Angiogenesis In Hindlimb Ischemia Model Research Articles

Abstract 14078: Ros-dependent Sumoylation of Cu Chaperone Atox1 Drives Its Nuclear Translocation to Promote Inflammatory Angiogenesis Induced by Ischemic Injury

Introduction: Neovascularization in response to ischemia depends on inflammation, angiogenesis and reactive oxygen species (ROS). Copper (Cu) is implicated in inflammation and angiogenesis. We reported that cytosolic Cu chaperone Atox1 activates secretory Cu enzymes lysyl oxidase (LOX), while nuclear Atox1 functions as a Cu-dependent transcription factor to promote ROS/NFkB-dependent inflammation in endothelial cells (ECs). However, mechanism of Atox1 nuclear translocation as well as role of endothelial Atox1 in inflammatory angiogenesis in vivo remain unknown. SUMOylation and its deSUMOylation by SENPs regulates transcription factor function. Silica analysis identified a conserved putative SUMOylation motif at Lys(K3) of Atox1. Results: Atox1 expression was dramatically increased in angiogenic ECs in mice hindlimb ischemia model. EC-specific Atox1-deficient mice significantly reduced angiogenesis (CD31+, 67%) and Mac+ inflammatory cells in ischemic tissues. In cultured ECs, inflammatory cytokine TNFα or hypoxia promoted Atox1 nuclear translocation and Atox1 SUMOylation (3.6-fold), which were inhibited by antioxidant NAC or overexpression of “SUMO-dead” Atox1K3R. Mechanistically, TNFα induced Cys603 oxidation/inactivation of SENP1 in cytosol, which in turn increased Atox1 SUMOylation and nuclear translocation. Functionally, siAtox1or Atox1K3R inhibited TNFα-induced inflammatory/angiogenic genes VCAM/ICAM, IL-15 and RANTES. In nucleus with reduced state, ChIP assay using SUMO-Atox1 revealed that Atox1 deSUMOylation by nuclear SENP1 increases Atox1 transcriptional activity for inflammatory genes. In parallel, Atox1K3R which maintains Cu chaperone function inhibited TNFα-induced EC permeability by activating LOX. In vivo, Atox1 SUMOylation was increased after hindlimb ischemia while CRISPR/Cas9-generated SUMO-dead Atox1K3R knock-in mice showed impaired angiogenesis in hindlimb ischemia model. Conclusion: Atox1 SUMOylation via oxidative/inactivation of SENP1 in cytosol promotes: 1) its translocation to nucleus where deSUMOylated Atox1 can function as Cu-dependent transcription factor to drive inflammatory angiogenesis and 2) EC barrier dysfunction in inflamed/hypoxic ECs after ischemic injury.

Roles of calcitonin gene-related peptide in ehancement of angiogenesis

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide produced by tissue- specific alternative splicing of the primary transcript of the calcitonin/CGRP gene. CGRP is widely distributed in the central and peripheral neuronal systems and exhibits numerous biological activities in mammals. Recently, it was clarified that endogenous CGRP released from primary afferent neurons facilitates the neovascularization indispensable to tumor growth. Angiogenesis in surgical sponge models that mimics tumor stromal reaction was inhibited by a CGRP antagonist, CGRP8-37. The unilateral sciatic nerves (L1-5) of mice were cut, and Lewis lung carcinoma (LLC) cells were implanted into the subcutaneous tissues of the legs. Tumor growth was significantly reduced in the sites of denervation of these mice, compared with the sham-operated mice. In CGRP knockout mice, the tumor growth and tumor-associated angiogenesis of implanted LLC cells were significantly reduced compared with those in wild-type mice. In LLC-bearing wild-type mice, CGRP precursor mRNA levels in the dorsal root ganglion were increased compared with those in non-LLC bearing mice. This increase was abolished by denervation. In a co-culture system using human umbilical vein endothelial cells (HUVEC) and fibroblasts, CGRP increased tube formation by endothelial cells. VEGF expression was up-regulated in the tumor implantation models in a CGRP-dependent manner. These recent results suggest that sensory nerves facilitate tumor-associated angiogenesis and tumor growth during tumor development via a release of CGRP. The same was true in the processes of wound healing and gastric ulcer healing. Further, CGRP was also reported to enhance the angiogenesis in hind-limb ischemia model. These recent results indicate CGRP may become a novel therapeutic target for controlling angiogenesis.