ACE2 Protein Levels Research Articles

ACE2 and TMPRSS2 in human kidney tissue and urine extracellular vesicles with age, sex, and COVID-19.

SARS-CoV-2 virus infects cells by engaging with ACE2 requiring protease TMPRSS2. ACE2 is highly expressed in kidneys. Predictors for severe disease are high age and male sex. We hypothesized that ACE2 and TMPRSS2 proteins are more abundant (1) in males and with increasing age in kidney and (2) in urine and extracellular vesicles (EVs) from male patients with COVID-19 and (3) SARS-CoV-2 is present in urine and EVs during infection. Kidney cortex samples from patients subjected to cancer nephrectomy (male/female; < 50years/˃75years, n = 24; ˃80years, n = 15) were analyzed for ACE2 and TMPRSS2 protein levels. Urine from patients hospitalized with SARS-CoV-2 infection was analyzed for ACE2 and TMPRSS2. uEVs were used for immunoblotting and SARS-CoV-2 mRNA and antigen detection. Tissue ACE2 and TMPRSS2 protein levels did not change with age. ACE2 was not more abundant in male kidneys in any age group. ACE2 protein was associated with proximal tubule apical membranes in cortex. TMPRSS2 was observed predominantly in the medulla. ACE2 was elevated significantly in uEVs and urine from patients with COVID-19 with no sex difference compared with urine from controls w/wo albuminuria. TMPRSS2 was elevated in uEVs from males compared to female. ACE2 and TMPRSS2 did not co-localize in uEVs/apical membranes. SARS-CoV-2 nucleoprotein and mRNA were not detected in urine. Higher kidney ACE2 protein abundance is unlikely to explain higher susceptibility to SARS-CoV-2 infection in males. Kidney tubular cells appear not highly susceptible to SARS-CoV-2 infection. Loss of ACE2 into urine in COVID could impact susceptibility and angiotensin metabolism.

ACE2 deficiency inhibits thoracic aortic dissection by enhancing SIRT3 mediated inhibition of inflammation and VSCMs phenotypic switch

BackgroundThoracic aortic dissection (TAD) is an irreversible cardiovascular disorder with high mortality and morbidity. However, the molecular mechanisms remain elusive. Thus, identifying an effective therapeutic target to prevent TAD is especially critical. The purpose of this study is to elucidate the potential mechanism of inflammation and vascular smooth muscle cell (VSMCs) phenotypic switch in β-aminopropionitrile fumarate (BAPN)-induced TAD.MethodsA mouse model of TAD induced by BAPN and IL-1β -stimulated HVSMCs in vivo and in vitro models, respectively. ACE2 Knockdown mice treated with BAPN or without, and the TAD mouse model was treated with or without AAV-ACE2. Transthoracic ultrasound was conducted for assessment the maximum internal diameter of the thoracic aorta arch. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Western blot were used to detect the expression of MMP2, MMP9, ACE2, SIRT3, OPN, SM22α and other inflammatory markers. The circulating levels of ACE2 was measured by ELISA assay. Histological changes of thoracic aorta tissues were assessed by H&E, EVG and IHC analysis.ResultsWe found that circulating levels of and the protein levels of ACE2 were increased in the TAD mouse model and in patients with TAD. For further evidence, ACE2 deficiency decelerated the formation of TAD. However, overexpression of ACE2 aggravated BAPN-induced aortic injury and VSMCs phenotypic switch via lowered SIRT3 expression and elevated inflammatory cytokine expression.ConclusionACE2 deficiency prevented the development of TAD by inhibiting inflammation and VSMCs phenotypic switch in a SIRT3-dependent manner, suggesting that the ACE2/SIRT3 signaling pathway played a pivotal role in the pathological process of TAD and might be a potential therapeutical target.

Hypoxia modifies levels of the SARS-CoV-2 cell entry proteins, angiotensin-converting enzyme 2, and furin in fetal human brain endothelial cells

