Expression of insulin-like growth factor I by activated hepatic stellate cells reduces fibrogenesis and enhances regeneration after liver injury.

BACKGROUND/AIM: Hepatic stellate cells (HSCs) express alpha-smooth muscle actin (alphaSMA) and acquire a profibrogenic phenotype upon activation by noxious stimuli. Insulin-like growth I (IGF-I) has been shown to stimulate HSCs proliferation in vitro, but it has been reported to reduce liver damage and fibrogenesis when given to cirrhotic rats. METHODS: The authors used transgenic mice (SMP8-IGF-I) expressing IGF-I under control of alphaSMA promoter to study the influence of IGF-I synthesised by activated HSCs on the recovery from liver injury. RESULTS: The transgene was expressed by HSCs from SMP8-IGF-I mice upon activation in culture and in the livers of these animals after CCl4 challenge. Twenty four hours after administration of CCl4 both transgenic and wild type mice showed similar extensive necrosis and increased levels of serum transaminases. However at 72 hours SMP8-IGF-I mice exhibited lower serum transaminases, reduced hepatic expression of alphaSMA, and improved liver morphology compared with wild type littermates. Remarkably, at this time all eight CCl4 treated wild type mice manifested histological signs of liver necrosis that was severe in six of them, while six out of eight transgenic animals had virtually no necrosis. In SMP8-IGF-I mice robust DNA synthesis occurred earlier than in wild type animals and this was associated with enhanced production of HGF and lower TGFbeta1 mRNA expression in the SMP8-IGF-I group. Moreover, Colalpha1(I) mRNA abundance at 72 hours was reduced in SMP8-IGF-I mice compared with wild type controls. CONCLUSIONS: Targeted overexpression of IGF-I by activated HSCs restricts their activation, attenuates fibrogenesis, and accelerates liver regeneration. These effects appear to be mediated in part by upregulation of HGF and downregulation of TGFbeta1. The data indicate that IGF-I can modulate the cytokine response to liver injury facilitating regeneration and reducing fibrosis.

Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease.

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.

IGF-I and procollagen alpha1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease.

This study tested the hypothesis that insulin-like growth factor I (IGF-I) expression is increased at sites of fibrosis in diseased intestine of patients with Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection assay in uninvolved and involved intestine of 13 CD patients (10 ileum, 3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ hybridization histochemistry compared the localization of IGF-I and procollagen alpha1(I) mRNAs. Masson's trichrome staining and immunohistochemistry for IGF-I precursor, alpha-smooth muscle actin (A), vimentin (V), desmin (D), and c-kit were used to examine the mesenchymal cell subtypes that express IGF-I and collagen in uninvolved and involved ileum and colon of CD patients and "normal" ileum and colon from noninflammatory controls. IGF-I mRNA was elevated in involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen alpha1(I) mRNA showed overlapping distribution within fibrotic submucosa and muscularis propria of involved CD ileum and colon. In involved CD intestine, increased IGF-I precursor expression localized to mesenchymal cells in regions of tissue disorganization and fibrosis in muscularis mucosa, submucosa, and muscularis propria. In these regions, there were increased numbers of V(+) cells relative to normal or uninvolved intestine. Increased IGF-I expression was localized to cells with a phenotype typical of fibroblasts (V(+)/A(-)/D(-)), myofibroblasts (V(+)/A(+)/D(+)), and, to a lesser extent, cells with normal enteric smooth muscle phenotype (V(-)/A(+)/D(+)). We conclude that increased IGF-I expression in multiple mesenchymal cell subtypes and increased numbers of cells with fibroblast/myofibroblast phenotype are involved in fibrosis associated with CD.

Fibrogenesis. IV. Fibrosis and inflammatory bowel disease: cellular mediators and animal models.

