Urokinase Plasminogen Activator: A Potential Thrombolytic Agent for Ischaemic Stroke

Stroke continues to be one of the leading causes of mortality and morbidity worldwide. Restoration of cerebral blood flow by recombinant plasminogen activator (rtPA) with or without mechanical thrombectomy is considered the most effective therapy for rescuing brain tissue from ischaemic damage, but this requires advanced facilities and highly skilled professionals, entailing high costs, thus in resource-limited contexts urokinase plasminogen activator (uPA) is commonly used as an alternative. This literature review summarises the existing studies relating to the potential clinical application of uPA in ischaemic stroke patients. In translational studies of ischaemic stroke, uPA has been shown to promote nerve regeneration and reduce infarct volume and neurological deficits. Clinical trials employing uPA as a thrombolytic agent have replicated these favourable outcomes and reported consistent increases in recanalisation, functional improvement and cerebral haemorrhage rates, similar to those observed with rtPA. Single-chain zymogen pro-urokinase (pro-uPA) and rtPA appear to be complementary and synergistic in their action, suggesting that their co-administration may improve the efficacy of thrombolysis without affecting the overall risk of haemorrhage. Large clinical trials examining the efficacy of uPA or the combination of pro-uPA and rtPA are desperately required to unravel whether either therapeutic approach may be a safe first-line treatment option for patients with ischaemic stroke. In light of the existing limited data, thrombolysis with uPA appears to be a potential alternative to rtPA-mediated reperfusive treatment due to its beneficial effects on the promotion of revascularisation and nerve regeneration

The secretome of endothelial progenitor cells: a potential therapeutic strategy for ischemic stroke

Ischemic stroke continues to be a leading cause of mortality and morbidity in the world. Despite recent advances in the field of stroke medicine, thrombolysis with recombinant tissue plasminogen activator remains as the only pharmacological therapy for stroke patients. However, due to short therapeutic window (4.5 hours of stroke onset) and increased risk of hemorrhage beyond this point, each year globally less than 1% of stroke patients receive this therapy which necessitate the discovery of safe and efficacious therapeutics that can be used beyond the acute phase of stroke. Accumulating evidence indicates that endothelial progenitor cells (EPCs), equipped with an inherent capacity to migrate, proliferate and differentiate, may be one such therapeutics. However, the limited availability of EPCs in peripheral blood and early senescence of few isolated cells in culture conditions adversely affect their application as effective therapeutics. Given that much of the EPC-mediated reparative effects on neurovasculature is realized by a wide range of biologically active substances released by these cells, it is possible that EPC-secretome may serve as an important therapeutic after an ischemic stroke. In light of this assumption, this review paper firstly discusses the main constituents of EPC-secretome that may exert the beneficial effects of EPCs on neurovasculature, and then reviews the currently scant literature that focuses on its therapeutic capacity

MicroRNA: An Emerging Predictive, Diagnostic, Prognostic and Therapeutic Strategy in Ischaemic Stroke

Stroke continues to be the third-leading cause of death and disability worldwide. The limited availability of diagnostic tools approved therapeutics and biomarkers that help monitor disease progression or predict future events remain as the major challenges in the field of stroke medicine. Hence, attempts to discover safe and efficacious therapeutics and reliable biomarkers are of paramount importance. MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression. Since miRNAs also play important roles in key mechanisms associated with the pathogenesis of stroke, including energy failure, inflammation and cell death, it is possible that miRNAs may serve as reliable blood-based markers for risk prediction, diagnosis and prognosis of ischaemic stroke. Discovery of better neurological outcome and smaller cerebral infarcts in animal models of ischaemic stroke treated with miRNA agomirs or antagomirs indicate that miRNAs may also play a cerebrovascular protective role after an ischaemic stroke. Nonetheless, further evidences on the optimum time for treatment and route of administration are required before effective translation of these findings into clinical practice. Bearing these in mind, this paper reviews the current literature discussing the involvement of miRNAs in major pathologies associated with ischaemic stroke and evaluates their value as reliable biomarkers and therapeutics for ischaemic stroke

