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Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD. [ABSTRACT FROM AUTHOR]

Abstract Aims/Introduction We investigated the relationship of circulating TSP‐1 mRNA and miR‐194 with diabetic kidney disease’s degree. Materials and Methods We enrolled 167 hospitalized type 2 diabetes patients in the endocrinology department. Patients were split into three groups according to urinary microalbumin: A, B and C. The control group comprised healthy outpatients (n = 163). The quantities of microribonucleic acid (miR)‐194 and thrombospondin‐1 (TSP‐1) messenger ribonucleic acid (mRNA) in the participants’ circulation were measured using a quantitative real‐time polymerase chain reaction. Results Circulating TSP‐1 mRNA (P = 0.024) and miR‐194 (P = 0.029) expressions significantly increased in type 2 diabetes patients. Circulating TSP‐1 mRNA (P = 0.040) and miR‐194 (P = 0.007) expression levels differed significantly among the three groups; circulating TSP‐1 mRNA expression increased with urinary microalbumin. However, miR‐194 declined in group B and increased in group C. Circulating TSP‐1 mRNA was positively correlated with cystatin‐c (r = 0.281; P = 0.021) and microalbumin/creatinine ratio (UmALB/Cr; r = 0.317; P = 0.009); miR‐194 was positively correlated with UmALB/Cr (r = 0.405; P = 0.003). Stepwise multivariate linear regression analysis showed cystatin‐c (β = 0.578; P = 0.021) and UmALB/Cr (β = 0.001; P = 0.009) as independent factors for TSP‐1 mRNA; UmALB/Cr (β = 0.005; P = 0.028) as an independent factor for miR194. Areas under the curve for circulating TSP‐1 mRNA and miR194 were 0.756 (95% confidence interval 0.620–0.893; sensitivity 0.69 and specificity 0.71, P

Background: Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic disorder leading to frequent bleeding in several organs. As HHT diagnosis is demanding and depends on clinical criteria, liquid biopsy would be beneficial. Exosomes from biofluids are nano-sized vesicles for intercellular communication. Their cargo and characteristics represent biomarkers for many diseases. Here, exosomes of HHT patients were examined regarding their biosignature. Methods: Exosomes were isolated from the plasma of 20 HHT patients and 17 healthy donors (HDs). The total exosomal protein was quantified, and specific proteins were analyzed using Western blot and antibody arrays. Human umbilical vein endothelial cells (HUVECs) co-incubated with exosomes were functionally examined via immunofluorescence, proliferation, and scratch assay. Results: The levels of the angiogenesis-regulating protein Thrombospondin-1 were significantly higher in HHT compared to HD exosomes. Among HHT, but not HD exosomes, a negative correlation between total exosomal protein and soluble Endoglin (sENG) levels was found. Other exosomal proteins (ALK1, ALK5) and the particle concentration significantly correlated with disease severity parameters (total consultations/interventions, epistaxis severity score) in HHT patients. Functionally, HUVECs were able to internalize both HD and HHT exosomes, inducing a similar change in the F-Actin structure and a reduction in migration and proliferation. Conclusions: This study provided first insights into the protein cargo and function of HHT-derived exosomes. The data indicate changes in sENG secretion via exosomes and reveal exosomal Thrombospondin-1 as a potential biomarker for HHT. Several exosomal characteristics were pointed out as potential liquid biomarkers for disease severity, revealing a possible new way of diagnosis and prognosis of HHT. [ABSTRACT FROM AUTHOR]

Aims/Introduction: We investigated the relationship of circulating TSP‐1 mRNA and miR‐194 with diabetic kidney disease's degree. Materials and Methods: We enrolled 167 hospitalized type 2 diabetes patients in the endocrinology department. Patients were split into three groups according to urinary microalbumin: A, B and C. The control group comprised healthy outpatients (n = 163). The quantities of microribonucleic acid (miR)‐194 and thrombospondin‐1 (TSP‐1) messenger ribonucleic acid (mRNA) in the participants' circulation were measured using a quantitative real‐time polymerase chain reaction. Results: Circulating TSP‐1 mRNA (P = 0.024) and miR‐194 (P = 0.029) expressions significantly increased in type 2 diabetes patients. Circulating TSP‐1 mRNA (P = 0.040) and miR‐194 (P = 0.007) expression levels differed significantly among the three groups; circulating TSP‐1 mRNA expression increased with urinary microalbumin. However, miR‐194 declined in group B and increased in group C. Circulating TSP‐1 mRNA was positively correlated with cystatin‐c (r = 0.281; P = 0.021) and microalbumin/creatinine ratio (UmALB/Cr; r = 0.317; P = 0.009); miR‐194 was positively correlated with UmALB/Cr (r = 0.405; P = 0.003). Stepwise multivariate linear regression analysis showed cystatin‐c (β = 0.578; P = 0.021) and UmALB/Cr (β = 0.001; P = 0.009) as independent factors for TSP‐1 mRNA; UmALB/Cr (β = 0.005; P = 0.028) as an independent factor for miR194. Areas under the curve for circulating TSP‐1 mRNA and miR194 were 0.756 (95% confidence interval 0.620–0.893; sensitivity 0.69 and specificity 0.71, P < 0.01) and 0.584 (95% confidence interval 0.421–0.748; sensitivity 0.54 and specificity 0.52, P < 0.01), respectively. Conclusions: Circulating TSP‐1 mRNA and miR‐194 expressions significantly increased in type 2 diabetes patients. The microalbumin group had lower levels of miR‐194 (a risk factor that is valuable for type 2 diabetes kidney disease evaluation). [ABSTRACT FROM AUTHOR]

Focal segmental glomerulosclerosis (FSGS), a common cause of primary glomerulonephritis, has a poor prognosis and is pathologically featured by tubulointerstitial injury. Thrombospondin-1 (TSP-1) is an extracellular matrix protein that acts in combination with different receptors in the kidney. Here, we analyzed the tubular expression of TSP-1 and its receptor integrin b3 (ITGB3) in FSGS. Previously the renal interstitial chip analysis of FSGS patients with tubular interstitial injury showed that the expression of TSP-1 and ITGB3 were upregulated. We found that the expression of TSP-1 and ITGB3 increased in the tubular cells of FSGS patients. The plasma level of TSP-1 increased and was correlated to the degree of tubulointerstitial lesions in FSGS patients. TSP-1/ ITGB3 signaling induced renal tubular injury in HK-2 cells exposure to bovine serum albumin and the adriamycin (ADR)- induced nephropathy model. THBS1 KO ameliorated tubular injury and renal fibrosis in ADR-treated mice. THBS1 knockdown decreased the expression of KIM-1 and caspase 3 in the HK-2 cells treated with bovine serum albumin, while THBS1 overexpression could induce tubular injury. In vivo, we identified cyclo-RGDfK as an agent to block the binding of TSP-1 to ITGB3. Cyclo-RGDfK treatment could alleviate ADR-induced renal tubular injury and interstitial fibrosis in mice. Moreover, TSP-1 and ITGB3 were colocalized in tubular cells of FSGS patients and ADR-treated mice. Taken together, our data showed that TSP-1/ITGB3 signaling contributed to the development of renal tubulointerstitial injury in FSGS, potentially identifying a new therapeutic target for FSGS. [ABSTRACT FROM AUTHOR]

Background: Fine particulate matter (PM2.5) induces ocular surface toxicity through pyroptosis, oxidative stress, autophagy, and inflammatory responses. However, the precise molecular pathways through which PM2.5 causes corneal damage remain unclear. This study aims to investigate the underlying mechanisms by exposing human corneal epithelial cells (HCECs) to PM2.5. Methods: After the morphology and chemical composition analysis of the PM samples, we conducted both in vivo and in vitro experiments to investigate PM2.5-induced corneal epithelial damage. We assessed corneal barrier function in HCECs using transepithelial electrical resistance (TEER) assays. To explore the molecular mechanisms of PM2.5-induced corneal epithelial damage, we performed whole-transcriptome resequencing, quantitative RT-PCR, and western blotting in vitro. In addition, we analyzed mouse corneas exposed to concentrated ambient PM2.5 through immunofluorescence staining to observe the resulting changes in corneal epithelial protein expression in vivo. Results: Our results showed significant impairment of corneal epithelial barrier function in PM2.5-treated HCECs, as indicated by decreased TEER values. The expression of thrombospondin-1 (THBS1) and claudin-1, both key factors for maintaining corneal epithelial barrier integrity, was markedly reduced at the gene and protein levels in both in vitro and in vivo PM2.5 exposure models. Moreover, the levels of tight junction-associated proteins, including occludin, zonula occludens-1 (ZO-1) and ZO-2, essential components of the corneal epithelial barrier, were significantly diminished in PM2.5-treated HCECs. Conclusion: PM2.5 exposure leads to corneal epithelium damage by disrupting tight junction proteins and THBS1 expression. These findings provide insight into potential pathways for PM2.5-induced ocular toxicity and underscore the need for protective strategies against such environmental pollutants.

