Promising Strategies for Preserving Adult Endothelium Health and Reversing Its Dysfunction: From Liquid Biopsy to New Omics Technologies and Noninvasive Circulating Biomarkers.

The endothelium has multiple functions, ranging from maintaining vascular homeostasis and providing nutrition and oxygen to tissues to evocating inflammation under adverse conditions and determining endothelial barrier disruption, resulting in dysfunction. Endothelial dysfunction represents a common condition associated with the pathogenesis of all diseases of the cardiovascular system, as well as of diseases of all of the other systems of the human body, including sepsis, acute respiratory distress syndrome, and COVID-19 respiratory distress. Such evidence is leading to the identification of potential biomarkers and therapeutic targets for preserving, reverting, or restoring endothelium integrity and functionality by promptly treating its dysfunction. Here, some strategies for achieving these goals are explored, despite the diverse challenges that exist, necessitating significant bench work associated with an increased number of clinical studies.

Prostacyclin in Intubated Patients with COVID-19 and Severe Endotheliopathy: A Multicenter, Randomized Clinical Trial.

Rationale: The mortality in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who require mechanical ventilation remains high, and endotheliopathy has been implicated. Objectives: To determine the effect of prostacyclin infusion in mechanically ventilated patients infected with SARS-CoV-2 with severe endotheliopathy. Methods: We conducted a multicenter, randomized clinical trial in adults infected with coronavirus disease (COVID-19) who required mechanical ventilation and had a plasma level of thrombomodulin >4 ng/ml; patients were randomized to 72-hour infusion of prostacyclin 1 ng/kg/min or placebo. Measurements and Main Results: The main outcome was the number of days alive and without mechanical ventilation within 28 days. Key secondary outcomes were 28-day mortality and serious adverse events within 7 days. Eighty patients were randomized (41 prostacyclin and 39 placebo). The median number of days alive without mechanical ventilation at 28 days was 16.0 days (SD, 12) versus 5.0 days (SD, 10) (difference of the medians, 10.96 days; 95% confidence interval [CI], -5 to 21; P = 0.07) in the prostacyclin and the placebo groups, respectively. The 28-day mortality was 21.9% versus 43.6% in the prostacyclin and the placebo groups, respectively (risk ratio, 0.50; 95% CI, 0.24 to 0.96; P = 0.06). The incidence of serious adverse events within 7 days was 2.4% versus 12.8% (risk ratio, 0.19; 95% CI, 0.001 to 1.11; P = 0.10) in the prostacyclin and the placebo groups, respectively. Conclusions: Prostacyclin was not associated with a significant reduction in the number of days alive and without mechanical ventilation within 28 days. The point estimates, however, favored the prostacyclin group in all analyses, including 28-day mortality, warranting further investigation in larger trials. Clinical trial registered with www.clinicaltrials.gov (NCT04420741); EudraCT Identifier: 2020-001296-33.

Cerebral microvascular endothelial glycocalyx damage, its implications on the blood-brain barrier and a possible contributor to cognitive impairment.

The socio-economic impact of diseases associated with cognitive impairment is increasing. According to the Alzheimer's Society there are over 850,000 people with dementia in the UK, costing the UK £26 billion in 2013. Therefore, research into treatment of those conditions is vital. Research into the cerebral endothelial glycocalyx (CeGC) could offer effective treatments. The CeGC, consisting of proteoglycans, glycoproteins and glycolipids, is a dynamic structure covering the luminal side oftheendothelial cells of capillaries throughout the body. The CeGC is thicker in cerebral micro vessels, suggesting specialisation for its function as part of the blood-brain barrier (BBB). Recent research evidences that the CeGC is vital in protecting fragile parenchymal tissue and effective functioning of the BBB, as one particularly important CeGC function is to act as a protective barrier and permeability regulator. CeGC degradation is one of the factors which can lead to an increase in BBB permeability. It occurs naturally in aging, nevertheless, premature degradationhas beenevidencedin multipleconditions linked to cognitive impairment, such as inflammation,brain edema, cerebral malaria, Alzheimer's and recently Covid-19. Increasing knowledge of the mechanisms of CeGC damage has led to research into preventative techniques showing that CeGC is a possible diagnostic marker and a therapeutic target. However, the evidence is relatively new, inconsistent and demonstrated mainly in experimental models. This review evaluates the current knowledge of the CeGC, its structure, functions, damage and repair mechanisms and the impact of its degeneration on cognitive impairment in multiple conditions, highlighting the CeGC as a possible diagnostic marker and a potential target for therapeutic treatment.

