Annual Changes in Serum Syndecan-1 as a Therapeutic Target and a Biomarker for the Severity Classication of COVID-19

Background: Coronavirus disease 2019 (COVID-19) pneumonitis associated with severe respiratory failure is associated with high mortality. The pathogenesis of COVID-19 is associated with microemboli or microvascular endothelial injuries. Here, we report that serum syndecan-1 (SDC-1), a component of the endothelial glycocalyx, may be a biomarker of severity classication for COVID-19 related to endothelial injury. Methods and analysis: We analyzed the data of COVID-19 patients for one year from February 2020 at Yokohama City University Hospital and Yokohama City University Medical Center Hospital. We selected COVID-19 patients who required admission care, including intensive care, and analyzed the classication of severe and critical COVID-19 retrospectively, using various clinical data and laboratory data with serum SDC-1 by ELISA. Results: We analyzed clinical and laboratory data with SDC-1 in ve severe COVID-19 and ten critical COVID-19 patients. In the two groups, their backgrounds were almost the same. In laboratory data, the LDH, CHE, and CRP levels showed signicant differences in each group (P=0.032, P <0.0001, and P = 0.007, respectively) with no signicant differences in coagulation-related factors (platelet, PT-INR, D-dimer, ISTH score; P=0.200, 0.277, 0.655, and 0.36, respectively). For the clinical data, the SOFA score was signicantly different from admission day to day 14 of admission (p<0.0001). The SDC-1 levels of critical COVID-19 patients were signicantly higher on admission day and all-time course compared with the levels of severe COVID-19 patients (P=0.009 and P <0.0001, respectively). Conclusions: Annual changes in serum SDC-1 levels closely reect the severity of COVID-19, therefore, serum SDC-1 may be a therapeutic target and a biomarker for the severity classication of Covid-19.


Introduction
The novel coronavirus disease 2019 (COVID-19) pandemic, as declared by the World Health Organization, is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) (1,2) with an overall mortality rate of approximately 31% of those admitted to an Intensive Care Unit (ICU) (3). Recent studies have reported a high prevalence of thrombotic events in COVID-19, similar to the high venous thromboembolism (VTE) rates observed in patients with other viral pneumonias, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS-CoV) (4)(5)(6)(7). Based on the changes in the plasma levels of d-dimers, brinogen-degradation products, and antithrombin, a prothrombotic state in COVID-19 has been suggested (6). Furthermore, an elevated d-dimer level at hospital admission has also been associated with increased odds of in-hospital death (8). Thrombotic complications and micro-thrombosis in the pulmonary vasculature have also been observed on autopsies on COVID-19 patients (9,10). While the mechanisms underlying thrombotic risk in COVID-19 patients are unclear, thrombosis developed in critically ill patients through the dysregulation of coagulation and/or endothelial injury (11,12). Therefore, venous abnormalities have emerged as important considerations in the management of hospitalized patients with COVID-19.
Microvascular endothelial cell injury also precipitates thrombosis (11), with or without coagulation abnormalities, particularly in the alveolar capillary where COVID-19 pneumonia and lung injury are observed clinically (10). Finally, platelet adhesion to the microvasculature is largely inhibited by the glycocalyx, a gel-like substance that coats the luminal surface of endothelial cells (13,14). The glycocalyx modulates leukocyte-endothelial interactions, thrombus formation, and other processes that lead to microcirculatory dysfunction and critical organ injury in sepsis. It also acts as a regulator of vascular permeability and contains mechano-sensors as well as receptors for growth factors and anticoagulants. In ammation-induced degradation of the glycocalyx is thought to contribute to microvascular pathology and thrombosis formation in sepsis of various etiologies. In a component of the endothelial glycocalyx, we focused on syndecan-1 (SDC-1) because the role of SDC-1 can be induced on the surface of endothelial cells in the context of wound healing and in ammation, suggesting a role for syndecan-1 in the interaction of leukocytes with endothelial cells, The overall aim of this hypothesis-generating study was to characterize the changes serum SDC-1, during the hospitalization of COVID-19 patients and it usefulness as a biomarker for the severity classi cation of COVID-19 in terms of endothelial injury.

