damage. This may be the case for kinins and FGF-2. Various other factors and their receptors may be upregulated during the early stages of the angiogenic process and become increasingly important as vascular growth continues. Such factors may either sustain angiogenesis, or may ensure survival and maturation of newly formed vessels. Peptides that are released into tissues during an acute inflammatory response, including bradykinin, substance P, CGRP, and angiotensin II, may well initiate angio-genesis. Other vasoactive peptides and growth factors that are upregulated during chronic inflammation may maintain the angiogenic process once initiated. Different tumors may use any one or more of these pathways to facilitate their vascularization and subsequent growth and metastasis either by producing angiogenic factors themselves or by inducing an inflammatory response.

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