Diet-induced obesity impairs endothelium-derived hyperpolarization via altered potassium channel signaling mechanisms

Background

The vascular endothelium plays a critical role in the control of blood flow. Altered endothelium-mediated vasodilator and vasoconstrictor mechanisms underlie key aspects of cardiovascular disease, including those in obesity. Whilst the mechanism of nitric oxide (NO)-mediated vasodilation has been extensively studied in obesity, little is known about the impact of obesity on vasodilation to the endothelium-derived hyperpolarization (EDH) mechanism; which predominates in smaller resistance vessels and is characterized in this study.

Methodology/Principal Findings

Membrane potential, vessel diameter and luminal pressure were recorded in 4^th order mesenteric arteries with pressure-induced myogenic tone, in control and diet-induced obese rats. Obesity, reflecting that of human dietary etiology, was induced with a cafeteria-style diet (∼30 kJ, fat) over 16–20 weeks. Age and sexed matched controls received standard chow (∼12 kJ, fat). Channel protein distribution, expression and vessel morphology were determined using immunohistochemistry, Western blotting and ultrastructural techniques. In control and obese rat vessels, acetylcholine-mediated EDH was abolished by small and intermediate conductance calcium-activated potassium channel (SK_Ca/IK_Ca) inhibition; with such activity being impaired in obesity. SK_Ca-IK_Ca activation with cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) and 1-ethyl-2-benzimidazolinone (1-EBIO), respectively, hyperpolarized and relaxed vessels from control and obese rats. IK_Ca-mediated EDH contribution was increased in obesity, and associated with altered IK_Ca distribution and elevated expression. In contrast, the SK_Ca-dependent-EDH component was reduced in obesity. Inward-rectifying potassium channel (K_ir) and Na⁺/K⁺-ATPase inhibition by barium/ouabain, respectively, attenuated and abolished EDH in arteries from control and obese rats, respectively; reflecting differential K_ir expression and distribution. Although changes in medial properties occurred, obesity had no effect on myoendothelial gap junction density.

Conclusion/Significance

In obese rats, vasodilation to EDH is impaired due to changes in the underlying potassium channel signaling mechanisms. Whilst myoendothelial gap junction density is unchanged in arteries of obese compared to control, increased IK_Ca and Na⁺/K⁺-ATPase, and decreased K_ir underlie changes in the EDH mechanism.     

Benzalkonium Chloride Suppresses Rabbit Corneal Endothelium Intercellular Gap Junction Communication

Purpose

Gap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.

Methods

Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco''s Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity.

Results

Topical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.

Conclusions

The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.     

Probiotics modulate intestinal expression of nuclear receptor and provide counter-regulatory signals to inflammation-driven adipose tissue activation

Background

Adipocytes from mesenteric white adipose tissue amplify the inflammatory response and participate in inflammation-driven immune dysfunction in Crohn''s disease by releasing proinflammatory mediators. Peroxisome proliferator-activated receptors (PPAR)-α and -γ, pregnane x receptor (PXR), farnesoid x receptor (FXR) and liver x-receptor (LXR) are ligand-activated nuclear receptor that provide counter-regulatory signals to dysregulated immunity and modulates adipose tissue.

Aims

To investigate the expression and function of nuclear receptors in intestinal and adipose tissues in a rodent model of colitis and mesenteric fat from Crohn''s patients and to investigate their modulation by probiotics.

Methods

Colitis was induced by TNBS administration. Mice were administered vehicle or VSL#3, daily for 10 days. Abdominal fat explants obtained at surgery from five Crohn''s disease patients and five patients with colon cancer were cultured with VSL#3 medium.

Results

Probiotic administration attenuated development of signs and symptoms of colitis, reduced colonic expression of TNFα, IL-6 and IFNγ and reserved colonic downregulation of PPARγ, PXR and FXR caused by TNBS. Mesenteric fat depots isolated from TNBS-treated animals had increased expression of inflammatory mediators along with PPARγ, FXR, leptin and adiponectin. These changes were prevented by VSL#3. Creeping fat and mesenteric adipose tissue from Crohn''s patients showed a differential expression of PPARγ and FXR with both tissue expressing high levels of leptin. Exposure of these tissues to VSL#3 medium abrogates leptin release.

