SARS-CoV-2 (MA10) Infection Aggravates Cerebrovascular Pathology in Endothelial Nitric Oxide Synthase-Deficient Mice

Abstract

SARS-CoV-2 can cause neurological issues, including cognitive dysfunction in COVID-19 survivors. Endothelial dysfunction, a key mechanism in COVID-19, is also a risk factor for vascular dementia (VaD). Reduced nitric oxide (NO) bioavailability is a pathogenic factor of endothelial dysfunction and platelet aggregation in COVID-19 patients, and endothelial NO synthase (eNOS) levels decline with advancing age, a risk factor for both COVID-19 morbidity and VaD. SARS-CoV-2 also induces cellular senescence and senescence-associated secretory phenotype (SASP). We hypothesized that eNOS deficiency would worsen neuroinflammation, senescence, blood-brain barrier (BBB) permeability, and hypercoagulability in eNOS-deficient mice. Six-month-old eNOS^+/- (pre-cognitively impaired experimental VaD) and wild-type (WT) male mice were infected with mouse-adapted (MA10) SARS-CoV-2. Mice were evaluated for weight loss, viral load, and markers of inflammation and senescence 3 days post-infection. eNOS^+/- mice showed more weight loss (~15%) compared to WT mice (~5%) and increased inflammatory markers (Ccl2, Cxcl9, Cxcl10, IL-1β, and IL-6) and senescence markers (p53 and p21). They also exhibited higher microglial activation (Iba1) and increased plasma coagulation and BBB permeability, despite comparable lung viral loads and absence of virus in the brain. This is the first experimental evidence demonstrating that eNOS deficiency exacerbates SARS-CoV-2-induced morbidity, neuroinflammation, and brain senescence, linking eNOS to COVID-19-related neuropathology.

Keywords: SARS-CoV-2; endothelial nitric oxide synthase; neuroinflammation; senescence; vascular dementia.

Figure 1

eNOS^+/− mice show intense disease severity compared to WT mice following mouse-adapted SARS-CoV-2 (MA10) infection. Six-month-old male eNOS^+/− and WT mice were inoculated with 1 × 10⁴ pfu via the intranasal route with mock (PBS) or MA10 strain of SARS-CoV-2. (A) MA10 infection induced a more significant decrease in body weight in eNOS^+/− mice compared to infected WT mice. ^^ p < 0.01 vs. WT Mock, ^### p < 0.001 vs. WT MA10, ^# p < 0.05 vs. WT MA10, ** p < 0.01 vs. eNOS^+/− Mock. Upon 3 days post-infection (3 dpi), mice were euthanized, and RNA was isolated from the lung and brain samples and analyzed for (B) genomic (N) and (C) subgenomic (SgN) viral copies in the lungs. There was no significant difference in genomic or subgenomic viral copies between MA10-infected WT and eNOS^+/− mice. Immunofluorescence analysis of (D,E) viral nucleocapsid showed that there were more positive cells in the lungs of eNOS^+/− mice than WT mice following infection. Data are represented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3–6/group. Scale bar = 100 µm.

Figure 2

MA10 infection increases pulmonary proinflammatory response in eNOS^+/− mice more than in WT. MA10 infection induced a significant increase in (A) Ccl2, (B) Cxcl9, (C) Cxcl10, (D) Il6, and (E) Ifn-γ mRNA in WT and eNOS^+/− mice compared to WT mock mice at 3 dpi. Data are represented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 5–6/group.

Figure 3

eNOS^+/− mice showed an increased neuroinflammatory response compared to WT mice following MA10 infection. (A) Il-6, (B) Il1β, (C) Ccl2, (D) Cxcl9, (E) Cxcl10, and (F) Vcam1 mRNA expression in the brain at 3 dpi with MA10 or mock. Immunofluorescence analysis of Iba1-positive signal in the prefontal cortex (G,H) of eNOS^+/− and WT mice. MA10 infection induced increased Il-6, Il1β, Ccl2, Cxcl9, Cxcl10, and Vcam1 mRNA expression and microglial activation in eNOS^+/− mice > WT mice, as also compared to WT mock and eNOS^+/− mock mice, respectively. Scale bar = 50 µM (20×). Data are presented as mean ± SEM. p values represent mock vs. SARS-CoV-2 (MA10) challenged groups n = 4–5/group; * p < 0.05. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

MA10 infection increases brain senescence in eNOS^+/− mice compared to WT. (A) MA10 infection-induced p53 mRNA is significantly higher in eNOS^+/− mice compared to MA10-infected WT and WT and eNOS^+/− mock mice. (B) The expression of p21 mRNA is significantly increased in MA10-infected WT and eNOS^+/− mice compared to WT and eNOS^+/− mock mice. MA10 significantly increased p53 mRNA in infected eNOS^+/− mice compared to WT MA10-infected mice. Mean ± SEM, * p < 0.05, **** p < 0.0001, ns, not significant, n = 5–6/group.

Figure 5

MA10 induces hypercoagulability in eNOS^+/− mice. Plasma analysis using the i-QATT technique showed that (A) clotting rate and (B) maximum clot firmness are elevated in MA10-infected eNOS^+/− mice compared to infected WT mice. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3–5/group.

Figure 6

MA10 infection increases BBB permeability in eNOS^+/− mice more than in WT mice. Six-month-old male eNOS^+/− and WT mice were inoculated with SARS-CoV-2. (A) MA10. (B) IF analysis of fibrinogen (orange) and lectin (green). Quantification of fibrinogen leak showed that MA10 induced more vascular leak in eNOS^+/− than in WT mice. Data are mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001 n = 5/group. Scale bar = 100 µm.