Timing matters in the use of renin-angiotensin system modulators and COVID-related cognitive and cerebrovascular dysfunction

Abstract

Renin-angiotensin system (RAS) modulators, including Angiotensin receptor blockers (ARB) and angiotensin-converting enzyme inhibitors (ACEI), are effective medications for controlling blood pressure. Cognitive deficits, including lack of concentration, memory loss, and confusion, were reported after COVID-19 infection. ARBs or ACEI increase the expression of angiotensin-converting enzyme-2 (ACE-2), a functional receptor that allows binding of SARS-CoV-2 spike protein for cellular invasion. To date, the association between the use of RAS modulators and the severity of COVID-19 cognitive dysfunction is still controversial.

Purpose: This study addressed the following questions: 1) Does prior treatment with RAS modulator worsen COVID-19-induced cerebrovascular and cognitive dysfunction? 2) Can post-treatment with RAS modulator improve cognitive performance and cerebrovascular function following COVID-19? We hypothesize that pre-treatment exacerbates COVID-19-induced detrimental effects while post-treatment displays protective effects.

Methods: Clinical study: Patients diagnosed with COVID-19 between May 2020 and December 2022 were identified through the electronic medical record system. Inclusion criteria comprised a documented medical history of hypertension treated with at least one antihypertensive medication. Subsequently, patients were categorized into two groups: those who had been prescribed ACEIs or ARBs before admission and those who had not received such treatment before admission. Each patient was evaluated on admission for signs of neurologic dysfunction. Pre-clinical study: Humanized ACE-2 transgenic knock-in mice received the SARS-CoV-2 spike protein via jugular vein injection for 2 weeks. One group had received Losartan (10 mg/kg), an ARB, in their drinking water for two weeks before the injection, while the other group began Losartan treatment after the spike protein injection. Cognitive functions, cerebral blood flow, and cerebrovascular density were determined in all experimental groups. Moreover, vascular inflammation and cell death were assessed.

Results: Signs of neurological dysfunction were observed in 97 out of 177 patients (51%) taking ACEIs/ARBs prior to admission, compared to 32 out of 118 patients (27%) not receiving ACEI or ARBs. In animal studies, spike protein injection increased vascular inflammation, increased endothelial cell apoptosis, and reduced cerebrovascular density. In parallel, spike protein decreased cerebral blood flow and cognitive function. Our results showed that pretreatment with Losartan exacerbated these effects. However, post-treatment with Losartan prevented spike protein-induced vascular and neurological dysfunctions.

Conclusion: Our clinical data showed that the use of RAS modulators before encountering COVID-19 can initially exacerbate vascular and neurological dysfunctions. Similar findings were demonstrated in the in-vivo experiments; however, the protective effects of targeting the RAS become apparent in the animal model when the treatment is initiated after spike protein injection.

Fig 1. Losartan increased ACE-2 expression, while SARS-CoV-2 spike protein decreased ACE-2 expression in hACE2 brains.

hACE2 KI mice were treated only with Losartan or intravenously injected with SARS-CoV-2 spike protein (SP, 4 μg/ animal) with or without Losartan (10 mg/kg body weight) for 2 weeks. Brain homogenate was assessed for ACE-2 expression. a) RT-PCR analysis showing the effect of Losartan and spike protein on ACE-2 gene expression. Losartan treatment increased ACE-2 gene expression, while spike protein significantly reduced ACE-2 gene expression. b) Western blot analysis for ACE-2 expression in brain homogenate. Our results showed that Losartan treatment increased ACE-2 expression while spike protein decreased ACE2 expression. Treatment of Losartan after spike protein restored ACE-2 expression in hACE2 mice treated with spike protein. (One-way ANOVA, *P<0.05, n = 3–4).

Fig 2. Only post-losartan treatment prevented SARS-CoV-2 spike protein-induced inflammation/apoptosis but not pre-losartan treatment.

hACE2 KI mice were injected with recombinant SARS-CoV-2 spike protein (4 mg/animal). Losartan treatment (10 mg/kg) began either two weeks before the spike protein injection (Losartan + SP) or immediately after the injection (SP + Losartan). Whole brain homogenate was assessed for inflammatory markers and apoptotic markers. a) RT-PCR analysis of inflammatory markers (TNF-α and Il-6) in hACE-2 brains. Our results show that spike protein injection caused a significant increase in TNF-α and Il-6 gene expression. Pre-Losartan treatment showed a similar increase in TNF-α and Il-6 gene expression. Post-Losartan treatment inhibited spike protein-induced increased inflammation. b) Western Blot analysis for apoptotic marker, cleaved caspase-3. Our results showed that spike protein and pre-losartan treatment increased caspase activation, while post-losartan treatment showed a protective effect against spike protein-induced cell death. (One-way ANOVA, *P<0.05, n = 4).

Fig 3. Post-losartan treatment prevented SARS-CoV-2 Spike protein-induced vascular rarefaction but not pre-losartan treatment.

hACE2 KI mice were injected with recombinant SARS-CoV-2 spike protein (4 mg/animal). Losartan treatment (10 mg/kg) began either two weeks before the spike protein injection (Losartan + SP) or immediately after the injection (SP + Losartan). Brains were isolated and sectioned 30–40 um. The brain section was stained for vasculature. 3D confocal images were reconstructed to assess vascular density using FIJI software. a) Representative images of the brain cortex. b) Image analysis showing that SARS-CoV-2 caused vascular rarefaction and significantly reduced vascular density. Pre-Losartan treatment showed decreased vascular density. Only post-losartan treatment showed a protective effect and restored vascular density compared to control. (One-way ANOVA, *P<0.05, n = 5).

Fig 4. Pre-losartan treatment reduced cerebral blood flow after SARS-CoV-2 spike injection but not post-losartan treatment.

hACE2 KI mice were injected with recombinant SARS-CoV-2 spike protein (4 mg/animal). Losartan treatment (10 mg/kg) began either two weeks before the spike protein injection (Losartan + SP) or immediately after the injection (SP + Losartan). a) Representative images of cerebral blood flow were measured at baseline and 2 weeks after spike protein injection. b) Percent change in cerebral blood flow after 15 days compared to baseline. Our results showed cerebral blood flow was reduced in pre-Losartan treatment group compared to the control (*P<0.05). Post-Losartan treatment group was similar to the control. (One-way ANOVA, P<0.05, n = 3–7).

Fig 5. Post-losartan treatment prevented SARS-CoV-2 spike protein-induced decreased cognitive impairment but not pre-losartan treatment.

a) Learning and memory functions were assessed using Y-maze. Spike protein and pre-losartan treatment showed significant impairment in cognitive function, as seen by reduced time spent in the new arm. Only post-losartan treatment showed improvement in cognitive functions after spike protein injection. b) The total distance traveled was measured to detect any motor disability with spike protein injection. No significant results were observed in the total distance traveled between groups. (One-way ANOVA, *P<0.05, n = 5).