FK-binding protein 5: Possible relevance to the pathogenesis of metabolic dysfunction and alcohol-associated liver disease.

The FK506-binding protein (FKBP5) plays significant roles in mediating stress responses by interacting with glucocorticoids, participating in adipogenesis, and influencing various cellular pathways throughout the body. In this review, we described the potential role of FKBP5 in the pathogenesis of two common chronic liver diseases, metabolic dysfunction-associated steatotic liver disease (MASLD), and alcohol-associated liver disease (ALD). We provided an overview of the FK-binding protein family and elucidated their roles in cellular stress responses, metabolic diseases, and adipogenesis. We explored how FKBP5 may mechanistically influence the pathogenesis of MASLD and ALD and provided insights for further investigation into the role of FKBP5 in these two diseases.

Telomere length in patients with alcohol-associated liver disease: a brief report.

The intact telomere structure is essential for the prevention of the chromosome end-to-end fusions and maintaining genomic integrity. The maintenance of telomere length is critical for cellular homeostasis. The shortening of telomeres has been reported in patients with chronic liver diseases. The telomere length has not been systemically studied in patients with alcohol-associated liver disease (ALD) at different stages, such as alcoholic hepatitis and alcoholic cirrhosis. In this brief report, we observed evidence of telomere shortening without changes in the telomerase activity in the liver of patients with alcoholic hepatitis and alcoholic cirrhosis when compared with controls. The alterations in the genes associated with telomere binding proteins were only observed in patients with alcoholic cirrhosis. Future studies are required to determine the mechanism of how alcohol affects the length of the telomere and if the shortening impacts the disease progression in ALD.

Transcriptomic Analysis Reveals the Messenger RNAs Responsible for the Progression of Alcoholic Cirrhosis.

Alcohol-associated liver disease is the leading cause of chronic liver disease. We hypothesized that the expression of specific coding genes is critical for the progression of alcoholic cirrhosis (AC) from compensated to decompensated states. For the discovery phase, we performed RNA sequencing analysis of 16 peripheral blood RNA samples, 4 healthy controls (HCs) and 12 patients with AC. The DEGs from the discovery cohort were validated by quantitative polymerase chain reaction in a separate cohort of 17 HCs and 48 patients with AC (17 Child-Pugh A, 16 Child-Pugh B, and 15 Child-Pugh C). We observed that the numbers of differentially expressed messenger RNAs (mRNAs) were more pronounced with worsening disease severity. Pathway analysis for differentially expressed genes for patients with Child-Pugh A demonstrated genes involved innate immune responses; those in Child-Pugh B belonged to genes related to oxidation and alternative splicing; those in Child-Pugh C related to methylation, acetylation, and alternative splicing. We found significant differences in the expression of heme oxygenase 1 (HMOX1) and ribonucleoprotein, PTB binding 1 (RAVER1) in peripheral blood of those who died during the follow-up when compared to those who survived. Conclusion: Unique mRNAs that may implicate disease progression in patients with AC were identified by using a transcriptomic approach. Future studies to confirm our results are needed, and comprehensive mechanistic studies on the implications of these genes in AC pathogenesis and progression should be further explored.

Serum metabolomic analysis reveals several novel metabolites in association with excessive alcohol use - an exploratory study.

