Ager Deletion Enhances Ischemic Muscle Inflammation, Angiogenesis, and Blood Flow Recovery in Diabetic Mice.

OBJECTIVE: Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block angiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. APPROACH AND RESULTS: Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated angiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1 (early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, angiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. CONCLUSIONS: These findings indicate that RAGE attenuates adaptive inflammation in hindlimb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic peripheral vascular disease.

Formin mDia1 mediates vascular remodeling via integration of oxidative and signal transduction pathways.

RATIONALE: The mammalian diaphanous-related formin (mDia1), governs microtubule and microfilament dynamics while functioning as an effector for Rho small GTP-binding proteins during key cellular processes such as adhesion, cytokinesis, cell polarity, and morphogenesis. The cytoplasmic domain of the receptor for advanced glycation endproducts binds to the formin homology 1 domain of mDia1; mDia1 is required for receptor for advanced glycation endproducts ligand-induced cellular migration in transformed cells. OBJECTIVE: Because a key mechanism in vascular remodeling is the induction of smooth muscle cell migration, we tested the role of mDia1 in this process. METHODS AND RESULTS: We report that endothelial denudation injury to the murine femoral artery significantly upregulates mDia1 mRNA transcripts and protein in the injured vessel, particularly in vascular smooth muscle cells within the expanding neointima. Loss of mDia1 expression significantly reduces pathological neointimal expansion consequent to injury. In primary murine aortic smooth muscle cells, mDia1 is required for receptor for advanced glycation endproducts ligand-induced membrane translocation of c-Src, which leads to Rac1 activation, redox phosphorylation of AKT/glycogen synthase kinase 3ß, and consequent smooth muscle cell migration. CONCLUSIONS: We conclude that mDia1 integrates oxidative and signal transduction pathways triggered, at least in part, by receptor for advanced glycation endproducts ligands, thereby regulating pathological neointimal expansion.

PKCß promotes vascular inflammation and acceleration of atherosclerosis in diabetic ApoE null mice.

OBJECTIVE: Subjects with diabetes mellitus are at high risk for developing atherosclerosis through a variety of mechanisms. Because the metabolism of glucose results in production of activators of protein kinase C (PKC)ß, it was logical to investigate the role of PKCß in modulation of atherosclerosis in diabetes mellitus. APPROACH AND RESULTS: ApoE(-/-) and PKCß(-/-)/ApoE(-/-) mice were rendered diabetic with streptozotocin. Quantification of atherosclerosis, gene expression profiling, or analysis of signaling molecules was performed on aortic sinus or aortas from diabetic mice. Diabetes mellitus-accelerated atherosclerosis increased the level of phosphorylated extracellular signal-regulated kinase 1/2 and Jun-N-terminus kinase mitogen-activated protein kinases and augmented vascular expression of inflammatory mediators, as well as increased monocyte/macrophage infiltration and CD11c(+) cells accumulation in diabetic ApoE(-/-) mice, processes that were diminished in diabetic PKCß(-/-)/ApoE(-/-) mice. In addition, pharmacological inhibition of PKCß reduced atherosclerotic lesion size in diabetic ApoE(-/-) mice. In vitro, the inhibitors of PKCß and extracellular signal-regulated kinase 1/2, as well as small interfering RNA to Egr-1, significantly decreased high-glucose-induced expression of CD11c (integrin, alpha X 9 complement component 3 receptor 4 subunit]), chemokine (C-C motif) ligand 2, and interleukin-1ß in U937 macrophages. CONCLUSIONS: These data link enhanced activation of PKCß to accelerated diabetic atherosclerosis via a mechanism that includes modulation of gene transcription and signal transduction in the vascular wall, processes that contribute to acceleration of vascular inflammation and atherosclerosis in diabetes mellitus. Our results uncover a novel role for PKCß in modulating CD11c expression and inflammatory response of macrophages in the development of diabetic atherosclerosis. These findings support PKCß activation as a potential therapeutic target for prevention and treatment of diabetic atherosclerosis.

