Incubation Of Human Coronary Artery Endothelial Cells Research Articles

Target induction of heme oxygenase-1 attenuates anti-HLA I antibody induced transplant vasculopathy in an allogeneic aortic transplantation model in mice

Abstract Introduction After solid organ transplantation every second patient develops transplant vasculopathy (TV), which still remains the major obstacle for long-term graft survival. The main risk factor for developing TV are de novo synthesized donor-specific antibodies binding molecules of the MHC complex (HLA complex in humans, respectively) on the surface of endothelial cells (EC). EC become activated, vascular smooth muscle cells proliferate, and monocytes are recruited, contributing the development of the neointima a hallmark of TV. We have demonstrated that the anti-HLA I antibody (w6/32) significantly increases transmigration of monocytes across an endothelial monolayer in vitro. These interactions were dependent on modulating the anti-inflammatory enzyme heme oxygenase (HO)-1. Purpose The aim of the project is to investigate which adhesion molecule might mediate the antibody-induced transmigration of monocytes sensitive to HO-1 modulation and whether this modulation has beneficial effects in an allogenic aortic transplantation model in mice. Methods Before w6/32 stimulation, HO-1 was modulated by pretreating Human Coronary Artery Endothelial Cells (HCAEC) either with CoPPIX as a HO-1 activator or with statins to test for a therapeutic approach. Expression of a variety of established adhesion receptors in HCAEC was screened after HO-1 modulation and w6/32 stimulation. Antibody-induced transmigration and adhesion of THP-1 were quantified in the presence of CD62E-blocking antibodies. Thoracic aorta was transplanted into the abdominal aorta between fully MHC mismatched mice and applying the corresponding anti-MHC I antibody (SF1-1.1) induced TV. Mice were treated with CoPPIX, statins or CORM to evaluate the effects of HO-1 modulation or its metabolite CO on TV development. Statistical evaluation was performed with t-test or ANOVA. Results HO-1 modulation in HCAEC diminished w6/32-induced THP-1 transmigration. CD62E-expression in HCAECs was enhanced after w6/32 stimulation on mRNA and protein level, but preincubation with CoPPIX or statins reduced these effects. Incubation of HCAEC with a CD62E-blocking antibody after w6/32 stimulation significantly diminished the rate of transmigration normalized to isotype-stimulated HCAEC (isotype 100%, w6/32 + IgG 226% vs. w6/32 + CD62E-block 138%, p<0.05.). Treatment of mice with CoPPIX resulted in a significant reduction of the SF1-1.1-induced number of transmigrated monocytes (SF1-1.1: 10 vs. CoPPIX: 5.4; p<0.05). Application of statins (SF1-1.1: 10 vs. statin 6.6; p<0.05) or CORM (CORM: 6.1 vs. iCORM: 11.3, p<0.05) significantly reduces monocyte number as well. In all treatment groups, the SF1-1.1-induced neointima formation was ameliorated. Conclusion w6/32-induced monocyte transmigration is CD62E-dependent. SF1-1.1-induced monocytic infiltrate and intimal hyperplasia in a murine aortic transplantation model is also CD62E dependent and amenable to HO-1 modulation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This sudy was funded by the German Research Foundation to Jan Larmann

Induction of decay accelerating factor and membrane cofactor protein by resveratrol attenuates complement deposition in human coronary artery endothelial cells

