Hyperglycaemia perturbs blood-brain barrier integrity through its effects on endothelial cell characteristics and function.

Breakdown of blood-brain barrier (BBB) represents a key pathology in hyperglycemia-mediated cerebrovascular damage after an ischemic stroke. As changes in the level and nature of vasoactive agents released by endothelial cells (ECs) may contribute to BBB dysfunction, this study first explored the specific impact of hyperglycemia on EC characteristics and secretome. It then assessed whether secretome obtained from ECs subjected to normoglycaemia or hyperglycemia might regulate pericytic cytokine profile differently. Using a triple cell culture model of human BBB, composed of brain microvascular EC (BMEC), astrocytes and pericytes, this study showed that exposure to hyperglycemia (25 mM D-glucose) for 72 h impaired the BBB integrity and function as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux of sodium fluorescein. Dissolution of zonula occludens-1, a tight junction protein, and appearance of stress fibers appeared to play a key role in this pathology. Despite elevations in angiogenin, endothelin-1, interleukin-8 and basic fibroblast growth factor levels and a decrease in placental growth factor levels in BMEC subjected to hyperglycemia vs normoglycaemia (5.5 mM D-glucose), tubulogenic capacity of BMECs remained similar in both settings. Similarly, pericytes subjected to secretome obtained from hyperglycemic BMEC released higher quantities of macrophage migration inhibitory factor and serpin and lower quantities of monocyte chemoattractant protein-1, intercellular adhesion molecule, interleukin-6 and interleukin-8. Taken together these findings indicate the complexity of the mechanisms leading to BBB disruption in hyperglycemic settings and emphasize the importance of endothelial cell-pericyte axis in the development of novel therapeutic strategies.

The Role of Stem Cells as Therapeutics for Ischaemic Stroke.

Stroke remains one of the leading causes of death and disability worldwide. Current reperfusion treatments for ischaemic stroke are limited due to their narrow therapeutic window in rescuing ischaemic penumbra. Stem cell therapy offers a promising alternative. As a regenerative medicine, stem cells offer a wider range of treatment strategies, including long-term intervention for chronic patients, through the reparation and replacement of injured cells via mechanisms of differentiation and proliferation. The purpose of this review is to evaluate the therapeutic role of stem cells for ischaemic stroke. This paper discusses the pathology during acute, subacute, and chronic phases of cerebral ischaemic injury, highlights the mechanisms involved in mesenchymal, endothelial, haematopoietic, and neural stem cell-mediated cerebrovascular regeneration, and evaluates the pre-clinical and clinical data concerning the safety and efficacy of stem cell-based treatments. The treatment of stroke patients with different types of stem cells appears to be safe and efficacious even at relatively higher concentrations irrespective of the route and timing of administration. The priming or pre-conditioning of cells prior to administration appears to help augment their therapeutic impact. However, larger patient cohorts and later-phase trials are required to consolidate these findings.

Analysis of endothelial progenitor cell subtypes as clinical biomarkers for elderly patients with ischaemic stroke.

Endothelial progenitor cells (EPCs), expressing markers for stemness (CD34), immaturity (CD133) and endothelial maturity (KDR), may determine the extent of post-stroke vascular repair. Given the prevalence of stroke in elderly, this study explored whether variations in plasmatic availability of certain EPC subtypes could predict the severity and outcome of disease in older patients. Blood samples were collected from eighty-one consented patients (≥ 65 years) at admission and days 7, 30 and 90 post-stroke. EPCs were counted with flow cytometry. Stroke severity and outcome were assessed using the National Institutes of Health Stroke Scale, Barthel Index and modified Rankin Scale. The levels of key elements known to affect EPC characteristics were measured by ELISA. Diminished total antioxidant capacity and CD34 + KDR + and CD133 + KDR + counts in early phases of stroke were associated with disease severity and worse functional outcome at day 90 post-stroke. Baseline levels of angiogenic agent PDGF-BB, but not VEGF, positively correlated with CD34 + KDR + numbers at day 90. Baseline LDL-cholesterol levels were inversely correlated with CD34 + KDR+, CD133 + KDR + and CD34 + CD133 + KDR + numbers at day 90. Close correlation between baseline CD34 + KDR + and CD133 + KDR + counts and the outcome of stroke proposes these particular EPC subtypes as potential prognostic markers for ischaemic stroke.

