Hypoxia-treated Vascular Smooth Muscle Cells Research Articles

Long Non-Coding RNA MEG8 Suppresses Hypoxia-Induced Excessive Proliferation, Migration and Inflammation of Vascular Smooth Muscle Cells by Regulation of the miR-195-5p/RECK Axis

It has been recognized that rebalancing the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) helps relieve vascular injury. Presently, we aim to investigate whether long non-coding RNA (lncRNA) maternally expressed 8 (MEG8) plays a role in affecting the excessive proliferation and migration of VSMCs following hypoxia stimulation. A percutaneous transluminal angioplasty balloon dilatation catheter was adopted to establish vascular intimal injury, the levels of MEG8 and miR-195-5p in the carotid artery were tested by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Hypoxia was used to stimulate VSMCs, then the cell counting kit-8 (CCK-8) assay, Transnwell assay, and wound healing assay were conducted to evaluate the proliferation, and migration of VSMCs. The protein levels of RECK (reversion inducing cysteine rich protein with kazal motifs), MMP (matrix metalloproteinase) 3/9/13, COX2 (cytochrome c oxidase subunit II), macrophage inflammatory protein (MIP)-1beta, VCAM-1 (vascular cell adhesion molecule 1), ICAM-1 (intercellular adhesion molecule 1), and HIF-1α (hypoxia inducible factor 1 subunit alpha) were determined by western blot or cellular immunofluorescence. As the data showed, MEG8 was down-regulated in the carotid artery after balloon injury in rats and hypoxia-treated VSMCs, and miR-195-5p was overexpressed. Forced MEG8 overexpression or inhibiting miR-195-5p attenuated hypoxia-promoted cell proliferation and migration of VSMCs. In addition, miR-195-5p up-regulation reversed MEG8-mediated effects. Hypoxia hindered the RECK expression while boosted MMP3/9/13 levels, and the effect was markedly reversed with MEG8 up-regulation or miR-195-5p down-regulation. Mechanistically, MEG8 functioned as a competitive endogenous (ceRNA) by sponging miR-195-5p which targeted RECK. Moreover, the HIF-1α inhibitor PX478 prevented hypoxia-induced proliferation, and migration of VSMCs, upregulated MEG8, and restrained miR-195-5p expression. Overall, lncRNA MEG8 participated in hypoxia-induced excessive proliferation, inflammation and migration of VSMCs through the miR-195-5p/RECK axis.

The Calcilytic Drug Calhex-231 Ameliorates Vascular Hyporesponsiveness in Traumatic Hemorrhagic Shock by Inhibiting Oxidative Stress and miR-208a-Mediated Mitochondrial Fission

Background The calcium-sensing receptor (CaSR) plays a fundamental role in extracellular calcium homeostasis in humans. Surprisingly, CaSR is also expressed in nonhomeostatic tissues and is involved in regulating diverse cellular functions. The objective of this study was to determine if Calhex-231 (Cal), a negative modulator of CaSR, may be beneficial in the treatment of traumatic hemorrhagic shock (THS) by improving cardiovascular function and investigated the mechanisms. Methods Rats that had been subjected to THS and hypoxia-treated vascular smooth muscle cells (VSMCs) were used in this study. The effects of Cal on cardiovascular function, animal survival, hemodynamics, and vital organ function in THS rats and the relationship to oxidative stress, mitochondrial fusion-fission, and microRNA (miR-208a) were investigated. Results Cal significantly improved hemodynamics, elevated blood pressure, increased vital organ blood perfusion and local oxygen supply, and markedly improved the survival outcomes of THS rats. Furthermore, Cal significantly improved vascular reactivity after THS in vivo and in vitro. Cal also restored the THS-induced decrease in myosin light chain (MLC) phosphorylation (the key element for VSMC contraction). Inhibition of MLC phosphorylation antagonized the Cal-induced restoration of vascular reactivity following THS. Cal suppressed oxidative stress in THS rats and hypoxic-VSMCs. Meanwhile, THS induced expression of mitochondrial fission proteins Drp1 and Fis1 and decreased expression of mitochondrial fusion protein Mfn1 in vascular tissues. Cal reduced expression of Drp1 and Fis1. In hypoxic-VSMCs, Cal inhibited mitochondrial fragmentation and preserved mitochondrial morphology. In addition, miR-208a mimic decreased Fis1 expression, and miR-208a inhibitor prevented Cal-induced Fis1 downregulation in hypoxic-VSMCs. Conclusion Calhex-231 exhibits outstanding potential for effective therapy of traumatic hemorrhagic shock, and the beneficial effects result from its protection of vascular function via inhibition of oxidative stress and miR-208a-mediated mitochondrial fission.

