Inflammatory Process In Endothelial Cells Research Articles

Vitexin Suppresses High-Glucose-upregulated Adhesion Molecule Expression in Endothelial Cells through Inhibiting NF-κB Signaling Pathway.

Vascular damage is one of the significant complications of diabetes mellitus (DM). Central to this damage is endothelial damage, especially under high-glucose conditions, which promotes inflammation via the NF-κB signaling pathway. Inflammatory processes in endothelial cells directly contribute to endothelial dysfunction, such as promoting inflammatory cytokine release and activation of adhesion molecules. Vitexin, a compound found in many medicinal plants, shows promise in countering oxidative stress in diabetic contexts and modulating blood glucose. However, its effect on high-glucose-induced endothelial cell activation has not yet been studied. This research explores vitexin's potential role in this process, focusing on its influence on the NF-κB pathway in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with 30 mM glucose (high glucose, HG) with or without vitexin treatment for 24 h. Western blotting assay was conducted for the NF-κB pathway and p-p38. Adhesion molecules (ICAM-1, VCAM-1, E-selectin, and MCP-1) were studied using flow cytometry, while pro-inflammatory cytokines were investigated using ELISA. Monocyte adhesion and vascular permeability tests were conducted to confirm the protective effect of vitexin under HG exposure. This study confirms vitexin's capacity to suppress p38 MAPK and NF-κB activation under HG conditions, reducing HG-elevated adhesion molecules and pro-inflammatory cytokine secretion. Additionally, vitexin mitigates HG-stimulated vascular permeability and monocyte adhesion. In conclusion, this study shows the therapeutic potential of vitexin against hyperglycemia-related vascular complications via p38 MAPK/NF-κB inhibition.

Unraveling the Pharmacological Potential of Lichen Extracts in the Context of Cancer and Inflammation With a Broad Screening Approach.

Lichen-forming fungi are symbiotic organisms that synthesize unique natural products with potential for new drug leads. Here, we explored the pharmacological activity of six lichen extracts (Evernia prunastri, Pseudevernia furfuracea, Umbilicaria pustulata, Umbilicaria crustulosa, Flavoparmelia caperata, Platismatia glauca) in the context of cancer and inflammation using a comprehensive set of 11 functional and biochemical in vitro screening assays. We assayed intracellular Ca2+ levels and cell migration. For cancer, we measured tumor cell proliferation, cell cycle distribution and apoptosis, as well as the angiogenesis-associated proliferation of endothelial cells (ECs). Targeting inflammation, we assayed leukocyte adhesion onto ECs, EC adhesion molecule expression, as well as nitric oxide production and prostaglandin (PG)E2 synthesis in leukocytes. Remarkably, none of the lichen extracts showed any detrimental influence on the viability of ECs. We showed for the first time that extracts of F. caperata induce Ca2+ signaling. Furthermore, extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca reduced cell migration. Interestingly, F. caperata extracts strongly decreased tumor cell survival. The proliferation of ECs was significantly reduced by E. prunastri, P. furfuracea, and F. caperata extracts. The extracts did not inhibit the activity of inflammatory processes in ECs. However, the pro-inflammatory activation of leukocytes was inhibited by extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca. After revealing the potential biological activities of lichen extracts by an array of screening tests, a correlation analysis was performed to evaluate particular roles of abundant lichen secondary metabolites, such as atranorin, physodic acid, and protocetraric acid as well as usnic acid in various combinations. Overall, some of the lichen extracts tested in this study exhibit significant pharmacological activity in the context of inflammation and/or cancer, indicating that the group lichen-forming fungi includes promising members for further testing.

Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice.

