Patent Application
20230194530
This disclosure generally relate to the field of COVID-19 diagnostic risk assessment, and in particular diagnostic methods using biomarker panels, as well as treatments of Covid-19.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious betacoronavirus, which results in coronavirus disease 2019 (COVID-19). While the majority of infected individuals manifest mild to moderate illness, 14-31% of symptomatic unvaccinated patients eventually require hospitalization, with intensive care unit (ICU) admission rates ranging from 2-26% among those hospitalized. Select populations, particularly older individuals and those with underlying comorbidities (including cardiovascular disease [CVD]) have high rates of morbidity and mortality. Substantially higher rates of morbidity and mortality have been observed as variants of concern become more predominant. While diagnostic testing has allowed for the rapid identification of COVID-19 cases, the lack of post-diagnosis risk assessment metrics, especially among the highest-risk subgroups, have undermined the cascade and allocation of care.
The use of existing cardiovascular and respiratory parameters could serve as a metric of risk prediction. However, case-fatality rates of those with comorbidities remain particularly high (e.g., preexisting CVD at ˜10.5%) with cardiorespiratory the aforementioned markers may have limited utility. In this regard, while standard metrics including measures of cardiac damage (e.g., troponin values above the 99th percentile), the extent of inflammatory activation (e.g., C-reactive protein expression), and cardiovascular imaging have elucidated the spectrum of COVID-19 complications, they may have only modestly elucidated the risk of adverse in-hospital outcomes and may often provide limited insight into disease mechanism. Albeit encouraging, some COVID-19 therapeutics focus on stemming aberrant immune responses and controlling viral reproduction (e.g., tocilizumab and remdesevir, respectively), which may neglect key elements of the host response contributing to outcomes. From this perspective, clinical data and autopsy studies revealing endotheliitis and thrombosis have raised the possibility that endothelial dysfunction, particularly fluctuations in vascular integrity and coagulative capacity, could be a driver of clinical outcomes. Thrombosis, fluid extravasation, and microangiopathy observed in the small vessels and capillaries of the lungs may suggest that an intense vascular reaction takes place in those with severe disease.
Approaches suggest that viral tropism towards angiotensin-converting enzyme 2 and acetylated sialic acid residues, which are highly expressed by vascular endothelial cells (EC), could instigate cardiovascular dysfunction. Other approaches have alternatively suggested that SARS-CoV-2 may have limited infectious potential and replicative ability in ECs. In this respect, mechanisms secondary to direct infection, such as cascading immunological activation may instead be the driving factor behind the observed endothelial dysfunction; particularly among the populations with coexisting conditions where EC dysfunction is already evident. In fact, select endothelium-related biomarkers such as thrombomodulin, von Willebrand Factor, angiopoietin-2 (Ang-2), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) may show utility in prognostication, being associated with both disease severity and in-hospital mortality. While markers of endothelial function may aid in prognostication, it seems possible that a simple combination of markers can provide insight significant enough to adjudicate the level of care a patient will need, nor is it apparent that these markers associate specifically with COVID-19 pathology. To date, the integration of clinical data with broad omics technologies has opened up new avenues for efficiently delineating complex patient phenotypes and their associations with clinical outcomes. Circulating profiles of plasma microRNAs (miRNA), in particular, have been shown to be tightly associated with disease, and capable of providing not only detailed prognostic information but also mechanistic insight.
The following seeks to characterize markers of endothelial function and inflammation and to develop for the first time an atlas of miRNA expression across both a spectrum of individuals diagnosed with COVID-19 as well as SARS-CoV-2-negative patients from the ICU. To enhance clinical utility, the following focuses on understanding how these markers are associated with in-hospital mortality of high-risk patients, particularly those requiring the highest levels of care.
In accordance with an aspect, there is provided a method of determining Coronavirus Disease 2019 (COVID-19) severity in a subject. The method including obtaining a circulating blood sample from the subject, and obtaining a biomarker panel comprising one or more of angiopoietin-2 (Ang-2), endothelin-1 (ET-1), soluble intercellular adhesion molecule (sICAM), soluble vascular cell adhesion molecule (sVCAM), soluble E-selectin (sE-selectin), triggering receptor expressed on myeloid cells-1 (sTREM-1), interkeulin-6 (IL-6), interleukin-8 (IL-8), myeloperoxidase (MPO), and high-sensitivity cardiac troponin (hs-cTnI). A different level of at least one biomarker in the biomarker panel compared to a reference panel from a SARS-CoV-2-negative subject is indicative of COVID-19 severity.
In accordance with a further aspect, the biomarker panel may include Ang-2.
In accordance with a further aspect, the biomarker panel may include Ang-2, and MPO.
In accordance with a further aspect, the biomarker panel may include Ang-2, ET-1, sICAM-1, sVCAM-1, sE-Selectin, sTREM-1, IL-6, IL-8, MPO and hs-cTnI.
In accordance with a further aspect, an elevated level of at least one biomarker may be indicative of COVID-19 severity.
In accordance with an aspect, there is provided a method of determining mortality risk of a subject infected with SARS-CoV-2. The method including detecting in a circulating blood sample from the subject one or more circulating microRNA (miR) biomarkers selected from the group consisting of: one or more miR from miR-30 family, miR-181a-5p, miR-199a-3p, miR-4793-5p, miR-6080, and miR-6750-5p.
In accordance with a further aspect, the one or more biomarkers may be selected from the group consisting of: miR-30b, miR-30c, miR-30e, miR-181a-5p, miR-199a-3p, and miR-6080.
In accordance with a further aspect, the method may include amplifying and detecting a target miR in the blood sample using polymerase chain reaction (PCR).
In accordance with a further aspect, the method may include capturing and sequencing the detected miR.
In accordance with a further aspect, the method may include sequencing the detected miR using next-generation sequencing. In some embodiments, the next-generation sequencing comprises Illumina™, Roche 454™, or Ion Torrent Sequencing™.
In accordance with a further aspect, sequencing the detected miR may include a nuclease protection assay. Exemplary sequencing technique for miRNA includes nuclease protection assays that are coupled to sequencing, such as miRNA whole transcriptome assays (https://www.htgmolecular.com/assays/mirna-wta, the entire content of which is incorporated herein by reference).
In accordance with a further aspect, the method further including measuring the expression level of at least one of the one or more circulating miR biomarkers.
In accordance with a further aspect, the method may include measuring the expression level of two or more of the circulating miR biomarkers.
In accordance with a further aspect, the method may include measuring the miR expression level using a multiplex assay.
In accordance with a further aspect, the multiplex assay may be a 5-plex assay.
In accordance with a further aspect, detecting the one or more circulating miR biomarkers may include detecting miR using a colorimetic or a CRISPR-based biosensor. Exemplary CRISPR-based biosensor includes CRISPR/Cas13a-powered electrochemical microfluidic biosensors (https://onlinelibrary.wiley.com/doi/full/10.1002/adma.201905311, the entire content of which is incorporated herein by reference).
In accordance with an aspect, there is provided a kit including one or more antibodies specific to angiopoietin-2 (Ang-2), endothelin-1 (ET-1), soluble intercellular adhesion molecule (sICAM), soluble vascular cell adhesion molecule (sVCAM), soluble E-selectin (sE-selectin), triggering receptor expressed on myeloid cells-1 (sTREM-1), interkeulin-6 (IL-6), IL-8, myeloperoxidase (MPO), or high-sensitivity cardiac troponin (hs-cTnI).
In accordance with a further aspect, the kit may include an antibody specific to Ang-2.
In accordance with a further aspect, the kit may include a first antibody specific to Ang-2, and a second antibody specific to MPO.
In accordance with a further aspect, the kit may further include one or more antibodies specific to a biomarker selected from ET-1, sICAM-1, sVCAM-1, sE-Selectin, sTREM-1, IL-6, IL-8, and hs-cTnI.
In accordance with an aspect, there is provided a kit including one or more probes for binding a circulating microRNA (miR) biomarker selected from the group consisting of one or more miR from miR-30 family, miR-181a-5p, miR-199a-3p, miR-4793-5p, miR-6080, and miR-6750-5p.
In accordance with a further aspect, the biomarker may be selected from the group consisting of miR-30b, miR-30c, miR-30e, miR-181a-5p, miR-199a-3p, and miR-6080.
In accordance with a further aspect, the kit may be for an assay.
In accordance with a further aspect, the one or more probes may be amplification probes.
In accordance with a further aspect, the one or more probes may be sequencing probes.
In accordance with an aspect, there is provided a method of treating Coronavirus Disease 2019 (COVID-19) in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject. The method including administering said subject with Slit2, or a variant or fragment thereof.
In accordance with a further aspect, the method may include administering the subject with a full-length Slit2.
