Stromal Cells in the Pathogenesis of Inflammatory Bowel Disease.

Up till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells also play an important and significant role in IBD pathogenesis. Stromal cells in the intestines regulate both intestinal epithelial and immune cell homeostasis. Different subsets of stromal cells have been found to play a role in other inflammatory diseases [e.g. rheumatoid arthritis], and these various stromal subsets now appear to carry out also specific functions in the inflamed gut in IBD. Novel potential therapies for IBD utilize, as well as target, these pathogenic stromal cells. Injection of mesenchymal stromal cells [MSCs] into fistula tracts of Crohn's disease patients is already approved and used in clinical settings. In this review we discuss the current knowledge of the role of stromal cells in IBD pathogenesis. We further outline recent attempts to modify the stromal compartment in IBD with agents that target or replace the pathogenic stroma.

Pitfalls in cytokine measurements - Plasma TGF-ß1 in chronic fatigue syndrome.

BACKGROUND: Serum TGF-ß1 concentrations are reported to be elevated in chronic fatigue syndrome (CFS). However, measurement of circulating cytokines is a complex procedure and control of pre-analytical procedures is essential. The objective of the current study was to measure circulating TGF-ß1 concentrations in CFS patients compared to healthy controls, taking into account differences in pre-analytical procedures. METHODS: Two cohorts of female CFS patients were included. In both studies patients were asked to bring a healthy, age-matched control. At baseline, TGF-ß1 levels were measured in plasma and additionally P-selectin, a marker of platelet activity, was determined in a subgroup of participants. RESULTS: 50 patients and 48 controls were included in cohort I, and 90 patients and 29 controls in cohort II. Within the cohorts there were no differences in TGF-ß1 concentrations. However, between the cohorts there was a large discrepancy, which appeared to be caused by differences in g-force of the centrifuges used. The lower g-force used in cohort II (1361 g) caused more platelet activation, reflected by higher p-selectin concentrations, compared to cohort I (p < 0.0001), which was confirmed in a second independent experiment. There was a correlation between TGF-ß1 and p-selectin concentrations (r 0.79, p < 0.0001). CONCLUSION: These results demonstrate that control of pre-analytical procedures is an essential aspect when measuring circulating cytokines. No evidence for enhanced TGF-ß1 in patients with CFS was found.

Endoglin targeting inhibits tumor angiogenesis and metastatic spread in breast cancer.

Endoglin, a transforming growth factor-ß co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical trials for anti-angiogenic therapy. In this project, the redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis and the effects of targeting both pathways on breast cancer metastasis were explored. In patient samples, increased endoglin signaling after VEGF inhibition was observed. In vitro TRC105, an endoglin-neutralizing antibody, increased VEGF signaling in endothelial cells. Moreover, combined targeting of the endoglin and VEGF pathway, with the VEGF receptor kinase inhibitor SU5416, increased antiangiogenic effects in vitro and in a zebrafish angiogenesis model. Next, in a mouse model for invasive lobular breast cancer, the effects of TRC105 and SU5416 on tumor growth and metastasis were explored. Although TRC105 and SU5416 decreased tumor vascular density, tumor volume was unaffected. Strikingly, in mice treated with TRC105, or TRC105 and SU5416 combined, a strong inhibition in the number of metastases was seen. Moreover, upon resection of the primary tumor, strong inhibition of metastatic spread by TRC105 was observed in an adjuvant setting. To confirm these data, we assessed the effects of endoglin-Fc (an endoglin ligand trap) on metastasis formation. Similar to treatment with TRC105 in the resection model, endoglin-Fc-expressing tumors showed strong inhibition of distant metastases. These results show, for the first time, that targeting endoglin, either with neutralizing antibodies or a ligand trap, strongly inhibits metastatic spread of breast cancer in vivo.

Fibulin-4 deficiency increases TGF-ß signalling in aortic smooth muscle cells due to elevated TGF-ß2 levels.

