The neuroprotective effect of melatonin in glutamate excitotoxicity of R28 cells and mouse retinal ganglion cells.

Glaucoma is the leading cause of irreversible blindness. The progressive degeneration of retinal ganglion cells (RGCs) is the major characteristic of glaucoma. Even though the control of intraocular pressure could delay the loss of RGCs, current clinical treatments cannot protect them directly. The overactivation of N-methyl-D-aspartic acid (NMDA) receptors by excess glutamate (Glu) is among the important mechanisms of RGC death in glaucoma progression. Melatonin (MT) is an indole neuroendocrine hormone mainly secreted by the pineal gland. This study aimed to investigate the therapeutic effect of MT on glutamate excitotoxicity of mouse RGCs and R28 cells. The Glu-induced R28 cell excitotoxicity model and NMDA-induced retinal injury model were established. MT was applied to R28 cells and the vitreous cavity of mice by intravitreal injection. Cell counting kit-8 assay and propidium iodide/Hoechst were performed to evaluate cell viability. Reactive oxygen species and glutathione synthesis assays were used to detect the oxidative stress state of R28 cells. Retina immunofluorescence and hematoxylin and eosin staining were applied to assess RGC counts and retinal structure. Flash visual-evoked potential was performed to evaluate visual function in mice. RNA sequencing of the retina was performed to explore the underlying mechanisms of MT protection. Our results found that MT treatment could successfully protect R28 cells from Glu excitotoxicity and decrease reactive oxygen species. Also, MT rescued RGCs from NMDA-induced injury and protected visual function in mice. This study enriches the indications of MT in the treatment of glaucoma, providing practical research ideas for its comprehensive prevention and treatment.

Nuclear factor erythroid 2-related factor 2 agonist protects retinal ganglion cells in glutamate excitotoxicity retinas.

OBJECTIVE: To investigate whether tert-Butylhydroquinone (TBHQ) can ameliorate oxidative stress and inflammation induced by glutamate excitotoxicity, and mediate retinal ganglion cell (RGC) damage by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and inhibiting the nuclear factor kappa B (NF-κB) signaling pathway. MATERIALS AND METHODS: TBHQ was used to treat a glutamate excitotoxicity model of retinal cell line 28 and C57 mice. Damage to RGCs and visual function were assessed using flash visual evoked potential (FVEP), immunofluorescence, propidium iodide staining, and hematoxylin and eosin staining. Knockdown of Nrf2 used Nrf2 shRNA. The expression levels of related proteins were detected using western blot and immunofluorescence. RESULTS: Glutamate excitotoxicity down-regulated Nrf2 expression in vitro and in vivo. Nuclear factor erythroid2-related factor 2 activation by TBHQ reduced the damage to retinal ganglion cells, reduced the thinning of the whole retina and the ganglion cell complex, and shortened the latency of the FVEP forward wave after injury. In addition, the levels of NAD(P)H quinone dehydrogenase 1 (NQO1), heme oxygenase 1 (HO-1), and Nrf2 increased significantly, and those of cyclooxygenase-2 (COX2) and NF-κB decreased significantly, after TBHQ treatment. Compared with TBHQ treatment group, the expression level of p-p65 in shRNA transfected group was increased, but still lower than that in Glu group. CONCLUSION: The protective effect of TBHQ on RGC loss under glutamate excitotoxicity might be related to the activation of the Nrf2 signaling pathway, anti-oxidative stress, inhibition of NF-κB activation, and inhibition of retinal inflammation. Thus, TBHQ might be used to treat glutamate excitotoxicity -related retinopathy.

Heat Shock Proteins 70 Regulate Cell Motility and Invadopodia-Associated Proteins Expression in Oral Squamous Cell Carcinoma.

