Role and therapeutic potential of vascular stem/progenitor cells in pathological neovascularisation during chronic portal hypertension.

OBJECTIVE: Pathological neovascularisation is intimately involved in portal hypertension (PH). Here, we determined the contribution of vascular stem/progenitor cells (VSPCs) to neovessel growth in PH and whether the RNA-binding protein cytoplasmic polyadenylation element binding protein-4 (CPEB4) was behind the mechanism controlling VSPC function. DESIGN: To identify and monitor VSPCs in PH rats (portal vein-ligated), we used a combinatorial approach, including sphere-forming assay, assessment of self-renewal, 5-bromo-2'-desoxyuridine label retention technique, in vitro and in vivo stem/progenitor cell (SPC) differentiation and vasculogenic capability, cell sorting, as well as immunohistochemistry, immunofluorescence and confocal microscopy expression analysis. We also determined the role of CPEB4 on VSPC proliferation using genetically engineered mouse models. RESULTS: We demonstrated the existence in the mesenteric vascular bed of VSPCs displaying capability to form cellular spheres in suspension culture, self-renewal ability, expression of molecules commonly found in SPCs, slow-cycling features, in addition to other cardinal properties exhibited by SPCs, like capacity to differentiate into endothelial cells and pericytes with remarkable vasculogenic activity. Such VSPCs showed, after PH induction, an early switch in proliferation, and differentiated in vivo into endothelial cells and pericytes, contributing, structurally and functionally, to abnormal neovessel formation. Quantification of VSPC-dependent neovessel formation in PH further illustrated the key role played by VSPCs. We also demonstrated that CPEB4 regulates the proliferation of the activated VSPC progeny upon PH induction. CONCLUSIONS: These findings demonstrate that VSPC-derived neovessel growth (ie, vasculogenesis) and angiogenesis cooperatively stimulate mesenteric neovascularisation in PH and identify VSPC and CPEB4 as potential therapeutic targets.

Antiangiogenic and antifibrogenic activity of pigment epithelium-derived factor (PEDF) in bile duct-ligated portal hypertensive rats.

OBJECTIVE: Antiangiogenic strategies have been proposed as a promising new approach for the therapy of portal hypertension and chronic liver disease. Pigment epithelium-derived factor (PEDF) is a powerful endogenous angiogenesis inhibitor whose role in portal hypertension remains unknown. Therefore, we aimed at determining the involvement of PEDF in cirrhotic portal hypertension and the therapeutic efficacy of its supplementation. DESIGN: PEDF expression profiling and its relationship with vascular endothelial growth factor (VEGF), neovascularisation and fibrogenesis was determined in bile duct-ligated (BDL) rats and human cirrhotic livers. The ability of exogenous PEDF overexpression by adenovirus-mediated gene transfer (AdPEDF) to inhibit angiogenesis, fibrogenesis and portal pressure was also evaluated in BDL rats, following prevention and intervention trials. RESULTS: PEDF was upregulated in cirrhotic human and BDL rat livers. PEDF and VEGF protein expression and localisation in mesentery and liver increased in parallel with portal hypertension progression, being closely linked in time and space with mesenteric neovascularisation and liver fibrogenesis in BDL rats. Furthermore, AdPEDF increased PEDF bioavailability in BDL rats, shifting the net balance in the local abundance of positive (VEGF) and negative (PEDF) angiogenesis drivers in favour of attenuation of portal hypertension-associated pathological neovascularisation. The antiangiogenic effects of AdPEDF targeted only pathological angiogenesis, without affecting normal vasculature, and were observed during early stages of disease. AdPEDF also significantly decreased liver fibrogenesis (through metalloproteinase upregulation), portosystemic collateralisation and portal pressure in BDL rats. CONCLUSIONS: This study provides compelling experimental evidence indicating that PEDF could be a novel therapeutic agent worthy of assessment in portal hypertension and cirrhosis.

Disruption of negative feedback loop between vasohibin-1 and vascular endothelial growth factor decreases portal pressure, angiogenesis, and fibrosis in cirrhotic rats.

