The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.     

Differential roles of tumor necrosis factor-alpha and interferon-gamma in mouse hypermetabolic and anorectic responses induced by LPS

Lipopolysaccharide (LPS)-induced effects on energy balance are characterized by alterations in energy expenditure (hypermetabolism) and food intake (anorexia). To study the role of tumour necrosis factor alpha (TNF-alpha) on some of these metabolic responses to endotoxin, we have used transgenic mice expressing soluble tumour necrosis factor receptor-1 IgG fusion protein (TNFR1-IgG) as well as TNF-alpha knockout (KO), lymphotoxin-alpha (LT-alpha) KO, and interferon-gamma receptor (IFN-gamma R) KO mice. The results from TNFR1-IgG transgenic mice suggest that the hypermetabolic and anorectic responses induced by LPS are independently regulated since, in the absence of TNF-alpha or LT-alpha, the LPS-induced hypermetabolism is almost prevented but not the anorexia. The anorectic response shows the strongest association with IFN-gamma since both IFN-gamma R KO mice and mice treated with anti-IFN-gamma antibody showed marked reduction in the LPS-induced anorexia compared to other mice. IFN-gamma R KO mice also have an attenuated thermogenic response to endotoxin. Anti-Asialo GM1 antibody treatment attenuated both the hypermetabolic and anorectic responses to LPS, to an extent comparable to that observed in IFN-gamma R KO mice. This finding suggests that natural killer cells (lymphocytic subsets) may be involved in IFN-gamma production and play an important role in the metabolic alterations induced by LPS. We also showed that the hypermetabolic response of control mice is associated with an upregulation of cytokine expression within the brain and an increase in permeability of the blood brain barrier. LPS-induced anorexia appears to involve peripheral cytokine expression.     

Lung endothelial heparan sulfates mediate cationic peptide-induced barrier dysfunction: a new role for the glycocalyx

The endothelial glycocalyx is believed to play a major role in microvascular permeability. We tested the hypothesis that specific components of the glycocalyx, via cytoskeletal-mediated signaling, actively participate in barrier regulation. With the use of polymers of arginine and lysine as a model of neutrophil-derived inflammatory cationic proteins, we determined size- and dose-dependent responses of cultured bovine lung microvascular endothelial cell permeability as assessed by transendothelial electrical resistance (TER). Polymers of arginine and lysine >11 kDa produced maximal barrier dysfunction as demonstrated by a 70% decrease in TER. Monomers of l-arginine and l-lysine did not alter barrier function, suggesting a cross-linking requirement of cell surface "receptors". To test the hypothesis that glycosaminoglycans (GAGs) are candidate receptors for this response, we used highly selective enzymes to remove specific GAGs before polyarginine (PA) treatment and examined the effect on TER. Heparinase III attenuated PA-induced barrier dysfunction by 50%, whereas heparinase I had no effect. To link changes in barrier function with structural alterations, we examined actin organization and syndecan localization after PA. PA induced actin stress fiber formation and clustering of syndecan-1 and syndecan-4, which were significantly attenuated by heparinase III. PA-induced cytoskeletal rearrangement and barrier function did not involve myosin light chain kinase (MLCK) or p38 MAPK, as ML-7, a specific MLCK inhibitor, or SB-20358, a p38 MAPK inhibitor, did not alter PA-induced barrier dysfunction. In summary, lung endothelial cell heparan sulfate proteoglycans are key participants in inflammatory cationic peptide-induced signaling that links cytoskeletal reorganization with subsequent barrier dysfunction.     