Beneficial effect of cyclosporine A on traumatic hemorrhagic shock

BackgroundVascular hyporeactivity plays an important role in severe trauma and shock. We investigated the beneficial effect of cyclosporine A (CsA) on traumatic shock and its relationship to vascular reactivity improvement and mitochondrial permeability transition pore (MPTP). Materials and methodsSodium pentobarbital-anesthetized rats were used to induce traumatic hemorrhagic shock by left femur fracture and hemorrhage, the beneficial effects of CsA (1, 5, and 10 mg/kg, intravenously) on animal survival, cardiovascular function, tissue blood perfusion, and mitochondrial function of vital organs were observed. In addition, hypoxia-treated vascular smooth muscle cells from normal rats were used to investigate the relationship of this beneficial effect of CsA to Rho-associated serine/threonine kinase (ROCK) and protein kinase C. ResultsCsA prolonged the survival time and increased the 24-h survival rate of traumatic hemorrhagic shock (31%, 56%, and 56% in 1, 5, and 10 mg/kg CsA group versus 25% in lactated Ringer solution group). Five milligrams per kilogram of CsA had the best effect, which stabilized and improved the hemodynamics, increased the tissue blood flow, and improved the liver and kidney function including its mitochondrial function in shock rats. CsA had no significant influences on the production of inflammatory mediators and cardiac output after traumatic hemorrhagic shock. Further results indicated that CsA significantly improved the vascular constriction and dilation reactivity of superior mesenteric artery to norepinephrine and acetylcholine, which was antagonized by ROCK inhibitor, Y27632, but not by protein kinase C inhibitor, staurosporine. Further studies showed that CsA restored hypoxia-induced decrease of ROCK activity and inhibited the opening of MPTP in hypoxia-treated vascular smooth muscle cells. ConclusionsCsA is beneficial for the treatment of traumatic hemorrhagic shock. The mechanism is mainly through improving the vascular reactivity, stabilizing the hemodynamics, and increasing tissue perfusion. This beneficial effect of CsA is related to the inhibitory effect of CsA on MPTP opening. ROCK is an important regulator molecule in this process.

Beneficial effects of platelet-derived growth factor on hemorrhagic shock in rats and the underlying mechanisms.

Studies have shown that local application of platelet-derived growth factor (PDGF) can be used for the treatment of acute and chronic wounds. We investigated if systemic application of PDGF has a protective effect on acute hemorrhagic shock in rats in the present study. Using hemorrhagic shock rats and isolated superior mesenteric arteries, the effects of PDGF-BB on hemodynamics, animal survival, and vascular reactivity as well as the roles of the gap junction proteins connexin (Cx)40 and Cx43, PKC, and Rho kinase were observed. PDGF-BB (1–15 μg/kg iv) significantly improved the hemodynamics and blood perfusion to vital organs (liver and kidney) as well as vascular reactivity and improved the animal survival in hemorrhagic shock rats. PDGF recovering shock-induced vascular hyporeactivity depended on the integrity of the endothelium and myoendothelial gap junction. Cx43 antisense oligodeoxynucleotide abolished these improving effects of PDGF, whereas Cx40 oligodeoxynucleotide did not. Further study indicated that PDGF increased the activity of Rho kinase and PKC as well as vascular Ca2+ sensitivity, whereas it did not interfere with the intracellular Ca2+ concentration in hypoxia-treated vascular smooth muscle cells. In conclusion, systemic application of PDGF-BB may exert beneficial effects on hemorrhagic shock, which are closely related to the improvement of vascular reactivity and hemodynamics. The improvement of PDGF-BB in vascular reactivity is vascular endothelium and myoendothelial gap junction dependent. Cx43, Rho kinase, and PKC play very important role in this process. These findings suggest that PDGF may be a potential measure to treat acute clinical critical diseases such as severe trauma, shock, and sepsis.

