Inhibition of GPR39 restores defects in endothelial cell-mediated neovascularization under the duress of chronic hyperglycemia: Evidence for regulatory roles of the sonic hedgehog signaling axis.

Impaired endothelial cell (EC)-mediated angiogenesis contributes to critical limb ischemia in diabetic patients. The sonic hedgehog (SHH) pathway participates in angiogenesis but is repressed in hyperglycemia by obscure mechanisms. We investigated the orphan G protein-coupled receptor GPR39 on SHH pathway activation in ECs and ischemia-induced angiogenesis in animals with chronic hyperglycemia. Human aortic ECs from healthy and type 2 diabetic (T2D) donors were cultured in vitro. GPR39 mRNA expression was significantly elevated in T2D. The EC proliferation, migration, and tube formation were attenuated by adenovirus-mediated GPR39 overexpression (Ad-GPR39) or GPR39 agonist TC-G-1008 in vitro. The production of proangiogenic factors was reduced by Ad-GPR39. Conversely, human ECs transfected with GPR39 siRNA or the mouse aortic ECs isolated from GPR39 global knockout (GPR39KO) mice displayed enhanced migration and proliferation compared with their respective controls. GPR39 suppressed the basal and ligand-dependent activation of the SHH effector GLI1, leading to attenuated EC migration. Coimmunoprecipitation revealed that the GPR39 direct binding of the suppressor of fused (SUFU), the SHH pathway endogenous inhibitor, may achieve this. Furthermore, in ECs with GPR39 knockdown, the robust GLI1 activation and EC migration were abolished by SUFU overexpression. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, the GPR39KO mice demonstrated a faster pace of revascularization from hind limb ischemia and lower incidence of tissue necrosis than GPR39 wild-type (GPR39WT) counterparts. These findings have provided a conceptual framework for developing therapeutic tools that ablate or inhibit GPR39 for ischemic tissue repair under metabolic stress.

The Impact of Obesity on Diabetes Onset and Neovascularization in Mouse Models of Metabolic Stress.

Animal models of metabolic disorders are essential to studying pathogenic mechanisms and developing therapies for diabetes, but the induction protocols vary, and sexual dimorphism often exists. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, blood glucose and lipid profiles were measured. The high-fat (HF) diet damaged insulin sensitivity and increased triglycerides, total cholesterol, LDL-cholesterol, HDL-cholesterol, and liver lipid deposition. STZ increased blood glucose and liver fibrosis with less effects on blood lipids or liver lipid deposition. The combination of DIO and STZ treatments led to significant liver lipid deposition and fibrosis. Female mice showed delayed body weight gain on HF diet and resisted STZ-induced hyperglycemia. However, once they developed DIO, which occurs around 26 weeks of HF diet, the female mice were prone to STZ-induced hyperglycemia. In hindlimb ischemia, male mice in the DIO-STZ group showed significantly worse neovascularization compared with DIO or STZ groups. The DIO-STZ females showed significantly worse recovery than the DIO-STZ males. Our observations suggest that DIO-STZ is a plausible model for studying metabolic and cardiovascular disorders in obesity and diabetes. Moreover, the findings in female animals stress the need to assess sexual dimorphism and investigate the underlying mechanisms that contribute to the worse vasculopathy manifestations in females in metabolic models.

A novel imidazolinone metformin-methylglyoxal metabolite promotes endothelial cell angiogenesis via the eNOS/HIF-1α pathway.