Absence of glutathione peroxidase-1 exacerbates cerebral ischemia-reperfusion injury by reducing post-ischemic microvascular perfusion

Mice deficient in the anti-oxidant enzyme glutathione peroxidase-1 (Gpx1) have a greater susceptibility to cerebral injury following a localized ischemic event. Much of the response to ischemia-reperfusion is caused by aberrant responses within the microvasculature, including inflammation, diminished endothelial barrier function (increased vascular permeability), endothelial activation, and reduced microvascular perfusion. However, the role of Gpx1 in regulating these responses has not been investigated. Wild-type and Gpx1-/- mice underwent focal cerebral ischemia via mid-cerebral artery occlusion followed by measurement of cerebral perfusion via laser Doppler and intravital microscopy. Post-ischemic brains in wild-type mice displayed significant deficit in microvascular perfusion. However, in Gpx1-/- mice, the deficit in cerebral blood flow was significantly greater than that in wild-type mice, and this was associated with significant increase in infarct size and increased vascular permeability. Ischemia-reperfusion also resulted in expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. The absence of Gpx1 was associated with marked increase in pro-MMP-9 expression as well as potentiated MMP-9 activity. Pre-treatment of Gpx1-/- mice with the anti-oxidant ebselen restored microvascular perfusion, limited the induction and activation of MMP-9, and attenuated the increases in infarct size and vascular permeability. These findings demonstrate that the anti-oxidant function of Gpx1 plays a critical role in protecting the cerebral microvasculature against ischemia-reperfusion injury by preserving microvascular perfusion and inhibiting MMP-9 expression.     

Adenosine receptor A2b on hematopoietic cells mediates LPS-induced migration of PMNs into the lung interstitium

Uncontrolled transmigration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lungs (intravascular, interstitial, alveolar) is a critical event in the early stage of acute lung injury and acute respiratory distress syndrome. Adenosine receptor A(2b) is highly expressed in the inflamed lungs and has been suggested to mediate cell trafficking. In a murine model of LPS-induced lung inflammation, we investigated the role of A(2b) on migration of PMNs into the different compartments of the lung. In A(2b)(-/-) mice, LPS-induced accumulation of PMNs was significantly higher in the interstitium, but not in the alveolar space. In addition, pulmonary clearance of PMNs was delayed in A(2b)(-/-) mice. Using chimeric mice, we identified A(2b) on hematopoietic cells as crucial for PMN migration. A(2b) did not affect the release of relevant chemokines into the alveolar space. LPS-induced microvascular permeability was under the control of A(2b) on both hematopoietic and nonhematopoietic cells. Activation of A(2b) on endothelial cells also reduced formation of LPS-induced stress fibers, highlighting its role for endothelial integrity. A specific A(2b) agonist (BAY 60-6583) was effective in decreasing PMN migration into the lung interstitium and microvascular permeability. In addition, in vitro transmigration of human PMNs through a layer of human endothelial or epithelial cells was A(2b) dependent. Activation of A(2b) on human PMNs reduced oxidative burst activity. Together, our results demonstrate anti-inflammatory effects of A(2b) on two major characteristics of acute lung injury, with a distinct role of hematopoietic A(2b) for cell trafficking and endothelial A(2b) for microvascular permeability.     

Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis,oxidative stress,and inflammation in pulmonary endothelial cells

Acute respiratory distress syndrome (ARDS) is a heterogenous syndrome characterised by diffuse alveolar damage, with an increase in lung endothelial and epithelial permeability. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses antiapoptotic and antioxidative effects in distinct situations. In the present study, the protective effects and potential molecular mechanisms of LBP against lipopolysaccharide (LPS)-induced ARDS were investigated in the mice and in the human pulmonary microvascular endothelial cells (HPMECs). The data indicated that pretreatment with LBP significantly attenuated LPS-induced lung inflammation and pulmonary oedema in vivo. LBP significantly reversed LPS-induced decrease in cell viability, increase in apoptosis and oxidative stress via inhibiting caspase-3 activation and intracellular reactive oxygen species (ROS) production in vitro. Moreover, the scratch assay verified that LBP restored the dysfunction of endothelial cells (ECs) migration induced by LPS stimulation. Furthermore, LBP also significantly suppressed LPS-induced NF-κB activation, and subsequently reversed the release of cytochrome c. These results showed the antiapoptosis and antioxidant LBP could partially protect against LPS-induced ARDS through promoting the ECs survival and scavenging ROS via inhibition of NF-κB signalling pathway. Thus, LBP could be potentially used for ARDS against pulmonary inflammation and pulmonary oedema.     

