Abstract 3595: Chloride Intracellular Channel-4 Determines Native Collateral Density

Abstract

The density of native (pre-existing) collaterals in healthy tissue and their capacity to enlarge in occlusive disease (arteriogenesis) are major determinants of tissue injury in ischemia. Mechanisms regulating arteriogenesis remain unclear. Moreover, nothing is known about what specifies formation of native collaterals, except our report that VEGF contributes to native collateral formation (Clayton et al FASEB J 21:LB352, 2007). Recently, chloride intracellular channel proteins (CLICs) have been implicated in cellular processes including cell hollowing. CLIC1 and CLIC4 are expressed by endothelial cells, and VEGF-induced cell hollowing is critical for vascular formation in the embryo and angiogenesis in ischemia. Therefore, we examined CLIC1−/− and CLIC4−/− (null) mice for effects on native collateral density in healthy tissues and on arteriogenesis and angiogenesis in a hindlimb model of ischemia. Responses of CLIC1−/−mice did not differ from wild-type in any experiment. In contrast, CLIC4−/− showed a greater reduction in perfusion immediately after femoral ligation (p<0.05 here and all statements below). Since baseline capillary density and collateral diameter were not different from wild-type, this suggested CLIC4−/− have reduced native collateral density. This was confirmed angiographically. In agreement, during 10 days after ligation CLIC4−/− mice had worse recovery of perfusion, greater ischemia and atrophy, and more impaired limb use, whereas collateral remodeling was unaffected. While ischemia increased expression of CLIC1 and CLIC4 similarly in wild-type mice, CLIC1 was augmented 3-fold in CLIC4−/−, suggesting CLIC1 may compensate for absence of CLIC4. Despite greater ischemia in CLIC4−/− mice, HIF1α and VEGF increased less in CLIC4−/−, suggesting CLIC4 may reside upstream of Hif1α-VEGF signaling. Native collateral density in the cerebral circulation of CLIC4−/− mice (but not CLIC1−/−) was also greatly reduced, resulting in 4-fold larger infarctions. This reduced collateral density in adults was associated with reduced formation and stabilization of nascent collaterals in the newborn. Hence, CLIC4 represents the second gene identified, besides VEGF, that specifies formation of the native collateral circulation.