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1.
FASEB J ; 36(1): e22052, 2022 01.
Article in English | MEDLINE | ID: covidwho-1550589

ABSTRACT

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.


Subject(s)
COVID-19 , Endothelial Cells , Endothelium, Vascular , Glycocalyx , SARS-CoV-2/metabolism , COVID-19/metabolism , COVID-19/pathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Glycocalyx/metabolism , Glycocalyx/pathology , Glycocalyx/virology , Humans , Oxidative Stress , Sulfotransferases/metabolism
2.
Immunity ; 54(11): 2632-2649.e6, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1549842

ABSTRACT

The incidence and severity of sepsis is higher among individuals of African versus European ancestry. We found that genetic risk variants (RVs) in the trypanolytic factor apolipoprotein L1 (APOL1), present only in individuals of African ancestry, were associated with increased sepsis incidence and severity. Serum APOL1 levels correlated with sepsis and COVID-19 severity, and single-cell sequencing in human kidneys revealed high expression of APOL1 in endothelial cells. Analysis of mice with endothelial-specific expression of RV APOL1 and in vitro studies demonstrated that RV APOL1 interfered with mitophagy, leading to cytosolic release of mitochondrial DNA and activation of the inflammasome (NLRP3) and the cytosolic nucleotide sensing pathways (STING). Genetic deletion or pharmacological inhibition of NLRP3 and STING protected mice from RV APOL1-induced permeability defects and proinflammatory endothelial changes in sepsis. Our studies identify the inflammasome and STING pathways as potential targets to reduce APOL1-associated health disparities in sepsis and COVID-19.


Subject(s)
Apolipoprotein L1/genetics , COVID-19/genetics , Genetic Predisposition to Disease/genetics , Sepsis/genetics , Animals , Apolipoprotein L1/blood , COVID-19/pathology , DNA, Mitochondrial/metabolism , Endothelial Cells/metabolism , Humans , Inflammation/genetics , Inflammation/pathology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Mitophagy/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Risk Factors , Sepsis/pathology , Severity of Illness Index , /statistics & numerical data
3.
Int J Mol Sci ; 22(21)2021 Nov 03.
Article in English | MEDLINE | ID: covidwho-1502439

ABSTRACT

The 2019 novel coronavirus, known as severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) or coronavirus disease 2019 (COVID-19), is causing a global pandemic. The virus primarily affects the upper and lower respiratory tracts and raises the risk of a variety of non-pulmonary consequences, the most severe and possibly fatal of which are cardiovascular problems. Data show that almost one-third of the patients with a moderate or severe form of COVID-19 had preexisting cardiovascular comorbidities such as diabetes mellitus, obesity, hypertension, heart failure, or coronary artery disease. SARS-CoV2 causes hyper inflammation, hypoxia, apoptosis, and a renin-angiotensin system imbalance in a variety of cell types, primarily endothelial cells. Profound endothelial dysfunction associated with COVID-19 can be the cause of impaired organ perfusion that may generate acute myocardial injury, renal failure, and a procoagulant state resulting in thromboembolic events. We discuss the most recent results on the involvement of endothelial dysfunction in the pathogenesis of COVID-19 in patients with cardiometabolic diseases in this review. We also provide insights on treatments that may reduce the severity of this viral infection.


Subject(s)
COVID-19/pathology , Endothelial Cells/metabolism , COVID-19/complications , COVID-19/virology , Cytokine Release Syndrome/etiology , Endothelial Cells/cytology , Endothelial Cells/virology , Heart Failure/etiology , Humans , Renal Insufficiency/etiology , Renin-Angiotensin System/physiology , SARS-CoV-2/isolation & purification , Thrombosis/etiology
4.
Clin Appl Thromb Hemost ; 27: 10760296211042940, 2021.
Article in English | MEDLINE | ID: covidwho-1484251

ABSTRACT

The world is in a hard battle against COVID-19. Endothelial cells are among the most critical targets of SARS-CoV-2. Dysfunction of endothelium leads to vascular injury following by coagulopathies and thrombotic conditions in the vital organs increasing the risk of life-threatening events. Growing evidences revealed that endothelial dysfunction and consequent thrombotic conditions are associated with the severity of outcomes. It is not yet fully clear that these devastating sequels originate directly from the virus or a side effect of virus-induced cytokine storm. Due to endothelial dysfunction, plasma levels of some biomarkers are changed and relevant clinical manifestations appear as well. Stabilization of endothelial integrity and supporting its function are among the promising therapeutic strategies. Other than respiratory, COVID-19 could be called a systemic vascular disease and this aspect should be scrutinized in more detail in order to reduce related mortality. In the present investigation, the effects of COVID-19 on endothelial function and thrombosis formation are discussed. In this regard, critical players, laboratory findings, clinical manifestation, and suggestive therapies are presented.