LPS induces cardiomyocyte injury through calcium-sensing receptor

The development of atherosclerosis (AS) is a multifactorial process in which elevated plasma cholesterol levels play a central role. As a new class of players involved in AS, the regulation and function of microRNAs (miR) in response to AS remain poorly understood. This study analyzed the effects of miR-1 (antagomir and mimic) on endothelial permeability and myosin light chain kinase (MLCK) expression and activity in the artery wall of apoE knock-out mice after feeding them a high-cholesterol diet. Further, we tested to determine whether that effects are involved in ERK phosphorylation. Here, we show that a high-cholesterol diet induces a significant decrease of miR-1 expression. Histopathologic examination demonstrated that miR-1 antagomir enhances endothelial permeability induced by high cholesterol and miR-1 mimic attenuated endothelial barrier dysfunction. Consistent with endothelial permeability, Western blotting, qPCR, and γ-³²P-ATP phosphate incorporation showed that MLCK expression and activity were further increased in miR-1 antagomir-treated mice and decreased in miR-1 mimic-treated mice compared with those of mice receiving control miR. Further mechanistic studies showed that high-cholesterol-induced extracellular signal regulated kinase (ERK) activation was enhanced by miR-1 antagomir and attenuated by miR-1 mimic. Collectively, those results indicate that miR-1 contributes to endothelial barrier function via mechanisms involving not only MLCK expression and activity but also ERK phosphorylation.     

Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability

Neutrophil-induced microvascular leakage is an early event in ischemic and inflammatory heart diseases. The specific signaling paradigm by which neutrophils increase microvascular permeability is not yet established. We investigated whether the small GTPase RhoA and its downstream effector Rho kinase mediate neutrophil-stimulated endothelial hyperpermeability. We assessed the effect of neutrophils on Rho activity in bovine coronary venular endothelial cells (CVEC) with a Rho-GTP pull-down assay. Permeability to FITC-albumin was evaluated using CVEC monolayers. We then tested the role of Rho kinase in the permeability response to neutrophils using two structurally distinct pharmacological inhibitors: Y-27632 and HA-1077. Furthermore, neutrophil-stimulated changes in endothelial F-actin organization were examined with fluorescence microscopy. The results show that C5a-activated neutrophils induced an increase in permeability coupled with RhoA activation in CVEC. Inhibition of Rho kinase with either Y-27632 or HA-1077 attenuated the hyperpermeability response. Rho kinase inhibition also attenuated increases in permeability stimulated by the neutrophil supernatant. In addition, activated neutrophils caused actin stress fiber formation in CVEC, which was diminished by either Y-27632 or HA-1077. These findings suggest that RhoA and Rho kinase are involved in the mediation of neutrophil-induced endothelial actin reorganization and barrier dysfunction.     

Protective role of vascular endothelial growth factor in endotoxin-induced acute lung injury in mice

Background

Vascular endothelial growth factor (VEGF), a substance that stimulates new blood vessel formation, is an important survival factor for endothelial cells. Although overexpressed VEGF in the lung induces pulmonary edema with increased lung vascular permeability, the role of VEGF in the development of acute lung injury remains to be determined.

Methods

To evaluate the role of VEGF in the pathogenesis of acute lung injury, we first evaluated the effects of exogenous VEGF and VEGF blockade using monoclonal antibody on LPS-induced lung injury in mice. Using the lung specimens, we performed TUNEL staining to detect apoptotic cells and immunostaining to evaluate the expression of apoptosis-associated molecules, including caspase-3, Bax, apoptosis inducing factor (AIF), and cytochrome C. As a parameter of endothelial permeability, we measured the albumin transferred across human pulmonary artery endothelial cell (HPAEC) monolayers cultured on porous filters with various concentrations of VEGF. The effect of VEGF on apoptosis HPAECs was also examined by TUNEL staining and active caspase-3 immunoassay.

