Nosirudeen Quadri

Deletion of the Receptor for Advanced Glycation End Products Reduces Glomerulosclerosis and Preserves Renal Function in the Diabetic OVE26 Mouse

OBJECTIVE Previous studies showed that genetic deletion or pharmacological blockade of the receptor for advanced glycation end products (RAGE) prevents the early structural changes in the glomerulus associated with diabetic nephropathy. To overcome limitations of mouse models that lack the progressive glomerulosclerosis observed in humans, we studied the contribution of RAGE to diabetic nephropathy in the OVE26 type 1 mouse, a model of progressive glomerulosclerosis and decline of renal function. RESEARCH DESIGN AND METHODS We bred OVE26 mice with homozygous RAGE knockout (RKO) mice and examined structural changes associated with diabetic nephropathy and used inulin clearance studies and albumin:creatinine measurements to assess renal function. Transcriptional changes in the Tgf-β1 and plasminogen activator inhibitor 1 gene products were measured to investigate mechanisms underlying accumulation of mesangial matrix in OVE26 mice. RESULTS Deletion of RAGE in OVE26 mice reduced nephromegaly, mesangial sclerosis, cast formation, glomerular basement membrane thickening, podocyte effacement, and albuminuria. The significant 29% reduction in glomerular filtration rate observed in OVE26 mice was completely prevented by deletion of RAGE. Increased transcription of the genes for plasminogen activator inhibitor 1, Tgf-β1, Tgf-β–induced, and α1-(IV) collagen observed in OVE26 renal cortex was significantly reduced in OVE26 RKO kidney cortex. ROCK1 activity was significantly lower in OVE26 RKO compared with OVE26 kidney cortex. CONCLUSIONS These data provide compelling evidence for critical roles for RAGE in the pathogenesis of diabetic nephropathy and suggest that strategies targeting RAGE in long-term diabetes may prevent loss of renal function.

Glutaminolysis and Transferrin Regulate Ferroptosis

Ferroptosis has emerged as a new form of regulated necrosis that is implicated in various human diseases. However, the mechanisms of ferroptosis are not well defined. This study reports the discovery of multiple molecular components of ferroptosis and its intimate interplay with cellular metabolism and redox machinery. Nutrient starvation often leads to sporadic apoptosis. Strikingly, we found that upon deprivation of amino acids, a more rapid and potent necrosis process can be induced in a serum-dependent manner, which was subsequently determined to be ferroptosis. Two serum factors, the iron-carrier protein transferrin and amino acid glutamine, were identified as the inducers of ferroptosis. We further found that the cell surface transferrin receptor and the glutamine-fueled intracellular metabolic pathway, glutaminolysis, played crucial roles in the death process. Inhibition of glutaminolysis, the essential component of ferroptosis, can reduce heart injury triggered by ischemia/reperfusion, suggesting a potential therapeutic approach for treating related diseases.

Aldose reductase mediates myocardial ischemia-reperfusion injury in part by opening mitochondrial permeability transition pore.

Aldose reductase (AR), a member of the aldo-keto reductase family, has been demonstrated to play a central role in mediating myocardial ischemia-reperfusion (I/R) injury. Recently, using transgenic mice broadly overexpressing human AR (ARTg), we demonstrated that AR is an important component of myocardial I/R injury and that inhibition of this enzyme protects heart from I/R injury (20-22, 48, 49, 56). To rigorously delineate mechanisms by which AR pathway influences myocardial ischemic injury, we investigated the role played by reactive oxygen species (ROS), antioxidant enzymes, and mitochondrial permeability transition (MPT) pore opening in hearts from ARTg or littermates [wild type (WT)] subjected to I/R. MPT pore opening after I/R was determined using mitochondrial uptake of 2-deoxyglucose ratio, while H2O2 was measured as a key indicator of ROS. Myocardial 2-deoxyglucose uptake ratio and calcium-induced swelling were significantly greater in mitochondria from ARTg mice than in WT mice. Blockade of MPT pore with cyclosphorin A during I/R reduced ischemic injury significantly in ARTg mice hearts. H2O2 measurements indicated mitochondrial ROS generation after I/R was significantly greater in ARTg mitochondria than in WT mice hearts. Furthermore, the levels of antioxidant GSH were significantly reduced in ARTg mitochondria than in WT. Resveratrol treatment or pharmacological blockade of AR significantly reduced ROS generation and MPT pore opening in mitochondria of ARTg mice hearts exposed to I/R stress. This study demonstrates that MPT pore opening is a key event by which AR pathway mediates myocardial I/R injury, and that the MPT pore opening after I/R is triggered, in part, by increases in ROS generation in ARTg mice hearts. Therefore, inhibition of AR pathway protects mitochondria and hence may be a useful adjunct for salvaging ischemic myocardium.

