ACE2 Expression Research Articles

Angiotensin-Converting Enzyme 2 Enhances Autophagy via the Consumption of miR-326 in a Mouse Model of Acute Lung Injury.

Angiotensin-converting enzyme 2 (ACE2) has been reported to exert a protective effect in acute lung injury (ALI), though its underlying mechanism remains incompletely understood. In this study, ACE2 expression was found to be upregulated in a mouse model of ALI induced by lipopolysaccharide (LPS) injection. ACE2 knockdown modulated the severity of ALI, the extent of autophagy, and the mTOR pathway in this model. ACE2 regulated liver kinase B1 (LKB1) gene expression by sequestering miR-326, thereby alleviating ALI severity through enhanced autophagy. In cell-based experiments, miR-326 was shown to regulate ACE2 and LKB1 expression and autophagy. Overexpression of ACE2 disrupted miR-326's regulatory effect on LKB1, suggesting that LKB1 may function as an endogenous sponge for miR-326. These findings imply that elevated ACE2 expression in lung could play enhance the autophagy via the consumption of miR-326.

In vitro gastrointestinal digestion simulation screening of novel ACEI peptides from broccoli: mechanism in high glucose-induced VSMCs dysfunction

Many natural angiotensin-converting enzyme inhibitory (ACEI) peptides have been widely studied. However, their stability in vivo is poor in most cases. In this study, peptides were initially digested from broccoli in vitro, and absorption was simulated by Caco2 cells transport and then analyzed by Peptideomics and molecular docking. Subsequently, the mechanisms were verified using a high glucose-induced vascular smooth muscle cells (VSMCs) dysfunction model. Results showed that ACEI activity of broccoli crude peptide increased by 70.73 ± 1.42% after digestion. The enzymatic hydrolysates of crude broccoli peptides before and after digestion were detected by HPLC. The digested crude peptides were highly stable (with a stability level > 90%) in the intestine and possessed a strong absorptive potential. Five peptides with high stability and strong permeability were first identified, including HLEVR, LTEVR, LEHGF, HLVNK, and LLDGR, which exhibited high activity with IC50 values of 3.19 ± 0.23 mM, 17.07 ± 1.37 mM, 0.64 ± 0.02 mM, 0.06 ± 0.01 mM, and 2.81 ± 0.12 mM, respectively. When the VSMCs model was exposed to Ang II, the expressions of PCNA, MMP2, and Bcl2 were increased, while the expression of BAX was inhibited. When the VSMCs was exposed to high glucose (HG), the Ang II concentration significantly increased. This indicates that HG elevated Ang II levels. Finally, five peptides significantly attenuated Ang II-induced VSMCs proliferation and migration by down-regulating AT1R expression and inhibiting ERK and p38 MAPK phosphorylation. Notably, in exploring VSMCs dysfunction on a high glucose-induced model, ACEI peptides resulted in down-regulation of ACE and up-regulation of ACE2 expression. Therefore, it can be further referenced for the functional food against hypertension and cardiovascular diseases.

Characterizing SV40-hTERT Immortalized Human Lung Microvascular Endothelial Cells as Model System for Mechanical Stretch-Induced Lung Injury.

Drug development for human disease relies on preclinical model systems such as human cell cultures and animal experiments before therapeutic treatments can ultimately be tested on humans in clinical studies. We here describe the generation of a novel human cell line (HLMVEC/SVTERT289) that we generated by transfection of microvascular endothelial cells from healthy donor lung tissue with the catalytic domain of telomerase and the SV40 large T/small t-antigen. These cells exhibited satisfactory growth characteristics and largely maintained their native characteristics, including morphology, cell surface marker expression, angiogenic potential and the protein composition of secreted extracellular vesicles. In order to test their suitability as a disease model, we simulated mechanical stress induced by cyclic stretch as encountered in ventilator-induced lung injury using the FlexCell® system and compared their performance to primary lung endothelial cells. In this setting, HLMVEC/SVTERT289 cells exhibited significantly higher neprilysin activity on the cell surface and extracellular vesicles secreted from the cell line exhibited higher Tissue Factor and ACE2 expression but lower ACE expression and ACE activity than vesicles released from the primary cells. This study provides an unprecedented and detailed characterization of the HLMVEC/SVTERT289 cell line, which should help to appraise its suitability in different molecular studies.