Appropriate screening tool for excessive alcohol use (EAU) is clinically important as it may help providers encourage early intervention and prevent adverse outcomes. We hypothesized that patients with excessive alcohol use will have distinct serum metabolites when compared to healthy controls. Serum metabolic profiling of 22 healthy controls and 147 patients with a history of EAU was performed. We employed seemingly unrelated regression to identify the unique metabolites and found 67 metabolites (out of 556), which were differentially expressed in patients with EAU. Sixteen metabolites belong to the sphingolipid metabolism, 13 belong to phospholipid metabolism, and the remaining 38 were metabolites of 25 different pathways. We also found 93 serum metabolites that were significantly associated with the total quantity of alcohol consumption in the last 30 days. A total of 15 metabolites belong to the sphingolipid metabolism, 11 belong to phospholipid metabolism, and 7 metabolites belong to lysolipid. Using a Venn diagram approach, we found the top 10 metabolites with differentially expressed in EAU and significantly associated with the quantity of alcohol consumption, sphingomyelin (d18:2/18:1), sphingomyelin (d18:2/21:0,d16:2/23:0), guanosine, S-methylmethionine, 10-undecenoate (11:1n1), sphingomyelin (d18:1/20:1, d18:2/20:0), sphingomyelin (d18:1/17:0, d17:1/18:0, d19:1/16:0), N-acetylasparagine, sphingomyelin (d18:1/19:0, d19:1/18:0), and 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1). The diagnostic performance of the top 10 metabolites, using the area under the ROC curve, was significantly higher than that of commonly used markers. We have identified a unique metaboloic signature among patients with EAU. Future studies to validate and determine the kinetics of these markers as a function of alcohol consumption are needed.

The role of SHP/REV-ERBα/CYP4A axis in the pathogenesis of alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp-/- mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp-/- mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα-/- hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.

Transcriptomic Analysis Reveals the MicroRNAs Responsible for Liver Regeneration Associated With Mortality in Alcohol-Associated Hepatitis.

BACKGROUND AND AIMS: We conducted a comprehensive serum transcriptomic analysis to explore the roles of microRNAs (miRNAs) in alcohol-associated hepatitis (AH) pathogenesis and their prognostic significance. APPROACH AND RESULTS: Serum miRNA profiling was performed in 15 controls, 20 heavy drinkers without liver disease, and 65 patients with AH and compared to publicly available hepatic miRNA profiling in AH patients. Among the top 26 miRNAs, expression of miR-30b-5p, miR-20a-5p, miR-146a-5p, and miR-26b-5p were significantly reduced in both serum and liver of AH patients. Pathway analysis of the potential targets of these miRNAs uncovered the genes related to DNA synthesis and cell-cycle progression pathways, including ribonucleotide reductase regulatory subunit M2 (RRM2), cyclin D1 (CCND1), cyclin D2 (CCND2), MYC proto-oncogene (MYC), and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1). We found a significant increase in the protein expression of RRM2, CCND1, and CCND2, but not MYC and PMAIP1, in AH patients who underwent liver transplantation; miR-26b-5p and miR-30b-5p inhibited the 3'-UTR (untranslated region) luciferase activity of RRM2 and CCND2, and miR-20a-5p reduced the 3'-UTR luciferase activity of CCND1 and CCND2. During a median follow-up of 346 days, 21% of AH patients died; these patients had higher body mass index (BMI), Model for End-Stage Liver Disease (MELD), and serum miR-30b-5p, miR-20a-5p, miR-146a-5p, and miR-26b-5p than those who survived. Cox regression analysis showed that BMI, MELD score, miR-20a-5p, miR-146a-5p, and miR-26b-5p predicted mortality. CONCLUSIONS: Patients with AH attempt to deal with hepatocyte injury by down-regulating specific miRNAs and up-regulating genes responsible for DNA synthesis and cell-cycle progression. Higher expression of these miRNAs, suggestive of a diminished capacity in liver regeneration, predicts short-term mortality in AH patients.

Hepatic Histopathology Among Excessive Drinkers Without Advanced Liver Disease.

