Angiotensin converting enzyme 2 gene expression and markers of oxidative stress are correlated with disease severity in patients with COVID-19.

BACKGROUND: Oxidative stress is thought to play a significant role in the pathogenesis and severity of COVID-19. Additionally, angiotensin converting enzyme 2 (ACE2) expression may predict the severity and clinical course of COVID-19. Accordingly, the aim of the present study was to evaluate the association of oxidative stress and ACE2 expression with the clinical severity in patients with COVID-19. METHODS AND RESULTS: The present study comprised 40 patients with COVID-19 and 40 matched healthy controls, recruited between September 2021 and March 2022. ACE 2 expression levels were measured using Hera plus SYBR Green qPCR kits with GAPDH used as an internal control. Serum melatonin (MLT) levels, serum malondialdehyde (MDA) levels, and total antioxidant capacity (TAC) were estimated using ELISA. The correlations between the levels of the studied markers and clinical indicators of disease severity were evaluated. Significantly, lower expression of ACE2 was observed in COVID-19 patients compared to controls. Patients with COVID-19 had lower serum levels of TAC and MLT but higher serum levels of MDA compared to normal controls. Serum MDA levels were correlated with diastolic blood pressure (DBP), Glasgow coma scale (GCS) scores, and serum potassium levels. Serum MLT levels were positively correlated with DBP, mean arterial pressure (MAP), respiratory rate, and serum potassium levels. TAC was correlated with GCS, mean platelet volume, and serum creatinine levels. Serum MLT levels were significantly lower in patients treated with remdesivir and inotropes. Receiver operating characteristic curve analysis demonstrates that all markers had utility in discriminating COVID-19 patients from healthy controls. CONCLUSIONS: Increased oxidative stress and increased ACE2 expression were correlated with disease severity and poor outcomes in hospitalized patients with COVID-19 in the present study. Melatonin supplementation may provide a utility as an adjuvant therapy in decreasing disease severity and death in COVID-19 patients.

Role of Vitamin D, ACE2 and the Proteases as TMPRSS2 and Furin on SARS-CoV-2 Pathogenesis and COVID-19 Severity.

BACKGROUND: COVID-19, the 21st century pandemic disease caused by SARS-CoV-2, has shown a wide clinical spectrum ranging from asymptomatic to deadly serious pneumonia. OBJECTIVE: In our study, the relationship between the pathogenesis and clinical severity of COVID-19 and vitamin D, ACE2, Furin and TMPRSS2 was investigated. METHODS: Serum 25(OH)D, 1,25(OH)2D and ACE2 protein were measured in 85 COVID-19 cases, divided into 5 groups, according to disease severity, from asymptomatic to severe and including a healthy control group. Expression levels of ACE2, VDR, TMPRSS2 and Furin mRNAs in PBMC were also measured. The relationship of the parameters within each group, the severity of the disease and the effect on the patients' fate were investigated. RESULTS: Statistically significant differences were found between the severity of COVID-19 and all study parameters, except for serum 25(OH)D. A strong negative correlation was found between serum ACE2 protein, 1,25(OH)2D, and ACE2 mRNA, and disease severity, length of hospital stay and death/survival rate. Vitamin D deficiency increased the death risk by 5.6-fold (95% CI 0.75-41.47), and the levels of 1,25(OH)2D lower than 1 ng/mL increased the risk of death by 3.8-fold (95% CI 1.07-13.30). CONCLUSION: This study suggests that vitamin D supplementation could be beneficial in the treatment and/or prevention of COVID-19.

Effects of Varying Glucose Concentrations on ACE2's Hypothalamic Expression and Its Potential Relation to COVID-19-Associated Neurological Dysfunction.