Endothelial cells play an essential role in regulating an immune response through promoting leukocyte adhesion and cell migration and production of cytokines such as TNFα. Regulation of endothelial cell immune function is tightly regulated and recent studies suggest that extracellular vesicles (EVs) are prominently involved in this process. However, the importance of EVs in regulating endothelial activation in the context of a bacterial infection is poorly understood. To begin addressing this knowledge gap we characterized the endothelial cell response to EVs released from Mycobacterium tuberculosis (Mtb) infected macrophages. Our result showed increased macrophage migration through the monolayer when endothelial cells were pretreated with EVs isolated from Mtb-infected macrophages. Transcriptome analysis showed a significant upregulation of genes involved in cell adhesion and the inflammatory process in endothelial cells treated with EVs. These results were validated by quantitative PCR and flow cytometry. Pathway analysis of these differentially expressed genes indicated that several immune response-related pathways were up-regulated. Endothelial cells were also treated with EVs isolated from the serum of Mtb-infected mice. Interestingly, EVs isolated 14 days but not 7 or 21 days post-infection showed a similar ability to induce endothelial cell activation suggesting a change in EV function during the course of an Mtb infection. Immunofluorescence microscopy result indicated that NF-κB and the Type 1 interferon pathways were involved in endothelial activation by EVs. In summary, our data suggest that EVs can activate endothelial cells and thus may play an important role in modulating host immune responses during an Mtb infection.

APOC3 induces endothelial dysfunction through TNF-α and JAM-1.

BackgroundThe fatality rate for cardiovascular disease (CVD) has increased in recent years and higher levels of triglyceride have been shown to be an independent risk factor for atherosclerotic CVD. Dysfunction of endothelial cells (ECs) is also a key factor of CVD. APOC3 is an important molecule in lipid metabolism that is closely associated with hyperlipidemia and an increased risk of developing CVD. But the direct effects of APOC3 on ECs were still unknown. This study was aimed at determining the effects of APOC3 on inflammation, chemotaxis and exudation in ECs.MethodsELISA, qRT-PCR, immunofluorescence, flow cytometry and transwell assays were used to investigate the effects of APOC3 on human umbilical vein endothelial cells (HUVECs). SiRNA-induced TNF-α and JAM-1 silencing were used to observe how APOC3 influenced the inflammatory process in the ECs.ResultsOur results showed that APOC3 was closely associated with the inflammatory process in ECs, and that this process was characterized by the increased expression of TNF-α. Inflammatory processes further disrupted the tight junctions (TJs) between HUVECs by causing increased expression of JAM-1. JAM-1 was involved in maintaining the integrity of TJs, and it promoted the assembly of platelets and the exudation of leukocytes. Changes in its expression promoted chemotaxis and the exudation of ECs, which contributed to atherosclerosis. While the integrity of the TJs was disrupted, the adhesion of THP-1 cells to HUVECs was also increased by APOC3.ConclusionsIn this study, we describe the mechanism by which APOC3 causes inflammation, chemotaxis and the exudation of ECs, and we suggest that controlling the inflammatory reactions that are caused by APOC3 may be a new method to treat CVD.

0180 : Role of non-muscular myosin light chain kinase (nmMLCK) in the inflammation associated with a model of intermittent hypoxia

Obstructive Sleep Apnea Syndrome (OSAS) is defined by repetitive obstructions of upper airway during sleep inducing intermittent decrease in oxygen saturation or intermittent hypoxia (IH). IH alters endothelial function favoring inflammation and could accelerate atherosclerosis-induced cardiovascular diseases. A protein that may play a role in this process is the nonmuscular myosin light chain kinase (nmMLCK), protein involved in the regulation of endothelial permeability. The aim of this study was to analyze the implication of nmMLCK in inflammation induced by IH on endothelial cells. Human aortic endothelial cells (HAoECs) were exposed to 6h of IH, including 30min of hypoxia (O2 5%) followed by 30min of normoxia (O2 21%), in absence or presence of ML-7 (5μM), a nmMLCK inhibitor, and in absence or presence of interleukin-6 (IL-6, 40ng/ml), an inflammatory cytokine found in OSAS patients. After stimulation, we investigated inflammatory process by evaluating p65-NF-κB pathway and the release of several inflammatory cytokines by HAoECs. Activation of p65-NF-κB inflammatory pathway is increased in response to IH, as well as secretion of certain inflammatory cytokines such as IL-6 and CXCL-1 in HAoECs. While nmMLCK inhibition by ML-7 did not prevent p65-NF-κB activation, it partially decreased IL-6 and CXCL-1 secretion induced by IH. Moreover, to mimic the pro-inflammatory environment already established in OSAS patients, we costimulated HAoECs with IL-6 and IH. Under these conditions, IL-6 alone increased activation of STAT3 but did not potentiate the effects of IH regarding p65-NF-κB activation and cytokine secretion. These results suggest that nmMLCK may participate to the IH-induced inflammatory process in endothelial cells by modulating IH-induced cytokine secretion. Furthermore, IH is sufficient to induce inflammatory process independently of the proinflammatory environment. The author hereby declares no conflict of interest

