Thrombospondin-1 Expression Research Articles

Profile of fibrosis-related gene transcripts and megakaryocytic changes in the bone marrow of myelodysplastic syndromes with fibrosis.

Bone marrow fibrosis (MF) in myelodysplastic syndromes (MDS) is associated with an adverse prognosis. It is likely that molecular changes similar to those in primary myelofibrosis (PMF) lead to MDS-MF, but gene expression profiling has not yet been carried out. We analysed bone marrow biopsy samples by PCR, qPCR (45 transcripts per sample), and immunohistochemistry from MDS patients with fibrosis (n = 70/119; including 19/70 MF0 > MF follow-up cases), MDS without fibrosis (n = 49/119), and 33 controls. SRSF2 and JAK2 mutations were detectable in up to 13% including 3/19 follow-up cases with evidence of clonal evolution during MF progression. MDS-MF showed increased expression of thrombospondin 1 (THBS1), TIMP metallopeptidase inhibitor 1 (TIMP1), transforming growth factor beta 1 (TGFB1), matrix metallopeptidases 2 and 14 (MMP2, MMP14), SMAD family members 3 and 4 (SMAD3, SMAD4), and miR-146b. Paralleling MF progression, a subfraction of follow-up cases showed megakaryocytic changes with increased CD42b+ pro-platelet deposition in the bone marrow. In summary, fibrosis in MDS-MF and PMF shows many molecular and morphological similarities.

Role of Thrombospondin-1 and Nuclear Factor-κB Signaling Pathways in Antiangiogenesis of Infantile Hemangioma

Propranolol is the first-line drug for treatment of infantile hemangioma. However, its mechanism of action remains unclear. Nuclear factor-κB is highly expressed in tumors, directly or indirectly promoting angiogenesis. Thrombospondin-1 is the most important antiangiogenesis protein in vivo. These proteins mediate signaling pathways, probably playing an important role in hemangioma treatment. This study explored the synergistic regulation of thrombospondin-1 and nuclear factor-κB signaling pathways in the treatment of hemangioma with propranolol. The hemangioma-derived endothelial cells were sorted out from the specimens of proliferative hemangioma by flow cytometry. Furthermore, a BALB/c nude mouse hemangioma model was established. Viability and proliferation of hemangioma-derived endothelial cells and the role of thrombospondin-1 and nuclear factor-κB signaling pathways were observed after propranolol administration in vitro and in vivo. The expression of thrombospondin-1 and its receptor CD36 in hemangioma-derived endothelial cells gradually increased with the increase in propranolol concentration, whereas the expression of nuclear factor-κBp65, phosphorylated inhibitor of κB alpha (p-IκBα), and phosphorylated inhibitor of nuclear factor-κB kinase beta (p-IκKβ) weakened gradually (p < 0.05). In vivo, the tumors shrank gradually after propranolol treatment, with an increase in thrombospondin-1 and CD36 and a decrease in nuclear factor-κBp65, p-IκBα, and p-IκKβ (p < 0.05). Glucocorticoid improved the antiangiogenesis mediated by thrombospondin-1/CD36 and inhibited the angiogenesis mediated by nuclear factor-κB/IκB (p < 0.05). Negative regulation occurred between the two signaling pathways. The treatment of infantile hemangioma with propranolol is promising to promote thrombospondin-1-mediated antiangiogenesis and to block nuclear factor-κB-mediated angiogenesis.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six days post-fertilization (6dpf) human embryos were co-cultured with Ishikawa cells for 12, 24 (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 145 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expressions were validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. None. This in vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. A.A. was supported by a grant from the 'Instituut voor Innovatie door Wetenschap en Technologie' (IWT, 121716, Flanders, Belgium). This work was supported by the 'Wetenschappelijk Fonds Willy Gepts' (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

