Chloride Intracellular Channel-4 Is a Determinant of Native Collateral Formation in Skeletal Muscle and Brain

Abstract

The capacity of the collateral circulation to lessen injury in occlusive vascular disease depends on the density and caliber of native (preexisting) collaterals, as well as their ability to outwardly remodel in ischemia. Native collateral conductance varies widely among healthy individuals, yet little is known about what specifies collateral formation. Chloride intracellular channel (CLIC)4 protein is required for endothelial cell hollowing, a process necessary for vessel formation during embryogenesis and ischemia. Whether CLIC4 has other physiological roles in vascular biology is uncertain. We studied collateral formation and remodeling in mice deficient in CLIC1 and CLIC4. Vascular responses to femoral artery ligation were similar in Clic1(-/-) and wild-type mice. In contrast, immediately after ligation perfusion dropped more in Clic4(-/-) than wild-type mice, suggesting fewer preexisting collaterals, a finding confirmed by angiography, greater ischemia, and worse recovery of perfusion; however, collateral remodeling was unaffected. Likewise, native cerebral collateral density in Clic4(-/-) (but not Clic1(-/-)) mice was reduced, resulting in severe infarctions. This was associated with impaired perinatal formation and stabilization of nascent collaterals. Clic4 hemizygous mice had intermediate deficits in the above parameters, suggesting a gene-dose effect. Ischemia augmented CLIC1 and CLIC4 expression similarly in wild-type mice. However, CLIC1 increased 3-fold more in Clic4(-/-) mice, suggesting compensation. Despite greater ischemia in Clic4(-/-) mice, hypoxia-inducible factor-1alpha, vascular endothelial growth factor (VEGF) and angiopoietin-2 increased less compared to wild-type, suggesting CLIC4 exerts influences upstream of hypoxia-inducible factor-1alpha-VEGF signaling. Hence, CLIC4 represents the second gene that, along with VEGF shown by us previously, specifies native collateral formation.