The cellular mediators of intestinal fibrosis and the relationship between fibrosis and normal repair are not understood. Identification of the types of intestinal mesenchymal cells that produce collagen during normal healing and fibrosis is vital for elucidating the answers to these questions. Acute injury may cause normal mesenchymal cells to convert to a fibrogenic phenotype that is not maintained during normal healing but may lead to fibrosis when inappropriately sustained. Proliferation of normal or fibrogenic mesenchymal cells may lead to muscularis overgrowth associated with fibrosis. The presence of increased numbers of vimentin-positive cells within fibrotic, hypertrophied muscularis in Crohn's disease suggests that changes in mesenchymal cell phenotype and number may indeed be associated with fibrosis. Fibrosis is induced in rats by peptidoglycan polysaccharides or trinitrobenzene sulfonic acid-ethanol administration, but inducing fibrosis in mice has been technically challenging. The development of current mouse models of colitis, such as dextran sodium sulfate or trinitrobenzene sulfonic acid-ethanol administration, into models of fibrosis will allow us to use genetic manipulation to study molecular mediators of fibrosis.

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver.

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.

Autocrine and paracrine actions of intestinal fibroblast-derived insulin-like growth factors.

Paracrine and autocrine actions of the insulin-like growth factors (IGFs) are inferred by local expression within the bowel. CCD-18Co cells, IEC-6 cells, and immunoneutralization were used to analyze whether IGFs have direct autocrine or paracrine effects on proliferation of cultured intestinal fibroblasts and epithelial cells. Growth factor expression was analyzed by ribonuclease protection assay and RT-PCR. Extracellular matrix (ECM) was analyzed for effects on cell proliferation. CCD-18Co cells express IGF-II mRNAs and low levels of IGF-I mRNA. Conditioned medium from CCD-18Co cells (CCD-CM) stimulated proliferation of IEC-6 and CCD-18Co cells. Neutralization of IGF immunoreactivity in CCD-CM reduced but did not abolish this effect. RT-PCR and immunoneutralization demonstrated that other growth factors contribute to mitogenic activity of CCD-CM. Preincubation of CCD-CM with ECM prepared from IEC-6 or CCD-18Co cells reduced its mitogenic activity. ECM from CCD-18Co cells enhanced growth factor-dependent proliferation of IEC-6 cells. IEC-6 cell ECM inhibited IGF-I action on CCD-18Co cells. We conclude that IGF-II is a potent autocrine mitogen for intestinal fibroblasts. IGF-II interacts with other fibroblast-derived growth factors and ECM to stimulate proliferation of intestinal epithelial cells in a paracrine manner.

Insulin-like growth factor-I and epidermal growth factor interact to regulate growth and gene expression in IEC-6 intestinal epithelial cells.

Epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) exert trophic effects on bowel mucosa. Each growth factor uses a distinct tyrosine kinase receptor but the receptors share some common signal transduction pathways. In other systems, regulation of cell growth involves interactions among multiple growth factors. We used IEC-6 cells, an epithelial cell line established from rat small intestine, to test whether EGF and IGF-I interact to regulate intestinal epithelial cell growth. EGF and IGF-I alone each stimulated DNA synthesis in IEC-6 cells. EGF was more potent than IGF-I, and effects of the two growth factors in combination were synergistic. Characterization of the IGF system [IGF-I, IGF-II, type 1 IGF receptor, and six IGF binding proteins (IGFBPs) 1-6] revealed that IEC-6 cells express high levels of type 1 IGF receptor mRNA, low or undetectable levels of IGF-I and IGF-II mRNAs, and mRNA for only one of the six IGFBPs, IGFBP2. IGF-I decreases expression of type 1 IGF receptor mRNA in IEC-6 cells and EGF attenuates this effect. EGF and IGF-I both reduce IGFBP2 mRNA expression, and inhibitory effects of EGF and IGF-I in combination are additive. EGF reduces IGFBP2 accumulated in conditioned medium relative to levels observed with IGF-I alone. These effects of EGF on type 1 IGF receptor expression and on levels of IGFBP2 mRNA and IGFBP2 in medium may contribute to synergistic mitogenic effects with IGF-I by promoting IGF-I responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of insulin-like growth factor-I (IGF-I) in protein-restricted rats is associated with differential regulation of IGF-binding protein messenger ribonucleic acids in liver and kidney, and peptides in liver and serum.