Conclusions

Mesenteric adipose tissue from rodent colitis and Crohn''s disease is metabolically active and shows inflammation-driven regulation of PPARγ, FXR and leptin. Probiotics correct the inflammation-driven metabolic dysfunction.     

TGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells

Background

Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E₂) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.

Methodology/Principal Findings

Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E₂ as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E₂ secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E₂ and TGF-β1, and E₂ treatment in turn restored the inhibition of TGF-β1 on GJIC.

Conclusions

Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E₂ secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.     

Inhibition of the K+ Channel KCa3.1 Reduces TGF-β1-Induced Premature Senescence,Myofibroblast Phenotype Transition and Proliferation of Mesangial Cells

Objective

K_Ca3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K_Ca3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

Methods & Materials

Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml) and TGF-β1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K_Ca3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K_Ca3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05.

Results

K_ca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G₀-G₁ phase and the expression of K_ca3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01).

Conclusion

Targeted disruption of K_Ca3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.     

Statins and Selective Inhibition of Rho Kinase Protect Small Conductance Calcium-Activated Potassium Channel Function (KCa2.3) in Cerebral Arteries

Background

In rat middle cerebral and mesenteric arteries the K_Ca2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, K_Ca2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain K_Ca2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA).

Methods

MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence.

Results

Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of K_Ca2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. K_Ca2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation.

Conclusions

Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K_Ca2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of K_Ca2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.     

Noninvasive Ultrasound Molecular Imaging of the Effect of Statins on Endothelial Inflammatory Phenotype in Early Atherosclerosis

Background/Objectives

Inflammatory changes on the endothelium are responsible for leukocyte recruitment to plaques in atherosclerosis. Noninvasive assessment of treatment-effects on endothelial inflammation may be of use for managing medical therapy and developing novel therapies. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) with contrast enhanced ultrasound (CEU) could assess treatment effects on endothelial phenotype in early atherosclerosis.

Methods

Mice with atherosclerosis produced by gene deletion of the LDL-receptor and Apobec-1-editing protein were studied. At 12 weeks of age, mice received 8 weeks of regular chow or atorvastatin-enriched chow (10 mg/kg/day). At 20 weeks, CEU molecular imaging for aortic endothelial VCAM-1 expression was performed with VCAM-1-targeted (MB_VCAM) and control microbubbles (MB_Ctr). Aortic wall thickness was assessed with high frequency ultrasound. Histology, immunohistology and Western blot were used to assess plaque burden and VCAM-1 expression.

Results

Plaque burden was reduced on histology, and VCAM-1 was reduced on Western blot by atorvastatin, which corresponded to less endothelial expression of VCAM-1 on immunohistology. High frequency ultrasound did not detect differences in aortic wall thickness between groups. In contrast, CEU molecular imaging demonstrated selective signal enhancement for MB_VCAM in non-treated animals (MB_VCAM 2±0.3 vs MB_Ctr 0.7±0.2, p<0.01), but not in statin-treated animals (MB_VCAM 0.8±0.2 vs MB_Ctr 1.0±0.2, p = ns; p<0.01 for the effect of statin on MB_VCAM signal).

Conclusions

Non-invasive CEU molecular imaging detects the effects of anti-inflammatory treatment on endothelial inflammation in early atherosclerosis. This easily accessible, low-cost technique may be useful in assessing treatment effects in preclinical research and in patients.     

NOX4 Mediates BMP4-Induced Upregulation of TRPC1 and 6 Protein Expressions in Distal Pulmonary Arterial Smooth Muscle Cells

Rationale

Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4) mediated, elevated expression of canonical transient receptor potential (TRPC) largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs). In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.

Methods

We employed recombinant human BMP4 (rhBMP4) to determine the effects of BMP4 on NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4) and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca²⁺ determination in PASMCs.

Results

In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13) %, and the mean ROS level was (123.65±1.62) % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca²⁺]_i and SOCE. Furthermore, compared with control group (0.21±0.001), the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001) (P<0.01). However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H₂O₂ 100 µM, 24 h) rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca²⁺]_i) and store-operated calcium entry (SOCE), suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.

