Overexpression Of Thrombospondin-1 Research Articles

Promoter hypermethylation-mediated downregulation of PAX6 promotes tumor growth and metastasis during the progression of liver cancer

BackgroundThe progression of liver cancer is a complicated process that involves genetic and epigenetic changes. Paired box 6 (PAX6) is a critical transcription factor for embryonic development. PAX6 is abnormally methylated in human cancer. The role of the PAX6 gene in the pathogenesis of hepatocellular carcinoma (HCC) is still unclear.MethodsTranscriptional silencing of PAX6 mediated by promoter methylation was confirmed using quantitative methylation-specific polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. Then we conducted gain-and-loss of function approaches to evaluate the function of PAX6 in HCC progression in vitro. Moreover, we designed xenograft mouse models to assess the effect of PAX6 on tumor growth and metastasis. Finally, we used RNA sequencing (RNA-seq) strategy and phenotypic rescue experiments to identify potential targets of PAX6 performing tumor-suppressive function.ResultsConstitutive expression of PAX6 suppressed anchorage-independent growth and cell invasion in vitro as well as tumor growth and metastasis in xenograft mouse models. In contrast, the inhibition of PAX6 using knockout and knockdown strategies increased tumor growth both in vitro and in vivo. Downregulation of PAX6 by doxycycline depletion partially reversed the malignant phenotypes of HCC cells induced by PAX6. Moreover, we identified E-cadherin (CDH1) and thrombospondin-1 (THBS1) as targets of PAX6. Ultimately, we demonstrated that the knockdown of CDH1 and overexpression of THBS1 in PAX6-expressing HCC cells partly reversed the tumor-suppressive effect.ConclusionPAX6 functions as a tumor suppressor partly through upregulation of CDH1 and downregulation of THBS1. Promoter hypermethylation-mediated suppression of PAX6 reduces the tumor suppressor function in the progression of liver cancer.

Stromal thrombospondin 1 suppresses angiogenesis in oral submucous fibrosis.

A decline in mucosal vascularity is a histological hallmark of oral submucous fibrosis (OSF), a premalignant disease that is largely induced by betel quid chewing. However, the lack of available models has challenged studies of angiogenesis in OSF. Here, we found that the expression of thrombospondin 1 (THBS1), an endogenous angiostatic protein, was elevated in the stroma of tissues with OSF. Using a fibroblast-attached organoid (FAO) model, the overexpression of THBS1 in OSF was stably recapitulated in vitro. In the FAO model, treatment with arecoline, a major pathogenic component in areca nuts, enhanced the secretion of transforming growth factor (TGF)-β1 by epithelial cells, which then promoted the expression of THBS1 in fibroblasts. Furthermore, human umbilical vein endothelial cells (HUVECs) were incorporated into the FAO to mimic the vascularized component. Overexpression of THBS1 in fibroblasts drastically suppressed the sprouting ability of endothelial cells in vascularized FAOs (vFAOs). Consistently, treatment with arecoline reduced the expression of CD31 in vFAOs, and this effect was attenuated when the endothelial cells were preincubated with neutralizing antibody of CD36, a receptor of THBS1. Finally, in an arecoline-induced rat OSF model, THBS1 inhibition alleviated collagen deposition and the decline in vascularity in vivo. Overall, we exploited an assembled organoid model to study OSF pathogenesis and provide a rationale for targeting THBS1.

