THBS1 Research Articles

CAF-macrophage crosstalk in tumour microenvironments governs the response to immune checkpoint blockade in gastric cancer peritoneal metastases

BackgroundPeritoneal metastasis is the most common metastasis pattern of gastric cancer. Patients with gastric cancer peritoneal metastasis (GCPM) have a poor prognosis and respond poorly to conventional treatments. Recently, immune...

Circulating thrombospondin 2 as a predictor of hepatocellular carcinoma in hepatitis B patients undergoing nucleos(t)ide analog therapy

Thrombospondin 2 (TSP2) plays a vital role in collagen/fibrin formation, bone growth, vascular density regulation, hemostasis, and cell adhesion. Close associations of serum TSP2 with histological severity in non-alcoholic fatty liver disease and chronic hepatitis C were reported. The present study investigated the significance of circulating TSP2 in chronic hepatitis B patients. Eighty-seven biopsy-proven chronic hepatitis B patients were analyzed in cross-sectional Study 1 to search for correlations between serum TSP2 levels prior to liver biopsy and clinicopathological parameters. In longitudinal Study 2, 51 chronic hepatitis B patients with long-term follow-up (mean: 7.5 years) were examined for changes in serum TSP2 levels during nucleos(t)ide analog (NA) therapy along with trends in hepatocarciongenesis. In Study 1, serum TSP2 levels were not significantly associated with portal inflammation or fibrosis. Study 2 revealed that serum TSP2 was significantly decreased after 48 weeks of NA therapy (P < 0.001). Notably, TSP2 levels at 48 weeks of NA administration (TSP2-48W) were significantly higher in the hepatocellular carcinoma (HCC) (+) group than in the HCC (−) group (P = 0.043). Kaplan–Meier analysis showed that higher TSP2-48W (≥ 24 ng/mL) was associated with future HCC development (P = 0.030). Serum TSP2 levels may be a potential predictor of HCC development in hepatitis B patients receiving NA therapy. Longitudinal prospective studies are necessary to validate our findings.

Thrombospondin 2 as a Predictive Biomarker for HCC inHepatitis C Patients: A Longitudinal Study Following DAA Therapy.

This multicentre study investigated the dynamics of thrombospondin 2 (TSP2) levels during direct-acting antiviral (DAA) therapy in hepatitis C virus (HCV) infected patients and evaluated TSP2's potential as a predictive marker for hepatocellular carcinoma (HCC). All 134 participants achieved sustained virological response at 12 weeks (SVR12) with DAA therapy, and serum TSP2 levels significantly decreased from before and after treatment (p < 0.001). During the median follow-up period of 6.0 years, HCC after DAA therapy was observed in 16 patients (11.9%). Patients with serum TSP2 High (≥ 32 ng/mL) at SVR12 had a significantly higher cumulative occurrence of HCC than did those without (26.5% vs. 7.0%, p = 0.0033). A multivariate Cox proportional hazards model identified male gender (HR 4.84, p = 0.005), HCC history (HR 4.61, p = 0.017) and TSP2 High (HR 3.93, p = 0.009) as significant independent predictors of HCC occurrence after DAA therapy. The model had a high concordance index of 0.878. Additionally, combining TSP2 High and FIB-4 High (≥ 3.538) at SVR12 yielded high specificity and negative predictive value (0.941 and 0.917, respectively) for predicting HCC. Kaplan-Meier analysis showed a higher HCC incidence in the TSP2 High + FIB-4 High group (log-rank p < 0.0001). In conclusion, TSP2 may be a promising biomarker for personalised HCC surveillance in DAA-treated hepatitis C patients.

Targeting hypoxia and thrombospondin-2 in diabetic wound healing.

Impaired wound healing in diabetic patients is the leading cause of diabetes-associated hospitalizations and approximately 50% of lower limb amputations. This is due to multiple factors, including elevated glucose, sustained hypoxia, and cell dysfunction. Previously, diabetic wounds were found to contain excessive levels of the matricellular protein thrombospondin-2 (TSP2) and genetic ablation of TSP2 in diabetic mice or treatment of wounds with a hydrogel derived from TSP2-null mouse skin improved healing. Previously, TSP2 has been shown to be repressed by hypoxia, but in the present study we observed sustained hypoxia and overlapping TSP2 deposition in diabetic wounds. We determined this observation was due to the insufficient HIF-1α activation verified by western blot and immunofluorescent analysis of wound tissues and invitro hypoxia experiments. Application of Dimethyloxalylglycine (DMOG), which can stabilize HIF-1α, inhibited TSP2 expression in diabetic fibroblasts in hypoxic conditions. Therefore, we prepared DMOG-containing TSP2KO hydrogel and applied it to the wounds of diabetic mice. In comparison to empty TSP2KO hydrogel or DMOG treatment, we observed improved wound healing associated with a reduction of TSP2, reduced hypoxia, and increased neovascularization. Overall, our findings shed light on the intricate interplay between hyperglycemia, hypoxia, and TSP2 in the complex environment of diabetic wounds.