Preeclampsia (PE) is considered as a disease of placental origin. However, the specific mechanism of placental abnormalities remains elusive. This study identified thrombospondin‐1 (THBS1) is downregulated in preeclamptic placentae and negatively correlated with blood pressure. Functional studies show that THBS1 knockdown inhibits proliferation, migration, and invasion and increases the cycle arrest and apoptosis rate of HTR8/SVneo cells. Importantly, THBS1 silencing induces necroptosis in HTR8/SVneo cells, accompanied by the release of damage‐associated molecular patterns (DAMPs). Necroptosis inhibitors necrostatin‐1 and GSK′872 restore the trophoblast survival while pan‐caspase inhibitor Z‐VAD‐FMK has no effect. Mechanistically, the results show that THBS1 interacts with transforming growth factor B‐activated kinase 1 (TAK1), which is a central modulator of necroptosis quiescence and affects its stability. Moreover, THBS1 silencing up‐regulates the expression of neuronal precursor cell‐expressed developmentally down‐regulated 4 (NEDD4), which acts as an E3 ligase of TAK1 and catalyzes K48‐linked ubiquitination of TAK1 in HTR8/SVneo cells. Besides, THBS1 attenuates PE phenotypes and improves the placental necroptosis in vivo. Taken together, the down‐regulation of THBS1 destabilizes TAK1 by activating NEDD4‐mediated, K48‐linked TAK1 ubiquitination and promotes necroptosis and DAMPs release in trophoblast cells, thus participating in the pathogenesis of PE. [ABSTRACT FROM AUTHOR]

Abstract Aims/Introduction Thrombospondin‐1 (TSP‐1) participates in a series of physiological and pathological processes by binding to various receptors regulating cell proliferation, adhesion and apoptosis. Elevated circulating TSP‐1 is linked with diabetic vascular complications (DVC). This study aimed to determine the relationship between circulating TSP‐1 levels and DVC. Materials and Methods A comprehensive search of PubMed, Embase, Web of Science and CNKI databases was carried out. A meta‐analysis was carried out to compare circulating TSP‐1 levels between diabetes patients without vascular complications (DNVC), diabetes patients with DVC and non‐diabetes patients. The correlation between TSP‐1 and metabolic parameters was also analyzed. Subgroup analysis was carried out according to complication type, defined as diabetic retinopathy, diabetic nephropathy and diabetic cardiovascular disease (DCVD). Results A total of eight studies were included. Compared with non‐diabetes patients, diabetic patients, including DNVC and DVC, had significantly higher circulating TSP‐1 levels (standardized mean difference [SMD] 2.660, 95% CI 1.17–4.145, P = 0.000). DNVC had significantly higher circulating TSP‐1 levels than non‐diabetes patients (SMD 3.613, 95% CI 1.607–5.619, P = 0.000). DVC had significantly higher TSP‐1 levels than DNVC (SMD 0.568, 95% CI 0.100–1.036, P = 0.017). TSP‐1 was significantly positively correlated with fasting plasma glucose (overall Fisher's z = 0.696, 95% CI 0.559–0.833) and HbA1c (overall Fisher's z = 0.849, 95% CI 0.776–0.923). Conclusions Elevated circulating TSP‐1 levels are closely related to DVC, especially in diabetic nephropathy and diabetic cardiovascular disease. Circulating TSP‐1 detection might be helpful in the timely diagnosis and treatment of DVC.

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Extended liver resection carries the risk of post-surgery liver failure involving thrombospondin-1-mediated aggravation of hepatic epithelial plasticity and function. Mesenchymal stromal cells (MSCs), by interfering with thrombospondin-1 (THBS1), counteract hepatic dysfunction, though the mechanisms involved remain unknown. Herein, two-thirds partial hepatectomy in mice increased hepatic THBS1, downstream transforming growth factor-β3, and perturbation of liver tissue homeostasis. All these events were ameliorated by hepatic transfusion of human bone marrow-derived MSCs. Treatment attenuated platelet and macrophage recruitment to the liver, both major sources of THBS1. By mitigating THBS1, MSCs muted surgery-induced tissue deterioration and dysfunction, and thus supported post-hepatectomy regeneration. After liver surgery, patients displayed increased tissue THBS1, which is associated with functional impairment and may indicate a higher risk of post-surgery complications. Since liver dysfunction involving THBS1 improves with MSC treatment in various animal models, it seems feasible to also modulate THBS1 in humans to impede post-surgery acute liver failure. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a vision threatening disease in older adults. Anti-VEGF treatment is effective for the majority of neovascular AMD (nAMD) patients, although approximately 30% of nAMD patients have an incomplete response for unknown reasons. Here we assessed the contribution of single nucleotide polymorphisms (SNPs) in key angioinflammatory regulatory genes in nAMD patients with an incomplete response compared to those responsive to anti-VEGF treatment. A total of 25 responsive and 30 nAMD patients with an incomplete response to anti-vascular endothelial growth factor (anti-VEGF) treatment were examined for known SNPs that impact the structure and function of thromobospondin-1 (TSP1), Bcl-2-interacting mediator of cell death (BIM) and complement factor H (CFH). Plasma levels of C-C motif chemokine ligand 2 (CCL2/MCP1), TSP1 and VEGF were assessed by ELISA. Patients responsive to anti-VEGF treatment showed a significant increase in the TSP1 rs2228262 AA allele and a trend for the BIM (rs724710) CT allele. Consistent with previous reports, 42% of the patients responsive to anti-VEGF expressed the CC allele for CFH rs1061170. Although the CFH TT allele had similarly low prevalence in both groups, the TC allele tended to be more prevalent in patients with an incomplete response. Patients with an incomplete response also had increased plasma CCL2/MCP1 levels, consistent with the role increased inflammation has in the pathogenesis of nAMD. Our studies point to new tools to assess the potential responsiveness of nAMD patients to anti-VEGF treatment and suggest the potential use of anti-CCL2 for treatment of nAMD patients with an incomplete response to anti-VEGF. [ABSTRACT FROM AUTHOR]