Subject(s)
Blood Coagulation , COVID-19/virology , Endothelial Cells/virology , Endothelium, Vascular/virology , SARS-CoV-2/pathogenicity , Thrombosis/virology , Animals , COVID-19/blood , COVID-19/pathology , COVID-19/physiopathology , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiopathology , Host-Pathogen Interactions , Humans , Signal Transduction , Thrombosis/blood , Thrombosis/pathology , Thrombosis/physiopathology
5.
Methods Mol Biol ; 2311: 185-193, 2021.
Article in English | MEDLINE | ID: covidwho-1482181

ABSTRACT

Studies of blood-brain barrier (BBB) require developing of a novel and convenient in vitro endothelial cell model. We isolated primary human and rodent brain microvascular endothelial cells and developed methods for culturing, characterization, and high-efficiency transfection of endothelial cells. Here, we describe the improved methods to obtain in vitro human and rodent BBB models to study expression of endogenous and exogenous genes of interest.


Subject(s)
Blood-Brain Barrier/physiology , Brain/blood supply , Cell Separation , Endothelial Cells/physiology , Microvessels/cytology , Transfection , Animals , Blood-Brain Barrier/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Cells, Cultured , Endothelial Cells/metabolism , Fetus , Gestational Age , Humans , Mice , Rats
6.
Viruses ; 13(10)2021 10 08.
Article in English | MEDLINE | ID: covidwho-1463838

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease (COVID-19), is currently infecting millions of people worldwide and is causing drastic changes in people's lives. Recent studies have shown that neurological symptoms are a major issue for people infected with SARS-CoV-2. However, the mechanism through which the pathological effects emerge is still unclear. Brain endothelial cells (ECs), one of the components of the blood-brain barrier, are a major hurdle for the entry of pathogenic or infectious agents into the brain. They strongly express angiotensin converting enzyme 2 (ACE2) for its normal physiological function, which is also well-known to be an opportunistic receptor for SARS-CoV-2 spike protein, facilitating their entry into host cells. First, we identified rapid internalization of the receptor-binding domain (RBD) S1 domain (S1) and active trimer (Trimer) of SARS-CoV-2 spike protein through ACE2 in brain ECs. Moreover, internalized S1 increased Rab5, an early endosomal marker while Trimer decreased Rab5 in the brain ECs. Similarly, the permeability of transferrin and dextran was increased in S1 treatment but decreased in Trimer, respectively. Furthermore, S1 and Trimer both induced mitochondrial damage including functional deficits in mitochondrial respiration. Overall, this study shows that SARS-CoV-2 itself has toxic effects on the brain ECs including defective molecular delivery and metabolic function, suggesting a potential pathological mechanism to induce neurological signs in the brain.


Subject(s)
Blood-Brain Barrier/metabolism , Brain/pathology , COVID-19/pathology , Endothelial Cells/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Brain/metabolism , Brain/virology , Endothelial Cells/virology , Humans , Mice , Mitochondria/metabolism , Protein Domains , SARS-CoV-2/metabolism , rab5 GTP-Binding Proteins/metabolism
7.
mBio ; 12(4): e0157221, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1349194

ABSTRACT

Tissue- and cell-specific expression patterns are highly variable within and across individuals, leading to altered host responses after acute virus infection. Unraveling key tissue-specific response patterns provides novel opportunities for defining fundamental mechanisms of virus-host interaction in disease and the identification of critical tissue-specific networks for disease intervention in the lung. Currently, there are no approved therapeutics for Middle East respiratory syndrome coronavirus (MERS-CoV) patients, and little is understood about how lung cell types contribute to disease outcomes. MERS-CoV replicates equivalently in primary human lung microvascular endothelial cells (MVE) and fibroblasts (FB) and to equivalent peak titers but with slower replication kinetics in human airway epithelial cell cultures (HAE). However, only infected MVE demonstrate observable virus-induced cytopathic effect. To explore mechanisms leading to reduced MVE viability, donor-matched human lung MVE, HAE, and FB were infected, and their transcriptomes, proteomes, and lipidomes were monitored over time. Validated functional enrichment analysis demonstrated that MERS-CoV-infected MVE were dying via an unfolded protein response (UPR)-mediated apoptosis. Pharmacologic manipulation of the UPR in MERS-CoV-infected primary lung cells reduced viral titers and in male mice improved respiratory function with accompanying reductions in weight loss, pathological signatures of acute lung injury, and times to recovery. Systems biology analysis and validation studies of global kinetic transcript, protein, and lipid data sets confirmed that inhibition of host stress pathways that are differentially regulated following MERS-CoV infection of different tissue types can alleviate symptom progression to end-stage lung disease commonly seen following emerging coronavirus outbreaks. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe atypical pneumonia in infected individuals, but the underlying mechanisms of pathogenesis remain unknown. While much has been learned from the few reported autopsy cases, an in-depth understanding of the cells targeted by MERS-CoV in the human lung and their relative contribution to disease outcomes is needed. The host response in MERS-CoV-infected primary human lung microvascular endothelial (MVE) cells and fibroblasts (FB) was evaluated over time by analyzing total RNA, proteins, and lipids to determine the cellular pathways modulated postinfection. Findings revealed that MERS-CoV-infected MVE cells die via apoptotic mechanisms downstream of the unfolded protein response (UPR). Interruption of enzymatic processes within the UPR in MERS-CoV-infected male mice reduced disease symptoms, virus-induced lung injury, and time to recovery. These data suggest that the UPR plays an important role in MERS-CoV infection and may represent a host target for therapeutic intervention.