The involvement of complement activation in various forms of cardiovascular disease renders it an important factor for disease progression and therapeutic intervention. The protective effect of resveratrol against cardiovascular disease via moderate red wine consumption has been established but the exact mechanisms are still under investigation. The current study utilised human coronary artery endothelial cells (HCAECs) in order to assess the extent to which the protective effect of resveratrol, at concentrations present in red wine, can be attributed to the upregulation of complement regulatory proteins through heme-oxygenase (HO)-1 induction. Resveratrol at concentrations as low as 0.001 μΜ increased HO-1 expression as well as membrane cofactor protein (MCP, CD46) and decay-accelerating factor (DAF, CD55) expression with no-effect on CD59. Silencing of HO-1 expression by HO-1 siRNAs abrogated both DAF and MCP protein expression with no effect on CD59. Resveratrol-mediated induction of DAF and MCP reduced C3b deposition following incubation of HCAECs with 10% normal human serum or normal rat serum as a source of complement. Incubation of HCAECs, with either a DAF blocking antibody or following transfection with HO-1 siRNAs, in the presence of 10% normal rat serum increased C3b deposition, indicating that both DAF and HO-1 are required for C3b reduction. These observations support a novel mechanism for the protective effect of resveratrol against cardiovascular disease and confirm the important role of HO-1 in the regulation of the complement cascade.

The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells.

Tissue factor (TF)-positive microvesicles from various sources can promote cellular proliferation or alternatively induce apoptosis, but the determining factors are unknown. In this study the hypothesis that the ratio of fVIIa:TF within microvesicles determines this outcome was examined. Microvesicles were isolated from HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7 cell lines and microvesicle-associated fVIIa and TF antigen and activity levels were measured. Human coronary artery endothelial cells (HCAECs) were incubated with these purified microvesicles, or with combinations of fVIIa-recombinant TF, and cell proliferation/apoptosis was measured. Additionally, by expressing mCherry-PAR2 on HCAEC surface, PAR2 activation was quantified. Finally, the activation of PAR2 on HCAEC or the activities of TF and fVIIa in microvesicles were blocked prior to addition of microvesicles to cells. The purified microvesicles exhibited a range of fVIIa:TF ratios with HepG2 and 786-O cells having the highest (54:1) and lowest (10:1) ratios, respectively. The reversal from proapoptotic to proliferative was estimated to occur at a fVIIa:TF molar ratio of 15:1, but HCAEC could not be rescued at higher TF concentrations. The purified microvesicles induced HCAEC proliferation or apoptosis according to this ruling. Blocking PAR2 activation on HCAEC, or inhibiting fVIIa or TF-procoagulant function on microvesicles prevented the influence on HCAEC. Finally, incubation of HCAEC with recombinant TF resulted in increased surface exposure of fVII. The induction of cell proliferation or apoptosis by TF-positive microvesicles is dependent on the ratio of fVIIa:TF and involves the activation of PAR2. At lower TF concentrations, fVIIa can counteract the proapoptotic stimulus and induce proliferation.

Adipocytokine resistin induces tissue factor expression in human coronary artery endothelial cells via NF kB-dependent pathway

Objective: Atherosclerosis is characterized by endothelial inflammation and dysfunction. Adipose tissue has been increasingly recognized as an active endocrine organ secreting so-called adipokines, and among them resistin, recently described and not yet extensively studied, has been defined as a novel inflammatory marker in atherosclerosis. The pathophysiology underlying this interplay, however, remains not completely characterized. Aim of the study is to determine whether resistin might affect prothrombotic characteristics of human coronary artery endothelial cells (HCAECs). Methods and results: Incubation of HCAECs with resistin caused up-regulation of the Tissue Factor (TF) expression as demonstrated by FACS analysis. Moreover, TF activity was induced in a dose dependent manner, as shown by real time PCR and by a colorimetric assay. Resistin-induced TF expression was mediated by oxygen free radicals through the activation of the transcription factor, nuclear factor-kappa B (NF-kB), as demonstrated by electrophoretic mobility shift assay and by suppression of TF expression by superoxide dismutase, catalase, and by NF-kB inhibitors, PDTC and BAY 11-7082. ![Figure][1] Conclusions: These data confirm the hypothesis that resistin may contribute to athero-thrombosis exerting direct effects on HCAECs by promoting TF expression, thus representing an effector molecule able to induce a pro-thrombotic phenotype in cells present in the vessel wall. [1]: pending:yes

Abstract 328: High-Density Lipoproteins Increase miR-223 Levels in Human Endothelial Cells, Which Regulate Scavenger Receptor Class B Member 1

