SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model

doi:10.21203/rs.3.rs-4649614/v1

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Research Article

SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model

https://doi.org/10.21203/rs.3.rs-4649614/v1

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published 02 Oct, 2024

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COVID-19 increases the risk for acute ischemic stroke, yet the molecular mechanisms are unclear and remain unresolved medical challenges. We hypothesize that the SARS-CoV-2 spike protein exacerbates stroke and cerebrovascular complications by increasing coagulation and decreasing fibrinolysis by disrupting the renin-angiotensin-aldosterone system (RAAS). A thromboembolic model was induced in humanized ACE2 knock-in mice after one week of SARS-CoV-2 spike protein injection. hACE2 mice were treated with Losartan, an angiotensin receptor (AT₁R) blocker, immediately after spike protein injection. Cerebral blood flow and infarct size were compared between groups. Vascular-contributes to cognitive impairments and dementia was assessed using a Novel object recognition test. Tissue factor-III and plasminogen activator inhibitor-1 were measured using immunoblotting to assess coagulation and fibrinolysis. Human brain microvascular endothelial cells (HBMEC) were exposed to hypoxia with/without SARS-CoV-2 spike protein to mimic ischemic conditions and assessed for inflammation, RAAS balance, coagulation, and fibrinolysis. Our results showed that the SARS-CoV-2 spike protein caused an imbalance in the RAAS that increased the inflammatory signal and decreased the RAAS protective arm. SARS-CoV-2 spike protein increased coagulation and decreased fibrinolysis when coincident with ischemic insult, which was accompanied by a decrease in cerebral blood flow, an increase in neuronal death, and a decline in cognitive function. Losartan treatment restored RAAS balance and reduced spike protein-induced effects. SARS-CoV-2 spike protein exacerbates inflammation and hypercoagulation, leading to increased neurovascular damage and cognitive dysfunction. However, the AT₁R blocker, Losartan, restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications.

SARS-CoV-2 spike protein

RAAS balance

COVID-19-induced thromboembolic complications

Vascular-contributes to cognitive impairments and dementia

The Severe Acute Respiratory Syndrome Corona virus-2 (SARS-CoV-2) is a single-stranded RNA virus responsible for the COVID-19 pandemic. As of 2024, SARS-CoV-2 had killed over a million Americans. COVID-19 causes acute respiratory distress syndrome (ARDS) [1, 2]. ARDS symptoms include severe pneumonia, hypoxemia, and elevated cytokines, known as a cytokine storm. Together, these can result in higher mortality rates, especially in patients with a concomitant cardiovascular disorder such as hypertension, diabetes, or obesity.

Many studies reported that COVID-19 causes neurological symptoms [3, 4]. COVID-19-induced neurological symptoms range from headaches and loss of taste and smell to encephalopathy, cognitive impairments, and stroke [5]. The mechanisms of these neurovascular disorders are unclear and represent a knowledge gap. COVID-19 caused life-threatening coagulopathies such as stroke and deep vein thrombosis [6]. Several reports indicate that these negative thrombotic events are associated with up to one-third of COVID-19 patients [7–9]. The molecular mechanisms behind increased hyper-coagulopathies and impaired fibrinolysis are unclear and require further investigation [10]. The development of vaccination against the SARS-CoV-2 virus has decreased mortality rates. However, long-term COVID-19-induced hyper-coagulopathy and cognitive dysfunction represent a significant medical challenge that requires intensive studies [11].