The coronavirus disease 2019 (COVID-19) pandemic has negatively impacted millions of lives, despite several vaccine interventions and strict precautionary measures. The main causative organism of this disease is the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which infects the host via two key players: the angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2). Some reports revealed that patients with glycemic dysregulation could have increased susceptibility to developing COVID-19 and its related neurological complications. However, no previous studies have looked at the involvement of these key molecules within the hypothalamus, which is the central regulator of glucose in the brain. By exposing embryonic mouse hypothalamic neurons to varying glucose concentrations, we aimed to investigate the expression of ACE2 and TMPRSS2 using quantitative real time polymerase chain reaction and western blotting. A significant and time-dependent increase and decrease was observed on the viability of hypothalamic neurons with increasing and decreasing glucose concentrations, respectively (p < 0.01 and p < 0.001, respectively). Under the same increasing and decreasing glucose conditions, the expression of hypothalamic ACE2 also revealed a significant and time-dependent increase (p < 0.01). These findings suggest that SARS-CoV-2 invades the hypothalamic circuitry. In addition, it highlights the importance of strict glycemic control for COVID-19 in diabetic patients.

Effects of vitamin C and D on the mRNA expression of angiotensin converting enzyme 2 receptor, cathepsin L, and transmembrane serine protease in the mouse lungs.

Vitamins (Vit) C and D are widely used as immunogenic supplements among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. The SAR-CoV-2 virus enters into the pulmonary endothelial cells through attachment to angiotensin converting enzyme 2 receptor (Ace2) and the proteolytic activity of Cathepsin L (Ctsl) and transmembrane serine protease 2 (Tmprss2) enzymes. This study aimed to determine the influence of Vit C and D on the mRNA expression of Ace2, Tmprss2, and Ctsl genes in the mouse lungs. Vitamins C and D were administrated to different groups of mice through intra-peritoneal route in doses equivalent to human for 30 days. Then, the mRNA expression of SARS-CoV-2 entry gene was analyzed using qRT-PCR. It is found that Vit D, but not C, upregulated significantly (P < 0.05) the mRNA expression of Ace2 by more than six folds, while downregulated the expression of Ctsl and Tmprss2 genes by 2.8 and 2.2 folds, respectively. It can be concluded from this study that Vit D alters the mRNA expression of Ace2, Tmprss, and Ctsl genes in the mouse lungs. This finding can help us in understanding, at least in part, the molecular influence of Vit D on genes involved in the entry of SARS-CoV-2 into the cells.

Antihypertensive Effects of Dehydroabietic and 4-Epi-Trans-Communic Acid Isolated from Pinus densiflora.

Korean red pine needle (RPN) exhibits various biological and pharmacological activities. Among the various compounds of RPN, we isolated dehydroabietic and 4-epi-trans-communic acid. At first, we confirmed that two compounds inhibited angiotensin converting enzyme (ACE) and induced p-Akt in human umbilical vein endothelial cells (HUVEC). RPN extract powder significantly reduced systolic blood pressure in spontaneous hypertensive rats (SHRs) through the reduced expression of ACE and angiotensin type I receptors in the lungs of SHRs. The Lineweaver-Burk plots suggested that the two compounds were noncompetitive inhibitors of ACE. Using docking analysis, we found that two compounds showed the best returned pose at ACE active sites, and formed hydrogen and hydrophobic bonds with ACE residues. These results demonstrate that RPNs may be a source of compounds effective for preventing hypertension and may be useful in the development of antihypertensive drugs.

Recovery of Ggt7 and Ace Expressions in the Colon Alleviates Collagen-Induced Arthritis in Rats by Specific Bioactive Polysaccharide Intervention.

Rheumatoid arthritis (RA) causes swollen joints and irreversible joint damage and may even elevate cancer risks. Several bioactive nonstarch polysaccharides (NSPs) were reported to alleviate RA, but the key colonic genes accountable for this alleviation were elusive. Using collagen-induced arthritis as an RA model, colonic candidate genes related to RA were selected by transcriptome and methylome. The key genes were determined by comparing the transcriptome, methylome, and quantitative reverse transcription polymerase chain reaction profiles in RA rats with and without Lycium barbarum polysaccharides' treatment and further validated using Angelica sinensis polysaccharides and Astragalus propinquus polysaccharides for comparison. Both colonic genes γ-glutamyltransferase 7 (Ggt7) and angiotensin-I-converting enzyme (Ace) were downregulated by RA, and they were upregulated after L. barbarum polysaccharides' and A. sinensis polysaccharides' intervention that reduced the RA-caused hypermethylation status in nucleotide sites in the exon/promoter region of the two genes. However, the A. propinquus polysaccharides' intervention barely reduced the hypermethylation in the corresponding sites, failing to recover the expressions of these two genes and improve RA. Therefore, the colonic Ggt7 and Ace can be considered as key genes accountable for RA alleviation by bioactive NSP intervention. This study provides a more comprehensive insight into diet intervention to improve RA.

