key: cord-0960748-dc5j41ks
authors: Oliveira, Murilo Rezende; Back, Guilherme Dionir; da Luz Goulart, Cássia; Domingos, Bianca; Arena, Ross; Borghi-Silva, Audrey
title: The endothelial function Provides early prognostic Information in patients with COVID-19: A cohort study
date: 2021-05-23
journal: Respir Med
DOI: 10.1016/j.rmed.2021.106469
sha: b8595f484af47ad71f956e89eb483711ce0f46ca
doc_id: 960748
cord_uid: dc5j41ks

BACKGROUND: The prothrombotic phenotype and diffuse intravascular coagulation observed in COVID-19 reflect endothelial dysfunction, which is linked to blood flow delivery deficiencies and cardiovascular risk. Assessments of detect vascular deficiencies among newly diagnosed and hospitalized patients due to COVID-19 have yet to be determined. OBJECTIVE: To assess endothelial function characteristics in relation to length of hospitalization and mortality in patients diagnosed with COVID-19 and compare to patients without COVID-19. METHODS: A prospective observational study involving 180 patients with confirmed COVID-19 (COVID-19 group) or suspected and ruled out COVID-19 (Non-COVID-19 group). Clinical evaluation and flow mediated vasodilation (FMD) were performed between the first 24-48 hours of hospitalization. Patients were followed until death or discharge. RESULTS: We evaluated 98 patients (COVID-19 group) and 82 (Non-COVID-19 group), COVID-19 group remained hospitalized longer and more deaths occurred compared to the Non-COVID-19 group (p = 0.01; and p<0.01). Patients in COVID-19 group also had a significantly greater reduction in both FMDmm and FMD% (p<0.01 in both). We found that absolute FMD<0.26mm and relative FMD<3.43% were the ideal cutoff point to predict mortality and longer hospital stay. In Kaplan Meyer's analysis patients had a high probability of death within a period of up to 10 days of hospitalization. CONCLUSION: Patients hospitalized for COVID-19 present endothelial vascular dysfunction early, remained hospitalized longer and had a higher number of deaths, when compared with patients without COVID-19.

Coronavirus disease 2019 , caused by SARS-CoV-2, was first reported in December 2019 in Wuhan city, China, was first reported in December 2019 in the city of Wuhan, China (1) . The route of entry is the respiratory tract where the type 2 angiotensin converting enzyme (ACE2) is a functional receptor sequestered by SARS-CoV-2 for entry into the host cell (2) . ACE2 is a protein expressed in the lungs, heart, kidneys, vascular endothelium and intestine, suggesting broad clinical consequences of SARS-CoV-2 infection that make COVID-19 a multiorgan disease (3) . The disease progresses to a severe form in 10-30% of patients infected, requiring hospitalization and potential intensive care unit (ICU) treatment (4) .

In this sense, endothelial cells have recently been implicated as a primary source for the initiation and spread of Acute Respiratory Discomfort Syndrome (ARDS) caused by SARS-CoV-2, resulting in severe endothelial injury and generalized thrombosis (5) . It has been proposed that the loss of endothelial ACE2 function is related to lung injury, where negative regulation and the fall of this enzyme can lead to dysfunction of the reninangiotensin system and potentially impair vascular function among individuals with COVID-19 infection (6) .

The prothrombotic phenotype and diffuse intravascular coagulation observed in COVID-19 reflect endothelial dysfunction, which stimulates thrombosis, leading to the exposure of pro-thrombotic subendothelial material, platelet aggregation, regulation of coagulation cascades, activation of thrombin and fibrin production, as well as changes in vascular muscle tone and blood flow (5, 7) . In addition, the association between a positive diagnosis for SARS-CoV-2 and risk of stroke (8) , as well as myocardial infarction (9) J o u r n a l P r e -p r o o f suggests a link between blood flow delivery deficiencies and cardiovascular risk during the acute stages of COVID-19 infection.