Angiotensin-converting enzyme-2 (ACE-2) is an enzyme that plays a crucial role in the RAAS balance. ACE-2 degrades angiotensin II, the bioactive form, which can bind to the angiotensin type receptor 1 (AT₁R) or the angiotensin type receptor 2 (AT₂R). AT₁R is more abundant than AT₂R, and its activation leads to physiological effects such as vasoconstriction, proinflammatory, thrombosis, and generation of reactive oxygen species [12–14]. Activation of the Ang II/AT₁R axis pathway increases hyper-coagulations via upregulation of the tissue factor (TF), thrombin activation, and platelet aggregation [15, 16]. In contrast, Ang II/AT₂R axis opposes the effects of AT₁R activation through vasodilation and anti-inflammatory responses and maintains hemostasis [17–19]. ACE-2 binds to the SARS-CoV-2 spike protein and acts as a functional receptor for the spike protein to internalize the viral particle [20]. SARS-CoV-2 downregulates the ACE-2 receptor [4], which decreases Ang II degradation, therefore increasing the RAAS destructive arm with increased activation of the abundant AT₁R over AT₂R [21, 22].

This study aims to investigate one of the possible mechanisms by which SARS-CoV-2 Spike protein alters the coagulation and fibrinolysis hemostasis, leading to stroke and neurovascular complications. We hypothesize that the SARS-CoV-2 spike protein exacerbates stroke and neurovascular complications by increasing coagulation and decreasing fibrinolysis via disrupting the RAAS balance. We examined the effect of SARS-CoV-2 spike protein in a mouse model of humanized ACE-2 knock-in (hACE2 KI) mice. Spike protein was injected for seven days before the induction stroke. Stroke is induced through distal middle cerebral artery (MCA) thromboembolism using ferric chloride (FeCl₃). We examined cerebral blood flow, infarct-sized and vascular contribution to cognitive impairments, and dementia (VCID) in stroked animals after one week of SARS-CoV-2 spike protein injection.

Our previous studies showed that SARS-CoV-2 spike protein disrupts RAAS balance and increases brain inflammation [4]. Here, our results provide novel evidence that SARS-CoV-2 Spike protein increased coagulation and decreased fibrinolysis in hACE2 KI mice. These effects were accompanied by decreased cerebral blood flow, increased neuronal death, and increased cognitive dysfunctions. Our results showed that using the AT₁R blocker, Losartan restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications.

Animals

Transgenic humanized ACE2 KI mice (hACE2 KI), B6.129S2 (Cg)-Ace2^{tm1(ACE2)Dwnt}/J mice were purchased from Jackson Laboratory (Jax lab: stock no. 035000, Ellsworth, Maine, USA). hACE2 KI mice have human ACE2 cDNA replacing the endogenous mouse Ace2 sequences. The endogenous Ace2 regulatory elements direct expression of human ACE2, the receptor used for cellular entry by several coronaviruses, including severe acute respiratory syndrome coronavirus-1 (SARS-CoV) and − 2 (SARS-CoV-2) [4, 23]. ARRIVE guidelines 2.0 were followed in conducting animal studies in accordance with the ethical guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mercer University Institutional Animal Care and Use Committee (IACUC) is accredited by the American Association for Accreditation of Laboratory Animal Care, and has approved the animal protocols. A standard mice chow diet was provided to the animals, as was unlimited access to tap water. Twelve-hour cycles of light and dark were used for the mice.

Animal Treatment

hACE2 mice were randomly assigned to four groups: 1) sham, 2) stroke, 3) stroke + SARSCoV-2 spike protein injection, and 4) stroke + SARSCoV-2 spike protein injection + Losartan. A recombinant protein for SARS-CoV-2 nucleoprotein/spike protein (4ug/animal, Invitrogen, USA, Cat. No. RP-87706) was injected intravenously via jugular vein injection 7 days prior to MCA/FeCl₃ thromboembolic surgery. Sham animals were exposed to the MCA/FeCl₃ thromboembolic surgery without FeCl₃ treatment.

Losartan (10 mg/kg body weight, Tokyo Chemical Industry, Tokyo, Japan, Cat. No. L0232) was added to the water supply of the animal cages. Losartan was first administered immediately after the injection of the recombinant spike protein for SARS-CoV-2.

