Extracellular glucose and dysfunctional insulin receptor signaling independently upregulate arterial smooth muscle TMEM16A expression.

Diabetes alters the function of ion channels responsible for regulating arterial smooth muscle membrane potential, resulting in vasoconstriction. Our prior research demonstrated an elevation of TMEM16A in diabetic arteries. Here, we explored the mechanisms involved in Transmembrane protein 16A (TMEM16A) gene expression. Our data indicate that a Snail-mediated repressor complex regulates arterial TMEM16A gene transcription. Snail expression was reduced in diabetic arteries while TMEM16A expression was upregulated. The TMEM16A promoter contained three canonical E-box sites. Electrophoretic mobility and super shift assays revealed that the -154 nt E-box was the binding site of the Snail repressor complex and binding of the repressor complex decreased in diabetic arteries. High glucose induced a biphasic contractile response in pressurized nondiabetic mouse hindlimb arteries incubated ex vivo. Hindlimb arteries incubated in high glucose also showed decreased phospho-protein kinase D1 and TMEM16A expression. In hindlimb arteries from nondiabetic mice, administration of a bolus dose of glucose activated protein kinase D1 signaling to induce Snail degradation. In both in vivo and ex vivo conditions, Snail expression exhibited an inverse relationship with the expression of protein kinase D1 and TMEM16A. In diabetic mouse arteries, phospho-protein kinase D1 increased while Akt2 and pGSK3ß levels declined. These results indicate that in nondiabetic mice, high glucose triggers a transient deactivation of the Snail repressor complex to increase arterial TMEM16A expression independently of insulin signaling. Conversely, insulin resistance activates GSK3ß signaling and enhances arterial TMEM16A channel expression. These data have uncovered the Snail-mediated regulation of arterial TMEM16A expression and its dysfunction during diabetes.NEW & NOTEWORTHY The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

Inhibition of microRNA-34c reduces detrusor ROCK2 expression and urinary bladder inflammation in experimental cystitis.

Interstitial cystitis (IC), also called painful bladder syndrome (PBS), is 2 to 5 times more common in women than in men, yet its cause and pathogenesis remain unclear. In our study using the cyclophosphamide (CYP)-induced mouse model of cystitis, histological evaluation of the urinary bladder (UB) lamina propria (LP) showed immune cell infiltrations, indicating moderate to severe inflammation. In this study, we noticed a differential expression of a subset of microRNAs (miRs) in the UB cells (UBs) of CYP-induced cystitis as compared to the control. UB inflammatory scores and inflammatory signaling were also elevated in CYP-induced cystitis as compared to control. We identified eight UBs miRs that exhibited altered expression after CYP induction and are predicted to have a role in inflammation and smooth muscle function (miRs-34c-5p, -34b-3p, -212-3p, -449a-5p, -21a-3p, -376b-3p, -376b-5p and - 409-5p). Further analysis using ELISA for inflammatory markers and real-time PCR (RT-PCR) for differentially enriched miRs identified miR-34c as a potential target for the suppression of UB inflammation in cystitis. Blocking miR-34c by antagomir ex vivo reduced STAT3, TGF-ß1, and VEGF expression in the UBs, which was induced during cystitis as compared to control. Interestingly, miR-34c inhibition also downregulated ROCK2 but elevated ROCK1 expression in bladder and detrusor cells. Thus, the present study shows that targeting miR-34c can mitigate the STAT3, TGF-ß, and VEGF, inflammatory signaling in UB, and suppress ROCK2 expression in UBs to effectively suppress the inflammatory response in cystitis. This study highlights miR-34c as a potential biomarker and/or serves as the basis for new therapies for the treatment of cystitis.

Diabetic Endothelial Cell Glycogen Synthase Kinase 3ß Activation Induces VCAM1 Ectodomain Shedding.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1's role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1's release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3ß, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3ß activation in control ECs increased ADAM10/17 and VCAM1. A GSK3ß inhibitor reduced active GSK3ß and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3ß activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3ß inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.

