Toll-like Receptor2/4 Play Important Roles In The Inflammatory Responses In Conorary Artery Endothelial Cells

BackgroundGrowing evidence supports an important role for vascular local inflammation in the development and progress of atherosclerosis. As the first cardiac cells that can come in contact with endogenous and exogenous agents, endothelial cells are important in immune and inflammatory responses, and inflammatory activation of the endothelial cells is a critical step in the development of many diseases. Further, inflammatory activation of the endothelial cells is a critical step in the recrument and infiltration of monocytes and macrophages in the tissue I/R injury, and the pathophysiological of atherosclerosis.Toll-like receptors (TLRs) are pathogen pattern recognition receptors that recognize bacterial and viral products, and other pathogens. Activation TLR2 or TLR4 by microbial ligands induce a cascade of intracellular signaling events, culminating in the production of pro-inflammatory mediators. Tissue I/R causes the release of endogenous TLR activators including heat shock protein and high mobility group box 1 (HMGB1), released from stressed and/or injured cells. Recent studies by our group and others indicate that myocardial tissue TLR2/4 are critical for leukocyte infiltration into the ischemic myocardium and contribute to the mechanisms of myocardial I/R injury. It is not determined that the roles of TLRs play in the inflammatory response in endothelial cells, and which plays in myocardial ischemia.Gender difference in cardiovascular diseases is well recognized in humans. Women are known to benefit from the cardioprotective actions of sex hormones which discontinue with the onset of menopause. This gender difference in cardiovascular conditions is also demonstrated in animal studies. Several studies confirmed that female mice have better cardiac function after myocardial infarction (MI) that is associated with attenuated early inflammation, augmented fibrotic response and less extent of LV adverse remodeling. Furthermore, female mice exhibit myocardial resistance to TNFR1 signaling during ischemia. Female sex hormones appear to be anti-inflammatory and are responsible for cardiovascular protection in females.Toll-like receptors (TLRs) are innate immune receptors that sense the presence of pathogen-associated molecular patterns. Accumulated evidence demonstrates that TLR2 and TLR4 play important roles in the signaling mechanisms that contribute to post-ischemic myocardial inflammatory response and injury. Several studies found that TLR2 and TLR4 also respond to endogenous proteins, such as heat shock proteins (HSP) and high mobility group box 1 (HMGB1), released from stressed and/or injured cells. TLR 2-deficient mice are also protected against post-ischemic coronary endothelial dysfunction. In addition, reduced myocardial fibrosis in TLR2 knock out (TLR2 KO) mice after ischemia indicates TLR2 is involved in maladaptive LV remodeling. Thus, TLR2 appears to play an important role in post-ischemic myocardial injury and cardiac dysfunction. However, these previous studies determined the role of TLR2 only in male mice. It is unknown whether TLR2 has a similar role in females. Sex hormones are important modifiers of tissue and cellular inflammatory response to injury. Several studies indicate the interaction of sex hormones with TLRs. LPS-induced inflammation in murine airway was reduced in female, but was exaggerated in females administered testosterone. In addition,17beta-estradiol inhibits IL-8 release induced by TLR agonists via ERbeta in cystic fibrosis bronchial epithelial cells. However, removal of endogenous estrogens has been shown to decrease the production of both pro- and anti-inflammatory cytokines, with a concomitant reduction in cell surface expression of TLR4 in macrophages. Similarly, testosterone is found to reduce macrophage expression of TLR4. Although these studies suggest that sex hormones could alter TLR expression and the TLR-mediated inflammatory responses, it remains unknown whether the TLR2-mediated myocardial inflammatory responses to ischemia are gender-dependent.In the Type 1 diabetes (T1D) population, atherogenesis occurs in younger ages and advances faster. However the underlying mechanisms are incompletely understood. A number of studies demonstrate that these two major innate immune receptors play a mechanistic role in the development of atherosclerosis. In addition, TLR2 ligand peptidoglycan (PGN) and TLR4 ligand lipopolysaccharide (LPS) have been found in vessels with early atherosclerotic lesions. While these bacterial agents induce the production of multiple pro-inflammatory mediators in mononuclear cells, their effects on the inflammatory responses in coronary artery endothelial cells (CAECs) remain to be determined. Investigation of the effect of T1D on CAEC inflammatory responses to TLR2/4 stimulation could provide insights into the mechanisms underlying the pro-atherogenic phenotype associated with this disease.TLR2 and TLR4 have also been implicated in the pathophysiology of T1D. In an experimental T1D model, TLR2 is involved in the autoimmune inflammation in the pancreatic islet. The expression of TLR2, as well as TLR3, TLR4 and TLR5 in bone marrow-derived macrophages is increased in diabetic NOD mice. Insulin is found to suppress the expression of TLR2 in mononuclear cells at the transcriptional level. In addition, altered TLR4 function is involved in the inflammation in B cells from diabetes mellitus patients by two mechanisms:elevation of pro-inflammatory IL-8 and lack of anti-inflammatory/protective IL-10 production. While these studies indicate altered cellular TLR expression and responses associated with T1D, it remains unclear whether TLR2/4 levels and the inflammatory responses to TLR2/4 agonists are altered in CAECs from T1D patients.In this study, we hypothized that TLR activation could induce inflammatory response in endothelial cells, the