Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determine the role of PNUTS in endothelial cell aging. We confirm that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. Findings are validate in vivo using endothelial-specific inducible PNUTS-deficient mice (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice present severe multiorgan failure and vascular leakage. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice reveal that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restores barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs. PNUTS-deficient mice exhibit multiorgan failure and vascular leakage. PNUTS regulates SEMA3B, suggesting a target for endothelial aging.

High gestational weight gain (GWG) is a cardiovascular risk factor and may disturb neonatal endothelial function. Long non‐coding RNAs (lncRNAs) regulate gene expression epigenetically and can modulate endothelial function. Endothelial colony forming cells (ECFCs), circulating endothelial precursors, are a recruitable source of endothelial cells and sustain endothelial function, vascular growth and repair. We here investigated whether higher GWG affects neonatal ECFC function and elucidated the role of lncRNAs herein. Wound healing of umbilical cord blood‐derived ECFCs after pregnancies with GWG <13 kg versus >13 kg was determined in a scratch assay and based on monolayer impedance after electric wounding (electric cell‐substrate impedance sensing, ECIS). LncRNA expression was analysed by RNA sequencing. The function of killer cell lectin‐like receptor K1 antisense RNA (KLRK1‐AS1) was investigated after siRNA‐based knockdown. Closure of the scratch was delayed by 25% (P = 0.041) in the higher GWG group and correlated inversely with GWG (R = −0.538, P = 0.012) in all subjects (n = 22). Similarly, recovery of the monolayer barrier after electric wounding was delayed (−11% after 20 h; P = 0.014; n = 15). Several lncRNAs correlated with maternal GWG, the most significant one being KLRK1‐AS1 (log2 fold change = −0.135, P < 0.001, n = 35). KLRK1‐AS1 knockdown (n = 4) reduced barrier recovery after electric wounding by 21% (P = 0.029) and KLRK1‐AS1 expression correlated with the time required for wound healing for both scratch (R = 0.447, P = 0.033) and impedance‐based assay (R = 0.629, P = 0.014). Higher GWG reduces wound healing of neonatal ECFCs, and lower levels of the lncRNA KLRK1‐AS1 may underlie this.

HomeCirculation ResearchVol. 132, No. 5COVID19 Impairs Cardiac Function via Endothelial H19 and IL6 Signaling Open AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialOpen AccessLetterPDF/EPUBCOVID19 Impairs Cardiac Function via Endothelial H19 and IL6 Signaling Rio P. Juni, Philippa G. Phelp, Diewertje I. Bink, Veerle Kremer, Anke S. van Bergen, Pedro Espinosa Gonzalez, Karlijn B. Rombouts, Karl J. Harber, Daan A.F. Heister, Kak K. Yeung, Jan Van den Bossche, Diederik W.D. Kuster, Amsterdam UMC COVID-19 Biobank Study Group Paul A.J. Krijnen, Hans W.M. Niessen and Reinier A. Boon Rio P. JuniRio P. Juni https://orcid.org/0000-0002-5850-2869 Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. , Philippa G. PhelpPhilippa G. Phelp Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Diewertje I. BinkDiewertje I. Bink https://orcid.org/0000-0003-1389-1677 Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Veerle KremerVeerle Kremer Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Anke S. van BergenAnke S. van Bergen https://orcid.org/0000-0002-1843-5657 Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Pedro Espinosa GonzalezPedro Espinosa Gonzalez Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Karlijn B. RomboutsKarlijn B. Rombouts Department of Vascular Surgery (K.B.R., K.K.Y.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Karl J. HarberKarl J. Harber Department of Molecular Cell Biology and Immunology (K.J.H., D.A.F.H., J.V.d.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Diabetes and Metabolism, the Netherlands (K.J.H., D.A.F.H., J.V.d.B.). , Daan A.F. HeisterDaan A.F. Heister Department of Molecular Cell Biology and Immunology (K.J.H., D.A.F.H., J.V.d.