TSP-1 increases autophagy level in cartilage by upregulating HSP27 which delays progression of osteoarthritis

This study aimed to determine whether Thrombospondin-1 (TSP-1) can be used as a biomarker to diagnose early osteoarthritis (OA) and whether it has a chondroprotective effect against OA. We examined TSP-1 expression in cartilage, synovial fluid, and serum at different time points after anterior cruciate ligament transection (ACLT) surgery in rats. Subsequently, TSP-1 was overexpressed or silenced to detect its effects on extracellular matrix (ECM) homeostasis, autophagy level, proliferation and apoptosis in chondrocytes. Adenovirus encoding TSP-1 was injected into the knee joints of ACLT rats to test its effect against OA. Combined with proteomic analysis, the molecular mechanism of TSP-1 in cartilage degeneration was explored. Intra-articular injection of an adenovirus carrying the TSP-1 sequence showed chondroprotective effects against OA. Moreover, TSP-1 expression decreases with OA progression and can effectively promote cartilage proliferation, inhibit apoptosis, and helps to sustain the balance between ECM anabolism and catabolism. Overexpression of TSP-1 also can increase autophagy by upregulating Heat Shock Protein 27 (HSP27, hspb1), thereby enhancing its effect as a stimulator of autophagy. TSP-1 is a hopeful strategy for OA treatment.

Investigation of a UPR-Related Gene Signature Identifies the Pro-Fibrotic Effects of Thrombospondin-1 by Activating CD47/ROS/Endoplasmic Reticulum Stress Pathway in Lung Fibroblasts.

Unfolded protein response (UPR) signaling and endoplasmic reticulum (ER) stress have been linked to pulmonary fibrosis. However, the relationship between UPR status and pulmonary function and prognosis in idiopathic pulmonary fibrosis (IPF) patients remains largely unknown. Through a series of bioinformatics analyses, we established a correlation between UPR status and pulmonary function in IPF patients. Furthermore, thrombospondin-1 (TSP-1) was identified as a potential biomarker for prognostic evaluation in IPF patients. By utilizing both bulk RNA profiling and single-cell RNA sequencing data, we demonstrated the upregulation of TSP-1 in lung fibroblasts during pulmonary fibrosis. Gene set enrichment analysis (GSEA) results indicated a positive association between TSP-1 expression and gene sets related to the reactive oxygen species (ROS) pathway in lung fibroblasts. TSP-1 overexpression alone induced mild ER stress and pulmonary fibrosis, and it even exacerbated bleomycin-induced ER stress and pulmonary fibrosis. Mechanistically, TSP-1 promoted ER stress and fibroblast activation through CD47-dependent ROS production. Treatment with either TSP-1 inhibitor or CD47 inhibitor significantly attenuated BLM-induced ER stress and pulmonary fibrosis. Collectively, these findings suggest that the elevation of TSP-1 during pulmonary fibrosis is not merely a biomarker but likely plays a pathogenic role in the fibrotic changes in the lung.

Enterolactone and trabectedin suppress epithelial ovarian cancer synergistically via upregulating THBS1.

Epithelial ovarian cancer (EOC) is the most common and fatal subtype of ovarian malignancies, with no effective therapeutics available. Our previous studies have demonstrated extraordinary suppressive efficacy of enterolactone (ENL) on EOC. A chemotherapeutic agent, trabectedin (Trabe), is shown to be effective on ovarian cancer, especially when combined with other therapeutics, such as pegylated liposomal doxorubicin or oxaliplatin. Thrombospondin 1 (THBS1), a kind of matrix glycoprotein, plays important roles against cancer development through inhibiting angiogenesis but whether it is involved in the suppression of EOC by ENL or Trabe remains unknown. To test combined suppressive effects of ENL and Trabe on EOC and possible involvement of THBS1 in the anticancer activities of ENL and Trabe. The EOC cell line ES-2 was transfected with overexpressed THBS1 by lentivirus vector. We employed tube formation assay to evaluate the anti-angiogenesis activity of ENL and of its combined use with Trabe after THBS1 overexpression and established drug intervention and xenograft nude mouse cancer models to assess the in vivo effects of the hypothesized synergistic suppression between the agents and the involvement of THBS1. Mouse fecal samples were collected for 16S rDNA sequencing and microbiota analysis. We detected strong inhibitory activities of ENL and Trabe against the proliferation and migration of cancer cells and observed synergistic effects between ENL and Trabe in suppressing EOC. ENL and Trabe, given either separately or in combination, could suppress the tube formation capability of human microvascular endothelial cells, and this inhibitory effect became even stronger with THBS1 overexpression. In the ENL plus Trabe combination group, the expression of tissue inhibitor of metalloproteinases 3 and cluster of differentiation 36 was both upregulated, whereas matrix metalloproteinase 9, vascular endothelial growth factor, and cluster of differentiation 47 were all decreased. With the overexpression of THBS1, the results became even more pronounced. In animal experiments, combined use of ENL and Trabe showed superior inhibitory effects to either single agent and significantly suppressed tumor growth, and the overexpression of THBS1 further enhanced the anti-cancer activities of the drug combination group. ENL and Trabe synergistically suppress EOC and THBS1 could remarkably facilitate the synergistic anticancer effects of ENL and Trabe.