Rho kinase acts as a downstream molecule to participate in protein kinase Cε regulation of vascular reactivity after hemorrhagic shock in rats.

Our previous study demonstrated that Rho kinase and protein kinase C (PKC) played important parts in the regulation of vascular reactivity after shock. Using superior mesenteric arteries (SMAs) from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), relationship of PKCε regulation of vascular reactivity to Rho kinase, as well as the signal transduction after shock, was investigated. The results showed that inhibition of Rho kinase with the Rho kinase-specific inhibitor Y-27632 antagonized the PKCε-specific agonist carbachol and highly expressed PKCε-induced increase of vascular reactivity in SMAs and VSMCs, whereas inhibition of PKCε with its specific inhibitory peptide did not antagonize the Rho kinase agonist (U-46619)-induced increase of vascular reactivity in SMAs and VSMCs. Activation of PKCε or highly expressed PKCε upregulated the activity of Rho kinase and the phosphorylation of PKC-dependent phosphatase inhibitor 17 (CPI-17), zipper interacting protein kinase (ZIPK), and integrin-linked kinase (ILK), whereas activation of Rho kinase increased only CPI-17 phosphorylation. The specific neutralization antibodies of ZIPK and ILK antagonized PKCε-induced increases in the activity of Rho kinase, but CPI-17 neutralization antibody did not antagonize this effect. These results suggested that Rho kinase takes part in the regulation of PKCε on vascular reactivity after shock. Rho kinase is downstream of PKCε. Protein kinase Cε activates Rho kinase via ZIPK and ILK; CPI-17 is downstream of Rho kinase.

Regulatory Effect of Rac1 on Vascular Reactivity After Hemorrhagic Shock in Rats

We used isolated superior mesenteric arteries (SMAs) from hemorrhagic-shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs; mimicking the shock state) to observe the effects of platelet-derived growth factor (PDGF; Rac1 stimulator) and NSC23766 (Rac1 antagonist) on vascular reactivity and the relationship with the Rho kinase-myosin light-chain phosphatase (MLCP) and p21-activated kinase (PAK)-myosin light-chain kinase (MLCK) signal pathway. The results indicated that the contractile responses of the SMAs and VSMCs were significantly increased at early shock or after transient hypoxia. NSC23766 (Rac1 antagonist) further increased, whereas PDGF (Rac1 stimulator) decreased the contractile responses of SMAs and VSMCs. In the late period of shock or prolonged hypoxia, the contractile responses of SMAs and VSMCs were significantly decreased; NSC23766 increased (whereas PDGF further decreased) the contractile response of the SMAs and VSMCs. Activation of Rac1 with PDGF significantly increased the activity of PAK and MLCP, and decreased Rho kinase and MLCK activity and 20-kDa myosin light-chain phosphorylation in VSMCs. The PAK inhibitor PAK-18 significantly antagonized the PDGF-induced decrease in MLCK activity, whereas the Rho kinase antagonist Y-27632 further enforced the PDGF-induced increase in MLCP activity. Simple fluid resuscitation did not improve but in combination with NSC23766 significantly improved vascular reactivity and animal survival at 24 hours. This suggested that Rac1 has an inhibitory effect on vasoreactivity after shock. Rac1-mediated regulation of vascular reactivity is mainly through activation of PAK, inhibition of MLCK and inhibition of Rho kinase, unpack the inhibition of Rho kinase to MLCP. Rac1 may be a potential target to treat vascular hyporeactivity in many critical conditions.