UNLABELLED: Pathological angiogenesis represents a critical hallmark for chronic liver diseases. Understanding the mechanisms regulating angiogenesis is essential to develop new therapeutic strategies that specifically target pathological angiogenesis without affecting physiological angiogenesis. Here we investigated the contribution and therapeutic impact of the endogenous angioinhibitor vasohibin-1 in portal hypertension and cirrhosis. The spatiotemporal expression profiling of vasohibin-1 and its relationship with vascular endothelial growth factor (VEGF), angiogenesis, and fibrogenesis was determined through the analysis of human cirrhotic liver specimens, widely accepted in vivo animal models of portal hypertension and cirrhosis, and in vitro angiogenesis assays. Effects of vasohibin-1 overexpression by adenoviral-mediated gene transfer on angiogenesis, fibrogenesis, and portal hypertension-associated hemodynamic alterations were also studied in rats. We found that vasohibin-1 and VEGF are up-regulated, in mesentery and liver, in cirrhotic and precirrhotic portal hypertensive rats and cirrhosis patients. Our results are consistent with vasohibin-1/VEGF cascades being spatially and temporally coordinated through a negative-feedback loop driving pathological angiogenesis. Paradoxically, further overexpression of vasohibin-1 by adenoviral gene transfer exerts multifold beneficial effects in portal hypertension and cirrhosis: reduction of pathologic angiogenesis, attenuation of liver fibrogenesis partly mediated through inhibition of hepatic stellate cell activation, and significant decreases in portocollateralization, splanchnic blood flow, portohepatic resistance, and portal pressure. The explanation for this apparent contradiction is that, unlike endogenous vasohibin-1, the ectopic overexpression is not regulated by VEGF and therefore disrupts the negative-feedback loop, thus generating constant, but lower levels of VEGF synthesis sufficient to maintain vascular homeostasis but not pathological angiogenesis. CONCLUSION: Our study provides evidence that vasohibin-1 regulates portal hypertension-associated pathological angiogenesis and highlights that increasing vasohibin-1 might be a promising novel therapeutic strategy for portal hypertension and cirrhosis.

RSK4 inhibition results in bypass of stress-induced and oncogene-induced senescence.

p90 Ribosomal S6 kinase (RSK) 4 is a serine-threonine kinase that belongs to the p90RSK family. RSK4 has been proposed as a tumor suppressor gene, related with anti-invasive activity, inhibition of the RAS-mitogen-activated protein kinase (MAPK) pathway and induction of senescence. Despite the related findings, little is known about RSK4 effectors. In human tumors, RSK4 is downregulated even in some benign lesions, such as colon adenomas and breast papillomas, indicating that RSK4 inhibition could be an early event in cellular transformation. For cells to achieve immortality and transformation, it is believed that they must override senescence. In the present study, we found that when RSK4 is inhibited in vitro using short hairpin RNA technology, cells can bypass stress-induced senescence and oncogene-induced senescence: normal human fibroblasts grew following oxidative stress, induction of DNA damage and KRAS(V12) or BRAF(E600) overexpression. To investigate the RSK4 effectors, we used short hairpin RNA or inhibitor molecules against major senescence mediators. We found that RSK4-induced senescence is mediated through p21, but is independent of p16, p38MAPKs and induction of reactive oxygen species, delimiting RSK4 signaling. These data support the importance of RSK4 for regulating senescence and indicate that downregulation of this kinase could be an important element in facilitating cell transformation.

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes.

dDsk2 is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. Here we report that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1, a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications.

Association between LRRK2 and 4E-BP1 protein levels in normal and malignant cells.

Translational control is a crucial component of cancer development and progression. Eukaryotic initiation factor (eIF) 4E mediates eIF4F association with the mRNA 5' cap structure to stimulate cap-dependent translation initiation. The eIF4E-binding protein, 4E-BP1, regulates cap-dependent translation through its phosphorylation at multiple sites. It has been described that some human carcinomas present a high level of p-4E-BP1, not always associated with high levels of p-mTOR. These previous observations suggest that other kinases could be involved in 4E-BP1 phosporylation. Investigation in new kinases that could be implicated in 4E-BP1 phosphorylation and mechanisms that affect 4E-BP1 stability is important to understand the role of eIF4E in cell transformation. In this study, we examined 48 kinases that could be involved in 4E-BP1 phosphorylation and stability. The screening study was based on analysis of 4E-BP1 status after inhibition of these kinases in a breast carcinoma cell line. Several kinases affecting 4E-BP1 stability (LRRK2, RAF-1, p38γ, GSK3ß, AMPKα, PRKACA and PRKACB) and 4E-BP1 phosphorylation (CDK1, PDK1, SRC, PRKCB1, PAK2, p38ß, PRKCA and CaMKKB) were identified. These findings provide evidence that 4E-BP1 can be regulated and stabilized by multiple kinases implicated in several cell signaling pathways. We focus on the finding that LRRK2 down-regulation was associated with a clearly decreased 4E-BP1 protein (and not with mRNA down-regulation). Importantly, knockdown of LRRK2 associated with high proliferative rate in normal cells and treatment with rapamycin and/or proteosome inhibition suppressed 4E-BP1 protein degradation. These results offer new insights into the regulation of total and phosphorylated 4E-BP1.