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Diabetes and Metabolism, the Netherlands (K.J.H., D.A.F.H., J.V.d.B.). , Kak K. YeungKak K. Yeung https://orcid.org/0000-0002-8455-286X Department of Vascular Surgery (K.B.R., K.K.Y.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Jan Van den BosscheJan Van den Bossche Department of Molecular Cell Biology and Immunology (K.J.H., D.A.F.H., J.V.d.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Diabetes and Metabolism, the Netherlands (K.J.H., D.A.F.H., J.V.d.B.). , Diederik W.D. KusterDiederik W.D. Kuster https://orcid.org/0000-0003-1498-6862 Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). , Amsterdam UMC COVID-19 Biobank Study Group , Paul A.J. KrijnenPaul A.J. Krijnen https://orcid.org/0000-0002-5399-5617 Department of Pathology (P.A.J.K., H.W.M.N.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. , Hans W.M. NiessenHans W.M. Niessen https://orcid.org/0000-0001-9404-9822 Department of Pathology (P.A.J.K., H.W.M.N.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. and Reinier A. BoonReinier A. Boon Correspondence to: Reinier A. Boon, PhD, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, Department of Physiology, O|2 Research building, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Email: E-mail Address: [email protected] https://orcid.org/0000-0001-7944-9748 Department of Physiology (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., D.W.D.K., R.A.B.), Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, the Netherlands. Amsterdam Cardiovascular Sciences, Microcirculation, the Netherlands (R.P.J., P.G.P., D.I.B., V.K., A.S.v.B., P.E.G., K.B.R., K.K.Y., D.W.D.K., R.A.B.). Institute for Cardiovascular Regeneration, Centre for Molecular Medicine, Goethe University Frankfurt am Main, Germany (R.A.B.). German Centre for Cardiovascular Research, Partner Site Frankfurt Rhein/Main (R.A.B.). Originally published14 Feb 2023https://doi.org/10.1161/CIRCRESAHA.122.322166Circulation Research. 2023;132:648–651is related toMeet the First AuthorsOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 2, 2023: Previous Version of Record February 14, 2023: Ahead of Print Cardiac complications occur in 40% of hospitalized patients with COVID-19, and individuals with COVID-19 are at increased risk for cardiovascular postacute sequela.1 Although direct cardiac SARS-COV-2 invasion is a plausible underlying mechanism, evidence shows little or no presence of SARS-COV-2 genome in premortem or postmortem cardiac biopsies,2 in line with the low incidence of virus particles found in the circulation.3 Cardiac microvascular endothelial cells (CMECs), which form the capillaries adjacent to cardiomyocytes in the heart, are important in regulating cardiac function.4 In the present study, we show that COVID-19 reduces the expression of long noncoding RNA H19-a known anti-inflammatory transcript in endothelium,5 leading to cardiac endothelial dysfunction and subsequent impairment of myocardial contractility, independent of viral invasion to the heart.Meet the First Author, see p 544We discovered that H19 expression was significantly lower in cardiac biopsies from patients with COVID-19 (Table S1) as compared to non-COVID-19 controls (Figure [A], left), despite the undetected levels of SARS-COV-2 genome (data not shown). Using single-cell resolution in situ hybridization, we found that H19 was enriched in cardiac endothelium (Figure [A], middle, left) as it was expressed along the endothelial-specific marker vascular endothelial cadherin (VE-cadherin) (Figure [A], middle, right), and confirmed the reduced expression of H19 in cardiac endothelium in COVID-19 heart biopsies (Figure [A], middle, left). H19 is important for endothelial function as its silencing (siH19) in primary human CMECs induced endothelial stress, shown as irregular and increased adherens junction area stained by VE-cadherin, and reduced endothelial NOS3 (nitric oxide synthase 3) expression (Figure [B]). In line with our previous study,5 H19 silencing induced STAT3 (signal transducer and activator of transcription 3) activation