Abstract 230: Matrix Regulation of Lymphangiogenesis in Atherosclerosis

Background: Atherosclerosis is a consequence of an imbalance of cellular uptake of lipoproteins versus removal of cholesterol from the arterial wall. The lymphatic vasculature represents the primary route of cholesterol removal from atherosclerotic vessels. Previous studies have linked matrix protein thrombospondin-1 (TSP1), a well-known inhibitor of angiogenesis, with human atherosclerotic disease; however the mechanisms by which TSP1 contributes to atherosclerosis and its effect on arterial lymphangiogenesis remain unknown. Methods: The effect of TSP1 on human lymphatic endothelial cell (LEC) proliferation, migration and tube formation was investigated in vitro . TSP1 protein expression was analyzed in human and murine (ApoE -/- , 12 weeks Western diet) atherosclerotic aortic tissue and lesion free segments using Western blotting. The role of TSP1 and its cognate receptor CD47 in the pathogenesis of atherosclerosis was investigated using knockout animals. Results: TSP1 expression was significantly increased in both human and ApoE -/- atherosclerotic arteries compared to plaque free segments in human and wild type aortic tissue. TSP1 at physiologically relevant concentrations (11-22 nM, 24 hrs) inhibited LEC proliferation, migration, and tube formation. RT-PCR data demonstrated that CD47 is the predominant TSP1 receptor expressed in LEC (&gt; 50-fold over CD36 mRNA levels). Finally, genome-wide deficiency in TSP1 and CD47 receptor attenuated atherosclerotic lesion formation in the aorta and brachiocephalic artery by ~50% compared to wild type controls. Importantly, there was no difference in plasma cholesterol levels, body weight, fat and lean mass, fasting plasma glucose, and blood pressure between the experimental groups. Conclusions: The present study suggests that matrix protein TSP1 via CD47 receptor contributes to the pathogenesis of atherosclerosis. Inhibition of arterial lymphangiogenesis by TSP1-CD47 signaling may contribute to arterial lipid accumulation and atherosclerosis.

Elevated Thrombospondin‐2 Contributes to Delayed Wound Healing in Diabetes

Impaired wound healing is a major complication of diabetes and can lead to the development of chronic wounds in a significant portion of diabetes patients. Despite the risks posed by impaired healing, treatment strategies for diabetic wounds remain limited due to an incomplete understanding of the underlying pathological mechanisms. Previous studies have demonstrated that overexpression of thrombospondin‐2 (TSP2), a matricellular protein released after tissue injury, is associated with significant delays in dermal healing in various mouse models. Consistently, wounds lacking TSP2 (TSP2 KO) heal faster than wounds in wild‐type mice or mice with elevated TSP2. Thus, the present study aimed to examine the role of TSP2 in delayed healing in diabetes.First, we evaluated TSP2 expression in human wounds, and found that TSP2 is elevated in wounds from diabetes patients. Then, to determine TSP2's contribution to impaired healing in diabetes, we developed a novel diabetic TSP2 KO mouse model. Though these db/db TSP2 KO mice develop obesity and hyperglycemia comparable to db/db mice, db/db TSP2 KO mice exhibit significantly improved healing. Moreover, primary fibroblasts isolated from db/db TSP2 KO mice exhibit improved migration, providing mechanistic insight into the accelerated healing in these mice. We also studied TSP2 expression in fibroblasts, the major source of TSP2 in the wound, to explore the mechanisms leading to its overexpression in diabetes. Our results showed that TSP2 expression is increased in hyperglycemia, through increased activation of the hexosamine biosynthesis pathway and subsequent O‐GlcNAc modification of transcriptional elements. Overall, the results of this study indicate for the first time that: 1) TSP2 expression is elevated in diabetes and hyperglycemia and 2) TSP2 contributes to impaired healing in diabetes. These results represent a novel role for TSP2 in diabetes complications and a novel target for improving wound healing in diabetes.Support or Funding InformationNIH HL107205, NIH GM 072194, Gruber Science FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Syndecan-1 inhibits early stages of liver fibrogenesis by interfering with TGFβ1 action and upregulating MMP14

