The Mechanism Of Endothelial Dysfunction With Advancing Age In Rat Mesenteric Artery

Objective Shear stress induced by blood flow acts through the endothelial cells(ECs) to regulate vascular tone and diameter. The flow-stimulated intracellular Ca2+ concentration([Ca2+]i) rise in ECs serves as an important early event in flow-induced vessel dilation. Several studies have demonstrated an involvement of the transient receptor potential vanilloid subtype 4(TRPV4), a Ca2+ permeable cation channel, in facilitating this Ca2+ increase. In this study, pressure myography was utilized to detect that the alteration of both flow- and TRPV4 activator 4α-PDD-induced vessel dilation of mesenteric arteries in aged rats compared to young rats. Using a Leica TCS SP5 confocal laser system to detect flow- and 4α-PDD-induced [Ca2+]i rise in primary cultured mesenteric artery endothelial cells(MAECs) from young aged rats. The expression levels of TRPV4 in MAECs of aged and young rats were showed by Western blotting and immunostainings.Methods 1. Grouping Adult male Sprague-Dawley rats were divided into two groups.Young rats 3~4 months,aged rats 20~22months.2. Primary culturing mesenteric artery endothelial cells(MAECs) The mesenteric artery endothelial cells were isolated with 0.02 % collagenase type I in phosphate-buffered saline(PBS) for 45 min at 37 ℃in the Controlled Crystal Oscillator. The pelleted cells were plated in dishes and cultured at 37 with 5℃ %CO2 for 3- 5 days for the subsequent experiments.3. Ca2+ measurements MAECs were loaded with 10 mol/L Fluo-8/AM and 0.02 % pluronic F-127 for 30 min. [Ca2+]i changes were displayed as a ratio of fluorescence(F1/F0).Fluorescence signals were recorded and analyzed using a Leica TCS SP5 confocal laser system.Treat the cells by TG to deplete intracellular Ca2+ stores, add RN1734, TRPV4 antagonist,and test the Ca2+ rise. Regulate the pressure on two sides to observe the flow-induced Ca2+ rise in young and aged MAECs.4. Flow-induced vessel dilation in young and aged rats. The isolated mesenteric arteries segments, were mounted on a pressure myograph(Danish Myo Technology, Denmark, model 110P).Measure the Flow-induced endothelium dependent vascular relaxation of superior mesenteric artery in aged and young rats.Use RN1734 to block TRPV4,and observe the change of Flow-induced endothelium dependent vascular relaxation;Teatment with 4α-PDD,which is a TRPV4-specific agonist to elicit vessel dilation in phenylephrine-precontracted small mesenteric artery segments of young and aged rats.5. Western Blot and Immunostaining. The expression levels of TRPV4 in MACEs from young and aged rats were observed by western blot immunofluorescence methods with TRPV4 antibody.6. Statistical method. All experimental data was analyzed statistically by sigmaplot software and showed with mean±standard deviation. Student’s t-test was used for statistical comparison, with a probability of P < 0.05 deemed significant.Results 1. Flow-induced vessel dilation and endothelial cell [Ca2+]i rise in young and aged rats. The dilation greatly reduced in mesenteric arteries from aged rats relative to those from young rats and flow induced [Ca2+]i rise was significantly decreased in aged MAECs relative to those in young rats.2. 4α-PDD-induced vessel dilation and MAECs [Ca2+]i rise in young and aged rats.4α-PDD-induced dilation largely decreased in arteries from aged rats. In congruence with vessel dilation,4α-PDD-induced [Ca2+]i rise was also remarkably reduced in aged primary MAECs.3. To prove the act of TRPV4 on flow-induced mesenteric arteries dilation and [Ca2+]i rise.Compared with the controlled group, RN1734 pretreatment greatly reduced in mesenteric arterie dilation and [Ca2+]i rise.4. Western Blot and Immunostaining. The Western blot results showed a reduced TRPV4 expression in aged MAECs. Moreover, the immunofluorence staining results further confirmed these findings.Conclusion TRPV4 played a critical role in mediating [Ca2+]i rise in endothelial cell-dependent flow response, with an impairment of this signaling contributing