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Transcription and translation of human F11R gene are required for an initial step of atherogenesis induced by inflammatory cytokines.

Azari BM, Marmur JD, Salifu MO, Ehrlich YH, Kornecki E, Babinska A - J Transl Med (2011)

Bottom Line: Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction.Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cardiology, Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, USA.

ABSTRACT

Background: The F11 Receptor (F11R; aka JAM-A, JAM-1) is a cell adhesion protein present constitutively on the membrane surface of circulating platelets and within tight junctions of endothelial cells (ECs). Previous reports demonstrated that exposure of ECs to pro-inflammatory cytokines causes insertion of F11R molecules into the luminal surface of ECs, ensuing with homologous interactions between F11R molecules of platelets and ECs, and a resultant adhesion of platelets to the inflamed ECs. The main new finding of the present report is that the first step in this chain of events is the de-novo transcription and translation of F11R molecules, induced in ECs by exposure to inflammatory cytokines.

Methods: The experimental approach utilized isolated, washed human platelet suspensions and cultured human venous endothelial cells (HUVEC) and human arterial endothelial cells (HAEC) exposed to the proinflammatory cytokines TNF-alpha and/or IFN-gamma, for examination of the ability of human platelets to adhere to the inflamed ECs thru the F11R. Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.

Results: Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction. Transfection of ECs with F11R siRNAs caused complete inhibition of the cytokine-induced upregulation of F11R mRNA and inhibition of detection of the newly- translated F11R molecules in cytokine-inflamed ECs. The functional consequence of the inhibition of F11R transcription and translation was the significant blockade of the adhesion of human platelets to inflamed ECs.

Conclusion: These results prove that de novo synthesis of F11R in ECs is required for the adhesion of platelets to inflamed ECs. Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

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De novo expression of F11R mRNA in inflamed endothelial cells: blockade of F11R mRNA expression in endothelial cells treated with TNFα and IFNγ by the RNA synthesis inhibitor, actinomycin. Confluent monolayers of HUVEC were maintained under Untreated conditions, or pretreated with the RNA synthesis inhibitor, actinomycin D (ActD) (5 μg/mL), in growth supplement-free media for 1 hr at 37°C. The response of HUVEC maintained in the presence of ActD alone is shown in histogram labeled ActD. The response of HUVEC treated with TNFα alone(100 u/ml) is shown in Figure 3a, and the response of HUVEC treated with IFNγ alone(200 u/ml) for 24 hrs is shown in Figure 3b. The response of HUVEC pretreated with ActD prior to 24 hr exposure to either TNFα (100 u/mL) or IFNγ (200 u/mL), is shown in the histograms labeled TNFα & ActD (see Figure 3a) or IFNγ & ActD (see Figure 3b). The F11R mRNA levels were measured by Real-Time PCR in triplicate for each condition. Values are the mean ± SEM. * P < 0.05 significant differences in F11R mRNA observed between cells exposed to TNFα or IFNγ alone vs ECs treated (or not treated) with ActD alone or ECs treated with ActD followed by their exposure to either TNFα or IFNγ.
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Figure 3: De novo expression of F11R mRNA in inflamed endothelial cells: blockade of F11R mRNA expression in endothelial cells treated with TNFα and IFNγ by the RNA synthesis inhibitor, actinomycin. Confluent monolayers of HUVEC were maintained under Untreated conditions, or pretreated with the RNA synthesis inhibitor, actinomycin D (ActD) (5 μg/mL), in growth supplement-free media for 1 hr at 37°C. The response of HUVEC maintained in the presence of ActD alone is shown in histogram labeled ActD. The response of HUVEC treated with TNFα alone(100 u/ml) is shown in Figure 3a, and the response of HUVEC treated with IFNγ alone(200 u/ml) for 24 hrs is shown in Figure 3b. The response of HUVEC pretreated with ActD prior to 24 hr exposure to either TNFα (100 u/mL) or IFNγ (200 u/mL), is shown in the histograms labeled TNFα & ActD (see Figure 3a) or IFNγ & ActD (see Figure 3b). The F11R mRNA levels were measured by Real-Time PCR in triplicate for each condition. Values are the mean ± SEM. * P < 0.05 significant differences in F11R mRNA observed between cells exposed to TNFα or IFNγ alone vs ECs treated (or not treated) with ActD alone or ECs treated with ActD followed by their exposure to either TNFα or IFNγ.