Background: Scleral extracellular matrix (ECM) remodeling plays a crucial role in the development of myopia, particularly in ocular axial elongation. Thrombospondin-1 (THBS1), also known as TSP-1, is a significant cellular protein involved in matrix remodeling in various tissues. However, the specific role of THBS1 in myopia development remains unclear. Method: We employed the HumanNet database to predict genes related to myopic sclera remodeling, followed by screening and visualization of the predicted genes using bioinformatics tools. To investigate the potential target gene Thbs1, we utilized lens-induced myopia models in male C57BL/6J mice and performed Western blot analysis to detect the expression level of scleral THBS1 during myopia development. Additionally, we evaluated the effects of scleral THBS1 knockdown on myopia development through AAV sub-Tenon's injection. The refractive status and axial length were measured using a refractometer and SD-OCT system. Results: During lens-induced myopia, THBS1 protein expression in the sclera was downregulated, particularly in the early stages of myopia induction. Moreover, the mice in the THBS1 knockdown group exhibited alterations in myopia development in both refraction and axial length changed compared to the control group. Western blotting analysis confirmed the effectiveness of AAV-mediated knockdown, demonstrating a decrease in COLA1 expression and an increase in MMP9 levels in the sclera. Conclusion: Our findings indicate that sclera THBS1 levels decreased during myopia development and subsequent THBS1 knockdown showed a decrease in scleral COLA1 expression. Taken together, these results suggest that THBS1 plays a role in maintaining the homeostasis of scleral extracellular matrix, and the reduction of THBS1 may promote the remodeling process and then affect ocular axial elongation during myopia progression. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Thrombospondin‐1 (TSP‐1) participates in a series of physiological and pathological processes by binding to various receptors regulating cell proliferation, adhesion and apoptosis. Elevated circulating TSP‐1 is linked with diabetic vascular complications (DVC). This study aimed to determine the relationship between circulating TSP‐1 levels and DVC. Materials and Methods: A comprehensive search of PubMed, Embase, Web of Science and CNKI databases was carried out. A meta‐analysis was carried out to compare circulating TSP‐1 levels between diabetes patients without vascular complications (DNVC), diabetes patients with DVC and non‐diabetes patients. The correlation between TSP‐1 and metabolic parameters was also analyzed. Subgroup analysis was carried out according to complication type, defined as diabetic retinopathy, diabetic nephropathy and diabetic cardiovascular disease (DCVD). Results: A total of eight studies were included. Compared with non‐diabetes patients, diabetic patients, including DNVC and DVC, had significantly higher circulating TSP‐1 levels (standardized mean difference [SMD] 2.660, 95% CI 1.17–4.145, P = 0.000). DNVC had significantly higher circulating TSP‐1 levels than non‐diabetes patients (SMD 3.613, 95% CI 1.607–5.619, P = 0.000). DVC had significantly higher TSP‐1 levels than DNVC (SMD 0.568, 95% CI 0.100–1.036, P = 0.017). TSP‐1 was significantly positively correlated with fasting plasma glucose (overall Fisher's z = 0.696, 95% CI 0.559–0.833) and HbA1c (overall Fisher's z = 0.849, 95% CI 0.776–0.923). Conclusions: Elevated circulating TSP‐1 levels are closely related to DVC, especially in diabetic nephropathy and diabetic cardiovascular disease. Circulating TSP‐1 detection might be helpful in the timely diagnosis and treatment of DVC. [ABSTRACT FROM AUTHOR]

Alterations in angiogenic properties play a pivotal role in the manifestation and onset of various pathologies, including vascular diseases and cancer. Thrombospondin-1 (TSP1) protein is one of the master regulators of angiogenesis. This study unveils a novel aspect of TSP1 regulation through reversible phosphorylation. The silencing of the B55α regulatory subunit of protein phosphatase 2A (PP2A) in endothelial cells led to a significant decrease in TSP1 expression. Direct interaction between TSP1 and PP2A-B55α was confirmed via various methods. Truncated TSP1 constructs were employed to identify the phosphorylation site and the responsible kinase, ultimately pinpointing PKC as the enzyme phosphorylating TSP1 on Ser93. The biological effects of B55α–TSP1 interaction were also analyzed. B55α silencing not only counteracted the increase in TSP1 expression during wound closure but also prolonged wound closure time. Although B55α silenced cells initiated tube-like structures earlier than control cells, their spheroid formation was disrupted, leading to disintegration. Cells transfected with phosphomimic TSP1 S93D exhibited smaller spheroids and reduced effectiveness in tube formation, revealing insights into the effects of TSP1 phosphorylation on angiogenic properties. In this paper, we introduce a new regulatory mechanism of angiogenesis by reversible phosphorylation on TSP1 S93 by PKC and PP2A B55α. [ABSTRACT FROM AUTHOR]

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47−/− mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47−/− spleens but significantly depleted in Thbs1−/− spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119−CD34+ progenitors and Ter119+CD34− committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1−/− spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Abstract Preeclampsia (PE) is considered as a disease of placental origin. However, the specific mechanism of placental abnormalities remains elusive. This study identified thrombospondin‐1 (THBS1) is downregulated in preeclamptic placentae and negatively correlated with blood pressure. Functional studies show that THBS1 knockdown inhibits proliferation, migration, and invasion and increases the cycle arrest and apoptosis rate of HTR8/SVneo cells. Importantly, THBS1 silencing induces necroptosis in HTR8/SVneo cells, accompanied by the release of damage‐associated molecular patterns (DAMPs). Necroptosis inhibitors necrostatin‐1 and GSK′872 restore the trophoblast survival while pan‐caspase inhibitor Z‐VAD‐FMK has no effect. Mechanistically, the results show that THBS1 interacts with transforming growth factor B‐activated kinase 1 (TAK1), which is a central modulator of necroptosis quiescence and affects its stability. Moreover, THBS1 silencing up‐regulates the expression of neuronal precursor cell‐expressed developmentally down‐regulated 4 (NEDD4), which acts as an E3 ligase of TAK1 and catalyzes K48‐linked ubiquitination of TAK1 in HTR8/SVneo cells. Besides, THBS1 attenuates PE phenotypes and improves the placental necroptosis in vivo. Taken together, the down‐regulation of THBS1 destabilizes TAK1 by activating NEDD4‐mediated, K48‐linked TAK1 ubiquitination and promotes necroptosis and DAMPs release in trophoblast cells, thus participating in the pathogenesis of PE.

Background and Purpose: Physiological angiogenesis and homeostasis of blood vessels is a complex process that is regulated at a high level by the balance between positive proteins such as follistatin-like factor-1 (FSTL-1) and negative proteins such as thrombospondin-1 (TSP-1). Although, there is considerable evidence about the role of positive factors in angiogenesis, the role of negative factors is not well defined. In physiological conditions, such as resistance training as one of the effective training methods to improve cardiovascular function, the role of these factors is not properly known. The aim of this study was to investigate the changes in thrombospondin-1 and follistatin-like factor-1 of heart tissue following six weeks of resistance training in obese male rats. Materials and Methods: In an experimental study, 20 male Wistar rats were divided into two equal groups of control and experimental based on their body weight. Prior to the exercise protocol, the rats received a high-calorie diet. The experimental group participated in a six-week training protocol with a frequency of three training sessions per week, and each session included 10 repetitions with a 90-second rest interval, climbing a resistance training ladder with a height of one meter and an 85-degree incline with a weight attached to the base of the tail (based on the the maximum weight carrying capacity of each rat). Fourty-eight hours after the last training session, TSP-1 and FSTL-1 values in the heart tissue were evaluated using ELISA technique. Between-group comparisons were made by using the independent t-test at a significance level of P

Abstract Thrombospondin-1 (TSP1) contributes to obesity-associated inflammation via activating Toll-like receptor 4 (TLR4). The regulatory role of vitamin D on this pathway has been suggested. This study aimed to investigate the relationship between TSP1/TLR4 pathway and vitamin D in obese and normal weight subjects with different metabolic phenotypes. Thirty obese and thirty normal weight men were selected. Anthropometric parameters and serum TSP1, TLR4, TNF-α, vitamin D, and metabolic profile were determined. Metabolic phenotypes of obese and normal weight subjects were determined. Findings revealed enhanced TSP1/TLR4/TNF-α levels and reduced 25(OH)D levels in obese compared to normal weight subjects and metabolically unhealthy compared to metabolically healthy subjects. TSP1 correlated positively with parameters of unhealthy metabolic profile. TSP1, TLR4 and TNF-α levels significantly negatively correlated with vitamin D levels. In conclusion, vitamin D might exert a regulatory role on TSP1/TLR4 pathway, providing a potential mechanism that links hypovitaminosis D with risk of metabolic dysfunction.