MCA/FeCl₃ Induced Thromboembolic Model

Eight-week-old male hACE2 KI mice weighing 20-25g were randomly assigned for surgery. Mice were anesthetized using a mixture of Ketamine and Xylazine intraperitoneal (IP) injection (100mg/kg ketamine and 10 mg/kg Xylazine). Long-acting Buprenorphine (0.1 mg/kg) IP injection was also given before surgery to decrease postoperative pain. A vertical incision was made between the eye and ear to locate the temporal muscle. A horizontal incision is then made on the lateral ridge of the frontal bone, and a vertical incision from the rostral-most point of the parietal bone is made to expose the skull. The distal trunk of the middle cerebral artery is then located and exposed by drilling through the mouse skull. The meninges were removed to expose the artery, and the transient focal ischemia model was initiated by using fresh FeCl₃ (20%) soaked filter paper for five minutes. After removal, sterile warm saline was applied to the area for 10 minutes and then dried with sterile gauze. The temporal muscle was then placed back, the mouse was sutured, and the surgical area was disinfected with a povidone-iodine solution. The mouse was then monitored under a heat lamp until recovery.

Assessment of Cerebral Blood Flow

Cerebral blood flow was measured with the RFLSI Ⅲ Laser Speckle Imaging System (RWD, San Diego, CA, USA). Mice were anesthetized using isoflurane, and a vertical incision was made from the sagittal suture to the interferential suture on the mouse’s skull. RWD laser speckle imaging was used to record the cerebral blood flow. LSCI software (Version 5.0) was used to measure the perfusion per area at hours 1, 2, 3, 6, and 24. Perfusion was represented as a percentage ratio of the stroke to the non-stroke hemisphere of the brain.

Assessment of Infarct Volume

hACE2 mice brains were collected 24 hours after MCA/FeCl₃ thromboembolic model surgery. These brains were sectioned into 5 coronal 2mm thick slices using an acrylic brain matrix. Coronal brain slices were examined by staining with 2% 2,3,5-triphenyl tetrazolium chloride (TTC) and incubating for 15 min in a humidified incubator (37%C, 5% CO2). Sections were photographed with units of measure to be quantified blindly using Image-J software.

Assessment of Cognitive Functioning

Memory and learning functions were assessed at baseline, before SARS-CoV-2 spike protein injection, and 24 hours after MCA/FeCl₃ thromboembolic surgery using Novel Object Recognition testing (NOR) [24, 25].

The NOR test is a 3-stage test used to examine recognition memory. The first stage involves habituation, with the mouse being exposed to its testing environment. The second is the familiarization stage, which involves the mouse being exposed to two identical objects. The final phase is the testing phase, which involves the replacement of one of the familiar objects with a novel object. The testing phase is recorded using ANY-maze software. Time spent around the novel object, the ratio of time spent investigating the novel object to the familiar object, and the total distance traveled were measured using the ANY-maze software recording and analyzed blindly.

Cell Culture

Primary human brain microvascular endothelial cells (HBMECs, from Angio-Proteomie, Boston, MA, USA, Cat. No. cAP-0002) were grown to confluency in complete media (MCDB-131 Complete, VEC Technologies, Inc., Rensselaer, NY, USA). HBMECs were switched to serum-reduced media and treated with SARS-CoV-2 recombinant spike protein (100 µM) with or without losartan (100 µM) for 24 hrs. The Next day, HBMECs were either kept under normoxia conditions (room air 21% oxygen) or placed in a hypoxia chamber (0.1% oxygen, BioSpherix, ProOx Model 110) for 6 hours. Cells were collected immediately once removed from the hypoxia chamber.

Polymerase Chain Reaction

RT-PCR was performed as described previously [4]. RNA was isolated using Triazole (Thermo-Fisher, USA, Cat. No. AC345480250) and quantified using Thermo Scientific NanoDrop 2000C Spectrophotometer (Thermo Scientific, USA). cDNA was prepared from isolated RNA using SuperScript IV VILO Master mix with an ezDNase kit (Invitrogen, USA, Cat. No. 11766050). The Quant Studio™ 3 Real-Time PCR System (Applied Biosystems, Thermo Scientific, USA) was utilized to run qRT-PCR using PowerUp SyBR Green Master mix (Applied Biosystems, Thermo Scientific, USA, Cat No. A25742). All primer sequences used are detailed in (Table 1). GAPDH was consistently used as the reference gene for normalization.

