In a mouse model of transient middle cerebral artery (MCA) ischemia, the authors from the United States investigated the possible mechanisms through which SARS-CoV-2 spike (S) protein influences acute ischemic stroke. Since the SARS-CoV-2 S protein binds only to human angiotensin-converting enzyme-2 (ACE-2) receptors, researchers chose the humanized ACE-2 knock-in (hACE2 KI) mice, expressing humanized ACE2 receptors. The same research group in their previous studies showed that injection of SARS-CoV-2 S protein in hACE2 KI mice downregulated brain expression of ACE2, increased brain inflammation, and disrupted the renin-angiotensin-aldosterone system (RAAS) balance. They, therefore, postulated that SARS-CoV-2 S protein could exacerbate stroke and cerebrovascular complications by disrupting the RAAS balance, increasing coagulation, and reducing fibrinolysis.
The authors stressed the crucial role of the ACE-2 enzyme in the RAAS balance. ACE-2 degrades the bioactive form angiotensin II (Ang II), which can bind to the angiotensin type receptor 1 (AT1R), which is more abundant, or the angiotensin type receptor 2 (AT2R). Activation of the Ang II/AT1R axis pathway leads to vasoconstriction, thrombosis, and generation of reactive oxygen species. The Ang II/AT2R axis opposes the effects of AT1R activation through vasodilation, anti-inflammatory responses, and maintenance of hemostasis. SARS-CoV-2 spike protein binds to ACE-2 and downregulates the ACE-2 receptor. This decreases Ang II degradation and increases activation of the AT1R, which is more abundant than AT2R and activates the RAAS destructive arm.
About the study
The authors examined the effect of SARS-CoV-2 S protein in humanized ACE-2 knock-in (hACE2 KI) mice, which express humanized ACE2 receptors to replace mice Ace2. Transient focal ischemia was induced by chemical thromboembolic occlusion of the middle cerebral artery (MCA) with ferric chloride (MCA/FeCl3model), where a clot forms and spontaneously recanalizes within a few hours. The recombinant SARS-CoV-2 S protein was injected intravenously seven days before stroke induction. Losartan, an AT1R blocker, was administered immediately after the injection of the recombinant S protein (10 mg/kg body weight), one week before the stroke induction. The relatively higher lipophilicity of losartan increases its ability to cross the blood-brain barrier.
hACE2 KI mice were randomly assigned to four groups: sham, stroke, stroke + SARS-CoV-2 S protein injection, and stroke +  SARS-CoV-2 S protein injection + losartan. Sham animals were exposed to the MCA thromboembolic surgery without FeCl3 treatment.
Animal recognition memory and learning functions were assessed by Novel Object Recognition testing before SARSCoV-2 S protein injection and 24 hours after MCA/FeCl3 thromboembolic occlusion. Cerebral blood flow was measured with the Laser Speckle Imaging System at 1, 2, 3, 6, and 24 hours after MCA occlusion. Infarct volume was measured in mice’s brains 24 hours after transient focal ischemia.
Tissue factor III (TF-III) and plasminogen activator inhibitor-1 (PAI-1) were measured by immunoblotting. TF-III is expressed and activated by endothelial cells, vascular smooth muscle cells, and monocytes. It is commonly released after the disruption of the endothelial matrix and activates the coagulation cascade, leading to thrombin activation. PAI-1 is located in the extracellular matrix and in the extracellular exosome. It inhibits the activation of the endogenous tissue-type plasminogen activator and the conversion of plasminogen to plasmin, thus hindering fibrin degradation.
The effects of SARS-CoV-2 S protein were also studied in cultures of primary human brain microvascular endothelial cells (HBMECs) treated with recombinant SARS-CoV-2 S protein (100 µM) with or without losartan (100 µM) for 24 hours. After 24 hours, HBMECs were either maintained under normoxic conditions or placed in a hypoxia chamber for 6 hours.