Peripheral arterial disease (PAD) is one of the major complications of diabetes due to an impairment in angiogenesis. Since there is currently no drug with satisfactory efficacy to enhance blood vessel formation, discovering therapies to improve angiogenesis is critical. An imidazolinone metabolite of the metformin-methylglyoxal scavenging reaction, (E)-1,1-dimethyl-2-(5-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl) guanidine (IMZ), was recently characterized and identified in the urine of type-2 diabetic patients. Here, we report the pro-angiogenesis effect of IMZ (increased aortic sprouting, cell migration, network formation, and upregulated multiple pro-angiogenic factors) in human umbilical vein endothelial cells. Using genetic and pharmacological approaches, we showed that IMZ augmented angiogenesis by activating the endothelial nitric oxide synthase (eNOS)/hypoxia-inducible factor-1 alpha (HIF-1α) pathway. Furthermore, IMZ significantly promoted capillary density in the in vivo Matrigel plug angiogenesis model. Finally, the role of IMZ in post-ischemic angiogenesis was examined in a chronic hyperglycemia mouse model subjected to hind limb ischemia. We observed improved blood perfusion, increased capillary density, and reduced tissue necrosis in mice receiving IMZ compared to control mice. Our data demonstrate the pro-angiogenic effects of IMZ, its underlying mechanism, and provides a structural basis for the development of potential pro-angiogenic agents for the treatment of PAD.

Methylglyoxal triggers human aortic endothelial cell dysfunction via modulation of the KATP/MAPK pathway.

Endothelial dysfunction is a key risk factor in diabetes-related multiorgan damage. Methylglyoxal (MGO), a highly reactive dicarbonyl generated primarily as a by-product of glycolysis, is increased in both type 1 and type 2 diabetic patients. MGO can rapidly bind with proteins, nucleic acids, and lipids, resulting in structural and functional changes. MGO can also form advanced glycation end products (AGEs). How MGO causes endothelial cell dysfunction, however, is not clear. Human aortic endothelial cells (HAECs) from healthy (H-HAECs) and type 2 diabetic (D-HAECs) donors were cultured in endothelial growth medium (EGM-2). D-HAECs demonstrated impaired network formation (on Matrigel) and proliferation (MTT assay), as well as increased apoptosis (caspase-3/7 activity and TUNEL staining), compared with H-HAECs. High glucose (25 mM) or AGEs (200 ng/ml) did not induce such immediate, detrimental effects as MGO (10 µM). H-HAECs were treated with MGO (10 µM) for 24 h with or without the ATP-sensitive potassium (KATP) channel antagonist glibenclamide (1 µM). MGO significantly impaired H-HAEC network formation and proliferation and induced cell apoptosis, which was reversed by glibenclamide. Furthermore, siRNA against the KATP channel protein Kir6.1 significantly inhibited endothelial cell function at basal status but rescued impaired endothelial cell function upon MGO exposure. Meanwhile, activation of MAPK pathways p38 kinase, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) (determined by Western blot analyses of their phosphorylated forms, p-JNK, p-p38, and p-ERK) in D-HAECs were significantly enhanced compared with those in H-HAECs. MGO exposure enhanced the activation of all three MAPK pathways in H-HAECs, whereas glibenclamide reversed the activation of p-stress-activated protein kinase/JNK induced by MGO. Glyoxalase-1 (GLO1) is the endogenous MGO-detoxifying enzyme. In healthy mice that received an inhibitor of GLO1, MGO deposition in aortic wall was enhanced and endothelial cell sprouting from isolated aortic segment was significantly inhibited. Our data suggest that MGO triggers endothelial cell dysfunction by activating the JNK/p38 MAPK pathway. This effect arises partly through activation of KATP channels. By understanding how MGO induces endothelial dysfunction, our study may provide useful information for developing MGO-targeted interventions to treat vascular disorders in diabetes.

Deficiency of the Mitochondrial NAD Kinase Causes Stress-Induced Hepatic Steatosis in Mice.

