miR-137 and its target T-type CaV 3.1 channel modulate dedifferentiation and proliferation of cerebrovascular smooth muscle cells in simulated microgravity rats by regulating calcineurin/NFAT pathway.

OBJECTIVES: Postflight orthostatic intolerance has been regarded as a major adverse effect after microgravity exposure, in which cerebrovascular adaptation plays a critical role. Our previous finding suggested that dedifferentiation of vascular smooth muscle cells (VSMCs) might be one of the key contributors to cerebrovascular adaptation under simulated microgravity. This study was aimed to confirm this concept and elucidate the underlying mechanisms. MATERIALS AND METHODS: Sprague Dawley rats were subjected to 28-day hindlimb-unloading to simulate microgravity exposure. VSMC dedifferentiation was evaluated by ultrastructural analysis and contractile/synthetic maker detection. The role of T-type CaV 3.1 channel was revealed by assessing its blocking effects. MiR-137 was identified as the upstream of CaV 3.1 channel by luciferase assay and investigated by gain/loss-of-function approaches. Calcineurin/nuclear factor of activated T lymphocytes (NFAT) pathway, the downstream of CaV 3.1 channel, was investigated by detecting calcineurin activity and NFAT nuclear translocation. RESULTS: Simulated microgravity induced the dedifferentiation and proliferation in rat cerebral VSMCs. T-type CaV 3.1 channel promoted the dedifferentiation and proliferation of VSMC. MiR-137 and calcineurin/NFATc3 pathway were the upstream and downstream signalling of T-type CaV 3.1 channel in modulating the dedifferentiation and proliferation of VSMCs, respectively. CONCLUSIONS: The present work demonstrated that miR-137 and its target T-type CaV 3.1 channel modulate the dedifferentiation and proliferation of rat cerebral VSMCs under simulated microgravity by regulating calcineurin/NFATc3 pathway.

BMAL1 Disrupted Intrinsic Diurnal Oscillation in Rat Cerebrovascular Contractility of Simulated Microgravity Rats by Altering Circadian Regulation of miR-103/CaV1.2 Signal Pathway.

The functional and structural adaptations in cerebral arteries could be one of the fundamental causes in the occurrence of orthostatic intolerance after space flight. In addition, emerging studies have found that many cardiovascular functions exhibit circadian rhythm. Several lines of evidence suggest that space flight might increase an astronaut's cardiovascular risks by disrupting circadian rhythm. However, it remains unknown whether microgravity disrupts the diurnal variation in vascular contractility and whether microgravity impacts on circadian clock system. Sprague-Dawley rats were subjected to 28-day hindlimb-unweighting to simulate the effects of microgravity on vasculature. Cerebrovascular contractility was estimated by investigating vasoconstrictor responsiveness and myogenic tone. The circadian regulation of CaV1.2 channel was determined by recording whole-cell currents, evaluating protein and mRNA expressions. Then the candidate miRNA in relation with Ca2+ signal was screened. Lastly, the underlying pathway involved in circadian regulation of cerebrovascular contractility was determined. The major findings of this study are: (1) The clock gene BMAL1 could induce the expression of miR-103, and in turn modulate the circadian regulation of CaV1.2 channel in rat cerebral arteries at post-transcriptional level; and (2) simulated microgravity disrupted intrinsic diurnal oscillation in rat cerebrovascular contractility by altering circadian regulation of BMAL1/miR-103/CaV1.2 signal pathway.

Focal adhesions are involved in simulated-microgravity-induced basilar and femoral arterial remodelling in rats.

Recent studies have suggested that microgravity-induced arterial remodelling contributes to post-flight orthostatic intolerance and that multiple mechanisms are involved in arterial remodelling. However, the initial mechanism by which haemodynamic changes induce arterial remodelling is unknown. Focal adhesions (FAs) are dynamic protein complexes that have mechanotransduction properties. This study aimed to investigate the role of FAs in simulated-microgravity-induced basilar and femoral arterial remodelling. A 4-week hindlimb-unweighted (HU) rat model was used to simulate the effects of microgravity, and daily 1-hour intermittent artificial gravity (IAG) was used to prevent arterial remodelling. After 4-week HU, wall thickness, volume of smooth muscle cells (SMCs) and collagen content were increased in basilar artery but decreased in femoral artery (P < 0.05). Additionally, the expression of p-FAK Y397 and p-Src Y418 was increased and reduced in SMCs of basilar and femoral arteries, respectively, by HU (P < 0.05). The number of FAs was increased in basilar artery and reduced in femoral artery by HU (P < 0.05). Furthermore, daily 1-hour IAG prevented HU-induced differential structural adaptations and changes in FAs of basilar and femoral arteries. These results suggest that FAs may act as mechanosensors in arterial remodelling by initiating intracellular signal transduction in response to altered mechanical stress induced by microgravity.

