Introduction
Klotho-deficient mice have extensive vascular calcification. However, the mechanism involved has not been elucidated. We analyzed the endothelial cells of Klotho-deficient mice (Klotho-ECs) and found a novel functional role for the Klotho-protein in the maintenance of vascular homeostasis.
Methods and Results
Primary-cultured endothelial cells from the aorta of wild-type- (WT) and Klotho- deficient mice were loaded with fura-2 and stimulated with VEGF. In WT-ECs, intracellular Ca(2+) concentration ([Ca(2+)]i) elevated to the peak level, and it decreased to the basal level in +5 minutes. Whereas, in Klotho-ECs, after similar peak level to WT-ECs, the elevation of [Ca(2+)]i was sustained at ~35% of the peak level for more than 10 minutes. In Ca(2+) free medium, this sustained elevation was abolished. Immunostaining revealed that, in Klotho-ECs, VEGF-induced co-internalization of the Ca(2+) channel; TRPC-1, with VEGF-receptor-2 (VEGFR-2) was abolished, thereby, at 30 minutes after VEGF-stimulation, expression levels of TRPC-1 in the plasmamembrane was 2.2-fold higher than in WT-ECs. Klotho-protein constitutively bound to VEGFR-2 and TRPC-1, strengthened their association, and promoted their co-internalization. VEGF-induced activation of Rac-1 and reorganization of F-actin, which are involved in internalization, were impaired in Klotho-ECs, and restored by the replacement of Klotho-protein. VEGF-induced phosphorylation of VEGFR-2 and its downstream signaling molecules, including Src, Akt, PLCγ, and Caveolin1, was attenuated. As the biological consequence of sustained increase in [Ca(2+)]i, activation of calpain and caspase-3 followed by apoptosis was promoted in Klotho-ECs in vivo and vitro. Also, expression of VE-cadherin in the plasmamembrane was reduced and intimal hyperpermeability was observed in the aorta of Klotho-deficient mice, which presumably lead to exudation of the Ca(2+)/phosphor -rich serum into the vessel walls, followed by extensive vascular calcification.
Conclusion
Thus, Klotho-protein regulates ligand-induced Ca(2+) influx and subsequent biological functions involved in vascular calcification.
Vasculogenesis and Angiogenesis in VEGF Receptor-1 Deficient Mice