Recent insights into the cellular biology of atherosclerosis.

Atherosclerosis occurs in the subendothelial space (intima) of medium-sized arteries at regions of disturbed blood flow and is triggered by an interplay between endothelial dysfunction and subendothelial lipoprotein retention. Over time, this process stimulates a nonresolving inflammatory response that can cause intimal destruction, arterial thrombosis, and end-organ ischemia. Recent advances highlight important cell biological atherogenic processes, including mechanotransduction and inflammatory processes in endothelial cells, origins and contributions of lesional macrophages, and origins and phenotypic switching of lesional smooth muscle cells. These advances illustrate how in-depth mechanistic knowledge of the cellular pathobiology of atherosclerosis can lead to new ideas for therapy.

Role of mitochondria in reactive oxygen species production and inflammatory processes in endothelial cells

Role of mitochondria in reactive oxygen species production and inflammatory processes in endothelial cells

0398: Role of non-muscular myosin light chain kinase (nmMLCK) in the inflammation associated with intermittent hypoxia

Intermittent hypoxia (IH) alters endothelial function favoring inflammation and could accelerate atherosclerosis-induced cardiovascular diseases. A protein that may play a role in this process is the non-muscular myosin light chain kinase (nmMLCK). The deficiency of this kinase protects mice from death in septic choc models and prevents the atherosclerosis in mice fed with a high fat dietary. The aim of this study was to analyze the implication of nmMLCK in the vascular effects and the inflammation induced by IH. Human aortic endothelial cells (HAoECs) and endothelial cells isolated from wild type or deficient nmMLCK mice were exposed to 6 h of IH, which include 30 min of hypoxia (O2 5%) followed by 30 min of normoxia (O2 21%), in the absence or the presence of ML-7 (5 μM), a nmMLCK inhibitor. After the stimulation, we evaluated the production of the superoxide anion, nitric oxide (NO), the pro-inflammatory cytokine IL-6, and also the activation of the NF-κB pathway. IH treatment increased superoxide, NO and IL-6 production in HAoECs. However, while the nmMLCK inhibitor ML-7 had no effect on the IH-induced superoxide anion increase, it decreased both NO and IL-6 production. Furthermore, p65-NF-κB pathway was activated by IH in a ML-7-insensitive manner in HAoECs. By using aortic endothelial cells from nmMLCK deficient mice, we showed that nmMLCK deletion abolished the increase of superoxide anion induced by IH indicating that the presence and not the kinase activity of nmMLCK is crucial to regulate oxidative stress induced by IH. In conclusion, IH induced oxidative and nitrative stresses as well as changes in inflammatory secretome of human endothelial cells. In this experimental model of IH, both nitrative and pro-inflammatory cytokine secretion are sensitive to inhibition of the activity of nmMLCK and oxidative stress require its presence. These results suggest strength the notion that nmMLCK participates to the IH-induced inflammatory process in endothelial cells.