Conclusion

These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.     

Immunoregulatory mechanisms underlying prevention of colitis-associated colorectal cancer by probiotic bacteria

Background

Inflammatory bowel disease (IBD) increases the risk of colorectal cancer. Probiotic bacteria produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-carcinogenic effects. This study aimed to investigate the cellular and molecular mechanisms underlying the efficacy of probiotic bacteria in mouse models of cancer.

Methodology/Principal Findings

The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in mouse models of inflammation-driven colorectal cancer. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen and colonic lamina propria lymphocytes (LPL) were phenotypically and functionally characterized. Mice treated with CLA or VSL#3 recovered faster from the acute inflammatory phase of disease and had lower disease severity in the chronic, tumor-bearing phase of disease. Adenoma and adenocarcinoma formation was also diminished by both treatments. VSL#3 increased the mRNA expression of TNF-α, angiostatin and PPAR γ whereas CLA decreased COX-2 levels. Moreover, VSL#3-treated mice had increased IL-17 expression in MLN CD4+ T cells and accumulation of Treg LPL and memory CD4+ T cells.

Conclusions/Significance

Both CLA and VSL#3 suppressed colon carcinogenesis, although VSL#3 showed greater anti-carcinogenic and anti-inflammatory activities than CLA. Mechanistically, CLA modulated expression of COX-2 levels in the colonic mucosa, whereas VSL#3 targeted regulatory mucosal CD4+ T cell responses.     

Increased constitutive αSMA and Smad2/3 expression in idiopathic pulmonary fibrosis myofibroblasts is KCa3.1-dependent

Background

Idiopathic pulmonary fibrosis is a common and invariably fatal disease with limited therapeutic options. Ca²⁺-activated K_Ca3.1 potassium channels play a key role in promoting TGFβ1 and bFGF-dependent profibrotic responses in human lung myofibroblasts (HLMFs). We hypothesised that K_Ca3.1 channel-dependent cell processes regulate HLMF αSMA expression via Smad2/3 signalling pathways.

Methods

In this study we have compared the phenotype of HLMFs derived from non-fibrotic healthy control lungs (NFC) with cells derived from IPF lungs. HLMFs grown in vitro were examined for αSMA expression by immunofluorescence (IF), RT-PCR and flow cytommetry. Basal Smad2/3 signalling was examined by RT-PCR, western blot and immunofluorescence. Two specific and distinct K_Ca3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on HLMF differentiation and the Smad2/3 signalling pathways.

Results

IPF-derived HLMFs demonstrated increased constitutive expression of both α-smooth muscle actin (αSMA) and actin stress fibres, indicative of greater myofibroblast differentiation. This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation. The increased Smad2/3 nuclear localisation was inhibited by removing extracellular Ca²⁺ or blocking K_Ca3.1 ion channels with selective K_Ca3.1 blockers (TRAM-34, ICA-17043). This was accompanied by de-differentiation of IPF-derived HLMFs towards a quiescent fibroblast phenotype as demonstrated by reduced αSMA expression and reduced actin stress fibre formation.

Conclusions

Taken together, these data suggest that Ca²⁺- and K_Ca3.1-dependent processes facilitate “constitutive” Smad2/3 signalling in IPF-derived fibroblasts, and thus promote fibroblast to myofibroblast differentiation. Importantly, inhibiting K_Ca3.1 channels reverses this process. Targeting K_Ca3.1 may therefore provide a novel and effective approach for the treatment of IPF and there is the potential for the rapid translation of K_Ca3.1-directed therapy to the clinic.     

HIF-1α Inhibition Reduces Nasal Inflammation in a Murine Allergic Rhinitis Model

Background

Hypoxia-inducible factor 1α (HIF-1α) is an important regulator of immune and inflammatory responses. We hypothesized that nasal allergic inflammation is attenuated by HIF-1α inhibition and strengthened by HIF-1α stabilization.

Objective

To elucidate the role of HIF-1α in a murine model of allergic rhinitis (AR).