AIMS: Alcohol-associated liver disease represents a spectrum of histopathological changes from steatosis to advanced fibrosis and cirrhosis. The major goals of this retrospective study were to characterize the histologic features in patients with excessive alcohol use who presented with an abnormal hepatic panel and/or abnormal radiographic imaging and did not meet the clinical diagnosis of alcoholic hepatitis or cirrhosis. METHODS: We performed a retrospective study to describe hepatic histology of 62 and 83 excessive drinkers with normal and abnormal serum aspartate transaminase, respectively. The types of inflammatory cells in the liver were characterized by immunohistochemistry for CD4, CD8, CD20, CD68 and myeloperoxidase. RESULTS: Among 62 patients with aspartate aminotransferase (AST) ≤ 50 U/L, 37% had histological evidence of steatosis. Of these, we found evidence of hepatocyte ballooning (21%), lobular inflammation (50%), portal inflammation (52%) and fibrosis (14%). For those with AST > 50 U/L, the presence of hepatic steatosis, lobular inflammation and portal inflammation was observed in 29, 60 and 69% of patients, respectively. Fibrosis was found in 33%, four with bridging fibrosis, and one with cirrhosis. We observed the aggregation of CD68+ macrophages, rather than normally distributed with minimal neutrophilic infiltration. Lobular and portal lymphocytic infiltrations are primarily CD8+ T cells. CONCLUSION: Abnormal hepatic histopathology occurs in excessive drinkers with normal transaminase activity. Future studies to determine the diagnostic modalities to detect such abnormalities and to better understand its clinical implications and long-term outcome are needed.

Stress-Responsive Gene FK506-Binding Protein 51 Mediates Alcohol-Induced Liver Injury Through the Hippo Pathway and Chemokine (C-X-C Motif) Ligand 1 Signaling.

BACKGROUND AND AIMS: Chronic alcohol drinking is a major risk factor for alcohol-associated liver disease (ALD). FK506-binding protein 51 (FKBP5), a cochaperone protein, is involved in many key regulatory pathways. It is known to be involved in stress-related disorders, but there are no reports regarding its role in ALD. This present study aimed to examine the molecular mechanism of FKBP5 in ALD. APPROACH AND RESULTS: We found a significant increase in hepatic FKBP5 transcripts and protein expression in patients with ALD and mice fed with chronic-plus-single binge ethanol. Loss of Fkbp5 in mice protected against alcohol-induced hepatic steatosis and inflammation. Transcriptomic analysis revealed a significant reduction of Transcriptional enhancer factor TEF-1 (TEA) domain transcription factor 1 (Tead1) and chemokine (C-X-C motif) ligand 1 (Cxcl1) mRNA in ethanol-fed Fkbp5-/- mice. Ethanol-induced Fkbp5 expression was secondary to down-regulation of methylation level at its 5' untranslated promoter region. The increase in Fkbp5 expression led to induction in transcription factor TEAD1 through Hippo signaling pathway. Fkbp5 can interact with yes-associated protein (YAP) upstream kinase, mammalian Ste20-like kinase 1 (MST1), affecting its ability to phosphorylate YAP and the inhibitory effect of hepatic YAP phosphorylation by ethanol leading to YAP nuclear translocation and TEAD1 activation. Activation of TEAD1 led to increased expression of its target, CXCL1, a chemokine-mediated neutrophil recruitment, causing hepatic inflammation and neutrophil infiltration in our mouse model. CONCLUSIONS: We identified an FKBP5-YAP-TEAD1-CXCL1 axis in the pathogenesis of ALD. Loss of FKBP5 ameliorates alcohol-induced liver injury through the Hippo pathway and CXCL1 signaling, suggesting its potential role as a target for the treatment of ALD.

Long noncoding RNA H19 - a new player in the pathogenesis of liver diseases.

The liver is a vital organ that controls glucose and lipid metabolism, hormone regulation, and bile secretion. Liver injury can occur from various insults such as viruses, metabolic diseases, and alcohol, which lead to acute and chronic liver diseases. Recent studies have demonstrated the implications of long noncoding RNAs (lncRNAs) in the pathogenesis of liver diseases. These newly discovered lncRNAs have various functions attributing to many cellular biological processes via distinct and diverse mechanisms. LncRNA H19, one of the first lncRNAs being identified, is highly expressed in fetal liver but not in adult normal liver. Its expression, however, is increased in liver diseases with various etiologies. In this review, we focused on the roles of H19 in the pathogenesis of liver diseases. This comprehensive review is aimed to provide useful perspectives and translational applications of H19 as a potential therapeutic target of liver diseases.

