Neflamapimod inhibits endothelial cell activation, adhesion molecule expression, leukocyte attachment and vascular inflammation by inhibiting p38 MAPKα and NF-κB signaling.

Neflamapimod, a selective inhibitor of the alpha isoform of p38 mitogen-activated protein kinase (MAPKα), was investigated for its potential to inhibit lipopolysaccharide (LPS)-induced activation of endothelial cells (ECs), adhesion molecule induction, and subsequent leukocyte attachment to EC monolayers. These events are known to contribute to vascular inflammation and cardiovascular dysfunction. Our results demonstrate that LPS treatment of cultured ECs and rats leads to significant upregulation of adhesion molecules, both in vitro and in vivo, which can be effectively inhibited by neflamapimod treatment. Western blotting data further reveals that neflamapimod inhibits LPS-induced phosphorylation of p38 MAPKα and the activation of NF-κB signaling in ECs. Additionally, leukocyte adhesion assays demonstrate a substantial reduction in leukocyte attachment to cultured ECs and the aorta lumen of rats treated with neflamapimod. Consistent with vascular inflammation, LPS-treated rat arteries exhibit significantly diminished vasodilation response to acetylcholine, however, arteries from rats treated with neflamapimod maintain their vasodilation capacity, demonstrating its ability to limit LPS-induced vascular inflammation. Overall, our data demonstrate that neflamapimod effectively inhibits endothelium activation, adhesion molecule expression, and leukocyte attachment, thereby reducing vascular inflammation.

Detection and Diagnosis of Cardiac Amyloidosis in Egypt.

Cardiac amyloidosis is a life-threatening disease that occurs when amyloid proteins, most commonly immunoglobulin light chain or transthyretin, mutate or become unstable, misfold, deposit as amyloid fibrils, and accumulate in the myocardium. Early diagnosis of cardiac amyloidosis is hindered by insufficient awareness, specifically regarding clinical red flags and diagnostic pathways. Cardiac amyloidosis diagnosis comprises two important phases, clinical suspicion (phase one) followed by definitive diagnosis (phase two). Each phase is associated with specific clinical techniques. For example, clinical features, electrocardiography, echocardiography, and cardiac magnetic resonance imaging serve to raise suspicion of cardiac amyloidosis and facilitate early diagnosis, whereas laboratory tests (i.e., blood or urine electrophoresis with immunofixation), biopsy, scintigraphy-based nuclear imaging, and genetic testing provide a definitive diagnosis of cardiac amyloidosis. In Egypt, both the lack of cardiac amyloidosis awareness amongst healthcare providers and the unavailability of clinical expertise for the use of diagnostic techniques must be overcome to improve the prognosis of cardiac amyloidosis in the region. Previously published diagnostic algorithms for cardiac amyloidosis have amalgamated techniques that can raise clinical suspicions of cardiac amyloidosis with those that definitively diagnose cardiac amyloidosis. Though such algorithms have been successful in developed countries, diagnostic tools like echocardiography, scintigraphy, and cardiac magnetic resonance imaging are not ubiquitously available across Egyptian facilities. This review presents the current state of knowledge regarding cardiac amyloidosis in Egypt and outlines a new diagnostic algorithm which leverages regional nuclear imaging expertise. Importantly, the proposed diagnostic algorithm guides accurate amyloid-typing to mitigate misdiagnosis and erroneous treatment selection and improve the cardiac amyloidosis diagnostic accuracy in Egypt.

Diagnostic algorithms are useful tools for guiding clinical diagnosis by summarizing diagnostic approaches and defining the patient pathway. The diagnostic algorithms for cardiac amyloidosis amalgamate techniques that raise suspicion of the disease with those that can definitively diagnose the disease. These algorithms, for the early detection and diagnosis of cardiac amyloidosis, are designed in accordance with developed healthcare systems that have the resources and infrastructure for diagnostic equipment and clinical expertise. There are limited financial resources across healthcare facilities in Egypt for diagnostic equipment like echocardiograms (ECHO), scintigraphy, and cardiac magnetic resonance imaging (cMRI), and the required clinical training for the diagnosis of cardiac amyloidosis. This reduces the possibility of early diagnosis of the disease and subsequent early intervention. Evidently, there is a significant unmet clinical need to develop an algorithm for the diagnosis of cardiac amyloidosis in accordance with the Egyptian healthcare system. This review article details the current awareness regarding the diagnosis of cardiac amyloidosis and the associated challenges in Egypt. Accordingly, a diagnostic algorithm that leverages nuclear imaging expertise to guide accurate amyloid-typing in order to mitigate misdiagnosis and erroneous treatment, and also improve the diagnostic accuracy of cardiac amyloidosis, has been proposed.