Background and Purpose: The expansion of fat tissue depends on its angiogenesis rate, and exercise affect on various angiogenic factors in this tissue. With the expansion of fat tissue and adipogenesis, a dense and closed capillary network is formed in this tissue, which can suppress angiogenic factors. If there is a wide capillary network and angiogenic factors such as VEGF-A in this tissue, the expansion of fat tissue can be prevented. The aim of this study was to investigate and compare the effect of a variety of endurance training on the expression of angiogenic genes of vascular endothelial growth factor (VEGF-A) and thrombospondin-1 (TSP-1) in the subcutaneous fat tissue of rats. Materials and Methods: In this experimental study, 32 male Wistar rats aged eight weeks and weighing 237±33 grams were randomly divided into four equal groups of control, high-intensity progressive continuous aerobic training (HIT), moderate-intensity continuous aerobic training (MIT) and, high-intensity interval aerobic training (HIIT). The treadmill training protocols were conducted for eight weeks, five sessions per week, where the HIT training consisted of running at a speed of 20 m/min or at an intensity of 65% of the maximum oxygen consumption (VO2max), with a gradually increasing slope for 30 minutes. MIT training consisted of running at an intensity of 65% VO2max for 37 minutes, and HIIT training consisted of four high-intensity intervals with four minutes of running at an intensity of 90-100% VO2max and four low-intensity intervals with a duration of three minutes at 50-60% VO2max (28 minutes in total). Finally, 48 hours after the last training session, the subcutaneous fat tissue was isolated and the expression of VEGF-A and TSP-1 genes in the subcutaneous fat tissue was measured by RT-qPCR technique. The data were analyzed using a one-way analysis of variance and Tukey's post hoc test, and the significance level was considered P≤0.05. Results: Statistical analyses showed that all three exercise interventions significantly increased the expression of the VEGF-A gene in subcutaneous fat tissue. However, only HIT training could significantly reduce the expression of TSP-1 in this tissue (P˂0.05). In addition, the increase in VEGF-A expression was significantly higher after HIT training compared to HIIT (P˂0.05). Conclusion: According to the findings of this study, high-intensity progressive continuous aerobic training is probably the most desirable training to increase angiogenesis in subcutaneous fat tissue. These types of exercises can be effective in reducing fat mass by improving blood supply to fat tissue. Keywords: Exercise Training, Angiogenesis, Adipose Tissue, Thrombospondin-1

Objective To investigate the effects of thrombospondin1 (THBS1) gene knockout on immune microenvironment and tumor progression of colorectal cancer. Methods THBS1 gene knockout (THBS1-/-) mice were constructed and its genotypes were determined by PCR and agarose gel electrophoresis. Subcutaneous tumor bearing and intraperitoneal implantation metastasis models of colorectal cancer were constructed in wild type (WT) and THBS1-/- mice to observe the effect of THBS1 on tumor growth. Immune cells in epididymal adipose of tumor bearing mice of both types were detected by flow cytometry. Results Compared with the WT mice, THBS1-/- mice showed no difference in tumor growth in colorectal cancer subcutaneous tumor bearing model (P>0.05), but significantly inhibited tumor growth in intraperitoneal implantation metastasis model (P < 0.05). Subsequently, flow cytometry indicated that the proportion of total macrophages in the epididymal adipose tissue was increased in the THBS1-/- mice than the WT mice (P < 0.01), with the proportion of M1-like macrophages (typical or proinflammatory type) increased (P < 0.01) and that of M2-like macrophages (alternately activated or anti-inflammatory type) decreased (P < 0.01). The proportion of myeloid-derived suppressor cells (MDSC) was decreased (P < 0.05), that of Ly6C subgroup was increased (P < 0.01), that of Ly6G subgroup was decreased (P < 0.05), that of CD8+ T cells was significantly increased (P < 0.01), and that of CD4+ T cells was decreased (P < 0.01). There were no changes in the proportions of total macrophages, M1-like macrophages and M2-like macrophages in the peripheral blood of intraperitoneal implantation metastasis model mice (P>0.05). The proportions of MDSC and ly6C subgroup were not changed (P>0.05), while that of ly6G subgroup were decreased (P < 0.05). The proportion of CD8+ T cells was significantly decreased (P < 0.05), while that of CD4+ T cells was not changed (P>0.05). Conclusion THBS1 gene knockout inhibits the intraperitoneal metastasis of colorectal cancer cells by enhancing tumor immunity.

The use of checkpoint-blockade antibodies is still restricted in several malignancies due to the modest efficacy, despite considerable success in anti-tumor immunotherapy. The poor response of cancer cells to immune destruction is an essential contributor to the failure of checkpoint therapy. We hypothesized that combining checkpoint therapy with natural-product chemosensitizer could enhance immune response. Herein, a targeted diterpenoid derivative was integrated with the checkpoint blockade (anti-CTLA-4) to improve immunotherapy using thermosensitive liposomes as carriers. In vivo, the liposomes enabled the co-delivery of the two drug payloads into the tumor. Consequently, the regulatory T cell proliferation was restrained, the cytotoxic T cell infiltration was enhanced, and the profound immunotherapeutic effect was achieved. In addition, the immunotherapeutic effect of another clinically used checkpoint antibody, anti-PD-1, also benefited from the diterpenoid derivative. Of note, our mechanism study revealed that the targeted diterpenoid derivative increased the sensitivity of cancer cells to immune attack via THBS1 downregulation and the resultant destruction of THBS1-CD47 interaction. Collectively, co-delivering THBS1 inhibitor and checkpoint blockade is promising to boost cancer immunotherapy. We first time discovered that THBS1 suppression could strengthen checkpoint therapy.

Introduction: Diabetic tendinopathy is a common invalidating and challenging disease that may be treated using stem cells. However, the effects of adipose-derived mesenchymal stem cell conditioned medium (ASC-CM) in diabetic tendinopathy have never been explored. Objectives: The present study evaluated the effects of ASC-CM on morphology, cell viability, structure, and scratch wound closure of human tenocytes (HTNC) exposed to high glucose (HG). Design: Experimental study. Methods: HTNC were exposed to HG (25 mM) for 7, 14 and 21 days with or without ASC-CM for the last 24 h. CM was collected from 4 × 105 ASCs, centrifuged for 10 min at 200 g and sterilized with 0.22 μm syringe filter. Results: At 7 days, HG-HTNC had decreased cell viability [72 ± 2%, p < 0.01 versus normal glucose (NG)] compared to NG-HTNC (90 ± 5%). A further decrement was detected after 14 and 21 days (60 ± 4% and 60 ± 5%, both, p < 0.01 versus NG and p < 0.01 versus HG7). While NG-HTNC evidenced a normal fibroblast cell-like elongated morphology, HG-HTNC showed increased cell roundness. In contrast, HG-HTNC exposed to ASC-CM showed a significant increase in cell viability, an improved cell morphology and higher scratch wound closure at all HG time points. Moreover, the exposure to ASC-CM significantly increased thrombospondin 1 and transforming growth factor beta 1 (TGF-β1) content in HG-HTNC. The TGF-β1 elevation was paralleled by higher Collagen I and Vascular Endothelial Growth Factor in HG-HTNC. Conclusion: ASC-CM may restore the natural morphology, cell viability and structure of HTNC, promoting their scratch wound closure through TGF-β1 increase. [ABSTRACT FROM AUTHOR]

Thrombophilia in venous thromboembolism (VTE) is multifactorial. Von Willebrand factor (vWF) plays a major role in primary hemostasis. While elevated vWF levels are well documented in VTE, findings related to its cleaving protease (ADAMTS-13) are contradicting. The aim of this study was to determine vWF, ADAMTS-13, and the multifactorial Thrombospondin-1 (TSP-1) protein levels in patients after 3-6 months following an unprovoked VTE episode. We also explored a possible association with factor V Leiden (FVL) mutation. vWF, ADAMTS-13 and TSP-1 were analyzed using ELISA kits in 60 VTE patients and 60 controls. Patients had higher levels of vWF antigen (P =.021), vWF collagen-binding activity (P =.008), and TSP-1 protein (P <.001) compared to controls. ADAMTS-13 antigen was lower in patients (P =.046) compared to controls but ADAMTS-13 activity was comparable between the two groups (P =.172). TSP-1 showed positive correlation with vWF antigen (rho = 0.303, P =.021) and negative correlation with ADAMTS-13 activity (rho = −0.244, P =.033) and ADAMTS-13 activity/vWF antigen ratio (rho = −0.348, P =.007). A significant association was found between the presence of FVL mutation and VTE (odds ratio (OR): 9.672 (95% confidence interval (CI) 2.074-45.091- P =.004), but no association was found between the mutation and the studied proteins (P >.05). There appears to be an imbalance between vWF and ADAMTS-13 in VTE patients even after 3-6 months following the onset of VTE. We report that the odds of developing VTE in carriers of FVL mutation are 9.672 times those without the mutation, but the presence of this mutation is not associated with the studied proteins. [ABSTRACT FROM AUTHOR]