In accordance with a further aspect, the method may include administering the subject with a N-terminal domain of Slit2 (Slit2-N).
In accordance with an aspect, there is provided a method of treating Coronavirus Disease 2019 (COVID-19) in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject. The method including upregulating the Slit2 signaling pathway in the subject.
In accordance with a further aspect, the method may include administering the subject with one or more factors or polypeptides from the Slit2 signaling pathway.
In accordance with a further aspect, the one or more factors or polypeptides from the Slit2 signaling pathway may be GIT1, ARF6, Ab1, GTPase, Ena, Myo9b, or Hakai.
In accordance with an aspect there is provided a use of Slit2 or a variant or fragment thereof in the treatment or inhibition of the development of Coronavirus Disease 2019 (COVID-19) or a sequelae resulting from COVID-19 in a subject.
In accordance with an aspect there is provided a use of Slit2 or a variant or fragment thereof in the manufacture of a medicament for the treatment or inhibition of the development of Coronavirus Disease 2019 (COVID-19) or a sequelae resulting from COVID-19 in a subject.
In accordance with a further aspect, the use may include use of a full-length Slit2.
In accordance with a further aspect, the use may include use of a N-terminal domain of Slit2 (Slit2-N).
In accordance with an aspect there is provided use of one or more factors or polypeptides from the Slit2 signaling pathway for the treatment of Coronavirus Disease 2019 (COVID-19) in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject by upregulating the Slit2 signaling pathway in the subject.
In accordance with an aspect there is provided use of one or more factors or polypeptides from the Slit2 signaling pathway for the manufacture of a medicament for the treatment of Coronavirus Disease 2019 (COVID-19) in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject.
Markers of endothelial dysfunction at presentation, while indicative of poor outcomes in COVID-19-positive patients, reflect systemic vascular dysfunction in critically ill patients and are not specific to SARS-CoV-2 infection.
Generation of a plasma microRNA atlas uncovers COVID-19-specific prognostic markers and multiple disease-specific pathways of interest, including endothelial barrier dysfunction.
Synthesis of electronic health record data with clinically relevant multi-omic datasets using a machine learning approach provide substantially better metrics by which mortality can be estimated in patients with severe COVID-19.
Targeted stabilization of the endothelial barrier with Slit2-N present novel therapeutic avenues that should be explored in COVID-19 patients.
Endothelial cell (EC) activation, endotheliitis, vascular permeability, and thrombosis have been observed in patients with severe COVID-19, indicating that the vasculature may be affected during the acute stages of SARS-CoV-2 infection. Prior to the present disclosure, it was unknown whether circulating vascular markers can be sufficient to predict clinical outcomes, unique to COVID-19 related pathology, and if vascular permeability can be therapeutically targeted.
Evaluating the prevalence of circulating inflammatory, cardiac, EC activation, and the development of a microRNA atlas in 241 patients with suspected SARS-CoV-2 infection, allowed their prognostic value to be assessed by a Random Forest model machine learning approach. Subsequent ex vivo experiments were performed to assess EC permeability responses to patient plasma and derive modulated gene regulatory networks from which rational therapeutic design was inferred.
Multiple inflammatory and EC activation biomarkers were associated with mortality in COVID-19 patients and in severity-matched SARS-CoV-2-negative patients. In contrast, dysregulation of specific microRNAs at presentation was specific for poor COVID-19-related outcomes and revealed disease-relevant pathways. Integrating the datasets using a machine learning approach further enhanced clinical risk prediction for in-hospital mortality. Exposure of ECs to COVID-19 patient plasma resulted in severity-specific gene expression responses and EC barrier dysfunction which was ameliorated using angiopoietin-1 mimetic or recombinant Slit2-N.
Integration of multi-omics data reveal microRNA and vascular biomarkers prognostic of in-hospital mortality in COVID-19 patients and that vascular stabilizing therapies should be explored as a treatment for the endothelial dysfunction of COVID-19, and other severe diseases where endothelial dysfunction has a central role in pathogenesis.
The induction of substantial acute respiratory distress phenotypes may be driven by a mismatched inflammatory response together with broad vascular dysfunction. Approaches implementing multi-omic approaches have provided insight into the immune cell phenotypes involved in these processes, risk stratifying markers specific to COVID-19 and the vasculature have not been explored. A comprehensive, multi-omics-based description of the molecular antecedents to COVID-19 mortality is herein presented, yielding new insights pertaining to the vasculature while highlighting the urgent need for clinical translation of novel biomarkers for disease prognosis.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious betacoronavirus, which results in coronavirus disease 2019 (COVID-19).(1) While the majority of infected individuals manifest mild to moderate illness, 14-31% of symptomatic unvaccinated patients eventually require hospitalization, with intensive care unit (ICU) admission rates ranging from 2-26% among those hospitalized.(2) Select populations, particularly older individuals and those with underlying comorbidities (including cardiovascular disease [CVD]) have high rates of morbidity and mortality.(3) Substantially higher rates of morbidity and mortality have been observed as variants of concern become more predominant.(4) While diagnostic testing has allowed for the rapid identification of COVID-19 cases, the lack of post-diagnosis risk assessment metrics, especially among the highest-risk subgroups, have undermined the cascade and allocation of care.
The use of existing cardiovascular and respiratory parameters could serve as a metric of risk prediction.(5, 6) However, case-fatality rates of those with comorbidities remain particularly high (e.g., preexisting CVD at ˜10.5%) with cardiorespiratory the aforementioned markers may have limited utility.(7) In this regard, while standard metrics including measures of cardiac damage (e.g., troponin values above the 99th percentile), the extent of inflammatory activation (e.g., C-reactive protein expression), and cardiovascular imaging have elucidated the spectrum of COVID-19 complications, they may have only modestly elucidated the risk of adverse in-hospital outcomes and may often provide limited insight into disease mechanism.(8-10) Albeit encouraging, some COVID-19 therapeutics focus on stemming aberrant immune responses and controlling viral reproduction (e.g., tocilizumab and remdesevir, respectively), which may neglect key elements of the host response contributing to outcomes.(11) From this perspective, clinical data and autopsy studies revealing endotheliitis and thrombosis have raised the possibility that endothelial dysfunction, particularly fluctuations in vascular integrity and coagulative capacity, could be a driver of clinical outcomes.(12) Thrombosis, fluid extravasation, and microangiopathy observed in the small vessels and capillaries of the lungs may suggest that an intense vascular reaction takes place in those with severe disease.(13)
Viral tropism towards angiotensin-converting enzyme 2 and acetylated sialic acid residues, which are highly expressed by vascular endothelial cells (EC), could instigate cardiovascular dysfunction.(14-19) However, SARS-CoV-2 may have limited infectious potential and replicative ability in ECs.(20) In this respect, mechanisms secondary to direct infection, such as cascading immunological activation may instead be the driving factor behind the observed endothelial dysfunction; particularly among the populations with coexisting conditions where EC dysfunction is already evident.(21, 22) In fact, select endothelium-related biomarkers such as thrombomodulin(23), von Willebrand Factor(24), angiopoietin-2 (Ang-2)(25), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1)(26) may show utility in prognostication, being associated with both disease severity and in-hospital mortality. While markers of endothelial function may aid in prognostication, it seems possible that a simple combination of markers can provide insight significant enough to adjudicate the level of care a patient will need, nor is it apparent that these markers associate specifically with COVID-19 pathology. To date, the integration of clinical data with broad omics technologies has opened up new avenues for efficiently delineating complex patient phenotypes and their associations with clinical outcomes.(27, 28) Circulating profiles of plasma microRNAs (miRNA), in particular, have been shown to be tightly associated with disease, and capable of providing not only detailed prognostic information but also mechanistic insight.(29, 30)
The following seeks to characterize markers of endothelial function and inflammation and to develop for the first time an atlas of miRNA expression across both a spectrum of individuals diagnosed with COVID-19 as well as SARS-CoV-2-negative patients from the ICU. To enhance clinical utility, the following focuses on understanding how these markers are associated with in-hospital mortality of high-risk patients, particularly those requiring the highest levels of care.