Higher circulating ACE2 and DPP3 but reduced ACE and angiotensinogen in hyperreninemic sepsis patients.

Sepsis and septic shock are global healthcare problems associated with high mortality rates. Activation of the renin-angiotensin-aldosterone system (RAAS) is an early event in sepsis, and elevated renin may be predictive of worse outcomes. In a subset of sepsis patients enrolled in the Vitamin C, Thiamine and Steroids in Sepsis (VICTAS) trial, elevated levels of active renin (median value > 189 pg/mL or 5.1 pM) at baseline (day 0) were strongly associated with mortality; however, corresponding plasma levels of the vasopressor hormone Angiotensin II were not substantially increased nor was Angiotensin II associated with disease severity. The current study assessed RAAS components that may impact the Angiotensin II response in control subjects, normal renin sepsis (NRS, renin < 5.1 pM) and high renin sepsis (HRS, renin > 5.1 pM) patients. NRS and HRS subjects exhibited a similar reduction in ACE (40%), but increased levels of ACE2 and DPP3. The ACE to DPP3 ratio was higher in controls but this relationship was reversed in both NRS and HRS subjects. Intact angiotensinogen was 50% lower in the HRS than control or NRS subjects, whereas the intact angiotensinogen to renin ratio was <10% of control or NRS subjects. We conclude that altered expression of ACE, ACE2, DPP3 and angiotensinogen may attenuate the expected increase in Angiotensin II, particularly in sepsis subjects with high renin concentrations.

Rescuing ACE2-Deficiency-Mediated Nucleus Pulposus Senescence and Intervertebral Disc Degeneration by a Nanotopology-Enhanced RNAi System.

Nucleus pulposus cell (NPC) senescence contributes to intervertebral disc degeneration (IVDD). However, the underlying molecular mechanisms are not fully understood. In this study, it is demonstrated that angiotensin-converting enzyme 2 (ACE2) counteracted the aging of NPCs and IVDD at the cellular and physiological levels. The expression of ACE2 correlates negatively with the degree of NPC senescence and IVDD. Using both loss- and gain-of-function mouse models, it is revealed that ACE2 deficiency increased the senescence of NPCs and exacerbated injury- or instability-induced IVDD, whereas ACE2 overexpression counteracted these detrimental effects. Mechanistically, integrated analysis of single-cell and bulk transcriptomics shows that ACE2 deficiency results in the activation of TGFβ2/Smads signaling pathway and the transcription of Serpine1, ultimately triggering NPC senescence and IVDD. A nanomedical delivery system (virus-like nanovectors, VNs) composed of nanovectors with a virus-like surface topology and small interfering RNA targeting Serpine1 (VN-siSer) is developed. With nanotopology-enhanced transfection efficiency, RNA-interfering treatment by VN-siSer effectively alleviated NPC senescence and IVDD at both the cellular and animal levels. Overall, the data reveal the underlying mechanisms of ACE2 in NPC senescence and IVDD pathogenesis and propose a distinct paradigm of precise nanomedical senescence-blockade RNAi for IVDD treatment.

Association of antihypertensive drug target genes with alzheimer’s disease: a mendelian randomization study