Vascular endotheliitis associated with infections: Its pathogenetic role and therapeutic implication.

Vascular endothelial cells are major participants in and regulators of immune responses and inflammation. Vascular endotheliitis is regarded as a host immune-inflammatory response of the endothelium forming the inner surface of blood vessels in association with a direct consequence of infectious pathogen invasion. Vascular endotheliitis and consequent endothelial dysfunction can be a principle determinant of microvascular failure, which would favor impaired perfusion, tissue hypoxia, and subsequent organ failure. Emerging evidence suggests the role of vascular endotheliitis in the pathogenesis of coronavirus disease 2019 (COVID-19) and its related complications. Thus, once initiated, vascular endotheliitis and resultant cytokine storm cause systemic hyperinflammation and a thrombotic phenomenon in COVID-19, leading to acute respiratory distress syndrome and widespread organ damage. Vascular endotheliitis also appears to be a contributory factor to vasculopathy and coagulopathy in sepsis that is defined as life-threatening organ dysfunction due to a dysregulated response of the host to infection. Therefore, protecting endothelial cells and reversing vascular endotheliitis may be a leading therapeutic goal for these diseases associated with vascular endotheliitis. In this review, we outline the etiological and pathogenic importance of vascular endotheliitis in infection-related inflammatory diseases, including COVID-19, and possible mechanisms leading to vascular endotheliitis. We also discuss pharmacological agents which may be now considered as potential endotheliitis-based treatment modalities for those diseases.

COVID-19 and the endothelium.

There is growing evidence that COVID-19 not only affects the lungs but beyond that the endothelial system. Recent studies showed that this can lead to microcirculatory impairments and in consequence to functional disorders of all inner organs. The combination of endothelial dysfunction with a generalized inflammatory state and complement elements may together contribute to the overall pro-coagulative state described in COVID-19 patients leading to venular as well as to arteriolar occlusions.

What is the impact of microvascular complications of diabetes on severe COVID-19?

Evidence suggests severe coronavirus disease-19 (COVID-19) infection is characterised by pulmonary and systemic microvasculature dysfunction, specifically, acute endothelial injury, hypercoagulation and increased capillary permeability. Diabetes, which is also characterised by vascular injury in itself, confers an increased risk of adverse COVID-19 outcomes. It has been suggested that pre-existing endothelial dysfunction and microvascular disease in diabetes will exacerbate the vascular insults associated with COVID-19 and thus lead to increased severity of COVID-19 infection. In this article, we evaluate the current evidence exploring the impact of microvascular complications, in the form of diabetic retinopathy and nephropathy, in individuals with COVID-19 and diabetes. Future insights gained from exploring the microvascular injury patterns and clinical outcomes may come to influence care delivery algorithms for either of these conditions.

Endothelial injury in COVID-19 and septic patients.

Systemic inflammatory response, as observed in sepsis and severe COVID-19, may lead to endothelial damage. Therefore, we aim to compare the extent of endothelial injury and its relationship to inflammation in both diseases. We included patients diagnosed with sepsis (SEPSIS group, n = 21), mild COVID-19 (MILD group, n = 31), and severe COVID-19 (SEVERE group, n = 24). Clinical and routine laboratory data were obtained, circulating cytokines (INF-γ, TNF-α, and IL-10) and endothelial injury markers (E-Selectin, Tissue Factor (TF) and von Willebrand factor (vWF)) were measured. Compared to the SEPSIS group, patients with severe COVID-19 present similar clinical and laboratory data, except for lower circulating IL-10 and E-Selectin levels. Compared to the MILD group, patients in the SEVERE group showed higher levels of TNF-α, IL-10, and TF. There was no clear relationship between cytokines and endothelial injury markers among the three studied groups; however, in SEVERE COVID-19 patients, there is a positive relationship between INF-γ with TF and a negative relationship between IL-10 and vWF. In conclusion, COVID-19 and septic patients have a similar pattern of cytokines and endothelial dysfunction markers. These findings highlight the importance of endothelium dysfunction in COVID-19 and suggest that endothelium should be better evaluated as a therapeutic target for the disease.