Upregulated TSP1 and CD47 expression in the lung in nitrofen-induced congenital diaphragmatic hernia.

Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). The secreted glycoprotein thrombospondin-1 (TSP1), a ligand for receptor CD47, is widely expressed on both systemic and pulmonary vascular cells. TSP1-CD47 signaling has been reported to be one of the pathogeneses of pulmonary hypertension (PH). After creating a nitrofen-induced CDH rat model, fetuses were sacrificed on D17, D19 and D21 and divided into a control group and a CDH group. Quantitative real-time polymerase chain reaction was performed to determine the pulmonary gene expression of TSP1, CD47 and Runx3 (a regulator of TSP1). An immunofluorescence study was performed to evaluate the expression and localization of TSP1, CD47 and Runx3. The relative mRNA expression of pulmonary TSP1, CD47 and Runx3 on D21 was significantly increased in the CDH group (p=0.005, p=0.001, p=0.046, and p=0.002, respectively). The immunofluorescence study also confirmed the overexpression of TSP1, CD47 and Runx3 in the CDH group. Our results provide evidence that TSP1-CD47 signaling is involved in the pathogenesis of PH in a nitrofen-induced CDH model. Our data suggest that anti-CD47 antibodies can be novel therapeutic targets for the treatment of PH in CDH.

Genetic regulation of THBS1 methylation in diabetic retinopathy.

Diabetic retinopathy (DR) is a common and serious microvascular complication of diabetes mellitus (DM), but its pathological mechanism, especially the formation mechanism of new blood vessels remains unclear. Thrombospondin-1 (THBS1) is a potent endogenous inhibitor of angiogenesis and it was found over expressed in DR in our previous study. Our study aimed to determine whether overexpression of THBS1 is associated with its promoter methylation level, and whether methylation of THBS1 is regulated by genetic variants in DR. Patients diagnosed with DR and DM patients without retinal problems were included in the case-control study. DNA methylation detection of THBS1 by bisulfite sequencing and genotyping of specific SNPs by MassARRAY analysis were performed in the patients recruited from 2019-2020. Real time quantitative PCR was performed to obtain mRNA expression of THBS1 in the patients recruited from August to October 2022. The differentially methylated CpG loci of THBS1 were identified by logistic regression, and associations between 13 SNPs and methylation levels of CpG loci were tested by methylation quantitative trait loci (meQTLs) analysis. Mediation analysis was applied to determine whether CpG loci were intermediate factors between meQTLs and DR. 150 patients diagnosed with DR and 150 DM patients without retinal complications were enrolled in the first recruitment, seven DR patients and seven DM patients were enrolled in the second recruitment. The patients with DR showed promoter hypomethylation of THBS1 (P value = 0.002), and six out of thirty-nine CpG sites within two CpG islands (CGIs) showed hypomethylation(P value < 0.05). THBS1 mRNA expression in peripheral blood was significantly higher in DR patients than in DM patients. Five out of thirteen cis-meQTLs were identified to be associated with CpG sites: rs13329154, rs34973764 and rs5812091 were associated with cis-meQTLs of CpG-4 (P value=0.0145, 0.0095, 0.0158), rs11070177 and rs1847663 were associated with cis-meQTLs of CpG-2 and CpG-3 respectively (P value=0.0201, 0.0275). CpG-4 methylation significantly mediated the effect of the polymorphism rs34973764 on DR (B=0.0535, Boot 95%CI: 0.004~0.1336). THBS1 overexpression is related to THBS1 hypomethylation in patients with DR. DNA methylation may be genetically controlled in DR.