It is not known whether human fetal brain endothelial cells (hfBECs) that form the blood-brain barrier (BBB) express ACE2, TMPRSS2 and Furin; SARS-CoV-2 cell entry proteins. It is also unclear whether hypoxia, commonly observed during severe maternal COVID-19, can modify their level of expression. We hypothesized that hfBECs isolated from early- and mid-gestation brain microvessels express ACE2, TMPRSS2 and Furin. We also hypothesized that hypoxia modifies their expression levels in a gestational-age and time of exposure-dependent manner. To investigate whether early- and mid-gestation hfBECs express ACE2, TMPRSS2 and Furin SARS-CoV-2 associated cell entry proteins. We also determined the effects of hypoxia on ACE2, TMPRSS2 and Furin expression levels in hfBECs. This is a prospective study where hfBECs isolated from early- (12.4 ± 0.7 weeks), and mid-gestation (17.9 ± 0.5) fetal brain microvessels (N=6/group) were exposed to different oxygen tensions (20-, 5- and 1%-oxygen) for 6-, 24- and 48-h. ACE2, TMPRSS2 and Furin mRNA and protein levels and localization were assessed using qPCR, Western blot and immunofluorescence. ACE2, TMPRSS2 and Furin co-localize with the endothelial cell marker von Willebrand factor (vWF) in hfBECS isolated from early- and mid-pregnancy. In early-gestation, TMPRSS2 mRNA expression was decreased by 5% oxygen compared to 20% oxygen after 6h of exposure (p<0.05). In mid-gestation, 5% oxygen downregulated ACE2 mRNA compared to 20% oxygen after 24h (p<0.05). Furin mRNA expression was decreased under 5% and 1% oxygen compared to 20% oxygen (p<0.05) after 24h. In mid-gestation, ACE2 protein levels were decreased under 5% and 1% oxygen (p<0.001) after 24h. In contrast, Furin protein levels were increased under 1% oxygen compared to 20% oxygen after 24h (p<0.05). At 48h, 1% oxygen increased ACE2 protein levels compared to 20% oxygen (p<0.01). Hypoxia modifies the expression of selected SARS-CoV-2 cell entry proteins in hfBECs, in a gestational-age- and time of exposure-dependent manner. Since severe COVID-19 illness may lead to maternal hypoxia, an altered expression of these proteins in the developing human BBB could potentially lead to altered SARS-CoV-2 brain invasion, and neurological sequelae in neonates born to pregnancies complicated by SARS-CoV-2 infection.

Circulating ACE2 level and zinc/albumin ratio as potential biomarkers for a precision medicine approach to COVID-19.

Highly mutable influenza is successfully countered based on individual susceptibility and similar precision-like medicine approach should be effective against SARS-COV-2. Among predictive markers to bring precision medicine to COVID-19, circulating ACE2 has potential features being upregulated in both severe COVID-19 and predisposing comorbidities. Spike SARS-CoVs were shown to induce ADAM17-mediated shedding of enzymatic active ACE2, thus accounting for its increased activity that has also been suggested to induce positive feedback loops leading to COVID-19-like manifestations. For this reason, pre-existing ACE2 activity and inhibition of ACE2/ADAM17 zinc-metalloproteases through zinc chelating agents have been proposed to predict COVID-19 outcome before infection and to protect from COVID-19, respectively. Since most diagnostic laboratories are not equipped for enzymatic activity determination, other potential predictive markers of disease progression exploitable by diagnostic laboratories were explored. Concentrations of circulating albumin, zinc, ACE2 protein and its activity were investigated in healthy, diabetic (COVID-19-susceptible) and SARS-CoV-2-negative COVID-19 individuals. ACE2 both protein levels and activity significantly increased in COVID-19 and diabetic patients. Abnormal high levels of ACE2 characterised a subgroup (16-19%) of diabetics, while COVID-19 patients were characterised by significantly higher zinc/albumin ratios, pointing to a relative increase of albumin-unbound zinc species, such as free zinc ones. Data on circulating ACE2 levels are in line with the hypothesis that they can drive susceptibility to COVID-19 and elevated zinc/albumin ratios support the therapeutic use of zinc chelating inhibitors of ACE2/ADAM17 zinc-metalloproteases in a targeted therapy for COVID-19.

ACE2 ve TMPRSS2 Genlerinin Farklı Hücre Hatlarındaki İfade Düzeyleri

Objective: ACE2 and TMPRSS2 proteins have received increased attention gained emphasis together with the pandemic COVID-19. These proteins have roles in respiratory and hypertension disorders as well as cardiovascular and renal diseases. The objective of this work was to examine the mRNA and protein levels of ACE2 and TMPRSS2 in cell lines derived from various tissue origins.&#x0D; Methods: After the growth of 14 different cell lines, protein and mRNA were isolated from the cell pellets. The amounts of mRNAs and proteins were then determined and quantified using RT-PCR and ELISA.&#x0D; Results: Findings showed that VERO, HEK293T, and VERO E6 cell lines significantly differed from others in the mRNA levels of both the ACE2 and TMPRSS2 genes. In protein levels obtained using ELISA, PNT1A cell line had the highest level of ACE2 protein expression, while for TMPRSS2, A549 had the highest level of protein expression. &#x0D; Conclusion: It was showed in this study how the expressions of ACE2 and TMPRSS2 depend on the cell type. This may be an explanation for why virulence entrance differs in different types of tissues. It is thought that HEK293T cells with high levels of both genes may be a suitable option for studies at the RNA level by using these two genes. MCF7 may be a good option for studies at the protein level. Given the high levels of mRNA expression of both genes, it may be inferred that cells derived from the kidney were among those that were most susceptible to virus entry.

