Hindlimb Unweighting Rats Research Articles

Estrogen receptor α regulates phenotypic switching and proliferation of vascular smooth muscle cells through the NRF1-OMI-mitophagy signaling pathway under simulated microgravity.

Vascular remodeling during microgravity exposure results in postflight cardiovascular deconditioning and orthostatic intolerance in astronauts. To clarify the underlying mechanism, we investigated whether estrogen receptor α (ERα)-NRF1-OMI-mitophagy signaling was involved in the dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) under simulated microgravity. Phenotypic markers, mtDNA copy number and mitochondrial biogenesis, mitochondrial dynamics and mitophagy in rat thoracic artery smooth muscle cells were examined. Four-week hindlimb unweighting (HU) was used to simulate microgravity in rats and 10% serum was used to induce VSMCs dedifferentiation in vitro. The effects of ERα-NRF1-OMI signaling on mitophagy, phenotypic switching and proliferation of VSMCs, and cerebrovascular remodeling in HU rats were studied by genetic manipulation and chronic drug intervention. We found that ERα is positively associated with contractile phenotype switching but inversely correlated with synthetic phenotype switching and proliferation of VSMCs both in vivo and in vitro. During the dedifferentiation process of VSMCs, reduced mtDNA copy number, disturbed mitochondrial biogenesis and respiration, and perturbed fission-fusion-mitophagy signaling were detected, which were reversed by ERα overexpression. Mechanistically, the ERα downstream protein OMI preserved the mitochondrial Parkin level by increasing its protein stability, thereby protecting mitophagy. In line with this, we found that activating ERα signaling by propyl pyrazole triol (PPT) could alleviate the synthetic phenotype switching and proliferation of HU rat cerebral VSMCs by reestablishing fission-fusion-mitophagy hemostasis. The current study clarified a novel mechanism by which inhibited ERα-NRF1-OMI-mitophagy signaling resulted in synthetic phenotype switching and proliferation of VSMCs and cerebrovascular remodeling under simulated microgravity.

The OPG/RANKL/RANK system modulates calcification of common carotid artery in simulated microgravity rats by regulating NF-κB pathway.

Functional and structural adaptation of common carotid artery could be one of the important causes of postflight orthostatic intolerance after microgravity exposure, the mechanisms of which remain unclear. Recent evidence indicates that long-term spaceflight increases carotid artery stiffness, which might present a high risk to astronaut health and postflight working ability. Studies have suggested that vascular calcification is a common pathological change in cardiovascular diseases that is mainly manifested as an increase in vascular stiffness. Therefore, this study investigated whether simulated microgravity induces calcification of common carotid artery and to elucidate the underlying mechanisms. Four-week-old hindlimb-unweighted (HU) rats were used to simulate the deconditioning effects of microgravity on cardiovascular system. We found that simulated microgravity induced vascular smooth muscle cell (VSMC) osteogenic differentiation and medial calcification, increased receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) and RANK expression, and enhanced NF-κB activation in rat common carotid artery. In vitro activation of the RANK pathway with exogenous RANKL, a RANK ligand, increased RANK and osteoprotegerin (OPG) expression in HU rats. Moreover, the expression of osteogenic markers and activation of NF-κB in HU rats were further enhanced by exogenous RANKL but suppressed by the RANK inhibitor osteoprotegerin fusion protein (OPG-Fc). These results indicated that the OPG/RANKL/RANK system modulates VSMC osteogenic differentiation and medial calcification of common carotid artery in simulated microgravity rats by regulating the NF-kB pathway.