Thrombospondin-1 overexpression stimulates loss of Smad4 and accelerates malignant behavior via TGF-β signal activation in pancreatic ductal adenocarcinoma

IntroductionPancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma and cancer-associated fibroblasts (CAFs) provide a favorable tumor microenvironment. Smad4 is known as tumor suppressor in several types of cancers including PDAC, and loss of Smad4 triggers accelerated cell invasiveness and metastatic potential. The thrombospondin-1 (TSP-1) can act as a major activator of latent transforming growth factor-β (TGF-β) in vivo. However, the roles of TSP-1 and the mediator of Smad4 loss and TGF-β signal activation during PDAC progression have not yet been addressed. The aim is to elucidate the biological role of TSP-1 in PDAC progression. Methods and resultsHigh substrate stiffness stimulated TSP-1 expression in CAFs, and TSP-1 knockdown inhibited cell proliferation with suppressed profibrogenic and activated stroma-related gene expressions in CAFs. Paracrine TSP-1 treatment for PDAC cells promoted cell proliferation and epithelial mesenchymal transition (EMT) with activated TGF-β signals such as phosphorylated Akt and Smad2/3 expressions. Surprisingly, knockdown of DPC4 (Smad4 gene) induced TSP-1 overexpression with TGF-β signal activation in PDAC cells. Interestingly, TSP-1 overexpression also induced downregulation of Smad4 expression and enhanced cell proliferation in vitro and in vivo. Treatment with LSKL peptide, which antagonizes TSP-1-mediated latent TGF-β activation, attenuated cell proliferation, migration and chemoresistance with enhanced apoptosis in PDAC cells. ConclusionsTSP-1 derived from CAFs stimulates loss of Smad4 expression in cancer cells and accelerates malignant behavior by TGF-β signal activation in PDAC. TSP-1 could be a novel therapeutic target, not only for CAFs in stiff stroma, but also for cancer cells in the PDAC microenvironment.

A TMT-based shotgun proteomics uncovers overexpression of thrombospondin 1 as a contributor in pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome

Hepatic sinusoidal obstruction disease (HSOS) is a rare but life-threatening vascular liver disease. However, its underlying mechanism and molecular changes in HSOS are largely unknown, thus greatly hindering the development of its effective treatment. Hepatic sinusoidal endothelial cells (HSECs) are the primary and essential target for HSOS. A tandem mass tag-based shotgun proteomics study was performed using primary cultured HSECs from mice with HSOS induced by senecionine, a representative toxic pyrrolizidine alkaloid (PA). Dynamic changes in proteome were found at the initial period of damage and the essential role of thrombospondin 1 (TSP1) was highlighted in PA-induced HSOS. TSP1 over-expression was further confirmed in human HSECs and liver samples from patients with PA-induced HSOS. LSKL peptide, a known TSP1 inhibitor, protected mice from senecionine-induced HSOS. In addition, TSP1 was found to be covalently modified by dehydropyrrolizidine alkaloids in human HSECs and mouse livers upon senecionine treatment, thus to form the pyrrole-protein adduct. These findings provide useful information on early changes in HSECs upon PA treatment and uncover TSP1 overexpression as a contributor in PA-induced HSOS.Graphical abstract

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP.