(323) Mechanistic Insights into a Rare Genetic Cause of COVID Orchitis

Abstract Introduction To date, biomedical efforts against the SARS-CoV-2 virus have mainly focused on mitigating spread and primary infection mortality. While significant strides have been made in community response and vaccine production, post-acute COVID-19 syndromes (Long COVID) are growing in prevalence and becoming an increasing burden on public health. Current evidence suggests COVID-19 can affect the testis and cases of orchitis (testicular pain) appear to present in up to 20% of men with COVID-19. With an aim of uncovering causal mechanisms of COVID-19 sequela outside of pulmonary symptomology, a recent study from our group has identified a nonsynonymous non-frameshift mutation in the NACAD protein associated with COVID-19 orchitis and decreased ACE2 serum levels. NACAD is thought to prevent inappropriate targeting of non-secreted peptides to the endoplasmic reticulum. Objective We hypothesized that a defect in NACAD function/processing will decrease ACE2 serum levels by disrupting extracellular transport and endoplasmic reticulum interaction. We explored whether intracellular levels of ACE2 as well as TMPRSS2 were altered and if cell membrane protein deposition was increased, which may result in more severe effects to SARS-CoV-2 infection. Methods We obtained testis biopsies from men undergoing sperm retrieval for infertility. After siRNA NACAD knockdown, we analyzed total ACE2 expression using Western blot and qPCR as well as immunofluorescence/H&amp;E staining for colocalization (with NACAD). Membrane-anchored ACE2 (mACE2) levels were quantified using immunostaining and flowcytometry while secreted ACE2 (sACE2) was analyzed by ELISA and Western blot. Results NACAD and ACE2 co-localize in both the Leydig and germ cells. When NACAD is knocked down, primary testes cells show 80% decreased mRNA and 50% total protein levels of ACE2. The secreted ACE2 was also reduced by nearly 50%. However, when we specifically measured the level of mACE2 on the cell surface, we found that the knockdown of NACAD did not lead to reduction. Conclusions NACAD likely specifically affects the level of sACE2, which is speculated to act as a protection factor by retaining the virus in serum and reducing cellular entry. While the exact reason for the differential effects of NACAD on mACE2 and sACE2 remain unclear, our data is consistent to other studies, which found a reduction of total ACE2 expression and sACE2 in patients is coupled with worse outcomes in COVID-19. We hope that establishing a relationship between NACAD and ACE2 expression will prove useful not only to elucidate why some men with COVID develop orchitis but, ultimately, why some men develop multiorgan failure with COVID infection whereas most do not. Disclosure No

Vitamin C promotes ACE2 degradation and protects against SARS-CoV-2 infection.

ACE2 is a major receptor for cellular entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2 binding, strategies to flexibly and sufficiently reduce ACE2 levels for the prevention of SARS-CoV-2 infection have not been explored. Here, we reveal vitamin C (VitC) administration as a potent strategy to prevent SARS-CoV-2 infection. VitC reduces ACE2 protein levels in a dose-dependent manner, while even a partial reduction in ACE2 levels can greatly inhibit SARS-CoV-2 infection. Further studies reveal that USP50 is a crucial regulator of ACE2 levels. VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination of ACE2 at Lys788 and subsequent degradation of ACE2 without affecting its transcriptional expression. Importantly, VitC administration reduces host ACE2 levels and greatly blocks SARS-CoV-2 infection in mice. This study reveals that ACE2 protein levels are down-regulated by an essential nutrient, VitC, thereby enhancing protection against infection of SARS-CoV-2 and its variants.

TGF-β1 Inhibition of ACE2 Mediated by miRNA Uncovers Novel Mechanism of SARS-CoV-2 Pathogenesis

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, utilizes receptor binding domain (RBD) of spike glycoprotein to interact with angiotensin (Ang)-converting enzyme 2 (ACE2). Altering ACE2 levels may affect entry of SARS-CoV-2 and recovery from COVID-19. Decreased cell surface density of ACE2 leads to increased local levels of Ang II and may contribute to mortality resulting from acute lung injury and fibrosis during COVID-19. Studies published early during the COVID-19 pandemic reported that people with cystic fibrosis (PwCF) had milder symptoms, compared to people without CF. This finding was attributed to elevated ACE2 levels and/or treatment with the high efficiency CFTR modulators. Subsequent studies did not confirm these findings reporting variable effects of CFTR gene mutations on ACE2 levels. Transforming growth factor (TGF)-β signaling is essential during SARS-CoV-2 infection and dominates the chronic immune response in severe COVID-19, leading to pulmonary fibrosis. TGF-β1 is a gene modifier associated with more severe lung disease in PwCF but its effects on the COVID-19 course in PwCF is unknown. To understand whether TGF-β1 affects ACE2 levels in the airway, we examined miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in response to TGF-β1. Small RNAseq and micro(mi)RNA profiling identified pathways uniquely affected by TGF-β1, including those associated with SARS-CoV-2 invasion, replication, and the host immune responses. TGF-β1 inhibited ACE2 expression by miR-136-3p and miR-369-5p mediated mechanism in CF and non-CF bronchial epithelial cells. ACE2 levels were higher in two bronchial epithelial cell models expressing the most common CF-causing mutation in CFTR gene F508del, compared to controls without the mutation. After TGF-β1 treatment, ACE2 protein levels were still higher in CF, compared to non-CF cells. TGF-β1 prevented the modulator-mediated rescue of F508del-CFTR function while the modulators did not prevent the TGF-β1 inhibition of ACE2 levels. Finally, TGF-β1 reduced the interaction between ACE2 and the recombinant spike RBD by lowering ACE2 levels and its binding to RBD. Our data demonstrate novel mechanism whereby TGF-β1 inhibition of ACE2 in CF and non-CF bronchial epithelial cells may modulate SARS-CoV-2 pathogenicity and COVID-19 severity. By reducing ACE2 levels, TGF-β1 may decrease entry of SARS-CoV-2 into the host cells while hindering the recovery from COVID-19 due to loss of the anti-inflammatory and regenerative effects of ACE2. The above outcomes may be modulated by other, miRNA-mediated effects exerted by TGF-β1 on the host immune responses, leading to a complex and yet incompletely understood circuitry.