TNF-α evokes blood-brain barrier dysfunction through activation of Rho-kinase and neurokinin 1 receptor.

Ischaemic stroke, accompanied by neuroinflammation, impairs blood-brain barrier (BBB) integrity through a complex mechanism involving activation of both RhoA/Rho kinase/myosin light chain-2 and neurokinin 1 receptor (NK1R). Using an in vitro model of human BBB composed of brain microvascular endothelial cells (BMEC), astrocytes and pericytes, this study examined the potential contributions of these elements to BBB damage induced by elevated availability of pro-inflammatory cytokine, TNF-α. Treatment of human BMECs with TNF-α significantly enhanced RhoA activity and the protein expressions of Rho kinase and phosphorylated Ser19MLC-2 while decreasing that of NK1R. Pharmacological inhibition of Rho kinase by Y-27632 and NK1R by CP96345 neutralised the disruptive effects of TNF-α on BBB integrity and function as ascertained by reversal of decreases in transendothelial electrical resistance and increases in paracellular flux of low molecular weight permeability marker, sodium fluorescein, respectively. Suppression of RhoA activation, mitigation of actin stress fibre formation and restoration of plasma membrane localisation of tight junction protein zonula occludens-1 appeared to contribute to the barrier-protective effects of both Y-27632 and CP96345. Attenuation of TNF-α-mediated increases in NK1R protein expression in BMEC by Y-27632 suggests that RhoA/Rho kinase pathway acts upstream to NK1R. In conclusion, specific inhibition of Rho kinase in cerebrovascular conditions, accompanied by excessive release of pro-inflammatory cytokine TNF-α, helps preserve endothelial cell morphology and inter-endothelial cell barrier formation and may serve as an important therapeutic target.

Vascular Ageing: Mechanisms, Risk Factors, and Treatment Strategies.

Ageing constitutes the biggest risk factor for poor health and adversely affects the integrity and function of all the cells, tissues, and organs in the human body. Vascular ageing, characterised by vascular stiffness, endothelial dysfunction, increased oxidative stress, chronic low-grade inflammation, and early-stage atherosclerosis, may trigger or exacerbate the development of age-related vascular diseases, which each year contribute to more than 3.8 million deaths in Europe alone and necessitate a better understanding of the mechanisms involved. To this end, a large number of recent preclinical and clinical studies have focused on the exponential accumulation of senescent cells in the vascular system and paid particular attention to the specific roles of senescence-associated secretory phenotype, proteostasis dysfunction, age-mediated modulation of certain microRNA (miRNAs), and the contribution of other major vascular risk factors, notably diabetes, hypertension, or smoking, to vascular ageing in the elderly. The data generated paved the way for the development of various senotherapeutic interventions, ranging from the application of synthetic or natural senolytics and senomorphics to attempt to modify lifestyle, control diet, and restrict calorie intake. However, specific guidelines, considering the severity and characteristics of vascular ageing, need to be established before widespread use of these agents. This review briefly discusses the molecular and cellular mechanisms of vascular ageing and summarises the efficacy of widely studied senotherapeutics in the context of vascular ageing.

Substance P reversibly compromises the integrity and function of blood-brain barrier.

