CD40 ligand increases expression of its receptor CD40 in human coronary artery endothelial cells

doi:10.1016/j.surg.2006.03.016

Surgery

Volume 140, Issue 2, August 2006, Pages 236-242

Presented at the 67^th Annual Meeting of the Society of University Surgeons, First Annual Academic Surgical Congress, February 7-11, 2006, San Diego, California.

https://doi.org/10.1016/j.surg.2006.03.016 Get rights and content

Background

Recently, CD40 ligand (CD40L) and its receptor CD40 have been implicated in atherosclerosis. Clinical data showed that elevated CD40L levels are associated with a high risk of cardiovascular events. The aim of this study was to investigate whether CD40L could affect the expression of its membrane receptor CD40 as a feedback mechanism by which CD40L could enhance its functions in human coronary artery endothelial cells (HCAECs).

Methods

The HCAECs were treated with human soluble CD40L, and the messenger RNA (mRNA) and protein levels of CD40 were determined by real-time polymerase chain reaction and Western blot analysis, respectively. The specific effect of CD40L was confirmed by a blocking experiment with antibody against CD40L. Involvements of oxidative stress and mitogen-activated protein kinases (MAPKs) were also studied with antioxidant seleno-L-methionine (SeMet) and MAPK inhibitors such as extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor.

Results

When HCAECs were cultured with CD40L (5 μg/mL) for 24 hours, CD40 mRNA levels were increased by 79% compared with controls (P < .05). Similarly, Western blot analysis showed an 80% increase in CD40 protein levels (P < .05). The CD40L-induced increase in CD40 mRNA levels were blocked specifically by anti-CD40L antibody. Antioxidant SeMet and specific ERK1/2 inhibitor (PD98059) also effectively blocked CD40L-induced CD40 mRNA increase.

Conclusions

These data demonstrate that clinically relevant concentration of CD40L increased the expression of its receptor CD40 in HCAECs. The CD40L-induced upregulation of CD40 may be mediated by oxidative stress and ERK1/2 activation. This study suggests a new mechanism by which CD40L could enhance its biologic functions in the vascular system and contribute to endothelial dysfunction and vascular disease.

Section snippets

Chemicals and reagents

Recombinant human soluble CD40L was obtained from PeproTech (Rocky Hill, NJ). The endotoxin level in CD40L is less than 0.1 ng/μg (1EU/μg). TriReagent kit, trypsin/EDTA, SeMet, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), rabbit IgG1 (isotype control immunoglobulin), and μ-actin monoclonal antibody were obtained from Sigma Chemical Co (St. Louis, Mo). Antibody against human CD40L was obtained from Abcam (Cambridge, Mass). The enhanced chemiluminescence kit (ECL-plus) was

CD40L increases the expression of CD40 in HCAECs

The HCAECs were treated with 5 μg/mL of CD40L for 24 hours. CD40 mRNA and protein levels were detected with the use of real-time PCR (n = 3) and Western blot analysis (n = 3), respectively. We did not see any change in cell morphology and viability before and after treatment. After treatment, CD40 mRNA levels were increased significantly from 0.0017 in controls to 0.0030 in CD40L-treated groups (79%, P < .05, t test). Western blot analysis also showed an 80% increase in CD40 compared with

Discussion

In the current study, we have tested our hypothesis that CD40L may upregulate expression of its receptor CD40, thereby enhancing its biologic functions in human endothelial cells. Our findings demonstrate that recombinant human soluble CD40L at the clinically relevant concentration induces overexpression of CD40 in HCAECs. This overexpression is confirmed at both mRNA and protein levels. In addition, an antioxidant and a specific inhibitor of ERK1/2 effectively block this CD40L-induced

