Klotho, a regulatory factor implicated in countering the aging process, has been reported to ameliorate endothelial dysfunction in vivo. To clarify whether Klotho protein directly affects endothelial cell function, we studied the effects of membrane-form Klotho on manganese superoxide dismutase (Mn-SOD) expression and nitric oxide production in human umbilical vein endothelial cells (HUVEC). We incubated HUVEC with conditioned medium from COS-1 cells transfected with expression vector, pCAGGS-klotho (Klotho-CM) or a recombinant, purified 6His-tagged Klotho protein. Both Klotho-CM and 6His-tagged Klotho protein enhanced Mn-SOD expression by approximately two-fold, partially via activation of the cAMP signaling pathway. Furthermore, Klotho-CM increased nitric oxide production, which also contributed to the up-regulation of Mn-SOD. Using the oxidation-sensitive dye dihydroethidium, we found that Klotho inhibited angiotensin II-induced reactive oxygen species production in HUVEC. These findings provide new insights into the mechanisms of Klotho action and support the therapeutic potential of membrane-form Klotho to regulate endothelial function.
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