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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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Related in: MedlinePlus

Expression of the F11R protein in inflamed endothelial cells: silencing of the F11R gene in HAEC and HUVEC using F11R siRNA. (a). Immunoblots demonstrate the detection of the F11R protein retained in cells (cell lysates) and released into the media of inflamed HAEC and HUVEC. Both aortic and umbilical vein endothelial cells were transfected with either the control, non-targeting siRNA or by the specific F11R targeting siRNA (as detailed in the Material and Methods section). Subsequently, the cells were treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs, followed by SDS-PAGE and immunoblotting utilizing F11R antibody (arrows point to F11R), and tubulin, as the protein loading control, of 50 kDa. Lanes 1 and 3 depict the F11R protein as detected in cytokine-treated HAEC or HUVEC transfected with the nontargeting siRNA. Lanes 2 and 4 depict the F11R protein as detected in cytokine-treated HAEC and HUVEC transfected with the specific targeting F11R siRNA.(b). Quantitation of immunoblots of the immunostained F11R protein, detected in the cell culture media of HAEC and HUVEC endothelial cells transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, followed by the exposure of transfected HAEC and HUVEC to a combination of the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. The values for F11R were normalized to tubulin levels by dividing the integrated density of the specific band by the integrative density of the tubulin band. ANOVA statistical analysis was performed on the normalized values. All values are the average of three immunoblots ± SEM. (c). Quantitation of the immunostained F11R protein within the cell lysates of HAEC and HUVEC transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, and further treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. F11R-immunostained protein bands were quantified by normalization to tubulin using image J. The F11R values were normalized to tubulin. ANOVA was performed on the normalized value (n = 3). Values depict the mean ± SEM, * p < 0.005.
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Figure 6: Expression of the F11R protein in inflamed endothelial cells: silencing of the F11R gene in HAEC and HUVEC using F11R siRNA. (a). Immunoblots demonstrate the detection of the F11R protein retained in cells (cell lysates) and released into the media of inflamed HAEC and HUVEC. Both aortic and umbilical vein endothelial cells were transfected with either the control, non-targeting siRNA or by the specific F11R targeting siRNA (as detailed in the Material and Methods section). Subsequently, the cells were treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs, followed by SDS-PAGE and immunoblotting utilizing F11R antibody (arrows point to F11R), and tubulin, as the protein loading control, of 50 kDa. Lanes 1 and 3 depict the F11R protein as detected in cytokine-treated HAEC or HUVEC transfected with the nontargeting siRNA. Lanes 2 and 4 depict the F11R protein as detected in cytokine-treated HAEC and HUVEC transfected with the specific targeting F11R siRNA.(b). Quantitation of immunoblots of the immunostained F11R protein, detected in the cell culture media of HAEC and HUVEC endothelial cells transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, followed by the exposure of transfected HAEC and HUVEC to a combination of the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. The values for F11R were normalized to tubulin levels by dividing the integrated density of the specific band by the integrative density of the tubulin band. ANOVA statistical analysis was performed on the normalized values. All values are the average of three immunoblots ± SEM. (c). Quantitation of the immunostained F11R protein within the cell lysates of HAEC and HUVEC transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, and further treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. F11R-immunostained protein bands were quantified by normalization to tubulin using image J. The F11R values were normalized to tubulin. ANOVA was performed on the normalized value (n = 3). Values depict the mean ± SEM, * p < 0.005.

Mentions: A comparison of the effects of transfection of endothelial cells on F11R levels in arterial (HAEC) and venous (HUVEC) endothelial cells transfected either by a nonspecific siRNA or a specific F11R siRNA is shown in Figure 6a. As shown in lane 1, the utilization of a nonspecific siRNA in the transfection of TNFα and IFNγ-inflamed arterial endothelial cells(HAEC) did not block the enhancement of the synthesis of the F11R protein which was identified both in the lysate of these arterial cells as well as in their media (see Figure 6a, HAEC, lane 1). In contrast, as shown in Lane 2, the transfection of arterial endothelial cells (HAEC) by the specific-F11R targeting siRNA resulted in the inhibition of F11R synthesis - the F11R protein was neither expressed in lysates nor detected in the media of TNFα and IFNγ-treated arterial endothelial cells (HAEC, see lane 2). Similar to the results obtained with inflamed arterial cells transfected with a non-targeting siRNA, the synthesis of the F11R protein was not blocked following the transfection of inflamed venous endothelial cells (HUVEC) by the non-targeting siRNA (see Figure 6a, HUVEC, lane 3). However, as shown in Lane 4, the F11R protein was neither expressed in the lysate nor detected in the media of TNFα and IFNγ-inflamed venous endothelial cells following the transfection of HUVEC by the specific-F11R targeting siRNA (HUVEC, lane 4). Quantitation of the F11R protein (immunostained 37 kDa) revealed that the transfection of inflamed arterial (HAEC) and inflamed venous (HUVEC) endothelial cells by specific interfering F11R siRNA resulted in a significant inhibition in the synthesis and release/shedding of the F11R protein. As shown in Figure 6b, almost 100% decrease of F11R occurred in media of F11R siRNA-transfected HAEC; an 80% decrease of F11R in the media of F11R siRNA-transfected HUVEC was observed. Furthermore, the targeted transfection of TNFα and IFNγ-treated HAEC and HUVEC by F11R siRNA resulted in the complete inhibition of F11R expression in the cell lysates of these inflamed arterial and venous endothelial cells as (shown in Figure 6c).