Neuroprotection of benzoinum in cerebral ischemia model rats via the ACE-AngI-VEGF pathway.

AIMS: Stroke is a devastating event with a limited choice of intervention. Benzoinum is frequently used to treat stroke in traditional Chinese medicine. Our team has found that the neuroprotection of benzoinum may related to angiogenesis, but the exact biological mechanism is unclear. The objective of this study was to explore its biological mechanism of angiogenesis in cerebral ischemia model rats. MAIN METHODS: First, network pharmacology and molecular docking were performed to predict the possible targets and mechanisms of benzoinum in treating ischemic stroke. The best dose was then selected according to pharmacodynamic indexes such as those for neurological deficit, cerebral infarction rate, and brain histopathology in middle cerebral artery occlusion (MCAO) model rats. Finally, RT-PCR, Western Blot and immunohistochemical analysis were applied to verify the prediction results from molecular docking. KEY FINDINGS: Network pharmacology and molecular docking demonstrated that the targets of treating cerebral ischemia were PDE4D, ACE and TTR, and the mechanism may be related to the ACE-AngI-VEGF signaling pathway. Experimental verification results suggested that 0.50 g/kg and 1.00 g/kg benzoinum could significantly protect against neurological deficit and reduce cerebral infarction rate in the cerebral cortex and hippocampus in MCAO model rats. At an optimal dose, benzoinum could significantly up-regulate VEGF, SHH and ANG-1, yet down-regulate ACE expression in MCAO model rats. SIGNIFICANCE: Balsamic acid is the active ingredient of benzoinum that protects against ischemic stroke and the possible mechanism is related to the promotion of angiogenesis via regulating ACE-AngI-VEGF pathway.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

Network pharmacology and molecular docking analysis on molecular targets and mechanisms of Huashi Baidu formula in the treatment of COVID-19.

PURPOSE: Huashi Baidu formula (HSBDF) was developed to treat the patients with severe COVID-19 in China. The purpose of this study was to explore its active compounds and demonstrate its mechanisms against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through network pharmacology and molecular docking. METHODS: All the components of HSBDF were retrieved from the pharmacology database of TCM system. The genes corresponding to the targets were retrieved using UniProt and GeneCards database. The herb-compound-target network was constructed by Cytoscape. The target protein-protein interaction network was built using STRING database. The core targets of HSBDF were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The main active compounds of HSBDF were docked with SARS-CoV-2 and angiotensin converting enzyme II (ACE2). RESULTS: Compound-target network mainly contained 178 compounds and 272 corresponding targets. Key targets contained MAPK3, MAPK8, TP53, CASP3, IL6, TNF, MAPK1, CCL2, PTGS2, etc. There were 522 GO items in GO enrichment analysis (p < .05) and 168 signaling pathways (p < .05) in KEGG, mainly including TNF signaling pathway, PI3K-Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, and HIF-1 signaling pathway. The results of molecular docking showed that baicalein and quercetin were the top two compounds of HSBDF, which had high affinity with ACE2. CONCLUSION: Baicalein and quercetin in HSBDF may regulate multiple signaling pathways through ACE2, which might play a therapeutic role on COVID-19.

Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment.

COVID-19, caused by SARS-CoV-2, is a virulent pneumonia, with >4,000,000 confirmed cases worldwide and >290,000 deaths as of May 15, 2020. It is critical that vaccines and therapeutics be developed very rapidly. Mice, the ideal animal for assessing such interventions, are resistant to SARS-CoV-2. Here, we overcome this difficulty by exogenous delivery of human ACE2 with a replication-deficient adenovirus (Ad5-hACE2). Ad5-hACE2-sensitized mice developed pneumonia characterized by weight loss, severe pulmonary pathology, and high-titer virus replication in lungs. Type I interferon, T cells, and, most importantly, signal transducer and activator of transcription 1 (STAT1) are critical for virus clearance and disease resolution in these mice. Ad5-hACE2-transduced mice enabled rapid assessments of a vaccine candidate, of human convalescent plasma, and of two antiviral therapies (poly I:C and remdesivir). In summary, we describe a murine model of broad and immediate utility to investigate COVID-19 pathogenesis and to evaluate new therapies and vaccines.

