Thrombospondin-1 Protein Research Articles

Chronic Delivery of a Thrombospondin-1 Mimetic Decreases Skeletal Muscle Capillarity in Mice

Angiogenesis is an essential process for normal skeletal muscle function. There is a growing body of evidence suggesting that thrombospondin-1 (TSP-1), a potent antiangiogenic protein in tumorigenesis, is an important regulator of both physiological and pathological skeletal muscle angiogenesis. We tested the hypothesis that chronic exposure to a TSP-1 mimetic (ABT-510), which targets the CD36 TSP-1 receptor, would decrease skeletal muscle capillarity as well as alter the balance between positive and negative angiogenic proteins under basal conditions. Osmotic minipumps with either ABT-510 or vehicle (5% dextrose) were implanted subcutaneously in the subscapular region of C57/BL6 mice for 14 days. When compared to the vehicle treated mice, the ABT-510 group had a 20% decrease in capillarity in the superficial region of the gastrocnemius (GA), 11% decrease in the plantaris (PLT), and a 35% decrease in the soleus (SOL). ABT-510 also decreased muscle protein expression of vascular endothelial growth factor (VEGF) in both the GA (−140%) and SOL (−62%); however there was no change in VEGF in the PLT. Serum VEGF was not altered in ABT-510 treated animals. Endogenous TSP-1 protein expression in all muscles remained unaltered. Tunnel staining revealed no difference in muscle apoptosis between ABT-510 and vehicle treated groups. These data provide evidence that the anti-angiogenic effects of TSP-1 are mediated, at least in part, via the CD36 receptor. It also suggests that under physiologic conditions the TSP-1/CD36 axis plays a role in regulating basal skeletal muscle microvessel density.

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.

Thrombospondin-1 Silencing Down-Regulates Integrin Expression Levels in Human Anaplastic Thyroid Cancer Cells with BRAFV600E: New Insights in the Host Tissue Adaptation and Homeostasis of Tumor Microenvironment

Background and Rationale: Anaplastic thyroid cancer (ATC) is characterized by pleomorphic cells, has a poor prognosis, is highly devastating disease, and is not curable. No reliable biomarkers of metastatic potential, helpful for early diagnosis of ATC and therapeutic response have been found yet. Thrombospondin-1 (TSP-1) plays a fundamental role in cancer progression by regulating cell stromal cross-talk in the tumor microenvironment.Goals: Our goal was to understand whether TSP-1 could affect protein levels of its integrin receptors (e.g., ITGα3, α6, and β1) and cell morphology in BRAFV600E-ATC cells in vitro and in vivo.Experimental Design: Anaplastic thyroid cancer-derived cell cultures and western blotting were used to assess integrin protein expression upon TSP-1 silencing. Immunohistochemistry was performed on orthotopic primary human ATC and metastatic ATC in lung tissue to compare TSP-1 and integrin protein expression levels.Results: TSP-1 knock-down down-regulates ITGα3, α6, and β1 in BRAFV600E-human ATC cells. BRAFV600E-ATC cells with TSP-1 knock-down were rounded compared to control cells, which displayed a spread morphology. TSP-1 knock-down also reduced TSP-1, ITGα3, α6, and β1 protein expression levels in vivo in the ATC microenvironment, which is enriched in stromal and inflammatory cells.Conclusion: TSP-1 silencing causes changes in ITG levels and ATC cell morphology. The assessment of TSP-1 and ITG levels might contribute to earlier metastatic potential of BRAFV600E-positive aggressive thyroid cancers, and allow improved patient selection for clinical trials.