Inhibitor of Apoptosis Proteins as Novel Targets in Inflammatory Processes

Inhibitor of apoptosis proteins (IAPs), such as X-linked or cellular IAP 1/2 (XIAP, cIAP1/2), are important regulators of apoptosis. IAP antagonists are currently under clinical investigation as anticancer agents. Interestingly, IAPs participate in the inflammation-associated TNF receptor signaling complex and regulate NFκB signaling. This raises the question about the role of IAPs in inflammation. Here, we investigated the anti-inflammatory potential of IAP inhibitors and the role of IAPs in inflammatory processes of endothelial cells. In mice, the small molecule IAP antagonist A-4.10099.1 (ABT) suppressed antigen-induced arthritis, leukocyte infiltration in concanavalin A-evoked liver injury, and leukocyte transmigration in the TNFα-activated cremaster muscle. In vitro, we observed an attenuation of leukocyte-endothelial cell interaction by downregulation of the intercellular adhesion molecule-1. ABT did not impair NFκB signaling but decreased the TNFα-induced activation of the TGF-β-activated kinase 1, p38, and c-Jun N-terminal kinase. These effects are based on the proteasomal degradation of cIAP1/2 accompanied by an altered ratio of the levels of membrane-localized TNF receptor-associated factors 2 and 5. Our results reveal IAP antagonism as a profound anti-inflammatory principle in vivo and highlight IAPs as important regulators of inflammatory processes in endothelial cells.

The oxidative stress of hyperglycemia and the inflammatory process in endothelial cells

Hyperglycemia and insulin resistance are common in many critically ill patients. Hyperglycemia increases the production of reactive oxygen species in cells, stimulates the production of the potent proinflammatory cytokines IL-8 and TNF-alpha, and enhances the expression of haem oxygenase-1, an inducible stress protein. It has been shown that administration of insulin and the semi-essential amino acid glutamine have been beneficial to the septic patient. The aim of our study is to test whether these two molecules, glutamine and insulin used in combination attenuate the proinflammatory responses in endothelial cells which have been triggered by hyperglycaemia. Our results demonstrate that a combination of insulin and glutamine are significantly more effective in reducing the expression of IL-8, TNF-alpha and HO-1 than insulin or glutamine alone.

Dehydroepiandrosterone inhibits CD40/CD40L expression on human umbilical vein endothelial cells induced by interferon gamma

Many studies indicated that the CD40/CD40 ligand (CD40L) pathway plays an important role in the pathogenesis of atherosclerosis. It has been demonstrated a protective role of dehydroepiandrosterone (DHEA) against atherosclerosis. The major purpose of our present work was to assess whether DHEA could decrease the expression of CD40 and CD40L on human umbilical vein endothelial cells (HUVECs) induced by interferon gamma (IFN-gamma). We found that DHEA inhibited IFN-γ-induced expression of CD40 and CD40L in a dose-dependent manner. Moreover, DHEA inhibited IFN-γ-induced activation of extracellular signal regulated kinase (ERK1/2). The important role of ERK1/2 in DHEA effect was further confirmed by using ERK1/2 inhibitor U0126. These findings suggest that DHEA can inhibit the expression of molecules involved in the inflammatory process in endothelial cells activated with IFN-gamma. Such antagonism is at least partially mediated through the modulation of ERK1/2 pathway. Therefore, DHEA may be considered as a potential preventive intervention for atherosclerosis.

Dehydroepiandrosterone Delays LDL Oxidation In Vitro and Attenuates Several oxLDL-Induced Inflammatory Responses in Endothelial Cells

Dehydroepiandrosterone (DHEA) has a protective role against atherosclerosis, most likely mediating an anti-inflammatory action. In order to understand the mechanisms involved in this protection, we evaluated the effects of DHEA on several molecules involved in the inflammatory response. Reactive oxygen species (ROS), expression of adhesion molecules, activation of the NF-kappaB/IkappaB-alpha pathway and of the AP-1 transcription factor were evaluated in human umbilical vein endothelial cells (HUVECs) treated with oxidized low density lipoproteins (oxLDL) and DHEA. We also determined if DHEA affected LDL oxidation in vitro. 100 microM DHEA-treatment inhibited the oxLDL-induced expression of ICAM-1, VCAM-1, PECAM-1, ROS production, and U937 cells adhesion to HUVECs. DHEA also delayed the kinetics of LDL oxidation in vitro. While DHEA did not affect the translocation of NF-kappaB neither the degradation IkappaB-alpha, it led to an increased translocation of AP-1. Our results suggest that DHEA inhibits the expression of molecules involved in the inflammatory process in endothelial cells activated with oxLDL, therefore its potential anti-inflammatory properties should be evaluated for the treatment of chronic inflammatory diseases such as atherosclerosis.

