Circulating factors induced by caloric restriction in the nonhuman primate Macaca mulatta activate angiogenic processes in endothelial cells

Abstract

Moderate caloric restriction (CR) without malnutrition increases healthspan in virtually every species studied, including nonhuman primates. In mice, CR exerts significant microvascular protective effects resulting in increased microvascular density in the heart and the brain, which likely contribute to enhanced tolerance to ischemia and improved cardiac performance and cognitive function. Yet, the underlying mechanisms by which CR confer microvascular protection remain elusive. To test the hypothesis that circulating factors triggered by CR regulate endothelial angiogenic capacity, we treated cultured human endothelial cells with sera derived from Macaca mulatta on long-term (over 10 years) CR. Cells treated with sera derived from ad-libitum-fed control monkeys served as controls. We found that factors present in CR sera upregulate vascular endothelial growth factor (VEGF) signaling and stimulate angiogenic processes, including endothelial cell proliferation and formation of capillary-like structures. Treatment with CR sera also tended to increase cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing [ECIS] technology) and adhesion to collagen. Collectively, we find that circulating factors induced by CR promote endothelial angiogenic processes, suggesting that increased angiogenesis may be a potential mechanism by which CR improves cardiac function and prevents vascular cognitive impairment.

Figure 1.

Effect of treatment with sera collected from caloric-restricted (CR) Macaca mulatta on vascular endothelial growth factor (VEGF)-induced adhesion of human coronary arterial endothelial cells (CAECs). VEGF (100ng/mL)-stimulated cell adhesion was monitored by electric cell–substrate impedance sensing (ECIS) technology (see the Methods section). (A) Time course of changes of capacitance (at 32kHz) after addition of CAECs to collagen-coated wells. Hundred percent change corresponds to the maximum level of cell coverage reached on the active electrode. Data are mean ± SEM. (n = 5 in each group). We calculated the time constant (τ) from each individual data set, which was used as an index of adhesiveness. (B) The summary data for τ_adhesion in CAECs treated with sera from control and CR animals are depicted here. Data are means ± SEM. (n = 5 in each group). There was a trend for a shorter τ in cells treated with CR sera, indicating that circulating factors triggered by CR tend to increase the adhesiveness of CAECs. (C) To assay barrier function, the time course of changes in capacitance (measured at 32kHz) and resistance (measured at 1,000 Hz) were monitored in parallel. (D) The time course of increases in resistance after capacitance has reached its minimum is indicative of barrier function associated with the formation of intercellular junctions.

Figure 2.

Treatment with sera collected from caloric-restricted (CR) Macaca mulatta significantly increases proliferation capacity of human coronary arterial endothelial cells (CAECs). Cell proliferation capacity was assessed in CAEC stimulated with vascular endothelial growth factor (VEGF; 100ng/mL) using the flow cytometry–based Guava Cell Growth assay (see Methods section). The inverse of the fluorescence intensity of the indicator dye carboxyfluorescein diacetate succinimidyl ester (CFSE) was used as an index of proliferation capacity of the cells. Data are means ± SEM (n = 5 in each group), *p < .05 versus control.

Figure 3.

Effect of treatment with sera collected from caloric-restricted (CR) Macaca mulatta on migration capacity of human coronary arterial endothelial cells (CAECs). Vascular endothelial growth factor (VEGF; 100ng/mL)-stimulated cell migration was monitored by electric cell–substrate impedance sensing (ECIS) technology in a wound-healing assay (see Methods section). (A) Representative figure showing the time course of resistance recovery after wounding (arrow indicates the time, when an electric pulse of 5 mA for 20 seconds at 100kHz was applied; 100% represents prewounding levels of resistance measured at 4000 Hz). Resistance (at 4000 Hz) was monitored for every 160 seconds. Time to reach 50% resistance recovery (corresponding to 50% confluence on the active electrode) was determined for CAECs treated with control sera or CR sera, and this parameter and the known physical dimensions of the electrode were used to calculate the migration rate (expressed as µm/h). (B) The summary data for migration rate in CAECs treated with control sera or CR sera are depicted. Data are means ± SEM (n = 5 in each group).

Figure 4.

(A) Treatment with sera collected from caloric-restricted (CR) Macaca mulatta significantly increases the formation of capillary-like structures by human coronary arterial endothelial cells (CAECs). CAECs were plated on Geltrex-coated wells, and tube formation was induced by treating CAECs with vascular endothelial growth factor (VEGF; 100ng/mL, for 24 hours). Representative examples of capillary-like structures are shown in (A). Summary data, expressed as total tube length per total area scanned (µm tube/mm²), are shown in (B). Data are means ± SEM, (n = 5 in each group), *p < .05 versus control.

Figure 5.

(A) Treatment with sera collected from caloric restricted (CR) Macaca mulatta significantly inhibits apoptosis in human coronary arterial endothelial cells (CAECs). Apoptotic cell death was assessed by measuring caspase 3/7 activity in cell lysates. *p < .05 versus control serum treated. Data are mean ± SEM. (n = 5 for each group). (B) In cultured CAECs treatment with CR sera did not alter cellular NO production, as compared with cells grown in the presence of sera obtained from control animals. Cellular NO production was assessed using the triazolofluorescein (DAF-2T) fluorescence method. Data are mean ± SEM. (n = 5 for each group).

Figure 6.

(A) Reporter gene assay showing the effects of treatment with sera collected from caloric-restricted (CR) Macaca mulatta on Nrf2/ARE reporter activity in cultured primary human coronary arterial endothelial cells (CAECs). Cells were transiently cotransfected with ARE-driven firefly luciferase and CMV-driven renilla luciferase constructs. At the end of the culture, period cells were then lysed and subjected to luciferase activity assay. After normalization, relative luciferase activity was obtained from four to six independent transfections. Data are mean ± SEM (n = 5 for each group). (B–E). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) data showing the effect of treatment with CR sera on mRNA expression of NFE2L2 (Nrf2), NQO1, HMOX1, and GCLC in cultured primary CAECs. Data are mean ± SEM. (n = 5 in each group). (F) Effect of treatment with CR sera on mitochondrial production in CAECs, assessed using the MitoSOX Red fluorescence method. Data are mean ± SEM. (n = 5 for each group).

Figure 7.

Treatment with sera collected from caloric restricted (CR) Macaca mulatta upregulates vascular endothelial growth factor (VEGF) signaling in human coronary arterial endothelial cells (CAECs). The time course for phosphorylation of SAPK/JNK (A), p38 MAPK (B), Akt (C), and HSP27 (D) in CAECs exposed to 100ng/mL VEGF for 5, 15, and 30 minutes was shown. Protein phosphorylation was analyzed using a magnetic multiplex bead array (Millipore; see the Methods section).

Figure 8.

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) data showing the effect of treatment with CR sera on mRNA expression of VEGFR2 (A) and VEGFR1 (B) in cultured primary coronary artery endothelial cells (CAECs). Data are mean ± SEM (n = 5 in each group).

Figure 9.

Treatment with sera collected from caloric-restricted (CR) Macaca mulatta upregulates vascular endothelial growth factor (VEGF; 100ng/mL)-induced expression of its target genes in human coronary artery endothelial cells (CAECs). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) data show the effect of treatment with CR sera on VEGF-induced mRNA expression of IL-8 (A) , NDRG1 (B), MEF2C (C), ANGPT2 (D), DNAJB9 (E), KLF4 (F), IGFBP3 (G), and RCAN1 (H) in cultured primary CAECs. Data are mean ± SEM (n = 5 in each group). *p < .05 versus control sera treated and #p < .05 versus no VEGF.