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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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Expression of F11R mRNA in human aortic endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC) exposed to proinflammatory cytokines TNFα and/or IFNγ: time course. Real-time PCR was performed in cultured HAEC (top panels) treated for 0, 3, 6, 12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL), and in cultured HUVEC (bottom panels) treated for 0,4,8,12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL). Real-time PCR was performed three times in triplicate for each time point. Values represent the mean ± SEM. *P < 0.05 indicates the level of significance determined at a specific interval of time of cytokine- treatment of ECs in comparison to the zero time points.
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Figure 1: Expression of F11R mRNA in human aortic endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC) exposed to proinflammatory cytokines TNFα and/or IFNγ: time course. Real-time PCR was performed in cultured HAEC (top panels) treated for 0, 3, 6, 12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL), and in cultured HUVEC (bottom panels) treated for 0,4,8,12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL). Real-time PCR was performed three times in triplicate for each time point. Values represent the mean ± SEM. *P < 0.05 indicates the level of significance determined at a specific interval of time of cytokine- treatment of ECs in comparison to the zero time points.

Mentions: The expression of F11R mRNA was examined both in arterial HAEC and venous HUVEC following their exposure to the pro-inflammatory cytokines TNFα and IFNγ. As shown in Figure 1, a time-dependent increase in F11R mRNA expression was observed following the exposure of arterial and venous cells to TNFα or IFNγ, or their combination. Arterial endothelial cells (top panels) demonstrated a slow, significant increase in the level of F11R mRNA at 12 hrs of exposure to either TNFα or IFNγ. Although a further increase was observed with TNFα for a subsequent 12 hr period, further exposure of cells to INFγ resulted in a drop in the F11R mRNA level. The simultaneous treatment of cells with TNFα and IFNγ resulted in a shortening in response time, with maximal F11R mRNA levels observed already at 3 hrs of cytokine-exposure. Similarly, venous endothelial cells (lower panels) demonstrated a gradual enhancement (also significant at 12 hrs) of F11R mRNA expression following the application of cytokines, alone or in combination.


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)

Expression of F11R mRNA in human aortic endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC) exposed to proinflammatory cytokines TNFα and/or IFNγ: time course. Real-time PCR was performed in cultured HAEC (top panels) treated for 0, 3, 6, 12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL), and in cultured HUVEC (bottom panels) treated for 0,4,8,12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL). Real-time PCR was performed three times in triplicate for each time point. Values represent the mean ± SEM. *P < 0.05 indicates the level of significance determined at a specific interval of time of cytokine- treatment of ECs in comparison to the zero time points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142510&req=5

Figure 1: Expression of F11R mRNA in human aortic endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC) exposed to proinflammatory cytokines TNFα and/or IFNγ: time course. Real-time PCR was performed in cultured HAEC (top panels) treated for 0, 3, 6, 12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL), and in cultured HUVEC (bottom panels) treated for 0,4,8,12, and 24 hrs with TNFα (100 u/mL) and/or IFNγ (200 u/mL). Real-time PCR was performed three times in triplicate for each time point. Values represent the mean ± SEM. *P < 0.05 indicates the level of significance determined at a specific interval of time of cytokine- treatment of ECs in comparison to the zero time points.
Mentions: The expression of F11R mRNA was examined both in arterial HAEC and venous HUVEC following their exposure to the pro-inflammatory cytokines TNFα and IFNγ. As shown in Figure 1, a time-dependent increase in F11R mRNA expression was observed following the exposure of arterial and venous cells to TNFα or IFNγ, or their combination. Arterial endothelial cells (top panels) demonstrated a slow, significant increase in the level of F11R mRNA at 12 hrs of exposure to either TNFα or IFNγ. Although a further increase was observed with TNFα for a subsequent 12 hr period, further exposure of cells to INFγ resulted in a drop in the F11R mRNA level. The simultaneous treatment of cells with TNFα and IFNγ resulted in a shortening in response time, with maximal F11R mRNA levels observed already at 3 hrs of cytokine-exposure. Similarly, venous endothelial cells (lower panels) demonstrated a gradual enhancement (also significant at 12 hrs) of F11R mRNA expression following the application of cytokines, alone or in combination.

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