Synthesis Of Thrombospondin Research Articles

Protective effect of alizarin on vascular endothelial dysfunction via inhibiting the type 2 diabetes-induced synthesis of THBS1 and activating the AMPK signaling pathway

BackgroundIn this study, we investigated the protective effects of alizarin (AZ) on endothelial dysfunction (ED). AZ has inhibition of the type 2 diabetes mellitus (T2DM)-induced synthesis of thrombospondin 1 (THBS1). Adenosine 5‘-monophosphate- activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. PurposeThe aim of this study was to investigate the ameliorative effect of AZ on vascular injury caused by T2DM and to reveal the potential mechanism of AZ in high glucose (HG)-stimulated human umbilical vein endothelial cells (HUVECs) and diabetic model rats. Study designHUVECs, rats and AMPK-/- transgenic mice were used to investigate the mitigating effects of AZ on vascular endothelial dysfunction caused by T2DM and its in vitro and in vivo molecular mechanisms. MethodsIn type 2 diabetes mellitus rats and HUVECs, the inhibitory effect of alizarin on THBS1 synthesis was verified by immunohistochemistry (IHC), immunofluorescence (IF) and Western blot (WB) so that increase endothelial nitric oxide synthase (eNOS) content in vitro and in vivo. In addition, we verified protein interactions with immunoprecipitation (IP). To probe the mechanism, we also performed AMPKα2 transfection. AMPK's pivotal role in AZ-mediated prevention against T2DM-induced vascular endothelial dysfunction was tested using AMPKα2-/- mice. ResultsWe first demonstrated that THBS1 and AMPK are targets of AZ. In T2DM, THBS1 was robustly induced by high glucose and inhibited by AZ. Furthermore, AZ activates the AMPK signaling pathway, and recoupled eNOS in stressed endothelial cells which plays a protective role in vascular endothelial dysfunction. ConclusionsThe main finding of this study is that AZ can play a role in different pathways of vascular injury due to T2DM. Mechanistically, alizarin inhibits the increase in THBS1 protein synthesis after high glucose induction and activates AMPKα2, which increases NO release from eNOS, which is essential in the prevention of vascular endothelial dysfunction caused by T2DM.

Transcriptional and Post-Transcriptional Regulation of Thrombospondin-1 Expression: A Computational Model.

Hypoxia is an important physiological stress signal that drives angiogenesis, the formation of new blood vessels. Besides an increase in the production of pro-angiogenic signals such as vascular endothelial growth factor (VEGF), hypoxia also stimulates the production of anti-angiogenic signals. Thrombospondin-1 (TSP-1) is one of the anti-angiogenic factors whose synthesis is driven by hypoxia. Cellular synthesis of TSP-1 is tightly regulated by different intermediate biomolecules including proteins that interact with hypoxia-inducible factors (HIFs), transcription factors that are activated by receptor and intracellular signaling, and microRNAs which are small non-coding RNA molecules that function in post-transcriptional modification of gene expression. Here we present a computational model that describes the mechanistic interactions between intracellular biomolecules and cooperation between signaling pathways that together make up the complex network of TSP-1 regulation both at the transcriptional and post-transcriptional level. Assisted by the model, we conduct in silico experiments to compare the efficacy of different therapeutic strategies designed to modulate TSP-1 synthesis in conditions that simulate tumor and peripheral arterial disease microenvironment. We conclude that TSP-1 production in endothelial cells depends on not only the availability of certain growth factors but also the fine-tuned signaling cascades that are initiated by hypoxia.

Chemopreventive apigenin controls UVB-induced cutaneous proliferation and angiogenesis through HuR and thrombospondin-1.

