Caloric restriction confers persistent anti-oxidative, pro-angiogenic, and anti-inflammatory effects and promotes anti-aging miRNA expression profile in cerebromicrovascular endothelial cells of aged rats

Abstract

In rodents, moderate caloric restriction (CR) without malnutrition exerts significant cerebrovascular protective effects, improving cortical microvascular density and endothelium-dependent vasodilation, but the underlying cellular mechanisms remain elusive. To elucidate the persisting effects of CR on cerebromicrovascular endothelial cells (CMVECs), primary CMVECs were isolated from young (3 month old) and aged (24 month old) ad libitum (AL)-fed and aged CR F344xBN rats. We found an age-related increase in cellular and mitochondrial oxidative stress, which is prevented by CR. Expression and transcriptional activity of Nrf2 are both significantly reduced in aged CMVECs, whereas CR prevents age-related Nrf2 dysfunction. Expression of miR-144 was up-regulated in aged CMVECs and overexpression of miR-144 significantly decreased expression of Nrf2 in cells derived from both young animals and aged CR rats. Overexpression of a miR-144 antagomir in aged CMVECs significantly decreases expression of miR-144 and up-regulates Nrf2. We found that CR prevents age-related impairment of angiogenic processes, including cell proliferation, adhesion to collagen and formation of capillary-like structures and inhibits apoptosis in CMVECs. CR also exerts significant anti-inflammatory effects, preventing age-related increases in the transcriptional activity of NF-kappaB and age-associated pro-inflammatory shift in the endothelial secretome. Characterization of CR-induced changes in miRNA expression suggests that they likely affect several critical functions in endothelial cell homeostasis. The predicted regulatory effects of CR-related differentially expressed miRNAs in aged CMVECs are consistent with the anti-aging endothelial effects of CR observed in vivo. Collectively, we find that CR confers persisting anti-oxidative, pro-angiogenic and anti-inflammatory cellular effects preserving a youthful phenotype in rat cerebromicrovascular endothelial cells, suggesting that through these effects CR may improve cerebrovascular function and prevent vascular cognitive impairment.