To investigate the influence of the insulin-like growth factor binding-proteins (IGFBPs) on the nutritional regulation of IGF-I's actions, we compared the gene expression of IGF-I and the six IGFBPs in liver and kidney of protein-restricted (P5) and normally fed (P15) young rats. Using poly(A)+ Northern blot analysis, we observed a decrease in IGF-I messenger RNA (mRNA) at steady state in liver (-50%) and kidney (-60%). The increases in IGFBP-1 mRNA were parallel in these two tissues (liver, 5.7-fold; kidney, 4-fold). In contrast, the expression of the other IGFBP genes exhibited organ-specific regulation during protein restriction; although IGFBP-2 mRNA increased in liver in the P5 group (3-fold), it decreased slightly in kidney (-15%). IGFBP-3 mRNA declined by 30% in liver and was unchanged in kidney. IGFBP-4 mRNA increased by 50-88% in liver and was not modified in kidney. IGFBP-5 mRNA was not detected in liver and was identical in kidney of P15 and P5 rats. IGFBP-6 mRNA was not changed in either liver or kidney during protein restriction. To determine whether the changes in IGFBP mRNAs induced by protein restriction were associated with changes in the respective peptides, IGFBPs in supernatants of liver homogenates and in serum of the same rats were measured by ligand blot analyses. IGFBP-1 and IGFBP-2 Western immunoblot analyses were also performed in serum. By ligand blot, a 45,000 mol wt (M(r)) band (IGFBP-3) decreased in liver and serum of P5 rats, paralleling the changes in liver IGFBP-3 mRNA. A 30,000 M(r) band, consistent with IGFBP-1 and/or IGFBP-2, increased in liver. By immunoblot in serum, IGFBP-1 was only detectable in P5 rats, whereas IGFBP-2 decreased in the P5 group. By ligand blot, a 24,000 M(r) band (IGFBP-4) declined slightly in serum (not detected in liver). Our study shows that protein restriction regulates the expression of four of six IGFBPs in rats, and this regulation is organ specific. The nutritional regulation of IGFBP peptides in biological fluids, in particular serum, seems to involve additional mechanisms.

Differential regulation of insulin-like growth factor I (IGF-I) and IGF binding protein-1 messenger ribonucleic acids by amino acid availability and growth hormone in rat hepatocyte primary culture.

To determine the mechanisms involved in the nutritional regulation of insulin-like growth factor I (IGF-I) production and action, we studied the regulation of IGF-I and IGF-Binding Protein 1 (IGFBP-1) gene expression by GH and amino acid availability in rat hepatocyte primary culture. Hepatocytes were isolated by in situ collagenase perfusion and cultured on Matrigel in serum-free medium containing insulin and hydrocortisone. Rat GH (500 ng/ml) increased IGF-I messenger RNA (mRNA) abundance 6.9-fold at 24 h, as measured by Northern Blot using an IGF-I-specific riboprobe. In contrast, IGFBP-1 mRNA levels were decreased by 41% after 24 h of rat GH treatment. Hepatocytes were incubated for 24 h in three media differing in their amino acid concentrations: 0.2X, 1X, and 5X the normal rat plasma concentration. Amino acid deprivation (0.2X) decreased the abundance of IGF-I mRNAs (-56% after 24 h), whereas amino acid excess (5X) increased it (+70%) in comparison to the 1X medium. In contrast, amino acid deprivation increased IGFBP-1 mRNA abundance (+69%), whereas excess decreased it (-75%). Studies of the interaction between GH and amino acids, accomplished by the simultaneous manipulation of the two, suggest that each factor modulates the IGF-I mRNA and the IGFBP-1 mRNA and protein response to the other. We conclude that the IGF-I and IGFBP-1 genes are regulated in opposite ways by GH and amino acid availability. Our observations suggest that amino acids and GH regulate the production of IGF-I directly and exert indirect effects on IGF-I action by regulating the production of IGFBP-1.

The effect of dietary protein supplementation on insulin-like growth factors (IGFs) and IGF-binding proteins in children with shigellosis.