Results
Cognitive impairment
The Novel Object Recognition Test, which assesses the memory and learning ability of hACE2 KI mice, showed no differences between the groups in terms of total distance traveled. However, the mice with transient focal ischemia showed a significant decrease in the number of entries into the zone containing the novel object and the time spent interacting with the novel object. Pretreatment with SARS-CoV-2 S protein further decreased the time spent interacting with the novel object. In contrast, the AT1R blocker losartan, administered immediately after injection of recombinant S protein, significantly improved cognitive function in hACE2 KI mice, as evidenced by increases in the time spent with the novel object and in the number of entries into the zone containing the novel object.
RAAS Imbalance
The main finding of this study is that the SARS-CoV-2 S protein disrupted the renin-angiotensin-aldosterone system (RAAS) balance in the brain vasculature.
Ischemic insult increased the AT1R expression in the brain by 2-fold. SARS-CoV-2 S protein injection given to hACE2 KI mice seven days before transient focal brain ischemia increased AT1R expression to 4-fold and downregulated the ACE2 gene expression and the RAAS protective arm AT2R. AT1R blocker losartan, administered immediately after S protein injection, restored ACE2 gene expression downregulated by the recombinant S protein.
These results were confirmed in cultures of primary human brain microvascular endothelial cells treated with recombinant SARS-CoV-2 S protein and exposed to hypoxia. Even under normoxic conditions, S protein increased AT1R expression and decreased AT2R expression. These effects were further enhanced under hypoxic conditions. According to these results, the S protein increased AT1R expression in the brain’s endothelial cells at the expense of the protective AT2R. Losartan reduced effects induced by SARS-CoV-2 S protein, but this result was insignificant.
Brain inflammation, infarct volume, and cerebral blood flow
The transient focal ischemia increased inflammation in both, the affected brain hemisphere and the contralateral unaffected hemisphere. Importantly, pretreatment with recombinant S protein significantly increased infarct volume and inflammation, as evidenced by increased nuclear factor kappa B (NF-kB), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 gene expression in the brain. The AT1R blocker losartan, administered immediately after the recombinant S protein injection, significantly reduced infarct volume.
In cultures of primary human brain microvascular endothelial cells, treatment with recombinant SARS-CoV-2 S protein significantly increased the expression of TNF-α under normoxic conditions and further increased inflammation under hypoxic conditions.
Cerebral blood flow was decreased in the brain hemisphere affected by transient focal ischemia compared to the contralateral unaffected hemisphere, but it was restored within six hours after transient thromboembolic occlusion of the MCA. Treatment with SARS-CoV-2 S protein seven days before stroke induction significantly intensified a decrease in cerebral blood flow and delayed recanalization to over 6 hours. The AT1R blocker losartan significantly decreased the effects of S protein by reducing recanalization time.
Coagulation
Transient focal ischemia upregulated TF−III and PAI-1 expression in brain homogenate of hACE2 KI mice. Treatment with S protein before stroke further increased TF−III and PAI-1 expression.
These results were confirmed in cultures of primary human brain microvascular endothelial cells treated with recombinant SARS-CoV-2 S protein. The S protein favored clot formation by increasing coagulation and decreasing fibrinolysis in cell cultures exposed to hypoxic conditions. Losartan decreased hypercoagulation induced by the S protein. According to these results, the S protein increased coagulation by increasing TTF-III and PAI-1 expression.
Conclusion
This study provides new evidence that SARS-CoV-2 S protein injection given to hACE2 KI mice seven days before transient focal brain ischemia disrupted the RAAS balance by increasing Ang II/AT1R signaling in the brain’s cells at the expense of the Ang II/AT2R protective arm. The RAAS imbalance increased coagulation and decreased fibrinolysis, which worsened ischemic stroke outcomes, as evidenced by increased infarct size, reduced cerebral blood flow, and increased cognitive impairment. Losartan, an AT1R blocker, restored RAAS balance and reduced spike protein-induced effects.
This article was published in Translational Stroke Research.
Journal Reference
Heath SP, Hermanns VC, Coucha M, Abdelsaid M. SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model. Translational Stroke Research. Published online on October 2, 2024. https://doi.org/10.1007/s12975-024-01301-5