The present disclosure provides methods for determining Coronavirus Disease 2019 (COVID-19) severity. In some embodiments, methods for determining COVID-19 severity in a subject comprise obtaining a circulating blood sample from the subject; and obtaining a biomarker panel comprising one or more of angiopoietin-2 (Ang-2), endothelin-1 (ET-1), soluble intercellular adhesion molecule (sICAM), soluble vascular cell adhesion molecule (sVCAM), soluble E-selectin (sE-selectin), triggering receptor expressed on myeloid cells-1 (sTREM-1), interkeulin-6 (IL-6), interleukin-8 (IL-8), myeloperoxidase (MPO), and high-sensitivity cardiac troponin (hs-cTnI); wherein a different level of at least one biomarker in the biomarker panel compared to a reference panel from a SARS-CoV-2-negative subject is indicative of COVID-19 severity. In one embodiment, the biomarker panel comprises two or more of the biomarkers. In one embodiment, the biomarker panel comprises three or more of the biomarkers. In one embodiment, the biomarker panel comprises four or more of the biomarkers. In one embodiment, the biomarker panel comprises five or more of the biomarkers. In one embodiment, the biomarker panel comprises six or more of the biomarkers. In one embodiment, the biomarker panel comprises seven or more of the biomarkers. In one embodiment, the biomarker panel comprises eight or more of the biomarkers. In one embodiment, the biomarker panel comprises nine or more of the biomarkers. In one embodiment, the biomarker panel comprises all of the biomarkers.
In some embodiments, elevated levels of at least one biomarker is indicative of COVID-19 severity. In one embodiment, elevated levels of at least two biomarkers are indicative. In one embodiment, elevated levels of at least three biomarkers are indicative. In one embodiment, elevated levels of at least four biomarkers are indicative. In one embodiment, elevated levels of at least five biomarkers are indicative. In one embodiment, elevated levels of at least six biomarkers are indicative. In one embodiment, elevated levels of at least seven biomarkers are indicative. In one embodiment, elevated levels of at least eight biomarkers are indicative. In one embodiment, elevated levels of at least nine biomarkers are indicative. In one embodiment, elevated levels of all biomarkers are indicative.
In one embodiment, the biomarker panel comprises Ang-2, preferably Ang-2, and MPO. In one preferred embodiment, the biomarker panel comprises Ang-2, ET-1, sICAM-1, sVCAM-1, sE-Selectin, sTREM-1, IL-6, IL-8, MPO and hs-cTnI. Elevated levels of these biomarkers are indicative of COVID-19 severity.
In some embodiments, methods for determining mortality risk of a subject infected with SARS-CoV-2 comprise detecting in a circulating blood sample from the subject one or more circulating microRNA (miR) biomarkers selected from the group consisting of: one or more miR from miR-30 family, miR-181a-5p, miR-199a-3p, miR-4793-5p, miR-6080, and miR-6750-5p. In one embodiment, two or more of these miR biomarkers are detected. In one embodiment, three or more of these miR biomarkers are detected. In one embodiment, four or more of these miR biomarkers are detected. In one embodiment, five or more of these miR biomarkers are detected. In one embodiment, all of these miR biomarkers are detected. In one embodiment, the miR biomarkers are selected from miR-30b, miR-30c, miR-30e, miR-181a-5p, miR-199a-3p, and miR-6080.
In some embodiments, the circulating microRNA miR is amplified, detected, captured, and/or sequenced using polymerase chain reaction (PCR), CRISPR, colorimetry, next-generation sequencing (i.e. isothermal amplification, loop-mediated amplification, Illumina™, pyrosequencing, real-time sequencing, parallel sequencing, nanopore sequencing), and/or nucleotide assays (i.e. nuclease protection assay, multiplex assay, 5-plex multiplex assay). Various other amplified, detected, captured, and/or sequenced methods are available and applicable to the present methods.
Kits for ducting the methods and diagnoses disclosed herein are also provided, containing one or more of: one or more antibodies specific to the one or more biomarkers described herein, buffers, probes (amplification probes, sequencing probes, capture probes, and/or detection probes), assay reagents, and test tubes.
In some embodiments, subjects diagnosed or predicted to have COVID-19, severe COVID-19, moderate COVID-19, or mild COVID-19 are treated by administering said subject with Slit2, or a variant or fragment thereof. In some embodiments, subjects diagnosed for COVID-19 severity with the methods described herein are treated by administering said subject with Slit2, or a variant or fragment thereof. In some embodiments, methods are provided for treating COVID-19 in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject are provided comprising administering said subject with Slit2, or a variant or fragment thereof.
In one embodiment, a variant of Slit2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with wild-type Slit2 protein, and has comparable biological activity. In one embodiment, a fragment of Slit2 comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a full length Slit2 protein. In one embodiment, the fragment is a N-terminal domain of Slit2.
In some embodiments, subjects diagnosed or predicted to have COVID-19, severe COVID-19, moderate COVID-19, or mild COVID-19 are treated by upregulating the Slit2 signaling pathway in the subject. In some embodiments, subjects diagnosed for COVID-19 severity with the methods described herein are treated by upregulating the Slit2 signaling pathway in the subject. In some embodiments, methods are provided for treating COVID-19 in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject are provided comprising upregulating the Slit2 signaling pathway in the subject.
In one embodiment, upregulating the Slit2 signaling pathway comprises administering said subject with one or more factors or polypeptides from the Slit2 signaling pathway from the following list: GIT1, ARF6, Ab1, GTPase, Ena, Myo9b, or Hakai. In one embodiment, two or more of these factors or polypeptides are administered. In one embodiment, three or more of these factors or polypeptides are administered. In one embodiment, four or more of these factors or polypeptides are administered. In one embodiment, five or more of these factors or polypeptides are administered. In one embodiment, six or more of these factors or polypeptides are administered. In one embodiment, all of these factors or polypeptides are administered.
EXAMPLES. The following is an exemplary, non-limiting embodiment of the ideas described herein.
Nonstandard Abbreviations and Acronyms
Methods
Reagent and Resource Sharing: Transcriptomic data (i.e., messenger RNA and miRNA sequencing) have been deposited at the Gene Expression Omnibus and the RStudio analysis pipeline is contained within the Supplement Section.
Synopsis of Study Design, Patient Demographics, and Clinical Severity: Two hundred and forty-one patients with suspected, community-acquired SARS-CoV-2 (acute infection) were enrolled prospectively in the emergency departments or upon admission at two urban, quaternary-care hospitals in Toronto, Canada (University Health Network and St. Michael's Hospital, Table 1 and
The cohort of 241 patients was categorized into three groups that reflected conventional concepts of COVID-19 severity (National Institutes of Health, ‘Clinical Spectrum of SARS-CoV-2 Infection’), as well as two analogous symptom/severity matched control groups.(31) The resulting five groups were: SARS-CoV-2 negative patients presenting to outpatient clinics with symptoms consistent with a respiratory tract illness (n=30, “mild negative”); SARS-CoV-2 positive patients presenting to outpatient clinics with symptoms consistent with a respiratory tract illness (n=27, “mild COVID-19”); admitted SARS-CoV-2 positive patients requiring supplemental oxygenation (n=39, “moderate COVID-19”); SARS-CoV-2 positive patients requiring high-level in-patient ICU care (n=76, “severe COVID-19”); and SARS-CoV-2 negative patients who exhibited symptoms of a severe respiratory disease requiring high-level in-patient ICU care (n=69, “severe negative”). Patients testing negative by nasopharyngeal SARS-CoV-2 polymerase chain reaction had immunological history (i.e., antibody reactivity) ascertained through spike antigen cross-reactivity using a Federal Drug Administration approved enzyme-linked immunosorbent sandwich assay (FIG. II).
Data Visualization and Statistical Analysis: Descriptive Analysis—Clinical characteristics were characterized using summary statistics. Continuous variables were described using median and inter-quartile range (IQR), and dichotomous or polytomous variables were described using frequencies. Between-group differences were evaluated using Wilcoxon rank-sum tests for continuous variables and Fisher's exact tests for dichotomous/polytomous variables. Correlation between continuous variables were quantified using Spearman rank correlation. Descriptive outcome analysis—The Kaplan-Meier survival method was applied to assess the in-hospital death, and the between-group differences in the freedom from death were evaluated using log-rank tests. The length of hospitalization/ICU was characterized using competing risk models in terms of cumulative incidence rate function. Univariable Cox proportional hazard regression were applied to assess and quantify the association of the baseline clinical characteristics with in-hospital/ICU death. The associations of continuous variables were modeled using natural cubic splines. Biomarker Analysis—Comparisons between two independent groups were made using t-tests for normally distributed continuous variables or Wilcoxon rank-sum tests non-normally distributed continuous variables. When more than two groups were compared, either a one-way ANOVA with a Tukey or Bonferroni post-hoc test (where appropriate) for multiple testing correction, Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison correction. Two-way ANOVA was used to estimate how the mean quantitative variable changes according to time and group differences in leak experiments. Where appropriate, Benjamini-Hochberg false discovery rate (FDR) was utilized with adjusted P values (or Q value where stated) of <0.05 being considered statistically significant and indicated in the graphs as reported by the analysis software with significance thresholds of P<0.05, P<0.01, P<0.001, and P<0.0001 indicated as *, **, ***, **** respectively. MiRNA pathway analysis was conducted using BioCarta/KEGG/Reactome databases and tested for enrichment by a hypergeometric test with adjustment for multiple comparisons using the Benjamini-Hochberg FDR, with P ≤1.05 considered to be statistically enriched in a gene set of interest.(32-34) Although many hypotheses were tested throughout the manuscript, no experiment-wide multiple test correction was applied. Unless indicated otherwise, graphs depict averaged values of independent data points with technical replicates and have error bars displayed as mean +/−standard deviation (±S.D.). Data were analyzed with GraphPad Prism 9.0.0 for MacOS (GraphPad Software, Inc., La Jolla, Calif., USA; Biomarker Multiple Comparisons), R(35) (v4.0.3; Spearman Correlation Plots), and FIJI(36) (v2.1.0/1.53c; Quantifying Image Intensities). Final figures were assembled for publication purposes using Adobe Illustrator (v25.4.1).
