pVHL Function Is Essential for Endothelial Extracellular

The tumor suppressor von Hippel-Lindau protein (pVHL) is critical for cellular molecular oxygen sensing, acting to target degradation of the hypoxia-inducible factor alpha transcription factor subunits under normoxic conditions. We have found that independent of its function in regulating hypoxic response, the VHL gene plays a critical role in embryonic endothelium development through regulation of vascular extracellular matrix assembly. We created mice lacking the VHL gene in endothelial cells; these conditional null mice died at the same stage as homozygous VHL-null mice, with similar vascular developmental defects. These included defective vasculogenesis in the placental labyrinth, a collapsed endocardium, and impaired vessel network patterning. The defects in embryonic vascularization were correlated with a diminished vascular fibronectin deposition in vivo and defective endothelial extracellular fibronectin assembly in vitro. We found that the impaired migration and adhesion of VHL-null endothelial cells can be partially rescued by the addition of back exogenous fibronectin, which indicates that pVHL regulation of fibronectin deposition plays an important functional role in vascular patterning and maintenance of vascular integrity. The von Hippel-Lindau (VHL) hereditary cancer syndrome is caused by germ line mutation of the VHL tumor suppressor gene (26). Clear-cell carcinomas of the kidney (renal cell cancers), angiomas of the retina, and hemangioblastomas of the central nervous system are very common in these patients. Other tumors, such as pheochromocytomas, endolymphatic sac tumors, islet cell tumors of the pancreas, and vascular tumors in non-centralnervous-system sites are also found in these patients (26). Individuals with VHL disease typically have inherited a defective VHL allele. The remaining wild-type (wt) VHL allele is either inactivated or lost in tumors, obeying the Knudsen two-hit hypothesis model for tumor suppressor function. The von Hippel-Lindau tumor suppressor protein (pVHL) plays an important role in the oxygen-sensing pathway. At a molecular level, pVHL forms complexes with elongin C, elongin B, and cullin-2, which structurally resemble SCF-like ubiquitin ligases in yeast (10, 29, 45). In the presence of oxygen, two proline residues of hypoxia-inducible factor alpha (HIF-) subunits are hydroxylated by prolyl hydroxylases (6, 13, 27). This facilitates pVHL complex binding and targets HIF- subunits for degradation by the proteasome (33). Under hypoxic conditions, the prolyl hydroxylation of HIF- subunits is inhibited, which allows HIF- subunits to escape ubiquitin-mediated proteolysis. Thus, HIF- subunits accumulate, translocate to the nucleus, and then form heterodimeric transcription factors with the aryl hydrocarbon receptor nuclear translocator (also called HIF-1). Many hypoxia-inducible genes, including vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), and erythropoietin, are regulated by HIF- at a transcriptional level (19, 24, 32, 43). Several groups have shown that cells lacking pVHL are unable to degrade HIF- subunits in the presence of oxygen, which in turn leads to excessive transcrip