Mitochondrial Oxidative Stress Enhances Vasoconstriction by Altering Calcium Homeostasis in Cerebrovascular Smooth Muscle Cells under Simulated Microgravity

ObjectiveExposure to microgravity results in postflight cardiovascular deconditioning in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process. To elucidate the mechanism for this condition, we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted (HU) rat cerebral arteries. MethodsThree-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca2+ distribution, inositol 1,4,5-trisphosphate receptor (IP3R) abundance, and the activities of voltage-gated K+ channels (KV) and Ca2+-activated K+ channels (BKCa) were examined in rat cerebral vascular smooth muscle cells (VSMCs). ResultsAn increase of cytoplasmic Ca2+ and a decrease of mitochondrial/sarcoplasmic reticulum (SR) Ca2+ were observed in HU rat cerebral VSMCs. The abundance of fusion proteins (mitofusin 1/2 [MFN1/2]) and fission proteins (dynamin-related protein 1 [DRP1] and fission-mitochondrial 1 [FIS1]) was significantly downregulated and upregulated, respectively in HU rat cerebral VSMCs. The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats, and IP3 R protein/mRNA levels were significantly upregulated. The current densities and open probabilities of KV and BKCa decreased and increased, respectively. Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1. It also decreased IP3R expression levels and restored the activities of the KV and BKCa channels. MitoTEMPO restored the Ca2+ distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries. ConclusionThe present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.

Regulation of the cerebrovascular smooth muscle cell phenotype by mitochondrial oxidative injury and endoplasmic reticulum stress in simulated microgravity rats via the PERK-eIF2α-ATF4-CHOP pathway

Microgravity exposure results in vascular remodeling and cardiovascular dysfunction. Here, the effects of mitochondrial oxidative stress on vascular smooth muscle cells (VSMCs) in rat cerebral arteries under microgravity simulated by hindlimb unweighting (HU) was studied. Endoplasmic reticulum (ER)-resident transmembrane sensor proteins and phenotypic markers of rat cerebral VSMCs were examined. In HU rats, CHOP expression was increased gradually, and the upregulation of the PERK-eIF2α-ATF4 pathway was the most pronounced in cerebral arteries. Furthermore, PERK/p-PERK signaling, CHOP, GRP78 and reactive oxygen species were augmented by PERK overexpression but attenuated by the mitochondria-targeting antioxidant MitoTEMPO. Meanwhile, p-PI3K, p-Akt and p-mTOR protein levels in VSMCs were increased in HU rat cerebral arteries. Compared with the control, HU rats exhibited lower α-SMA, calponin, SM-MHC and caldesmon protein levels but higher OPN and elastin levels in cerebral VSMCs. The cerebral VSMC phenotype transition from a contractile to synthetic phenotype in HU rats was augmented by PERK overexpression and 740Y-P but reversed by MitoTEMPO and the ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). In summary, mitochondrial oxidative stress and ER stress induced by simulated microgravity contribute to phenotype transition of cerebral VSMCs through the PERK-eIF2a-ATF4-CHOP pathway in a rat model.

BKCa compensates impaired coronary vasoreactivity through RhoA/ROCK pathway in hind-limb unweighted rats.

Previous studies have demonstrated cardiac and vascular remodeling induced by microgravity exposure. Yet, as the most important branch of vasculatures circulating the heart, the coronary artery has been seldomly studied about its adaptations under microgravity conditions. Large-conductance Ca2+-activated potassium channel (BKCa) and the Ras homolog family member A (RhoA)/Rho kinase (ROCK) pathway play key roles in control of vascular tone and mediation of microgravity-induced vascular adjustments. Therefore, we investigated the adaptation of coronary vasoreactivity to simulated microgravity and the role of BKCa and the RhoA/ROCK pathway in it. Four-week-old hind-limb unweighted (HU) rats were adopted to simulate effects of microgravity. Right coronary artery (RCA) constriction was measured by isometric force recording. The activity and expression of BKCa and the RhoA/ROCK pathway were examined by Western blot, patch-clamp recordings, and immunoprecipitation. We found HU significantly decreased RCA vasoconstriction to KCl, serotonin, and U-46619, but increased protein expression and current densities of BKCa, inhibition of which by iberiotoxin (IBTX) further decreased RCA vasoconstriction (P < 0.05). Expression of RhoA and ROCK as well as active RhoA and phosphorylation of myosin light chain (MLC) at Ser19 and MLC phosphatase target-1 at Thr696 were significantly increased by HU, and ROCK inhibitor Y-27632 exerted greater suppressing effect on HU RCA vasoconstriction than that of control (P < 0.05). BKCa opener NS1619 increased HU RCA vasoconstriction, which was blocked by both RhoA and ROCK inhibitor, similar to the effect of IBTX. These results indicate that HU impairs coronary vasoconstriction but enhances BKCa activity acting as a protective mechanism avoiding excessive decrease of coronary vasoreactivity through activation of the RhoA/ROCK pathway.-Wu, Y., Yue, Z., Wang, Q., Lv, Q., Liu, H., Bai, Y., Li, S., Xie, M., Bao, J., Ma, J., Zhu, X., Wang, Z. BKCa compensates impaired coronary vasoreactivity through RhoA/ROCK pathway in hind-limb unweighted rats.