Although recent studies have suggested that endothelial biomarker and vascular function tests should be performed (10), we have not yet identified any early assessments of vascular deficiencies among newly diagnosed and hospitalized patients with SARS-CoV-2. In this sense, the flow-mediated dilation (FMD) technique of the brachial artery is a noninvasive method used to assess systemic vascular function (11) , which strongly correlates with coronary vascular function (12) and is predictive of future cardiovascular events (13) .

Therefore, the aim of the current study was to assess the relationship between FMD measures of endothelial function on hospitalization days and mortality in patients diagnosed with COVID-19, as well as compare FMD measurements and outcomes to patients without COVID-19. The hypothesis of our study is that patients with COVID-19 may present with early endothelial dysfunction compared to patients without COVID-19 and this finding will be related to differences in days of hospitalization and mortality risk.

This is an observational, cohort prospective study, performed in hospitals of São Carlos -SP. This study adhered to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) (14) . The study was approved by the local ethics committees (protocol number: 33265220.9.0000.5504) of Federal University of Sao Carlos (UFSCAR) and adhered to Resolution 466/2012 of the National Health Council and followed the ethical guidelines of the Declaration of Helsinki (1975) (15) .

All patients included in this study were over 18 years of age and were of both sexes, hospitalized (i.e., ward or ICU) at the University Hospital of São Carlos and Santa Casa of Misericordia, from July to December 2020. Measurements included in this study occurred between 24-48 hours after hospitalization. Patients with positive diagnosis for COVID-19 by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) from the nasopharyngeal swab (16) were included in the COVID-19 group. Patients without a negative diagnosis for COVID-19 RT-PCR, but with hospitalized with similar signs and respiratory symptoms (e.g., cough, fever, runny nose, pain in the body and throat) comprised the Non-COVID-19 group.

It should be noted that in hospitals where patients were evaluated, specific wards were created for patients with respiratory symptoms. When these patients arrived at the hospital and were assessed urgently for suspected COVID-19, they were admitted to the special ward or ICU. A positive or negative COVID-19 diagnosis was confirmed within 48 hours of hospitalization.

Exclusion criteria of the present study consisted of: 1) Patients who did not agree to participate in the study and/or did not sign the Informed Consent Form; 2) severe forms of COVID-19 in the initial phase of hospitalization that culminated with sedation and intubation with invasive mechanical ventilation; 3) patients in palliative care; and 4) cases of readmission.

Patients were screened and recruited by members of the research team who visited the hospital on a daily basis. A brief evaluation of the patient's medical record was carried out to confirm the potential for eligibility. In the first face-to-face meeting, the patients or responsible family members received a description of the study and informed consent for J o u r n a l P r e -p r o o f them to read, have any questions answered and, if they agreed to participate, sign the Informed Consent. Patients who consented were then familiarized with the procedures and data collection was initiated. Members of the research team were properly equipped with personal protective equipment (PPE), including a waterproof apron, goggles, latex gloves, mask n95, disposable mask, disposable cap and face shield, in accordance with World Health Organization recommendations (WHO) (17) .

All assessments were carried out at the same time of the day (afternoon), avoiding different physiological responses due to the influence of the circadian cycle. The evaluations took place at the bedside as soon as possible following hospital admission, always within the first 24-48 hours of admission. It is also noteworthy that all assessments were carried out with a minimum time of 1 hour after patient feeding or any intervention by the staff, avoiding any confounding factor in the endothelial function. The following evaluations were carried out:

Clinical data, including age, sex, weight, height, race, comorbidities, medication use and oxygen supplementation were collected from the medical records to characterize the sample.

Blood count, arterial blood gas analysis and vital signs were analyzed from medical records. Hemoglobin (g/dL), Leukocytes (N/mm 3 

Endothelium-dependent brachial vascular function was evaluated using ultrasonography (Sonosite turbo M, Fujifilme, Bothell, WA, USA). Briefly, patients were asked to rest for 20 minutes in the supine position. An inflatable cuff was placed 1-3 cm distal to the antecubital fossa on the forearm. Baseline artery diameter was measured along with pulsed Doppler signals for flow velocity analysis. The brachial artery was imaged in the longitudinal plane, 5-11 cm proximal to the medial epicondyle using a high frequency MHz) linear-array probe (18) .