BACKGROUND & AIMS: The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice. METHODS: We generated MNADK knockout (KO) mice on a C57BL/6NTac background; mice with a wild-type Mnadk gene were used as controls. Some mice were placed on an atherogenic high-fat diet (16% fat, 41% carbohydrate, and 1.25% cholesterol supplemented with 0.5% sodium cholate) or given methotrexate intraperitoneally. We measured rates of fatty acid oxidation in primary hepatocytes using radiolabeled palmitate and in mice using indirect calorimetry. We measured levels of reactive oxygen species in mouse livers and primary hepatocytes. Metabolomic analyses were used to quantify serum metabolites, such as amino acids and acylcarnitines. RESULTS: The KO mice had metabolic features of MNADK-deficient patients, such as increased serum concentrations of lysine and C10:2 carnitine. When placed on the atherogenic high-fat diet, the KO mice developed features of nonalcoholic fatty liver disease and had increased levels of reactive oxygen species in livers and primary hepatocytes, compared with control mice. During fasting, the KO mice had a defect in fatty acid oxidation. MNADK deficiency reduced the activation of cAMP-responsive element binding protein-hepatocyte specific and peroxisome proliferator-activated receptor alpha, which are transcriptional activators that mediate the fasting response. The activity of mitochondrial sirtuins was reduced in livers of the KO mice. Methotrexate inhibited the catalytic activity of MNADK in hepatocytes and in livers in mice with methotrexate injection. In mice given injections of methotrexate, supplementation of a diet with nicotinamide riboside, an NAD precursor, replenished hepatic NADP and protected the mice from hepatotoxicity, based on markers such as increased level of serum alanine aminotransferase. CONCLUSION: MNADK facilitates fatty acid oxidation, counteracts oxidative damage, maintains mitochondrial sirtuin activity, and prevents metabolic stress-induced non-alcoholic fatty liver disease in mice.

100 m/500 Mbps underwater optical wireless communication using an NRZ-OOK modulated 520 nm laser diode.

In this paper, we proposed and experimentally demonstrated a long-distance high-speed underwater optical wireless communication (UOWC) system in a laboratory environment by using a low-cost green laser diode (LD) and power-efficient non-return-to-zero on-off keying (NRZ-OOK) modulation. The system successfully achieved a data rate of 500 Mbps through a 100 m tap-water channel by using a pigtailed single-mode fiber 520 nm green LD. The tap water was measured to have an attenuation coefficient comparable to pure seawater. The measured system bit error rate (BER) value of 2.5 × 10-3 was below the forward error correction (FEC) limit of 3.8 × 10-3 with 7% overhead. The distance can be extended if the received optical power is allowed to reduce to the minimum power to meet the data rate requirement. Based on the measured minimum required power and the power decay model in the water channel, the transmission performance was predicted to be 146 m/500 Mbps and 174 m/100 Mbps.

Disruption of GPR35 Exacerbates Dextran Sulfate Sodium-Induced Colitis in Mice.

BACKGROUND: G protein-coupled receptor 35 (GPR35) is an orphan receptor and is vastly expressed in immune cells and gastrointestinal cells, suggesting the potential physiological importance of GPR35 in these cells. Here, we tested the hypothesis that the lack of GPR35 expression in the colon mucosa exacerbates the severity of dextran sulfate sodium (DSS)-induced experimental colitis in mice. METHODS: Colitis was induced in GPR35 wild-type (GPR35+/+) and GPR35 knockout (GPR35-/-) mice through the administration of DSS in drinking water for 5 days followed by regular facility water for 1 day. Induction of colitis was evaluated by measuring relative body weight loss, clinical illness scores, and morphological changes in the colon. Abolition of Gpr35 gene expression in the colon mucosa of GPR35-/- mice was confirmed by quantitative real-time PCR (qPCR). Gene expressions of inflammatory and tissue remodeling cytokines were detected by qPCR. Human colorectal epithelial Caco cells were transfected with siRNA against GPR35 before treated with 1% DSS in vitro. Protein expressions were measured using Western blot. RESULTS: GPR35-/- mice receiving DSS showed a significantly worsened colitis disease with profound loss of body weight and a considerable amount of severe clinical illness compared to GPR35+/+ mice that received DSS. The histology of colon sections from GPR35-/- mice showed extensive pathological changes including submucosal edema, diffuse ulcerations, and evidence of complete loss of crypts compared to wild-type mice. The mean histopathological score was significantly higher in GPR35-/- mice as compared to GPR35+/+ mice. The qPCR data revealed significant expression of pro-inflammatory and tissue remodeling cytokines in GPR35-/- colon mucosa, including IL-1ß, CXCL1, CXCL2, CCL2, HMGB1, TGFß1, TGFß3, MMP1/9/12. The protein expressions of Zonula occludens-1, E-cadherin, Claudin1 were decreased upon knocking down GPR35 with or without 1% DSS treatment. CONCLUSIONS: Our experimental data suggest that lack of GPR35 resulted in worsened disease outcome in DSS-induced experimental colitis, indicating that GPR35 could play a crucial role in protecting from colonic inflammation and serve as a therapeutic target.