Background: Many studies have shown that diabetes is often closely related to oral squamous cell carcinoma (OSCC) occurrence and metastasis. Heat shock protein 70 (Hsp70) is a molecular chaperone related to diabetes complications. This study aims to investigate the role of Hsp70 in OSCC in expression of invadopodia-associated proteins. Methods: The expressions and correlation of HSP70, Hif1α, MMP2, MMP14, and cortactin were examined using bioinformatics analysis and verified by OSCC tissue microarrays. Assay in vitro was performed to analyze cell migration capacity after treatment with or without the HSP70 inhibitor. Results: The expressions of invadopodia-associated proteins were enhanced in OSCC tissues compared with paracarcinoma tissues and partially correlated with HSP70. Inhibiting HSP70 significantly decreased the cell viability, proliferation, and migration of OSCC cells. Conclusions: HSP70 may be involved in invadopodia-associated proteins in OSCC cells, which provides a promising method for treatment of OSCC metastasis.

Molecular investigation of candidate genes for pyroptosis-induced inflammation in diabetic retinopathy.

Background: Diabetic retinopathy is a diabetic microvascular complication. Pyroptosis, as a way of inflammatory death, plays an important role in the occurrence and development of diabetic retinopathy, but its underlying mechanism has not been fully elucidated. The purpose of this study is to identify the potential pyroptosis-related genes in diabetic retinopathy by bioinformatics analysis and validation in a diabetic retinopathy model and predict the microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) interacting with them. Subsequently, the competing endogenous RNA (ceRNA) regulatory network is structured to explore their potential molecular mechanism. Methods: We obtained mRNA expression profile dataset GSE60436 from the Gene Expression Omnibus (GEO) database and collected 51 pyroptosis-related genes from the PubMmed database. The differentially expressed pyroptosis-related genes were obtained by bioinformatics analysis with R software, and then eight key genes of interest were identified by correlation analysis, Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) network analysis. Then, the expression levels of these key pyroptosis-related genes were validated with quantitative real-time polymerase chain reaction (qRT-PCR) in human retinal endothelial cells with high glucose incubation, which was used as an in vitro model of diabetic retinopathy. Western blot was performed to measure the protein levels of gasdermin D (GSDMD), dasdermin E (GSDME) and cleaved caspase-3 in the cells. Moreover, the aforementioned genes were further confirmed with the validation set. Finally, the ceRNA regulatory network was structured, and the miRNAs and lncRNAs which interacted with CASP3, TLR4, and GBP2 were predicted. Results: A total of 13 differentially expressed pyroptosis-related genes were screened from six proliferative diabetic retinopathy patients and three RNA samples from human retinas, including one downregulated gene and 12 upregulated genes. A correlation analysis showed that there was a correlation among these genes. Then, KEGG pathway and GO enrichment analyses were performed to explore the functional roles of these genes. The results showed that the mRNA of these genes was mainly related to inflammasome complex, interleukin-1 beta production, and NOD-like receptor signaling pathway. In addition, eight hub genes-CASP3, TLR4, NLRP3, GBP2, CASP1, CASP4, PYCARD, and GBP1-were identified by PPI network analysis using Cytoscape software. High glucose increased the protein level of GSDMD and GSDME, as critical effectors of pyroptosis, in retinal vascular endothelial cells. Verified by qRT-PCR, the expression of all these eight hub genes in the in vitro model of diabetic retinopathy was consistent with the results of the bioinformatics analysis of mRNA chip. Among them, CASP4, GBP1, CASP3, TLR4, and GBP2 were further validated in the GSE179568 dataset. Finally, 20 miRNAs were predicted to target three key genes-CASP3, GBP2, and TLR4, and 22 lncRNAs were predicted to potentially bind to these 20 miRNAs. Then, we constructed a key ceRNA network that is expected to mediate cellular pyroptosis in diabetic retinopathy. Conclusion: Through the data analysis of the GEO database by R software and verification by qRT-PCR and validation set, we successfully identified potential pyroptosis-related genes involved in the occurrence of diabetic retinopathy. The key ceRNA regulatory network associated with these genes was structured. These findings might improve the understanding of molecular mechanisms underlying pyroptosis in diabetic retinopathy.