Background: MicroRNAs (miRNAs) are recently discovered small non-encoding RNAs that control and regulate gene expression. We and others have recently demonstrated that miRNAs play key roles in lipoprotein metabolism. Here we aim to determine if miR-223 regulates scavenger receptor class B member 1 (SR-B1) in human endothelial cells. Methods and results: In silico target prediction studies suggest that SR-BI harbors a miR-223 target site within its 3’ untranslated region (3’UTR). This possibility was investigated by determining the miRNA profile of human coronary artery endothelial cells (HCAECs) using TaqMan arrays. Mature miR-223 levels were found in HCAECs and confirmed by real-time PCR. Here we demonstrate that incubation of HCAECs with native HDL (1 mg protein/ml) for 16 h increased miR-223 levels by 4-fold increase (p<0.007) as assessed by TaqMan Assay and decreased SR-B1 mRNA levels by 29±6% (p<0.05) as measured by real-time PCR. To determine if endogenous levels of miR-223 in HCAECs are sufficient to repress SR-BI mRNA, we used hairpin inhibitors (100 nM) to block miR-223 activity. Inhibition of miR-223 resulted in an increase in SR-B1 mRNA levels by 33±11% (p<0.05). Furthermore, transfection of HCAECs with miR-223 mimic (100 nM) decreased SR-B1 mRNA levels by 27±4% (p<0.01). Transient transfection of HCAECs with miR-223 mimics also reduced SR-BI protein levels by 30±9%. Using gene reporter (luciferase) assays, we determined that miR-223 directly targets the 3’UTR of SR-B1. Conclusion: miR-223 directly targets and represses endothelial cell SR-B1 levels, and may regulate HDL-induced cholesterol efflux and lipoprotein metabolism.

Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells

Summary Histamine is a well-recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)-induced production of prostaglandin I(2) (PGI(2)), PGE(2) and interleukin-6 (IL-6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine -induced expression of Toll-like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in two-fold to fourfold increases in H1R mRNA expression in a time-dependent and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS-induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20-24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS-treated cells. The specific binding of [(3)H]pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was threefold higher than the untreated cells. The LPS-induced H1R expression was mediated through TLR4 as gene silencing by TLR4-siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre-treated with LPS for 24 hr, washed and challenged with histamine, 17-, 10- and 15-fold increases in PGI(2), PGE(2) and IL-6 production, respectively, were noted. Histamine-induced enhancement of the synthesis of PGI(2), PGE(2) and IL-6 by LPS-primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up-regulates the expression and function of H1R and amplifies histamine-induced inflammatory responses in HCAEC.

Tissue Factor Is Induced by Resistin in Human Coronary Artery Endothelial Cells by the NF-ĸB-Dependent Pathway

Objective: Atherosclerosis is characterized by endothelial inflammation and dysfunction. Adipose tissue has increasingly been recognized as an active endocrine organ secreting so-called adipokines. Among these, resistin – recently described, but not yet extensively studied – has been defined as a novel inflammatory marker in atherosclerosis. The pathophysiology underlying this interplay, however, remains to be fully characterized. The aim of the study is to determine whether resistin might affect prothrombotic characteristics of human coronary artery endothelial cells (HCAECs). Methods and Results: Incubation of HCAECs with resistin caused upregulation of tissue factor (TF) expression as demonstrated by FACS analysis. Moreover, TF activity was induced in a dose-dependent manner, as shown by real-time PCR and colorimetric assay. Resistin-induced TF expression was mediated by oxygen free radicals through the activation of the transcription factor nuclear factor-ĸB (NF-ĸB), as demonstrated by electrophoretic mobility shift assay and by suppression of TF expression by superoxide dismutase, catalase, and the NF-ĸB inhibitors PDTC and BAY 11-7082. Conclusions: These data confirm the hypothesis that resistin may contribute to atherothrombosis, exerting direct effects on HCAECs by promoting TF expression; thus, it represents an effector molecule able to induce a prothrombotic phenotype in cells present in the vessel wall.