Subject(s)
Acute Lung Injury/pathology , Apoptosis/physiology , Coronavirus Infections/pathology , Unfolded Protein Response/physiology , Acute Lung Injury/virology , Animals , Cell Line , Endothelial Cells/metabolism , Endothelial Cells/virology , Female , Fibroblasts/metabolism , Fibroblasts/virology , Humans , Male , Mice , Middle East Respiratory Syndrome Coronavirus/immunology
8.
Biol Pharm Bull ; 44(10): 1371-1379, 2021.
Article in English | MEDLINE | ID: covidwho-1445700

ABSTRACT

The vascular permeability of the endothelium is finely controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions. In the majority of normal adult tissues, endothelial cells in blood vessels maintain vascular permeability at a relatively low level, while in response to inflammation, they limit vascular barrier function to induce plasma leakage and extravasation of immune cells as a defense mechanism. Thus, the dynamic but also simultaneously tight regulation of vascular permeability by endothelial cells is responsible for maintaining homeostasis and, as such, impairments of its underlying mechanisms result in hyperpermeability, leading to the development and progression of various diseases including coronavirus disease 2019 (COVID-19), a newly emerging infectious disease. Recently, increasing numbers of studies have been unveiling the important role of Rap1, a small guanosine 5'-triphosphatase (GTPase) belonging to the Ras superfamily, in the regulation of vascular permeability. Rap1 enhances VE-cadherin-mediated endothelial cell-cell junctions to potentiate vascular barrier functions via dynamic reorganization of the actin cytoskeleton. Importantly, Rap1 signaling activation reportedly improves vascular barrier function in animal models of various diseases associated with vascular hyperpermeability, suggesting that Rap1 might be an ideal target for drugs intended to prevent vascular barrier dysfunction. Here, we describe recent progress in understanding the mechanisms by which Rap1 potentiates VE-cadherin-mediated endothelial cell-cell adhesions and vascular barrier function. We also discuss how alterations in Rap1 signaling are related to vascular barrier dysfunction in diseases such as acute pulmonary injury and malignancies. In addition, we examine the possibility of Rap1 signaling as a target of drugs for treating diseases associated with vascular hyperpermeability.


Subject(s)
Antigens, CD/metabolism , Cadherins/metabolism , Capillary Permeability , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Intercellular Junctions/metabolism , rap1 GTP-Binding Proteins/metabolism , Animals , Humans
9.
Ophthalmic Res ; 64(5): 785-792, 2021.
Article in English | MEDLINE | ID: covidwho-1443682

ABSTRACT

INTRODUCTION: In December 2019, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic broke out. The virus rapidly spread globally, resulting in a major world public-health crisis. The major disease manifestation occurs in the respiratory tract. However, further studies documented other systemic involvement. This study investigates histopathologic eye changes in postmortem material of coronavirus disease 2019 (COVID-19) patients. METHODS: Sections of formalin-fixed, paraffin-embedded eyes from 5 patients (10 eyes) who died of COVID-19 at the University Hospital in Basel were included. Gross examination and histological evaluation were performed by 3 independent ophthalmopathologists. Immunohistochemical staining was performed using antibodies against fibrin, cleaved caspase 3, and ACE-2. Five enucleated eyes of patients not infected with SARS-CoV-2 served as control group. All cases have been studied for presence of SARS-CoV-2 RNA by means of reverse transcription PCR and RNA in situ hybridization (ISH). The choroidal vessels of one case were analyzed with electron microscope. RESULTS: Ophthalmopathologically, 8 eyes from 4 patients displayed swollen endothelial cells in congested choroidal vessels. No further evidence of specific eye involvement of SARS-CoV-2 was found in any of the patients. In the 8 eyes with evidence of changes due to SARS-CoV-2, immunohistochemical staining demonstrated fibrin microthrombi, apoptotic changes of endothelial and inflammatory cells. In control eyes, ACE-2 was detectable in the conjunctiva, cornea, retina, and choroidea and displayed significantly lower amounts of stained cells as in COVID-19 eyes. SARS-CoV-2 RNA was detectable in both bulbi of 2/5 patients, yet ISH failed to visualize viruses. Electron microscopy showed no significant results due to the artifacts. DISCUSSION/CONCLUSION: As already described in other organs of COVID-19 patients, the ophthalmological examination revealed-microthrombi, that is, hypercoagulation and vasculopathy most probably due to endothelial damage. A possible viral spread to the endothelial cells via ACE-2 provides one pathophysiological explanation. The expression of ACE-2 receptors in the conjunctiva hints toward its susceptibility to infection. To what extend eyes, function is disrupted by SARS-CoV-2 is subject to further studies, especially in the clinic.