Simulated microgravity increases myocardial susceptibility to ischemia-reperfusion injury via a deficiency of AMP-activated protein kinase.

Gravitation is an important factor in maintaining cardiac contractility. Our study investigated whether simulated microgravity increases myocardial susceptibility to ischemia-reperfusion (IR) injury. Using the Langendorff-perfused heart model with 300 beats/min pacing, 4-week tail suspension (SUS) and control (CON) male Sprague-Dawley rats (n = 10 rats/group) were subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 120 min of reperfusion. Left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), creatine kinase (CK) and lactate dehydrogenase (LDH) activity, and infarct size were assessed. Data demonstrated that there were significantly increased LVEDP, CK, LDH, and infarct size in SUS compared with CON (P < 0.05), accompanied by decreased LVESP (P < 0.05). Furthermore, TUNEL-positive cardiomyocytes were higher in SUS than that in CON (P < 0.01), and AMP-activated protein kinase (AMPK) phosphorylation and Bcl-2/Bax in SUS were less compared with CON (P < 0.05). Similarly, isolated hearts pre-treated with A-769662 exhibited better recovery of cardiac function, increased AMPK phosphorylation, and reduced necrosis and apoptosis. Furthermore, AMPKα protein showed a significant suppression in 4-week hindlimb unweighting rats. These results suggest that AMPK deficiency increases myocardial susceptibility to IR injury in rats subjected to simulated microgravity.

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats.

The roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established. We investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats. ROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats. These results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravit.

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats

ObjectiveThe roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established. MethodsWe investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats. ResultsROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats. ConclusionThese results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravity.

Simulated microgravity promotes monocyte adhesion to rat aortic endothelium via nuclear factor-κB activation.

Microgravity-induced vascular remodelling may play an important role in post-spaceflight orthostatic intolerance. In this study, we aimed to investigate the effects of simulated microgravity on monocyte adhesion to aortic endothelium in hindlimb unweighted rats and to elucidate the underlying mechanisms associated with this event. Sprague-Dawley rats were subjected to 4-week hindlimb unweighting to simulate microgravity. The recruitment of monocytes to the abdominal aorta was investigated by en face immunofluorescence staining and monocyte binding assays. The expression of the adhesion molecules E-selectin and vascular cell adhesion molecule-1 as well as the cytokine monocyte chemoattractant protein (MCP)-1 was evaluated by immunohistochemical staining, western blot, and quantitative reverse transcription polymerase chain reaction analyses. Additionally, nuclear factor-κB (NF-κB) activation and the messenger RNA expression levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 were assessed with the administration of an NF-κB inhibitor, pyrrolidine dithiocarbamate. Results showed that simulated microgravity significantly increased monocyte recruitment to the aortic endothelium, protein expression of E-selectin and MCP-1, and NF-κB activation in the abdominal aorta of rats. Pyrrolidine dithiocarbamate treatment not only significantly inhibited NF-κB activity but also reduced the messenger RNA levels of E-selectin, vascular cell adhesion molecule-1, and MCP-1 as well as monocyte recruitment in the abdominal aorta of hindlimb unweighted rats. These results suggest that simulated microgravity increases monocyte adhesion to rat aortic endothelium via the NF-κB-mediated expression of the adhesion molecule E-selectin and the cytokine MCP-1. Therefore, an NF-κB-mediated inflammatory response may be one of the cellular mechanisms responsible for arterial remodelling during exposure to microgravity.