Transcriptional up-regulation of the signaling regulatory protein LNK in activated endothelial cells1

A better understanding of inflammatory processes in endothelial cells (ECs) might reveal new ways of controlling inflammation and graft rejection. This study investigates EC genes regulated in response to human tumor necrosis factor (TNF)-alpha and xenogeneic natural antibodies (XNAs) that contribute to endothelial activation during transplantation. Gene expression between resting and activated ECs was investigated by RNA differential display reverse-transcriptase polymerase chain reaction and confirmed by reverse-Northern blot. Forty-five cDNA fragments corresponding to genes up-regulated in activated ECs were identified. Our findings show that TNF-alpha-mediated EC activation was associated with increased levels of mRNA for the adaptor protein Lnk, the nuclear protein RED, and the initiation factor eIF4G. We further show that Lnk and eIF4G were also up-regulated in response to XNA binding to ECs. Our data suggest that TNF-alpha and XNAs could share common signaling pathways involving Lnk and eIF4G but may also drive specific transcriptional events.

Activation and regulation of Hsp32 and Hsp70.

Endothelial cells (EC) play a key role in the propagation of inflammatory responses. Better understanding of inflammatory processes in EC might provide new ways of controlling inflammation. We report here that the known antioxidant pyrrolidinedithiocarbamate (PDTC) leads to time and dose dependent activation of heat-shock protein 70 (Hsp70) as well as Hsp32 in EC. We further demonstrate that PDTC activates heat-shock factor 1 (HSF1), one of several transcription factor involved in the upregulation of heat-shock proteins. And more importantly, we demonstrate that Hsp32 as well as Hsp70 can be upregulated independently of the transcription factor nuclear factor kappaB (NF-kappaB). The presented data provide further insight into the mechanism of Hsp32 and Hsp70 regulation, as well as further distinguishing these genes from other so called 'protective genes' whose upregulation depends on the activation of NF-kappaB. These findings indicate that Hsp32 and Hsp70 might be ideal candidates among protective genes. Hsp32 and Hsp70 provide many desirable protective effects but, being independent of NF-kappaB, would leave open the option to interfere with the upregulation of proinflammatory genes by modulating the activation of NF-kappaB.

Immunopharmacological activities and clinical development of muramyl peptides with particular emphasis on murabutide

Certain immunopharmacological activities of muramyl peptides have been associated with inflammatory and undesirable side-effects typically observed following the administration of the prototype molecule muramyl dipeptide. This activity is now demonstrated not to be linked to a direct activation of inflammatory processes in endothelial cells. Neither MDP nor other structural derivatives were able to induce inflammatory cytokines release or E-selectin gene expression in cultured human umbilical vein endothelial cells. However, oral administration of muramyl peptides has been reported to induce certain biological effects, including the downregulation of anamnestic, antigen-specific IgE responses, which are not observed following parenteral administration. We elaborate on these findings and extend them to show the efficacy of a new muramyl peptide in suppressing polyclonally induced serum IgE levels in anti-IgD-treated mice. The comparative effects of muramyl peptides, selected for clinical development, on the induction of cytokines in human whole blood are then presented at the level of mRNA accumulation and protein secretion. Moreover, the cytokine profile induced in vitro and in vivo by the combination of the safe immunostimulant, Murabutide, with interferon-α is examined. This combination reveals a selective and beneficial synergistic activity and induces anti-inflammatory cytokines in the absence of synergistic toxicity. The potential and the implications for the use of a therapeutic combination of an immunostimulant with a cytokine are discussed.