SARS-CoV-2 infection causes fibrotic pathogenesis through deregulating mitochondrial beta-oxidation

doi:10.21203/rs.3.rs-2557548/v1

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SARS-CoV-2 infection causes fibrotic pathogenesis through deregulating mitochondrial beta-oxidation

https://doi.org/10.21203/rs.3.rs-2557548/v1

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published 26 May, 2023

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The global high prevalence of COVID-19 is a major challenge for health professionals and patients. SARS-CoV-2 virus mutate predominantly in the spike proteins, whilst the other key viral components remain stable. Previous studies have shown that the human oral cavity can potentially act as reservoir of the SARS-CoV-2 virus. COVID-19 can cause severe oral mucosa lesions and is likely to be connected with poor periodontal conditions. However, the consequence of SARS-CoV-2 viral infection on human oral health has not been systematically examined. In this research, we aimed to study the pathogenicity of SARS-CoV-2 viral components on human periodontal tissues and cells. We found that by exposing to SARS-CoV-2, especially to the viral envelope and membrane proteins, the human periodontal fibroblasts could develop fibrotic pathogenic phenotypes, including hyperproliferation that was concomitant induced together with increased apoptosis and senescence. The fibrotic degeneration was mediated by a down-regulation of mitochondrial β-oxidation in the fibroblasts. Fatty acid β-oxidation inhibitor, etomoxir treatment could mirror the same pathological consequence on the cells, similar to SARS-CoV-2 infection. Our results therefore provide novel mechanistic insights into how SARS-CoV-2 infection can affect human periodontal health at the cell and molecular level with potential new therapeutic targets for COVID-19 induced fibrosis.

Biological sciences/Cell biology/Mechanisms of disease

Biological sciences/Cell biology/Cell signalling/Stress signalling

COVID-19

SARS-CoV-2

Tooth

Periodontal ligament

Fibrosis

Mitochondria

Since its outbreak, the coronavirus disease 2019 (COVID-19) pandemic has infected over 63 million people globally (https://covid19.who.int) and has been a major challenge to human health. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 virus) has multiple transmission routes such as through respiratory fluids, therefore is highly infectious (1). It has been reported that there were ever increasing “long COVID” (2) and repeated infection cases (3), with the UK alone already having more than 2 million long COVID cases (4). Although COVID-19 has been initially connected with acute inflammatory disease, which causes progressive pulmonary fibrosis (5), increasing evidence have suggested that COVID-19 does affect other organs such as heart, skin, kidneys and brain (6). The human oral cavity and saliva have also been demonstrated to be an important reservoir of the SARS-CoV-2 virus (7), and saliva have been used for effective diagnosis of COVID-19 (8). Periodontal tissues are highly vulnerable to different infectious diseases and COVID-19 has been reported to be potentially connected with poor periodontal health (9). Furthermore, severe oral mucosa lesions including detachment of oral epithelium with inflammation and fibrosis have been reported in the COVID-19 patients (10, 11). However, the molecular mechanisms under the potential pathological consequence of SARS-CoV-2 infection on human oral health have not been systematically investigated.

The evolution of SARS-CoV-2 has generated different variants that are responsible for infection speeds and symptoms (https://www.who.int/activities/tracking-SARS-CoV-2-variants). SARS-CoV-2 has four different structural protein components: envelope, membrane, nucleocapsid and spike (Fig. 1A) (12). The mutations of SARS-CoV-2 predominantly occur in the spike proteins, particularly the receptor-binding domain (RBD), whilst the other key viral components remain stable (13) (14). A key step of SARS-CoV-2 infection is the binding of spike RBD to angiotensin-converting enzyme 2 (ACE2) receptor on the target cells (15) and co-receptor transmembrane serine protease 2 (TMPRSS2) to trigger viral internalization together with the molecular downstream cascades (16). Currently, most of the COVID-19 related research and vaccine development have focused on the spike protein, leaving the other structural proteins’ pathological functions remain to be elucidated.

As part of a series of research on the biological connection of COVID-19 with oral health, this study intended to apply human periodontal tissue and cells as examples to dissect the direct pathological effects of SARS-CoV-2 viral structural components.

