Endothelin-1 is increased in the plasma of patients hospitalised with Covid-19


 Virus induced endothelial dysregulation is a well-recognised feature of severe Covid-19 infection. Endothelin-1 (ET-1) is the most highly expressed peptide in endothelial cells and a potent vasoconstrictor, thus representing a potential therapeutic target.
 ET-1 plasma levels were measured in a cohort of 194 Covid-19 patients stratified according to the clinical severity of their illness. Hospitalised patients, including those who died and those developing acute myocardial or kidney injury, had significantly elevated ET-1 plasma levels during the acute phase of infection.
 The results support the hypothesis that endothelin receptor antagonists may provide clinical benefit for certain Covid-19 patients.


The Coronavirus disease 2019  pandemic continues to place a devastating strain on healthcare services worldwide and there remains an ongoing requirement for new therapies [1].
SARS-CoV-2, the causative agent of Covid-19, infects host cells by exploiting ACE2, a surface receptor expressed by epithelial cells and the vascular endotheliumthe layer of cells lining all blood vessels [1]. There is emerging consensus that in progressive severe Covid-19 disease, virus-induced endothelial damage may result in a syndrome of excessive vasoconstriction, inflammation and thrombosis [2,3].
Endothelin-1 (ET-1), being the most highly expressed peptide in endothelial cells and potent vasoconstrictor of human blood vessels [9,10], represents a potential therapeutic target. The benefit of endothelin receptor antagonists is already well established in pulmonary arterial hypertension [11] hence these medications may be suitable for accelerated regulatory approval. ET-1 is released from endothelial cells via a continuous constitutive pathway and supplemented by ET-1 release from Weibel-Palade bodies (the unique storage granules of endothelial cells) in response to extra-cellular stimuli including inflammatory cytokines [10].

Willems et al. have recently reported elevated ET-1 levels in patients 3 months post
Covid-19 infection [12]; however the association of ET-1 with clinical outcomes likely J o u r n a l P r e -p r o o f Journal Pre-proof to impact the provision of healthcare resources, such as hospitalisation, has not been investigated. In the present study, we tested the hypothesis that elevated levels of plasma ET-1 in the acute phase of Covid-19 infection would be associated with more severe disease. levels between patient categories A-C and controls, between different time-points for categories A-C, and between subgroups defined by clinical endpoints was undertaken using the Independent Samples Kruskal-Wallis Test. To assess for potential confounding effects when comparing ET-1 levels between patient categories A-C and controls as well as between all Covid-19 infected patients with controls, univariate analysis of co-variance was performed for each of: age, gender, ethnicity, hypertension, ischemic heart disease, diabetes, congestive cardiac failure and chronic kidney disease. To assess the association of baseline ET-1 with hospitalisation amongst infected patients, a binary logistic regression model was calculated adjusted for recognised confounders of baseline ET-1: hypertension, ischemic heart disease, diabetes, congestive cardiac failure and chronic kidney disease. in Group B and 32.1% in Group C. The frequency of underlying comorbidities was higher in Groups B and C compared with Group A and controls ( Table 1). Despite heterogeneity in clinical and demographic characteristics, differences in baseline ET-1 between infected versus non-infected patients and all patient categories remained significant (p<0.05) in corrected models for age, gender, ethnicity, hypertension, ischemic heart disease, diabetes, congestive cardiac failure and chronic kidney disease using between-subjects effect analysis of co-variance (      J o u r n a l P r e -p r o o f  the lack of prognostic utility of C-terminal proendothelin-1 (proET-1) to predict mortality in Covid-19 [14]. Compared with ET-1 measured in the present study, proET-1 is metabolised differently and is an inactive peptide that does not cause vasoconstriction therefore significant correlation between the two biomarkers is unlikely in vivo.

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Higher plasma levels of ET-1 are a recognised feature of pulmonary arterial hypertension (PAH) and increased expression of ET-1 in pulmonary endothelial cells has been shown to correlate with increased pulmonary vascular resistance in this condition [11]. The magnitude of the ET-1 elevation we have observed in hospitalised Covid-19 patients was similar to that reported in the early studies comparing ET-1 levels in PAH patients with normal subjects [15] [16]. Multiple large clinical trials have now established the prognostic benefit of ET receptor antagonists in idiopathic and connective tissue disease associated PAH [11]. Outside of PAH, increased plasma levels of ET-1 are also associated with increased coronary [17] and systemic vasoconstriction [18] in coronary microvascular dysfunction, and endothelial dysfunction in vasospastic angina [19]  ). Due to the skewed distribution of ET-1, no potential or extreme outliers have been excluded from analysis. Outliers were noted to mostly occur in patient category C (hospitalised and requiring supplemental oxygen/assisted ventilation) or amongst subgroups with clinical complications of Covid-19 infection therefore we have assumed these values reflect true biological variability in the study population. Comparison of ET-1 between patient categories was performed using the Independent Samples Kruskal-Wallis Test and also between time-points for each patient category. In the latter case, an independent samples non-parametric test was chosen to increase power given the low number of paired corresponding samples available due to patient drop-out at 28 and 90 days.
Reasons for loss of patients to follow up were in many cases unavoidable including patient death, persisting disability and patients being repatriated outside our local area having been referred to our centre for specialist tertiary care during their initial illness.
No demographic or clinical endpoint data was available for 3/39 patients in category B otherwise clinical variables were available for all other enrolled patients.

Binary Logistic Regression
Binary Logistic Regression was used to calculate a model predicting hospitalisation for Covid-19 infected patients in our cohort using baseline ET-1, hypertension, diabetes, ischemic heart disease, congestive cardiac failure and chronic kidney disease (step 1).

Omnibus Tests of Model Coefficients
Chi-square df Sig.
Step Chi-Square Test indicates the adjusted model incorporating all 6 covariates was significantly better than the unadjusted logistic regression model.   The endothelial peptide, ET-1 is a potential therapeutic target.

Classification
 Higher circulating ET-1 levels are associated with more severe infection.
 Endothelin receptor antagonists may be of clinical benefit in Covid-19.
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