The ultrasound measured the arterial diameter (B-mode) continuously in 60 second segments and Doppler (blood flow velocity) at each time point. After baseline recordings, reactive hyperemia was induced by inflation of a blood pressure cuff to a pressure of 220 mmHg for 5 minutes (19) . Arterial diameter was subsequently measured immediately following blood pressure cuff release for 3 minutes. The analyses were performed with Brachial Analyzer software (Medical Imaging Applications LLC, Iowa, USA). Before and after starting each evaluation, the equipment was cleaned with 3% hydrogen peroxide, according to the manufacturer's guidelines, in order to avoid cross-contamination of the virus.

The variables analyzed at baseline and immediately post-hyperemia were Absolute FMD (mm) (diameter before cuffblood vessel diameter after cuff), percentage (%) FMD [(peak diameter − baseline diameter)/ baseline diameter) × 100] (18) .

After the evaluations, the research team followed patient's outcomes through medical record review, verifying days of hospitalization, outcome of hospital discharge, and deaths.

The results are presented as mean and standard deviation (SD) for continuous variables and percentages for categorical variables. The Kolmogorov-Smirnov test was used to verify the distribution of the data. The Student's t-test and chi-square test were used for comparison between COVID-19 and Non-COVID-19 groups.

Analysis of the receiver's operating characteristic curve (ROC) was used to identify the ideal threshold values of the FMD on hospitalization days and mortality in patients diagnosed with COVID-19. Both FMD mm and FMD% optimal predictive cut points were determined in patients with COVID-19. The 95% confidence interval (95% CI) were also calculated with the lower limit being greater than 0.50. Subsequently, the cutoff points of the variables that obtained significant areas under the ROC curve were identified, with the respective values of sensitivity and specificity. Survival was calculated using the Kaplan-Meyer's analysis and the log-rank test, using as exposures FMDmm, deaths number and hospitalization days. Cox proportional univariate and multivariate regression models (adjusted for FMD mm, age, sex, comorbidities and BMI) were performed, with associations expressed as risk ratios (HRs) and 95% CIs. All tests were made in Graphpad Prism 8.0 (GraphPad Software, California, USA) with statistical significance set at p≤0.05. 

Clinical data, comorbidities, number of deaths, hospitalization days, medication use, blood count and FMD results are listed in Table 1 . The ROC analysis showed that an absolute FMD<0.26mm and relative FMD<3.43%

were the ideal cutoff point to predict mortality and longer hospital stay for patients with COVID-19, showing a sensitivity of 75% and 85% and specificity of 73% and 84 %, respectively (Table 2 and Figure 4) . We emphasize that we performed the prediction cutoff points for mortality and longer hospital stay also for Non-COVID-19 patients, however, due to the low number of deaths (4), there was no difference. The groups with the worst FMD outcomes (<3.43% and <0.26mm) remained in the hospital longer (p = 0.04 and p= 0.01), required more oxygen supplementation (p = 0.03 and p= 0.02) and had a higher mortality rate (p = <0.01 in both) compared with the groups with more favorable FMD measures (>3.43% and>0.26).

As for the blood count, there was an increase in the number of troponin-1 and total CK in patients with COVID-19 who had an FMD<0.26mm when compared to patients with an Modeling FMD<0.26mm as a principal variable in the Cox regression model, we found a significant difference for the risk of mortality (95% CI, 0.41 to 0.60; p=<0.01) and body mass index (BMI) (95% CI, 1.12 to 7.04; p=0.02). For sex, age, hypertension and diabetes our model was not significant (Table 4) . Therefore, the Cox regression model showed that there was no influence of comorbidities on endothelial dysfunction, which was a consequence of the COVID-19 infection. (22) . From these studies, we can say that the mortality rate in our cohort was lower, and may thus impact the prognostic ability of FMD in other cohorts hospitalized with COVID-19 with differing mortality rates.