Berberine reduced blood pressure and improved vasodilation in diabetic rats.

Hyperglycemia and hypertension are considered to be the two leading risk factors for vascular disease in diabetic patients. However, few pharmacologic agents could provide a combinational therapy for controlling hyperglycemia and hypertension at the same time in diabetes. The objectives of this study are to investigate whether berberine treatment could directly reduce blood pressure and identify the molecular mechanism underlying the vascular protection of berberine in diabetic rats. Berberine was intragastrically administered with different dosages of 50, 100 and 200 mg/kg/day to diabetic rats for 8 weeks since the injection of streptozotocin. The endothelium-dependent/-independent relaxation in middle cerebral arteries was investigated. The activity of large-conductance Ca2+-activated K+ channel (BKCa) was investigated by recording whole-cell currents, analyzing single-channel activities and assessing the expressions of α- and ß1-subunit at protein or mRNA levels. Results of the study suggest that chronic administration of 100 mg/kg/day berberine not only lowered blood glucose but also reduced blood pressure and improved vasodilation in diabetic rats. Furthermore, berberine markedly increased the function and expression of BKCa ß1-subunit in cerebral vascular smooth muscle cells (VSMCs) isolated from diabetic rats or when exposed to hyperglycemia condition. The present study provided initial evidences that berberine reduced blood pressure and improved vasodilation in diabetic rats by activation of BKCa channel in VSMCs, which suggested that berberine might provide a combinational therapy for controlling hyperglycemia and blood pressure in diabetes. Furthermore, our work indicated that activation of BKCa channel might be the underlying mechanism responsible for the vascular protection of berberine in diabetes.

Salidroside contributes to reducing blood pressure and alleviating cerebrovascular contractile activity in diabetic Goto-Kakizaki Rats by inhibition of L-type calcium channel in smooth muscle cells.

BACKGROUND: Vascular disease is a common and often severe complication in diabetes mellitus. Hyperglycemia and hypertension are considered to be two of the leading risk factors for vascular complications in diabetic patients. However, few pharmacologic agents could provide a combinational therapy for controlling hyperglycemia and blood pressure in diabetic patients at the same time. Salidroside (SAL) is the major active ingredient derived from Rhodiola. Recently, it has been reported that SAL have an obvious hypoglycemic effect in diabetes and show a beneficial activity in diabetic vascular dysfunction. However, it remains unknown whether or not SAL treatment could directly reduce blood pressure in diabetes. Furthermore, it is not clear what is the molecular mechanism underlying the vascular protection of SAL treatment in diabetes. METHODS: Male diabetic Goto-Kakizaki (GK) and non-diabetic control Wistar-Kyoto (WKY) rats were administrated with different dosages of SAL (50, 100 and 200 mg/kg/day) for 4 weeks. Contractile responsiveness of cerebral artery to KCl or 5-HT was investigated by Pressure Myograph System. The activity of CaL channel was investigated by recording whole-cell currents, assessing the expressions of CaL channel α1C-subunit and its downstream kinase, MLCK, at protein or mRNA levels. RESULTS: We showed that administration of 100 mg/kg/day SAL for 4 weeks not only lowered blood glucose, but also reduced blood pressure and alleviated cerebrovascular contractile activity in diabetic GK rats, which suggested that SAL treatment may provide a combinational therapy for lowering blood glucose and reducing blood pressure in diabetes at the same time. Furthermore, SAL treatment markedly inhibited the function and expression of CaL channel in cerebral VSMCs isolated from diabetic GK rats or when exposed to hyperglycemia condition, which may be the underlying mechanism responsible for the vascular protection of SAL in diabetes. CONCLUSIONS: The present study provided evidences that SAL contributes to reducing blood pressure and alleviating cerebrovascular contractile activity in diabetic GK rats by inhibition of CaL channel in smooth muscle cells, which may provide a novel approach to treat vascular complications in diabetic patients.

