Pathophysiological role of RhoA/Rho-kinase under oxygen-glucose deprivation/reperfusion and hyperglycaemia

Introduction: Oxygen-glucose deprivation (OGD)±reperfusion and hyperglycaemia exacerbate the ischaemic cerebral injuries during or after a stroke. The key biochemical events associated with these pathologies include excessive cytoskeletal remodelling, modulation of tight junction proteins and the induction of oxidative stress. Recently, the overactivities of protein kinase C (PKC), RhoA/Rho-kinase, and pro-oxidant NADPH oxidase have been shown to account for the development of these events and the consequent disruption of human blood-brain barrier (BBB) integrity. Objectives: This thesis focused on the putative roles of RhoA/Rho-kinase signalling in OGD and OGD+reperfusion-evoked modulation of cytoskeletal remodelling, tight junction proteins and oxidative stress in human brain microvascular endothelial cells (HBMEC). The effects of hyperglycaemia-mediated PKC overactivities in modulating the RhoA/Rho-kinase pathway with reference to the aforementioned parameters i.e. cytoskeletal remodelling and tight junction protein expression and localisation have also been the focus of this thesis. Methods: For the OGD studies, the HBMEC were exposed to normoxia (controls), OGD (4, 20 hours) alone and followed by reperfusion (20 hours). The HBMEC-human astrocyte (HA) cocultures were established to mimic human BBB before exposing them to the experimental conditions. The integrity and function of HBMEC-HA cocultures were measured by transendothelial electrical resistance (TEER) and flux of permeability markers sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA), respectively. For the hyperglycaemia studies, the HBMEC monolayers and the cocultures were exposed to normoglycaemia (5.5 mM D-glucose), hyperglycaemia (25 mM D-glucose), and hyperglycaemia with inhibitors of Rho-kinase, PKC, PKC-α, PKC-β, PKC-βII, PKC-δ; and the BBB integrity and function were measured by the TEER and flux studies, respectively. Fold differences in the protein expression or activity of RhoA, Rho-kinase-2, mono- and di-phosphorylated myosin light chain-2 (MLC2), total MLC2, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5, zonula occludens-1 (ZO-1), β-catenin, and vinculin were either measured by in-cell or ordinary Western analyses. Results from the OGD studies: OGD compromised the barrier integrity as observed by decreases in TEER values and concomitant increases in flux of EBA and NaF across the cocultures. Transfection of HBMEC with constitutively active RhoA also decreased the TEER and increased the NaF paracellular permeability, whereas inactivation of RhoA by anti-RhoA-IgG electroporation exerted barrier protective effects. Moreover, OGD alone and after constitutively active RhoA transfection introduced stress fibres in HBMEC, which were abrogated by inactivation of RhoA and the specific inhibition of its main effector Rho-kinase by Y-27632. In addition, dramatic increases in the protein expressions of RhoA-GTP, Rho-kinase-2, gp91-phox, and antioxidant catalase were observed in HBMEC exposed to OGD+reperfusion conditions. These along with increases in the NADPH oxidase activity and total superoxide anion levels confirmed the oxidative stress in HBMEC under these experimental conditions. A marked rise in the protein expressions of claudin-5 and β-catenin observed after OGD (20 hours) alone and followed by reperfusion may represent the effects of oxidative stress on tight and adherens junction proteins stability, respectively. These results also concurred with marked decreases in TEER and concomitant increases in the flux of EBA across the in vitro models of human BBB exposed to OGD±reperfusion conditions when compared with the controls. Cotreatment with Y-27632 under OGD±reperfusion normalised the protein expressions of RhoA, Rho-kinase-2, gp91-phox, claudin-5, catalase; activities of RhoA and NADPH oxidase; and total superoxide anions levels, alongside improving the expression of occludin and the coculture integrity under the OGD±reperfusion conditions. Results from the hyperglycaemia studies: Hyperglycaemia also increased RhoA-GTP, Rho-kinase-2, mono- and di-phosphorylated MLC2 protein levels and total PKC activity. These changes were consistent with the actin stress fibre formations, ZO-1 and occludin redistribution from HBMEC periphery. Hyperglycaemia-mediated endothelial-barrier dysfunction was further characterised by reduction in TEER and elevation in flux of EBA. Glucose normalisation, RhoA neutralisation by anti-RhoA-IgG electroporation and Rho-kinase-2 inhibition by Y-27632 normalised all abovementioned protein expressions, restored actin and tight junction protein localisations and barrier integrity. Cotreatment of HBMEC with hyperglycaemia and a general PKC inhibitor namely, bisindolylmaleimide-I normalised the Rho-kinase-2, mono- and di-phosphorylated MLC2 levels. Moreover, specific inhibitors of PKC-α (Ro-32-0432), PKC-β (LY333531), PKC-βII (CGP53353) attenuated the PKC overactivity, normalised all protein expressions, restored actin localisation and improved barrier integrity. In addition, the PKC-α and PKC-β siRNA transfections mimicked the effects of the specific inhibitors and attenuated the hyperglycaemia-evoked RhoA-GTP, mono- and di-phosphorylated MLC2 protein levels and stress fibre formations. Conclusions: The RhoA/Rho-kinase overactivities compromise the endothelial-barrier integrity, in part, by modulating the cytoskeletal remodelling and inducing the NADPH oxidase-evoked oxidative stress under OGD±reperfusion pathology. Moreover, hyperglycaemia-mediated increases in PKC-α and PKC-β activities exacerbate the endothelial-barrier dysfunction by modulating RhoA/Rho-kinase signalling pathway. Summary: These findings support the hypothesis that OGD±reperfusion and hyperglycaemia perturb BBB integrity through regulation of RhoA/Rho-kinase activity and modulation of cytoskeletal reorganisation, oxidative stress and tight junction protein expressions or localisations

