IntroductionCoronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still a global pandemic1Wu F Zhao S Yu B Chen Y Wang W Song Z et al.A new coronavirus associated with human respiratory disease in China.. Angiotensin converting enzyme 2 (ACE2) is the major recognized cell-surface receptor enabling cellular entry of SARS-CoV-2 and SARS-CoV2HLi W Michael JM Natalya V Sui J Swee KW Michael AB et al.Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus., 3Zhou P Yang X Wang X Hu B Zhang L Zhang W et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin., widely expressed in lung, cardiovascular system, gut, kidney, central nervous system and adipose tissue4Mahmoud G Wang K Anissa V Quynh N Zhong J Anthony JT et al.Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2.. As a zinc metalloproteinase, ACE2 plays an important role in maintaining the homeostasis of renin-angiotensin system (RAS) 5Donoghue M Hsieh F Baronas E Godbout K Gosselin M Stagliano N et al.A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9., 6Tipnis SR Hooper NM Hyde R Karran E Christie G Turner AJ A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase.. Angiotensin converting enzyme (ACE) cleaves angiotensin (Ang) I to generate Ang II7Imai Y Kuba K Penninger JM Angiotensin-converting enzyme 2 in acute respiratory distress syndrome., while ACE2 cleaves Ang I to generate Ang 1-9 and Ang II to generate Ang 1-7. The ACE-Ang II-AT1R axis leads to vasoconstriction, water and sodium retention, inflammatory response, oxidative stress and cardiovascular remodeling8Vaibhav BP Zhong J Maria BG Gavin YO Role of the ACE2/Angiotensin 1-7 Axis of the Renin-Angiotensin System in Heart Failure.. In contrast, ACE2-Ang1-7-MAS axis antagonized the ACE-AngII-AT1R axis, thus exerting the functions of vasodilation, natriuretic, diuretic, anti-inflammatory, anti-oxidative stress and anti-cardiovascular remodeling9Molecular and cellular mechanisms of the inhibitory effects of ACE-2/ANG1-7/Mas axis on lung injury..SARS-CoV-2 infection has been reported to activate RAS system and destroy endothelial cells, inducing cardiovascular dysfunction, oxidative stress and thrombosis10Toshiaki I Jean MC, Jerrold HL The coagulopathy, endotheliopathy, and vasculitis of COVID-19.. Several clinical cohort studies have also found that cardiovascular disease like myocardial damage were more common in COVID-19 patients11Coronavirus Disease-2019 (COVID-19) and Cardiovascular Complications., 12Luo I Zhu X Jian J Chen X Yin K Cardiovascular disease in patients with COVID-19: evidence from cardiovascular pathology to treatment., 13Zeng J Liu Y Yuan J Wang F Wu W Li J et al.First case of COVID-19 complicated with fulminant myocarditis:a case report and insights.. Increased circulating Ang II was also found in the serum of COVID-19 patients than that in healthy controls14Liu Y Yang Y Zhang C Huang F Wang F Yuan J et al.Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury.. These findings suggest that SARS-CoV-2 infection more usually generates the cardiovascular diseases in COVID-19 patients, while the underlying mechanisms remain unclear.

Given the versatility of ACE2 in SARS-CoV-2 infections, we investigate the crucial roles of ACE2 in the cardiovascular pathology of COVID-19. Here, we show that SARS-CoV-2 Spike protein downregulates ACE2 expression, thus causing cardiovascular stress by failure to cleave Ang II and subsequently leading to lung injury and cardiovascular complication-associated mortality.