Background and Purpose: Physiological angiogenesis and homeostasis of blood vessels is a complex process that is regulated at a high level by the balance between positive proteins such as follistatin-like factor-1 (FSTL-1) and negative proteins such as thrombospondin-1 (TSP-1). Although, there is considerable evidence about the role of positive factors in angiogenesis, the role of negative factors is not well defined. In physiological conditions, such as resistance training as one of the effective training methods to improve cardiovascular function, the role of these factors is not properly known. The aim of this study was to investigate the changes in thrombospondin-1 and follistatin-like factor-1 of heart tissue following six weeks of resistance training in obese male rats. Materials and Methods: In an experimental study, 20 male Wistar rats were divided into two equal groups of control and experimental based on their body weight. Prior to the exercise protocol, the rats received a high-calorie diet. The experimental group participated in a six-week training protocol with a frequency of three training sessions per week, and each session included 10 repetitions with a 90-second rest interval, climbing a resistance training ladder with a height of one meter and an 85-degree incline with a weight attached to the base of the tail (based on the the maximum weight carrying capacity of each rat). Fourtyeight hours after the last training session, TSP-1 and FSTL-1 values in the heart tissue were evaluated using ELISA technique. Between-group comparisons were made by using the independent t-test at a significance level of P<0.05. Results: Data analyses revealed that six weeks of resistance training in the experimental group compared to the control group caused a significant increase in FSTL-1 values (P=0.0001) and a non-significant decrease in TSP-1 values (P=0.09). Conclusion: Based on the findings of the present study it could be concluded that exercise in the form of resistance exercises can have positive effects on the development of angiogenesis process in heart tissue and it seems that six weeks of resistance training, despite the non-significant decrease in TSP-1 as an inhibitory factor in the angiogenesis process and an increase in FSTL-1 values as an angiogenesis stimulating factor, may have positive effect on improving blood supply to the heart muscle in obese subjects. The data of the present study refer to the role of resistance training in facilitating intracellular signaling through, modulating some inhibitory factors and stimulatory factors of angiogenesis in heart tissue of obese rats. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Thrombospondin-1 (TSP1) contributes to obesity-associated inflammation via activating Toll-like receptor 4 (TLR4). The regulatory role of vitamin D on this pathway has been suggested. This study aimed to investigate the relationship between TSP1/TLR4 pathway and vitamin D in obese and normal weight subjects with different metabolic phenotypes. Thirty obese and thirty normal weight men were selected. Anthropometric parameters and serum TSP1, TLR4, TNF-α, vitamin D, and metabolic profile were determined. Metabolic phenotypes of obese and normal weight subjects were determined. Findings revealed enhanced TSP1/TLR4/TNF-α levels and reduced 25(OH)D levels in obese compared to normal weight subjects and metabolically unhealthy compared to metabolically healthy subjects. TSP1 correlated positively with parameters of unhealthy metabolic profile. TSP1, TLR4 and TNF-α levels significantly negatively correlated with vitamin D levels. In conclusion, vitamin D might exert a regulatory role on TSP1/TLR4 pathway, providing a potential mechanism that links hypovitaminosis D with risk of metabolic dysfunction. [ABSTRACT FROM AUTHOR]

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic TH2 disorder complicated by tissue fibrosis and loss of esophageal luminal patency. The fibrostenotic esophagus does not respond well to therapy, but profibrotic therapeutic targets are largely unclear. OBJECTIVE: Our aim was to utilize proteomics and primary cells as a novel approach to determine relevant profibrotic factors. METHODS: We utilized primary esophageal EoE and normal fibroblasts, their derivative extracellular matrixes (ECMs), an approach of fibroblast culture on autologous versus nonautologous ECM, and proteomics to elucidate EoE ECM proteins that dysregulate cellular function. RESULTS: We cultured esophageal fibroblasts from normal esophagi and esophagi from patients with severe EoE on autologous versus nonautologous ECM. The EoE ECM proteome shifted normal esophageal fibroblast protein expression. Proteomic analysis demonstrated that thrombospondin-1 is detected only in the EoE ECM, is central in the EoE ECM protein-protein interactome, is found at significantly elevated levels in biopsy specimens from patients with active EoE, and induces fibroblast collagen I production. CONCLUSION: Fibroblasts from patients with EoE secrete a unique ECM proteome that reflects their in vivo state and induces collagen I and α-smooth muscle actin protein expression from normal fibroblasts. Thrombospondin-1 is a previously unappreciated profibrotic molecule in EoE.

Roles of thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) in tissue repair and inflammation are well-documented, but the association of their serum expressions with the pathogenesis of COVID-19 remains unclear. We investigate the roles of TSP-1 and TSP-2 in COVID-19.

Objective To investigate the effect of thrombospondin-1(TSP-1) knockout on radiation-induced pulmonary fibrosis and explore its underlying mechanism. Methods Male wild type C57BL/6 mice (6~8 weeks old, WT mice) and TSP-1 knockout mice (TSP-1-/- mice) were subjected and received 16 Gy 60Co γ-ray whole lung irradiation (IR) to induce radiation pulmonary fibrosis, and then, the mice were assigned into WT+IR group and TSP-1-/-+IR group, respectively, with 6 mice in each group.In 16 weeks after irradiation, HE staining was performed to observe the morphological changes in lung tissues, and Sirius red staining was used to observe the collagen expression in lung tissues.qRT-PCR was employed to detect the mRNA expression of TGF-β1 and Collagen Ⅰ in lung tissues.Micro-CT scanning was adopted to evaluate lung injury and substantial lung changes.FlexiVent system was employed to assess pulmonary function.Immunofluorescence staining was conducted to measure the protein levels of LC-3 and p62 in lung tissues.Transmission electron microscopy (TEM) was used to observe the cellular autophagic vesicles in lung tissues. Results Compared with the WT+IR group, the TSP-1-/-+IR group had reduced lung injury and collagen deposition in lung tissue, significantly lower mRNA levels of TGF-β1 and Collagen Ⅰ in lung tissue (P < 0.01), and improved pulmonary function.Immunofluorescence assay and TEM showed that autophagy was observed in the mice of TSP-1-/-+IR group. Conclusion TSP-1 knockout can alleviate radiation-induced pulmonary fibrosis, which may be related to activation of autophagy.

Dupuytren's disease (DD) is a fibroproliferative disorder affecting the palmar fascia, causing functional restrictions of the hand and thereby limiting patients' daily lives. The disturbed and excessive myofibroblastogenesis, causing DD, is mainly induced by transforming growth factor (TGF)-β1. But, the extent to which impaired TGF-β1 release or TGF-β signal degradation is involved in pathologically altered myofibroblastogenesis in DD has been barely examined. Therefore, the complex in which TGF-β1 is secreted in the extracellular matrix to elicit its biological activity, and proteins such as plasmin, integrins, and matrix metalloproteinases (MMPs), which are involved in the TGF-β1 activation, were herein analyzed in DD-fibroblasts (DD-FBs). Additionally, TGF-β signal degradation via caveolin-1 was examined with 5-fluoruracil (5-FU) in detail. Gene expression analysis was performed via Western blot, PCR, and immunofluorescence analyses. As a surrogate parameter for disturbed myofibroblastogenesis, -smooth-muscle-actin (-SMA) expression was evaluated. It was demonstrated that latency-associated peptide (LAP)-TGF-β and latent TGF-β-binding protein (LTBP)-1 involved in TGF-β-complex building were significantly upregulated in DD. Plasmin a serinprotease responsible for the TGF-β release was significantly downregulated. The application of exogenous plasmin was able to inhibit disturbed myofibroblastogenesis, as measured via -SMA expression. Furthermore, a reduced TGF-β1 degradation was also involved in the pathological phenotype of DD, because caveolin-1 expression was significantly downregulated, and if rescued, myofibroblastogenesis was also inhibited. Therefore, our study demonstrates that a deficient release and degradation of TGF-β1 are important players in the pathological phenotype of DD and should be addressed in future research studies to improve DD therapy or other related fibrotic conditions. [ABSTRACT FROM AUTHOR]

