Image_1_Role of Hippocampal Lipocalin-2 in Experimental Diabetic Encephalopathy.TIF

Diabetic encephalopathy is a severe diabetes-related complication in the central nervous system (CNS) that is characterized by degenerative neurochemical and structural changes leading to impaired cognitive function. While the exact pathophysiology of diabetic encephalopathy is not well-understood, it is likely that neuroinflammation is one of the key pathogenic mechanisms that cause this complication. Lipocalin-2 (LCN2) is an acute phase protein known to promote neuroinflammation via the recruitment and activation of immune cells and glia, particularly microglia and astrocytes, thereby inducing proinflammatory mediators in a range of neurological disorders. In this study, we investigated the role of LCN2 in multiple aspects of diabetic encephalopathy in mouse models of diabetes. Here, we show that induction of diabetes increased the expression of both Lcn2 mRNA and protein in the hippocampus. Genetic deficiency of Lcn2 significantly reduced gliosis, recruitment of macrophages, and production of inflammatory cytokines in the diabetic mice. Further, diabetes-induced hippocampal toxicity and cognitive decline were both lower in Lcn2 knockout mice than in the wild-type animals. Taken together, our findings highlight the critical role of LCN2 in the pathogenesis of diabetic encephalopathy.</p

Image_5_Role of Hippocampal Lipocalin-2 in Experimental Diabetic Encephalopathy.TIF

Diabetic encephalopathy is a severe diabetes-related complication in the central nervous system (CNS) that is characterized by degenerative neurochemical and structural changes leading to impaired cognitive function. While the exact pathophysiology of diabetic encephalopathy is not well-understood, it is likely that neuroinflammation is one of the key pathogenic mechanisms that cause this complication. Lipocalin-2 (LCN2) is an acute phase protein known to promote neuroinflammation via the recruitment and activation of immune cells and glia, particularly microglia and astrocytes, thereby inducing proinflammatory mediators in a range of neurological disorders. In this study, we investigated the role of LCN2 in multiple aspects of diabetic encephalopathy in mouse models of diabetes. Here, we show that induction of diabetes increased the expression of both Lcn2 mRNA and protein in the hippocampus. Genetic deficiency of Lcn2 significantly reduced gliosis, recruitment of macrophages, and production of inflammatory cytokines in the diabetic mice. Further, diabetes-induced hippocampal toxicity and cognitive decline were both lower in Lcn2 knockout mice than in the wild-type animals. Taken together, our findings highlight the critical role of LCN2 in the pathogenesis of diabetic encephalopathy.</p

Microwave Annealing Effect for Highly Reliable Biosensor: Dual-Gate Ion-Sensitive Field-Effect Transistor Using Amorphous InGaZnO Thin-Film Transistor

We used a microwave annealing process to fabricate a highly reliable biosensor using amorphous-InGaZnO (a-IGZO) thin-film transistors (TFTs), which usually experience threshold voltage instability. Compared with furnace-annealed a-IGZO TFTs, the microwave-annealed devices showed superior threshold voltage stability and performance, including a high field-effect mobility of 9.51 cm²/V·s, a low threshold voltage of 0.99 V, a good subthreshold slope of 135 mV/dec, and an outstanding on/off current ratio of 1.18 × 10⁸. In conclusion, by using the microwave-annealed a-IGZO TFT as the transducer in an extended-gate ion-sensitive field-effect transistor biosensor, we developed a high-performance biosensor with excellent sensing properties in terms of pH sensitivity, reliability, and chemical stability

Image_4_Role of Hippocampal Lipocalin-2 in Experimental Diabetic Encephalopathy.TIF

Diabetic encephalopathy is a severe diabetes-related complication in the central nervous system (CNS) that is characterized by degenerative neurochemical and structural changes leading to impaired cognitive function. While the exact pathophysiology of diabetic encephalopathy is not well-understood, it is likely that neuroinflammation is one of the key pathogenic mechanisms that cause this complication. Lipocalin-2 (LCN2) is an acute phase protein known to promote neuroinflammation via the recruitment and activation of immune cells and glia, particularly microglia and astrocytes, thereby inducing proinflammatory mediators in a range of neurological disorders. In this study, we investigated the role of LCN2 in multiple aspects of diabetic encephalopathy in mouse models of diabetes. Here, we show that induction of diabetes increased the expression of both Lcn2 mRNA and protein in the hippocampus. Genetic deficiency of Lcn2 significantly reduced gliosis, recruitment of macrophages, and production of inflammatory cytokines in the diabetic mice. Further, diabetes-induced hippocampal toxicity and cognitive decline were both lower in Lcn2 knockout mice than in the wild-type animals. Taken together, our findings highlight the critical role of LCN2 in the pathogenesis of diabetic encephalopathy.</p