Identification and Validation of Autophagy-Related Genes in Diabetic Retinopathy.

Background: Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes, which is associated with damage of blood-retinal barrier and ischemia of retinal vasculature. It devastates visual acuity due to leakage of retinal vessels and aberrant pathological angiogenesis in diabetic patients. The etiology of DR is complex, accumulated studies have shown that autophagy plays an important role in the pathogenesis of DR, but its specific mechanism needs to be further studied. Methods: This study chose the online Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE146615 to carry on the research. Autophagy-related genes that were potentially differentially expressed in DR were screened by R software. Then, the differentially expressed autophagy-related genes were analyzed by correlation analysis, tissue-specific gene expression, gene-ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction (PPI) network analysis. Finally, retinal pigment epithelial cell line (ARPE-19) incubated with high glucose (HG) was used to mimic the DR model, and the mRNA level of key genes was verified by quantitative real-time polymerase chain reaction (qRT-PCR) in vitro. Results: A total of 23 differentially expressed autophagy-related genes (9 up-regulated genes and 14 down-regulated genes) were identified by differential expression analysis. The analysis of tissue-specific gene expression showed that these differentially expressed autophagy-related genes were enriched in the retina. GO and KEGG enrichment analysis showed that differentially expressed autophagy-related genes were significantly enriched in autophagy-related pathways such as regulation of autophagy and macroautophagy. Then 10 hub genes were identified by PPI network analysis and construction of key modules. Finally, qRT-PCR confirmed that the expression of MAPK3 in the DR model was consistent with the results of bioinformatics analysis of mRNA chip. Conclusion: Through bioinformatics analysis, we identified 23 potential DR autophagy-related genes, among which the down-regulated expression of MAPK3 may affect the occurrence and development of DR by regulating autophagy. It provides a novel insight into the pathogenesis of DR.

Long-Term Oral Administration of Salidroside Alleviates Diabetic Retinopathy in db/db Mice.

Diabetic retinopathy (DR), a microvascular complication of diabetes mellitus, is the leading cause of vision loss in the working-age population worldwide. Unfortunately, current clinical treatments cannot completely prevent the occurrence and development of DR. Salidroside (Sal) is a medicinal supplement that has antioxidative and cytoprotective properties. This study aimed to investigate the therapeutic effect of Sal on DR. Briefly, Sal treatment was applied to wide-type mice and db/db mice (a widely used diabetic mice) at 25 mg/kg by oral gavage once daily from 8 weeks to 20 weeks. Mice's bodyweight, blood glucose, total cholesterol, triglyceride, high density lipoprotein and low density lipoprotein were recorded and analyzed. Retinal trypsin digestion and evans blue dye assay were used to detect retinal microvessel changes and function. Retinal glutathione and malondialdehyde content measurements were applied to assess retinal oxidative stress. Full-length transcriptome analysis was performed to explore the underlying mechanisms of Sal protection. Our results found that Sal treatment could successfully relieve blood glucose and blood lipid abnormalities, and reduce retinal oxidative stress level in diabetic mice. Also, Sal treatment repaired the abnormal transcriptome caused by diabetes, alleviated the microvascular lesion of the fundus in diabetic mice, and protected retinal normal barrier function. This study enriches the indications of Sal in the treatment of diabetic diseases, providing practical research ideas for the comprehensive preventions and treatments of DR.

DNMT1-mediated PPARα methylation aggravates damage of retinal tissues in diabetic retinopathy mice.