Toll-like Receptor (TLR) Signaling Interacts with CREBH to Modulate High-density Lipoprotein (HDL) in Response to Bacterial Endotoxin.

Bacterial endotoxin can induce inflammatory and metabolic changes in the host. In this study, we revealed a molecular mechanism by which a stress-inducible, liver-enriched transcription factor, cAMP-responsive element-binding protein hepatic-specific (CREBH), modulates lipid profiles to protect the liver from injuries upon the bacterial endotoxin lipopolysaccharide (LPS). LPS challenge can activate CREBH in mouse liver tissues in a toll-like receptor (TLR)/MyD88-dependent manner. Upon LPS challenge, CREBH interacts with TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase that functions as a key mediator of TLR signaling, and this interaction relies on MyD88. Further analysis demonstrated that TRAF6 mediates K63-linked ubiquitination of CREBH to facilitate CREBH cleavage and activation. CREBH directly activates expression of the gene encoding Apolipoprotein A4 (ApoA4) under LPS challenge, leading to modulation of high-density lipoprotein (HDL) in animals. CREBH deficiency led to reduced production of circulating HDL and increased liver damage upon high-dose LPS challenge. Therefore, TLR/MyD88-dependent, TRAF6-facilitated CREBH activation represents a mammalian hepatic defense response to bacterial endotoxin by modulating HDL.

MiR-27b augments bone marrow progenitor cell survival via suppressing the mitochondrial apoptotic pathway in Type 2 diabetes.

Bone marrow-derived progenitor cells (BMPCs) are potential candidates for autologous cell therapy in tissue repair and regeneration because of their high angiogenic potential. However, increased progenitor cell apoptosis in diabetes directly limits their success in the clinic. MicroRNAs are endogenous noncoding RNAs that regulate gene expression at the posttranscriptional level, but their roles in BMPC-mediated angiogenesis are incompletely understood. In the present study, we tested the hypothesis that the proangiogenic miR-27b inhibits BMPC apoptosis in Type 2 diabetes. Bone marrow-derived EPCs from adult male Type 2 diabetic db/db mice and their normal littermates db/+ mice were used. MiR-27b expression (real-time PCR) in EPCs was decreased after 24 h of exposure to methylglyoxal (MGO) or oxidized low-density lipoprotein but not high glucose, advanced glycation end products, the reactive oxygen species generator LY83583, or H2O2 The increase in BMPC apoptosis in the diabetic mice was rescued following transfection with a miR-27b mimic, and the increased apoptosis induced by MGO was also rescued by the miR-27b mimic. p53 protein expression and the Bax/Bcl-2 ratio in EPCs (Western blot analyses) were significantly higher in db/db mice, both of which were suppressed by miR-27b. Furthermore, mitochondrial respiration, as measured by oxygen consumption rate, was enhanced by miR-27b in diabetic BMPCs, with concomitant decrease of mitochondrial Bax/Bcl-2 ratio. The 3' UTR binding assays revealed that both Bax, and its activator RUNX1, were direct targets of miR-27b, suggesting that miR-27b inhibits Bax expression in both direct and indirect manners. miR-27b prevents EPC apoptosis in Type 2 diabetic mice, at least in part, by suppressing p53 and the Bax/Bcl-2 ratio. These findings may provide a mechanistic basis for rescuing BMPC dysfunction in diabetes for successful autologous cell therapy.

HMGB1-Driven Inflammation and Intimal Hyperplasia After Arterial Injury Involves Cell-Specific Actions Mediated by TLR4.