The use of treatments, such as programmed death protein 1 (PD1) or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies, that loosen the natural checks upon immune cell activity to enhance cancer killing have shifted clinical practice and outcomes for the better. Accordingly, the number of antibodies and engineered proteins that interact with the ligand–receptor components of immune checkpoints continue to increase along with their use. It is tempting to view these molecular pathways simply from an immune inhibitory perspective. But this should be resisted. Checkpoint molecules can have other cardinal functions relevant to the development and use of blocking moieties. Cell receptor CD47 is an example of this. CD47 is found on the surface of all human cells. Within the checkpoint paradigm, non-immune cell CD47 signals through immune cell surface signal regulatory protein alpha (SIRPα) to limit the activity of the latter, the so-called trans signal. Even so, CD47 interacts with other cell surface and soluble molecules to regulate biogas and redox signaling, mitochondria and metabolism, self-renewal factors and multipotency, and blood flow. Further, the pedigree of checkpoint CD47 is more intricate than supposed. High-affinity interaction with soluble thrombospondin-1 (TSP1) and low-affinity interaction with same-cell SIRPα, the so-called cis signal, and non-SIRPα ectodomains on the cell membrane suggests that multiple immune checkpoints converge at and through CD47. Appreciation of this may provide latitude for pathway-specific targeting and intelligent therapeutic effect. [ABSTRACT FROM AUTHOR]

Matricellular proteins comprise several families of secreted proteins that function in higher animals at the interface between cells and their surrounding extracellular matrix. Targeted gene disruptions that result in loss of viability in mice have revealed critical roles for several matricellular proteins in murine embryonic development, including two members of the cellular communication network (CCN) gene family. In contrast, mice lacking single or multiple members of the thrombospondin (THBS) gene family remain viable and fertile. The frequency of loss of function mutants, identified using human deep exome sequencing data, provided evidence that some of the essential genes in mice, including Ccn1, are also essential genes in humans. However, a deficit in loss of function mutants in humans indicated that THBS1 is also highly loss-intolerant. In addition to roles in embryonic development or adult reproduction, genes may be loss-intolerant in humans because their function is needed to survive environmental stresses that are encountered between birth and reproduction. Laboratory mice live in a protected environment that lacks the exposures to pathogens and injury that humans routinely face. However, subjecting Thbs1−/− mice to defined stresses has provided valuable insights into functions of thrombospondin-1 that could account for the loss-intolerance of THBS1 in humans. [ABSTRACT FROM AUTHOR]

Secretory and membrane proteins are vital for cell activities, including intra- and intercellular communication. Therefore, protein quality control in the endoplasmic reticulum (ER) is an essential and crucial process for eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) targets misfolded proteins during the protein maturation process in the ER and leads to their disposal. This process maintains the ER productive function and prevents misfolded protein stress (i.e., ER stress). The ERAD-stimulating factor ER degradation-enhancing α mannosidase-like 1 protein (EDEM1) acts on misfolded proteins to accelerate ERAD, thereby maintaining the productivity of the ER. However, the detail mechanism underlying the function of EDEM1 in ERAD is not completely understood due to a lack of established physiological substrate proteins. In this study, we attempted to identify substrate proteins for EDEM1 using siRNA. The matrix component thrombospondin-1 (TSP1) and epidermal growth factor receptor (EGFR) were identified as candidate targets of EDEM1. Their protein maturation status and cellular localization were markedly affected by knockdown of EDEM1. We also showed that EDEM1 physically associates with EGFR and enhances EGFR degradation via ERAD. Our data highlight the physiological role of EDEM1 in maintaining specific target proteins and provide a potential approach to the regulation of expression of clinically important proteins. [ABSTRACT FROM AUTHOR]

The use of checkpoint-blockade antibodies is still restricted in several malignancies due to the modest efficacy, despite considerable success in anti-tumor immunotherapy. The poor response of cancer cells to immune destruction is an essential contributor to the failure of checkpoint therapy. We hypothesized that combining checkpoint therapy with natural-product chemosensitizer could enhance immune response. Herein, a targeted diterpenoid derivative was integrated with the checkpoint blockade (anti-CTLA-4) to improve immunotherapy using thermosensitive liposomes as carriers. In vivo , the liposomes enabled the co-delivery of the two drug payloads into the tumor. Consequently, the regulatory T cell proliferation was restrained, the cytotoxic T cell infiltration was enhanced, and the profound immunotherapeutic effect was achieved. In addition, the immunotherapeutic effect of another clinically used checkpoint antibody, anti-PD-1, also benefited from the diterpenoid derivative. Of note, our mechanism study revealed that the targeted diterpenoid derivative increased the sensitivity of cancer cells to immune attack via THBS1 downregulation and the resultant destruction of THBS1-CD47 interaction. Collectively, co-delivering THBS1 inhibitor and checkpoint blockade is promising to boost cancer immunotherapy. We first time discovered that THBS1 suppression could strengthen checkpoint therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Serum protein biomarkers are used to diagnose, monitor treatment response, and to differentiate various forms of chronic enteropathies (CE) in humans. The utility of liquid biopsy proteomic approaches has not been examined in cats. Hypothesis/Objectives: To explore the serum proteome in cats to identify markers differentiating healthy cats from cats with CE. Animals: Ten cats with CE with signs of gastrointestinal disease of at least 3 weeks duration, and biopsy‐confirmed diagnoses, with or without treatment and 19 healthy cats were included. Methods: Cross‐sectional, multicenter, exploratory study with cases recruited from 3 veterinary hospitals between May 2019 and November 2020. Serum samples were analyzed and evaluated using mass spectrometry‐based proteomic techniques. Results: Twenty‐six proteins were significantly (P <.02, ≥5‐fold change in abundance) differentially expressed between cats with CE and controls. Thrombospondin‐1 (THBS1) was identified with >50‐fold increase in abundance in cats with CE (P < 0.001) compared to healthy cats. Conclusions and Clinical Importance: Damage to the gut lining released marker proteins of chronic inflammation that were detectable in serum samples of cats. This early‐stage exploratory study strongly supports THBS1 as a candidate biomarker for chronic inflammatory enteropathy in cats. [ABSTRACT FROM AUTHOR]

Fibrosis is present in various physiologic and pathologic conditions of salivary glands (SGs). This study aimed to identify novel biomarkers of SG fibrosis by next-generation sequencing. We established the SG fibrosis mouse model by excretory main duct ligation. Next-generation sequencing, differentially expressed gene (DEG) analysis, and gene set enrichment analysis was performed by comparing ligated and control SGs. We used algorithms of Cytohubba, molecular complex detection, Lasso logistic regression, and support vector machine to identify the key biomarkers. Selected key biomarkers were verified by the polymerase chain reaction and immunohistochemistry. We also retrieved and analyzed the key gene expression in the fibrosis of the heart, liver, lung, and kidney to ensure the generalization of key biomarkers in SG fibrosis. Both interlobular and intralobular fibrosis was confirmed in the ligated SGs, with improved expressions of collagen I and transforming growth factor β. Next-generation sequencing identified 2666 upregulated DEGs and 336 downregulated DEGs, which were highly enriched in the extracellular matrix-related pathways. Multiple algorithms identified 15 key biomarkers in SG fibrosis, including Thrombospondin-1 (THBS1) and Prolyl 4-Hydroxylase Subunit Alpha 3 (P4HA3). The mRNA and protein expression of THBS1 and P4HA3 was verified in mice. THBS1 was also highly expressed in lung and kidney fibrosis, whereas P4HA3 was upregulated in liver fibrosis. THBS1 and P4HA3 may be potential biomarkers for SG fibrosis. They may be also applicable in the diagnosis of multi-organ fibrosis. [ABSTRACT FROM AUTHOR]