BackgroundEpidemiological and genetic studies have elucidated associations between antihypertensive medication and Alzheimer’s disease (AD), with the directionality of these associations varying upon the specific class of antihypertensive agents.MethodsGenetic instruments for the expression of antihypertensive drug target genes were identified using expression quantitative trait loci (eQTL) in blood, which are associated with systolic blood pressure (SBP). Exposure was derived from existing eQTL data in blood from the eQTLGen consortium and in the brain from the PsychENCODE and subsequently replicated in GTEx V8 and BrainMeta V2. We performed two-sample Mendelian randomization (MR) to estimate the potential effect of different antihypertensive drug classes on AD using summary statistics from a meta-analysis (111,326 cases and 677,663 controls) and further replicated in FinnGen cohorts (9301 cases and 367,976 controls). The reverse causality detection, assessing horizontal pleiotropy, Bayesian co-localization, phenotype scanning, and protein quantitative trait loci (pQTL) analysis were implemented to consolidate the MR findings further.ResultsA 1-standard deviation (SD) lower expression of the angiotensin-converting enzyme (ACE) gene in blood was associated with a lower SBP of 3.92 (95% confidence interval (CI), 2.69–5.15) mmHg but an increased risk of AD (odds ratio (OR), 2.46; 95% CI, 1.82–3.33). A similar direction of association was also observed between ACE expression in prefrontal cortex (OR, 1.19; 95% CI, 1.10–1.28), frontal cortex (OR, 1.19; 95% CI, 1.11–1.27), cerebellum (OR, 1.13; 95% CI, 1.09–1.17), cortex (OR, 1.59; 95% CI, 1.33–1.28) and ACE protein levels in plasma (OR, 1.13; 95% CI, 1.09–1.17) and AD risk. Colocalization supports these results. Similar results were found in external validation. We found no evidence for an association between genetically estimated blood pressure (BP) and AD risk.ConclusionsThere findings suggest an adverse association of lower ACE messenger RNA and protein levels with an elevated risk of AD, irrespective of its BP-lowering effects. These findings warrant greater pharmacovigilance and further investigation into the effect of ACE inhibitors, particularly those that are centrally acting, on neurodegenerative symptoms in patients with AD.

Scent of COVID-19: Whole-Genome Sequencing Analysis Reveals the Role of ACE2, IFI44, and NDUFAF4 in Long-Lasting Olfactory Dysfunction.

COVID-19-related persistent olfactory dysfunction (OD) presents remarkable interindividual differences, and little is known about the host genetic factors that are involved in its etiopathogenesis. The goal of this study was to explore the genetic factors underpinning COVID-19-related OD through the analysis of Whole Genome Sequencing data of 153 affected subjects, focusing on genes involved in antiviral response regulation. An innovative approach was developed, namely the assessment of the association between a "gene score", defined as the ratio of the number of homozygous alternative variants within the gene to its length, and participants' olfactory function. The analysis highlighted how an increased gene score in the ACE2 gene is associated with a worse olfactory performance, while an increased gene score in the IFI44 and NDUFAF4 genes is associated with a better olfactory function. Considering the physiological role of the proteins encoded by these genes, it can be hypothesized that a reduced expression of ACE2 may be associated with a protracted and severe inflammatory response in the olfactory epithelium, thus worsening patients' smell abilities. Conversely, an increased gene score in IFI44 and NDUFAF4 might be associated with a decreased inflammatory response, thus correlating with a better olfactory performance. Overall, this study identified new host genetic factors that may play a pivotal role in determining COVID-19-related OD heterogeneity, possibly enabling more personalized and effective clinical management for affected individuals.

Genetic determinants of COVID-19 severity and mortality: ACE1 Alu 287 bp polymorphism and ACE1, ACE2, TMPRSS2 expression in hospitalized patients.

The angiotensin-converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2) are central human molecules in the SARS-CoV-2 virus-host interaction. Evidence indicates that ACE1 may influence ACE2 expression. This study aims to determine whether ACE1, ACE2, and TMPRSS2 mRNA expression levels, along with the ACE1 Alu 287 bp polymorphism (rs4646994), contribute to the severity and mortality of COVID-19. Swabs were collected in two Brazilian cities in 2020: Belo Horizonte (n=134) and Rio de Janeiro (n=41). A swab of mild patients in Rio de Janeiro who were not hospitalized (n=172) was also collected. All analyzed biological material was obtained from residual diagnostic samples in 2020, prior to the emergence of SARS-CoV-2 variants of concern. ACE1, ACE2, TMPRSS2, and B2M (reference gene) expression levels were evaluated in 40 cycles of quantitative PCR. ACE1 Alu 287 bp polymorphism was genotyped using the FastStart Universal SYBR Green Master kit. The median age differed between clinical sites (p=0.016), but no difference in median days of hospitalization was observed (p=0.329). Age was associated with severity (p=0.014) and mortality (p=0.014) in the Belo Horizonte cohort. No alteration in ACE1, ACE2 and TMPRSS2 expression was associated with severity or mortality. ACE1 polymorphism rs4646994 did not influence the likelihood of either outcome. A meta-analysis including available data from the literature showed significant effects: the D-allele conferred risk (OR = 1.39; 95% CI [1.12-1.72]).