The Global Phosphorylation Landscape of SARS-CoV-2 Infection.

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.

COVID-19: A Concern for Cardiovascular Disease Patients.

Coronavirus disease 2019 (COVID-19) is declared as a pandemic that has spread worldwide, affecting 205 countries. The disease affected 1, 40, 43, 176 individuals and caused 5, 97, 583 deaths around the globe. The organism responsible for the cause of disease is Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). SARS-CoV-2 enters into the cell via receptors present on the cell surface named angiotensin-converting enzyme 2 (ACE2) receptor. Notwithstanding ACE2 receptors acts as a gateway for infection, and most of the cardiovascular patients are treated with the ACE inhibitors. Thus, the role of ACE inhibitors or angiotensin receptor blockers may play a critical role in the severity or outcome of disease. Also, the effect of ACE inhibitors varies with the polymorphism in ACE2 receptors present in the individuals. Hence, it is the need of the hour to investigate the mechanisms which could better aid in the treatment of COVID-19-infected cardiovascular disease (CVD) patients.

The Yin and Yang of ACE/ACE2 Pathways: The Rationale for the Use of Renin-Angiotensin System Inhibitors in COVID-19 Patients.

The article describes the rationale for inhibition of the renin-angiotensin system (RAS) pathways as specific targets in patients infected by SARS-CoV-2 in order to prevent positive feedback-loop mechanisms. Based purely on experimental studies in which RAS pathway inhibitors were administered in vivo to humans/rodents, a reasonable hypothesis of using inhibitors that block both ACE and ACE2 zinc metalloproteases and their downstream pathways in COVID-19 patients will be proposed. In particular, metal (zinc) chelators and renin inhibitors may work alone or in combination to inhibit the positive feedback loops (initially triggered by SARS-CoV-2 and subsequently sustained by hypoxia independently on viral trigger) as both arms of renin-angiotensin system are upregulated, leading to critical, advanced and untreatable stages of the disease.

COVID-19 Coronavirus spike protein analysis for synthetic vaccines, a peptidomimetic antagonist, and therapeutic drugs, and analysis of a proposed achilles' heel conserved region to minimize probability of escape mutations and drug resistance.

This paper continues a recent study of the spike protein sequence of the COVID-19 virus (SARS-CoV-2). It is also in part an introductory review to relevant computational techniques for tackling viral threats, using COVID-19 as an example. Q-UEL tools for facilitating access to knowledge and bioinformatics tools were again used for efficiency, but the focus in this paper is even more on the virus. Subsequence KRSFIEDLLFNKV of the S2' spike glycoprotein proteolytic cleavage site continues to appear important. Here it is shown to be recognizable in the common cold coronaviruses, avian coronaviruses and possibly as traces in the nidoviruses of reptiles and fish. Its function or functions thus seem important to the coronaviruses. It might represent SARS-CoV-2 Achilles' heel, less likely to acquire resistance by mutation, as has happened in some early SARS vaccine studies discussed in the previous paper. Preliminary conformational analysis of the receptor (ACE2) binding site of the spike protein is carried out suggesting that while it is somewhat conserved, it appears to be more variable than KRSFIEDLLFNKV. However compounds like emodin that inhibit SARS entry, apparently by binding ACE2, might also have functions at several different human protein binding sites. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 is again argued to be a convenient model pharmacophore perhaps representing an ensemble of targets, and it is noted that it occurs both in lung and alimentary tract. Perhaps it benefits the virus to block an inflammatory response by inhibiting the dehydrogenase, but a fairly complex web involves several possible targets.

Potential of Flavonoid-Inspired Phytomedicines against COVID-19.