Thrombospondin-1 expression in melanoma is blocked by methylation and targeted reversal by 5-Aza-deoxycytidine suppresses angiogenesis

BackgroundReversibility of aberrant methylation via pharmacological means is an attractive target for therapies through epigenetic reprogramming. To establish that pharmacologic reversal of methylation could result in functional inhibition of angiogenesis, we undertook in vitro and in vivo studies of thrombospondin-1 (TSP1), a known inhibitor of angiogenesis. TSP1 is methylated in several malignancies, and can inhibit angiogenesis in melanoma xenografts. We analyzed effects of 5-Aza-deoxycytidine (5-Aza-dC) on melanoma cells in vitro to confirm reversal of promoter hypermethylation and restoration of TSP1 expression. We then investigated the effects of TSP1 expression on new blood vessel formation and tumor growth in vivo. Finally, to determine potential for clinical translation, the methylation status of TSP1 promoter regions of nevi and melanoma tissues was investigated. Results5-Aza-dC reduced DNA (cytosine-5)-methyltransferase 1 (DNMT1) protein, reversed promoter hypermethylation, and restored TSP1 expression in five melanoma cell lines, while having no effect on TSP1 protein levels in normal human melanocytes. In in vivo neovascularization studies, mice were implanted with melanoma cells (A375) either untreated or treated with 5Aza-dC. Vessels at tumor sites were counted by an observer blinded to treatments and the number of tumor vessels was significantly decreased at pretreated tumor sites. This difference occurred before a significant difference in tumor volumes was seen, yet in further studies the average tumor volume in mice treated in vivo with 5-Aza-dC was decreased by 55% compared to untreated controls. Knockdown of TSP1 expression with shRNA enhanced tumor-induced angiogenesis by 68%. Analyses of promoter methylation status of TSP1 in tumors derived from untreated and treated mice identified 67% of tumors from untreated and 17% of tumors from treated mice with partial methylation consistent with the methylation specific PCR analysis of A375 cells. Examination of methylation patterns in the promoter of TSP1 and comparison of aberrantly methylated TSP1 in melanoma with non-malignant nevi identified a significantly higher frequency of promoter methylation in tumor samples from melanoma patients. ConclusionsPharmacological reversal of methylation silenced TSP1 had functional biological consequences in enhancing angiogenesis inhibition and inducing antitumor effects to decrease murine melanoma growth. Angiogenesis inhibition is an additional mechanism by which epigenetic modulators can have antitumor effects.

Autocrine stimulation of clear-cell renal carcinoma cell migration in hypoxia via HIF-independent suppression of thrombospondin-1

Thrombospondin-1 is a matricellular protein with potent antitumour activities, the levels of which determine the fate of many different tumours, including renal carcinomas. However, the factors that regulate this protein remain unclear. In renal carcinomas, hypoxic conditions enhance the expression of angiogenic factors that help adapt tumour cells to their hostile environment. Therefore, we hypothesized that anti-angiogenic factors should correspondingly be dampened. Indeed, we found that hypoxia decreased the thrombospondin-1 protein in several clear cell renal carcinoma cell lines (ccRCC), although no transcriptional regulation was observed. Furthermore, we proved that hypoxia stimulates multiple signals that independently contribute to diminish thrombospondin-1 in ccRCC, which include a decrease in the activity of oxygen-dependent prolylhydroxylases (PHDs) and activation of the PI3K/Akt signalling pathway. In addition, thrombospondin-1 regulation in hypoxia proved to be important for ccRCC cell migration and invasion.

Endogenous Thrombospondin-1 Regulates Leukocyte Recruitment and Activation and Accelerates Death from Systemic Candidiasis

Disseminated Candida albicans infection results in high morbidity and mortality despite treatment with existing antifungal drugs. Recent studies suggest that modulating the host immune response can improve survival, but specific host targets for accomplishing this goal remain to be identified. The extracellular matrix protein thrombospondin-1 is released at sites of tissue injury and modulates several immune functions, but its role in C. albicans pathogenesis has not been investigated. Here, we show that mice lacking thrombospondin-1 have an advantage in surviving disseminated candidiasis and more efficiently clear the initial colonization from kidneys despite exhibiting fewer infiltrating leukocytes. By examining local and systemic cytokine responses to C. albicans and other standard inflammatory stimuli, we identify a crucial function of phagocytes in this enhanced resistance. Subcutaneous air pouch and systemic candidiasis models demonstrated that endogenous thrombospondin-1 enhances the early innate immune response against C. albicans and promotes activation of inflammatory macrophages (inducible nitric oxide synthase+, IL-6high, TNF-αhigh, IL-10low), release of the chemokines MIP-2, JE, MIP-1α, and RANTES, and CXCR2-driven polymorphonuclear leukocytes recruitment. However, thrombospondin-1 inhibited the phagocytic capacity of inflammatory leukocytes in vivo and in vitro, resulting in increased fungal burden in the kidney and increased mortality in wild type mice. Thus, thrombospondin-1 enhances the pathogenesis of disseminated candidiasis by creating an imbalance in the host immune response that ultimately leads to reduced phagocytic function, impaired fungal clearance, and increased mortality. Conversely, inhibitors of thrombospondin-1 may be useful drugs to improve patient recovery from disseminated candidiasis.