ACE2 Inhibits Dermal Regeneration Through Ang II in Tissue Expansion.

Tissue expansion is a widely employed technique in reconstructive surgery aimed at addressing considerable skin defects. Nevertheless, matters like inadequate expansion capability and the potential for skin breakage due to the fragility of the expanded tissue present notable hurdles in enhancing skin regeneration during this process. Angiotensin-converting enzyme 2 (ACE2) is recognized for its essential role in facilitating tissue renewal and regeneration. However, its precise impact on skin renewal during tissue expansion remains underexplored. This study seeks to elucidate ACE2's contribution to skin regeneration, specifically examining its role in collagen synthesis. This study evaluated the expression and distribution of ACE2 in expanded skin using samples derived from both rats and human patients. Additionally, we investigated ACE2 expression in stretched keratinocytes invitro. ACE2 knockout keratinocytes were transfected with small interfering RNA (siRNA) and cocultured with fibroblasts to observe fibroblast proliferation and migration. MLN-4760 was utilized to inhibit the ACE2 enzymatic activity. Additionally, we analyzed parameters such as the size of expanded skin, dermal thickness, and the levels of collagen I (COL I), collagen III (COL III), and transforming growth factor β (TGF-β) to elucidate the role of ACE2 in the context of expanded skin. The thinning of the expanded dermis was linked with elevated ACE2 expression. Enzymatic activity and ACE2 expression were both increased by mechanical stress. Additionally, ACE2 utilized Ang II to activate the migration and proliferation of human dermal fibroblasts. Invivo, the ACE2 inhibitor MLN-4760 promoted skin regeneration and reduced dermal thinning by elevating COL I, COL III, and TGF-β during expansion. This finding suggest that mechanical stretch increases ACE2 expression, which in turn promotes the regeneration of expanded skin. The basis for using ACE2 in clinical settings to increase tissue expansion efficacy is provided by this work.

Involvement of metformin and aging in salivary expression of ACE2 and TMPRSS2.

SARS-CoV-2-related proteins, ACE2 and TMPRSS2, are determinants of SARS-CoV-2 infection. Although these proteins are expressed in oral-related tissues, their expression patterns and modulatory mechanisms in the salivary glands remain unknown. We herein showed that full-length ACE2, which has both a fully functional enzyme catalytic site and high-affinity SARS-CoV-2 spike S1-binding sites, was more highly expressed in salivary glands than in oral mucosal epithelial cells and the lungs. Regarding TMPRSS2, zymogen and the cleaved form were both expressed in the salivary glands, whereas only zymogen was expressed in murine lacrimal glands and the lungs. Metformin, an AMPK activator, increased stimulated saliva secretion and full-length ACE2 expression and decreased cleaved TMPRSS2 expression in the salivary glands, and exerted the same effects on soluble ACE2 (sACE2) and sTMPRSS2 in saliva. Moreover, metformin decreased the expression of beta-galactosidase, a senescence marker, and ADAM17, a sheddase of ACE2 to sACE2, in the salivary glands. In aged mice, the expression of ACE2 was decreased in the salivary glands, whereas that of sACE2 was increased in saliva, presumably by the up-regulated expression of ADAM17. The expression of TMPRSS2 in the salivary glands and sTMPRSS2 in saliva were both increased. Collectively, these results suggest that the protein expression patterns of ACE2 and TMPRSS2 in the salivary glands differ from those in other oral-related cells and tissues, and also that metformin and aging affect the salivary expression of ACE2 and TMPRSS2, which have the potential as targets for preventing the transmission of SARS-CoV-2.

Blockade of PVN neuromedin B receptor alleviates inflammation via the RAS/ROS/NF-κB pathway in spontaneously hypertensive rats.