The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1 (TSP1) in human renal tubular epithelial cells (HK-2 cells). HK-2 cells were exposed to high concentrations of glucose (HG). The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis. The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry. Western blotting was used to detect the apoptosis- and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA. HG treatment induced high cell apoptosis, abundantly expressed TSP1 level and restrained viability in HK-2 cells. Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions. Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression. 4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers, such as PERK, ATF4, ATF6, p-eIF2α, IRE1, CHOP and XBP1, suggesting that the administration of 4-PBA was successful. Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis. TSP1 restrained cell proliferation, and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

Thrombospondin-1 induced programmed death-ligand 1-mediated immunosuppression by activating the STAT3 pathway in osteosarcoma.

Thrombospondin‐1 (TSP1) is generally assumed to suppress the growth of osteosarcoma through inhibiting angiogenesis; however, it is unclear whether TSP1 could affect the antitumor immunity against osteosarcoma. We aimed to explore the immune‐related tumor‐promoting effects of TSP1 and decipher its underlying mechanism. First, we identified that TSP1 regulated programmed death‐ligand 1 (PD‐L1) expression, which was related to the CD8+ T cells anergy in osteosarcoma cells. The exact role of PD‐L1 in the immunosuppressive effect of TSP1 was then further confirmed by the addition of the PD‐L1 neutralizing Ab. With the addition of PD‐L1 neutralizing Abs during cocultivation, the inhibition of CD8+ T cells was abolished to a certain extent. Further mechanistic investigations showed that TSP1‐induced PD‐L1 upregulation was achieved by activation of the signal transducer and activator of transcription 3 (STAT3) pathway. In vivo experiments also indicated that TSP1 overexpression could promote the growth of primary lesions, whereas TSP1 knockdown effectively inhibits the growth of the primary lesion as well as lung metastasis by restoring the antitumor immunity. Thrombospondin‐1 knockdown combined with PD‐L1 neutralizing Ab achieved a more pronounced antitumor effect. Taken together, our study showed that TSP1 upregulates PD‐L1 by activating the STAT3 pathway and, therefore, impairs the antitumor immunity against osteosarcoma.

Apatinib inhibits glioma cell malignancy in patient-derived orthotopic xenograft mouse model by targeting thrombospondin 1/myosin heavy chain 9 axis

We determined the antitumor mechanism of apatinib in glioma using a patient-derived orthotopic xenograft (PDOX) glioma mouse model and glioblastoma (GBM) cell lines. The PDOX mouse model was established using tumor tissues from two glioma patients via single-cell injections. Sixteen mice were successfully modeled and randomly divided into two equal groups (n = 8/group): apatinib and normal control. Survival analysis and in vivo imaging was performed to determine the effect of apatinib on glioma proliferation in vivo. Candidate genes in GBM cells that may be affected by apatinib treatment were screened using RNA-sequencing coupled with quantitative mass spectrometry, data mining of The Cancer Genome Atlas, and Chinese Glioma Genome Atlas databases, and immunohistochemistry analysis of clinical high-grade glioma pathology samples. Quantitative reverse transcription-polymerase chain reaction (qPCR), western blotting, and co-immunoprecipitation (co-IP) were performed to assess gene expression and the apatinib-mediated effect on glioma cell malignancy. Apatinib inhibited the proliferation and malignancy of glioma cells in vivo and in vitro. Thrombospondin 1 (THBS1) was identified as a potential target of apatinib that lead to inhibited glioma cell proliferation. Apatinib-mediated THBS1 downregulation in glioma cells was confirmed by qPCR and western blotting. Co-IP and mass spectrometry analysis revealed that THBS1 could interact with myosin heavy chain 9 (MYH9) in glioma cells. Simultaneous THBS1 overexpression and MYH9 knockdown suppressed glioma cell invasion and migration. These data suggest that apatinib targets THBS1 in glioma cells, potentially via MYH9, to inhibit glioma cell malignancy and may provide novel targets for glioma therapy.

