Vascular smooth muscle (VSM) cell phenotypic expression and autophagic state are dynamic responses to stress. Vascular pathologies, such as hypoxemia and ischemic injury induce a synthetic VSM phenotype and autophagic flux resulting in a loss of vascular integrity and VSM cell death respectfully. Both clinical pilot and experimental stroke studies demonstrate that sphingosine-1-phosphate receptor (S1PR) modulation improves stroke outcome; however, specific mechanisms associated with a beneficial outcome at the level of the cerebrovasculature have not been clearly elucidated. We hypothesized that ozanimod, a selective S1PR type 1 ligand, will attenuate VSM synthetic phenotypic expression and autophagic flux in primary human brain VSM cells following acute hypoxia plus glucose deprivation (HGD; in vitro ischemic-like injury) exposure. Cells were treated with ozanimod and exposed to normoxia or HGD. Crystal violet staining, standard immunoblotting, and immunocytochemical labeling techniques assessed cellular morphology, vacuolization, phenotype, and autophagic state. We observed that HGD temporally decreased VSM cell viability and concomitantly increased vacuolization, both of which ozanimod reversed. HGD induced a simultaneous elevation and reduction in levels of pro- and anti-autophagic proteins respectfully, and ozanimod attenuated this response. Protein levels of VSM phenotypic biomarkers, smoothelin and SM22, were decreased following HGD. Furthermore, we observed an HGD-induced epithelioid and synthetic morphological appearance accompanied by disorganized cytoskeletal filaments which was rescued by ozanimod. Thus, we conclude that ozanimod, a selective S1PR1 ligand, protects against acute HGD-induced phenotypic switching and promotes cell survival, in part, by attenuating HGD-induced autophagic flux thus improving vascular patency in response to acute ischemia-like injury.

Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular injury is elevated levels of oxidized low‐density lipoprotein (OxLDL). Via the lectin‐like OxLDL receptor 1 (LOX‐1), OxLDL contributes to mechanisms associated with vascular dysfunction and inflammation. The impact of OxLDL/LOX‐1 on cerebrovascular endothelial dysfunction and inflammation in the setting of AIS remains to be elucidated. The aim of this study is to investigate the effect of OxLDL via LOX‐1 on endothelial proinflammatory mediator and integral barrier protein levels during in vitro ischemic‐like conditions. We hypothesized that acute ischemic injury would increase endothelial barrier dysfunction as well as inflammation and that OxLDL would exacerbate these responses in a LOX‐1 dependent manner. Primary male human brain microvascular endothelial cells (HBMEC) were preconditioned with human OxLDL (50μg/dL; 12h) or vehicle using a serum dose reported in AIS patients. Next cells were treated with BI‐0115 (selective LOX‐1 inhibitor; 10μM) or vehicle (<0.1% DMSO) for 0.5h prior to being exposed to normoxia (21% O2) or hypoxia plus glucose deprivation (HGD; 1% O2) for 6h in the continued presence of OxLDL or vehicle. Levels of HBMEC barrier protein (ZO‐1), adhesion molecule (VCAM‐1), and inflammatory proteins (iNOS, IL‐1β) were examined using FIJI mediated immunocytochemical analysis. In terms of barrier protein levels, we observed that ZO‐1 levels were decreased following HGD. Contrary to our hypothesis, OxLDL plus HGD resulted in increased levels of ZO‐1; and moreover, we observed a greater increase in ZO‐1 levels when treated with BI‐0115 alone or BI‐0115 plus OxLDL. During normoxic conditions OxLDL increased levels of ZO‐1 and this response was LOX‐1 independent. Concomitantly, HGD increased levels of the adhesion molecule, VCAM‐1. The presence of HGD plus OxLDL or OxLDL plus BI‐0115 had no effect on VCAM‐1 expression. During normoxic conditions, OxLDL increased VCAM‐1 levels in a LOX‐1 dependent manner. We next observed that HGD increased the levels of proinflammatory mediators, iNOS and IL‐1β. OxLDL under normoxic conditions significantly increased the levels of iNOS in a LOX‐1 dependent manner but had no effect on the levels of IL‐1β. HGD plus OxLDL had no effect on iNOS levels; however, treatment with BI‐0115 alone or BI‐0115 plus OxLDL significantly attenuated HGD‐mediated increases in iNOS levels. Moreover during HGD, OxLDL increased levels of IL‐1β in a LOX‐1 dependent manner suggesting that OxLDL acting through LOX‐1 leads to an increase in a central mediator of vascular inflammation and permeability. In conclusion, the beneficial effect of LOX‐1 inhibition on cerebrovascular endothelial permeability and inflammation suggests that this receptor may be a novel, viable therapeutic target in the treatment of AIS.

