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

OBJECTIVE: Exposure 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. METHODS: Three-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca 2+ distribution, inositol 1,4,5-trisphosphate receptor (IP 3R) abundance, and the activities of voltage-gated K + channels (K V) and Ca 2+-activated K + channels (BK Ca) were examined in rat cerebral vascular smooth muscle cells (VSMCs). RESULTS: An increase of cytoplasmic Ca 2+ and a decrease of mitochondrial/sarcoplasmic reticulum (SR) Ca 2+ 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 IP 3R protein/mRNA levels were significantly upregulated. The current densities and open probabilities of K V and BK Ca 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 IP 3R expression levels and restored the activities of the K V and BK Ca channels. MitoTEMPO restored the Ca 2+ distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries. CONCLUSION: The present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.

Earlier application of loading doses of aspirin and clopidogrel decreases rate of recurrent cardiovascular ischemic events for patients undergoing percutaneous coronary intervention.

BACKGROUND: Aspirin and clopidogrel resistance plays a significant role in the development of cardiovascular ischemic events for ninety patients undergoing percutaneous coronary intervention. Recent studies have indicated that increasing the dose of antiplatelet drugs maybe a potent method to improve the inhibition of platelet aggregation. METHODS: Thrombelastograph (TEG) determinations were used to evaluate the effect of antiplatelet therapy. According to the results, 90 patients were divided into three groups and given different doses of aspirin and clopidogrel. Thirty patients with both an inhibition rate of aspirin > 50% and an inhibition rate of clopidogrel > 50% were defined as the control group. Sixty patients with an inhibition rate for aspirin < 50% and an inhibition rate for clopidogrel < 50% were defined as the resistance group. Patients in resistance group were randomly assigned to be given a routine dose (100 mg aspirin plus 75 mg clopidogrel per day, which we called a resistance plus routine dose group, R + R) and a loading dose (200 mg aspirin and 150 mg clopidogrel per day, which we called resistance plus loading dose group, R + L) of antiplatelet therapy. A 12-month follow-up was observed to examine the change of inhibition rate of antiplatelet therapy and to estimate the relationship between inhibition rate and the occurrence of cardiovascular ischemic events. RESULTS: After 6 months of antiplatelet therapy, the inhibition rate of aspirin in the R + L group increased from (31.4 ± 3.7)% to (68.6 ± 7.1)%, which was significantly higher than that in R + R group, (51.9 ± 8.2)% (P < 0.01). The inhibition rate of clopidogrel in the R + L group increased from (22.1 ± 3.8)% to (60.2 ± 7.4)%, which was significantly higher than in the R + R group, (45.9 ± 4.3)% (P < 0.01). The occurrence rates of cardiovascular ischemic events, stent thrombosis, recurrent unstable angina and myocardial infarction in the R + R group were 20%, 36% and 17%, respectively. Occurrence was significantly increased compared with that in the control group, 3%, 10% and 1%, respectively (P < 0.01). In contrast, the occurrence rates in the R + L group (10%, 23% and 6%, respectively) were attenuated compared with those in the R + R group (P < 0.01), although still higher than in the control group (P < 0.01). CONCLUSIONS: Almost all of the cardiovascular ischemic events occurred in the first six months after percutaneous coronary intervention. According to the result of TEG determinations, earlier application of a loading dose of aspirin and clopidogrel can decrease the rate of recurrent cardiovascular ischemic events.

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)

[Time-course changes of myogenic tone of mesenteric small artery in spontaneously hypertensive rat].

To investigate the time-course changes of myogenic tone in mesenteric small artery (MSA) of spontaneously hypertensive rat (SHR), thirty-two 7-week aged SHR rats were randomly divided into four groups (8, 16, 24, 32 weeks of age), and 32 sex- and age-matched Wistar-Kyoto (WKY) rats were assigned to control groups (CON). On the day of the study, segments of MSA were isolated and then cannulated to the two pipettes. Vascular diameters in response to the increased intraluminal pressure (from 0 mmHg to 150 mmHg, by 25 mmHg steps) of isolated MSA under no-flow conditions were recorded by a Pressure Myograph System both in physiologic salt solution (PSS) (active diameter, Da) and calcium-free PSS (passive diameter, Dp). The myogenic tone was calculated by (Dp - Da)/Dp × 100%. The tail artery pressure and vascular myogenic tone in SHR rats were significantly higher than those of the CON rats. Before 24 weeks, the vascular myogenic tone of MSA in SHR group increased monotonically, but at the end of 32 weeks, the vascular myogenic tone decreased in comparison with that in 24-week group, but was significantly higher than that in CON group. The tail artery pressure in SHR group slowly increased monotonically with increasing weeks of age, and the tail arterial pressure in 32-week group remained significantly higher than that in 24-week group. Vascular myogenic tone may participate in the whole process of hypertension. Early in the development of hypertension, because of the compensatory role of vascular tone, the vascular function has been partially compensated, thus guaranteeing adequate blood supply to organs. Late in the development of hypertension, because of the decompensation of myogenic tone, the vascular function is damaged, leading to the occurrence of severe vascular disease.

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.

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.

[Comparison of biomechanical behavior of cerebral and mesenteric small arteries of simulated microgravity rats].

