Role of miR-124 and miR-141 in the regulation of vascular reactivity and the relationship to RhoA and Rac1 after hemorrhage and hypoxia.

Recent studies show that hypoxia can alter expression levels of microRNAs (miRNAs). Whether hypoxia or hemorrhage-induced vascular hyporeactivity is related to miRNAs and the underlying mechanisms of this process is not clear. Using hypoxia-treated superior mesenteric arteries (SMAs) and vascular smooth muscle cells (VSMCs) of rats that underwent hemorrhage, we observed the regulatory effects of miR-124/miR-141 on vascular reactivity, the relationship of these miRNAs to RhoA and Rac1, and the mutual regulation of miR-124 and miR-141. The contractile responses of SMAs and VSMCs showed an increase in early stages and a decrease in late stages of hypoxia and hemorrhage. Forty-five miRNAs appeared to have been significantly changed in SMAs after hypoxia, and miR-124 and miR-141 underwent the most change. Overexpressed miR-124 or miR-141 and their antisenses appeared to alter both vascular reactivity and expression of the proteins RhoA and Rac1 after hypoxia. miR-124 inhibited Rac1 by acting at the Rac1 mRNA 3'-untranslated region (UTR), but it led to an increase in RhoA by inhibiting miR-141. miR-141 inhibited RhoA by acting at the RhoA mRNA 3'-UTR, but it led to an increase in Rac1 by inhibiting miR-124. Further study found that miR-124 inhibited miR-141 via transcription factor early growth response gene-1 (Egr-1), whereas miR-141 inhibited miR-124 via transcription of nuclear factor erythroid 2-related factor 2 (Nrf-2). These results suggest that miR-124 and miR-141 participate in the regulation of vascular reactivity after hypoxia and hemorrhage by regulating expression of the RhoA and Rac1 proteins, and in doing so, miR-124 and miR-141 are mutually regulated. These findings provide potential targets for restoring vascular function as part of the treatment protocol for hemorrhagic shock and some other critical illness.

4-Phenylbutyric Acid Reveals Good Beneficial Effects on Vital Organ Function via Anti-Endoplasmic Reticulum Stress in Septic Rats.

OBJECTIVES: Sepsis and septic shock are the common complications in ICUs. Vital organ function disorder contributes a critical role in high mortality after severe sepsis or septic shock, in which endoplasmic reticulum stress plays an important role. Whether anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to sepsis and the underlying mechanisms are not known. DESIGN: Laboratory investigation. SETTING: State Key Laboratory of Trauma, Burns and Combined Injury. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Using cecal ligation and puncture-induced septic shock rats, lipopolysaccharide-treated vascular smooth muscle cells, and cardiomyocytes, effects of 4-phenylbutyric acid on vital organ function and the relationship with endoplasmic reticulum stress and endoplasmic reticulum stress-mediated inflammation, apoptosis, and oxidative stress were observed. MEASUREMENTS AND MAIN RESULTS: Conventional treatment, including fluid resuscitation, vasopressin, and antibiotic, only slightly improved the hemodynamic variable, such as mean arterial blood pressure and cardiac output, and slightly improved the vital organ function and the animal survival of septic shock rats. Supplementation of 4-phenylbutyric acid (5 mg/kg; anti-endoplasmic reticulum stress), especially administered at early stage, significantly improved the hemodynamic variables, vital organ function, such as liver, renal, and intestinal barrier function, and animal survival in septic shock rats. 4-Phenylbutyric acid application inhibited the endoplasmic reticulum stress and endoplasmic reticulum stress-related proteins, such as CCAAT/enhancer-binding protein homologous protein in vital organs, such as heart and superior mesenteric artery after severe sepsis. Further studies showed that 4-phenylbutyric acid inhibited endoplasmic reticulum stress-mediated cytokine release, apoptosis, and oxidative stress via inhibition of nuclear factor-κB, caspase-3 and caspase-9, and increasing glutathione peroxidase and superoxide dismutase expression, respectively. CONCLUSIONS: Anti-endoplasmic reticulum stress with 4-phenylbutyric acid is beneficial to septic shock. This beneficial effect of 4-phenylbutyric acid is closely related to the inhibition of endoplasmic reticulum stress-mediated oxidative stress, apoptosis, and cytokine release. This finding provides a potential therapeutic measure for clinical critical conditions, such as severe sepsis.

Age and sex differences in vascular responsiveness in healthy and trauma patients: contribution of estrogen receptor-mediated Rho kinase and PKC pathways.