Cerebrovascular pathologies contribute significantly to acute ischemic stroke (AIS) outcome. Sphingosine‐1‐phosphate receptor (S1PR) modulators show beneficial effects against hypoxia plus glucose deprivation (HGD; in vitro ischemic‐like model)‐induced brain microvascular endothelial dysfunction and demonstrate clinical promise in improving cognitive AIS outcome in patients. However, the impact of S1PR modulation on vascular smooth muscle (VSM) health during ischemic injury has not been clearly elucidated. Therefore, the aim of this study was to investigate human brain VSM cell viability and autophagic responses following HGD. Adult male brain VSM cells were treated with ozanimod (selective S1PR type 1 ligand; 0.5nM) at P7 and exposed to normoxia (21% O2) or HGD (1% O2) for 9h, a timepoint based on cell survival studies conducted using trypan blue exclusion staining following HGD 3, 6, and 9h. Crystal violet staining determined vacuole presence and autophagic protein levels were examined using standard immunoblotting. Localization of autophagic markers were assessed via immunocytochemical labeling. We observed that HGD temporally decreased VSM cell viability and concomitantly increased vacuolization, and these responses were reversed by ozanimod. To characterize whether these vacuoles were of autophagosome phenotype, localization and punctation quantification of the upstream ubiquitin‐like complex I mediator, Atg5 and its associated downstream autophagic marker, LC3B, were assessed. Following HGD insult, increased density of Atg5 and LC3B punctate clusters were visualized and expression levels were increased. In the presence of ozanimod, HGD‐induced LC3B levels were attenuated suggesting that S1PR1modulation may regulate HGD‐induced vacuole formation during autophagic flux. Concomitantly, basal levels of the anti‐apoptotic and anti‐autophagic protein Bcl‐2 were significantly decreased following HGD, and ozanimod reversed this response. Further investigation revealed that while HGD did not alter LC3A/B‐I (non‐autophagic) levels, HGD‐induced LC3A/B‐II (autophagic) levels were increased but were attenuated by ozanimod. Autophagic protein levels of beclin‐1 and Atg7 were not altered following HGD. However, in the presence of HGD plus ozanimod, we observed a decrease in Atg7 levels suggesting that ozanimod may have a protective effect against autophagy during HGD. While we did not observe alterations in beclin‐1 protein levels across treatment groups, when combined with the decreased Bcl‐2 levels, the availability of beclin‐1 to mediate initiation of autophagy is enhanced following HGD via suppression of Bcl‐2 inhibition. The rescue of Bcl‐2 suggests that ozanimod may be suppressing the ability of beclin‐1 to mediate initiation of autophagy following HGD. In conclusion, S1PRtype 1 ligands promote cell survival in part by attenuating HGD‐induced autophagic flux in human brain VSM.