Long non-coding RNAs in liver diseases: Focusing on nonalcoholic fatty liver disease, alcohol-related liver disease, and cholestatic liver disease.

Long non-coding RNAs (lncRNAs), a class of transcribed RNA molecules with the lengths exceeding 200 nucleotides, are not translated into protein. They can modulate protein-coding genes by controlling transcriptional and posttranscriptional processes. The dysregulation of lncRNAs has been related to various pathological disorders. In this review, we summarized the current knowledge of lncRNAs and their implications in the pathogenesis of three common liver diseases: nonalcoholic fatty liver disease, alcohol-related liver disease, and cholestatic liver disease. Future studies to further define the role of lncRNAs and their mechanisms in various types of liver diseases should be explored. An improved understanding from these studies will provide us a useful perspective leading to mechanism-based intervention by targeting specific lncRNAs for the treatment of liver diseases.

Role of microRNA-7 in liver diseases: a comprehensive review of the mechanisms and therapeutic applications.

MicroRNA-7 (miR-7) is a small non-coding RNA, which plays critical roles in regulating gene expression of multiple key cellular processes. MiR-7 exhibits a tissue-specific pattern of expression, with abundant levels found in the brain, spleen, and pancreas. Although it is expressed at lower levels in other tissues, including the liver, miR-7 is involved in both the development of organs and biological functions of cells. In this review, we focus on the mechanisms by which miR-7 controls cell growth, proliferation, invasion, metastasis, metabolism, and inflammation. We also summarize the specific roles of miR-7 in liver diseases. MiR-7 is considered as a tumor suppressor miRNA in hepatocellular carcinoma and is involved in the pathogenesis of hepatic steatosis and hepatitis. Future studies to further define miR-7 functions and its mechanism in association with other types of liver diseases should be explored. An improved understanding from these studies will provide us a useful perspective leading to mechanism-based intervention by targeting miR-7 for the treatment of liver diseases.

Alcohol Metabolizing Enzymes, Microsomal Ethanol Oxidizing System, Cytochrome P450 2E1, Catalase, and Aldehyde Dehydrogenase in Alcohol-Associated Liver Disease.

Once ingested, most of the alcohol is metabolized in the liver by alcohol dehydrogenase to acetaldehyde. Two additional pathways of acetaldehyde generation are by microsomal ethanol oxidizing system (cytochrome P450 2E1) and catalase. Acetaldehyde can form adducts which can interfere with cellular function, leading to alcohol-induced liver injury. The variants of alcohol metabolizing genes encode enzymes with varied kinetic properties and result in the different rate of alcohol elimination and acetaldehyde generation. Allelic variants of these genes with higher enzymatic activity are believed to be able to modify susceptibility to alcohol-induced liver injury; however, the human studies on the association of these variants and alcohol-associated liver disease are inconclusive. In addition to acetaldehyde, the shift in the redox state during alcohol elimination may also link to other pathways resulting in activation of downstream signaling leading to liver injury.

H19 Is Expressed in Hybrid Hepatocyte Nuclear Factor 4α+ Periportal Hepatocytes but Not Cytokeratin 19+ Cholangiocytes in Cholestatic Livers.