Increased expression of syndecan-1 is a characteristic feature of human liver cirrhosis. However, no data are available on the significance of this alteration. To address this question we designed a transgenic mouse strain that driven by albumin promoter, expresses human syndecan-1 in the hepatocytes. Liver cirrhosis was induced by thioacetamide in wild type and hSDC1+/+ mice of the identical strain. The process of fibrogenesis, changes in signal transduction and proteoglycan expression were followed. In an in vitro experiment, the effect of syndecan-1 overexpression on the action of TGFβ1 was determined. Human syndecan-1 and TGFβ1 levels were measured by ELISA in the circulation. Without challenge, no morphological differences were observed between wild type and transgenic mice livers, although significant upregulation of phospho-Akt and FAK was observed in the latter. Fibrogenesis in the transgenic livers, characterized by picrosirius staining, collagen type I, and SMA levels, lagged behind that of control in the first and second months. Changes in signal transduction involved in the process of fibrogenesis, as SMAD, MAPK, Akt and GSK, pointed to the decreased effect of TGFβ1, and this was corroborated by the decreased mRNA expression of TIEG and the growth factor itself. In vitro experiments exposing the LX2 hepatic stellate cell line to conditioned media of wild type and syndecan-1 transfected Hep3B cell lines proved that medium with high syndecan-1 content inhibits TGFβ1-induced upregulation of SMA, TIEG, collagen type I and thrombospondin-1 expression. Detection of liver proteoglycans and heparan sulfate level revealed that their amounts are much higher in control transgenic liver, than that in the wild type. However, it decreases dramatically as a result of shedding after hepatic injury. Shedding is likely promoted by the upregulation of MMP14. As syndecan-1 can bind thrombospondin-1, and as our result demonstrated that the same is true for TGFβ1, shed syndecan-1 can remove the growth factor and its activator together into the systemic circulation.Taking together, our results indicate that the effect of syndecan-1 is accomplished on two levels: a, the shedded syndecan can bind, inhibit and remove TGFβ1; b, interferes with the activation of TGFβ1 by downregulation and binding thrombospondin-1, the activator of the growth factor. However, by the end of the fourth month the protective effect was lost, which is explained by the considerable decrease of syndecan-1 and the almost complete loss of heparan sulfate from the surface of hepatocytes.

Human expression patterns: qualitative and quantitative analysis of thrombospondin-1 under physiological and pathological conditions.

Thrombospondin‐1 (TSP‐1), a matricellular protein and one of the first endogenous anti‐angiogenic molecules identified, has long been considered a potent modulator of human diseases. While the therapeutic effect of TSP‐1 to suppress cancer was investigated in both research and clinical settings, the mechanisms of how TSP‐1 is regulated in cancer remain elusive, and the scientific answers to the question of whether TSP‐1 expressions can be utilized as diagnostic or prognostic marker for patients with cancer are largely inconsistent. Moreover, TSP‐1 plays crucial functions in angiogenesis, inflammation and tissue remodelling, which are essential biological processes in the progression of many cardiovascular diseases, and therefore, its dysregulated expressions in such conditions may have therapeutic significance. Herein, we critically analysed the literature pertaining to TSP‐1 expression in circulating blood and pathological tissues in various types of cancer as well as cardiovascular and inflammation‐related diseases in humans. We compare the secretion rates of TSP‐1 by different cancer and non‐cancer cells and discuss the potential connection between the expression changes of TSP‐1 and vascular endothelial growth factor (VEGF) observed in patients with cancer. Moreover, the pattern and emerging significance of TSP‐1 profiles in cardiovascular disease, such as peripheral arterial disease, diabetes and other related non‐cancer disorders, are highlighted. The analysis of published TSP‐1 data presented in this review may have implications for the future exploration of novel TSP‐1‐based treatment strategies for cancer and cardiovascular‐related diseases.

TIPE2 is negatively correlated with tissue factor and thrombospondin-1 expression in patients with bronchial asthma.

The interaction between inflammatory processes and a hypercoagulant state may aggravate the severity of asthma and stimulate the airway remodeling of asthma. The aim of the current study was to evaluate the association between the negative inflammatory regulator tumor necrosis factor α induced protein-8 like-2 (TIPE2) and the coagulating substances tissue factor (TF) and thrombospondin-1 (TSP-1) in patients with bronchial asthma. Compared with healthy controls, TIPE2 expression was significantly downregulated, whereas TF expression was upregulated in the peripheral blood mononuclear cells (PBMCs) of patients with bronchial asthma. In addition, levels of TF and TSP-1 in the sera were up-regulated in patients with asthma compared with healthy controls. TIPE2 expression was negatively correlated with TF in the PBMCs and sera and was negatively correlated with TSP-1 levels in the sera of patients with bronchial asthma. The results of the current study indicated that anti-inflammatory TIPE2 levels are associated with levels of the coagulation substances TF and TSP-1. However, further studies are required to determine whether TIPE2 participates in the pathogenesis of asthma by interacting with the coagulation substances TF and TSP-1.

