Peritumoral adipose tissue as a source of inflammatory and angiogenic factors in colorectal cancer.

PURPOSE: Obesity is a risk factor for the development of human colorectal cancer (CC). The aim of this work is to report the inflammatory and angiogenic scenario in lean (BMI < 25 kg/m2) and obese (BMI > 30 kg/m2) patients with and without CC and to assess the role of peritumoral adipose tissue in CC-induced inflammation. MATERIAL AND METHODS: Patients were divided in four experimental groups: obese patients with CC (OB-CC), lean patients with CC (LEAN-CC), obese patients without CC (OB), and lean patients without CC (LEAN). RESULTS: Plasma levels of pro-inflammatory cytokines (interleukin (IL)-6, IL-4, IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in OB-CC patients. Peritumoral adipose tissue (TF) explants and cultured mature adipocytes secreted higher amounts of nitrites and nitrates than did control and non-tumoral (NTF) adipose tissue both alone and in response to lipopolysaccharide (LPS). Nitrite and nitrate secretion was also increased in TF explants from OB-CC patients compared with that from LEAN-CC patients. Gene expression of adiponectin, tumor necrosis factor alpha (TNF-α), insulin-like growth factor type I (IGF-I), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor γ (PPAR-γ) was increased in TF explants from CC patients. LPS increased the gene expression of IL-6, IL-10, TNF-α, vascular endothelial growth factor (VEGF), and COX-2 in OB and in TF explants from OB-CC patients. COX-2 and PPAR-γ inhibition further increased LPS-induced release of nitrites and nitrates in TF explants and adipocytes from OB-CC patients. CONCLUSIONS: In conclusion, OB-CC patients have increased plasma levels of pro-inflammatory and angiogenic factors. TF from OB-CC patients shows an increased secretion of inflammatory markers compared with both TF from LEAN-CC and non-tumoral adipose tissue (AT) through a COX-2- and PPAR-γ-independent mechanism.

Angiotensin II stimulates alpha3(IV) collagen production in mouse podocytes via TGF-beta and VEGF signalling: implications for diabetic glomerulopathy.

BACKGROUND: The podocyte is bathed in an angiotensin II (AngII)-rich ultrafiltrate, but the impact of AngII on podocyte pathobiology is not well known. Because podocytes play a direct role in the glomerular basement membrane (GBM) thickening of diabetes, the alpha3(IV) collagen chain was examined. Podocyte expression of alpha3(IV) collagen may involve the transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF) systems. METHODS: Cultured mouse podocytes were treated with various doses of AngII for selected periods of time, with or without inhibitors of TGF-beta and VEGF signalling, SB-431542 and SU5416, respectively. TGF-beta1 and VEGF were assayed by enzyme-linked immunosorbent assay (ELISA); alpha3(IV) collagen, TGF-beta type II receptor and phospho-Smad2 were assayed by immunoblotting. RESULTS: AngII >or=10(-10) M was found to stimulate the production of alpha3(IV) collagen significantly in as short a time as 3 h. The expression of alpha3(IV) collagen was influenced by the TGF-beta system, but AngII did not increase the podocyte's production of TGF-beta1 ligand; rather, it increased the expression of the TGF-beta type II receptor and activated the TGF-beta signalling system through Smad2. Despite the TGF-beta receptor upregulation, synergy between AngII and TGF-beta1 to boost alpha3(IV) collagen production was not observed. However, blockade of TGF-beta signalling with SB-431542 prevented AngII from stimulating alpha3(IV) collagen production. Podocyte expression of alpha3(IV) collagen was also increased by the autocrine activity of VEGF. Podocytes were stimulated to secrete VEGF by 10(-10) M or higher AngII after 48 h. Blockade of the endogenous VEGF activity by SU5416 prevented AngII-stimulated alpha3(IV) collagen production. CONCLUSIONS: AngII stimulates the podocyte to produce alpha3(IV) collagen protein via mechanisms involving TGF-beta and VEGF signalling. Alterations in alpha3(IV) collagen production may contribute to GBM thickening and perhaps proteinuria in diabetes.

Retinoids as a potential treatment for experimental puromycin-induced nephrosis.

