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The endothelium in COVID-19 complications and in the regulation of vascular tone

  • Author / Creator
    Maheux, Alexia C
  • Background: This thesis encompasses two distinct projects. 1) von Willebrand factor (VWF) is a highly adhesive multimeric glycoprotein found in the Weibel-Palade bodies of endothelial cells and in ⍺-granules of megakaryocytes and platelets. At the site of vascular injury, VWF is essential for recruitment and activation of platelets and is the key mediator of blood clot formation. SARS-CoV-2 is a respiratory virus that infects host cells by binding to angiotensin converting enzyme 2 (ACE2) on host cell surfaces. In addition to respiratory complications, inflammation and an increase in thrombotic markers (such as VWF) are also markers of severe SARS-CoV-2 infection. Since VWF is released from endothelial cells and these cells also express ACE2, I hypothesized that engagement of ACE2 by the SARS-CoV-2 spike protein on endothelial cells induces upregulation and release of VWF. 2) Resistance arteries can actively regulate their diameter through endothelial and smooth muscle mechanisms that rely on signalling molecules such as calcium (Ca2+) or inositol triphosphate (IP3). Connexins (Cx) make up the gap junctions between adjacent endothelial and smooth muscle cells that allow for the spread of electrical and chemical signalling along the vessel wall via rapid transfer of ions and small molecules. Changes in gap junction function are associated with cardiovascular diseases such as hypertension but the relative importance of various Cx to vascular control is unclear. I examined vascular responses of mesenteric arteries from rats lacking Cx40 globally to test the hypothesis that arteries from Cx40 deficient rats will show impaired responses to the endothelium-dependent vasodilator acetylcholine (ACh).
    Results: In cultured human umbilical vein endothelial cells (HUVECs), exposure to SARS-CoV-2 spike protein did not significantly change VWF mRNA expression levels but caused a significant increase in released VWF after five minutes. VWF release in cells that were treated again 72 hours
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    after their first treatment with spike protein was also significantly increased compared to the control and initial treatment groups. Immunofluorescence imaging confirmed the presence of ACE2 on the surface and in the cytoplasm of endothelial cells; additionally, VWF and ACE2 expression in HUVECs was detectable using western blot analysis. I used a combination of inhibitors of endothelium-dependent vasodilator pathways to investigate the impact of loss of Cx40 on responses of mesenteric resistance arteries to the endothelium-dependent vasorelaxant ACh and to the ⍺1-adrenoceptor agonist phenylephrine. The effects of the inhibitors on the responses to ACh were significantly different in arteries from female rats of all genotypes and male wild type (WT) and knockout (KO) rats but not male heterozygous (HET) rats. However, in arteries from female rats, addition of the inhibitors together revealed a difference between the genotypes. A difference in the genotypes was also seen in the male control group. Phenylephrine-induced tone was enhanced in arteries from both male and female KO rats relative to responses of arteries from WT rats.
    Conclusions: Exposure of endothelial cells to SARS-CoV-2 spike protein alone increases VWF release. I confirmed that HUVECs express ACE2, so it is possible that this effect is due to a spike protein-ACE2 interaction. However, more work is needed to confirm that the observed increase is due to this interaction and to elucidate the mechanism through which engagement of ACE2 leads to increased VWF release. Loss of Cx40 impacted responses to the endothelium-dependent relaxant ACh in male but not female rats but there was no obvious effect on the relative contributions of NO and EDH to ACh-evoked vasorelaxation. However, the enhancement of phenylephrine-induced tone in arteries from Cx40 KO rats of both sexes indicates that Cx40 may play a role in smooth muscle to endothelial

  • Subjects / Keywords
  • Graduation date
    Fall 2022
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/r3-8g3k-1047
  • License
    This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.