Endothelial dysfunction and thrombotic complications associated with SARS-CoV-2 in adults: a systematic review.

Introduction: During the pandemic, occurrences of thromboembolic complications and endothelial dysfunctions associated with COVID-19 were observed. Thus, the pathophysiology of the mechanisms of action are not fully elucidated. Methods: Aiming at a better understanding of the associated mechanisms of endothelial function due to COVID-19, a systematic review was developeded out using index databases and a selection process in which the inclusion criteria were articles that addressed the mechanism of thrombotic events by endothelium dysfunction and publications that associate COVID-19 with thrombotic complications in adults up to 59 years of age. Conclusion: SARS CoV 2 is associated with altered endothelial function by endothelial mechanisms induced by direct viral infection and injury, leading to changes in the angiotensin II/AT1 axis and inflammatory response.


Introduction
Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by the SARS-CoV-2 coronavirus that has reached pandemic proportions since December 2019, with more than 3 million confirmed cases worldwide and more than 260,000 deaths in just 5 months (Róman et al., 2020).
This way, when being notified of the hypothesis of the outbreak in Wuhan, China, having been caused by a new strain of the coronavirus, the World Health Organization (WHO) declared a public health emergency of international concern (WHO, 2020).
All systemic manifestations of COVID-19, including thrombotic complications, are considered to be caused by direct endothelial dysfunction in infected patients, as human endothelial cells express ACE2, CD147, sialic acid receptor and transmembrane serine protease 2. Cofactors linked by the COVID-19 virus to access host cells. In addition, the risk of a large-vessel and cardiogenic cerebrovascular accident (CVA) has already been described in the literature, even without the classic factors for this in individuals with an infected respiratory tract (Amoah et al., 2020).
Angiotensin converting enzyme 2 (ACE2) combines with the spike surface protein of host cells when SARS-CoV-2 invades them. This enzyme is widely expressed in arterial and venous endothelial cells, epithelial cells, neurocortex, brainstem, neural cortex, alveolar cells, muscle cells of organs and enterocytes of the small intestine. (Fei et al., 2020).
So much that it is believed that diffuse endotheliitis is caused by endothelial damage arising from this mechanism between ACE2 and SARS-CoV-2, as damage to endothelial cells can lead to excessive host immune activation, thrombosis and a procoagulant/hypercoagulable state that eventually evolves for diffuse intravascular coagulation, leading to embolism and multi-organ failure. In addition, microvascular thrombosis occurs systematically and can also affect various organs (Chen & Pan, 2021).

Methodology
This is a systematic review with a protocol registered at OSF (https://osf.io/r425d). Inclusion criteria were articles that addressed the mechanism of thrombotic events through endothelial dysfunction and publications that associated COVID-19 with thrombotic complications in adults up to 59 years of age. There was no restriction on date of publication or language.

Study selection process
The studies collected from the indexers were entered into Mendeley Desktop. From then on, the selection was carried out manually by two independent evaluators, divided into three stages: 1. Pre-analysis: verification of texts through title and abstract in order to verify their eligibility based on the criteria described in the methodology; 2. Exploration of the material: the articles were selected based on their ability to answer the research question; 3. Treatment of the results: critical analysis of the articles selected for the conclusion of this study.

Data extraction and synthesis
After selecting the articles used for the study, data such as: name of the main author; purpose of the study; kind of study; outcome; Thrombotic complications and complications after endothelial dysfunction were entered manually into a table generated by the Microsoft Word 2016© program (Table 2).

Carmeliet (2021)
To research the viral relationship of SARS-CoV-2 with endothelial cells and inflammatory changes.

Cohort study
Emerging evidence suggests that the virus alters vessel barrier integrity, promoting a procoagulative state, inducing inflammation (endothelitis) and mediating inflammatory cell infiltration.

Freda et al. (2021)
To investigate how the prothrombotic and pro-inflammatory responses of endothelial cells are altered after exposure to the spike, nucleocap-sid and membraneenvelope proteins of SARS-CoV-2.
Cohort study Significant increases in the expression of thrombotic and inflammatory markers were found, with no change in cell culture parameters.

Gencer et al. (2020)
To examine mechanisms that may explain how viral entry and endothelial cell activation by SARS-CoV-2 can give rise to systemic inflammation, thrombosis and microvascular dysfunction.

Cohort study
Recognizing comorbidities and potential organ damage over the course of severe COVID-19 is crucial in the clinical management of patients that affect treatment approaches and recovery rate.

Agrati et al. (2021)
The objective of the study was to investigate, in critically ill patients hospitalized with COVID-19, the plasma concentration of P-selectin as a biomarker of endothelial dysfunction and platelet activation.

Case-control study
Elevated P-selectin suggests a central role for platelet endothelial interaction as part of the multifaceted pathogenic mechanism of COVID-19, leading to local activation of the hemostatic system forming pulmonary thrombi.

