Thrombospondin 1 in Metabolic Diseases.

The thrombospondin family comprises of five multifunctional glycoproteins, whose best-studied member is thrombospondin 1 (TSP1). This matricellular protein is a potent antiangiogenic agent that inhibits endothelial migration and proliferation, and induces endothelial apoptosis. Studies have demonstrated a regulatory role of TSP1 in cell migration and in activation of the latent transforming growth factor beta 1 (TGFß1). These functions of TSP1 translate into its broad modulation of immune processes. Further, imbalances in immune regulation have been increasingly linked to pathological conditions such as obesity and diabetes mellitus. While most studies in the past have focused on the role of TSP1 in cancer and inflammation, recently published data have revealed new insights about the role of TSP1 in physiological and metabolic disorders. Here, we highlight recent findings that associate TSP1 and its receptors to obesity, diabetes, and cardiovascular diseases. TSP1 regulates nitric oxide, activates latent TGFß1, and interacts with receptors CD36 and CD47, to play an important role in cell metabolism. Thus, TSP1 and its major receptors may be considered a potential therapeutic target for metabolic diseases.

Expression of WIF-1 in inflammatory bowel disease.

The WNT/ß-catenin cellular network has been extensively studied in numerous diseases including inflammatory bowel disease (IBD). IBD is a condition that increases the risk of developing colorectal cancer. WIF-1 is an inhibitory protein that acts by blocking the interactions of WNT with its receptor complex, thus leading to downregulation of end products of this pathway. While WIF-1 has been characterized in several cancers, its relationship with IBD has yet to be elucidated. In this study, the expression of WIF-1 in patients with IBD was analyzed in order to provide insights into the pathophysiology and rationale for alternative therapies. Biopsies of both normal and inflamed colonic mucosa from patients with Crohn's disease or ulcerative colitis were histologically examined for the degree of morphologic changes, immune cell infiltration and presence of WIF-1 through immunohistochemistry. No differences were observed in WIF-1 expression linked to a particular condition, but WIF-1 stain was significantly enhanced in the crypts and lamina propria as inflammation increased in biopsies from patients with both, ulcerative colitis and Crohn's disease. These findings could give guidance to new therapeutic applications of the WNT/ß-catenin system and WIF-1 in IBD.

Thrombospondin-1 in a Murine Model of Colorectal Carcinogenesis.

Colorectal Cancer (CRC) is one of the late complications observed in patients suffering from inflammatory bowel diseases (IBD). Carcinogenesis is promoted by persistent chronic inflammation occurring in IBD. Understanding the mechanisms involved is essential in order to ameliorate inflammation and prevent CRC. Thrombospondin 1 (TSP-1) is a multidomain glycoprotein with important roles in angiogenesis. The effects of TSP-1 in colonic tumor formation and growth were analyzed in a model of inflammation-induced carcinogenesis. WT and TSP-1 deficient mice (TSP-1-/-) of the C57BL/6 strain received a single injection of azoxymethane (AOM) and multiple cycles of dextran sodium sulfate (DSS) to induce chronic inflammation-related cancers. Proliferation and angiogenesis were histologically analyzed in tumors. The intestinal transcriptome was also analyzed using a gene microarray approach. When the area containing tumors was compared with the entire colonic area of each mouse, the tumor burden was decreased in AOM/DSS-treated TSP-1-/- versus wild type (WT) mice. However, these lesions displayed more angiogenesis and proliferation rates when compared with the WT tumors. AOM-DSS treatment of TSP-1-/- mice resulted in significant deregulation of genes involved in transcription, canonical Wnt signaling, transport, defense response, regulation of epithelial cell proliferation and metabolism. Microarray analyses of these tumors showed down-regulation of 18 microRNAs in TSP-1-/- tumors. These results contribute new insights on the controversial role of TSP-1 in cancer and offer a better understanding of the genetics and pathogenesis of CRC.

Thrombospondin peptide ABT-898 inhibits inflammation and angiogenesis in a colitis model.

