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Postnatal neovascularization is triggered by tissue hypoxia and the hypoxia-inducible transcription factor dependent upregulation of vascular growth factors. At the same time hypoxia is associated with replication stress and can induce a cellular DNA repair response including the phosphorylation of histone H2AX. Recent findings point to a role of H2AX in endothelial cell proliferation under hypoxia and thereby in hypoxia-driven neovascularization.
The development and growth of blood vessels is essential for embryonic organ development and consists of vasculogenesis, which is the formation of a primitive vascular network from endothelial cells, as well as of angiogenesis, a process requiring branching, remodeling and interactions of endothelial cells with pericytes and resulting in the generation of a mature vasculature. In the adult organism, endothelial cells are quiescent, however, during wound healing or under pathologic conditions, endothelial cells can proliferate and neovascularization can be triggered predominantly in response to a hypoxia stimulus 1;2. Adult neovascularization contributes to inflammatory, malignant and eye pathologies. In tumors or in proliferative retinopathies, such as the diabetic retinopathy or the retinopathy of prematurity (ROP), hypoxia-driven endothelial cell proliferation and the neovascularization response are exuberant 1-3. For instance, in the course of proliferative retinopathies, the retinal tissue hypoxia due to vascular regression and reduced vascularization results in excessive abnormal vessel growth that leaves behind non-functional vascular structures and is responsible for vision-threatening complications, such as hemorrhages 3. The common denominator of angiogenesis in tumors, retinopathies or other pathologies is hypoxia and the master regulator of the hypoxia response is the transcription factor hypoxia-inducible factor (HIF), which is activated in hypoxic tissues and increases the expression of multiple growth factors, including the vascular endothelial growth factor (VEGF) 4.
Besides upregulating HIF, hypoxia is also biologically significant as a replication-associated cellular stress 5. Although not entirely clear how hypoxia may affect cell proliferation, hypoxia may activate a cellular DNA repair response 5. The DNA repair pathway is typically the response to DNA lesions, for example by ionizing or ultraviolet radiation, or the collapse of replication forks 6. A central molecular component of the DNA repair response is the activation of the kinases, ataxia teleangiectasia mutated kinase (ATM), ATM- and Rad3-related kinase (ATR) and DNA-dependent protein kinase that phosphorylate numerous targets that regulate cell fate 7. An important event upon DNA damage is the γ-phosphorylation of histone H2AX at Ser139 (designated γ-H2AX) that besides being a sensitive marker of activation of the DNA repair machinery, also acts to promote DNA repair by maintaining repair factors at the vicinity of the DNA lesion 8-12. Hypoxia has been reported to induce γ-H2AX and to activate a cellular DNA repair response including the activation of ATR and/or ATM 13;14. The hypoxia-triggered DNA repair response may be due to the HIF-mediated repression of the expression of DNA repair factors, such as NBS1 15. Alternatively, hypoxia may not really induce DNA damage itself but hypoxia could simply accentuate the low levels of damage occurring during replication 16.
During hypoxia-driven angiogenesis, endothelial cells are stimulated to proliferate despite being subject to hypoxia. We recently demonstrated that H2AX and an efficient DNA repair response are important for endothelial cell proliferation under hypoxia and thereby for pathologic hypoxia-related angiogenesis 17. In primary endothelial cells, hypoxia induced a replication-stress related generation of γ-H2AX, as indicated by the finding that nuclear foci of H2AX phoshorylation were predominantly observed in proliferating cells that were positive for proliferating cell nuclear antigen. Moreover, hypoxia-induced γ-H2AX foci in endothelial cells colocalized with replication protein A 17, which is bound to persistent single-stranded DNA and is considered a marker of stalled replication forks 18. Experiments using siRNA-mediated knockdown of the major DNA repair signaling kinases, ATR and ATM, revealed that the phosphorylation of H2AX upon hypoxia in endothelial cells was dependent on ATR activity 17, which is consistent with the function of ATR to monitor DNA replication 7. In the ROP mouse model, in which obliteration of the developing retina vascular network is induced by incubation of 7 day old pups in 75% oxygen for 5 days, resulting in retina hypoxia after day 12 when pups are brought back to normal room air, thereby inducing excessive hypoxia-driven angiogenesis, γ-H2AX generation was higher in the retinas during the hypoxic phase (days 13-15). Moreover, γ-H2AX foci were predominantly localized to proliferating (BrDU-positive) vascular endothelial cells including endothelial nuclei of neo-vascular tufts 17. The newly formed vessels and tufts may be non-perfused or partially perfused, thus, although a large part of γ-H2AX generation in the endothelial cell nuclei of newly formed vessels may be the result of the hypoxia stimulus, it cannot be excluded that reoxygenation after reperfusion contributes to formation of the observed γ-H2AX foci.