Methods

Mice were pretreated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2) or the HIF-1α inducer cobalt chloride (CoCl₂) in an established AR murine model using ovalbumin (OVA)-sensitized BALB/c mice. HIF-1α and vascular endothelial growth factor (VEGF) expression in nasal mucosa was measured and multiple parameters of allergic responses were evaluated.

Results

HIF-1α and VEGF levels were locally up-regulated in nasal mucosa during AR. Inflammatory responses to OVA challenge, including nasal symptoms, inflammatory cell infiltration, eosinophil recruitment, up-regulation of T-helper type 2 cytokines in nasal lavage fluid, and serum OVA-specific IgE levels were present in the OVA-challenged mice. 2ME2 effectively inhibited HIF-1α and VEGF expression and attenuated the inflammatory responses. Stabilization of HIF-1α by CoCl₂ facilitated nasal allergic inflammation. HIF-1α protein levels in nasal airways correlated with the severity of AR in mice.

Conclusions

HIF-1α is intimately involved in the pathogenesis of nasal allergies, and the inhibition of HIF-1α may be useful as a novel therapeutic approach for AR.     

VSL#3 Resets Insulin Signaling and Protects against NASH and Atherosclerosis in a Model of Genetic Dyslipidemia and Intestinal Inflammation

Background

Signals generated by the inflammed intestine are thought to contribute to metabolic derangement. The intestinal microbiota contributes to instructing the immune system beyond the intestinal wall and its modulation is a potential target for treating systemic disorders.

Aims

To investigate the pathogenetic role of low grade intestinal inflammation in the development of steatohepatitis and atherosclerosis in a model of genetic dyslipidemia and to test the therapeutic potential of a probiotics intervention in protecting against development of these disorders.

Results

ApoE^−/− mice were randomized to receive vehicle or VSL#3, a mixture of eight probiotics, at the dose of 20×10⁹ colony-forming units/kg/day for three months alone or in combination with 0.2% of dextran sulfate sodium (DSS) in drinking water. Administering DSS to ApoE^−/− mice failed to induce signs and symptoms of colitis but increased intestinal permeability to dextran FITC and, while had no effect on serum lipids, increased the blood levels of markers of liver injury and insulin resistance. DSS administration associated with low level inflammation of intestinal and mesenteric adipose tissues, caused liver histopathology features of steatohepatitis and severe atherosclerotic lesions in the aorta. These changes were prevented by VSL#3 intervention. Specifically, VSL#3 reversed insulin resistance, prevented development of histologic features of mesenteric adipose tissue inflammation, steatohepatitis and reduced the extent of aortic plaques. Conditioned media obtained from cultured probiotics caused the direct transactivation of peroxisome proliferator-activated receptor-γ, Farnesoid-X-receptors and vitamin D receptor.

Conclusions

Low grade intestinal inflammation drives a transition from steatosis to steatohepatitis and worsens the severity of atherosclerosis in a genetic model of dyslipidemia. VSL#3 intervention modulates the expression of nuclear receptors, corrects for insulin resistance in liver and adipose tissues and protects against development of steatohepatitis and atherosclerosis.     

Cardiac Overexpression of Constitutively Active Galpha q Causes Angiotensin II Type1 Receptor Activation,Leading to Progressive Heart Failure and Ventricular Arrhythmias in Transgenic Mice

Background

Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gα_q-TG) exhibt progressive heart failure and ventricular arrhythmias after the initiating stimulus of transfected constitutively active Gα_q becomes undetectable. However, the mechanisms are still unknown. We examined the effects of chronic administration of olmesartan on heart failure and ventricular arrhythmia in Gα_q-TG mice.

Methodology/Principal Findings

Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gα_q-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan administration prevented the severe reduction of left ventricular fractional shortening, and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gα_q-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gα_q-TG but in none of 10 olmesartan-treated Gα_q-TG. The collected QT interval and monophasic action potential duration in the left ventricle were significantly shorter in olmesartan-treated Gα_q-TG than in vehicle-treated Gα_q-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased and olmesartan significantly decreased the expression of these genes in Gα_q-TG mouse ventricles. The expression of canonical transient receptor potential (TRPC) 3 and 6 channel and angiotensin converting enzyme (ACE) proteins but not angiotensin II type 1 (AT₁) receptor was increased in Gα_q-TG ventricles compared with NTG mouse ventricles. Olmesartan significantly decreased TRPC6 and tended to decrease ACE expressions in Gα_q-TG. Moreover, it increased AT₁ receptor in Gα_q-TG.