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The role of CD40L and VEGF in the modulation of angiogenesis and inflammation
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Recently, there has been growing interest in deciphering the role of angiogenesis in the progression of atherogenesis. Importantly, CD40–CD40L interactions are of significant relevance because of their involvement in both angiogenesis and atherosclerotic development. Previously, we have shown that recombinant soluble CD40 ligand (rsCD40L) stimulates auto-inflammatory CD40L synthesis and reactive oxygen species (ROS) generation in vascular cells. In the current study, we demonstrate that redox-mediated CD40–CD40L interaction can enhance vascular endothelial growth factor (VEGF)-induced angiogenesis, endothelial migration, and actin polymerization processes. Interestingly, the addition of exogenous VEGF leads to cleavage of de novo CD40L produced in endothelial cells following rsCD40L treatment. Using inhibitor and silencing RNA-based experiments, it was observed that VEGF-induced protease, calpain 2, was responsible for the cleavage of de novo CD40L. While our in vivo experiments using a matrigel plug assay indicate a VEGF and CD40L induced enhancement of angiogenesis, our studies also identify a novel mechanism by which VEGF can abrogate CD40L-mediated inflammation. Together, these studies reveal a new pathway by which VEGF–CD40L interactions can regulate the angiogenic and inflammatory process depending on the specific environment.
Soluble CD40 ligand induces human coronary artery smooth muscle cells proliferation and migration
2009, Surgery
Citation Excerpt :
Human sCD40L levels usually range from picograms per milliliter (pg/mL) to nanograms per milliliter (ng/mL). In the current study, we used much greater concentrations (1 or 5 μg/mL), which were similar to the concentrations used by our previous investigation on endothelial cells and studies by others.11,15,18,19 Our data showed that sCD40L at 5 μg/mL increased both proliferation and migration of human VSMCs.
Clinical data have shown that an increased level of serum soluble CD40 ligand (sCD40L) is associated with atherosclerogenesis. We hypothesize that sCD40L induces proliferation and migration of vascular smooth muscle cells (VSMCs) through activation of matrix metalloproteinases (MMPs).
Human VSMCs were treated with sCD40L (1 or 5 μg/mL). Cell proliferation and migration were studied using a nonradioactive cell proliferation assay (MTT) and a modified Boyden chamber combined with a scrape-wound assay, respectively. Messenger RNA (mRNA) and protein levels of MMP-2 and MMP-9 were measured with real-time polymerase chain reaction and enzyme-linked immunosorbent assays. Neutralizing antibodies against MMP-2 or MMP-9 were used to evaluate their effects on sCD40L–induced cell proliferation and migration.
MTT assay showed a 35% increase in cell proliferation in the high-dose (5 μg/mL) sCD40L-treated group. Cell migration was also increased by 33% (Transwell assay) to 3-fold (scrape-wound assay) after high-dose sCD40L treatment. When cells were treated with 5 μg/mL of sCD40L for 24 hours, significant decreases in MMP-2 and increases in MMP-9 mRNA and protein levels were observed. Neutralizing antibodies against MMP-9 effectively blocked sCD40L–induced cell proliferation and migration.
This study suggests that sCD40L increases VSMC proliferation and migration through the MMP-9 pathway, which may be a potential mechanism through which sCD40L induces intimal hyperplasia and atherosclerosis.
Soluble CD40 ligand induces endothelial dysfunction in human and porcine coronary artery endothelial cells
2008, Blood
Citation Excerpt :
In the present study, we demonstrate that p38 and ERK2 are activated in response to sCD40L stimulation in human endothelial cells, and p38 inhibitor SB239063 and ERK1/2 inhibitor PD98059 effectively block sCD40L-induced eNOS down-regulation at both mRNA and protein levels. These new data are consistent with our previous report that sCD40L-induced up-regulation of CD40 in HCAECs is mediated by enhanced O2− production and ERK1/2 activation.36 However, a recent study showed that sCD40L was also able to activate JNK in HUVECs.
