Recombinant VEGF-C (Cys156Ser) improves mesenteric lymphatic drainage and gut immune surveillance in experimental cirrhosis.

Background & Aims: Lymphatic vessels (LVs) are crucial for maintaining abdominal fluid homoeostasis and immunity. In cirrhosis, mesenteric LVs (mLVs) are dilated and dysfunctional. Given the established role of vascular endothelial growth factor-C (VEGF-C) in improving LVs, we hypothesised that VEGF-C treatment could ameliorate the functions of mLVs in cirrhosis. Methods: In this study, we developed a nanoformulation comprising LV-specific growth factor, recombinant human VEGF-C (Cys156Ser) protein (E-VEGF-C) and delivered it orally in different models of rat cirrhosis to target mLVs. Cirrhotic rats were given nanoformulation without VEGF-C served as vehicles. Drainage of mLVs was analysed using tracer dye. Portal and systemic physiological assessments and computed tomography were performed to measure portal pressures and ascites. Gene expression and permeability of primary mesenteric lymphatic endothelial cells (LyECs) was studied. Immune cells in mesenteric lymph nodes (MLNs) were quantified by flow cytometry. Endogenous and exogenous gut bacterial translocation to MLNs was examined. Results: In cirrhotic rats, mLVs were dilated and leaky with impaired drainage. Treatment with E-VEGF-C induced proliferation of mLVs, reduced their diameter, and improved functional drainage. Ascites and portal pressures were significantly reduced in E-VEGF-C rats compared with vehicle rats. In MLNs of E-VEGF-C animals, CD8+CD134+ T cells were increased, whereas CD25+ regulatory T cells were decreased. Both endogenous and exogenous bacterial translocation were limited to MLNs in E-VEGF-C rats with reduced levels of endotoxins in ascites and blood in comparison with those in vehicle rats. E-VEGF-C treatment upregulated the expression of vascular endothelial-cadherin in LyECs and functionally improved the permeability of these cells. Conclusions: E-VEGF-C treatment ameliorates mesenteric lymph drainage and portal pressure and strengthens cytotoxic T-cell immunity in MLNs in experimental cirrhosis. It may thus serve as a promising therapy to manage ascites and reduce pathogenic gut bacterial translocation in cirrhosis. Impact and Implications: A human recombinant pro-lymphangiogenic growth factor, VEGF-C, was encapsulated in nanolipocarriers (E-VEGF-C) and orally delivered in different models of rat liver cirrhosis to facilitate its gut lymphatic vessel uptake. E-VEGF-C administration significantly increased mesenteric lymphatic vessel proliferation and improved lymph drainage, attenuating abdominal ascites and portal pressures in the animal models. E-VEGF-C treatment limited bacterial translocation to MLNs only with reduced gut bacterial load and ascitic endotoxins. E-VEGF-C therapy thus holds the potential to manage ascites and portal pressure and reduce gut bacterial translocation in patients with cirrhosis.

Long non-coding RNA in Non-alcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic disease of worldwide impact. The disease process begins with steatosis, i.e., fat accumulation in the liver, and proceeds to nonalcoholic steatohepatitis (NASH). Liver biopsy is the gold standard for NASH diagnosis, but the procedure is invasive, expensive, error prone and poses considerable risk. Unfortunately, there are currently no precise FDA-approved therapies for NAFLD, the only options being lifestyle change and symptomatic treatment. Recently, much research has focused on the identification of molecular mechanisms that could be translated into novel diagnostics and therapeutics. With the advent of high throughput genomics and transcriptomics, noncoding RNAs, including long non-coding RNAs (lncRNAs) have been identified as key players of NAFLD pathogenesis and have accordingly attracted much attention as potential diagnostics and therapeutics. In this chapter, we reviewed various lncRNAs and their functions at different stages of NAFLD. We also highlighted how these unique molecules can be developed as stage-specific non-invasive diagnostic biomarkers for NAFLD.

Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH.

BACKGROUND: Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH). METHODS: We performed in vivo targeted silencing of the RUNX1 gene in liver sinusoidal endothelial cells (LSECs) by using vegfr3 antibody tagged immunonano-lipocarriers encapsulated RUNX1 siRNA (RUNX1 siRNA) in murine models of methionine choline deficient (MCD) diet-induced NASH. MCD mice given nanolipocarriers-encapsulated negative siRNA were vehicle, and mice with standard diet were controls. RESULTS: Liver RUNX1 expression was increased in the LSECs of MCD mice in comparison to controls. RUNX1 protein expression was decreased by 40% in CD31-positive LSECs of RUNX1 siRNA mice in comparison to vehicle, resulting in the downregulation of adhesion molecules, ICAM1 expression, and VCAM1 expression in LSECs. There was a marked decrease in infiltrated T cells and myeloid cells along with reduced inflammatory cytokines in the liver of RUNX1 siRNA mice as compared to that observed in the vehicle. CONCLUSIONS: In vivo LSEC-specific silencing of RUNX1 using immunonano-lipocarriers encapsulated siRNA effectively reduces its expression of adhesion molecules, infiltrate on of immune cells in liver, and inflammation in NASH.