OBJECTIVE: Endoluminal vascular interventions such as angioplasty initiate a sterile inflammatory response resulting from local tissue damage. This response drives the development of intimal hyperplasia (IH) that, in turn, can lead to arterial occlusion. We hypothesized that the ubiquitous nuclear protein and damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1), is one of the endogenous mediators that activates processes leading to IH after endoluminal injury to the arterial wall. The aim of this study is to investigate whether approaches that reduce the levels of HMGB1 or inhibit its activity suppresses IH after arterial injury. APPROACH AND RESULTS: Here, we show that HMGB1 regulates IH in a mouse carotid wire injury model. Induced genetic deletion or neutralization of HMGB1 prevents IH, monocyte recruitment, and smooth muscle cell growth factor production after endoluminal carotid artery injury. A specific inhibitor of HMGB1 myeloid differentiation factor 2-toll-like receptor 4 (TLR4) interaction, P5779, also significantly inhibits IH. HMGB1 deletion is mimicked in this model by global deletion of TLR4 and partially replicated by myeloid-specific deletion of TLR4 but not TLR2 or receptor for advanced glycation endproducts deletion. The specific HMGB1 isoform known to activate TLR4 signaling (disulfide HMGB1) stimulates smooth muscle cell to migrate and produce monocyte chemotactic protein 1/CCL2) via TLR4. Macrophages produce smooth muscle cell mitogens in response to disulfide HMGB1 also in a TLR4/myeloid differentiation primary response gene (88)/Trif-dependent manner. CONCLUSIONS: These findings place HMGB1 and its receptor, TLR4 as critical regulators of the events that drive the inflammation leading to IH after endoluminal arterial injury and identify this pathway as a possible therapeutic target to limit IH to attenuate damage-associated molecular pattern molecule-mediated vascular inflammatory responses.

Hypoxic trophoblast HMGB1 induces endothelial cell hyperpermeability via the TRL-4/caveolin-1 pathway.

High mobility group box 1 (HMGB1) plays an important role in the pathologic processes of endothelial permeability under oxidative stress. Trophoblast oxidative stress has been implicated in the pathophysiology of preeclampsia (PE). HMGB1 serum levels are increased in PE. However, the potential roles of HMGB1 in endothelial permeability in PE remain unclear. We assessed the effects of the hypoxic trophoblast on the permeability of the endothelial monolayer. Our results showed that the hypoxic trophoblast displayed higher HMGB1 mRNA, intracellular HMGB1 protein, and HMGB1 in conditioned medium than those of the normoxic trophoblast did. The hypoxic trophoblast conditioned medium increased the endothelial monolayer permeability and increased TLR 4 and caveolin-1 (CAV-1) protein expression in endothelial cells, which was inhibited by glycyrrhizic acid and HMGB1 small interfering RNA transfection to trophoblasts before hypoxia. The increased endothelial permeability induced by hypoxic trophoblast conditioned medium could be inhibited with TLR4 or CAV-1 gene silencing in endothelial cells. Immunoprecipitation showed that CAV-1 and TLR4 are colocalized in HUVECs and C57BL/6 mouse kidney. TLR4 small interfering RNA suppressed CAV-1 protein expression in endothelial cells upon stimulation of hypoxic trophoblast conditioned medium or HMGB1. We conclude that hypoxic trophoblasts play an important role in the mechanism of general edema (including protein urine) in PE via increasing endothelial monolayer permeability through the HMGB1/TLR4/CAV-1 pathway.

Inhibition of PKR impairs angiogenesis through a VEGF pathway.

