Glioblastoma remodelling of human neural circuits decreases survival.

Gliomas synaptically integrate into neural circuits1,2. Previous research has demonstrated bidirectional interactions between neurons and glioma cells, with neuronal activity driving glioma growth1-4 and gliomas increasing neuronal excitability2,5-8. Here we sought to determine how glioma-induced neuronal changes influence neural circuits underlying cognition and whether these interactions influence patient survival. Using intracranial brain recordings during lexical retrieval language tasks in awake humans together with site-specific tumour tissue biopsies and cell biology experiments, we find that gliomas remodel functional neural circuitry such that task-relevant neural responses activate tumour-infiltrated cortex well beyond the cortical regions that are normally recruited in the healthy brain. Site-directed biopsies from regions within the tumour that exhibit high functional connectivity between the tumour and the rest of the brain are enriched for a glioblastoma subpopulation that exhibits a distinct synaptogenic and neuronotrophic phenotype. Tumour cells from functionally connected regions secrete the synaptogenic factor thrombospondin-1, which contributes to the differential neuron-glioma interactions observed in functionally connected tumour regions compared with tumour regions with less functional connectivity. Pharmacological inhibition of thrombospondin-1 using the FDA-approved drug gabapentin decreases glioblastoma proliferation. The degree of functional connectivity between glioblastoma and the normal brain negatively affects both patient survival and performance in language tasks. These data demonstrate that high-grade gliomas functionally remodel neural circuits in the human brain, which both promotes tumour progression and impairs cognition.

Targeting TSP-1 decreased periodontitis by attenuating extracellular matrix degradation and alveolar bone destruction.

An important factor in periodontitis pathogenesis relates to a network of interactions of various cytokines. Thrombospondin-1 (TSP-1) is upregulated in several inflammatory diseases. We previously found that Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS)-induced TSP-1 production, and that TSP-1 simultaneously and effectively elevated inflammatory cytokines in THP-1 macrophages. This suggests that TSP-1 plays an important role in the pathology of periodontitis. However, the function of TSP-1 on oral cells is largely unknown. This study aimed to elucidate the underlying molecular mechanisms of TSP-1 in human periodontal fibroblasts (hPDLFs). We demonstrated that TSP-1 is highly expressed in the gingival crevicular fluid of patients with chronic periodontitis and in the inflammatory gingival tissues of rats. TSP-1 overexpression or treatment with recombinant human TSP-1(rTSP-1) promoted the expression of MMP-2, MMP-9 and RANKL/OPG in hPDLFs, while anti-TSP-1 inhibited cytokines production from P. gingivalis LPS-treated hPDLFs. Additional experiments showed that SB203580 (a special p38MAPK inhibitor) inhibited MMP-2, MMP-9 and RANKL/OPG expression induced by rTSP-1. Thus, TSP-1 effectively promoted P. gingivalis LPS-induced periodontal tissue (extracellular matrix (ECM) and alveolar bone) destruction by the p38MAPK signalling pathway, indicating that it may be a potential therapeutic target against periodontitis.

Inhibition of thrombospondin-1 reduces glutathione activity and worsens acute liver injury during acetaminophen hepatotoxicity in mice.

Acetaminophen (N-Acetyl-p-Aminophenol or APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the United States and Western Europe. Previous studies have shown that TGFß1 is elevated during APAP-induced hepatotoxicity and promotes liver injury by reducing liver regeneration while inducing hepatocyte senescence. At this time, little is known about the role of proteins that activate latent TGFß1 and their effects during APAP-induced hepatotoxicity. Thrombospondin-1 (TSP1) is a homotrimeric protein that can not only activate latent TGFß1 but can also interact with other proteins including Nrf2 to induce antioxidant signaling. The aim of the current study was to assess the role of thrombospondin-1 (TSP1) in both TGFß1 activation and its contribution to APAP-induced liver injury. C57Bl/6 mice or TSP1 null mice (TSP1-/-) were administered 300 mg/kg or 600 mg/kg of APAP. TGFß1 signaling, TSP1 expression, measures of hepatic injury, Nrf2 expression, measures of oxidative/nitrosative stress and GSH metabolism were assessed. The expression of TGFß1, TSP1 and phosphorylation of SMAD proteins increased in APAP-treated mice compared to controls. TSP1-/- mice had reduced TGFß1 expression and phosphorylation of SMAD proteins but increased liver injury. Hepatocyte cell death was increased in TSP1-/- mice and this was associated with decreased Nrf2 activity, decreased GSH levels and increased oxidative stress in comparison to wild-type C57Bl/6 mice. Together, these data demonstrate that elimination of TSP1 protein in APAP-treated mice reduces TGFß1 signaling but leads to increased liver injury by reducing Nrf2 expression and GSH activity, ultimately resulting in increased cell death.