The aim of the present study was to further elucidate the mechanisms of vascular adaptation to microgravity and its gravity-based countermeasure by a biomechanical approach. Active (the dissected vessel segment was superfused with PPS) and passive (while it was superfused with Ca(2+)-free PPS) biomechanical properties of mesenteric third-order small arteries and middle cerebral arteries isolated from 3-day simulated microgravity (SUS), countermeasure (STD, daily 1 h of -G(x) gravitation), and control (CON) groups of rats were studied. The following mechanical parameters were calculated: the overall stiffness parameter of passive vessels (beta), circumferential stress (sigma(theta))-strain (epsilon(theta)) relationship, and pressure-dependent incremental elastic modulus (E(inc,p)) of both active and passive vessels, and vascular smooth muscle (VSM) activity-dependent incremental modulus (E(inc,a)). Results from the analysis of active biomechanical properties revealed the contribution of vascular smooth muscle (VSM) tone during the early adaptation to microgravity: (1) For mesenteric small arteries, active circumferential sigma(theta) -epsilon(theta) curve of SUS group was comparable with that of the passive vessels, indicating that the function of VSM to restore the normal stress distribution is compromised; however, this mal-adaptation was fully prevented by the countermeasure of daily 1 h of -G(x) gravitation; (2) For the middle cerebral arteries, active circumferential sigma(theta) -epsilon(theta) relation of SUS group was shifted to the left side of the passive curve and epsilon(theta) was kept at a nearly constant level with the corresponding sigma(theta) being at its normal range; furthermore, the enhanced myogenic tone responsiveness was not prevented by daily short-duration -G(x). Analysis of the passive biomechanical properties has suggested remodeling changes in matrix components of different types of vessels, which might be significant if the exposure duration was further prolonged. In brief, studies of vascular biomechanics are of particular importance in elucidating the mechanisms underlying vascular adaptation to microgravity and its gravity-based countermeasure.

[Differential effect of simulated microgravity on myogenic tone of middle cerebral and mesenteric small arteries in rats].

The aim of the present study was to investigate the effect of a short-term (3-day) simulated microgravity with and without daily dorsoventral gravitation (-G(x)) for 1 h on myogenic tone and vasoconstrictor responsiveness of the middle cerebral artery and mesenteric third-order small artery in rats. The tail-suspension (SUS) model was used to simulate cardiovascular deconditioning due to microgravity. Daily restoring to normal standing (STD) posture for 1 h was adopted to provide -G(x) as the countermeasure. Segments of middle cerebral artery and mesenteric third-order small artery were isolated and cannulated. Vascular diameters in response to increased intraluminal pressure (from 20 mmHg to 120 mmHg, by 20 mmHg steps) of isolated arteries under no-flow conditions were recorded by a Pressure Myograph System in both physiologic salt solution (PSS) (active diameter, Da) and calcium-free PSS (passive diameter, Dp). The myogenic tone was calculated by (Dp-Da)/Dpx100%. Vasoconstrictor responsiveness of the isolated middle cerebral artery to serotonin and that of small mesenteric artery to phenylephrine were assessed in the PSS under an intraluminal pressure of 40 mmHg. The results showed that SUS induced an enhancement of the myogenic tone and vasoconstrictor responsiveness in the isolated middle cerebral artery but a depression of those in the small mesenteric artery. Daily STD for 1 h prevented the depression of myogenic tone and vasoconstrictor responsiveness in the small mesenteric artery, but did not prevent the functional enhancement in the middle cerebral artery. These data suggest that a short-term simulated microgravity may result in different alterations in the function of the cerebral artery and the resistance vessel in the hind-body. Moreover, only the decrease of function in these resistance vessels, not in the cerebral arteries, can be prevented by such a countermeasure of daily STD for 1 h.

Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats.

Previous studies have demonstrated activation of the local renin-angiotensin system in hindlimb unweighting (HU) rat vasculature. The present study intended to identify the effects of blockade of angiotensin II (ANG II) type 1 (AT(1)) receptors with losartan on vascular reactivity, nitric oxide synthase (NOS) expression, and superoxide anion (O(2)(*-)) levels in 3-wk HU rat cerebral and carotid arteries. Three weeks later, vasoconstriction, vasodilatation, endothelial NOS (eNOS) and inducible NOS (iNOS) protein, as well as O(2)(*-) levels in rat cerebral and carotid arteries were examined. We found that HU enhanced maximal response to KCl/5-hydroxytryptamine (P < 0.01) in basilar arteries and KCl/phenylephrine (P < 0.05) in common carotid arteries from HU rats. Acetylcholine induced concentration-dependent vasodilatation in all the artery rings, but with significantly smaller amplitude in basilar (P < 0.01) and common carotid (P < 0.05) arteries from HU rats than those from control rats. Chronic treatment with losartan partially restored response to vasoconstrictors and acetylcholine-induced vasodilatation in basilar (P < 0.01) and common carotid (P < 0.05) arteries from losartan-treated HU rats. Furthermore, iNOS content in cerebral arteries and eNOS/iNOS content in carotid arteries were significantly (P < 0.01) increased in HU rats. Meanwhile, HU increased O(2)(*-) levels in all the layers of these arteries. However, losartan restored NOS content and O(2)(*-) levels toward normal. These results suggested that the HU-induced enhancement of vasoconstriction and reduction in endothelium-dependent relaxation involved alterations in O(2)(*-) and NOS content through an ANG II/AT(1) receptor signaling pathway.