BACKGROUND: Substance P (SP) plays a role in vasodilatation and tissue integrity through its receptor, neurokinin 1 (NK1R). However, its specific effect on blood-brain barrier (BBB) remains unknown. METHODS: The impact of SP on the integrity/function of human BBB model in vitro, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, was assessed by measurements of transendothelial electrical resistance and paracellular flux of sodium fluorescein (NaF), respectively in the absence/presence of specific inhibitors targeting NK1R (CP96345), Rho-associated protein kinase (ROCK; Y27632) and nitric oxide synthase (NOS; N(G)-nitro-L-arginine methyl ester). Sodium nitroprusside (SNP), a NO donor, was employed as a positive control. The levels of tight junction proteins, zonula occludens-1, occludin and claudin-5 alongside RhoA/ROCK/myosin regulatory light chain-2 (MLC2) and extracellular signal­regulated protein kinase (Erk1/2) proteins were detected by western analyses. Subcellular localisations of F-actin and tight junction proteins were visualized by immunocytochemistry. Flow cytometry was used to detect transient calcium release. RESULTS: Exposure to SP increased RhoA, ROCK2 and phosphorylated serine-19 MLC2 protein levels and Erk1/2 phosphorylation in BMECs which were abolished by CP96345. These increases were independent of the changes in intracellular calcium availability. SP perturbed BBB in a time-dependent fashion through induction of stress fibres. Changes in tight junction protein dissolution or relocalisation were not involved in SP-mediated BBB breakdown. Inhibition of NOS, ROCK and NK1R mitigated the effect of SP on BBB characteristics and stress fibre formation. CONCLUSION: SP promoted a reversible decline in BBB integrity independent of tight junction proteins expression or localisation.

Evaluation of Endothelial Progenitor Cell Characteristics as Clinical Biomarkers for Elderly Patients with Ischaemic Stroke.

Ageing impairs endothelial function and predisposes the person to ischaemic stroke (IS). Endothelial progenitor cells (EPCs) repair endothelial damage and induce post-ischaemic neovascularisation. Given the prevalence of IS in older population, this study explored whether changes in EPC number and function may reliably predict the type or outcome of stroke in patients ≥ 65 years of age. For this, blood samples were collected once from healthy volunteers (HVs, n = 40) and four times (admission and days 7, 30 and 90 post-stroke) from participants with lacunar (n = 38) or cortical (n = 43) stroke. EPCs were counted with flow cytometry and defined as non-haematopoietic cells (CD45-) expressing markers for stemness (CD34 +), immaturity (CD133 +) and endothelial maturity (KDR +). Clonogenesis, tubulogenesis, migration and proliferation assays were performed as measures of EPC functionality. Biochemical profile of plasma inflammatory and angiogenic agents were studied using specific ELISAs. Primary outcome was disability or dependence on day 90 post-stroke, assessed by the modified Rankin Scale (mRS). Compared to HVs, EPC numbers were higher in stroke patients at all time points studied, reaching significance at baseline and day 30. No differences in EPC counts and functionality were observed between lacunar and cortical stroke groups at any time. Plasma endostatin, PDGF-BB, TNF-α and VEGF levels were higher in stroke patients vs HVs. Patient outcome, evaluated by mRS on day 90 post-stroke, did not correlate with EPC count or functionality. Baseline EPC counts may serve as a diagnostic marker for stroke but fail to distinguish between different stroke subtypes and predict post-stroke outcome.

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.

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-ß-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.

Effects of substance P on human cerebral microvascular endothelial cell line hCMEC/D3 are mediated exclusively through a truncated NK-1 receptor and depend on cell confluence.

The neuropeptide substance P (SP) mediates pain transmission, immune modulation, vasodilation and neurogenic inflammation. Its role in the peripheral nervous system has been well characterised. However, its actions on the blood-brain barrier (BBB) are less clear and warrant further study. The aim of this study was to characterise the effect of SP on the brain microvascular endothelial cells using the immortalized human brain microvascular endothelial cell line hCMEC/D3. As part of our studies, we have evaluated changes in expression, at mRNA and protein levels, of genes involved in the function of the blood-brain barrier such as occludin, induced by exposure to SP. We show that the effect of SP is dependent on cell confluence status. Thus, at low confluence but not at full confluence, SP treatment reduced occludin expression. The expression of the SP receptor, neurokinin-1 receptor (NK-1R) (the truncated form of the receptor expressed exclusively in this cell line) was also modulated in a similar pattern. SP treatment stimulated extracellular signal-regulated kinase (Erk2) phosphorylation which was not associated to changes in Interleukin-6 (IL-6), Interleukin-8 (IL-8), or Intercellular Adhesion Molecule 1 (ICAM-1) protein expression. In addition, SP treatment effectively recovered nitric oxide production on cells exposed to tumour necrosis factor alpha (TNF-α). SP did not trigger intracellular calcium release in hCMEC/D3 cells. We conclude that hCMEC/D3 cells are partially responsive to SP, that the effects are mediated through the truncated form of the receptor and are dependent on the confluence status of these cells.