Patients And Methods
In this retrospective study, we used the data of COVID-19 patients (except for those without symptoms or mild symptoms) who underwent standard treatment, including intensive care at our department of the Yokohama City University Hospital and Yokohama City Hospital Center Hospital from February 2020 to December 2020, to analyze their clinical and biological features. Risk factors, morbidity, and mortality outcomes were also analyzed. Comorbidities were obtained for each patient, and outcome data were obtained at the follow-up in our hospital. This study was approved by the Ethics Committee of the Yokohama City University School of Medicine. Written informed consent was obtained from each patient or family before treatment.
Disease severity was categorized into four stages, that is, mild, moderate, severe, and critical, based on previously published guidelines on the diagnosis and treatment of novel coronavirus.(15) Brie y, mild cases were de ned as no symptoms and no need of oxygen without pneumonia in computed tomography (CT) scan. Moderate cases were de ned as mild respiratory symptoms, radiological evidence of pneumonia, and 93%< SpO 2 < 96%. Severe cases were de ned as SpO 2 ≤ 94%, requiring oxygen support. Critical was de ned as requiring heart-lung machine or extracorporeal membrane oxygenation (ECMO) support for the acute respiratory distress syndrome (ARDS). We combined this with severe and critical, referred to as critical in our study.
We collected blood samples daily, starting at admission depending on the clinical condition of the severe COVID-19 patients who needed oxygen and the critical ones who needed intubation management. The severity classi cation of treatment strategies for the patients with critical COVID-19 are shown in Table 1.
Upon admission to our hospital, we made a diagnosis of COVID-19 using a positive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for SARS-CoV-2 in the respiratory tract and laryngeal swab samples tested by a designated diagnostic laboratory.

Sample Collection
Blood Draws Standard operating procedures were used to ensure that all the samples were treated rapidly and equally. Blood was obtained from critically ill ICU patients via indwelling catheters daily in the morning and placed immediately on ice. Once transferred to a negative pressure hood, blood was centrifuged and plasma was isolated, aliquoted at 500μL, and frozen at -80°C. All the samples remained frozen until use, and the freeze/thaw cycles were minimized. All the plasma analytes were measured in duplicate with enzyme-linked immunosorbent assays (ELISA) as per the manufacturer's recommendation.

Results
We investigated ve patients with severe symptoms and 10 patients with critical symptoms with a positive diagnosis of COVID-19 (median age, 73.0 [IQR, 67-77] years). Serum samples that could be tested retrospectively were extracted from the admission day to day14 after admission because all the clinical and test data were collected at an accurate time point. Three critical patients were managed with mechanical ventilation, four ECMO were patients, and three death patients. Baseline demographic characteristics, admission day after onset, comorbidities, and APACHE II score on admission day are reported in Table 2. All patients had typical COVID-19 pneumonia ndings on CT scans, such as bilateral grand glass opacities (GGOs), and consolidations caused respiratory insu ciency with associated hypoxemia in both groups. The day of admission after onset was signi cantly different in both groups (P = 0.0160). All the other reported baseline measures were nonsigni cant between the patients. Regarding hemostatic parameters, platelet counts over time during the observational period were not signi cantly different between the two groups (Ps: 0.200, 0.056, 0.902, for group, time differences, and their interaction, respectively) (Fig. 1A), and the severe group had consistently higher platelet counts from the beginning of hospitalization than the critical group. Similarly, there were no signi cant differences in PT-INR (Ps: 0.227, 0.686, 0746, for group, time differences, and their interaction, respectively) and D-dimer levels (Ps: 0.655, 0.695, 0.220, for group, time differences, and their interaction, respectively) between the two groups (Fig. 1B, 1C). Therefore, the hemostatic parameters were not signi cantly different between the two groups in our study.
