Thrombospondin-1 Expression Research Articles

Circ_0058063 regulates cell vitality and proliferation in oesophageal squamous-cell carcinomas.

Oesophageal squamous-cell carcinoma (ESCC) is a malignant tumor of the digestive system with a poor prognosis. Recent studies have shown the promoting effect of hsa_circ_0058063 (circ_0058063) on ESCC, but the potential regulatory mechanisms of circ_0058063 in ESCC remain largely unclear. The levels of circ_0058063, microRNA-4319 (miR-4319) and mRNA of thrombospondin-1 (THBS1) were indicated by quantitative real-time polymerase chain reactionin ESCC tissues and cells. Meanwhile, the level of THBS1 was quantified by western blot analysis. In addition, the cell functions were examined by CCK8 assay, Edu assay, flow cytometry assay and transwell assay. Furthermore, the interplay between miR-4319 and circ_0058063 or THBS1 was detected by dual-luciferase reporter assay. Finally, anin vivo experiment was implemented to confirm the effect of circ_0058063. The level of circ_0058063 and THBS1 were increased, and the miR-4319 level was decreased in ESCC tissues in contrast to that in normal tissues and cells. For functional analysis, circ_0058063 deficiency inhibited cell vitality, cell proliferation, migration and invasion in ESCC cells, whereas promoted cell apoptosis. Moreover, miR-4319 was confirmed to repress the progression of ESCC cells by suppressing THBS1. In mechanism, circ_0058063 acted as a miR-4319 sponge to regulate the level of THBS1. Besides, circ_0058063 knockdown also attenuated tumour growth in vivo.Circ_0058063 facilitates the development of ESCC through increasing THBS1 expression by regulating miR-4319, which also offered an underlying targeted therapy for ESCC treatment.

Relationship of thrombospondin-1 and thrombospondin-2 with hematological, biochemical and inflammatory markers in COVID-19 patients

Abstract Objectives 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. Methods 106 SARS-CoV-2 infected patients and 23 healthy people were enrolled in our study. COVID-19 patients were divided into two groups as non-severe and severe. TSP-1 and TSP-2 concentrations were measured with an enzyme-linked Immunosorbent Assay, and blood markers were analyzed with routine laboratory techniques. Results COVID-19 patients had significantly higher TSP-1 and TSP-2 levels than healthy controls. TSP-1 and TSP-2 positively correlated with inflammatory markers, including ESR, CRP, PCT, ferritin, and biochemical parameters such as ALT, AST, BUN, CK, and LDH. In addition, TSP-1 and TSP-2 were negatively correlated with hematological markers such as LYM, EOS, and HGB. Receiver operating characteristic analyses revealed that COVID-19 may be predicted with TSP-1 levels over 189.94 ng/mL and TSP-2 levels higher than 0.70 ng/mL. Conclusions Our analysis suggests that TSP-1 and TSP-2 expressions at the systemic level may have clinical importance for COVID-19.

Depleted uranium induces thyroid damage through activation of ER stress via the thrombospondin 1-PERK pathway

Depleted uranium (DU) can cause damage to the body, but its effects on the thyroid are unclear. The purpose of this study was to investigate the DU-induced thyroid damage and its potential mechanism in order to find new targets for detoxification after DU poisoning. A model of acute exposure to DU was constructed in rats. It was observed that DU accumulated in the thyroid, induced thyroid structure disorder and cell apoptosis, and decreased the serum T4 and FT4 levels. Gene screening showed that thrombospondin 1 (TSP-1) was a sensitive gene of DU, and the expression of TSP-1 decreased with the increase of DU exposure dose and time. TSP-1 knockout mice exposed to DU had more severe thyroid damage and lower serum FT4 and T4 levels than wild-type mice. Inhibiting the expression of TSP-1 in FRTL-5 cells aggravated DU-induced apoptosis, while exogenous TSP-1 protein alleviated the decreased viability in FRTL-5 cells caused by DU. It was suggested that DU may caused thyroid damage by down-regulating TSP-1. It was also found that DU increased the expressions of PERK, CHOP, and Caspase-3, and 4-Phenylbutyric (4-PBA) alleviated the DU-induced FRTL-5 cell viability decline and the decrease levels of rat serum FT4 and T4 caused by DU. After DU exposure, the PERK expression was further up-regulated in TSP-1 knockout mice, and the increased expression of PERK was alleviated in TSP-1 over-expressed cells, as well as the increased expression of CHOP and Caspase-3. Further verification showed that inhibition of PERK expression could reduce the DU-induced increased expression of CHOP and Caspase-3. These findings shed light on the mechanism that DU may activate ER stress via the TSP 1-PERK pathway, thereby leading to thyroid damage, and suggest that TSP-1 may be a potential therapeutic target for DU-induced thyroid damage.

CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis.

TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 (cluster of differentiation) and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis. Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC Cd47 in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms. Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF (vascular endothelial growth factor)-C-induced AKT and eNOS activation in LEC and attenuated NO (nitric oxide) production. Further, CD47 silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV (adeno-associated virus) 8-PCSK9-injected LEC-specific Cd47 knockout mice (Cd47ΔLEC) had reduced atherosclerosis in both aorta and aortic root compared with control mice (Cd47ΔWT). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in Cd47ΔLEC mice in comparison to controls. These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC Cd47 attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.

Thrombospondin-2 Couples Pressure-Promoted Chondrogenesis through NF-κB Signaling.

Our previous studies found that the mechanical stimulation promote chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), along with up-regulation of thrombospondin-2 (TSP-2). The aim of this study was to explore the effect of thrombospondin-2 (TSP-2) on the mechanical pressure-stimulated chondrogenic differentiation of BMSCs and the possible role of NF-κB signaling in the mechano-chemical coupling regulation toward chondrogenesis. Rat BMSCs were isolated, cultured and identified. The time-dependent expressions of TSP-2 and Sox9 in BMSCs under a dynamic mechanical pressure of 0-120kPa at 0.1Hz for 1h were tested by qPCR and Western blotting. The role of TSP-2 in chondrogenic differentiation of BMSCs under mechanical pressure was validated by using small interfering RNA. Theimpact of TSP-2 andmechanicalpressureonchondrogenesisweredetectedandthedownstreamsignalingmolecules were explored using Western blotting. Mechanical pressure stimulation of 0-120kPa for 1h significantly upregulated the expression of TSP-2 in BMSCs. The expression of the chondrogenesis markers Sox9, Aggrecan, and Col-II were all upregulated under dynamic mechanical pressure or TSP-2 stimulation. Additional exogenous TSP-2 may potentiate the chondrogenic effect of mechanical stimulation. After knock down TSP-2, the upregulation of Sox9, Aggrecan and Col-II under mechanical pressure was inhibited. The NF-κB signaling pathway responded to both dynamic pressure and TSP-2 stimulation, and the cartilage-promoting effect was blocked by an NF-κB signaling inhibitor. TSP-2 plays an essential role in the chondrogenic differentiation of BMSCs under mechanical pressure. NF-κB signaling is involved in the mechano-chemical coupling of TSP-2 and mechanical pressure for the chondrogenic differentiation of BMSCs.

Inhibition of lysophosphatidic acid receptor 1 relieves PMN recruitment in CNS via LPA1/TSP1/CXCR2 pathway and alleviates disruption on blood-brain barrier following intracerebral haemorrhage in mice

BackgroudThe frequencies of morbidity and impairment associated with spontaneous intracerebral haemorrhage (ICH) are comparatively high. Blood-brain barrier (BBB) integrity was compromised due to subsequent brain injury induced by ICH, which is crucial for a poor prognosis. Polymorphonuclear leukocyte (PMN) strongly modulate the disruption of BBB in the central nervous system (CNS). The lysophosphatidic acid receptor 1 (LPA1) mediated thrombospondin-1 (TSP1) regulation in astrocytes, which induce macrophage inflammatory protein 2(MIP2) secretion. MIP2 enhance PMN recruitment through CXC chemokine type 2 (CXCR2) activation. The purpose of this study was to investigate whether the LPA1-mediated inhibition of PMN recruitment and BBB protection after ICH is regulated by TSP1 and CXCR2 networks.MethodsICH induction was performed in CD1 mice using collagenase administration. AM966, a targeted LPA1 antagonist, was orally administered 1 and 12 h following ICH. further identify possible LPA1-mediated BBB protection mechanisms, we intracerebroventricularly (ICV) administered a CXCR2 ligand MIP2, as well as TSP1 CRISPR activation (ACT) with AM966. Consequently, we performed neurobehavioral, brain water content (BWC), Evans blue staining (EBS), immunofluorescence (IF), and western blot (WB) analyses.ResultsAfter ICH, astrocytes showed signs of LPA1, which peaked after 24 h, while PMN\\ displayed evidence of CXCR2. The AM966-mediated LPA1 suppression relieved PMN recruitment, diminished brain oedema, demonstrated extravasation (as evidenced by EBS), protected BBB integrity, and enhanced neurologic activity following ICH. AM966 treatment strongly reduced TSP1, CXCR2, Occludin, and Claudin-5 expressions and PMN recruitment following ICH, and their expressions were restored by MIP2 and TSP1 CRISPR (ACT).ConclusionsThis study shows that LAP1 suppression reduced PMN recruitment after ICH in mice via TSP1/CXCR2 signalling, which minimized BBB disruption and improved the CNS’s neurobehavioral functioning. Hence, LPA1 is a strong candidate for therapy to reduce PMN recruitment and offer protection of BBB integrity after ICH.

