Bindu Pillai

Peroxynitrite Mediates Retinal Neurodegeneration by Inhibiting Nerve Growth Factor Survival Signaling in Experimental and Human Diabetes

OBJECTIVE—Recently we have shown that diabetes-induced retinal neurodegeneration positively correlates with oxidative stress and peroxynitrite. Studies also show that peroxynitrite impairs nerve growth factor (NGF) survival signaling in sensory neurons. However, the causal role of peroxynitrite and the impact of tyrosine nitration on diabetes-induced retinal neurodegeneration and NGF survival signaling have not been elucidated. RESEARCH DESIGN AND METHODS—Expression of NGF and its receptors was examined in retinas from human and streptozotocin-induced diabetic rats and retinal ganglion cells (RGCs). Diabetic animals were treated with FeTPPS (15 mg · kg−1 · day−1 ip), which catalytically decomposes peroxynitrite to nitrate. After 4 weeks of diabetes, retinal cell death was determined by TUNEL assay. Lipid peroxidation and nitrotyrosine were determined using MDA assay, immunofluorescence, and Slot-Blot analysis. Expression of NGF and its receptors was determined by enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoprecipitation, and Western blot analyses. RESULTS—Analyses of retinal neuronal death and NGF showed ninefold and twofold increases, respectively, in diabetic retinas compared with controls. Diabetes also induced increases in lipid peroxidation, nitrotyrosine, and the pro-apoptotic p75NTR receptor in human and rat retinas. These effects were associated with tyrosine nitration of the pro-survival TrkA receptor, resulting in diminished phosphorylation of TrkA and its downstream target, Akt. Furthermore, peroxynitrite induced neuronal death, TrkA nitration, and activation of p38 mitogen-activated protein kinase (MAPK) in RGCs, even in the presence of exogenous NGF. FeTPPS prevented tyrosine nitration, restored NGF survival signal, and prevented neuronal death in vitro and in vivo. CONCLUSIONS—Together, these data suggest that diabetes-induced peroxynitrite impairs NGF neuronal survival by nitrating TrkA receptor and enhancing p75NTR expression.

Peroxynitrite mediates diabetes-induced endothelial dysfunction: possible role of Rho kinase activation.

Endothelial dysfunction is characterized by reduced bioavailability of NO due to its inactivation to form peroxynitrite or reduced expression of eNOS. Here, we examine the causal role of peroxynitrite in mediating diabetes-induced endothelial dysfunction. Diabetes was induced by STZ-injection, and rats received the peroxynitrite decomposition catalyst (FeTTPs, 15 mg/Kg/day) for 4 weeks. Vasorelaxation to acetylcholine, oxidative-stress markers, RhoA activity, and eNOS expression were determined. Diabetic coronary arteries showed significant reduction in ACh-mediated maximal relaxation compared to controls. Diabetic vessels showed also significant increases in lipid-peroxides, nitrotyrosine, and active RhoA and 50% reduction in eNOS mRNA expression. Treatment of diabetic animals with FeTTPS blocked these effects. Studies in aortic endothelial cells show that high glucose or peroxynitrite increases the active RhoA kinase levels and decreases eNOS expression and NO levels, which were reversed with blocking peroxynitrite or Rho kinase. Together, peroxynitrite can suppress eNOS expression via activation of RhoA and hence cause vascular dysfunction.

Thioredoxin interacting protein is a novel mediator of retinal inflammation and neurotoxicity

BACKGROUND AND PURPOSE Up‐regulation of thioredoxin interacting protein (TXNIP), an endogenous inhibitor of thioredoxin (Trx), compromises cellular antioxidant and anti‐apoptotic defences and stimulates pro‐inflammatory cytokines expression, implying a role for TXNIP in apoptosis. Here we have examined the causal role of TXNIP expression in mediating retinal neurotoxicity and assessed the neuroprotective actions of verapamil, a calcium channel blocker and an inhibitor of TXNIP expression.

Compound 21 is pro-angiogenic in the brain and results in sustained recovery after ischemic stroke.

Introduction: Angiotensin II type 2 receptor (AT2R) stimulation is neuroprotective after experimental stroke. However, the therapeutic utility of AT2R stimulation has been hampered by the lack of a specific agonist with favourable bioavailability. Compound 21 (C21) – the first non-peptide AT2R agonist – offers a potential option to enhance stroke recovery. This study aimed to investigate the effect of C21 administration on early and late stroke outcomes, and the molecular mediators involved. Methods: Rats were subjected to 3 h or 90 min of middle cerebral artery occlusion (MCAO) and randomized to intraperitoneal C21 (0.03 mg/kg) or saline at reperfusion. Animals were sacrificed at 24 h or 7 days and brains were collected for molecular analysis and immunostaining, respectively. Functional outcome at days 1, 4 and 7 was assessed blindly. C21 angiogenic potential was assessed in vitro. Results: After 3 h of MCAO, C21 treatment reduced infarct size and improved behavioural outcome at 24 h without affecting blood pressure. Co-administration of the AT2R antagonist (PD123319) blocked these effects. On the molecular level, C21 decreased brain haemoglobin content, down-regulated apoptotic and oxidative markers, and increased pro-survival molecules in the brain. After 90 min of MCAO, C21 treatment resulted in sustained functional improvement at 7 days, together with increased vascular density in the ischemic penumbra. In vitro, C21 showed a pro-angiogenic effect that was blocked with brain-derived neurotrophic factor neutralization. Conclusion: These findings demonstrate that a single dose of C21 is neurovascular-protective and improves stroke outcome possibly through increasing neurotrophin activity, mitigating brain inflammation, and promoting antioxidant and pro-angiogenic effects.