Human periodontal tissues and fibroblasts express SARS-CoV-2 receptors

To achieve robust evidence of the presence of SARS-CoV-2 receptors: ACE2 and TMPRSS2 in human periodontal tissue and cells, using immunofluorescence analysis, we confirmed that both ACE2 and TMPRSS2 were notably expressed in the gingiva epithelium, as well as in the periodontal ligament (PDL) cells (Fig. 1B and C). By analyzing the cultured HPLF, we could also confirm the presence of the two receptors by immunofluorescence (Fig. 1D) and western blotting analysis in those cells (Fig. 1E). With an established collagen gel based 3D human gingiva tissue equivalent using gingival epithelial cells and HPLF (Appendix Fig. 1A), we also observed consistent clear expression of ACE2 and TMPRSS2 both in the epithelial cells and PDL fibroblasts (Fig. 1F).

SARS-CoV-2 has high affinity to PDL fibroblasts

We next explored the potentiality of SARS-CoV-2 in infecting PDL fibroblasts. By treating the cells with His Tag conjugated spike protein, followed by immunofluorescent analysis of the spike protein location using an anti-His Tag APC conjugated antibody (Appendix Fig. 1B), we could observe clear association of the spike protein with the cells either under natural growing condition (Fig. 1G), or in lentiviral mediated ACE2 overexpression condition (Fig. 1H). The affinity of spike protein to the cells could be further validated by fluorescence activated cell sorting (FACS) analysis (Fig. 1I and J). Therefore, we confirmed that SARS-CoV-2 indeed could infect PDL fibroblasts directly.

SARS-CoV-2 envelope and membrane proteins induce fibrotic pathogenic phenotypes in PDL fibroblasts

A key pathological hallmark of COVID-19 infection is fibrosis in the lung and potentially also in the other tissues (5). To evaluate the consequence of COVID-19 infection on the PDL, we either adopted lentiviral mediated SARS-CoV-2 infection for envelope, membrane or nucleocapsid proteins (17), or applied recombinant spike proteins at 500ng/ml or 5ug/ml, to cultured human PDL fibroblasts. We simulated acute and long infection by infecting or treating PDLs for 6 hours or 48 hours. Cell proliferation was evaluated using Bromodeoxyuridine (BrdU) incorporation followed by immunostaining for anti-BrdU antibodies. The results indicated that under our tested conditions, only membrane protein significantly increased cell proliferation at 6 hours, while at 48 hours, the envelope and membrane proteins could both elevate the BrdU incorporation index (Fig. 2A and B; Appendix Fig. 2A). Spike protein, on the contrary, could not modulate cell proliferation instead (Fig. 2C and D; Appendix Fig. 2B).

With Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), we then evaluated apoptosis status in the cells. The results indicated that at 48 hours, again only the envelope and membrane proteins, but not the nucleocapsid nor spike proteins could increase apoptosis (Fig. 2E-H; Appendix 3A and B). In the meanwhile, for cellular senescence status in the tested conditions, the results showed that at 6 and 48 hours, only the envelope and membrane protein groups resulted in a significant increase in senescent cells (Fig. 2I-L; Appendix 4A and B).

We then investigated extracellular matrix production by focusing on Collagen I and MMP1, the two key components and enzymes responsible for PDL tissue integrity. Interestingly, western blotting analysis revealed that all the SARS-CoV-2 structure proteins could induce Collagen I production (Fig. 3A-F), and reduce MMP1 production, spike protein elevated MMP1 expression under one of the two tested doses (Fig. 3A-F). Real time RT-PCR analysis suggested the Collagen I expression induction and MMP1 downregulation were modulated at transcription levels (Fig. 3G and H). By applying a hydrogel based 3D culture system, we evaluated Collagen I and MMP1 production and deposition at three dimensions. The results showed that Collagen I deposition were again highly elevated, especially in the envelope and membrane protein groups (Fig. 3I and J). And membrane and nucleocapsid proteins could bring down the MMP1 expression (Fig. 3K and L).

Together, with the evidence of elevated fibroblast proliferation, apoptosis and senescence concomitantly, alongside increased matrix deposition and reduced metalloprotease production, our results pointed out that the SARS-CoV-2’s envelope and membrane were the most potent components to induce a fibrotic degeneration phenotype in PDL cells.