Table 1

PCR primers.
Primer	Species	FWD sequence	REV sequence
ACE2	Human	5’-TCC ATT GGT CTT CTG TCA CCC G-3’	5’-AGA CCA TCC ACC TCC ACT TCT C-3’
ACE2	Mouse	5’-TCC ATT GGT CTT CTG CCA TCC G-3’	5’-AGA CCA TCC ACC TCC ACT TCT C-3’
AT₁R	Human	5’-CAG CGT CAG TTT CAA CTT GTA CG-3’	5’-GCA GGT GAC TTT GGC TAC AAG C-3’
AT₁R	Mouse	5’-GCC ATT GTC CAC CCG ATG AAG T-3’	5’-ACA CAT TTC GGT GGA TGA CGG C-3’
AT₂R	Human	5’-CCA TGT TCT GAC CTT CCT GGA TG-3’	5’-CGG ATT AAC GCA GCT GTT GGT G-3’
AT₂R	Mouse	5’-CGT GAC CAA GTC CTG AAG ATG G-3’	5’-GGA AGT GCC AGG TCA ATG ATG ACT G-3’
GAPDH	Human	5’-GTC TCC TCT GAC TTC AAC AGC G-3’	5’-ACC ACC CTG TTG CTG TAG CCA A-3’
GAPDH	Mouse	5’-CAT CAC TGC CAC CCA GAA GAC TG-3’	5’-ATG CCA GTG AGC TTC CCG TTC AG-3’
Il-1β	Human	5’-CCA CAG ACC TTC CAG GAG AAT G-3’	5’-GTG CAG TTC AGT GAT CGT ACA GG-3’
Il-1β	Mouse	5’-TGG ACC TTC CAG GAT GAG GAC A-3’	5’-GTT CAT CTC GGA GCC TGT AGT G-3’
I-6	Human	5’-AGA CAG CCA CTC ACC CTCT TAC G-3’	5’-TTC TGC CAG TGC CTC TTT GCT G-3’
I-6	Mouse	5’-TAC CAC TTC ACA AGT CGG AGG C-3’	5’-CTG CAA GTG CAT CAT CGT TGT TC-3’
MASR	Human	5’-CAG CAC CAT CTT GGT CGT GAA G-3’	5’-CAG CAG GTA AAG GAG TCT CAT GG-3’
MASR	Mouse	5’-CTG ACA GCC ATC AGT GTG GAG A-3’	5’-GTG GTC ACC AAG CAC GAA AGT G-3’
NFκB	Mouse	5’-GCT GCC AAA GAA GGA CAC GAC A-3’	5’-GGC AGG CTA TTG CTC ATC ACA G-3’
TNFα	Human	5’-CTC TTC TGC CTG CTG CAC TTT G-3’	5’-ATG GGC TAC AGG CTT GTC ACT C-3’
TNFα	Mouse	5’-GGT GCC TAT GTC TCA GCC TCT T-3’	5’-GCC ATA GAA CTG ATG AGA GGG AG-3’

Immunoblotting

RIPA buffer (Millipore, Billerica, MA, USA, Cat# 3P 20188) was used to extract protein from the hACE2 brain and HBMECs. Equal protein loads were separated on 10% SDS-polyacrylamide gel utilizing the Mini PROTEAN Tetra Cell SDS-PAGE Gel electrophoresis kit (Biorad Laboratories Inc, Hercules, CA). Gels were transferred onto nitrocellulose membranes using the Bio-Rad Trans-Blot Turbo (Biorad Laboratories Inc, Hercules, CA, USA). Membranes were blocked with 5% milk and incubated overnight with primary antibodies. Membranes were washed and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody. Membranes were reacted with Western chemiluminescent HRP Substrate (Millipore, USA) and imaged using the iBright Imaging system (Invitrogen Thermo Fisher Scientific, USA, Model FL1500). Image-J software (Version 1.54g) was used to measure band intensity. β-actin was used for normalization. All antibodies used are listed in (Table 2).

