The main finding of the present study is that both biomarkers (NIHSS and sVCAM-1) obtained within 24 h of a patient's hospital admission and diagnosed with IS performed well in predicting short-term mortality. When used together, these two biomarkers correctly classified 86.5% of the IS patients regarding their short-term mortality. This study highlights the role of NIHSS and sVCAM-1 in the prediction of mortality following the IS.
The application of the NIHSS is well-established in the literature and in IS therapy guidelines. It's even used as a criterion for deciding on reperfusion therapy and monitoring the patient's progress (Powers, 2020). Previous research indicates that NIHSS scores, whether or not they are related to other indicators, may be effective in predicting functional prognosis and death following IS (Chen et al., 2021; Kusvuran Ozkan et al., 2013; Saber; Saver, 2020; Zhao et al., 2018; Lehmann et al. 2022). According to Lehman et al. (2021), high NIHSS scores at admission strongly predict short-term disability as well as short-term mortality after IS.
Given the close pathophysiological relationship between neuroinflammatory mediators and processes related to poor outcome in IS, the role of molecules that are part of different inflammatory pathways, such as acute phase proteins, pro-inflammatory and anti-inflammatory cytokines, and endothelial dysfunction as prognostic biomarkers of IS may be expected. CAMs play a key role in leukocyte infiltration into active endothelia and their expression increases after ischemic injury. CAMs mediate the molecular interactions between endothelium and leukocytes, such as rolling, adherence, and transendothelial migration of leukocytes that are required for these cells to pass through the BBB and reach the ischemic brain (Krieglstein; Granger, 2001; Ramiro et al., 2018; Yang et al., 2019). The immunoglobulin gene superfamily (ICAM-1 and 2, VCAM-1, and PECAM-1), selectins (E-, P-, and L-selectin), and integrins are types of CAMs. These molecules have been investigated as biomarkers and treatment targets for IS (Krieglstein; Granger, 2001; Ramiro et al., 2018; Yang et al., 2019).
VCAM-1 is a protein expressed on the surface of endothelial cells. Its expression is minimal on unstimulated endothelium but is upregulated by a number of pro-inflammatory cytokines, such as IL-1β and TNF-a. The adherence of lymphocytes and monocytes in inflamed vascular beds is mediated by the VCAM-1 (Krieglstein; Granger, 2001; Ramiro et al., 2018; Yang et al., 2019).
Previous research indicates that factors such as age and smoking influence VCAM-1 levels. Cavusoglu et al. (2004) found levels of sVCAM-1 in smokers are substantially higher than in nonsmokers. Smoking possibly causes an increase in sVCAM-1, which could be another mechanism for cigarette smoking's negative effects on the atherosclerotic process and its consequences.
Regarding the positive effect of age on the sVCAM-1, our result is in agreement with previous studies. Purschwitz et al. (2001) and Miles et al. (1997) reported an age-dependent increase in sVCAM-1 and sICAM-1 in human subjects. Age-related accumulations of ROS and reactive nitrogen species (RNS) are followed by a decrease in total thiol content, resulting in a net increase in oxidative stress. Consequently, vascular endothelial cells generate activation/dysfunction with elevated levels of sE-selectin, sP-selectin, sVCAM-1, and sICAM, which contributes to an increase in the incidence of vascular disorders with aging (Zou et al. 2004). These authors demonstrated that older animals had the highest levels of CAMs, indicating a higher vulnerability to the inflammatory stimuli with age and highlighting the crucial role that inflammation plays in age-related changes of CAMs (Zou et al., 2004). On the other hand, Morisaki et al. (1997) showed that sVCAM-1, but not sICAM-1, was positively correlated with age; moreover, a negative correlation between sE-selectin, sICAM-1, and sVCAM-1 levels and age was suggested (Nash et al., 1996).
Other biomarker that exerted a role in the CAMs levels in the present study was homocysteine. Homocysteine has also been studied as a factor that stimulates the expression of VCAM-1 and affects the progression of atherosclerotic lesions. Silverman et al. (2002) demonstrated increased adhesion of monocytes to VCAM-1-dependent endothelial cells after treatment of aortic endothelial cells with homocysteine but without explaining the underlying mechanism. Caluccio et al. (2007) demonstrated that homocysteine affects the expression of CAMs, mainly VCAM-1, by increasing their gene expression. Our findings are in agreement with these studies in which age, homocysteine, and smoking had an effect on CAMs levels, revealed by the correlation between age and homocysteine with sVCAM-1 levels.
