Thrombospondin-1 Expression Research Articles

CD47 and thrombospondin-1 contribute to immune evasion by Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative anaerobic bacterium linked to periodontal disease. Remarkably, P. gingivalis thrives in an inflamed environment rich in activated neutrophils. Toll-like receptor 2 (TLR2) recognition is required for P. gingivalis to evade innate immune killing; however, the mechanisms through which P. gingivalis uncouples host inflammation from bactericidal activity are only partially known. Since integrin activation and alternative signaling are implicated in P. gingivalis TLR2-mediated immune escape, we explored the role of CD47, a widely expressed integrin-associated protein known to suppress phagocytosis and implicated as an interacting partner with other innate immune receptors. We found that CD47 associates with TLR2, and blocking CD47 leads to decreased intracellular P. gingivalis survival in macrophages in a manner dependent on the bacterial major fimbria. In vivo, CD47 knock-out mice cleared P. gingivalis more efficiently than wild-type mice. Next, we found increased expression and secretion of the CD47 ligand thrombospondin-1 (TSP-1) following P. gingivalis infection. Secreted TSP-1 broadly protected P. gingivalis and other periodontitis-associated bacterial species from neutrophil bactericidal activity. Therefore, CD47-TLR2 cosignaling in response to P. gingivalis induces TSP-1 that in turn suppresses neutrophil activity, an effect that can explain how species such as P. gingivalis survive in an inflamed environment and cause dysbiosis.

Targeting hypoxia and thrombospondin-2 in diabetic wound healing.

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

MiR-335-3p attenuates transforming growth factor beta 1-induced fibrosis by suppressing Thrombospondin 1.

Pulmonary fibrosis is characterized by excessive extracellular matrix (ECM) accumulation caused by detrimental stimuli. The progressive impairment in lung functions is chronic and highly fatal, presenting itself as a global health challenge. Because of the lack of efficacious treatments, the underlying mechanism should be investigated. The progression of fibrosis involves transforming growth factor-beta 1 (TGF-β1), which accelerates ECM production via epithelial-mesenchymal transition and cell invasion. As microRNAs (miRNAs) serve as regulators of disease development and progression, this study aimed to investigate the interaction of miRNAs and target genes that could contribute to pulmonary fibrosis when exposed to TGF-β1. Differentially expressed mRNA and miRNA were identified in respiratory epithelial cells via transcriptome analysis by using the constructed TGF-β1-induced fibrosis model. Our results revealed a significant increase in the expression of thrombospondin 1 (THBS1), which participates in TGF-β1 activation, where THBS1 was identified as a core gene in protein interactions analyzed through bioinformatics. The expression of miR-335-3p, which targets 3'-UTR of THBS1, substantially decreased upon TGF-β1 treatment. The TGF-β1 downstream signal was suppressed by inhibiting the interaction between TGF-β1 and THBS1, consequently alleviating fibrosis. When the miR-335-3p mimic was transfected in TGF-β1-treated respiratory epithelial cells, THBS1 and fibrosis markers were downregulated, while the introduction of miR-335-3p inhibitor exhibited a reverse phenomenon. Our findings demonstrated that TGF-β1 exposure to respiratory epithelial cells led to a decrease in miR-335-3p expression, resulting in the upregulation of THBS1 and ultimately exacerbating fibrosis. This study provides insights into TGF-β1-induced pulmonary fibrosis, suggesting new therapeutic targets and mechanisms.

The TβRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells

TGFβ potently modifies the extracellular matrix (ECM), which is thought to favor tumor cell invasion. However, the mechanism whereby the cancer cells employ the ECM proteins to facilitate their motility is largely unknown. In this study we used RNA-seq and proteomic analysis to examine the proteins secreted by castration-resistant prostate cancer (CRPC) cells upon TGFβ treatment and found that thrombospondin 1 (THBS1) was observed to be one of the predominant proteins. The CRISPR Cas9, or siRNA techniques was used to downregulate TGFβ type I receptor (TβRI) to interfere with TGFβ signaling in various cancer cells in vitro. The interaction of ECM proteins with the TβRI in the migratory prostate cancer cells in response to TGFβ1 was demonstrated by several different techniques to reveal that THBS1 mediates cell migration by interacting with integrin subunit alpha V (ITGAV) and TβRI. Deletion of TβRI or THBS1 in cancer cells prevented their migration and invasion. THBS1 belongs to a group of tumorigenic ECM proteins induced via TGFβ signaling in CRPC cells, and high expression of THBS1 in human prostate cancer tissues correlated with the degree of malignancy. TGFβ-induced production of THBS1 through TβRI facilitates the invasion and metastasis of CRPC cells as shown in vivo xenograft animal experiments.