HGF regulates the activation of TGF‐β1 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-β1). Specifically, we tested whether HGF decreases the levels of active TGF-β1, and whether such decrease depends on the predominantly hepatocyte-secreted protease plasmin, and whether it depends on the TGF-β1 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-β1, 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-β1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP-1 protein cleavage; and decreased the levels of active TGF-β1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF-β1 activation and Collagen I. Meanwhile, the TSP-1-specific peptide inhibitor, LSKL, reduced TGF-β1 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-β1 activation. Therefore, HGF can decrease TGF-β1 activation and TGF-β1-dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP-1-dependent activation of TGF-β1.

Thrombospondin-1 Regulates Blood Flow via CD47 Receptor–Mediated Activation of NADPH Oxidase 1

Although the matricellular protein thrombospondin-1 (TSP1) is highly expressed in the vessel wall in response to injury, its pathophysiological role in the development of vascular disease is poorly understood. This study was designed to test the hypothesis that TSP1 stimulates reactive oxygen species production in vascular smooth muscle cells and induces vascular dysfunction by promoting oxidative stress. Nanomolar concentrations of TSP1 found in human vascular disease robustly stimulated superoxide (O(2)(•-)) levels in vascular smooth muscle cells at both cellular and tissue level as measured by cytochrome c and electron paramagnetic resonance. A peptide mimicking the C terminus of TSP1 known to specifically bind CD47 recapitulated this response. Transcriptional knockdown of CD47 and a monoclonal inhibitory CD47 antibody abrogated TSP1-triggered O(2)(•-) in vitro and ex vivo. TSP1 treatment of vascular smooth muscle cells activated phospholipase C and protein kinase C, resulting in phosphorylation of the NADPH oxidase organizer subunit p47(phox) and subsequent Nox1 activation, leading to impairment of arterial vasodilatation ex vivo. Further, we observed that blockade of CD47 and NADPH oxidase 1 gene silencing in vivo in rats improves TSP1-induced impairment of tissue blood flow after ischemia reperfusion. Our data suggest a highly regulated process of reactive oxygen species stimulation and blood flow regulation promoted through a direct TSP1/CD47-mediated activation of Nox1. This is the first report, to our knowledge, of a matricellular protein acting as a ligand for NADPH oxidase activation and through specific engagement of integrin-associated protein CD47.

The Absence of CD47 Promotes Nerve Fiber Growth from Cultured Ventral Mesencephalic Dopamine Neurons

In ventral mesencephalic organotypic tissue cultures, two timely separated sequences of nerve fiber growth have been observed. The first appearing nerve fiber pattern is a long-distance outgrowth that occurs before astrocytes start to proliferate and migrate to form an astrocytic monolayer that finally surrounds the tissue slice. These long-distance growing nerve fibers are retracted as the astrocytes migrate, and are followed by a secondary outgrowth. The secondary outgrowth is persistent in time but reaches short distances, comparable with outgrowth seen from a dopaminergic graft implanted to the brain. The present study was focused on the interaction between the astrocytes and the long-distance growing non-glial associated nerve fibers. Cross talk between astroglia and neurite formation might occur through the integrin-associated protein CD47. CD47 serves as a ligand for signal regulatory protein (SIRP) α and as a receptor for the extracellular matrix protein thrombospondin-1 (TSP-1). Embryonic day 14 ventral mesencephalic tissue from CD47+/+ and CD47−/− mice was used to investigate astrocytic migration and the tyrosine hydroxylase (TH) –positive outgrowth that occurred remote from the astrocytes. TH-immunohistochemistry demonstrated that the non-glial-associated nerve fiber outgrowth in CD47−/− cultures reached significantly longer distances and higher density compared to nerve fibers formed in CD47+/+ cultures at 14 days in vitro. These nerve fibers often had a dotted appearance in CD47+/+ cultures. No difference in the astrocytic migration was observed. Further investigations revealed that the presence of CD47 in control culture did neither hamper non-glial-associated growth through SIRPα nor through TSP-1 since similar outgrowth was found in SIRPα mutant cultures and in CD47+/+ cultures treated with blocking antibodies against the TSP-1, respectively, as in the control cultures. In conclusion, long-distance growing nerve fiber formation is promoted by the absence of CD47, even though the presence of astrocytes is not inhibited.

