Wael Eldahshan

Molecular mechanisms of diabetic retinopathy: Potential therapeutic targets

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults in United States. Research indicates an association between oxidative stress and the development of diabetes complications. However, clinical trials with general antioxidants have failed to prove effective in diabetic patients. Mounting evidence from experimental studies that continue to elucidate the damaging effects of oxidative stress and inflammation in both vascular and neural retina suggest its critical role in the pathogenesis of DR. This review will outline the current management of DR as well as present potential experimental therapeutic interventions, focusing on molecules that link oxidative stress to inflammation to provide potential therapeutic targets for treatment or prevention of DR. Understanding the biochemical changes and the molecular events under diabetic conditions could provide new effective therapeutic tools to combat the disease.

Brain-Derived Neurotrophic Factor Knockdown Blocks the Angiogenic and Protective Effects of Angiotensin Modulation After Experimental Stroke

Angiotensin type 1 receptor blockers (ARBs) have been shown to be neuroprotective and neurorestorative in experimental stroke. The mechanisms proposed include anti-inflammatory, antiapoptotic effects, as well as stimulation of endogenous trophic factors leading to angiogenesis and neuroplasticity. We aimed to investigate the involvement of the neurotrophin, brain-derived neurotrophic factor (BDNF), in ARB-mediated functional recovery after stroke. To achieve this aim, Wistar rats received bilateral intracerebroventricular (ICV) injections of short hairpin RNA (shRNA) lentiviral particles or nontargeting control (NTC) vector, to knock down BDNF in both hemispheres. After 14xa0days, rats were subjected to 90-min middle cerebral artery occlusion (MCAO) and received the ARB, candesartan, 1xa0mg/kg, or saline IV at reperfusion (one dose), then followed for another 14xa0days using a battery of behavioral tests. BDNF protein expression was successfully reduced by about 70xa0% in both hemispheres at 14xa0days after bilateral shRNA lentiviral particle injection. The NTC group that received candesartan showed better functional outcome as well as increased vascular density and synaptogenesis as compared to saline treatment. BDNF knockdown abrogated the beneficial effects of candesartan on neurobehavioral outcome, vascular density, and synaptogenesis. In conclusion, BDNF is directly involved in candesartan-mediated functional recovery, angiogenesis, and synaptogenesis.

Within the Brain: The Renin Angiotensin System

For many years, modulators of the renin angiotensin system (RAS) have been trusted by clinicians for the control of essential hypertension. It was recently demonstrated that these modulators have other pleiotropic properties independent of their hypotensive effects, such as enhancement of cognition. Within the brain, different components of the RAS have been extensively studied in the context of neuroprotection and cognition. Interestingly, a crosstalk between the RAS and other systems such as cholinergic, dopaminergic and adrenergic systems have been demonstrated. In this review, the preclinical and clinical evidence for the impact of RAS modulators on cognitive impairment of multiple etiologies will be discussed. In addition, the expression and function of different receptor subtypes within the RAS such as: Angiotensin II type I receptor (AT1R), Angiotensin II type II receptor (AT2R), Angiotensin IV receptor (AT4R), Mas receptor (MasR), and Mas-related-G protein-coupled receptor (MrgD), on different cell types within the brain will be presented. We aim to direct the attention of the scientific community to the plethora of evidence on the importance of the RAS on cognition and to the different disease conditions in which these agents can be beneficial.

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 0.06u2009mg/kg/d) or saline (orally) for five days, with the first-dose given IV at 3u2009h post-eMCAO. For the time-window study, the optimal-dose of C21 was initiated at 3, 6 or 24u2009h post-eMCAO and continued for five days. For the combinatorial study, animals received IV-tissue plasminogen activator (tPA) at either 2 or 4u2009h, with IV-C21 (0.01u2009mg/kg) or saline at 3u2009hu2009post-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 (0.01u2009mg/kg) of C21. In the time-window study, this same dose resulted in improvements when given 6u2009h and 24u2009h post-eMCAO. Moreover, C21-treated animals performed better on the novel object recognition test. Neither the single treatment with C21 or tPA (4u2009h) 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.