Peripheral artery disease (PAD) is a common clinical problem, and its pathophysiological mechanisms are incompletely understood. Double-stranded RNA-activated protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. Although PKR has been reported in antivirus and the immune system, the role of PKR in vascular function, especially in angiogenesis, is still unclear. PKR(-/-) mice were used in our experiments. Blood flow recovery was significantly delayed in PKR(-/-) vs. WT mice (Laser Doppler detection, n = 9, P < 0.01), accompanied by 34% reduced CD31-positive stain in ischemic muscle 28 days after procedure (immunohistochemistry, n = 9, P < 0.05). PKR expression decreased in the first 12 h and increased to peak at 24 h in human umbilical vein endothelial cells (HUVECs) in response to hypoxia (Western blot analyses, n = 3, P < 0.05). Accordingly, phospho-PKR expression increased in HUVECs 24 h after treatment with hypoxia (Western blot analyses, n = 3, P < 0.05). Inhibition of PKR (siRNA transfection) reduced microtubule formation (Matrigel tube formation, n = 3, vs. control siRNA, P < 0.05) and migration (wound healing, n = 3, vs. control siRNA, P < 0.05) by 33 and 59%, respectively. Vascular endothelial growth factor (VEGF) expression in ischemic muscle from PKR(-/-) mice was significantly decreased by 54% 1 day after procedure (n = 3, P < 0.05, vs. WT) and by 63% 7 days after procedure (n = 3, P < 0.01, vs. WT), respectively. At the same time, VEGF expression in HUVECs decreased by 21% (n = 3, P < 0.05, PKR siRNA vs. control siRNA). These findings demonstrate that PKR mediates angiogenesis through a VEGF pathway, which may form the basis for future intervention of PAD.

Exposure to fine airborne particulate matters induces hepatic fibrosis in murine models.

BACKGROUND & AIMS: Hepatic fibrosis, featured by the accumulation of excessive extracellular matrix in liver tissue, is associated with metabolic disease and cancer. Inhalation exposure to airborne particulate matter in fine ranges (PM2.5) correlates with pulmonary dysfunction, cardiovascular disease, and metabolic syndrome. In this study, we investigated the effect and mechanism of PM2.5 exposure on hepatic fibrogenesis. METHODS: Both inhalation exposure of mice and in vitro exposure of specialized cells to PM2.5 were performed to elucidate the effect of PM2.5 exposure on hepatic fibrosis. Histological examinations, gene expression analyses, and genetic animal models were utilized to determine the effect and mechanism by which PM2.5 exposure promotes hepatic fibrosis. RESULTS: Inhalation exposure to concentrated ambient PM2.5 induces hepatic fibrosis in mice under the normal chow or high-fat diet. Mice after PM2.5 exposure displayed increased expression of collagens in liver tissues. Exposure to PM2.5 led to activation of the transforming growth factor ß-SMAD3 signaling, suppression of peroxisome proliferator-activated receptor γ, and expression of collagens in hepatic stellate cells. NADPH oxidase plays a critical role in PM2.5-induced liver fibrogenesis. CONCLUSIONS: Exposure to PM2.5 exerts discernible effects on promoting hepatic fibrogenesis. NADPH oxidase mediates the effects of PM2.5 exposure on promoting hepatic fibrosis.

MicroRNA miR-27b rescues bone marrow-derived angiogenic cell function and accelerates wound healing in type 2 diabetes mellitus.

OBJECTIVE: Vascular precursor cells with angiogenic potentials are important for tissue repair, which is impaired in diabetes mellitus. MicroRNAs are recently discovered key regulators of gene expression, but their role in vascular precursor cell-mediated angiogenesis in diabetes mellitus is unknown. We tested the hypothesis that the microRNA miR-27b rescues impaired bone marrow-derived angiogenic cell (BMAC) function in vitro and in vivo in type 2 diabetic mice. APPROACH AND RESULTS: BMACs from adult male type 2 diabetic db/db and from normal littermate db/+ mice were used. miR-27b expression was decreased in db/db BMACs. miR-27b mimic improved db/db BMAC function, including proliferation, adhesion, tube formation, and delayed apoptosis, but it did not affect migration. Elevated thrombospondin-1 (TSP-1) protein in db/db BMACs was suppressed on miR-27b mimic transfection. Inhibition of miR-27b in db/+ BMACs reduced angiogenesis, which was reversed by TSP-1 small interfering RNA (siRNA). miR-27b suppressed the pro-oxidant protein p66(shc) and mitochondrial oxidative stress, contributing to its protection of BMAC function. miR-27b also suppressed semaphorin 6A to improve BMAC function in diabetes mellitus. Luciferase binding assay suggested that miR-27b directly targeted TSP-1, TSP-2, p66(shc), and semaphorin 6A. miR-27b improved topical cell therapy of diabetic BMACs on diabetic skin wound closure, with a concomitant augmentation of wound perfusion and capillary formation. Normal BMAC therapy with miR-27b inhibition demonstrated reduced efficacy in wound closure, perfusion, and capillary formation. Local miR-27b delivery partly improved wound healing in diabetic mice. CONCLUSIONS: miR-27b rescues impaired BMAC angiogenesis via TSP-1 suppression, semaphorin 6A expression, and p66shc-dependent mitochondrial oxidative stress and improves BMAC therapy in wound healing in type 2 diabetic mice.