Abstract 279: Intricate Roles Of Myeloid-derived Thrombospondin-1 In Aortic Aneurysm And Rupture

Introduction: Abdominal aortic aneurysm (AAA) is the progressive weakening and dilation of the aorta. Aneurysm rupture is the major cause of mortality among AAA patients, but its molecular mechanism is still unclear. We previously reported elevated expression of thrombospondin-1 (TSP1), a matricellular protein, in human and mouse AAA. We also identified Mφs as the major source of TSP1. In this study, we investigated the role of myeloid-derived TSP1 in AAA and rupture. Methods and Results: Male myeloid-specific TSP1 deficient mice ( Thbs1 Δ Mφ ) and wildtype littermates ( Thbs1 wt ) were challenged with angiotensin II (Ang II) coupled with hypercholesterolemia. Sixty-one percent of Thbs1 Δ Mφ died from AAA rupture, an incidence that was 2.6 times higher than Thbs1 wt (n=23 for Thbs1 wt , n=30 for Thbs1 Δ Mφ ). Intriguingly, Thbs1 Δ Mφ that survived to the end of 28-day Ang II infusion had less aortic dilation than Thbs1 wt . Majority of the rupture occurred between 7 to 14 days of Ang II infusion. Ultrasound imaging at early time points (baseline, 3 or 7 days after AAA induction) showed no difference in the aortic diameters between genotypes. To understand the molecular mechanisms underlying the rupture-prone phenotype of Thbs1 Δ Mφ , we conducted single-cell RNA sequencing on the aortic tissues 7 days after AAA induction. Thbs1 Δ Mφ showed increased percentages of smooth muscle cells (SMCs), and decreased Mφs and fibroblasts. Six functional SMC subclusters were identified, which were contractile, fibroblast-like, proliferative, macrophage-like, hemoglobin positive, and intermediate SMCs. Thbs1 Δ Mφ specifically increased the intermediate SMCs, all the other subclusters were decreased. Pathway analysis revealed an extended reduction of extracellular matrix-related genes. Histological analysis confirmed the predicted matrix weakening caused by the myeloid-specific TSP1 deficiency. Conclusions: Myeloid TSP1 is critical for Mφ infiltration, which fuels aneurysm growth. However, Mφs, likely through signaling to SMCs and fibroblasts via TSP1, may also contribute to the vascular remodeling at least in the Ang II-induced aortopathy. Therefore, Thbs1 Δ Mφ provides a unique model to investigate the complex interplays between Mφs and vascular cells in AAA and rupture.

Evaluation of GREM1 and THBS2 as prognostic markers in in non-small cell lung cancer.