Mentions: We examined whether the observed increases in the level of F11R mRNA in inflamed endothelial cells resulted from the de novo expression of F11R by conducting experiments involving the pretreatment of endothelial cells with the RNA synthesis inhibitor actinomycin D (5 μg/ml). Endothelial cells were pretreated (or not pretreated) with actinomycin D for a period of 1 hr at 37°C prior to their exposure to either TNFα or IFNγ. Cells that were not pretreated with actinomycin (ActD) demonstrated a significant increase in the level of F11R mRNA following their exposure to TNFα, as shown in Figure 3a (TFNα), whereas cells pretreated with ActD were unable to demonstrate the induced increase in the level of F11R mRNA induced by TNFα treatment, and a complete inhibition was observed (see TNFα & ActD). Pretreatment of cells with actinomycin D alone did not produce a decrease in basal levels of F11RmRNA (see ActD) as identical values to the basal levels measured in untreated cells were obtained. Similar to the results observed with TNFα, venous cells treated with IFNγ (200 u/ml) (as shown in Figure 3b, IFNγ) demonstrated a significant rise in their level of F11R mRNA; such an increase in F11R mRNA level could be completely blocked by the presence of ActD (see Figure 3b, IFNγ & ActD),


Transcription and translation of human F11R gene are required for an initial step of atherogenesis induced by inflammatory cytokines.

Azari BM, Marmur JD, Salifu MO, Ehrlich YH, Kornecki E, Babinska A - J Transl Med (2011)

De novo expression of F11R mRNA in inflamed endothelial cells: blockade of F11R mRNA expression in endothelial cells treated with TNFα and IFNγ by the RNA synthesis inhibitor, actinomycin. Confluent monolayers of HUVEC were maintained under Untreated conditions, or pretreated with the RNA synthesis inhibitor, actinomycin D (ActD) (5 μg/mL), in growth supplement-free media for 1 hr at 37°C. The response of HUVEC maintained in the presence of ActD alone is shown in histogram labeled ActD. The response of HUVEC treated with TNFα alone(100 u/ml) is shown in Figure 3a, and the response of HUVEC treated with IFNγ alone(200 u/ml) for 24 hrs is shown in Figure 3b. The response of HUVEC pretreated with ActD prior to 24 hr exposure to either TNFα (100 u/mL) or IFNγ (200 u/mL), is shown in the histograms labeled TNFα & ActD (see Figure 3a) or IFNγ & ActD (see Figure 3b). The F11R mRNA levels were measured by Real-Time PCR in triplicate for each condition. Values are the mean ± SEM. * P < 0.05 significant differences in F11R mRNA observed between cells exposed to TNFα or IFNγ alone vs ECs treated (or not treated) with ActD alone or ECs treated with ActD followed by their exposure to either TNFα or IFNγ.
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Figure 3: De novo expression of F11R mRNA in inflamed endothelial cells: blockade of F11R mRNA expression in endothelial cells treated with TNFα and IFNγ by the RNA synthesis inhibitor, actinomycin. Confluent monolayers of HUVEC were maintained under Untreated conditions, or pretreated with the RNA synthesis inhibitor, actinomycin D (ActD) (5 μg/mL), in growth supplement-free media for 1 hr at 37°C. The response of HUVEC maintained in the presence of ActD alone is shown in histogram labeled ActD. The response of HUVEC treated with TNFα alone(100 u/ml) is shown in Figure 3a, and the response of HUVEC treated with IFNγ alone(200 u/ml) for 24 hrs is shown in Figure 3b. The response of HUVEC pretreated with ActD prior to 24 hr exposure to either TNFα (100 u/mL) or IFNγ (200 u/mL), is shown in the histograms labeled TNFα & ActD (see Figure 3a) or IFNγ & ActD (see Figure 3b). The F11R mRNA levels were measured by Real-Time PCR in triplicate for each condition. Values are the mean ± SEM. * P < 0.05 significant differences in F11R mRNA observed between cells exposed to TNFα or IFNγ alone vs ECs treated (or not treated) with ActD alone or ECs treated with ActD followed by their exposure to either TNFα or IFNγ.
Mentions: We examined whether the observed increases in the level of F11R mRNA in inflamed endothelial cells resulted from the de novo expression of F11R by conducting experiments involving the pretreatment of endothelial cells with the RNA synthesis inhibitor actinomycin D (5 μg/ml). Endothelial cells were pretreated (or not pretreated) with actinomycin D for a period of 1 hr at 37°C prior to their exposure to either TNFα or IFNγ. Cells that were not pretreated with actinomycin (ActD) demonstrated a significant increase in the level of F11R mRNA following their exposure to TNFα, as shown in Figure 3a (TFNα), whereas cells pretreated with ActD were unable to demonstrate the induced increase in the level of F11R mRNA induced by TNFα treatment, and a complete inhibition was observed (see TNFα & ActD). Pretreatment of cells with actinomycin D alone did not produce a decrease in basal levels of F11RmRNA (see ActD) as identical values to the basal levels measured in untreated cells were obtained. Similar to the results observed with TNFα, venous cells treated with IFNγ (200 u/ml) (as shown in Figure 3b, IFNγ) demonstrated a significant rise in their level of F11R mRNA; such an increase in F11R mRNA level could be completely blocked by the presence of ActD (see Figure 3b, IFNγ & ActD),