Ligand-Activated Peroxisome Proliferator-Activated Receptor δ Attenuates Vascular Oxidative Stress by Inhibiting Thrombospondin-1 Expression

Thrombospondin-1 (TSP-1) is implicated in vascular diseases associated with oxidative stress, such as abdominal aortic aneurysms, ischemia-reperfusion injury, and atherosclerosis. However, the regulatory mechanisms underlying TSP-1 expression are not fully elucidated. In this study, we found that peroxisome proliferator-activated receptor δ (PPARδ) inhibited oxidative stress-induced TSP-1 expression and migration in vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly attenuated hydrogen peroxide (H₂O₂)-induced expression of TSP-1 in VSMCs. Small interfering RNA-mediated knockdown of PPARδ and treatment with GSK0660, a selective PPARδ antagonist, reversed the effect of GW501516 on H₂O₂-induced expression of TSP-1, suggesting that PPARδ is associated with GW501516 activity. Furthermore, JNK (c-Jun N-terminal kinase), but not p38 and ERK (extracellular signal-regulated kinase), mediated PPARδ-dependent inhibition of TSP-1 expression in VSMCs exposed to H₂O₂. GW501516- activated PPARδ also reduced the H₂O₂-induced generation of reactive oxygen species, concomitant with inhibition of VSMC migration. In particular, TSP-1 contributed to the action of PPARδ in the regulation of H₂O₂-induced interleukin-1β expression. These results suggest that PPARδ-modulated downregulation of TSP-1 is associated with reduced cellular oxidative stress, thereby inhibiting H₂O₂-induced pheno­typic changes in vascular cells.

Impact of glutathione peroxidase 4 on cell proliferation, angiogenesis and cytokine production in hepatocellular carcinoma.

Insufficient supplementation with the micronutrient selenium and persistent hepatic inflammation predispose to hepatocellular carcinoma (HCC). Inflammation-associated reactive oxygen species attack membrane lipids and form lipid hydroperoxides able to propagate oxidative hepatic damage. Selenium-containing enzyme glutathione peroxidase 4 (GPx4) antagonizes this damage by reducing lipid hydroperoxides to respective hydroxides. However, the role of GPx4 in HCC remains elusive.We generated two human HCC cell lines with stable overexpression of GPx4, performed xenotransplantation into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) host mice and characterized the tumors formed. The experimental data were verified using gene expression data from two independent HCC patient cohorts.GPx4 overexpression protected from oxidative stress and reduced intracellular free radical level. GPx4-overexpressing cells displayed impaired tumor growth, reduced proliferation, altered angiogenesis and decreased expression of clinically relevant cytokine interleukin-8 and C-reactive protein. Moreover, GPx4 overexpression impaired migration of endothelial cells in vitro, and enhanced expression of thrombospondin 1, an endogenous inhibitor of angiogenesis. In patients, GPx4 expression in tumors positively correlated with survival and was linked to pathways which regulate cell proliferation, motility, tissue remodelling, immune response and M1 macrophage polarization. The patient data largely confirmed experimental findings especially in a subclass of poor prognosis tumors with high proliferation.GPx4 suppresses formation and progression of HCC by inhibition of angiogenesis and tumor cell proliferation as well as by immune-mediated mechanisms. Modification of GPx4 expression may represent a novel tool for HCC prevention or treatment.

Thrombospondin-1 secreted by human umbilical cord blood-derived mesenchymal stem cells rescues neurons from synaptic dysfunction in Alzheimer\u2019s disease model

Alzheimer’s disease (AD) is an incurable neurodegenerative disease characterised clinically by learning and memory impairments. Amyloid beta (Aβ) peptide-induced synaptic dysfunction is a pathological process associated with early-stage AD. Here, we show that paracrine action of human umbilical cord blood-derived-mesenchymal stem cells (hUCB-MSCs) protects the hippocampus from synaptic-density loss in in vitro and in vivo AD models. To identify paracrine factors underlying this rescue effect, we analysed hUCB-MSCs’ secretome co-cultured with Aβ42-treated mouse hippocampal neurons. Thrombospondin-1 (TSP-1), a protein secreted by hUCB-MSCs in in vitro and 5XFAD AD mouse models, was selected for study. Treatment with exogenous recombinant TSP-1 or co-cultures with hUCB-MSCs significantly increased expression of synaptic-density markers, such as synaptophysin (SYP) and post-synaptic density protein-95 (PSD-95) in Aβ42-treated mouse hippocampal neurons. Knockdown of TSP-1 expression in hUCB-MSCs through small interfering RNA (siRNA) abolished the reversal of Aβ42-induced hippocampal synaptic-density loss. We demonstrate that the rescue effect of hUCB-MSC-secreted TSP-1 was mediated by neuroligin-1 (NLGN1) or α2δ-1 receptors. Interestingly, NLGN1 and α2δ-1 expression, which was reduced in Aβ42-treated hippocampal neurons, increased in co-cultures with hUCB-MSCs or exogenous TSP-1. Together, these findings suggest that hUCB-MSCs can attenuate Aβ42-induced synaptic dysfunction by regulating TSP-1 release, thus providing a potential alternative therapeutic option for early-stage AD.