Several medical conditions exhibit age- and sex-based differences. Whether or not traumatic shock exhibits such differences with regard to vascular responsiveness is not clear. In a cohort of 177 healthy subjects and 842 trauma patients (21-82 years) as well as different ages (4, 8, 10, 14, 18, and 24 wk; 1 and 1.5 years) and sexes of Sprague-Dawley normal and traumatic shock rats, the age- and sex-based differences of vascular responsiveness and the underlying mechanisms were investigated. Middle-aged and young women as well as female rats of reproductive age had higher vascular responsiveness in the normal condition and a lower decrease in vascular responsiveness after traumatic shock than older men and male rats of identical age. Exogenous supplementation of 17ß-estrdiol increased vascular reactivity in both male and femal rats of 8-24 wk and preserved vascular responsiveness in rats following traumatic shock. No effect was observed in rats 1 to 1.5 years. These protective effects of estrogen were closely related to G protein-coupled receptor (GPR)30, estrogen receptor-mediated Rho kinase, and PKC pathway activation. Vascular responsiveness exhibits age- and sex-based differences in healthy subjects and trauma patients. Estrogen and its receptor (GPR30) mediated activation of Rho kinase and PKC using genomic and nongenomic mechanisms to elicit protective effects in vascular responsiveness. This finding is important for the personalized treatment for several age- and sex-related diseases involving estrogen.

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 (1­15 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.

Role of non-MLC20 phosphorylation pathway in the regulation of vascular reactivity during shock.

BACKGROUND: Studies have shown that shock-induced vascular hyporeactivity is associated with the decrease in 20-kDa myosin light chain (MLC20) phosphorylation. Whether and how a non-MLC20 phosphorylation pathway participates in the regulation of vascular reactivity after shock is not known. METHODS: With superior mesentery artery (SMA) obtained from rats in hemorrhagic shock and hypoxia-treated SMA, the regulatory effect of platelet-derived growth factor (PDGF) on vascular reactivity and the roles of caldesmon, 27-kDa heat shock protein (HSP27), extracellular signal-regulated protein kinase (Erk), and p38 mitogen-activated protein kinase (MAPK), the main molecules that are involved in the non-MLC20 phosphorylation pathway of the regulation of smooth-muscle contraction, were investigated. RESULTS: PDGF (40-100 ng/mL) increased the vascular reactivity after shock in a dose-dependent manner, whereas it did not increase the MLC20 phosphorylation in a dose-dependent manner. PDGF with concentration more than 60 ng/mL did not further increase the MLC20 phosphorylation, whereas upregulated the phosphorylation of HSP27, Erk, and p38MAPK, and the activity of myosin adenosine triphosphatase in SMAs, and downregulated the phosphorylation of caldesmon. p38MAPK antagonist, SB203580, not only antagonized PDGF-induced increase in the phosphorylation of HSP27, but also antagonized PDGF-induced decrease in the phosphorylation of caldesmon, whereas Erk antagonist, PD98059, only antagonized PDGF-induced decrease in the phosphorylation of caldesmon. CONCLUSIONS: These findings suggested that a non-MLC20 phosphorylation pathway participated in the regulation of vascular reactivity after shock. Caldesmon- and HSP27-mediated change in myosin adenosine triphosphatase activity and Erk and p38MAPK played an important role in this process. These findings may provide some potential targets for the treatment of vascular hyporeactivity after shock.

Pericytes protect rats and mice from sepsis-induced injuries by maintaining vascular reactivity and barrier function: implication of miRNAs and microvesicles.

BACKGROUND: Vascular hyporeactivity and leakage are key pathophysiologic features that produce multi-organ damage upon sepsis. We hypothesized that pericytes, a group of pluripotent cells that maintain vascular integrity and tension, are protective against sepsis via regulating vascular reactivity and permeability. METHODS: We conducted a series of in vivo experiments using wild-type (WT), platelet-derived growth factor receptor beta (PDGFR-ß)-Cre + mT/mG transgenic mice and Tie2-Cre + Cx43flox/flox mice to examine the relative contribution of pericytes in sepsis, either induced by cecal ligation and puncture (CLP) or lipopolysaccharide (LPS) challenge. In a separate set of experiments with Sprague-Dawley (SD) rats, pericytes were depleted using CP-673451, a selective PDGFR-ß inhibitor, at a dosage of 40 mg/(kg·d) for 7 consecutive days. Cultured pericytes, vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were used for mechanistic investigations. The effects of pericytes and pericyte-derived microvesicles (PCMVs) and candidate miRNAs on vascular reactivity and barrier function were also examined. RESULTS: CLP and LPS induced severe injury/loss of pericytes, vascular hyporeactivity and leakage (P < 0.05). Transplantation with exogenous pericytes protected vascular reactivity and barrier function via microvessel colonization (P < 0.05). Cx43 knockout in either pericytes or VECs reduced pericyte colonization in microvessels (P < 0.05). Additionally, PCMVs transferred miR-145 and miR-132 to VSMCs and VECs, respectively, exerting a protective effect on vascular reactivity and barrier function after sepsis (P < 0.05). miR-145 primarily improved the contractile response of VSMCs by activating the sphingosine kinase 2 (Sphk2)/sphingosine-1-phosphate receptor (S1PR)1/phosphorylation of myosin light chain 20 pathway, whereas miR-132 effectively improved the barrier function of VECs by activating the Sphk2/S1PR2/zonula occludens-1 and vascular endothelial-cadherin pathways. CONCLUSIONS: Pericytes are protective against sepsis through regulating vascular reactivity and barrier function. Possible mechanisms include both direct colonization of microvasculature and secretion of PCMVs.