Fibroblast-specific TGF-β signaling mediates cardiac dysfunction, fibrosis, and hypertrophy in obese diabetic mice.

Transforming growth factor (TGF)-β is up-regulated in the diabetic myocardium and may mediate fibroblast activation. We aimed at examining the role of TGF-β-induced fibroblast activation in the pathogenesis of diabetic cardiomyopathy. We generated lean and obese db/db mice with fibroblast-specific loss of TbR2, the Type 2 receptor-mediating signaling through all three TGF-β isoforms, and mice with fibroblast-specific Smad3 disruption. Systolic and diastolic function, myocardial fibrosis, and hypertrophy were assessed. Transcriptomic studies and in vitro experiments were used to dissect mechanisms of fibroblast activation. Fibroblast-specific TbR2 loss attenuated systolic and diastolic dysfunction in db/db mice. The protective effects of fibroblast TbR2 loss in db/db mice were associated with attenuated fibrosis and reduced cardiomyocyte hypertrophy, suggesting that in addition to their role in fibrous tissue deposition, TGF-β-stimulated fibroblasts may also exert paracrine actions on cardiomyocytes. Fibroblast-specific Smad3 loss phenocopied the protective effects of fibroblast TbR2 loss in db/db mice. Db/db fibroblasts had increased expression of genes associated with oxidative response (such as Fmo2, encoding flavin-containing monooxygenase 2), matricellular genes (such as Thbs4 and Fbln2), and Lox (encoding lysyl oxidase). Ingenuity pathway analysis (IPA) predicted that neurohumoral mediators, cytokines, and growth factors (such as AGT, TGFB1, and TNF) may serve as important upstream regulators of the transcriptomic profile of diabetic mouse fibroblasts. IPA of scRNA-seq data identified TGFB1, p53, MYC, PDGF-BB, EGFR, and WNT3A/CTNNB1 as important upstream regulators underlying fibroblast activation in db/db hearts. Comparison of the transcriptome of fibroblasts from db/db mice with fibroblast-specific Smad3 loss and db/db Smad3 fl/fl controls identified Thbs4 [encoding thrombospondin-4 (TSP-4), a marker of activated fibroblasts] as a candidate diabetes-induced fibrogenic mediator. However, in vitro experiments showed no significant activating effects of matricellular or intracellular TSP-4 on cardiac fibroblasts. Fibroblast-specific TGF-β/Smad3 signaling mediates ventricular fibrosis, hypertrophy, and dysfunction in Type 2 diabetes.

The role and mechanism of thrombospondin-4 in pulmonary arterial hypertension associated with congenital heart disease

BackgroundDue to a special hemodynamic feature, pulmonary vascular disease in pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD) has two stages: reversible and irreversible. So far, the mechanism involved in the transition from reversible to irreversible stage is elusive. Moreover, no recognized and reliable assessments to distinguish these two stages are available. Furthermore, we found that compared with control and reversible PAH, thrombospondin-4 (THBS4) was significantly upregulated in irreversible group by bioinformatic analysis. Hence, we further verify and investigate the expression and role of THBS4 in PAH-CHD.MethodsWe established the monocrotaline plus aorto-cava shunt-induced (MCT-AV) rat model. We measured the expression of THBS4 in lung tissues from MCT-AV rats. Double immunofluorescence staining of lung tissue for THBS4 and α-SMA (biomarker of smooth muscle cells) or vWF (biomarker of endothelial cells) to identify the location of THBS4 in the pulmonary artery. Primary pulmonary artery smooth muscle cells (PASMCs) were cultivated, identified, and used in this study. THBS4 was inhibited and overexpressed by siRNA and plasmid, respectively, to explore the effect of THBS4 on phenotype transformation, proliferation, apoptosis, and migration of PASMCs. The effect of THBS4 on pulmonary vascular remodeling was evaluated in vivo by adeno-associated virus which suppressed THBS4 expression. Circulating level of THBS4 in patients with PAH-CHD was measured by ELISA.ResultsTHBS4 was upregulated in the lung tissues of MCT-AV rats, and was further upregulated in severe pulmonary vascular lesions. And THBS4 was expressed mainly in PASMCs. When THBS4 was inhibited, contractile markers α-SMA and MYH11 were upregulated, while the proliferative marker PCNA was decreased, the endothelial-mensenchymal transition marker N-cad was downregulated, proapototic marker BAX was increased. Additionally, proliferation and migration of PASMCs was inhibited and apoptosis was increased. Conversely, THBS4 overexpression resulted in opposite effects. And the impact of THBS4 on PASMCs was probably achieved through the regulation of the PI3K/AKT pathway. THBS4 suppression attenuated pulmonary vascular remodeling. Furthermore, compared with patients with simple congenital heart disease and mild PAH-CHD, the circulating level of THBS4 was higher in patients with severe PAH-CHD.ConclusionsTHBS4 is a promising biomarker to distinguish reversible from irreversible PAH-CHD before repairing the shunt. THBS4 is a potential treatment target in PAH-CHD, especially in irreversible stage.