Abstract 368: Dynamic Receptor-Ligand Activation of Nox1 Regulates Blood Flow via a Novel TSP1-CD47 Signaling Axis

Reactive oxygen species (ROS) play an important role in ischemia-reperfusion injury but the mechanisms controlling this phenomenon remain elusive. Our previous studies demonstrated that matrix protein thrombospondin-1 (TSP1) at concentrations found in human disease (2.2 nM) is a rapid and potent stimulator of superoxide (O2•–) production in vascular smooth muscle cells (VSMC). However, the mechanism by which TSP1 mediates this response and its role in reperfusion-induced impairment of tissue perfusion are unknown. Here, we show that blockade of integrin-associated CD47 signaling (pmol O2•–/mg protein/min: 6.7±0.6, 10.2±0.7*, 6.0±0.5, and 7.1±0.7 for isotype control + vehicle, isotype control + TSP1, CD47 blocking antibody + vehicle and CD47 blocking antibody + TSP1; *p<0.05 vs isotype control + vehicle) and gene silencing of NADPH oxidase 1 (Nox1) abrogated TSP1-stimulated O2•– in VSMC. By luciferase reporter array, we identified myocyte enhancer factor 2 (MEF2) as a downstream effector of TSP1 and show that knockdown of MEF2A suppresses TSP1-stimulated O2•– (pmol O2•–/mg protein/min: 4.2±0.3, 6.3±0.4*, 4.1±0.3, and 4.6±0.3 for scrambled siRNA + vehicle, scrambled siRNA + TSP1, MEF2A siRNA + vehicle and MEF2A siRNA + TSP1;*p<0.05 vs scrambled siRNA + vehicle). Further, TSP1 rapidly phosphorylated the Nox organizer subunit p47phox in VSMC. Electron paramagnetic resonance demonstrated that TSP1 stimulates O2•– in wild type (WT), but not in Nox1y/-, endothelium-free aortic rings (CM• radical formation (x10 3): 20.9±11.1, 86.8±20.5*, 20.2±28.6 and 29.6±22.2 for WT + vehicle, WT + TSP1, Nox1y/- + vehicle, and Nox1y/- + TSP1; *p<0.05 vs WT + vehicle). Finally, intravenous TSP1 acutely decreased recovery of hindlimb blood flow following ischemia-reperfusion as measured by real time laser Doppler in control, but not in Nox1 gene silenced, rats. Our findings suggest a highly-regulated role of matricellular TSP1 as a stimulator of a novel CD47-MEF2A-Nox1 signaling axis and ROS and support a role for the matrix protein as a blood flow attenuator in cardiovascular disease.

Inhibitory signaling through signal regulatory protein-α is not sufficient to explain the antitumor activities of CD47 antibodies

CD47 is a receptor for the secreted protein thrombospondin-1 and a counterreceptor for two members of the signal regulatory protein (SIRP) family. These interactions play important roles in hemostasis, cardiovascular pathophysiology, ischemic injuries, inflammation, radiation injuries, and cancer. Correspondingly, antibodies that block thrombospondin-1 or SIRPα binding and antisense oligonucleotide analogs that suppress CD47 expression have shown efficacy in treating rodent and porcine models of these diseases.