Hindlimb unweighting does not alter vasoconstrictor responsiveness and nitric oxide-mediated inhibition of sympathetic vasoconstriction.

Physical inactivity increases the risk of cardiovascular disease and may alter sympathetic nervous system control of vascular resistance. Hindlimb unweighting (HU), a rodent model of physical inactivity, has been shown to diminish sympathetic vasoconstrictor responsiveness and reduce NO synthase expression in isolated skeletal muscle blood vessels. Our understanding of the effects of HU on sympathetic vascular regulation in vivo is very limited. The present findings demonstrate that HU did not alter sympathetic vasoconstrictor responsiveness and NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. This study suggests that short-term physical inactivity does not alter in vivo sympathetic vascular control in the skeletal muscle vascular bed at rest and during contraction. We tested the hypothesis that physical inactivity would increase sympathetic vasoconstrictor responsiveness and diminish NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. Sprague-Dawley rats (n=33) were randomly assigned to sedentary time control (S) or hindlimb unweighted (HU) groups for 21days. Following the intervention, rats were anaesthetized and instrumented for measurement of arterial blood pressure and femoral artery blood flow and stimulation of the lumbar sympathetic chain. The percentage change of femoral vascular conductance (%FVC) in response to sympathetic chain stimulation delivered at 2 and 5Hz was determined at rest and during triceps surae muscle contraction before (control) and after NO synthase blockade with l-NAME (5mgkgi.v.). Sympathetic vasoconstrictor responsiveness was not different (P>0.05) in S and HU rats at rest (S, 2Hz, -26±8% and 5Hz, -46±12%; and HU, 2Hz, -29±9% and 5Hz, -51±10%) and during contraction (S, 2Hz, -10±7% and 5Hz, -23±11%; and HU, 2Hz, -9±5% and 5Hz, -22±7%). Nitric oxide synthase blockade caused a similar increase (P>0.05) in sympathetic vasoconstrictor responsiveness in HU and S rats at rest (S, 2Hz, -41±7% and 5Hz, -58±8%; and HU, 2Hz, -43±6% and 5Hz, -63±8%) and during muscle contraction (S, 2Hz, -15±6% and 5Hz, -31±11%; and HU, 2Hz, -12±5% and 5Hz, -29±8%). Skeletal muscle NO synthase expression and ACh-mediated vasodilatation were also not different between HU and S rats. These data suggest that HU does not alter sympathetic vasoconstrictor responsiveness and NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle.

28-Day hindlimb unweighting reduces expression of Rho kinase and inhibits its effects in femoral artery of rat.

Previous studies have demonstrated inconsistent roles of Rho kinase (ROCK) in the decreased vasoconstriction of rat hindquarter vessels induced by hindlimb unweighting (HU). The present study was designed to determine the unclear role of ROCK in the mediation of HU-induced decreased femoral arterial vasoconstriction. 28-day HU rat was adopted as the animal model. With or without Y-27632, a ROCK inhibitor, isometric force of femoral artery was measured. The expression of ROCK and its effects on downstream targets were also examined. Results showed that (1) HU caused a significant decrease of the phenylephrine (PE)-evoked and potassium chloride (KCl)-evoked femoral arterial vasoconstriction (P < 0.05), confirming the functional findings by previous studies. (2) Inhibition of ROCK with Y-27632 produced an equal reduction of the vasoconstriction in CON and HU. (3) HU significantly decreased ROCK II expression and the effects of ROCK on myosin light-chain phosphatase (MLCP) and MLC (P < 0.05), but increased p65 nuclear translocation (P < 0.05) and inducible nitric oxide synthase (iNOS) expression (P < 0.05). (4) HU significantly (P < 0.05) increased NO production in femoral arteries, with Y-27632 significantly (P < 0.01) amplifying this effect. These findings have revealed that 28-day HU reduced the expression and effects of ROCK on downstream targets both directly (MLCP and MLC) and possibly indirectly (NF-κB/iNOS/NO pathway) related to vasoconstriction in femoral arteries.