To analyze the correlation between bone morphogenetic protein (BMP) antagonist Gremlin 1 (GREM1), Thrombospondin-2 (THBS2) and immune cell infiltration in non-small cell lung cancer (NSCLC) and the related clinical significance. A total of 150 NSCLC patients admitted to our hospital during May 2019-January 2022 were picked. The expression of GREM1 and THBS2 and the infiltration of immune cells in tumor tissues were detected through immunohistochemistry (IHC). These objects were graded as GREM1-positive group (n = 97), GREM1-negative group (n = 53), THBS2-positive group (n = 102) and THBS2-negative group (n = 48) according to the expression of GREM1 and THBS2. The correlation between the expression of GREM1 and THBS2 with immune cell infiltration and clinicopathological indicators was analyzed. Kaplan-Meier survival analysis was adopted to analyze the relationship between the expression of GREM1 and THBS2 and the prognosis in NSCLC tissues. The overall progression-free survival (PFS) of the two groups were compared by log-rank test. The results of IHC showed that the positive expression rate of GREM1 was 64.67% (97/150) in cancer tissues and 36.00% (54/150) in adjacent tissues. The positive expression rate of THBS2 was 68.00% (102/150) in cancer tissues and 25.33% (38/150) in adjacent tissues. The positive expression rate of GREM1 and THBS2 in cancer tissues was both much higher than that in adjacent tissues (P < 0.01). GREM1-positive group had much higher proportion of tumor diameter ≥ 2cm, stage III-IV and lymph-node metastasis than GREM1-negative group (P < 0.05). THBS2-positive group had markedly higher proportion of tumor diameter ≥ 2cm, stage III-IV, lymph-node metastasis and high differentiation than THBS2-negative group (P < 0.01). GREM1-positive group had much higher level of CD3 + T, and sharply lower level of CD8 + T and CD11c + DCs than GREM1-negative group (P < 0.01). THBS2-positive group had much higher level of CD3 + T, and sharply lower level of CD8 + T and CD11c + DCs than THBS2-negative group (P < 0.01). According to Kaplan-Meier survival analysis, the overall median progression-free survival (PFS) was 7.45months. Log-rank test showed that NSCLC patients with positive GREM1 and THBS2 had much shorter PFS than negative patients (P < 0.01). Log-rank test showed that the expression of GREM1 and THBS2 was related to the PFS of patients (P < 0.01). GREM1 and THBS2 were highly expressed in NSCLC tissues and acted as pro-oncogenes in the development and progression of NSCLC, which aggravated the disease by mediating immune cell infiltration.

Abstract 5165: In vivo therapeutic validation of NRT-YHD_001, a novel macrophage checkpoint inhibitor, in liver cancer

Abstract Previously, we demonstrated that recovery of Histone deacetylase 6 (HDAC6) elicited let-7i-5p suppression to de-repress the thrombospondin-1 (TSP1) expression, and thereby it occupied CD47 receptor to block CD47-SIRPα mediated anti-phagocytosis of macrophage in liver cancer. NRT-YHD_001 is a modified antisense miRNA of let-7i-5p for liver cancer treatment. NRT-YHD_001 was substituted with O-methoxyethyl and phosphothioate for stability, and N-acetylgalactosamine was attached for specific delivery to the liver. In in vitro phagocytosis assay, NRT-YHD_001 showed macrophage phagocytosis activity with similar or better results compared to non-modified antisense let-7i-5p (AS_let-7i-5p). In addition, it was confirmed that NRT-YHD_001 inhibited endogenous let-7i-5p of liver cancer cells and activated macrophage phagocytosis at 4°C or 25°C for up to 2 weeks. When NRT-YHD_001 activity was investigated in vivo after 1 week, 2 weeks, and 4 weeks after administration of single dose NRT-YHD_001 to the tail vein of a mouse, NRT-YHD_001 was not degraded, and let-7i-5p was inhibited while maintaining activity in liver tissue for 2 weeks after injection. To investigate the in vivo therapeutic effect, a mouse spontaneous (Ras-transgenic) liver cancer model was used and compared with the sorafenib-administered group. After administration of NRT-YHD_001 and sorafenib each week at the time when liver mass was detected, the tumor size and growth rate were compared. As a result, the NRT-YHD_001 administration group showed significant therapeutic effect compared to sorafenib. These results showed that NRT-YHD_001 had a strong anticancer effect by converting the macrophage of don't eat me into eat me signal in the tumor microenvironment of liver cancer, thereby removing or inhibiting liver cancer cells. Keywords: let-7i-5p, macrophage, phagocytosis, liver cancer, NRT-YHD_001 Citation Format: Suk Woo Nam, Sangyean Kim, Min Jeong Na, Eunbi Shin, Jin Woong Ha, Soyoung Jeon, Jeong Hwan Yoon. In vivo therapeutic validation of NRT-YHD_001, a novel macrophage checkpoint inhibitor, in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5165.