Study Approval: This is a multicenter, secondary analysis of a prospectively recruited longitudinal cohort study enrolling consecutive patients with suspected SARS-CoV-2 infection who were referred to two Canadian quaternary care networks in Toronto, Canada from May 2020 to December 2020: University Health Network and St. Michael's Hospital. All participants who were 18 years of age or older, provided either direct written informed consent or were consented into the study by a lawfully entitled substitute decision-maker on behalf of a participant when lacking the capacity to make the decision. The study, and consenting, was conducted in accordance with protocols approved by the Research Ethics Board (REB) of the University Health Network (REB #: 20-5453.6; Cardiovascular Disease and Outcomes among Patients with SARS-CoV-2 Infection During Admission and Post-Discharge [The COVID study]) or St. Michael's Hospital (REB #: 20-078; COVID-19 Longitudinal Biomarkers in Lung Injury [COLOBILI]). SARS-CoV-2-negative patients with severe respiratory illness symptoms were enrolled within the COLOBILI study.
Detailed materials and methods can be found in the Supplement Section and the Major Resource Tables (Tables I and II).
Results
The baseline characteristics for the cohort are indicated in Table 1. The median age (interquartile range [IQR]) of the entire cohort (n=241) was 61 [51-72] years (155 male patients [64.3%]), and of those 201 were admitted, having a median hospital stay of 18 (IQR, 8-40) days within which 54 (26.9%) died (Table 1). Regardless of admission or SARS-CoV-2 status, co-existing medical conditions were common amongst the population including 83 (out of a total of 234; 35.5%) with diabetes mellitus, 80 (34.4%) with underlying CVD, 31 (12.9%) having chronic kidney disease, and 159 (66.0%) patients having more than one co-existing condition. In this respect, there were significant differences in age (P<0.0001), and the frequency of obesity (P=0.0006), smoking history (P<0.0001), chronic kidney disease (P=0.0204), diabetes mellitus (P<0.0001), dyslipidemia (P=0.0266), gastroesophageal reflux disease (P=0.0019), hypertension (P=0.0105), calcium channel blockers (P=0.0338), and diuretics (P=0.0088) between groups (Table 1). Although a small proportion of patients had documented smoking status, there were 40 (total 138; 29.0%) current or former smokers, with 33 (total 234; 14.1%) patients having chronic obstructive pulmonary disease.
On admission to hospital, clinical lab data highlighted disparities between the groups with leukocytosis and lymphopenia seen amongst more severe groups (e.g., white blood cell count [P<0.0001]; lymphocytes [P<0.0023, table 111]). Amongst cardiovascular markers, there were significant differences in the frequency of elevated creatine kinase (P<0.0001) and D-dimers (P<0.0001). Although the etiology of admissions was predominantly extracardiac in nature, high-sensitivity troponin 1 (hs-CnTl) values at t0-1 were detectable (>3 pg/mL) in 157 patients (65.1%), with values significantly elevated (>15 pg/mL in males; >10 pg/mL in females) in 90 patients (37.3%, Table 111), predominantly in the severe groups (P<0.0001)
COVID-19 Outcomes During Hospital Admission: Among admitted patients with COVID-19, 33 died, with Kaplan-Meier survival curves indicating that patients with severe disease higher mortality risk than did patients with less severe phenotypes (P<0.001,
Associations of Cardiovascular and Inflammatory Biomarkers with Outcomes: The vascular endothelium acts as the crucial interface between blood components and tissues, displaying a series of properties that maintain homeostasis (i.e., maintenance of vascular barrier, coagulative capacity, and modulation of immunological responses).(22, 37, 38) While these functions participate in the dynamic regulation of cardiovascular function and coordinate many host defense mechanisms, proinflammatory cytokines may elicit a change in endothelial phenotype, promoting thrombosis, local tissue injury, propagating inflammation and potentially contributing to mortality.(39) In order to test the hypothesis that metrics of cardiac damage and inflammatory endothelial dysfunction/activation may better reflect COVID-19 severity and subsequent mortality than standard clinical metrics, a custom Simple Plex assay for markers robustly associated with inflammation and endothelial dysfunction was performed (i.e., Ang-2, endothelin-1 [ET-1], soluble intercellular adhesion molecule [sICAM], soluble vascular cell adhesion molecule [sVCAM], soluble E-Selectin [sE-selectin], sTREM-1, interleukin-6 [IL], and IL-8), along with standalone assays measuring myeloperoxidase (MPO) and high-sensitivity cardiac troponin (hs-cTnI). Correlation analysis revealed that Ang-2, sE-Selectin, and sICAM had moderate correlations with numerous other inflammatory markers, with correlation coefficients ranging from −0.89 (IL-6:MPO) to 0.89 (sICAM-1:Ang-2;
Amongst all patients admitted with COVID-19, univariable analysis revealed that only Ang-2 was associated with mortality (P=0.015; Table IV), suggesting higher concentrations are associated with higher mortality, while both Ang-2 (P=0.020) and sVCAM-1 (P=0.012) were associated with mortality when looking specifically at the severe COVID-19 patients (
Plasma MiRNA Atlas of SARS-CoV-2 Infection May Reveal Markers Specific to COVID-19 and Mortality: While select inflammatory/EC activation markers were informative for ICU mortality, they may lack the ability to distinguish between COVID-19 and non-COVID-19 pathology. Assessment of the circulating miRNA transcriptome may provide further precision with respect to patient subgroups, since miRNA profiling (in contrast to circulating protein markers) has been shown to effectively differentiate complex disease etiologies.(40, 41) Using whole transcriptome miRNA sequencing (2,083 mature miRNAs) plasma obtained from the differing groups of disease severity was screened to identify meaningful differences in miRNA composition (n=30, negative mild; n=14, mild COVID-19; n=15, moderate COVID-19; n=36, severe COVID-19; n=33, negative severe). Comparative analyses indicated that there were substantially higher numbers of differentially expressed miRNA as disease severity progressed (
Predictive Power of Clinical, Protein, and miRNA Data on Mortality using Machine Learning: Given the high dimensionality of the dataset generated, the utility of models developed were examined using machine learning to predict in-hospital mortality of COVID-19 patients at admission, based on common clinical data, protein expression data, and miRNA expression data. 250 experiments were performed using repeated randomized stratified sub-sampling cross-validation into disjoint sets of 80% training and 20% testing, to train a set of Random Forest models.(42) Model performance was assessed by the area under the receiver operating characteristic (AUROC) calculated on the testing sets. Aggregate statistics on AUROC were calculated across the 250 experiments. A low AUROC was observed using only clinical features available at the time of admission (AUROC 0.44, 95% CI 0.22-0.69,
Identification of miRNAs Associated with Severe COVID-19 Mortality: Since miRNAs may have provided superior specificity for COVID-19 mortality compared to protein biomarkers (
Endothelial Barrier Disruption may be Rapidly Induced During Co-incubation with Plasma from Patients With Moderate to Severe COVID-19: With studies showing conflicting evidence that SARS-CoV-2 can directly infect the endothelium in a physiologically meaningful fashion(12, 50, 51), it was reasoned that the modulation of the extracellular milieu (i.e., changes to circulating plasma components such as sTREM-1, Ang-2, and MPO) as a result of the systemic immune response may be a driving factor behind the observed endothelial dysfunction. In this context, data from previous studies conducted in systemic inflammatory response syndrome and sepsis have underscored the impact of endothelial barrier disruption on disease outcomes.(52) The data presented so far suggested that dysfunction of the endothelium may also accompany COVID-19 and may be associated with poor outcomes. To further examine this hypothesis, two validated permeability platforms using an endothelial monolayer model were used: continuous monitoring of transendothelial electrical resistance (
Modulation of Inflammatory and Cytoskeletal Processes may be Coincident with Barrier Dysfunction: To gain a better understanding of how the endothelium is modulated by plasma components, the gene-level changes using RNA-sequencing of ECs after six hours of co-incubation with patient-derived plasma was investigated. On the transcriptome level (16,285 total quantified genes, having ≥10 reads in at least five samples), biological replicates within groups were tightly correlated (Pearson r=0.975-0.988, n=4-5), suggesting robust intra-group clustering even among a heterogeneous patient group. Principal component analysis of the normalized transcriptome showed segregation between experimental groups, with the severe group being clearly distinct from the mild and moderate groups (
Vascular Barrier Stabilizing Drugs May Prevent EC Permeability Induced by COVID-19 Patient Plasma Exposure in vitro: Two principal structural contributors to the EC barrier were examined, vascular endothelial-cadherin (VE-Cadherin) and Claudin-5. VE-cadherin can be an essential adherens junction protein that regulates cell-cell junctional stability, and Claudin-5 can be a tight junction protein that regulates size-dependent paracellular permeability pathways.(54) Immunostaining confirmed significant disruptions to both VE-cadherin and Claudin-5 expression as well as junctional localization, particularly in ECs exposed to the moderate and severe COVID-19 plasma (