To this point, a study by Ratchford et al. is the only other analysis using FMD assessment in patients with COVID-19. However, this study only included 20 young adults without chronic diseases and relatively healthy, assessed up to 4 weeks following infection (i.e., after the acute process of infection) (23) . Even so, the results indicated that COVID-19 may have detrimental effect on the systemic vasculature in young adults. Thus, we emphasize the novelty of our work, as we know that the highest prevalence of poor outcomes in COVID-19 is in elderly patients and in those with chronic diseases. Therefore, identifying noninvasive methods that can assist in the treatment of these patients is of great importance (23).

In relation to blood counts, patients with poorer FMD outcomes had higher values for troponin-1 and total CK. As in our study, Lombardi et al. elevated troponin levels were an independent predictor of hospital mortality and increased risk of cardiovascular and noncardiovascular complications in a multicenter study including 614 patients hospitalized for COVID-19 (24) . As for total CK, Ponti et al. and Guzik et al. also found hospitalized patients with COVID-19 had elevated levels of this marker. CK parameters correlate with severe prognosis and poor outcomes in patients with COVID-19 and can be used as a predictive biomarker, helping to stratify patients positive for COVID-19 into risk categories, which would be extremely important in the clinical setting and in therapeutic management (25, 26) .

Coronavirus is known to invade the human cell directly after the glycoprotein binds to the ectodomain of ACE2, with ACE2 being the main input mechanism for COVID-19 (27, 28) . The endocytosis of ACE2 linked to the virus particle reduces the number of ACE2 enzymes on the cell surface and therefore weakens tissue protection, thereby impacting cardiac and vascular changes and dysfunction (28) . Thus, when SARS-CoV-2 is bound to the ACE2 receptor, the expression of the receptor is unregulated, which in turn induces vascular endothelial dysfunction, which activates the prothrombotic cascade and ends up leading to vascular thrombosis and risks of heart disease, already observed in patients with COVID-19 (29) , supporting our findings. Therefore, it is extremely important to identify these changes early, which can be done through the cutoff points outlined in our FMD study (<3.43% and <0.26 mm). In this context, the previous pro-inflammatory state associated with SARS-CoV-2 infection can directly cause inactivation of the ACE2 receptor, which is associated with higher death rates and days of hospitalization ( Table 1) . A previous study by our group showed that endothelial dysfunction is present in patients with sepsis (30) , which is related to higher death rates. In this line of evidence, noninvasive measurements of endothelial function 

The study has some limitations that must be considered. First, it must be recognized that the hospital environment, in which evaluations were carried out, is not the best J o u r n a l P r e -p r o o f environment for a controlled research study, as factors such as noise, light and stress of patients during hospitalization were not controlled.

In second place, the lack of baseline FMD results before COVID-19 infection makes it difficult to compare with the results during COVID-19 infection, as these patients have comorbidities and advanced age. As it is the first study to assess endothelial function in this population, there is still no data to compare the results, but hypotheses based on reviews. It is also noteworthy that we obtained a low mortality rate among to the number of cases of patients evaluated. However, fortunately, number of deaths confirmed in the São Carlos hospitals was lower compared to other cities of the country (131 deaths for 9.639 confirmed cases of the COVID-19 until February of 2021).

Third, in relation to blood count, arterial blood gas analysis and vital signs, we can see that some variables did not have differences or did not demonstrate the values expected according to some epidemiological studies (21, 35) . This may be due to the fact that our sample is smaller in relation to these studies and because they are not severely ill patients, but rather mild to moderate cases at the beginning of hospitalization. In addition, these variables did not influence our main outcomes (FMDmm and FMD%).

Our fourth limitation refers to the lack of tests that could confirm our results of endothelial dysfunction, such as platelet function and NO levels. Finally, the most severe patients, who arrived at the hospital already in need of invasive ventilatory support and sedation in 24-48h, were excluded. As our study has the potential to use FMD as an early event predictor, sedation and the use of invasive ventilatory support may be a potential confounding factor in our findings. Thus, the results of the present study are limited to patients with mild to moderate degree of respiratory dysfunction in the first 48 hours and who required hospitalization for clinical stability.