The online version of the original article can be found at https://doi.org/10.1186/1477-7827-12-21.Correction: Reprod Biol Endocrinol 12, 21 (2014)https://doi.org/10.1186/1477-7827-12-21Following publication of the original article [[1]], the authors identified an error in Fig. 1a. The correct Fig. 1a is given below.Incorrect Fig. 1aGraphCorrect Fig. 1aGraphPublisher's noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.By Kata Osz; Michelle Ross and Jim PetrikReported by Author; Author; Author [Extracted from the article]

Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival. [ABSTRACT FROM AUTHOR]

The present study focuses on investigating the expression of thrombospondin-1 (TSP-1), a natural inhibitor of neovascularization. Immunofluorescent staining was used to detect the expression of TSP-1 in rabbit corneal tissue with vascularization induced by limbectomy. TSP-1 was detected in healthy and Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) grafted rabbit corneas. TSP-1 was not detected in diseased corneas. Rabbit and human primary oral mucosal and corneal epithelial cells were cultured and treated with proteasome inhibitor (PI) in vitro. Changes in the expression of TSP-1, HIF-1 alpha and 2 alpha, VEGF-A, and VEGF receptor were analyzed by Western blotting. Neovascularization developed in rabbits’ corneas as early as 1 month after limbectomy and was stable for at least 3 months. HIF-1 alpha and VEGF-A expression was reduced in CAOMECS grafted corneas, as compared to sham corneas. While TSP-1 expression was decreased in injured corneas, it was expressed in CAOMECS grafted corneas, but still less expressed compared to healthy corneas. PI treatment, of human oral mucosal and corneal epithelial cells increased TSP-1 expression and reduced VEGF-A expression. The results showed that TSP-1 expression was lost in injured corneal surface and that CAOMECS grafting restored TSP-1 expression to certain extent. Proteasome inhibition treatment increased TSP-1 and decreased VEGF-A expression in human oral mucosal and corneal epithelial cells. The result suggests that corneal neovascularization could be managed with the inhibition of the proteasome after CAOMECS grafting and increase corneal transparency.

Peri-implantitis is a major factor affecting implant prognosis, and the specific anatomy of the peri-implant area makes it more vulnerable to the local hypoxic environment caused by inflammation. N6-methyladenosine (m6A) plays a vital role in a multitude of biological processes, and its main "reader" Yth m6A RNA-binding protein 1 (YTHDF1) is suggested to affect osteogenic differentiation. However, the mechanism underlying the effect of YTHDF1 on osteogenic differentiation under hypoxic conditions remains unclear. To address this question, we examined the expression of YTHDF1 under hypoxia and observed that hypoxia suppressed osteogenic differentiation but promoted the expression of YTHDF1. Then we knocked down YTHDF1 and found decreased levels of osteogenic-related markers, alkaline phosphatase (ALP) activity, and alizarin red staining (ARS) under normoxia or hypoxia treatment. Bioinformatics analysis identified Thrombospondin-1 (THBS1) might be a downstream factor of YTHDF1. The results revealed that YTHDF1 enhanced the stability of THBS1 mRNA, and immunofluorescence assays found co-localization with YTHDF1 and THBS1 under hypoxia. Loss of function studies showed knocking down YTHDF1 or THBS1 exacerbated the osteogenic inhibition caused by hypoxia. All data imply that hypoxia suppresses osteogenic differentiation and promotes the expression of YTHDF1, which translationally regulates THBS1 in an m6A-dependent manner, potentially counteracting hypoxia-induced osteogenic inhibition through the YTHDF1/THBS1 pathway. The results of this study reveal for the first time the molecular mechanism of the regulation of osteogenic differentiation by YTHDF1 under hypoxia and suggest that YTHDF1, together with its downstream factor THBS1, may be critical targets to counteract osteogenic inhibition under hypoxic conditions, providing promising therapeutic strategy for the hypoxia-induced bone loss in peri-implantitis. [ABSTRACT FROM AUTHOR]

Thrombospondin-1 (TSP1) is a matricellular protein with many important roles in mediating carcinogenesis, fibrosis, leukocyte recruitment, and metabolism. We have previously shown a role of diet in the absence of TSP1 in liver metabolism in the context of a colorectal cancer model. However, the metabolic implications of TSP1 regulation by diet in the liver metabolism are currently understudied. Therefore Discrete correlation summation (DCS) was used to re-interrogate data and determine the metabolic alterations of TSP1 deficiency in the liver, providing new insights into the role of TSP1 in liver injury and the progression of liver pathologies such as nonalcoholic fatty liver disease (NAFLD). DCS analysis provides a straightforward approach to rank covariance and data clustering when analyzing complex data sets. Using this approach, our previous liver metabolite data was re-analyzed by comparing wild-type (WT) and Thrombospondin-1 null (Thbs1−/−) mice, identifying changes driven by genotype and diet. Principal component analysis showed clustering of animals by genotype regardless of diet, indicating that TSP1 deficiency alters metabolite handling in the liver. High-fat diet consumption significantly altered over 150 metabolites in the Thbs1−/− livers versus approximately 90 in the wild-type livers, most involved in amino acid metabolism. The absence of Thbs1 differentially regulated tryptophan and tricarboxylic acid cycle metabolites implicated in the progression of NAFLD. Overall, the lack of Thbs1 caused a significant shift in liver metabolism with potential implications for liver injury and the progression of NAFLD. [ABSTRACT FROM AUTHOR]

Background: Fine particulate matter (PM 2.5 ) induces ocular surface toxicity through pyroptosis, oxidative stress, autophagy, and inflammatory responses. However, the precise molecular pathways through which PM 2.5 causes corneal damage remain unclear. This study aims to investigate the underlying mechanisms by exposing human corneal epithelial cells (HCECs) to PM 2.5 ., Methods: After the morphology and chemical composition analysis of the PM samples, we conducted both in vivo and in vitro experiments to investigate PM 2.5 -induced corneal epithelial damage. We assessed corneal barrier function in HCECs using transepithelial electrical resistance (TEER) assays. To explore the molecular mechanisms of PM 2.5 -induced corneal epithelial damage, we performed whole-transcriptome resequencing, quantitative RT-PCR, and western blotting in vitro. In addition, we analyzed mouse corneas exposed to concentrated ambient PM 2.5 through immunofluorescence staining to observe the resulting changes in corneal epithelial protein expression in vivo., Results: Our results showed significant impairment of corneal epithelial barrier function in PM 2.5 -treated HCECs, as indicated by decreased TEER values. The expression of thrombospondin-1 (THBS1) and claudin-1, both key factors for maintaining corneal epithelial barrier integrity, was markedly reduced at the gene and protein levels in both in vitro and in vivo PM 2.5 exposure models. Moreover, the levels of tight junction-associated proteins, including occludin, zonula occludens-1 (ZO-1) and ZO-2, essential components of the corneal epithelial barrier, were significantly diminished in PM 2.5 -treated HCECs., Conclusion: PM 2.5 exposure leads to corneal epithelium damage by disrupting tight junction proteins and THBS1 expression. These findings provide insight into potential pathways for PM 2.5 -induced ocular toxicity and underscore the need for protective strategies against such environmental pollutants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Pancreatic 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 results: High 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. Conclusions: TSP-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.