Chemotherapy induces ACE2 expression in breast cancer via the ROS-AKT-HIF-1α signaling pathway: a potential prognostic marker for breast cancer patients receiving chemotherapy

BackgroundAngiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system and a well-known functional receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells. The COVID-19 pandemic has brought ACE2 into the spotlight, and ACE2 expression in tumors and its relationship with SARS-COV-2 infection and prognosis of cancer patients have received extensive attention. However, the association between ACE2 expression and tumor therapy and prognosis, especially in breast cancer, remains ambiguous and requires further investigation. We have previously reported that ACE2 is elevated in drug-resistant breast cancer cells, but the exact function of ACE2 in drug resistance and progression of this malignant disease has not been explored.MethodsThe expression of ACE2 and HIF-1α in parental and drug-resistant breast cancer cells under normoxic and hypoxic conditions was analyzed by Western blot and qRT-PCR methods. The protein levels of ACE2 in plasma samples from breast cancer patients were examined by ELISA. The relationship between ACE2 expression and breast cancer treatment and prognosis was analyzed using clinical specimens and public databases. The reactive oxygen species (ROS) levels in breast cancer cells were measured by using a fluorescent probe. Small interfering RNAs (siRNAs) or lentivirus-mediated shRNA was used to silence ACE2 and HIF-1α expression in cellular models. The effect of ACE2 knockdown on drug resistance in breast cancer was determined by Cell Counting Kit 8 (CCK-8)-based assay, colony formation assay, apoptosis and EdU assay.ResultsACE2 expression is relatively low in breast cancer cells, but increases rapidly and specifically after exposure to anticancer drugs, and remains high after resistance is acquired. Mechanistically, chemotherapeutic agents increase ACE2 expression in breast cancer cells by inducing intracellular ROS production, and increased ROS levels enhance AKT phosphorylation and subsequently increase HIF-1α expression, which in turn upregulates ACE2 expression. Although ACE2 levels in plasma and cancer tissues are lower in breast cancer patients compared with healthy controls, elevated ACE2 in patients after chemotherapy is a predictor of poor treatment response and an unfavorable prognostic factor for survival in breast cancer patients.ConclusionACE2 is a gene in breast cancer cells that responds rapidly to chemotherapeutic agents through the ROS-AKT-HIF-1α axis. Elevated ACE2 modulates the sensitivity of breast cancer cells to anticancer drugs by optimizing the balance of intracellular ROS. Moreover, increased ACE2 is not only a predictor of poor response to chemotherapy, but is also associated with a worse prognosis in breast cancer patients. Thus, our findings provide novel insights into the spatiotemporal differences in the function of ACE2 in the initiation and progression of breast cancer.

MECHANISTIC INSIGHTS INTO A RARE MUTATION IN NACAD AS A POSSIBLE CAUSE OF COVID ORCHITIS

Aiming to uncover causal mechanisms of COVID-19 sequela outside of pulmonary symptomology, a recent study from our group identified a variant of the NACAD protein (120bp coding sequence deletion) associated with COVID-19 orchitis, a risk factor for male infertility. Interestingly, these patients had decreased ACE2 serum levels. NACAD is proposed to prevent inappropriate targeting of non-secreted peptides to the endoplasmic reticulum. We hypothesized that a defect in NACAD function/processing will decrease ACE2 serum levels by disrupting extracellular transport and endoplasmic reticulum interaction. We explored whether intracellular levels of ACE levels were altered and if cell membrane protein deposition was increased, which may result in more severe effects to SARS-CoV-2 infection. We obtained testis biopsies from men undergoing sperm retrieval for infertility. After siRNA NACAD knockdown, we analyzed total ACE2 expression using Western blot and qPCR as well as immunofluorescence/H&E staining for co-localization (with NACAD). Membrane-anchored ACE2 (mACE2) levels were quantified using immunostaining and flowcytometry while secreted ACE2 (sACE2) was analyzed by ELISA and Western blot. NACAD and ACE2 co-localize in both the Leydig and germ cells. When NACAD is knocked down, primary testes cells show 80% decreased mRNA and 50% total protein levels of ACE2. The secreted ACE2 was also reduced by nearly 50%. However, when we specifically measured the level of mACE2 on the cell surface, we found that the knockdown of NACAD did not lead to reduction. NACAD likely specifically affects the level of sACE2, which is speculated to act as a protection factor by retaining the virus in serum and reducing cellular entry. While the exact reason for the differential effects of NACAD on mACE2 and sACE2 remain unclear, our data is consistent to other studies, which found a reduction of total ACE2 expression and sACE2 in patients is coupled with worse outcomes in COVID-19. We hope that establishing a relationship between NACAD and ACE2 expression will prove useful not only to elucidate why some men with COVID develop orchitis but, ultimately, why some men develop multiorgan failure with COVID infection whereas most do not.