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARα) is associated with diabetic retinopathy (DR), and the underlying mechanism is still unclear. Aim of this work was to investigate the mechanism of PPARα in DR. METHODS: Human retinal capillary pericytes (HRCPs) were treated with high glucose (HG) to induce DR cell model. DR mouse model was established by streptozotocin injection, and then received 5-Aza-2-deoxycytidine (DAC; DNA methyltransferase inhibitor) treatment. Hematoxylin-eosin staining was performed to assess retinal tissue damage. PPARα methylation was examined by Methylation-Specific PCR. Flow cytometry and DCFH-DA fluorescent probe was used to estimate apoptosis and reactive oxygen species (ROS). The interaction between DNA methyltransferase-1 (DNMT1) and PPARα promoter was examined by Chromatin Immunoprecipitation. Quantitative real-time PCR and western blot were performed to assess gene and protein expression. RESULTS: HG treatment enhanced the methylation levels of PPARα, and repressed PPARα expression in HRCPs. The levels of apoptotic cells and ROS were significantly increased in HRCPs in the presence of HG. Moreover, DNMT1 was highly expressed in HG-treated HRCPs, and DNMT1 interacted with PPARα promoter. PPARα overexpression suppressed apoptosis and ROS levels of HRCPs, which was rescued by DNMT1 up-regulation. In DR mice, DAC treatment inhibited PPARα methylation and reduced damage of retinal tissues. CONCLUSION: DNMT1-mediated PPARα methylation promotes apoptosis and ROS levels of HRCPs and aggravates damage of retinal tissues in DR mice. Thus, this study may highlight novel insights into DR pathogenesis.

Peroxisome Proliferator-Activated Receptor α Activation Protects Retinal Ganglion Cells in Ischemia-Reperfusion Retinas.

Background and Objective: Retinal ischemia-reperfusion (IR) leads to massive loss of retinal ganglion cells (RGC) and characterizes several blind-causing ophthalmic diseases. However, the mechanism related to retinal IR is controversial, and a drug that could prevent the RGC loss caused by IR is still lacking. This study aimed to investigate the role of endogenous retinal peroxisome proliferator-activated receptor (PPAR)α and the therapeutic effect of its agonist, fenofibric acid (FA), in IR-related retinopathy. Materials and Methods: Fenofibric acid treatment was applied to the Sprague-Dawley rats with IR and retinal cell line 28 cells with oxygen-glucose deprivation (OGD) (an in vitro model of IR). Western blotting, real-time PCR, and immunofluorescence were used to examine the expression levels of PPARα, glial fibrillary acidic protein (GFAP), and cyclooxygenase-2 (COX2). Hematoxylin and eosin (HE) staining, propidium iodide (PI) staining, retrograde tracing, and flash visual-evoked potential (FVEP) were applied to assess RGC injury and visual function. Results: Retinal IR down-regulated PPARα expression in vitro and in vivo. Peroxisome proliferator-activated receptor α activation by FA promoted survival of RGCs, mitigated thinning of the ganglion cell complex, and decreased the latency of positive waves of FVEPs after IR injury. Further, FA treatment enhanced the expression of endogenous PPARα and suppressed the expression of GFAP and COX2 significantly. Conclusion: Peroxisome proliferator-activated receptor α activation by FA is protective against RGC loss in retinal IR condition, which may occur by restoring PPARα expression, inhibiting activation of glial cells, and suppressing retinal inflammation. All these findings indicate the translational potential of FA in treating IR-related retinopathy.

DNMT1-mediated PPARα methylation aggravates damage of retinal tissues in diabetic retinopathy mice

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARα) is associated with diabetic retinopathy (DR), and the underlying mechanism is still unclear. Aim of this work was to investigate the mechanism of PPARα in DR. METHODS: Human retinal capillary pericytes (HRCPs) were treated with high glucose (HG) to induce DR cell model. DR mouse model was established by streptozotocin injection, and then received 5-Aza-2-deoxycytidine (DAC; DNA methyltransferase inhibitor) treatment. Hematoxylin-eosin staining was performed to assess retinal tissue damage. PPARα methylation was examined by Methylation-Specific PCR. Flow cytometry and DCFH-DA fluorescent probe was used to estimate apoptosis and reactive oxygen species (ROS). The interaction between DNA methyltransferase-1 (DNMT1) and PPARα promoter was examined by Chromatin Immunoprecipitation. Quantitative real-time PCR and western blot were performed to assess gene and protein expression. RESULTS: HG treatment enhanced the methylation levels of PPARα, and repressed PPARα expression in HRCPs. The levels of apoptotic cells and ROS were significantly increased in HRCPs in the presence of HG. Moreover, DNMT1 was highly expressed in HG-treated HRCPs, and DNMT1 interacted with PPARα promoter. PPARα overexpression suppressed apoptosis and ROS levels of HRCPs, which was rescued by DNMT1 up-regulation. In DR mice, DAC treatment inhibited PPARα methylation and reduced damage of retinal tissues. CONCLUSION: DNMT1-mediated PPARα methylation promotes apoptosis and ROS levels of HRCPs and aggravates damage of retinal tissues in DR mice. Thus, this study may highlight novel insights into DR pathogenesis.