Long noncoding RNA (lncRNA) H19 is abundantly expressed in fetal liver. Its expression is significantly diminished in adult healthy liver but is re-induced in chronic liver diseases, including cholestasis. In this study, we developed a new method with combined in situ hybridization (ISH) and immunofluorescence (IF) colabeling to establish an H19 expression profile with both parenchymal and nonparenchymal cell-specific markers in the livers of cholestatic mouse models and patients with cholestasis. H19RNA+ cells showed no colocalization with biliary epithelial cell marker cytokeratin 19 (CK19)+ cholangiocytes but were immediately adjacent to biliary structures in bile duct ligation (BDL), 3,5-diethoxycarbony1-1,4-dihydrocollidine (DDC), and multidrug-resistant gene 2 knockout ( Mdr2 -/- ) mouse models and in human primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) liver specimens. In contrast, double-positive H19RNA+/sex-determining region Y (SRY)-box 9 (SOX9)+ ductal progenitor cells, H19RNA+/hepatocyte nuclear factor 4α (HNF4α)+ hepatocytes, H19RNA+/F4/80+ Kupffer cells, HNF4α+/SOX9+ hybrid hepatocytes, as well as triple-positive H19 RNA+/HNF4α+/SOX9+ periportal hepatocytes were identified. In addition, H19 RNA could not be detected in mesenchymal cell marker desmin+ cells. Furthermore, H19 RNA was predominately detected in cytoplasm with a small amount at the interspace with neighboring cells. Conclusion: H19RNA is localized in HNF4α+ periportal hepatocytes, SOX9+ ductal progenitor cells, and F4/80+ Kupffer cells but not in CK19+ cholangiocytes and desmin+ stellate cells in cholestatic livers.

EGN model of non-linear fiber propagation.

The GN-model has been proposed as an approximate but sufficiently accurate tool for predicting uncompensated optical coherent transmission system performance, in realistic scenarios. For this specific use, the GN-model has enjoyed substantial validation, both simulative and experimental. Recently, however, it has been pointed out that its predictions, when used to obtain a detailed picture of non-linear interference (NLI) noise accumulation along a link, may be affected by a substantial NLI overestimation error, especially in the first spans of the link. In this paper we analyze in detail the GN-model errors. We discuss recently proposed formulas for correcting such errors and show that they neglect several contributions to NLI, so that they may substantially underestimate NLI in specific situations, especially over low-dispersion fibers. We derive a complete set of formulas accounting for all single, cross, and multi-channel effects, This set constitutes what we have called the enhanced GN-model (EGN-model). We extensively validate the EGN model by comparison with accurate simulations in several different system scenarios. The overall EGN model accuracy is found to be very good when assessing detailed span-by-span NLI accumulation and excellent when estimating realistic system maximum reach. The computational complexity vs. accuracy trade-offs of the various versions of the GN and EGN models are extensively discussed.

Endothelial NADPH oxidase 4 mediates vascular endothelial growth factor receptor 2-induced intravitreal neovascularization in a rat model of retinopathy of prematurity.

PURPOSE: NADPH oxidase-generated reactive oxygen species (ROS) are implicated in angiogenesis. Isoforms of NADPH oxidase NOX1, NOX2, and NOX4 are reported to be expressed in endothelial cells (ECs). Of these, NOX1 and NOX2 have been reported to contribute to intravitreal neovascularization (IVNV) in oxygen-induced retinopathy (OIR) models. In this study, we tested the hypothesis that the isoform NOX4 in ECs contributed to vascular endothelial growth factor (VEGF)-induced angiogenesis and IVNV. METHODS: Isoforms of NADPH oxidase MRNA were measured in several types of cultured vascular ecs: human retinal microvascular ECs (hRMVECs), choroidal ECs (CECs), and human umbilical vascular ECs (HUVECs) using real-time PCR. Newborn rat pups and dams were placed into an OIR model that cycled oxygen concentration between 50% and 10% every 24 h for 14 days, and then were placed in room air (RA) for an additional 4 days (rat OIR model). NOX4 expression in retinal lysates from the RA-raised pups at postnatal day 0 (P0), P14, and P18 was determined with western blots. STAT3 activation was determined as the ratio of phosphorylated STAT3 to total STAT3 with western blot analysis of retinal lysates from pups raised in RA or from the rat OIR model at P18. Semiquantitative assessment of the density of NOX4 colabeling with lectin-stained retinal ECs was determined by immunolabeling of retinal cryosections from P18 pups in OIR or in RA. In hRMVECs transfected with NOX4 siRNA and treated with VEGF or control, 1) ROS generation was measured using the 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester fluorescence assay and 2) phosphorylated VEGF receptor 2 and STAT3, and total VEGFR2 and STAT3 were measured in western blot analyses. VEGF-stimulated hRMVEC proliferation was measured following transfection with NOX4 siRNA or STAT3 siRNA, or respective controls. RESULTS: NOX4 was the most prevalent isoform of NADPH oxidase in vascular ECs. NOX4 expression in retinal lysates was significantly decreased during development in RA. Compared to RA, the expression of retinal NOX4 increased at P18. At p18 OIR, semiquantitative assessment of the density of lectin and NOX4 colabeling in retinal vascular ECs was greater in retinal cryosections and activated STAT3 was greater in retinal lysates when compared to the RA-raised pups. In cultured hRMVECs, knockdown of NOX4 by siRNA transfection inhibited VEGF-induced ROS generation. VEGF induced a physical interaction of phosphorylated-VEGFR2 and NOX4. Knockdown of NOX4: 1) reduced VEGFR2 activation but did not abolish it and 2) abolished STAT3 activation in response to VEGF. Knockdown of either NOX4 or STAT3 inhibited VEGF-induced EC proliferation. CONCLUSIONS: Our data suggest that in a model representative of human retinopathy of prematurity, NOX4 was increased at a time point when IVNV developed. VEGF-activated NOX4 led to an interaction between VEGF-activated VEGFR2 and NOX4 that mediated EC proliferation via activation of STAT3. Altogether, our results suggest that NOX4 may regulate VEGFR2-mediated IVNV through activated STAT3.