The immunocytochemical study of brain tissue from patients who died after IS revealed intense expression of VCAM-1 by endothelial cells and infarct astrocytes (Ramiro et al., 2018; Zaremba; Losy, 2002). When VCAM-1 levels were compared between IS patients and controls, several authors found that the IS group had higher levels (Bitsch et al., 1998; Blann, 1999; Licata et al., 2009; Tuttolomondo et al., 2009). The increase in sVCAM-1 was also observed by Blann et al. (1999) when compared healthy controls and individuals with carotid atherosclerosis, and by Fassbender et al. (1995) when compared healthy controls and individuals with vascular risk factors. This change could be due to VCAM-1 upregulation in the acute phase of IS, which does not occur in individuals who do not have ischemia. VCAM-1 levels remained elevated even after three month-follow-up according to Blann et al. (1999). These findings suggest the possibility that VCAM-1 may be expressed in both the acute and chronic phases of ischemic injury and that it may play a role in both infarction and tissue repair. Other study also showed that patients with IS had a persistent increase of sVCAM (Fassbender et al., 1995).
According to Bitsch et al (1998), soluble CAMs levels exhibit substantial variation in the kinetics after the IS. While sICAM-1 levels peaked within 24 hours, sVCAM-1 levels peaked after five days, and sE-selectin levels declined after five days. These authors also showed that there was no correlation of soluble CAMs levels with infarct volume or clinical disability. However, their negative result could be explained by the small number of IS patients (n=26) included in the study and the large interindividual variability of the CAMs levels.
Other studies also failed to find association between CAMs and IS. Compared with control subjects, sP-selectin and sE-selectin were significantly elevated in acute stage of IS, but also in symptomatic carotid stenosis; however, sICAM-1 and sVCAM-1 were not increased in these patients (Frijns et al.,1997). Supanc et al. (2011) found no significant difference between the levels of sVCAM-1 and sICAM-1 when IS patients and controls were compared, with marked biological interindividual variability in all groups. Only the levels of sVCAM-1 were significantly higher in patients with the CEI subtype than in controls. Moreover, these authors found no significant correlation between the levels of sVCAM-1 and sICAM-1 and IS severity and disability.
On the other hand, increased plasma levels of sICAM-1, sVCAM-1 and sE-selectin were associated with IS, independent of age, sex and other conventional risk factors for IS (Simundic et al., 2004). Moreover, sICAM-1 and sVCAM-1 levels were significantly higher in IS patients who non-survived compared to those who survived; however, after adjusting for various confounding factors, only sICAM-1 levels were independently associated with early death (Rallidis et al., 2009).
Regarding the association of CAMs with IS prognosis, our results are in agreement with the MITICO study, which showed that patients with higher sVCAM-1 levels at admission had a higher probability of IS recurrence than those with lower sVCAM-1 levels (Castillo et al., 2009). In another investigation (Richard et al., 2015), high level of sVCAM-1 in the second to third week after IS was associated with a worse prognosis after three months. Corroborating our findings, the Cox regression analysis of the present study, hierarchical regression models confirmed the association of NIHSS and sVCAM-1 with mortality after three-month follow-up, and patients with higher sVCAM-1 and NIHSS scores died earlier than the others of the present cohort.
Licata et al. (2009) and Tuttololondo et al. (2009) found no difference between IS subtypes in the TOAST classification despite evidence of higher sVCAM-1 levels in IS patients compared to controls. In our study, when the CAMs were analyzed in a dichotomized group, LAAS versus non-LAAS, the LAAS subtype was associated with higher levels of sVCAM-1 when compared to the non-LAAS. However, after adjusting by age, this association did not remain significant. As a result, age is a more critical determinant for sVCAM-1 levels in our study than the IS subtype.