Cerebrovascular endothelial cells are among the first cells to be impacted by acute ischemic stroke (AIS) which poses them as important therapeutic targets pivotal to the maintenance of blood brain barrier (BBB) integrity. Following AIS, matrix metalloproteinase 9 (MMP‐9) proteolytic activity contributes to the loss in BBB integrity, increased hemorrhagic risk, and worse outcomes. We have previously shown that sphingosine‐1‐phosphate receptor (S1PR) ligands attenuated hypoxia plus glucose deprivation (HGD)‐induced decreases in brain microvascular endothelial trans‐endothelial resistance (TEER), suggesting a potential role for S1PR ligands on barrier function during AIS. Endothelial vascular cell adhesion molecule 1 (VCAM‐1) is an inflammatory mediator and adhesion molecule that contributes to the loss of endothelial barrier integrity by serving as a scaffold for immune cell extravasation as well as a mediator for intracellular signals involved in MMP‐9 activation. Cell surface adhesion receptor, CD44, likewise plays a key role in facilitating MMP‐9 activity via co‐localization at the endothelial surface. Thusly, we sought to investigate protein expression levels of VCAM‐1, MMP‐9, tight junctional proteins, (ZO‐1 and claudin‐5), as well as to characterize CD44 protein expression in cerebrovascular endothelial cells following HGD. Pediatric male human brain microvascular endothelial cells (HBMECs) were treated with ozanimod (selective S1PR type 1 ligand; 0.5 nM) at P7 and immediately exposed to normoxia (21% O2) or HGD (1% O2) for 3h, a timepoint determined from our previous TEER studies which revealed deterioration of the endothelial barrier as early as 3h. To verify S1PR type 1 dependency, selective blockade with antagonist W146 was applied to select experiments. We observed through standard immunoblotting that HGD induced a simultaneous decrease in ZO‐1 and claudin‐5 in parallel to increased expression of VCAM‐1. Additionally, intracellular pro‐MMP‐9 (inactive enzymatic form) levels decreased following HGD and the ratio of active MMP‐9 to pro‐MMP‐9 was increased. Ozanimod did not alter these responses. Using immunocytochemistry, anti‐CD44 fluorescence was increased following HGD exposure, and ozanimod attenuated this response. S1PR type 1 blockade demonstrated receptor dependence. In conclusion, HGD induced endothelial activation characterized by increased adhesion molecule levels and involvement of MMP‐9 activity potentially contributing to tight junction protein loss. Furthermore, S1PR type 1 ligands may in part attenuate HGD‐induced BBB integrity loss by modulating the bioavailability of MMP‐9 to disrupt endothelial barrier integrity. Mechanism(s) by which S1PR type 1 ligands regulate MMP‐9 activity and its associated cell surface adhesion proteins (e.g. CD44 and VCAM‐1) following ischemic‐like injury warrants further investigation.

The loss of vascular integrity at the level of the blood brain barrier leads to a vicious cascade of secondary injuries following acute ischemic stroke (AIS). Elevated MMP‐9 activity within the cerebral vasculature has been implicated with severe degradation of the vascular basement membrane leading to abnormal cerebrovascular permeability and detrimental stroke outcome. Sphingosine‐1‐phosphate receptor (S1PR) modulation improves stroke outcome in AIS patients, however the influence of selective S1PR1ligands, such as ozanimod, on brain endothelial health and MMP‐9 activity following AIS has not been investigated. Thus, the aim of this study was to determine the impact of acute ischemic injury on MMP‐9 activity in both the rat and the human cerebrovasculature as well as the vascular specific role of ozanimod on human endothelial health and MMP‐9 activity. Using an in vivo thromboembolic stroke model, cerebral vessels were isolated from male Wistar rats that underwent a right middle cerebral artery (MCA) thrombin injection. Sham‐operated animals underwent the same surgical procedure; however, nothing was injected. Vascular MMP‐9 enzymatic activity of was evaluated at 3, 6 and 24h post injury using zymography. Using an in vitro ischemic injury model (HGD; hypoxia plus glucose deprivation), male human brain microvascular endothelial cells (HBMECs; P7) were treated with ozanimod (0.5nM) or vehicle (DMSO) and then exposed to normoxia (21% O2) or HGD (1% O2). In some experiments, W146 (a selective antagonist), verified S1PR1 dependence. Morphology and vacuole formations were assessed using crystal violet staining. Enzymatic activity of MMP‐9 was evaluated via zymography and the extracellular H2O2 concentration, inducer of MMP‐9 activity, was measured using a colorimetric assay. Following in vivo thromboembolic occlusion, ipsilateral MMP‐9 activity increased at 6h post injury and returned to baseline when compared to sham. In comparison to the contralateral side, thromboembolic occlusion induced a time dependent modulation of ipsilateral MMP‐9 activity compared to the contralateral side with the highest activity peaking at 6h. In our in vitro HBMEC stroke model, MMP‐9 enzymatic activity was increased following 3h HGD exposure, and this response was attenuated by ozanimod. Concomitantly, during HGD, H2O2 production, a partial inducer of MMP‐9 activity, was increased in a time dependent manner when compared to normoxic controls. Furthermore, HGD induced an increase in HBMEC vacuole formations and a decrease in cell viability at 3h. Loss of cell viability was rescued with ozanimod. In conclusion, increased MMP‐9 activity, in part due to H2O2, is an acute response to ischemic injury in the cerebrovasculature. Specifically, ozanimod's ability to attenuate endothelial MMP‐9 activity may play an important beneficial role in mitigating blood brain barrier integrity loss following an acute ischemic injury.