RESUMEN

Liver fibrosis is the common outcome of many chronic liver diseases, resulting from altered cell-cell and cell-matrix interactions that promote hepatic stellate cell (HSC) activation and excessive matrix production. This study aimed to investigate functions of cellular communication network factor 2 (CCN2)/Connective tissue growth factor (CTGF), an extracellular signaling modulator of the CYR61/CTGF/Nov (CCN) family, in liver fibrosis. Tamoxifen-inducible conditional knockouts in mice and hepatocyte-specific deletion of this gene in rats were generated using the Cre-lox system. These animals were subjected to peri-central hepatocyte damage caused by carbon tetrachloride. Potential crosstalk of this molecule with a new profibrotic pathway mediated by the Slit2 ligand and Roundabout (Robo) receptors was also examined. We found that Ccn2/Ctgf was highly upregulated in periportal hepatocytes during carbon tetrachloride-induced hepatocyte damage, liver fibrosis and cirrhosis in mice and rats. Overexpression of this molecule was observed in human hepatocellular carcinoma (HCC) that were surrounded with fibrotic cords. Deletion of the Ccn2/Ctgf gene significantly reduced expression of fibrosis-related genes including Slit2, a smooth muscle actin (SMA) and Collagen type I during carbon tetrachloride-induced liver fibrosis in mice and rats. In addition, Ccn2/Ctgf and its truncated mutant carrying the first three domains were able to interact with the 7th -9th epidermal growth factor (EGF) repeats and the C-terminal cysteine knot (CT) motif of Slit2 protein in cultured HSC and fibrotic murine livers. Ectopic expression of Ccn2/Ctgf protein upregulated Slit2, promoted HSC activation, and potentiated fibrotic responses following chronic intoxication by carbon tetrachloride. Moreover, Ccn2/Ctgf and Slit2 synergistically enhanced activation of phosphatidylinositol 3-kinase (PI3K) and AKT in primary HSC, whereas soluble Robo1-Fc chimera protein could inhibit these activities. These observations demonstrate conserved cross-species functions of Ccn2/Ctgf protein in rodent livers. This protein can be induced in hepatocytes and contribute to liver fibrosis. Its novel connection with the Slit2/Robo signaling may have therapeutic implications against fibrosis in chronic liver disease.

RESUMEN

Yes-associated protein (YAP), a central effector in the Hippo pathway, is involved in the regulation of organ size, stem cell self-renewal, and tissue regeneration. In this study, we observed YAP activation in patients with alcoholic steatosis, hepatitis, and cirrhosis. Accumulation of this protein in the nucleus was also observed in murine livers that were damaged after chronic-plus-single binge or moderate ethanol ingestion combined with carbon tetrachloride intoxication (ethanol/CCl4 ). To understand the role of this transcriptional coactivator in alcohol-related liver injury, we knocked out the Yap1 gene in hepatocytes of floxed homozygotes through adeno-associated virus (AAV8)-mediated deletion utilizing Cre recombinase. Yap1 hepatocyte-specific knockouts (KO) exhibited hemorrhage, massive hepatic necrosis, enhanced oxidative stress, elevated hypoxia, and extensive infiltration of CD11b+ inflammatory cells into hepatic microenvironments rich for connective tissue growth factor (Ctgf) during ethanol/CCl4 -induced liver damage. Analysis of whole-genome transcriptomics indicated upregulation of genes involved in hypoxia and extracellular matrix (ECM) remodeling, whereas genes related to hepatocyte proliferation, progenitor cell activation, and ethanol detoxification were downregulated in the damaged livers of Yap1 KO. Acetaldehyde dehydrogenase (Aldh)1a1, a gene that encodes a detoxification enzyme for aldehyde substrates, was identified as a potential YAP target because this gene could be transcriptionally activated by a hyperactive YAP mutant. The ectopic expression of the human ALDH1A1 gene caused increase in hepatocyte proliferation and decrease in hepatic necrosis, oxidative stress, ECM remodeling, and inflammation during ethanol/CCl4 -induced liver damage. Taken together, these observations indicated that YAP was crucial for liver repair during alcohol-associated injury. Its regulation of ALDH1A1 represents a new link in liver regeneration and detoxification.