Neflamapimod induces vasodilation in resistance mesenteric arteries by inhibiting p38 MAPKα and downstream Hsp27 phosphorylation.

Neflamapimod, a selective inhibitor of p38 mitogen activated protein kinase alpha (MAPKα), is under clinical investigation for its efficacy in Alzheimer's disease (AD) and dementia with Lewy Bodies (DLB). Here, we investigated if neflamapimod-mediated acute inhibition of p38 MAPKα could induce vasodilation in resistance-size rat mesenteric arteries. Our pressure myography data demonstrated that neflamapimod produced a dose-dependent vasodilation in mesenteric arteries. Our Western blotting data revealed that acute neflamapimod treatment significantly reduced the phosphorylation of p38 MAPKα and its downstream target heat-shock protein 27 (Hsp27) involved in cytoskeletal reorganization and smooth muscle contraction. Likewise, non-selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation, and induced vasodilation. Endothelium denudation or pharmacological inhibition of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) had no effect on such vasodilation. Neflamapimod-evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell K+ channels. Altogether, our data for the first time demonstrates that in resistance mesenteric arteries, neflamapimod inhibits p38 MAPKα and phosphorylation of its downstream actin-associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which neflamapimod is being investigated.

Mechanisms of Preconditioning Exercise-Induced Neurovascular Protection in Stroke.

Ischemic stroke is a leading cause of death and disability. Tissue plasminogen activator is the only U.S. Food and Drug Administration approved thrombolytic therapy for ischemic stroke patients till date. However, its use is limited due to increased risk of bleeding and narrow therapeutic window. Most of the preclinically tested pharmacological agents failed to be translated to the clinic. This drives the need for alternative therapeutic approaches that not only provide enhanced neuroprotection, but also reduce the risk of stroke. Physical exercise is a sort of preconditioning that provides the body with brief ischemic episodes that can protect the body from subsequent severe ischemic attacks like stroke. Physical exercise is known to improve cardiovascular health. However, its role in providing neuroprotection in stroke is not clear. Clinical observational studies showed a correlation between regular physical exercise and reduced risk and severity of ischemic stroke and better outcomes after stroke. However, the underlying mechanisms through which prestroke exercise can reduce the stroke injury and improve the outcomes are not completely understood. The purpose of this review is to: demonstrate the impact of exercise on stroke outcomes and show the potential role of exercise in stroke prevention and recovery; uncover the underlying mechanisms through which exercise reduces the neurovascular injury and improves stroke outcomes aiming to develop novel therapeutic approaches.

Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis.

It is a clinically well-established fact that patients with diabetes have very poor stroke outcomes. Yet, the underlying mechanisms remain largely unknown. Our previous studies showed that male diabetic animals show greater hemorrhagic transformation (HT), profound loss of cerebral vasculature in the recovery period, and poor sensorimotor and cognitive outcomes after ischemic stroke. This study aimed to determine the impact of iron chelation with deferoxamine (DFX) on (1) cerebral vascularization patterns and (2) functional outcomes after stroke in control and diabetic rats. After 8 weeks of type 2 diabetes induced by a combination of high-fat diet and low-dose streptozotocin, male control and diabetic animals were subjected to thromboembolic middle cerebral artery occlusion (MCAO) and randomized to vehicle, DFX, or tPA/DFX and followed for 14 days with behavioral tests. Vascular indices (vascular volume and surface area), neurovascular remodeling (AQP4 polarity), and microglia activation were measured. Brain microvascular endothelial cells (BMVEC) from control and diabetic animals were evaluated for the impact of DFX on ferroptotic cell death. DFX treatment prevented vasoregression and microglia activation while improving AQP4 polarity as well as blood-brain barrier permeability by day 14 in diabetic rats. These pathological changes were associated with improvement of functional outcomes. In control rats, DFX did not have an effect. Iron increased markers of ferroptosis and lipid reactive oxygen species (ROS) to a greater extent in BMVECs from diabetic animals, and this was prevented by DFX. These results strongly suggest that (1) HT impacts post-stroke vascularization patterns and recovery responses in diabetes, (2) treatment of bleeding with iron chelation has differential effects on outcomes in comorbid disease conditions, and (3) iron chelation and possibly inhibition of ferroptosis may provide a novel disease-modifying therapeutic strategy in the prevention of post-stroke cognitive impairment in diabetes.