Establishment of an In Vitro Model of Human Blood-Brain Barrier to Study the Impact of Ischemic Injury.

The blood-brain barrier (BBB), mainly composed of brain microvascular endothelial cells, astrocyte end-feet, and pericytes, serves as a physical and biochemical barrier that selectively limits the passage of circulating molecules into the brain parenchyma. The disruption of its integrity and function is a major cause of increased mortality and disability among ischemic stroke patients. Hence, scrutiny of the cellular and molecular mechanisms that alter BBB permeability following an ischemic injury remains of paramount importance. In this context, establishment of an in vitro model of BBB that closely simulates human cerebral barrier may offer an easy, inexpensive, and straightforward approach to identify signaling pathways involved in BBB breakdown and may help to discover new therapeutic targets to restore its damage. This chapter describes a sequential method pertaining to establishment of a triple culture model of human BBB consisting of the three main cellular components of the cerebral barrier. It also documents how the integrity and function of this barrier are evaluated through measurements of transendothelial electrical resistance (TEER) and paracellular flux of permeability marker and sodium fluorescein (NaF, 376 Da), respectively, both in normal and experimental conditions mimicking ischemic injury.

Protein kinase C-ß 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-ß (PKC-ß) 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-ß 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 4 h of oxygen-glucose deprivation with/out LY333531 (0.05 µM). The inhibition of PKC-ß 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-ß signalling pathway in ischaemic settings requires close attention while using OECs as cellular therapeutic.

Inhibition of oxidative stress delays senescence and augments functional capacity of endothelial progenitor cells.

Ageing is characterised by a progressive loss of vascular endothelial function and integrity. Endothelial progenitor cells (EPCs) play an integral role in endothelial regeneration but are prone to age-dependent changes which may accelerate their senescence and diminish their availability and functionality. Considering these, we firstly investigated the quantity of circulating EPCs in older (73.3 ± 7.2 years) and younger (40.2 ± 14.3 years) healthy volunteers and showed sharp declines in the number of EPCs expressing stemness markers (CD34 + and/or CD133 + ) in older people. These coincided with the decreases in total anti-oxidant capacity (TAC) and concomitant increases in plasma levels of pro-inflammatory cytokine, TNF-α and anti-angiogenic factor, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional properties of outgrowth endothelial cells (OECs), the functional subtype of EPCs, showed that chronological ageing, mimicked by replicative senescence, profoundly impaired proliferation, migration, tubulogenesis, and blood-brain barrier (BBB)-forming capacity of these cells. Similar to those seen in the clinical observational studies, senescent OECs also manifested decreased TAC and increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using structurally and functionally distinct anti-oxidants, namely vitamin C or VAS2870, an NADPH oxidase inhibitor, delayed OEC senescence and restored their tubulogenic and BBB-forming capacities. In conclusion, the enhanced oxidative stress level that develops during physiological ageing may promote EPC senescence and evoke endothelial dysfunction. Effective control of oxidative stress using either compound somewhat delays both phenomena and augments EPC functionality.

Outgrowth endothelial progenitor cells restore cerebral barrier function following ischaemic damage: The impact of NOX2 inhibition.