CTGF, FN1, IL-6, THBS1, and WISP1 genes and PI3K-Akt signaling pathway as prognostic and therapeutic targets in gastric cancer identified by gene network modeling

ObjectiveGastric cancer (GC) is one of the most common malignancies worldwide and it is considered the fourth most common cause of cancer death. This study aimed to find critical genes/pathways in GC pathogenesis to be used as biomarkers or therapeutic targets.MethodsDifferentially expressed genes were explored between human gastric cancerous and noncancerous tissues, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analyses were done. Hub genes were identified based on the protein–protein interaction network constructed in the STRING database with Cytoscape software. The hub genes were selected for further investigation using GEPIA2 and DrugBank databases.ResultsTen overexpressed hub genes in GC were identified in the current study, including FN1, TP53, IL-6, CXCL5, ELN, ADAMTS2, WISP1, MMP2, CTGF, and THBS1. The study demonstrated the PI3K-Akt pathway's central involvement in GC, with pronounced alterations in essential components. Survival analysis revealed significant correlations between CTGF, FN1, IL-6, THBS1, and WISP1 overexpression and reduced overall survival times in GC patients.ConclusionA mutual interplay emerged, where PI3K-Akt signaling could upregulate certain genes, forming feedback loops and intensifying cancer phenotypes. The interconnected overexpression of genes and the PI3K-Akt pathway fosters gastric tumorigenesis, suggesting therapeutic potential. DrugBank analysis identified limited FDA-approved drugs, advocating for further exploration while targeting these hub genes could reshape GC treatment. The identified genes could be novel diagnostic/prognostic biomarkers or potential therapeutic targets for GC, but further clinical validation is required.

Bone-derived extracellular matrix hydrogel from thrombospondin-2 knock-out mice for bone repair

Bone extracellular matrix (ECM) has been shown to mimic aspects of the tissue's complex microenvironment, suggesting its potential role in promoting bone repair. However, current ECM-based therapies suffer from limitations such as inefficient scale-up, lack of mechanical integrity, and sub-optimal efficacy. Here, we fabricated hydrogels from decellularized ECM (dECM) from wild type (WT) and thrombospondin-2 knock-out (TSP2KO) mouse bones. TSP2KO bone ECM hydrogel was found to have distinct mechanical properties and collagen fibril assembly from WT. Furthermore, TSP2KO hydrogel promoted mesenchymal stem cell (MSC) attachment, spreading, and invasion in vitro. Similarly, it promoted formation of tube-like structures by human umbilical vein endothelial cells (HUVECs). When applied to a murine calvarial defect model, TSP2KO hydrogel enhanced repair, in part, due to increased angiogenesis. Our study suggests the pro-angiogenic therapeutic potential of TSP2KO bone ECM hydrogel in bone repair. Statement of significanceThe study describes the first successful preparation of a novel hydrogel made from decellularized bones from wild-type mice and mice lacking thrombospondin-2 (TSP2). Hydrogels from TSP2 knock-out (TSP2KO) bones have unique characteristics in structure and biomechanics. These gels interacted well with cells in vitro and helped repair damaged bone in a mouse model. Therefore, TSP2KO bone-derived hydrogel has translational potential for accelerating repair of bone defects that are otherwise difficult to heal. This study not only creates a new material with promise for accelerated healing, but also validates tunability of native biomaterials by genetic engineering.