SARS-CoV-2 envelope and membrane proteins down-regulate mitochondrial β-oxidation

To understand the molecular regulation mechanisms behind the observed pathological consequence of SARS-CoV-2 infection, we performed proteomic analysis on the treated PDL fibroblasts, by focusing on the effects of the envelope, membrane and nucleocapsid proteins. We then identified a group of proteins that were both suppressed at 6 and 48 hours post infection (Fig. 4A; Appendix Table 1). Among the significantly down-regulated proteins, the trifunctional enzyme subunit alpha, isoform 2 of very long-chain specific acyl-CoA dehydrogenase, cytochrome c oxidase subunit 2 and isoform cytoplasmic of fumarate hydratase are all essential enzymes in mitochondria functions and mitochondrial β-oxidation (Fig. 4A). Particularly the trifunctional enzyme subunit alpha (HADHA), isoform 2 of very long-chain specific acyl-CoA dehydrogenase were connected with mitochondrial fatty acid β-oxidation. In addition, western blotting analysis could further confirm that HADHA was indeed down-regulated by SARS-CoV-2 structural protein infection (Fig. 4B-E).

We therefore conducted Seahorse Mito stress test (Appendix Fig. 5) for evaluating if and how the mitochondria fatty acid pathway’s function could be affected by SARS-CoV-2 infection. The results showed that indeed, both SARS-CoV-2 envelope and membrane protein, could inhibit fatty acid β-oxidation at both 6 hours and 48 hours’ time points (Fig. 4F and G).

Chemical inhibition of mitochondrial β-oxidation can mirror the fibrotic pathological consequence of SARS-CoV-2 infection

To validate the effect of fatty acid β-oxidation pathway inhibition, we next treated the cells with etomoxir, a specific inhibitor of the pathway through inhibiting carnitine palmitoyltransferase I (CPT1), a key regulatory enzyme for fatty acid to be imported into mitochondria (Fig. 5A). The results indicated that etomoxir treatment could indeed mirror the same pathological consequence on the fibroblasts, similar to SARS-CoV-2 infection. We again observed significantly increased cell proliferation (Fig. 5B; Appendix Fig. 6), apoptosis (Fig. 5C; Appendix Fig. 6), and senescence (Fig. 5D and E), together with elevated Collagen I production (Fig. 5F and G). Similarly, in the hydrogel based three dimension PDL fibroblasts culture, Collagen I deposition was highly increased in the etomoxir treated samples (Fig. 5H and I).

COVID-19 infection can cause a series of symptoms. Among them, fibrosis particularly in lung tissues has evoked significant attention due to the severe consequence on patient life and health quality. Although the current dominant SARS-CoV-2 variants (such as the Omicron) induce milder symptoms in human bodies, the exact pathological consequence of COVID-19 to different human tissues and organs, particularly for oral cavity tissues are still missing, possibly due to the difficulty of distinguishing conventional periodontal diseases from COVID-19 connections. Current concepts of COVID-19 etiology suggest lung fibrosis caused by SARS-CoV-2 infection can be mainly due to damages on lung epithelial cells that trigger acute inflammation followed by fibroblast hyperproliferation (18). However, as SARS-CoV-2 infection is rapid and it is difficult to distinguish if the deeper cells (such as fibroblasts) can also be infected directly, such as in oral cavity the epithelial barrier could be rapidly disrupted upon infection (10). We therefore cannot neglect the potential direct infection consequence of SARS-CoV-2 on fibroblasts. In particular, PDL is one of the most vulnerable human tissues that extrinsic virus and bacteria can often enter PDL directly in pathological conditions (19). Consistent with this concept, previous reports have shown that ACE2 expression were elevated in the periodontal fibroblasts under gingivitis and periodontitis conditions(20), and could be induced by lipopolysaccharide, inflammatory cytokines etc. (21). As such, it is reasonable to postulate that SARS-CoV-2 can also directly infect PDL fibroblasts in the already damaged PDL, and can induce further pathological changes or enhance existing periodontal lesions. Attentions therefore should be made by dental clinicians to the patients who got COVID-19 and appeared to be diagnosed with periodontal disease at the same time, particularly for those long COVID and repeated infected cases.