Upregulation of THBS1 is Related to Immunity and Chemotherapy Resistance in Gastric Cancer.

PurposeThrombospondin 1 (THBS1) is an endogenous inhibitor of angiogenesis, but it also promotes tumor invasion, metastasis, and immune response in the tumor environment. Previous research has found that THBS1 is highly expressed in many tumors and has a negative correlation with tumor prognosis. However, research on the relationship between THBS1 and immune infiltration in GC is less well documented, and the objective of our study was to investigate the role of THBS1 expression in GC.Patients and MethodsThe expression of THBS1 in GC was analyzed by Oncomine, TIMER, TGCA, GEO and IHC staining. Analysis of the signaling pathways associated with THBS1 expression in GC uses GSEA. The relationship between THBS1 expression and immune infiltration was analyzed by the ESTIMATE algorithm, single-cell transcriptome analysis, TIMER2 database and CIBERSORT algorithm. Finally, the relationship between THBS1 expression and drug sensitivity was analyzed by the CellMiner database.ResultsTHBS1 was overexpressed in GC and was associated with poor prognosis, and high THBS1 expression was an independent risk factor. GSEA results showed that high THBS1 expression in GC was associated with tumorigenesis, adhesion, and significant immune enrichment. THBS1 expression was most strongly correlated with tumor-associated macrophages (TAMs), M2 macrophages and cancer-associated fibroblast (CAFs) in GC. THBS1 expression positively correlates with most immune checkpoint members, suggesting that THBS1 may play an important role in the tumor microenvironment. THBS1 overexpression was negatively correlated with some drug sensitivities, such as Oxaliplatin.ConclusionUpregulation of THBS1 was positively correlated with poor prognosis and immunosuppression in GC and negatively correlated with anticancer drug sensitivity, suggesting that THBS1 may serve as a potential target for the treatment of GC.

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.

Contribution of Thrombospondin-1 and -2 to Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome.

Thrombospondin (TSP) proteins have been shown to impact T-cell adhesion, migration, differentiation, and apoptosis. Thrombospondin-1 (TSP-1) is specifically upregulated in several inflammatory diseases and can effectively promote lipopolysaccharide- (LPS-) induced inflammation. In contrast, thrombospondin-2 (TSP-2) has been associated with activation of “anti-inflammatory” T-regulatory cells (Tregs). In this study, we investigated the effects of both TSP-1 and TSP-2 overexpression on macrophage polarization and activation in vitro and in vivo. We analyzed the effects of TSP-1 and TSP-2 on inflammation, vascular endothelial permeability, edema, ultrastructural morphology, and apoptosis in lung tissues of an ARDS mouse model and cultured macrophages. Our results demonstrated that TSP-2 overexpression effectively attenuated LPS-induced ARDS in vivo and promoted M2 macrophage phenotype polarization in vitro. Furthermore, TSP-2 played a role in regulating pulmonary vascular barrier leakage by activating the PI3K/Akt pathway. Overall, our findings indicate that TSP-2 can modulate inflammation and could therefore be a potential therapeutic target against LPS-induced ARDS.

Mild hyperbaric oxygen exposure attenuates rarefaction of capillary vessels in streptozotocin-induced diabetic soleus muscle in rats.

We examined the effects of mild hyperbaric oxygen (mHBO) exposure on capillary rarefaction in skeletal muscles of rats with diabetes. Streptozotocin (100 mg/kg) was administered to male Wistar rats via the tail vein to prepare a diabetic model. These rats were divided into 2 groups: the group with mHBO exposure (1.25 atmospheres absolute (ATA) with 36% oxygen; 3 h/day) and the group without mHBO exposure. Age-matched rats were used as the control group. Eight weeks later, the soleus of the rats was removed and then analyzed. With the onset of diabetes mellitus, capillary number, diameter, and volume in the soleus of the rats with diabetes decreased compared with those of the rats in the control group. In addition, increased anti-angiogenic thrombospondin-1 (TSP-1) and decreased pro-angiogenic murine double minute 2 (MDM-2) protein expressions were observed in the rats with diabetes. Alternatively, mHBO exposure attenuated the decrease in capillary diameter and volume in skeletal muscles of rats with diabetes, suppressed the overexpression of TSP-1, and restored the MDM-2 expression. These results indicate the exposure of mHBO partially attenuates capillary rarefaction in diabetic soleus muscle.