Simultaneous inhibition of PI3K and PAK in preclinical models of neurofibromatosis type 2-related schwannomatosis.

Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.

The Oncolytic Activity of Zika Viral Therapy in Human Neuroblastoma In Vivo Models Confers a Major Survival Advantage in a CD24-dependent Manner.

Neuroblastoma is the most common extracranial tumor, accounting for 15% of all childhood cancer-related deaths. The long-term survival of patients with high-risk tumors is less than 40%, and MYCN amplification is one of the most common indicators of poor outcomes. Zika virus (ZIKV) is a mosquito-borne flavivirus associated with mild constitutional symptoms outside the fetal period. Our published data showed that high-risk and recurrent neuroblastoma cells are permissive to ZIKV infection, resulting in cell type-specific lysis. In this study, we assessed the efficacy of ZIKV as an oncolytic treatment for high-risk neuroblastoma using in vivo tumor models. Utilizing both MYCN-amplified and non-amplified models, we demonstrated that the application of ZIKV had a rapid tumoricidal effect. This led to a nearly total loss of the tumor mass without evidence of recurrence, offering a robust survival advantage to the host. Detection of the viral NS1 protein within the tumors confirmed that a permissive infection preceded tissue necrosis. Despite robust titers within the tumor, viral shedding to the host was poor and diminished rapidly, correlating with no detectable side effects to the murine host. Assessments from both primary pretreatment and recurrent posttreatment isolates confirmed that permissive sensitivity to ZIKV killing was dependent on the expression of CD24, which was highly expressed in neuroblastomas and conferred a proliferative advantage to tumor growth. Exploiting this viral sensitivity to CD24 offers the possibility of its use as a prognostic target for a broad population of expressing cancers, many of which have shown resistance to current clinical therapies. SIGNIFICANCE: Sensitivity to the tumoricidal effect of ZIKV on high-risk neuroblastoma tumors is dependent on CD24 expression, offering a prognostic marker for this oncolytic therapy in an extensive array of CD24-expressing cancers.

Impaired slow axonal transport in diabetic peripheral nerve is independent of RAGE.

Diabetic peripheral nerve dysfunction is a common complication occurring in 30-50% of long-term diabetic patients. The pathogenesis of this dysfunction remains unclear but growing evidence suggests that it might be attributed, in part, to alteration in axonal transport. Our previous studies demonstrated that RAGE (Receptor for Advanced Glycation Endproducts) contributes to the pathogenesis of diabetic peripheral neuropathy and impairs nerve regeneration consequent to sciatic nerve crush, particularly in diabetes. We hypothesize that RAGE plays a role in axonal transport impairment via the interaction of its cytoplasmic domain with mammalian Diaphanous 1 (mDia1) - actin interacting molecule. Studies showed that mDia1-RAGE interaction is necessary for RAGE-ligand-dependent cellular migration, AKT phosphorylation, macrophage inflammatory response and smooth muscle migration. Here, we studied RAGE, mDia1 and markers of axonal transport rates in the peripheral nerves of wild-type C57BL/6 and RAGE null control and streptozotocin-injected diabetic mice at 1, 3 and 6 h after sciatic nerve crush. The results show that in both control and diabetic nerves, the amount of RAGE accumulated at the proximal and distal side of the crush area is similar, indicating that the recycling rate for RAGE is very high and that it is evenly transported from and towards the neuronal cell body. Furthermore, we show that slow axonal transport of proteins such as Neurofilament is affected by diabetes in a RAGE-independent manner. Finally, our study demonstrates that mDia1 axonal transport is impaired in diabetes, suggesting that diabetes-related changes affecting actin binding proteins occur early in the course of the disease.

Opposing roles of RAGE and Myd88 signaling in extensive liver resection.