Our results also suggested SARS-CoV-2 infection indeed can directly induce fibrotic disease phenotypes in fibroblasts through distinct pathways. Mitochondrial fatty acid β-oxidation is the major pathway responsible for fatty acids degradation, hence is essential for human body energy homeostasis. Impeding the pathway can cause different disorders (22). Very recent studies have observed mitochondrial dysregulation in COVID-19 patient blood cells (23, 24). Interestingly, the dysfunction of the fatty acid oxidation has been previously connected with fibrosis particularly in the lung and kidney (25, 26){Merritt, 2018 #12}. For the first time, our findings further confirmed that in the fibroblasts, SARS-CoV-2 could induce fibrotic degeneration directly, through down-regulating fatty acid β-oxidation, particularly by the virus’ envelope and membrane proteins.

Among the SARS-CoV-2’s structural proteins: envelope, membrane, nucleocapsid and spike, the first three proteins are stable structure proteins for all the reported variants, while so far all of the identified mutations happen inside the spike proteins (14). Although spike protein is still the target especially for COVID-19 vaccine development, increasing evidence suggest the other SARS-CoV-2 structural proteins might have unexpected important roles in inducing COVID-19 symptoms. Previous structural analysis of the envelope protein suggested it might be important for virus pathogenicity (27). The envelope protein can also physically increase intra-Golgi pH and forms cation channel(28), and biochemically modulate spike protein in the meantime (29). The most abundant protein: the membrane protein in the SARS-CoV-2 virus, is rationally important for virus assembly (30). Our results further confirmed that the envelope and membrane proteins are actually responsible for the fibrosis phenotypes in the cells by down regulating the key fatty acid β-oxidation regulator such as the trifunctional enzyme subunit alpha. The molecular mechanism behind this regulation axis would require further biochemical analysis.

In this study, our results provide novel mechanistic insights into how SARS-CoV-2 infection can affect human health, particularly for inducing fibrosis, at the cell and molecular level. The findings could be possibly extended to the other body systems to explain and explore the fibrosis pathology and treatment.

Cell culture

Human periodontal ligament fibroblasts (HPLF) were purchased from ScienCell (Cat. 2630) and cultured in DMEM/F12 ((Gibco, 31331-028) containing 20% Fetal bovine serum (FBS) (Sigma, F7524), 1% penicillin-streptomycin (Hyclone, SV30079.01). Human gingival epithelial cells (HGEPp) were cultured in CnT-57 (CELLnTEC, CnT-57).

Viral Infection

Plasmids for SARS-CoV-2 structural proteins were purchased from Addgene (Appendix Table 2). Lentiviral supernatant was collected according to the manual using 293FT cells. HPLFs were infected with lentiviruses carrying target sequences above with 10 µg/ml polybrene (Merck, TR-1003). After 2 h, dishes were topped up with fresh culture medium. Samples were collected at 6 h or 48 h. For overexpressing ACE2 (Addgene, Appendix Table 2) in HPLFs, the cells were infected with lentiviruses carrying target sequences above with 10 µg/ml polybrene (Merck). After 2 h, dishes were topped up with fresh normal culture medium. Samples were collected according to different time points. Infected cells were selected with 1 µg/ml puromycin (Thermo Scientific, 10781691) for 7–10 days.

Recombinant SARS-CoV-2 spike protein treatment

Recombinant SARS-CoV-2 spike protein with His-tag (Biotechne, 10549-CV) was diluted with culture medium to 500 ng/ml or 5 µg/ml and added into the cell culture medium. Samples were collected at 6 h or 48 h.

Mitochondria β-oxidation inhibition assay.

Etomoxir (Sigma, E1905) was diluted with culture medium to aimed concentration then added into cell culture medium. Samples were collected at 6 h or 48 h.

Human periodontal tissue 3D equivalents

5*10^4 of HPLFs were mixed with 150 µl gel mixture of rat tail collagen (Fisher, 11519816), DMEM (Fisher, 21969-035) and FBS (Ratio of volume 9:1:1) homogenously on ice. 2.3 µl 1M Sodium hydroxide solution (NaOH, Sigma 71687) was added into the mixture for neutralization, 150 µl gel was pipetted into a 0.4 µm culture insert (Greiner, 662641) incubated at 37°C for 1 h then fresh culture medium was added into the insert. Culture medium was replaced by CnT-57 before seeding 5*10^5 of HGEPp on top of the gel. HGEPp was cultured for 72 h before airlifting. Culture medium inside the insert was removed every day and the medium outside was changed every 2 days. Samples were frozen directly in OCT (Agar Scientific, AGR1180) at day 14 and sectioned at 20 µm for further analysis.