The RP11-417E7.1/THBS2 signaling pathway promotes colorectal cancer metastasis by activating the Wnt/β-catenin pathway and facilitating exosome-mediated M2 macrophage polarization

BackgroundMetastasis is the major cause of colorectal cancer (CRC) mortality. Emerging evidence suggests that long noncoding RNAs (lncRNAs) drive cancer metastasis and that their regulatory pathways could be targeted for preventing metastasis. However, the underlying mechanisms of lncRNAs in CRC metastasis remain poorly understood.MethodsMicroarray analysis was used to screen for differentially expressed lncRNAs. Transwell assays, fibronectin cell adhesion assays, and mouse metastasis models were utilized to evaluate the metastatic capacities of CRC in vitro and in vivo. Chromatin isolation by RNA purification, chromatin immunoprecipitation and chromosome conformation capture were applied to investigate the underlying mechanism involved. qRT‒PCR and transmission electron microscopy were performed to confirm macrophage polarization and the presence of cancer-derived exosomes.ResultsThe lncRNA RP11-417E7.1 was screened and identified as a novel metastasis-associated lncRNA that was correlated with a poor prognosis. RP11-417E7.1 enhances the metastatic capacity of CRC cells in vivo and in vitro. Mechanistically, RP11-417E7.1 binding with High mobility group A1 (HMGA1) promotes neighboring thrombospondin 2 (THBS2) transcription via chromatin loop formation between its promoter and enhancer, which activates the Wnt/β-catenin signaling pathway and facilitates CRC metastasis. Furthermore, exosomes derived from CRC cells transport THBS2 into macrophages, thereby inducing the M2 polarization of macrophages to sustain the prometastatic microenvironment. Notably, netropsin, a DNA-binding drug, suppresses chromatin loop formation mediated by RP11-417E7.1 at the THBS2 locus and significantly inhibits CRC metastasis in vitro and in vivo.ConclusionsThis study revealed the novel prometastatic function and mechanism of the lncRNA RP11-417E7.1, which provides a potential prognostic indicator and therapeutic target in CRC.

The long noncoding RNA lncMPD2 inhibits myogenesis by targeting the miR-34a-5p/THBS1 axis

Long noncoding RNAs (lncRNAs) participate in regulating skeletal muscle development. However, little is known about their role in regulating chicken myogenesis. In this study, we identified a novel lncRNA, lncMPD2, through transcriptome sequencing of chicken myoblasts at different developmental stages. Functionally, gain- and loss-of-function experiments showed that lncMPD2 inhibited myoblast proliferation and differentiation. Mechanistically, lncMPD2 directly bound to miR-34a-5p, and miR-34a-5p promoted myoblasts proliferation and differentiation and inhibited the mRNA and protein expression of its target gene THBS1. THBS1 inhibited myoblast proliferation and differentiation in vitro and delayed muscle regeneration in vivo. Furthermore, rescue experiments showed that lncMPD2 counteracted the inhibitory effects of miR-34a-5p on THBS1 and myogenesis-related gene mRNA and protein expression. In conclusion, lncMPD2 regulates the miR-34a-5p/THBS1 axis to inhibit the proliferation and differentiation of myoblasts and skeletal muscle regeneration. This study provides more insight into the molecular regulatory network of skeletal muscle development, identifying novel potential biomarkers for improving chicken quality and increasing chicken yield. In addition, this study provides a potential goal for breeding strategies that minimize muscle damage in chickens.

Acupuncture modulation of the ACE/Ang II/AT1R and ACE2/Ang(1-7)/MasR pathways in the rostral ventrolateral medulla reduces sympathetic output and prevents cardiac injury caused by SHR hypertension.

Acupuncture can reduce blood pressure, heart rate (HR), and ameliorate cardiac damage by modulating the excitability of the sympathetic nervous system, but the exact mechanism of this effect remains unclear. This study investigated the potential mechanisms of acupuncture in the treatment of cardiac damage in hypertension. Spontaneously hypertensive rats (SHR) were used as the hypertension model with Wistar-Kyoto rats as the control. Manual acupuncture, electroacupuncture, and metoprolol were used as interventions. Systolic and diastolic blood pressure (SBP, DBP) plus HR were monitored with cardiac structure determined using Masson staining. Angiotensin II (Ang II) and norepinephrine in myocardium were detected with ELISA as was Ang(1-7) and gamma aminobutyric acid (GABA) in the rostral ventrolateral medulla (RVLM). Expression of mRNA for collagen type I (Col-I), Col-III, actin α1 (ACTA1), and thrombospondin 4 (THBS4) in myocardium was detected using real-time PCR. Expression of angiotensin converting enzyme (ACE), Ang II, angiotensin II type 1 receptor (AT1R), ACE2, and Mas receptor (MasR) proteins in RVLM was monitored using western blot. After manual acupuncture and electroacupuncture treatment, SHRs showed decreased SBP, DBP and HR, reduced myocardial damage. There was decreased expression of the ACE/Ang II/AT1R axis, and increased expression of the ACE2/Ang(1-7)/MasR axis within the RVLM. GABA levels were increased within the RVLM and norepinephrine levels were decreased in myocardial tissue. Metoprolol was more effective than either manual acupuncture or electroacupuncture. Acupuncture directed against hypertensive cardiac damage may be associated with regulation of ACE/Ang II/AT1R and the ACE2/Ang(1-7)/MasR pathway within the RLVM to reduce cardiac sympathetic excitability.