Berberine alleviates the cerebrovascular contractility in streptozotocin-induced diabetic rats through modulation of intracellular Ca²âº handling in smooth muscle cells.

BACKGROUND: Vascular dysfunction is a distinctive phenotype in diabetes mellitus. Current treatments mostly focus on the tight glycemic control and few of these treatments have been designed to directly recover the vascular dysfunction in diabetes. As a classical natural medicine, berberine has been explored as a possible therapy for DM. In addition, it is reported that berberine has an extra-protective effect in diabetic vascular dysfunction. However, little is known whether the berberine treatment could ameliorate the smooth muscle contractility independent of a functional endothelium under hyperglycemia. Furthermore, it remains unknown whether berberine affects the arterial contractility by regulating the intracellular Ca(2+) handling in vascular smooth cells (VSMCs) under hyperglycemia. METHODS: Sprague-Dawley rats were used to establish the diabetic model with a high-fat diet plus injections of streptozotocin (STZ). Berberine (50, 100, and 200 mg/kg/day) were intragastrically administered to control and diabetic rats for 8 weeks since the injection of STZ. The intracellular Ca(2+) handling of isolated cerebral VSMCs was investigated by recording the whole-cell L-type Ca(2+) channel (CaL) currents, assessing the protein expressions of CaL channel, and measuring the intracellular Ca(2+) in response to caffeine. Our results showed that chronic administration of 100 mg/kg/day berberine not only reduced glucose levels, but also inhibited the augmented contractile function of cerebral artery to KCl and 5-hydroxytryptamine (5-HT) in diabetic rats. Furthermore, chronic administration of 100 mg/kg/day berberine significantly inhibited the CaL channel current densities, reduced the α1C-subunit expressions of CaL channel, decreased the resting intracellular Ca(2+) ([Ca(2+)]i) level, and suppressed the Ca(2+) releases from RyRs in cerebral VSMCs isolated from diabetic rats. Correspondingly, acute application of 10 µM berberine could directly inhibit the hyperglycemia-induced CaL currents and suppress the hyperglycemia-induced Ca(2+) releases from RyRs in cerebral VSMCs isolated from normal control rats. CONCLUSIONS: Our study indicated that berberine alleviated the cerebral arterial contractility in the rat model of streptozotocin-induced diabetes via regulating the intracellular Ca(2+) handling of smooth muscle cells.

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

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.

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.

Simulated microgravity induces an inflammatory response in the common carotid artery of rats.

Post-spaceflight orthostatic intolerance is one of the most important adverse effects after exposure to space microgravity, and there are still no effective countermeasures. It has been considered that arterial remodeling may play an important role in the occurrence of post-spaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. In this study, we investigated whether an inflammatory response exists in the common carotid artery of rats exposed to simulated microgravity. For this, Sprague-Dawley rats were subjected to 4 weeks of hindlimb unweighting to simulate microgravity. The expression levels of the adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and the cytokine monocyte chemoattractant protein-1 (MCP-1) in the common carotid artery of simulated microgravity rats were evaluated by immunohistochemical staining, quantitative RT-PCR, and Western blot analyses. The recruitment of monocytes in the common carotid artery of rats exposed to simulated microgravity was investigated by en face immunofluorescence staining and monocyte binding assays. Our results provided convincing evidence that there is an inflammatory response in the common carotid artery of rats exposed to simulated microgravity. Our work suggests that the inflammatory response may be a novel cellular mechanism that is responsible for the arterial remodeling that occurs during exposure to microgravity.

Mechanics and composition of middle cerebral arteries from simulated microgravity rats with and without 1-h/d -Gx gravitation.