Hypoxia induces purinergic receptor signaling to disrupt endothelial barrier function.

Blood-brain-barrier permeability is regulated by endothelial junctional proteins and is vital in limiting access to and from the blood to the CNS. When stressed, several cells, including endothelial cells, can release nucleotides like ATP and ADP that signal through purinergic receptors on these cells to disrupt BBB permeability. While this process is primarily protective, unrestricted, uncontrolled barrier disruption during injury or inflammation can lead to serious neurological consequences. Purinergic receptors are broadly classified into two families: the P1 adenosine and P2 nucleotide receptors. The P2 receptors are further sub-classified into the P2XR ion channels and the P2YR GPCRs. While ATP mainly activates P2XRs, P2YRs have a broader range of ligand selectivity. The P2Y1R, essential for platelet function, is reportedly ubiquitous in its expression. Prior studies using gene knockout and specific antagonists have shown that these approaches have neuroprotective effects following occlusive stroke. Here we investigated the expression of P2Y1R in primary cultured brain endothelial cells and its relation to the maintenance of BBB function. Results show that following in vitro hypoxia and reoxygenation, P2Y1R expression is upregulated in both control and diabetic cells. At the same time, endothelial junctional markers, ZO-1 and VE-cadherin, were downregulated, and endothelial permeability increased. siRNA knockdown of P2Y1R and MRS 2500 effectively blocked this response. Thus, we show that P2Y1R signaling in endothelial cells leads to the downregulation of endothelial barrier function.

Rab GTPases as Modulators of Vascular Function.

Rab GTPases, the largest family of small GTPases, are ubiquitously expressed proteins that control various aspects of cellular function, from cell survival to exocytosis. Rabs cycle between the GDP-bound inactive form and the GTP-bound active form. When activated, specific Rab GTPase-positive vesicles mediate cellular networks involved in intracellular trafficking, recycling, and/or exocytosis of cargo proteins. Dysfunctional Rab signaling pathways have been implicated in various disease processes. The precise cellular functions of several members of the Rab GTPase family are still unknown. A lack of pharmacological tools and the lethality of gene knockouts have made more detailed characterizations of their protein interaction networks difficult. Nevertheless, available evidence suggests that these proteins are vital for normal cell function. Endothelial and smooth muscle cells control vascular lumen diameter and modulate blood flow. Endothelial cells also secrete several pro- and antithrombotic factors and vasoactive substances to coordinate local inflammatory responses and angiogenesis. Rab GTPase function in endothelial cells has been relatively well-explored, while only a handful of reports are available on these proteins in vascular smooth muscle. This review summarizes the present knowledge on Rab GTPases in the vasculature.

Histamine Potentiates SARS-CoV-2 Spike Protein Entry Into Endothelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes coronavirus disease (COVID-19) is one of the most serious global health crises in recent history. COVID-19 patient symptoms range from life-threatening to mild and asymptomatic, which presents unique problems in identifying, quarantining, and treating the affected individuals. The emergence of unusual symptoms among survivors, now referred to as "Long COVID", is concerning, especially since much about the condition and the treatment of it is still relatively unknown. Evidence so far also suggests that some of these symptoms can be attributed to vascular inflammation. Although famotidine, the commonly used histamine H2 receptor (H2R) blocker, was shown to have no antiviral activity, recent reports indicate that it could prevent adverse outcomes in COVID-19 patients. Histamine is a classic proinflammatory mediator, the levels of which increase along with other cytokines during COVID-19 infection. Histamine activates H2R signaling, while famotidine specifically blocks H2R activation. Investigating the effects of recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike) on ACE2 expression in cultured human coronary artery endothelial cells, we found that the presence of histamine potentiated spike-mediated ACE2 internalization into endothelial cells. This effect was blocked by famotidine, protein kinase A inhibition, or by H2 receptor protein knockdown. Together, these results indicate that histamine and histamine receptor signaling is likely essential for spike protein to induce ACE2 internalization in endothelial cells and cause endothelial dysfunction and that this effect can be blocked by the H2R blocker, famotidine.