Thrombospondin-1 in maladaptive aging responses: a concept whose time has come.

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth, including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.

Blockade of thrombospondin-1 ameliorates high glucose-induced peritoneal fibrosis through downregulation of TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) is a profibrotic cytokine which induces mesothelial cell mesothelial-to-mesenchymal transition (MMT) and peritoneal fibrosis in patients receiving treatment of peritoneal dialysis. Because thrombospondin-1 (TSP-1) is able to activate latent TGF-ß1 in vivo, we investigated whether blockade of TSP-1 could modulate mesothelial cell MMT and ameliorate peritoneal fibrosis. METHODS: Human pleural mesothelial cells (Met-5A cells) were treated with TSP-1 and addition of TGF-ß1 neutralizing antibody to assess the effect of TSP-1 on MMT. Furthermore, TSP-1 blocking peptide Leu-Ser-Lys-Leu (LSKL) was applied to Met-5A cells treated with 4.25% d-glucose to determine its function in high glucose-induced MMT. Consequently, a uremic dialysate injection rat model was set up to confirm the results in vivo. RESULTS: Exposure of Met-5A cells to TSP-1 increased TGF-ß1 secretion, expression and bioactivity, triggered Smad3 phosphorylation, upregulated the expression of mesenchymal molecules including fibronectin, collagen type III, α-smooth muscle actin, Snail, and decreased calretinin expression. The effect was partially attenuated by TGF-ß1 neutralizing antibody. TSP-1 expression in Met-5A cells was increased by 4.25% d-glucose, followed by increased secretion and bioactivity of TGF-ß1, the onset of Smad3 phosphorylation and induction of MMT. LSKL significantly attenuated high glucose-mediated mesothelial cell MMT and ameliorated peritoneal fibrosis in uremic rats receiving dextrose dialysate injection. CONCLUSIONS: Taken together, these data demonstrated that TSP-1 contributes to mesothelial cell MMT by activating TGF-ß1/Smad3 signaling pathway and blockade of TSP-1 attenuates high glucose-mediated mesothelial cell MMT and peritoneal fibrosis.

Far upstream element-binding protein 1 facilitates hepatocellular carcinoma invasion and metastasis.

Previous research suggests that far upstream element-binding protein 1 (FUBP1) plays an important role in various tumors including epatocellular carcinoma (HCC). However, the role of FUBP1 in liver cancer remains controversial, and the regulatory pathway by FUBP1 awaits to be determined. This study aims to identify the role of FUBP1 in HCC progression. Our result shows that the high level of FUBP1 expression in HCC predicts poor prognosis after surgery. Overexpression of FUBP1 promotes HCC proliferation, invasion, and metastasis by activating transforming growth factor-ß (TGF-ß)/Smad pathway and enhancing epithelial-mesenchymal transition (EMT) in vitro and in vivo. Inhibitor of Thrombospondin-1 (LSKL) could inhibit HCC proliferation and invasion in vitro and in vivo by blocking the activation of TGF-ß/Smad pathway mediated by thrombospondin-1 (THBS1). Our study identified the critical role of FUBP1-THBS1-TGF-ß signaling axis in HCC and provides potentially new therapeutic modalities in HCC.

Selenium nanoparticles act as an intestinal p53 inhibitor mitigating chemotherapy-induced diarrhea in mice.