and increased level of IL (interleukin)-6 (Figure [B]), indicating proinflammatory activation of cardiac endothelial cells upon H19 depletion.Download figureDownload PowerPointFigure. Dysregulation of H19 in COVID-19 induces cardiac endothelial dysfunction, leading to impairment of cardiomyocyte (CM) function. A, Left, Quantitative real-time PCR (qRT-PCR) analysis of H19 in postmortem cardiac biopsies from control (non-COVID-19, n=8) vs patients with COVID-19 (n=14 donors, unpaired t test). Middle, Single-cell resolution in situ hybridization on tissues (SCRINSHOT) of H19 (green, arrows, left) and sequential slides of immunofluorescent (IF) staining of endothelial marker vascular endothelial cadherin (VE-cadherin) (yellow, right, lining the cardiac microvasculature [dashed line]) in the cardiac biopsies (DAPI: blue; cTNT: green, right). Right, Staining overview. Top, IF of VE-cadherin (Cell Signaling; No. 2500), cTNT (Abcam; No. ab8295), and DAPI of low (left) and high magnification (right) of a cardiac tissue. Bottom, SCRINSHOT negative (no H19 probe) and positive control (H19 probe added) from sequential slides of the same cardiac biopsy. Most representative images were shown. B, IF of VE-cadherin (green; DAPI: blue) and adherens junction area in human cardiac microvascular endothelial cells (CMECs) upon siRNA-mediated H19 silencing (siH19) vs siRNA control (sictr); qRT-PCR of NOS3 (nitric oxide synthase 3); Western blot of phospho-STAT3 (Y705, No. 9145; Cell Signaling) over total STAT3 (Cell Signaling; No. 4904); and ELISA of IL-6 (interleukin-6) (Thermo Fisher Scientific; No. EH2IL6) in CMECs (n=4 biological replicates/different donors, Mann-Whitney U test). C, Coculture of CMECs and adult rat ventricular CMs. CMECs were transfected with siH19 or sictr for 48 hours and subsequently cocultured with CMs. Measurement of CM contraction (% sarcomere shortening) and relaxation (return velocity and tau) upon field stimulation using video-based MultiCell system (CytoCypher; n=6 independent experiments, 30–40 CMs were measured per condition per experiment, ANOVA followed by Tukey multiple comparison test). D, qRT-PCR of H19 in CMECs treated with plasma from patients with COVID-19 from ward or intensive care unit (ICU) (n=5 plasma donors per condition, Kruskal-Wallis test). Coculture of CMs with CMECs pretreated for 6 hours with the collected plasma (the plasma was washed out, and the medium was refreshed before CMECs were cocultured with CMs). Measurement of CM contraction and relaxation after coculture (n=5 independent experiments, Kruskal-Wallis test). E, qRT-PCR of IL-6 in cardiac biopsies from control (non-COVID-19, n=8) vs patients with COVID-19 (n=14, unpaired t test). Measurement of CM function after coculture with CMECs (+CMEC) transfected with siH19, in the presence or absence of tocilizumab (tcz) administered in the CM compartment (n=6 independent experiments, ANOVA). F, Measurement of CM function after coculture with CMECs (+CMEC) pretreated with plasma from patients with COVID-19 before and after the patients received tocilizumab (n=5 independent experiments, Kruskal-Wallis test). Data are presented as mean±SD. Differences were considered significant when P was <0.05.To underline the functional effect of endothelial H19 silencing on cardiomyocyte contractility, we cocultured CMECs devoid of H19 with cardiomyocytes (Figure [C]) and measured cardiomyocyte contractile function.4 Silencing of H19 in CMECs impaired the beneficial effects of endothelial cells on cardiomyocyte contraction (shown as reduced percentage of shortening) and relaxation (shown as reduced return velocity and longer constant time of relaxation, tau), as compared with CMECs with no H19 silencing (siRNA control; Figure [C]).As patients with COVID-19 are characterized by hyperinflammatory response indicated by elevated levels of circulating proinflammatory cytokines, such as TNFα (tumor necrosis factor alpha), IL-1, and IL-6, we wondered whether this proinflammatory state impairs cardiac endothelial control of cardiomyocyte function. Interestingly, COVID-19 plasma reduced endothelial