Subject(s)
COVID-19/pathology , Choroid Diseases/pathology , Eye Infections, Viral/pathology , RNA, Viral/genetics , Retinal Diseases/pathology , SARS-CoV-2/genetics , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , COVID-19 Nucleic Acid Testing , Caspase 3/metabolism , Choroid/blood supply , Choroid/pathology , Choroid Diseases/virology , Ciliary Body/blood supply , Ciliary Body/pathology , Conjunctiva/metabolism , Cornea/metabolism , Endothelial Cells/metabolism , Eye Infections, Viral/virology , Female , Fibrin/metabolism , Humans , Immunohistochemistry , In Situ Hybridization , Male , Real-Time Polymerase Chain Reaction , Retinal Diseases/virology , Retinal Vessels/pathology , Thrombosis/metabolism , Thrombosis/pathology
10.
J Virol ; 95(23): e0139621, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1434896

ABSTRACT

Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiorgan failure in patients with coronavirus disease 2019 (COVID-19). However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved in endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkably, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HUB1-CoV, and influenza virus H1N1 did not activate endothelial cells. These findings are consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy and suggests that simvastatin, an FDA-approved lipid-lowering drug, may help prevent the pathogenesis and improve the outcome of COVID-19 patients. IMPORTANCE Coronavirus disease 2019 (COVID-19), caused by the betacoronavirus SARS-CoV-2, is a worldwide challenge for health care systems. The leading cause of mortality in patients with COVID-19 is hypoxic respiratory failure from acute respiratory distress syndrome (ARDS). To date, pulmonary endothelial cells (ECs) have been largely overlooked as a therapeutic target in COVID-19, yet emerging evidence suggests that these cells contribute to the initiation and propagation of ARDS by altering vessel barrier integrity, promoting a procoagulative state, inducing vascular inflammation and mediating inflammatory cell infiltration. Therefore, a better mechanistic understanding of the vasculature is of utmost importance. In this study, we screened the SARS-CoV-2 viral proteins that potently activate human endothelial cells and found that nucleocapsid protein (NP) significantly activated human endothelial cells through TLR2/NF-κB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Our results provide insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/virology , SARS-CoV-2 , Signal Transduction , Simvastatin/pharmacology , COVID-19/virology , Cell Line , Human Umbilical Vein Endothelial Cells , Humans , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Toll-Like Receptor 2/metabolism
11.
Viruses ; 13(9)2021 09 17.
Article in English | MEDLINE | ID: covidwho-1430976

ABSTRACT

Human leukocyte antigen (HLA) is a group of molecules involved in inflammatory and infective responses. We evaluated blood sHLA-E and sHLA-G levels in hospitalized COVID-19 patients with respiratory failure and their relationship with clinical evolution, changes in endothelial activation biomarker profile, and neutrophil adhesion. sHLA-E, sHLA-G, and endothelial activation biomarkers were quantified by ELISA assay in plasma samples. Neutrophil adhesion to endothelium was assessed in the presence/absence of patients' plasma samples. At admission, plasma levels of sHLA-G and sHLA-E were significantly higher in COVID-19 patients with respiratory failure compared to controls. COVID-19 clinical improvement was associated with increased sHLA-G plasma levels. In COVID-19, but not in control patients, an inverse correlation was found between serum sICAM-1 and E-selectin levels and plasma sHLA-G values. The in vitro analysis of activated endothelial cells confirmed the ability of HLA-G molecules to control sICAM-1 and sE-selectin expression via CD160 interaction and FGF2 induction and consequently neutrophil adhesion. We suggest a potential role for sHLA-G in improving COVID-19 patients' clinical condition related to the control of neutrophil adhesion to activated endothelium.