In extensive liver resection secondary to primary or metastatic liver tumors, or in living donor liver transplantation, strategies to quell deleterious inflammatory responses and facilitate regeneration are essential. The receptor for advanced glycation endproducts (RAGE) and myeloid differentiating factor 88 (Myd88) are implicated in the inflammatory response. To establish the contributions of RAGE vs. Myd88 signaling in extensive liver resection, we probed the effect of RAGE and/or Myd88, the latter primarily a key transducer of major toll-like receptors and also implicated in interleukin-1 (Il1) signaling, in a murine model of extensive (85%) hepatectomy. We report that, although Myd88 is thoroughly essential for survival via regulation of NF-κB and TNF-α, deletion of RAGE significantly improved survival compared to wild-type, Myd88-null, or RAGE-null/Myd88-null mice. RAGE opposes Myd88 signaling at multiple levels: by suppression of p65 levels, thereby reducing activation of NF-κB and consequent production of cyclin D1, and by suppression of Il6-mediated phosphorylation of Stat3, thereby down-regulating Pim1 and suppressing the hyperplastic response. Further, RAGE-dependent suppression of glyoxalase1, a detoxification pathway for pre-AGEs, enhances AGE levels and suppresses Il6 action. We conclude that blockade of RAGE may rescue liver remnants from the multiple signals that preclude adaptive proliferation triggered primarily by Myd88 signaling pathways.

Simultaneous Inhibition of PI3K and PAK in Preclinical Models of Neurofibromatosis Type 2-related Schwannomatosis.

Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.

Aldose reductase modulates cardiac glycogen synthase kinase-3ß phosphorylation during ischemia-reperfusion.

Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3ß (p-GSK3ß). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3ß inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3ß was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3ß was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3ß and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3ß inhibitors improved p-GSK3ß expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3ß was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/ß inhibitor displayed significant increases in p-Akt and p-GSK3ß expression, and resulted in decreased LDH release. In summary, AR mediates changes in p-GSK3ß, in part, via PKCα/ß and Akt during I/R.

Activation of the ROCK1 branch of the transforming growth factor-beta pathway contributes to RAGE-dependent acceleration of atherosclerosis in diabetic ApoE-null mice.

RATIONALE: The multiligand RAGE (receptor for advanced glycation end products) contributes to atherosclerosis in apolipoprotein (Apo)E-null mice. OBJECTIVE: To delineate the specific mechanisms by which RAGE accelerated atherosclerosis, we performed Affymetrix gene expression arrays on aortas of nondiabetic and diabetic ApoE-null mice expressing RAGE or devoid of RAGE at nine weeks of age, as this reflected a time point at which frank atherosclerotic lesions were not yet present, but that we would be able to identify the genes likely involved in diabetes- and RAGE-dependent atherogenesis. METHODS AND RESULTS: We report that there is very little overlap of the genes that are differentially expressed both in the onset of diabetes in ApoE-null mice, and in the effect of RAGE deletion in diabetic ApoE-null mice. Pathway-Express analysis revealed that the transforming growth factor-beta pathway and focal adhesion pathways might be expected to play a significant role in both the mechanism by which diabetes facilitates the formation of atherosclerotic plaques in ApoE-null mice, and the mechanism by which deletion of RAGE ameliorates this effect. Quantitative polymerase chain reaction studies, Western blotting, and confocal microscopy in aortic tissue and in primary cultures of murine aortic smooth muscle cells supported these findings. CONCLUSIONS: Taken together, our work suggests that RAGE-dependent acceleration of atherosclerosis in ApoE-null mice is dependent, at least in part, on the action of the ROCK1 (rho-associated protein kinase 1) branch of the transforming growth factor-beta pathway.

Human aldose reductase expression accelerates atherosclerosis in diabetic apolipoprotein E-/- mice.

OBJECTIVE: There are several pathways that mediate the aberrant metabolism of glucose and that might induce greater vascular damage in the setting of diabetes. The polyol pathway mediated by aldose reductase (AR) has been postulated to be one such pathway. However, it has been reported that AR reduces toxic lipid aldehydes and, under some circumstances, might be antiatherogenic. METHODS AND RESULTS: Atherosclerosis development was quantified in 2 lines of transgenic mice expressing human AR (hAR) crossed on the apolipoprotein E knockout background. The transgenes were used to increase the normally low levels of this enzyme in wild-type mice. Both generalized hAR overexpression and hAR expression via the Tie 2 promoter increased lesion size in streptozotocin diabetic mice. In addition, pharmacological inhibition of AR reduced lesion size. CONCLUSIONS: Although in some settings AR expression might reduce levels of toxic aldehydes, transgenic expression of this enzyme within the artery wall leads to greater atherosclerosis.