MicroRNAs related to angiogenesis are dysregulated in endometrioid endometrial cancer

Which is the role of microRNAs (miRNAs) related to several angiogenesis regulators such as VEGF-A (Vascular endothelial growth factor-A) and TSP-1 (Thrombospondin-1) in endometrial cancer? A dysregulated expression of miRNAs related to angiogenesis and an increase in the VEGF-A levels were observed in endometrial cancer in comparison with control. The different expression of miRNAs could modulate the expression of angiogenic and antiangiogenic factors, which may play an important role in the pathogenesis of endometrial cancer. Dysregulated miRNA expression has been previously evaluated in endometrial adenocarcinoma. To the best of our knowledge, there are no studies on the relationship between angiogenic factors and miRNAs in endometrial cancer. Case-control study: 41 patients with histologically proven endometrioid endometrial cancer and 56 women without endometrial cancer. RNAs isolated from tissue samples were analyzed using the GeneChip miRNA 2.0 Array platform (Affymetrix). TaqMan qRT-PCR was used to assess the expression of the selected miRNAs related to angiogenesis (miR-15b, -16, -17-5p, -20a, -21, -125a, -200b, -210, -214*, -221, -222 and -424), and VEGF-A and TSP-1 mRNAs were assessed by qRT-PCR using SYBR Green. Protein levels were quantified by ELISAs. Compared with the miRNAs in the control endometrium, eight miRNAs (miR-15b, -17-5p, -20a, -125a, -214*, -221, -222 and -424) were significantly down-regulated and two miRNAs (miR-200b and -210) were significantly up-regulated in the cancerous endometrium. A significant increase in VEGF-A mRNA and protein expression and in TSP-1 protein levels (P <0.01) was observed in endometrial cancer. Moreover, significant inverse correlations between VEGF-A protein levels and miR-20a, -125a, -214*, -221, -222 and -424 were detected. In contrast, a positive correlation was observed between VEGF-A and miR-200b and -210. Furthermore, stage IB endometrial cancer was associated with a higher VEGF-A protein/mRNA ratio and lower miR-214*, -221 and -222 expression in comparison with stage IA. Future functional studies (e.g. miRNA inhibition or ectopic overexpression) in cell culture models are needed to confirm the VEGF targeting by the miRNAs found in the present study. The findings of the present study have potential implications for diagnostics and therapeutics of endometrial carcinoma. This work was supported by research grants from the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, PI080185, PI0110091) and Red RECAVA (RD06/0014/0004), by Consellería de Sanidad (AP-141/11) and Consellería de Educación (PROMETEO/2011/027), Generalitat Valenciana, by Beca Fibrinolisis 2009 and Becario 2010, 2011 from Fundación Española de Trombosis y Hemostasia and by the Fundación Investigación Hospital La Fe, Spain. None of the authors have any conflicts of interest.

Expression of apoptosis associated proteins Survivin, Livin and Thrombospondin‐1 in Burkitt lymphoma

Burkitt lymphoma (BL) accounts for 80% of Non-Hodgkin lymphoma in Uganda. The tumour is fast growing with a very short doubling time. Inhibition of apoptosis, achieved through the apoptosis inhibitor family of proteins enhances survival of cancer cells. The aim of this study was to explore the expression of apoptosis related proteins Survivin, Livin and Thrombospondin-1 (TSP-1) in BL and comparing it with the expression in reactive follicular hyperplasia. Among the BL cases, survivin was positive in 37 (75%) and negative in 12 (25%). Livin staining was positive in 23 (48%) and negative in 25 (52%) of the BL cases. TSP-1 was positive in 40 (87%) and negative in 6 (13%) of the BL cases. All the 10 reactive lymphoid hyperplasia (RLH) were positive for survivin, livin expression was found in 4 (40%), whereas TSP-1 was positive in 7 (70%) of the RLH cases. Among the BL, there was no statistically significant association between survivin and livin expression (p = 0.6), livin and TSP-1 (p = 1.0) or survivin and TSP-1 (p = 0.6). Likewise no statistical association was found between the expressions of the proteins in RLH. There was no statistically significant difference between the expression frequency of the apoptosis associated proteins examined in BL and RLH. In conclusion, we have demonstrated the expression of inhibitor of apoptosis proteins, survivin and Livin and the pro-apoptotic protein TSP-1 in endemic BL. No significant difference was found between BL and RLH.