Table 2

Antibodies
Primary Antibody	Company	CAT/ Lot/ Prod	Recommended Concentration	Dilution Immunoblot	Secondary Antibody
AT₁R	Invitrogen	PA5-20812	1 mg/mL	1:1000	Rabbit
AT₂R	Novus	NBP1-60097	1 mg/mL	1:1000	Rabbit
Β-actin	R&D	MAB8929	1 mg/mL	1:1000	Mouse
PAI-1	Biotechne	MAB-17861-100	1 mg/mL	1:1000	Rabbit
TNFα	NOVUS	NBP1-19532	1 mg/mL	1:500	Rabbit
TF-III	R&D	MAB-3178	1 mg/mL	1:1000	Mouse

Statistical Analysis

GraphPad prism version 10 or higher was employed for all data analysis. For animal studies, the sample size was determined from our previous work [4]. One-way ANOVA was utilized to assess mean differences between 1) sham, 2) stroke, 3) stroke + SARS-CoV-2 spike protein injection, and 4) stroke + SARS-CoV-2 spike protein injection + Losartan. Significance was determined at P < 0.05. Data is presented as mean ± standard deviation. A Tukey’s post-hoc test was used to adjust for the multiple comparisons to assess significant interaction effects from all analyses.

SARS-CoV-2 spike protein intensifies RAAS imbalance after ischemic insult.

We assessed the effect of SARS-CoV-2 spike protein on the gene expression of both RAAS arms in hACE2 KI mice brain and human microvascular endothelial cells (HBMECs) subjected to ischemic insult. We analyzed the gene expression of ACE2 in brain homogenate. Moreover, we measured AT₁R, AT₂R, and MASR in both the contralateral and ipsilateral brain homogenate using qRT-PCR. Singh et al. showed that a transient MCA occlusion stroke model increases ACE2 expression in the lung of mice [26]. Here, we showed that the MCA/FeCl₃ transient stroke model increases the ACE2 expression in the brain of hACE2 KI mice (Fig. 1.A). We previously showed that SARS-CoV-2 spike protein downregulated ACE2 expression in hACE2 KI mice brains [4]. In the current study, a similar finding was reported. SARS-CoV-2 spike protein downregulates ACE2 gene expression in hACE2 KI brains after the thromboembolic occlusion model (Fig. 1.A). Losartan treatment restored ACE2 gene expression after SARS-CoV-2 spike protein injection with the thromboembolic occlusion model. Next, we evaluated the AT₁R, AT₂R, and MASR in both stroke and non-stroke hemispheres. Our results showed that ischemic insult increases the expression of AT₁R by 2-fold. Pre-injection of SARS-CoV-2 spike protein before the ischemic insult further increases the expression of AT₁R to 4-fold in the brain of hACE2 KI mice (Fig. 1.B). In contrast, SARS-CoV-2 spike protein decreased the gene expression of the RAAS protective arms AT₂R and MASR (Fig. 1.C-D). These results were confirmed in HBMECs that were treated with SARS-CoV-2 spike protein and subjected to hypoxia to mimic the ischemic insult. Our results showed that SARS-CoV-2 spike protein increases AT₁R expression and decreases AT₂R expression under normoxia control conditions. The addition of ischemic insult further augments SARS-CoV-2 spike protein-induced effects. Losartan treatment reduced spike protein-induced effects. (Fig. 1.E-F).

Increased inflammation after a double hit, SARS-CoV-2 spike protein followed by ischemic insult exacerbate inflammation in hACE2 brains.