Exendin-4 stimulates proliferation of human coronary artery endothelial cells through eNOS-, PKA- and PI3K/Akt-dependent pathways and requires GLP-1 receptor

Endothelial cells have a robust capacity to proliferate and participate in angiogenesis, which underlies the maintenance of intimal layer integrity. We previously showed the presence of the GLP-1 receptor in human coronary artery endothelial cells (HCAECs) and the ameliorative actions of GLP-1 on endothelial dysfunction in type 2 diabetic patients. Here, we have studied the effect of exendin-4 on cell proliferation and its underlying mechanisms in HCAECs. Incubation of HCAECs with exendin-4 resulted in a dose-dependent increase in DNA synthesis and an increased cell number, associated with an enhanced eNOS and Akt activation, which were inhibited by PKA, PI3K, Akt or eNOS inhibitors and abolished by a GLP-1 receptor antagonist. Similar effects were obtained by applying GLP-1 (7–36) or GLP-1 (9–36). Co-incubation of exendin-4 and GLP-1 did not show additive effects. Our results suggest that exendin-4 stimulates proliferation of HCAECs through PKA-PI3K/Akt-eNOS activation pathways via a GLP-1 receptor-dependent mechanism.

Regulation of the expression of cyclooxygenases and production of PGI2 and PGE2 in human coronary artery endothelial cells by curcumin (142.5)

Abstract Curcumin is a hydrophobic polyphenol present in turmeric (Curcuma longa). PGE2 and PGI2 are produced from arachidonic acid (AA) by the cyclooxygenases (COX1, COX2) and PGE2 synthase (PGE2S) and PGI2S. We evaluated the in vitro effect of CUR on COX1, COX2, PGI2S and PGE2S expression, and PGE2 and PGI2 production in human coronary artery endothelial cells (HCAEC). HCAEC were incubated with CUR (1.25-5.0 μM), and mRNA and protein expression were determined by qRT-PCR and Western blots, respectively. PGI2 and PGE2 levels in the culture supernatants were quantified by EIA. Incubation of HCAEC with CUR led to concentration-dependent increases in COX2 mRNA with a small but significant decrease in COX1 mRNA expression. CUR also induced 2- to 3-fold increase in PGI2S and PGE2S mRNA expression. Although CUR stimulated COX2, PGI2S and PGE2S mRNA, no increase in PGI2 or PGE2 production was noted. Addition of exogenous AA dose-dependently increased prostanoid production by both resting and CUR-treated cells. However, CUR-treated HCAEC produced significantly higher PGE2 when AA concentration was raised to 25µM. 14C-AA uptake by CUR-treated HCAEC was 30% lower than controls suggesting that CUR may inhibit exogenous AA uptake. These results show that CUR acts both as an inducer of COX2, PGI2S and PGE2S genes and a suppressor of COX1 gene. Taken together, these results suggest that CUR regulates AA metabolism and prostanoid homeostasis at both the gene and enzyme levels.

Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells (35.10)

Abstract Histamine (HIS) is a well-recognized modulator of vascular inflammation. We have shown that HIS, acting via H1 receptors (H1R), synergizes LPS-induced production of IL-6, IL-8, PGE2 and PGI2 by endothelial cells. The synergy between HIS and LPS was partly attributed to HIS-induced expression of TLR4. In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) and enhances HIS responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in 2- to 4-fold increases in H1R mRNA expression in a time and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. LPS-induced H1R expression was maximum at 4h after LPS treatment which returned to the basal level by 8h. The LPS-induced H1R gene expression was mediated through TLR4 because gene silencing by TLR4-siRNA inhibited LPS effect. The specific binding of [3H]-pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was 3-fold higher than the untreated cells. When HCAEC were pretreated with LPS for 24h, washed and subsequently challenged with HIS, 15-, 17- and 10-fold increases in IL-6, PGI2 and PGE2 production, respectively, were noted. HIS-induced syntheses of IL-6, PGE2 and PGI2 by LPS-primed HCAEC were completely blocked by the H1R antagonist cetirizine. These results demonstrate that LPS upregulates the expression and function of H1R in HCAEC leading to agonist-induced increase in IL-6, PGI2 and PGE2 production.