Short-Term Acute Exercise Preconditioning Reduces Neurovascular Injury After Stroke Through Induced eNOS Activation.

Physical exercise is known to reduce cardiovascular risk but its role in ischemic stroke is not clear. It was previously shown that an acute single bout of exercise reduced increased eNOS activation in the heart and reduced myocardial infarction. However, the impact of a single bout or short-term exercise on eNOS-induced neuroprotection after stroke was not previously studied. Accordingly, this study was designed to test the hypothesis that short-term acute exercise can provide "immediate neuroprotection" and improve stroke outcomes through induced eNOS activation. Male Wistar rats (300 g) were subjected to HIIT treadmill exercise for 4 days (25 min/day), break for 2 days, and then one acute bout for 30 min. Exercised animals were subjected to thromboembolic stroke 1 h, 6 h, 24 h, or 72 h after the last exercise session. At 24 h after stroke, control (sedentary) and exercised rats were tested for neurological outcomes, infarct size, and edema. The expression of active eNOS (p-S1177-eNOS) and active AMPK (p-T172-AMPK) was measured in the brain, cerebral vessels, and aorta. In an additional cohort, animals were treated with the eNOS inhibitor, L-NIO (I.P, 20 mg/kg), and stroked 1 h after exercise and compared with non-exercise animals. Acute exercise significantly reduced infarct size, edema, and improved functional outcomes, and significantly increased the expression of peNOS and pAMPK in the brain, cerebral vessels, and aorta. eNOS inhibition abolished the exercise-induced improvement in outcomes. Short-term acute preconditioning exercise reduced the neurovascular injury and improved functional outcomes after stroke through eNOS activation.

Review of Diabetic Polyneuropathy: Pathogenesis, Diagnosis and Management According to the Consensus of Egyptian Experts.

Diabetic polyneuropathy (DPN) is a complex and multifactorial entity in which various factors besides hyperglycemia play an important role. Symptoms of DPN are sensory, motor or autonomic. Intensive research proved that oxidative stress is the common denominator for the four major destructive pathways of hyperglycemia including increased hexosamine pathway flux, activation of Protein kinase-C (PKC) pathway, increased Advanced Glycated End-products (AGEs) formation, and increased Polyol Pathway flux. National data in Egypt confirms that more than 60% of Egyptian diabetic patients suffer from neuropathy. The most common complications of DPN are Cardiac Autonomic Neuropathy (CAN), diabetic foot and ulcers, neuromuscular disability, and anxiety. In addition, DPN affects the Quality of Life (QoL). According to common clinical practice, the common diagnostic tools are bed-side diagnosis and electrophysiological tests. Early diagnosis is critical to improve the prognosis of DPN and therapeutic intervention in the early phase. In this review, we provide a clear understanding of the pathogenesis, early diagnosis and the good management of DPN. Since the pathogenesis of DPN is multifactorial, its management is based on combination therapy of symptomatic; either pharmacological or non-pharmacological treatments, and pathogenic treatment. Alpha Lipoic Acid (ALA) is a potent anti-oxidant that has several advantages as a pathogenic treatment of DPN. So, in clinical practice, ALA may be prescribed for patients with early neuropathic deficits and symptoms. Patient education has an important role in the managemement of DPN.

Endothelial stromelysin1 regulation by the forkhead box-O transcription factors is crucial in the exudative phase of acute lung injury.

Enhanced vascular permeability is associated with inflammation and edema in alveoli during the exudative phase of acute respiratory distress syndrome (ARDS). Mechanisms leading to the endothelial contribution on the early exudative stage of ARDS are not precise. We hypothesized that modulation of endothelial stromelysin1 expression and activity by Akt1-forkhead box-O transcription factors 1/3a (FoxO1/3a) pathway could play a significant role in regulating pulmonary edema during the initial stages of acute lung injury (ALI). We utilized lipopolysaccharide (LPS)-induced mouse ALI model in vivo and endothelial barrier resistance measurements in vitro to determine the specific role of the endothelial Akt1-FoxO1/3a-stromelysin1 pathway in ALI. LPS treatment of human pulmonary endothelial cells resulted in increased stromelysin1 and reduced tight junction claudin5 involving FoxO1/3a, associated with decreased trans-endothelial barrier resistance as determined by electric cell-substrate impedance sensing technology. In vivo, LPS-induced lung edema was significantly higher in endothelial Akt1 knockdown (EC-Akt1-/-) compared to wild-type mice, which was reversed upon treatment with FoxO inhibitor (AS1842856), stromelysin1 inhibitor (UK356618) or with shRNA-mediated FoxO1/3a depletion in the mouse lungs. Overall, our study provides the hope that targeting FoxO and styromelysin1 could be beneficial in the treatment of ALI.