Protein kinase C-β distinctly regulates blood-brain barrier-forming capacity of Brain Microvascular endothelial cells and outgrowth endothelial cells

Outgrowth endothelial cells (OECs) provide an endogenous repair mechanism and thus maintain endothelial barrier integrity. As inhibition of protein kinase C-β (PKC-β) activity has been shown to attenuate endothelial damage in various pathological conditions including hyperglycaemia and ischaemic injury, the present study comparatively assessed the effect of LY333531, a PKC-β inhibitor, on the cerebral barrier integrity formed by OECs or human brain microvascular endothelial cells (HBMECs). To this end, an in vitro model of human BBB established by co-culture of astrocytes and pericytes with either OECs or HBMECs was exposed to 4h of oxygen-glucose deprivation with/out LY333531 (0.05 µM). The inhibition of PKC-β protected the integrity and function of the BBB formed by HBMECs, as evidenced by increases in transendothelial electrical resistance and decreases in sodium fluorescein flux. It also attenuated ischaemia-evoked actin cytoskeleton remodelling, oxidative stress, and apoptosis in HBMECs. In contrast, treatments with LY333531 exacerbated the deleterious effect of ischaemia on the integrity and function of BBB formed by OECs while augmenting the levels of oxidative stress, apoptosis, and cytoskeletal reorganisation in OECs. Interestingly, the magnitude of damage in all aforementioned parameters, notably oxidative stress, was lower with low dose of LY333531 (0.01 µM). It is therefore possible that the therapeutic concentration of LY333531 (0.05 µM) may neutralise the activity of NADPH oxidase and thus trigger a negative feedback mechanism which in turn exacerbate the detrimental effects of ischaemic injury. In conclusion, targeting PKC-β signalling pathway in ischaemic settings requires close attention while using OECs as cellular therapeutic

Therapeutic hypothermia augments the restorative effects of PKC-β and Nox2 inhibition on an in vitro model of human blood–brain barrier

To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-β (PKC-β) and Nox2 inhibition on an in vitro model of human blood–brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-β inhibitor (LY333531, 0.05 μM) or a Nox2 inhibitor (gp91ds-tat, 50 μM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-β or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-β or Nox2 pathways

Outgrowth Endothelial Cell Conditioned Medium Negates TNF-α-Evoked Cerebral Barrier Damage: A Reverse Translational Research to Explore Mechanisms

Improved understanding of the key mechanisms underlying cerebral ischemic injury is essential for the discovery of efficacious novel therapeutics for stroke. Through detailed analysis of plasma samples obtained from a large number of healthy volunteers (n = 90) and ischemic stroke patients (n = 81), the current study found significant elevations in the levels of TNF-α at baseline (within the first 48 h of stroke) and on days 7, 30, 90 after ischaemic stroke. It then assessed the impact of this inflammatory cytokine on an in vitro model of human blood–brain barrier (BBB) and revealed dramatic impairments in both barrier integrity and function, the main cause of early death after an ischemic stroke. Co-treatment of BBB models in similar experiments with outgrowth endothelial cell-derived conditioned media (OEC-CM) negated the deleterious effects of TNF-α on BBB. Effective suppression of anti-angiogenic factor endostatin, stress fiber formation, oxidative stress, and apoptosis along with concomitant improvements in extracellular matrix adhesive and tubulogenic properties of brain microvascular endothelial cells and OECs played an important role in OEC-CM-mediated benefits. Significant increases in pro-angiogenic endothelin-1 and monocyte chemoattractant protein-1 in OEC-CM compared to the secretomes of OEC and HBMEC, detected by proteome profiling assay, accentuate the beneficial effects of OEC-CM. In conclusion, this reverse translational study identifies TNF-α as an important mediator of post-ischemic cerebral barrier damage and proposes OEC-CM as a potential vasculoprotective therapeutic strategy by demonstrating its ability to regulate a wide range of mechanisms associated with BBB function. Clinical trial registration NCT02980354

