key: cord-0890468-kk6amyk0 authors: Lambadiari, Vaia; Mitrakou, Asimina; Kountouri, Aikaterini; Thymis, John; Katogiannis, Konstantinos; Korakas, Emmanouil; Varlamos, Charalampos; Andreadou, Ioanna; Tsoumani, Maria; Triantafyllidi, Helen; Bamias, Aristotelis; Thomas, Konstantinos; Kazakou, Pinelopi; Grigoropoulou, Sotiria; Kavatha, Dimitra; Antoniadou, Anastasia; Dimopoulos, Meletios A; Ikonomidis, Ignatios title: Association of COVID‐19 with impaired endothelial glycocalyx, vascular function and myocardial deformation four months after infection date: 2021-08-20 journal: Eur J Heart Fail DOI: 10.1002/ejhf.2326 sha: fd6922d8dc1dc5594a8b5138a9bd0748073c4a9a doc_id: 890468 cord_uid: kk6amyk0 AIMS: SARS‐CoV‐2 infection may lead to endothelial and vascular dysfunction. We investigated alterations of arterial stiffness, endothelial coronary and myocardial function markers four months after COVID‐19 infection. METHODS AND RESULTS: In a case‐control prospective study, we included 70 patients four months after COVID‐19 infection, 70 age‐ and sex‐matched untreated hypertensive patients (positive control) and 70 healthy individuals. We measured a) perfused boundary region (PBR) of the sublingual arterial microvessels (increased PBR indicates reduced endothelial glycocalyx thickness), b) flow‐mediated dilation (FMD), c) coronary Flow Reserve (CFR) by Doppler echocardiography d) pulse wave velocity (PWV) e) global left (LV) and right (RV) ventricular longitudinal strain (GLS) and f) malondialdehyde (MDA), an oxidative stress marker, thrombomodulin and von Willebrand factor (vWF) as endothelial biomarkers. COVID‐19 patients had similar CFR and FMD with hypertensives (2.48±0.41 vs 2.58±0.88, p=0.562, 5.86±2.82% vs 5.80±2.07%, p=0.872 respectively) but lower values than controls (3.42±0.65, p=0.0135, 9.06±2.11%, p=0.002 respectively). Compared to controls, both COVID‐19 and hypertensives had greater PBR5‐25 (2.07±0.15μm and 2.07±0.26μm p=0.8 vs 1.89±0.17μm, p=0.001), higher PWV (PWVc‐f 12.09±2.50 vs 11.92±2.94, p=0.7 vs 10.04±1.80m/sec, p=0.036) and impaired LV and RV GLS (‐19.50 ±2.56% vs −19.23±2.67%, p=0.864 vs −21.98±1.51%, p=0.020 and ‐16.99±3.17% vs ‐18.63±3.20%, p=0.002 vs ‐20.51±2,.28%, p<0.001). MDA and thrombomodulin were higher in COVID‐19 patients than both hypertensives and controls (10.67±2.75 vs 1.76± 0.30, p=0.003 vs 1.01±0.50nmole/L, p=0.001 and 3716.63±188.36 vs 3114.46±179.18, p=0.017 vs 2590.02±156.51pg/ml, p<0.001). Residual cardiovascular symptoms at 4 months were associated with oxidative stress and endothelial dysfunction markers. CONCLUSIONS: SARS‐CoV‐2 may cause endothelial and vascular dysfunction linked to impaired cardiac performance four months after infection. This article is protected by copyright. All rights reserved. Coronavirus disease , a newly recognized infectious disease with a rapid spread worldwide, is caused by a novel enveloped RNA beta-coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1, 2] . Despite the growing number of publications regarding the epidemiological and clinical characteristics of SARS-CoV-2 [3], the underlying pathophysiological mechanisms of the disease remain unclear [4] . Although COVID-19 infection primarily affects the respiratory system, numerous patients display manifestations from cardiovascular system including acute myocardial injury, arrythmias and myocarditis [5] . According to research evidence, SARS-CoV-2 affects cardiovascular system through multiple mechanisms. Firstly, in vitro studies showed that angiotensin-converting enzyme 2 (ACE2) receptors, which are expressed in various human tissues, are the main targets for SARS-CoV-2 [6]. Therefore, this raises the possibility that the virus may directly damage endothelial and cardiac cells. Secondly, the overproduction of proinflammatory cytokines during disease progression is possibly associated with endothelial derangement and myocardial injury [7, 8, 9] . Recent guidelines suggest that the evaluation of endothelial biomarkers may contribute to the risk stratification of COVID-19 patients. [10] . Carotid-Femoral Pulse Wave Velocity (PWV) and central blood pressure are reliable markers of aortic elastic properties and have been suggested as valuable prognostic markers for cardiovascular events [11] . Glycocalyx damage, impaired artery flowmediated dilation (FMD) and coronary flow reserve (CFR) as well as measurement of circulating thrombomodulin and von Willebrand factor (vWF) may represent early manifestations of endothelial dysfunction [12, 13, 14] . Global left (LV) and right This article is protected by copyright. All rights reserved. criteria: mild, moderate, severe [20] . Furthermore, we recorded the presence of postinfection symptoms related to cardiovascular system (dyspnea, fatigue, cough, chest pain) four months post-infection. Inclusion criteria for the study were age>18 years old and a recent diagnosis of SARS-CoV-2 proven by PCR. Exclusion served as control group. This study was approved by the institutional ethical board of University General Hospital "Attikon". All participants signed an informed consent prior to any procedure included in the study protocol. All methods were conducted according to relevant guidelines and regulations (Declaration of Helsinki). In all patients we recorded