BACKGROUND: To elucidate further from the biomechanical aspect whether microgravity-induced cerebral vascular mal-adaptation might be a contributing factor to postflight orthostatic intolerance and the underlying mechanism accounting for the potential effectiveness of intermittent artificial gravity (IAG) in preventing this adverse effect. METHODOLOGY/PRINCIPAL FINDINGS: Middle cerebral arteries (MCAs) were isolated from 28-day SUS (tail-suspended, head-down tilt rats to simulate microgravity effect), S+D (SUS plus 1-h/d -Gx gravitation by normal standing to simulate IAG), and CON (control) rats. Vascular myogenic reactivity and circumferential stress-strain and axial force-pressure relationships and overall stiffness were examined using pressure arteriography and calculated. Acellular matrix components were quantified by electron microscopy. The results demonstrate that myogenic reactivity is susceptible to previous pressure-induced, serial constrictions. During the first-run of pressure increments, active MCAs from SUS rats can strongly stiffen their wall and maintain the vessels at very low strains, which can be prevented by the simulated IAG countermeasure. The strains are 0.03 and 0.14 respectively for SUS and S+D, while circumferential stress being kept at 0.5 (106 dyn/cm2). During the second-run pressure steps, both the myogenic reactivity and active stiffness of the three groups declined. The distensibility of passive MCAs from S+D is significantly higher than CON and SUS, which may help to attenuate the vasodilatation impairment at low levels of pressure. Collagen and elastin percentages were increased and decreased, respectively, in MCAs from SUS and S+D as compared with CON; however, elastin was higher in S+D than SUS rats. CONCLUSIONS: Susceptibility to previous myogenic constrictions seems to be a self-limiting protective mechanism in cerebral small resistance arteries to prevent undue cerebral vasoconstriction during orthostasis at 1-G environment. Alleviating of active stiffening and increasing of distensibility of cerebral resistance arteries may underlie the countermeasure effectiveness of IAG.

Mechanical properties and composition of mesenteric small arteries of simulated microgravity rats with and without daily -G(x) gravitation.

The aim of the present study was to evaluate the active and passive mechanical properties and wall collagen and elastin contents of mesenteric small arteries (MSAs) isolated from rats of 28-day simulated microgravity (SUS), countermeasure [S + D: SUS plus 1 h/d -G(x) to simulate intermittent artificial gravity (IAG)] and control (CON) groups. Three mechanical parameters were calculated: the overall stiffness (ß), circumferential stress (σ(θ))-strain (ε(θ)) relationship and pressure-dependent incremental elastic modulus (E(inc,p)). Vessel wall collagen and elastin percentage were quantified by electron microscopy. The results demonstrate that the active mechanical behavior of MSAs differs noticeably among the three groups: the active stress-strain curve of SUS vessels is very close to the passive curve, whereas the active σ(θ)-ε(θ) curves of CON and S + D vessels are shifted leftward and display a parabolic shape, indicating that for MSAs isolated from S + D, but not those from SUS rats, the pressure-induced myogenic constriction can effectively stiffen the vessel wall as the CON vessels. The passive mechanical behavior of MSAs does not show significant differences among the three groups. However, the percentage of collagen is decreased in the wall of SUS and S + D compared with CON vessels in the following order: SUS < S + D < CON. Thus, the relationship between passive mechanical behavior and compositional changes may be complex and yet depends on factors other than the quantity of collagen and elastin. These findings have provided biomechanical data for the understanding of the mechanism of postflight orthostatic intolerance and its gravity-based countermeasure.

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

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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 (AU)

[In-vivo and ex-vivo studies on region-specific remodeling of large elastic arteries due to simulated weightlessness and its prevention by gravity-based countermeasure].