Effects of the Balance in Activity of RhoA and Rac1 on the Shock-Induced Biphasic Change of Vascular Reactivity in Rats

To investigate the effects of the balance in activity of RhoA and Rac1 on the shock-induced biphasic change in vascular reactivity and the related mechanism. Vascular reactivity after hemorrhagic shock shows a biphasic change. RhoA and Rac1 are the main members of a family of Rho GTPases; whether and how they participate in the regulation of the biphasic change in vascular reactivity after shock is not known. The relationship of the balance of the activity RhoA and Rac1 with the changes in vascular reactivity after hemorrhagic shock, the effects of artificially changing the balance of RhoA and Rac1 activity on vascular reactivity, and the roles of Rho kinase and p21-activated kinase (PAK) in RhoA/Rac1 regulation of vascular reactivity were observed in isolated superior mesenteric arteries (SMAs) from hemorrhagic shocked rats and hypoxia-treated vascular smooth muscle cells (VSMCs). The reactivity of SMAs and VSMCs to norepinephrine after shock or hypoxia was positively correlated with changes in the RhoA and Rac1 activity ratio. Artificially changing the balance in activity of RhoA and Rac1 significantly changed the shock-induced biphasic response of vascular reactivity. Specific antagonist of Rho kinase and PAK (Y-27632 and PAK-18) respectively abolished the effect of activation of RhoA and Rac1. Activation of RhoA significantly increased the activity of Rho kinase and inhibited the activity of Rac1 in SMAs. Rac1 activation significantly increased the activity of PAK and decreased the activity of RhoA. The balance in the activity of RhoA and Rac1 participated in the biphasic vascular reactivity seen after hemorrhagic shock. RhoA and Rac1 regulation of the vascular reactivity after shock are closely related to Rho kinase and the PAK pathway. RhoA regulates vascular reactivity mainly through activation of Rho kinase and inhibition of Rac1. Rac1 regulates vascular reactivity mainly through inhibition of RhoA and activation of PAK. These findings have potential significance for the treatment of vascular hyporesponsiveness.

Protein Kinase C Isoforms Responsible for the Regulation of Vascular Calcium Sensitivity and Their Relationship to Integrin-Linked Kinase Pathway After Hemorrhagic Shock

The aim of this study was to investigate the protein kinase C (PKC) isoforms involved in the regulation of vascular calcium sensitivity after hemorrhagic shock, the related mechanism, and the role of integrin-linked kinase (ILK). Using superior mesenteric artery from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells, the effects of PKC isoforms agonists and antagonists on vascular calcium sensitivity, their relationship with myosin light chain phosphatase (MLCP), myosin light chain (MLC20) phosphorylation, and ILK were observed. The results indicated that PKCα and ε agonists, thymelea toxin and carbachol, restored shock-induced decrease of vascular calcium sensitivity; PKCα antagonist, Gö-6976 and PKCε pseudosubstrate inhibition peptide, aggravated shock-induced calcium desensitization, whereas the agonists and antagonists of PKCδ and ζ had no effects on shock-induced calcium desensitization. PKCα and ε agonists reversed the increased MLCP activity and the decreased MLC20 phosphorylation induced by shock or hypoxia. ILK inhibitor abolished the effects of PKCα and ε agonists on vascular calcium sensitivity, MLCP activity, and MLC20 phosphorylation. These findings suggested that PKCα and ε may be the main isoforms responsible for the regulation of vascular calcium sensitivity after hemorrhagic shock, which enforce their regulation through MLCP-MLC20 pathway, and ILK may be a downstream molecule of PKCα and ε.

The mechanism by which RhoA regulates vascular reactivity after hemorrhagic shock in rats