Novel Role for Aldose Reductase in Mediating Acute Inflammatory Responses in the Lung

Exaggerated inflammatory responses and the resultant increases in alveolar-capillary permeability underlie the pathogenesis of acute lung injury during sepsis. This study examined the functions of aldose reductase (AR) in mediating acute lung inflammation. Transgenic mice expressing human AR (ARTg) were used to study the functions of AR since mice have low intrinsic AR activity. In a mild cecal ligation and puncture model, ARTg mice demonstrated an enhanced AR activity and a greater inflammatory response as evaluated by circulating cytokine levels, neutrophil accumulation in the lungs, and activation of Rho kinase in lung endothelial cells (ECs). Compared with WT lung cells, ARTg lung cells produced more IL-6 and showed augmented JNK activation in response to LPS stimulation ex vivo. In human neutrophils, AR activity was required for fMLP-included CD11b activation and up-regulation, respiratory burst, and shape changes. In human pulmonary microvascular ECs, AR activity was required for TNF-α-induced activation of the Rho kinase/MKK4/JNK pathway and IL-6 production, but not p38 activation or ICAM-1 expression. Importantly, AR activity in both human neutrophils and ECs was required for neutrophil adhesion to TNF-α-stimulated ECs. These data demonstrate a novel role for AR in regulating the signaling pathways leading to neutrophil-EC adhesion during acute lung inflammation.

Acute Administration of n-3 Rich Triglyceride Emulsions Provides Cardioprotection in Murine Models after Ischemia-Reperfusion

Dietary n-3 fatty acids (FAs) may reduce cardiovascular disease risk. We questioned whether acute administration of n-3 rich triglyceride (TG) emulsions could preserve cardiac function and decrease injury after ischemia/reperfusion (I/R) insult. We used two different experimental models: in vivo, C57BL/6 mice were exposed to acute occlusion of the left anterior descending coronary artery (LAD), and ex-vivo, C57BL/6 murine hearts were perfused using Langendorff technique (LT). In the LAD model, mice treated with n-3 TG emulsion (1.5g/kg body weight), immediately after ischemia and 1h later during reperfusion, significantly reduced infarct size and maintained cardiac function (p<0.05). In the LT model, administration of n-3 TG emulsion (300mgTG/100ml) during reperfusion significantly improved functional recovery (p<0.05). In both models, lactate dehydrogenase (LDH) levels, as a marker of injury, were significantly reduced by n-3 TG emulsion. To investigate the mechanisms by which n-3 FAs protects hearts from I/R injury, we investigated changes in key pathways linked to cardioprotection. In the ex-vivo model, we showed that n-3 FAs increased phosphorylation of AKT and GSK3β proteins (p<0.05). Acute n-3 TG emulsion treatment also increased Bcl-2 protein level and reduced an autophagy marker, Beclin-1 (p<0.05). Additionally, cardioprotection by n-3 TG emulsion was linked to changes in PPARγ protein expression (p<0.05). Rosiglitazone and p-AKT inhibitor counteracted the positive effect of n-3 TG; GSK3β inhibitor plus n-3 TG significantly inhibited LDH release. We conclude that acute n-3 TG injection during reperfusion provides cardioprotection. This may prove to be a novel acute adjunctive reperfusion therapy after treating patients with myocardial infarction.

Carbon monoxide form of PEGylated hemoglobin protects myocardium against ischemia/reperfusion injury in diabetic and normal mice

Abstract We investigated the pre-clinical utility of carbon monoxide form of PEGylated hemoglobin (PEG-Hb also named SANGUINATE™) in myocardial infarction (MI) and in particular the response of diabetic tissues to superimposed ischemia/reperfusion injury. SANGUINATE™ was evaluated in diabetic and normal mice subjected to 30 min of coronary artery ligation followed by either 48 h or 28 days of reperfusion. Our results demonstrate that SANGUINATE™ was effective in reducing infarct size when administered either prior to left anterior descending coronary artery (LAD) occlusion or during reperfusion. This finding is an important step in exploring the efficacy of a pharmacoinvasive strategy using SANGUINATE™ in patients with acute coronary syndromes.

