IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain.
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.
Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15261-0001, USA. rjb8@pitt.edu
Richard J
RJ
Bodnar
Cecelia C
CC
Yates
Alan
A
Wells
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D054357:Chemokine CXCL10; D019743:Chemokines, CXC / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D005260:Female; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors*
C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D019743:Chemokines, CXC; C515188:Cxcr3 protein, mouse; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine; D042461:Vascular Endothelial Growth Factor A; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002154:Calpain