Fibulins are extracellular matrix proteins associated with elastic fibres. Homozygous Fibulin-4 mutations lead to life-threatening abnormalities such as aortic aneurysms. Aortic aneurysms in Fibulin-4 mutant mice were associated with upregulation of TGF-ß signalling. How Fibulin-4 deficiency leads to deregulation of the TGF-ß pathway is largely unknown. Isolated aortic smooth muscle cells (SMCs) from Fibulin-4 deficient mice showed reduced growth, which could be reversed by treatment with TGF-ß neutralizing antibodies. In Fibulin-4 deficient SMCs increased TGF-ß signalling was detected using a transcriptional reporter assay and by increased SMAD2 phosphorylation. Next, we investigated if the increased activity was due to increased levels of the three TGF-ß isoforms. These data revealed slightly increased TGF-ß1 and markedly increased TGF-ß2 levels. Significantly increased TGF-ß2 levels were also detectable in plasma from homozygous Fibulin-4(R/R) mice, not in wild type mice. TGF-ß2 levels were reduced after losartan treatment, an angiotensin-II type-1 receptor blocker, known to prevent aortic aneurysm formation. In conclusion, we have shown increased TGF-ß signalling in isolated SMCs from Fibulin-4 deficient mouse aortas, not only caused by increased levels of TGF-ß1, but especially TGF-ß2. These data provide new insights in the molecular interaction between Fibulin-4 and TGF-ß pathway regulation in the pathogenesis of aortic aneurysms.

The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC.

BACKGROUND: Constitutive Wnt activation is essential for colorectal cancer (CRC) initiation but also underlies the cancer stem cell phenotype, metastasis and chemosensitivity. Importantly Wnt activity is still modulated as evidenced by higher Wnt activity at the invasive front of clonal tumours termed the ß-catenin paradox. SMAD4 and p53 mutation status and the bone morphogenetic protein (BMP) pathway are known to affect Wnt activity. The combination of SMAD4 loss, p53 mutations and BMP signalling may integrate to influence Wnt signalling and explain the ß-catenin paradox. METHODS: We analysed the expression patterns of SMAD4, p53 and ß-catenin at the invasive front of CRCs using immunohistochemistry. We activated BMP signalling in CRC cells in vitro and measured BMP/Wnt activity using luciferase reporters. MTT assays were performed to study the effect of BMP signalling on CRC chemosensitivity. RESULTS: Eighty-four percent of CRCs with high nuclear ß-catenin staining are SMAD4 negative and/or p53 aberrant. BMP signalling inhibits Wnt signalling in CRC only when p53 and SMAD4 are unaffected. In the absence of SMAD4, BMP signalling activates Wnt signalling. When p53 is lost or mutated, BMP signalling no longer influences Wnt signalling. The cytotoxic effects of 5-FU are influenced in a similar manner. CONCLUSIONS: The BMP signalling pathway differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. The use of BMPs in cancer therapy, as has been proposed by previous studies, should be targeted to individual cancers based on the mutational status of p53 and SMAD4.

Interaction with colon cancer cells hyperactivates TGF-ß signaling in cancer-associated fibroblasts.

The interaction between epithelial cancer cells and cancer-associated fibroblasts (CAFs) has a major role in cancer progression and eventually in metastasis. In colorectal cancer (CRC), CAFs are present in high abundance, but their origin and functional interaction with epithelial tumor cells has not been elucidated. In this study we observed strong activation of the transforming growth factor-ß (TGF-ß)/Smad signaling pathway in CRC CAFs, accompanied by decreased signaling in epithelial tumor cells. We evaluated the TGF-ß1 response and the expression of target genes including matrix metalloproteinases (MMPs) and plasminogen activator inhibitor (PAI)-1 of various epithelial CRC cell lines and primary CAFs in vitro. TGF-ß1 stimulation caused high upregulation of MMPs, PAI-1 and TGF-ß1 itself. Next we showed that incubation of CAFs with conditioned medium (CM) from epithelial cancer cells led to hyperactivation of the TGF-ß signaling pathway, enhanced expression of target genes like PAI-1, and the expression of α-smooth muscle actin (α-SMA). We propose that the interaction of tumor cells with resident fibroblasts results in hyperactivated TGF-ß1 signaling and subsequent transdifferentiation of the fibroblasts into α-SMA-positive CAFs. In turn this leads to cumulative production of TGF-ß and proteinases within the tumor microenvironment, creating a cancer-promoting feedback loop.

The prognostic role of TGF-ß signaling pathway in breast cancer patients.

