Assessment of response to beta-blockers by expression of ßArr2 and RhoA/ROCK2 in antrum mucosa in cirrhotic patients.

BACKGROUND & AIMS: Non-selective beta-blockers (NSBB) are first choice for prevention of variceal bleeding. But possible deleterious effects in refractory ascites and frequent non-response are clinical drawbacks. Since levels of vasoactive proteins in antrum mucosa reflect vascular dysfunction in cirrhosis, these expression levels might also reflect hemodynamic response to NSBB. METHODS: Biopsies from the gastric and duodenal mucosa of 25 patients with cirrhosis were collected and the hepatic venous pressure gradient (HVPG) was measured before and after an acute propranolol challenge. Transcription and protein expression of Ras homolog family member A (RhoA), Rho-kinase (ROCK)2, beta-arrestin2 (ßArr2), endothelial nitric oxide synthase (eNOS) and the phosphorylation of downstream effectors VASP and moesin were analyzed using PCR and Western blot. Further 21 patients on NSBB were evaluated on their follow up for events of variceal bleeding defined as non-response. RESULTS: Ten patients showed HVPG <10mmHg, further seven patients showed significant hemodynamic response to NSBB, whereas eight patients were non-responders. The mucosal transcription of vasoactive proteins was higher in antrum mucosa compared to corpus and duodenum. The transcriptional levels of vasoactive proteins were higher in patients with HVPG >10mmHg and HVPG >16mmHg. Interestingly, mRNA levels of RhoA and ROCK2 were lower in patients with large varices at endoscopy. Moreover, RhoA and ROCK2 transcription correlated with the decrease of HVPG after acute NSBB challenge. Finally, acute and long-term non-responders showed lower expression of ßArr2 in antrum mucosa. CONCLUSION: This study shows for the first time that the expression of ßArr2 in antrum mucosa biopsies might reflect the hemodynamic response to NSBB and their long-term protective effect. This finding might offer an easy approach at upper endoscopy to facilitate the decision to treat with NSBB if varices are present.

ROCK1 in AgRP neurons regulates energy expenditure and locomotor activity in male mice.

Normal leptin signaling is essential for the maintenance of body weight homeostasis. Proopiomelanocortin- and agouti-related peptide (AgRP)-producing neurons play critical roles in regulating energy metabolism. Our recent work demonstrates that deletion of Rho-kinase 1 (ROCK1) in the AgRP neurons of mice increased body weight and adiposity. Here, we report that selective loss of ROCK1 in AgRP neurons caused a significant decrease in energy expenditure and locomotor activity of mice. These effects were independent of any change in food intake. Furthermore, AgRP neuron-specific ROCK1-deficient mice displayed central leptin resistance, as evidenced by impaired Signal Transducer and Activator of Transcription 3 activation in response to leptin administration. Leptin's ability to hyperpolarize and decrease firing rate of AgRP neurons was also abolished in the absence of ROCK1. Moreover, diet-induced and genetic forms of obesity resulted in reduced ROCK1 activity in murine arcuate nucleus. Of note, high-fat diet also impaired leptin-stimulated ROCK1 activity in arcuate nucleus, suggesting that a defect in hypothalamic ROCK1 activity may contribute to the pathogenesis of central leptin resistance in obesity. Together, these data demonstrate that ROCK1 activation in hypothalamic AgRP neurons is required for the homeostatic regulation of energy expenditure and adiposity. These results further support previous work identifying ROCK1 as a key regulator of energy balance and suggest that targeting ROCK1 in the hypothalamus may lead to development of antiobesity therapeutics.

A novel lentivirus for quantitative assessment of gene knockdown in stem cell differentiation.

Loss of gene function is a valuable tool for screening genes in cellular processes including stem cell differentiation differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis and real-time, dynamic information is lacking. To overcome these limitations, we engineered a shRNA encoding LentiViral Dual Promoter vector (shLVDP) that enabled real-time monitoring of mesenchymal stem (MSC) differentiation and simultaneous gene knockdown. In this vector, the activity of the alpha-smooth muscle actin (αSMA) promoter was measured by the expression of a destabilized green fluorescent protein, and was used as an indicator of myogenic differentiation; constitutive expression of discosoma red fluorescent protein was used to measure transduction efficiency and to normalize αSMA promoter activity; and shRNA was encoded by a doxycycline (Dox)-regulatable H1 promoter. Importantly, the normalized promoter activity was independent of lentivirus titer allowing quantitative assessment of gene knockdown. Using this vector, we evaluated 11 genes in the TGF-ß1 or Rho signaling pathway on SMC maturation and on MSC differentiation along the myogenic lineage. As expected, knockdown of genes such as Smad2/3 or RhoA inhibited myogenic differentiation, while knocking down the myogenic differentiation inhibitor, Klf4, increased αSMA promoter activity significantly. Notably, some genes for example, Smad7 or KLF4 showed differential regulation of myogenic differentiation in MSC from different anatomic locations such as bone marrow and hair follicles. Finally, Dox-regulatable shRNA expression enabled temporal control of gene knockdown and provided dynamic information on the effect of different genes on myogenic phenotype. Our data suggests that shLVDP may be ideal for development of lentiviral microarrays to decipher gene regulatory networks of complex biological processes such as stem cell differentiation or reprogramming.