Evolutionary conservation of long non-coding RNAs in non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of conditions ranging from hepatic steatosis to steatohepatitis (NASH) to fibrosis in the absence of alcohol consumption. Its pathogenesis involves both genetic and environmental factors with a multitude of underlying molecular mechanisms and mediators at each stage. Recent transcriptomic-based studies have led to the identification and association of long non-coding RNAs (lncRNAs) with disease pathology in NAFLD patients and in vivo rodent models. However, the knowledge of function of most of the lncRNAs in NAFLD pathology remains obscure. In the current review, we give a comprehensive catalogue of well reported lncRNAs in NAFLD and classify them using sequence and synteny-based evolutionary conservation across rodents, nonhuman primate and human species. The conserved lncRNAs across all the three species may be dissected in larger clinical studies of NAFLD and can be explored as biomarkers and therapeutic targets. In addition, we also review and analyse single nucleotide polymorphisms (SNPs) in these lncRNAs. It adds another facet to the regulatory role of NAFLD-associated lncRNAs and underscores the significance of a novel genetic landscape of non-coding genome in determining the genetic susceptibility of NAFLD.

Increased Expression of RUNX1 in Liver Correlates with NASH Activity Score in Patients with Non-Alcoholic Steatohepatitis (NASH).

Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF- significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH.

Endothelial progenitor cells from aged subjects display decreased expression of sirtuin 1, angiogenic functions, and increased senescence.

Studies have demonstrated that aging is associated with a substantial decline in numbers and angiogenic activity of endothelial progenitor cells (EPCs). In view of senescence being an important regulator of age-related cell survival and function, in the current study, we correlated EPCs numbers and functions with their senescence status and mechanisms in young and elderly subjects. Healthy young subjects (n = 30, below 60 y) and old subjects (n = 30, equal to or above 60 y) participated in the study. Subjects had no significant disease or risk factors of disease and aging was the only risk factor in the aged subjects. Enumeration of CD34-vegfr2 dual positive EPCs was performed. The ex vivo culture of EPCs was done to study colony formation, migration, and senescence-associated beta-galactosidase activity. The expression of cell cycle and senescence regulatory proteins including, p53, p21, and sirtuin 1 (SIRT1), a deacetylase protein was studied in cultured EPCs by RT-PCR and immunofluorescence staining. In vivo proliferation, ex vivo colonies, migration, and secretory ability of EPCs was significantly higher in young subjects than that in elderly subjects. EPCs in old subjects showed enhanced senescence and decreased expression of SIRT1 in comparison to that observed in young subjects. An inhibition of SIRT1 in EPCs of young subjects led to significant increase in senescence and reduction of cell differentiation. The study suggests that EPCs have decreased proliferation and functions in aged subjects due to increased senescence which may be attributable to decreased expression of SIRT1.

Correlation Between Intron 4 Polymorphism of the Endothelial Nitric Oxide Synthase Gene and Cardiovascular Risk with the Numbers of Circulating Endothelial Progenitor Cells in Healthy Subjects.

In the present study, we investigated the relationship between an important 27 bp repeat polymorphism in intron 4 of eNOS and numbers of circulating EPCs in presence of cardiovascular disease (CVD) risk factors in a group of healthy human volunteers. The study comprised of 45 healthy subjects (30-50 years). These subjects had various degrees of CVD risk but no history of CVD. The repeat polymorphism of eNOS was detected by polymerase chain reaction and EPC levels were analyzed by flow cytometry. We observed a good association between the intronic 4 mutant a/b genotype and the combined Framingham risk factor score in our subjects (χ2 = 3.2, P = 0.07). EPC numbers in subjects with mutant eNOS a/b genotype were also less than those observed in subjects with normal eNOS b/b genotype (P = 0.06). Interestingly, subjects with eNOS a/b genotype showed a significant inverse correlation between framingham risk score and EPC numbers (R = -0.57, P < 0.05). The study suggests that the presence of CVD risk factors in subjects with eNOS intron 4 polymorphism results in reduced number of circulating EPCs, which may significantly predispose them to CVD and aberrant endothelial repair.