Effect Of High Fat Diet On Aortic Endothelial Structure And Function In Hypoxic Rats

Backgrounds and objectives: With the development of the western area of China, the people’s health in high altitude has been drawn more and more attention. Cardiovascular diseases as the leading cause of death in the world cause to the economic burden. Cardiovascular diseases are the main diseases affect people’s health in plateau as well.Vascular endothelium is an important target for cardiovascular disease and its risk factors. The vascular endothelium has barrier function. At the same time, vascular endothelium as the largest endocrine system of the body can secrete a variety of factors to keep the normal vascular structure and function, including nitric oxide(NO) which is an important index of clinical evaluation of endothelial function. NO is a gas produced by nitric oxide synthase(NOS) enzymes catalyzing substrate L-arginine. NO plays its cardiovascular protective roles such as vasodilatation, inhibition of platelet aggregation and activation, inhibition of neutrophils and monocytes infiltration and adsorption to vascular wall, inhibition of vascular smooth muscle cell proliferation and resisting thrombosis, regulation vascular tone and maintaining steady state and vascular endothelial function. Hyperlipidemia and other cardiovascular risk factors can induce dysregulation of vascular structure and impairment of endothelium dependent vasorelaxation-vascular endothelial dysfunction(VED) by reducing the NO content in the blood circulation, and then make the occurrence and development of cardiovascular diseases. Moreover, NO is also a crucial factor for high altitude adaptation and acclimatization. The levels of circulating NO are increased in high altitude natives and migrants that being adaptation or acclimatization to hypoxia. Higher levels of plasma NO can maintain normal function of body to compensate the influence of hypoxia by dilating blood vessels, reducing the peripheral resistance, increasing blood flow and the oxygen delivery to tissues. Along with the high altitude tourism is booming, there are increasing number of visitors from low altitude to high altitude; Couple with the high incidence of chronic respiratory disease, not only high altitude natives, residents and migrants, but also more and more people are affected by hypoxia. For high altitude people, hypoxia is an irreversible factor of natural environment which has the extensive influence tissues and organs. Moreover, chronic hypoxia can cause hyperlipidemia. At the same time, the eating habit has changed-high fat diet being widespread because of the cold environment of high altitude, and thereby the incidence of hyperlipidemia is increasing in high altitude. The threat by hyperlipidemia for high altitude people is increasing.Hyperlipidemia is a common risk factor for cardiovascular diseases, which can cause vascular endothelial dysfunction, and initiate the occurrence of cardiovascular disease. Whether a high fat diet or hyperlipidemia causes the vascular endothelial dysfunction in hypoxic setting? Is a high fat diet or hyperlipidemia a risk factor for hypoxic setting(people in high altitude hypoxic environment or chronic hypoxic disease such as coronary heart disease, chronic obstructive pulmonary disease)? It remains unclear. Therefore, our study investigate the effects of high fat diet on vascular endothelial function in SD rat exposed to long-term and continuous hypo baric hypoxia by testing the levels of endothelial nitric oxide synthase(e NOS) m RNA and protein expression, induced nitric oxide synthase(i NOS) m RNA expression, plasma NO content and endothelium dependent relaxing function of aortic vascular ring in vitro. Methods: Male Sprague-Dawley(SD) rats(n=72) were purchased from China Pharmaceutical University in Nanjing. Rats were randomly divided into three groups: a hypoxia(H) group with regular chow, a combined hypoxia and high fat diet(H+HFD) group, and for comparison, rats maintained in normoxic environment(Nanjing, altitude of 10 meters), and regular chow conditions were set as baseline(BL) group. As the aim of our study was to investigate the effects of high fat diet on VED in SD rats of exposure to hypoxia, other than the interaction effect of hypoxia and high fat diet, so a group of combined normoxia and high fat diet wasn’t set. The samples of BL group were collected in Nanjing right away. The rats of H+HFD group and H group were transported to Xining and maintained in hypobaric hypoxic chamber in laboratory in Xining. The environment of altitude of 5000 meters was simulated