Curcumin stimulates cyclooxygenase-2 (COX-2), prostaglandin I2 synthase (PGI2S) and PGE2 synthase (PGE2S) gene expression in human coronary artery endothelial cells (93.19)

Abstract Curcumin is reported to decrease atherosclerosis progression in Apo-E/LDL receptor knockout mice. PGI2 and PGE2 are generated from arachidonic acid (AA) by the COX-PGI2S/PGE2S pathway and are known to have important roles in cardiovascular function. In this study, we evaluated the in vitro effect of curcumin on the expression and function of COX-2, PGI2S and PGE2S in human coronary artery endothelial cells (HCAEC). HCAEC monolayers were incubated with curcumin (1.25-10 μM), and the mRNA and protein expressions were monitored by qRT-PCR and Western blots, respectively. PGI2 and PGE2 levels in the culture supernatants were quantified by EIA. Incubation of HCAEC with curcumin led to concentration-dependent increases in COX-2 mRNA and protein expression without change in COX-1 expression. Curcumin also induced 2-fold increases in PGI2S and PGE2S gene expression. Despite the increase in COX-2, PGI2S and PGE2S gene expression, curcumin failed to increase PGI2 or PGE2 production. Furthermore, curcumin inhibited LPS-induced PGE2 and PGI2 production by HCAEC and exogenous AA could not reverse the inhibitory effect. These results show that curcumin acts both as an inducer of COX-2, PGI2S and PGE2S gene expression and as an inhibitor of stimulated PGE2 and PGI2 synthesis. The ability of curcumin to regulate prostanoid biosynthesis in HCAEC indicates its therapeutic potential for cardiovascular disease. (Supported by NIH R01-HL070101 and the Carey Arthritis Fund).

Histamine‐induced Cyclooxygenase‐2 Expression in Human Coronary Artery Endothelial Cells is Mediated Through MAP Kinases

Previous studies from our laboratory showed that treatment of human coronary artery endothelial cells (HCAEC) with histamine led to overexpression of cyclooxygenase‐2 (COX‐2) gene and protein (but not COX‐1), and increased production of its products PGE2 and PGI2 (Tan et al, J.Immunol, 179: 7899–7906, 2007). In this study, we investigated the involvement of MAP kinases in the signaling mechanisms leading to the histamine‐mediated COX‐2 overexpression in HCAEC. Treatment of HCAEC with histamine (10μg/ml) resulted in multi‐fold increases in the phosphorylation of p38 MAPK, ERK and JNK. Histamine‐induced phosphorylation of these proteins continued for 10–15 minutes post treatment, after which the phosphorylation levels returned to the basal levels. Incubation of HCAEC with pharmacological inhibitors of the individual MAP Kinases inhibited histamine‐induced COX‐2 gene expression as quantified by real time PCR. The inhibitors also abrogated phosphorylation of specific MAP Kinases confirming the involvement of MAP Kinase pathway in COX‐2 overexpression. In addition, inhibition of the MAP Kinases also inhibited HCAEC generation of prostacyclin (PGI2) confirming that histamine mediated phosphorylation of MAP Kinases is involved in the PGI2 production in HCAEC. These results indicate that histamine‐mediated overexpression of COX‐2 and the resultant production of PGI2 require p38, ERK, and JNK activation, and inhibition of any of these MAP kinases can decrease the expression of COX‐2 and the production of PGI2. (Supported by NIH R01‐HL070101 and Carey Arthritis Fund)