1 Puromycin aminonucleoside (PAN)-induced nephrosis is a model of human minimal change disease. In rats, PAN induces nephrotic-range proteinuria, renal epithelial cell (podocyte) damage, infiltration of mononuclear leukocytes, and apoptosis of several renal cell types. 2 Retinoic acid (RA) modulates a wide range of biological processes, such as inflammation and apoptosis. Since renal damage by PAN is characterized by inflammatory infiltration and epithelial cell death, the effect of treatment with all-trans RA (tRA) was examined in the PAN nephrosis model and in the cultured differentiated podocyte. 3 Treatment with tRA 4 days after PAN injection did not inhibit the proteinuria peak but reversed it significantly. However, treatment with tRA both before and 2 days after the injection of PAN protected the glomerular epithelial cells, diminishing the cellular edema and diffuseness of the foot process effacement. Preservation of the podocyte architecture correlated with the inhibition of proteinuria. The anti-inflammatory effect of tRA was evidenced by the inhibition of PAN-induced interstitial mononuclear cell infiltration and the decreased renal expression of two molecules involved in monocyte infiltration: fibronectin and monocyte chemoattractant protein-1. TUNEL assays showed that tRA inhibited the PAN-induced apoptosis of cultured differentiated mouse podocytes. 4 We conclude that tRA treatment may prevent proteinuria by protecting the podocytes from injury and diminishing the interstitial mononuclear infiltrate in the model of PAN nephrosis. Retinoids are a potential new treatment for kidney diseases characterized by proteinuria and mononuclear cell infiltration.

The key role of the transforming growth factor-beta system in the pathogenesis of diabetic nephropathy.

Progressive renal injury in diabetes mellitus leads to major morbidity and mortality. The manifestations of diabetic nephropathy may be a consequence of the actions of certain cytokines and growth factors. Prominent among these is transforming growth factor-beta (TGF-beta) because it promotes renal cell hypertrophy and stimulates extracellular matrix accumulation, the two hallmarks of diabetic renal disease. In cell culture, high ambient glucose increases TGF-beta mRNA and protein in proximal tubular, glomerular epithelial, and mesangial cells. Neutralizing anti-TGF-beta antibodies prevent the hypertrophic and matrix stimulatory effects of high glucose in these cells. In experimental and human diabetes mellitus, several reports describe overexpression of TGF-beta in the glomeruli and tubulointerstitium. We demonstrate that short-term treatment of diabetic mice with neutralizing monoclonal antibodies against TGF-beta significantly reduces kidney weight and glomerular hypertrophy and attenuates the increase in extracellular matrix mRNAs. Long-term treatment of diabetic mice further improves the renal pathology and also ameliorates the functional abnormalities of diabetic nephropathy. Finally, we provide evidence that the renal TGF-beta system is significantly up-regulated in human diabetes. The kidney of a diabetic patient actually elaborates TGF-beta1 protein into the circulation whereas the kidney of a non-diabetic subject extracts TGF-beta1 from the circulation. The data we review here strongly support the hypothesis that elevated production or activity of the TGF-beta system mediates diabetic renal hypertrophy and extracellular matrix expansion.

Increased glomerular and tubular expression of transforming growth factor-beta1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse.

Activation of the renal transforming growth factor-beta (TGF-beta) system likely mediates the excess production of extracellular matrix in the diabetic kidney. To establish the role of the TGF-beta system in type 2 diabetic nephropathy, we examined the intrarenal localization and expression of the TGF-beta1 isoform, the TGF-beta type II receptor, and the Smad signaling pathway in the 16-week-old db/db mouse, a genetic model of type 2 diabetes that exhibits mesangial matrix expansion, glomerular basement membrane thickening, and renal insufficiency that closely resemble the human disease. Compared with its nondiabetic db/m littermate, the db/db mouse showed significantly increased TGF-beta1 mRNA expression by in situ hybridization in both glomerular and tubular compartments. Likewise, TGF-beta1 protein, by immunohistochemical staining, was increased in both renal compartments, but the fractional expression of TGF-beta1 protein was less than that of the mRNA in the glomerulus. In situ hybridization and immunohistochemical staining for the TGF-beta type II receptor revealed concordant and significant increases of both mRNA and protein in the glomerular and tubular compartments of diabetic animals. Finally, immunohistochemistry showed preferential accumulation of Smad3 in the nuclei of glomerular and tubular cells in diabetes. The complementary technique of Southwestern histochemistry using a labeled Smad-binding element demonstrated increased binding of nuclear proteins to Smad-binding element, indicating active signaling downstream of the TGF-beta stimulus. We therefore propose that the TGF-beta system is up-regulated at the ligand, receptor, and signaling levels throughout the renal cortex in this animal model of type 2 diabetes. Our findings suggest that the profibrotic effects of TGF-beta may underlie the progression to glomerulosclerosis and tubulointerstitial fibrosis that characterize diabetic nephropathy.

Hydrogen peroxide increases extracellular matrix mRNA through TGF-beta in human mesangial cells.