Suppression of Akt1- β-catenin pathway in advanced prostate cancer promotes TGFβ1-mediated epithelial to mesenchymal transition and metastasis

Akt1 is essential for the oncogenic transformation and tumor growth in various cancers. However, the precise role of Akt1 in advanced cancers is conflicting. Using a neuroendocrine TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model, we first show that the genetic ablation or pharmacological inhibition of Akt1 in mice blunts oncogenic transformation and prostate cancer (PCa) growth. Intriguingly, triciribine (TCBN)-mediated Akt inhibition in 25-week old, tumor-bearing TRAMP mice and Akt1 gene silencing in aggressive PCa cells enhanced epithelial to mesenchymal transition (EMT) and promoted metastasis to the lungs. Mechanistically, Akt1 suppression leads to increased expression of EMT markers such as Snail1 and N-cadherin and decreased expression of epithelial marker E-cadherin in TRAMP prostate, and in PC3 and DU145xa0cells. Next, we identified that Akt1 knockdown in PCa cells results in increased production of TGFβ1 and its receptor TGFβ RII, associated with a decreased expression of β-catenin. Furthermore, treatment of PCa cells with ICG001 that blocks nuclear translocation of β-catenin promoted EMT and N-cadherin expression. Together, our study demonstrates a novel role of the Akt1-β-catenin-TGFβ1 pathway in advanced PCa.

Deletion of TXNIP Mitigates High-Fat Diet-Impaired Angiogenesis and Prevents Inflammation in a Mouse Model of Critical Limb Ischemia

Background: Previous work demonstrated that high-fat diet (HFD) triggered thioredoxin-interacting protein (TXNIP) and that silencing TXNIP prevents diabetes-impaired vascular recovery. Here, we examine the impact of genetic deletion of TXNIP on HFD-impaired vascular recovery using hind limb ischemia model. Methods: Wild type mice (WT, C57Bl/6) and TXNIP knockout mice (TKO) were fed either normal chow diet (WT-ND and TKO-ND) or 60% high-fat diet (WT-HFD and TKO-HFD). After four weeks of HFD, unilateral hind limb ischemia was performed and blood flow was measured using Laser doppler scanner at baseline and then weekly for an additional three weeks. Vascular density, nitrative stress, infiltration of CD68+ macrophages, and expression of inflammasome, vascular endothelial growth factor (VEGF), VEGF receptor-2 were examined by slot blot, Western blot and immunohistochemistry. Results: By week 8, HFD caused similar increases in weight, cholesterol and triglycerides in both WT and TKO. At week 4 and week 8, HFD significantly impaired glucose tolerance in WT and to a lesser extent in TKO. HFD significantly impaired blood flow and vascular density (CD31 labeled) in skeletal muscle of WT mice compared to ND but not in TKO. HFD and ischemia significantly induced tyrosine nitration, and systemic IL-1β and infiltration of CD68+ cells in skeletal muscle from WT but not from TKO. HFD significantly increased cleaved-caspase-1 and IL-1 β compared to ND. Under both ND, ischemia tended to increase VEGF expression and increased VEGFR2 activation in WT only but not TKO. Conclusion: Similar to prior observation in diabetes, HFD-induced obesity can compromise vascular recovery in response to ischemic insult. The mechanism involves increased TXNIP-NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome activation, nitrative stress and impaired VEGFR2 activation. Deletion of TXNIP restored blood flow, reduced nitrative stress and blunted inflammasome-mediated inflammation; however, it did not impact VEGF/VEGFR2 in HFD. Targeting TXNIP-NLRP3 inflammasome can provide potential therapeutic target in obesity-induced vascular complication.

Artery reopening is required for the neurorestorative effects of angiotensin modulation after experimental stroke

Blood flow restoration with fibrinolysis and thrombectomy is recommended to limit injury in stroke patients with proximal artery occlusion. Angiotensin receptor blockers have been shown to be neuroprotective in models of permanent and temporary occlusion, but the benefits on expression of trophic factors have been seen only when the artery is reopened. It is possible that early artery opening with endovascular intervention may increase the likelihood of identifying an effective combination therapy for patients. Normotensive male Wistar rats were subjected to mechanical middle cerebral artery occlusion (either temporary or permanent), followed by randomization to receive candesartan (0.3xa0mg/kg IV) or saline. Functional outcome, infarct size, and biochemical changes were assessed 24xa0h after ischemia induction. Lack of reperfusion blunted candesartan induced neuroprotection (pxa0<xa00.05) and reduced the improvement of functional outcome (pxa0<xa00.05). With reperfusion, candesartan increased mature BDNF expression in the contralateral hemisphere (pxa0<xa00.05) and activated prosurvival (Akt-GSK3-β) signaling (pxa0<xa00.05). Without reperfusion, candesartan significantly reduced VEGF expression and MMP activation and increased NOGO A expression, creating an environment hostile to recovery. Candesartan induced pro-recovery effects are dependent on the presence of reperfusion.BackgroundBlood flow restoration with fibrinolysis and thrombectomy is recommended to limit injury in stroke patients with proximal artery occlusion. Angiotensin receptor blockers have been shown to be neuroprotective in models of permanent and temporary occlusion, but the benefits on expression of trophic factors have been seen only when the artery is reopened. It is possible that early artery opening with endovascular intervention may increase the likelihood of identifying an effective combination therapy for patients.MethodsNormotensive male Wistar rats were subjected to mechanical middle cerebral artery occlusion (either temporary or permanent), followed by randomization to receive candesartan (0.3xa0mg/kg IV) or saline. Functional outcome, infarct size, and biochemical changes were assessed 24xa0h after ischemia induction.ResultsLack of reperfusion blunted candesartan induced neuroprotection (pxa0<xa00.05) and reduced the improvement of functional outcome (pxa0<xa00.05). With reperfusion, candesartan increased mature BDNF expression in the contralateral hemisphere (pxa0<xa00.05) and activated prosurvival (Akt-GSK3-β) signaling (pxa0<xa00.05). Without reperfusion, candesartan significantly reduced VEGF expression and MMP activation and increased NOGO A expression, creating an environment hostile to recovery.ConclusionCandesartan induced pro-recovery effects are dependent on the presence of reperfusion.