Neuromedin B (NMB) has potentially great impacts on the development of cardiovascular diseases by promoting hypertensive and sympatho-excitation effects. However, studies regarding the NMB function in paraventricular nucleus (PVN) are lacking. With selective neuromedin B receptor (NMBR) antagonist, BIM-23127, we aim to determine whether the blockade of NMB function in PVN could alleviate central inflammation and attenuate hypertensive responses. Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were chronically infused with BIM-23127 in the PVN for 6 weeks. Mean arterial pressure (MAP) was assessed with tail cuff and electrophysiological acquisition systems. PVN tissues were collected to analyze expressions of Fra-LI, inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-10, and IL-4), renin-angiotensin system (angiotensin-converting enzyme (ACE), ACE2, and AT1-reporter (AT1-R)) and oxidative stress (reactive oxygen species (ROS), superoxide dismutase (SOD)1, NADPH oxidase (NOX)2, and NOX4). ELISA was used to detect inflammation indices, norepinephrine (NE), and nuclear factor κB (NF-κB) p65 in plasma and PVN tissue homogenate. Compared to WKY, SHR exhibited higher mean arterial pressure (MAP), plasma NE, and pro-inflammatory cytokines (PICs). Higher PVN levels of Fra-LI, PICs, ACE, AT1-R, ROS, NOX2, NOX4, and NF-κB p65, while lower central levels of anti-inflammatory cytokines (AICs), ACE2, and SOD1 were observed in SHR. Administration of BIM-23127 in PVN reversed all these changes in SHR. In SHR, blockade of NMBR in the PVN inhibited sympatho-excitation and attenuated hypertensive response. The attenuation mechanism may involve reducing inflammation and the RAS/ROS/ NF-κB pathways in PVN.

BMY 7378, a selective α1D-adrenoceptor antagonist, is a new angiotensin converting enzyme inhibitor: In silico, in vitro and in vivo approach

BMY 7378 is a multitarget drug primarily known for its selective antagonism of α1D-adrenoceptors (α1D-AR), exhibiting both hypotensive effects and the ability to prevent or reverse angiotensin II-induced vascular hypertrophy. Notably, BMY 7378 contains a phenylpiperazine moiety, a structural feature associated with angiotensin-converting enzyme (ACE) inhibition. This study aimed to investigate ACE inhibition as a potential pharmacological mechanism of BMY 7378. Using an in silico approach we predicted BMY 7378 interactions with the ACE active site, followed by in vitro activity assays. Additionally, ACE protein expression in the heart was analyzed following four weeks of BMY 7378 treatment in 7–8-month-old spontaneously hypertensive rats (SHR). All assays were benchmarked against captopril, a standard ACE inhibitor. In silico results showed that BMY 7378 binds to the ACE active site, though with reduced interaction with Zn701 (73.7 % compared to captopril), likely due to the pKa of its amino group. The inhibitory concentration 50 (IC50) for BMY 7378 was 136 μM, lower than other reported phenylpiperazine derivatives. Furthermore, BMY 7378 significantly increased ACE expression in the hearts of SHR, with an increase of 8.5-fold compared to captopril. In conclusion, BMY 7378 exhibits dual activity as an α1D-AR antagonist and an ACE inhibitor, making it a promising pharmacological tool for investigating and potentially treating hypertension and its associated cardiovascular complications.

Secondary metabolites of Alternaria alternate appraisal of their SARS-CoV-2 inhibitory and anti-inflammatory potentials.