Stroke is known to increase brain inflammation [27]. We have previously shown that intravenous injection of SARS-CoV-2 spike protein increases inflammation in the brain of hACE2 KI mice [4]. Here, we examined the effect of SARS-CoV-2 spike protein injection coincident with ischemic insult on brain inflammation. Our results showed that SARS-CoV-2 spike protein pre-injection significantly intensifies inflammation after induction of thromboembolic occlusion model. Ischemic insults increased inflammation in both ipsilateral and contralateral brain hemispheres. Pre-injection of SARS-CoV-2 spike protein significantly increased NF_ϏB, TNF-α, Il-1β, and Il-6 gene expression in the brains of hACE2 KI mice. Losartan showed a modest but significant effect in reducing the SARS-CoV-2 spike protein-induced effect (Fig. 2.A-D). Our results showed similar findings when we examined the impact of SARS-CoV-2 spike protein on HBMCE subjected to hypoxia. SARS-CoV-2 spike protein significantly increased the expression of TNF-α under normoxia conditions. Pretreatment of HBMECs with SARS-CoV-2 spike protein prior to hypoxia further significantly increased endothelial cell inflammation (Fig. 2.E).

SARS-CoV-2 spike protein disrupts coagulation homeostasis

We examined the effect of SARS-CoV-2 spike protein on the prothrombotic state. The disruption of the endothelial matrix leads to the release of tissue factor III (TF-III), which activates the coagulation cascade, resulting in thrombin activation. Our results showed that the thromboembolic stroke model exacerbated TF-III protein expression in hACE2 KI mice brain homogenate. The injection of spike protein before stroke further escalated TF-III expression. (Fig. 3.A-C). PAI-1 inhibits the conversion of plasminogen to plasmin by urokinase-plasminogen activator and tissue-type plasminogen activator (tPA), thereby hindering fibrin degradation. Our results showed that the thromboembolic stroke model increased PAI-1, and the expression of PAI-1 was further increased by the injection of the spike protein seven days before the stroke. (Fig. 3.A-C). Similar findings were observed in the HBMECs when exposed to hypoxic conditions with and without SARS-CoV-2 spike protein. Spike protein favors clot formation by increasing coagulation and decreasing fibrinolysis. Losartan treatment decreased spike protein-induced hypercoagulation. (Fig. 3. D-E).

SARS-CoV-2 spike protein decreases cerebral blood flow following stroke.

The thromboembolic occlusion was induced in hACE2 KI mice using the MCA/FeCl₃ model. SARS-CoV-2 spike protein was injected intravenously one week before stroke induction. Laser speckle imaging was employed to measure cerebral blood flow at 1, 2, 3, 6, and 24 hours post-surgery. Our results showed that the thromboembolic model decreased cerebral blood flow in the stroke hemisphere compared to the contralateral hemisphere. The spontaneous recanalization occurred, and cerebral blood flow was restored within six hours of the embolic model. The pre-treatment with SARS-COV-2 spike protein showed a significant decrease in cerebral blood flow and increased vascular recanalization time to over 6 hrs. Treatment with Losartan helped in faster restoration of cerebral blood flow and decreased recanalization time compared to spike protein. (Fig. 4.A-B).

SARS-CoV-2 spike protein increases infarction volume following stroke.

ACE2 KI mice were injected with SARS-CoV-2 spike protein one week before thromboembolic model induction. Brains were isolated and stained with TTC stain to detect dead infarct volume. Our results showed that pre-treatment with SARS-CoV-2 spike protein significantly increased infarct volume compared to stroke. Treatment with the AT₁R blocker, Losartan, significantly reduced infarct volume (Fig. 5, A-B).

SARS-CoV-2 spike protein aggravates cognitive dysfunction after thromboembolic occlusion model.