gender-disparity of TLR2, and T1D could enhance the inflammatory responses induced by TLR activation in endothelial cells. We determined the inflammatory response in the mouse MVECs and human coronary endothelial cells treated with PGN and LPS, the gender disparity of TLR2 in the ischemia myocardium in the LAD ligation model.Chapter 1:TLR activation induced inflammatory responses in microvascular endothelial cellsObjectiveTo determine the role and possible mechanism of TLR2 and TLR4 activation induce microvascular inflammatory responses induced by PGN and LPS, respectively.MethodsMicrovascular endothelial cells (MVECs) were isolated from mice and treated with TLR2 ligand PGN and TLR4 ligand LPS, respectively. ICAM-1 mRNA was analyzed with qPCR, IL-6 and IL-8 were assessed by ELISA, expression of ICAM-1 and NF- B activation by immunoblotting. ResultsPGN and LPS couldn't induce ICAM-1 expression with TLR2 and TLR4 dysfunction, respectively. TLR2 and TLR4 activation could induce the expression of ICAM-1 mRNA and protein, IL-6 and IL-8 peptide, with increase of NF- B phosphorylation.ConclusionsPGN and LPS induce inflammatory response by activation of TLR2 and TLR4 in MVECs, respectively. TLR2 and TLR4 activation could induce the inflammatory response in MVECs, which maybe due to the NF- B phosphorylation.Chapter 2:Gender disparity in the role of Toll-like receptor 2 in post-ischemic myocardial inflammatory response and cardiac failureObjectiveThe main purpose was to determine whether TLR2 KO suppresses myocardial inflammatory response and improves early left ventricular remodeling after permanent ischemia in both male and female mice.MethodsPermanent ischemia was induced in male and female C57BL/6J (WT) and TLR2 KO mice. Infarct size and LV function were analyzed at day 7. Myocardial levels of MCP-1 and ICAM-1, as well as neutrophil infiltration, were assessed at day 3, and mononuclear cell accumulation was determined at day 7.ResultsWe found lower MCP-1 and ICAM-1 levels, and reduced leukocyte accumulation in male TLR2 KO mice. Suppression of myocardial inflammatory response by TLR2 KO in male mice correlated with smaller infarct size and improved LV function. Female WT mice exhibited attenuated myocardial inflammatory response and injury, and TLR2 KO in females did not provide a protective effect on either myocardial inflammatory response or injury although myocardial TLR2 levels in female WT mice is unaltered, and their cardiac cells respond to bacterial TLR2 agonist properly.ConclusionsThese results showed that TLR2 KO in male mice suppresses post-ischemic myocardial inflammatory response, reduces infarct size and improves early LV remodeling. However, TLR2 KO is not beneficial in female mice. The gender disparity in the role of TLR2 in post-ischemic myocardial inflammatory response and injury suggests that interception with TLR2 signaling may have therapeutic potentials only in males.Chapter 3:The mechanism underlying Toll-like receptor 2/4 stimulation in human coronary artery endothelial cellsBackgroundEndothelial inflammatory responses mediated by Toll-like receptors (TLRs), particularly TLR2 and TLR4, play an important role in atherogenesis. While Type 1 diabetes (T1D) promotes the development and progression of atherosclerosis, the effect of T1D on TLR2/4-mediated inflammatory responses in coronary artery endothelial cells (CAECs) remains unclear.MethodsWe tested the hypothesis that diabetic CAECs have enhanced inflammatory responses to TLR2/4 stimulation. Non-diabetic and diabetic CAECs were treated with TLR2 agonist peptidoglycan and TLR4 agonist lipopolysaccharide. The expression of ICAM-1, IL-6 and IL-8 were analyzed by real-time PCR, immunoblotting and ELISA, and NF-κB activation by immunoblotting and immunostaining. In additional experiments, insulin was added before TLR stimulation to determine whether insulin deficiency alone is responsible for the alteration of TLR2/4-mediated inflammatory responses.ResultsStimulation of TLR2 or TLR4 induced NF-κB activation, and the expression of ICAM-1, IL-6 and IL-8. Interestingly, the expression of inflammatory mediators was significantly enhanced in diabetic cells. The enhanced inflammatory responses correlated with augmented NF-κB activation in the absence of a change in TLR2 or TLR4 protein levels. Further, pretreatment of diabetic cells with insulin failed to suppress the enhanced inflammatory responses.ConclusionsDiabetic CAECs have enhanced inflammatory responses to stimulation of TLR2 or TLR4, and insulin alone is insufficient to correct the hyper-inflammatory responses. The mechanism underlying the enhanced inflammatory responses appears to be augmentation of pro-inflammatory signaling, rather than up-regulation of levels of TLR2 and TLR4. These findings suggest that diabetic CAECs adopt a hyper-inflammatory phenotype and that this endothelial phenotypic change may predispose coronary artery to atherogenesis.Total Conclusion1. TLR2 and TLR4 activation could induce inflammatory response in the myocardiac microvascular cells by PGN and LPS, respectively;2. TLR2 and TLR4 activation could activate NF- B phosphorylation which plays an important role in the inflammatory response;3. TLR2 KO suppresses myocardial inflammatory responses, reduces infarct size and improves early LV remodeling in male mice after permanent myocardial ischemia;4. Female mice have attenuated myocardial inflammatory responses and myocardial injury, and are not benefited from TLR2 KO;5. The lack of TLR2 KO in females effect is not due to myocardial TLR2 deficiency;6. Stimulation of TLR2 and TLR4 induces greater expression of IL-6, IL-8 and ICAM-1 in T1D CAECs;7. The enhanced inflammatory responses to TLR2 and TLR4 agonists in diabetic CAECs correlate with augmented NF-κB activation in the absence of an alteration of cellular TLR2 and TLR4 protein levels;8. Insulin alone is insufficient to suppress the hyper-inflammatory responses to both TLR2 and TLR4 agonists in diabetic CAECs.