Results

Exogenous VEGF significantly decreased LPS-induced extravascular albumin leakage and edema formation. Treatment with anti-VEGF antibody significantly enhanced lung edema formation and neutrophil emigration after intratracheal LPS administration, whereas extravascular albumin leakage was not significantly changed by VEGF blockade. In lung pathology, pretreatment with VEGF significantly decreased the numbers of TUNEL positive cells and those with positive immunostaining of the pro-apoptotic molecules examined. VEGF attenuated the increases in the permeability of the HPAEC monolayer and the apoptosis of HPAECs induced by TNF-α and LPS. In addition, VEGF significantly reduced the levels of TNF-α- and LPS-induced active caspase-3 in HPAEC lysates.

Conclusion

These results suggest that VEGF suppresses the apoptosis induced by inflammatory stimuli and functions as a protective factor against acute lung injury.     

Cigarette smoke-induced pulmonary inflammation and emphysema are attenuated in CCR6-deficient mice

Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, and is characterized by an increase in inflammatory cells in the airways and pulmonary tissue. The chemokine receptor CCR6 and its ligand MIP-3alpha/CCL20 may be involved in the recruitment of these inflammatory cells. To investigate the role of CCR6 in the pathogenesis of COPD, we analyzed the inflammatory responses of CCR6 knockout (KO) and wild-type mice upon cigarette smoke (CS) exposure. Both subacute and chronic exposure to CS induced an increase in cells of the innate and adaptive immune system in the bronchoalveolar lavage, both in CCR6 KO and wild-type mice. However, the accumulation of dendritic cells, neutrophils, and T lymphocytes, which express CCR6, was significantly attenuated in the CCR6 KO mice, compared with their wild-type littermates. In the lung tissue of CCR6 KO mice, there was an impaired increase in dendritic cells, activated CD8(+) T lymphocytes, and granulocytes. Moreover, this attenuated inflammatory response in CCR6 KO mice offered a partial protection against pulmonary emphysema, which correlated with an impaired production of MMP-12. Importantly, protein levels of MIP-3alpha/CCL20, the only chemokine ligand of the CCR6 receptor, and MCP-1/CCL2 were significantly increased upon CS exposure in wild-type, but not in CCR6 KO mice. In contrast, CCR6 deficiency had no effect on the development of airway wall remodeling upon chronic CS exposure. These results indicate that the interaction of CCR6 with its ligand MIP-3alpha contributes to the pathogenesis of CS-induced pulmonary inflammation and emphysema in this murine model of COPD.     

LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor

Lipopolysaccharide (LPS) is known to stimulate the circulation and local production of angiotensin II (Ang II). To assess whether Ang II plays a role in LPS-induced acute lung injury, rats were injected with LPS, the microvascular endothelial permeability injury was evaluated by histological changes, increased pulmonary wet/dry weight ratio, and pulmonary microvascular protein leak. Besides, increased rat pulmonary microvascular endothelial cell monolayer permeability coefficient (K(f)) was measured after treatment with LPS and/or Ang II, respectively. LPS/Ang II, treatment resulted in a significant increase in K(f). Ang II cooperates with LPS to further increase K(f). Hence, LPS increases pulmonary microvascular endothelial permeability both in vitro and in vivo. Local lung Ang II was increased in response to LPS challenge, and elevated Ang II ulteriorly exacerbates LPS-induced endothelium injury. [Sar(1),Ile(8)]Ang II, a selective block of Ang II type 1 (AT(1)) receptors, eliminated these changes significantly. Our conclusion is that the LPS-induced lung injury may be mediated by the AT(1) receptor.     

New molecular mechanisms of the unexpectedly complex role of VEGF in ulcerative colitis

The effects of VEGF on endothelial cells are mediated by different intracellular signaling cascades (e.g., Erk1/2, Akt, Src). VEGF plays a recently recognized role in ulcerative colitis (UC) pathogenesis, mostly by increasing vascular permeability and promoting the infiltration of inflammatory cells. We hypothesized that the excessive activation of signal transduction pathways, which is responsible for VEGF/VEGFR-2-mediated endothelial permeability (Src, Akt), is a new element in the pathogenesis of chronic UC. We demonstrated increased expression of pro-angiogenic growth factor VEGF and its receptor VEGFR-2 in colonic tissue during acute 6% iodoacetamide-induced UC in rats and chronic spontaneously developed UC in IL-10 knockout mice (IL-10 KO). Development of acute 6% iodoacetamide-induced UC in rats was accompanied by activation of Erk1/2 and Src kinase, while expression of total proteins Erk1/2 and Src was unchanged. During chronic colitis phosphorylation (i.e., activation) of Erk1/2 was significantly decreased in IL-10 KO mice vs. wild-type mice. Levels of total Erk1/2 proteins were unchanged, but the expression of total Src protein as well as its phosphorylated form was significantly increased in IL-10 KO vs. wild-type mice. There were no changes in total Akt proteins, while levels of activated Akt (pAkt) were slightly increased in IL-10 KO vs. wild-type mice. We conclude that VEGF/VEGFR-2-associated signal transduction pathways, that mediate increased vascular permeability (Src, Akt), might play a central role in perpetuation of chronic experimental UC.     