Subject(s)
Biomarkers , COVID-19/immunology , COVID-19/virology , HLA-G Antigens/immunology , Neutrophils/immunology , SARS-CoV-2/immunology , Aged , Alleles , COVID-19/epidemiology , Cell Adhesion/immunology , Endothelial Cells/immunology , Endothelial Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Gene Frequency , HLA-G Antigens/blood , Humans , Male , Middle Aged , Models, Biological , Neutrophils/metabolism
12.
Redox Biol ; 46: 102099, 2021 10.
Article in English | MEDLINE | ID: covidwho-1401817

ABSTRACT

The outbreak of COVID-19 has remained uncontained with urgent need for robust therapeutics. We have previously reported sex difference of COVID-19 for the first time indicating male predisposition. Males are more susceptible than females, and more often to develop into severe cases with higher mortality. This predisposition is potentially linked to higher prevalence of cigarette smoking. Nonetheless, we found for the first time that cigarette smoking extract (CSE) had no effect on angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) expression in endothelial cells. The otherwise observed worse outcomes in smokers is likely linked to baseline respiratory diseases associated with chronic smoking. Instead, we hypothesized that estrogen mediated protection might underlie lower morbidity, severity and mortality of COVID-19 in females. Of note, endothelial inflammation and barrier dysfunction are major mediators of disease progression, and development of acute respiratory distress syndrome (ARDS) and multi-organ failure in patients with COVID-19. Therefore, we investigated potential protective effects of estrogen on endothelial cells against oxidative stress induced by interleukin-6 (IL-6) and SARS-CoV-2 spike protein (S protein). Indeed, 17ß-estradiol completely reversed S protein-induced selective activation of NADPH oxidase isoform 2 (NOX2) and reactive oxygen species (ROS) production that are ACE2-dependent, as well as ACE2 upregulation and induction of pro-inflammatory gene monocyte chemoattractant protein-1 (MCP-1) in endothelial cells to effectively attenuate endothelial dysfunction. Effects of IL-6 on activating NOX2-dependent ROS production and upregulation of MCP-1 were also completely attenuated by 17ß-estradiol. Of note, co-treatment with CSE had no additional effects on S protein stimulated endothelial oxidative stress, confirming that current smoking status is likely unrelated to more severe disease in chronic smokers. These data indicate that estrogen can serve as a novel therapy for patients with COVID-19 via inhibition of initial viral responses and attenuation of cytokine storm induced endothelial dysfunction, to substantially alleviate morbidity, severity and mortality of the disease, especially in men and post-menopause women. Short-term administration of estrogen can therefore be readily applied to the clinical management of COVID-19 as a robust therapeutic option.


Subject(s)
COVID-19 , Estrogens/therapeutic use , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/genetics , COVID-19/drug therapy , COVID-19/metabolism , Chemokine CCL2/genetics , Endothelial Cells/metabolism , Female , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Male , NADPH Oxidase 2 , Reactive Oxygen Species/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Up-Regulation
13.
Cells ; 10(9)2021 09 04.
Article in English | MEDLINE | ID: covidwho-1403545

ABSTRACT

Stroke is the third leading cause of mortality in women and it kills twice as many women as breast cancer. A key role in the pathophysiology of stroke plays the disruption of the blood-brain barrier (BBB) within the neurovascular unit. While estrogen induces vascular protective actions, its influence on stroke remains unclear. Moreover, experiments assessing its impact on endothelial cells to induce barrier integrity are non-conclusive. Since pericytes play an active role in regulating BBB integrity and function, we hypothesize that estradiol may influence BBB by regulating their activity. In this study using human brain vascular pericytes (HBVPs) we investigated the impact of estradiol on key pericyte functions known to influence BBB integrity. HBVPs expressed estrogen receptors (ER-α, ER-ß and GPER) and treatment with estradiol (10 nM) inhibited basal cell migration but not proliferation. Since pericyte migration is a hallmark for BBB disruption following injury, infection and inflammation, we investigated the effects of estradiol on TNFα-induced PC migration. Importantly, estradiol prevented TNFα-induced pericyte migration and this effect was mimicked by PPT (ER-α agonist) and DPN (ER-ß agonist), but not by G1 (GPR30 agonist). The modulatory effects of estradiol were abrogated by MPP and PHTPP, selective ER-α and ER-ß antagonists, respectively, confirming the role of ER-α and ER-ß in mediating the anti-migratory actions of estrogen. To delineate the intracellular mechanisms mediating the inhibitory actions of estradiol on PC migration, we investigated the role of AKT and MAPK activation. While estradiol consistently reduced the TNFα-induced MAPK and Akt phosphorylation, only the inhibition of MAPK, but not Akt, significantly abrogated the migratory actions of TNFα. In transendothelial electrical resistance measurements, estradiol induced barrier function (TEER) in human brain microvascular endothelial cells co-cultured with pericytes, but not in HBMECs cultured alone. Importantly, transcriptomics analysis of genes modulated by estradiol in pericytes showed downregulation of genes known to increase cell migration and upregulation of genes known to inhibit cell migration. Taken together, our findings provide the first evidence that estradiol modulates pericyte activity and thereby improves endothelial integrity.