Hydrogel based 3D matrix production assay

1*10^6 of HPLFs were mixed with 100 µl bioink (CELLINK, CELLINK SKIN+) slowly and gently. Gels were pipetted into 6 well plate then 1.5 ml crosslinking agent (CELLINK) was added to cover the gel at RT for 5 min. Crosslinking agent was removed and 1.5 ml fresh culture media was added which was replaced every 2 days. Samples were frozen directly in OCT at day 7 and sectioned at 30 µm for further analysis.

Immunohistochemistry

For the details of immunostaining, BrdU incorporation assay, Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and Senescence assay details please see Appendix materials & methods.

Flow Cytometry Analysis

HPLFs and ACE2 overexpressed HPLFs were harvested and fixed in 2% PFA solution in 10 mM PBS for 10 min then washed with FACS buffer (1% BSA in PBS). 1*10^6 cells were resuspended in 500 µl flow cytometry permeabilization buffer (0.1% Tween-20 in PBS) for 15 min then washed with FACS buffer again. Cells were resuspended in 100 µl FACS buffer and APC His-Tag conjugated antibody was added, incubated for 2 h at room temperature then kept in dark at 4 °C degree overnight. Samples were analyzed using the BD FACSAria™ II (BD Biosciences). Data was acquired using red laser (633-640nm) for APC signal. Results were analyzed using the FlowJo software (Tree Star Inc., Version 10.8.1). Gates and regions were placed around populations of cells with common characteristics based on SSC and APC.

Western Blotting

A NuPage® Electrophoresis System (Thermo Fisher Scientific), 4–12% Bis-Tris gradient gel (Thermo Fisher Scientific, NP0335BOX), and MOPS buffer (Thermo Fisher Scientific, NP0001), 25–40 µg protein were used for protein separation. Transfer of protein samples onto a 0.45 µm PVDF membrane (Thermo Fisher Scientific, LC2005) was carried out using a NuPage® XCell II Blot Module, and transfer buffer (Thermo Fisher Scientific, NP0006) with 10% methanol (Sigma, 322415). The iBind™ Western System (Thermo Fisher Scientific) was used for blocking, primary and secondary antibody incubations which details could be found above. A C-Digit scanner (LI-COR) was used for band detection with Image studio software (LI-COR, Version 3.1).

Proteomic Analysis

Sample preparation, in-gel digestion, sample cleanup and mass spectrometric analysis was carried out as described previously (Dunn, J et al, 2018). For details please see Appendix material & methods.

Real-time PCR and data analysis

Real-time RT-PCR analysis was performed on a LightCycler 480 Real-Time system (Roche) for 45 cycles, using a SYBR Green I MasterMix (Roche, 04887352001) and primers. 36b4 gene was used as housekeeping gene. Analyses were performed using three technical replicates using the 2-ΔΔCt method.

Seahorse Mito Stress Test

3*10^3 of HPLFs per well were seeded into Seahorse XFe96 well plate (Agilent Technologies, 200941) for overnight. After 6 h and 48 h of viral infection, the medium was removed but a nominal 20 µl per well was left. Each well was washed twice with pre-warmed assay medium (Agilent Technologies, 103680). 80µl assay medium were added into each well, and cells were then incubated for 1 h at 37°C. Meanwhile, effector working solutions were prepared and loaded into ports of the XFe96 cartridge which was already rehydrated in XF buffer at 37°C overnight. The cartridge and the utility plate were inserted into XFe96 instrument to calibrate probes. Once the calibration was finished, the utility plate was replaced by cell plate and continued with the assay as indicated. When the progress was completed, cell plate was removed from the instrument. Medium was aspirated slowly from wells and all wells were gently washed with warmed assay buffer, Plates were stored at -20°C or continued with DNA assay for further normalization. Data were analyzed by Seahorse Analytics website and Wave software (Agilent Technologies, Version 2.6.3).