Thrombospondin 2, matrix Gla protein and digital analysis identified distinct fibroblast populations in fibrostenosing Crohn’s disease

Fibrosis is an important complication in inflammatory bowel diseases. Previous studies suggest an important role of matrix Gla protein (MGP) and thrombospondin 2 (THBS2) in fibrosis in various organs. Our aim was to analyse their expression together with regulatory miRNAs in submucosal and subserosal fibroblasts in ulcerative colitis (UC) and Crohn’s disease (CD) using immunohistochemistry and qPCR. Digital pathology was used to compare collagen fibre characteristics of submucosal and subserosal fibrosis. Immunohistochemistry showed expression of MGP, but not THBS2 in submucosa in UC and CD. In the subserosa, there was strong staining for both proteins in CD but not in UC. qPCR showed significant upregulation of THBS2 and MGP genes in CD subserosa compared to the submucosa. Digital pathology analysis revealed higher proportion of larger and thicker fibres that were more tortuous and reticulated in subserosal fibrosis compared to submucosal fibrosis. These results suggest distinct fibroblast populations in fibrostenosing CD, and are further supported by image analysis showing significant differences in the morphology and architecture of collagen fibres in submucosal fibrosis in comparison to subserosal fibrosis. Our study is the first to describe differences in submucosal and subserosal fibroblast populations, contributing to understanding of the pathogenesis of fibrostenosis in CD.

Nattokinase attenuates endothelial inflammation through the activation of SRF and THBS1

Natto contains a potent fibrinolytic enzyme called nattokinase (NK), which has thrombolytic, antihypertensive, antiatherosclerotic and lipid-lowering effects. Although NK has been recognized for its beneficial effect on humans with atherosclerotic cardiovascular disease (ASCVD), the underlying mechanisms involved in vascular inflammation-atherosclerosis development remain largely unknown. The current study aimed to explore the effects of NK on gene regulation, autophagy, necroptosis and inflammasome in vascular inflammation. The transcriptional profiles of NK in endothelial cells (ECs) by RNA sequencing (RNA-seq) revealed that NK affected THBS1, SRF and SREBF1 mRNA expression. In Q-PCR analysis, SRF and THBS1 were upregulated but SREBF1 was unaffected in ECs treated with NK. NK treatment induced autophagy and inhibited NLRP3 inflammasome and necroptosis in ECs. Furthermore, the inhibition of SRF or THBS1 by siRNA suppressed autophagy and enhanced the NLRP3 inflammasome and necroptosis. In a mouse model, NK reduced vascular inflammation by activating autophagy and inhibiting NLRP3 inflammasome and necroptosis. Our findings provide the first evidence that NK upregulates SRF and THBS1 genes, subsequently increasing autophagy and decreasing necroptosis and NLRP3 inflammasome formation to reduce vascular inflammation. Therefore, NK could serve as nutraceuticals or adjuvant therapies to reduce vascular inflammation and possible atherosclerosis progression.

A proteomics-based survey reveals thrombospondin-4 as a ligand regulated by the mannose receptor in the injured lung