DiscussionClinical studies have found that cardiovascular disease is more common in COVID-19 patients22Cardiovascular disease and COVID-19., while hypertension and cardiovascular patients are more likely to develop severe disease after contracting the SARS-CoV-223Guo T Fan Y Chen M Wu X Zhang L He T et al.Cardiovascular Implications of Fatal Outcomes of Patients With Coronavirus Disease 2019 (COVID-19).. The pivotal mechanisms mediating the detrimental the effects of SARS-CoV-2 on cardiovascular disease remain largely unknown. Our study shows that SARS-CoV-2 Spike not only binds to ACE2 to mediate virus entry but also down-regulated the expression of ACE2, indicating that the downregulation of ACE2 potentially links COVID-19 its imbalance of RAS.ACE2 downregulation has also been observed in some other viral infections, such as influenza and SARS-CoV 24Liu X Yang N Tang J Liu S Luo D Duan Q et al.Downregulation of angiotensin-converting enzyme 2 by the neuraminidase protein of influenza A (H1N1) virus., 25Glowacka I Bertram S Herzog P Pfefferle S Steffen I Muench MO Differential downregulation of ACE2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus NL63.. A recent study also found that SARS-CoV-2 infection down-regulated ACE2 expression, and found that ACE2 links COVID-19 to its metabolic complications, while ACE2 activators Imatinib and methazolamide improve metabolic disorders under SARS-CoV-2 infection26Li Z Peng M Chen P Liu C Hu A Zhang Y et al.Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry.. However, these studies do not explain the molecular mechanism of ACE2 downregulation. A recent study found that Spike pseudovirus infected hamsters and PAEC cells reduced ACE2 expression27Lei Y Zhang J Cara R Schiavon CR He M Chen L Shen H et al.SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE2.. But we were unable to replicate that the expression of ACE2 can be down-regulated after Spike pseudovirus infection in HeLa-ACE2 cells. No significant changes of ACE2 protein expression were also observed in Spike protein treated HeLa-ACE2 cells. This may be because the cell lines we used are different. Our study found that SARS-CoV-2 Spike protein activates intracellular signals to reduce ACE2 RNA, possibly altering ACE2 RNA modification to affect its stability. More detailed mechanisms require future studies. Moreover, the decrease of ACE2 was verified in SARS-CoV-2 infected patient and mice. Finally, the decrease of ACE2 would lead to an increase of Ang II secretion, which affected the normal endothelial cell function and aggravated cardiovascular disease (Fig. 6).

Figure 6The abstract overview of this study. SARS-CoV-2 Spike destroys the renin-angiotensin system in the lungs by down-regulating the expression of ACE2.

Our previous study found that SARS-COV-2 infected lung epithelial cells induced high expression of inflammatory cytokines, and cytokines such as IFN and IL-6 can up-regulate ACE2 expression28Xu G Li Y Zhang S Peng H Wang Y Li D et al.SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation.. In contrast, the expression of ACE2 was down-regulated in SARS-COV-2 infected hypoinflammatory cells such as Vero-E6 and Hela-ACE2, suggesting that ACE2 was down-regulated in the truly infected cells, while the expression of ACE2 was up-regulated in bystander cells. Thus, the bystander cells expressing high levels of ACE2 were more susceptible to SARS-CoV-2. While SARS-CoV-2 hijacks ACE2 to facilitate its entry into host cells and then destroys host RAS by down-regulating ACE2 in the infected target cells.As the main receptor mediating SARS-CoV-2 entry, targeting ACE2 has become one of the therapeutic strategies for COVID-1929Ross CL Xing E Adam DK Zhang X Jasmine AT Li J et al.Rationally Designed ACE2-Derived Peptides Inhibit SARS-CoV-2.. ACE2-targeting monoclonal antibody 3E8 has been shown to broadly block the invasion of multiple coronaviruses, including SARS-CoV, SARS-CoV-2 mutant variants, without markedly affecting the physiological activities of ACE2 or causing severe toxicity in ACE2 transgenic mice30Chen Y Zhang Y Yan R Wang G Zhang Y Zhang Z et al.ACE2-targeting monoclonal antibody as potent and broad-spectrum coronavirus blocker.. APN01, a recombinant soluble form of human ACE2 (rhACE2), was reported to be effective in blocking viral infection and protecting the lung from pathological damage31Robert HS Reynold APJ Steven KL Howard SH Norman RW Paul TW et al.Development of a novel, pan-variant aerosol intervention for COVID-19.. This peptide drug was slated for a multicenter trial in Europe (NCT04335136). A similar rhACE2 drug was applied for clinical trials in China, but it was subsequently withdrawn (NCT04287686)32Andrew MS Debra ID Mark CC COVID-19, ACE2, and the cardiovascular consequences.. Overall, ACE2 based drugs represent a double-edged sword in coronavirus infection33Chidinma LO Zhang Y Thomas C Christopher EH De LF William DF ACEI/ARB therapy in COVID-19: the double-edged sword of ACE2 and SARSCoV-2 viral docking.. Low expression of ACE2 promote the progression of Systemic hypotension, hypokalemia and lung injury, aggravating the severity of the disease34Kuba K Imai Y Rao S Gao H Guo F Guan B et al.A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury., 35Yang P Gu H Zhao Z Wang W Cao B Lai C et al.Angiotensin-converting enzyme 2 (ACE2) mediates influenza H7N9 virus-induced acute lung injury., 36Zou Z Yan Y Shu Y Gao R Sun Y Li X et al.Angiotensin-converting enzyme 2 protects from lethal avian influenza A H5N1 infections., 37Imai Y Kuba K Rao S Huan Y Guo F Guan B et al.Angiotensin-converting enzyme 2 protects from severe acute lung failure., 38Silhol F Sarlon G Deharo JC Vaïsse B Downregulation of ACE2 induces overstimulation of the renin–angiotensin system in COVID-19: should we block the renin–angiotensin system?.. These data suggested that possible dysregulation of patient's RAS should be noted when targeting ACE2 for COVID-19 therapy.Materials and MethodsEthics statement