to endothelial dysfunction with advancing age.Objective Edothelial dysfunction is one of the main risk factors for the development of cardiovascular diseases, and it is associated with complex structural and functional alterations in the vasculature. Dysfunction of both the vascular endothelium and smooth muscle cells appears to occur as age progresses. Transduction pathways and communication between endothelial cells and smooth muscle cells are also altered. Healthy endothelial function is primarily regulated by several factors including endothelium-dependent relaxing factors(EDRFs), endothelium-dependent contracting factors(EDCFs) and endothelium-dependent hyperpolarizing factor(EDHF). The aging process is accompanied by deterioration in the balance between vasodilator and vasoconstriction substances produced by the endothelium.This imbalanceis mainly characterized by a progressive reduction of the bioavailability of nitric oxide(NO) and an increase in the production of cyclooxygenase(COX)-derived vasoconstrictor factors. These age-related alterations may be also affected by an increased expression of pro-inflammatory cytokines that accompanies the process of senescence and that frequently lead to the alteration of the expression and/or activity of certain enzymes[6,7].Stor-operated Ca2+ entry(SOCE)is a major mechanism for Ca2+ infux in many cells types including vascular endothelial cell[8,9]. For SOCE Ca2+ entery is activated in response to depletion of intracellar Ca2+ store[8]. SOCE serves to replenish intracellular Ca2+ stores,and more importantly functions as key signal to regulate diverse celluar processes[8]. In vascular endothelial cells, SOCE is involved in the control of vascular tone,vascular permeability and many other functions[10,11]. However, the role of SOCE in ECs and subsequent SOCE induced vasodialation are largely uncharacterized. In the present study, we investigated the mechanism of the store operated Ca2+ entery(SOCE) in rat superior mesenteric artery endothelial cells(MAECs) and its effects on regulating the vasodilation.Methods 1. Grouping Adult male Sprague-Dawley rats(SPF grade) were divided into two groups.Young rats 3-4 months, aged rats 20-22 months.2. Primary culturing mesenteric artery endothelial cells(MAECs) The mesenteric artery endothelial cells were isolated with 0.02 % collagenase type I in phosphate-buffered saline(PBS) for 45 min at 37 ℃ in the Controlled Crystal Oscillator.the pelleted cells were plated in dishes and cultured at 37 ℃ with 5 % CO2 for 3- 5 days for the subsequent experiments.3. Ca2+ measurements MAECs were loaded with 10 mol/L Fluo-8/AM and 0.02 % pluronic F-127 for 30 min. [Ca2+]i changes were displayed as a ratio of fluorescence(F1/F0). Fluorescence signals were recorded and analyzed using a Leica TCS SP5 confocal laser system.Treat the cells by TG and BK to deplete intracellular Ca2+ stores,and t to compare SOCE.4. To messure vessel dilation in young and aged rats. The isolated mesenteric arteries segments, were mounted on a pressure myograph(Danish Myo Technology, Denmark, model 110P) and measure the vasodilation of superior mesenteric artery in aged and young rats caused by Bradykinin(BK).5. Immunostaining. The expression levels of Orai 1 or stromal interaction factor 1(STIM 1) were observed by immunofluorescence method.6. Statistical method. All experimental data was analyzed statistically by sigmaplot software and showed with mean±standard deviation. Student’s t-test was used for statistical comparison, with a probability of P < 0.05 deemed significant.Results 1. Vessedilation in young and aged rats. Compared with the young rats, vasodialation-induced by BK in aged rats declined.2. Ca2+ measurements. SOCE-induced by BK and TG attenuated in primary cultured MAECs of aged rats.3. Immunostaining. The expression of SOCE components Orai 1 and STIM 1 decreased obviously in aged MAECs.Conclusion SOCE in MAECs and SOCE induced vasodilation significantly attenuated in aged rats. The decreasing of Orai 1 and STIM 1 expression level may contribute to this alteration.