Receptor-mediated cellular uptake of specific ligands constitutes an important step in the dynamic regulation of individual protein levels in extracellular fluids. With a focus on the inflammatory lung, we here performed a proteomics-based search for novel ligands regulated by the mannose receptor (MR), a macrophage-expressed endocytic receptor. Wildtype and MR-deficient mice were exposed to lipopolysachharide (LPS), after which the protein content in their lung epithelial lining fluid (ELF) was compared by tandem mass tag-based mass spectrometry. More than 1200 proteins were identified in the ELF using this unbiased approach, but only six showed a statistically different abundance. Among these, an unexpected potential new ligand, thrombospondin-4 (TSP-4), displayed a striking 17-fold increased abundance in the MR-deficient mice. Experiments using exogenous addition of TSP-4 to MR-transfected CHO cells or MR-positive alveolar macrophages confirmed that TSP-4 is a ligand for MR-dependent endocytosis. Similar studies revealed that the molecular interaction with TSP-4 depends on both the lectin activity and the fibronectin type-II domain of MR and that a closely related member of the thrombospondin family, TSP-5, is also efficiently internalized by the receptor. This was unlike the other members of this protein family, including TSPs -1 and -2, which are ligands for a close MR homologue known as uPARAP. Our study shows that MR takes part in the regulation of TSP-4, an important inflammatory component in the injured lung, and that two closely related endocytic receptors, expressed on different cell types, undertake the selective endocytosis of distinct members of the thrombospondin family.

Abstract 2497: Extracellular vesicle based ALPPL2 and THBS2 as biomarkers for disease monitoring in patients with pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge in the realm of oncology, characterized by its aggressive nature and resistance to treatment. Several therapeutic regimens have been shown to extend patient survival, but the majority of the patients become refractory to the treatments in a short period of time. Hence, it is critical to monitor a patient’s response to treatment in a timely manner so that different treatment regimens can be considered promptly. Currently, CA19-9 is the most common blood-based marker being used to monitor disease burden in patients with PDAC. However, 15-20% of patients with PDAC do not have elevated blood CA19-9 levels. In this study, we sought to determine the utility of two circulating extracellular vesicle (EV) based protein biomarkers, ALPPL2 (Alkaline phosphatase, placental-like 2) and THBS2 (Thrombospondin 2), for disease monitoring in patients with PDAC. We first established and optimized EV assays using the ExoViewTM platform for detecting and quantifying the number of ALPPL2+ or THBS2+ EVs in serum samples. We then determined the concentrations of ALPPL2+ or THBS2+ EVs in samples from healthy individuals and longitudinal samples from patients with Stage IV PDAC undergone treatment. The longitudinal samples were from 26 patients of which 16 were CA19-9 secretors (defined as &amp;gt;35 U/ml) and 10 were CA19-9 non-secretors (defined as &amp;lt;35 U/ml) that were treated with various regimens for 4-24 months with monthly blood sample collection and CT scans (a total of 305 samples). We found that the concentrations of ALPPL2+ and THBS2+ EVs are on average 1,737 and 1,113 times higher in PDAC patients than in the healthy controls, respectively. Correlation analysis using the mixed linear model showed that the concentrations of both ALPPL2+ and THBS2+ EVs significantly correlate with changes in tumor size (based on RECIST measurements) in the longitudinal samples of CA19-9 non-secretors or secretors. In CA19-9 non-secretors the correlation p values are 0.003 and 0.006 for ALPPL2+ and THBS2+ EVs, respectively, whereas in CA19-9 secretors, the p values are 0.014 and 0.016 for ALPPL2+ and THBS2+ EVs, respectively. Our data indicates that EV based ALPPL2 and THBS2 could potentially serve as biomarkers for disease monitoring in patients with PDAC. (This work was supported in part by the Dorrance Family Fund and the Flinn Foundation) Citation Format: Kuntal Halder, Gayle Jameson, Erkut Borazanci, Wei Lin, Dereck Cridebring, Daniel Von Hoff, Haiyong Han. Extracellular vesicle based ALPPL2 and THBS2 as biomarkers for disease monitoring in patients with pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2497.

Abstract 7623: Biomarker study to personalized neoadjuvant chemotherapy using preoperative gastric cancer specimens of biopsy from the COMPASS trial, a phase II randmazed controlled trial

Abstract Background and Aim: The results of COMPASS, a randomized phase II trial, suggest that preoperative adjuvant chemotherapy (NAC) regimen or course for locally advanced gastric cancer (GC) does not affect overall survival (OS). However, we hypothesized that some NAC regimens may be more effective for OS on a case-by-case basis. Furthermore, we searched for biomarkers, assuming that further improvement in the outcome of locally advanced GC could be expected if appropriate NAC regimens could be selected using biomarkers before treatment initiation. Materials and Methods: RNA was extracted from endoscopic biopsy specimens of primary tumors collected prior to NAC administration in COMPASS patients, and real-time polymerase chain reaction (PCR) of 127 genes was performed to identify genes with significant (P &amp;lt; 0.05) interaction P values for survival with each regimen and stratified OS with each regimen. Genes were identified and biomarkers were searched at the protein level using immunohistochemical analysis. Results: The THBS1, MSI1, and IGF2BP3 genes were identified as genes whose expression levels significantly (P &amp;lt; 0.05) stratified survival for each regimen and had significant (P &amp;lt; 0.05) interaction P values. Furthermore, a strong correlation was observed between each gene and THBS1, MSI1, and IGF2BP protein expression by immunohistochemical analysis, confirming that protein expression levels by immunohistochemical analysis are also useful as biomarkers. Conclusion: Using endoscopic biopsy specimens prior to NAC for locally advanced GC, biomarkers were identified to select NAC regimens over predicted OS. The results of this study may guide the conduct of clinical trials of individualized treatment of NAC using biomarkers. Citation Format: Takashi Oshima. Biomarker study to personalized neoadjuvant chemotherapy using preoperative gastric cancer specimens of biopsy from the COMPASS trial, a phase II randmazed controlled trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7623.