Subject(s)
Brain/blood supply , Cell Movement/drug effects , Estradiol/pharmacology , Gene Expression Profiling , Pericytes/cytology , Cell Movement/genetics , Cell Proliferation/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gene Expression Regulation/drug effects , Humans , Mitogen-Activated Protein Kinases/metabolism , Pericytes/drug effects , Pericytes/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Estrogen/metabolism , Tumor Necrosis Factor-alpha/metabolism
14.
Proteomics ; 21(2): e2000246, 2021 01.
Article in English | MEDLINE | ID: covidwho-1384281

ABSTRACT

The genome of coronaviruses, including SARS-CoV-2, encodes for two proteases, a papain like (PLpro ) protease and the so-called main protease (Mpro ), a chymotrypsin-like cysteine protease, also named 3CLpro or non-structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that Mpro proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS-CoV, SARS-CoV-2, and hCoV-NL63 coronviruses, an LC-MS based N-terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS-CoV-2 Mpro , 445 cleavage events from more than 300 proteins are identified, while 151 and 331 Mpro derived cleavage events are identified for SARS-CoV and hCoV-NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.


Subject(s)
COVID-19/virology , Coronavirus 3C Proteases/metabolism , Coronavirus NL63, Human/enzymology , Peptide Fragments/analysis , SARS Virus/enzymology , SARS-CoV-2/enzymology , Viral Proteins/metabolism , COVID-19/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/virology , Epithelial Cells/metabolism , Epithelial Cells/virology , Eukaryotic Initiation Factor-4G/metabolism , Host-Pathogen Interactions , Humans , Lung/metabolism , Lung/virology , Substrate Specificity
15.
CNS Neurosci Ther ; 27(1): 36-47, 2021 01.
Article in English | MEDLINE | ID: covidwho-1388231

ABSTRACT

The blood-brain barrier (BBB) is an important physiological barrier that separates the central nervous system (CNS) from the peripheral circulation, which contains inflammatory mediators and immune cells. The BBB regulates cellular and molecular exchange between the blood vessels and brain parenchyma. Normal functioning of the BBB is crucial for the homeostasis and proper function of the brain. It has been demonstrated that peripheral inflammation can disrupt the BBB by various pathways, resulting in different CNS diseases. Recently, clinical research also showed CNS complications following SARS-CoV-2 infection and chimeric antigen receptor (CAR)-T cell therapy, which both lead to a cytokine storm in the circulation. Therefore, elucidation of the mechanisms underlying the BBB disruption induced by peripheral inflammation will provide an important basis for protecting the CNS in the context of exacerbated peripheral inflammatory diseases. In the present review, we first summarize the physiological properties of the BBB that makes the CNS an immune-privileged organ. We then discuss the relevance of peripheral inflammation-induced BBB disruption to various CNS diseases. Finally, we elaborate various factors and mechanisms of peripheral inflammation that disrupt the BBB.


Subject(s)
Blood-Brain Barrier/metabolism , Brain/metabolism , COVID-19/metabolism , Inflammation Mediators/metabolism , Animals , Blood-Brain Barrier/immunology , Blood-Brain Barrier/pathology , Brain/immunology , Brain/pathology , COVID-19/immunology , COVID-19/pathology , Endothelial Cells/immunology , Endothelial Cells/metabolism , Humans , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Inflammation Mediators/immunology
16.
Sci Adv ; 6(48)2020 11.
Article in English | MEDLINE | ID: covidwho-1388431

ABSTRACT

Acute respiratory distress syndrome is associated with a robust inflammatory response that damages the vascular endothelium, impairing gas exchange. While restoration of microcapillaries is critical to avoid mortality, therapeutic targeting of this process requires a greater understanding of endothelial repair mechanisms. Here, we demonstrate that lung endothelium possesses substantial regenerative capacity and lineage tracing reveals that native endothelium is the source of vascular repair after influenza injury. Ablation of chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TF2) (Nr2f2), a transcription factor implicated in developmental angiogenesis, reduced endothelial proliferation, exacerbating viral lung injury in vivo. In vitro, COUP-TF2 regulates proliferation and migration through activation of cyclin D1 and neuropilin 1. Upon influenza injury, nuclear factor κB suppresses COUP-TF2, but surviving endothelial cells ultimately reestablish vascular homeostasis dependent on restoration of COUP-TF2. Therefore, stabilization of COUP-TF2 may represent a therapeutic strategy to enhance recovery from pathogens, including H1N1 influenza and SARS-CoV-2.