Outgrowth Endothelial Cells (OECs): Cell-Based Therapy, Cell-Free Strategy, and Role of Senescence

Background: Ischaemic stroke, emerging from an interference of blood supply leading to or within the brain, continues to be one of the main causes of mortality and disability worldwide. Since the disruption of the blood–brain barrier (BBB), primarily formed by brain microvascular endothelial cells (BMECs), and ensuing brain oedema have been associated with the enhanced risk of death in the early stages of ischaemic stroke, the discovery of novel agent(s) that can effectively repair endothelial integrity and overall BBB function is of paramount importance to mitigate stroke-related damages. In this regard, endothelial progenitor cells (EPCs), which are capable of replacing the dead or dying endothelial cells through directly differentiating into mature endothelial cells or indirectly via releasing various active compounds, may represent such agent. Methods: To assess the cerebral barrier-reparative effect of outgrowth endothelial cells (OECs), the functional subtype of EPCs, an in vitro model of human BBB was established by co-culture of human BMECs (HBMECs), astrocytes, and pericytes before exposure to oxygen-glucose deprivation alone or followed by reperfusion (OGD±R). Using a rodent model of middle cerebral artery occlusion (MCAO), this thesis subsequently assessed the therapeutic potential of OECs in vivo. Since the availability and functionality of EPCs may be adversely affected by age, the thesis then investigated the level of circulating EPCs and several elements known to regulate their mobilisation and survivability in elderly (n=40, 73.3±7.2 years) and young (n=50, 40.2±14.3 years) healthy individuals. An experimental model of chronological ageing, mimicked by repetitive culture of the cells up to passage 14, was employed to assess the effects of ageing on the morphology and functional properties of OECs. To elucidate the key mechanism underlies the limited functional capacity of aged OECs, so-called replicative senescence, the cells were treated either with specific inhibitor of NADPH oxidase, VAS2870 (5 M), or broad-spectrum anti-oxidant, vitamin C (0.5 M), starting at passage 12. To understand the pathological mechanisms involved in ischemic stroke and to provide a strong foundation for therapeutic target, a reverse translational research, which analysed the biochemical alteration of 90 ischaemic stroke patients compared to 81 healthy controls, was employed. The thesis eventually assessed the capacity of OEC-derived conditioned medium (OEC-CM), produced by exposure of OECs to hypoxic injury, to neutralise these changes using the aforementioned in vitro BBB model. Results Part I: Similar to HBMECs, OECs possessed classical endothelial characteristics, as observed by their typical cobblestone morphology and capacity to bind with FITC-UEA-1 and Dil-Ac-LDL. These cells also equipped with remarkable proliferative, migratory, and anti-oxidant capacity. They can integrate with resident brain endothelial cells and form a tight and functional BBB. Even so, exposing OECs to OGD±R impaired their function to a similar degree as HBMECs. Exogenous addition of OECs during OGD±R effectively repaired the integrity and function of an in vitro BBB model, as ascertained by the increases of transendothelial electrical resistance (TEER) and decreases of sodium fluorescein flux, respectively. Similar to these results, treatment with OECs also restored the scratch damage induced on the endothelial layer of the BBB model in serum-free conditions, and protected overall cerebral barrier integrity and function. These barrier-reparative effects have also been replicated in in vivo settings, with the intravenous administration of OECs 24 hours after induction to MCAO markedly decreasing the brain infarct volume in the ipsilateral hemisphere brain assessed on day 3 after treatment. The mechanistic studies showed that the suppression of oxidative stress and apoptosis of resident cerebral endothelial cells may mediate this barrier-reparative effect of OECs. Part II: An investigation to quantify the level of circulating EPCs in older versus younger healthy participants showed insignificant differences in the level of EPCs (CD34+CD133+KDR+) between these two groups. However, the number of cells exclusively expressing stemness markers and known to possess unique capacity to differentiate into mature endothelial cells (CD34+ and/or CD133+) sharply declines in the elderly. This phenomenon was followed by a decrease in total anti-oxidant capacity and simultaneous increases in plasma levels of inflammatory mediators, TNF-, and anti-angiogenic factors, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional phenotypes of OECs showed that chronological ageing profoundly perturbed the critical functions of these cells, and induced the appearance of multiple typical signs of replicative senescence. In line with the findings from clinical observational studies, senescent OECs also manifested decreased total anti-oxidant capacity along with the increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using anti-oxidants compounds, namely vitamin C or VAS2870, somewhat delayed OEC senescence and repaired their tubulogenic and BBB-forming capacities. Part III: A comprehensive analysis of plasma samples acquired from a large number of healthy volunteers and ischaemic stroke patients showed significant increases in the levels of TNF- during acute, subacute, and chronic phases of stroke. Further analysis from this study also showed that the level of this inflammatory mediator was significantly higher in healthy volunteers with endothelial dysfunction associated risk factors, such as hypertension, diabetes mellitus, and hyperlipidaemia than those without. The subsequent experimental studies also showed the dramatic impairment on the integrity and function of an in vitro BBB model exposed to high concentration of TNF-(10 ng/mL, 6 hours), as observed by the decreases in TEER value and concomitant increases in sodium fluorescein flux, respectively. Co-treatment with OEC-CM effectively negated the detrimental effects of TNF- on the BBB. The remarkable suppression on endostatin level, oxidative stress, apoptosis, stress fibre formation as well as the improvements in HBMEC and OEC viability, tubulogenic, and adhesion properties appeared to contribute to this protective effect. Conclusion: Treatment with OECs effectively repaired BBB damages in both in vitro and in vivo model of ischaemic stroke through suppressing oxidative stress and apoptosis of resident brain endothelial cells. While OECs provide the endogenous repair mechanism to counteract on-going brain endothelial injury, advanced age ultimately evokes senescence and cellular dysfunction. Nonetheless, regulating oxidative stress level appears to delay the appearance of senescence phenotype and protect overall stem cell function. An alternative cell-free strategy using OEC-CM effectively negated the detrimental effects of TNF-, an important inflammatory cytokine that was remarkably elevated in all phases of ischaemic stroke, on BBB integrity and function by simultaneously modulating a variety of mechanisms