WP1066 induces cell death in a schwannomatosis patient-derived schwannoma cell line.

Schwannomatosis is a rare genetic disorder that predisposes individuals to development of multiple schwannomas mainly in spinal and peripheral nerves and to debilitating chronic pain often unrelated to any schwannoma. Pathogenic variants of two genes, SMARCB1 and LZTR1, are causal in familial cases. However, many schwannomatosis patients lack mutations in these genes. Surgery is the standard treatment for schwannomas but leaves patients with increasing neurological deficits. Pain management is a daily struggle controlled by the use of multiple analgesic and anti-inflammatory drugs. There is a need for both nonsurgical treatment to manage tumor growth and nonaddictive, nonsedative pain control. Because standard clinical trials are exceedingly difficult for patients with rare disorders, precision medicine approaches offer the possibility of bespoke therapeutic regimens to control tumor growth. As a proof of principle, we obtained a bio-specimen of paraspinal schwannoma from a schwannomatosis patient with a germline point mutation in the SMARCB1/INI gene. We created an hTERT immortalized cell line and tested the ability of targeted small molecules with efficacy in neurofibromatosis type 2-related schwannomas to reduce cell viability and induce cell death. We identified WP1066, a STAT3 inhibitor, currently in phase 2 clinical trials for pediatric and adult brain tumors as a lead compound. It reduced cell viability and STAT-3 phosphorylation and induced expression of markers for both necroptosis and caspase-dependent cell death. The results demonstrate feasibility in creating patient-derived cell lines for use in precision medicine studies.

CUDC907, a dual phosphoinositide-3 kinase/histone deacetylase inhibitor, promotes apoptosis of NF2 Schwannoma cells.

Neurofibromatosis Type 2 (NF2) is a rare tumor disorder caused by pathogenic variants of the merlin tumor suppressor encoded by NF2. Patients develop vestibular schwannomas (VS), peripheral schwannomas, meningiomas, and ependymomas. There are no approved drug therapies for NF2. Previous work identified phosphoinositide-3 kinase (PI3K) as a druggable target. Here we screened PI3K pathway inhibitors for efficacy in reducing viability of human schwannoma cells. The lead compound, CUDC907, a dual histone deacetylase (HDAC)/PI3K inhibitor, was further evaluated for its effects on isolated and nerve-grafted schwannoma model cells, and primary VS cells. CUDC907 (3 nM IG50) reduced human merlin deficient Schwann cell (MD-SC) viability and was 5-100 fold selective for MD over WT-SCs. CUDC907 (10 nM) promoted cell cycle arrest and caspase-3/7 activation within 24 h in human MD-SCs. Western blots confirmed a dose-dependent increase in acetylated lysine and decreases in pAKT and YAP. CUDC907 decreased tumor growth rate by 44% in a 14-day treatment regimen, modulated phospho-target levels, and decreased YAP levels. In five primary VS, CUDC907 decreased viability, induced caspase-3/7 cleavage, and reduced YAP levels. Its efficacy correlated with basal phospho-HDAC2 levels. CUDC907 has cytotoxic activity in NF2 schwannoma models and primary VS cells and is a candidate for clinical trials.

Metabolic adjustment and regulation of gene expression are essential for increased resistance to severe water deficit and resilience post-stress in soybean.