Plant flavonoid apigenin prevents and inhibits UVB-induced carcinogenesis in the skin and has strong anti-proliferative and anti-angiogenic properties. Here we identify mechanisms, by which apigenin controls these oncogenic events. We show that apigenin acts, at least in part, via endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1). TSP1 expression by the epidermal keratinocytes is potently inhibited by UVB. It inhibits cutaneous angiogenesis and UVB-induced carcinogenesis. We show that apigenin restores TSP1 in epidermal keratinocytes subjected to UVB and normalizes proliferation and angiogenesis in UVB-exposed skin. Importantly, reconstituting TSP1 anti-angiogenic function in UVB-irradiated skin with a short bioactive peptide mimetic representing exclusively its anti-angiogenic domain reproduced the anti-proliferative and anti-angiogenic effects of apigenin. Cox-2 and HIF-1α are important mediators of angiogenesis. Both apigenin and TSP1 peptide mimetic attenuated their induction by UVB. Finally we identified the molecular mechanism, whereby apigenin did not affect TSP1 mRNA, but increased de novo protein synthesis. Knockdown studies implicated the RNA-binding protein HuR, which controls mRNA stability and translation. Apigenin increased HuR cytoplasmic localization and physical association with TSP1 mRNA causing de novo TSP1 synthesis. HuR cytoplasmic localization was, in turn, dependent on CHK2 kinase. Together, our data provide a new mechanism, by which apigenin controls UVB-induced carcinogenesis.

Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.

A cytokine‐controlled mechanism for integrated regulation of T‐lymphocyte motility, adhesion and activation

The co-ordination of T-cell motility, adhesion and activation remains poorly understood. It is also unclear how these functions are co-ordinated with external stimuli. Here we unveil a series of molecular interactions in cis at the surface of T lymphocytes with potent effects on motility and adhesion in these cells, and communicating with proliferative responses. These interactions were controlled by the signature cytokines of T helper subsets interleukin-2 (IL-2) and IL-4. Low-density lipoprotein receptor-related protein 1 (LRP1) was found to play a key role for T-cell motility by promoting development of polarized cell shape and cell movement. Endogenous thrombospondin-1 (TSP-1) enhanced cell surface expression of LRP1 through CD47. Cell surface expressed LRP1 induced motility and processing of TSP-1 while inhibiting adhesion to intercellular adhesion molecule 1 and fibronectin. Interleukin-2, but not IL-4, stimulated synthesis of TSP-1 and motility through TSP-1 and LRP1. Stimulation of the T-cell receptor (TCR)/CD3 complex inhibited TSP-1 expression. Inhibitor studies indicated that LRP1 regulated TSP-1 expression and promoted motility through JAK signalling. This LRP1-mediated motogenic signalling was connected to CD47/Gi protein signalling and IL-2-induced signalling through TSP-1. The motogenic TSP-1/LRP1 mechanism antagonized TCR/CD3-induced T-cell proliferation. These results indicate that LRP1 in collaboration with TSP-1 directs a counter-adhesive and counter-proliferative motogenic cascade. T cells seem programmed to prioritize movement before adhesion through this cascade. In conclusion, vital decision-making in T lymphocytes regulating motility, adhesive interactions and proliferation, are integrated through a molecular mechanism connecting different cell surface receptors and their signalling pathways.

Effects of PI3-k/Akt short hairpin RNA on proliferation, fibronectin production and synthesis of thrombospondin-1 and transforming growth factor-beta1 in glomerular mesangial cells induced by sublytic C5b-9 complexes.

To explore proliferation of glomerular mesangial cells (GMC) and secretion of extracellular matrix (fibronectin induced by sublytic C5b-9 complexes), and then ascertain the role of phosphatidylinositol 3-kinase (PI3-k)/Akt signal pathway in these processes, by using small hairpin RNAs. The expression of cyclin D(2), (3)H-thymidine into DNA and production of fibronectin including thrombospondin-1 and transforming growth factor-beta(1) in the GMCs stimulated by sublytic C5b-9 or transfected with expression vectors of PI3-k and Akt short hairpin RNA or LY294002 (PI3-k inhibitor) were measured by Real-time quantitative polymerase chain reaction (PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and (3)H-thymidine incorporation ((3)H-TdR), respectively. The expression of cyclin D(2), (3)H-thymidine into DNA and fibronectin in the GMCs stimulated by sublytic C5b-9 could all be increased, and the elevations of these parameters mentioned above were also markedly reduced in the GMCs transfected with vectors of PI3-k and Akt short hairpin RNA or LY294002, respectively. These data indicate that sublytic C5b-9 can promote proliferation of GMCs and secretion of fibronectin as well as synthesis of thrombospondin-1 and transforming growth factor-beta(1). The PI3-k/Akt signal pathway in these reactions, mediated by sublytic C5b-9 complexes, may play at least a partial role.