In the biomarkers of each organ, WBC had no signi cant difference between the two groups (Ps: 0.219, 0.299, 0.163, for group, time differences, and their interaction, respectively) (Fig. 1D), but the C-reactive protein (CRP), reported as a prognostic factor, was signi cantly different between the two groups (Ps: 0.007, 0.257, 0.257, for group, time differences, and their interaction, respectively) (Fig. 1E). Regarding organ biomarkers, there were almost no signi cant differences between the two groups. However, the LDH (Ps: 0.032, 0.949, 0.784, for group, time differences, and their interaction, respectively) and CHE (Ps: <0.0001, 0.002, 0.138, for group, time differences, and their interaction, respectively) levels were signi cantly different between the two groups ( Fig. 1F, 1G).
In terms of severity of illness and the coagulation scores, the SOFA scores were signi cantly different between the two groups (Ps: <0.0001, 0.236, 0.163, for group, time differences, and their interaction, respectively) ( Fig. 2A) because critical patients needed to reduce their levels of consciousness with sedation to enable intensive care with mechanical ventilation for respiratory disorders and; moreover, use vasopressors for the hypotension caused by sedation as a counter-measure, compared to severe patients. In contrast, the ISTH scores were not signi cantly different between the two groups (Ps: 0.360, 0.041, 0.608, for group, time differences, and their interaction, respectively) (Fig. 2B).
We measured SDC-1, one of the components of the glycocalyx as an endothelial cell injury marker in serum using ELISA. Figure 3 shows that the SDC-1 levels were elevated signi cantly in the critical COVID-19 patients compared with the severe COVID-19 patients, with signi cant differences on admission day and over time in both groups (Ps: <0.0001, < 0.0001, 0.009, for group, time differences, and their interaction, respectively). The data suggests that SDC-1levels re ect the severity of disease and the main pathology of vasculitis in the lungs.

Discussion
In this study, we analyzed the relationship between the severity of COVID-19 and SDC-1, focusing on one of the components of the glycocalyx as an indicator of endothelial injury with coagulopathy and as a biomarker for the severity of COVID-19. Previous reports that have been extensively cited (16, 17), have suggested that the hyperactivation of the in ammatory cascade, leading to cytokine storm, is a critical biological response in patients with severe COVID-19. From regular blood tests, D-dimer, LDH, CHE, and CRP levels have been reported previously as potential biomarkers for severity (16, 17). In our study, although LDH, CHE, and CRP levels were signi cantly different between patients with severe and critical COVID-19 indicating their potential as biomarkers for severity, the D-dimer levels were not signi cantly different. Signi cantly elevated levels of the in ammatory cytokines TNF-α, IL-1, IL-6, IFN-λ3, IL-6, IP-10, and CXCL9 have been documented in severe COVID-19 compared to non-severe disease cases (18-22).
In our study, we did not focus on in ammatory cytokines for the COVID-19 classi cation of severity.
It has been reported that COVID-19 respiratory distress may be due to micro-emboli or endothelial injuries associated with respiratory deterioration and death (8, 23). In addition, many critical COVID-19 patients developed venous thromboembolism, which appeared to be related to coagulopathy (6, 24). In particular, VTE emerged as an important consideration in the management of hospitalized patients with COVID-19.