H19 levels (Figure [D]). We then pretreated CMECs with plasma from hospitalized patients with COVID-19 (Table S1) and subsequently cocultured them with cardiomyocytes (Figure [D], experiment setup). Interestingly, COVID-19 plasma impaired the ability of CMECs to enhance cardiomyocyte contraction and relaxation (Figure [D]), the effects that were more pronounced in the intensive care unit (ICU) than in the ward patient group. These indicate that the proinflammatory state induced in patients with COVID-19 affects cardiac endothelial H19 levels, leading to cardiac endothelial dysfunction, which impairs cardiomyocyte function.In line with increased endothelial IL-6 level upon H19 silencing in CMECs (Figure [B]), we found increased IL-6 expression in cardiac biopsies of patients with COVID-19 (Figure [E]), indicating that IL-6 can act as an endothelial-derived factor released upon H19 reduction that impairs cardiomyocyte function. Interestingly, using the IL-6 receptor inhibitor tocilizumab in our coculture experiments (Figure [E], experiment setup), we showed that inhibition of IL-6 pathway on cardiomyocytes can restore cardiomyocyte contractile function upon coculture with CMECs devoid of H19 (Figure [E]), underlining the role of endothelial-derived IL-6 in mediating the detrimental effect of H19 depletion on cardiomyocyte function.Lastly, to further translate these findings to the clinical situation, we treated CMECs with plasma samples collected from ICU patients with COVID-19 before and after the patients received tocilizumab treatment (Figure [F]). We demonstrated that exposure to the plasma post-tocilizumab treatment restored endothelial enhancement of cardiomyocyte function (Figure [F]), as compared with the exposure to the plasma pre-tocilizumab treatment, further underlining the contribution of IL-6 in the COVID-19–induced cardiac contractile impairment.In conclusion, we demonstrated that H19-a cardiac endothelium-enriched long noncoding RNA—is downregulated in the heart of patients with COVID-19. H19 silencing in CMECs induces cardiac endothelial dysfunction and impairs cardiomyocyte function. Exposure of CMECs to COVID-19 plasma reduces H19 levels and impairs endothelial regulation of cardiomyocyte function. Mechanistically, H19 silencing induced cardiac endothelial STAT3 activation with increased IL-6 expression, which was also observed in the cardiac biopsies from patients with COVID-19, suggesting that reduced H19 level causes endothelial-derived IL-6 release, inducing impairment of cardiomyocyte function. Interestingly, the IL-6 receptor blocker tocilizumab restored impairment of cardiomyocyte function upon coculture with CMECs devoid of H19. Moreover, exposure to plasma from patients with COVID-19 post-tocilizumab treatment rescued the impairment of endothelial enhancement of cardiomyocyte function. Our findings provide the first proof that COVID-19 induces dysregulation of long noncoding RNA H19, which results in cardiac endothelial dysfunction, leading to impairment of cardiac function—a novel mechanism underlying the pathogenesis of cardiac sequela in COVID-19. Further, we showed that the IL-6 pathway interference with tocilizumab can rescue the blunted cross talk between cardiac endothelium and cardiomyocytes, highlighting the potential of this pathway as a target therapy in patients with COVID-19 with cardiac complications.Article InformationAcknowledgmentsThe authors acknowledge Valentijn Jansen, MSc, for excellent technical assistance.Author ContributionsR.P. Juni and R.A. Boon designed the study; R.P. Juni, P.G. Phelp, D.I. Bink, V. Kremer, A.S. van Bergen, K.B. Rombouts, P.E. Gonzalez, K.J. Harber, and D.A.F. Heister performed the experiments; P.A.J. Krijnen and H.W.M. Niessen provided cardiac biopsies from COVID-19 patients; Amsterdam UMC COVID-19 Biobank provided plasma samples from COVID-19 patients; R.P. Juni, P.G. Phelp, D.I. Bink, and V. Kremer analyzed the data; R.P. Juni made the figures; R.P. Juni drafted the manuscript; R.P. Juni, P.G. Phelp, D.I. Bink, A.S. van Bergen, D.W.D. Kuster, P.A.J. Krijnen, H.W.M. Niessen, K.K. Yeung, J. Van