Mitochondrial regulation of NADPH oxidase in hindlimb unweighting rat cerebral arteries.

Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.

Simulated microgravity‐induced mitochondrial dysfunction in rat cerebral arteries

Exposure to microgravity results in cardiovascular deconditioning, and cerebrovascular oxidative stress injury has been suggested to occur. To elucidate the mechanism for this condition, we investigated whether simulated microgravity induces mitochondrial dysfunction in rat arteries. Four-week hindlimb unweighting (HU) was used to simulate microgravity in rats. Mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), mitochondrial permeability transition pore (mPTP) opening, mitochondrial respiratory control ratio (RCR), MnSOD/GPx activity and expression, and mitochondrial malondialdehyde (MDA) were examined in rat cerebral and mesenteric VSMCs. Compared with the control rats, mitochondrial ROS levels, mPTP opening, and MDA content increased significantly (P<0.001, P<0.01, and P<0.01, respectively), Δψm, RCR, MnSOD/GPx activity (P<0.001 for Δψm and RCR; P<0.05 for MnSOD; and P<0.001 for GPx activity) and protein abundance of mitochondrial MnSOD/GPx-1 decreased (P<0.001 for MnSOD and GPx-1) in HU rat cerebral but not mesenteric arteries. Chronic treatment with NADPH oxidase inhibitor apocynin and mitochondria-targeted antioxidant mitoTempol promoted recovery of mitochondrial function in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, simulated microgravity resulted in cerebrovascular mitochondrial dysfunction, and crosstalk between NADPH oxidase and mitochondria participated in the process.

NAD(P)H oxidase inhibiting with apocynin improved vascular reactivity in tail-suspended hindlimb unweighting rat

Recent studies suggested that reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase is of functional importance in modulating vascular tone, and we have previously detected excessive superoxide production in tail-suspended hindlimb unweighting (HU) rat cerebral and carotid arteries. HU rat was a widely used model to simulate physiological effects on the vasculature. The present study tended to investigate whether NAD(P)H oxidase inhibition with apocynin influences vasoconstriction, endothelium-dependent relaxation, and nitrite/nitrate (NOx) content in HU rat cerebral and carotid arteries. Vascular contractile and dilate responses were assessed in a myograph organ bath. NOx content in cerebral and carotid arteries was measured. We found enhanced maximal contractile response and impaired endothelium-dependent relaxation in HU rat basilar (P < 0.01) and common carotid artery (P < 0.05); however, chronic treatment of apocynin (50 mg/kg/day) partially reversed abnormal vascular response. Furthermore, 21-day HU increased arterial NOx content (P < 0.01) in cerebral and carotid arteries compared with control rats; however, apocynin treatment restored it toward near-normal values. These data demonstrated that NAD(P)H oxidase-derived oxidative stress mediated abnormal vasoreactivity though nitric oxide mechanism in the settings of simulated microgravity.