SARS‐CoV‐2 spike protein enhances MAP4K3/GLK‐induced ACE2 stability in COVID‐19

ACE2 on epithelial cells is the SARS‐CoV‐2 entry receptor. Single‐cell RNA‐sequencing data derived from two COVID‐19 cohorts revealed that MAP4K3/GLK‐positive epithelial cells were increased in patients. SARS‐CoV‐2‐induced GLK overexpression in epithelial cells was correlated with COVID‐19 severity and vesicle secretion. GLK overexpression induced the epithelial cell‐derived exosomes containing ACE2; the GLK‐induced exosomes transported ACE2 proteins to recipient cells, facilitating pseudovirus infection. Consistently, ACE2 proteins were increased in the serum exosomes from another COVID‐19 cohort. Remarkably, SARS‐CoV‐2 spike protein‐stimulated GLK, and GLK stabilized ACE2 in epithelial cells. Mechanistically, GLK phosphorylated ACE2 at two serine residues (Ser776, Ser783), leading to the dissociation of ACE2 from its E3 ligase UBR4. Reduction in UBR4‐induced Lys48‐linked ubiquitination at three lysine residues (Lys26, Lys112, Lys114) of ACE2 prevented its degradation. Furthermore, SARS‐CoV‐2 pseudovirus or live virus infection in humanized ACE2 mice induced GLK and ACE2 protein levels, and ACE2‐containing exosomes. Collectively, ACE2 stabilization by SARS‐CoV‐2‐induced MAP4K3/GLK may contribute to the pathogenesis of COVID‐19.

Exercise-induced myokines downregulates the ACE2 level in bronchial epithelial cells: Implications for SARS-CoV-2 prevention.

BackgroundThe Covid-19 pandemic has emerged as the leading public health challenge of our time (20th century). While vaccinations have finally blunted the death rate, concern has remained about more virulent forms highlighting the need for alternative approaches. Epidemiological studies indicate that physical activity has been shown to decrease the risk of infection of some respiratory viruses. Part of the salutary effects of exercise is believed to be through the elaboration of cytokines by contracting skeletal muscles (termed myokines). The objective of this study was to investigate whether exercise-induced myokines would mitigate the SARS-CoV-2 infectivity of the bronchial epithelium through modulating the SARS-CoV-2 Covid-19 receptor (angiotensin-converting enzyme 2 -ACE2) its priming enzyme, transmembrane serine protease 2 (TMPRSS2).MethodsWe utilized a cell culture model of exercise to generate myokines by differentiating C2C12 cells into myotubules and inducing them to contract via low-frequency electric pulse stimulation. Condition media was concentrated via centrifugation and applied to human immortalized human bronchial epithelium cell line (6HBE14o) along with conditioned media from unstimulated myotubules as controls. Following exposure to myokines, the 16HBE14o cells were harvested and subjected to quantitative RT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) for assessment of mRNA and protein levels of ACE2 and TMPRSS2, respectively. Pilot proteomic data was performed with isotope barcoding and mass spectroscopy.ResultsQuantitative Real-Time PCR of 16HBE14o with 48 h treated unstimulated vs. stimulated myokine treatment revealed a reduction of ACE2 and TMPRSS2 mRNA by 32% (p<2.69x10-5) and 41% (p<4.57x10-5), respectively. The high sensitivity of ELISAs showed downregulation of ACE2 and TMPRSS2 protein expression in 16HBE14o cells by 53% (p<0.01) and 32% (p<0.03) respectively with 48 h treated. For rigor, this work was replicated in the human lung cancer cell line A549, which mirrored the downregulation. Proteomic analysis showed dramatic alteration in myokine profile between contracted and uncontracted C2C12 tubules.ConclusionsThe current study explores a novel approach of a modified exercise cell culture system and uses ACE2 and TMPRSS2 as a surrogate marker of SARS-CoV-2 infectivity. In conclusion, we demonstrated biological data supporting exercise’s protective effect against Covid-19. These further strengthen myokines’ beneficial role as potential therapeutic targets against SARS-CoV-2 and similar viruses albeit these preliminary cell culture studies will require future validation in animal models.