den Bossche, and R.A. Boon revised the manuscript; all authors approved the final version of the manuscript.Data AvailabilityNo next generation sequencing, proteomics or similar high throughput data was generated for this study. The data that was generated for this study is available upon request.Sources of FundingThis work was supported by the European Union (ERC, project number 101002599), the Netherlands Organisation for Scientific Research (NWO Vidi), the European Union (Horizon 2020 Grant No. 825670), and the Deutsche Forschungsgemeinschaft (TRR267).Nonstandard Abbreviations and AcronymsCMECcardiac microvascular endothelial cellILinterleukinNOS3nitric oxide synthase 3TNFαtumor necrosis factor alphaDisclosures None.FootnotesFor Sources of Funding and Disclosures, see page xxx.Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCRESAHA.122.322166.Correspondence to: Reinier A. Boon, PhD, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, Department of Physiology, O|2 Research building, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Email: r.a.[email protected]nlReferences1. Xie Y, Xu E, Bowe B, Al-Aly Z. Long-term cardiovascular outcomes of COVID-19.Nat Med. 2022; 28:583–590. doi: 10.1038/s41591-022-01689-3CrossrefMedlineGoogle Scholar2. Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, Liu S, Zhao P, Liu H, Zhu L, et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome.Lancet Respir Med. 2020; 8:420–422. doi: 10.1016/S2213-2600(20)30076-XCrossrefMedlineGoogle Scholar3. Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, Tan W. Detection of SARS-CoV-2 in different types of clinical specimens.JAMA. 2020; 323:1843–1844. doi: 10.1001/jama.2020.3786CrossrefMedlineGoogle Scholar4. Juni RP, Kuster DWD, Goebel M, Helmes M, Musters RJP, van der Velden J, Koolwijk P, Paulus WJ, van Hinsbergh VWM. Cardiac microvascular endothelial enhancement of cardiomyocyte function is impaired by inflammation and restored by empagliflozin.JACC Basic Transl Sci. 2019; 4:575–591. doi: 10.1016/j.jacbts.2019.04.003CrossrefMedlineGoogle Scholar5. Hofmann P, Sommer J, Theodorou K, Kirchhof L, Fischer A, Li Y, Perisic L, Hedin U, Maegdefessel L, Dimmeler S, et al. Long non-coding RNA H19 regulates endothelial cell aging via inhibition of STAT3 signalling.Cardiovasc Res. 2019; 115:230–242. doi: 10.1093/cvr/cvy206CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMeet the First AuthorsCirculation Research. 2023;132:542-544 March 3, 2023Vol 132, Issue 5 Advertisement Article InformationMetrics © 2023 The Authors. Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.https://doi.org/10.1161/CIRCRESAHA.122.322166PMID: 36786206 Originally publishedFebruary 14, 2023 Keywordsinterleukinsmyocytes, cardiacendothelial cellsCOVID-19RNA, long noncodingPDF download Advertisement SubjectsContractile FunctionEndothelium/Vascular Type/Nitric OxideInflammationInflammatory Heart DiseaseVascular Biology

Abstract Background COVID19 is accompanied by cardiac complications. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of cardiovascular diseases. However, their contribution to the cardiac manifestation of COVID19 is unknown. Methods and results We discovered that endothelial-enriched lncRNA H19 is downregulated in the heart of patients with COVID19 (∼2 fold, p<0.01). H19 was highly expressed in cardiac microvascular endothelial cells (CMEC) as compared to the other endothelial cell types (∼10 fold, p<0.05), suggesting its cardiac enrichment. H19 silencing in CMEC induced endothelial stress phenotype and a reduction in endothelial markers VE-cadherin and eNOS (∼1.5 fold, p<0.01), indicating its importance in endothelial physiology. Using the endothelial-cardiomyocyte co-culture system we previously developed, we showed that H19 silencing in CMEC reduced cardiomyocyte (CM) relaxation and contraction (∼1.5 fold, p<0.01). Interestingly, exposure to plasma from COVID19 patients decreased endothelial H19 level and impaired endothelial enhancement of CM function. Mechanistically, reduced level of H19 increased endothelial IL6 expression (∼1.5 fold, p<0.01). Further, exposure of CMs