Selenium, at high-dose levels approaching its toxicity, protects tissues from dose-limiting toxicities of many cancer chemotherapeutics without compromising their therapeutic effects on tumors, there by allowing the delivery of higher chemotherapeutic doses to achieve increased cure rate. In this regard, selenium nanoparticles (SeNPs), which show the lowest toxicity among extensively investigated selenium compounds including methylselenocysteine and selenomethionine, are more promising for application. The key issue remains to be resolved is whether low-toxicity SeNPs possess a selective protective mechanism. p53 or p53-regulated thrombospondin-1 has each been confirmed to be an appropriate target for therapeutic suppression to reduce side effects of anticancer therapy. The present study demonstrated that SeNPs transiently suppressed the expression of many intestinal p53-associated genes in healthy mice. SeNPs did not interfere with tumor-suppressive effect of nedaplatin, a cisplatin analogue; however, effectively reduced nedaplatin-evoked diarrhea. Nedaplatin-induced diarrhea was associated with activation of intestinal p53 and high expression of intestinal thrombospondin-1. The preventive effect of SeNPs on nedaplatin-induced diarrhea was correlated with a powerful concomitant suppression of p53 and thrombospondin-1. Moreover, the high-dose SeNPs used in the present study did not suppress growth nor caused liver and kidney injuries as well as alterations of hematological parameters in healthy mice. Overall, the present study reveals that chemotherapeutic selectivity conferred by SeNPs involves a dual suppression of two well-documented targets, the p53 and thrombospondin-1, providing mechanistic and pharmacologic insights on low-toxicity SeNPs as a potential chemoprotectant for mitigating chemotherapy-induced diarrhea.

Deciphering the complex role of thrombospondin-1 in glioblastoma development.

We undertook a systematic study focused on the matricellular protein Thrombospondin-1 (THBS1) to uncover molecular mechanisms underlying the role of THBS1 in glioblastoma (GBM) development. THBS1 was found to be increased with glioma grades. Mechanistically, we show that the TGFß canonical pathway transcriptionally regulates THBS1, through SMAD3 binding to the THBS1 gene promoter. THBS1 silencing inhibits tumour cell invasion and growth, alone and in combination with anti-angiogenic therapy. Specific inhibition of the THBS1/CD47 interaction using an antagonist peptide decreases cell invasion. This is confirmed by CD47 knock-down experiments. RNA sequencing of patient-derived xenograft tissue from laser capture micro-dissected peripheral and central tumour areas demonstrates that THBS1 is one of the gene with the highest connectivity at the tumour borders. All in all, these data show that TGFß1 induces THBS1 expression via Smad3 which contributes to the invasive behaviour during GBM expansion. Furthermore, tumour cell-bound CD47 is implicated in this process.

Anti-inflammatory activity of CKC-containing cationic emulsion eye drop vehicles.

Purpose: Preservative-free cationic emulsion-based artificial tears (ATs) or drug vehicles are innovative eye drop formulations with tear film stabilization and drug delivery properties, and valuable in vivo anti-inflammatory and wound healing properties. These ATs have recently reached the market as ATs for the management of dry eye disease (DED) symptoms (i.e., Cationorm) or as a drug vehicle for cyclosporine (Ikervis). The aim of the present study was to explore the mechanism of action underlying the intrinsic anti-inflammatory and wound-healing efficacies harbored by the cationic emulsions of cetalkonium chloride (CE-CKC). Methods: The anti-inflammatory activity of two CE-CKC (0.002% and 0.005% CKC) emulsions was evaluated by assessing the expression of proinflammatory genes and the secretion of various markers in the following human cell types stressed by different agents: peripheral blood mononuclear cells (PBMCs; stimulation with anti-CD3/anti-CD28 or lipopolysaccharide (LPS)), CD4+ T lymphocytes (TCD4; stimulation with anti-CD3/anti-CD28), and a human corneal epithelial cell line (HCE-2; stimulation with LPS). The cells were incubated for 30 min with a 10% dilution of CE-CKC emulsions and then cultured without the emulsions for 24 h or 72 h in the presence of the various challenging agents. The supernatant was collected, and the secreted markers quantitated with flow cytometry or an enzyme-linked immunosorbent assay (ELISA). Gene expression of inflammatory markers was evaluated only in the PBMCs and HCE-2 cells stimulated with LPS. The in vitro protein kinase C (PKC) binding assay for IC50 determination was performed using standard procedures. Results: The CE-CKC emulsions decreased inflammatory gene expression in LPS-stimulated PBMCs (IFN-γ, IL-17A, CXCL-9, and TNFα) and LPS-stimulated HCE-2 cells (THBS1 and CCL2). Both CE-CKC emulsions inhibited the secretion of IL-17 (from anti-CD3/anti-CD28-stimulated TCD4), TNFα, IFN-γ, and IL-2 (from anti-CD3-/anti-CD28-stimulated PBMCs), and IL-6 and IL-8 (from LPS-stimulated HCE-2). The in vitro PKC binding assay revealed that CKC, the cationic agent, is a specific PKCα inhibitor. In addition, tyloxapol, another excipient, showed some anti-inflammatory activity on IL-6 and IL-8 in the LPS-stimulated HCE-2 cells. Conclusions: This study indicates that the CE-CKC emulsions are able to directly modulate the secretion and expression of proinflammatory cytokines and chemokines. The results also suggest that CKC and tyloxapol are pharmacologically active excipients with potentially beneficial effects in vivo. These data shed new light on the efficacy observed on the DED signs of these CE-CKC emulsions in clinical trials.