We have previously shown that SARS-CoV-2 causes cognitive dysfunction in hACE2 KI mice [4]. Here, we assessed the effect of SARS-CoV-2 spike protein on cognitive function after induction of the thromboembolic model. hACE2 KI mice were injected with SARS-CoV-2 spike protein one week before thromboembolic model induction. The Novel Object Recognition test was used to assess memory and learning in hACE2 KI mice. hACE2 KI mice were familiarized with two identical objects. On test day, one of the objects was replaced with a novel object. Time spent investigating the novel object indicates memory and learning cognition in the mouse. We assessed the total distance traveled for each animal to exclude motor dysfunction. Our results showed no significant changes between groups in the total distance traveled (Fig. 6.A). There was a significant decrease in both the number of entries into the novel object zone and the time spent interacting with the novel object in the mice subjected to stroke. A similar decrease in the number of entries was observed in the group that received a prior spike protein injection before stroke. However, the spike protein pre-injection further exacerbated the reduction in time spent with the novel object. Treatment with Losartan significantly improved hACE2 KI mice’s cognitive function, as evidenced by the increase in both the time spent with the novel object and the number of entries into the novel object zone. (Fig. 6.B-D)

The COVID-19 pandemic caused over one million American deaths. Survivors suffered a wide range of cerebrovascular complications, including stroke and cognitive impairments. The potential mechanisms underlying these disorders are not fully understood. Our study tests one of the possible mechanisms by which SARS-CoV-2 disrupts coagulation hemostasis and increases cerebrovascular thromboembolic complications. The main finding of our study is that the SARS-CoV-2 spike protein disrupts the renin-angiotensin-aldosterone system (RAAS) balance in the brain vasculature. The SARS-CoV-2 spike protein increases Ang II/AT₁R signaling in the brain’s endothelial cells at the expense of the Ang II/AT₂R protective arm, which increases brain inflammation. Moreover, our results showed that RAAS imbalance contributes to increased coagulation and decreased fibrinolysis, exacerbating stroke, and vascular contribution to cognitive impairments and dementia (VCID). Lastly, restoration of RAAS balance using AT₁R blocker, Losartan, decreased SARS-CoV-2 spike protein-induced thromboembolic cerebrovascular complications.

With the development of effective COVID-19 vaccines and the reduction of COVID-19 mortality, many COVID-19-induced neurovascular complications are more clinically visible. COVID-19 causes a wide range of neurological disorders [28–31]. These neurological disorders ranged from headaches, loss of smell, and altered mental status to encephalitis and ischemic stroke. COVID-19 not only increased the ischemic stroke rates in the general population but also increased mortality and severity in stroke patients. COVID-19 infections worsen stroke outcomes, especially in patients with a prevalence of vascular risk factors, including age, male gender, hypertension, hyperlipidemia, ischemic heart disease, and diabetes mellitus [4, 11]. There are multiple hypotheses that account for COVID-19’s increased thromboembolic events in patients, including increased vascular inflammation and cytokine storms, endothelial dysfunction, pericyte loss, blood-brain barrier dysfunction, and neuroinflammation [32–36]. Here, we hypothesized that SARS-CoV-2 spike protein exacerbates stroke and cerebrovascular complications by increasing coagulation and decreasing fibrinolysis via disrupting the RAAS balance.

SARS-CoV-2 spike protein binds with the ACE-2 receptor as one of the binding sites to achieve cell entry. ACE-2 plays a crucial role in the degradation of Ang II, the bioactive form of the RAAS, to Ang 1–7. We have previously shown that SARS-CoV-2 spike protein decreases ACE-2 expression and increases Ang II/AT₁R downstream inflammatory signaling and endothelial cell apoptosis in the brain of humanized ACE2 knock-in mice [4]. Our study also showed that spike protein significantly downregulated the RAAS protective arm with decreased AT₂R and MAS receptor expression ³. Singh et al. showed that transient MCA occlusion increases ACE-2 expression in mice, which might increase the binding affinity to SARS-CoV-2 spike protein [26]. In the present study, we provide novel evidence that the SARS-CoV-2 spike protein-induced RAAS imbalance increases coagulation and decreases fibrinolysis, which worsens ischemic stroke outcomes in a distal middle cerebral artery (MCA) thromboembolic model.

