High-salt Diet-induced Upregulation Of Caveolin-1 Contributes To Aggravated Endothelial Dysfunction In Diabetic Rats

Background Diabetes mellitus(DM) is characterized by hyperglycemia and development of vascular pathology, and its associated cardiovascular complications represent a growing and major global health problem. Hypertension constitutes one of the most prevalent cardiovascular risk factors worldwide and is particularly associated with diabetes mellitus. Hypertension is more common in individuals with DM than in the general population, with estimates of the prevalence of hypertension in DM ranging from 40% to 80%. The diabetic hypertensive patients appear to accelerate the development of microvascular and macrovascular complications and increase the risk of stroke, end stage renal diseases and other cardiovascular diseases, compared with that in patients suffering from hypertension or diabetes alone.The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized, but the mechanism by which excess salt intake elevates blood pressure has puzzled researchers. High dietary sodium may promote vasoconstriction by increased nuclear expression of vasoconstrictive stimuli such as endothelin-1, activation of the sodium/calcium exchange mechanisms, increased reactive oxygen species generation and reduced nitric oxide(NO) bioavailability. Spontaneously hypertensive rats show impaired insulin-induced and NO-mediated relaxation and reduced insulin-induced activation of Akt/endothelial NO synthase(e NOS). High salt diet also played an important role in the control of blood pressure in diabetes mellitus. Significant salt-sensitive hypertension was observed in the salt-loaded Spontaneously Diabetic Torii-Lepr(fa) fatty rats. In addition to intake of excess fat and carbohydrate, salt in excess contributes much to our Western diet and the high incident rate of diabetes. However, the mechanisms involved in the pathogenesis of high-salt diet-induced endothelial dysfunction and hypertension in diabetes are poorly understood. Caveolae/lipid rafts are specialized membrane microdomains where multimolecular signaling molecule complexes are compartmentalized, in part, via interacting with caveolin1(Cav-1). Cav-1 is most abundantly expressed in many cells types, including endothelial cells, vascular smooth muscle(VSM), adipocytes and fibroblasts. Caveolae have also been proposed as the vesicle carriers responsible for transcellular transport in endothelial cells. Evidence suggests a role of cav-1 in the anchoring of e NOS to caveolae, thus limiting its translocation and phosphoactivation, maintaining the e NOS in its inactive conformation. Experimental evidence suggests increased cav-1 expression in diabetes. However, it still remains unknown whether interaction between e NOS and cav-1 plays a role in the endothelial dysfunction in diabetes.Aims This study was aimed to investigate the underlying mechanisms responsible for endothelial dysfunction of type 1 diabetic rats fed with high salt diet.Methods1. Type 1 diabetes(DM) was induced by intraperitoneal injection of streptozotocin(STZ, 70 mg?kg-1) and only rats with blood glucose levels ≥ 16.7 m M were included. Normal or diabetic rats were randomly fed a high salt food(HS, 8% Na Cl) or a standard food for 6 weeks. Fasting blood glucose and blood pressure(BP) was measured at intervals of one week for 6 weeks. 2. Isometric tension of mesenteric arteries was measured using the Power Lab Chart v 7.2.1 programme(model 610M). Production of NO was determinated. 3. Artery was stained with Hematoxylin-eosin(H&E). 4. Double staining immunofluorescent detection of e NOS and Cav-1 in arteries. Co-immunoprecipitation of e NOS with caveolin-1 in mesenteric arteries. DHE staining detection of ROS of arteries. 5. The expressions and phosphorylations of Akt, e NOS and Cav-1 proteins were measured using Western blot. 6. Human umbilical vein endothelial cells(HUVEC) were grown in culture and transfected with wortmannin,L-NAME or Cav-1 si RNA. The expressions of e NOS and Cav-1 proteins were measured using Western blot.Results 1. After 6 weeks of the experimental diets, Both HS(143 ± 10 mm Hg) and DM+HS(169 ± 11 mm Hg) groups displayed significantly higher systolic blood pressure(s BP) levels than those in CON group(112 ± 12 mm Hg, P<0.01). After 6 weeks, s BP levels in DM group were similar to those in CON. At 3 weeks of treatment, the DM+HS group(151 ± 11 mm Hg) showed the levels in s BP were significantly higher than HS group(128 ± 8 mm Hg, P<0.01). 2. After 3 weeks of treatment, DM showed an impairment of endothelium-dependent relaxation to ACh(10-6mol/L) and insulin(10-6mol/L) that was similar to that observed in HS. After 6 weeks, DM+HS exhibited a more pronounced impairment of vasorelaxation(32.5 ± 2.8%) compared with DM(56.8 ± 3.5%, P<0.01) and HS(42.1 ± 3.4%, n=8, P<0.05). DM showed an impairment of endothelium-dependent relaxation to insulin(10-6 mol/L) that was severer than that observed in HS. Moreover, DM+HS exhibited a significant impairment ofvasorelaxation(9.7 ± 1.9%) compared with DM(42.0 ± 4.5%, P<0.01) and HS(22.3 ± 4.1%, P<0.05). DM+HS showed an impairment of endothelium-independent relaxation to SNP(10-4 mol/L) was severer than other groups(P<0.01). NO concentration in DM+HS was lower than that in DM(6.9 ± 1.1 μmol/L vs. 10.3 ± 1.3 μmol/L, n=6, P <0.05). NO level was decreased, ROS was increased. 3. The thickness of intima and media was decreased in DM+HS compared to that of CON and HS(P<0.01). The elastic fibers of mesenteric artery wall were disorderly in DM+HS. 4. Co-location of cav-1 and e NOS in mesenteric artery was enhanced in DM+HS group. Increased interaction between vascular Cav-1 and e NOS in diabetes with high-salt diet. 5. After 3 or 6 weeks, Akt expressions were similar in four groups. But a lower level of e NOS expressions was observed in HS and DM+HS, and DM expressed was similar to that observed in CON. The Akt/e NOS phosphorylation levels in HS were significantly higher than in DM, while the Akt/e NOS phosphorylation levels in DM+HS were significantly lower than in DM. The levels of Cav-1 in DM+HS were significantly higher than that in CON, DM and HS. 6. Akt-e NOS pathway was impaired and increased Cav-1 expression of DM+HS. In the presence of Cav-1 si RNA, the e NOS phosphorylations in HUVEC were significantly increased compared with that in cells treated with control si RNA, Cav-1 was not significantly lower in HUVEC cultured with high glucose and high salt buffer solution in wortmannin and L-NAME.Conclusions 1. Diabetic rats fed with high-salt diet decreases endothelium-dependent vasorelaxation. 2. Impaired endothelial Akt-e NOS pathway and high-salt diet-induced upregulation of caveolin-1 exert as negative regulations of e NOS phosphorylation leading to a decreased NO-dependent vasorelaxation in diabetes.