Lysoplasmenylcholine increases neutrophil adherence to human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of human coronary artery endothelial cells (HCAEC) results in release of choline lysophospholipids [lysophosphatidylcholine (lysoPtdCho) and lysoplasmenylcholine (lysoPlsCho)]. These amphiphilic metabolites have been implicated in arrhythmogenesis following the onset of myocardial ischemia, but studies examining their direct effects on the vasculature remain limited. We and others have shown that thrombin and lysoPtdCho can increase cell surface adhesion molecules and adherence of circulating inflammatory cells to the endothelium. This study supports our hypothesis that these changes may be mediated, at least in part, by lysoPlsCho, thus implicating this metabolite as an inflammatory mediator in the coronary vasculature and a modulator of the progression of atherosclerosis. Apical stimulation of HCAEC with thrombin resulted in the production and release of choline lysophospholipids from the apical surface of the HCAEC monolayer. Basolateral stimulation had no effect on choline lysophospholipid production or release from either the apical or basolateral surface of the HCAEC monolayer. Incubation of HCAEC with lysoPlsCho or lysoPtdCho resulted in similar increases in HCAEC surface expression of P-selectin and E-selectin. Furthermore, lysoPlsCho increased cell surface expression of P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 with a time course similar to that of thrombin stimulation. Increased presence of cell surface adhesion molecules may contribute to the significant increase in adherence of neutrophils to either thrombin- or lysoPlsCho-stimulated HCAEC. These results demonstrate that the presence of thrombin at sites of vascular injury in the coronary circulation, resulting in increased choline lysophospholipid release from the HCAEC apical surface, has the potential to propagate vascular inflammation by upregulation of adhesion molecules and recruitment of circulating inflammatory cells to the endothelium.

Calcium-independent phospholipase A2 is regulated by a novel protein kinase C in human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 +/- 0.1 vs. 5.3 +/- 0.4 nmol PO4/mg of protein) and plasmenylethanolamine (7.5 +/- 1.0 vs. 12.0 +/- 0.9 nmol PO4/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 +/- 0.3 nmol PO4/mg of protein) and plasmenylethanolamine (6.0 +/- 1.0 nmol PO4/mg of protein). Incubation of HCAEC with the selective iPLA2 inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 +/- 0.3 vs. 1.1 +/- 0.1%) and increased production of lysoplasmenylcholine (1.4 +/- 0.2 vs. 0.6 +/- 0.1 nmol PO4/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 +/- 0.6 to 0.5 +/- 0.1%) and lysoplasmenylcholine production (2.0 +/- 0.1 to 0.2 +/- 0.1 nmol PO4/mg of protein). These data suggest that PKC activates iPLA2 in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production.

Histamine-Induced Production of Interleukin-6 and Interleukin-8 by Human Coronary Artery Endothelial Cells Is Enhanced by Endotoxin and Tumor Necrosis Factor-α

In this study, we tested the synergy between histamine and LPS, and histamine and TNF-α, on endothelial cell production of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). Human coronary artery endothelial cells (HCAEC) were cultured in vitro with histamine (0.1 to 1000 μM) in the presence or absence of LPS or TNF-α for 24 h, and the secreted IL-6, IL-8 and MCP-1 were quantified. Unactivated HCAEC produced minimal levels of IL-6, IL-8, or MCP-1. The incubation of HCAEC with histamine resulted in low level induction of IL-6 and IL-8 production, which was dose-dependent and attained a plateau at a concentration of 10 μM. On the other hand, histamine failed to induce MCP-1 production. Stimulation of HCAEC with LPS or TNF-α caused dose-dependent increase in cytokine production. In the presence of all stimulatory concentrations of LPS and TNF-α tested, histamine was shown to further enhance IL-6 and IL-8 production. The effect of histamine on endothelial cell production of cytokines was completely inhibited by the H-1 receptor antagonist, diphenhydramine, and not by the H-2 antagonist, famotidine. Electrophoretic mobility shift assays of nuclear proteins extracted from HCAEC treated with histamine and LPS, or histamine and TNF-α, revealed amplified translocation of NF-κB proteins to the nuclei. Since both LPS and TNF-α potentiated histamine-induced cytokine production, it is possible that these activators stimulate H-1 receptor expression and/or augment the signal transduction pathways leading to the expression of IL-6 and IL-8. These results indicate the importance of synergy between histamine and other inflammatory stimuli on endothelial cell activation and implicate their cooperative participation in vascular leak and inflammation.