Baicalin inhibited both the Furin/TGFβ1/Smad3/TSP-1 pathway in endothelial cells and the AKT/Ca2+/ROS pathway in platelets to ameliorate inflammatory coagulopathy

Inflammatory coagulopathy is resulted from endothelial dysfunction and platelet hyperactivation in inflammatory diseases. In this study, the effects of baicalin, an active component of the traditional Chinese medicine Huangqin, on inflammatory coagulopathy were observed both in vivo and in vitro. In LPS-induced rats, baicalin ameliorated coagulation indexes, inhibited platelet hyperactivation and decreased the expression of thrombospondin-1 (TSP-1) in vessels. In cultured endothelial cells, baicalin decreased the expression of TSP-1 and collagen as well as the TNF-α-induced increase in the levels of TSP-1 and ICAM-1. Baicalin could significantly decrease the platelet adhesion on endothelial cells treated with TNF-α. Baicalin also could inhibit the increase of ROS level and the activation of the NLRP3/Caspase-1/GSDMD pathway in TNF-α-induced endothelial cells. Furin was found to be the direct target of baicalin in HUVECs. Knockdown of Furin using siRNA could ameliorate the effects of baicalin on the activation of TGFβ1/Smad3 pathway, TSP-1 expression and the adhesion of platelets on TNF-α-treated endothelial cells. At the same time, baicalin inhibited platelet aggregation induced by collagen or combination of collagen and TSP-1 peptide. Collagen-induced Ca2+ mobilization, ROS level increase, AKT1 phosphorylation, platelet degranulation and TSP-1 release could be all inhibited by baicalin. In all, baicalin ameliorated endothelial dysfunction by inhibiting Furin/TGFβ1/Smad3/TSP-1 pathway and also ameliorated platelet activation by inhibiting AKT-related pathway. Both the inhibiting effects of baicalin on endothelial dysfunction and platelet activation might contribute to its ameliorating effects on inflammatory coagulopathy.

RNF7 Induces Skeletal Muscle Cell Apoptosis and Arrests Cell Autophagy via Upregulation of THBS1 and Inactivation of the PI3K/Akt Signaling Pathway in a Rat Sepsis Model.

Recently, long noncoding RNAs (lncRNAs) have been highlighted for extensive functionality in sepsis. In this study, we aimed to explore the role of RNF7 in the progression of sepsis. We initially established a rat model of sepsis through cecal ligation and puncture induction, whereupon RNF7 expression was determined by RT-qPCR. Following adenovirus infection, the role of RNF7 in muscle injury, skeletal muscle protein metabolism, oxidative stress, and inflammation in sepsis rats was analyzed. Then, downstream mechanisms of RNF7 were identified and validated. Further, lipopolysaccharide was applied to treat myoblast to further demonstrate the in vitro role of RNF7. Our results showed that RNF7 expression was upregulated during sepsis. Overexpression of RNF7 worsened the sepsis-induced skeletal muscle injury, induced skeletal muscle protein metabolism, oxidative stress, and inflammation in sepsis rats. Meanwhile, overexpression of RNF7 elevated thrombospondin-1 (THBS1) expression. Silencing of RNF7 inhibited THBS1 and activated the PI3K/Akt signaling pathway, arresting the release of inflammatory factors and oxidative stress levels in skeletal muscle cells. Altogether, RNF7 may promote skeletal muscle cell apoptosis while simultaneously inhibiting cell autophagy through the promotion of THBS1 and inactivation of the PI3K/Akt signaling pathway.

Expression of CD47 and its ligands in pregnant mice infected with Toxoplasma gondii during pregnancy

Expression of CD47 and its ligands in pregnant mice infected with Toxoplasma gondii during pregnancy

LYN kinase programs stromal fibroblasts to facilitate leukemic survival via regulation of c-JUN and THBS1

Microenvironmental bystander cells are essential for the progression of chronic lymphocytic leukemia (CLL). We have discovered previously that LYN kinase promotes the formation of a microenvironmental niche for CLL. Here we provide mechanistic evidence that LYN regulates the polarization of stromal fibroblasts to support leukemic progression. LYN is overexpressed in fibroblasts of lymph nodes of CLL patients. LYN-deficient stromal cells reduce CLL growth in vivo. LYN-deficient fibroblasts show markedly reduced leukemia feeding capacity in vitro. Multi-omics profiling reveals that LYN regulates the polarization of fibroblasts towards an inflammatory cancer-associated phenotype through modulation of cytokine secretion and extracellular matrix composition. Mechanistically, LYN deletion reduces inflammatory signaling including reduction of c-JUN expression, which in turn augments the expression of Thrombospondin-1, which binds to CD47 thereby impairing CLL viability. Together, our findings suggest that LYN is essential for rewiring fibroblasts towards a leukemia-supportive phenotype.