Nagashima et al. (2020)
To identify tissue biomarkers associated with endothelial activation/dysfunction and the pyroptosis pathway in lung samples from patients with COVID-19.

Case-control study
The results demonstrated endothelial dysfunction and suggested the participation of the pyroptosis pathway in lung samples.

Schmaier et al. (2021)
To assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and changes in the Tie2/angiopoietin axis.

Cohort study
In conclusion, by the dysfunction of procoagulant endothelial cells, the degree of which increases in parallel with the severity of the COVID-19 disease. Elevated levels of Angpt-2 may potentiate endothelial cell dysfunction through inhibition of Tie2 antithrombotic signaling.

Zhang et al,
To assess platelet and coagulation changes in patients with COVID-19.
Case-control study SARS-CoV-2-induced platelet activation may participate in thrombus formation and inflammatory responses in patients with COVID-19.

Results and Discussion
At the end of the searches, 1124 articles were collected, of which 104 were duplicates. During the pre-analysis phase 1020 were evaluated and 950 excluded as reported in the study selection process based on title and abstract. During the period of exploration of the material, 61 studies out of 70 were excluded because they did not answer the main question of the research. The flowchart of the results built according to the PRISMA method, which can be viewed as shown in Figure 1.

Endothelial dysfunction and thrombotic complications associated with SARS-CoV-2 in
adults: a systematic review. Pamplona et. al. Brazilian Journal of Implantology and Health Sciences Volume 5, Issue 4 (2023), Page xx-xx. Among the cases of thromboembolism identified in the study (9/9), complications could also be evidenced, such as venous thromboembolism and hypoxia that occurred in 5/9 (55.56%) of the cases, followed by Disseminated Intravascular Coagulation -DIC that corresponds to 44.43% of the cases and DVT which was evidenced in 33.32%. The results can also be analyzed in Table 3. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to pneumocytes and contributes to the release of inflammatory cytokines that subsequently damage the endothelium. Furthermore, endothelial cells express ACE-2 (angiotensin-converting enzyme) which is associated with SARS-CoV-2 infection (Nagashima et. al., 2020). Excessive production of oxidative stress is triggered by functional ACE-2. Thus, it induces TNF-α and stimulates NADPH-oxidase in serial clinical examination, further regulating the expression of endothelial adhesion molecules, such as ICAM-1, and increasing cell permeability (Ma et al., 2022).
A cohort of lung autopsy samples from 98 patients with mild, moderate, or severe COVID-19 was assessed using circulating endothelial markers and revealed deep endothelial dysfunction indicative of a prothrombotic state. In addition, a prothrombotic endothelial signature was found, evidenced by an increase in Von Willebrand factor and loss of anticoagulant proteins (Schamaier et. al., 2021).
Anticoagulant and antiplatelet molecules are observed in the endothelial cell. This is covered by a glycocalyx that serves as a barrier against platelets and blood cells. Compromise of the glycocalyx would lead to endothelial activation, endothelitis and thrombotic events (endotheliopathy or endothelial dysfunction) (Nagashima et al., 2020).
Under prothrombotic conditions, capillary-alveolar endothelial cells receive a shower of cytokines from infected SARS-CoV-2 pneumocytes, and breakdown of the glycocalyx promotes endothelial dysfunction and thrombosis. In part, the higher levels of IL-6 and TNF-α in the COVID-19 group are responsible for the endothelial dysfunction (Nagashima et al., 2020).
Furthermore, endothelial cells contribute to the onset and spread of acute respiratory distress syndrome (ARDS), according to emerging evidence, by altering the integrity of the vascular barrier, inducing vascular inflammation (endothelitis), promoting a procoagulative state, and mediating the inflammatory cell infiltration (Carmeliet, 2021).
The protein angiotensin converting enzyme 2 (ACE2) is a central regulator of the renin-angiotensin aldosterone system (RAAS), a hormonal system that influences the maintenance of blood pressure and fluids. Imbalance can lead to increased inflammation and thrombosis. ACE2 is used by SARS CoV 2 to Endothelial dysfunction and thrombotic complications associated with SARS-CoV-2 in adults: a systematic review. Pamplona et. al. Brazilian Journal of Implantology and Health Sciences Volume 5, Issue 4 (2023), Page xx-xx.
invade host cells. Its expression is low in cardiomyocytes and high and specific for pericytes (PER) (Gencer et. al., 2020). These are central nervous system (CNS) cells that make up the blood-brain barrier (BBB) and endothelium of cerebral blood vessels.
Angiotensin II and its type I receptor (AT1) promote vascular wall inflammation, NF kB-mediated adhesion molecule expression, cytokine and chemokine release, and increased oxidative stress. These factors contribute to endothelial dysfunction and the recruitment of arterial leukocytes in the development of atherosclerotic plaques (Gencer et. al., 2020).
Endothelial dysfunction can be promoted by SARS CoV2 by shifting the balance in the Renin Angiotensin Aldosterone System (RAAS) to the angiotensin II/AT1 axis, in such a way as to elevate oxidative stress and inflammation. Endothelial dysfunction is characterized by decreased nitric oxide levels as a consequence of impaired endothelial nitric oxide synthase function. Nitric oxide is a vasodilator and its deficiency leads to hypertension by constricting blood vessels, which can trigger even more thrombosis and vascular inflammation. In addition to RAAS-mediated effects, emerging evidence has revealed that SARS CoV 2 can also directly cause endothelial dysfunction by infecting endothelial cells (Gencer et. al., 2020).
Activation of the inflammatory and thrombotic mechanism by SARS-CoV-2. The infection is related to an increase in inflammatory cytokines and coagulation disorders, with a predisposition to thrombus formation. PAR: protease-activated receptor; TLR4: Toll-like receptor 4; ttp: activated partial thromboplastin time; PT: prothrombin time; IL: interleukin; TNFα; tumor necrosis factor-α ( Figure 2).