RhoA, an important member of the Rho family of GTPases, has been implicated in many cellular processes. Our pilot study found that RhoA participated in the regulation of vascular reactivity after shock, but the mechanism was incompletely understood. Whether RhoA regulates vascular reactivity through the Rho kinase-myosin light-chain phosphatase (MLCP) and Rac1-p21-activated kinase (PAK)-myosin light-chain kinase (MLCK) signaling pathway needs investigation. With isolated, superior mesenteric arteries from hemorrhagic-shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), the effects of U-46619 (RhoA agonist) and C3 transferase (RhoA antagonist) on vascular reactivity, and the relationship to the Rho kinase-MLCP and Rac1-PAK-MLCK signaling pathways were observed. The vascular reactivity of the superior mesenteric artery and the contractile response of VSMCs to norepinephrine after prolonged hemorrhagic shock and hypoxia (2 h) were significantly decreased. Activation of RhoA with U-46619 significantly increased shock or hypoxia-induced decreased vascular reactivity. These effects of U-46619 were abolished by Y-27632 (Rho kinase inhibitor) and PDGF (Rac1 stimulator). Y-27632 had a stronger antagonistic effect than PDGF. U-46619 increased the activity of Rho kinase and MLCK, enhanced the phosphorylation of 20-kDa myosin light chain, and decreased the activity of Rac1, PAK, and MLCP in VSMCs after hypoxia. Y-27632-antagonized U-46619 induced the decrease of MLCP activity and the increase of 20-kDa myosin light chain phosphorylation. PDGF-antagonized U-46619 induced decrease of PAK activity and increase of MLCK activity. RhoA has an important role in the regulation of vascular reactivity after hemorrhagic shock. The Rho kinase-MLCP and Rac1-PAK-MLCK signal pathways participate in the regulatory process of RhoA. Rho kinase-MLCP may be the main signaling pathway by which RhoA regulates vascular reactivity.

INVOLVEMENT OF CPI-17 AND ZIPPER-INTERACTING PROTEIN KINASE IN THE REGULATION OF PROTEIN KINASE C-α, PROTEIN KINASE C-ε ON VASCULAR CALCIUM SENSITIVITY AFTER HEMORRHAGIC SHOCK

Our previous studies have found that protein kinase C (PKC) alpha and epsilon take part in the regulation of calcium sensitization and vascular reactivity after hemorrhagic shock. However, the regulatory mechanism is still unclear. The aim of this study was to investigate the role of PKC-potentiated inhibitory protein for protein phosphatase 1 of 17 kDa (CPI-17) and zipper-interacting protein kinase (ZIPK) in the regulation of PKC-alpha and PKC-epsilon on vascular calcium sensitivity after hemorrhagic shock. Using superior mesenteric artery from hemorrhagic shock rats (at 40 mmHg for 2 h), the effects of PKC-alpha and PKC-epsilon agonists on calcium sensitivity and the phosphorylation of 20-kDa myosin light chain (MLC20), and the modulating effects of CPI-17 and ZIPK in this process were observed. In hypoxia-treated vascular smooth muscle cells (VSMCs), the effects of PKC-alpha and PKC-epsilon agonists on protein expression and activity of CPI-17 and ZIPK and the modulating effects of CPI-17 and ZIPK on the regulation of PKC-alpha and PKC-epsilon on the activity of myosin light chain phosphatase (MLCP) were observed. The results indicated that PKC-alpha and PKC-epsilon agonists thymelea toxin and carbachol seemed to increase the calcium sensitivity and MLC20 phosphorylation of superior mesenteric artery after hemorrhagic shock and antagonize the increase of MLCP activity in VSMC after hypoxia. The effects of PKC-alpha and PKC-epsilon agonists were abolished by CPI-17 and ZIPK neutralizing antibody. The protein expression and activity of CPI-17 and ZIPK in VSMC were suppressed after hypoxia but increased by PKC-alpha and PKC-epsilon agonists. Immunoprecipitation experiment showed that PKC-alpha and PKC-epsilon, especially PKC-epsilon, may directly bind with ZIPK but not CPI-17 in VSMC, and ZIPK may directly bind with CPI-17. These results suggest that CPI-17 and ZIPK participate in the regulation of PKC-alpha and PKC-epsilon on vascular calcium sensitivity after hemorrhagic shock. Protein kinase C alpha and epsilon regulation of MLCP-MLC20 phosphorylation pathway in the process of VSMC contraction is mainly through ZIPK; CPI-17 may play an indirect modulating role.