Delay of endothelial cell senescence protects cerebral barrier against age-related dysfunction: role of senolytics and senomorphics

Accumulation of senescent cells in cerebrovasculature is thought to play an important role in age-related disruption of blood–brain barrier (BBB). Using an in vitro model of human BBB, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, this study explored the so-called correlative link between BMEC senescence and the BBB dysfunction in the absence or presence of functionally distinct senotherapeutics. Replicative senescence was deemed present at passage ≥19 where BMECs displayed shortened telomere length, reduced proliferative and tubulogenic potentials and increased NADPH oxidase activity, superoxide anion production (markers of oxidative stress), S-β-galactosidase activity and γ-H2AX staining. Significant impairments observed in integrity and function of a model of BBB established with senescent BMECs, ascertained successively by decreases in transendothelial electrical resistance and increases in paracellular flux, revealed a close correlation between endothelial cell senescence and BBB dysfunction. Disruptions in the localization or expression of tight junction proteins, zonula occludens-1, occludin, and claudin-5 in senescent BMECs somewhat explained this dysfunction. Indeed, treatment of relatively old BMEC (passage 16) with a cocktail of senolytics (dasatinib and quercetin) or senomorphics targeting transcription factor NF-κB (QNZ), p38MAPK signaling pathway (BIRB-796) or pro-oxidant enzyme NADPH oxidase (VAS2870) until passage 20 rendered these cells more resistant to senescence and totally preserved BBB characteristics by restoring subcellular localization and expression of tight junction proteins. In conclusion, attempts that effectively mitigate accumulation of senescent endothelial cells in cerebrovasculature may prevent age-related BBB dysfunction and may be of prophylactic or therapeutic value to extend lifelong health and wellbeing