Chrysin Attenuates Fructose-Induced Nonalcoholic Fatty Liver in Rats via Antioxidant and Anti-Inflammatory Effects: The Role of Angiotensin-Converting Enzyme 2/Angiotensin (1-7)/Mas Receptor Axis.

Aim Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, and if untreated, it may propagate into end-stage liver disease. The classical arm of the renin-angiotensin system (RAS) has a fundamental role in triggering oxidative stress and inflammation, which play potential roles in the pathogenesis of NAFLD. However, the nonclassical alternative axis of RAS, angiotensin- (Ang-) converting enzyme 2 (ACE2)/Ang (1-7)/Mas receptor, opposes the actions of the classical arm, mitigates the metabolic dysfunction, and improves hepatic lipid metabolism rendering it a promising protective target against NAFLD. The current study is aimed at investigating the impact of chrysin, a well-known antioxidant flavonoid, on this defensive RAS axis in NAFLD. Methods Rats were randomly distributed and treated daily for eight weeks as follows: the normal control, chrysin control (50 mg/kg, p.o), NAFLD group (received 20% fructose in drinking water), and treated groups (25 and 50 mg/kg chrysin given orally and concomitantly with fructose). Diminazene aceturate (DIZE) (15 mg/kg, s.c.) was used as a reference ACE2 activator. Key Findings. High fructose induced significant weight gain, hepatocyte degeneration with fat accumulation, and inflammatory cell infiltration (as examined by H&E staining). This was accompanied by a substantial increase in liver enzymes, glucose, circulating and hepatic triglycerides, lipid peroxides, inflammatory cytokines, and Ang II (the main component of classical RAS). At the same time, protein levels of ACE2, Ang (1-7), and Mas receptors were markedly reduced. Chrysin (25 and 50 mg/kg) significantly ameliorated these abnormalities, with a prominent effect of the dose of 50 mg/kg over DIZE and the lower dose in improving ACE2, Ang (1-7), and Mas. Significance. Chrysin is a promising efficient protective remedy against NAFLD; mechanisms include the activation of ACE2/Ang (1-7)/Mas axis.

Plasmatic renin-angiotensin system in normotensive and hypertensive patients hospitalized with COVID-19.

BackgroundBesides its counterbalancing role of the renin-angiotensin system (RAS), angiotensin-converting enzyme (ACE) 2 is the receptor for the type 2 coronavirus that causes severe acute respiratory syndrome, the etiological agent of COVID-19. COVID-19 is associated with increased plasmatic ACE2 levels, although conflicting results have been reported regarding angiotensin (Ang) II and Ang-(1−7) levels. We investigated plasmatic ACE2 protein levels and enzymatic activity and Ang II and Ang-(1−7) levels in normotensive and hypertensive patients hospitalized with COVID-19 compared to healthy subjects.MethodsAng II and Ang-(1−7), and ACE2 activity and protein levels were measured in 93 adults (58 % (n = 54) normotensive and 42 % (n = 39) hypertensive) hospitalized with COVID-19. Healthy, normotensive (n = 33) and hypertensive (n = 7) outpatient adults comprised the control group.ResultsCOVID-19 patients displayed higher ACE2 enzymatic activity and protein levels than healthy subjects. Within the COVID-19 group, ACE2 activity and protein levels were not different between normotensive and hypertensive-treated patients, not even between COVID-19 hypertensive patients under RAS blockade treatment and those treated with other antihypertensive medications. Ang II and Ang-(1−7) levels significantly decreased in COVID-19 patients. When COVID-19 patients under RAS blockade treatment were excluded from the analysis, ACE2 activity and protein levels remained higher and Ang II and Ang-(1−7) levels lower in COVID-19 patients compared to healthy people.ConclusionsOur results support the involvement of RAS in COVID-19, even when patients under RAS blockade treatment were excluded. The increased circulating ACE2 suggest higher ACE2 expression and shedding.

Dietary Empagliflozin Regulates Angiotensin Converting Enzyme 2 (ACE2) and Transmembrane Serine Protease 2 (TMPRSS2) in Kidney Cortex of Mice