The induction of substantial acute respiratory distress phenotypes may be driven by a mismatched inflammatory response together with broad vascular dysfunction.(58, 59) While several detailed reports implementing multi-omic approaches have provided insight into the immune cell phenotypes involved in these processes(60-62), risk stratifying immune markers specific to COVID-19 have not been fully elucidated. A comprehensive, multi-omics-based description of the molecular antecedents to COVID-19 mortality, yielding new insights pertaining to the vasculature is provided. The above study further delineates the gradient of vascular dysfunction observed in patients across the spectrum of COVID-19 severity, particularly among those with severe illness. While the findings are consistent with smaller cohort studies examining single markers of cardiovascular dysfunction(26, 63), the results described herein is the first to use proper, disease-negative controls, which asked etiological questions. The analysis may have revealed that although markers of cardiovascular dysfunction (such as Ang-2 and sVCAM-1,
Circulating miRNAs have emerged as exquisite biomarkers for complex pathological conditions, including influenza and sepsis.(64, 65) The inherent stability of plasma microRNAs under harsh conditions and reproducible quantification makes them attractive candidates for use as noninvasive biomarkers.(30) Incorporating whole transcriptome sequencing as an added metric may allow for the effective differentiation between severities of COVID-19 and severe SARS-CoV-2 negative controls using samples gathered on admission. Through this study, further empirical evidence for the value of data from novel biomarkers over metrics traditionally utilized in healthcare systems (i.e., clinical demographics and laboratory data) may have been provided. Notably, the data from electronic medical records fails to adequately capture the risk of in-hospital mortality. Using a Random Decision Forest machine learning model on the multi-omic biologically relevant datasets an exploratory prognostic risk prediction model that incorporates markers that are COVID-19 and vascular specific was developed. Importantly, there are several platforms that allow the interrogation of candidate miRNAs within clinically relevant timeframes.(66, 67) While still investigational, given the extent of pre-clinical and clinical research on miRNAs(40), reasonable to expect miRNA datasets will soon be amenable for clinical implementation. As an example of the importance of a multi-omic approach that includes clinically relevant disease negative controls, miR-1 (which has been intensively studied as a cardiac enriched miRNA and associated with numerous cardiac etiologies(41, 43, 44, 68)) failed to show an association with in-hospital mortality in the study. If the promising miRs-30b/c/e, -6080, -181a-5p, -199a-3p, and -339 identified here are validated in larger populations they would represent new biomarkers that could be utilized for rapid in-hospital risk assessment. As miRNAs represent functional biomarkers, having active roles in gene regulation, they also present an important opportunity to understand the pathophysiological relevance of endothelial-based processes affected by SARS-CoV-2 infection.
Given that the endothelium is the gatekeeper of vascular permeability, which is a main pathophysiological process in systemic inflammatory conditions, there is intense research on their role in ARDS, systemic inflammatory response syndrome, and sepsis (among others).(69-71) Several lines of evidence support the hypothesis that endothelial function is a major determinant of COVID-19 outcome.(63, 72) Endothelial derangements (i.e., pathological sprouting angiogenesis), increased endothelial apoptosis, modified metabolism, and a strong correlation between underlying CVD and mortality may show COVID-19 severity increases.(23, 25, 69) Often forgotten, endothelial dysfunction—associated molecular patterns are broad and strong activators of innate immune responses, leading to innate immunity-mediated organ injury. To this end, plasma from patients with moderate and severe COVID-19 induced profound barrier disruption, as assessed through the modulation of VE-Cadherin and Claudin-5 at EC-EC junctions was observed. Transcriptomic analysis revealed that COVID-19 plasma from moderate to severe patients appears to preferentially induce pro-inflammatory immune gene processes, while plasma from mild patients induces an interferon (IFN) response. This is in line with several studies on the immune response of mild vs severe COVID-19 patients, and confirmed by the over-representation of type I IFN genes in ECs exposed to mild COVID-19 plasma.(63, 73, 74) Type I IFNs are critical mediators of the antiviral immune response and subverting the early type I IFN response has been shown repeatedly to be a contributor to coronavirus infection.(73) Looking at the moderate and severe phenotypes where endothelial pro-inflammatory responses (e.g., TNF) and histone deacetylase dysregulation occurred, both epigenetic modifications as well as TNF responses have been documented in COVID-19.(63, 74)
Increased pro-inflammatory cytokine expression, specifically TNF and IL-6, has been shown to upregulate trypsin, resulting in the loss of endothelial tight junctions, subsequently inducing vascular hyperpermeability.(75, 76) Based on the above study, recombinant Slit2-N, may effectively prevent the induction of endothelial permeability in vitro. Additionally, measurement of the levels of endogenous sSlit2 in SARS-CoV-2 patient plasma revealed a significant upregulation only in patients with severe disease suggesting a possible compensatory mechanism and furthering the biological relevance of Slit2-N treatment (Figure XXI). Therefore, as a proof-of-concept, these data support the hypothesis that disruption of endothelial barrier function, particularly as a result of loss of adherens and tight junctions, is induced during COVID-19. These findings support a model in which systemic induction of pro-inflammatory processes, rather than direct infection, may be the primary driver of systemic endothelial perturbations observed in COVID-19 patients, and that relative to current treatments aimed at dampening immune responses, preserving endothelial barrier may be a viable adjuvant to reduce the mortality of SARS-CoV-2.
It will nonetheless be important to determine whether pre-existing and potentially chronic endothelial permeability—layered on top of a permeabilizing stimuli such as infection—can be reversed through biologics that interact directly at the endothelial interface. If the significant induction of endothelial permeability can be overridden, even in patients with an ongoing inflammatory insult, then pharmacological intervention at the level of the vasculature may be an effective adjuvant therapy for patients at a higher risk of adverse events. Pharmacological treatments that enhance vascular resiliency in the face of an aberrant host immune response may be a therapeutically feasible approach for managing vascular dysfunction without compromising immunity. Although stabilizing the vasculature may be more practical than attenuating individual cytokines, it remains to be determined whether severe vascular damage can be reversed in a clinically meaningful way. Adrenomedullin, a peptide hormone capable of endothelial stabilization, may have high in vivo efficacy and positive results from both Phase I and II trials in patients with sepsis.(80, 81) A prospective study comparing endothelial function in critical illness of non-viral etiology and viral etiology may provide clearer insights as to the translatability of these findings.
Taken together, herein is provided the first highly salient comparison of a diverse group of cardiovascular markers between COVID-19 positive and negative patients, highlighting that well-known markers of inflammation, cardiac damage, and endothelial dysfunction are not specific to COVID-19 pathology. Notably, with a targeted miRNA transcriptomic approach, specific markers that may show better discrimination between these two groups were discerned. Using machine learning, incorporation of protein and miRNA markers improved the prediction of in-hospital mortality over baseline clinical variables. While exploratory, this is a clinically feasible approach that has the advantage of using pathophysiologically relevant SARS-CoV-2's markers, as opposed to the surrogate markers used in most other published risk stratification models.(10, 26) Finally, the data provide several lines of evidence supporting the notion that endothelial barrier function is affected in a SARS-CoV-2 specific manner, that is distinct from the pathways involved in critically ill patients with non-COVID-19 severe respiratory illnesses. The data reinforce the idea that barrier dysfunction is likely independent of direct viral infection and instead secondary to yet undiscovered mediators in the plasma. These observations were further pursued using assays of EC function using an in vitro model, which served as a platform for rational therapeutic choice. Here, EC barrier may be reduced by the addition of COVID-19 patient plasma in a disease severity-dependent manner and that this can be prevented by stabilization with Slit2-N was shown. Together, the work provides biological insight into the role of the endothelium in SARS-CoV-2 infection, the importance of miRNA as disease- and pathway-specific biomarkers, and the exciting possibility that endothelial barrier stabilizing treatments might hold promise in COVID-19. Moreover, the work provides insights into the use of this approach to find therapeutic options that might prove useful in other critical illnesses and emerging infectious diseases where endothelial permeability is central to disease pathophysiology.