Thrombospondin-1 regulation of latent TGF-ß activation: A therapeutic target for fibrotic disease.

Transforming growth factor-ß (TGF-ß) is a central player in fibrotic disease. Clinical trials with global inhibitors of TGF-ß have been disappointing, suggesting that a more targeted approach is warranted. Conversion of the latent precursor to the biologically active form of TGF-ß represents a novel approach to selectively modulating TGF-ß in disease, as mechanisms employed to activate latent TGF-ß are typically cell, tissue, and/or disease specific. In this review, we will discuss the role of the matricellular protein, thrombospondin 1 (TSP-1), in regulation of latent TGF-ß activation and the use of an antagonist of TSP-1 mediated TGF-ß activation in a number of diverse fibrotic diseases. In particular, we will discuss the TSP-1/TGF-ß pathway in fibrotic complications of diabetes, liver fibrosis, and in multiple myeloma. We will also discuss emerging evidence for a role for TSP-1 in arterial remodeling, biomechanical modulation of TGF-ß activity, and in immune dysfunction. As TSP-1 expression is upregulated by factors induced in fibrotic disease, targeting the TSP-1/TGF-ß pathway potentially represents a more selective approach to controlling TGF-ß activity in disease.

Matricellular TSP-1 as a target of interest for impeding melanoma spreading: towards a therapeutic use for TAX2 peptide.

Thrombospondin-1 (TSP-1) is a matricellular glycoprotein known for being highly expressed within a tumor microenvironment, where it promotes an aggressive phenotype particularly by interacting with the CD47 cell-surface receptor. While it originates from the stromal compartment in many malignancies, melanoma is an exception as invasive and metastatic melanoma cells overexpress TSP-1. We recently demonstrated that a new molecular agent that selectively prevents TSP-1 binding to CD47, called TAX2, exhibits anti-cancer properties when administered systemically by decreasing viable tumor tissue within subcutaneous B16 melanoma allografts. At the same time, emerging evidence was published suggesting a contribution of TSP-1 in melanoma metastatic dissemination and resistance to treatment. Through a comprehensive systems biology approach based on multiple genomics and proteomics databases analyses, we first identified a TSP-1-centered interaction network that is overexpressed in metastatic melanoma. Then, we investigated the effects of disrupting TSP-1:CD47 interaction in A375 human malignant melanoma xenografts. In this model, TAX2 systemic administrations induce tumor necrosis by decreasing intra-tumoral blood flow, while concomitantly making tumors less infiltrative. Besides, TAX2 treatment also drastically inhibits B16F10 murine melanoma cells metastatic dissemination and growth in a syngeneic experimental model of lung metastasis, as demonstrated by histopathological analyses as well as longitudinal and quantitative µCT follow-up of metastatic progression. Altogether, the results obtained by combining bioinformatics and preclinical studies strongly suggest that targeting TSP-1/CD47 axis may represent a valuable therapeutic alternative for hampering melanoma spreading.

Thrombospondin-1 Affects Bovine Luteal Function via Transforming Growth Factor-Beta1-Dependent and Independent Actions.