AIM: To evaluate the efficacy of the improved thrombospondin mimetic peptide ABT-898 in a murine model of ulcerative colitis. METHODS: The dextran sodium sulfate (DSS) was used for the induction of colitis in both TSP-1 deficient (TSP-1(-/-)) and wild type (WT) mice during 7 d. While mice were receiving the DSS dissolved in the drinking water, the ABT-898 peptide was dissolved in sterile 5% glucose solution and delivered using mini pumps subcutaneously implanted. Plasma samples were analyzed for interleukin (IL)-6 by ELISA assay and colonic tissues were harvested, fixed and processed for histological evaluation. Immunohistochemistry using antibodies for the detection of CD31 and MECA in endothelial cells was performed. Inflammation was graded in colonic sections and the number of microvessels in each lesion was assessed. Activation of signal transducer and activator of transcription 3 (STAT3) in colonic samples was quantified by immunohistochemistry and Western blotting using antibodies against total STAT3 and phosphorylated STAT3 (pSTAT3) (Ser727). RESULTS: Treatment with ABT-898 considerably diminished the inflammatory response in WT and TSP-1(-/-) mice (P < 0.0001 in both groups vs control). Identification of blood vessels highlighted by CD31/MECA immunohistochemistry, showed significantly reduced vessel counts in colitic lesions of WT and TSP-1(-/-) mice treated with ABT898 (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0002; WT controls/WT treated, P = 0.0005). Consistently, IL-6 was significantly diminished in plasma samples of TSP-1(-/-) and WT treated with the peptide when compared to the control mice (P = 0.0002 and P = 0.0148, respectively). pSTAT3 positive cells were quantified in WT and TSP-1(-/-) treated with ABT-898. A significant decrease in positive cells for pSTAT3 was observed in treated mice (TSP-1(-/-) controls/TSP-1(-/-) treated, P = 0.0089; WT/WT treated, P = 0.0110). These results were confirmed by Western blotting analyses showing lower levels of pSTAT3 in colitic lesions from mice treated with the peptide ABT-898. CONCLUSION: These findings indicate that the new peptide ABT-898 ameliorates inflammation and angiogenesis and might be a therapeutic alternative in IBD and inflammatory diseases.

Thrombospondin-1 type 1 repeats in a model of inflammatory bowel disease: transcript profile and therapeutic effects.

Thrombospondin-1 (TSP-1) is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR) domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS) to induce colitis in wild-type (WT) mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1) the three type 1 repeats of the TSP-1 (3TSR), (2) the second type 1 repeat (TSR2), or (3) TSR2 with the RFK sequence (TSR2+RFK). Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE) genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease.

Thrombospondin-1: multiple paths to inflammation.

Inflammation is a defensive process against tissue injury. Once this self-protective strategy is initiated, an effective resolution of the process is crucial to avoid major and unnecessary tissue damage. If the underlying event inducing inflammation is not addressed and homeostasis is not restored, this process can become chronic and lead to angiogenesis and carcinogenesis. Thrombospondin-1 (TSP-1) is a matricellular protein involved in angiogenesis, cancer, and inflammation. The effects of TSP-1 have been studied in many preclinical tumor models, and mimetic peptides are being tested in cancer clinical trials. However, the molecular mechanisms explaining its role in inflammatory processes are not well understood. This paper will discuss the role of TSP-1 in inflammation and its interaction with key receptors that may explain its functions in that process. Recent literature will be reviewed showing novel mechanisms by which this multifaceted protein could modulate the inflammatory process and impact its resolution.

The role of thrombospondin 1 on intestinal inflammation and carcinogenesis.

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) quite common in the United States and other Western countries. Patients suffering IBD are at greater risk of developing colorectal adenocarcinoma than the general population. Both, the adenoma-carcinoma and the inflammation-carcinogenesis processes are characterized by active angiogenesis. Recent studies also have shown that anti-angiogenesis might be a novel therapeutic approach for IBD. Thrombospondin 1 (TSP1) is an extracellular protein well known for its anti-angiogenic properties. TSP1 also has key functions in inflammation, which is assumed to be the primary cause for carcinogenesis in IBD. This review is focused on the role of TSP1 in colorectal carcinogenesis. The therapeutic effects of TSP derived-peptides on inhibiting the inflammationcarcinogenesis progression are also discussed.

Thrombospondin 1 and its mimetic peptide ABT-510 decrease angiogenesis and inflammation in a murine model of inflammatory bowel disease.