This study identifies the secondary metabolites from Alternaria alternate and evaluates their ACE-2: Spike RBD (SARS-CoV-2) inhibitory activity confirmed via immunoblotting in human lung microvascular endothelial cells. In addition, their in vitro anti-inflammatory potential was assessed using a cell-based assay in LPS-treated RAW 264.7 macrophage cells. Two novel compounds, altenuline (1), phthalic acid bis (7'/7'' pentyloxy) isohexyl ester (2), along with 1-deoxyrubralactone (3) alternariol-5-O-methyl ether (4) and alternariol (5) were identified. Molecular docking and in vitro studies showed that compounds 2 and 4 were promising to counteract SARS-CoV-2 attachment to human ACE-2. Thus, they are considered promising natural anti-viral agents. SwissADME in silico analysis was conducted to predict the drug-like potential. Immunoblotting analysis confirmed that the tested compounds (1-4) demonstrated downregulation of ACE-2 expression in the endothelial cells from the lungs with variable degrees. Furthermore, the tested compounds (1-4) showed promising anti-inflammatory activities through TNF-α: TNFR2 inhibitory activity and their inhibitory effect on the proinflammatory cytokines (TNF-α and IL-6) in LPS-stimulated monocytes. In conclusion, our study, for the first time, provides beneficial experimental confirmation for the efficiency of the A. alternate secondary metabolites for the treatment of COVID-19 as they hinder SARS-CoV-2 infection and lower inflammatory responses initiated by SARS-CoV-2. A. alternate and its metabolites are considered in developing preventative and therapeutic tactics for COVID-19.

Enhancing human ACE2 expression in mouse models to improve COVID-19 research.

Mice are one of the most common biological models for laboratory use. However, wild-type mice are not susceptible to COVID-19 infection due to the low affinity of mouse ACE2, the entry protein for SARS-CoV-2. Although mice with human ACE2 (hACE2) driven by Ace2 promoter reflect its tissue specificity, these animals exhibit low ACE2 expression, potentially limiting their fidelity in mimicking COVID-19 manifestations and their utility in viral studies. Here, we created and compared hACE2 mouse models generated with different strategies. Our findings show that distinct β-globin insertion within hACE2 cassette significantly influences its expression, with downstream placement enhancing transcription. Moreover, optimizing hACE2 codons (opt-hACE2) improves translation efficiency in multiple tissues. Notably, opt-hACE2 mice displayed more active immune responses and severe COVID-19 phenotypes following SARS-CoV-2 challenge compared to other models. Our study demonstrates the dual regulatory role of β-globin element in transgene transcription and suggests that opt-hACE2 mice might serve as valuable tools for SARS-CoV-2 research.

Daphnetin may protect from SARS-CoV-2 infection by reducing ACE2

To combat the SARS-CoV-2 pandemic, innovative prevention strategies are needed, including reducing ACE2 expression on respiratory cells. This study screened approved drugs in China for their ability to downregulate ACE2. Daphnetin (DAP) was found to significantly reduce ACE2 mRNA and protein levels in PC9 cells. DAP exerts its inhibitory effects on ACE2 expression by targeting HIF-1α and JAK2, thereby impeding the transcription of the ACE2 gene. The SARS-CoV-2 pseudovirus infection assay confirmed that DAP-treated PC9 cells exhibited decreased susceptibility to viral infection. At therapeutic doses, DAP effectively lowers ACE2 expression in the respiratory systems of mice and humans. This suggests that DAP, already approved for other conditions, could be a new preventive measure against SARS-CoV-2, offering a cost-effective and accessible way to reduce SARS-CoV-2 spread.

IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by infection with the enveloped RNA betacoronavirus, SARS-CoV-2, led to a global pandemic involving over 7 million deaths. Macrophage inflammatory responses impact COVID-19 severity; however, it is unclear whether macrophages are infected by SARS-CoV-2. We sought to identify mechanisms regulating macrophage expression of ACE2, the primary receptor for SARS-CoV-2, and to determine if macrophages are susceptible to productive infection. We developed a humanized ACE2 (hACE2) mouse whereby hACE2 cDNA was cloned into the mouse ACE2 locus under control of the native promoter. We validated susceptibility of hACE2 mice to SARS-CoV-2 infection relative to wild-type mice and an established K18-hACE2 model of acute fulminating disease. Intranasal exposure to SARS-CoV-2 led to pulmonary consolidations with cellular infiltrate, edema, and hemorrhage, consistent with pneumonia, yet unlike the K18-hACE2 model, hACE2 mice survived and maintained stable weight. Infected hACE2 mice also exhibited a unique plasma chemokine, cytokine, and growth factor inflammatory signature relative to K18-hACE2 mice. Infected hACE2 mice demonstrated evidence of viral replication in infiltrating lung macrophages, and infection of macrophages in vitro revealed a transcriptional profile indicative of altered RNA and ribosomal processing machinery as well as activated cellular antiviral defense. Macrophage IL-1β-driven NF-κB transcription of ACE2 was an important mechanism of dynamic ACE2 upregulation, promoting macrophage susceptibility to infection. Experimental models of COVID-19 that make use of native hACE2 expression will allow for mechanistic insight into factors that can either promote host resilience or increase susceptibility to worsening severity of infection.