to IL6 also impaired CM relaxation and contraction, suggesting that endothelial cells devoid of H19 release IL6 which represses CM function. Interestingly, we found increased IL6 levels in the heart of COVID19 patients (∼2 fold, p<0.05). Indeed, the impairment of endothelial enhancement of CM function upon H19 silencing in CMEC was restored in the presence of tocilizumab, an IL6 receptor antagonist (∼1.5 fold, p<0.01). Furthermore, the impairment of the endothelial control on CM function upon exposure to COVID19 plasma was mitigated when the patients were treated with tocilizumab (∼1.5 fold, p<0.01). Conclusion COVID19 reduces cardiac endothelial H19 level and induces impairment of endothelial enhancement of CM function via increased release of endothelial-derived IL6, the effect that can be rescued in the presence of IL6 receptor blocker tocilizumab. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC

Aging is accompanied by many physiological changes. These changes can progressively lead to many types of cardiovascular diseases. During this process blood vessels lose their ability to maintain vascular homeostasis, ultimately resulting in hypertension, stroke, or myocardial infarction. Increase in DNA damage is one of the hallmarks of aging and can be repaired by the DNA signaling and repair system. In our study we show that long non-coding RNA Aerrie (linc01013) contributes to the DNA signaling and repair mechanism. Silencing of Aerrie in endothelial cells impairs angiogenesis, migration, and barrier function. Aerrie associates with YBX1 and together they act as important factors in DNA damage signaling and repair. This study identifies Aerrie as a novel factor in genomic stability and as a binding partner of YBX1 in responding to DNA damage.

Endothelial cells can acquire a mesenchymal phenotype through a process called Endothelial-to-Mesenchymal transition (EndMT). This event is found in embryonic development, but also in pathological conditions. Blood vessels lose their ability to maintain vascular homeostasis and ultimately develop atherosclerosis, pulmonary hypertension, or fibrosis. An increase in inflammatory signals causes an upregulation of EndMT transcription factors, mesenchymal markers, and a decrease in endothelial markers. In our study, we show that the induction of EndMT results in an increase in long non-coding RNA AERRIE expression. JMJD2B, a known EndMT regulator, induces AERRIE and subsequently SULF1. Silencing of AERRIE shows a partial regulation of SULF1 but showed no effect on the endothelial and mesenchymal markers. Additionally, the overexpression of AERRIE results in no significant changes in EndMT markers, suggesting that AERRIE is marginally regulating mesenchymal markers and transcription factors. This study identifies AERRIE as a novel factor in EndMT, but its mechanism of action still needs to be elucidated.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH ( OXCT1-AS1 ) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah , which is downregulated by aging, as a regulator of cardiomyocyte survival.

Blood vessels are constantly exposed to shear stress, a biomechanical force generated by blood flow. Normal shear stress sensing and barrier function are crucial for vascular homeostasis and are controlled by adherens junctions (AJs). Here we show that AJs are stabilized by the shear stress-induced long non-coding RNA LASSIE (linc00520). Silencing of LASSIE in endothelial cells impairs cell survival, cell-cell contacts and cell alignment in the direction of flow. LASSIE associates with junction proteins (e.g. PECAM-1) and the intermediate filament protein nestin, as identified by RNA affinity purification. The AJs component VE-cadherin showed decreased stabilization, due to reduced interaction with nestin and the microtubule cytoskeleton in the absence of LASSIE. This study identifies LASSIE as link between nestin and VE-cadherin, and describes nestin as crucial component in the endothelial response to shear stress. Furthermore, this study indicates that LASSIE regulates barrier function by connecting AJs to the cytoskeleton.