Pathophysiological role of RhoA/Rho-kinase under oxygen-glucose deprivation/reperfusion and hyperglycaemia

Introduction: Oxygen-glucose deprivation (OGD)±reperfusion and hyperglycaemia exacerbate the ischaemic cerebral injuries during or after a stroke. The key biochemical events associated with these pathologies include excessive cytoskeletal remodelling, modulation of tight junction proteins and the induction of oxidative stress. Recently, the overactivities of protein kinase C (PKC), RhoA/Rho-kinase, and pro-oxidant NADPH oxidase have been shown to account for the development of these events and the consequent disruption of human blood-brain barrier (BBB) integrity. Objectives: This thesis focused on the putative roles of RhoA/Rho-kinase signalling in OGD and OGD+reperfusion-evoked modulation of cytoskeletal remodelling, tight junction proteins and oxidative stress in human brain microvascular endothelial cells (HBMEC). The effects of hyperglycaemia-mediated PKC overactivities in modulating the RhoA/Rho-kinase pathway with reference to the aforementioned parameters i.e. cytoskeletal remodelling and tight junction protein expression and localisation have also been the focus of this thesis. Methods: For the OGD studies, the HBMEC were exposed to normoxia (controls), OGD (4, 20 hours) alone and followed by reperfusion (20 hours). The HBMEC-human astrocyte (HA) cocultures were established to mimic human BBB before exposing them to the experimental conditions. The integrity and function of HBMEC-HA cocultures were measured by transendothelial electrical resistance (TEER) and flux of permeability markers sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA), respectively. For the hyperglycaemia studies, the HBMEC monolayers and the cocultures were exposed to normoglycaemia (5.5 mM D-glucose), hyperglycaemia (25 mM D-glucose), and hyperglycaemia with inhibitors of Rho-kinase, PKC, PKC-α, PKC-β, PKC-βII, PKC-δ; and the BBB integrity and function were measured by the TEER and flux studies, respectively. Fold differences in the protein expression or activity of RhoA, Rho-kinase-2, mono- and di-phosphorylated myosin light chain-2 (MLC2), total MLC2, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5, zonula occludens-1 (ZO-1), β-catenin, and vinculin were either measured by in-cell or ordinary Western analyses. Results from the OGD studies: OGD compromised the barrier integrity as observed by decreases in TEER values and concomitant increases in flux of EBA and NaF across the cocultures. Transfection of HBMEC with constitutively active RhoA also decreased the TEER and increased the NaF paracellular permeability, whereas inactivation of RhoA by anti-RhoA-IgG electroporation exerted barrier protective effects. Moreover, OGD alone and after constitutively active RhoA transfection introduced stress fibres in HBMEC, which were abrogated by inactivation of RhoA and the specific inhibition of its main effector Rho-kinase by Y-27632. In addition, dramatic increases in the protein expressions of RhoA-GTP, Rho-kinase-2, gp91-phox, and antioxidant catalase were observed in HBMEC exposed to OGD+reperfusion conditions. These along with increases in the NADPH oxidase activity and total superoxide anion levels confirmed the oxidative stress in HBMEC under these experimental conditions. A marked rise in the protein expressions of claudin-5 and β-catenin observed after OGD (20 hours) alone and followed by reperfusion may represent the effects of oxidative stress on tight and adherens junction proteins stability, respectively. These results also concurred with marked decreases in TEER and concomitant increases in the flux of EBA across the in vitro models of human BBB exposed to OGD±reperfusion conditions when compared with the controls. Cotreatment with Y-27632 under OGD±reperfusion normalised the protein expressions of RhoA, Rho-kinase-2, gp91-phox, claudin-5, catalase; activities of RhoA and NADPH oxidase; and total superoxide anions levels, alongside improving the expression of occludin and the coculture integrity under the OGD±reperfusion conditions. Results from the hyperglycaemia studies: Hyperglycaemia also increased RhoA-GTP, Rho-kinase-2, mono- and di-phosphorylated MLC2 protein levels and total PKC activity. These changes were consistent with the actin stress fibre formations, ZO-1 and occludin redistribution from HBMEC periphery. Hyperglycaemia-mediated endothelial-barrier dysfunction was further characterised by reduction in TEER and elevation in flux of EBA. Glucose normalisation, RhoA neutralisation by anti-RhoA-IgG electroporation and Rho-kinase-2 inhibition by Y-27632 normalised all abovementioned protein expressions, restored actin and tight junction protein localisations and barrier integrity. Cotreatment of HBMEC with hyperglycaemia and a general PKC inhibitor namely, bisindolylmaleimide-I normalised the Rho-kinase-2, mono- and di-phosphorylated MLC2 levels. Moreover, specific inhibitors of PKC-α (Ro-32-0432), PKC-β (LY333531), PKC-βII (CGP53353) attenuated the PKC overactivity, normalised all protein expressions, restored actin localisation and improved barrier integrity. In addition, the PKC-α and PKC-β siRNA transfections mimicked the effects of the specific inhibitors and attenuated the hyperglycaemia-evoked RhoA-GTP, mono- and di-phosphorylated MLC2 protein levels and stress fibre formations. Conclusions: The RhoA/Rho-kinase overactivities compromise the endothelial-barrier integrity, in part, by modulating the cytoskeletal remodelling and inducing the NADPH oxidase-evoked oxidative stress under OGD±reperfusion pathology. Moreover, hyperglycaemia-mediated increases in PKC-α and PKC-β activities exacerbate the endothelial-barrier dysfunction by modulating RhoA/Rho-kinase signalling pathway. Summary: These findings support the hypothesis that OGD±reperfusion and hyperglycaemia perturb BBB integrity through regulation of RhoA/Rho-kinase activity and modulation of cytoskeletal reorganisation, oxidative stress and tight junction protein expressions or localisations