Peroxynitrite-Induced Tyrosine Nitration Contributes to Matrix Metalloprotease-3 Activation: Relevance to Hyperglycemic Ischemic Brain Injury and Tissue Plasminogen Activator.

Matrix metalloprotease-3 (MMP3) activation mediates the tissue plasminogen activator (tPA)-induced hemorrhagic transformation after stroke. Hyperglycemia (HG) further exacerbates this outcome. We have recently shown that HG increases MMP3 activity in the brain after stroke. However, the combined HG-tPA effect on MMP3 activation, and the mechanisms through which MMP3 is activated were not previously reported. Accordingly, this study tested the hypothesis that tPA and HG increases MMP3 activity in the brain after stroke through peroxynitrite induced tyrosine nitration. Normoglycemic and mildly hyperglycemic male Wistar rats were subjected to middle cerebral artery suture occlusion for 90 min or thromboembolic occlusion, and up to 24 h reperfusion, with and without tPA. MMP3 activity and tyrosine nitration were evaluated in brain homogenates at 24 h. Brain microvascular endothelial cells (BMVEC) were subjected to either 3 h hypoxia or 6 h OGD under either normal or high glucose conditions with or without tPA, with or without peroxynitrite scavenger, FeTPPs. MMP3 activity and MMP3 tyrosine nitration were assessed at 24 h. HG and tPA significantly increased activity and tyrosine nitration of MMP3 in the brain. In BMVECs, tPA but not HG increased MMP3 activity. Treating BMVEC with FeTPPs significantly reduced the tPA-induced increase in MMP3 activity and nitration. Augmented oxidative and nitrative stress may be potential mechanisms contributing to MMP3 activation in hyperglycemic stroke, especially with tPA administration. Peroxynitrite may be playing a critical role in mediating MMP3 activation through tyrosine nitration in hyperglycemic stroke.

Chronic Remote Ischemic Conditioning Is Cerebroprotective and Induces Vascular Remodeling in a VCID Model.

Vascular contributions to cognitive impairment and dementia (VCID) make up 50% of the cases of dementia. The purpose of this study was to determine the effect of chronic remote ischemic conditioning (C-RIC) on improving long-term (6 months) outcomes and cerebral blood flow (CBF) and collateral formation in a mouse model of VCID. Adult C57BL/6J male mice (10 weeks) were randomly assigned to four different groups: (1) sham-bilateral carotid artery stenosis (BCAS), (2) BCAS + sham RIC, (3) BCAS+C-RIC for 1 month (1MO), and (4) BCAS+C-RIC-4 months (4MO). CBF, cognitive impairment, and functional outcomes were performed up for 6 months after BCAS surgery. The expression of CD31, α-SMA, and myelin basic protein (MBP) was assessed by immunohistochemistry (IHC). Additional set of mice were randomized to sham, BCAS, and BCAS+C-RIC. The cerebrovascular angioarchitecture was studied with micro-CT. RIC therapy for either 1 or 4 months significantly improved CBF, new collateral formation, functional and cognitive outcomes, and prevented white matter damage. There was no difference between C-RIC for 1 or 4 months; IHC studies at 6 months showed an increase in brain CD31 and α-SMA expression indicating increased angiogenesis and MBP indicating preservation of white matter in animals receiving RIC. One month of daily RIC is as effective as 4 months of daily RIC in improving CBF, angiogenesis, and long-term functional outcomes (6 months) in a VCID model. This suggests that 1 month of RIC is sufficient to reduce cognitive impairment and induce beneficial cerebrovascular remodeling.

Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes.