Flavonoids are widely used as phytomedicines. Here, we report on flavonoid phytomedicines with potential for development into prophylactics or therapeutics against coronavirus disease 2019 (COVID-19). These flavonoid-based phytomedicines include: caflanone, Equivir, hesperetin, myricetin, and Linebacker. Our in silico studies show that these flavonoid-based molecules can bind with high affinity to the spike protein, helicase, and protease sites on the ACE2 receptor used by the severe acute respiratory syndrome coronavirus 2 to infect cells and cause COVID-19. Meanwhile, in vitro studies show potential of caflanone to inhibit virus entry factors including, ABL-2, cathepsin L, cytokines (IL-1ß, IL-6, IL-8, Mip-1α, TNF-α), and PI4Kiiiß as well as AXL-2, which facilitates mother-to-fetus transmission of coronavirus. The potential for the use of smart drug delivery technologies like nanoparticle drones loaded with these phytomedicines to overcome bioavailability limitations and improve therapeutic efficacy are discussed.

Gender and Age Differences Reveal Risk Patterns in COVID-19 Outbreak.

By April 28th 2020, the global number of people that were viciously infected with the newfound novel corona virus (COVID-19) stood at a staggering 3 077 133 cases, as per the confirmed data released by the WHO. It has been reported that women from the Chinese Han population are associated with essential hypertension due to their relation with the 5 SNPs, namely, rs1514283, rs4646155, rs4646176, rs2285666, and rs879922, which belong to the ACE2 gene. The level of ACE2 activity was very low in normal healthy younger persons, and was reported to be increased in patients with cardiovascular diseases. Thus, there might be severe myocarditis, that may result in acute heart failure and cardiac complexities in the elderly subjects.

EZH2-mediated H3K27me3 inhibits ACE2 expression.

The outbreak of corona virus disease 2019 (COVID-19) caused by SARS-CoV-2 infection is spreading globally and quickly, leading to emerging health issues. SARS-CoV-2 enters into and infects host cells through its spike glycoprotein recognizing the cell receptor Angiotensin-converting enzyme II (ACE2). Here, we noticed that ACE2 was further enhanced by SARS-CoV-2 infection. Human germ cells and early embryos express high level of ACE2. Notably, RNA-seq result showed that reduction of H3K27me3, but not H3K4/9/36me3, led to upregulation of Ace2 expression in mouse germ cell line GC-2. In agreement with this result, we found in human embryonic stem cells that ACE2 expression was significantly increased in absence of EZH2, the major enzyme catalyzing H3K27me3. ChIP-seq analysis further confirmed decrease of H3K27me3 signal and increase of H3K27ac signal at ACE2 promoter upon EZH2 knockout. Therefore, we propose that EZH2-mediated H3K27me3 at ACE2 promoter region inhibits ACE2 expression in mammalian cells. This regulatory pattern may also exist in other human cells and tissues. Our discovery provides clues for pathogenesis and targeted drug therapy towards ACE2 expression for prevention and adjuvant therapy of COVID-19.

Mechanism Analysis of a Novel Angiotensin-I-Converting Enzyme Inhibitory Peptide from Isochrysis zhanjiangensis Microalgae for Suppressing Vascular Injury in Human Umbilical Vein Endothelial Cells.

Microalgae are primary producers with multiple nutrients in aquatic environments and mostly have applications in biological feed and fuel industry. There are few studies assessing the angiotensin-I-converting enzyme (ACE) inhibition potential of Isochrysis zhanjiangensis, other than its antioxidant potential. In this study, we evaluated a peptide from I. zhanjiangensis (PIZ, FEIHCC) and its vascular endothelial factors and mechanism in human umbilical vein endothelial cells (HUVEC). The results reveal that PIZ (IC50 = 61.38 µM) acts against ACE in a non-competitive binding mode. In addition, PIZ inhibits angiotensin II (Ang II)-induced vascular factor secretion and expression by blocking inflammation and apoptosis through nuclear factor κB (NF-κB), nuclear erythroid 2-related factor 2 (Nrf2), mitogen-activated protein kinases (MAPKs), and the serine/threonine kinase (Akt) signal pathways. This study reveals that PIZ has potential to be developed as a therapeutic agent for hypertension and provides a new method of high-value utilization of I. zhanjiangensis.