Targets of vascular protection in acute ischemic stroke differ in type 2 diabetes

Hemorrhagic transformation is an important complication of acute ischemic stroke, particularly in diabetic patients receiving thrombolytic treatment with tissue plasminogen activator, the only approved drug for the treatment of acute ischemic stroke. The objective of the present study was to determine the effects of acute manipulation of potential targets for vascular protection [i.e., NF-κB, peroxynitrite, and matrix metalloproteinases (MMPs)] on vascular injury and functional outcome in a diabetic model of cerebral ischemia. Ischemia was induced by middle cerebral artery occlusion in control and type 2 diabetic Goto-Kakizaki rats. Treatment groups received a single dose of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III), the nonspecific NF-κB inhibitor curcumin, or the broad-spectrum MMP inhibitor minocycline at reperfusion. Poststroke infarct volume, edema, hemorrhage, neurological deficits, and MMP-9 activity were evaluated. All acute treatments reduced MMP-9 and hemorrhagic transformation in diabetic groups. In addition, acute curcumin and minocycline therapy reduced edema in these animals. Improved neurological function was observed in varying degrees with treatment, as indicated by beam-walk performance, modified Bederson scores, and grip strength; however, infarct size was similar to untreated diabetic animals. In control animals, all treatments reduced MMP-9 activity, yet bleeding was not improved. Neuroprotection was only conferred by curcumin and minocycline. Uncovering the underlying mechanisms contributing to the success of acute therapy in diabetes will advance tailored stroke therapies.

Role of interleukin-10 in the neuroprotective effect of the Angiotensin Type 2 Receptor agonist, compound 21, after ischemia/reperfusion injury.

Introduction We and others have shown that the angiotensin type 2 (AT2) receptor agonist, compound 21 (C21), provides neuroprotection and enhances recovery in rodent stroke models yet the mechanism involved is not known. Moreover, C21 treatment is associated with an anti‐inflammatory response. Here we tested the hypothesis that C21 mediates neuroprotection by upregulating the neuroprotective and anti‐inflammatory cytokine, interleukin (IL)−10. Methods Wistar rats were subjected to 3 h‐middle cerebral artery suture occlusion and treated at reperfusion with C21 (0.03 mg/kg)±IL‐10 neutralizing antibody (0.1 mg/kg) both given i.p. Infarct size, behavioral outcomes, and molecular analysis were performed at 24 h post‐injury. Primary rat neurons were used to test the direct neuroprotective effect of C21 in vitro. Results C21 treatment reduced infarct size, improved functional outcome and decreased the pro‐inflammatory cytokine, tumor necrosis factor alpha (TNF‐&agr;) in the ischemic hemisphere compared to saline. Anti‐IL‐10 co‐treatment blocked the C21‐induced reduction in infarct size and inflammation, and the improvement in behavioral outcome. In vitro, C21 treatment increased neuron survival and reduced cell apoptosis after oxygen glucose deprivation (OGD) and OGD/reoxygenation. These effects were mediated through AT2R stimulation. Conclusion C21 provides direct neuroprotection as well as indirect protection through IL‐10.

Role of Matrix Metalloproteinase Activity in the Neurovascular Protective Effects of Angiotensin Antagonism

Background and Purpose. Oxidative stress and matrix metalloproteinase (MMP) activity have been identified as key mediators of early vascular damage after ischemic stroke. Somewhat surprisingly, the angiotensin II type 1 receptor (AT1) blocker, candesartan, has been shown to acutely increase MMP activity while providing neurovascular protection. We aimed to determine the contribution of MMP and nitrative stress to the effects of angiotensin blockade in experimental stroke. Methods. Wistar rats (n = 9–14/group; a total of 99) were treated in a factorial design with candesartan 1 mg/kg IV, alone or in combination with either a peroxynitrite decomposition catalyst, FeTPPs, 30 mg/kg IP or GM6001 50 mg/kg IP (MMP inhibitor). Neurological deficit, infarct, size and hemorrhagic transformation (HT) were measured after 3 h of middle cerebral artery occlusion (MCAO) and 21 h of reperfusion. MMP activity and nitrotyrosine expression were also measured. Results. Candesartan reduced infarct size and HT when administered alone (P = 0.0011) and in combination with FeTPPs (P = 0.0016). GM6001 did not significantly affect HT when administered alone, but the combination with candesartan caused increased HT (P < 0.0001) and worsened neurologic score (P = 0.028). Conclusions. Acute administration of candesartan reduces injury after stroke despite increasing MMP activity, likely by an antioxidant mechanism.