Limitations
The results of this study should be viewed in the context of its design, sample size, and socio-geographical context (i.e., access to advanced care life support). Variation between studies may represent temporal differences in circulating viral strains or vaccination status (notably, this study was done in a timeframe exclusive of B.1.1.7 [Alpha], B.1.351 [Beta], B.1.617.2 [Delta], and P.1 [Gamma] circulating strains and prior to public vaccination programs), which can impact severity and mortality. Findings from the miRNA atlas and machine learning algorithms should be validated in additional cohorts before implementation in clinical practice. The in vitro system used in this manuscript represents a reductionist approach to disease modelling, and as such, vascular cell co-culture or examination of specific endothelial beds (i.e., coronary or pulmonary) may better inform microenvironment changes in COVID-19. Putative therapies could be further assessed in co-culture models (e.g., organ on a chip) that better mimic tissue complexity and will require testing in animal models. Nevertheless, the findings provide important knowledge relating to the pathophysiology and risk stratification of betacoronavirus infection and provide avenues for future research in infectious disease.
Perspectives (Clinical Competencies/Translational Outlook)
Bedside microRNA-based diagnostics could significantly enhance clinical care of those with acute care diseases.
Vascular stabilizing therapies represent an attractive supplemental modality for treating critical illnesses and emerging infectious diseases where endothelial permeability is central to disease pathophysiology and should be further explored.
Primary Figure Legends
Primary Tables
†Body mass index is the weight in kilograms divided by the square of the height in meters.
‡Obesity is classified according to World Health Organization guidelines (i.e., >25).
§Smoker is defined as an aggregate of self-reported current and previous smokers.
Supplemental Section
Supplemental Abbreviations and Acronyms
Supplemental Methods
Rationale and Design: Coronavirus Disease 2019 (COVID-19) is a leading infectious etiology currently present throughout the world, for which there are limited therapeutic interventions. Understanding the pathobiology of COVID-19 outcomes can enable personalized patient management protocols, improve survival, and aid in the preparation of diagnostic tools for future pandemics. Due to the dynamic nature of clinical care guidelines, clinical care did not follow a standardized protocol but rather was determined by individual providers and patient needs. Plasma of enrolled patients was taken at presentation and at set intervals (days 2-3 [t2-3], 4-5 [t4-5], and 6-7 [t6-7]) following obtainment of consent. Comprehensive, integrated analysis of plasma transcriptome data was performed to prioritize COVID-19 outcome signals.
Patient Categorization: After retrospective clinical adjudications of medical records and triaging protocols, patients were assigned to pre-defined clinical groups, centered around the National Institutes of Health, ‘Clinical Spectrum of SARS-CoV-2 Infection’′, those being: (i) severe acute respiratory syndrome coronavirus (SARS-CoV-2) negative patients with mild acute respiratory disease, (ii) mild COVID-19, (iii) moderate COVID-19, (iv) severe COVID-19, and (v) SARS-CoV-2 negative patients requiring intensive care unit (ICU) level management for severe respiratory illness. These assignments were made solely on the basis of information available in the medical record and were blind to any novel biomarker data, which had not yet been generated. For biomarker studies, where appropriate, patients were matched for age, sex, body mass index (BMI), and co-existing conditions with non-infected controls as reference groups.
Study Population: This is a multicenter, secondary analysis of a prospectively recruited longitudinal cohort study enrolling consecutive patients with suspected SARS-CoV-2 infection who were referred to two Canadian quaternary care networks in Toronto, Canada from May 2020 to December 2020: University Health Network and St. Michael's Hospital. All participants who were 18 years of age or older, provided either direct written informed consent or were consented into the study by a lawfully entitled substitute decision-maker on behalf of a participant when lacking the capacity to make the decision. The study, and consenting, was conducted in accordance with protocols approved by the Research Ethics Board (REB) of the University Health Network (REB #: 20-5453.6; Cardiovascular Disease and Outcomes among Patients with SARS-CoV-2 Infection During Admission and Post-Discharge [The COVID study]) or St. Michael's Hospital (REB #: 20-078; COVID-19 Longitudinal Biomarkers in Lung Injury [COLOBILI]). SARS-CoV-2-negative patients with severe respiratory illness symptoms were enrolled within the COLOBILI study. Diagnoses of COVID-19 were confirmed through real-time reverse transcription-polymerase chain reaction assays of nasopharyngeal swabs according to the Public Health Ontario guidelines for SARS-CoV-2 testing.2 The timeframe for recruitment largely excludes community level spread for the variants of concern (i.e., B.1.1.7 [Alpha], B.1.351 [Beta], B.1.617.2 [Delta], and P.1 [Gamma]), with the assumption that all patients harbored one of the predominant unmutated G strains (i.e., GR, GH, and GV).3 Samples were collected prior to the initiation of public vaccination programs in Ontario, with all patients assumed to be unvaccinated. Admitted patients were followed up after their COVID-19 diagnosis, with all causes of in-hospital mortality, complications, and therapeutic regimen ascertained until discharge. Patients' data were extracted from the in-hospital electronic medical records, de-identified, and assigned random identification numbers which were used throughout the project. Information on sex, age, and pertinent clinical parameters for the cohorts are provided (Table 1). The laboratory parameters were collected as reported by the individual centers, with standard international reference ranges applied to decide the cutoff point for abnormal levels.
Clinical Data Collection: Clinical characteristics, medical history, therapeutics administered during admission, complications, and outcomes were obtained and extracted from electronic medical records by clinical coordinators. Participant disease severity was quantified according to the National Institutes of Health Clinical Spectrum of SARS-CoV-2 infection which is largely based on respiratory parameters. Complete follow-up was available only for those requiring admission to hospital. Obesity was defined as BMI 30 kg/m2 in line with World Health Organization guidelines.4 Preexisting cardiovascular disease (i.e., atrial fibrillation, arterial hypertension, coronary artery disease, dyslipidemia, diabetes mellitus, chronic obstructive pulmonary disease, or heart failure), myocardial infarction, and concurrent active malignancy were defined and recorded from the electronic medical records at the discretion of the clinical coordinators upon availability from previously recorded histories. Smoking was collected as a self-reported variable, with social smoking being defined as less than <4 times per week, and avid smoker being anything greater than that. Cardiovascular complications were defined as a surrogate of new-onset atrial or ventricular arrhythmia (atrial fibrillation, atrial flutter, non-sustained ventricular tachycardia, sustained ventricular tachycardia), acute coronary syndrome, clinical heart failure, right ventricular failure, left ventricular failure, biventricular failure, moderate or greater pulmonary embolism, tamponade, stroke, acute non-coronary ischemia due to hypercoagulable state, or intracardiac thrombus. In patients admitted to the ICU the Acute Physiologic Assessment and Chronic Health Evaluation II and the Sequential Organ Failure Assessment scores were used.5,6 These are validated methods for grading the severity of illness in critically ill patients based upon point-scoring systems associated with the degree of dysfunction with specific physiologic variables.
Processing of Bloodwork: Peripheral blood samples were collected synchronously with standard of care bloodwork at the University Health Network or St. Michael's Hospital between May 2020 and January 2021. Peripheral blood samples (10 mL) were drawn from the cubital vein into BD Vacutainer® Blood Collection Tubes (BD Bioscience, Franklin Lakes, N.J.) containing K2EDTA and processed within three hours. Plasma was separated from whole blood through centrifugation (2,000×g, 24° C., 15 min) and stored at −80° C. until downstream processing. At no time during the process was the plasma subjected to temperatures below 4° C. or above 25° C. Samples were thawed on ice and the plasma was subjected to sequential centrifugation of (2,500×g, 4° C., 25 min) to reduce platelets and large particulate. Hemolysis was examined prior to downstream analysis by measuring the absorbance at 414 nm using a DS-11+Spectrophotometer (DeNovix, Wilmington, Del., United States).