Activated CD47 regulates multiple vascular and stress responses: implications for acute kidney injury and its management.

Ischemia-reperfusion injury (IRI) remains a significant source of early and delayed renal transplant failure. Therapeutic interventions have yet to resolve this ongoing clinical challenge although the reasons for this remain unclear. The cell surface receptor CD47 is widely expressed on vascular cells and in tissues. It has one known soluble ligand, the stress-released matricellular protein thrombospondin-1 (TSP1). The TSP1-CD47 ligand receptor axis controls a number of important cellular processes, inhibiting survival factors such as nitric oxide, cGMP, cAMP, and VEGF, while activating injurious pathways such as production of reactive oxygen species. A role of CD47 in renal IRI was recently revealed by the finding that the TSP1-CD47 axis is induced in renal tubular epithelial cells (RTEC) under hypoxia and following IRI. The absence of CD47 in knockout mice increases survival, mitigates RTEC damage, and prevents subsequent kidney failure. Conversely, therapeutic blockade of TSP1-CD47 signaling provides these same advantages to wild-type animals. Together, these findings suggest an important role for CD47 in renal IRI as a proximate promoter of injury and as a novel therapeutic target.

Thrombospondin-1 and Transforming Growth Factor-β1 Levels in Prolactinoma and their Clinical Significance

To investigate the roles of angiogenesis, thrombospondin-1 (TSP-1) and transforming growth factor-β1 (TGF-β1) in invasive and noninvasive prolactinoma. TSP-1 and TGF-β1 protein were examined using immunohistochemistry and Western blotting in 81 prolactinomas. Angiogenesis was assessed by measuring microvessel density via CD34 immunostaining. Microvessel density was significantly higher in invasive prolactinomas than in noninvasive prolactinomas. Immunohistochemistry demonstrated that significantly fewer invasive prolactinomas were positive for TSP-1 compared with noninvasive prolactinomas (17.9% versus 50.0%, respectively), and significantly higher numbers of invasive prolactinomas were positive for TGF-β1 compared with noninvasive prolactinomas (82.1% versus 42.9%, respectively). Microvessel density was significantly lower in TSP-1-positive prolactinomas than in TSP-1-negative prolactinomas, and significantly higher in TGF-β1-positive prolactinomas than in TGF-β1-negative prolactinomas. These results suggest a close relationship between angiogenesis and tumour invasiveness in prolactinoma. TSP-1 and TGF-β1 may play important roles in the progression of prolactinoma, by affecting angiogenesis.

Thrombospondin-4 Contributes to Spinal Sensitization and Neuropathic Pain States

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Myocardial Protection by Salvia Miltiorrhiza Injection in Streptozotocin-Induced Diabetic Rats through Attenuation of Expression of Thrombospondin-1 and Transforming Growth Factor-β1

To investigate the myocardially protective effects of Salvia miltiorrhiza injection in streptozotocin-induced diabetic rats and the possible mechanisms involved. Adult male Sprague-Dawley rats were randomized into three groups (n = 10 per group): diabetes, no treatment (Sm-); diabetes, S. miltiorrhiza injection (Sm+); control (no diabetes; saline treatment). After model induction and 4 weeks' treatment, heart function of five rats from each group was tested by Langendorff isolated in vivo heart perfusion. In the remaining rats, pathological changes of the myocardium were observed by haematoxylin and eosin staining, and protein levels of thrombospondin-1 (TSP-1) and transforming growth factor-β1 (TGF-β1) were assessed by immunohistochemistry. Left ventricular systolic end pressure and left ventricular developed pressure were significantly improved in the Sm+ group compared with the Sm- group. Pathological changes were ameliorated through significantly reduced TSP-1 and TGF-β1 protein levels. S. miltiorrhiza injection may improve the heart function of diabetic rats and protect against cardiomyopathy by downregulating TSP-1 and TGF-β1 in myocardial tissue.