Enhanced hemostatic performance of tranexamic acid-loaded chitosan/alginate composite microparticles.

Novel microparticles based on chitosan and sodium alginate were prepared using emulsification and cross-linking technologies. The spherical microparticles had a porous surface and a diameter of 2 ~ 40 µm. In simulated body fluid, these microparticles quickly swelled but gradually degraded. The results of the MTT assay revealed that a slight inhibition of cell proliferation was observed on day 2 and then gradually decreased afterward. No cell morphology changes were observed. By loading tranexamic acid, the hemostatic performance of the microparticles was obviously improved. Using fast-acting styptic powder (Flashclot) as the control, the hemostatic efficiency was investigated in rabbits using a liver transection bleeding model. It was found that both Flashclot and the microparticles achieved hemostasis in 3.07 ± 0.84 min and 2.48 ± 0.88 min, respectively; however, the tranexamic acid-loaded microparticles stopped the bleeding in 1.90 ± 0.75 min (P < 0.05). Additionally, Flashclot resulted in heat injury to the experimental livers, while the microparticles did not. Thus, with their biodegradability, safety, and superior hemostatic efficiency, tranexamic acid-loaded microparticles might be a promising new powdered hemostatic agent with a wide range of potential applications.

[Study on the haemostatic efficiency of composite bio-particles].

A porous composite particle (CP) was fabricated by the methods of emulsification and cross-link based on chitosan, alginate and collagen protein, and the tranexamic acid-loaded composite particles (TACP) was prepared by immersing the composite particle into the solution of tranexamic acid and by freeze drying. In the hepatic and splenic hemorrhage model of rabbits, CP and TACP were randomly used as haemostatic agents, and the Suxiaozhixuefen (Flashclot) was used as control. The corresponding hemostatic time and bleeding amount were observed respectively. The hemostatic time of CP and Flashclot were (2.48 +/- 0.88) min and (3.07 +/- 0.84) min, respectively, no significant difference was observed. However, the hemostatic time of TACP was (1.90 +/- 0.75) min, which was significantly shorter than that of CP and Flashclot (P < 0.05). In the splenic bleeding model of rabbits, similar results were obtained with these three kinds of hemostatics. These results indicated that the CP based on chitosan, alginate and collagen protein displayed similar hemostatic efficiency to Flashclot. However, the TACP might be one of promising haemostatic powders due to its more excellent hemostatic efficiency.

Role of resveratrol in protecting vasodilatation function in septic shock rats and its mechanism.

