Background: A non-negligible number of patients develop up to 15 month after coronavirus disease 2019 (COVID-19) a significant endothelial dysfunction that is accompanied by a residual risk for perioperative cardiovascular complications due to suboptimal tissue oxygen delivery. Severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) persistence has been proposed to be associated with post-infectious disorders. Since augmented levels of endothelial von Willebrand Factor (vWF) lead to endotheliopathy and persist even longer in long COVID patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) spreading to endothelial cells through angiotensin converting enzyme 2 (ACE2). Method: Gene silencing of vWF or ACE2 was performed in human umbilical vein endothelial cells (HUVEC) by RNA interference (siRNA), while vWF overexpression was obtained by transient pcDNA3.1-WT-VWF plasmid transfection of HUVEC. QRT-PCR evaluated expression levels of ACE2 and vWF in endothelial cells. Immunofluorescent labeling detected changes of surface localization of both endothelial proteins. Wild type and transfected endothelial cells were incubated with SARS-CoV-2 for one hour, and the level of cell infection was assessed by qRT-PCR of viral RNA after 24 / 48 hours. Results: Nearly 90 ± 2% (p<0.0001) silencing of vWF in viable HUVEC was achieved with vWF-specific siRNA and we observed a significant reduction of SARS-CoV-2 infection of vWF-silenced cells (-56% SARS-CoV-2 RNA, p=0.0058). Interestingly, surface vWF knockdown associated with a significant downregulation of ACE2 expression on the same cells (-55% mRNA expression, p=0.0032). Conversely, vWF overexpression (+130-fold, p=7.4 ·10-7) determined a significant upregulation of ACE2 (+24-fold p=0.028) and increased susceptibility of HUVEC to SARS-CoV-2 infection (+18-fold, p=0.0009). Conclusion: Endothelial surface vWF knockdown hampered SARS-CoV-2 spreading to endothelial cells through downregulation of surface ACE2 expression. Our findings reveal a hitherto unsuspected role of vWF in preventing of endothelial SARS-CoV-2 infection and might be helpful to design new therapeutic strategy to improve perioperative tissue oxygen delivery in long COVID patients. This is particularly attractive in light of the increasing number of viral mutants that may persist in vessels of different vital tissues.
Gene silencing of endothelial Von Willebrand factor hampering SARS-COV-2 infection of human endothelial cells as an innovative way to improve perioperative outcome in long covid patients
Furini G.;Lionetti V.
2022-01-01
Abstract
Background: A non-negligible number of patients develop up to 15 month after coronavirus disease 2019 (COVID-19) a significant endothelial dysfunction that is accompanied by a residual risk for perioperative cardiovascular complications due to suboptimal tissue oxygen delivery. Severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) persistence has been proposed to be associated with post-infectious disorders. Since augmented levels of endothelial von Willebrand Factor (vWF) lead to endotheliopathy and persist even longer in long COVID patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) spreading to endothelial cells through angiotensin converting enzyme 2 (ACE2). Method: Gene silencing of vWF or ACE2 was performed in human umbilical vein endothelial cells (HUVEC) by RNA interference (siRNA), while vWF overexpression was obtained by transient pcDNA3.1-WT-VWF plasmid transfection of HUVEC. QRT-PCR evaluated expression levels of ACE2 and vWF in endothelial cells. Immunofluorescent labeling detected changes of surface localization of both endothelial proteins. Wild type and transfected endothelial cells were incubated with SARS-CoV-2 for one hour, and the level of cell infection was assessed by qRT-PCR of viral RNA after 24 / 48 hours. Results: Nearly 90 ± 2% (p<0.0001) silencing of vWF in viable HUVEC was achieved with vWF-specific siRNA and we observed a significant reduction of SARS-CoV-2 infection of vWF-silenced cells (-56% SARS-CoV-2 RNA, p=0.0058). Interestingly, surface vWF knockdown associated with a significant downregulation of ACE2 expression on the same cells (-55% mRNA expression, p=0.0032). Conversely, vWF overexpression (+130-fold, p=7.4 ·10-7) determined a significant upregulation of ACE2 (+24-fold p=0.028) and increased susceptibility of HUVEC to SARS-CoV-2 infection (+18-fold, p=0.0009). Conclusion: Endothelial surface vWF knockdown hampered SARS-CoV-2 spreading to endothelial cells through downregulation of surface ACE2 expression. Our findings reveal a hitherto unsuspected role of vWF in preventing of endothelial SARS-CoV-2 infection and might be helpful to design new therapeutic strategy to improve perioperative tissue oxygen delivery in long COVID patients. This is particularly attractive in light of the increasing number of viral mutants that may persist in vessels of different vital tissues.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