Small Molecule Inhibition of Ligand-Stimulated RAGE-DIAPH1 Signal Transduction

The receptor for advanced glycation endproducts (RAGE) binds diverse ligands linked to chronic inflammation and disease. NMR spectroscopy and x-ray crystallization studies of the extracellular domains of RAGE indicate that RAGE ligands bind by distinct charge- and hydrophobicity-dependent mechanisms. The cytoplasmic tail (ct) of RAGE is essential for RAGE ligand-mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE signaling requires interaction of ctRAGE with the intracellular effector, mammalian diaphanous 1 or DIAPH1. We screened a library of 58,000 small molecules and identified 13 small molecule competitive inhibitors of ctRAGE interaction with DIAPH1. These compounds, which exhibit in vitro and in vivo inhibition of RAGE-dependent molecular processes, present attractive molecular scaffolds for the development of therapeutics against RAGE-mediated diseases, such as those linked to diabetic complications, Alzheimer’s disease, and chronic inflammation, and provide support for the feasibility of inhibition of protein-protein interaction (PPI).

Morphological Changes and Immunohistochemical Expression of RAGE and its Ligands in the Sciatic Nerve of Hyperglycemic Pig (Sus Scrofa)

The aim of our project was to study the effect of streptozotocin (STZ)–-induced hyperglycemia on sciatic nerve morphology, blood plasma markers and immunohistochemical expression of RAGE (the Receptor for Advanced Glycation End-products), and its ligands–-S100B and Carboxymethyl Lysine (CML)-advanced glycation endproduct (AGE) in the laboratory pig. Six months after STZ–-injections, blood plasma measurements, morphometric analysis of sciatic nerve fiber density, immunofluorescent distribution of potential molecular neuropathy contributors, ELISA measurement of plasma AGE level and HPLC analysis of sciatic nerve levels of one of the pre-AGE and the glycolysis intermediate products–-methylglyoxal (MG) were performed. The results of our study revealed that STZ–-injected animals displayed elevated levels of plasma glucose, gamma glutamyl transferase (GGT) and triglycerides. The sciatic nerve of STZ-injected pigs revealed significantly lower numbers of small-diameter myelinated fibers, higher immunoreactivity for RAGE and S100B and increased levels of MG as compared to control animals. Our results correspond to clinical findings in human patients with hyperglycemia/diabetes-evoked peripheral neuropathy and suggest that the domestic pig may be a suitable large animal model for the study of mechanisms underlying hyperglycemia-induced neurological complications in the peripheral nerve and may serve as a relevant model for the pre-clinical assessment of candidate drugs in neuropathy.

Aldose Reductase Acts as a Selective Derepressor of PPARγ and the Retinoic Acid Receptor

Histone deacetylase 3 (HDAC3), a chromatin-modifying enzyme, requires association with the deacetylase-containing domain (DAD) of the nuclear receptor corepressors NCOR1 and SMRT for its stability and activity. Here, we show that aldose reductase (AR), the rate-limiting enzyme of the polyol pathway, competes with HDAC3 to bind the NCOR1/SMRT DAD. Increased AR expression leads to HDAC3 degradation followed by increased PPARγ signaling, resulting in lipid accumulation in the heart. AR also downregulates expression of nuclear corepressor complex cofactors including Gps2 and Tblr1, thus affecting activity of the nuclear corepressor complex itself. Though AR reduces HDAC3-corepressor complex formation, it specifically derepresses the retinoic acid receptor (RAR), but not other nuclear receptors such as the thyroid receptor (TR) and liver X receptor (LXR). In summary, this work defines a distinct role for AR in lipid and retinoid metabolism through HDAC3 regulation and consequent derepression of PPARγ and RAR.

The Formin, DIAPH1, is a Key Modulator of Myocardial Ischemia/Reperfusion Injury

The biochemical, ionic, and signaling changes that occur within cardiomyocytes subjected to ischemia are exacerbated by reperfusion; however, the precise mechanisms mediating myocardial ischemia/reperfusion (I/R) injury have not been fully elucidated. The receptor for advanced glycation end-products (RAGE) regulates the cellular response to cardiac tissue damage in I/R, an effect potentially mediated by the binding of the RAGE cytoplasmic domain to the diaphanous-related formin, DIAPH1. The aim of this study was to investigate the role of DIAPH1 in the physiological response to experimental myocardial I/R in mice. After subjecting wild-type mice to experimental I/R, myocardial DIAPH1 expression was increased, an effect that was echoed following hypoxia/reoxygenation (H/R) in H9C2 and AC16 cells. Further, compared to wild-type mice, genetic deletion of Diaph1 reduced infarct size and improved contractile function after I/R. Silencing Diaph1 in H9C2 cells subjected to H/R downregulated actin polymerization and serum response factor-regulated gene expression. Importantly, these changes led to increased expression of sarcoplasmic reticulum Ca2 + ATPase and reduced expression of the sodium calcium exchanger. This work demonstrates that DIAPH1 is required for the myocardial response to I/R, and that targeting DIAPH1 may represent an adjunctive approach for myocardial salvage after acute infarction.