BACKGROUND: Reactive oxygen species (ROS) are excessively produced in pathologic states, including many renal diseases. Transforming growth factor-beta (TGF-beta) may mediate renal fibrotic injury, and ROS may act through the TGF-beta pathway to exert a profibrotic effect. METHODS: The expression of TGF-beta1 and extracellular matrix (ECM) components were assessed in cultured human mesangial cells (HMCs) incubated with glucose oxidase (GO), an enzyme that continuously generates hydrogen peroxide from glucose. A neutralizing anti-TGF-beta antibody was added to test the hypothesis that hydrogen peroxide acts through activation of the TGF-beta pathway to stimulate ECM expression. RESULTS: Northern blot analysis revealed significantly increased steady-state levels of TGF-beta1 and ECM proteins (collagen types I, III, and IV, and fibronectin) by approximately twofold. While no significant effect on mRNA stability after treatment with GO was observed, other studies employing promoter-reporter assays, competitive-quantitative reverse transcription-polymerase chain reaction, mink lung epithelial cell proliferation assay, and TGF-beta1 enzyme-linked immunosorbent assay all demonstrated significant stimulation by GO (>1.5-fold) of TGF-beta1 promoter activity, mRNA level, bioactivity, and protein production, respectively. Catalase pretreatment prevented the GO-induced stimulation of TGF-beta1 mRNA. When incubations were performed with a panselective neutralizing anti-TGF-beta antibody, the GO-stimulated expression of ECM molecules was prevented. CONCLUSIONS: GO-induced hydrogen peroxide production induces TGF-beta1 synthesis and thereby increases ECM gene expression in cultured HMCs. These cellular responses may underlie the development and progression of renal diseases characterized by oxidative stress.

Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice.

Emerging evidence suggests that transforming growth factor-beta (TGF-beta) is an important mediator of diabetic nephropathy. We showed previously that short-term treatment with a neutralizing monoclonal anti-TGF-beta antibody (alphaT) in streptozotocin-diabetic mice prevents early changes of renal hypertrophy and increased matrix mRNA. To establish that overactivity of the renal TGF-beta system mediates the functional and structural changes of the more advanced stages of nephropathy, we tested whether chronic administration of alphaT prevents renal insufficiency and glomerulosclerosis in the db/db mouse, a model of type 2 diabetes that develops overt nephropathy. Diabetic db/db mice and nondiabetic db/m littermates were treated intraperitoneally with alphaT or control IgG, 300 microgram three times per week for 8 wk. Treatment with alphaT, but not with IgG, significantly decreased the plasma TGF-beta1 concentration without decreasing the plasma glucose concentration. The IgG-treated db/db mice developed albuminuria, renal insufficiency, and glomerular mesangial matrix expansion associated with increased renal mRNAs encoding alpha1(IV) collagen and fibronectin. On the other hand, treatment with alphaT completely prevented the increase in plasma creatinine concentration, the decrease in urinary creatinine clearance, and the expansion of mesangial matrix in db/db mice. The increase in renal matrix mRNAs was substantially attenuated, but the excretion of urinary albumin factored for creatinine clearance was not significantly affected by alphaT treatment. We conclude that chronic inhibition of the biologic actions of TGF-beta with a neutralizing monoclonal antibody in db/db mice prevents the glomerulosclerosis and renal insufficiency resulting from type 2 diabetes.

Phenotypic modifications of human mesangial cells by extracellular matrix: the importance of matrix in the contractile response to reactive oxygen species.

The progression of chronic renal diseases is characterized by the accumulation of extracellular matrix proteins in the glomerulus. The present experiments were designed to analyze the effect of hydrogen peroxide on the contractile and proliferative phenotypes of human mesangial cells grown on different culture substrates: plastic, collagen type I, and collagen type IV. Contraction was analyzed by measuring planar cell surface area and myosin light chain phosphorylation, whereas proliferation was studied by [(3)H]thymidine incorporation. No changes were detected in the proliferation rate of human mesangial cells grown on different culture substrates, neither under basal conditions nor in the presence of fetal calf serum or H(2)O(2). Cells grown on plastic or collagen did not contract in the presence of H(2)O(2), but cells grown on collagen I elicited a significant contraction with H(2)O(2). Platelet-activating factor induced contraction of human mesangial cells on the three culture substrates. The different contractile responses observed were not due to different degradation rates of H(2)O(2). The present experiments support the importance of extracellular matrix in the response to exogenous stimuli and point to the possibility that patients with changes in the mesangial matrix as a result of chronic renal diseases may have an increased susceptibility to the pathological actions of reactive oxygen species.