Bottom Line: Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction.Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cardiology, Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203, USA.

ABSTRACT

Background: The F11 Receptor (F11R; aka JAM-A, JAM-1) is a cell adhesion protein present constitutively on the membrane surface of circulating platelets and within tight junctions of endothelial cells (ECs). Previous reports demonstrated that exposure of ECs to pro-inflammatory cytokines causes insertion of F11R molecules into the luminal surface of ECs, ensuing with homologous interactions between F11R molecules of platelets and ECs, and a resultant adhesion of platelets to the inflamed ECs. The main new finding of the present report is that the first step in this chain of events is the de-novo transcription and translation of F11R molecules, induced in ECs by exposure to inflammatory cytokines.

Methods: The experimental approach utilized isolated, washed human platelet suspensions and cultured human venous endothelial cells (HUVEC) and human arterial endothelial cells (HAEC) exposed to the proinflammatory cytokines TNF-alpha and/or IFN-gamma, for examination of the ability of human platelets to adhere to the inflamed ECs thru the F11R. Our strategy was based on testing the effects of the following inhibitors on this activity: general mRNA synthesis inhibitors, inhibitors of the NF-kappaB and JAK/STAT pathways, and small interfering F11R-mRNA (siRNAs) to specifically silence the F11R gene.

Results: Treatment of inflamed ECs with the inhibitors actinomycin, parthenolide or with AG-480 resulted in complete blockade of F11R- mRNA expression, indicating the involvement of NF-kappaB and JAK/STAT pathways in this induction. Transfection of ECs with F11R siRNAs caused complete inhibition of the cytokine-induced upregulation of F11R mRNA and inhibition of detection of the newly- translated F11R molecules in cytokine-inflamed ECs. The functional consequence of the inhibition of F11R transcription and translation was the significant blockade of the adhesion of human platelets to inflamed ECs.

Conclusion: These results prove that de novo synthesis of F11R in ECs is required for the adhesion of platelets to inflamed ECs. Because platelet adhesion to an inflamed endothelium is crucial for plaque formation in non-denuded blood vessels, we conclude that the de-novo translation of F11R is a crucial early step in the initiation of atherogenesis, leading to atherosclerosis, heart attacks and stroke.

Show MeSH
Related in: MedlinePlus