Docetaxel (D) plus prednisone (P) in combination with metronomic cyclophosphamide (CTX) and celecoxib (C) as first line chemotherapy in metastatic hormone refractory prostate cancer (HRPC): Phase II clinical trial with pharmacodynamic evaluation

16084 Background: Docetaxel based chemotherapy has became the standard of care for metastatic HRPC, moreover, low-dose metronomic antiangiogenic CTX and C have demonstrated a significant activity in preclinical and clinical studies without relevant toxicities. Combination of such strategies could be of interest in prostate cancer patients. Methods: A total of 19 patients with metastatic HRPC received D 60 mg/sqm iv day 1 every 3 weeks up to 12 cycles and from day 2 in a continuous fashion: P 5 mg po BID, CTX 50 mg po daily and C 200 mg po BID. Primary end point was PFS; secondary were: activity (PSA reduction >50%); objective responses, toxicities (NCI-CTC criteria), OS, evaluation of plasma levels of thrombospondin-1 (TSP-1), VEGF, sVEGFR-2, VE-cadherin mRNA; expression and synthesis of TSP-1 and VEGF in peripheral blood mononuclear cells. Patients (pts) characteristics: median age 69,5 years (59–78), median PS 1 (0–2), median baseline PSA 48,3 ng/ml (10–444); main sites of disease: bone 17 pts (90%), lymphnodes 1 pt (5%), liver 1 pt (5%). Results: 14 pts were evaluable for toxicity and response. Median number of docetaxel cycles administered was 8,5 (1–12) and median duration of metronomic CTX plus P and C was 183 days (18–636 days). Overall PSA decrease ≥ 50% was found in 11 pts (78%), with 6 patients that achieved a PSA complete response (43%). Three pts showed stable disease (21%). Up today, only 4 pts (28%) experienced disease progression. Median time to PSA progression was 7,7 months (3,7–10,3 months). Main grade 3 hematologic or non-hematologic toxicities were: neutropenia (1 pt; 5%) and diarrhoea (1 pt, 5%). No grade 4 toxicities have been observed. Conclusions: combination of docetaxel plus metronomic chemotherapy with CTX and C was feasible and showed only mild toxicity, preliminary activity was also interesting. The evaluation of biologic and pharmacodynamic effects of the combination is ongoing. No significant financial relationships to disclose.

The role of PI3‐k/Akt signaling pathway in sublytic C5b‐9 complexes‐induced synthesis of thrombospondin‐1 (Tsp‐1), transforming growth factor‐β1 (TGF‐β1) and proliferation of glomerular mesangial cells (GMCs) including secretion of extracelluar matrix (ECM)

Sublytic complement C5b‐9 complexes can cause many forms of immune‐mediated diseases. But the role and relative signal pathway of sublytic C5b‐9 on the pathologic changes of glomerular mesangial cells (GMCs) has not been demonstrated. In this study, we first explored the effects of sublytic C5b‐9 on the synthesis of thrombospondin‐1 (Tsp‐1) and transforming growth factor‐β1 (TGF‐β1) including proliferation of GMCs and secretion of extracelluar matrix (ECM) i.e. fibronectin (FN), and then ascertained the roles of phosphatidylinositol 3‐kinase (PI3‐k)/Akt signal pathway in these processes by using small hairpin RNAs (shRNA). The results showed that the expression of Tsp‐1, TGF‐β1, cyclin D2 and 3H‐thymidine into DNA as well as FN in the GMCs stimulated by sublytic C5b‐9 complexes could be increased, and the elevation of these parameters mentioned‐above was also markedly reduced in the GMCs transfected with PI3‐k and Akt shRNA expression vectors or LY294002 (PI3‐k inhibitor) respectively. These data indicate that sublytic C5b‐9 can induce the sythesis of Tsp‐1, TGF‐β1 and promote the proliferation of GMCs and secretion of FN, and PI3‐k/Akt signal pathway plays a partial role in these reactions mediated by sublytic C5b‐9 complexes at least.