Asunto(s)

Familia de Aldehído Deshidrogenasa 1/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Etanol/toxicidad , Regeneración Hepática , Retinal-Deshidrogenasa/metabolismo , Proteínas Señalizadoras YAP/fisiología , Familia de Aldehído Deshidrogenasa 1/genética , Animales , Proliferación Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Retinal-Deshidrogenasa/genética , Transducción de Señal

RESUMEN

The liver possesses an extraordinary ability to regenerate after injury. Hepatocyte-driven liver regeneration is the default pathway in response to mild-to-moderate acute liver damage. When replication of mature hepatocytes is blocked, facultative hepatic progenitor cells (HPCs), also referred to as oval cells (OCs) in rodents, are activated. HPC/OCs have the ability to proliferate clonogenically and differentiate into several lineages including hepatocytes and bile ductal epithelia. This is a conserved liver injury response that has been studied in many species ranging from mammals (rat, mouse, and human) to fish. In addition, improper HPC/OC activation is closely associated with fibrotic responses, characterized by myofibroblast activation and extracellular matrix production, in many chronic liver diseases. Matrix remodeling and metalloprotease activities play an important role in the regulation of HPC/OC proliferation and fibrosis progression. Thus, understanding molecular mechanisms underlying HPC/OC activation has therapeutic implications for rational design of anti-fibrotic therapies.

RESUMEN

During progression to type 1 diabetes, insulin-producing ß-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered ß-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.

Asunto(s)

Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas/metabolismo , Animales , Línea Celular , Femenino , Glucagón/análisis , Glucagón/metabolismo , Antígenos HLA-D/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Hiperglucemia/metabolismo , Islotes Pancreáticos/química , Masculino , Ratones , Ratones Endogámicos NOD , Proteínas/análisis , Proteínas/antagonistas & inhibidores , Transactivadores/metabolismo

RESUMEN

miRNAs are involved in liver regeneration, and their expression is dysregulated in hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF), a direct target of miR-133b, is crucial in the ductular reaction (DR)/oval cell (OC) response for generating new hepatocyte lineages during liver injury in the context of hepatotoxin-inhibited hepatocyte proliferation. Herein, we investigate whether miR-133b regulation of CTGF influences HCC cell proliferation and migration, and DR/OC response. We analyzed miR-133b expression and found it to be down-regulated in HCC patient samples and induced in the rat DR/OC activation model of 2-acetylaminofluorene with partial hepatectomy. Furthermore, overexpression of miR-133b via adenoviral system in vitro led to decreased CTGF expression and reduced proliferation and Transwell migration of both HepG2 HCC cells and WBF-344 rat OCs. In vivo, overexpression of miR-133b in DR/OC activation models of 2-acetylaminofluorene with partial hepatectomy in rats, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine in mice, led to down-regulation of CTGF expression and OC proliferation. Collectively, these results show that miR-133b regulation of CTGF is a novel mechanism critical for the proliferation and migration of HCC cells and OC response.

Asunto(s)

Factor de Crecimiento del Tejido Conjuntivo/metabolismo , MicroARNs/fisiología , 2-Acetilaminofluoreno/farmacología , Adenoviridae/genética , Anciano , Animales , Carcinógenos/farmacología , Carcinoma Hepatocelular/fisiopatología , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Femenino , Vectores Genéticos , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/fisiopatología , Masculino , Ratones , MicroARNs/metabolismo , Persona de Mediana Edad , Ratas , Transfección