The role of glia in the dysregulation of neuronal spinogenesis in Ube3a-dependent ASD

Overexpression of the Ube3a gene and the resulting increase in Ube3a protein are linked to autism spectrum disorder (ASD). However, the cellular and molecular processes underlying Ube3a-dependent ASD remain unclear. Using both male and female mice, we find that neurons in the somatosensory cortex of the Ube3a 2× Tg ASD mouse model display reduced dendritic spine density and increased immature filopodia density. Importantly, the increased gene dosage of Ube3a in astrocytes alone is sufficient to confer alterations in neurons as immature dendritic protrusions, as observed in primary hippocampal neuron cultures. We show that Ube3a overexpression in astrocytes leads to a loss of astrocyte-derived spinogenic protein, thrombospondin-2 (TSP2), due to a suppression of TSP2 gene transcription. By neonatal intraventricular injection of astrocyte-specific virus, we demonstrate that Ube3a overexpression in astrocytes in vivo results in a reduction in dendritic spine maturation in prelimbic cortical neurons, accompanied with autistic-like behaviors in mice. These findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels. Significance statementIncreased gene dosage of Ube3a is tied to autism spectrum disorders (ASDs), yet cellular and molecular alterations underlying autistic phenotypes remain unclear. We show that Ube3a overexpression leads to impaired dendritic spine maturation, resulting in reduced spine density and increased filopodia density. We find that dysregulation of spine development is not neuron autonomous, rather, it is mediated by an astrocytic mechanism. Increased gene dosage of Ube3a in astrocytes leads to reduced production of the spinogenic glycoprotein thrombospondin-2 (TSP2), leading to abnormalities in spines. Astrocyte-specific Ube3a overexpression in the brain in vivo confers dysregulated spine maturation concomitant with autistic-like behaviors in mice. These findings indicate the importance of astrocytes in aberrant neurodevelopment and brain function in Ube3a-depdendent ASD.

CxsAssociation study between single nucleotide polymorphism in thrombospondins THBS1 gene and THBS2 gene and coronary artery disease in Indian population

BackgroundCoronary artery disease (CAD) is a complex medical condition characterized by atherosclerotic plaque accumulation in coronary arteries, leading to narrowed blood vessels and impaired blood flow. Endothelial dysfunction, smooth muscle cell proliferation, and various risk factors contribute to CAD development. Matricellular proteins, including thrombospondins (THBS), play crucial roles in vascular processes and cardiac function.MethodsA case–control study was conducted among 296 participants from Era's Lucknow Medical College and Hospital, India, to investigate genetic variations in THBS1 (N700S) and THBS2 (3′ UTR T → G) in relation to CAD. Genomic DNA was isolated, and PCR–RFLP was employed for genotyping. Clinical and biochemical parameters were assessed, and statistical analyses were performed using SPSS software.ResultsThe study revealed that age, serum cholesterol, HDL, VLDL, and LDL were significantly associated with CAD in the Indian population. However, no statistically significant associations were found between triglyceride and serum creatinine levels, as well as the studied THBS1 and THBS2 genetic polymorphisms, and CAD. The analysis of genotypic and allelic frequencies did not indicate significant associations with CAD risk.ConclusionsThis study suggests that specific genetic variations in THBS1 and THBS2 may not be strongly linked to the development or risk of CAD in the studied Indian population. The associations observed between age, lipid profiles, and CAD highlight the multifactorial nature of CAD susceptibility. Further research with larger sample sizes and diverse populations is warranted to validate these findings and explore additional genetic factors contributing to CAD in specific populations.