This study was reviewed and approved by the Medical Ethical Committee of Shenzhen Third People's Hospital (2021-030)Patient

One COVID-19 patient in this study was from the Shenzhen Third People's Hospital. Control lung tissues were from normal tissues far from the tumor of lung adenocarcinoma patients. Series plasma were obtained from COVID-19 patients who had positive RT-PCR test results during hospitalization.

Cells, plasmids and reagents

Vero-E6 cells, hACE2-HeLa, hACE2-293T and HEK-293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% FBS (Gibco), 100U/mL Penicillin-Streptomycin. Calu3 cells were cultured in Modified Eagle Medium (MEM, Gibco) supplemented with 20% FBS (Gibco), 100U/mL Penicillin-Streptomycin (Gibco). HPAEC were purchased from ATCC and cultured in Medium 200 supplemented with Low Serum Growth Supplement (LSGS) containing FBS.

SARS-CoV-2 plasmids (NSP4, NSP5, NSP7, NSP9, NSP11, ORF6, ORF7A, ORF8, ORF9B, ORF9C, ORF10, E, NSP12, NSP2, NSP14, NSP10, NSP1, NSP8, GP, ORF3A, NSP6, NSP15, N, NSP13) were kindly provided by professor Nevan J. Krogan (University of California San Francisco). SARS-CoV-2 Spike (VG40589-NF), Spike 1 (VG40591-CH), Spike 2 (VG40590-CY) were purchased from Sino Biological. MG132 (S2619) and E64D (S7393) were obtained from Selleck Chemicals. Angiotensin II (ALX-151-039-M005) was purchased from Cayman.

SARS-CoV-2 preparation and infection

All experiments involving SARS-CoV-2 infection were carried out in the biosafety level-3 (BLS-3) laboratory of Shenzhen Third People's Hospital. SARS-CoV-2 isolate SZTH-003 was sourced from a COVID-19 patient. The genomic sequence of SZTH-003 has been deposited in the Global Initiative of Sharing All Influenza Data (GISAID, EPI_ISL_406594). SARS-CoV-2 was amplified once in Vero-E6 cells and viral stocks were contained at -80°C. For Vero-E6 cells and hACE2-HeLa cells infection, virus (MOI=0.3) was poured on cells directly and incubated for 1 hour for viral adsorption at 37°C 5% CO2. Subsequently, infection media was removed and cells were incubated in 2% FBS media in 37°C 5% CO2 condition and harvested for analysis at indicated time points.

For titration of the 50% tissue culture infectious dose (TCID50) of SARS-CoV-2, Vero-E6 cells were plated in 96-well plates at 20, 000 cells per well in DMEM containing 5% FBS, Penicillin/Streptomycin, L-Glutamine, and 15 mM HEPES (Life Technologies). The cells were incubated overnight in a 5% CO2 environment at 37 °C, washed once with PBS and then cultured in serum-free DMEM containing Penicillin/Streptomycin, L-Glutamine, 15 mM HEPES and 1 μg/mL TPCK-treated trypsin. A 10-fold initial dilution of samples with one freeze-thaw cycle was made in octuplicate wells of the 96-well plates followed by 6 serials 10-fold dilutions. The last row served as negative control without addition of any sample. After a 4-day incubation, the plates were observed for the presence of cytopathogenic effect (CPE) using an inverted optical microscope. Any sign of CPE was categorized as a positive result. The endpoint titers were calculated by means of a simplified Reed & Muench method.

Mice

The animal experiments were conducted in the BLS-3 animal facility approved for the studies of SARS-CoV-2. The experiment protocol has been approved by the Animal Ethics Committee of the Second Military Medical University. AC70 CAG-hACE2 transgenic mice (Shanghai Model Organisms, NM-TG-200002) (6-8 weeks of age, male) were intranasal infected with 20 μl of SARS-CoV-2 (1 × 104 PFU). On the 4th day of infection, mice in each group were sacrificed and their lungs were removed and fixed with formalin for 48 h and prepared for paraffin embedding and subsequent IHC staining.