Syndecan-1, an indicator of endothelial glycocalyx degradation, predicts outcome of patients admitted to an ICU with COVID-19.

BACKGROUND: We investigated the feasibility of two biomarkers of endothelial damage (Syndecan-1 and thrombomodulin) in coronavirus disease 2019 (COVID-19), and their association with inflammation, coagulopathy, and mortality. METHODS: The records of 49 COVID-19 patients who were admitted to an intensive care unit (ICU) in Wuhan, China between February and April 2020 were examined. Demographic, clinical, and laboratory data, and outcomes were compared between survivors and non-survivors COVID-19 patients, and between patients with high and low serum Syndecan-1 levels. The dynamics of serum Syndecan-1 levels were also analyzed. RESULTS: The levels of Syndecan-1 were significantly higher in non-survivor group compared with survivor group (median 1031.4 versus 504.0 ng/mL, P = 0.002), and the levels of thrombomodulin were not significantly different between these two groups (median 4534.0 versus 3780.0 ng/mL, P = 0.070). Kaplan-Meier survival analysis showed that the group with high Syndecan-1 levels had worse overall survival (log-rank test: P = 0.023). Patients with high Syndecan-1 levels also had significantly higher levels of thrombomodulin, interleukin-6, and tumor necrosis factor-α. Data on the dynamics of Syndecan-1 levels indicated much greater variations in non-survivors than survivors. CONCLUSIONS: COVID-19 patients with high levels of Syndecan-1 develop more serious endothelial damage and inflammatory reactions, and have increased mortality. Syndecan-1 has potential for use as a marker for progression or severity of COVID-19. Protecting the glycocalyx from destruction is a potential treatment for COVID-19.

Pathogenesis of COVID-19 described through the lens of an undersulfated and degraded epithelial and endothelial glycocalyx.

The glycocalyx surrounds every eukaryotic cell and is a complex mesh of proteins and carbohydrates. It consists of proteoglycans with glycosaminoglycan side chains, which are highly sulfated under normal physiological conditions. The degree of sulfation and the position of the sulfate groups mainly determine biological function. The intact highly sulfated glycocalyx of the epithelium may repel severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) through electrostatic forces. However, if the glycocalyx is undersulfated and 3-O-sulfotransferase 3B (3OST-3B) is overexpressed, as is the case during chronic inflammatory conditions, SARS-CoV-2 entry may be facilitated by the glycocalyx. The degree of sulfation and position of the sulfate groups will also affect functions such as immune modulation, the inflammatory response, vascular permeability and tone, coagulation, mediation of sheer stress, and protection against oxidative stress. The rate-limiting factor to sulfation is the availability of inorganic sulfate. Various genetic and epigenetic factors will affect sulfur metabolism and inorganic sulfate availability, such as various dietary factors, and exposure to drugs, environmental toxins, and biotoxins, which will deplete inorganic sulfate. The role that undersulfation plays in the various comorbid conditions that predispose to coronavirus disease 2019 (COVID-19), is also considered. The undersulfated glycocalyx may not only increase susceptibility to SARS-CoV-2 infection, but would also result in a hyperinflammatory response, vascular permeability, and shedding of the glycocalyx components, giving rise to a procoagulant and antifibrinolytic state and eventual multiple organ failure. These symptoms relate to a diagnosis of systemic septic shock seen in almost all COVID-19 deaths. The focus of prevention and treatment protocols proposed is the preservation of epithelial and endothelial glycocalyx integrity.

Endothelial contribution to COVID-19: an update on mechanisms and therapeutic implications.

