Roles Of Local Renin-Angiotensin System In Rat Vascular Function Remodeling In The Settings Of Simulated Microgravity

Long-term exposure to microgravity leads to cardiovascular adaptation in the astronauts and cardiovascular symptoms such as orthostatic hypotension, palpitation, short breath and syncope will occur when they return to 1 G on earth. Although much progress has been made in the past half century on the mechanism of postflight cardiovascular deconditioning, the present results cannot fully interpret the abnormal cardiovascular changes in the settings of microgravity. Therefore, clarifying the underlying mechanism of cardiovascular dysfunction makes for interpreting and preventing the occurrence and development of postflight cardiovascular diseases in the astronauts.Microgravity changes normal hydrostatic pressure gradient and therefore results in a redistribution of transmural pressure within the vasculature in human: the arteries in the upper and lower body regions experience"hypertension"and"hypotension"due to increased and decreased vascular transmural pressure respectively, which is also the case in simulated microgravity rat by hindlimb unweighting, a widely used animal model to simulate the effects of microgravity on cardiovascular system. The differentiated changes of blood pressure lead to vascular structural and functional adaptation to simulated microgravity in rat: vascular hypertrophy and enhanced response to vasoconstrictors in the cerebral arteries as well as vascular atrophy and attenuated response to vasoconstrictors in the hindquarter arteries. Previous studies suggested that the changes in the function of ion channels and the expression of NO synthase were the possible participants of vascular adaptation to simulated microgravity. Furthermore, recent studies demonstrated that local renin-angiotensin system (L-RAS) was differentiatedly activated in simulated microgravity rat vasculature: the key components of L-RAS were upregulated and downregulated in the cerebral and hindquarter arteries respectively. However, whether the characteristic changes of L-RAS participate in vascular structure and function remodeling in the settings of microgravity or simulated microgravity is unclear. Angiotensin II, the major mediators of L-RAS, has been confirmed not only a potent vasoconstrictor but also an indirect regulator of vascular reactivity by inducing vascular oxidative stress and inflammation injury, through which L-RAS is involved in the etiology of cardiovascular diseases. Nevertheless, no evidence of oxidative stress and inflammation in simulated microgravity rat vasculature has been provided before. Otherwise, whether L-RAS regulates vascular reactivity in the conditions of simulated microgravity by inducing arterial oxidative stress and inflammation is unclear. Now that NO is an important determinant of vascular reactivity, can L-RAS regulate vascular reactivity through modulating the expression of NO synthase (NOS) protein? We will try to provide the plausible answers to the above mentioned concerns.Hindlimb unweighting (HU) rat was applied in the present study to simulate the physiological effects of microgravity on the cardiovascular system. Losartan (30 mg/kg/day) and apocynin (50 mg/kg/day) were used to specificly block angiotensin II type 1 (AT1) receptors and inhibit the activity of NAD(P)H oxidase respectively. In the morphological protocols, immunohistochemistry analysis was used to localize the expression of vascular cell adhesion molecule-1 (VCAM-1) in the basilar, common carotid, abdominal aorta and femoral arteries. Superoxide levels in these arteries were also detected by laser confocal microscopy. In the physiological protocols, isolated artery rings were used to measure the development of isometric force in the presence of vasoconstrictors and vasodilators. In the molecular biological protocols, Western Blot was applied to measure the expression of endothelial NOS (eNOS), inducible NOS (iNOS) and VCAM-1 protein; commercial kit was used to identify the arterial content of nitrate and nitrite (NOx).The major findings of the present study are the following:1. L-RAS participates in vascular adaptation to simulated microgravity by modulating the expression of NOS protein in rat.The data from the isolated artery rings showed that 3-week hindlimb unweighting significantly enhanced the vasoconstriction to KCl/phenylephrine (in carotid arteries) or 5-hydroxytryptamine (in basilar arteries) in the basilar and common carotid arteries as well attenuated the vasoconstriction to KCl/phenylephrine in the abdominal aorta and femoral arteries compared with control. The endothelium-dependent relaxation to acetylcholine was significantly impaired in all these arteries from HU rats. Chronic treatment of losartan (30 mg/kg/day) partially renormalized vascular response to the vasoconstrictors and acetylcholine in the basilar and carotid arteries from losartan-treated HU rat, but did not influence vascular reactivity of abdominal and femoral arteries. Western blot analysis revealed that the expression of iNOS protein was significantly upregulated in HU rat cerebral and carotid arteries; the expression of eNOS was significantly upregulated and downregulated in HU rat carotid arteries and abdominal aorta/femoral arteries respectively. Treatment with losartan restored the expression of eNOS and iNOS protein in the cerebral and carotid arteries from losartan-treated HU rat, but exerted no significant influence on the expression of NOS in abdominal aorta and femoral arteries. The present data suggested that L-RAS might regulate vascular reactivity by modulating the expression of eNOS/iNOS protein in HU rat cerebral and carotid arteries.2. L-RAS participates in vascular adaptation to simulated microgravity by modulating the expression of NOS protein through the mechanism of oxidative stress injury.The data from laser confocal microscopy showed increased and decreased superoxide levels in 3-week HU rat basilar/carotid arteries and abdominal aorta/femoral arteries respectively. Treatment with losartan renormalized superoxide production in the basilar and carotid arteries from losartan-treated HU rats. Similar to the previous section, enhanced contractile response and impaired endothelium-dependent relaxation in HU rat basilar and carotid arteries were significantly improved in the condition of NAD(P)H oxidase inhibition with apocynin (50 mg/kg/day). Furthermore, chronic treatment with apocynin also restored the expression of eNOS/iNOS protein in apocynin-treated HU rat cerebral and carotid arteries. The NOx content significantly increased and decreased in 3-week HU cerebral/carotid arteries and abdominal aorta/femoral arteries respectively compared with control, but apocynin renormalized NOx content in the cerebral and carotid arteries from apocynin-treated HU rats. The present data suggested that oxidative stress might be involved in the regulation of vascular reactivity in HU rat cerebral and carotid arteries induced by L-RAS.3. Inflammation coexists with L-RAS-induced abnormal vascular reactivity: a possible alternate mechanism.The data from immunohistochemistry analysis showed the localization of VCAM-1 protein on the surface of endothelial cells in HU rat basilar and common carotid arteries, which was further confirmed by Western Blot analysis; however, it is not the case in HU rat abdominal aorta and femoral arteries. Also, the data obtained by Western Blot showed downregulated expression of VCAM-1 protein in losartan-treated HU rat cerebral and carotid arteries compared with these arteries from HU rats. The present data suggested that arterial inflammation might be an alternate mechanism in the course of L-RAS-induced vascular adaptation to simulated microgravity.In summary, we provided the first evidence that arterial oxidative stress and inflammation also existed in 3-week simulated microgravity rat cerebral and carotid arteries, which provided further information of the effects of microgravity on cardiovascular system. Also, the present study firstly reported that L-RAS might participate in vascular adaptation to simulated microgravity by modulating the expression of NOS protein through the mechanisms of arterial oxidative stress and inflammation. Here we provided the functional evidence of differentially activated L-RAS in simulated microgravity rat vasculature. Due to the potentially pathological roles of RAS in the occurrence and development of cardiovascular diseases such as hypertension and artherosclerosis, the present study will be valuable in preventing the etiology of postflight cardiovascular diseases in the astronauts.