A Thrombospondin-Dependent Pathway for a Protective ER Stress Response

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

Activity of neoadjuvant bevacizumab at the time of surgery after 6 weeks of preoperative cessation: A translational study.

e14099 Background: The use of bevacizumab (BV), a monoclonal antibody against VEGF, together with an increase in hepatic resection has improved the outcome of colorectal cancer (CRC) patients with liver metastasis. However, despite the frequent use of BV, its accurate timing prior to liver resection to avoid surgical complications and to allow for hepatic regeneration remains discussed controversially. In this context, it is unclear to which extent VEGF is complexed by BV at the time of surgery. Methods: 42 CRC patients with liver metastases, potentially curable by resection, were included. Patients received neoadjuvant chemotherapy (CTx) either with or without concomitant biweekly BV. The last cycle of therapy did not include BV, resulting in a median 6 weeks between the last administration of BV and surgery. Plasma and wound fluid were obtained perioperatively. Immunoprecipitation was applied to determine the proportion of BV-bound VEGF. Alternative angiogenesis factors were evaluated. Results: No increased perioperative complications in BV treated patients were observed. At the time of surgery, the majority of plasma VEGF was inactivated by BV. In wound fluid, the actual site of wound healing and hepatic regeneration, VEGF was remarkably low and also complexed by BV. The anti-angiogenic protein thrombospondin-1 was reduced in BV treated patients. Conclusions: These data document that after the clinical standard cessation time of 6 weeks, BV is still fully active and binds to the majority of circulating and local (wound fluid) VEGF. Despite the potent VEGF inactivation at the time of liver resection no substantial increase in perioperative morbidity after neoadjuvant BV therapy has been documented in our study and the current literature. Compensatory mechanisms, such as thrombospondin-1 reduction during BV treatment may enable wound healing.

Do the Fibrin Architecture and Leukocyte Content Influence the Growth Factor Release of Platelet Concentrates? An Evidence-based Answer Comparing a Pure Platelet-Rich Plasma (P-PRP) Gel and a Leukocyte- and Platelet-Rich Fibrin (L-PRF)

Platelet concentrates for surgical use are tools of regenerative medicine designed for the local release of platelet growth factors into a surgical or wounded site, in order to stimulate tissue healing or regeneration. Leukocyte content and fibrin architecture are 2 key characteristics of all platelet concentrates and allow to classify these technologies in 4 families, but very little is known about the impact of these 2 parameters on the intrinsic biology of these products. In this demonstration, we highlight some outstanding differences in the growth factor and matrix protein release between 2 families of platelet concentrate: Pure Platelet-Rich Plasma (P-PRP, here the Anitua's PRGF - Preparation Rich in Growth Factors - technique) and Leukocyte- and Platelet-Rich Fibrin (L-PRF, here the Choukroun's method). These 2 families are the extreme opposites in terms of fibrin architecture and leukocyte content. The slow release of 3 key growth factors (Transforming Growth Factor β1 (TGFβ1), Platelet-Derived Growth Factor AB (PDGF-AB) and Vascular Endothelial Growth Factor (VEGF)) and matrix proteins (fibronectin, vitronectin and thrombospondin-1) from the L-PRF and P-PRP gel membranes in culture medium is described and discussed. During 7 days, the L-PRF membranes slowly release significantly larger amounts of all these molecules than the P-PRP gel membranes, and the 2 products display different release patterns. In both platelet concentrates, vitronectin is the sole molecule to be released almost completely after only 4 hours, suggesting that this molecule is not trapped in the fibrin matrix and not produced by the leukocytes. Moreover the P-PRP gel membranes completely dissolve in the culture medium after less than 5 days only, while the L-PRF membranes are still intact after 7 days. This simple demonstration shows that the polymerization and final architecture of the fibrin matrix considerably influence the strength and the growth factor trapping/release potential of the membrane. It also suggests that the leukocyte populations have a strong influence on the release of some growth factors, particularly TGFβ1. Finally, the various platelet concentrates present very different biological characteristics, and an accurate definition and characterization of the different families of product is a key issue for a better understanding and comparison of the reported clinical effects of these surgical adjuvants.