Thrombospondin-1 (THBS1) and transforming growth factor-beta1 (TGFB1) are specifically up-regulated by prostaglandin F2alpha in mature corpus luteum (CL). This study examined the relationship between the expression of THBS1 and TGFB1 and the underlying mechanisms of their actions in luteal endothelial cells (ECs). TGFB1 stimulated SMAD2 phosphorylation and SERPINE1 levels in dose- and time-dependent manners in luteal EC. THBS1 also elevated SERPINE1; this effect was abolished by TGFB1 receptor-1 kinase inhibitor (SB431542). The findings here further imply that THBS1 activates TGFB1 in luteal ECs: THBS1 increased the effects of latent TGFB1 on phosphorylated SMAD (phospho-SMAD) 2 and SERPINE1. THBS1 silencing significantly decreased SERPINE1 and levels of phospho-SMAD2. Lastly, THBS1 actions on SERPINE1 were inhibited by LSKL peptide (TGFB1 activation inhibitor); LSKL also counteracted latent TGFB1-induced phospho-SMAD2. We found that TGFB1 up-regulated its own mRNA levels and those of THBS1. Both compounds generated apoptosis, but THBS1 was significantly more effective (2.5-fold). Notably, this effect of THBS1 was not mediated by TGFB1. THBS1 and TGFB1 also differed in their activation of p38 mitogen-activated protein kinase. Whereas TGFB1 rapidly induced phospho-p38, THBS1 had a delayed effect. Inhibition of p38 pathway by SB203580 did not modulate TGFB1 effect on cell viability, but it amplified THBS1 actions. THBS1-stimulated caspase-3 activation coincided with p38 phosphorylation, suggesting that caspase-induced DNA damage initiated p38 phosphorylation. The in vitro data suggest that a feed-forward loop exists between THBS1, TGFB1, and SERPINE1. Indeed all these three genes were similarly induced in the regressing CL. Their gene products can promote vascular instability, apoptosis, and matrix remodeling during luteolysis.

Thrombospondin-1 repression is mediated via distinct mechanisms in fibroblasts and epithelial cells.

Tumor-associated angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate here that the critical step in establishing the angiogenic capability of human tumor cells is the repression of a key secreted anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. In transformed epithelial cells, a signaling pathway leading from Ras to Tsp-1 repression induces the sequential activation of PI3 kinase, Rho and ROCK, leading to activation of Myc through phosphorylation, thereby enabling Myc to repress Tsp-1 transcription. In transformed fibroblasts, however, the repression of Tsp-1 can be achieved by an alternative mechanism involving inactivation of both p53 and pRb. We thus describe novel mechanisms by which the activation of oncogenes in epithelial cells and the inactivation of tumor suppressors in fibroblasts permits angiogenesis and, in turn, tumor formation.

A peptide antagonist of thrombospondin-1 promotes abdominal aortic aneurysm progression in the angiotensin II-infused apolipoprotein-E-deficient mouse.

OBJECTIVE: Interaction of the activating sequence in thrombospondin-1 (TSP-1) with the conserved sequence (leucine-serine-lysine-leucine [LSKL]) in the latency-associated peptide region of latent transforming growth factor (TGF)-ß complex is important in regulating TGF-ß1 activity. We aimed to assess the effect of blocking peptide LSKL on the progression of pre-established abdominal aortic aneurysm in angiotensin II-infused apolipoprotein E-deficient (ApoE(-/-)) mice. APPROACH AND RESULTS: Abdominal aortic aneurysm was established in 3-month-old male ApoE(-/-) mice with subcutaneous infusion of angiotensin II for 28 days. After this, mice received LSKL peptide or control SLLK (serine-leucine-leucine-lysine) peptide (4 mg/kg) via daily intraperitoneal injection for an additional 2 weeks. Administration of LSKL peptide promoted larger suprarenal aortic diameter, as determined by ultrasound and morphometric analysis, and stimulated more severe atherosclerosis within the aortic arch. In addition, mice receiving LSKL peptide exhibited elevated circulating proinflammatory cytokine levels and greater inflammatory cells within the suprarenal aorta compared with controls. Mice receiving LSKL peptide showed low plasma TGF-ß1 activity and low levels of aortic tissue phosphorylated to total Smad2/3. Aortic gene expression of TGF-ß receptor 1 (TGFBRI) and receptor 2 (TGFBRII), but not TGF-ß1 and thrombospondin-1, were lower in mice receiving LSKL peptide than controls. LSKL peptide administration was associated with greater aortic elastin fragmentation and lower expression and activity of the TGF-ß1-target gene lysyl oxidase like 1 (LOXL1). CONCLUSIONS: Attenuation of thrombospondin-1-directed activation of TGF-ß1 promotes abdominal aortic aneurysm and atherosclerosis progression in the angiotensin II-infused ApoE(-/-) mouse model.

Functions and transcriptional regulation of thrombospondins and their interrelationship with fibroblast growth factor-2 in bovine luteal cells.