Fibroblast growth factor-2 and transforming growth factor-beta1 oppositely regulate miR-221 that targets thrombospondin-1 in bovine luteal endothelial cells.

Thrombospondin-1 (THBS1) affects corpus luteum (CL) regression. Highly induced during luteolysis, it acts as a natural anti-angiogenic, proapoptotic compound. THBS1 expression is regulated in bovine luteal endothelial cells (LECs) by fibroblast growth factor-2 (FGF2) and transforming growth factor-beta1 (TGFB1) acting in an opposite manner. Here we sought to identify specific microRNAs (miRNAs) targeting THBS1 and investigate their possible involvement in FGF2 and TGFB1-mediated THBS1 expression. Several miRNAs predicted to target THBS1 mRNA (miR-1, miR-18a, miR-144, miR-194, and miR-221) were experimentally tested. Of these, miR-221 was shown to efficiently target THBS1 expression and function in LECs. We found that this miRNA is highly expressed in luteal cells and in mid-cycle CL. Consistent with the inhibition of THBS1 function, miR-221 also reduced Serpin Family E Member 1 [SERPINE1] in LECs and promoted angiogenic characteristics of LECs. Plasminogen activator inhibitor-1 (PAI-1), the gene product of SERPINE1, inhibited cell adhesion, suggesting that PAI-1, like THBS1, has anti-angiogenic properties. Importantly, FGF2, which negatively regulates THBS1, elevates miR-221. Conversely, TGFB1 that stimulates THBS1, significantly reduces miR-221. Furthermore, FGF2 enhances the suppression of THBS1 caused by miR-221 mimic, and prevents the increase in THBS1 induced by miR-221 inhibitor. In contrast, TGFB1 reverses the inhibitory effect of miR-221 mimic on THBS1, and enhances the upregulation of THBS1 induced by miR-221 inhibitor. These data support the contention that FGF2 and TGFB1 modulate THBS1 via miR-221. These in vitro data propose that dynamic regulation of miR-221 throughout the cycle, affecting THBS1 and SERPINE1, can modulate vascular function in the CL.

ADAMTS1-mediated targeting of TSP-1 by PPARδ suppresses migration and invasion of breast cancer cells.

Migration and invasion of cancer cells into surrounding tissue is a key stage of cancer metastasis. Here, we show that peroxisome proliferator-activated receptor (PPAR) δ regulates migration and invasion of human breast cancer cells via thrombospondin-1 (TSP-1) and its degrading protease, a disintegrin and metalloprotease domains with thrombospondin motifs 1 (ADAMTS1). Activation of PPARδ by GW501516, a specific ligand for PPARδ, led to marked inhibition in the cell migration and TSP-1 expression of breast cancer. These effects were suppressed by small interfering RNA-mediated knock-down of ADAMTS1, indicating that ADAMTS1 is involved in PPARδ-mediated inhibition of migration and TSP-1 expression in breast cancer cells. In addition, ligand-activated PPARδ upregulated expression of ADAMTS1 at the transcriptional level via binding of PPARδ to a direct repeat-1 site within the ADAMTS1 gene promoter. Furthermore, ligand-activated PPARδ suppressed invasion of breast cancer cells in an ADAMTS1-dependent manner. Taken together, these results demonstrate that PPARδ suppresses migration and invasion of breast cancer cells by downregulating TSP-1 in a process mediated by upregulation of ADAMTS1.

Metformin accelerates wound healing in type 2 diabetic db/db mice.