Anti-inflammatory therapy ameliorates leukocyte adhesion and microvascular flow abnormalities in transgenic sickle mice

In sickle cell disease, inflammatory activation of vascular endothelium and increased leukocyte-endothelium interaction may play an important role in the occurrence of vasoocclusion. In sickle mouse models, inflammatory stimuli (e.g., hypoxia-reoxygenation and cytokines) result in increased leukocyte recruitment and can initiate vasoocclusion, suggesting that anti-inflammatory therapy could be beneficial in management of this disease. We have tested the hypothesis that inhibition of endothelial activation in a transgenic mouse model by anti-inflammatory agents would lead to reduced leukocyte recruitment and improved microvascular blood flow in vivo. In transgenic sickle mice, hypoxia-reoxygenation resulted in greater endothelial oxidant production than in control mice. This exaggerated inflammatory response in transgenic mice, characterized by increased leukocyte recruitment and microvascular flow abnormalities, was significantly attenuated by antioxidants (allopurinol, SOD, and catalase). In contrast, control mice exhibited a muted response to antioxidant treatment. In addition, hypoxia-reoxygenation induced activation of NF-kappaB in transgenic sickle mice but not in control mice. In transgenic sickle mice, sulfasalazine, an inhibitor of NF-kappaB activation and endothelial activation, attenuated endothelial oxidant generation, as well as NF-kappaB activation, accompanied by a marked decrease in leukocyte adhesion and improved microvascular blood flow. Thus targeting oxidant generation and/or NF-kappaB activation may constitute promising therapeutic approaches in sickle cell disease.     

Depletion of tissue plasminogen activator attenuates lung ischemia-reperfusion injury via inhibition of neutrophil extravasation

Ischemia-reperfusion (IR) injury following lung transplantation remains a major source of early morbidity and mortality. Histologically, this inflammatory process is characterized by neutrophil infiltration and activation. We previously reported that lung IR injury was significantly attenuated in plasminogen activator inhibitor-1-deficient mice. In this study, we explored the potential role of tissue plasminogen activator (tPA) in a mouse lung IR injury model. As a result, tPA knockout (KO) mice were significantly protected from lung IR injury through several mechanisms. At the cellular level, tPA KO specifically blocked neutrophil extravasation into the interstitium, and abundant homotypic neutrophil aggregation (HNA) was detected in the lung microvasculature of tPA KO mice after IR. At the molecular level, inhibition of neutrophil extravasation was associated with reduced expression of platelet endothelial cell adhesion molecule-1 mediated through the tPA/ LDL receptor-related protein/NF-κB signaling pathway, whereas increased P-selectin triggered HNA. At the functional level, tPA KO mice incurred significantly decreased vascular permeability and improved lung function following IR. Protection from lung IR injury in tPA KO mice occurs through a fibrinolysis-independent mechanism. These results suggest that tPA could serve as an important therapeutic target for the prevention and treatment of acute IR injury after lung transplantation.     

Role of CPI-17 in the regulation of endothelial cytoskeleton

We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99-L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-alpha abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-alpha isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.     

Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent

Protein kinase C (PKC) is activated in response to various inflammatory mediators and contributes significantly to the endothelial barrier breakdown. However, the mechanisms underlying PKC-mediated permeability regulation are not well understood. We prepared microvascular myocardial endothelial cells from both wild-type (WT) and caveolin-1-deficient mice. Activation of PKC by phorbol myristate acetate (PMA) (100 nM) for 30 min induced intercellular gap formation and fragmentation of VE-cadherin immunoreactivity in WT but not in caveolin-1-deficient monolayers. To test the effect of PKC activation on VE-cadherin-mediated adhesion, we allowed VE-cadherin-coated microbeads to bind to the endothelial cell surface and probed their adhesion by laser tweezers. PMA significantly reduced bead binding to 78±6% of controls in WT endothelial cells without any effect in caveolin-1-deficient cells. In WT cells, PMA caused an 86±18% increase in FITC-dextran permeability whereas no increase in permeability was observed in caveolin-1-deficient monolayers. Inhibition of PKC by staurosporine (50 nM, 30 min) did not affect barrier functions in both WT and caveolin-1-deficient MyEnd cells. Theses data indicate that PKC activation reduces endothelial barrier functions at least in part by the reduction of VE-cadherin-mediated adhesion and demonstrate that PKC-mediated permeability regulation depends on caveolin-1.     

MicroRNA-21 Knockout Improve the Survival Rate in DSS Induced Fatal Colitis through Protecting against Inflammation and Tissue Injury

Background

MicroRNA-21 (miR-21) is overexpressed in most inflammatory diseases, but its physiological role in gut inflammation and tissue injury is poorly understood. The goal of this work is to understand the role of miR-21 in colitis and damage progression of intestine in a genetically modified murine model.

Methods

Experimental colitis was induced in miR-21 KO and wild-type (WT) mice by 3.5% dextran sulphate sodium (DSS) administration for 7 days. Disease activity index(DAI), blood parameters, intestinal permeability, histopathologic injury, cytokine and chemokine production, and epithelial cells apoptosis were examined in colons of miR-21 KO and WT mice.

Results

miR-21 was overexpressed in intestine of inflammatory bowel diseases (IBD) and acute intestinal obstruction (AIO) patients when compared with normal intestinal tissues. Likewise, miR-21 was up-regulated in colon of IL-10 KO mice when compared with control mice. WT mice rapidly lost weight and were moribund 5 days after treatment with 3.5% DSS, while miR-21 KO mice survived for at least 6 days. Elevated leukocytes and more severe histopathology were observed in WT mice when compared with miR-21 KO mice. Elevated levels of TNF-α and macrophage inflammatory protein-2(MIP-2) in colon culture supernatants from WT mice exhibited significant higher than miR-21 KO mice. Furthermore, CD3 and CD68 positive cells, intestinal permeability and apoptosis of epithelial cells were significantly increased in WT mice when compared with miR-21 KO mice. Finally, we found that miR-21 regulated the intestinal barrier function through modulating the expression of RhoB and CDC42.

Conclusion

Our results suggest that miR-21 is overexpressed in intestinal inflammation and tissue injury, while knockout of miR-21 in mice improve the survival rate in DSS-induced fatal colitis through protecting against inflammation and tissue injury. Therefore, attenuated expression of miR-21 in gut may prevent the onset or progression of inflammatory bowel disease in patients.     

Association of aorta intima permeability with myosin light chain kinase expression in hypercholesterolemic rabbits

The development of hypercholesterolemia is a multifactorial process in which elevated plasma cholesterol levels play a central role. This study analyzed the variability of the expression and activity of myosin light chain kinase (MLCK) and endothelial permeability in the artery wall of rabbits after feeding the animals with a normal or a high-cholesterol diet. Hypercholesterolemia was induced by a high-cholesterol diet for 4 weeks. Aortas were removed and analyzed for endothelial permeability and MLCK expression. Samples of the arterial media were analyzed for MLCK activity and expression. A selective MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7) were used in hypercholesterolemia rabbit (1 mg/kg body weight). The aortas of high-cholesterol diet rabbits showed an increase in MLCK expression and activity (nearly threefold compare with control) as well as endothelial permeability. ML7 inhibit MLC phosphorylation and MLCK activity (nearly twofold compare with control) and endothelial permeability stimulated by cholesterol. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and activity through which endothelial permeability is increased.