The purpose of this study was to determine the effects and mechanisms of sCD40L on endothelial dysfunction in both human coronary artery endothelial cells (HCAECs) and porcine coronary artery rings. HCAECs treated with sCD40L showed significant reductions of endothelial nitric oxide synthase (eNOS) mRNA and protein levels, eNOS mRNA stability, eNOS enzyme activity, and cellular NO levels, whereas superoxide anion (O₂⁻) production was significantly increased. sCD40L enhanced eNOS mRNA 3′UTR binding to cytoplasmic molecules and induced a unique expression pattern of 95 microRNAs. sCD40L significantly decreased mitochondrial membrane potential, and catalase and SOD activities, whereas it increased NADPH oxidase (NOX) activity. sCD40L increased phosphorylation of MAPKs p38 and ERK1/2 as well as IκBα and enhanced NF-κB nuclear translocation. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and eNOS mRNA levels, whereas it increased O₂⁻ levels. Antioxidant seleno-l-methionine; chemical inhibitors of p38, ERK1/2, and mitochondrial complex II; as well as dominant negative mutant forms of IκBα and NOX4 effectively blocked sCD40L-induced eNOS down-regulation in HCAECs. Thus, sCD40L reduces eNOS levels, whereas it increases oxidative stress through the unique molecular mechanisms involving eNOS mRNA stability, 3′UTR-binding molecules, microRNAs, mitochondrial function, ROS-related enzymes, p38, ERK1/2, and NF-κB signal pathways in endothelial cells.
Increased expression of inflammation-related co-stimulatory molecules by HUVECs from newborns with a strong family history of myocardial infarction stimulated with TNF-α and oxLDL
2007, Immunology Letters
Citation Excerpt :
Besides, CD40 can also mediate induction of chemotactic mediators such as CXCL8 and monocyte chemotactic protein-1 in endothelial cells [28]. It has recently been demonstrated that soluble CD40L enhances the expression of CD40 on endothelial cells by oxidative stress and ERK1/2 activation [29], thus reinforcing the importance of CD40/CD40L interaction in the progression of atherosclerosis and other vascular diseases. Nevertheless, we acknowledge that the increased adhesion of Jurkat and U-937 cells to HUVECs, which represent a venous endothelium, may not be entirely representative of the response of other endothelia, since other endothelial cells have different phenotypic and functional characteristics as well as expression levels of adhesion molecules under basal and cytokine-stimulated conditions [30].
Recent findings indicate that atherosclerosis, a chronic inflammatory process, might start during childhood. Nevertheless, the expression of inflammation-related molecules of endothelial cell isolated from healthy neonates with a strong family history of myocardial infarction (SFHMI) has been rarely analyzed.
Human umbilical vein endothelial cells (HUVECs) from children with SFHMI were assessed for the expression of CD40 and CD40L, in the presence of TNF-α and oxLDL. The intracellular content of CD80, CXCL8 and tissue factor by HUVECs stimulated with a CD40 agonist monoclonal antibody as well as monocytes/lymphocyte adhesion to TNF-α-stimulated HUVECs was also evaluated.
The basal expression of CD40 and CD40L was higher in SFHMI-positive HUVECs in comparison to controls. TNF-α and oxLDL upregulated the expression of CD40 and CD40L in SFHMI versus control HUVECs (p < 0.001). The intracellular expression of CXCL8, tissue factor and CD80 was also higher than in controls, and the adhesion of lymphocyte- and monocyte-like cells augmented upon TNF-α stimulation.
It is possible that the modifications observed in the SFHMI-positive HUVECs, all of them relevant to the atherosclerosis process, may lead to early inflammatory reactions, thus contributing to the premature initiation of atherosclerotic lesions in these children.
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: Supported in part by research grants from the National Institutes of Health (DE15543 and AT003094 to Q.Y.; HL076345 to P.H.L.; HL65916, HL72716, EB-002436, and HL083471 to C.C.).

View full text

Society of University SurgeonCD40 ligand increases expression of its receptor CD40 in human coronary artery endothelial cells

Background

Methods

Results

Conclusions

Section snippets

Chemicals and reagents

CD40L increases the expression of CD40 in HCAECs

Discussion

Cell Immunol

Am J Pathol

J Surg Res

Trends Biochem Sci

CD40-tumor necrosis factor receptor-associated factor (TRAF) interactionsregulation of CD40 signaling through multiple TRAF binding sites and TRAF hetero-oligomerization

Biochemistry

Reactive oxygen species play roles on B cell surface receptor CD40-mediated proximal and distal signaling eventseffects of an antioxidant, N-acetyl-L-cysteine treatment

Mol Cell Biochem

The CD40/CD154 receptor/ligand dyad

Cell Mol Life Sci

CD40 liganda novel target in the fight against cardiovascular disease

Ann Pharmacother

CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells

Nature

Platelet-derived CD40Lthe switch-hitting player of cardiovascular disease

Circulation

p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes

J Immunol

Post-transcriptional inhibition of CD40 gene expression in microglia by transforming growth factor-beta

Eur J Immunol

Society of University Surgeon
CD40 ligand increases expression of its receptor CD40 in human coronary artery endothelial cells