VEGFA activates erythropoietin receptor and enhances VEGFR2-mediated pathological angiogenesis.

Clinical and animal studies implicate erythropoietin (EPO) and EPO receptor (EPOR) signaling in angiogenesis. In the eye, EPO is involved in both physiological and pathological angiogenesis in the retina. We hypothesized that EPOR signaling is important in pathological angiogenesis and tested this hypothesis using a rat model of oxygen-induced retinopathy that is representative of human retinopathy of prematurity. We first determined that EPOR expression and activation were increased and that activated EPOR was localized to retinal vascular endothelial cells (ECs) in retinas at postnatal day 18 (p18), when pathological angiogenesis in the form of intravitreal neovascularization occurred. In human retinal microvascular ECs, EPOR was up-regulated and activated by VEGF. Lentiviral-delivered shRNAs that knocked down Müller cell-expressed VEGF in the retinopathy of prematurity model also reduced phosphorylated EPOR (p-EPOR) and VEGFR2 (p-VEGFR2) in retinal ECs. In human retinal microvascular ECs, VEGFR2-activated EPOR caused an interaction between p-EPOR and p-VEGFR2; knockdown of EPOR by siRNA transfection reduced VEGF-induced EC proliferation in association with reduced p-VEGFR2 and p-STAT3; however, inhibition of VEGFR2 activation by siRNA transfection or semaxanib (SU5416) abolished VEGFA-induced proliferation of ECs and phosphorylation of VEGFR2, EPOR, and STAT3. Our results show that VEGFA-induced p-VEGFR2 activates EPOR and causes an interaction between p-EPOR and p-VEGFR2 to enhance VEGFA-induced EC proliferation by exacerbating STAT3 activation, leading to pathological angiogenesis.

Targeting Müller cell-derived VEGF164 to reduce intravitreal neovascularization in the rat model of retinopathy of prematurity.