N-Methyl-d-Aspartate-Induced Excitotoxicity and Its Impact on the Renin-Angiotensin System in Retinal Tissue.

Purpose: Renin-angiotensin system (RAS) is expressed in neuronal tissue and plays a role in neurodegenerative diseases involving excitotoxicity as a pathophysiological mechanism. In retina, excessive excitatory neurotransmission via N-methyl-d-aspartate (NMDA) receptors underlies neuronal apoptosis in conditions like glaucoma. However, it is not known if NMDA-mediated excitotoxicity alters retinal RAS expression. Hence, this study investigated the effect of NMDA exposure on the expression of RAS in rat retinas. Methods: Two groups of Sprague-Dawley rats received either phosphate buffer saline or NMDA (160 nmol). On day 7 posttreatment, retinal expression of RAS components including renin, angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II), Ang 1-7, Ang 1-9, MAS receptor, angiotensin II type 1 receptor (AT1R), ACE2, and aldosterone was measured using enzyme-linked immunosorbent assay and polymerase chain reaction. Morphometric studies were done to assess morphological alterations. Results: Following the exposure to NMDA, an upregulation of ACE expression was noted at both the protein (2.03-folds; P < 0.001) and mRNA (1.86-folds; P < 0.01) levels in rat retinas. AT1R protein and mRNA expression were greater by 1.73 (P < 0.0001) and 2.28-folds (P < 0.0001), respectively. However, mRNA expression for ACE2, Ang 1-7, and Ang 1-9, showed a 1.51-(P < 0.05), 2.41-(P < 0.001), and 2.37-(P < 0.0001) fold decrease. Ganglion cell layer (GCL) thickness and linear cell density in GCL were significantly lower in the NMDA-treated group (P < 0.05). Conclusions: NMDA exposure increases expression of the classical RAS and suppresses that of alternate RAS in rat retinas. These alterations are associated with retinal morphological changes indicating significant loss of neuronal cells in the GCL of rat retinas.

Bioinformatic characterization of ENPEP, the gene encoding a potential cofactor for SARS-CoV-2 infection.

Research on SARS-CoV-2, the viral pathogen that causes COVID-19, has identified angiotensin converting enzyme 2 (ACE2) as the primary viral receptor. Several genes that encode viral cofactors, such as TMPRSS2, NRP1, CTSL, and possibly KIM1, have since been discovered. Glutamyl aminopeptidase (APA), encoded by the gene ENPEP, is another cofactor candidate due to similarities in its biological role and high correlation with ACE2 and other human coronavirus receptors, such as aminopeptidase N (APN) and dipeptidyl peptidase 4 (DPP4). Recent studies have proposed a role for ENPEP as a viral receptor in humans, and ENPEP and ACE2 are both closely involved in the renin-angiotensin-aldosterone system proposed to play an important role in SARS-CoV-2 pathophysiology. We performed bioinformatic analyses using publicly available bulk (>17,000 samples from 49 distinct tissues) and single-cell (>2.5 million cells) RNA-Seq gene expression datasets to evaluate the expression and function of the ENPEP gene. We also investigated age- and sex-related changes in ENPEP expression. Overall, expression of ENPEP was highest in the small intestine enterocyte brush border and the kidney cortex. ENPEP is widely expressed in a subset of vascular smooth muscle cells (likely pericytes) in systemic vasculature, the heart, and the brain. ENPEP is expressed at low levels in the lower respiratory epithelium. In the lung, ENPEP is most highly expressed in para-alveolar fibroblasts. Single-cell data revealed ENPEP expression in a substantial fraction of ependymal cells, a finding not reported before in humans. Age increases ENPEP expression in skeletal muscle and the prostate, while decreasing it in the heart and aorta. Angiogenesis was found to be a central biological function associated with the ENPEP gene. Tissue-specific roles, such as protein digestion and fat metabolism, were also identified in the intestine. In the liver, the gene is linked to the complement system, a connection that has not yet been thoroughly investigated. Expression of ENPEP and ACE2 is strongly correlated in the small intestine and renal cortex. Both overall and in blood vessels, ENPEP and ACE2 have a stronger correlation than many other genes associated with SARS-CoV-2, such as TMPRSS2, CTSL, and NRP1. Possible interaction between glutamyl aminopeptidase and SARS-CoV-2 should be investigated experimentally.