RESUMEN

AIM: To study the binding of connective tissue growth factor (CTGF) to cystine knot-containing ligands and how this impacts platelet-derived growth factor (PDGF)-B signaling. METHODS: The binding strengths of CTGF to cystine knot-containing growth factors including vascular endothelial growth factor (VEGF)-A, PDGF-B, bone morphogenetic protein (BMP)-4, and transforming growth factor (TGF)-ß1 were compared using the LexA-based yeast two-hybrid system. EYG48 reporter strain that carried a wild-type LEU2 gene under the control of LexA operators and a lacZ reporter plasmid (p80p-lacZ) containing eight high affinity LexA binding sites were used in the yeast two-hybrid analysis. Interactions between CTGF and the tested growth factors were evaluated based on growth of transformed yeast cells on selective media and colorimetric detection in a liquid ß-galactosidase activity assay. Dissociation constants of CTGF to VEGF-A isoform 165 or PDGF-BB homo-dimer were measured in surface plasma resonance (SPR) analysis. CTGF regulation in PDGF-B presentation to the PDGF receptor ß (PDGFRß) was also quantitatively assessed by the SPR analysis. Combinational effects of CTGF protein and PDGF-BB on activation of PDGFRß and downstream signaling molecules ERK1/2 and AKT were assessed in rabbit corneal fibroblast cells by Western analysis. RESULTS: In the LexA-based yeast two-hybrid system, cystine knot motifs of tested growth factors were fused to the activation domain of the transcriptional factor GAL4 while CTGF was fused to the DNA binding domain of the bacterial repressor protein LexA. Yeast co-transformants containing corresponding fusion proteins for CTGF and all four tested cystine knot motifs survived on selective medium containing galactose and raffinose but lacking histidine, tryptophan, and uracil. In liquid ß-galactosidase assays, CTGF expressing cells that were co-transformed with the cystine knot of VEGF-A had the highest activity, at 29.88 ± 0.91 fold above controls (P < 0.01). Cells containing the cystine knot of BMP-4 expressed the second most activity, with a 24.77 ± 0.47 fold increase (P < 0.01). Cells that contained the cystine knot of TGF-ß1 had a 3.80 ± 0.66 fold increase (P < 0.05) and the ones with the cystine knot of PDGF-B had a 2.64 ± 0.33 fold increase of ß-galactosidase activity (P < 0.01). Further SPR analysis showed that the association rate between VEGF-A 165 and CTGF was faster than PDGF-BB and CTGF. The calculated dissociation constant (KD) of CTGF to VEGF165 and PDGF-BB was 1.8 and 43 nmol/L respectively. PDGF-BB ligand and PDGFRß receptor formed a stable complex with a low dissociation constant 1.4 nmol/L. Increasing the concentration of CTGF up to 263.2 nmol/L significantly the ligand/receptor binding. In addition, CTGF potentiated phosphorylation of PDGFRß and AKT in rabbit corneal fibroblast cells stimulated by PDGF-BB in tissue culture condition. In contrast, CTGF did not affect PDGF-B induced phosphorylation of ERK1/2. CONCLUSION: CTGF has a differential binding affinity to VEGF-A, PDGF-B, BMP-4, and TGF-ß. Its weak association with PDGF-B may represent a novel mechanism to enhance PDGF-B signaling.

Asunto(s)

Diferenciación Celular , Células Estrelladas Hepáticas , Regeneración Hepática , Hígado/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales

RESUMEN

Hepatic progenitor/oval cell (OC) activation occurs when hepatocyte proliferation is inhibited and is tightly associated with the fibrogenic response during severe liver damage. Connective tissue growth factor (CTGF) is important for OC activation and contributes to the pathogenesis of liver fibrosis. By using the Yeast Two-Hybrid approach, we identified a disintegrin and metalloproteinase with thrombospondin repeat 7 (ADAMTS7) as a CTGF binding protein. In vitro characterization demonstrated CTGF binding and processing by ADAMTS7. Moreover, Adamts7 mRNA was induced during OC activation, after the implantation of 2-acetylaminofluorene with partial hepatectomy in rats or on feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet in mice. X-Gal staining showed Adamts7 expression in hepatocyte nuclear factor 4α(+) hepatocytes and desmin(+) myofibroblasts surrounding reactive ducts in DDC-treated Adamts7(-/-) mice carrying a knocked-in LacZ gene. Adamts7 deficiency was associated with higher transcriptional levels of Ctgf and OC markers and enhanced OC proliferation compared to Adamts7(+/+) controls during DDC-induced liver injury. We also observed increased α-smooth muscle actin and procollagen type I mRNAs, large fibrotic areas in α-smooth muscle actin and Sirius red staining, and increased production of hepatic collagen by hydroxyproline measurement. These results suggest that ADAMTS7 is a new protease for CTGF protein and a novel regulator in the OC compartment, where its absence causes CTGF accumulation, leading to increased OC activation and biliary fibrosis.

Asunto(s)

Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Desintegrinas/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Nicho de Células Madre/fisiología , Trombospondinas/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS7 , Animales , Factor de Crecimiento del Tejido Conjuntivo/genética , Desintegrinas/genética , Hígado/patología , Cirrosis Hepática/patología , Regeneración Hepática/fisiología , Ratones , Ratones Noqueados , Trombospondinas/genética