Increase in transforming growth factor-β didnot affect trombospondin1 in preeclampsia placentas.

The abnormalities of the placental growth process are a theory causing pre-eclampsia. Antiangiogenic factors contributed to it, such as thrombospondin-1 (TSp-1) that could stimulate transforming growth factor-beta (TGF-β), or vice versa. Some research showed that an increase in TGF-β did not always figurized its signaling. Therefore, we conducted a study to examine the TGF-β signaling proteins through its receptors and TSp-1 expression in preeclampsia placentas. This observational study used 33 normal and 33 pre-eclampsia placental storaged samples, for examination of TGF-β and TGF-βR 1 and 2, SMAD2 using ELISA, and SMAD2 and TSp-1 mRNA using the reverse transcription polymerase chain reaction method. Data were analyzed using SPSS version 20.0, normality test by Kolmogorov-Smirnov, and significancy was analyzed using nonparametric Mann-Whitney test, or t-test for parametric, with confidence interval 95%. Spearman correlation was used for non-parametric data, besides the Pearson correlation for parametric data. Results showed that there were significant differences between preeclampsia and normal placenta in TGF-β, its receptors, SMAD2, and TSp-1 mRNA. Normal-TGF-β=1.19 (0.713-2.051) pg/mg; preeclampsia-TGFB=2.69 (0.906-10.252) pg/mg; p=0.001; normal-TGFBR1=1.025 (0.622-1.402) ng/mg; preeclampsia-TGFBR1=1.223 (0.372-2.553) ng/mg; p=0.004; Normal-TGF-βR2=0.959 (0.644-1.634) pg/mg; preeclampsia-TGFBR2=1.490 (0.775-3.645) pg/mg; p=0.0001; normal-SMAD2=2.087 (1.279-4.300) ng/mg; preeclampsia-SMAD2=3.508 (1.842-22.489) ng/mg; p=0.0001. The SMAD2 mRNA relative expression (Livax) in the normal placenta was=0.71 (0.03-7.25); pre-eclampsia placenta (PE)=0.49 (0.01-40.71); p=0.075, the normal TSp-1 mRNA expression=1.08 (0.09-5.31); PE=0.21 (0.002-24.06); p=0.002. The correlation test showed a strong correlation between TGF-β with TGFBR1 and 2 in the normal placenta, conversely, there was no correlation in the preeclampsia placenta. There was also no correlation between SMAD2 and TSp-1 mRNA in both normal and pre-eclampsia. TGF-β signaling in the preeclampsia placenta was changed due to the increased of the protein signaling it self without correlation between TGF-β to its receptors and TSp-1 relative expression.

Latexin regulates sex dimorphism in hematopoiesis via gender-specific differential expression of microRNA 98-3p and thrombospondin 1

Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate to all blood cell types. HSCs and their differentiated progeny show sex/gender differences. The fundamental mechanisms remain largely unexplored. We previously reported that latexin (Lxn) deletion increased HSC survival and repopulation capacity in female mice. Here, we find no differences in HSC function and hematopoiesis in Lxn knockout (Lxn-/-) male mice under physiologic and myelosuppressive conditions. We further find that Thbs1, a downstream target gene of Lxn in female HSCs, is repressed in male HSCs. Male-specific high expression of microRNA 98-3p (miR98-3p) contributes to Thbs1 suppression in male HSCs, thus abrogating the functional effect of Lxn in male HSCs and hematopoiesis. These findings uncover a regulatory mechanism involving a sex-chromosome-related microRNA and its differential control of Lxn-Thbs1 signaling in hematopoiesis and shed light on the process underlying sex dimorphism in both normal and malignant hematopoiesis.