Statistics

Statistical analyses were performed using Prism software (GraphPad software, Version 9.4.1). Unpaired t-test was applied to all measurements. Data from Seahorse mito stress assay was analyzed by Seahorse Analytics website and Wave software (Agilent Technologies, Version 2.6.3). All quantification and real time RT-PCR results were presented using style of Mean and Standard Deviation (error bars). Observed differences were calculated for p-values: * p < 0.05; ** p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Acknowledgement

We would like to thank the helps provided by Dr Jane Carré for assisting Seahorse analysis, Dr Paul Waines for FACS operation, and Prof. Simon Whawell for critical reading. This study was supported by a research grant from the European Orthodontic Society to Bing Hu. Yan Gao received a fellowship from the Peninsula Dental Social Enterprise.

Author contributions

Yan Gao: Contributed to conception and design, acquisition, analysis, and interpretation, critically revised the manuscript.

Wai Ling Kok: Contributed to acquisition and analysis and critically revised the manuscript.

Vikram Sharma: Contributed to acquisition and analysis and critically revised the manuscript.

Charlotte Sara Illsley: Contributed to conception and design, and critically revised the manuscript.

Sally Hanks: Contributed to conception and design, and critically revised the manuscript.

Christopher Tredwin: Contributed to conception and design, and critically revised the manuscript.

Bing Hu: Contributed to conception and design, interpretation, and drafted and critically revised the manuscript.

All authors gave their final approval and agree to be accountable for all aspects of the work. The authors declare that there is no conflict of interest regarding the publication of this article.

Material and data availability statement

The materials used and datasets generated during and/or analyzed during the current study are available from Professor Bing Hu on reasonable request.