AIMS/HYPOTHESIS: Diabetes promotes cerebral neovascularisation via increased vascular endothelial growth factor (VEGF) angiogenic signalling. Roundabout-4 (ROBO4) protein is an endogenous inhibitor of VEGF signalling that stabilises the vasculature. Yet, how diabetes affects ROBO4 function remains unknown. We hypothesised that increased VEGF signalling in diabetes decreases ROBO4 expression and function via binding of ROBO4 with VEGF-activated ß3 integrin and that restoration of ROBO4 expression prevents/repairs cerebral neovascularisation in diabetes. METHODS: ROBO4 protein expression in a rat model of type 2 diabetes (Goto-Kakizaki [GK] rats) was examined by western blotting and immunohistochemistry. ROBO4 was locally overexpressed in the brain and in primary brain microvascular endothelial cells (BMVECs). GK rats were treated with SKLB1002, a selective VEGF receptor-2 (VEGFR-2) antagonist. Cerebrovascular neovascularisation indices were determined using a FITC vascular space-filling model. Immunoprecipitation was used to determine ROBO4-ß3 integrin interaction. RESULTS: ROBO4 expression was significantly decreased in the cerebral vasculature as well as in BMVECs in diabetes (p < 0.05). Silencing Robo4 increased the angiogenic properties of control BMVECs (p < 0.05). In vivo and in vitro overexpression of ROBO4 inhibited VEGF-induced angiogenic signalling and increased vessel maturation. Inhibition of VEGF signalling using SKLB1002 increased ROBO4 expression (p < 0.05) and reduced neovascularisation indices (p < 0.05). Furthermore, SKLB1002 significantly decreased ROBO4-ß3 integrin interaction in diabetes (p < 0.05). CONCLUSIONS/INTERPRETATION: Our study identifies the restoration of ROBO4 and inhibition of VEGF signalling as treatment strategies for diabetes-induced cerebral neovascularisation.

Impact of Comorbidities on Acute Injury and Recovery in Preclinical Stroke Research: Focus on Hypertension and Diabetes.

Human ischemic stroke is very complex, and no single preclinical model can comprise all the variables known to contribute to stroke injury and recovery. Hypertension, diabetes, and hyperlipidemia are leading comorbidities in stroke patients. The use of predominantly young adult and healthy animals in experimental stroke research has created a barrier for translation of findings to patients. As such, more and more disease models are being incorporated into the research design. This review highlights the major strengths and weaknesses of the most commonly used animal models of these conditions in preclinical stroke research. The goal is to provide guidance in choosing, reporting, and executing appropriate disease models that will be subjected to different models of stroke injury.

Matrix Metalloprotease 3 Exacerbates Hemorrhagic Transformation and Worsens Functional Outcomes in Hyperglycemic Stroke.

BACKGROUND AND PURPOSE: Acute hyperglycemia worsens the clinical outcomes and exacerbates cerebral hemorrhage after stroke. The mediators of hemorrhagic transformation (HT) in hyperglycemic stroke are not fully understood. Matrix metalloproteinase 3 (MMP3) plays a critical role in the tissue-type plasminogen activator-induced HT. However, the role of MMP3 in exacerbating the HT and worsening the functional outcomes in hyperglycemic stroke remains unknown. METHODS: Control/normoglycemic and hyperglycemic (blood glucose, 140-200 mg/dL) male Wistar rats were subjected to middle cerebral artery occlusion for 90 minutes and either 24 hours or 7 days reperfusion. MMP3 was inhibited pharmacologically (UK 356618, 15 mg/kg IV at reperfusion) or knocked down in the brain by shRNA lentiviral particles (injected intracerebroventricular). Neurovascular injury was assessed at 24 hours, and functional outcomes were assessed at 24 hours, day 3, and day 7. MMP3 activity was measured in brain homogenate and cerebral macrovessels. Localization of MMP3 within the neurovascular unit after hyperglycemic stroke was demonstrated by immunohistochemistry. RESULTS: Hyperglycemia significantly increased MMP3 activity in the brain after stroke, and this was associated with exacerbated HT and worsened functional outcomes. MMP3 inhibition significantly reduced HT and improved functional outcomes. CONCLUSIONS: MMP3 plays a critical role in mediating cerebrovascular injury in hyperglycemic stroke. Our findings point out MMP3 as a potential therapeutic target in hyperglycemic stroke.

Comparative Analysis of Different Methods of Ischemia/Reperfusion in Hyperglycemic Stroke Outcomes: Interaction with tPA.

