Jolanta B. Pucilowska

Bacterial cell wall polymers promote intestinal fibrosis by direct stimulation of myofibroblasts

Normal luminal bacteria and bacterial cell wall polymers are implicated in the pathogenesis of chronic intestinal inflammation. To determine the direct involvement of bacteria and their products on intestinal fibrogenesis, the effects of purified bacterial cell wall polymers on collagen and cytokine synthesis were evaluated in intestinal myofibroblast cultures established from normal fetal and chronically inflamed cecal tissues. In this study, the intestines of Lewis rats were intramurally injected with peptidoglycan-polysaccharide polymers. Collagen and transforming growth factor (TGF)-beta1 mRNA levels were measured and correlated with mesenchymal cell accumulation by immunohistochemistry. The direct effects of cell wall polymers on fibrogenic cytokine and collagen alpha1 (type I) expression were evaluated in intestinal myofibroblast cultures. We found that intramural injections of bacterial cell wall polymers induced chronic granulomatous enterocolitis with markedly increased collagen synthesis and concomitant increased TGF-beta1 and interleukin (IL)-6 expression. Intestinal myofibroblast cultures were established, which both phenotypically and functionally resemble the mesenchymal cells that are involved in fibrosis in vivo. Bacterial cell wall polymers directly stimulated collagen alpha1 (I), TGF-beta1, IL-1beta, and IL-6 mRNA expression in the intestinal myofibroblasts derived from both normal and inflamed cecum. Neutralization of endogenous TGF-beta1 inhibited in vitro collagen gene expression. From our results, we conclude that increased exposure to luminal bacterial products can directly activate intestinal mesenchymal cells, which accumulate in areas of chronic intestinal inflammation, thus stimulating intestinal fibrosis in genetically susceptible hosts.

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 α-smooth muscle actin (αSMA) 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 αSMA 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 αSMA, 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 TGFβ1 mRNA expression in the SMP8-IGF-I group. Moreover, Colα1(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 accelarates liver regeneration. These effects appear to be mediated in part by upregulation of HGF and downregulation of TGFβ1. The data indicate that IGF-I can modulate the cytokine response to liver injury facilitating regeneration and reducing fibrosis.

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.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.

Hypothalamic-pituitary-adrenal axis function and corticosterone receptor expression in behaviourally characterized young and aged Long-Evans rats: Glucocorticoids, memory dysfunction and ageing

In the current investigation, hypothalamic–pituitary–adrenal (HPA) axis function was examined in young and aged male Long‐Evans rats that were initially assessed on a version of the Morris water maze sensitive to cognitive impairment during ageing. In behaviourally characterized rats, a 1‐h restraint stress paradigm revealed that plasma corticosterone concentrations in aged cognitively impaired rats took significantly longer to return to baseline following the stressor than did those in young or aged cognitively unimpaired rats. No differences in basal or peak plasma corticosterone concentrations, however, were observed between young or aged rats, irrespective of cognitive status. Using ribonuclease protection assays and in situ hybridization, we evaluated mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA abundance in young and aged rats characterized on the spatial task. Abundance of MR mRNA was decreased as a function of age in stratum granulosum but not hippocampus proper, and the decrease in MR mRNA was largely unrelated to cognitive status. However, GR mRNA was significantly reduced in several hippocampal subfields (i.e. stratum granulosum and temporal hippocampus proper) and other related cortical structures (medial prefrontal and olfactory regions) of aged cognitively impaired rats compared to either young or aged cognitively unimpaired cohorts, and was significantly correlated with spatial learning ability among the aged rats in each of these brain regions. In agreement with previous stereological data from this ageing model, no changes were detected in neuron density in the hippocampus of the rats used in the in situ hybridization analysis. These data are the first to describe a coordinated decrease in GR mRNA in a functional brain system including hippocampus and related cortical areas that occurs in tandem with impairments of the HPA response to stress and cognitive decline in ageing.