BACKGROUND: The transforming growth factor-ß (TGF-ß) pathway has dual effects on tumor growth. Seemingly, discordant results have been published on the relation between TGF-ß signaling markers and prognosis in breast cancer. Improved prognostic information for breast cancer patients might be obtained by assessing interactions among TGF-ß signaling biomarkers. PATIENTS AND METHODS: The expression of nuclear Smad4, nuclear phosphorylated-Smad2 (p-Smad2), and the membranous expression of TGF-ß receptors I and II (TßRI and TßRII) was determined on a tissue microarray of 574 breast carcinomas. Tumors were stratified according to the Smad4 expression in combination with p-Smad2 expression or Smad4 in combination with the expression of both TGF-ß receptors. RESULTS: Tumors with high expression of TßRII, TßRI and TßRII, and p-Smad2 (P = 0.018, 0.005, and 0.022, respectively), and low expression of Smad4 (P = 0.005) had an unfavorable prognosis concerning progression-free survival. Low Smad4 expression combined with high p-Smad2 expression or low expression of Smad4 combined with high expression of both TGF-ß receptors displayed an increased hazard ratio of 3.04 [95% confidence interval (CI) 1.390-6.658] and 2.20 (95% CI 1.464-3.307), respectively, for disease relapse. CONCLUSIONS: Combining TGF-ß biomarkers provides prognostic information for patients with stage I-III breast cancer. This can identify patients at increased risk for disease recurrence that might therefore be candidates for additional treatment.

MMP-2 and MMP-9 in normal mucosa are independently associated with outcome of colorectal cancer patients.

BACKGROUND: Upregulation of the matrix metalloproteinases MMP-2 and MMP-9 in various cancers has been associated with worse survival of the patients. METHODS: We assessed MMP-2 and MMP-9 levels in normal colorectal mucosa from colorectal cancer patients in relation to the course of the disease. RESULTS: A high protein expression of MMP-2 as well as MMP-9 in normal mucosa was found to be correlated with worse 5-year survival. The combination of both parameters was an even stronger prognostic factor. These protein levels were found not to be related to the corresponding single nucleotide polymorphisms of MMP-2 (-1306C>T) and MMP-9 (-1562C>T). Multivariate analyses indicated that the MMP-2 and MMP-9 levels in normal mucosa are prognostic for survival, independent of TNM classification. CONCLUSION: MMP-2 and MMP-9 levels in normal mucosa are indicative of the course of disease in colorectal cancer patients.

MMP-2 geno-phenotype is prognostic for colorectal cancer survival, whereas MMP-9 is not.

The prognostic significance of single-nucleotide polymorphisms (SNPs) and tumour protein levels of MMP-2 and MMP-9 was evaluated in 215 colorectal cancer patients. Single-nucleotide polymorphism MMP-2(-1306T) and high MMP-2 levels were significantly associated with worse survival. Extreme tumour MMP-9 levels were associated with poor prognosis but SNP MMP-9(-1562C>T) was not. Tumour MMP levels were not determined by their SNP genotypes.

Tissue level, activation and cellular localisation of TGF-beta1 and association with survival in gastric cancer patients.

Transforming growth factor-beta1 (TGF-beta1), a tumour suppressing as well as tumour-promoting cytokine, is stored as an extracellular matrix-bound latent complex. We examined TGF-beta1 activation and localisation of TGF-beta1 activity in gastric cancer. Gastric tumours showed increased stromal and epithelial total TGF-beta1 staining by immunohistochemistry. Active TGF-beta1 was present in malignant epithelial cells, but most strongly in smooth muscle actin expressing fibroblasts. Normal gastric mucosa from the same patient showed some staining for total, and little for active TGF-beta1. Active TGF-beta1 levels were determined by ELISA on tissue homogenates, confirming a strong increase in active TGF-beta1 in tumours compared to corresponding normal mucosa. Moreover, high tumour TGF-beta1 activity levels were significantly associated with clinical parameters, including worse survival of the patients. Total and active TGF-beta1 levels were not correlated, suggesting a specific activation process. Of the different proteases tested, active TGF-beta1 levels were only correlated with urokinase activity levels. The correlation with urokinase activity suggests a role for plasmin in TGF-beta1 activation in the tumour microenvironment, resulting in transformation of resident fibroblasts to tumour promoting myofibroblasts. In conclusion we have shown localisation and clinical relevance of TGF-beta1 activity levels in gastric cancer.