Diabetic nephropathy execrates epithelial-to-mesenchymal transition (EMT) via miR-2467-3p/Twist1 pathway.

Although diabetic nephropathy (DN) is induced by a complicate interplay of multiple factors, the underlying mechanisms remain poorly characterized, even the treatment. Herein, we show that both of DN patients and STZ-induced type 1 diabetic rat exhibit the reduction both of urinary and circulating miR-2467-3p. We identify a negative correlation between miR-2467-3p levels and renal dysfunction. Administration of miR-2467-3p prevents diabetes-induced renal dysfunction and represses renal fibrosis in STZ-induced type 1 diabetic rats. Conversely, anti-miR-2467 overexpression exacerbates renal dysfunction and fibrosis in STZ-induced rats. In diabetic condition, the reduction of miR-2467-3p promotes expression of Twist1, inducing epithelial-to-mesenchymal transition (EMT), resulting in renal fibrosis and kidney dysfunction. Together, our study presents miR-2467/Twist1/EMT as a regulatory axis of renal dysfunction in DN.

The 3' Untranslated Region Protects the Heart from Angiotensin II-Induced Cardiac Dysfunction via AGGF1 Expression.

The messenger RNA (mRNA) 3' untranslated regions (3' UTRs), as cis-regulated elements bound by microRNAs (miRNAs), affect their gene translation. However, the role of the trans-regulation of 3' UTRs during heart dysfunction remains elusive. Compared with administration of angiogenic factor with G-patch and forkhead-associate domains 1 (Aggf1), ectopic expression of Aggf1 with its 3' UTR significantly suppressed cardiac dysfunction in angiotensin II-infused mice, with upregulated expression of both Aggf1 and myeloid cell leukemia 1 (Mcl1). Along their 3' UTRs, Mcl1 and Aggf1 mRNAs share binding sites for the same miRNAs, including miR-105, miR-101, and miR-93. We demonstrated that the protein-coding Mcl1 and Aggf1 mRNAs communicate and co-regulate each other's expression through competition for these three miRNAs that target both transcripts via their 3' UTRs. Our results indicate that Aggf1 3' UTR, as a trans-regulatory element, accelerates the cardioprotective role of Aggf1 in response to hypertensive conditions by elevating Mcl1 expression. Our work broadens the scope of gene therapy targets and provides a new insight into gene therapy strategies involving 3' UTRs.

The Anti-apoptotic Role of 3'-Untranslational Region in Response to Angiotensin II via Mcl1 Expression.

Myeloid cell leukemia 1 (Mcl1), an abundant protein in the myocardium, plays an essential role in fibrosis and anti-inflammation in cardiomyocytes to prevent heart failure. However, whether Mcl1 3'-untranslated regions (3'-UTR) has the cardio-protecting function remains unclear. Down-regulation of Mcl1 was observed in adult mice heart tissues after Angiotensin II (Ang II) treatment. Consistent with in vivo results, the reduction of Mcl1 expression was identified in Ang II-treated neonatal cardiomyocytes. Mechanistically, Mcl1 3'-UTR prevented Ang II-induced cardiac apoptosis via up-regulation of Mcl1 and an angiogenic factor with a G-patch domain and a forkhead-associated domain 1 (Aggf1), which plays cardiac-protective role. Our work broadens the scope of gene therapy targets and provides a new insight into gene therapy strategies involving mRNAs' 3'-UTRs application.