The global propagation of SARS-CoV-2 leads to an unprecedented public health emergency. Despite that the lungs are the primary organ targeted by COVID-19, systemic endothelial inflammation and dysfunction is observed particularly in patients with severe COVID-19, manifested by elevated endothelial injury markers, endotheliitis, and coagulopathy. Here, we review the clinical characteristics of COVID-19 associated endothelial dysfunction; and the likely pathological mechanisms underlying the disease including direct cell entry or indirect immune overreactions after SARS-CoV-2 infection. In addition, we discuss potential biomarkers that might indicate the disease severity, particularly related to the abnormal development of thrombosis that is a fatal vascular complication of severe COVID-19. Furthermore, we summarize clinical trials targeting the direct and indirect pathological pathways after SARS-CoV-2 infection to prevent or inhibit the virus induced endothelial disorders.

Indications of Persistent Glycocalyx Damage in Convalescent COVID-19 Patients: A Prospective Multicenter Study and Hypothesis.

The COVID-19 pandemic is caused by the SARS CoV-2 virus and can lead to severe lung damage and hyperinflammation. In the context of COVID-19 infection, inflammation-induced degradation of the glycocalyx layer in endothelial cells has been demonstrated. Syndecan-1 (SDC-1) is an established parameter for measuring glycocalyx injury. This prospective, multicenter, observational, cross-sectional study analyzed SDC-1 levels in 24 convalescent patients that had been infected with SARS-CoV-2 with mild disease course without need of hospitalization. We included 13 age-matched healthy individuals and 10 age-matched hospitalized COVID-19 patients with acute mild disease course as controls. In convalescent COVID-19 patients, significantly elevated SDC-1 levels were detected after a median of 88 days after symptom onset compared to healthy controls, whereas no difference was found when compared to SDC-1 levels of hospitalized patients undergoing acute disease. This study is the first to demonstrate signs of endothelial damage in non-pre-diseased, convalescent COVID-19 patients after mild disease progression without hospitalization. The data are consistent with studies showing evidence of persistent endothelial damage after severe or critical disease progression. Further work to investigate endothelial damage in convalescent COVID-19 patients should follow.

Coronavirus Disease 2019 (COVID-19) Coronary Vascular Thrombosis: Correlation with Neutrophil but Not Endothelial Activation.

Severe coronavirus disease 2019 (COVID-19) increases the risk of myocardial injury that contributes to mortality. This study used multiparameter immunofluorescence to extensively examine heart autopsy tissue of 7 patients who died of COVID-19 compared to 12 control specimens, with or without cardiovascular disease. Consistent with prior reports, no evidence of viral infection or lymphocytic infiltration indicative of myocarditis was found. However, frequent and extensive thrombosis was observed in large and small vessels in the hearts of the COVID-19 cohort, findings that were infrequent in controls. The endothelial lining of thrombosed vessels typically lacked evidence of cytokine-mediated endothelial activation, assessed as nuclear expression of transcription factors p65 (RelA), pSTAT1, or pSTAT3, or evidence of inflammatory activation assessed by expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), tissue factor, or von Willebrand factor (VWF). Intimal EC lining was also generally preserved with little evidence of cell death or desquamation. In contrast, there were frequent markers of neutrophil activation within myocardial thrombi in patients with COVID-19, including neutrophil-platelet aggregates, neutrophil-rich clusters within macrothrombi, and evidence of neutrophil extracellular trap (NET) formation. These findings point to alterations in circulating neutrophils rather than in the endothelium as contributors to the increased thrombotic diathesis in the hearts of COVID-19 patients.

COVID-19 and dys-regulation of pulmonary endothelium: implications for vascular remodeling.

Coronavirus disease-2019 (COVID-19), the disease caused by severe acute respiratory syndrome-coronavirus-2, has claimed more than 4.4 million lives worldwide (as of 20 August 2021). Severe cases of the disease often result in respiratory distress due to cytokine storm, and mechanical ventilation is required. Although, the lungs are the primary organs affected by the disease, more evidence on damage to the heart, kidney, and liver is emerging. A common link in these connections is the cardiovascular network. Inner lining of the blood vessels, called endothelium, is formed by a single layer of endothelial cells. Several clinical manifestations involving the endothelium have been reported, such as its activation via immunomodulation, endotheliitis, thrombosis, vasoconstriction, and distinct intussusceptive angiogenesis (IA), a unique and rapid process of blood-vessel formation by splitting a vessel into two lumens. In fact, the virus directly infects the endothelium via TMPRSS2 spike glycoprotein priming to facilitate ACE-2-mediated viral entry. Recent studies have indicated a significant increase in remodeling of the pulmonary vascular bed via intussusception in patients with COVID-19. However, the lack of circulatory biomarkers for IA limits its detection in COVID-19 pathogenesis. In this review, we describe the implications of angiogenesis in COVID-19, unique features of the pulmonary vascular bed and its remodeling, and a rapid and non-invasive assessment of IA to overcome the technical limitations in patients with COVID-19.