Outgrowth Endothelial Cells (OECs): Cell-Based Therapy, Cell-Free Strategy, and Role of Senescence

Background: Ischaemic stroke, emerging from an interference of blood supply leading to or within the brain, continues to be one of the main causes of mortality and disability worldwide. Since the disruption of the blood–brain barrier (BBB), primarily formed by brain microvascular endothelial cells (BMECs), and ensuing brain oedema have been associated with the enhanced risk of death in the early stages of ischaemic stroke, the discovery of novel agent(s) that can effectively repair endothelial integrity and overall BBB function is of paramount importance to mitigate stroke-related damages. In this regard, endothelial progenitor cells (EPCs), which are capable of replacing the dead or dying endothelial cells through directly differentiating into mature endothelial cells or indirectly via releasing various active compounds, may represent such agent. Methods: To assess the cerebral barrier-reparative effect of outgrowth endothelial cells (OECs), the functional subtype of EPCs, an in vitro model of human BBB was established by co-culture of human BMECs (HBMECs), astrocytes, and pericytes before exposure to oxygen-glucose deprivation alone or followed by reperfusion (OGD±R). Using a rodent model of middle cerebral artery occlusion (MCAO), this thesis subsequently assessed the therapeutic potential of OECs in vivo. Since the availability and functionality of EPCs may be adversely affected by age, the thesis then investigated the level of circulating EPCs and several elements known to regulate their mobilisation and survivability in elderly (n=40, 73.3±7.2 years) and young (n=50, 40.2±14.3 years) healthy individuals. An experimental model of chronological ageing, mimicked by repetitive culture of the cells up to passage 14, was employed to assess the effects of ageing on the morphology and functional properties of OECs. To elucidate the key mechanism underlies the limited functional capacity of aged OECs, so-called replicative senescence, the cells were treated either with specific inhibitor of NADPH oxidase, VAS2870 (5 M), or broad-spectrum anti-oxidant, vitamin C (0.5 M), starting at passage 12. To understand the pathological mechanisms involved in ischemic stroke and to provide a strong foundation for therapeutic target, a reverse translational research, which analysed the biochemical alteration of 90 ischaemic stroke patients compared to 81 healthy controls, was employed. The thesis eventually assessed the capacity of OEC-derived conditioned medium (OEC-CM), produced by exposure of OECs to hypoxic injury, to neutralise these changes using the aforementioned in vitro BBB model. Results Part I: Similar to HBMECs, OECs possessed classical endothelial characteristics, as observed by their typical cobblestone morphology and capacity to bind with FITC-UEA-1 and Dil-Ac-LDL. These cells also equipped with remarkable proliferative, migratory, and anti-oxidant capacity. They can integrate with resident brain endothelial cells and form a tight and functional BBB. Even so, exposing OECs to OGD±R impaired their function to a similar degree as HBMECs. Exogenous addition of OECs during OGD±R effectively repaired the integrity and function of an in vitro BBB model, as ascertained by the increases of transendothelial electrical resistance (TEER) and decreases of sodium fluorescein flux, respectively. Similar to these results, treatment with OECs also restored the scratch damage induced on the endothelial layer of the BBB model in serum-free conditions, and protected overall cerebral barrier integrity and function. These barrier-reparative effects have also been replicated in in vivo settings, with the intravenous administration of OECs 24 hours after induction to MCAO markedly decreasing the brain infarct volume in the ipsilateral hemisphere brain assessed on day 3 after treatment. The mechanistic studies showed that the suppression of oxidative stress and apoptosis of resident cerebral endothelial cells may mediate this barrier-reparative effect of OECs. Part II: An investigation to quantify the level of circulating EPCs in older versus younger healthy participants showed insignificant differences in the level of EPCs (CD34+CD133+KDR+) between these two groups. However, the number of cells exclusively expressing stemness markers and known to possess unique capacity to differentiate into mature endothelial cells (CD34+ and/or CD133+) sharply declines in the elderly. This phenomenon was followed by a decrease in total anti-oxidant capacity and simultaneous increases in plasma levels of inflammatory mediators, TNF-, and anti-angiogenic factors, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional phenotypes of OECs showed that chronological ageing profoundly perturbed the critical functions of these cells, and induced the appearance of multiple typical signs of replicative senescence. In line with the findings from clinical observational studies, senescent OECs also manifested decreased total anti-oxidant capacity along with the increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using anti-oxidants compounds, namely vitamin C or VAS2870, somewhat delayed OEC senescence and repaired their tubulogenic and BBB-forming capacities. Part III: A comprehensive analysis of plasma samples acquired from a large number of healthy volunteers and ischaemic stroke patients showed significant increases in the levels of TNF- during acute, subacute, and chronic phases of stroke. Further analysis from this study also showed that the level of this inflammatory mediator was significantly higher in healthy volunteers with endothelial dysfunction associated risk factors, such as hypertension, diabetes mellitus, and hyperlipidaemia than those without. The subsequent experimental studies also showed the dramatic impairment on the integrity and function of an in vitro BBB model exposed to high concentration of TNF-(10 ng/mL, 6 hours), as observed by the decreases in TEER value and concomitant increases in sodium fluorescein flux, respectively. Co-treatment with OEC-CM effectively negated the detrimental effects of TNF- on the BBB. The remarkable suppression on endostatin level, oxidative stress, apoptosis, stress fibre formation as well as the improvements in HBMEC and OEC viability, tubulogenic, and adhesion properties appeared to contribute to this protective effect. Conclusion: Treatment with OECs effectively repaired BBB damages in both in vitro and in vivo model of ischaemic stroke through suppressing oxidative stress and apoptosis of resident brain endothelial cells. While OECs provide the endogenous repair mechanism to counteract on-going brain endothelial injury, advanced age ultimately evokes senescence and cellular dysfunction. Nonetheless, regulating oxidative stress level appears to delay the appearance of senescence phenotype and protect overall stem cell function. An alternative cell-free strategy using OEC-CM effectively negated the detrimental effects of TNF-, an important inflammatory cytokine that was remarkably elevated in all phases of ischaemic stroke, on BBB integrity and function by simultaneously modulating a variety of mechanisms