Diabetic retinopathy: Focus on NADPH oxidase and its potential as therapeutic target.

Diabetic retinopathy is a common complication of diabetes that affects the retina due to a sustained high blood sugar level. Recent studies have demonstrated that high glucose-driven oxidative stress plays an important role in the microvascular complications of retina in diabetes. Oxidative stress occurs due to the excess of reactive oxygen species, which causes oxidative damage to retina, leading to the leak of tiny blood vessels, or acts as signaling molecules to trigger neovascularization, resulting in new fragile vessels. NADPH oxidase (NOX) is a key enzymatic source of reactive oxygen species in the retina, and it is involved in the early as well as the advanced stage of diabetic retinopathy. To date, at least 7 NOX isoforms, including NOX1 to NOX5, dual oxidase1 and dual oxidase 2, have been identified. It has been shown that NOX isoforms exert different roles in the pathogenesis of diabetic retinopathy. Intervention of NOX by its inhibitors or modulators shows beneficial effect on improving the retinal functions in the models of diabetic retinopathy in vivo or in vitro. Thereby, NOX might be a potential target for the therapy of diabetic retinopathy. The present review focuses on the role of NOX, particularly the NOX isoforms, in promoting the development of diabetic retinopathy. In addition, NOX isoforms as potential targets for therapy of diabetic retinopathy are also discussed.

Neuroprotective effects of PPARα in retinopathy of type 1 diabetes.

Diabetic retinopathy (DR) is a common neurovascular complication of type 1 diabetes. Current therapeutics target neovascularization characteristic of end-stage disease, but are associated with significant adverse effects. Targeting early events of DR such as neurodegeneration may lead to safer and more effective approaches to treatment. Two independent prospective clinical trials unexpectedly identified that the PPARα agonist fenofibrate had unprecedented therapeutic effects in DR, but gave little insight into the physiological and molecular mechanisms of action. The objective of the present study was to evaluate potential neuroprotective effects of PPARα in DR, and subsequently to identify the responsible mechanism of action. Here we reveal that activation of PPARα had a robust protective effect on retinal function as shown by Optokinetic tracking in a rat model of type 1 diabetes, and also decreased retinal cell death, as demonstrated by a DNA fragmentation ELISA. Further, PPARα ablation exacerbated diabetes-induced decline of visual function as demonstrated by ERG analysis. We further found that PPARα improved mitochondrial efficiency in DR, and decreased ROS production and cell death in cultured retinal neurons. Oxidative stress biomarkers were elevated in diabetic Pparα-/- mice, suggesting increased oxidative stress. Mitochondrially mediated apoptosis and oxidative stress secondary to mitochondrial dysfunction contribute to neurodegeneration in DR. Taken together, these findings identify a robust neuroprotective effect for PPARα in DR, which may be due to improved mitochondrial function and subsequent alleviation of energetic deficits, oxidative stress and mitochondrially mediated apoptosis.

Pulsatility Index as a Novel Parameter for Perfusion in Mouse Model of Hindlimb Ischemia.