Novel T-cell subsets as non-invasive biomarkers of vascular damage along the predialysis stages of chronic kidney disease

IntroductionCardiovascular disease is the major cause of premature death in chronic kidney disease (CKD) and vascular damage is often detected belatedly, usually evaluated by expensive and invasive techniques. CKD involves specific risk factors that lead to vascular calcification and atherosclerosis, where inflammation plays a critical role. However, there are few inflammation-related markers to predict vascular damage in CKD. This study aimed to investigate immune populations in pre-dialysis patients to (i) identify subset alterations, (ii) assess longitudinal changes, and (iii) evaluate their applicability as biomarkers of subclinical vascular indices.Methods43 pre-dialysis CKD patients in stages CKD-2 to CKD-5 and 38 controls were recruited at baseline and after 18-month follow-up. Aortic stiffness was determined by carotid-femoral pulse wave velocity (PWV) and abdominal aortic calcification was quantified by the Kauppila index on X-rays. Carotid intima-media thickness, the number of carotid plaques and adventitial neovascularization were evaluated by Superb Microvascular Imaging. Peripheral blood mononuclear cells were isolated and immune cell populations were assessed by flow cytometry: senescent T cells (CD4+CD28null), Tang (CD3+CD31+CD184+) and derived subsets, and monocyte subsets (classical, intermediate and non-classical; and ACE expression).ResultsSenescent T cells were increased in CKD. Despite Tang levels were unchanged compared to controls, this subset exhibited enhanced immunosenescence traits (CD28null and inverted CD4+CD8+ ratio) in CKD. Furthermore, Tang were negatively correlated with CKD progression. Slight alterations within monocyte subsets were observed. These findings were validated at the 18-month follow-up. Tang were correlated with several subclinical indices, and further analyses revealed an independent effect on PWV and their potential value as biomarkers. Intermediate monocytes were positively correlated with PWV.ConclusionPre-dialysis CKD stages are hallmarked by alterations in immune cell populations related to vascular homeostasis, including early T-cell immunosenescence traits and a stage-dependent Tang depletion, which was independently related to vascular stiffness. All these features were replicated upon follow-up, thus providing validation toward our results. Our findings pave the ground for future studies addressing the functional contribution of these cellular mediators at the local level, assessing their potential predictive value in the long-term and implementing preventive strategies in the clinical setting.

Cell-Sensing Analogue Nanopore for Rapid Detection of Protein-Related Targets.

Nanopores offer significant advantages in biosensing. Conventional nanopore sensors require probe modification within the pore, and there are two major obstacles: inhomogeneity of probe modification within the pore, and distortion of the detection signal due to the uncontrollable dynamics of the target within the pore. Here, we constructed a cell-sensing analogue nanopore (CeSa-nanopore), by coating the outer surface of the nanopore with cell membrane. The inhomogeneity of probe modification and the uncontrollable kinetics of target-probe binding were also addressed. Specific cells are selected to prepare CeSa-nanopore, for example, cells with high expression of angiotensin-converting enzyme 2 (ACE2) are used to achieve the detection of SARS-CoV-2. When SARS-CoV-2 binds to CeSa-nanopore the surface potential changes, causing a change in the ionic current, thus enabling its detection with a detection rate of 100 %. In addition, the detection of different proteins, such as follicle-stimulating hormone (FSH), can be achieved by changing the cell membrane coating. The identification of cancer cells in ascites can also be achieved by utilizing homologous targeting between cancer cells. Importantly, the use of CeSa-nanopores for the detection of these targets eliminates the need for pre-processing and significantly reduces detection time.