Cell surface GRP78 regulates TGFβ1-mediated profibrotic responses via TSP1 in diabetic kidney disease.

Introduction: Diabetic kidney disease (DKD) is the leading cause of kidney failure in North America, characterized by glomerular accumulation of extracellular matrix (ECM) proteins. High glucose (HG) induction of glomerular mesangial cell (MC) profibrotic responses plays a central role in its pathogenesis. We previously showed that the endoplasmic reticulum resident GRP78 translocates to the cell surface in response to HG, where it mediates Akt activation and downstream profibrotic responses in MC. Transforming growth factor β1 (TGFβ1) is recognized as a central mediator of HG-induced profibrotic responses, but whether its activation is regulated by cell surface GRP78 (csGRP78) is unknown. TGFβ1 is stored in the ECM in a latent form, requiring release for biological activity. The matrix glycoprotein thrombospondin 1 (TSP1), known to be increased in DKD and by HG in MC, is an important factor in TGFβ1 activation. Here we determined whether csGRP78 regulates TSP1 expression and thereby TGFβ1 activation by HG. Methods: Primary mouse MC were used. TSP1 and TGFβ1 were assessed using standard molecular biology techniques. Inhibitors of csGRP78 were: 1) vaspin, 2) the C-terminal targeting antibody C38, 3) siRNA downregulation of its transport co-chaperone MTJ-1 to prevent GRP78 translocation to the cell surface, and 4) prevention of csGRP78 activation by its ligand, active α2-macroglobulin (α2M*), with the neutralizing antibody Fα2M or an inhibitory peptide. Results: TSP1 transcript and promoter activity were increased by HG, as were cellular and ECM TSP1, and these required PI3K/Akt activity. Inhibition of csGRP78 prevented HG-induced TSP1 upregulation and deposition into the ECM. The HG-induced increase in active TGFβ1 in the medium was also inhibited, which was associated with reduced intracellular Smad3 activation and signaling. Overexpression of csGRP78 increased TSP-1, and this was further augmented in HG. Discussion: These data support an important role for csGRP78 in regulating HG-induced TSP1 transcriptional induction via PI3K/Akt signaling. Functionally, this enables TGFβ1 activation in response to HG, with consequent increase in ECM proteins. Means of inhibiting csGRP78 signaling represent a novel approach to preventing fibrosis in DKD.

Thrombospondin-1 and prolyl 4-hydroxylase subunit alpha 3 as potential biomarkers of salivary gland fibrosis

Background/purposeFibrosis 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. Materials and methodsWe 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. ResultsBoth 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. ConclusionTHBS1 and P4HA3 may be potential biomarkers for SG fibrosis. They may be also applicable in the diagnosis of multi-organ fibrosis.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Thrombospondin-1 induction and VEGF reduction by proteasome inhibition

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.

Lactic acidosis induces metabolic and phenotypic reprogramming in cholangiocarcinoma cells via the upregulation of thrombospondin-1.

The high glycolytic activity of cancer cells leads to lactic acidosis (LA) in the tumor microenvironment. LA is not merely a consequence of metabolic activities but also has functional roles in metabolic reprogramming and cancer progression. Cholangiocarcinoma (CCA) cells exhibit a high dependency on glycolysis for survival and growth, but the specific effects of LA on cellular characteristics remain unknown. Here, we demonstrate that long-term LA (LLA) reprograms the metabolic phenotype of CCA cells from glycolytic to oxidative and enhances their migratory activity. In CCA cell culture, short-term LA (24 h) showed a growth inhibitory effect, while extended LA exposure for more than 2 weeks (LLA) led to enhanced cell motility. Coincidentally, LLA enhanced the respiratory capacity with an increase in mitochondrial mass. Inhibition of mitochondrial function abolished LLA-induced cell motility, suggesting that metabolic remodeling affects the phenotypic outcomes. RNA-sequencing analysis revealed that LLA upregulated genes associated with cell migration and epithelial-mesenchymal transition (EMT), including thrombospondin-1 (THBS1), which encodes a pro-EMT-secreted protein. Inhibition of THBS1 resulted in the suppression of both LLA-induced cell motility and respiratory capacity. Moreover, high THBS1 expression was associated with poor survival in patients with CCA. Collectively, our study suggests that the increased expression of THBS1 by LLA promotes phenotypic alterations, leading to CCA progression.