Effects of α-difluoromethylornithine on thrombospondin-1 production by human breast cancer cells

We have previously observed that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) upregulates production of thrombospondin-1 (TSP-1), an extracellular matrix protein with potent anti-angiogenic and antimetastatic properties, by MDA-MB-435 human breast cancer cells in culture. The present experiments were designed to investigate the mechanisms by which DFMO regulates TSP-1 production in this system. 35S-methionine pulse chase experiments indicated that DFMO administration increased TSP-1 synthesis by approximately 6-fold, while it slightly but significantly decreased protein half-life from 35 to 28 min. DFMO treatment increased steady state TSP-1 mRNA levels by 2-fold in MDA-MB-435 cells. TSP-1 promoter reporter studies indicated that this increase was largely due to activation of transcription. Analysis of distribution of TSP-1 mRNA levels between non-polysomal, subpolysomal and polysomal fractions in control and DFMO-treated cells suggested a major stimulatory effect of the drug on TSP-1 translation. A similar increase in TSP-1 transcription and translation in response to DFMO treatment was also observed in vivo in MDA-MB-435 breast cancer xenografts. Surprisingly however, we failed to detect an increase in TSP-1 protein as assessed by Western blot analysis. The reason for this unexpected finding is unknown but may be due to DFMO-induced stimulation of TSP-1 secretion into the systemic circulation, thus preventing its accumulation within the tumor.

Atorvastatin Neutralizes the Up-Regulation of Thrombospondin-1 Induced by Thrombin in Human Umbilical Vein Endothelial Cells

Statins have been reported to affect blood vessel formation. Thrombospondin-1 (TSP-1) is a multifunctional protein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. This study was designed to investigate the effect of atorvastatin on TSP-1 synthesis in thrombin-stimulated human umbilical vein endothelial cells (HUVECs), and its regulation by mevalonate or its derivatives. The results showed that atorvastatin down-regulated TSP-1 expression in HUVECs. This effect was fully reversed by mevalonate, farnesylpyrophosphate (FPP), and gerarylgeranylpyrophosphate (GGPP). Furthermore, farnesyltransferase and geranylgeranyltransferase inhibitors decreased TSP-1expression. It was also found that thrombin increased TSP-1 expression in HUVECs. Atorvastatin (0.1, 1, and 10 muM) decreased TSP-1 in thrombin-stimulated cells (45%, 66%, and 80%). Mevalonate partially reversed this inhibitory effect of atorvastatin on TSP-1, whereas the presence of FPP and GGPP did not alter TSP-1. Rho-kinase inhibitor neutralized the up-regulation of TSP-1 induced by thrombin. In conclusion, atorvastatin inhibits TSP-1 expression in endothelial cells via the mevalonate pathway. Rho protein activation is necessary for up-regulation of TSP-1 synthesis induced by thrombin. Because FPP and GGPP are essential for the activity of Rho proteins, inhibition of these proteins may constitute the mechanism by which atorvastatin inhibits thrombin up-regulated TSP-1 expression.