RESUMEN

Obesity poses an increased risk of developing metabolic syndrome and closely associated nonalcoholic fatty liver disease, including liver cancer. Satiety hormone leptin-deficient (ob/ob) mice, considered paradigmatic of nutritional obesity, develop hepatic steatosis but are less prone to developing liver tumors. Sustained activation of peroxisome proliferator-activated receptor α (PPARα) in ob/ob mouse liver increases fatty acid oxidation (FAO), which contributes to attenuation of obesity but enhances liver cancer risk. To further evaluate the role of PPARα-regulated hepatic FAO and energy burning in the progression of fatty liver disease, we generated PPARα-deficient ob/ob (PPARα(Δ)ob/ob) mice. These mice become strikingly more obese compared to ob/ob littermates, with increased white and brown adipose tissue content and severe hepatic steatosis. Hepatic steatosis becomes more severe in fasted PPARα(Δ)ob/ob mice as they fail to up-regulate FAO systems. PPARα(Δ)ob/ob mice also do not respond to peroxisome proliferative and mitogenic effects of PPARα agonist Wy-14,643. Although PPARα(Δ)ob/ob mice are severely obese, there was no significant increase in liver tumor incidence, even when maintained on a diet containing Wy-14,643. We conclude that sustained PPARα activation-related increase in FAO in fatty livers of obese ob/ob mice increases liver cancer risk, whereas deletion of PPARα in ob/ob mice aggravates obesity and hepatic steatosis. However, it does not lead to liver tumor development because of reduction in FAO and energy burning.

Asunto(s)

Ácidos Grasos/metabolismo , Neoplasias Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , PPAR alfa/deficiencia , Animales , Modelos Animales de Enfermedad , Immunoblotting , Neoplasias Hepáticas/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Enfermedad del Hígado Graso no Alcohólico/etiología , Obesidad/complicaciones , Oxidación-Reducción , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

UNLABELLED: Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell-matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvß6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvß6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter-driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvß6 during liver injury induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen-inducible Cre-loxP system down-regulated integrin αvß6 in DDC-damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvß6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis-related genes, smaller areas of alpha-smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvß6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)-ß1 activation in vitro. CONCLUSIONS: CTGF and integrin αvß6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-ß1. CTGF and integrin αvß6 are potential therapeutic targets to control DRs and fibrosis in related liver disease.

Asunto(s)

Antígenos de Neoplasias/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Integrinas/metabolismo , Cirrosis Hepática/metabolismo , Células Madre Adultas/metabolismo , Animales , Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos , Adhesión Celular , Colangiocarcinoma/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Piridinas , Conejos , Ratas , Factor de Crecimiento Transformador beta1/metabolismo

RESUMEN

PURPOSE: This study aimed to elucidate the role of connective tissue growth factor (CTGF) in healthy eyes and wounded corneas of mice and rabbits. Conditional knockout mice were used to determine the role of CTGF in corneal healing. METHODS: CTGF expression was determined using transgenic mice carrying CTGF promoter driven-eGFP, quantitative RT-PCR, and immunofluorescent staining. Mice that carried two floxed CTGF alleles and a Cre/ERT2 transgene under the control of human ubiquitin C (ubc) promoter were used to conditionally delete CTGF gene in a tamoxifen-inducible manner. Phototherapeutic keratectomy (PTK) was used to generate an acute corneal wound and corneal re-epithelialization was assessed by fluorescein staining. RESULTS: Connective tissue growth factor expression was found in multiple ocular tissues with relatively high levels in the corneal endothelium, lens subcapsular epithelium, and in the vasculature of the iris and retina. Wounded corneas responded with an immediate upregulation of CTGF in the epithelium at the wound margin and a sustained CTGF induction during re-epithelialization. At the onset of haze formation, CTGF protein becomes more focused in the basal epithelium. Deletion of the CTGF gene caused a 40% reduction (P < 0.01) in the cornea re-epithelialization rate in knockout mice compared with wild-type mice. CONCLUSIONS: Connective tissue growth factor is expressed in the naïve cornea, lens, iris, and retina, and is expressed immediately after epithelial injury. Loss of CTGF impairs efficient re-epithelialization of corneal wounds.

Asunto(s)

Factor de Crecimiento del Tejido Conjuntivo/genética , Córnea/metabolismo , Lesiones Oculares/genética , Regulación de la Expresión Génica , ARN/genética , Cicatrización de Heridas/genética , Animales , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Lesiones de la Cornea , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Lesiones Oculares/metabolismo , Lesiones Oculares/patología , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

PURPOSE: The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes. METHODS: Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3' untranslated region (UTR) of transforming growth factor ß1 (TGFß1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative real-time PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration. RESULTS: At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at -14.33. Pre-miR-133b targeted the 3' UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor ß1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFß1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF. CONCLUSIONS: Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation.