After cerebral ischemia, cerebral endothelium is capable of expressing high levels of CAMs and recruiting large numbers of leukocytes and platelets. Initially, the predominant leukocytes are neutrophils followed by a more sustained increase in mononuclear cells and T lymphocyte cells. This recruitment is a highly coordinated and well-regulated two-step process that involves different CAMs expressed on vascular endothelium and circulating cells. In the first step, occurs low affinity binding manifested as rolling mediated by P-selectin upregulation as early as 15 minutes following an ischemic and E-selectin upregulation within 2 hours of ischemia. In the second step of recruitment, occurs high affinity interaction mediated by ICAM-1 that results in firm adhesion of leukocytes in cerebral microvessel. Lymphocytes and other mononuclear cells also express the α4 integrin named very late antigen-4 (VLA-4), which adheres the cell to the endothelium through its interaction with VCAM-1 (Yilmaz; Granger, 2008). Based on what is known about the expression of CAMs after IS, modulation of the VCAM-1/VLA-4 interaction may be a good strategy for reducing the post-ischemic inflammatory response than inhibition of other CAMs (Becker et al., 2001)
The importance of VCAM-1 was also demonstrated by a recent review based on results of protein-protein interaction network, enrichment, and annotation analyses. This study indicates that the activated immune response, as demonstrated by increased levels of cytokines (IL-6, IL-10, TNF-α), MACs (VCAM-1, E-selectin) and the positive acute phase protein (C-reactive protein) but lowered anti-inflammatory cytokine (TGF-β1) and negative acute phase protein (albumin), together with the activated hemostasis, thrombosis and coagulation pathways, as demonstrated by increased levels of Factor VIII, von Willebrand, and fibrinogen but reduced levels of protein C, Protein S, antithrombin, and albumin, are interrelated phenomena associated with short-term mortality after IS (Maes et al., 2021).
The main ligand of VCAM-1, the VLA-4, has been studied in the treatment of IS. Preclinical and monoclonal-clinical studies on VLA-4 that reduce leukocyte infiltration, reduce infarct volume, and improve stroke, though not in all animal models (Becker et al., 2001; Llovera et al., 2015). Researchers investigated the use of natalizumab, a monoclonal antibody that targets the α4-β1 integrin of VLA-4 in acute IS in the ACTION (Elkins et al., 2017) and ACTION II (Elkind et al., 2020) trials. Although no reduction was observed in focal infarct growth from day 1 to day 5 day 1 and day 5, as the primary study endpoint, a functional outcome improvement sustained over 90 days was observed particularly in subgroups of patients with smaller infarcts in ACTION trial (Elkins et al., 2017). Regarding the ACTION II trial, natalizumab administered ≤ 24 hours after IS did not improve patient outcomes (Elkind et al., 2020).
The discrepancies in results between studies of sVCAM-1 and other CAMs in IS may be explained by the differences in patient inclusion and exclusion criteria, control group definitions, time between IS ictus and sample collection, or laboratory methods. Previous studies (Bitsch et al., 1998; Frijns et al., 1997) determined the CAMs levels with enzyme immunoassay (ELISA), a less sensitive method compared to immunofluorimetric assay that was used in the present study. Our study excluded all patients with evidence of acute infections on admission, chronic infections, in addition to autoimmune and auto-inflammatory diseases, and the use of corticosteroids and immunosuppressive medications. As a result, the possibility of the increase in sVCAM-1 being caused by other systemic inflammation and not by acute ischemic brain injury was reduced.
The present study has some limitations and strengths. First, the rigid inclusion and exclusion criteria limited the number of samples; however, they allowed an analysis with few confounding factors. Second, the samples were collected in the first 24 hours after the admission on the emergency room of the university hospital, the most acute phase of the event. The obtention of samples during the follow-up period could show the kinetics of the CAMs levels after cerebral ischemia and their correlation with the outcome. On the other hand, the evaluation of the potential role of CAMs in IS patients from the Brazilian population is scares and necessary and our study proposed the evaluation of predictive models with a panel of combined biomarkers. Given the heterogeneity and dynamic nature of the cellular and molecular changes that occur after IS, a single biomarker will not accurately predict all adverse outcomes of the ischemic event. The most indicated will be the combination of different biomarkers of the different pathways involved in the pathophysiology of IS, in order to achieve more significant results.
Taken together, our results underscore the important role of CAMs in the pathophysiology of IS and highlight the potential use of NIHSS and sVCAM-1 as possible biomarkers of short-term mortality in IS patients. While systematic reviews of the role of blood biomarkers in the diagnosis of IS indicate that these tests cannot be recommended in clinical practice, it is highly desirable that new studies be conducted so that a faster diagnosis of IS may be possible, even before the patient arrives at the hospital, using biological markers of cerebral ischemia or inflammation (Gagliardi et al., 2005). Research on the role of the CAMs in the pathophysiology of IS might yield useful biomarkers and treatment targets. Therefore, interventions that impede lymphocyte trafficking into the brain and lymphocyte activation may be potential therapeutic approaches for reducing brain injury after IS. Thus, medications designed to minimize endothelial activation and specifically VCAM-1 may be a viable strategy for preventing poor outcome after IS.