Nitric oxide synthase activity in the abdominal aorta of rats is decreased after 4 weeks of simulated microgravity

1. The aim of the present study was to investigate the effects of simulated microgravity on the arterial dilatory responsiveness and L-arginine (L-Arg)-nitric oxide (NO)-cGMP pathway in the abdominal aorta of rats. 2. Twenty healthy male Sprague-Dawley were randomly divided into control and simulated microgravity groups. Rats in the simulated microgravity group were subjected to hindlimb unweighting (HU). After 4 weeks, arterial dilatory responsiveness was examined in vitro in isolated abdominal aortic rings. Western blotting was used to measure endothelial (e) and inducible (i) NO synthase (NOS) protein content. Total concentrations of nitrate and nitrite (NO(x)), the stable metabolites of NO, were determined by the chemiluminescence method. Nitric oxide synthase activity in the abdominal aorta was determined through the conversion of [(3)H]-L-Arg to [(3)H]-L-citrulline. 3. The data showed that the dilatory responses of the arterial rings to L-Arg and acetylcholine decreased in rats exposed to simulated microgravity, but the dilatory responses to sodium nitroprusside and 8-bromo-cGMP were similar in both simulated microgravity and control rats. The expression of eNOS and iNOS did not differ significantly between the two groups. The NO(x) concentration in the abdominal aorta of HU rats was significantly less than that in control rats. Nitric oxide synthase activity in the aorta decreased after 4 weeks of HU. 4. The data indicate that endothelium-dependent vasorelaxation in the abdominal aorta decreased due to 4 weeks of simulated microgravity in rats and that this impaired dilatory responsiveness may result from decreased NOS activity.

Differential vascular cell adhesion molecule-1 expression and superoxide production in simulated microgravity rat vasculature.

Exposure to microgravity leads to orthostatic intolerance in astronauts and differential vascular structural and functional adaptations have been implicated in its occurrence. The present study tended to clarify the characteristics of vascular inflammation and oxidative stress in hindlimb unweighting (HU) rat vasculature. Male Sprague-Dawley rats were randomly divided into control (CON) and hindlimb unweighting (HU) groups. Three weeks later, immunohistochemistry was used to localize the expression of vascular cell adhesion molecule-1 (VCAM-1) and laser scanning confocal microscope were used to detect superoxide production. Immunohistochemical results revealed positive staining of VCAM-1 on endothelial cells in HU rat basilar and carotid arteries compared with CON, but not in abdominal aorta and femoral arteries. Meanwhile, HU increased O2·- levels in all the layers of basilar and carotid arteries from HU rat but not in abdominal aorta and femoral arteries from HU rat. These data suggested that differential expression of VCAM-1 and O2·- production were concomitant with the vascular adaptations to simulated microgravity and whether they participate in vascular structure and function remodeling merits further investigation.

P59 Can we predict different phenotypes of preeclampsia in low risk nulliparous women using first trimester angiogenic/antiangiogenic biomarkers

The roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established.We investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats.ROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats.These results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravity.

Early changes in vasoreactivity after simulated microgravity are due to an upregulation of the endothelium-dependent nitric oxide/cGMP pathway

Emerging evidence suggests that nitric oxide (NO) plays a pivotal role in the mechanism of vascular hyporesponsiveness contributing to microgravity-induced orthostatic intolerance. The cellular and enzymatic source of the NO, however, remains controversial. In addition, the time course of the endothelial-dependent contribution remains unstudied. We tested the hypotheses that the change in vasoresponsiveness seen in acute (3-day) hindlimb unweighted (HLU) animals is due to an endothelium-dependent mechanism and that endothelial-dependent attenuation in vasoreactivity is due to endothelial nitric oxide synthase (NOS-3) dependent activation. Vasoreactivity was investigated in rat aortic rings following acute HLU treatment. Dose responsiveness to norepinepherine (NE) was depressed after 3-day HLU [1,338 +/- 54 vs. 2,325 +/- 58 mg at max (NE), HLU vs. C, P < 0.001]. However, removal of the endothelium restored the vascular contractility to that of C. In addition, 1H-oxadiazole quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, restored the reduced vasoconstrictor responses to phenylephrine (PE) seen in 3-day HLU rings (1.30 +/- 0.10 vs. 0.53 +/- 0.07 g, HLU + ODQ vs. HLU, P = 0.0001). Ca(+) dependent nitric oxide synthase (NOS) activity was increased, as was vascular NO products as a result of HLU. While NOS-3 expression was not increased in HLU rats, phosphorylation of NOS-3 at serine-1177 (an activator of NOS-3) was increased while phosphorylation of serine-495 (an inactivator of NOS-3) was decreased. These findings demonstrate that changes in vasoresponsiveness in the acute HLU model of microgravity are due to an upregulation of the endothelial-dependent NO/cGMP pathway through NOS phosphorylation.