The proangiogenic phenotype of human tumor-derived endothelial cells depends on thrombospondin-1 downregulation via phosphatidylinositol 3-kinase/Akt pathway

Tumor-derived endothelial cells (TEC) display increased survival and angiogenic properties in respect to normal endothelial cells. The aim of this study was to investigate the mechanism potentially involved in TEC proangiogenic phenotype. We found that thrombospondin-1 (TSP-1), a potent physiological inhibitor of angiogenesis, was significantly reduced in TEC in respect to normal endothelial cells. This reduction was confirmed by immunofluorescence in the intratumor vessels of clear cell renal carcinomas. As TEC were shown to display a basal upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we evaluated the possible regulation of TSP-1 by this pathway by using LY294002 and wortmannin, the PI3K inhibitors, and rapamycin, the mammalian target of rapamycin (mTOR) inhibitor. In addition, we developed negative dominant TEC for Akt. TSP-1 production by TEC was enhanced by the treatment with LY294002 and wortmannin and with rapamycin, suggesting a negative regulation of TSP-1 expression by the PI3K/Akt/mTOR pathway. In addition, downregulation of Akt activation in negative dominant Akt TEC enhanced TSP-1 expression and release. Administration of exogenous TSP-1 to TEC reduced their proangiogenic properties in vitro and in vivo. In parallel, blockade of TSP-1 with an anti-TSP-1 antibody in negative dominant Akt TEC restored their proangiogenic phenotype to levels similar to wild-type TEC. In conclusion, these results indicate that the upregulation of the PI3K/Akt/mTOR pathway is responsible for the inhibition of TSP-1 synthesis which is critical in determining the proangiogenic phenotype of TEC. Strategies aimed to inhibit the PI3K/Akt/mTOR pathway may restore a normal quiescent endothelial phenotype in TEC by promoting TSP-1 production.

Elevated glucose induction of thrombospondin-1 up-regulates fibronectin synthesis in proximal renal tubular epithelial cells through TGF-β1 dependent and TGF-β1 independent pathways

TGF-beta1 bioactivation, consequent to the interaction of latent TGF-beta1 with thrombospondin-1 (TSP-1), correlates with matrix accumulation in mesangial cells. Tubulointerstitial damage predicts poor renal survival. There is little data on TGF-beta1 bioactivation and matrix synthesis in human proximal renal tubular epithelial cells under the influence of high glucose concentrations. This study thus investigates the role of TSP-1 in mediating elevated glucose-induction of TGF-beta1 bioactivation and fibronectin (FN) synthesis in human proximal tubular epithelial cells. Human proximal renal tubular epithelial cells (HK-2 cells) were incubated with 5, 10, 20 or 30 mM D-glucose for up to 3 weeks either in the presence or absence of TSP-1 blocking peptide. In separate studies HK-2 cells were incubated with exogenous TSP-1 (0-10 ng/ml) or TGF-beta1 (0-10 ng/ml) for 24 h. Cell proliferation was assessed by [(3)H]-thymidine incorporation. TGF-beta1 transcript, secretion and bioactivity were investigated by quantitative real-time PCR, ELISA and the MLEC bioassay respectively. TSP-1 and FN synthesis were assessed by quantitative real-time PCR, ELISAs and Western blot analysis. Elevated glucose concentrations increased TSP-1 synthesis, which was associated with reduced cell proliferation, increased TGF-beta1 bioactivity, and stimulation of FN synthesis. The inclusion of TSP-1 blocking peptide to cells stimulated with elevated glucose concentration abrogated activation of TGF-beta1 and induction of FN secretion. Exogenous TSP-1 increased bioactive TGF-beta1 in HK-2 cells to initiate FN accumulation. Of interest is our observation that TSP-1 also increased matrix synthesis through a mechanism independent of TGF-beta1. TGF-beta1 in turn modulated TSP-1 synthesis, indicative of an autocrine loop between TSP-1 and TGF-beta1. TSP-1 plays an important role in the induction of matrix synthesis by high glucose concentrations in human proximal renal tubular epithelial cells, through TGF-beta1 dependent and TGF-beta1 independent pathways. Pharmacological intervention targeting increased TSP-1 expression may interrupt the pathogenesis of diabetic nephropathy.