OBJECTIVE: Vascular abnormalities and expression of proangiogenic factors have been repeatedly reported in inflammatory bowel disease (IBD). Thrombospondin 1 (TSP-1) is a protein well known for its antiangiogenic and anti-inflammatory properties. Using the dextran sulfate sodium (DSS) model, the role of TSP-1 in IBD has been investigated in vivo. METHODS: TSP-1-deficient mice (TSP-1-/-) and WT mice were treated with DSS for 7 days. Disease activity indices, myeloperoxidase activity (MPO) and histology were analyzed. Microvascular density (MVD) was quantified using immunohistochemistry (IMH) with CD31 antibody. TGF-beta(1), basic FGF, VEGF, TNF-alpha and MMPs protein levels were evaluated by IMH and enzyme-linked immunoabsorbent assay (ELISA). Mice were treated with ABT-510 (Abbott Laboratories), an antiangiogenic TSP peptide, using miniosmotic pumps for 7 days. RESULTS: TSP-1(-/-) mice had a worse clinical outcome and exhibited severe signs of rectal bleeding compared to the WT controls. The TSP-1-/- mice showed a higher level of crypt damage and deeper lesions. The grade of inflammation and the levels of MPO activity were also significantly higher in colons of TSP-1-/- mice. TSP-1-/- mice displayed higher MVD in focal areas of the colon after only 3 days of DSS treatment. Furthermore, clinical severity of the colitis and angiogenesis was significantly diminished when mice was treated with ABT-510. CONCLUSIONS: These findings directly link TSP-1 as a protective factor in IBD and suggest antiangiogenesis treatment, including compounds such as ABT-510 as an adjuvant therapy for IBD.

Lack of thrombospondin-1 increases angiogenesis in a model of chronic inflammatory bowel disease.

BACKGROUND AND AIMS: Vascular abnormalities and expression of pro-angiogenic factors are observed in inflammatory bowel diseases (IBD). In this study, the role of thrombospondin-1 (TSP-1), an antiangiogenic protein, was analyzed using the dextran sulfate sodium (DSS) model for IBD. MATERIALS AND METHODS: Wild-type (WT) and thrombospondin-1-deficient (TSP-1(-/-)) mice were subjected to four cycles (7 days) of DSS. Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGFbeta-1), and pro-apoptotic proteins such as Fas and its ligand (FasL) were determined by enzyme-linked immunosorbent assay. Double immunohistochemistry for cluster of differential 31 (CD31) and panendothelial cell antigen-32 antibodies was performed for detecting blood vessels. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay was also performed for identifying apoptotic cells. Inflammation, dysplasia, microvascular density (MVD), apoptotic indices (AI), protein 53 (p53), and beta-catenin expression were determined. RESULTS: VEGF and bFGF protein levels and MVD were higher in the TSP-1(-/-) mice (p = 0.0312, p = 0.0246, and p = 0.0085, respectively). AI in the endothelial cells (EC) and FasL levels were significantly lower in TSP-1(-/-) compared to WT mice (p = 0.0042 and p = 0.0362, respectively). Dysplasia was detected in 66% of TSP-1(-/-) mice compared to 14% in WT mice. Hscores of ss-catenin and areas overexpressing p53 were higher in TSP-1(-/-) mice (p = 0.0002 and p = 0.0339, respectively). CONCLUSION: TSP-1 may decrease angiogenesis by reducing the levels of pro-angiogenic factors and inducing apoptosis in EC through the Fas or FasL pathway. These findings, along with the increased overexpression of p53 and beta-catenin in TSP-1(-/-) mice, underline its role in carcinogenesis.

Fas ligand expression and its correlation with apoptosis and proliferation in Lobund-Wistar prostate carcinomas.

OBJECTIVE: The Fas (CD95) interaction with its receptor Fas ligand (FasL) is one of the main mechanisms of cell apoptosis. High expression of FasL has been consistently observed in a variety of human cancers. In this study, we evaluated FasL and its relationship with apoptosis and proliferation in Lobund-Wistar (L-W) cancers. The L-W rat strain develops spontaneous and induced adenocarcinomas in the anterior prostate and seminal vesicles. Although FasL expression has been observed in a subset of human prostate carcinomas, this multistage model allowed in vivo evaluation of subclones of malignant cells with a single genetic susceptibility. METHODS: Apoptosis was evaluated in spontaneous, induced and transplanted tumors as well as metastasis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique and transmission electron microscopy. Proliferating cell nuclear antigen (PCNA) and FasL expression were detected using immunohistochemistry and analyzed according to the number of positive cells and intensity of staining using a semiquantitive method. RESULTS: Apoptotic indexes were significantly higher in spontaneous tumors compared to induced (p < 0.008), transplanted tumors (p < 0.0112) and metastases (p < 0.009). TUNEL-positive cells were frequently observed in the leukocytic infiltrate of the stroma in transplanted carcinomas and metastases. These findings were confirmed by electron microscopy. FasL expression was not uniformly localized in L-W carcinomas and its highest expression was observed in transplanted tumors and metastasis (p < 0.005). Moreover, PCNA indices were directly correlated with cancers showing high FasL total scores (Hscores). CONCLUSIONS: In this model, high FasL expression was associated with cells displaying low apoptotic indexes and high PCNA index. Therefore, analysis of FasL may have clinical relevance in detecting the malignant potential of prostate cancers.