Thioredoxin Prevents Loss of UCP2 in Hyperoxia via MKK4-p38 MAPK-PGC1α Signaling and Limits Oxygen Toxicity.

Administration of high concentrations of oxygen (hyperoxia) is one of few available options to treat acute hypoxemia-related respiratory failure, as seen in the current coronavirus disease (COVID-19) pandemic. Although hyperoxia can cause acute lung injury through increased production of superoxide anion (O2•-), the choice of high-concentration oxygen administration has become a necessity in critical care. The objective of this study was to test the hypothesis that UCP2 (uncoupling protein 2) has a major function of reducing O2•- generation in the lung in ambient air or in hyperoxia. Lung epithelial cells and wild-type; UCP2-/-; or transgenic, hTrx overexpression-bearing mice (Trx-Tg) were exposed to hyperoxia and O2•- generation was measured by using electron paramagnetic resonance, and lung injury was measured by using histopathologic analysis. UCP2 expression was analyzed by using RT-PCR analysis, Western blotting analysis, and RNA interference. The signal transduction pathways leading to loss of UCP2 expression were analyzed by using IP, phosphoprotein analysis, and specific inhibitors. UCP2 mRNA and protein expression were acutely decreased in hyperoxia, and these decreases were associated with a significant increase in O2•- production in the lung. Treatment of cells with rhTrx (recombinant human thioredoxin) or exposure of Trx-Tg mice prevented the loss of UCP2 protein and decreased O2•- generation in the lung. Trx is also required to maintain UCP2 expression in normoxia. Loss of UCP2 in UCP2-/- mice accentuated lung injury in hyperoxia. Trx activates the MKK4-p38MAPK (p38 mitogen-activated protein kinase)-PGC1α (PPARγ [peroxisome proliferator-activated receptor γ] coactivator 1α) pathway, leading to rescue of UCP2 and decreased O2•- generation in hyperoxia. Loss of UCP2 in hyperoxia is a major mechanism of O2•- production in the lung in hyperoxia. rhTrx can protect against lung injury in hyperoxia due to rescue of the loss of UCP2.

SARS-CoV-2 Spike Protein Induces Degradation of Junctional Proteins That Maintain Endothelial Barrier Integrity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface to enter cells. Angiotensin converting enzyme 2 is present in many cell types including endothelial cells, where it functions to protect against oxidative damage. There is growing evidence to suggest that coronavirus disease (COVID-19) patients exhibit a wide range of post-recovery symptoms and shows signs related to cardiovascular and specifically, endothelial damage. We hypothesized that these vascular symptoms might be associated with disrupted endothelial barrier integrity. This was investigated in vitro using endothelial cell culture and recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike). Mouse brain microvascular endothelial cells from normal (C57BL/6 mice) and diabetic (db/db) mice were used. An endothelial transwell permeability assay revealed increased permeability in diabetic cells as well as after Spike treatment. The expression of VE-Cadherin, an endothelial adherens junction protein, JAM-A, a tight junctional protein, Connexin-43, a gap junctional protein, and PECAM-1, were all decreased significantly after Spike treatment in control and to a greater extent, in diabetic cells. In control cells, Spike treatment increased association of endothelial junctional proteins with Rab5a, a mediator of the endocytic trafficking compartment. In cerebral arteries isolated from control and diabetic animals, Spike protein had a greater effect in downregulating expression of endothelial junctional proteins in arteries from diabetic animals than from control animals. In conclusion, these experiments reveal that Spike-induced degradation of endothelial junctional proteins affects endothelial barrier function and is the likely cause of vascular damage observed in COVID-19 affected individuals.

Cardiovascular Impacts on COVID-19 Infected Patients.