The effect of p-4E-BP1 and p-eIF4E on cell proliferation in a breast cancer model.

Cell signaling pathways and protein translation are crucial for understanding malignant transformation. 4E-BP1 and the eIF4F complex regulate cap-dependent translation. We investigated how 4E-BP1 and eIF4E phosphorylation status affects in vitro and in vivo cell proliferation in a breast cancer model. Cells from 2 breast carcinoma lines (MDA-MB 231 and MDA-MB 468) and human fibroblasts (IMR90 cells) were infected in vitro with a retrovirus carrying a wild-type 4E-BP1 or a mutant 4E-BP1 unable to hyperphosphorylate. Overexpression of the mutant 4E-BP1 induced a significant decrease in cell proliferation in IMR90 and MDA-MB 468 cells, but not in MDA-MB 231 cells. A correlation was observed between baseline-phosphorylated eIF4E (p-eIF4E) levels and sensitivity to 4E-BP1 transduction. By co-immunoprecipitation, p-eIF4E seemed to present lower affinity for 4E-BP1 than total eIF4E in MDA-MB 468 cells. After treatment with CGP57380, the MAP kinase-interacting kinase (MNK) inhibitor, downregulation of p-eIF4E levels was associated with an increase of E-cadherin and ß-catenin protein expression. These results provide evidence that 4E-BP1 transduction leads to a decrease in cell proliferation, and that high p-eIF4E levels may counteract the suppressor effect of 4E-BP1. We propose that high p-4E-BP1 and p-eIF4E levels are central factors in cell signaling and reflect the oncogenic potential of cell signaling pathways in breast cancer.

Regulation of replicative and stress-induced senescence by RSK4, which is down-regulated in human tumors.

PURPOSE: The control of senescence and its biochemical pathways is a crucial factor for understanding cell transformation. In a large RNA interference screen, the RSK4 gene was found to be related to p53-dependent arrest. The purpose of the present study was to investigate the potential role of RSK4 as a tumor suppressor gene. EXPERIMENTAL DESIGN: RSK4 expression was determined by quantitative real-time PCR and immunoblot in 30 colon and 20 renal carcinomas, and in 7 colon adenomas. Two HCT116 colon carcinoma cell lines (p53 wt and p53 null), IMR90 human fibroblasts, and E1A-expressing IMR90 cells were infected with RSK4 cDNA and/or shRNA. RSK4 expression levels were analyzed in HCT116 p53 wt or p53 null and IMR90 after senescence induction by quantitative real-time PCR and Western blot. RESULTS: The RSK4 gene was down-regulated in 27 of 30 colon carcinomas (P < 0.001), 16 of 20 renal cell carcinomas (P < 0.01), and 6 of 7 colon adenomas (P < 0.01). In vitro overexpression of RSK4 induced cell arrest and senescence features in normal fibroblasts and malignant colon carcinoma cell lines. Interestingly, in these cell lines RSK4 mRNA levels were increased both in replicative and stress-induced senescence. Moreover, IMR90 partially immortalized by RSK4 shRNA and HCT116 with this short hairpin RNA were more resistant to cisplatin treatment. Finally, cells expressing E1A or Rb short interfering RNA were resistant to RSK4-mediated senescence. CONCLUSION: These results support the concept that RSK4 may be an important tumor suppressor gene by modulating senescence induction and contributing to cell proliferation control in colon carcinogenesis and renal cell carcinomas.