The present study was designed to test the hypothesis that a medium-term simulated microgravity can induce region-specific remodeling in large elastic arteries with their innermost smooth muscle (SM) layers being most profoundly affected. The second purpose was to examine whether these changes can be prevented by a simulated intermittent artificial gravity (IAG). The third purpose was to elucidate whether vascular local renin-angiotensin system (L-RAS) plays an important role in the regional vascular remodeling and its prevention by the gravity-based countermeasure. This study consisted of two interconnected series of in-vivo and ex-vivo experiments. In the in-vivo experiments, the tail-suspended, hindlimb unloaded rat model was used to simulate microgravity-induced cardiovascular deconditioning for 28 days (SUS group); and during the simulation period, another group was subjected to daily 1-hour dorso-ventral (-G(x)) gravitation provided by restoring to normal standing posture (S + D group). The activity of vascular L-RAS was evaluated by examining the gene and protein expression of angiotensinogen (Ao) and angiotensin II receptor type 1 (AT1R) in the arterial wall tissue. The results showed that SUS induced an increase in the media thickness of the common carotid artery due to hypertrophy of the four SM layers and a decrease in the total cross-sectional area of the nine SM layers of the abdominal aorta without significant change in its media thickness. And for both arteries, the most prominent changes were in the innermost SM layers. Immunohistochemistry and in situ hybridization revealed that SUS induced an up- and down-regulation of Ao and AT1R expression in the vessel wall of common carotid artery and abdominal aorta, respectively, which was further confirmed by Western blot analysis and real time PCR analysis. Daily 1-hour restoring to normal standing posture over 28 days fully prevented these remodeling and L-RAS changes in the large elastic arteries that might occur due to SUS alone. In the ex-vivo experiments, to elucidate the important role of transmural pressure in vascular regional remodeling and differential regulation of L-RAS activity, we established an organ culture system in which rat common carotid artery, held at in-vivo length, can be perfused and pressurized at varied flow and pressure for 7 days. In arteries perfused at a flow rate of 7.9 mL/min and pressurized at 150 mmHg, but not at 0 or 80 mmHg, for 3 days led to an augmentation of c-fibronectin (c-FN) expression, which was also more markedly expressed in the innermost SM layers, and an increase in Ang II production detected in the perfusion fluid. However, the enhanced c-FN expression and increased Ang II production that might occur due to a sustained high perfusion pressure alone were fully prevented by daily restoration to 0 or 80 mmHg for a short duration. These findings from in-vivo and ex-vivo experiments have provided evidence supporting our hypothesis that redistribution of transmural pressures might be the primary factor that initiates region-specific remodeling of arteries during microgravity and the mechanism of IAG is associated with an intermittent restoration of the transmural pressures to their normal distribution. And they also provide support to the hypothesis that L-RAS plays an important role in vascular adaptation to microgravity and its prevention by the IAG countermeasure.

Lysosome-membrane fusion mediated superoxide production in hyperglycaemia-induced endothelial dysfunction.

Lysosomal exocytosis and fusion to cellular membrane is critical in the oxidative stress formation of endothelium under apoptotic stimulus. We investigated the role therein of it in hyperglycaemia-induced endothelial dysfunction. The lysosome-membrane fusion was shown by the expression of lamp1, the lysosomal membrane marker, on cellular membrane and the transportation of lysosomal symbolic enzymes into cultural medium. We also examined the ceramide production, lipid rafts (LRs) clustering, colocalization of gp91(phox), a NADPH oxidase subunit (NOX) to LRs clusters, superoxide (O2·â») formation and nitric oxide (NO) content in human umbilical vein endothelial cells (HUVEC) and the endothelium-dependent NO-mediated vasodilation in isolated rat aorta. As compared to normal glucose (5.6 mmol/l, Ctrl) incubation, high glucose (22 mmol/l, HG) exposure facilitated the lysosome-membrane fusion in HUVEC shown by significantly increased quantity of lamp1 protein on cellular membrane and enhanced activity of lysosomal symbolized enzymes in cultural medium. HG incubation also elicited ceramide generation, LRs clustering and gp91(phox) colocalization to LRs clusters which were proved to mediate the HG induced O2·â» formation and NO depletion in HUVEC. Functionally, the endothelium-dependent NO-mediated vasodilation in aorta was blunted substantially after HG incubation. Moreover, the HG-induced effect including ceramide production, LRs clustering, gp91(phox) colocalization to LRs clusters, O2·â» formation and endothelial dysfunction could be blocked significantly by the inhibition of lysosome-membrane fusion. We propose that hyperglycaemia-induced endothelial impairment is closely related to the lysosome-membrane fusion and the following LRs clustering, LRs-NOX platforms formation and O2·â» production.

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.

[Short-term simulated weightlessness enhances response of L-type calcium channel to angiotensin II in cerebral vascular smooth muscle cells in rats].