Ferretti L, Wymant C, Kendall M, Zhao L, Nurtay A, Abeler-Dorner L, et al. Quantifying SARS-CoV-2 transmission suggests epidemic control with digital contact tracing. Science. 2020;368(6491).
Lopez-Leon S, Wegman-Ostrosky T, Perelman C, Sepulveda R, Rebolledo PA, Cuapio A, et al. More than 50 long-term effects of COVID-19: a systematic review and meta-analysis. Sci Rep. 2021;11(1):16144.
Wang J, Kaperak C, Sato T, Sakuraba A. COVID-19 reinfection: a rapid systematic review of case reports and case series. J Investig Med. 2021;69(6):1253–5.
Wise J. Covid-19: Two million people in the UK are estimated to be experiencing long covid, says ONS. BMJ. 2022;377:o1391.
Spagnolo P, Balestro E, Aliberti S, Cocconcelli E, Biondini D, Casa GD, et al. Pulmonary fibrosis secondary to COVID-19: a call to arms? Lancet Respir Med. 2020;8(8):750–2.
Wang T, Du Z, Zhu F, Cao Z, An Y, Gao Y, et al. Comorbidities and multi-organ injuries in the treatment of COVID-19. Lancet. 2020;395(10228):e52.
Huang N, Perez P, Kato T, Mikami Y, Okuda K, Gilmore RC, et al. SARS-CoV-2 infection of the oral cavity and saliva. Nat Med. 2021;27(5):892–903.
Baghizadeh Fini M. Oral saliva and COVID-19. Oral Oncol. 2020;108:104821.
Qi X, Northridge ME, Hu M, Wu B. Oral health conditions and COVID-19: A systematic review and meta-analysis of the current evidence. Aging Health Res. 2022;2(1):100064.
Henin D, Pellegrini G, Carmagnola D, Lanza Attisano GC, Lopez G, Ferrero S, et al. Morphological and Immunopathological Aspects of Lingual Tissues in COVID-19. Cells. 2022;11(7).
Pandiar D, Ramani P, Krishnan RP, Y D. Histopathological analysis of soft tissue changes in gingival biopsied specimen from patients with underlying corona virus disease associated mucormycosis (CAM). Med Oral Patol Oral Cir Bucal. 2022;27(3):e216-e22.
Huang Y, Yang C, Xu XF, Xu W, Liu SW. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin. 2020;41(9):1141–9.
Satarker S, Nampoothiri M. Structural Proteins in Severe Acute Respiratory Syndrome Coronavirus-2. Arch Med Res. 2020;51(6):482–91.
Harvey WT, Carabelli AM, Jackson B, Gupta RK, Thomson EC, Harrison EM, et al. SARS-CoV-2 variants, spike mutations and immune escape. Nat Rev Microbiol. 2021;19(7):409–24.
Ni W, Yang X, Yang D, Bao J, Li R, Xiao Y, et al. Role of angiotensin-converting enzyme 2 (ACE2) in COVID-19. Crit Care. 2020;24(1):422.
Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181(2):271–80 e8.
Gordon DE, Jang GM, Bouhaddou M, Xu J, Obernier K, O'Meara MJ, et al. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing. bioRxiv. 2020.
Merad M, Martin JC. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat Rev Immunol. 2020;20(6):355–62.
Kononen E, Gursoy M, Gursoy UK. Periodontitis: A Multifaceted Disease of Tooth-Supporting Tissues. J Clin Med. 2019;8(8).
Santos CF, Morandini AC, Dionisio TJ, Faria FA, Lima MC, Figueiredo CM, et al. Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue. PLoS One. 2015;10(8):e0134601.
Sena K, Furue K, Setoguchi F, Noguchi K. Altered expression of SARS-CoV-2 entry and processing genes by Porphyromonas gingivalis-derived lipopolysaccharide, inflammatory cytokines and prostaglandin E(2) in human gingival fibroblasts. Arch Oral Biol. 2021;129:105201.
Merritt JL, 2nd, Norris M, Kanungo S. Fatty acid oxidation disorders. Ann Transl Med. 2018;6(24):473.
Ajaz S, McPhail MJ, Singh KK, Mujib S, Trovato FM, Napoli S, et al. Mitochondrial metabolic manipulation by SARS-CoV-2 in peripheral blood mononuclear cells of patients with COVID-19. Am J Physiol Cell Physiol. 2021;320(1):C57-C65.
Guntur VP, Nemkov T, de Boer E, Mohning MP, Baraghoshi D, Cendali FI, et al. Signatures of Mitochondrial Dysfunction and Impaired Fatty Acid Metabolism in Plasma of Patients with Post-Acute Sequelae of COVID-19 (PASC). Metabolites. 2022;12(11).
Geng J, Liu Y, Dai H, Wang C. Fatty Acid Metabolism and Idiopathic Pulmonary Fibrosis. Front Physiol. 2021;12:794629.
Jang HS, Noh MR, Kim J, Padanilam BJ. Defective Mitochondrial Fatty Acid Oxidation and Lipotoxicity in Kidney Diseases. Front Med (Lausanne). 2020;7:65.
Mandala VS, McKay MJ, Shcherbakov AA, Dregni AJ, Kolocouris A, Hong M. Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers. Nat Struct Mol Biol. 2020;27(12):1202–8.
Cabrera-Garcia D, Bekdash R, Abbott GW, Yazawa M, Harrison NL. The envelope protein of SARS-CoV-2 increases intra-Golgi pH and forms a cation channel that is regulated by pH. J Physiol. 2021;599(11):2851–68.
Boson B, Legros V, Zhou B, Siret E, Mathieu C, Cosset FL, et al. The SARS-CoV-2 envelope and membrane proteins modulate maturation and retention of the spike protein, allowing assembly of virus-like particles. J Biol Chem. 2021;296:100111.
Zhang Z, Nomura N, Muramoto Y, Ekimoto T, Uemura T, Liu K, et al. Structure of SARS-CoV-2 membrane protein essential for virus assembly. Nat Commun. 2022;13(1):4399.

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You are reading this latest preprint version

SARS-CoV-2 infection causes fibrotic pathogenesis through deregulating mitochondrial beta-oxidation

Status:

Journal Publication

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Abstract

Figures

Introduction

Results

Human periodontal tissues and fibroblasts express SARS-CoV-2 receptors

SARS-CoV-2 has high affinity to PDL fibroblasts

SARS-CoV-2 envelope and membrane proteins induce fibrotic pathogenic phenotypes in PDL fibroblasts

SARS-CoV-2 envelope and membrane proteins down-regulate mitochondrial β-oxidation

Chemical inhibition of mitochondrial β-oxidation can mirror the fibrotic pathological consequence of SARS-CoV-2 infection

Discussion

Materials & Methods

Cell culture

Viral Infection

Recombinant SARS-CoV-2 spike protein treatment

Human periodontal tissue 3D equivalents

Hydrogel based 3D matrix production assay

Immunohistochemistry

Flow Cytometry Analysis

Western Blotting

Proteomic Analysis

Real-time PCR and data analysis

Seahorse Mito Stress Test

Statistics

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1