Treatment with outgrowth endothelial cells protects cerebral barrier against ischemic injury

Background and aims: We have previously reported that outgrowth endothelial cells (OECs) restore cerebral endothelial cell integrity through effective homing to the injury site. This study further investigates whether treatment with OECs can restore blood-brain barrier (BBB) function in both in vitro and in vivo settings of ischaemia-reperfusion injury. Methods: An in vitro model of human BBB was established by co-culture of astrocytes, pericytes, and human brain microvascular endothelial cells (HBMECs) before exposure to oxygen-glucose deprivation alone, or followed by reperfusion (OGD±R) in the absence or presence of exogenous OECs. Using a rodent model of middle cerebral artery occlusion (MCAO), we further assessed the therapeutic potential of OECs in vivo. Results: Owing to their prominent anti-oxidant, proliferative, and migratory properties, alongside their inherent capacity to incorporate into brain vasculature, treatments with OECs attenuated the extent of OGD±R injury on BBB integrity and function, as ascertained by increases in transendothelial electrical resistance and decreases in paracellular flux across the barrier. Similarly, intravenous delivery of OECs also led to better barrier protection in MCAO rats as evidenced by significant decreases in ipsilateral brain oedema volumes on day 3 after treatment. Mechanistic studies subsequently showed that treatment with OECs substantially reduced oxidative stress and apoptosis in HBMECs subjected to ischaemic damages. Conclusion: This experimental study has shown that OEC-based cell therapy restores BBB integrity in an effective manner by integrating into resident cerebral microvascular network, suppressing oxidative stress and cellular apoptosis