Facilitative interaction between angiotensin II and oxidised LDL in cultured human coronary artery endothelial cells.

Background Several studies have shown that angiotensin II (Ang II) and oxidised low-density lipoprotein (ox-LDL) are critical factors in atherosclerosis. In this study, we examined the molecular basis of mutually facilitative interactions between Ang II and ox-LDL in human coronary artery endothelial cells (HCAECs). Methods and results We observed that incubation of cultured HCAECs with Ang II (10-12 to 10-6 M) for 24 hours caused a concentration-dependent increase in the expression of mRNA and protein of a specialised receptor for ox-LDL (LOX-1). These effects of Ang II were completely blocked by pretreatment of HCAECs with candesartan (10-6 M), a specific AT1-receptor blocker, but not by PD 123319 (10-6 M), a specific AT2-receptor blocker. On the other hand, incubation of HCAECs with ox-LDL (10 and 40 µg/ml) for 24 hours progressively upregulated AT1-, but not AT 2-, receptor mRNA and protein. Pretreatment of cells with the anti-oxidant alpha-tocopherol (1-5 x 10-6 M) inhibited the upregulation of AT1-receptor expression induced by ox-LDL (p<0.05). To determine the significance of expression of AT1-receptors and LOX-1, we measured cell injury in response to Ang II and ox-LDL. Incubation of cells with both ox-LDL and Ang II synergistically increased cell injury, measured as cell viability and LDH release, compared with either ox-LDL or Ang II alone (both p<0.05). Alpha-tocopherol, as well as candesartan, attenuated cell injury in response to Ang II and ox-LDL (both p<0.05). Conclusions These observations show that Ang II upregulates a novel endothelial receptor for ox-LDL (LOX-1) gene expression and ox-LDL in turn upregulates Ang II AT 1receptor gene expression. This interaction between Ang II and ox-LDL further augments cell injury in HCAECs. These findings provide basis for the use of AT1-receptor blockers and anti-oxidants in designing therapy for atherosclerosis and myocardial ischaemia.

Effects of alpha-tocopherol on ox-LDL-mediated degradation of IkappaB and apoptosis in cultured human coronary artery endothelial cells.

Experimental studies have demonstrated that vitamin E (alpha-tocopherol) may provide significant cytoprotection during cell injury. In this study, we examined the effects of alpha-tocopherol on oxidized low-density lipoprotein (ox-LDL)-induced apoptosis in human coronary artery endothelial cells (HCAECs). In addition, we examined the activation of NF-kappaB pathway in this process. Cultured HCAECs were treated with ox-LDL for 24 h. Incubation of HCAECs with ox-LDL resulted in apoptosis of HCAECs in a concentration-dependent manner, as determined by TUNEL and DNA laddering. Ox-LDL degraded IkappaB and activated NF-kappaB in HCAECs, as determined by Western blot analysis. Treatment with alpha-tocopherol (10 and 50 microM) decreased ox-LDL-mediated apoptosis as well as degradation of IkappaB and activation of NF-kappaB in HCAECs. High concentration of alpha-tocopherol (50 microM) was more effective than the low concentration of alpha-tocopherol (10 microM). Thus, ox-LDL induces apoptosis of HCAECs, in concurrence with degradation of IkappaB and activation of NF-kappaB. Alpha-tocopherol markedly decreases ox-LDL-induced effects.