Background: Soybean is the main oilseed crop grown in the world; however, drought stress affects its growth and physiology, reducing its yield. The objective of this study was to characterize the physiological, metabolic, and genetic aspects that determine differential resistance to water deficit in soybean genotypes. Methods: Three soybean genotypes were used in this study, two lineages (L11644 and L13241), and one cultivar (EMBRAPA 48-C48). Plants were grown in pots containing 8 kg of a mixture of soil and sand (2:1) in a greenhouse under sunlight. Soil moisture in the pots was maintained at field capacity until the plants reached the stage of development V4 (third fully expanded leaf). At this time, plants were subjected to three water treatments: Well-Watered (WW) (plants kept under daily irrigation); Water Deficit (WD) (withholding irrigation until plants reached the leaf water potential at predawn of -1.5 ± 0.2 MPa); Rewatered (RW) (plants rehydrated for three days after reached the water deficit). The WW and WD water treatments were evaluated on the eighth day for genotypes L11644 and C48, and on the tenth day for L13241, after interruption of irrigation. For the three genotypes, the treatment RW was evaluated after three days of resumption of irrigation. Physiological, metabolic and gene expression analyses were performed. Results: Water deficit inhibited growth and gas exchange in all genotypes. The accumulation of osmolytes and the concentrations of chlorophylls and abscisic acid (ABA) were higher in L13241 under stress. The metabolic adjustment of lineages in response to WD occurred in order to accumulate amino acids, carbohydrates, and polyamines in leaves. The expression of genes involved in drought resistance responses was more strongly induced in L13241. In general, rehydration provided recovery of plants to similar conditions of control treatment. Although the C48 and L11644 genotypes have shown some tolerance and resilience responses to severe water deficit, greater efficiency was observed in the L13241 genotype through adjustments in morphological, physiological, genetic and metabolic characteristics that are combined in the same plant. This study contributes to the advancement in the knowledge about the resistance to drought in cultivated plants and provides bases for the genetic improvement of the soybean culture.

Glyphosate hormesis attenuates water deficit stress in safflower (Carthamus tinctorius L.) by modulating physiological and biochemical mediators.

Changes in photosynthetic machinery can induce physiological and biochemical damage in plants. Low doses of glyphosate have been shown to exert a positive effect in mitigating the deleterious effects of water deficit in plants. Here, the physiological and biochemical mechanisms of safflower plants (Carthamus tinctorius L.) were studied under conditions of water deficit mediated by the attenuating effect of low-dose glyphosate. The plants were divided into two groups of water regimes in soil, without water deficit (-10 kPa) and with water deficit (-70 kPa), and were exposed to different concentrations of glyphosate (0, 1.8, 3.6, 7.2, 18, 36, 72, 180, 360, and 720 g a.e. ha-1). Evident protective responses at the physiological and biochemical levels were obtained after applying low doses of glyphosate to plants under water deficit, with a limiting dose for the occurrence of hormesis (LDS) = 72 g a.e. ha-1. The water deficit in plants resulted in hydrogen peroxide (H2O2) accumulation and consequently lipid peroxidation (LPO) associated with the accumulation of shikimic acid and glyphosate in plants, which triggered an increase in the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) that act by dismuting the levels of reactive oxygen species (ROS), maintaining, and/or increasing the maximum quantum efficiency of photosystem II (Fv/Fm), effective quantum yield of photosystem II (ΦPSII), electron transport rate (ETR), photochemical extinction coefficient (qP), and non-photochemical extinction coefficient (NPQ). APX appears to be the main enzyme involved in eliminating H2O2. Low doses of glyphosate act as water deficit ameliorators, allowing the plant to maintain/increase metabolism at physiological and biochemical levels by activating antioxidant enzymes in the dismutation of ROS in safflower plants.

Advanced glycation end product (AGE)-receptor for AGE (RAGE) signaling and up-regulation of Egr-1 in hypoxic macrophages.

Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligand-binding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C betaII, ERK1/2, and c-Jun NH(2)-terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1.

Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE-/- mice.

Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE-/- mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall.

Increased soybean tolerance to water deficiency through biostimulant based on fulvic acids and Ascophyllum nodosum (L.) seaweed extract.