MiR-22 is mostly considered as a hepatic tumor-suppressor microRNA based on in vitro analyses. Yet, whether miR-22 exerts a tumor-suppressive function in the liver has not been investigated in vivo. Herein, in silico analyses of miR-22 expression were performed in hepatocellular carcinomas from human patient cohorts and different mouse models. Diethylnitrosamine-induced hepatocellular carcinomas were then investigated in lean and diet-induced obese miR-22-deficient mice. The proteome of liver tissues from miR-22-deficient mice prior to hepatocellular carcinoma development was further analyzed to uncover miR-22 regulated factors that impact hepatocarcinogenesis with miR-22 deficiency. MiR-22 downregulation was consistently observed in hepatocellular carcinomas from all human cohorts and mouse models investigated. The time of appearance of the first tumors was decreased and the number of tumoral foci induced by diethylnitrosamine was significantly increased by miR-22-deficiency in vivo, two features which were further drastically exacerbated with diet-induced obesity. At the molecular level, we provide evidence that the loss of miR-22 significantly affects the energetic metabolism and mitochondrial functions of hepatocytes, and the expression of tumor-promoting factors such as thrombospondin-1. Our study demonstrates that miR-22 acts as a hepatic tumor suppressor in vivo by restraining pro-carcinogenic metabolic deregulations through pleiotropic mechanisms and the overexpression of relevant oncogenes. [ABSTRACT FROM AUTHOR]

Aim: We aimed to evaluate the circulating thrombospondin-1 (TSP-1) and nuclear factor kappa B (NF-κB) in nonalcoholic fatty liver disease (NAFLD) in order to integrate these signaling pathways in the inflammatory and fibrogenic processes of this liver disorder. Methods: Ninety-five NAFLD patients were recruited in the study. The study also included 83 age-sex matched healthy controls. Results: The number of patients with metabolic syndrome (MetS) criteria was 57 (60%). TSP-1 level was found to be statistically significantly lower in the NAFLD group compared to the control group (p=0.037). However, NF-κB level was found to be significantly higher in the NAFLD group compared to the control group (p=0.004). There was a significant negative correlation between plasma TSP-1 levels with glucose (r=-0.235, p=0.022), alanine aminotransferase (r=-0.261, p=0.011) and aspartate transaminase (r=-0.328, p=0.001) levels. In addition, a significant negative correlation was found between plasma TSP-1 and NF-κB levels (r=-0.729, p<0.001). Conclusions: Our results suggest a close relationship between increased NF-κB and reduced TSP-1 in NAFLD. TSP-1 and NF-κB signaling pathways might have a role in the inflammatory and fibrogenic processes. Furthermore, they may be used as a noninvasive marker and could assist as a therapeutic target for NAFLD. [ABSTRACT FROM AUTHOR]

Matricellular proteins comprise a diverse group of molecular entities secreted into the extracellular space. They interact with the extracellular matrix (ECM), integrins, and other cell-surface receptors, and can alter matrix strength, cell attachment to the matrix, and cell-cell adhesion. A founding member of this group is thrombospondin-1 (TSP-1), a high molecular-mass homotrimeric glycoprotein. Given the importance of the matrix and ECM remodeling in the lung following injury, TSP-1 has been implicated in a number of lung pathologies. This review examines the role of TSP-1 as a damage controller in the context of lung inflammation, injury resolution, and repair in noninfectious and infectious models. This review also discusses the potential role of TSP-1 in human diseases as it relates to lung inflammation and injury. [ABSTRACT FROM AUTHOR]

Urethral stricture is related to scar tissue fibrosis, but its pathogenesis is still unclear. This study aims to explore the regulatory mechanism of circular RNA (circRNA) in the occurrence and development of urethral stricture. CircRNA microarray was employed to analyze circRNA expression profiles between human urethral scar tissue and normal urethral tissue. The results of circRNA microarray showed that there were 296 differentially expressed genes between urethral scar tissue and normal urethral tissue. The enrichment analysis of Kyoto encyclopedia of genes and genomes showed that these circRNAs were significantly correlated with ECM–receptor interaction. The first nine differentially expressed circRNA were selected to predict the circRNA–miRNA network. RT-qPCR results showed that circ_0047339 was upregulated considerably in urethral scar tissue. Urethral scar fibroblasts were isolated from human urethral scar tissue and cultured in vitro. After silencing circ_0047339, the proliferation of urethral scar cells decreased significantly, and the expressions of Collagen I (COL-1) and α-smooth muscle actin (α-SMA) also reduced. As a competing endogenous RNA, circ_0047339 could increase the expression of TSP-1 by competitively binding miR-4691-5p. In addition, miR-4691-5p mimic transfection could inhibit the proliferation of urethral scar fibroblasts and the presentation of thrombospondin-1 (TSP-1), α-SMA and COL-1, while circ_0047339 overexpression eliminated this inhibition. Our results showed that circ_0047339 might promote the growth and fibrosis of urethral scar fibroblasts through miR-4691-5p/TSP-1 axis, thus promoting the development of urethral stricture. [ABSTRACT FROM AUTHOR]

Abstract Objectives Atrial remodeling is the main developmental cause of atrial arrhythmias (AA), which may induce atrial fibrillation, atrial flutter, atrial tachycardia, and frequent premature atrial beats in acute myocardial infarction (AMI) patients. Thrombospondin-1 (TSP-1) has been shown to play an important role in inflammatory and fibrotic processes, but its role in atrial arrhythmias is not well described. The purpose of this study was to investigate the role of TSP-1 in AMI patients with atrial arrhythmias. Methods A total of 219 patients with AMI who underwent percutaneous coronary intervention and with no previous arrhythmias were included. TSP-1 were analyzed in plasma samples. Patients were classified into 2 groups, namely, with and without AA during the acute phase of MI. Continuous electrocardiographic monitoring was used for AA diagnosis in hospital. Results Twenty-four patients developed AA. Patients with AA had higher TSP-1 levels (29.01 ± 25.87 μg/mL vs 18.36 ± 10.89 μg/mL, p

BackgroundThrombospondin-1 (THBS1) derived from platelets and acted as a critical mediator of hemostasis promoting platelet activation in thrombus formation. The biological connection of genetic variants and mRNA expression of THBS1 with ischemic stroke (IS) warrants further validation with population-based evidence.ObjectiveTo evaluate the association of single nucleotide polymorphisms (SNPs) and mRNA expression of THBS1 with the risks of IS and long-term death after stroke.MethodsA case-control study consisted of 4,584 IS patients recruited from five hospitals in Jiangsu, China, and 4,663 age-gender-matched controls free of IS. A cohort study enrolled 4,098 participants free of stroke and lasted from 2009 to 2022. Early collected 3158 IS patients aged between 35 and 80 years were followed up an average of 5.86-year to follow up their long-term death outcomes. Two tagSNPs of the THBS1 gene, rs2236471 and rs3743125, were genotyped in all subjects and THBS1 mRNA expression of peripheral leukocyte was measured using RT-qPCR in 314 IS cases and 314 controls.ResultsThere is no significant difference in genotype and haplotype frequencies of rs2236741 and rs3743125 between IS cases and controls (all P > 0.05). Furthermore, the cohort studies did not observe significant associations between THBS1 variants and the risk of IS incidence or long-term death after IS (all P > 0.05). The THBS1 mRNA expression level (2–ΔΔCT) in IS cases was approximately equal to that in controls (1.01 vs. 0.99, P = 0.833). In addition, THBS1 mRNA expression had no significant association with all-cause death, stroke death, and IS death of IS patients (all P > 0.05).ConclusionTherefore, our study suggested that there is no significant association of THBS1 polymorphisms and mRNA expression level with the risk of IS and long-term death after IS.

Oxygen is a powerful trigger for cellular reactions, but there are few comparative investigations assessing the effects over a large range of partial pressures. We investigated a metabolic response to single exposures to either normobaric (10%, 15%, 30%, 100%) or hyperbaric (1.4 ATA, 2.5 ATA) oxygen. Forty-eight healthy subjects (32 males/16 females; age: 43.7 ± 13.4 years, height: 172.7 ± 10.07 cm; weight 68.4 ± 15.7 kg) were randomly assigned, and blood samples were taken before and 2 h after each exposure. Microparticles (MPs) expressing proteins specific to different cells were analyzed, including platelets (CD41), neutrophils (CD66b), endothelial cells (CD146), and microglia (TMEM). Phalloidin binding and thrombospondin-1 (TSP), which are related to neutrophil and platelet activation, respectively, were also analyzed. The responses were found to be different and sometimes opposite. Significant elevations were identified for MPs expressing CD41, CD66b, TMEM, and phalloidin binding in all conditions but for 1.4 ATA, which elicited significant decreases. Few changes were found for CD146 and TSP. Regarding OPB, further investigation is needed to fully understand the future applications of such findings. [ABSTRACT FROM AUTHOR]