Asunto(s)

Epitelio Corneal/lesiones , Fibroblastos/metabolismo , Láseres de Excímeros/uso terapéutico , MicroARNs/metabolismo , Cicatrización de Heridas/fisiología , Actinas/metabolismo , Análisis de Varianza , Animales , Colágeno Tipo I/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Epitelio Corneal/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Conejos , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología

RESUMEN

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham-operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.

Asunto(s)

Hígado/citología , Medicina Regenerativa/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Hígado/inmunología , Trasplante de Hígado/inmunología , Masculino , Ratas , Ratas Endogámicas F344 , Sus scrofa , Porcinos , Trasplante Heterólogo

RESUMEN

This review illustrates promising regenerative medicine technologies that are being developed for the treatment of gastrointestinal diseases. The main strategies under validation to bioengineer or regenerate liver, pancreas, or parts of the digestive tract are twofold: engineering of progenitor cells and seeding of cells on supporting scaffold material. In the first case, stem cells are initially expanded under standard tissue culture conditions. Thereafter, these cells may either be delivered directly to the tissue or organ of interest, or they may be loaded onto a synthetic or natural three-dimensional scaffold that is capable of enhancing cell viability and function. The new construct harbouring the cells usually undergoes a maturation phase within a bioreactor. Within the bioreactor, cells are conditioned to adopt a phenotype similar to that displayed in the native organ. The specific nature of the scaffold within the bioreactor is critical for the development of this high-function phenotype. Efforts to bioengineer or regenerate gastrointestinal tract, liver and pancreas have yielded promising results and have demonstrated the immense potential of regenerative medicine. However, a myriad of technical hurdles must be overcome before transplantable, engineered organs become a reality.

Asunto(s)

Enfermedades Gastrointestinales/cirugía , Medicina Regenerativa , Trasplante de Células Madre , Ingeniería de Tejidos/métodos , Bioingeniería/tendencias , Enfermedades Gastrointestinales/patología , Humanos , Enfermedades Intestinales/terapia , Fallo Hepático/cirugía , Regeneración Hepática , Trasplante de Hígado/métodos , Trasplante de Órganos , Enfermedades Pancreáticas/cirugía , Medicina Regenerativa/tendencias , Andamios del Tejido

RESUMEN

Gene therapy is suggested to be one of the most specific and efficient modulations for gene deficient diseases and extended to other diseases like cancer and inflammation, even though there are still challenges to be faced, such as specific and selective delivery, minimal to no toxicity, efficient metabolism, simplicity, and measurable efficiency. It is important to identify and validate drug-able disease-specific targets for molecular and cellular therapies, while it is equally important to have disease biomarkers to trace and define the biological effects of molecular and cellular therapies. The importance and significance of allostery in molecular and cellular therapies and "allosteric disease", "allosteric effect", and "allosteric drug" should be more carefully examined and validated. Cell therapy has been attracting an increasing amount of consideration in the development of new treatments for diseases. Molecular and Cellular Therapies (MCT) is a new, open-access journal, devoted to molecular mechanisms, preclinical and clinical research and development of gene-, peptide-, protein-, and cell-based therapies.

RESUMEN

Subtotal cystectomy (STC; surgical removal of ∼75% of the rat urinary bladder) elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU). Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP), muscularis propria (MP) and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA), to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh), Gli-1 and bone morphogenic factor-4 (BMP-4) were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends previous observations and further establishes the rodent bladder as an excellent model for studying novel aspects of mammalian organ regeneration.

Asunto(s)

Proliferación Celular , Regeneración/fisiología , Vejiga Urinaria , Animales , Cistectomía , Femenino , Humanos , Ratas , Vejiga Urinaria/citología , Vejiga Urinaria/crecimiento & desarrollo

RESUMEN

Blood vessels are formed during development and tissue repair through a plethora of modifiers that coordinate efficient vessel assembly in various cellular settings. Here we used the yeast 2-hybrid approach and demonstrated a broad affinity of connective tissue growth factor (CCN2/CTGF) to C-terminal cystine knot motifs present in key angiogenic regulators Slit3, von Willebrand factor, platelet-derived growth factor-B, and VEGF-A. Biochemical characterization and histological analysis showed close association of CCN2/CTGF with these regulators in murine angiogenesis models: normal retinal development, oxygen-induced retinopathy (OIR), and Lewis lung carcinomas. CCN2/CTGF and Slit3 proteins worked in concert to promote in vitro angiogenesis and downstream Cdc42 activation. A fragment corresponding to the first three modules of CCN2/CTGF retained this broad binding ability and gained a dominant-negative function. Intravitreal injection of this mutant caused a significant reduction in vascular obliteration and retinal neovascularization vs. saline injection in the OIR model. Knocking down CCN2/CTGF expression by short-hairpin RNA or ectopic expression of this mutant greatly decreased tumorigenesis and angiogenesis. These results provided mechanistic insight into the angiogenic action of CCN2/CTGF and demonstrated the therapeutic potential of dominant-negative CCN2/CTGF mutants for antiangiogenesis.