To meet the growing demand for soybean it is necessary to increase crop yield, even in low water availability conditions. To circumvent the negative effects of water deficit, application of biostimulants with anti-stress effect has been adopted, including products based on fulvic acids and Ascophyllum nodosum (L.) seaweed extracts. In this study, we determined which formulation and dosage of a biostimulant is more efficient in promoting the recovery of soybean plants after stress due to water deficit. The experiment was conducted in a greenhouse, in a double-factorial randomized block design with two additional factors, four repetitions and eleven treatments consisting of three biostimulant formulations (F1, F2 and F3), and three dosages (0.25; 0.50 and 1.0 kg ha-1); a control with water deficit and a control without water deficit. Soybean plants were kept at 50% of the pot's water capacity for three days, then rehydrated and submitted to the application of treatments with biostimulant. After two days of recovery, growth, physiological, biochemical and yield parameters were evaluated. All plants that received the application of the biostimulant produced more than the water-stressed control plants. The biostimulant provided higher photosynthetic rates, more efficient mechanisms for dissipating excess energy and higher activities of antioxidant enzymes. Plants treated with biostimulant were more efficient in the recovery of the metabolic activities after rewatering, resulting in increased soybean tolerance to water deficit and reduced yield losses. The best result obtained was through the application of formulation 2 of the biostimulant at a dosage of 0.25 kg ha-1.

Receptor for advanced glycation end product (RAGE)-dependent modulation of early growth response-1 in hepatic ischemia/reperfusion injury.

BACKGROUND/AIMS: We previously showed that blockade of RAGE significantly attenuates hepatic ischemia/reperfusion (I/R) injury in mice. Here, we identify that early growth response-1 (Egr-1) is a downstream target of RAGE in hepatic I/R injury. METHODS: Hepatic I/R was induced in male mice. Liver remnants were analyzed for induction of Egr-1 and cytokines, as well as regulation of apoptotic pathways after reperfusion. RESULTS: Egr-1 was upregulated in the liver remnants after hepatic I/R injury and was suppressed by administration of soluble RAGE or deletion of the RAGE gene. RAGE-mediated increased expression of Egr-1 upregulates a central downstream gene, MIP2. In contrast, RAGE-stimulated Egr-1-independent pathways regulate TNF-alpha production and apoptosis in response to I/R. Consistent with these findings, phospho-p44/42 and phospho-JNK MAPK and c-Jun were strikingly suppressed in RAGE(-/-) versus WT mice, but not in Egr-1(-/-) mice. RAGE ligand HMGB1 was upregulated after I/R in the liver remnants. In vitro, incubation of RAGE-expressing liver dendritic cells (DCs) with recombinant HMGB-1 resulted in increased Egr-1 transcripts, in a manner suppressed by RAGE gene deletion, soluble RAGE and inhibitors of p44/p42 or JNK MAP kinase. CONCLUSIONS: Suppression of Egr-1 may contribute to the protective mechanisms underlying the beneficial impact of RAGE blockade or deletion.

RAGE mediates podocyte injury in adriamycin-induced glomerulosclerosis.

In the kidney, the receptor for advanced glycation end products (RAGE) is principally expressed in the podocyte at low levels, but is upregulated in both human and mouse glomerular diseases. Because podocyte injury is central to proteinuric states, such as the nephrotic syndrome, the murine adriamycin nephrosis model was used to explore the role of RAGE in podocyte damage. In this model, administration of the anthracycline antibiotic adriamycin provokes severe podocyte stress and glomerulosclerosis. In contrast to wild-type animals, adriamycin-treated RAGE-null mice were significantly protected from effacement of the podocyte foot processes, albuminuria, and glomerulosclerosis. Administration of adriamycin induced rapid generation of RAGE ligands, and treatment with soluble RAGE protected against podocyte injury and glomerulosclerosis. In vitro, incubation of RAGE-expressing murine podocytes with adriamycin stimulated AGE formation, and treatment with RAGE ligands rapidly activated nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, via p44/p42 MAP kinase signaling, and upregulated pro-fibrotic growth factors. These data suggest that RAGE may contribute to the pathogenesis of podocyte injury in sclerosing glomerulopathies such as focal segmental glomerulosclerosis.