HGF as a multifunctional anti-fibrotic agent with high impact on gene therapy for renal interstitial fibrosis

Fibrotic processes in chronic kidney diseases are the leading cause of renal failure. Hepatocyte growth factor (HGF), effecting organ restructuring by its mitogenic, motogenic, morphogenic and anti-apoptotic activities, is one of the central mediators involved in tubular repair and regeneration after acute renal injury. In addition, HGF acts as an anti-inflammatory and anti-fibrotic factor antagonizing pro-fibrotic actions of transforming growth factor beta (TGF-beta). However, the molecular and cellular mechanisms underlying the anti-fibrotic function of HGF in chronic kidney disease are not well understood. Therefore, in the present study HGF signaling and HGF induced expression profiles were studied in renal interstitial fibroblasts that represent a central cell type in tubulointerstitial fibrosis due to their prominent production of extracellular matrix proteins. Furthermore, gene therapeutical HGF application using different serotypes of the adeno-associated viral vector (AAV), namely AAV2, AAV8 and AAV9, was tested in order to treat tubulointerstitial fibrosis in a COL4A3 knockout mouse model. Analyses of HGF signaling demonstrated that in agreement to signaling in epithelial cells HGF stimulation results in the activation of the Erk1/2 and the Akt pathway. However, the Stat3 signal transducer was not phosphorylated. Smad2/3 phosphorylation in response to Erk1/2 activation in HGF stimulated fibroblasts supports previous data showing the antagonistic interaction of the HGF- and TGF-beta-signaling. A comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridisation could further define the anti-fibrotic signals mediated by HGF. Functional cluster analyses and quantitative PCR assays indicated that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix roteins, various chemokines, and members of the CCN family. Interruption of the HGF signaling via the Akt pathway or support of the HGF signaling via the Erk1/2 pathway by RNA interference, using Akt-siRNA or Smad4-siRNA, proved that not only Erk1/2 activation but also Akt activation is responsible for anti-fibrotic signal transduction by HGF. These data clearly point out that the Akt signaling upon HGF stimulation acts as an auxiliary pathway in the anti-fibrotic function of HGF. In order to apply the anti-fibrotic effect of HGF to chronic kidney diseases, a gene therapeutical system was established, intended to reduce renal interstitial fibrosis by the use of HGF as transgene and the adeno-associated viral vector (AAV) as gene vehicle. COL4A3 knockout mice mimicking the human Alport syndrome served as model system for renal tubulointerstitial fibrosis. Different natural occurring AAV serotypes, namely AAV2, AAV8 and AAV9, were studied with regard to their capability to target renal epithelial cells compared to liver parenchyma. Furthermore, a mammalian promoter construct was generated that restricted transgene expression to the kidney and the liver for a combined endocrine and paracrine expression of HGF. Systemic application of AAV8 and AAV9 carrying HGF as transgene resulted in high serum levels of HGF in COL4A3 knockout mice, however, AAV9 achieved the highest HGF expression in both the liver and the kidney. HGF serum levels were associated with pronounced repression of fibrotic markers such as collagen1A1, PDGF-receptor-beta, and alpha-smooth-muscle actin. In addition, AAV mediated HGF expression resulted a remarkable reduction in the severity of fibrosis. In conclusion, HGF is a promising anti-fibrotic agent for the treatment of chronic kidney diseases. Additionally, this study established a proof-of-concept of AAV-based therapy as a promising vector platform to treat chronic kidney diseases

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. METHODOLOGY: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. PRINCIPAL FINDINGS: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. CONCLUSIONS: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively

Differentiation and Selection of Hepatocyte Precursors in Suspension Spheroid Culture of Transgenic Murine Embryonic Stem Cells

Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein-and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for live monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays

Vital staining of blood vessels and bile ducts with carboxyfluorescein diacetate succinimidyl ester: a novel tool for isolation of cholangiocytes

Background and aim: Current methods for visualization of the blood vasculature, biliary tree and for isolation of vital cholangiocytes are afflicted with a plethora of technical difficulties, especially in mice. In this project, we propose a novel, reliable and straightforward alternative technique for histological demonstration of blood- and biliary systems and derivation of vital cholangiocytes. Methods: Intravital retrograde perfusion of bile ducts was performed in twenty wild type mice. Liver and gallbladder were exposed by median laparotomy. Using a venous catheter, the gallbladder was cannulated, a few millimeters of the liver edge were cropped to allow free outflow of the perfusate, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) solution was retrogradely infused. Thereafter, formaldehyde solution was either injected through the same catheter, or the liver was immediately dissociated into a single-cell suspension for FACS-analysis. Intravital perfusion of the vascular system was performed in ten Lewis rats by direct intra-arterial injection of CFDA-SE into the abdominal aorta. The specificity and sensitivity of CFDA-SE labeling was controlled using Indian ink or cytokeratin 19 immunohistochemistry respectively. Results: Upon histomorphological analysis of cryoand paraffin sections, strong fluorescence was noted in large and small bile ducts throughout the entire liver and in the vascular system after infusion of the CFDA-SE solution. In preliminary FACS-experiments, we succeeded in separating cholangiocytes based on combined CFDA-SE-staining and cell size. Conclusions: Visualization of liver architecture and the isolation of cholangiocytes is feasible using a fast and cost-effective method of retrograde perfusion and vital fluorescent labeling of mouse bile duct epithelium and vascular endothelium with CFDA-SE

Glycogen storage and ICG up-take in spheroid-derived adherent colonies.

<p>(<b>A</b>) Glycogen-storing cells in a 12-day-old spheroid-derived adherent culture. (<b>B</b>) Cell cluster in the culture, reversibly up-taking ICG.</p

Expression of eGFP and hepatic proteins in spheroid-derived adherent cultures.

<p>(<b>A</b>) Cell colonies outgrowing on collagen Type I matrix. Down-regulation of eGFP fluorescence during the course of the colony growth is evident. (<b>B</b>) Expression of hepatic proteins, using fluorescent immunoassay. For better view of Ecad and lst-1 expression patterns across the cells, the corresponding images are displayed at a magnification ×400. Respective staining of primary hepatocytes (panels <i>a–c</i> and <i>e</i>) or of a liver tissue section (panel <i>d</i>) are shown for comparison. Alexa Fluor® 555-conjugated IgG served as a secondary antibody; cell nuclei were visualized by Hoechst 33342 staining. Merged Alexa Fluor® 555/Hoechst 33342 images are shown. Samples treated with an isotype control antibody instead of the primary antibody stained negatively (data not shown).</p

Engrafted spheroid-derived cells within recipient liver tissue.

<p>(<b>A</b>) eGFP-fluorescent cells and cell clusters observed in liver sections one and two weeks after spheroid transplantation. (<b>B</b>) Expression of Ecad in engrafted cells. “Live” eGFP and merged Alexa Fluor® 555/Hoechst 33342 images are shown.</p

Spheroid formation and growth in dynamic and static conditions.

<p>(<b>A</b>) Top panels: eGFP-expressing cell aggregates immediately after their separation from EBs cultured in a rotary or in a static condition. Bottom panels: rotary and static cultures of 2-day-old spheroids. (<b>B</b>) Comparative diagrams of spheroid growth in SFs and in Petri dishes. The mean of the spheroid diameter ± standard error of the mean (SEM) are plotted. The sign (***) indicates extremely statistically significant differences (p<0.001) in spheroid size between the dynamic and static cultures.</p