age, sex, comorbidities, and concomitant medications. All subjects were studied in the morning, having abstained from alcohol, caffeine and food for 8 h prior to the study; all vasoactive medications were withheld for 48 h before the study. The operators who performed the vascular and cardiac measurements were blinded for the history of the patients. Prior to the study procedures, each patient rested in a supine position for 10 minutes in a quiet room. For the evaluation of the brachial blood pressure and the heart rate we Accepted Article use an automated digital oscillometric sphygmomanometer (TensioMed, Budapest, Hungary). In each patient, blood pressure was measured three times with interval of two minutes. The operators who performed the examination were blinded for the history of the patients. We use the mean value for statistical analysis. Perfused boundary region (PBR) of the sublingual arterial microvessels with diameter ranged 5 -25 μm was assessed by the use of Sidestream Darkfield (SDF) imaging (Microscan, Glycocheck, Microvascular Health Solutions Inc., Salt Lake City, Utah, USA). The PBR represents the depth of penetration of red blood cells (RBC) into endothelial glycocalyx. Нigher PBR values are associated with increased penetration of RBC into the endothelium indicating an impaired endothelial glycocalyx [22] . The evaluation of endothelial glycocalyx using SDF imaging is a non-invasive, reproducible technique which lasts three minutes and provides recording and automated analysis of >3000 sublingual microvessel segments [22] . Therefore, the European Society of Cardiology Working Group on Peripheral Circulation suggest that the forementioned technique is valid for the assessment of endothelial function [12] . The operators who performed the examination were blinded for the history of the patients. In all patients we assessed the endothelium dependent FMD in the right brachial artery. We obtained images of the brachial artery and we record a resting scan with the use of Echo -Doppler Ultrasound system (Vivid Е95, GE Medical Systems, This article is protected by copyright. All rights reserved. Horten, Norway). Afterwards, we inflated a blood pressure cuff on the forearm at a pressure of 200 mmHg for 5 min and subsequently we deflated it, causing reactive hyperemia. Images form the brachial artery were obtained continuously 30 s before and 90 s after cuff deflation. Artery diameter measurements were performed using electronic calipers from the anterior to the posterior m-line. FMD was calculated as the percentage increase in arterial diameter during hyperemia as compared with the resting scan [23] . The operators who performed the assessment of FMD were blinded for the history of the patients. Carotid-femoral pulse wave velocity (PWV-m/s), central aortic pressures (systolic and diastolic) and central pulse pressure were measured using tonometry by Complior Studies were performed using a Vivid Е95 (GE Medical Systems, Horten, Norway) ultrasound system. All studies were digitally stored in a computerized station (EchoPac GE 203, Horten, Norway) and analyzed by two observers, who had no access to clinical and laboratory data. The operators who performed the echocardiography were blinded for the history of the patients. This article is protected by copyright. All rights reserved. velocity of the tricuspid annulus (S'RV) were obtained by Tissue Doppler Imaging (normal reference range of values is 13.32.5 cm/s). We quantified in all participants the circulating levels of vWF and Thrombomodulin to assess endothelial function. vWF was measured by ELISA using a commercial kit (Abcam, Human Von Willebrand Factor ELISA Kit (ab223864)), range: 469 ng/ml -30.000 ng/ml). Thrombomodulin was quantified by ELISA using a commercial kit Data are presented as mean ± SD/ Binary variables were compared using the χ2 test. This article is protected by copyright. All rights reserved. and severe disease at admission. The interaction terms between the variable of the three study groups and the other clinical co-variables (smoking, brachial blood pressure, heart rate and disease severity and symptoms) were also examined. The associations between measured markers were assessed using Pearson correlation and the respective correlation coefficient (r) and p values are reported. A p value <0.05 was considered statistically significantResults The general characteristics of study population are summarized on Table 1 . Twentyfour patients (34.28%) were diagnosed with mild disease and were not subsequently hospitalized at any time of the course of the disease. Furthermore, 23 (32.85%) patients were diagnosed to have moderate and 23 (32.85%) severe disease at initial clinical assessment and thus were admitted to hospital. None of the examined patients required mechanical ventilation and none required hospitalization for a period of more than 15 days. Twenty-six patients (37.87%) presented with post-infection symptoms four months after COVID-19 disease. Among the symptoms, fatigue was the most common and it was present in eleven patients (15,71 %) dyspnea in 9 (12, 8 %) cough in 3 (4,3 %) and chest pain in 3 (4,3 %). There was no significant difference among the patients with or without post-infection symptoms regarding clinical characteristics (age, BMI, data