Nutrient deficiency causes growth failure and decreases serum insulin-like growth factor-I (IGF-I) concentrations. Because IGFBPs modulate the concentrations and availability of IGFs in serum, IGF-binding proteins (IGFBPs) were measured along with IGF-I and IGF-II before and after 21 days of refeeding in 22 undernourished Bangladeshi children (2-4 yr of age) with shigellosis. The effects of a 150 Cal/kg.day diet with a normal protein (6%; n = 10) or high protein (15%; n = 12) content were studied. The results were compared with those of 25 age-matched healthy American children (controls). Body weight gain was better in patients receiving the high protein diet than in those receiving the normal protein diet. In both groups, initial IGF-I (32 +/- 6 and 24 +/- 7 ng/mL; mean +/- SD) and IGF-II (177 +/- 15, 174 +/- 45 ng/mL) concentrations were low compared to controls (100 +/- 12 and 542 +/- 29 ng/mL, respectively; P < 0.007). After refeeding, IGF-I increased to 160 +/- 26 ng/mL on the normal protein diet and to 322 +/- 41 ng/mL on the high protein diet, exceeding values in controls (P < 0.007). IGF-II increased more than 2-fold on each diet (P < 0.007), reaching control values. IGFBP-2 concentrations before refeeding were twice those in controls (750 +/- 200 vs. 317 +/- 33 ng/mL; P < 0.007) and normalized after refeeding in the high protein group (288 +/- 32 ng/mL; P = NS), but remained elevated in the normal protein group (526 +/- 77 ng/mL; P < 0.007). IGFBP-3 levels before refeeding were low and returned to normal on each diet. IGFBP-3 proteolytic activity in serum was initially increased and declined on the high protein diet. In conclusion, protein content in the refeeding diet differentially affects IGFs and IGFBPs in young undernourished children with infection. IGF-I and IGFBP-2 seem to be particularly sensitive to dietary protein alterations. We speculate that an increase in IGF-I concentrations, normalization of IGFBP levels, and a decrease in IGFBP-3 proteolytic activity in serum may all be involved in the improved recovery and catch-up growth observed with the high protein diet.

Insulin-like growth factor-binding protein-3 proteolysis is induced after elective surgery.

The insulin-like growth factors (IGFs) are bound to several binding proteins (IGFBPs) that appear to regulate IGF transport, receptor binding, and action. The concentrations of these peptides are altered by catabolic conditions. To determine if IGF-I and IGFBP levels change after surgery, sera were obtained from 16 patients before and after cholecystectomy. Immunoreactive IGF-I measured in plasma samples from which IGFBPs had been extracted did not change postoperatively. In contrast, IGF-I determined in unextracted samples increased roughly 3-fold postoperatively, presumably due to changes in IGFBPs. Two days postoperatively, IGFBP-3 levels, determined by ligand blot, averaged 36% of preoperative values, whereas levels of IGFBP-2 and a 24,000 mol wt IGFBP did not change significantly. Similarly, by immunoblot, intact IGFBP-3 was decreased 84.2 +/- 20.2%, and a 31,000 mol wt IGFBP-3 fragment increased 57.5 +/- 47.4% postoperatively. Coincubation of postoperative, but not preoperative, sera with control sera resulted in a significant decrease in IGFBP-3 and production of proteolytic fragments. IGFBP-3 proteolytic activity in postoperative sera was markedly inhibited by antipain, Na-p-tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonylfluoride, aprotinin, o-phenanthroline, and EDTA, but not by leupeptin or N-tosyl-L-phenylalanine chloromethyl ketone. This pattern of inhibition is consistent with a metal-dependent trypsin-like serine protease. We speculate that proteolysis of IGFBP-3 may alter tissue uptake of IGF-I and thereby help to counteract the catabolic state caused by surgery.

Increased serum clearance and degradation of 125I-labeled IGF-I in protein-restricted rats.

Dietary protein restriction in young rats stunts growth and decreases serum insulin-like growth factor I (IGF-I) concentrations. To investigate the possibility of a diet-induced accelerated clearance of IGF-I, the clearance of 125I-labeled IGF-I was determined by measuring the radioactivity in serum obtained at different intervals after bolus injection in 5-wk-old rats that had received either a normal 15% protein diet (P15; n = 6) or a 5% protein isocaloric diet (P5; n = 7) for 1 wk. The clearance and volume of distribution of 125I-IGF-I were increased in P5 rats by 58 and 75%, respectively, whereas the terminal elimination half-life was not changed. The calculated IGF-I production rate was decreased by 40% in P5 rats. The intermediate distribution phase (alpha) was twofold faster in P5 than in P15 animals, and 125I-IGF-I was preferentially bound to IGF binding proteins in the 40-kDa complex in P5 rats. These observations might explain the increased clearance and degradation of IGF-I in the P5 rats. Dietary protein restriction appears to lower serum IGF-I concentrations by both decreasing its production rate and increasing its clearance.