The SARS-CoV-2 virus has taken more than 2 million lives on a global scale. Over 10 million people were confirmed with COVID-19 infection. The well-known spot of primary infection includes the lungs and the respiratory system. Recently it has been reported that the cardiovascular system and coagulation mechanisms were the second major targets of biological system affected due to the viral replication. The replication mechanism of SARS-CoV-2 involves the angiotensin-converting enzyme 2- (ACE2) surface receptors of endothelial cells belonging to various organs which act as the binding site for the viral spike (S) protein of SARS-CoV-2. The COVID-19 virus has been recently listed as a primary risk factor for the following cardiovascular conditions such as pericarditis, myocarditis, arrhythmias, myocardial injury, cardiac arrest, heart failure and coagulation abnormalities in the patients confirmed with COVID-19 viral infection. Direct and indirect type of tissue damage were the two major categories detected with cardiovascular abnormalities. Direct myocardial cell injury and indirect damage to the myocardial cell due to inflammation were clinically reported. Few drugs were clinically administered to regulate the vital biological mechanism along with symptomatic treatment and supportive therapy.

TMEM16A channel upregulation in arterial smooth muscle cells produces vasoconstriction during diabetes.

The pathological involvement of anion channels in vascular dysfunction that occurs during type 2 diabetes (T2D) is unclear. Here, we tested the hypothesis that TMEM16A, a calcium-activated chloride (Cl-) channel, contributes to modifications in arterial contractility during T2D. Our data indicate that T2D increased TMEM16A mRNA in arterial smooth muscle cells and total and surface TMEM16A protein in resistance-size cerebral and hindlimb arteries of mice. To examine vascular cell types in which TMEM16A protein increased and the functional consequences of TMEM16A upregulation during T2D, we generated tamoxifen-inducible, smooth muscle cell-specific TMEM16A knockout (TMEM16A smKO) mice. T2D increased both TMEM16A protein and Cl- current density in arterial smooth muscle cells of control (TMEM16Afl/fl) mice. In contrast, T2D did not alter arterial TMEM16A protein or Cl- current density in smooth muscle cells of TMEM16A smKO mice. Intravascular pressure stimulated greater vasoconstriction (myogenic tone) in the arteries of T2D TMEM16Afl/fl mice than in the arteries of nondiabetic TMEM16Afl/fl mice. This elevation in myogenic tone in response to T2D was abolished in the arteries of T2D TMEM16A smKO mice. T2D also reduced Akt2 protein and activity in the arteries of T2D mice. siRNA-mediated knockdown of Akt2, but not Akt1, increased arterial TMEM16A protein in nondiabetic mice. In summary, data indicate that T2D is associated with an increase in TMEM16A expression and currents in arterial smooth muscle cells that produces vasoconstriction. Data also suggest that a reduction in Akt2 function drives these pathological alterations during T2D.NEW & NOTEWORTHY We investigated the involvement of TMEM16A channels in vascular dysfunction during type 2 diabetes (T2D). TMEM16A message, protein, and currents were higher in smooth muscle cells of resistance-size arteries during T2D. Pressure stimulated greater vasoconstriction in the arteries of T2D mice that was abolished in the arteries of TMEM16A smKO mice. Akt2 protein and activity were both lower in T2D arteries, and Akt2 knockdown elevated TMEM16A protein. We propose that a decrease in Akt2 function stimulates TMEM16A expression in arterial smooth muscle cells, leading to vasoconstriction during T2D.

Short-duration hyperoxia causes genotoxicity in mouse lungs: protection by volatile anesthetic isoflurane.