Wound healing impairment is increasingly recognized to be a consequence of hyperglycemia-induced dysfunction of endothelial precursor cells (EPCs) in type 2 diabetes mellitus (T2DM). Metformin exhibits potential for the improvement of endothelial function and the wound healing process. However, the underlying mechanisms for the observed beneficial effects of metformin application remain to be completely understood. The present study assessed whether metformin, a widely used therapeutic drug for T2DM, may accelerate wound closure in T2DM db/db mice. Genetically hyperglycemic db/db mice were used as the T2DM model. Metformin (250 mg/kg/day; intragastric) was administered for two weeks prior to EPC collection and wound model creation in db/db mice. Wound healing was evaluated by alterations in the wound area and the number of platelet endothelial cell adhesion molecule-positive cells. The function of the isolated bone marrow-derived EPCs (BM-EPCs) was assessed by a tube formation assay. The number of circulating EPCs, and the levels of intracellular nitric oxide (NO) and superoxide (O2−) were detected by flow cytometry. Thrombospondin-1 (TSP-1) expression was determined by western blot analysis. It was observed that treatment with metformin accelerated wound healing, improved angiogenesis and increased the circulating EPC number in db/db mice. In vitro, treatment with metformin reversed the impaired BM-EPC function reflected by tube formation, and significantly increased NO production while decreasing O2− levels in BM-EPCs from db/db mice. In addition, TSP-1 expression was markedly attenuated by treatment with metformin in cultured BM-EPCs. Metformin contributed to wound healing and improved angiogenesis in T2DM mice, which was, in part, associated with stimulation of NO, and inhibition of O2− and TSP-1 in EPCs from db/db mice.

Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations.

KRIT1 mutations are the most common cause of cerebral cavernous malformation (CCM). Acute Krit1 gene inactivation in mouse brain microvascular endothelial cells (BMECs) changes expression of multiple genes involved in vascular development. These changes include suppression of Thbs1, which encodes thrombospondin1 (TSP1) and has been ascribed to KLF2- and KLF4-mediated repression of Thbs1 In vitro reconstitution of TSP1 with either full-length TSP1 or 3TSR, an anti-angiogenic TSP1 fragment, suppresses heightened vascular endothelial growth factor signaling and preserves BMEC tight junctions. Furthermore, administration of 3TSR prevents the development of lesions in a mouse model of CCM1 (Krit1ECKO ) as judged by histology and quantitative micro-computed tomography. Conversely, reduced TSP1 expression contributes to the pathogenesis of CCM, because inactivation of one or two copies of Thbs1 exacerbated CCM formation. Thus, loss of Krit1 function disables an angiogenic checkpoint to enable CCM formation. These results suggest that 3TSR, or other angiogenesis inhibitors, can be repurposed for TSP1 replacement therapy for CCMs.

Potential role of sirtuin 1 in Müller glial cells in mice choroidal neovascularization.

This study investigated the potential role of sirtuin 1 in Müller glial cells in choroidal neovascularization. In the in vitro study, primary Müller glial cells were cultured and treated with resveratrol, a sirtuin 1 activator. Glial fibrillary acidic protein expression and angiogenesis-related gene expression were examined using quantitative polymerase chain reaction and phagocytosis, as a marker of Müller glial cell function; in addition, a latex bead assay was used to analyze cell function. For the in vivo study, choroidal neovascularization was induced in C57BL/6 mice via laser photocoagulation, and resveratrol was administered intravitreally. Eyecup whole mounts were created to measure choroidal neovascularization volumes on day 7. Immunohistochemical analysis with anti-glial fibrillary acidic protein antibody was used to detect Müller glial cell activation in eyes with choroidal neovascularization on day 1, 3, 5, and 7 after laser surgery. Resveratrol significantly promoted glial fibrillary acidic protein, anti-angiogenic factor, pigment epithelium-derived factor, and thrombospondin-1 expression in the cells as well as the phagocytic activities. Treatment of the choroidal neovascularization model with resveratrol resulted in early activation of Müller glial cells near choroidal neovascularization sites. Resveratrol-activated cells but not the controls migrated to the top of choroidal neovascularization sites and into the lesions from day 3. Resveratrol reduced the choroidal neovascularization size relative to controls. In conclusion, sirtuin 1 activation in Müller glial cells suppressed the development of choroidal neovascularization, and therefore, might be a therapeutic option.

Pancreatic β-cell protection from inflammatory stress by the endoplasmic reticulum proteins thrombospondin 1 and mesencephalic astrocyte-derived neutrotrophic factor (MANF)

Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, and human β-cells exposed to cytokines or thapsigargin-induced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure of β-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes.