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism.

Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p < 0.05), and increased THP-1 monocyte adhesion to ECs (p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19.

Increased Autotaxin Levels in Severe COVID-19, Correlating with IL-6 Levels, Endothelial Dysfunction Biomarkers, and Impaired Functions of Dendritic Cells.

Autotaxin (ATX; ENPP2) is a secreted lysophospholipase D catalyzing the extracellular production of lysophosphatidic acid (LPA), a pleiotropic signaling phospholipid. Genetic and pharmacologic studies have previously established a pathologic role for ATX and LPA signaling in pulmonary injury, inflammation, and fibrosis. Here, increased ENPP2 mRNA levels were detected in immune cells from nasopharyngeal swab samples of COVID-19 patients, and increased ATX serum levels were found in severe COVID-19 patients. ATX serum levels correlated with the corresponding increased serum levels of IL-6 and endothelial damage biomarkers, suggesting an interplay of the ATX/LPA axis with hyperinflammation and the associated vascular dysfunction in COVID-19. Accordingly, dexamethasone (Dex) treatment of mechanically ventilated patients reduced ATX levels, as shown in two independent cohorts, indicating that the therapeutic benefits of Dex include the suppression of ATX. Moreover, large scale analysis of multiple single cell RNA sequencing datasets revealed the expression landscape of ENPP2 in COVID-19 and further suggested a role for ATX in the homeostasis of dendritic cells, which exhibit both numerical and functional deficits in COVID-19. Therefore, ATX has likely a multifunctional role in COVID-19 pathogenesis, suggesting that its pharmacological targeting might represent an additional therapeutic option, both during and after hospitalization.

Further Findings Concerning Endothelial Damage in COVID-19 Patients.

Systemic vascular damage with micro/macro-thrombosis is a typical feature of severe COVID-19. However, the pathogenesis of this damage and its predictive biomarkers remain poorly defined. For this reason, in this study, serum monocyte chemotactic protein (MCP)-2 and P- and E-selectin levels were analyzed in 204 patients with COVID-19. Serum MCP-2 and P-selectin were significantly higher in hospitalized patients compared with asymptomatic patients. Furthermore, MCP-2 increased with the WHO stage in hospitalized patients. After 1 week of hospitalization, MCP-2 levels were significantly reduced, while P-selectin increased in patients in WHO stage 3 and decreased in patients in WHO stages 5-7. Serum E-selectin was not significantly different between asymptomatic and hospitalized patients. The lower MCP-2 levels after 1 week suggest that endothelial damage triggered by monocytes occurs early in COVID-19 disease progression. MCP-2 may also predict COVID-19 severity. The increase in P-selectin levels, which further increased in mild patients and reduced in severe patients after 1 week of hospitalization, suggests that the inactive form of the protein produced by the cleavage of the active protein from the platelet membrane is present. This may be used to identify a subset of patients that would benefit from targeted therapies. The unchanged levels of E-selectin in these patients suggest that endothelial damage is less relevant.

Autopsy-Based Pulmonary and Vascular Pathology: Pulmonary Endotheliitis and Multi-Organ Involvement in COVID-19 Associated Deaths.

