Various genetic data support the notion that the integrin a5b1 and its ECM ligand, fibronectin (FN), are absolutely essential for normal blood vessel function. Mouse embryos harboring an ablated a5 gene die by embryonic day (E) 10.5, displaying severe embryonic and extraembryonic vascular defects, as well as posterior trunk and somitic abnormalities. Additionally, a5-null embryonic stem (ES) cells injected into syngeneic mice form teratocarcinomas with reduced size and a poorly developed vasculature. Also, embryoid bodies derived from a5-null ES cells display delayed vascular differentiation and organization. In concordance with the a5 genetic data, deletion of the FN gene leads to embryonic lethality associated with severe vascular defects, and similar angiogenic abnormalities are observed in FN-null embryoid bodies .
Other experimental models also support a necessary role for a5b1 and FN in angiogenesis. For example, protein expression of a5b1 and FN is upregulated on angiogenic tumor blood vessels. Likewise, in the chick chorioallantoic membrane (CAM) assay, angiogenic growth factors upregu-late a5b1 expression. In both systems anti-a5b1 or anti-FN function-blocking antibodies or peptide antagonists perturb neovascularization .
a1b1 and a2b1 Integrins
The collagen-binding integrins a1b1 and a2b1 are both upregulated on angiogenic blood vessels following vascular endothelial growth factor (VEGF) stimulation (reviewed in Ref. ). Function-blocking anti-a1 and -a2 antibodies inhibit angiogenesis in vivo using Matrigel plugs or transplanted tumor models. Interestingly, a1-null mice are viable and fertile, yet are less susceptible to tumor growth. This resistance correlates with increased levels of circulating matrix metalloproteinase (MMp) activity involving MMp-2, MMP-7, and MMP-9. Elevated proteolysis in a1-null mice leads to the conversion of plasminogen to angiostatin, a potent inhibitor of endothelial cell neovascularization. Mice harboring a deletion of the a2 gene are viable and fertile and do not display any gross vascular abnormalities. Obviously, analysis of mice null for both a1 b1 and a2b1
Figure 1 The integrin family of ECM receptors. The 26 integrin a and b subunits, separated into three groups, are shown. Lines connecting sub-units indicate the 24 known heterodimeric pairs. The leukocyte-specific integrins are shown in blue. Integrins with proposed roles in various microvascular functions are highlighted in red. (see color insert)
expression should be useful in understanding the exact functions of these integrins during physiological and pathological angiogenesis.
The pi integrin subunit can pair with at least 12 a sub-units (Figure 1). Various pi-containing integrins are expressed in virtually all vertebrate cells and are involved in most aspects of development. Not surprisingly, pi genetic ablation leads to very early (E7.5) embryonic lethality, prior to the onset of vascular development. Analyses of pi-null ES cells, embryoid bodies, and teratocarcinomas reveal an essential role for p1 integrins in blood vessel formation. Whereas pi integrins are dispensable for endothelial cell differentiation, they are required for VEGF-induced endo-thelial cell proliferation and blood vessel elaboration.
As mentioned previously, integrins aipi and a2pi are also important for microvascular formation. It is likely that some of the defects observed in the absence of pi integrin can be explained by loss of aipi and a2pi function. However, ES cells or embryoid bodies null for pi integrin, a5 integrin, or fibronectin display many strikingly similar defects. Thus, many of the pi-null defects can be explained by loss of integrin a5pi. Again, these data emphasize that fibronectin and its primary receptor, a5pi integrin, play essential roles in the regulation of vascular development .
The av integrin subunit can pair with pi, p3, p5, p6, and p8, yielding five distinct integrin heterodimers (Figure i).
An extensive body of published data exists concerning avp3 and avp5 during various types of physiological and pathological angiogenesis. Various angiogenic growth factors or cytokines upregulate avp3 or avp5 protein expression on cultured endothelial cells or on vessels using the CAM assay. Likewise, many angiogenic blood vessels display increased levels of avp3 or avp5. Function-blocking antibodies or peptide antagonists inhibit this integrin-mediated neovascularization in these various systems, ultimately leading to endothelial cell death. Along these lines, avp3 has been intensely pursued as a potential anti-angiogenic target, and avp3 antagonists are currently being tested in human clinical trials.
Surprisingly, ablation of the p3 and/or p5 genes does not lead to angiogenic abnormalities. Mice null for both avp3 and/or avp5 are viable and fertile, and developmental vas-culogenesis and angiogenesis proceed normally in these mutants. Physiological angiogenesis occurs normally in p3-null or mice null for both p3 and p5. No experimental differences are observed using hypoxia-induced retinal angiogenesis or growth factor-mediated corneal neovascu-larization models. Interestingly, tumor growth and neovas-cularization are actually enhanced in p3-/- or p3/p5-null mice . Thus, it is difficult to infer the significance of avp3 and avp5 in neovascularization based on the antibody and peptide inhibition results versus the genetic ablation data.
One could argue that, in mice lacking p3 and/or p5 gene expression, other av-associated integrins are compensating for normal avp3 and/or avp5 functions. As mentioned earlier, av can indeed pair with five different p subunits. However, mice null for the av gene, and thus lacking the expression of all five av integrins, do not display general vascular defects. Approximately 70 percent of av-nulls survive to Ei0.0, having developed a normal vasculature, but die by Ei0.5 because of placental defects. The remaining av-null embryos that survive the mid-gestation crisis subsequently develop to term with a largely normal vasculature. However, they do develop cerebral hemorrhage and a cleft palate, and they die shortly after birth. The hemorrhage is not due to endothelial or pericyte defects, but rather involves defective associations between cerebral vessels and central nervous system glia . Interestingly, the cerebral hemorrhage in the av-nulls is specifically due to the loss of inte-grin avp8; mice null for the p8 gene display phenotypes that are essentially identical to those seen in the av-nulls .
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