Previously, we showed luteal stage-specific regulation of angiogenesis-modulating factors by prostaglandin F2 alpha (PGF2alpha). Fibroblast growth factor 2 (FGF2) and thrombospondins (THBSs) exhibited the most divergent profile of induction by PGF2alpha. We therefore examined the transcriptional regulation and roles of THBSs in luteal cells and studied their interaction with FGF2. THBSs and their receptors exhibited cell-specific expression: THBS1 was the predominant form in luteal endothelial cells (LEC), whereas luteinized granulosa cells (LGC) expressed mostly THBS2. CD36 was confined to LGC, but CD47 did not exhibit preferential expression between LEC and LGC. THBS1 and THBS2 were both stimulated in vitro by PGF2a and its analog in LGC. In contrast, luteinizing signals (LH and insulin) decreased the expression of THBS1, THBS2, and CD36. Importantly, LH increased FGF2 expression, suggesting that THBSs and FGF2 are conversely regulated. We found that FGF2 inhibited THBS1 and vice versa, and that THBS1 treatment decreased FGF2 expression, suggesting reciprocal inhibition. In agreement, ablation of THBS1 by specific small interference RNAs elevated FGF2 levels. THBS1 reduced LEC numbers and promoted apoptosis by activation of caspase-3. In contrast, FGF2 reduced basal and THBS1-induced caspase-3 levels. Consistent with these findings, small interference RNA silencing of THBS1 in luteal cells reduced the levels of active caspase-3 and improved the survival of cells when challenged with staurosporine. Taken together, these studies suggest that THBSs are suppressed during luteinization but are induced by PGF2alpha in luteolysis. THBS1 has antiangiogenic, proapoptotic properties; these, together with its ability to inhibit FGF2 expression and activity, can promote luteolysis.

[Role of thrombospondin-1 in the development of kidney diseases]. / Rôle de la thrombospondine-1 dans le développement des maladies rénales.

Thrombospondin-1 (TSP-1) is a 450-kDa matricellar glycoprotein. By its various domains, it can interact with various partners and exhibit anti-angiogenic, pro-apoptotic and immunomodulatory activities. TSP-1 is also a major endogenous activator of the pro-fibrotic growth factor TGF-ß. In healthy adult renal parenchyma, TSP-1 expression is very scarce and limited to Bowman's capsule and interstitium. During nephropathies, many cell types can express or secrete TSP-1 (mesangial, endothelial, smooth muscle, tubular cells, podocytes and fibroblasts) depending on the nature of injury and the evolutive stage of the disease. Inhibition of the different domains of TSP-1 using specific antibodies or peptides, blockade of TSP-1 expression by antisense oligonucleotides and use of knock-out mice, allowed to identify the role of TSP-1 in various models of experimental nephropathy. All these studies demonstrated a deleterious effect of TSP-1 on renal repair by inducing TGF-ß and fibrosis, decreasing VEGF and capillary density, and enhancing inflammatory cells recruitment. Thus, TSP-1 represents a potential therapeutic target for the management of chronic kidney diseases.

Blockade of CD47 increases survival of mice exposed to lethal total body irradiation.

Accidental or therapeutic total body exposure to ionizing radiation has profound pathophysiological consequences including acute radiation syndrome. Currently only investigational drugs are available in case of radiological or nuclear accidents or terrorism. Lack of selective radioprotectants for normal tissues also limits the therapeutic doses that can be delivered to treat cancers. CD47 is a receptor for the secreted protein thrombospondin-1. Blockade of thrombospondin-1 or CD47 provides local radioprotection of soft tissues and bone marrow. We now report that suppression of CD47 using an antisense morpholino increases survival of mice exposed to lethal total body irradiation. Increased survival is associated with increased peripheral circulating blood cell counts and increased proliferative capacity of bone marrow derived cells. Moreover, CD47 blockade decreased cell death while inducing a protective autophagy response in radiosensitive gastrointestinal tissues. Thus, CD47 is a new target for radiomitigation that prevents both hematopoietic and gastrointestinal radiation syndromes.

HGF regulates the activation of TGF-ß1 in rat hepatocytes and hepatic stellate cells.