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood–brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection

PKC-β exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway

Stroke patients with hyperglycemia (HG) develop higher volumes of brain edema emerging from disruption of blood–brain barrier (BBB). This study explored whether inductions of protein kinase C-β (PKC-β) and RhoA/Rho-kinase/myosin-regulatory light chain-2 (MLC2) pathway may account for HG-induced barrier damage using an in vitro model of human BBB comprising human brain microvascular endothelial cells (HBMEC) and astrocytes. Hyperglycemia (25 mmol/L D-glucose) markedly increased RhoA/Rho-kinase protein expressions (in-cell westerns), MLC2 phosphorylation (immunoblotting), and PKC-β (PepTag assay) and RhoA (Rhotekin-binding assay) activities in HBMEC while concurrently reducing the expression of tight junction protein occludin. Hyperglycemia-evoked in vitro barrier dysfunction, confirmed by decreases in transendothelial electrical resistance and concomitant increases in paracellular flux of Evan's blue-labeled albumin, was accompanied by malformations of actin cytoskeleton and tight junctions. Suppression of RhoA and Rho-kinase activities by anti-RhoA immunoglobulin G (IgG) electroporation and Y-27632, respectively prevented morphologic changes and restored plasma membrane localization of occludin. Normalization of glucose levels and silencing PKC-β activity neutralized the effects of HG on occludin and RhoA/Rho-kinase/MLC2 expression, localization, and activity and consequently improved in vitro barrier integrity and function. These results suggest that HG-induced exacerbation of the BBB breakdown after an ischemic stroke is mediated in large part by activation of PKC-β

Phase formation and stability of alloy phases in free nanoparticles: some insights

This paper explores phase formation and phase stability in free nanoparticles of binary alloys. A procedure for estimating the size and composition dependent free energies incorporating the contributions from the interfaces has been presented. Both single phase solid solution and two phase morphology containing interphase interfaces have been considered. A free energy scenario has been evaluated for two binary alloy systems Ag-Ni and Ag-Cu to predict the microstructure of the alloy nanoparticles at different size ranges as a function of composition. Both Ag-Cu and Ag-Ni systems exhibit wide bulk immiscibility. Ag-Ni nanoparticles were synthesized using the wet chemical synthesis technique whereas Ag-Cu nanoparticles were synthesized using laser ablation of a Ag-Cu target immersed in distilled water. Microstructural and compositional characterization of Ag-Ni and Ag-Cu nanoparticles on a single nanoparticle level was conducted using transmission electron microscopy. Nanoparticle microstructures observed from the microscopic investigation have been correlated with thermodynamic calculation results. It is shown that the observed two phase microstructure consisting of Ag-Ni solid solution in partial decomposed state coexisting with pure Ag phases in the case of Ag-Ni nanoparticles can be only be rationalized by invoking the tendency for phase separation of an initial solid solution with increase in nanoparticle size. Smaller sized Ag-Ni nanoparticles prefer a single phase solid solution microstructure. Due to an increase in particle size during the synthesis process the initial solid solution decomposes into an ultrafine scale phase separated microstructure. We have shown that it is necessary to invoke critical point phenomenon and wetting transition in systems showing a critical point that leads to phase separated Ag-Ni nanoparticles providing a catalytic substrate for the nucleation of equilibrium Ag over it. In the case of the Ag-Cu system, we report the experimental observation of a core shell structure at small sizes. This can be rationalized in terms of a metastable solid solution. It is argued that the nucleation barrier can prevent the formation of biphasic morphology with an internal interface. In such a situation, demixing of the solid solution can bring the system to a lower energy configuration. This has lead to the observed core-shell morphology in the Ag-Cu system during room temperature synthesis