BACKGROUND/AIMS: In clinical settings, the pulsatility index (PI) has become a widely used tool for monitoring obstetrics or other vascular diseases. It is based on the maximum Doppler shift waveform derived from ultrasonography. However, it remains unclear whether the PI levels are correctly predicted from the perfusion in mouse model of hindlimb ischemia. METHODS: To explore the relationship between PI and perfusion, we generated a unilateral hindlimb ischemia model in 8-week-old C57BL/6 male mice by ligation of the right common iliac artery and femoral artery. These mice were monitored with laser Doppler perfusion imaging (LDPI) and an ultrasound system (Vevo2100). Vessel densities in ischemic skeletal muscles were measured with vWF staining, which functions as a marker for endothelial cells. In order to further verify PI evaluation in other conditions, we performed therapeutic experiments using hindlimb ischemic mouse with PBS or FGF2 treatment. RESULTS: In the mouse model of hindlimb ischemia, the PI levels were continuously elevated and were accompanied by an increased ratio of perfusion to blood flow. 1 and 4 weeks after ischemia, the densities of vWF staining were correlated with PI values. Moreover, the PI index exactly reflected the perfusion in hindlimb ischemic mice after FGF2 treatment, while it indicated the condition of angiogenesis after therapeutic treatment based on the association between PI values and the number of vWF-positive stained cells in muscles. CONCLUSION: This study confirms the utility of a noninvasive and reproducible ultrasound index for a rapid evaluation of perfusion and blood recovery after hindlimb ischemia in vivo. PI, as one stable and comparable parameter, is correlated with angiogenesis in hindlimb ischemic mouse. Moreover, PI can exactly reflect perfusion and angiogenesis in therapeutic hindlimb ischemic mouse models. This study suggested that PI can serve as a novel index for relatively reproducible and repeatable blood flow recovery in the evaluation of emerging ischemic therapies and disease development in mouse models of hindlimb ischemia.

Renal Resistive Index as a Novel Indicator for Renal Complications in High-Fat Diet-Fed Mice.

BACKGROUND/AIMS: The renal resistive index (RI) is a novel candidate as a renal injury prognostic indicator, but it remains unclear how renal RI levels correspond to renal injury in diabetic nephropathy. METHODS: To examine this issue, we compared 8-week-old male C57BL/6 mice fed with high-fat diet (HFD) versus chow diet (CHD) for 16 weeks. At 8 and 12 weeks, the glomerular filtration rate (GFR), urinary albumin-to-creatinine ratio (UACR), and inflammatory factors (IL-1ß, IL-6, TNFα, and MCP-1) were measured, along with the increase in renal RI. RESULTS: Our study suggests RI values positively correlate with GFR for the first 12 weeks of HFD feeding. In contrast, the GFR of 16-week HFD feeding is lower than that of 12-week HFD feeding, whereas RI levels are significantly increased. Additionally, our study suggests RI values accurately indicate the renal fibrosis and renal injury in HFD-fed mice treated with lovastatin. CONCLUSION: This study seems to confirm the utility of a noninvasive and repeatable ultrasound parameter to rapidly evaluate renal fibrosis in a HFD-induced type 2 diabetic mouse model in vivo. This highly sensitive and comparable renal RI measurement could monitor the whole procedure of disease development in real-time. RI measurement of the renal artery is capable of differentiating responses to standard therapy with lovastatin in HFD-fed mice from the CHD group.

Interaction of PPARα With the Canonic Wnt Pathway in the Regulation of Renal Fibrosis.

Peroxisome proliferator-activated receptor-α (PPARα) displays renoprotective effects with an unclear mechanism. Aberrant activation of the canonical Wnt pathway plays a key role in renal fibrosis. Renal levels of PPARα were downregulated in both type 1 and type 2 diabetes models. The PPARα agonist fenofibrate and overexpression of PPARα both attenuated the expression of fibrotic factors, and suppressed high glucose-induced or Wnt3a-induced Wnt signaling in renal cells. Fenofibrate inhibited Wnt signaling in the kidney of diabetic rats. A more renal prominent activation of Wnt signaling was detected both in PPARα-/- mice with diabetes or obstructive nephropathy and in PPARα-/- tubular cells treated with Wnt3a. PPARα did not block the transcriptional activity of ß-catenin induced by a constitutively active mutant of lipoprotein receptor-related protein 6 (LRP6) or ß-catenin. LRP6 stability was decreased by overexpression of PPARα and increased in PPARα-/- tubular cells, suggesting that PPARα interacts with Wnt signaling at the Wnt coreceptor level. 4-Hydroxynonenal-induced reactive oxygen species production, which resulted in LRP6 stability, was suppressed by overexpression of PPARα and dramatically enhanced in PPARα-/- tubular cells. Diabetic PPARα-/- mice showed more prominent NADPH oxidase-4 overexpression compared with diabetic wild-type mice, suggesting that the inhibitory effect of PPARα on Wnt signaling may be ascribed to its antioxidant activity. These observations identified a novel interaction between PPARα and the Wnt pathway, which is responsible, at least partially, for the therapeutic effects of fenofibrate on diabetic nephropathy.