by hypobaric hypoxic chamber. Rats were gavaged with fat emulsion(H+HFD group, 10 ml/kg/d) or equal volume of saline solution(H group) once daily for 4 weeks. The samples of rats in H group and H+HFD group were collected at 1,2,3,4 week(n=8, each group at each time point). A noninvasive rat tail blood pressure cuff was used to assessed blood pressure(BP) and heart rate(HR). The rats were anesthetized with urethane, and the venous blood was collected. The aortas were separated from the anaesthetized rats, and then cut into 5 mm length to make aortic rings. Aortic rings were mounted on constant temperature organ bath connected to an isometric tension transducer. Then measure endothelium-dependent relaxation responses and endothelium-independent relaxation responses. Meanwhile, the effects of L-arginine(L-arg; a NOS substrate) and Nω-Nitro-L-Arginine Methyl Ester(L-NAME; a NOS inhibitor) on endothelium-dependent relaxation responses to acetylcholine(ACh) were assessed. An automatic blood cell analyzer was used to test the hemoglobin(HGB) concentration. A light microscopy was used analyze the aortic sections stained with hematoxylin-eosin(HE). Western blotting was used to test the expression of e NOS protein and real-time PCR was used to test the expression of e NOS m RNA and i NOS m RNA. WST-1 methods, TBA methods and Nitrale reduetase method were used to test the plasma superoxide dismutase(SOD) activity, the content of malondialdehyde(MDA) and nitrates and nitrites(NOx, the stable metabolite of NO). End point method and direct method were used to test the plasma triglyceride(TG), total cholesterol(TCH), high density lipoprotein(HDL) and low density lipoprotein(LDL) levels. Results: High fat diet significantly reduced the heart rates in hypoxia exposed rats from 2 week and increased the levels of plasma TCH and LDL at 2 week and 4 week(P<0.05). The plasma NOx levels in rats of H group were significantly higher than that in rats of BL group(P<0.05), and then gradually decreased. In contrast, high fat diet significantly reduced the plasma NOx levels in hypoxia exposed rats(P<0.05). In rats exposed to hypoxia only, aortas were relatively normal at week 1 and at week 2 with mild endothelial cells(EC) swelling and hyperplasia, smooth muscle cells(SMC) proliferation. These changes became more severe with intima layer desquamation from the vascular wall, rounded EC nuclei and disarranged SMCs with oval nuclei by 4 weeks. In comparison, the aortas of the H+HFD group exhibited more significant changes than H group with rounded EC nuclei and intima layer desquamation at 2 week. These alterations became more severe phenotypically by week 3 and week 4 with intima layer desquamation, SMC proliferation, round nuclei and vacuolar degeneration. Aortic rings from the H+HFD group showed impaired endothelium dependent vasorelaxation by ACh stimulation. And the ACh-mediated vasorelaxation was fully inhibited in all groups when pre-incubated the rings with the NOS inhibitor L-NAME, Meanwhile, impairments in endothelium dependent vasorelaxation from the H+HFD group were rescued when pre-incubated the rings with the NOS substrate L-arginie, and offset its difference from H group. The e NOS m RNA and protein levels of aorta were significantly increasing in H group compared with BL group. However, this adaptive increasing response was significantly blunted during the first two weeks in H+HFD group compared to H group. The expression of i NOS m RNA was similar among hypoxia alone and H+HFD groups during the first 3 weeks of treatment. Hypoxia alone resulted in significantly higher plasma MDA content from 2 weeks. However, hypoxia plus HFD didn’t increase the MDA content additionally and, MDA levels were significantly lower than that in hypoxia alone conditions by 4 weeks into treatment. The similar trends as MDA were showed in plasma levels of SOD activity.Conclusions: 1. High fat diet caused earlier and more severe changes in aortic endothelial structures and impaired endothelium-dependent vasorelaxation responses in continuous hypoxia exposed rats.2. High fat diet blunted adaptive increase in plasma NO in continuous hypoxia exposed rats is the main reason for vascular endothelial dysfunction.3. A high fat diet reduce the expression of e NOS m RNA and protein in aorta and the activity of L-arg in continuous hypoxia exposed rats may be account for the blunted increase in NO.4. A high fat diet did not exacerbate imbalance of oxidative and antioxidative system in continuous hypoxia exposed rats, which may be not the main reason for NO decreasing and vascular endothelial dysfunction.