Corneal epithelial-stromal constructs to study differences associated with diabetes mellitus

Diabetes mellitus (DM) is a common metabolic disease associated with severe macrovascular and microvascular complications that influence nearly every tissue in the body, including the anterior and posterior segments of the eye. In the cornea, DM is associated with recurrent epithelial erosion and reduced wound-healing capacity, which increases the risk of corneal scarring. We previously developed a co-culture model of the cornea consisting of immortalized human corneal epithelial cells (hCE-TJ) overlaying a self-assembled stromal layer generated by human corneal fibroblasts (hCFs) over a 4-week period. In this study, we investigated epithelial-stromal constructs generated from hCFs derived from subjects with Type 1 (T1DM) or 2 diabetes (T2DM) compared to controls. We found that T2DM constructs exhibited a disrupted epithelium and a thicker, stratified stromal layer compared to controls or T1DM. Both T1DM and T2DM stromal constructs expressed lower expression of thrombospondin-1 in isolated extracellular vesicles (EVs) compared to controls with no significant difference observed in the presence of epithelial cells, suggesting that reduced provisional matrix secretion in the corneal stroma may be a factor that promotes delayed corneal wound healing in diabetes. The tetraspanins are established extracellular vesicle (EV) markers and include CD63, CD81, and CD9, and were highly expressed by EVs in all three cell types. Control corneal stromal fibroblasts produced more and larger EVs when compared to T1DM and T2DM hCF-derived EVs, supporting a role for altered cell-cell communication in the context of DM. Further characterization of EVs and their cargo is expected to aid in the development of targeted treatments to improve corneal wound healing.

Circ_0079480 facilitates proliferation, migration and fibrosis of atrial fibroblasts in atrial fibrillation by sponing miR-338-3p to activate the THBS1/TGF-β1/Smad3 signaling

BackgroundAtrial fibrosis is associated with the pathogenesis of atrial fibrillation (AF). This study aims to discuss the function of circ_0079480 in atrial fibrosis and its underlying mechanism. MethodsIn vitro and in vivo models of atrial fibrosis were established by using angiotensin II (Ang II) to treat human atrial fibroblasts (HAFs) and C57/B6J mice. qRT-PCR and western blot were used to examine the mRNA and protein expression levels. CCK-8, EdU, cell strach, and transwell assays were performed to determine the proliferation and migration of HAFs. Dual-luciferase reporter and RIP/RNA pull-down assays were explored to identify the interaction of miR-338-3p and circ_0079480/THBS1. HE and Masson's trichrome staining experiments were performed to analyze the histopathological change in mice atrial tissues. ResultsCirc_0079480 expression was increased in AF patients' atrial tissues and Ang II-treated HAFs. Silencing circ_0079480 inhibited cell proliferation and migration and reduced fibrosis-associated gene expression in Ang II-treated HAFs. Circ_0079480 could target miR-338-3p to repress its expression. MiR-338-3p inhibitor blocked the inhibitory effects of circ_0079480 knockdown on HAFs proliferation, migration, and fibrosis. Thrombospondin-1 (THBS1) was confirmed as a downstream target of miR-338-3p, and circ_0079480 could sponge miR-338-3p to upregulate THBS1 expression. Moreover, silencing THBS1 suppressed Ang II-induced proliferation, migration, and fibrosis in HAFs. More importantly, depletion of circ_0079480 inactivated the THBS1/TGF-β1/Smad3 signaling by upregulating miR-338-3p. Mice experiments also confirmed the suppression of circ_0079480 knockdown on atrial fibrosis. ConclusionCirc_0079480 acts as a sponge of miR-338-3p to upregulate THBS1 expression and activate the TGF-β1/Smad3 signaling, finally promoting Ang II-induced atrial fibrosis.