Maggot excretions/secretions promote diabetic wound angiogenesis via miR18a/19a – TSP-1 axis

AimsThe impaired angiogenesis is one of the main factors affecting the healing of diabetic foot ulcer (DFU) wounds. Maggot debridement therapy (MDT) promotes granulation tissue growth and angiogenesis during DFU wound healing. Non-coding microRNAs can also promote local angiogenesis in DFU wounds by regulating wound repairing related gene expression. The purpose of this study was to investigate the mechanism of microRNAs in MDT promoting DFU wound angiogenesis. MethodsIn this study, we applied MDT to treat DFU wound tissue and detect the expression of the miR-17–92 cluster. In vitro experiments, human umbilical vein endothelial cells (HUVECs) were treated with maggot excretions/secretions (ES), the miR-17–92 cluster and the predicted target gene expression were measured. Tube formation assay and cell scratch assay were performed when inhibition of miR-18a/19a or overexpression of thrombospondin-1 (TSP-1) were used in this study. ResultsmiR-18a/19a transcription significantly up-regulated and TSP-1 expression down-regulated in patients wound tissue and in HUVECs. Inhibition of miR-18a/19a or overexpression of TSP-1 partially blocked the migration and tube formation ability stimulated by ES. ConclusionTargeted activation of miR-18a/19a transcription levels and subsequent regulation of TSP-1 expression may be a novel therapeutic strategy for DFU.

Enhanced thrombospondin-1 causes dysfunction of vascular endothelial cells derived from Fabry disease-induced pluripotent stem cells.

BackgroundFabry disease (FD) is a recessive X-linked lysosomal storage disorder caused by α-galactosidase A (GLA) deficiency. Although the mechanism is unclear, GLA deficiency causes an accumulation of globotriaosylceramide (Gb3), leading to vasculopathy.MethodsTo explore the relationship between the accumulation of Gb3 and vasculopathy, induced pluripotent stem cells generated from four Fabry patients (FD-iPSCs) were differentiated into vascular endothelial cells (VECs). Genome editing using CRISPR-Cas9 system was carried out to correct the GLA mutation or to delete Thrombospondin-1 (TSP-1). Global transcriptomes were compared between wild-type (WT)- and FD-VECs by RNA-sequencing analysis.FindingsHere, we report that overexpression of TSP-1 contributes to the dysfunction of VECs in FD. VECs originating from FD-iPSCs (FD-VECs) showed aberrant angiogenic functionality even upon treatment with recombinant α-galactosidase. Intriguingly, FD-VECs produced more p-SMAD2 and TSP-1 than WT-VECs. We also found elevated TSP-1 in the peritubular capillaries of renal tissues biopsied from FD patients. Inhibition of SMAD2 signaling or knock out of TSP-1 (TSP-1−/−) rescues normal vascular functionality in FD-VECs, like in gene-corrected FD-VECs. In addition, the enhanced oxygen consumption rate is reduced in TSP-1−/− FD-VECs.InterpretationThe overexpression of TSP-1 secondary to Gb3 accumulation is primarily responsible for the observed FD-VEC dysfunction. Our findings implicate dysfunctional VEC angiogenesis in the peritubular capillaries in some of the complications of Fabry disease.FundingThis study was supported by grant 2018M3A9H1078330 from the National Research Foundation of the Republic of Korea.