Protein Biomarker Analysis: Circulating levels of angiopoietin-2 (Ang-2; Lower limit of quantification [LLOQ] 9.91 pg/mL), soluble CD62 antigen-like family member E (sE-Selectin; LLOQ 4.22 pg/mL), soluble CD54/intercellular adhesion molecule-1 (sICAM-1; LLOQ 4.1 pg/mL), soluble CD106/vascular cell adhesion protein-1 (sVCAM-1; LLOQ 137 pg/mL), CD105/endoglin, endothelin-1 (ET-1; LLOQ 0.250 pg/mL), interleukin-6 ([IL]-6; LLOQ 0.41 pg/mL), IL-8 (LLOQ 0.19 pg/mL), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1; LLOQ 4.19 pg/mL) were quantified in platelet free plasma samples using the Simple Plex Ella (ProteinSimple, San Jose, Calif., USA) multiplex platform according to the manufacturer's instructions; all Simple Plex values are reported as the average of triplicate readings. Soluble Slit homolog 2 protein (sSLIT2) was quantified using an enzyme-linked immunosorbent sandwich assay (ELISA; LLOQ 0.10 ng/mL, Elabscience, Wuhan, China). Myeloperoxidase was additionally quantified through ELISA (LLOQ; 0.062 ng/mL; R&D Systems Minneapolis, Minn., USA). Circulating cardiac troponin I (cTnI) was quantified using a CLIA certified high sensitivity ELISA kit (LLOQ 0.92 pg/mL; Biomatik, Kitchener, ON, CAN). Cardiac injury was defined as plasma levels of high-sensitivity cTnI (hs-cTnI) greater than the 99th percentile of normal values, as per clinical guidelines. See Table I for reagents.
Cell Culture: Pooled human umbilical vein endothelial cells ([pHUVECs], Lonza, Basel, Switzerland) from multiple donors were cultured in EC Growth Basal Medium-2 (Lonza, Basel, Switzerland) containing the complete EC Growth Medium BulletKit™ (Lonza, Basel, Switzerland) at 37° C. in a 5% CO2 humidified incubator. Cells were maintained on 10 cm tissue culture plates (Corning, Costar, New York, N.Y., USA) coated with 0.1% (v/v) gelatin attachment factor (Thermo Fisher, Waltham, Mass., USA) and passaged every 2-4 days at 70%-80% confluency. For experimentation, pHUVECs at passages 3-7 were used. Cells tested negative for the presence of mycoplasma contamination (ThermoFisher, Waltham, Mass., USA).
xCelligence Real-Time Cell Analysis (Transendothelial Electrical Resistance [TEER]): Baseline resistance measurements were acquired using 150 μL EC Growth Basal Medium-2 for 15 minutes prior to experiment initiation using xCelligence Real-Time Cell Analyzer (RTCA, ACEA Biosciences, San Diego, Calif.). Following this, pHUVECs were seeded at densities of 4×104/well into multiple 16-well E-plates (ACEA Biosciences, San Diego, Calif.) in volumes of 150 μL EC Growth Basal Medium-2 and monitored on the xCelligence RTCA. Once the cells reached confluency, 20% v/v plasma or 2 U/mL thrombin (Sigma-Aldrich, St Louis, Mo.) was added, and the cells incubated at 37° C. in a 5% carbon dioxide atmosphere for six hours. To quantify the change in permeability, cell indexes (surrogate metric for change in resistance across the monolayer) were adjusted to the resistance of reference wells (i.e., untreated cells in growth media) and normalized to the time point immediately prior to the addition of the plasma. The net area between the curve of the normalized dataset was utilized to calculate the overall change in permeability, with an increased area representing increases in TEER and decreases representing declines in TEER (i.e., loss of endothelial barrier function). During therapeutic testing, cells were co-treated with 1280 ng/mL recombinant Slit2-N8, 50 μg/mL nangibotide9, 3 μM dexamethasone10, or a matching DMSO control with Dulbecco's Phosphate Buffered Saline without magnesium and calcium ([PBS−/−], Gibco, Gaithersburg, Md., USA.
Transwell Leak Assay: Endothelial monolayer leak assays were performed as previously described.11 Briefly, pHUVECs were seeded on 3 μm pore transwell inserts (Corning Life Sciences, Corning, N.Y., USA) coated with 0.1% (v/v) gelatin attachment factor (ThermoFisher, Waltham, Mass., USA). The cells were grown on the inserts for two days until reaching 90%-95% confluency. Cells were subsequently treated for either one hour (acutely) or six hours (sub-chronically) with either 20% (v/v) human plasma or control PBS−/−. Prior to leak quantification, cell media was changed to Hanks Buffered Salt Solution (ThermoFisher, Waltham, Mass., USA) and 1 mg/mL 40 kilodalton (kDa) fluorescein isothiocyanate—dextran (FITC-dextran, Sigma-Aldrich, St Louis, Mo.) was added to the top chamber with tracer flux subsequently being allowed to occur for one hour; 2 U/mL thrombin was added as a positive control to certain wells at the time of FITC addition. The experimenter was blinded to the grouping of each sample. The FITC accumulation in the bottom chamber was assessed in triplicate via excitation at 485 nm and emission at 535 nm using a Biotek Cytation 5 (Biotek, Winooski, Vt., USA).
Bulk RNA-Seq: Total RNA was isolated from treated pHUVEC samples (n=5 control [PBS−/−], n=5 mild SARS-CoV-2 negative, n=5 mild disease, n=5 moderate disease, and n=5 severe disease) at the six-hour timepoint using the RNeasy Plus Micro kit (Qiagen, Germantown, Md., USA), after washing twice with ice-cold PBS−/−. RNA quantities and quality were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada). All samples passed a quality control threshold (RNA integrity number 7.0) to proceed to library preparations and RNA-seq. A total amount of 20 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ipswich, Mass., USA) following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H−). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of ˜150-200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μL USER Enzyme (NEB, Ipswich, Mass., USA) was used with size-selected, adaptor-ligated cDNA at 37° C. for 15 minutes followed by five minutes at 95° C. before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. The resulting PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Sequencing was carried out on an Illumina NovaSeq® 6000 (NovoGene; Illumina, San Diego, Calif., United States), using paired-end 2×150 by chemistry at a depth of 20 million reads per sample.
Sequencing quality was examined using FastQC12 (v0.11.2), and adaptors were subsequently trimmed with Trimmomatic13 (v0.36) (in paired-end mode, parameters: TruSeq3-PE-2.fa:2:30:7:8:true LEADING:10 TRAILING:10 SLIDINGWINDOW:5:20 MINLEN:36). Trimmed fastq files were subsequently re-examined with FastQC again to ensure efficient adaptor and quality trimming. Reads were aligned to the hg38 genome (obtained through UCSC) with STAR14 (v2.7.8a) using the default parameters only for quality control purposes. Aligned reads were subjected to the following quality control metrics: (i) duplication rate assessment through Picard Markduplicates (v.2.18), (ii) rRNA content, genomic read distribution and 3′-5′ bias through RNA-SeQC (v.2.4.0). These metrics, together with the read alignment summary from STAR, were summarized using MultiQC15 (v1.9), resulting in the removal of one sample on the basis of abnormal read duplication rates (n=1 mild disease). Gene quantification was performed using Salmon16 (v1.3.0) with adaptor-trimmed reads and gene annotation obtained from GENCODE version 31 (parameters: —gcBias-validateMappings). Gene-level read counts were imported into R using R package Tximport13 (v.1.18.0) with transcript length adjustment. Read counts were first normalized by DESeq217 (v.1.30.0), with the svaseq( ) function from R package sva (v.3.38.0)18 being used to subsequently estimate hidden batch effects with n.sv=4. Sva-normalized read counts were obtained by regressing out covariates using the following function: https://github.com/LieberInstitute/jaffelab/blob/master/R/cleaningY.R and were subsequently used for PCA analysis and sample correlation analysis. R package fgsea (v 1.16.0)19 was used to perform Gene Set Enrichment Analysis with genes ranked based on fold change between the compared conditions. Gene set files were obtained from the Molecular Signatures Database (MSigDB, v7.0) and only C2: curated gene sets and C5: ontology gene sets were used. Pathway enrichment using significantly differentially expressed genes were performed with R package gProfilerR (v.0.7.0)2°. R scripts and essential data files to reproduce the mRNA-seq analysis are available (raw data not presented herein).