Nanoparticle-mediated expression of a Wnt pathway inhibitor ameliorates ocular neovascularization.

OBJECTIVE: The deficiency of very low-density lipoprotein receptor resulted in Wnt signaling activation and neovascularization in the retina. The present study sought to determine whether the very low-density lipoprotein receptor extracellular domain (VLN) is responsible for the inhibition of Wnt signaling in ocular tissues. APPROACH AND RESULTS: A plasmid expressing the soluble VLN was encapsulated with poly(lactide-co-glycolide acid) to form VLN nanoparticles (VLN-NP). Nanoparticles containing a plasmid expressing the low-density lipoprotein receptor extracellular domain nanoparticle were used as negative control. MTT, modified Boyden chamber, and Matrigel (™) assays were used to evaluate the inhibitory effect of VLN-NP on Wnt3a-stimulated endothelial cell proliferation, migration, and tube formation. Vldlr(-/-) mice, oxygen-induced retinopathy, and alkali burn-induced corneal neovascularization models were used to evaluate the effect of VLN-NP on ocular neovascularization. Wnt reporter mice (BAT-gal), Western blotting, and luciferase assay were used to evaluate Wnt pathway activity. Our results showed that VLN-NP specifically inhibited Wnt3a-induced endothelial cell proliferation, migration, and tube formation. Intravitreal injection of VLN-NP inhibited abnormal neovascularization in Vldlr(-/-), oxygen-induced retinopathy, and alkali burn-induced corneal neovascularization models, compared with low-density lipoprotein receptor extracellular domain nanoparticle. VLN-NP significantly inhibited the phosphorylation of low-density lipoprotein receptor-related protein 6, the accumulation of ß-catenin, and the expression of vascular endothelial growth factor in vivo and in vitro. CONCLUSIONS: Taken together, these results suggest that the soluble VLN is a negative regulator of the Wnt pathway and has antiangiogenic activities. Nanoparticle-mediated expression of VLN may thus represent a novel therapeutic approach to treat pathological ocular angiogenesis and potentially other vascular diseases affected by Wnt signaling.

Peroxisome proliferator-activated receptor α protects capillary pericytes in the retina.

Pericyte degeneration is an early event in diabetic retinopathy and plays an important role in progression of diabetic retinopathy. Clinical studies have shown that fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has robust therapeutic effects on diabetic retinopathy in type 2 diabetic patients. We evaluated the protective effect of PPARα against pericyte loss in diabetic retinopathy. In streptozotocin-induced diabetic mice, fenofibrate treatment significantly ameliorated retinal acellular capillary formation and pericyte loss. In contrast, PPARα(-/-) mice with diabetes developed more severe retinal acellular capillary formation and pericyte dropout, compared with diabetic wild-type mice. Furthermore, PPARα knockout abolished the protective effect of fenofibrate against diabetes-induced retinal pericyte loss. In cultured primary human retinal capillary pericytes, activation and expression of PPARα both significantly reduced oxidative stress-induced apoptosis, decreased reactive oxygen species production, and down-regulated NAD(P)H oxidase 4 expression through blockade of NF-κB activation. Furthermore, activation and expression of PPARα both attenuated the oxidant-induced suppression of mitochondrial O2 consumption in human retinal capillary pericytes. Primary retinal pericytes from PPARα(-/-) mice displayed more apoptosis, compared with those from wild-type mice under the same oxidative stress. These findings identified a protective effect of PPARα on retinal pericytes, a novel function of endogenous PPARα in the retina.

CD82 restrains pathological angiogenesis by altering lipid raft clustering and CD44 trafficking in endothelial cells.

BACKGROUND: Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. METHODS AND RESULTS: Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. CONCLUSIONS: Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.