Image_3_Role of Hippocampal Lipocalin-2 in Experimental Diabetic Encephalopathy.TIF

Diabetic encephalopathy is a severe diabetes-related complication in the central nervous system (CNS) that is characterized by degenerative neurochemical and structural changes leading to impaired cognitive function. While the exact pathophysiology of diabetic encephalopathy is not well-understood, it is likely that neuroinflammation is one of the key pathogenic mechanisms that cause this complication. Lipocalin-2 (LCN2) is an acute phase protein known to promote neuroinflammation via the recruitment and activation of immune cells and glia, particularly microglia and astrocytes, thereby inducing proinflammatory mediators in a range of neurological disorders. In this study, we investigated the role of LCN2 in multiple aspects of diabetic encephalopathy in mouse models of diabetes. Here, we show that induction of diabetes increased the expression of both Lcn2 mRNA and protein in the hippocampus. Genetic deficiency of Lcn2 significantly reduced gliosis, recruitment of macrophages, and production of inflammatory cytokines in the diabetic mice. Further, diabetes-induced hippocampal toxicity and cognitive decline were both lower in Lcn2 knockout mice than in the wild-type animals. Taken together, our findings highlight the critical role of LCN2 in the pathogenesis of diabetic encephalopathy.</p

Snail is upregulated under extracellular matrix (ECM)-mediated signals.

<p>(A) Western blot analysis showing Snail expression on immobilized ECM. After HUVECs were transfected with siCon or siSnail, the transfectants were reseeded and cultured on PLL (20 μg/mL)-, FN (20 μg/mL)-, or CI (20 μg/mL)-coated culture dishes for 2 h. PLL, poly-L-lysine; FN, fibronectin; CI, collagen type I. (B) Time-course expression pattern of Snail on immobilized ECM. Confluent HUVECs were reseeded and cultured on PLL-, FN- or CI-coated dishes for the indicated time points. Snail expression was evaluated by western blot (upper) and quantitative RT-PCR (lower) analyses. (C) Western blot analysis showing the induction of phosphorylated Akt (p-Akt) and phosphorylated extracellular-regulated kinase 1/2 (p-Erk1/2) in HUVECs that were cultured on FN-coated dishes. (D) Snail expression on immobilized ECM after MK2206 treatment. Confluent HUVECs or human retinal endothelial cells (HRECs) were pre-exposed to 10 μM PP2 (a Src kinase inhibitor) or 1 μg/mL MK2206 (an allosteric Akt inhibitor) for 1 h, followed by reseeding and culture on PLL-, FN-, or CI-coated dishes for 2 h (western blot) or 1 h (quantitative RT-PCR).</p

Snail upregulates VEGF receptor 3 (VEGFR3).

<p>(A) Western blot and RT-PCR analyses showing Snail, early growth response protein-1 (Egr-1), VEGF receptor 3 (VEGFR3), and VEGFR2 expression. HRECs were seeded at a density of 2–2.5×10⁴ cells/cm² on FN- (for western blot and RT-PCR) or PLL (for western blot)-coated dishes and cultured for the indicated time points. (B) Western blot analysis showing the effect of Snail knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siSnail on FN-coated dishes, and cultured for the indicated time. (C) Quantitative RT-PCR analysis showⁱng the effect of Snail knockdown on VEGFR3 expression. SiSnail-transfected ECs were reseeded and cultured on FN-coated dishes for 8 h. *, p<0.01. (D) Western blot and quantitative RT-PCR analyses showing the effect of Snail overexpression on VEGFR3. HUVECs were transfected with Snail. On the next day, the medium was changed, and the transfected cells were cultured for 8 h (quantitative RT-PCR; right) or 16 h (western blot; left). *, p<0.01.</p

Proposed model of capillary branching morphogenesis in postnatal mice.

