Functional Role of STIM-1 and Orai1 in Human Microvascular Aging

Abstract

The impact of aging on vascular function is heterogeneous depending on the vascular territories. Calcium regulation plays a key role in vascular function and has been implicated in aging-related hypercontractility of corpus cavernosum. We aimed to evaluate stromal interaction molecule (STIM)/Orai system involvement in aging-related vascular alterations in the human macro and microvasculature. Aortae specimens and mesenteric arteries (MA), obtained from 45 organ donors, were functionally evaluated in organ chambers and wire myographs. Subjects were divided into groups either younger or older than 65-years old. The expressions of STIM-1, Orai1, and Orai3 were determined by immunofluorescence in the aorta and MA, and by Western blot in the aorta homogenates. The inhibition of STIM/Orai with YM-58483 (20 μM) reversed adrenergic hypercontractility in MA from older subjects but did not modify aging-related hypercontractility in the aortic strips. Aging was related to an increased expression of Orai1 in human aorta, while Orai1 and STIM-1 were upregulated in MA. STIM-1 and Orai1 protein expressions were inversely correlated to endothelial function in MA. Circulating levels of Orai1 were correlated with the inflammatory factor TNF-α and with the endothelial dysfunction marker asymmetric dimethylarginine. Aging is associated with an increased expression of the STIM/Orai system in human vessels with functional relevance only in the microvascular territory, suggesting its role in aging-related microvascular dysfunction.

Keywords: Orai channel; STIM-1; aging; human mesenteric arteries; vascular function.

Figure 1

Aging-related hypercontractility to adrenergic stimulation in the human mesenteric artery is reversed by Orai channel inhibition. Effects of Orai channel inhibition with YM-58483 (20 μM) on norepinephrine (NE)-induced contraction in human mesenteric arteries (MA (A)) and human aortic strips (AS (B)) obtained from organ donors <65-years and >65-years old. Data are expressed as mean ± SEM of the percentage of contraction induced by 125 mM K⁺ before the addition of YM-58483 or the vehicle (0.2% DMSO). n indicates the number of subjects. *** p < 0.001 vs. <65-years old; ^† p < 0.05, ^††† p < 0.001 vs. respective responses without YM-58483 by two-factor ANOVA followed by Bonferroni’s correction. (C) Complete concentration–response curves of YM-58483-induced relaxation in human AS and MA precontracted with NE obtained from organ donors. Data are expressed as mentioned above. *** p < 0.001 vs. MA. Representative tracings of these responses in MA and AS are shown in (D,E), respectively.

Figure 2

Aging is associated with an overexpression of the Orai1 channel in human aortic strips. (A) Representative immunoblots for the detection of STIM-1, Orai1, and Orai3 and corresponding β-actin in aortic strip homogenates from subjects <65-years and >65-years old. Quantification of expression assays for STIM-1, Orai1, and Orai3 are displayed in panels (B–D), respectively. Data are expressed as mean ± SEM of band intensities normalized by respective β-actin band intensities. n indicates the number of subjects. ** p < 0.01 vs. <65-year-old group by unpaired t-test. (E,F) Representative immunofluorescence images for the detection of Orai1 (green fluorescence) in cryosections of aortic strips from a 36 year-old subject and a 73 year-old subject, respectively. Nuclei are counterstained in blue. Magnifications: ×200. (G) Simple regression analysis of individual values of the Orai1 expression in human aortic strip homogenates with respect to the age of the subject from which the tissue was obtained. (H) The association between adrenergic contraction determined as the maximum response (E_max) to norepinephrine (NE) expressed as the percentage of K⁺-induced contraction with respect to the age from the same subject. (I) Association between aortic Orai1 quantification and E_max to NE. Every point represents the averaged E_max value of the strips from one single subject. Coefficients of determination (r²) and p values are indicated for each association (in bold italic when significant).

Figure 3

STIM-1 and Orai1 are up-regulated in human aged mesenteric arteries. Upper panel shows the representative immunofluorescence images for detection (green fluorescence) of STIM-1 (A,B); Orai1 (C,D) and Orai3 (E,F) in cryosections of mesenteric arteries from organ donors younger than 65-years old (A,C,E) and older than 65-years old (B,D,F). Nuclei are counterstained in blue. Magnifications: ×100. Quantification of expression assays for STIM-1, Orai1, and Orai 3 are displayed in panels (G–I), respectively. Data are expressed as mean ± SEM of STIM/Orai arbitrary units of fluorescence intensities normalized by number of nuclei. n indicates the number of subjects from which the tissues were obtained for the experiments. * p < 0.05, *** p < 0.001 vs. <65-year-old group by unpaired t-test.

Figure 4

STIM1 and Orai1 are associated with vascular function in human mesenteric arteries. Simple regression analysis for STIM-1 (A), Orai1 (B), and Orai3 (C) fluorescence intensity normalized by the number of nuclei in the mesenteric arteries with respect to endothelium-dependent vasodilation, determined as –log molar of concentration required to obtain 50% relaxation (pEC₅₀) for bradykinin (BK) in isolated mesenteric arteries obtained from the same subject. Coefficients of determination (r²) and p values (in bold italic when significant) are indicated for each association. n indicates the number of subjects for the determinations.

Figure 5

Plasma Orai1 concentrations are associated with biomarkers of endothelial dysfunction and inflammation. Simple regression analysis for plasma concentrations of Orai1 with respect to the plasmatic levels of asymmetric dimethyl arginine (ADMA) (A) and tumor necrosis factor-α (TNF-α); (B) determined in the samples from the same subjects. Coefficients of determination (r²) and p values (in bold italic when significant) are indicated for each association. n indicates the number of subjects for determinations.