TMIC-46. GLIOMA-INDUCED SYNAPTOGENESIS IS ENRICHED WITHIN FUNCTIONAL CONNECTIVITY NETWORK HUBS AND INFLUENCES LANGUAGE PROCESSING IN ADULT IDH WT GLIOBLASTOMA

Abstract INTRODUCTION Little is known about the mechanisms by which gliomas integrate into functional neural networks and influence complex cognitive processes such as language. Glioma-neuron interactions are bidirectional, with increased neuronal activity promoting tumor growth and the latter in turn influencing neuronal excitability and synaptic connections. It remains unknown whether glioma-neuron interactions play a role in maintaining long-range neural networks subserving cognition in humans. We test the hypothesis that glioma-neuron interactions (“synaptogenic glioma cells”) are enriched within intratumoral high functional connectivity (FC) network hubs, thereby influencing language processing via release of synaptogenic factors into the tumor microenvironment. METHODS We employed magnetoencephalography imaginary coherence measures to identify intratumoral high (HFC) and low (LFC) functional connectivity network hubs in newly diagnosed glioblastoma patients. Primary patient samples and cultures from HFC and LFC sites were assessed for pre and post-synaptic marker expression (IF), cocultured with murine hippocampal neurons, and induced neuron organoids. ECOG Field recordings were performed on HFC/LFC tumors. Secreted proteins were measured from patient serum and LFC/HFC culture supernatant. Language assessments were performed to correlate task performance with FC measures. RESULTS Primary patient samples from HFC regions are enriched for glioblastoma cells with a synaptogenic profile as characterized by pre- and post-synaptic marker expression at both tissue and cellular level (coculture with mouse hippocampal neuron and organoid models). RNA sequencing and proteomic analyses from HFC samples revealed a neurogenic signature including thrombospondin 1 (TSP1). Overexpression of TSP1 in LFC primary patient cultures rescues the synaptogenic and proliferative phenotype. Importantly, we found a linear relationship between intratumoral HFC with patient serum TSP1 (ELISA) with a further correlation with language task performance. CONCLUSION An enriched population of synaptogenic glioma cells are organized within intratumoral high network connectivity regions. Glioma-induced neuronal synaptogenesis contributes to the microenvironment in support of network connectivity through secretion of TSP1.

TSP-1 is downregulated and inversely correlates with miR-449c expression in Cushing's disease.

The pathogenesis of Cushing's disease, which is caused by pituitary corticotroph adenoma, remains to be studied. Secreted angioinhibitory factor thrombospondin‐1 (TSP‐1) is an adhesive glycoprotein that mediates cell‐to‐cell and cell‐to‐matrix interactions and is associated with platelet aggregation, angiogenesis and tumorigenesis. We have found that the expression of TSP‐1 is significantly lower in human pituitary corticotroph tumours compared with normal adenohypophysis. This study aims to elucidate the role of TSP‐1 in regulating the tumour function of pituitary adenomas. Forced overexpression of TSP‐1 in a murine AtT20 pituitary corticotroph tumour cell line decreased corticotroph precursor hormone proopiomelanocortin (POMC) transcription and adrenocorticotropic hormone (ACTH) secretion. Functional studies showed that TSP‐1 overexpression in pituitary adenoma cells suppressed proliferation, migration and invasion. We have demonstrated that TSP‐1 is a direct target of miR‐449c. Further study showed that miR‐449c activity enhanced tumorigenesis by directly inhibiting TSP‐1 expression. Low expression of lncTHBS1, along with low expression of TSP‐1, was associated with the high expression of miR‐449c in Cushing's disease patients. Furthermore, RNA‐immunoprecipitation associates miR‐449c with lncTHBS1 suggesting that lncTHBS1 might be a negative regulator of miR‐449c. Taken together, this study has demonstrated that lncTHBS1 might function as competing endogenous RNA for miR‐449c, which could suppress the development of Cushing's disease.