Figure 2. Activation of the inflammatory and thrombotic mechanism by SARS-CoV-2
Source: . Current rates of thromboembolic events demonstrate the importance of post-discharge surveillance and, potentially, extended post-discharge thromboprophylaxis (Giannis et.al., 2020). The incidence of complications is approximately 30% of arterial and venous thromboembolic events of COVID-19. The hypercoagulability profile is composed of elevated levels of D-dimers and fibrinogen, prolonged prothrombin time (PT) and mild thrombocytopenia (Almyroudi & Dimopoulos, 2020). D-dimer being evidenced in 33.32% of the total articles analyzed.

Endothelial dysfunction and thrombotic complications associated with SARS-
A simple and inexpensive biomarker to identify a high-risk subpopulation with latent systemic microvascular damage, regardless of respiratory symptoms, is schistocytes. There was 87.5% evidence of myocardial injury in all with schistocytes ≥ 1%. Thus, the in vivo evaluation of diffuse endothelial damage with formation of fibrin thrombi can be performed through the presence of schistocytes (Della Rocca et al., 2021). At any stage of disease severity, regardless of pulmonary involvement, one can observe their presence and an increase in high-sensitivity troponin T (7 in 10; 70.0%). Because of this, hypercoagulability may explain the variety of clinical manifestations such as myocardial infarction with normal coronary arteries, neurological manifestations and purpura (Della Rocca et al., 2021).
SARS-CoV-2 and its Spike protein directly increase platelet activation, through the binding of PAC-1, expressed by CD62P secretes the α granule, causing the release of dense granules to occur and have platelet spread and clot retraction in vitro (Zhang et.al., 2021). Furthermore, increase the plasma concentration of P-selectin that can reduces significantly after the platelets removal (Agrati et. al.,2021).
The release of clotting factors, the secretion of inflammatory factors and the formation of leukocyteplatelet aggregates are facilitated by direct stimulation of SARS-CoV-2 and spike protein on platelets.

Figure 3. Parameters in critically ill patients with Covid-19
Endothelial dysfunction and thrombotic complications associated with SARS-CoV-2 in adults: a systematic review. Pamplona et. al. Brazilian Journal of Implantology and Health Sciences Volume 5, Issue 4 (2023), Page xx-xx.
TF cells (residual factor) and MVs (precoagulant microvesicles) in patients with COVID-19 were two to four times higher than HS (p < 0.0001). As well as P-selectin and PLA. The residual TG correlates with the severity of the disease (Canzano et al., 2020).
For Christopher (Freda et. al., 2021): The endothelial cell gC1qR, receptor of the globular head of C1q, acts in the regulation of inflammatory and thrombotic responses observed during vascular pathologies, it was decided to investigate how the structural proteins SARS-CoV-2 interact with the endothelial cells gC1qR. In the study, SARS-CoV-2 structural proteins together with endothelial cell culture and gC1qR expression show rapid and significant changes in the expression of molecules that are related to inflammatory and thrombotic progression. Our data also further support the role of the gC1qR, and specifically the C1q binding site on the gC1qR, as a docking receptor for viral proteins.

Conclusion
In conclusion, systemic endothelial cell injury driven by SARS CoV 2 increases the threat of multiorgan failure, and patients suffering from impaired endothelial function due to underlying conditions such as CVDs are at a much greater risk of serious complications from COVID 19 (Gencer et. al., 2020). SARS CoV 2 can impair endothelial function by several mechanisms, including endothelitis induced by direct viral infection and endothelial injury, leading to changes in the angiotensin II/AT1 axis and host inflammatory response.
For future work, we recommend further study of vaccines in their study phases, in addition to the pathophysiology and ways of interfering with the cytokine storm and the mechanisms that cause greater comorbidity and mortality from the virus, such as endothelial dysfunction. We also suggest that new drugs be studied to help prevent complications and sequelae of the disease.