BACKGROUND: Findings from autopsies have provided evidence on systemic microvascular damage as one of the underlying mechanisms of Coronavirus disease 2019 (CO-VID-19). The aim of this study was to correlate autopsy-based cause of death in SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive patients with chest imaging and severity grade of pulmonary and systemic morphological vascular pathology. METHODS: Fifteen SARS-CoV-2 positive autopsies with clinically distinct presentations (age 22-89 years) were retrospectively analyzed with focus on vascular, thromboembolic, and ischemic changes in pulmonary and in extrapulmonary sites. Eight patients died due to COVID-19 associated respiratory failure with diffuse alveolar damage in various stages and/or multi-organ failure, whereas other reasons such as cardiac decompensation, complication of malignant tumors, or septic shock were the cause of death in 7 further patients. The severity of gross and histopathological changes was semi-quantitatively scored as 0 (absent), 1 (mild), and 3 (severe). Severity scores between the 2 groups were correlated with selected clinical parameters, initial chest imaging, autopsy-based cause of death, and compared using Pearson χ2 and Mann-Whitney U tests. RESULTS: Severe pulmonary endotheliitis (p = 0.031, p = 0.029) and multi-organ involvement (p = 0.026, p = 0.006) correlated significantly with COVID-19 associated death. Pulmonary microthrombi showed limited statistical correlation, while tissue necrosis, gross pulmonary embolism, and bacterial superinfection did not differentiate the 2 study groups. Chest imaging at hospital admission did not differ either. CONCLUSIONS: Extensive pulmonary endotheliitis and multi-organ involvement are characteristic autopsy features in fatal CO-VID-19 associated deaths. Thromboembolic and ischemic events and bacterial superinfections occur frequently in SARS-CoV-2 infection independently of outcome.

Endothelial Dysfunction, Inflammation, and Oxidative Stress in COVID-19-Mechanisms and Therapeutic Targets.

The outbreak of the COVID-19 pandemic represents an ongoing healthcare emergency responsible for more than 3.4 million deaths worldwide. COVID-19 is the disease caused by SARS-CoV-2, a virus that targets not only the lungs but also the cardiovascular system. COVID-19 can manifest with a wide range of clinical manifestations, from mild symptoms to severe forms of the disease, characterized by respiratory failure due to severe alveolar damage. Several studies investigated the underlying mechanisms of the severe lung damage associated with SARS-CoV-2 infection and revealed that the respiratory failure associated with COVID-19 is the consequence not only of acute respiratory distress syndrome but also of macro- and microvascular involvement. New observations show that COVID-19 is an endothelial disease, and the consequent endotheliopathy is responsible for inflammation, cytokine storm, oxidative stress, and coagulopathy. In this review, we show the central role of endothelial dysfunction, inflammation, and oxidative stress in the COVID-19 pathogenesis and present the therapeutic targets deriving from this endotheliopathy.

Cell-Free Hemoglobin Does Not Attenuate the Effects of SARS-CoV-2 Spike Protein S1 Subunit in Pulmonary Endothelial Cells.

SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor angiotensin-converting enzyme 2 (ACE2), which binds to the S1 spike protein on the surface of the virus. To delineate the impact of S1 spike protein interaction with the ACE2 receptor, we incubated the S1 spike protein with human pulmonary arterial endothelial cells (HPAEC). HPAEC treatment with the S1 spike protein caused disruption of endothelial barrier function, increased levels of numerous inflammatory molecules (VCAM-1, ICAM-1, IL-1ß, CCL5, CXCL10), elevated mitochondrial reactive oxygen species (ROS), and a mild rise in glycolytic reserve capacity. Because low oxygen tension (hypoxia) is associated with severe cases of COVID-19, we also evaluated treatment with hemoglobin (HbA) as a potential countermeasure in hypoxic and normal oxygen environments in analyses with the S1 spike protein. We found hypoxia downregulated the expression of the ACE2 receptor and increased the critical oxygen homeostatic signaling protein, hypoxia-inducible factor (HIF-1α); however, treatment of the cells with HbA yielded no apparent change in the levels of ACE2 or HIF-1α. Use of quantitative proteomics revealed that S1 spike protein-treated cells have few differentially regulated proteins in hypoxic conditions, consistent with the finding that ACE2 serves as the host viral receptor and is reduced in hypoxia. However, in normoxic conditions, we found perturbed abundance of proteins in signaling pathways related to lysosomes, extracellular matrix receptor interaction, focal adhesion, and pyrimidine metabolism. We conclude that the spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in HPAEC, and that treatment with HbA failed to reverse the vast majority of these spike protein-induced changes.