H2AX phosphorylation was not solely an epiphenomenon of hypoxia-triggered neovascularization. H2AX was functionally involved for maintaining endothelial cell proliferation under hypoxic conditions, as assessed in vitro by utilizing H2AX-deficient endothelial cells, or in vivo during pathologic neovascularization. H2AX-/- mice as well as mice with endothelial-specific deletion of H2AX displayed reduced pathologic neovascularization in the ROP model, as compared to wild-type mice. Associated with this finding was that H2AX deficiency resulted in reduced endothelial cell proliferation and increased endothelial cell apoptosis in the course of the ROP model. In contrast, developmental retina angiogenesis was not affected by the absence of H2AX. In addition, H2AX deficiency decreased endothelial cell proliferation and neovascularization in the model of hind limb ischemia by ligation of the femoral artery. Furthermore, new vessel formation in the course of tumor angiogenesis was reduced upon H2AX deficiency, which was also accompanied by impaired growth of tumors implanted into H2AX deficient animals, as compared to wild-type animals 17.
The role H2AX plays in pathologic angiogenesis may be due to its function to mediate DNA repair and thereby help endothelial cells cope with hypoxia-related replication stress. This hypothesis would imply that further DNA repair factors that could interact functionally with H2AX, such as BRCA1, may also participate in pathologic hypoxia-driven angiogenesis 9;19. In addition, H2AX may act in other yet unknown ways in the endothelial cell response to hypoxia. For instance, whether the function of H2AX in hypoxia-driven angiogenesis is related to the hypoxia response pathways mediated by HIF is unclear and merits investigation. The regulation of the oncoprotein c-Myc by HIF is involved in modulating cell proliferation under hypoxia. HIF-1α and HIF-2α seem to differentially regulate c-Myc; while HIF-1α inhibits c-Myc expression and thereby cell cycle progression, HIF-2α enhances c-Myc activation 20;21. Intriguingly, in renal cancer, HIF-2α limited DNA damage accumulation, associated with reduced H2AX phosphorylation, and thereby enhanced proliferation 21. Interestingly, the acetylation of HIF-2α can be reversed by the SIRT1 deacetylase during hypoxia, which results in augmented HIF-2α-dependent signaling 22. In addition, the function of NBS1, a DNA repair factor that lies downstream of H2AX 9 is modulated by its acetylation status that can be modified by SIRT1 23. Strikingly, endothelial SIRT1 deficiency impaired postnatal ischemia-induced neovascularization without affecting developmental angiogenesis in mice. The function of SIRT1 in vessel growth could be attributed to negative regulation of the forkhead transcription factor Foxo1 that inhibits angiogenesis 24. Whether a crosstalk of this SIRT1-dependent pathway with the function of H2AX exists, requires further studies.
In conclusion, detailed studies are required to identify the exact mechanism underlying the function of H2AX in hypoxia-triggered neovascularization. Nevertheless these findings point to an interesting crosstalk between the DNA repair response and neovascularization.
Supported by the NIH Intramural Research Program, National Cancer Institute.