High concentrations of oxygen (hyperoxia) are routinely used during anesthesia, and supplemental oxygen is also administered in connection with several other clinical conditions. Although prolonged hyperoxia is known to cause acute lung injury (ALI), whether short-duration hyperoxia causes lung toxicity remains unknown. We exposed mice to room air (RA or 21% O2) or 60% oxygen alone or in combination with 2% isoflurane for 2 h and determined the expression of oxidative stress marker genes, DNA damage and DNA repair genes, and expression of cell cycle regulatory proteins using quantitative PCR and Western analyses. Furthermore, we determined cellular apoptosis using TUNEL assay and assessed the DNA damage product 8-hydroxy-2'-deoxyguanosine (8-Oxo-dG) in the urine of 60% hyperoxia-exposed mice. Our study demonstrates that short-duration hyperoxia causes mitochondrial and nuclear DNA damage and that isoflurane abrogates this DNA damage and decreases apoptosis when used in conjunction with hyperoxia. In contrast, isoflurane mixed with RA caused significant 8-Oxo-dG accumulations in the mitochondria and nucleus. We further show that whereas NADPH oxidase is a major source of superoxide anion generated by isoflurane in normoxia, isoflurane inhibits superoxide generation in hyperoxia. Additionally, isoflurane also protected the mouse lungs against ALI (95% O2 for 36-h exposure). Our study established that short-duration hyperoxia causes genotoxicity in the lungs, which is abrogated when hyperoxia is used in conjunction with isoflurane, but isoflurane alone causes genotoxicity in the lung when delivered with ambient air.

Protein Kinase Cθ Via Activating Transcription Factor 2-Mediated CD36 Expression and Foam Cell Formation of Ly6Chi Cells Contributes to Atherosclerosis.

BACKGROUND: Although the role of thrombin in atherothrombosis is well studied, its role in the pathogenesis of diet-induced atherosclerosis is not known. METHODS: Using a mouse model of diet-induced atherosclerosis and molecular biological approaches, here we have explored the role of thrombin and its G protein-coupled receptor signaling in diet-induced atherosclerosis. RESULTS: In exploring the role of G protein-coupled receptor signaling in atherogenesis, we found that thrombin triggers foam cell formation via inducing CD36 expression, and these events require Par1-mediated Gα12-Pyk2-Gab1-protein kinase C (PKC)θ-dependent ATF2 activation. Genetic deletion of PKCθ in apolipoprotein E (ApoE)-/- mice reduced Western diet-induced plaque formation. Furthermore, thrombin induced Pyk2, Gab1, PKCθ, and ATF2 phosphorylation, CD36 expression, and foam cell formation in peritoneal macrophages of ApoE-/- mice. In contrast, thrombin only stimulated Pyk2 and Gab1 but not ATF2 phosphorylation or its target gene CD36 expression in the peritoneal macrophages of ApoE-/-:PKCθ-/- mice, and it had no effect on foam cell formation. In addition, the aortic root cross-sections of Western diet-fed ApoE-/- mice showed increased Pyk2, Gab1, PKCθ, and ATF2 phosphorylation and CD36 expression as compared with ApoE-/-:PKCθ-/- mice. Furthermore, although the monocytes from peripheral blood and the aorta of Western diet-fed ApoE-/- mice were found to contain more of Ly6Chi cells than Ly6Clo cells, the monocytes from Western diet-fed ApoE-/-:PKCθ-/- mice were found to contain more Ly6Clo cells than Ly6Chi cells. It is interesting to note that the Ly6Chi cells showed higher CD36 expression with enhanced capacity to form foam cells as compared with Ly6Clo cells. CONCLUSIONS: These findings reveal for the first time that thrombin-mediated Par1-Gα12 signaling via targeting Pyk2-Gab1-PKCθ-ATF2-dependent CD36 expression might be playing a crucial role in diet-induced atherogenesis.

Protease-activated receptor 1 inhibits cholesterol efflux and promotes atherogenesis via cullin 3-mediated degradation of the ABCA1 transporter.