Acute hyperglycemia (HG) exacerbates reperfusion injury and aggravates tissue plasminogen activator (tPA)-induced hemorrhagic transformation (HT). Previous experimental hyperglycemic stroke studies employed very high blood glucose levels and exclusively used suture occlusion model to induce ischemia. Only few studies evaluated HG in embolic stroke and mostly involving the use of 10-fold higher dose of tPA than that is used in patients. However, the interaction between acute HG and low (human) dose tPA in different experimental models of stroke has never been reported. We first tested the impact of the severity of acute HG on stroke outcome. Building upon our findings, we then compared the impact of mild acute HG on neurovascular injury in rats subjected to suture or thromboembolic occlusion with and without low dose tPA. We assessed cerebral blood flow, neurobehavioral outcomes, infarction, hemorrhage, and edema. tPA did not change the infarct size in either control or hyperglycemic animals when compared to no tPA groups. HG increased HT and worsened functional outcomes in both suture and embolic occlusion models. The combination of HG and tPA exacerbated the vascular injury and worsened the neurological deficits more than each individual treatment in both models. Our findings show that the interaction between HG and even low dose tPA has detrimental effects on the cerebrovasculature and functional outcomes independent of the method of reperfusion.

SOD1 overexpression prevents acute hyperglycemia-induced cerebral myogenic dysfunction: relevance to contralateral hemisphere and stroke outcomes.

Admission hyperglycemia (HG) amplifies vascular injury and neurological deficits in acute ischemic stroke, but the mechanisms remain controversial. We recently reported that ischemia-reperfusion (I/R) injury impairs the myogenic response in both hemispheres via increased nitration. However, whether HG amplifies contralateral myogenic dysfunction and whether loss of tone in the contralateral hemisphere contributes to stroke outcomes remain to be determined. Our hypothesis was that contralateral myogenic dysfunction worsens stroke outcomes after acute hyperglycemic stroke in an oxidative stress-dependent manner. Male wild-type or SOD1 transgenic rats were injected with saline or 40% glucose solution 10 min before surgery and then subjected to 30 min of ischemia/45 min or 24 h of reperfusion. In another set of animals (n = 5), SOD1 was overexpressed only in the contralateral hemisphere by stereotaxic adenovirus injection 2-3 wk before I/R. Myogenic tone and neurovascular outcomes were determined. HG exacerbated myogenic dysfunction in contralateral side only, which was associated with infarct size expansion, increased edema, and more pronounced neurological deficit. Global and selective SOD1 overexpression restored myogenic reactivity in ipsilateral and contralateral sides, respectively, and enhanced neurovascular outcomes. In conclusion, our results show that SOD1 overexpression nullified the detrimental effects of HG on myogenic tone and stroke outcomes and that the contralateral hemisphere may be a novel target for the management of acute hyperglycemic stroke.

Metformin treatment in the period after stroke prevents nitrative stress and restores angiogenic signaling in the brain in diabetes.

Diabetes impedes vascular repair and causes vasoregression in the brain after stroke, but mechanisms underlying this response are still unclear. We hypothesized that excess peroxynitrite formation in diabetic ischemia/reperfusion (I/R) injury inactivates the p85 subunit of phosphoinositide 3-kinase (PI3K) by nitration and diverts the PI3K-Akt survival signal to the p38-mitogen-activated protein kinase apoptosis pathway. Nitrotyrosine (NY), Akt and p38 activity, p85 nitration, and caspase-3 cleavage were measured in brains from control, diabetic (GK), or metformin-treated GK rats subjected to sham or stroke surgery and in brain microvascular endothelial cells (BMVECs) from Wistar and GK rats subjected to hypoxia/reoxygenation injury. GK rat brains showed increased NY, caspase-3 cleavage, and p38 activation and decreased Akt activation. Metformin attenuated stroke-induced nitrative signaling in GK rats. GK rat BMVECs showed increased basal nitrative stress compared with controls. A second hit by hypoxia/reoxygenation injury dramatically increased the nitration of p85 and activation of p38 but decreased Akt. These effects were associated with impairment of angiogenic response and were restored by treatment with the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride or the nitration inhibitor epicatechin. Our results provide evidence that I/R-induced peroxynitrite inhibits survival, induces apoptosis, and promotes peroxynitrite as a novel therapeutic target for the improvement of reparative angiogenesis after stroke in diabetes.

Hyperglycemia, acute ischemic stroke, and thrombolytic therapy.