PURPOSE: To determine whether knockdown of Müller cell-derived VEGFA-splice variant, VEGF164, which is upregulated in the rat retinopathy of prematurity (ROP) model, safely inhibits intravitreal neovascularization (IVNV). METHODS: Short hairpin RNAs for VEGF164 (VEGF164.shRNAs) or luciferase.shRNA control were cloned into lentivectors with CD44 promoters that specifically target Müller cells. Knockdown efficiency, off-target effects, and specificity were tested in HEK reporter cell lines that expressed green fluorescent protein (GFP)-tagged VEGF164 or VEGF120 with flow cytometry or in rat Müller cells (rMC-1) by real-time PCR. In the rat oxygen-induced retinopathy (OIR) ROP model, pups received 1 µL subretinal lentivector-driven luciferase.shRNA, VEGFA.shRNA, or VEGF164.shRNA at postnatal day 8 (P8). Analyses at P18 and P25 included: IVNV and avascular retina (AVA); retinal and serum VEGF (ELISA); density of phosphorylated VEGFR2 (p-VEGFR2) in lectin-labeled retinal endothelial cells (ECs; immunohistochemistry); TUNEL staining and thickness of inner nuclear (INL) and outer nuclear layers (ONL) in retinal cryosections; and pup weight gain. RESULTS: In HEK reporter and in rMC-1 cells and in comparison to lucifferase.shRNA, VEGFA.shRNA reduced both VEGF120 and VEGF164, but VEGF164.shRNA only reduced VEGF164 and not VEGF120. Compared with luciferase.shRNA, VEGFA.shRNA and VEGF164.shRNA reduced retinal VEGF and IVNV without affecting AVA at P18 and P25. At P25, VEGF164.shRNA more effectively maintained IVNV inhibition than VEGFA.shRNA. VEGFA.shRNA and VEGF164.shRNA reduced pVEGFR2 in retinal ECs at P18, but VEGFA.shRNA increased it at P25. VEGFA.shRNA increased TUNEL+ cells at P18 and decreased ONL thickness at P18 and P25. VEGFA.shRNA and VEGF164.shRNA did not affect pup weight gain and serum VEGF. CONCLUSIONS: Short hairpin RNA to Müller cell VEGF164 maintained long-term inhibition of IVNV and limited cell death compared with shRNA to VEGFA.

Quantitative analyses of retinal vascular area and density after different methods to reduce VEGF in a rat model of retinopathy of prematurity.

PURPOSE: Targeted inhibition of Müller cell (MC)-produced VEGF or broad inhibition of VEGF with an intravitreal anti-VEGF antibody reduces intravitreal neovascularization in a rat model of retinopathy of prematurity (ROP). In this study, we compared the effects of these two approaches on retinal vascular development and capillary density in the inner and deep plexi in the rat ROP model. METHODS: In the rat model of ROP, pups received 1 µL of (1) subretinal lentivector-driven short hairpin RNA (shRNA) to knockdown MC-VEGFA (VEGFA.shRNA) or control luciferase shRNA, or (2) intravitreal anti-VEGF antibody (anti-VEGF) or control isotype goat immunoglobulin G (IgG). Analyses of lectin-stained flat mounts at postnatal day 18 (p18) included: vascular/total retinal areas (retinal vascular coverage) and pixels of fluorescence/total retinal area (capillary density) of the inner and deep plexi determined with the Syncroscan microscope, and angles between cleavage planes of mitotic vascular figures labeled with anti-phosphohistone H3 and vessel length. RESULTS: Retinal vascular coverage and density increased in both plexi between p8 and p18 in room air (RA) pups. Compared with RA, p18 ROP pups had reduced vascular coverage and density of both plexi. Compared with respective controls, VEGFA.shRNA treatment significantly increased vascular density in the deep plexus, whereas anti-VEGF reduced vascular density in the inner and deep plexi. Vascular endothelial growth factor-A.shRNA caused more cleavage angles predicting vessel elongation and fewer mitotic figures, whereas anti-VEGF treatment led to patterns of pathologic angiogenesis. CONCLUSIONS: Targeted treatment with lentivector-driven VEGFA.shRNA permitted physiologic vascularization of the vascular plexi and restored normal orientation of dividing vascular cells, suggesting that regulation of VEGF signaling by targeted treatment may be beneficial.

Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization.

Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2'-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b(-/-) mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.

Short hairpin RNA-mediated knockdown of VEGFA in Müller cells reduces intravitreal neovascularization in a rat model of retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Müller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30eembedded shRNA against VEGFA that targeted Müller cells. Reduction of VEGFA by lentivector VEGFA-shRNAetargeting Müller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Müller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Müller cells safely reduces IVNV in a relevant ROP model.