Disruption of blood-brain barrier (BBB), formed mainly by human brain microvascular endothelial cells (HBMECs), constitutes the major cause of mortality following ischaemic stroke. This study investigates whether OECs (outgrowth endothelial cells) can restore BBB integrity and function following ischaemic damage and how inhibition of NOX2, a main source of vascular oxidative stress, affects the characteristics of BBB established with OECs and HBMECs in ischaemic settings. In vitro models of human BBB were constructed by co-culture of pericytes and astrocytes with either OECs or HBMECs before exposure to oxygen-glucose deprivation (OGD) alone or followed by reperfusion (OGD + R) in the absence or presence of NOX2 inhibitor, gp91ds-tat. The function and integrity of BBB were assessed by measurements of paracellular flux of sodium fluorescein (NaF) and transendothelial electrical resistance (TEER), respectively. Treatment with OECs during OGD + R effectively restored BBB integrity and function. Compared to HBMECs, OECs possessed lower NADPH oxidase activity, superoxide anion levels and had greater total antioxidant capacity during OGD and OGD + R. Inhibition of NADPH oxidase during OGD and OGD + R restored the integrity and function of BBB established by HBMECs. This was evidenced by reductions in NADPH oxidase activity and superoxide anion levels. In contrast, treatment with gp91ds-tat aggravated ischaemic injury-induced BBB damage constructed by OECs. In conclusion, OECs are more resistant to ischaemic conditions and can effectively repair cerebral barrier following ischaemic damage. Suppression of oxidative stress through specific targeting of NOX2 requires close attention while using OECs as therapeutics.

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 settings of ischemia-reperfusion injury both in vitro and in vivo. 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 antioxidant, 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 edema volumes on day 3 after treatment. Mechanistic studies subsequently showed that treatment with OECs substantially reduced oxidative stress and apoptosis in HBMECs subjected to ischemic damages. CONCLUSION: This experimental study shows 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.

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.

Therapeutic hypothermia augments the restorative effects of PKC-ß 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-ß (PKC-ß) 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-ß 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-ß 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-ß or Nox2 pathways.

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.

Endothelial progenitor cells, potential biomarkers for diagnosis and prognosis of ischemic stroke: protocol for an observational case-control study.

Ischemic stroke is a devastating, life altering event which can severely reduce patient quality of life. Despite years of research there have been minimal therapeutic advances. Endothelial progenitor cells (EPCs), stem cells involved in both vasculogenesis and angiogenesis, may be a potential therapeutic target. After a stroke, EPCs migrate to the site of ischemic injury to repair cerebrovascular damage, and their numbers and functional capacity may determine patients' outcome. This study aims to determine whether the number of circulating EPCs and their functional aspects may be used as biomarkers to identify the type (cortical or lacunar) and/or severity of ischemic stroke. The study will also investigate if there are any differences in these characteristics between healthy volunteers over and under 65 years of age. 100 stroke patients (50 lacunar and 50 cortical strokes) will be recruited in this prospective, observational case-controlled study. Blood samples will be taken from stroke patients at baseline (within 48 hours of stroke) and days 7, 30 and 90. EPCs will be counted with flow cytometry. The plasma levels of pro- and anti-angiogenic factors and inflammatory cytokines will also be determined. Outgrowth endothelial cells will be cultured to be used in tube formation, migration and proliferation functional assays. Primary outcome is disability or dependence on day 90 after stroke, assessed by the modified Rankin Scale. Secondary outcomes are changes in circulating EPC numbers and/or functional capacity between patient and healthy volunteers, between patient subgroups and between elderly and young healthy volunteers. Recruitment started in February 2017, 167 participants have been recruited. Recruitment will end in November 2019. West Midlands - Coventry & Warwickshire Research Ethics Committee approved this study (REC number: 16/WM/0304) on September 8, 2016. Protocol version: 2.0. The Bayraktutan Dunhill Medical Trust EPC Study was registered in ClinicalTrials.gov (NCT02980354) on November 15, 2016. This study will determine whether the number of EPCs can be used as a prognostic or diagnostic marker for ischemic strokes and is a step towards discovering if transplantation of EPCs may aid patient recovery.

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.