The anti-ischemia agent ranolazine promotes the development of intestinal tumors in APC(Min/+) mice.

Ranolazine was shown to improve exercise parameters in patients with chronic angina. It works by switching myocardial energy metabolism from fatty acids to glucose, thus increasing the efficiency of ATP production under hypoxic conditions. Tumors are hypoxic and may also respond to ranolazine. We found that ranolazine caused a dose-dependent increase in tumor number in APC(Min/+) mice, a model of spontaneous intestinal tumorigenesis. Tumors from drug-treated mice were also more dysplastic and invasive than those from untreated mice. These findings have implications for the use of ranolazine in patients with a history of malignant neoplasms or adenomatous polyps.

Transcriptional profiling of the transition from normal intestinal epithelia to adenomas and carcinomas in the APCMin/+ mouse.

Mutations in the adenomatous polyposis coli (APC) gene that result in excessive beta-catenin-induced cell signaling are implicated in the risk of colon cancer. Although the mechanism of APC-mediated tumorigenesis is known, the pathways that translate beta-catenin signaling into tumor growth in vivo are undefined. To address this, gene expression profiles of normal intestinal epithelial cells were compared with those from adenomas and carcinomas from APC(Min/+) mice, a model of APC-related colorectal cancer. The gene expression profiles of adenomas and carcinomas were very similar, which is consistent with the theory that carcinomas progress from adenomas in this model system. Tumors had altered transcript abundance for members of several pathways that influence cell growth and proliferation including growth factors/receptors, molecules involved in apoptosis, and protein processing and catabolism enzymes. Comparison of gene expression between adenomas and carcinomas revealed nine differentially expressed transcripts. These included members of three growth-regulating pathways, and the results are consistent with the increased growth potential of carcinomas. SRY-box containing gene 17 (Sox 17), a negative regulator of beta-catenin signaling, and calbindin-D9K, a factor that enhances calcium transport, were more highly expressed in adenomas than carcinomas (approximately 4-fold and 15- to 22-fold, respectively). Transcript abundance for insulin-like growth factor binding protein 5, which mediates insulin-like growth factor function, was 2.6-fold greater in carcinomas. Because the changes in gene expression observed in this study are directly associated with a deficiency in APC, the data provide new insights into how loss of this important tumor suppressor translates into benign and malignant tumor growth.

Thrombospondin 1--a regulator of adenoma growth and carcinoma progression in the APC(Min/+) mouse model.

Thrombospondin 1 (TSP-1) is a multifunctional extracellular matrix protein that is an endogenous regulator of tumor angiogenesis. The effects of TSP-1 on adenoma formation and development into cancerous lesions has been evaluated in the Min(/+) (multiple intestinal neoplasia) mouse model. These mice develop multiple adenomas in the small intestine due to a mutation in the homologous APC (adenomatous polyposis coli) gene. As in its human counterpart, these adenomas may progress to carcinomas. Intestines of APC(Min/+) mice were dissected and histologic evaluation of adenomas was then conducted. Significant increases in vascularization and proliferation were observed in adenomatous, as compared with normal, mucosa. TSP-1 immunostaining revealed significant decreases in the number and intensity of positive cells in adenomas, as compared with normal mucosa. TSP-1 scores were inversely correlated with vascularity and proliferation rate. Cross breeding of mice homozygous for a deletion of the TSP-1 gene (TSP-1(-/-)) with mice heterozygous for the APC gene mutation (APC(Min/+)), resulted in animals that showed a significant increase in adenoma number and diameter. Also, histopathological examination of these adenomas showed accelerated dysplasic changes, carcinoma in situ and early invasion, compared with their APC(Min/+) littermates. Moreover, a significant decrease of TUNEL-positive cells was observed in intestinal adenomas of TSP-1(-/-)/APC(Min/+) mice. This study reports the first in vivo impact of TSP-1 during early stages of tumor initiation and development in an intestinal carcinogenesis model and demonstrates that TSP-1 affects both angiogenesis and tumor cell apoptosis.