Our results showed that SARS-CoV-2 spike protein increases coagulation via increased Tissue Factor III (TF-III) expression in brain endothelial cells. TF-III activates the extrinsic coagulation pathway that activates factor VII, which in turn catalyzes the conversion of the inactive factor X into the active factor Xa. In addition, SARS-CoV-2 spike protein increased the expression of Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that inhibits endogenous tissue-type plasminogen activator (tPA) activation and hence prevents fibrinolysis. Elevated PAI-1 is associated with thrombosis and atherosclerosis [37]. These effects were reversed with the use of Losartan, an AT₁R blocker. These findings were confirmed in human brain microvascular endothelial cells (HBMECs), in which spike protein increased TF-III and PAI-1 following exposure to hypoxic conditions in HBMECs exposed to hypoxia.

We used a mild chemically induced distal transient MCA thromboembolic model where a clot forms and spontaneously recanalizes within a few hours. However, the model outcomes were significantly changed when animals were pre-injected with SARS-CoV-2 spike protein. Our study showed that pre-injection of SARS-CoV-2 spike protein intensified the decrease in cerebral blood flow and delayed recanalization in the thromboembolic model. These effects may be the result of increased clot formation and reduction in fibrinolysis. Our results showed that SARS-CoV-2 spike protein increased TF-III and PAI expression. Moreover, increased clot formation and delayed recanalization were associated with significant neurological damage, as seen with increased brain infarct size in hACE2 KI mice preinjected with spike protein compared to stroke. Restoration of RAAS balance using AT₁R blocker, Losartan prevented SARS-CoV-2 spike protein-induced thromboembolic cerebrovascular complications.

Finally, we reported that vascular dysfunction contributes to cognitive impairment and dementia associated with SARS-CoV-2 spike protein-induced thromboembolic stroke. These results agree with Ahmed et al., who showed that the restoration of RAAS via AT₂R activation contributes to the improvement of cognitive impairments after stroke [38]. We showed that Losartan significantly improved cognitive functions after SARS-CoV-2 spike protein-induced thromboembolic ischemic stroke.

In conclusion, our study provides new evidence that SARS-CoV-2 spike protein increased coagulation and decreased fibrinolysis in hACE2 KI mice. These effects were accompanied by decreased cerebral blood flow, increased neuronal death, and increased cognitive dysfunctions. Our results showed that restoring RAAS balance using the AT₁R blocker, Losartan, restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications.

Funding:

This study was funded by the American Heart Association grant number 23AIREA1045073 to MA.

Author Contribution

M.A. and SP.H. wrote the main manuscript text and SP.H and VH. prepared figures. All authors reviewed the manuscript.

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SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Animal Treatment

MCA/FeCl₃ Induced Thromboembolic Model

Assessment of Cerebral Blood Flow

Assessment of Infarct Volume

Assessment of Cognitive Functioning

Cell Culture

Polymerase Chain Reaction

Immunoblotting

Statistical Analysis

Results

SARS-CoV-2 spike protein disrupts coagulation homeostasis

Discussion

Declarations

Funding:

Author Contribution

References

Additional Declarations

Status:

Journal Publication

Version 1

SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Animal Treatment

MCA/FeCl3 Induced Thromboembolic Model

Assessment of Cerebral Blood Flow

Assessment of Infarct Volume

Assessment of Cognitive Functioning

Cell Culture

Polymerase Chain Reaction

Immunoblotting

Statistical Analysis

Results

SARS-CoV-2 spike protein disrupts coagulation homeostasis

Discussion

Declarations

Funding:

Author Contribution

References

Additional Declarations

Status:

Journal Publication

Version 1

MCA/FeCl₃ Induced Thromboembolic Model