Asunto(s)

Factor de Crecimiento del Tejido Conjuntivo/fisiología , Motivos Nodales de Cisteina/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Animales , Carcinoma Pulmonar de Lewis/inducido químicamente , Motivos Nodales de Cisteina/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas de la Membrana/fisiología , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Vasos Retinianos/crecimiento & desarrollo , Técnicas del Sistema de Dos Híbridos

RESUMEN

OBJECTIVE: We have identified a novel protein in bone marrow-derived insulin-producing cells. Here we characterize this protein, hereby named islet homeostasis protein (IHoP), in the pancreatic islet. METHODS: Detection of IHoP mRNA and protein was performed using reverse transcriptase-polymerase chain reaction, immunocytochemistry, and in situ hybridization. Islet homeostasis protein functions were utilizing proliferation, insulin secretion by in vitro assays, and following small interfering RNA protocols for suppression of IHoP. RESULTS: We found that IHoP did not homolog with known pancreatic hormones. Islet homeostasis protein expression was seen in both bone marrow-derived insulin-producing cells and isolated pancreatic islets. Immunohistochemistry on pancreatic islet revealed that IHoP localized to the glucagon-synthesizing α cells. Inhibition of IHoP by small interfering RNA resulted in the loss of glucagon expression, which induced low blood glucose levels (63-85 mg/dL). Subsequently, cellular apoptosis was observed throughout the islet, including the insulin-producing ß cells. Islets of preonset diabetic patients showed normal expression of IHoP and glucagon; however, IHoP was lost upon onset of the disease. CONCLUSIONS: These data suggest that IHoP could be a new functional protein in the islet and may play a role in islet homeostasis.

Asunto(s)

Células Secretoras de Glucagón/metabolismo , Glucagón/biosíntesis , Islotes Pancreáticos/metabolismo , Proteínas/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Expresión Génica , Glucagón/farmacología , Glucosa/farmacología , Humanos , Inmunohistoquímica , Hibridación in Situ , Insulina/biosíntesis , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Proteínas/metabolismo , Interferencia de ARN , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

BACKGROUND: Somatostatin is a pleiotropic peptide, exerting a variety of effects through its receptor subtypes. Recently, somatostatin has been shown to act as a chemoattractant for haematopoietic progenitor cells and hepatic oval cells (HOC) via receptor subtype 2 and subtype 4 (SSTR4) respectively. AIMS: We investigated the in vivo effect of somatostatin/SSTR4 on HOC migration in the injured liver model of rats and the type of signalling molecules associated with the chemotactic function. METHODS: Migration assay, HOC transplantation and phosphatidylinositol-3-kinase (PI3K) signalling were assessed with or without somatostatin and an analogue of somatostatin (TT232) that specifically binds to SSTR4. RESULTS: TT232 was shown to have an antimigratory action on HOC induced by somatostatin in vitro. In HOC transplantation experiments, a lower number of donor-derived cells were detected in TT232-treated animals, as compared with control animals. Activation of PI3K was observed in HOC exposed to somatostatin, and this activation was suppressed by either SSTR4 antibody or TT232-pretreatment. In addition, a PI3K inhibitor abrogated the motility of HOC. CONCLUSION: Together, these data suggest that somatostatin stimulates the migration of HOC within injured liver through SSTR4, and this action appears to be mediated by the PI3K pathway.

Asunto(s)

Movimiento Celular/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Péptidos Cíclicos/farmacología , Somatostatina/farmacología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Trasplante de Células , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Hepatocitos/patología , Hígado/patología , Masculino , Péptidos Cíclicos/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Ratas , Ratas Endogámicas F344 , Receptores de Somatostatina/metabolismo , Transducción de Señal/efectos de los fármacos , Somatostatina/metabolismo

RESUMEN

BACKGROUND AND AIMS: Activation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade. METHODS: The established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver. RESULTS: Notch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21(Waf1)/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α. CONCLUSIONS: Taken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional for repair of the liver by oval cells.