MECHANISMS OF RHO KINASE REGULATION OF VASCULAR REACTIVITY FOLLOWING HEMORRHAGIC SHOCK IN RATS

Our previous research showed that Rho kinase took part in the regulation of vascular hyporeactivity after shock. The objective of the present study was to investigate its mechanism. With isolated superior mesenteric artery (SMA) from hemorrhagic shock rats, we studied the relationship of Rho kinase regulating vascular reactivity to calcium sensitivity and myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK). The vascular reactivity and calcium sensitivity of SMA were observed by measuring the contraction initiated by accumulative norepinephrine (NE) and calcium under depolarizing condition (120 mM K(+)) with an isolated organ perfusion system. Hypoxia-treated vascular smooth muscle cells (VSMCs) were used to study the effects of Rho kinase on the activity of MLCP and MLCK and the phosphorylation of 20-kDa myosin light chain (MLC(20)). Myosin light chain (20 kDa) phosphorylation of VSMC in mesenteric artery was detected by immunoprecipitation and Western blotting. The activity of MLCP and MLCK was assayed by enzymatic catalysis. The contractile response of VSMC was measured by the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin through transwell. The results indicated that the vascular reactivity and calcium sensitivity of SMA to NE and calcium following hemorrhagic shock and the contractile response of VSMC to NE following hypoxia were significantly decreased. Angiotensin II (Ang-II), the Rho kinase stimulator, significantly improved hypoxia or hemorrhagic shock-induced decrease of vascular reactivity and calcium sensitivity. These effects of Ang-II on vascular reactivity were abolished by Y-27632, the specific Rho kinase inhibitor. Calyculin A, the MLCP inhibitor, further enhanced Ang-II-induced increase of calcium sensitivity, but ML-9, the MLCK inhibitor, had no effect. Further studies showed Ang-II reversed the hypoxia-induced increase of MLCP activity and increased the hypoxia-induced decrease of MLC(20) phosphorylation in VSMC. It was suggested that Rho kinase played an important role in the regulation of vascular reactivity after hemorrhagic shock. The mechanisms may be related to its calcium sensitivity regulation. Rho kinase up-regulates calcium sensitivity of VSMC possibly through inhibiting the activity of MLCP and increasing the phosphorylation of MLC(20).

Regulatory role of sphingomyelin metabolites in hypoxia-induced vascular smooth muscle cell proliferation.

Vascular cell adaptive response to hypoxic stress includes enhanced production of sphingomyelin metabolites that regulate cell growth. Here, we examined the vascular smooth muscle (VSM) cell adaptive response to hypoxia (2 and 5% O(2)) and demonstrated that acute (</=16h) hypoxic stress significantly stimulated VSM cell growth compared to cells grown under normoxic (21% O(2)) conditions. This stimulatory effect of hypoxia on VSM cell growth was significantly inhibited by pretreatment of cells with D-erythro-N,N-dimethylsphingosine, an inhibitor of sphingosine kinase. These results suggest a mechanism by which sphingosine 1-phosphate (S-1-P), a promitogenic sphingolipid-derived second messenger, may play a key role in hypoxia-induced VSM cell growth. Supporting this, S-1-P formation was significantly increased in VSM cells subjected to hypoxia. The hypoxia-induced increase in S-1-P level correlated with the decrease in total cellular ceramide content, a sphingolipid metabolite associated with inhibition of cell growth. The activity of sphingomyelinase was also significantly inhibited in hypoxia-treated VSM cells, likely further contributing to a decrease in total intracellular content of ceramide. As a decrease in ceramide content may play a role in hypoxia-induced VSM growth, we next examined the effects of ceramide in VSM cell growth. Elevating intracellular ceramide content through exogenous (C(6)-ceramide) or endogenous (ceramidase inhibition) manipulations led to a decrease in hypoxia-induced VSM cell growth. In contrast, hypoxia-induced VSM cell growth was further enhanced by S-1-P treatment. Together, our study indicates that hypoxia-induced VSM cell growth may be modulated by sphingomyelin metabolism that results in reduction of total intracellular ceramide level with concomitant increase in S-1-P formation.