Expression changes of thrombospondin-1 and neuropeptide Y in myocardium of STZ-induced rats

Diabetic cardiomyopathy was the most dangerous diabetic complication facing diabetics, with its exact mechanisms remaining obscure. Our study was conducted to investigate the expression of thrombospondin-1 (TSP-1) and neuropeptide Y (NPY) in myocardium of streptozotocin (STZ)-induced diabetic rats. We employed streptozotocin (STZ)-induced diabetic rats to study the alteration of the TSP-1 and NPY expression in the left ventricle myocardium in diabetic and normal group by immunohistochemistry and immunofluorescence. The data of weight, blood sugar and urine sugar indicated no significant difference between the two groups before the animal model was induced. Four weeks after the induction of diabetes the weight of the diabeteic animals was 189.1 ± 18.4 g, plasma glucose was 23.7 ± 3.25 mmol/L and urine glucose was (++) to (+++); whereas the weight of the control animals was 260.5 ± 32.1 g, plasma glucose was 4.9 ± 0.5 mmol/L and urine glucose undetectable (−). The differences between the control and the diabetes group were distinct. A significant increase of the TSP-1 and NPY expression was also observed in the diabetic rat's heart. The number of the NPY positive myocardium and the light density of the positive myocardium in the left ventricle of the diabetic model were 17.3 ± 2.1 and 102.5 ± 9.3/mm 2, respectively, which were considered as increased when compared with the control that were 10.1 ± 2.6 and 61.2 ± 6.7, respectively. Our results support the view that high glucose conditions can induce an increased synthesis of TSP-1 through the PKC–TGF-β–TSP-1 pathway, which in turn facilitate TGF-β activation. Additionally, the activation of PKC may further lead to the over-expression of NPY. This may be involved in diabetic cardiomyopathy.

Expression of thrombospondin-1 in prostate-derived cell lines

Thrombospondin-1 (TSP-1) is a multifunctional matricellular glycoprotein involved in several mechanisms critical to the formation and progression of solid tumors including cell adhesion, proliferation, migration, invasion, and angiogenesis. However, work related to TSP-1 expression and functionality in prostate cancer is limited. Expression experiments in the present study demonstrated lower expression of TSP-1 in the prostate cancer cell lines DU 145 and LNCaP compared to SV40-immortalized prostatic epithelial cells PNT 1A. All three cell lines expressed the TSP-1 receptor CD36. Exogenously added TSP-1 modulated the cellular phenotype of LNCaP cells, which demonstrated decreased proliferation rate and partly entered apoptosis. Collectively, these data support the concept that partial or complete loss of TSP-1 synthesis may provide tumor cells with a proliferation advantage. In addition, TSP-1 located at the border between tumor and stroma as observed in primary prostate tumors may act as a barrier of tumor growth depending on the TSP-1 receptor repertoire of the tumor cells.

Role of stromal thrombospondin-1 in motility and proteolytic activity of oral squamous cell carcinoma cells

The immunohistochemical localization of thrombospondin-1 (TSP-1) in human normal oral mucosa and oral squamous cell carcinoma (SCC) was examined. The immunostaining of TSP-1 was mainly localized in the connective tissues adjacent to the epithelium of oral mucosa, whereas epithelial cells showed negligible immunoreactivity for TSP-1. TSP-1 expression was striking in the stroma of SCC. TSP-1 synthesis by SCC cells and fibroblasts in culture was examined by immunoprecipitation with anti-TSP-1 antibody. Fibroblasts produced a large amount of TSP-1, whereas SCC cells secreted little amount of TSP-1. Treatment of fibroblasts by the conditioned medium of SCC cells led to an increase in TSP-1 synthesis. TSP-1 induced haptotactic migration and stimulated the production of matrix metalloproteinase-9 (MMP-9) in SCC cells. These results suggest that TSP-1 in the stroma of oral SCC might be synthesized by mesenchymal cells but not by epithelial cells, and that the synthesis of TSP-1 might be regulated by interaction with SCC cells. TSP-1 accumulated in the stroma might promote the progression of oral SCC by enhancing the motility and proteolytic activity in a paracrine manner.

Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-β in Fibrotic Renal Disease in the Rat in Vivo

Specific treatment of chronic progressive renal disease is very limited. TGF-beta, considered as the major cytokine causing tissue scarring, must be activated extracellularly before it can bind to its receptors. Thrombospondin-1 (TSP1) has been identified as an activator of latent TGF-beta in in vitro systems and in pancreas and lung homeostasis in mouse pups in vivo, but whether this is also true in inflammatory fibrotic disease is unknown. We examined a rat model of mesangial proliferative glomerulonephritis, where TGF-beta has been demonstrated to mediate renal fibrosis. In this study, antisense phosphorothioate oligonucleotides against TSP1 were successfully transferred into almost all glomeruli of perfused diseased kidneys and markedly inhibited de novo synthesis of TSP1. This effect was accompanied by decreased activation but not expression of TGF-beta and by the inhibition of the TGF-beta-dependent smad-signaling pathway, as well as transcription of TGF-beta target genes such as EDA-fibronectin, resulting in a markedly suppressed accumulation of extracellular matrix. In sharp contrast, neither glomerular cell proliferation nor influx of macrophages was affected by this therapy in experimental mesangial proliferative glomerulonephritis. These results demonstrate that TSP1 is the major endogenous activator of TGF-beta in experimental inflammatory kidney disease.

High glucose levels enhance TGF-β1–thrombospondin-1 pathway in cultured human mesangial cells via mechanisms dependent on glucose-induced PKC activation

We have previously demonstrated that thrombospondin-1 (TSP-1) promotes activation of latent TGF-β1 in cultured human mesangial cells (MCs) [Nephron 79 (1998) 38.]. This study was performed to clarify the relationship between protein kinase C (PKC) activity and activation of TGF-β1 and TSP-1 production in cultured human MCs exposed to high glucose levels. MCs grown in 33 mmol/l glucose demonstrated a significant ( P<.01) increase in activation of TGF-β1 compared with those in 5 mmol/l glucose, which were evaluated by both an ELISA and a bioassay. High glucose-induced increase in active TGF-β1 was completely inhibited by coincubation with 5 μmol/l GFX, a PKC inhibitor, and was mimicked by the addition of 0.1 μmol/l phorbol 12,13-dibutyrate (PDBu). On the other hand, increased TSP-1 production from MCs stimulated by 0.1 nmol/l recombinant TGF-β1 and 0.1 μmol/l PDBu were significantly ( P<.01) reduced by the addition of 10 nmol/l latency-associated peptide (LAP), a specific inhibitor of TGF-β1 activity. The amount of TSP-1 secreted from MCs increased by a high ambient glucose. The glucose-induced increase in TSP-1 production was markedly attenuated by the treatment with GFX and LAP, while those agents did not affect TSP-1 production in low-glucose concentrations. Taken together, our results suggest that glucose-induced activation of TGF-β1 is dependent on PKC activity, leading to a sequential increase in TSP-1 synthesis in cultured human MCs. Thus, we propose that high glucose conditions induce an increase in PKC–TGF-β1 activity–TSP-1 pathway, and that glucose-induced increase in TSP-1 may synergistically facilitate TGF-β1 activation in an autocrine manner in MCs.

Expression of thrombospondin-1 in human hepatocarcinoma cell lines and its regulation by transcription factor Jun/AP-1.