<p>(A) Outline of Snail stabilization by ECM-mediated signaling. Snail is rapidly degraded by the GSK3β-dependent proteosomal system. On exposure of ECs to ECM, they activate Akt, which can suppress GSK3β-dependent system by phosphorylating GSK3β (pGSK3β). This process stabilizes Snail by releasing it from GSK3β system. Thereby, the formation of Snail-Egr-1 complex promotes VEGFR3 expression by binding to the <i>VEGFR3</i> promoter region to facilitate EC morphogenesis, such as EC sprouting, extension, and branching. pSnail, phosphorylated Snail by GSK3β; pAkt, Akt phosphorylation; E, Egr-1; EC, endothelial cell. (B) Capillary branching morphogenesis is controlled by Snail. In P7–P8 mice, venous ECs in the superficial plexus start to extend capillary branching toward the deep retina in response to tissue needs. The sprouting ECs at the border between the GCL and IPL are exposed to ECM, which subsequently contributes to Snail induction and stabilization, followed by enhanced VEGFR3 expression. Snail/VEGFR3-expressing ECs vertically migrate toward deep retina. At P9–P11 mice, vertically migrating ECs reach in the boundary of INL and turn sideways to form the deep capillary plexus in the OPL region. Snail knockdown attenuates the initiation of EC sprouting, which subsequently impairs the formation of the deep capillary plexus.</p

Snail upregulates VEGFR3 transcripts via cooperating with Egr-1.

<p>(A) <i>VEGFR3</i> promoter activity after the exposure of HRECs to immobilized FN. HRECs were transfected with the human <i>VEGFR3</i> promoter_luciferase (hVEGFR3_Luc) reporter (wildR3) and then reseeded at a density of 2–2.5×10⁴ cells/cm² on FN-coated dishes. (B) Schematic illustration of the location of putative Snail and the Egr-1-binding site in the human <i>VEGFR3</i> promoter. WildR3, human <i>VEGFR3</i> promoter_luciferase (hVEGFR3_Luc) reporter; mutR3(Snail), mutation in the putative E-box. Broken line, Egr-1-binding elements; thick line, putative E-box; Luc, luciferase. (C) Western blot analysis showing the effect of Egr-1 knockdown on VEGFR3. HRECs were reseeded after transfections with siCon or siEgr-1 on FN-coated dishes. Arrow, an Egr-1 band; *, a non-specific band. (D) <i>VEGFR3</i> promoter activity after the knockdown of Snail or Egr-1. HRECs were co-transfected with the indicated siRNA and the wildR3 reporter and then reseeded and cultured on FN-coated dishes for 16 h. (E) Mutant <i>VEGFR3</i> promoter activity. HRECs were transfected with the indicated wildR3 and mutR3 (Snail) reporters and then reseeded on FN-coated dishes for 16 h. (F) Immunoprecipitation assay demonstrating the complex association between Snail and Egr-1. HRECs were seeded on FN-coated dishes. After 2 h, the cell lysates were immunoprecipitated (IP) with immunoglobulin G (IgG) or anti-Egr-1 antibody (α-Egr-1). (G) Chromatin immunoprecipitation analysis of the <i>VEGFR3</i> promoter in HUVECs. HUVECs were transfected with flag-Snail (Snail) and immunoprecipitated using anti-Snail antibodies (α-Snail). PCR was performed to detect the <i>VEGFR3</i> promoter region containing the putative E box.</p

Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression

<div><p>Vascular branching morphogenesis is activated and maintained by several signaling pathways. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) signaling is largely presented in arteries, and VEGFR3 signaling is in veins and capillaries. Recent reports have documented that Snail, a well-known epithelial-to-mesenchymal transition protein, is expressed in endothelial cells, where it regulates sprouting angiogenesis and embryonic vascular development. Here, we identified Snail as a regulator of VEGFR3 expression during capillary branching morphogenesis. Snail was dramatically upregulated in sprouting vessels in the developing retinal vasculature, including the leading-edged vessels and vertical sprouting vessels for capillary extension toward the deep retina. Results from <i>in vitro</i> functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated <i>VEGFR3</i> mRNA by directly binding to the <i>VEGFR3</i> promoter via cooperating with early growth response protein-1. Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression. Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.</p></div