Generation of pseudotyped lentivirus

SARS-CoV-2 spike (S) glycoprotein plasmid were kindly provided by professor Ping Zhao (Second Military Medical University, Shanghai, China). HEK-293T cells were grown in DMEM containing 10% FBS and co-transfected with HIV Gag/Pol, HIV rev, plenti-EGFP and the SARS-CoV-2 spike expression plasmids using Lipofectamine 2000 reagent (Thermo Fisher). The supernatant with produced virus (Spike protein-pseudotyped (SPP) or (VSV-G lentivirus)) was harvested at 48 hours post transfection, clarified by centrifuging at 1, 200 rpm for 5 min at 4°C and then filtered with 0.22 μm membrane. The virus was stored at -80°C.

RT-qPCR

Total RNA was extracted with TRIzol™ Reagent in accordance with the manufacturer's instructions. Reverse transcription into cDNA was performed with a High-Capacity cDNA Reverse Transcription Kit (Takara, RR036A). The expression levels of indicated RNA were determined by qPCR analysis using Power SYBR Green PCR Master Mix (Vazyme, Q311-02). The primers used RT-qPCR were human ACE2-F:5’-GGAGTTGTGATGGGAGTGAT-3’; ACE2-R: 5’-GATGGAGGCATAAGGATTTT-3’; human GAPDH-F:5’-GGAGCGAGATCCCTCCAAAAT-3’; GAPDH-R: 5’-GGCTGTTGTCATACTTCTCATG-3’; SARS-CoV-2(Spike)-F: 5’-TGCAGGTATATGCGCTAGTTATCAG-3’; SARS-CoV-2(Spike)-R: 5’-CACCAAGTGACATAGTGTAGGCAAT-3’.

Western blotting

Cells were harvested and boiled in 2X lammili sample buffer containing 10% βME (Sigma, M3148). Cell lysates were separated by 9% SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, IPFL10100). After being blocked with 5% BSA in TBS buffer containing 0.05% Tween 20, the blot was probed with indicated first antibodies and the horseradish peroxidase (HRP)-conjugated secondary antibody sequentially. Protein bands were detected by SuperSignal West Pico Chemiluminescent substrate (Bio-Rad). The following antibodies were used: ACE2 (Abcam, ab108209), SARS-CoV-2 Nucleoprotein (Sino Biological, 40588-T62), SARS-CoV-2 Spike (Sino Biological, 40591-MM42), Actin (TransGen Biotech, HC201), GAPDH (TransGen Biotech, HC301), AT-1R (Abcam, ab18801), AT-2R (Abcam, ab92445), anti-mouse IgG (TransGen Biotech, HS201-01) and anti-rabbit IgG (TransGen Biotech, HS101-01).

Immunohistochemistry

Formalin-fixed paraffin-embedded Lung tissue was cut into 4 μm sections and mounted on frosted glass slides. After deparaffinization and rehydration, slides were submerged into pH 8.0 EDTA buffer and boiled for 2 minutes with high-pressure for antigenic retrieval. Slides were washed in PBS, incubated in 3% hydrogen peroxide for 10 minutes and then blocked with 1% bovine serum albumin (BSA) for 1 hour. The following primary antibodies were used: cleaved-Caspase3 (Cell Signaling Technology, 9661), ACE2 (Abcam, ab108209) and VWF (Cell Signaling Technology, 65707S). Slides were then washed in PBST (PBS plus 0.1% Tween 20), incubated with the biotinylated secondary antibody (Zymed, San Francisco, CA) and visualized with 3,3′-diaminobenzidine (DAB) under the microscopy.

Plasma angiotensin II measurement

The plasma samples from COVID-19 infected patients were separated from blood samples in the BSL-3 laboratory. The concentrations of plasma angiotensin II were detected by ELISA assay according to the manufacturer's instructions (Sigma, RAB0010).

RNA sequencing (RNA-seq) analysis

Differentially expressed genes (DEGs) are defined as the ratio of the infected group to the uninfected group (Mock group) by the rank-sum test. The P values were adjusted using the FDR method. Corrected P values of 0.05, and the value of FC greater than 1.5 or less than 1/1.5, were set as the threshold for significant differential expression. The differential expression results were displayed with the volcano map through the ggplot2 package of the R project.

Statistics analysis

All statistical analyses are performed using GraphPad Prism version 8.0.1. Paired t-test was used in qPCR analysis. Data were derived from the average of three biological replicate experiments, and calculated as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P <0.001.