Thrombospondin-1 (TSP-1) is a homotrimeric glycoprotein synthesized in a variety of normal and transformed cells, and secreted into the extracellular matrix. Based on its known effects on the tumor and endothelial cells, TSP-1 was implicated in the tumor growth and metastasis. In the present study, we have demonstrated the expression of TSP-1 in the human hepatocarcinoma cell lines. TSP-1 was detected in human hepatocarcinoma SK-HEP-1, Hep 3B and immortalized human liver Chang cells. Using two different cell lines, SK-HEP-1 and Hep 3B cells, we have studied effects of phorbol 12-myristate 13-acetate (PMA) on TSP-1 expression. TSP-1 synthesis was stimulated by PMA in both cell lines. When the cells were treated with PMA, the TSP-1 mRNA started to increase at 30 min and reached the maximal level at 6 h. TSP-1 induction by PMA was completely inhibited by the pre-treatment of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor. A TSP-1 promoter-luciferase reporter gene was transcriptionally activated by PMA, as well as by the expression of c-Jun. Among three putative AP-1 recognition sites on the TSP-1 promoter, a deletion of the 1st and 2nd sites caused loss of PMA-induced upregulation, while the 3rd site deletion showed no effect. In subsequent experiments, both the recombinant c-Jun and nuclear proteins induced by PMA have a stronger binding affinity for the 2nd AP-1 recognition site than the 1st and 3rd ones. Our study demonstrated that TSP-1 could be expressed and secreted by human hepatoma cell lines and its expression could be effectively regulated by PMA. We also suggest that AP-1 binding activity through the protein kinase C activation is a critical event for the TSP-1 gene expression and consequently affects production and processing of the protein.

Responsive site on the thrombospondin-1 promotor to down-regulation by phorbol 12-myristate 13-acetate in porcine aortic endothelial cells.

Thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, inhibits neovascularization and is implicated in the regression of tumor growth and metastasis. We found that the synthesis of TSP-1 in porcine aortic endothelial (PAE) cells was decreased in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) treatment in porcine aortic endothelial (PAE) cells. In this study, a responsive site on the TSP-1 promotor affected by PMA treatment in PAE was characterized. The level of TSP-1 mRNA was also decreased by PMA after 1 h and persisted that way for at least 24 h. PMA treatment and c-Jun overexpression suppressed the transcription of TSP-1 promotor-luciferase reporter gene. A deletion between -767 and -657 on the TSP-1 promotor neutralized the PMA-induced down-regulation. In addition, oligo a (-767 approximately -723) was responsive to PMA-induced repression, while oligo b (-734 approximately -689) and c (-700 approximately -656) was not. Electrophoretic mobility shift assays showed that this PMA responsive element specifically bound a nuclear protein and that the binding activity was diminished by PMA treatment in PAE cells but not in Hep 3B cells. In supershift assay, potential regulatory elements in this region, SP1 and GATA-1, were not responsive to the inhibition of TSP-1 expression by PMA. Our results suggest that the repression of TSP-1 synthesis by PMA is mediated by blocking a particular unknown nuclear protein binding to the responsive site (-767 approximately -735), which is regulated by c-Jun.

Repair in avascular tissues: fibrosis in the transparent structures of the eye and thrombospondin 1.

Wound repair is a process which is normally dependent on the vasculature of the damaged tissue. However, the transparent structures of the eye (e.g. central cornea, lens, vitreous) are avascular and yet are still subject to repair and fibrosis. Moreover, the resulting ophthalmic scars often remain avascular. Since this type of ocular scarring may result in blindness, it is the subject of intense research. An aspect of avascular ophthalmic fibrosis which has attracted attention is the question concerning early wound healing components that are usually derived from blood constituents. One such molecule is the glycoprotein thrombospondin 1. Thrombospondin 1 is thought to be a key regulator of cell behaviour in early wound repair and appears to be derived totally from platelet alpha-granules during repair of incisional skin wounds. It has been shown that the ocular cells involved in avascular repair processes, and which are thus responsible for healing in the absence of platelet-derived thrombospondin 1, are capable of synthesizing the protein themselves. It is suggested that cells involved in ophthalmic repair processes produce thrombospondin 1 in the absence of the platelet-derived molecule. Local synthesis of thrombospondin 1 may represent a therapeutic target in the management of ophthalmic fibrosis.