IP-10 Blocks Vascular Endothelial Growth Factor-Induced Endothelial Cell Motility and Tube Formation via Inhibition of Calpain

Abstract

Abstract-Angiogenesis plays a critical role in wound repair. Endothelial cells present CXC receptor 3 (CXCR3) for chemokines expressed late in wound regeneration. To understand the physiological role CXCR3 plays in regulating endothelial function, we analyzed the ability of a CXCR3 ligand, IP-10 (CXCL10), to influence endothelial cell tube formation. Treatment of endothelial cells with IP-10 in the presence of vascular endothelial growth factor (VEGF) inhibited tube formation on growth factor-reduced Matrigel and in a subcutaneous Matrigel plug. Furthermore, IP-10 significantly inhibited VEGF-induced endothelial motility, a response critical for angiogenesis. Previous work showed that CXCR3 ligandation initiates protein kinase A (PKA) phosphorylation-dependent inhibition of m-calpain, required for induced cell motility, in fibroblasts but not epithelial cells. Here we show that CXCR3 activation in endothelial cells induces an increase in cAMP and PKA activation. Treatment of endothelial cells with Rp-8-Br-cAMP, an inhibitor of PKA, or small interference RNA to PKA was able to reverse the inhibitory effects of IP-10 on VEGF-mediated tube formation and motility. Importantly, treatment of endothelial cells with VEGF induced the activation of m-calpain, but costimulation with IP-10 significantly decreased this activity. Using Rp-8-Br-cAMP, we show blocking PKA reversed the IP-10 inhibition of VEGF-induced m-calpain activity. These data indicate that the activation of CXCR3 inhibits endothelial tube formation through a PKA mediated inhibition of m-calpain. This provides a means by which late wound repair signals limit the angiogenesis driven early in the wound response process. Key Words: CXCL10 Ⅲ CXCR3 Ⅲ angiogenesis Ⅲ signal transduction Ⅲ cAMP Ⅲ receptor tyrosine kinase W ound healing is a dynamic complex biological event. 1 During the regenerative phase of wound healing angiogenesis stops, followed by involution during the remodeling phase. Key to both the initial proliferation of these vessels and the subsequent stasis and then involution of the vascular network is the response of the endothelial cells to signals from the surrounding tissues. At present it is not known whether the termination of angiogenesis occurs because of depletion of the proangiogenic or induction of antiangiogenic signals. Recently, we have found that during the regenerative phase of wound repair, chemokines are expressed to limit fibroblast immigration and motility. 2 Thus, we asked whether the same signals stopped angiogenic ingrowth.Two of the ELR (glutamic acid-leucine-arginine)-negative CXC chemokines appear late in the regenerative phase. IP-10 (interferon ␥-inducible protein 10, CXCL10) is produced by endothelial cells themselves late in the regenerative phase 3 and IP-9 (I-TAC, CXCL11) derives from redifferentiating keratinocytes. 2 These ELR-negative chemokines are of particular interest, because they have been reported to be angiostatic. [3][4][5] These chemokines bind to the common CXC chemokine receptor 3 (CXCR3), 6 which has...

PLCγ contributes to metastasis of in situ-occurring mammary and prostate tumors

“…This conditional, post-natal expression was required as constitutive ablation of PLCg is embryonic lethal (Ji et al, 1998b). Furthermore, by limiting expression to the target epithelial cells, we avoid issues of altering the microenvironment of the primary and metastatic tumor sites, as well as affecting tumor-associated angiogenesis (Bodnar et al, 2006). We chose a reversible, inducible system rather than conditional gene deletion (i.e.…”

Chemokines in Wound Healing and as Potential Therapeutic Targets for Reducing Cutaneous Scarring

“…40,[88][89][90][91] These ligandreceptor interactions reduce fibroblast and endothelial motility while increasing keratinocyte migration. 67,92,93 It is proposed that this signaling axis allows wounds to transition through remodeling resulting in the characteristic appearance of scar tissue with densely packed collagen bundles and reduced elastin. 94,95 Wound contraction secondary to myofibroblast action begins in the proliferative phase, but continues during remodeling.…”

Calpain and Atherosclerosis

“…1 and Table 1). It is likely that VEGF 35,36) and fluid shear stress 13,37,38) are representative activators of calpain systems in ECs under physiological conditions. ECs exhibit measurable calpain activity even under unstimulated conditions 39,40) , which may be due to spontaneous Ca 2＋ mobilization in the cells 41) .…”

“…Mechanistically, calpainmediated regulation of small GTPases 13,40) as well as calpain-induced proteolysis of focal adhesion proteins 37) during EC dynamics has been documented previously. Accordingly, calpain systems appear to play key roles in angiogenesis 36,38) , wound closure 39) and maintenance of barrier functions 13,40) in ECs. In addition to motility responses, it has been reported that nitric oxide (NO) production in ECs is modulated through calpain-induced proteolysis of HSP90 42) or calpain-regulated PI3K/AMPK signaling 35) .…”