Dose–response, therapeutic time-window and tPA-combinatorial efficacy of compound 21: A randomized, blinded preclinical trial in a rat model of thromboembolic stroke:

The aim of this translational, randomized, controlled, blinded preclinical trial was to determine the effect of compound 21 (C21) in embolic stroke. Rats were subjected to embolic-middle cerebral artery occlusion (eMCAO). They received C21 (0.01, 0.03 and 0.06 mg/kg/d) or saline (orally) for five days, with the first-dose given IV at 3 h post-eMCAO. For the time-window study, the optimal-dose of C21 was initiated at 3, 6 or 24 h post-eMCAO and continued for five days. For the combinatorial study, animals received IV-tissue plasminogen activator (tPA) at either 2 or 4 h, with IV-C21 (0.01 mg/kg) or saline at 3 h post-eMCAO and daily thereafter for five days. After performing the behavior tests, brains were collected for analyses. The dose–response study showed significant motor improvements with the lowest-dose (0.01 mg/kg) of C21. In the time-window study, this same dose resulted in improvements when given 6 h and 24 h post-eMCAO. Moreover, C21-treated animals performed better on the novel object recognition test. Neither the single treatment with C21 or tPA (4 h) nor the combination therapy was effective in reducing the hemorrhage or infarct size, although C21 alone lowered sensorimotor deficit scores post-eMCAO. Future studies should focus on the long-term cognitive benefits of C21, rather than acute neuroprotection.

Role of angiotensin system modulation on progression of cognitive impairment and brain MRI changes in aged hypertensive animals – A randomized double- blind pre-clinical study

HighlightsRAS modulators preserved cognition in aged SHRs with cerebral hypoperfusion.C21 reduced accumulation of A&bgr;1‐42 in the prefrontal cortex of aged SHRs.C21 improved cognitive function in aged SHRs, as did candesartan.Candesartan prevented progressive loss of brain volume in aged SHRs. ABSTRACT Growing evidence suggests that renin angiotensin system (RAS) modulators support cognitive function in various animal models. However, little is known about their long‐term effects on the brain structure in aged hypertensive animals with chronic cerebral hypoperfusion as well as which specific domains of cognition are most affected. Therefore, in the current study we examined the effects of Candesartan and Compound 21 (C21) (RAS modulators) on aspects of cognition known to diminish with advanced age and accelerate with hypertension and vascular disease. Outcome measures for sensorimotor and cognitive function were performed using a sequence of tests, all blindly conducted and assessed at baseline and after 4 and 8 weeks of chronic hypoxic hypoperfusion and treatment. Magnetic resonance imaging (MRI) was performed at the end of the 8 week study period followed by animal sacrifice and tissue collection. Both Candesartan and C21 effectively preserved cognitive function and prevented progression of vascular cognitive impairment (VCI) but only candesartan prevented loss of brain volume in aged hypertensive animals. Collectively, our findings demonstrate that delayed administration of RAS modulators effectively preserve cognitive function and prevent the development / progression of VCI in aged hypertensive animals with chronic cerebral hypoperfusion.

Mechanisms of acute neurovascular protection with AT1 blockade after stroke: Effect of prestroke hypertension

Stroke is a leading cause of adult disability worldwide. Improving stroke outcome requires an orchestrated interplay that involves up regulation of pro-survival pathways and a concomitant suppression of pro-apoptotic mediators. In this investigation, we assessed the involvement of eNOS in the AT1 blocker-mediated protective and pro-recovery effects in animals with hypertension. We also evaluated the effect of acute eNOS inhibition in hypertensive animals. To achieve these goals, spontaneously hypertensive rats (SHR) were implanted with blood pressure transmitters, and randomized to receive either an eNOS inhibitor (L-NIO) or saline one hour before cerebral ischemia induction. After 3 hours of ischemia, animals were further randomized to receive either candesartan or saline at the time of reperfusion and sacrificed either 24 hours or 7 days later. Candesartan induced an early protective effect that was independent of eNOS inhibition (50% improvement in motor function). However, the protective effect of candesartan was associated with about five fold up regulation of BDNF expression and about three fold reduction in ER stress markers, in an eNOS dependent manner. The early benefit of a single dose of candesartan, present at 24 hours after stroke, was diminished at 7 days, perhaps due to a failure to induce an angiogenic response in these hypertensive animals. In conclusion, our findings demonstrate an early prorecovery effect of candesartan at both functional and molecular levels. Candesartan induced prorecovery signaling was mediated through eNOS. This effect was not maintained at 7 days after experimental ischemia.