Oxidative stress-mediated thrombospondin-2 upregulation impairs bone marrow-derived angiogenic cell function in diabetes mellitus.

OBJECTIVE: Circulating angiogenic cells play an essential role in angiogenesis but are dysfunctional in diabetes mellitus characterized by excessive oxidative stress. We hypothesize that oxidative stress-mediated upregulation of thrombospondin-2 (TSP-2), a potent antiangiogenic protein, contributes to diabetic bone marrow-derived angiogenic cell (BMAC) dysfunction. APPROACH AND RESULTS: BMACs were isolated from adult male type 2 diabetic db/db mice and control db/+ (C57BLKS/J) mice. In Matrigel tube formation assay, angiogenic function was impaired in diabetic BMACs, accompanied by increased oxidative stress and nicotinamide adenine dinucleotide phosphate oxidase activity. BMAC angiogenic function was restored by overexpression of dominant negative Rac1 or by overexpression of manganese superoxide dismutase. TSP-2 mRNA and protein were both significantly upregulated in diabetic BMACs, mediated by increased oxidative stress as shown by a decrease in TSP-2 level after overexpression of dominant negative Rac1 or manganese superoxide dismutase. Silencing TSP-2 by its small interfering RNA in diabetic BMACs improved BMAC function in tube formation, adhesion, and migration assays. Notably, the upregulation of TSP-2 was also found in BMACs from streptozotocin-induced type 1 diabetic mice, and normal BMACs with high glucose treatment. let-7f, a microRNA which has been related to endothelial angiogenic function, is found to play key role in TSP-2 increase, but let-7f did not directly interact with TSP-2 mRNA. CONCLUSIONS: The upregulation of TSP-2 mediated by increased oxidative stress contributes to angiogenesis dysfunction in diabetic BMACs.

Thrombospondin-1/CD36 pathway contributes to bone marrow-derived angiogenic cell dysfunction in type 1 diabetes via Sonic hedgehog pathway suppression.

Refractory wounds in diabetic patients present a significant clinical problem. Sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes. Regulation of SHH in wound healing is poorly understood. We hypothesize that thrombospondin-1 (TSP-1), through its receptor CD36, contributes to the SHH signaling defect in bone marrow-derived angiogenic cells (BMACs) in type 1 diabetic mice. Isolated BMACs from TSP-1-knockout mice demonstrated improved tube formation, migration, and adhesion in parallel with active SHH signaling. BMACs from STZ-induced type 1 diabetic mice showed significantly impaired Matrigel tube formation (n = 5; P < 0.05 vs. control), which was rescued by TSP-1 depletion (n = 5; P < 0.05 STZ-TSP-1(-/-) vs. STZ-WT) or exogenous SHH (20 mg/l, 24 h, n = 4; P < 0.05 vs. STZ-control). The expression of CD36 was elevated in BMACs from STZ mice (n = 4; P < 0.05). SHH signaling was significantly higher in BMACs from TSP-1(-/-) mice and TSP-1 receptor CD36-knockout mice (n = 6; P < 0.05 vs. WT) but not CD47-knockout mice (n = 3; P > 0.05 vs. WT). The impairment of recombinant human TSP-1 (2.2 nM, 24 h) on BMAC Matrigel tube formation was delayed significantly by CD36 deletion (n = 5; P < 0.05). CD36(-/-) BMACs demonstrated better tube formation under both normal and diabetic conditions with active SHH signaling (n = 4; P < 0.05 vs. WT BMACs). In conclusion, The TSP-1/CD36 pathway contributes to the SHH signaling defect, resulting in BMAC dysfunction in type 1 diabetic mice.