In recent years, common pathways for venous thrombosis have been described, with in ammation and hypercoagulation being key factors in the mechanism of venous thrombotic events (25). These concerns should be balanced by emerging data that the incidence of VTE in hospitalized critical COVID-19 patients or in ICU settings were higher than that reported by historical data in similar patients, with an incidence of VTE of 27% in a previous study using standard thromboprophylaxis and an incidence of 25% in another study without prophylaxis (4,6,26). Although the mechanisms underlying vascular thrombosis in COVID- 19 have not yet been clearly de ned, several have been postulated. Activation of the complement system leads to endothelial cell injury and death with subsequent vascular denudation and exposure of the thrombogenic basement membrane, which drives the activation of clotting cascades. These events result in in ammation, microvascular thrombosis, vessel edema, and hemorrhagic sequelae, all of which are prominent features of lung pathology in patients with COVID-19-associated pneumonia (27). In an autopsy study of ten patients with COVID-19, small vessel thrombi formation in the lung peripheries were associated with foci of alveolar hemorrhages (28). Many researchers have focused on coagulopathy in COVID-19 by investigating coagulation-related factors. In our study, the platelet counts were signi cantly different between the two groups while the PT-INR and D-dimer levels were not signi cantly different. In addition, the ISTH score, as an index for evaluating coagulopathy, also showed no signi cant differences. Since we administered therapeutic doses of anticoagulant therapy targeting the micro-thrombi of COVID-19 in all the cases, many coagulant factors other than platelet counts may have been improved by anticoagulant therapy. Therefore, it may not be useful as a predictor of the severity of COVID-19. For anticoagulant therapy, while the World Health Organization (WHO) recommended therapeutic anticoagulation rather than intermediate dosing (29) the optimal thrombo-prophylactic strategy in the critically ill hospitalized COVID-19 patient population remains uncertain (conditional recommendation, very low certainty). However, the thrombotic tendencies in COVID-19 promote VTE formation in intensive care management, so we suggest that therapeutic anticoagulant doses are more appropriate than intermediate-dose anticoagulants.
In contrast, we focused on endothelial injury by SDC-1 as a part of the glycocalyx. Our data indicate increased serum SDC-1 and continuous changes in SDC-1, a core protein of the glycocalyx whose degradation indicates endothelial injury (30)(31)(32) in critical COVID-19 patients relative to severe COVID-19 patients at 2 weeks of admission. Serum SDC-1 level at 2 weeks of admission showed no signi cant differences in critical COVID-19 patients, under mechanical ventilation, ECMO management, and death.
Serum SDC-1 levels in critical COVID-19 patients have been reported to be related to respiratory disorders of COVID-19 (33)(34)(35). These reports showed that critically ill patients with COVID-19 had higher serum SDC-1 levels than healthy controls. Therefore, the relationship between treatment progress and serum SDC-1 levels in critically ill patients with COVID-19 remains controversial. Our data suggest that COVID-19 results in endothelial injury and the degradation of the endothelial glycocalyx. The SDC-1 levels were all elevated signi cantly on admission day in the plasma of COVID-19 patients and they remained elevated persistently, in the plasma up to day 14 after admission. Oda et al. reported that 78 healthy individuals receiving no treatment and with no relevant medical history or laboratory data reported a median serum SDC-1 concentration of 19.3 (36). As a preliminary experiment (data not shown), we analyzed the SDC-1 levels of COVID-19 negative pneumonia patients suspected of COVID-19 pneumonia when they presented at our department with some symptoms. Compared with a volunteer healthy control, there were signi cant differences in the serum SDC-1 levels between the COVID-19 positive patients and the healthy controls (SDC-1 concentration of healthy control 23.6 ng/ml; P = 0.043), nevertheless there were no signi cant differences with the COVID-19 negative pneumonia group (SDC-1 concentration of COVID-19 negative pneumonia patient 40.8 ng/ml; P = 0.238). The glycocalyx is a complex structure composed of glycosaminoglycans (e.g., hyaluronic acid and chondroitin sulfate), proteoglycans (e.g., syndecan-1 and heparan sulfate), and various plasma proteins (e.g., albumin and antithrombin). Disturbance of the glycocalyx, often due to the increased expression and release of proteinases and glycosidases (e.g., hyaluronidases, sheddases, and matrix metalloproteinases), and has profound consequences on vascular function (37). For example, loss of glycocalyx components decreases nitric oxide production and increases oxidant production, thereby facilitating ligand-receptor interactions and subsequent platelet recruitment to the vascular endothelium (38). SDC-1 is a proteoglycan containing both heparanand chondroitin-sulfate chains that mediate cellular responses to signaling molecules as well as cell-cell and cell-matrix interactions (39). During in ammation, the SDC-1 inhibits neutrophil adhesion and migration. The shedding of SDC-1 from the cell surface is initiated by the heparanase-dependent removal of the heparan-sulfate side chains (40), thereby instigating subsequent cleavage of the core SDC-1 protein by enzymes such as matrix metalloproteinases. Moreover, while moderate SDC-1 shedding is thought to aid in resolving in ammation, excessive shedding is likely pathogenic, as the complete loss of SDC-1 allows for increased leukocyte adhesion and recruitment across the endothelial monolayer, as well as enhanced platelet aggregation and adhesion. Platelet interaction with activated pulmonary endothelial cells, is at least in part due to the glycocalyx degradation and the subsequent decrease in nitric oxide production which promotes vascular occlusion, enhances in ammation, and drives viral pathogenesis. Inhibition of this interaction, through antiplatelet or thrombolytic therapies, can represent a potential therapeutic strategy (i.e., using reduced doses of recombinant tissue-type plasminogen activator over prolonged periods) for the treatment of severe viral infections, such as COVID-19. Therefore, our analysis indicated that serum SDC-1 may be a biomarker for the severity of severe COVID-19. In addition, if some treatment to remove glycocalyx from the endothelium can be established, it may contribute to a reduced oxygen administration period, and shortened mechanical ventilation and ECMO administration periods, thereby improving the prognosis, and reducing unexplained sequelae after COVID-19, including respiratory symptoms.
Our study identi ed a unique prothrombotic state in critically ill COVID-19 patients that may be amenable to therapeutic targeting. However, our study had several limitations. First, this study was performed in a single hospital with a small study population, since there are currently few con rmed and recovered cases of COVID-19 in Japan. Therefore there may have been selection bias, in that the clinical data of our selected cases were known and while their serum was collected according to the protocol from the day of admission to the 14th day of admission without intentional selection, we should have analyzed all patients in our institute according to strict protocol. Second, no therapeutic treatment for VTE in critical COVID-19 patients was available for use in a parallel control group. However, we believe that the credibility of the therapeutic effect is high, as our study provided a comprehensive examination, including clinical features, laboratory ndings, and physical ndings, in a single institution. These hypothesisgenerating results are valuable for future studies on antithrombotic therapies and clinical trials. Finally, we reported mortality as a clinical outcome in our COVID-19 patients; however, future studies with larger sample sizes can explore whether reported changes in thrombotic factors and endothelial injury markers correlate with additional clinical outcomes such as hospital stay or mortality. The bene cial effects of speci c therapeutic strategies may be diluted by patient and disease heterogeneity, suggesting that a personalized treatment approach is required. Our study revealed signi cantly elevated serum SDC-1 levels in COVID-19 patients, suggesting that therapies to coagulopathy and to protect/restore the glycocalyx may be therapeutically indicated. One possibility is that recombinant thrombomodulin (rhTM), which is used to treat DIC, may be effective as a therapeutic agent because rhTM treatment affects in ammation, cell proliferation/differentiation, and glycocalyx synthesis in serum and lung tissue, subsequently attenuating the ARDS caused by endothelial injury in animal experimental models. Further clinical studies are required to validate this concept.

Conclusion
We suggest that it is important to analyze the serum SDC-1 levels of COVID-19 patients to determine their severity and prognosis. Further studies on detailed and early laboratory, clinical, and imaging characterizations are required to better understand the pathophysiology of endothelial injury in COVID-19 patients. Thus, the inhibition of the glycocalyx degradation may be a therapeutic target for critical COVID-19 patients.