This is the first study to emphasize the importance of evaluating FMD in patients with COVID-19, we demonstrated through our cutoff point that this population may have early endothelial dysfunction. Therefore, strategies to minimize this dysfunction are of paramount importance, prioritizing the performance of aerobic and resistance exercises both for prevention and for the immediate admission of the patient to the hospital, associated with oxygen supplementation strategies and medications, thus being able to reduce the days hospitalization and mortality in this population. In this context, it is highly recommendable early endothelial function assessment in order that the staff initiates, when necessary, antithrombotic measures from the initial moment of hospitalization, in order to prevent thromboembolic events.

We conclude that patients hospitalized for COVID-19 present endothelial vascular dysfunction early, remained hospitalized longer and had a higher number of deaths, when compared with patients without COVID-19. In addition, it was the first study to establish cutoff points for FMD (FMD<3.43% and FMD<0.26mm) for patients infected with COVID- 19 . In addition, we identified that FMD<0.26mm is a strong predictor of mortality risk in a 10-day hospitalization period due to COVID-19.

Our data suggest that these patients are more likely to have endothelial dysfunction and consequently risks of cardiovascular diseases, but that, from the cutoff points established, the FMD assessed early can help in the recognition of these patients and in clinical treatment.

J o u r n a l P r e -p r o o f 

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan , China : a retrospective cohort study

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Incidence of thrombotic complications in critically ill ICU patients with COVID-19

COVID-19 and Cardiovascular Disease

Thrombotic Regulation From the Endothelial Cell Perspectives

Large-Vessel Stroke as a Presenting Feature of Covid-19 in the Young

Management of acute myocardial infarction during the <scp>COVID</scp> -19 pandemic

Endothelial dysfunction in COVID-19: a position paper of the ESC Working Group for Atherosclerosis and Vascular Biology, and the ESC Council of Basic Cardiovascular Science

Noninvasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis

Strong Relationship Between Vascular Function in the Coronary and Brachial Arteries

Predictive value of brachial flow-mediated dilation for incident cardiovascular events in a populationbased study: The multi-ethnic study of atherosclerosis

Iniciativa STROBE: subsídios para a comunicação de estudos observacionais

The 1975 Declaration of Helsinki and consent

Detection profile of SARS-CoV-2 using RT-PCR in different types of clinical specimens: A systematic review and metaanalysis

Rational use of personal protective equipment for COVID-19 and considerations during severe shortages: interim guidance

Expert consensus and evidence-based recommendations for the assessment of flowmediated dilation in humans

Guidelines for the Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery A Report of the International Brachial Artery Reactivity Task Force

Prevalence and severity of corona virus disease 2019 (COVID-19): A systematic review and meta-analysis

Baseline Characteristics and Outcomes of 1591 Patients Infected with SARS-CoV-2 Admitted to ICUs of the Lombardy Region, Italy

Association Between Hypoxemia and Mortality in Patients With COVID-19

Vascular Alterations Among Young Adults with SARS-CoV-2

Association of Troponin Levels with Mortality in Italian Patients Hospitalized with Coronavirus Disease 2019: Results of a Multicenter Study

Biomarkers associated with COVID-19 disease progression

COVID-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Endothelialitis plays a central role in the pathophysiology of severe COVID-19 and its cardiovascular complications

SARS-CoV-2 cell entry receptor ACE2 mediated endothelial dysfunction leads to vascular thrombosis in COVID-19 patients

Noninvasive measurements of hemodynamic, autonomic and endothelial function as predictors of mortality in sepsis: A prospective cohort study

Pulmonary rehabilitation in COVID-19 pandemic era: The need for a revised approach

Non-invasive ventilation improves exercise tolerance and peripheral vascular function after high-intensity exercise in COPD-HF patients

Effects of exercise modalities on arterial stiffness and wave reflection: A systematic review and metaanalysis of randomized controlled trials

Exercise training and endothelial function in patients with type 2 diabetes: A meta-analysis

Hematological findings and complications of COVID-19