Some studies suggest that the calcium channels and rennin-angiotensin system (RAS) play pivotal roles in the region-specific vascular adaptation due to simulated weightlessness. This study was designed to clarify if angiotensin II (Ang II) was involved in the adaptational change of the L-type calcium channel (Ca(L)) in the cerebral arterial vascular smooth muscle cells (VSMCs) under simulated weightlessness. Tail suspension (SUS) for 3 d was used to simulate immediate early cardiovascular changes to weightlessness. Then VSMCs in cerebral basilar artery were enzymatically isolated using papain, and Ca(L) current (barium instead of calcium as current carrier) in VSMCs was measured by whole-cell patch-clamp techniques. The results showed that 3-day simulated weightlessness significantly increased current density of Ca(L). However, I-V relationships of normalized peak current densities and steady-state activation curves of Ca(L) were not affected by simulated weightlessness. Although Ang II significantly increased current densities of Ca(L) in both SUS and control rats, the increase of Ca(L) current density in SUS rats was much more than that in control rats. These results suggest that 3-day simulated weightlessness induces the adaptational change of Ca(L) in cerebral VSMCs including increased response to Ang II, indicating that Ang II may play an important role in the adaptational change of cerebral arteries under microgravity.

Activation of BKCa channel is associated with increased apoptosis of cerebrovascular smooth muscle cells in simulated microgravity rats.

Cerebral arterial remodeling is one of the critical factors in the occurrence of postspaceflight orthostatic intolerance. We hypothesize that large-conductance calcium-activated K(+) (BK(Ca)) channels in vascular smooth muscle cells (VSMCs) may play an important role in regulating cerebrovascular adaptation during microgravity exposure. The aim of this work was to investigate whether activation of BK(Ca) channels is involved in regulation of apoptotic remodeling of cerebral arteries in simulated microgravity rats. In animal studies, Sprague-Dawley rats were subjected to 1-wk hindlimb unweighting to simulate microgravity. Alterations of BK(Ca) channels in cerebral VSMCs were investigated by patch clamp and Western blotting; apoptosis was assessed by electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL). To evaluate the correlation of BK(Ca) channel and apoptosis, channel protein and cell nucleus were double-stained. In cell studies, hSloalpha+beta1 channel was coexpressed into human embryonic kidney 293 (HEK293) cells to observe the effects of BK(Ca) channels on apoptosis. In rats, enhanced activities and expression of BK(Ca) channels were found to be correlated with increased apoptosis in cerebral VSMCs after simulated microgravity. In transfected HEK293 cells, activation of cloned BK(Ca) channel induced apoptosis, whereas inhibition of cloned BK(Ca) channel decreased apoptosis. In conclusion, activation of BK(Ca) channels is associated with increased apoptosis in cerebral VSMCs of simulated microgravity rats.

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.

Contrasting effects of simulated microgravity with and without daily -Gx gravitation on structure and function of cerebral and mesenteric small arteries in rats.

This study was designed to test the hypothesis that a 28-day tail suspension (SUS) could induce hypertrophy and enhanced myogenic and vasoconstrictor reactivity in middle cerebral arteries (MCAs), whereas atrophy and decreased myogenic and vasoconstrictor responses in mesenteric third-order arterioles (MSAs). Also, in addition to the functional enhancement in MCAs, structural changes in both kinds of arteries and functional decrement in MSAs could all be prevented by the intervention of daily 1-h dorsoventral (-G(x)) gravitation by restoring to standing posture. To test this hypothesis, vessel diameters to pressure alterations and nonreceptor- and receptor-mediated agonists were determined using a pressure arteriograph with a procedure to measure in vivo length and decrease hysteresis of vessel segments and longitudinal middlemost sections of vessels fixed at maximally dilated state were examined using electron microscopy and histomorphometry. Functional studies showed that 28-day tail-suspended, head-down tilt (SUS) resulted in enhanced and decreased myogenic tone and vasoconstrictor responses, respectively, in MCAs and MSAs. Histomorphometric data revealed that SUS-induced hypertrophic changes in MCAs characterized by increases in thickness (T) and cross-sectional area (CSA) of the media and the number of vascular smooth-muscle-cell layers (N(CL)), whereas in MSAs, it induced decreases in medial CSA and T and N(CL). Daily 1-h -G(x) over 28 days can fully prevent these differential structural changes in both kinds of small arteries and the functional decrement in MSAs, but not the augmented myogenic tone and increased vasoreactivity in the MCAs. These findings have revealed special features of small resistance arteries during adaptation to microgravity with and without gravity-based countermeasure.