Silencing VEGF-B Diminishes the Neuroprotective Effect of Candesartan Treatment After Experimental Focal Cerebral Ischemia

The pro-survival effect of VEGF-B has been documented in different in vivo and in vitro models. We have previously shown an enhanced VEGF-B expression in response to candesartan treatment after focal cerebral ischemia. In this study, we aimed to silence VEGF-B expression to assess its contribution to candesartan’s benefit on stroke outcome. Silencing VEGF-B expression was achieved by bilateral intracerebroventricular injections of lentiviral particles containing short hairpin RNA (shRNA) against VEGF-B. Two weeks after lentiviral injections, rats were subjected to either 90xa0min or 3xa0h of middle cerebral artery occlusion (MCAO) and randomized to intravenous candesartan (1xa0mg/kg) or saline at reperfusion. Animals were sacrificed at 24 or 72xa0h and brains were collected and analyzed for hemoglobin (Hb) excess and infarct size, respectively. Functional outcome at 24, 48 and 72xa0h was assessed blindly. Candesartan treatment improved neurobehavioral and motor function, and decreased infarct size and Hb. While silencing VEGF-B expression diminished candesartan’s neuroprotective effect, candesartan-mediated vascular protection was maintained even in the absence of VEGF-B suggesting that this growth factor is not the mediator of candesartan’s vascular protective effects. However, VEGF-B is a mediator of neuroprotection achieved by candesartan and represents a potential drug target to improve stroke outcome. Further studies are needed to elucidate the underlying molecular mechanisms of VEGF-B in neuroprotection and recovery after ischemic stroke.

RAS modulation prevents progressive cognitive impairment after experimental stroke: a randomized, blinded preclinical trial

BackgroundWith the aging population, the prevalence and incidence of cerebrovascular disease will continue to rise, as well as the number of individuals with vascular cognitive impairment/dementia (VCID). No specific FDA-approved treatments for VCID exist. Although clinical evidence supports that angiotensin receptor blockers (ARBs) prevent cognitive decline in older adults, whether ARBs have a similar effect on VCID after stroke is unknown. Moreover, these agents reduce BP, which is undesirable in the acute stroke period, so we believe that giving C21 in this acute phase or delaying ARB administration would enable us to achieve the neurovascular benefits without the risk of unintended and potentially dangerous, acute BP lowering.MethodsThe aim of our study was to determine the impact of candesartan (ARB) or compound-21 (an angiotensin type 2 receptor––AT2R––agonist) on long-term cognitive function post-stroke, in spontaneously hypertensive rats (SHRs). We hypothesized that AT2R stimulation, either directly with C21, or indirectly by blocking the angiotensin type 1 receptor (AT1R) with candesartan, initiated after stroke, would reduce cognitive impairment. Animals were subjected to a 60-min transient middle cerebral artery occlusion and randomly assigned to either saline/C21 monotherapy, for the full study duration (30xa0days), or given sequential therapy starting with saline/C21 (7xa0days) followed by candesartan for the remainder of the study (21xa0days). Outcome measures included sensorimotor/cognitive-function, amyloid-β determination, and histopathologic analyses.ResultsTreatment with RAS modulators effectively preserved cognitive function, reduced cytotoxicity, and prevented chronic-reactive microgliosis in SHRs, post-stroke. These protective effects were apparent even when treatment was delayed up to 7xa0days post-stroke and were independent of blood pressure and β-amyloid accumulation.ConclusionCollectively, our findings demonstrate that RAS modulators effectively prevent cognitive impairment after stroke, even when treatment is delayed.