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor-beta 1 (TGF-ß1). Specifically, we tested whether HGF decreases the levels of active TGF-ß1, and whether such decrease depends on the predominantly hepatocyte-secreted protease plasmin, and whether it depends on the TGF-ß1 activator thrombospondin-1 (TSP-1). With hepatocyte monocultures, we found HGF-induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI-1, TGF-ß1, and TIMP-2). With in vitro models of fibrotic liver (HSC-T6 hepatic stellate cells, or co-cultures of HSC-T6 and hepatocytes), we found high levels of fibrosis-associated proteins such as TSP-1, active TGF-ß1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP-1 protein cleavage; and decreased the levels of active TGF-ß1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF-ß1 activation and Collagen I. Meanwhile, the TSP-1-specific peptide inhibitor, LSKL, reduced TGF-ß1 to the same level as in the HGF-treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP-1 dependent pathway of TGF-ß1 activation. Therefore, HGF can decrease TGF-ß1 activation and TGF-ß1-dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP-1-dependent activation of TGF-ß1.

Molecular basis for the regulation of angiogenesis by thrombospondin-1 and -2.

Thrombospondins TSP-1 and TSP-2 are potent endogenous inhibitors of angiogenesis. They inhibit angiogenesis through direct effects on endothelial cell migration, proliferation, survival, and apoptosis and by antagonizing the activity of VEGF. Several of the membrane receptor systems and signal transduction molecules that mediate the effects of TSP-1 and TSP-2 have been elucidated. TSP-1 and TSP-2 exert their direct effects through CD36, CD47, and integrins. Recent data indicate that CD36 and ß1 integrins collaborate to transmit the signals that are initiated by TSP-1 and TSP-2. Furthermore, these receptors appear to associate with VEGFR2 to form a platform for the integration of positive and negative signals for angiogenesis. Cross talk between pro- and antiangiogenic signal transduction pathways may enable TSP-1 and TSP-2 to inhibit angiogenesis by antagonizing survival pathways while also activating apoptotic pathways. CD36 and CD47 are both involved in the suppression of nitric oxide (NO). Advances in understanding of the molecular regulation of angiogenesis by TSP have paved the way for innovations in experimental treatment of cancers and will likely continue to offer vast avenues for discovery in other disease processes as well.

GTP cyclohydrolase I/BH4 pathway protects EPCs via suppressing oxidative stress and thrombospondin-1 in salt-sensitive hypertension.

Endothelial progenitor cells (EPCs) are both reduced and dysfunctional in hypertension that correlates inversely with its mortality, but the mechanisms are poorly understood. Endothelial nitric oxide synthase (eNOS) critically regulates EPC mobilization and function but is uncoupled in salt-sensitive hypertension because of the reduced cofactor tetrahydrobiopterin (BH4). We tested the hypothesis that GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 de novo synthesis, protects EPCs and its function in deoxycorticosterone acetate (DOCA)-salt mice. EPCs were isolated from peripheral blood and bone marrow of wild-type (WT), WT DOCA-salt, endothelial-specific GTPCH transgenic (Tg-GCH), GTPCH transgenic DOCA-salt, and BH4-deficient hph-1 mice. In WT DOCA-salt and hph-1 mice, EPCs were significantly decreased with impaired angiogenesis and adhesion, which were restored in Tg-GCH DOCA-salt mice. Superoxide (O2⁻) and nitric oxide (NO) levels in EPCs were elevated and reduced, respectively, in WT DOCA-salt and hph-1 mice; both were rescued in Tg-GCH DOCA-salt mice. eNOS(-/-)/GCH(+/-) hybrid mice demonstrated that GTPCH preserved the circulating EPC number, reduced intracellular O2⁻ in EPCs, and ameliorated EPC dysfunction independent of eNOS in DOCA-salt hypertension. Secreted thrombospondin-1 (TSP-1; a potent angiogenesis inhibitor) from EPCs was elevated in WT DOCA-salt and hph-1 but not DOCA-salt Tg-GCH mice. In vitro treatment with BH4, polyethylene glycol-superoxide dismutase (PEG-SOD), or Nomega-nitro-L-arginine (L-NNA) significantly augmented NO and reduced TSP-1 and O2⁻ levels from EPCs of WT DOCA-salt mice. These results demonstrated, for the first time, that the GTPCH/BH4 pathway critically regulates EPC number and function in DOCA-salt hypertensive mice, at least in part, via suppressing TSP-1 expression and oxidative stress.