not shown). Τhe mean age of participants was 54.59±8.85 years and there was no significant difference among three groups regarding age (p=0.991) and sex (44 males and 26 females per group, p=1). By ANOVA, brachial systolic and diastolic blood pressure (BP) were different among the 3 study groups (F=9.51, p<0.001 and F=8.11, p=0.001 respectively). More and examined blood pressure markers or heart rate in the 3 study subgroups (p>0.05). (Table 2) . By ANOVA, FMD values were found different among the 3 study groups (F=8.71, p=0.001). More specifically, COVID-19 patients and hypertensives had similar FMD (5.86±2.82% vs 5.80±2.07%, p=0.872) while both groups had lower FMD values than control group (9.06±2.11%, p=0.002 and p=0.002 respectively) ( Figure 1A ). Likewise, a similar trend to that of FMD was observed for PBR5- 25 Figure 2C ). Finally, TAPSE and S' of RV were lower in COVID-19 patients compared to both hypertensives and controls (table 2, p<0.05) No significant interaction was found between disease severity (mild, moderate, severe) and LVGLS, RVGLS and RVFWS in the 3 study subgroups (p>0.05). By ANOVA, there were significant differences between the 3 study groups regarding the blood levels of MDA (F=9.6, p=0.001). Specifically, COVID-19 patients displayed much higher MDA levels than both hypertensives and healthy individuals (10.67±2.75 vs 1.76±0.30 vs 1.01±0.50 nmol/L, p=0.003 and p=0.001 respectively). No significant interaction was found between disease severity (mild, moderate, severe) and MDA in the 3 study subgroups (p>0.05) ( Figure 3A ). By ANOVA, there were significant differences between the three study groups This article is protected by copyright. All rights reserved. In COVID-19 group increased PBR5-25 was corelated positively with increased Our study supports that SARS-CoV-2 causes endothelial and vascular dysfunction that remain four months after initial infection and is, therefore, linked to reduced cardiac performance. In accordance with our hypothesis, we observed that COVID-19 induce vasculitis in multiple organs. [28, 29] . Finally, a recent study had shown that thrombomodulin and vWF markers of endothelial dysfunction were elevated in hospitalized COVID-19 patients and were associated with adverse in-hospital outcome [14] suggesting the presence of severe endotheliopathy. Extending the findings of this study, we found that thrombomodulin levels were higher in COVID-19 patients compared to both hypertensives and normal controls and vWF levels similar to those in hypertensives but higher than those in normal controls. This article is protected by copyright. All rights reserved. and controls Additionally, in accordance with our data, this evidence suggests significant alterations of the microcirculation and the endothelial glycocalyx in COVID-19 patients which are linked with the elevated thrombomodulin and vWf found in our COVID-19 patients in the present study [39] . Finally, the presence of vascular and cardiac dysfunction in COVID-19 patients was associated with presence of symptoms (dyspnea, cough chest pain or fatigue) in our study. In particular, impaired endothelial glycocalyx, FMD, and increased thrombomodulin were associated with presence of cardiovascular symptoms suggesting the contribution of residual endothelial dysfunction to lack of symptom resolution 4 months post-infection. Furthermore, the presence fatigue was linked with increased oxidative stress, arterial stiffness and impaired LV myocardial deformation. In our study markers of vascular dysfunction were associated with impaired LV myocardial dysfunction linking endothelial dysfunction with lack of symptom resolution likely related to a subclinical cardiac dysfunction. COVID-19 patients also displayed more deteriorated RV myocardial function than hypertensives which may be attributed to effects of the acute respiratory infection. In support of this mechanism, we found that COVID-19 patients with presence of residual dyspnea four months after infection presented more impaired RVGLS values compared to patients without this symptom. In our study the degree of oxidative stress was 10-fold higher in COVID-19 patients compared to hypertensives and normal controls and was associated with both impaired endothelial glycocalyx and coronary flow reserve. Moreover, in COVID-19 group, increased thrombomodulin was associated with increased PWVc-f a well This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. P<0.05, obtained by post hoc analysis between hypertensives and control group. Significant differences at p<0.05 level were not observed for comparisons of COVID-19 group and control group using post hoc analysis. This article is protected by copyright. All rights reserved. COVID-19 and heart failure: from infection to inflammation and angiotensin II stimulation. 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Ann Intensive Care Sublingual microcirculation in patients with SARS-CoV-2 undergoing veno-venous extracorporeal membrane oxygenation Microvascular dysfunction in COVID-19: the MYSTIC study The authors thank Professor Gerasimos Filippatos for his contribution to the study design and the critical review of the manuscript. The present study has been supported by the Hellenic Cardiology Society. Conflict of Interest: none declared.