Although signaling of thrombin via its receptor protease-activated receptor 1 (Par1) is known to occur in atherothrombosis, its link to the actual pathogenesis of this condition is less clear. To better understand the role of thrombin-Par1 signaling in atherosclerosis, here we have studied their effects on cellular cholesterol efflux in mice. We found that by activating Par1 and cullin 3-mediated ubiquitination and degradation of ABC subfamily A member 1 (ABCA1), thrombin inhibits cholesterol efflux in both murine macrophages and smooth muscle cells. Moreover, disruption of the Par1 gene rescued ABCA1 from Western diet-induced ubiquitination and degradation and restored cholesterol efflux in apolipoprotein E-deficient (ApoE-/-) mice. Similarly, the Par1 deletion diminished diet-induced atherosclerotic lesions in the ApoE-/- mice. These observations for the first time indicate a role for thrombin-Par1 signaling in the pathogenesis of diet-induced atherosclerosis. We identify cullin 3 as a cullin-RING ubiquitin E3 ligase that mediates ABCA1 ubiquitination and degradation and thereby inhibits cholesterol efflux. Furthermore, compared with peripheral blood mononuclear cells (PBMCs) from ApoE-/- mice, the PBMCs from ApoE-/-:Par1-/- mice exhibited decreased trafficking to inflamed arteries of Western diet-fed ApoE-/- mice. This finding suggested that besides inhibiting cholesterol efflux, thrombin-Par1 signaling also plays a role in the recruitment of leukocytes during diet-induced atherogenesis. Based on these findings, we conclude that thrombin-Par1 signaling appears to contribute to the pathogenesis of atherosclerosis by impairing cholesterol efflux from cells and by recruiting leukocytes to arteries.

Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function.

Resolvins are a novel class of lipid mediators that play an important role in the resolution of inflammation, although the underlying mechanisms are not very clear. To explore the anti-inflammatory mechanisms of resolvins, we have studied the effects of resolvin D1 (RvD1) on lipopolysaccharide (LPS)-induced endothelial barrier disruption as it is linked to propagation of inflammation. We found that LPS induces endothelial cell (EC) barrier disruption via xanthine oxidase (XO)-mediated reactive oxygen species (ROS) production, protein tyrosine phosphatase SHP2 inactivation and Fyn-related kinase (Frk) activation leading to tyrosine phosphorylation of α-catenin and VE-cadherin and their dissociation from each other affecting adherens junction (AJ) integrity and thereby increasing endothelial barrier permeability. RvD1 attenuated LPS-induced AJ disassembly and endothelial barrier permeability by arresting tyrosine phosphorylation of α-catenin and VE-cadherin and their dislocation from AJ via blockade of XO-mediated ROS production and thereby suppression of SHP2 inhibition and Frk activation. We have also found that the protective effects of RvD1 on EC barrier function involve ALX/FPR2 and GPR32 as inhibition or neutralization of these receptors negates its protective effects. LPS also increased XO activity, SHP2 cysteine oxidation and its inactivation, Frk activation, α-catenin and VE-cadherin tyrosine phosphorylation and their dissociation from each other leading to AJ disruption with increased vascular permeability in mice arteries and RvD1 blocked all these effects. Thus, RvD1 protects endothelial AJ and its barrier function from disruption by inflammatory mediators such as LPS via a mechanism involving the suppression of XO-mediated ROS production and blocking SHP2 inactivation.

The Advanced Lipoxidation end Product Precursor Malondialdehyde Induces IL-17E Expression and Skews Lymphocytes to the th17 Subset.

Malondialdehyde (MDA) is a highly reactive endogenous product of thromboxane synthesis in the prostagland and lipid peroxidation by reactive oxygen species. Elevated MDA levels occur in diabetes and atherosclerotic plaques. The aim of this study was to examine the molecular mechanisms of MDA-induced IL-17E cytokine expression and its effect on T-cell differentiation. Real-time PCR, RT-PCR and ELISA were used to assess the expression of IL-17 family cytokines in Jurkat T-cells and human peripheral blood lymphocytes (PBLCs) from diabetic subjects. Luciferase reporter assays were used for the promoter activation study. Pharmacological inhibitors were used for signaling pathway experiments. FACS analyses were used to measure the Th1, Th2 and Th17 subset levels. MDA induced significant (2- to 3-fold; p < 0.01) generation of IL-17E mRNA in a dose- and time-dependent manner in Jurkat T-cells and PBLCs. Elevated IL-17E mRNA levels were found in the lymphocytes from diabetic subjects. The increased IL-17E protein and mRNA levels correlate well with serum MDA levels from diabetic patients. Transient transfection of plasmid containing the minimum IL-17E promoter region (pIL-17E-Luc) showed a significant (2-fold; p < 0.01) increase in luciferase activity. Pretreatment of lymphocytes with pharmacological inhibitors showed the involvement of antioxidant, NF-ƙB, p38MAPK, PKC and ERK signaling pathways. Quantification of the Th1, Th2 and Th17 cell population in PBLCs via FACS analyses revealed an increase in the Th17 subset. These results show that MDA transcriptionally upregulates the expression of IL-17E in lymphocytes and alters lymphocyte differentiation towards the pathogenic Th17 subset.