MicroRNA-98 interferes with thrombospondin 1 expression in peripheral B cells of patients with asthma.

Thrombospondin 1 (TSP1)-producing B cells are an important immune regulatory cell fraction in the body, which are compromised in a number of immune diseases. miRs are involved in the immune regulation. The present study aims to elucidate the mechanism by which miR-98 interferes with the expression of TSP1 in B cells of the peripheral blood system. In the present study, peripheral blood samples were collected from patients with allergic asthma. The B cells were isolated from the blood samples to be analyzed for the expression of miR-98 and TSP1. The results showed that the levels of miR-98 were higher, the levels of TSP1 were lower, in B cells isolated from the peripheral blood in patients with asthma. A negative correlation was identified between the data of miR-98 and TSP1 in B cells. Exposure to T helper (Th) 2 (Th2) cytokine, interleukin (IL)-13, increased the expression of miR-98 and suppressed the expression of TSP1 in peripheral B cells, which was abolished by knocking down the miR-98 gene. In conclusion, miR-98 can suppress the expression of TSP1 in the peripheral B cells of patients with allergic asthma.

HIF-1α represses the expression of the angiogenesis inhibitor thrombospondin-2

Thrombospondin-2 (TSP2) is a potent inhibitor of angiogenesis whose expression is dynamically regulated following injury. In the present study, it is shown that HIF-1α represses TSP2 transcription. Specifically, in vitro studies demonstrate that the prolyl hydroxylase inhibitor DMOG or hypoxia decrease TSP2 expression in fibroblasts. This effect is shown to be via a transcriptional mechanism as hypoxia does not alter TSP2 mRNA stability and this effect requires the TSP2 promoter. In addition, the documented repressive effect of nitric oxide (NO) on TSP2 is shown to be non-canonical and involves stabilization of hypoxia inducible factor-1a (HIF-1α). The regulation of TSP2 by hypoxia is supported by the in vivo observation that TSP2 has spatiotemporal expression distinct from regions of hypoxia in gastrocnemius muscle following murine hindlimb ischemia (HLI). A role for TSP2 regulation by HIF-1α is supported by the dysregulation of TSP2 expression in SM22α-cre HIF-1α KO mice following HLI. Indeed, there is a reduction in blood flow recovery in the SM22a-cre HIF-1α KO mice compared to littermate controls following HLI surgery, associated with impaired recovery and increased TSP2 levels. Moreover, SM22α-cre HIF-1α KO smooth muscle cells mice have increased TSP2 mRNA levels that persist in hypoxia. These findings identify a novel, ischemia-induced pro-angiogenic mechanism involving the transcriptional repression of TSP2 by HIF-1α.

Tsp1 promotes alveolar stem cell proliferation and its down-regulation relates to lung inflammation in intralobar pulmonary sequestration.

An aberrant systemic artery supply results in recurrent infections in the abnormal lung lobe of intralobar pulmonary sequestration (ILS). The mechanisms underlying such persistent inflammation are unknown. Here, we hypothesize that alteration of an endothelial cell niche for alveolar epithelial cells results in the impaired proliferation potential of alveolar progenitor cells, leading to the defective defense mechanism in intralobar pulmonary sequestration. Paraffin sections of lung tissues from patients with intralobar pulmonary sequestration or from healthy controls were collected for analysis of alveolar epithelial alterations in intralobar pulmonary sequestration by quantitative RT-PCR or immunofluorescent staining. Differential transcripts were identified between human pulmonary artery endothelial cells and human aortic endothelial cells by microarray. Validation of microarray data by quantitative PCR analysis indicated that thrombospondin-1 expression level is low in near-lesion part but high in lesion part of ILS lobe as compared to healthy controls. In vitro 3-D matrigel culture was adopted to evaluate the regulation of alveolar progenitor cells by thrombospondin-1 and CD36. We found that the proliferative potential of alveolar type 2 stem/progenitor cells was impaired in intralobar pulmonary sequestration. Mechanistically, we discovered that endothelial thrombospondin-1 promotes alveolar type 2 cell proliferation through the interaction with CD36. These data demonstrate that alveolar stem cells are impaired in the abnormal lobe from patients with intralobar pulmonary sequestration and imply that restoring epithelial integrity can be beneficial for the future treatments of recurrent infections in lung pathologies.