Subject(s)
COUP Transcription Factor II/metabolism , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Influenza A Virus, H1N1 Subtype , Lung/cytology , Lung/physiology , Orthomyxoviridae Infections/metabolism , Regeneration/genetics , Animals , COUP Transcription Factor II/genetics , Cell Movement/genetics , Cell Proliferation/genetics , Disease Models, Animal , Female , Gene Knockout Techniques , HEK293 Cells , Humans , Male , Mice , Mice, Transgenic , Orthomyxoviridae Infections/virology , Transfection
17.
Am J Respir Cell Mol Biol ; 65(3): 300-308, 2021 09.
Article in English | MEDLINE | ID: covidwho-1381187

ABSTRACT

Endothelial dysfunction is implicated in the thrombotic events reported in patients with coronavirus disease (COVID-19), but the underlying molecular mechanisms are unknown. Circulating levels of the coagulation cascade activator PAI-1 are substantially higher in patients with COVID-19 with severe respiratory dysfunction than in patients with bacterial sepsis and acute respiratory distress syndrome. Indeed, the elevation of PAI-1 is recognized as an early marker of endothelial dysfunction. Here, we report that the rSARS-CoV-2-S1 (recombinant severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] viral envelope spike) glycoprotein stimulated robust production of PAI-1 by human pulmonary microvascular endothelial cells (HPMECs). We examined the role of protein degradation in this SARS-CoV-2-S1 induction of PAI-1 and found that the proteasomal degradation inhibitor bortezomib inhibited SARS-CoV-2-S1-mediated changes in PAI-1. Our data further show that bortezomib upregulated KLF2, a shear-stress-regulated transcription factor that suppresses PAI-1 expression. Aging and metabolic disorders are known to increase mortality and morbidity in patients with COVID-19. We therefore examined the role of ZMPSTE24 (zinc metallopeptidase STE24), a metalloprotease with a demonstrated role in host defense against RNA viruses that is decreased in older individuals and in metabolic syndrome, in the induction of PAI-1 in HPMECs by SARS-CoV-2-S1. Indeed, overexpression of ZMPSTE24 blunted enhancement of PAI-1 production in spike protein-exposed HPMECs. In addition, we found that membrane expression of the SARS-CoV-2 entry receptor ACE2 was reduced by ZMPSTE24-mediated cleavage and shedding of the ACE2 ectodomain, leading to accumulation of ACE2 decoy fragments that may bind SARS-CoV-2. These data indicate that decreases in ZMPSTE24 with age and comorbidities may increase vulnerability to vascular endothelial injury by SARS-CoV-2 viruses and that enhanced production of endothelial PAI-1 might play role in prothrombotic events in patients with COVID-19.


Subject(s)
COVID-19/virology , Endothelial Cells/pathology , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Pulmonary Artery/pathology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Aging , COVID-19/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/virology , Humans , Membrane Proteins/genetics , Metalloendopeptidases/genetics , Plasminogen Activator Inhibitor 1/genetics , Proteolysis , Pulmonary Artery/metabolism , Pulmonary Artery/virology , Spike Glycoprotein, Coronavirus/genetics
18.
Stem Cell Reports ; 16(10): 2459-2472, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1377840

ABSTRACT

The pathogenicity of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been attributed to its ability to enter through the membrane-bound angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, it has been heavily speculated that angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) therapy may modulate SARS-CoV-2 infection. In this study, exposure of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and human endothelial cells (hECs) to SARS-CoV-2 identified significant differences in protein coding genes involved in immunity, viral response, and cardiomyocyte/endothelial structure. Specifically, transcriptome changes were identified in the tumor necrosis factor (TNF), interferon α/ß, and mitogen-activated protein kinase (MAPK) (hPSC-CMs) as well as nuclear factor kappa-B (NF-κB) (hECs) signaling pathways. However, pre-treatment of hPSC-CMs or hECs with two widely prescribed antihypertensive medications, losartan and lisinopril, did not affect the susceptibility of either cell type to SARS-CoV-2 infection. These findings demonstrate the toxic effects of SARS-CoV-2 in hPSC-CMs/hECs and, taken together with newly emerging multicenter trials, suggest that antihypertensive drug treatment alone does not alter SARS-CoV-2 infection.