BACKGROUND: Vascular dysfunction is a major cause of sepsis-induced multiple-organ dysfunction. Resveratrol is a polyphenol compound with extensive pharmacological effects including anti-inflammation. The aim of this study was to determine the role and mechanism of resveratrol in protecting vascular function following sepsis. METHODS: The cecal ligation and puncture method was used to establish a septic shock rat model. Resveratrol (5 mg/kg and 10 mg/kg) was administered intravenously immediately and at 12 hours after cecal ligation and puncture, respectively. The effects of resveratrol on vasodilatation function, blood flow velocity, hemodynamics, and vital organ function and its relationship to Rac-1 and HIF-1α were observed. RESULTS: Vascular relaxation reactivity and blood flow velocity were significantly decreased after septic shock, both were significantly improved by resveratrol 5 mg/kg and 10 mg/kg, and the effect of 10 mg/kg was greater. The relaxation reactivity of the superior mesenteric artery to acetylcholine (Ach) was increased by 43.2%. The blood flow velocity of mesenteric arterioles and venules was increased by 47.1% and 51%, respectively, after resveratrol (10 mg/kg) administration compared with the septic shock group. The hemodynamics and both liver and kidney blood flow were significantly decreased after septic shock, which were significantly improved them by resveratrol, which enhanced the vascular relaxation reactivity in septic shock rats. The 72-hour survival rate of septic shock rats in the resveratrol group (62.5%) was significantly higher than that in the septic shock group (6.3%). Resveratrol significantly upregulated the expression of endothelial nitric oxide synthase (eNOS) and downregulated the expression of inducible NOS, Rac-1, and HIF-1α. Inhibitors of Rac-1 and HIF-1α significantly improved the expression of eNOS, and inhibition of eNOS (L-NAME, 5 mg/kg) antagonized the resveratrol-induced improvement in vascular relaxation reactivity and survival. CONCLUSION: Resveratrol was beneficial for vasodilatation function in rats with septic shock, which is the major contribution to resveratrol improving hemodynamics and organ perfusion. The mechanism involved resveratrol upregulating the expression of eNOS by inhibiting Rac-1 and HIF-1α.

Beneficial Effect of Intermedin 1-53 in Septic Shock Rats: Contributions of Rho Kinase and BKCA Pathway-Mediated Improvement in Cardiac Function.

OBJECTIVE: Intermedin (IMD) is a calcitonin gene-related peptide shown to have a protective effect on myocardial function in ischemia-reperfusion injury. Whether IMD has beneficial effect in severe sepsis and septic shock (and its underlying mechanisms) is not known. METHODS: We induced septic shock using cecal ligation and puncture (CLP). We focused on the potential beneficial effect of IMD1-53 on cardiac papillary muscle and cardiomyocytes against septic shock and its relationship with the protection of cardiac function. RESULTS: Early (immediately after CLP) and late (12 h after CLP) administration of IMD1-53 (0.5 µg/kg) improved animal survival significantly, increased cardiac contractility and function, and improved tissue perfusion and oxygen delivery. The effect of early administration of IMD1-53 was better than that of late administration. The Rho kinase/TnI and BKCa pathways participated in the protective effect of IMD1-53 on cardiac function in septic rats. An inhibitor of Rho kinase (Y-27632) or a BKCa opener (NS1619) abolished the protective effect of IMD1-53 on cardiac function. IMD1-53 increased expression of Rho kinase in cardiac muscle and inhibited TnI phosphorylation. IMD1-53 inhibited currents in BKCa channels and intracellular calcium concentration in cardiomyocytes. CONCLUSIONS: IMD1-53 is beneficial against severe sepsis/septic shock. IMD1-53 can improve cardiac contractility and cardiac function significantly, and then improve tissue perfusion and oxygen delivery. Rho kinase and the BKCa pathways have important roles in these effects. These findings provide a new treatment strategy for severe sepsis with cardiac dysfunction.

Hypotensive resuscitation in combination with arginine vasopressin may prolong the hypotensive resuscitation time in uncontrolled hemorrhagic shock rats.

BACKGROUND: The optimal resuscitation strategy for traumatic hemorrhagic shock is not completely determined. The objective of the present study was to investigate whether hypotensive resuscitation in combination with arginine vasopressin (AVP) can prolong the hypotensive resuscitation time by minimizing blood loss and stabilizing hemodynamics for uncontrolled hemorrhagic shock. METHODS: With an established rat model of uncontrolled hemorrhagic shock, we compared the beneficial effects of hypotensive resuscitation in combination with AVP to maintain blood pressure at 50 mm Hg for 3 hours to hypotensive resuscitation alone on animal survival, blood loss, and vital organ functions. RESULTS: Hypotensive resuscitation in combination with AVP maintenance for 3 hours significantly reduced total blood loss and fluid requirement during hypotensive resuscitation period and significantly improved the survival of shock rats as compared with hypotensive resuscitation alone. Among the four concentrations of AVP, 5 × 10 U/mL had the best effect: it significantly improved hemodynamics and increased cardiac function, oxygen delivery, as well as hepatic blood flow and hepatic function in the shock rats. However, renal blood flow in the hypotensive resuscitation + AVP group was lower than that in the hypotensive resuscitation alone group. CONCLUSION: Hypotensive resuscitation in combination with early application of AVP could prolong the tolerance time of hypotensive resuscitation and "buy" longer safe prehospital transport time by reducing blood loss and stabilizing hemodynamics. This strategy may be a promising strategy for the early management of trauma patients with active bleeding.

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.