Regulation and role of endogenously produced hydrogen sulfide in angiogenesis

Abstract

Recent studies have implicated endogenously produced H₂S in the angiogenic process. On one hand, pharmacological inhibition and silencing of the enzymes involved in H₂S synthesis attenuate the angiogenic properties of endothelial cells, including proliferation, migration and tube-like structure network formation. On the other hand, enhanced production of H₂S by substrate supplementation or over-expression of H₂S-producing enzymes leads to enhanced angiogenic responses in cultured endothelial cells. Importantly, H₂S up-regulates expression of the key angiogenic factor vascular endothelial growth factor (VEGF) and contributes to the angiogenic signaling in response to VEGF. The signaling pathways mediating H₂S-induced angiogenesis include mitogen-activated protein kinases, phosphoinositide-3 kinase, nitric oxide/cGMP-regulated cascades and ATP-sensitive potassium channels. Endogenously produced H₂S has also been shown to facilitate neovascularization in prototypical model systems in vivo, and to contribute to wound healing, post-ischemic angiogenesis in the heart and other tissues, as well as in tumor angiogenesis. Targeting of H₂S synthesizing enzymes might offer novel therapeutic opportunities for angiogenesis-related diseases.

Figure 1. Proposed interactions between VEGF and H₂S

VEGF binding to VEGFR2 causes enhanced H₂S production presumably by activating CSE in a calcium-dependent manner. Nucleophilic attack of the disulfide bond between Cys 1045-1024 by H₂S leads to a disulfide reduction and boosts VEGFR2 tyrosine kinase activity. H₂S generated by CBS increases the stability and transcriptional activity of Sp1, enhancing VEGFR2 transcription. H₂S causes an increase in HIF-1α levels, DNA binding and transcriptional activity. VEGFR2 (vascular endothelial growth factor receptor 2); VEGF (vascular endothelial growth factor); CSE (cystathionine-γ lyase); CBS (cystathionine-β synthase); HIF (hypoxia inducible factor); HRE (hypoxia response element); Sp1 (specificity protein 1); H₂S (hydrogen sulfide)

Figure 2. Interactions of H₂S and NO in angiogenesis

NO activates sGC to increase cGMP levels, while H₂S both facilitates cGMP production (by activating eNOS and keeping the sGC in a reduced NO-responsive state) and prevents cGMP breakdown by inhibiting PDE. This allows cGMP to reach the threshold levels required to trigger PKG angiogenic signaling. H₂S also promotes eNOS dimerization through eNOS persulfidation. PI3K (phosphoinositide 3-kinase); Akt (protein kinase B); PTEN (phosphatase and tensin homolog); 3,4,5 InsP3 (phosphatidylinositol-3,4,5-trisphosphate); 4,5 InsP2 (phosphatidylinositol-4,5-bisphosphate); eNOS (endothelial nitric oxide synthase); sGC (soluble guanylate cyclase); cGMP (cyclic guanosine monophosphate); GMP (guanosine monophosphate); PDE (phosphodiesterase); PKG (cGMP-dependent protein kinase), NO (nitric oxide); H₂S (hydrogen sulfide)

Figure 3

Representative photomicrographs of chorioallantoic membranes incubated with vehicle (control), PAG or BCA. A clear reduction in the vascular network is seen when CSE is inhibited (magnification × 2,5).

Figure 4. PAG inhibits VEGF-stimulated angiogenesis in vivo

C57BL/6 male mice 10–12 weeks old mice were randomized in 4 groups (4 mice/group): 1. CONTROL: 100μl water for injection (WFI) intra-peritoneally (i.p.) for 15 days, 2. PAG: 100μl of 50mg/kg in WFI PAG i.p. for 15 days, 3. VEGF:100μl WFI i.p. for 15 days and 4.PAG+VEGF: 100μl of 50mg/kg in WFI PAG i.p. for 15 days. The first day, mice were anesthetized by a 1–2 min exposure to isoflurane and 400μl 50:50 Matrigel® Matrix: 1xPBS with or without VEGF(100ng/ml) was infused subcutaneously. Mice were sacrificed 16 days after Matrigel® injection. (A) Macroscopic appearance and vessel infiltration in Matrigel plugs (B) Hemoglobin content in μg/mg *p<0.05 vs Control, ***p<0.01 vs Control, #p<0.05 vs VEGF. (C) Immunostaining for the expression of endothelial cell marker CD31 staining in 5μm sections of Matrigel® plugs. 40X magnification.