Modulation of intracellular calcium transient in response to β-adrenoceptor stimulation in the hearts of 4-wk-old rats during simulated weightlessness

Modulation of intracellular calcium ([Ca(2+)](i)) transient in response to beta-adrenoceptor stimulation in the hearts of hindlimb unweighted (HLU) rats during simulated weightlessness has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness. Effects of simulated microgravity on beta-adrenoceptor responsiveness were then studied. Mean arterial blood pressure, left ventricular pressure (LVP), systolic function [maximum positive change in pressure over time (+dP/dt(max))], and diastolic function [maximum negative change in pressure over time (-dP/dt(max))] were monitored during the in vivo experiment. beta-Adrenoceptor density was quantitated by radioactive ligand binding. Single rat ventricular myocyte was obtained by enzymatic dissociation method. +/-dP/dt(max), myocyte contraction, intracellular [Ca(2+)](i) transient, and L-type calcium current in response to beta-adrenoceptor stimulation with isoproterenol were measured. Compared with the control group, no significant changes were found in heart weight, body weight, and mean arterial blood pressure, whereas LVP and +/-dP/dt(max) were significantly reduced. LVP and +/-dP/dt(max) were significantly attenuated in the HLU group in response to isoproterenol administration. In the in vitro study, the beta-adrenoceptor density was unchanged. Effects of isoproterenol on electrically induced single-cell contraction and [Ca(2+)](i) transient in myocytes of ventricles in HLU rats were significantly attenuated. The enhanced L-type Ca(2+) current elicited by isoproterenol in cardiomyocytes was significantly decreased in the HLU group. The above results indicate that impaired function of L-type Ca(2+) current and decreased [Ca(2+)](i) transient cause the depressed responsiveness of the beta-adrenoceptor stimulation, which may be partially responsible for the depression of cardiac function.

Hindlimb unweighting induces changes in the p38MAPKcontractile pathway of the rat abdominal aorta

Hindlimb unweighting (HLU) of rats is a model used to mimic the cephalic fluid shift potentially involved in the orthostatic intolerance experienced by astronauts. Certain arteries in these rats exhibit a decreased contractile response to adrenergic agonists. It was shown previously that this may be caused by changes in thick filament regulation (Summers et al., Vascul Pharmacol 48: 208-214, 2008). In the present study, it was hypothesized that HLU also modifies thin filament regulation by effects on p38(MAPK) and ERK. Abdominal aorta rings from 20-day HLU rats and untreated controls were subjected to phenylephrine and phorbol 12,13-dibutyrate (PDBU) concentration response curves in the presence and absence of two inhibitors: the p38(MAPK) inhibitor SB-203580 and the MEK inhibitor U-0126. SB-203580 decreased control sensitivity to both agonists, but HLU sensitivity was not significantly affected. U-0126, which blocks enzymes immediately upstream of ERK, affected sensitivity to both agonists equally between control and HLU. Western blot analysis revealed no change in total levels of p38(MAPK) and its downstream target heat shock protein 27 but did reveal a decrease in phosphorylated levels of both after stimulation with PDBU and phenylephrine after HLU treatment. Neither total ERK nor phosphorylated levels after stimulation were affected by HLU. Total levels of caldesmon, a molecule downstream of both pathways, were decreased, but phosphorylated levels after stimulation were decreased by roughly twice as much. The results of this study demonstrate that HLU downregulates p38(MAPK), but not ERK, signaling. In turn, this may decrease actin availability for contraction.