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 mo old) and aged (24 mo old) ad libitum-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 upregulated 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 upregulates 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-κB 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.

Keywords: aging; angiogenesis; caloric restriction; dietary restriction; senescence.

Fig. 1.

Caloric restriction (CR) significantly decreases oxidative stress in aged cerebromicrovascular endothelial cells (CMVECs). A and B: effect of CR on cellular oxidative stress (A) and mitochondrial reactive oxygen species production (B) in CMVECs. Cellular peroxide production and mitochondrial O₂^·− production were assessed in cultured primary CMVECs derived from young and ad libitum (AL)- and CR-fed aged rats using the flow cytomeytry-based CM-H₂DCFDA fluorescence (A) and MitoSOX Red (B) method, respectively. Data are means ± SE (n = 5 for each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL). C: quantitative real time RT-PCR data showing the effect of aging and CR on mRNA expression of Nox4 in CMVECs. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL). DCF, dichlorofluorescein.

Fig. 2.

CR prevents age-related Nrf2 dysfunction in CMVECs. A: reporter gene assay showing the effects of CR on Nrf2/ARE reporter activity in cultured primary CMVECs. Cells derived from young and AL- and CR-fed aged rats were transiently cotransfected with ARE-driven firefly luciferase and CMV-driven renilla luciferase constructs. The cells were then lysed and subjected to luciferase activity assay. Data are means ± SE (n = 5 for each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL). B and C: quantitative real time RT-PCR data showing the effect of CR on mRNA expression of Nfe2l2 (Nrf2; B) and expression of miR-144 (C) in cultured primary CMVECs. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL). D: to assess the effect of miR-144 on Nrf2 expression, miR-144 was overexpressed in CMVECs derived from young and aged CR rats. In E, overexpression of miR-144 significantly decreased expression of Nrf2 in both group. Data are means ± SE. *P < 0.05 vs. respective controls. F and G: overexpression of a miR-144 antagomir in CMVECs derived from aged AL-fed rats significantly decreases expression of miR-144 (F) and upregulates Nrf2 (G). Data are means ± SE. *P < 0.05 vs. control.

Fig. 3.

CR significantly increases proliferation and adhesion capacity and inhibits apoptosis in aged CMVECs. A: cell proliferation capacity was assessed in primary CMVECs, derived from young and AL- and CR-fed aged rats, stimulated with VEGF (100 ng/ml) using the flow cytometry based Guava CellGrowth assay (see methods). The inverse of the fluorescence intensity of the indicator dye carboxyfluorescein diacetate succinimidyl ester was used as an index of proliferation capacity of the cells. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL). B: CR significantly inhibits apoptosis in aged CMVECs. Apoptotic cell death was assessed by measuring caspase 3/7 activity in cell lysates. *P < 0.05 vs. control; #P < 0.05 vs. aged (AL). Data are means ± SE (n = 5 for each group). C and D: effect of CR on VEGF-induced adhesion of CMVECs. VEGF (100 ng/ml)-stimulated cell adhesion was monitored by electric cell-substrate impedance sensing technology (see methods). C: time course of changes of capacitance (at 32 kHz) after addition of CMVECs to collagen-coated wells. One-hundred percent change corresponds to the maximum level of cell coverage reached on the active electrode. Data are means ± SE (n = 5 in each group). We calculated the time constant from each individual dataset, the inverse of which was used as an index of adhesiveness. In D, summary data for cell adhesion index in CMVECs from each experimental group are shown. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL). AU, arbitrary units.

Fig. 4.

CR significantly increases angiogenic capacity in aged CMVECs. A–C: ability to form capillary-like structures by primary CMVECs, derived from young and AL- and CR-fed aged rats, was assessed. CMVECs were plated on Geltrex-coated wells, and tube formation was induced by treatment of the cells with VEGF (100 ng/ml, for 24 h). Representative examples of capillary-like structures are shown in A–C. Summary data, expressed as total tube length per total area scanned (in μm tube/mm²), are shown in D. Data are means ± SE (n = 5 in each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL).

Fig. 5.

CR exerts anti-inflammatory effects. A: reporter gene assay showing that aging significantly increases transcriptional activity NF-κB in cultured primary CMVECs. Cells derived from CR-fed aged rats exhibited significantly decreased NF-κB activity compared with CMVECs derived from AL-fed animals. The inhibitory effect of CR on NF-κB activation was evident also after TNF-α stimulation. CMVECs were transiently cotransfected with NF-κB-driven firefly luciferase and CMV-driven renilla luciferase constructs. The cells were then lysed and subjected to luciferase activity assay. Data are means ± SE (n = 5 for each group). *P < 0.05 vs. control; #P < 0.05 vs. aged (AL); $P < 0.05 vs. no TNF-α. B: secretory profiles of CMVECs derived from young and AL- and CR-fed aged rats. Soluble monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, TNF-α, IL-6, IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), and MIP-2 secreted by these cells were detected by a magnetic multiplex bead array (see methods). Data are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. aged (AL).

Fig. 6.

CR-induced changes in miRNA expression profile in CMVECs. A: heat map is a graphic representation of normalized miRNA expression values in CMVECs derived from aged CR and AL-fed rats, depicted by color intensity, from highest (bright red) to lowest (bright green) expression. Values represent average miRNA expression levels [log2 (fold change, normalized to the respective control mean value)] in cells derived from 4 different animals in each group. B: key genes (blue) predicted to be regulated by the differentially expressed miRNAs. In green are upregulated miRNA in CMVECs derived from CR rats. In red are downregulated miRNA in CMVECs derived from CR rats. Note that CR-induced changes in TNF-α expression are predicted to be causally linked to changes in miRNA expression.