Ischemic stroke is a leading cause of disability and is considered now the fourth leading cause of death. Many clinical trials have shown that stroke patients with acute elevation in blood glucose at onset of stroke suffer worse functional outcomes, longer in-hospital stay, and higher mortality rates. The only therapeutic hope for these patients is the rapid restoration of blood flow to the ischemic tissue through intravenous administration of the only currently proven effective therapy, tissue plasminogen activator (tPA). However, even this option is associated with the increased risk of intracerebral hemorrhage. Nonetheless, the underlying mechanisms through which hyperglycemia (HG) and tPA worsen the neurovascular injury after stroke are not fully understood. Accordingly, this review summarizes the latest updates and recommendations about the management of HG and coadministration of tPA in a clinical setting while focusing more on the various experimental models studying (1) the effect of HG on stroke outcomes, (2) the potential mechanisms involved in worsening the neurovascular injury, (3) the different therapeutic strategies employed to ameliorate the injury, and finally, (4) the interaction between HG and tPA. Developing therapeutic strategies to reduce the hemorrhage risk with tPA in hyperglycemic setting is of great clinical importance. This can best be achieved by conducting robust preclinical studies evaluating the interaction between tPA and other therapeutics in order to develop potential therapeutic strategies with high translational impact.

Remote ischemic perconditioning is effective after embolic stroke in ovariectomized female mice.

Remote ischemic conditioning is neuroprotective in young male rodents after experimental stroke. However, it has never been tested in females whom remain at higher risk of stroke injury after menopause. We tested remote ischemic perconditioning therapy (RIPerC) at 2 h after embolic stroke in ovariectomized (OVX) female mice with and without intravenous tissue plasminogen activator (IV-tPA) treatment. We assessed cerebral blood flow (CBF), neurobehavioral outcomes, infarction, hemorrhage, edema, and survival. RIPerC therapy with and without IV-tPA improved the CBF and neurobehavioral outcomes and reduced the infarction, hemorrhage, and edema significantly. Late IV-tPA alone at 4 h post-stroke neither improved the neurobehavior nor reduced the infarction but aggravated hemorrhage and mortality in OVX mice. RIPerC therapy prevented the increased mortality during late IV-tPA. Our study demonstrates for the first time that RIPerC therapy is effective in OVX females.

Thioredoxin-interacting protein is required for endothelial NLRP3 inflammasome activation and cell death in a rat model of high-fat diet.

AIMS/HYPOTHESIS: Obesity and hypertension, known pro-inflammatory states, are identified determinants for increased retinal microvascular abnormalities. However, the molecular link between inflammation and microvascular degeneration remains elusive. Thioredoxin-interacting protein (TXNIP) is recognised as an activator of the NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome. This study aims to examine TXNIP expression and elucidate its role in endothelial inflammasome activation and retinal lesions. METHODS: Spontaneously hypertensive (SHR) and control Wistar (W) rats were compared with groups fed a high-fat diet (HFD) (W+F and SHR+F) for 8-10 weeks. RESULTS: Compared with W controls, HFD alone or in combination with hypertension significantly induced formation of acellular capillaries, a hallmark of retinal ischaemic lesions. These effects were accompanied by significant increases in lipid peroxidation, nitrotyrosine and expression of TXNIP, nuclear factor κB, TNF-α and IL-1ß. HFD significantly increased interaction of TXNIP-NLRP3 and expression of cleaved caspase-1 and cleaved IL-1ß. Immunolocalisation studies identified TXNIP expression within astrocytes and Müller cells surrounding retinal endothelial cells. To model HFD in vitro, human retinal endothelial (HRE) cells were stimulated with 400 µmol/l palmitate coupled to BSA (Pal-BSA). Pal-BSA triggered expression of TXNIP and its interaction with NLRP3, resulting in activation of caspase-1 and IL-1ß in HRE cells. Silencing Txnip expression in HRE cells abolished Pal-BSA-mediated cleaved IL-1ß release into medium and cell death, evident by decreases in cleaved caspase-3 expression and the proportion of live to dead cells. CONCLUSIONS/INTERPRETATION: These findings provide the first evidence for enhanced TXNIP expression in hypertension and HFD-induced retinal oxidative/inflammatory response and suggest that TXNIP is required for HFD-mediated activation of the NLRP3 inflammasome and the release of IL-1ß in endothelial cells.