Antisense to LOX-1 Inhibits Oxidized LDL–Mediated Upregulation of Monocyte Chemoattractant Protein-1 and Monocyte Adhesion to Human Coronary Artery Endothelial Cells

We have recently demonstrated a lectin-like receptor for oxidized (ox)-LDL (LOX-1) in human coronary artery endothelial cells (HCAECs). This receptor is upregulated by ox-LDL. The present study examined the significance of LOX-1 in monocyte adhesion to HCAECs and endothelial injury in response to ox-LDL. HCAECs were incubated in the presence of antisense oligodeoxynucleotides to the 5'-coding sequence of the human LOX-1 gene (0.5 microm/L). Basal LOX-1 mRNA and protein were suppressed by antisense LOX-1. Ox-LDL-mediated upregulation of LOX-1 was also suppressed by antisense LOX-1. Incubation of HCAECs with ox-LDL (40 microg/mL) for 24 hours markedly increased monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression as well as monocyte adhesion to HCAECs (P<0.01). After 48 hours of preincubation of HCAECs with antisense LOX-1, ox-LDL-mediated upregulation of MCP-1 and monocyte adhesion to HCAECs both were suppressed (P<0.01), whereas sense LOX-1 had no effect. Whereas antisense or sense LOX-1 alone (both 0.5 nmol/L) did not injure the cells, antisense LOX-1, but not sense LOX-1, reduced ox-LDL-mediated HCAEC injury, determined as LDH release (P<0.01). Activation of mitogen-activated protein kinase (MAPK) may play a critical role in signal transduction in ox-LDL-mediated alteration in MCP-1 expression, since antisense LOX-1, but not the sense LOX-1, completely inhibited the ox-LDL-induced MAPK activation. These observations with the first use of a specific antisense to human LOX-1 mRNA suggest that LOX-1 is a key factor in ox-LDL-mediated monocyte adhesion to HCAECs.

Upregulation of endothelial receptor for oxidized LDL (LOX-1) by oxidized LDL and implications in apoptosis of human coronary artery endothelial cells: evidence from use of antisense LOX-1 mRNA and chemical inhibitors.

A specific lectin-like endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL-induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 microg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL-mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL-mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL-mediated apoptosis of HCAECs. Nuclear factor (NF)-kappaB was markedly activated in ox-LDL-treated HCAECs. The critical role of NF-kappaB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL-mediated NF-kappaB activation. In this process, an NF-kappaB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL-mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL-induced apoptosis is mediated by the action of LOX-1. In this process, NF-kappaB activation may play an important role as a signal transduction mechanism.

Upregulation of endothelial receptor for oxidized low-density lipoprotein (LOX-1) in cultured human coronary artery endothelial cells by angiotensin II type 1 receptor activation.

Cross talk between oxidized LDL (ox-LDL) and angiotensin II (Ang II) may be relevant in atherosclerosis. In this study, we examined the presence of a specific endothelial receptor for ox-LDL (LOX-1) and Ang II receptors in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of Ang II on LOX-1 gene and protein expression. LOX-1 was consistently identified in HCAECs by reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA sequence, Western blot, and 125I-labeled ox-LDL binding assay (Bmax, 29.7 ng/mg protein). The HCAECs also exhibited Ang II receptors (AT1>AT2), as determined by RT-PCR and 125I-labeled Ang II binding assay (Bmax, 2.21 and 1.19 fmol/mg protein, respectively). Incubation of HCAECs with Ang II markedly increased LOX-1 mRNA (RT-PCR) and protein (Western blot) expression. The increase in LOX-1 expression was dependent on Ang II concentration (10(-12) to 10(-6) mol/L). Ang II caused a concentration-dependent increase in 125I-labeled ox-LDL uptake by HCAECs and enhanced ox-LDL-mediated cell injury, as evident from an increase in LDH release and a decrease in cell viability. These effects of Ang II were completely blocked by pretreatment of HCAECs with losartan, a specific AT1 blocker, but not by PD123319, a specific AT2 blocker. These observations indicate the following: (1) HCAECs possess abundant LOX-1 as well as Ang II (AT1>AT2) receptors, (2) Ang II upregulates LOX-1 receptor and ox-LDL uptake, (3) the effects of Ang II are mediated by AT1 activation, and (4) Ang II enhances ox-LDL-mediated injury to HCAECs.