Persons with type 2 diabetes mellitus (T2D) are at greater risk for poor prognosis, including renal pathology, when infected with Covid‐19 virus. A newer class of medications for T2D is the gliflozin class, including Empagliflozin (EMPA), which inhibits the renal proximal tubule sodium glucose cotransporter, type 2 (SGLT2) resulting in glucosuria and reduction in hyperglycemia. Whether these medications alter susceptibility and responses to Covid‐19, and similar infections, is not known. The angiotensin 2 converting enzyme (ACE2) is the receptor for Covid‐19, while transmembrane serine protease 2 (TMPRSS2) is a proteinase expressed on the surface of epithelial cells and facilitates virus uptake. In general, ACE2 is thought to be renoprotective, as it converts Ang I (Ang 1‐10) to Ang 1‐9 and Ang II (Ang 1‐8) to Ang 1‐7. Ang 1‐8, in particular, is a potent vasoconstrictor and natriferic agent. Our aim was to determine how chronic EMPA affected ACE2 and TMPRSS2 protein levels and ACE2 activity in the kidney cortex of mice. Adult male (M) and female (F) mice of three strains (n = 8/group), i.e., C57Bl6 (C57), Swiss Webster (SW), and Tally Ho/Jng (TH) were fed high‐fat control diet (C, 60% milk‐fat) or EMPA‐containing high‐fat diet (E, 0.01%) for 12 weeks. TH mice are insulin resistant and obese, with a tendency to develop T2D over time. SW are lean and the closest genetically related background strain to TH. EMPA increased glucosuria but not body weight (p = 0.38 for treatment, 3‐way ANOVA); however kidney weight (normalized to body weight) was 2‐22% increased (depending on group) by EMPA (p = 0.002 for treatment). Kidney cortex protein levels of ACE2 and TMPRSS2 were determined by conventional western blotting (to observe band size and antibody specificity) and by dot blotting (to compare all samples on the same blot). Dot blotting revealed significantly higher ACE2 protein in males (p < 0.0001) relative to females and in the TH and SW strains (p < 0.0001), relative to C57. There was a significant sex‐by‐strain interaction in that the TH females were not significantly lower than TH males. Similarly, cortical TMPRSS2 was lower in females (p < 0.0001) and in C57 mice (p < 0.0001), but also reduced by EMPA (p = 0.030). ACE2 activity was determined in 10 μg kidney cortex by calculating the slope (over 2 hours) of the difference in fluorescence generated in the presence of the ACE2 substrate (Mca‐YVADAPK(Dnp)‐OH) with the addition of the ACE2‐specific antagonist (MLN) minus fluorescence with addition of both captopril (ACE antagonist) plus MLN. Slopes were: (RFU/min, mean ± sem): MC57C‐ 19.9 ± 5.5; MC57E‐ 31.8 ± 5.2; MSWC‐ 20.2 ± 1.9; MSWE‐ 25.7 ± 3.6; MTHC‐ 18.8 ± 3.6; MTHE‐ 34.7 ± 11.3; FC57C‐ 4.7 ± 1.2; FC57E‐ 9.7 ± 3.4; FSWC‐ 4.9 ± 1.2; FSWE‐ 4.4 ± 1.8; FTHC‐ 9.0 ± 1.8; and FTHE‐ 16.2 ± 2.3. Three‐way ANOVA revealed significant effects of both sex (p < 0.0001) and treatment (p = 0.0052) in that Empagliflozin and male sex increased the slope (activity). Overall, male sex was the greatest determinant of differences in ACE2 protein and activity and TMPRSS2 protein. EMPA increased ACE2 activity, but reduced TMPRSS2 protein levels. In general, sex differences were attenuated in the TH strain. There is the potential for these relative increases to play a role in increased sensitivity of male, obese, and/or T2D subjects to pathology associated with Covid‐19; however, the beneficial effect of enhanced ACE2 to reduce Ang II may also provide countering protection.

The Traditional Chinese Medicine Formula Jing Guan Fang for Preventing SARS-CoV-2 Infection: From Clinical Observation to Basic Research.

COVID-19 is a global epidemic. Developing adjuvant therapies which could prevent the virus from binding to cells may impair viral infection. This study produces a traditional Chinese medicine formula, Jing Guan Fang (JGF), based on ancient medical texts, and examines the efficacy and the mechanism by which JGF prevents viral infections. JGF reduces COVID-19 like symptoms. Functional studies show that JGF inhibits the formation of syncytium and reduces the formation of viral plaque. JGF is not toxic in vitro and in vivo. Mechanistically, JGF induces lysosomal-dependent ACE2 degradation and suppresses mRNA and the protein levels of TMPRSS2 in human lung WI-38 and MRC-5 cells. Mice that inhale JGF exhibit reduced ACE2 and TMPRSS2 protein levels in lung tissues. Together, these findings suggest that JGF may improve the COVID-19 like symptoms and inhibit viral infection. Moreover, JGF may be applicable as an adjuvant preventive strategy against SARS-CoV-2 infection in addition to the use of vaccines.

COVID-19 Resulting in Potential Hearing Damage of Rodents

Abstract Objectives To find out the association between the sensorineural hearing loss and coronavirus disease 2019 (COVID-19), the expression of ACE2 and TMPRSS2 in hamsters and mice was detected. Design Using the public data from the National Center for Biotechnology Information and the Global Initiative on Sharing All Influenza Data, the expression of ACE2 and TMPRSS2 at the transcriptomic, DNA, and protein levels of ACE2 in the brain, inner ear, and muscle from the golden Syrian hamsters (Mesocricetus auratus) and mice (Mus musculus) was assessed. Results We identified ACE2 and TMPRSS2 expressed at different levels in the inner ear and brain at DNA and transcriptomic levels of both mice and hamsters. The protein expression from the brain and inner ear showed a similar pattern, while the expression of ACE2 from the inner ear was relatively higher than that from the muscle. Conclusion Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shows genetic potential to infect the hearing system of rodents and lead to sudden sensorineural hearing loss that can be used as a characteristic to detect asymptomatic patients of COVID-19.

