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 inflammatory 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 significantly different between patients with severe and critical COVID-19 indicating their potential as biomarkers for severity, the D-dimer levels were not significantly different. Significantly elevated levels of the inflammatory 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 inflammatory cytokines for the COVID-19 classification 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 inflammation 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 defined, 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 inflammation, 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 significantly different between the two groups while the PT-INR and D-dimer levels were not significantly different. In addition, the ISTH score, as an index for evaluating coagulopathy, also showed no significant 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 (30–32) 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 significant 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 (33–35). 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 significantly 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 significant 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 significant 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 heparan- and chondroitin-sulfate chains that mediate cellular responses to signaling molecules as well as cell-cell and cell-matrix interactions (39). During inflammation, 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 inflammation, 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 inflammation, 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 identified 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 confirmed 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 findings, and physical findings, in a single institution. These hypothesis-generating 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 beneficial effects of specific therapeutic strategies may be diluted by patient and disease heterogeneity, suggesting that a personalized treatment approach is required. Our study revealed significantly 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 inflammation, 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.