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 the F11R protein in inflamed endothelial cells: silencing of the F11R gene in HAEC and HUVEC using F11R siRNA. (a). Immunoblots demonstrate the detection of the F11R protein retained in cells (cell lysates) and released into the media of inflamed HAEC and HUVEC. Both aortic and umbilical vein endothelial cells were transfected with either the control, non-targeting siRNA or by the specific F11R targeting siRNA (as detailed in the Material and Methods section). Subsequently, the cells were treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs, followed by SDS-PAGE and immunoblotting utilizing F11R antibody (arrows point to F11R), and tubulin, as the protein loading control, of 50 kDa. Lanes 1 and 3 depict the F11R protein as detected in cytokine-treated HAEC or HUVEC transfected with the nontargeting siRNA. Lanes 2 and 4 depict the F11R protein as detected in cytokine-treated HAEC and HUVEC transfected with the specific targeting F11R siRNA.(b). Quantitation of immunoblots of the immunostained F11R protein, detected in the cell culture media of HAEC and HUVEC endothelial cells transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, followed by the exposure of transfected HAEC and HUVEC to a combination of the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. The values for F11R were normalized to tubulin levels by dividing the integrated density of the specific band by the integrative density of the tubulin band. ANOVA statistical analysis was performed on the normalized values. All values are the average of three immunoblots ± SEM. (c). Quantitation of the immunostained F11R protein within the cell lysates of HAEC and HUVEC transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, and further treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. F11R-immunostained protein bands were quantified by normalization to tubulin using image J. The F11R values were normalized to tubulin. ANOVA was performed on the normalized value (n = 3). Values depict the mean ± SEM, * p < 0.005.
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Figure 6: Expression of the F11R protein in inflamed endothelial cells: silencing of the F11R gene in HAEC and HUVEC using F11R siRNA. (a). Immunoblots demonstrate the detection of the F11R protein retained in cells (cell lysates) and released into the media of inflamed HAEC and HUVEC. Both aortic and umbilical vein endothelial cells were transfected with either the control, non-targeting siRNA or by the specific F11R targeting siRNA (as detailed in the Material and Methods section). Subsequently, the cells were treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs, followed by SDS-PAGE and immunoblotting utilizing F11R antibody (arrows point to F11R), and tubulin, as the protein loading control, of 50 kDa. Lanes 1 and 3 depict the F11R protein as detected in cytokine-treated HAEC or HUVEC transfected with the nontargeting siRNA. Lanes 2 and 4 depict the F11R protein as detected in cytokine-treated HAEC and HUVEC transfected with the specific targeting F11R siRNA.(b). Quantitation of immunoblots of the immunostained F11R protein, detected in the cell culture media of HAEC and HUVEC endothelial cells transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, followed by the exposure of transfected HAEC and HUVEC to a combination of the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. The values for F11R were normalized to tubulin levels by dividing the integrated density of the specific band by the integrative density of the tubulin band. ANOVA statistical analysis was performed on the normalized values. All values are the average of three immunoblots ± SEM. (c). Quantitation of the immunostained F11R protein within the cell lysates of HAEC and HUVEC transfected with either the non-targeting siRNA or the specific targeting F11R siRNA, and further treated with the proinflammatory cytokines TNFα (100 u/ml) and IFNγ (200 u/ml) for 24 hrs. F11R-immunostained protein bands were quantified by normalization to tubulin using image J. The F11R values were normalized to tubulin. ANOVA was performed on the normalized value (n = 3). Values depict the mean ± SEM, * p < 0.005.
Mentions: A comparison of the effects of transfection of endothelial cells on F11R levels in arterial (HAEC) and venous (HUVEC) endothelial cells transfected either by a nonspecific siRNA or a specific F11R siRNA is shown in Figure 6a. As shown in lane 1, the utilization of a nonspecific siRNA in the transfection of TNFα and IFNγ-inflamed arterial endothelial cells(HAEC) did not block the enhancement of the synthesis of the F11R protein which was identified both in the lysate of these arterial cells as well as in their media (see Figure 6a, HAEC, lane 1). In contrast, as shown in Lane 2, the transfection of arterial endothelial cells (HAEC) by the specific-F11R targeting siRNA resulted in the inhibition of F11R synthesis - the F11R protein was neither expressed in lysates nor detected in the media of TNFα and IFNγ-treated arterial endothelial cells (HAEC, see lane 2). Similar to the results obtained with inflamed arterial cells transfected with a non-targeting siRNA, the synthesis of the F11R protein was not blocked following the transfection of inflamed venous endothelial cells (HUVEC) by the non-targeting siRNA (see Figure 6a, HUVEC, lane 3). However, as shown in Lane 4, the F11R protein was neither expressed in the lysate nor detected in the media of TNFα and IFNγ-inflamed venous endothelial cells following the transfection of HUVEC by the specific-F11R targeting siRNA (HUVEC, lane 4). Quantitation of the F11R protein (immunostained 37 kDa) revealed that the transfection of inflamed arterial (HAEC) and inflamed venous (HUVEC) endothelial cells by specific interfering F11R siRNA resulted in a significant inhibition in the synthesis and release/shedding of the F11R protein. As shown in Figure 6b, almost 100% decrease of F11R occurred in media of F11R siRNA-transfected HAEC; an 80% decrease of F11R in the media of F11R siRNA-transfected HUVEC was observed. Furthermore, the targeted transfection of TNFα and IFNγ-treated HAEC and HUVEC by F11R siRNA resulted in the complete inhibition of F11R expression in the cell lysates of these inflamed arterial and venous endothelial cells as (shown in Figure 6c).

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