A198 RELATIONSHIP BETWEEN HEPATIC GENE EXPRESSION AND MICROBIOME ACCORDING TO DISEASE SEVERITY IN OBESE PATIENTS WITH NON-ALCOHOLIC FATTY LIVER DISEASE

Abstract Background Non-alcoholic fatty liver disease (NAFLD) ranges from steatosis to inflammation (steatohepatitis; NASH), fibrosis and cirrhosis. Development and progression of NAFLD/NASH is attributed to factors such as obesity and metabolic syndrome as well as intestinal microbiome (IM) but research is limited on the relationship between IM and hepatic gene expression. Aims Our objective was to assess in obese patients;1) the transcriptome signature of NAFLD and fibrosis and; 2) determine the relationship between the identified genes and IM. Methods Biochemical and anthropometric measurements as well fecal samples for IM shotgun metagenomic sequencing were collected in those with obesity undergoing bariatric surgery. Liver histology was assessed using Brunt scoring system and hepatic transcriptome was assessed using RNA-Seq. Differences in biochemical data and IM were determined using Kruskal-Wallis test followed by Wilcoxon ranked sum test. Differentially expressed genes (DEGs) in the liver were determined using DESeq2 package in R and had |log2 foldchange| ≥1 with adjusted p-value ampersand:003C0.05. Spearman correlation coefficients was used to evaluate the association between significant DEGs and significant bacteria and adjusted using Benjamini-Hochberg. Results 93 patients had both transcriptome and IM data. Of those, 27 had normal liver obese (NLO) and 66 had NAFLD (including 31 NASH). In those with NAFLD, 19 had no fibrosis (F0) and 47 had presence of fibrosis (F1-F4). We found 71 significantly different DEGs (e.g. PPP1R3G ) in NASH vs NLO and 12 (e.g. THBS2 and GLI2) in NAFLD with presence of fibrosis vs no fibrosis. Those with NASH generally had higher Escherichia coli, Blautia hansenii and lower Alistipees putredinis. Those with NAFLD and fibrosis had increased E. coli and reduced Eubacterium ventriosum and A. putredinis. In the preliminary analysis of NASH vs NLO, B. hansenii negatively correlated with glucose homeostasis-related PPP1R3G. In those with NAFLD, A. putredinis correlated negatively with THBS2 (i.e. Thrombospondin-2) and, in patients with NAFLD and fibrosis, E. coli correlated positively with GLI2 (related to activation of hepatic stellate cells). Conclusions DEGs were associated with specific bacteria in NASH or fibrosis. Next steps include analyzing the microbial metagenomic pathways as well as metabolites and determine the relationship between the IM, hepatic transcriptome and metabolites according to disease severity. Funding Agencies CIHRAmerican College of Gastroenterology Bridge Award, BBDC Tamarack Graduate Award in Diabetes Research

Proteomic associations with forced expiratory volume: a Mendelian randomisation study

BackgroundA decline in forced expiratory volume (FEV1) is a hallmark of respiratory diseases that are an important cause of morbidity among the elderly. While some data exist on biomarkers that are related to FEV1, we sought to do a systematic analysis of causal relations of biomarkers with FEV1.MethodsData from the population-based AGES-Reykjavik study were used. Serum proteomic measurements were done using 4782 DNA aptamers (SOMAmers). Data from 1479 participants with spirometric data were used to assess the association of SOMAmer measurements with FEV1 using linear regression. Bi-directional two-sample Mendelian randomisation (MR) analyses were done to assess causal relations of observationally associated SOMAmers with FEV1, using genotype and SOMAmer data from 5368 AGES-Reykjavik participants and genetic associations with FEV1 from a publicly available GWAS (n = 400,102).ResultsIn observational analyses, 530 SOMAmers were associated with FEV1 after multiple testing adjustment (FDR < 0.05). The most significant were Retinoic Acid Receptor Responder 2 (RARRES2), R-Spondin 4 (RSPO4) and Alkaline Phosphatase, Placental Like 2 (ALPPL2). Of the 257 SOMAmers with genetic instruments available, eight were associated with FEV1 in MR analyses. Three were directionally consistent with the observational estimate, Thrombospondin 2 (THBS2), Endoplasmic Reticulum Oxidoreductase 1 Beta (ERO1B) and Apolipoprotein M (APOM). THBS2 was further supported by a colocalization analysis. Analyses in the reverse direction, testing whether changes in SOMAmer levels were caused by changes in FEV1, were performed but no significant associations were found after multiple testing adjustments.ConclusionsIn summary, this large scale proteogenomic analyses of FEV1 reveals circulating protein markers of FEV1, as well as several proteins with potential causality to lung function.