Conclusions/Significance

These findings suggest that angiotensin II type 1 receptor activation plays an important role in the development of heart failure and ventricular arrhythmia in Gα_q-TG mouse model of heart failure.     

Probiotic Bacteria Regulate Intestinal Epithelial Permeability in Experimental Ileitis by a TNF-Dependent Mechanism

Background

We previously showed that the probiotic mixture, VSL#3, prevents the onset of ileitis in SAMP/YitFc (SAMP) mice, and this effect was associated with stimulation of epithelial-derived TNF. The aim of this study was to determine the mechanism(s) of VSL#3-mediated protection on epithelial barrier function and to further investigate the “paradoxical” effects of TNF in preventing SAMP ileitis.

Methods

Permeability was evaluated in SAMP mice prior to the onset of inflammation and during established disease by measuring transepithelial electrical resistance (TEER) on ex vivo-cultured ilea following exposure to VSL#3 conditioned media (CM), TNF or VSL#3-CM + anti-TNF. Tight junction (TJ) proteins were assessed by qRT-PCR, Western blot, and confocal microscopy, and TNFRI/TNFRII expression measured in freshly isolated intestinal epithelial cells (IEC) from SAMP and control AKR mice.

Results

Culture with either VSL#3-CM or TNF resulted in decreased ileal paracellular permeability in pre-inflamed SAMP, but not SAMP with established disease, while addition of anti-TNF abrogated these effects. Modulation of the TJ proteins, claudin-2 and occludin, occurred with a significant decrease in claudin-2 and increase in occludin following stimulation with VSL#3-CM or TNF. TNF protein levels increased in supernatants of SAMP ilea incubated with VSL#3-CM compared to vehicle, while IEC-derived TNFR mRNA expression decreased in young, and was elevated in inflamed, SAMP versus AKR mice.

Conclusions

Our data demonstrate that the previously established efficacy of VSL#3 in preventing SAMP ileitis is due to direct innate and homeostatic effects of TNF on the gut epithelium, modulation of the TJ proteins, claudin-2 and occludin, and overall improvement of intestinal permeability.     

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods

Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of α_vβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results

Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and α_vβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and α_vβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions

Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.     

Exendin-4 Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells

Objective

Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca²⁺-independent phospholipase A2.

Methods

We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder). We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca₂-independent phospholipase A2 and Ca²⁺-independent phospholipase A2 mRNA.

Results

The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05). The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05), and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca²⁺-independent phospholipase A2 activity was positively related to Exendin-4 activity.

Conclusion

Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca²-independent phospholipase A2.     

Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice

Background

It has recently been suggested that RhoA plays an important role in the enhancement of the Ca²⁺ sensitization of smooth muscle contraction. In the present study, a participation of RhoA-mediated Ca²⁺ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined.

Methods

Ovalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge. The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively.

Results

Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K⁺-depolarization. In α-toxin-permeabilized BSMs, ACh induced a Ca²⁺ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca²⁺ sensitization. Interestingly, the ACh-induced, RhoA-mediated Ca²⁺ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice. Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs.

Conclusion

These findings suggest that the augmentation of Ca²⁺ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.     

Regulatory effect of connexin 43 on basal Ca2+ signaling in rat ventricular myocytes

Background

It has been found that gap junction-associated intracellular Ca²⁺ [Ca²⁺]_i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca²⁺ signaling, in particular the basal [Ca²⁺]_i activities, is unclear.

Methods and Results

Global and local Ca²⁺ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca²⁺ transients and local Ca²⁺ sparks in monolayer NRVMs and Ca²⁺ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP₃ butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca²⁺ signal and LY uptake by gap uncouplers, whereas blockade of IP₃ receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca²⁺ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca²⁺ signaling regulation in cardiomyocytes.

Conclusions

These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca²⁺ signaling in normal ventricular myocytes, in which IP₃/IP₃ receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca²⁺ activities in cardiomyocytes.