Ligation of RAGE with ligand S100B attenuates ABCA1 expression in monocytes.

HYPOTHESIS: ATP Binding Cassette Transporter (ABC) A1 is one of the key regulators of HDL synthesis and reverse cholesterol transport. Activation of Receptors for Advanced Glycation End products (RAGE) is involved in the pathogenesis of diabetes, and its complications. The aim of the present study is to examine the effect of RAGE ligand S100B on ABCA1 expression. METHODS: S100B mediated regulation of LXR target genes like ABCA1, ABCG1, ABCG8, LXR-α and LXR-ß in THP-1 cells was analyzed by real-time PCR, RT-PCR and western blots. ABCA1 mRNA expression in monocytes from diabetic patients was studied. Effect of LXR ligand on S100B induced changes in LXR target genes was also studied. Luciferase reporter assay was used for S100B induced ABCA1 promoter regulation. RESULTS: S100B treatment resulted in a significant 2-3 fold reduction (p<0.01) in ABCA1 and ABCG1 mRNA in dose and time dependent manner in THP1 cells. ABCA1 protein level was also significantly (p<0.01) reduced. S100B-induced reduction on ABCA1 mRNA expression was blocked by treating THP-1 cell with anti-RAGE antibody. Reduced ABCA1 mRNA levels seen in peripheral blood monocytes from diabetes patients showed the in-vivo relevance of our in-vitro results. Effect of S100B on ABCA1 and ABCG1 expression was reversed by LXR ligand treatment. S100B treatment showed significant 2 fold (p<0.01) decrease in T1317 induced ABCA1 promoter activation. CONCLUSIONS: These results show for the first time that ligation of RAGE with S100B can attenuate the expression of ABCA1 and ABCG1 through the LXRs. This could reduce ApoA-I-mediated cholesterol efflux from monocytes.

Proinflammatory effects of malondialdehyde in lymphocytes.

Diabetes is an inflammatory disease promoted by alterations in immune cell function. Animal study indicates that T cells are important mediators of inflammation in diabetes. Lipid peroxidation by reactive oxygen species leads to the formation of highly reactive malondialdehyde (MDA), and extensive MDA is found in diabetes. However, the biological functions of MDA have not been studied yet. We hypothesized that increased MDA, as in diabetes, can regulate inflammatory cytokines via specific signaling pathways. This could then result in increased lymphocyte activation and skewing a particular inflammatory subset thereby exacerbates diabetes complications. Commercial cytokine antibody and RT(2)-PCR array profiling were performed with Jurkat T cells grown with or without MDA. Ingenuity pathways analysis (IPA) and pharmacological inhibitors were used for networks and signaling pathway identification, respectively. For validation, real-time PCR, RT-PCR, and Western blots were performed. MDA induced significant increases in 47 key proinflammatory molecules such as IL-25, IL-6, IL-8, ICAM-1, and light mRNA in Jurkat T cells and primary peripheral blood lymphocytes (PBLCs). A significant 2-fold increase in serum MDA also correlated the increased IL-25 and IL-8 mRNA in PBLCs of diabetic patients. Pharmacological inhibitor studies showed that MDA induced its effect via p38MAPK and protein kinase C pathways. Furthermore, IPA uncovered 5 groups of inflammatory networks and placed our candidate genes in canonical IL-6 and NF-κB signaling pathways and also suggested 5 toxic lists and 3 major toxic functions, namely cardiotoxicity, hepatotoxicity, and nephrotoxicity. These new results suggest that MDA can promote lymphocyte activation via induction of inflammatory pathways and networks.