Renin-angiotensin system (RAS) enzymes and placental trophoblast syncytialisation

Placental renin-angiotensin system (RAS) components; prorenin, angiotensinogen, and angiotensin (Ang) II type 1 receptor (AT1R) are upregulated during syncytialisation. This study examined whether angiotensin-converting enzyme (ACE), ACE2 and neprilysin (NEP) are also altered during syncytialisation. Two in vitro models of syncytialisation were used: forskolin-treated BeWo cells and spontaneously syncytialising primary human trophoblast cells. Term placentae and primary trophoblasts had the highest levels of ACE, ACE2 and NEP mRNA. In primary trophoblasts, ACE mRNA levels significantly increased with syncytialisation, ACE2 and NEP mRNA levels decreased. ACE, ACE2 and NEP protein levels and ACE2 activity did not change. Syncytialisation of primary trophoblasts decreased soluble (s)ACE and sNEP but not sACE2 levels. In primary trophoblasts, the balance between the enzymes controlling the two opposing pathways of the RAS was maintained. These findings were unable to be reproduced in BeWo cells. Future studies exploring placental levels of these enzymes in pregnancies complicated by placental insufficiency are warranted.

Tidal Volume-Dependent Activation of the Renin-Angiotensin System in Experimental Ventilator-Induced Lung Injury.

Ventilator-induced lung injury (VILI) is a major contributor to morbidity and mortality in critically ill patients. Mechanical damage to the lungs is potentially aggravated by the activation of the renin-angiotensin system (RAS). This article describes RAS activation profiles in VILI and discusses the effects of angiotensin (Ang) 1-7 supplementation or angiotensin-converting enzyme (ACE) inhibition with captopril as protective strategies. Animal study. University research laboratory. C57BL/6 mice. Anesthetized mice ( n = 12-18 per group) were mechanically ventilated with low tidal volume (LV T , 6 mL/kg), high tidal volume (HV T , 15 mL/kg), or very high tidal volume (VHV T , 30 mL/kg) for 4 hours, or killed after 3 minutes (sham). Additional VHV T groups received infusions of 60 μg/kg/hr Ang 1-7 or a single dose of 100 mg/kg captopril. VILI was characterized by increased bronchoalveolar lavage fluid levels of interleukin (IL)-6, keratinocyte-derived cytokine, and macrophage inflammatory protein-2 (MIP2). The Ang metabolites in plasma measured with liquid chromatography tandem mass spectrometry showed a strong activation of the classical (Ang I, Ang II) and alternative RAS (Ang 1-7, Ang 1-5), with highest concentrations found in the HV T group. Although the lung-tissue ACE messenger RNA expression was unchanged, its protein expression showed a dose-dependent increase under mechanical ventilation. The ACE2 messenger RNA expression decreased in all ventilated groups, whereas ACE2 protein levels remained unchanged. Both captopril and Ang 1-7 led to markedly increased Ang 1-7 plasma levels, decreased Ang II levels, and ACE activity (Ang II/Ang I ratio), and effectively prevented VILI. VILI is accompanied by a strong activation of the RAS. Based on circulating Ang metabolite levels and tissue expression of RAS enzymes, classical ACE-dependent and alternative RAS cascades were activated in the HV T group, whereas classical RAS activation prevailed with VHV T ventilation. Ang 1-7 or captopril protected from VILI primarily by modifying the systemic RAS profile.

Role of Human Antigen R (HuR) in the Regulation of Pulmonary ACE2 Expression.

Patients with COPD may be at an increased risk for severe illness from COVID-19 because of ACE2 upregulation, the entry receptor for SARS-CoV-2. Chronic exposure to cigarette smoke, the main risk factor for COPD, increases pulmonary ACE2. How ACE2 expression is controlled is not known but may involve HuR, an RNA binding protein that increases protein expression by stabilizing mRNA. We hypothesized that HuR would increase ACE2 protein expression. We analyzed scRNA-seq data to profile ELAVL1 expression in distinct respiratory cell populations in COVID-19 and COPD patients. HuR expression and cellular localization was evaluated in COPD lung tissue by multiplex immunohistochemistry and in human lung cells by imaging flow cytometry. The regulation of ACE2 expression was evaluated using siRNA-mediated knockdown of HuR. There is a significant positive correlation between ELAVL1 and ACE2 in COPD cells. HuR cytoplasmic localization is higher in smoker and COPD lung tissue; there were also higher levels of cleaved HuR (CP-1). HuR binds to ACE2 mRNA but knockdown of HuR does not change ACE2 protein levels in primary human lung fibroblasts (HLFs). Our work is the first to investigate the association between ACE2 and HuR. Further investigation is needed to understand the mechanistic underpinning behind the regulation of ACE2 expression.