HTG EdgeSeq MicroRNA (miRNA) Whole Transcriptome Assay (WTA) from Plasma: Lysis of t0-1 plasma aliquots was facilitated by combining 30 μL plasma with equivalent (v/v) amounts of HTG Plasma Lysis Buffer (HTG Molecular, Tucson, Ariz., USA) as well as 1/10th (v/v) amounts of Proteinase K (HTG Molecular, Tucson, Ariz., USA). The mixture was subsequently incubated for three hours at 50° C. shaking at 1,400 rpm. From each prepared sample, 35 μL were added per well to a 96-well sample plate. Human fetal brain RNA was added to one well at 25 ng/well to serve as an internal control. Samples were run on an HTG EdgeSeq Processor using the HTG EdgeSeq miRNA WTA (HTG Molecular, Tucson, Ariz., USA) to facilitate nuclease protection, whereby a pre-selected miRNA population is protected with proprietary protection probes, followed by degradation of all non-hybridized probes and non-targeted RNA by 51 nuclease. Following the processing, samples were individually barcoded (using a 16-cycle PCR reaction), individually purified using AMPure XP beads (Beckman Coulter, Brea, Calif., USA), and quantified using a KAPA Library Quantification kit (KAPA Biosystem, Wilmington, Mass., USA). The library was sequenced on a NextSeq (Illumina, Inc., San Diego, Calif.) using a V3 150-cycle kit with two index reads. PhiX (Roche, Mississauga, ON, CAN) was spiked into the library at 5%; this spike-in control is standard for Illumina sequencing libraries. Data were returned from the sequencer in the form of demultiplexed FASTQ files, with one file per original well of the assay. The HTG EdgeSeq Parser (v. 5.0.535.3181, HTG Molecular, Tucson, Ariz., USA) was used to align the FASTQ files to the probe list to collate the data. Data were provided as data tables of raw, quality control (QC) raw, counts per million, and median normalized.
HTG EdgeSeq MiRNA Analysis: Samples were initially analyzed using three QC metrics: (i) QC0, examining degraded sample; cut-off of positive % ≥14% as failure, (ii) QC1, insufficient read depth; read depth 500k as failure, (iii) QC2, minimal expression variability; relative standard deviation of reads 0.08 as failure. Of the 156 samples sent for sequencing, 12 exhibited failure at the level of QC2 and were excluded from all downstream analyses. Normalization of miRNA expression data on the remaining samples was performed using DeSeq217 (v. 1.14.1) in the HTG reveal software (v.3.0.0, HTG Molecular, Tucson, Ariz., USA). MiRNAs were considered detectable if they had expression levels of >5 counts per million in more than half of the samples. Expression counts were logarithmically scaled (log 10) for data visualization.
Simultaneous Multiplexed In Vitro Immunofluorescence: Cells were fixed in ice-cold methanol (Sigma-Aldrich, St Louis, Mo.) for five minutes at room temperature. Blocking was subsequently conducted in a solution containing 1% BSA (w/v, BioShop, Burlington, ON, CAN), 22.52 mg/mL glycine (ThermoFisher, Waltham, Mass., USA), and PBS−/− with Tween 20 (PBST, PBS−/−+0.1% Tween 20) for 30 minutes. Immunostaining was then conducted with diluted antibody (Table II) in 1% BSA PBST for 16 hours at 4° C. The cells were subsequently washed three times in PBS−/−, five minutes for each wash, and re-incubated with the concordant secondary antibodies in 1% BSA for one hour at room temperature in the dark. Post-washing, cells were counterstained with Vectashield Antifade Mounting Media with 4′,6-diamidino-2-phenylindole ([DAPI], Vector Laboratories, Burlingame, Calif., USA) and mounted with coverslips (VWR International, Mississauga, ON, CAN) and sealed with nail polish. Confocal images were taken using an Olympus Fluoview 1000 Confocal microscope Olympus IX81 inverted stand (Olympus, Calif., USA). Fluorochromes were excited using the following wavelengths: 405 nm for DAPI, 473 nm for Alexa Fluor 488, and 559 nm for Alexa Fluor 568 (ThermoFisher, Waltham, Mass., USA). A 20X/0.75NA UPLSAPO super apochromat objective was used to take the 20X images while a Plan Apo 40x/1.35 NA oil immersion objective was utilized for the 40X images. Image processing was done with the FV10-ASW 4.2 Viewer (Olympus, Calif., USA). Image intensities were calculated using FIJI21 (v2.1.0/1.53c), by examining the integrated density and dividing that by the number of DAPI positive cells within each image.
Data Visualization and Statistical Analysis: All data generated and analyzed which support the findings of this study are included in this article. Associated supplementary information files are available on a publicly accessible archive (see below). Descriptive Analysis—Clinical characteristics were characterized using summary statistics. Continuous variables were described using median and inter-quartile range (IQR), and dichotomous or polytomous variables were described using frequencies. Between-group differences were evaluated using Wilcoxon rank-sum tests for continuous variables and Fisher's exact tests for dichotomous/polytomous variables. Correlation between continuous variables were quantified using Spearman rank correlation. Descriptive outcome analysis—The Kaplan-Meier survival method was applied to assess the in-hospital death, and the between-group differences in the freedom from death were evaluated using log-rank tests. The length of hospitalization/ICU was characterized using competing risk models in terms of cumulative incidence rate function. Univariable Cox proportional hazard regression were applied to assess and quantify the association of the baseline clinical characteristics with in-hospital/ICU death. The associations of continuous variables were modeled using natural cubic splines. Biomarker Analysis—Comparisons between two independent groups were made using t-tests for normally distributed continuous variables or Wilcoxon rank-sum tests non-normally distributed continuous variables. When more than two groups were compared, either a one-way ANOVA with a Tukey or Bonferroni post-hoc test (where appropriate) for multiple testing correction, Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison correction. Two-way ANOVA was used to estimate how the mean quantitative variable changes according to time and group differences in leak experiments. Where appropriate, Benjamini-Hochberg false discovery rate (FDR) was utilized with adjusted P values (or Q value where stated) of <0.05 being considered statistically significant and indicated in the graphs as reported by the analysis software with significance thresholds of P<0.05, P<0.01, P<0.001, and P<0.0001 indicated as *, **, ***, **** respectively. MiRNA pathway analysis was conducted using BioCarta/KEGG/Reactome databases and tested for enrichment by a hypergeometric test with adjustment for multiple comparisons using the Benjamini-Hochberg FDR, with P<1.05 considered to be statistically enriched in a gene set of interest.22-24 Although many hypotheses were tested throughout the manuscript, no experiment-wide multiple test correction was applied. Unless indicated otherwise, graphs depict averaged values of independent data points with technical replicates and have error bars displayed as mean +/−standard deviation (±S.D.). Data were analyzed with GraphPad Prism 9.0.0 for MacOS (GraphPad Software, Inc., La Jolla, Calif., USA; Biomarker Multiple Comparisons), R25 (v4.0.3; Spearman Correlation Plots), and FIJI21 (v2.1.0/1.53c; Quantifying Image Intensities). Final figures were assembled for publication purposes using Adobe Illustrator (v25.4.1).
Risk Assessment Using Machine Learning: 250 experiments were performed using repeated randomized stratified sub-sampling cross-validation into 80% training and 20% testing using Python (v3.8.8) and scikit-learn26 (v.0.24.1). Categorical features were hot encoded, with missing variables recorded as additional categorical variables, having −1.0 for numerical features. For each experiment, a Random Decision Forest model was fit to the training dataset and evaluated on the independent testing set.27 Model performance was assessed by the area under the receiver operating characteristic (AUROC) calculated on the testing set. Average AUROC and 95% confidence intervals were calculated across the 250 runs using the percentile method. Feature importance was estimated using permutation feature importance and aggregated across the 250 runs. The microRNA model had a multiphase selection, whereby all microRNA were inputted into the first phase (feature selection via collinearity), upon which the remaining 102 microRNA were used to train the model.
Data Deposit: The data generated in this study have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus and are accessible through the GEO Series accession number (GSE; GSE178331, mRNA) and (GSE178246, miRNA).
Supplemental Tables
0.003
0.009
0.010
0.015
0.017
0.027
0.028
0.035
0.036
0.046
0.0071
0.0126
<0.0001
0.0158
<0.0001
<0.0001
0.0003
0.0126
0.0242
0.0013
0.0195
0.0068
†Body mass index is the weight in kilograms divided by the square of the height in meters.
‡Obesity is classified according to World Health Organization guidelines (i.e., >25).
<0.001
0.005
0.006
0.010
0.012
0.017
0.017
0.020
0.030
0.035
0.038
0.041
0.042
1. A method of treating Coronavirus Disease 2019 (COVID-19) in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject, the method comprising administering said subject with Slit2, or a variant or fragment thereof.
2. The method of 1 comprising administering said subject with a full-length Slit2.
3. The method of 1, comprising administering said subject with a N-terminal domain of Slit2 (Slit2-N).
4. A method of treating Coronavirus Disease 2019 (COVID-19) in a subject or inhibiting the development of COVID-19 or a sequelae resulting from COVID-19 in a subject, the method comprising upregulating the Slit2 signaling pathway in the subject.
5. The method of 4, comprising administering said subject with one or more factors or polypeptides from the Slit2 signaling pathway.
6. The method of 4, wherein the one or more factors or polypeptides from the Slit2 signaling pathway is GIT1, ARF6, Ab1, GTPase, Ena, Myo9b, or Hakai.
This application claims the benefit of U.S. Provisional Application No. 63/290,560, filed Dec. 16, 2021 and having same title, the entire content of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63290560 | Dec 2021 | US |