A grading method for Kayser Fleischer ring images based on ResNet.

The corneal K-F ring is the most common ophthalmic manifestation of WD patients. Early diagnosis and treatment have an important impact on the patient's condition. K-F ring is one of the gold standards for the diagnosis of WD disease. Therefore, this paper mainly focused on the detection and grading of the K-F ring. The aim of this study is three-fold. Firstly, to create a meaningful database, the K-F ring images are collected which contains 1850 images with 399 different WD patients, and then this paper uses the chi-square test and Friedman test to analyze the statistical significance. Subsequently, the all collected images were graded and labeled with an appropriate treatment approach, as a result, these images could be used to detect the corneal through the YOLO. After the detection of corneal, image segmentation was realized in batches. Finally, in this paper, different deep convolutional neural networks (VGG, ResNet, and DenseNet) were used to realize the grading of the K-F ring images in the KFID. Experimental results reveal that the entire pre-trained models obtain excellent performance. The global accuracies achieved by the six models i.e., VGG-16, VGG-19, ResNet18, ResNet34, ResNet50, and DenseNet are 89.88%, 91.89%, 94.18%, 95.31%, 93.59%, and 94.58% respectively. ResNet34 displayed the highest recall, specificity, and F1-score of 95.23%, 96.99%, and 95.23%. DenseNet showed the best precision of 95.66%. As such, the findings are encouraging, demonstrating the effectiveness of ResNet in the automatic grading of the K-F ring. Moreover, it provides effective help for the clinical diagnosis of HLD.

Oxidative Stress and MicroRNAs in Endothelial Cells under Metabolic Disorders.

Reactive oxygen species (ROS) are radical oxygen intermediates that serve as important second messengers in signal transduction. However, when the accumulation of these molecules exceeds the buffering capacity of antioxidant enzymes, oxidative stress and endothelial cell (EC) dysfunction occur. EC dysfunction shifts the vascular system into a pro-coagulative, proinflammatory state, thereby increasing the risk of developing cardiovascular (CV) diseases and metabolic disorders. Studies have turned to the investigation of microRNA treatment for CV risk factors, as these post-transcription regulators are known to co-regulate ROS. In this review, we will discuss ROS pathways and generation, normal endothelial cell physiology and ROS-induced dysfunction, and the current knowledge of common metabolic disorders and their connection to oxidative stress. Therapeutic strategies based on microRNAs in response to oxidative stress and microRNA's regulatory roles in controlling ROS will also be explored. It is important to gain an in-depth comprehension of the mechanisms generating ROS and how manipulating these enzymatic byproducts can protect endothelial cell function from oxidative stress and prevent the development of vascular disorders.

Boosting UPR transcriptional activator XBP1 accelerates acute wound healing.

Patients' suffering from large or deep wounds caused by traumatic and/or thermal injuries have significantly lower chances of recapitulating lost skin function through natural healing. We tested whether enhanced unfolded protein response (UPR) by expression of a UPR transcriptional activator, X-box-binding protein 1 (XBP1) can significantly promote wound repair through stimulating growth factor production and promoting angiogenesis. In mouse models of a second-degree thermal wound, a full-thickness traumatic wound, and a full-thickness diabetic wound, the topical gene transfer of the activated form of XBP1 (spliced XBP1, XBP1s) can significantly enhance re-epithelialization and increase angiogenesis, leading to rapid, nearly complete wound closure with intact regenerated epidermis and dermis. Overexpression of XBP1s stimulated the transcription of growth factors in fibroblasts critical to proliferation and remodeling during wound repair, including platelet-derived growth factor BB, basic fibroblast growth factor, and transforming growth factor beta 3. Meanwhile, the overexpression of XBP1s boosted the migration and tube formation of dermal microvascular endothelial cells in vitro. Our functional and mechanistic investigations of XBP1-mediated regulation of wound healing processes provide novel insights into the previously undermined physiological role of the UPR in skin injuries. The finding opens an avenue to developing potential XBP1-based therapeutic strategies in clinical wound care protocols.