Subject(s)
Antihypertensive Agents/pharmacology , COVID-19/drug therapy , Endothelial Cells/drug effects , Myocytes, Cardiac/drug effects , COVID-19/genetics , Cells, Cultured , Disease Susceptibility , Endothelial Cells/metabolism , Host-Pathogen Interactions/drug effects , Humans , Lisinopril/pharmacology , Losartan/pharmacology , Myocytes, Cardiac/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Transcriptome/drug effects
19.
Biochim Biophys Acta Mol Basis Dis ; 1867(12): 166260, 2021 12 01.
Article in English | MEDLINE | ID: covidwho-1377661

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection-induced inflammatory responses are largely responsible for the death of novel coronavirus disease 2019 (COVID-19) patients. However, the mechanism by which SARS-CoV-2 triggers inflammatory responses remains unclear. Here, we aimed to explore the regulatory role of SARS-CoV-2 spike protein in infected cells and attempted to elucidate the molecular mechanism of SARS-CoV-2-induced inflammation. METHODS: SARS-CoV-2 spike pseudovirions (SCV-2-S) were generated using the spike-expressing virus packaging system. Western blot, mCherry-GFP-LC3 labeling, immunofluorescence, and RNA-seq were performed to examine the regulatory mechanism of SCV-2-S in autophagic response. The effects of SCV-2-S on apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Western blot, and flow cytometry analysis. Enzyme-linked immunosorbent assay (ELISA) was carried out to examine the mechanism of SCV-2-S in inflammatory responses. RESULTS: Angiotensin-converting enzyme 2 (ACE2)-mediated SCV-2-S infection induced autophagy and apoptosis in human bronchial epithelial and microvascular endothelial cells. Mechanistically, SCV-2-S inhibited the PI3K/AKT/mTOR pathway by upregulating intracellular reactive oxygen species (ROS) levels, thus promoting the autophagic response. Ultimately, SCV-2-S-induced autophagy triggered inflammatory responses and apoptosis in infected cells. These findings not only improve our understanding of the mechanism underlying SARS-CoV-2 infection-induced pathogenic inflammation but also have important implications for developing anti-inflammatory therapies, such as ROS and autophagy inhibitors, for COVID-19 patients.


Subject(s)
COVID-19/metabolism , Inflammation/metabolism , Spike Glycoprotein, Coronavirus/immunology , Animals , Apoptosis/immunology , Autophagy/physiology , Cell Line , Chlorocebus aethiops , Endothelial Cells/metabolism , HEK293 Cells , Humans , Inflammation/immunology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , SARS-CoV-2/pathogenicity , Signal Transduction/immunology , Spike Glycoprotein, Coronavirus/metabolism , TOR Serine-Threonine Kinases/metabolism , Vero Cells
20.
Am J Physiol Lung Cell Mol Physiol ; 321(2): L477-L484, 2021 08 01.
Article in English | MEDLINE | ID: covidwho-1376529

ABSTRACT

Acute lung injury (ALI) leading to acute respiratory distress syndrome is the major cause of COVID-19 lethality. Cell entry of SARS-CoV-2 occurs via the interaction between its surface spike protein (SP) and angiotensin-converting enzyme-2 (ACE2). It is unknown if the viral spike protein alone is capable of altering lung vascular permeability in the lungs or producing lung injury in vivo. To that end, we intratracheally instilled the S1 subunit of SARS-CoV-2 spike protein (S1SP) in K18-hACE2 transgenic mice that overexpress human ACE2 and examined signs of COVID-19-associated lung injury 72 h later. Controls included K18-hACE2 mice that received saline or the intact SP and wild-type (WT) mice that received S1SP. K18-hACE2 mice instilled with S1SP exhibited a decline in body weight, dramatically increased white blood cells and protein concentrations in bronchoalveolar lavage fluid (BALF), upregulation of multiple inflammatory cytokines in BALF and serum, histological evidence of lung injury, and activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways in the lung. K18-hACE2 mice that received either saline or SP exhibited little or no evidence of lung injury. WT mice that received S1SP exhibited a milder form of COVID-19 symptoms, compared with the K18-hACE2 mice. Furthermore, S1SP, but not SP, decreased cultured human pulmonary microvascular transendothelial resistance (TER) and barrier function. This is the first demonstration of a COVID-19-like response by an essential virus-encoded protein by SARS-CoV-2 in vivo. This model of COVID-19-induced ALI may assist in the investigation of new therapeutic approaches for the management of COVID-19 and other coronaviruses.


Subject(s)
Acute Lung Injury/pathology , COVID-19/complications , Cell Membrane Permeability , Endothelial Cells/pathology , Lung/pathology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Acute Lung Injury/etiology , Acute Lung Injury/metabolism , Animals , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/virology , Humans , Lung/metabolism , Lung/virology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Subunits , Spike Glycoprotein, Coronavirus/genetics , Virus Replication
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