Activation of SIRT3 by Tanshinone IIA ameliorates renal fibrosis by suppressing the TGF-β/TSP-1 pathway and attenuating oxidative stress

Although doxorubicin (DOX) is a common chemotherapeutic drug, the serious nephrotoxicity caused by DOX-induced renal fibrosis remains a considerable clinical problem. Tanshinone IIA (Tan IIA), a compound extracted from Salvia miltiorrhiza, has been reported to have an anti-fibrotic effect. Therefore, this study investigated the molecular pathway whereby Tan IIA protects the kidneys from DOX administration. DOX (3 mg/kg body weight) was intraperitoneally administered every 3 d for a total of 7 injections (cumulative dose of 21 mg/kg) to induce nephrotoxicity. Then, Tan IIA (5 or 10 mg/kg/d) was administered by intraperitoneal injection for 28 d. In an in vitro study, 293 T cells were cultured and treated with DOX and Tan IIA for 24 h. Tan IIA reduced the blood urea nitrogen levels elevated by DOX while increasing superoxide dismutase activity, down-regulating reactive oxygen species, ameliorating renal-tubule thickening, and rescuing mitochondrial morphology. Additionally, Tan IIA reduced the renal collagen deposition, increased ATP production and complex-I activity, down-regulated transforming growth factor-β1 (TGF-β1) and thrombospondin-1 (TSP-1), and up-regulated sirtuin 3 (SIRT3). Tan IIA significantly increased cell viability. Additionally, RNA interference was employed to silence the expression of SIRT3, which eliminated the effect of Tan IIA in suppressing the expression of TGF-β1 and TSP-1. In conclusion, Tan IIA ameliorated DOX-induced nephrotoxicity by attenuating oxidative injury and fibrosis. The Tan IIA-induced rescue of mitochondrial morphology and function while alleviating renal fibrosis may be associated with the activation of SIRT3 to suppress the TGF-β/TSP-1 pathway.

Blocking TSP1 Ameliorates Diabetes Mellitus-Induced Erectile Dysfunction by Inhibiting the TGF-β/SMAD Pathway.

To examine the role and mechanism of thrombospondin-1 (TSP1) in the development of fibrosis in diabetes mellitus-induced erectile dysfunction (DMED). DMED was induced by intraperitoneal streptozotocin injection. All rats were categorized into three groups: control group (n=8), DMED group (n=8) and DMED+Leu-Ser-Lys-Leu (LSKL) group (n=8). After eight weeks following the induction of diabetes mellitus, the DMED+LSKL group was subjected to intraperitoneal injections of LSKL twice weekly for four weeks. To measure intracavernous pressure (ICP), a 25-gauge needle connected to a PE tube containing heparin was inserted into the corpus cavernosum (CC). Additionally, a needle was inserted into the carotid artery to measure mean arterial pressure (MAP). Sirius red staining and Masson trichrome staining were utilized to assess CC fibrosis. Moreover, high glucose (HG)-induced CC smooth muscle cells (CCSMCs) and CC fibroblasts (CCFs) were treated with or without LSKL. Western blotting and immunofluorescence were utilized to assess the phosphorylation and expression of related proteins. Compared with those in the control group, the ratio of the maximum ICP to the MAP markedly decreased in the DMED group, as did the ratio of smooth muscle to collagen and the ratio of collagen I to collagen III. These ratios were greater in the DMED+LSKL group than in the DMED group. TSP1 was highly expressed in the CC of DMED rats. In vitro experiments indicated that TSP1 expression significantly increased in the medium of CCSMCs and CCFs cultured in HG media and that the TGF-β pathway was activated in CCSMCs. Collagen IV was overexpressed in CCSMCs, indicating severe fibrosis was severe. Adding LSKL or knocking TSP1 down can prevent the activation of TGF-β signaling, as well as the overexpression of collagen IV in CCSMCs promoted by TSP1 secreted from CCSMCs itself or CCFs. TSP1 expression is increased in the CC of DMED rats. HG-induced TSP1 secretion via autocrine signaling from CCSMCs and/or paracrine signaling from CCFs to accelerate penile fibrosis. LSKL, an antagonist of TSP1, could improve erectile dysfunction by inhibiting the TGF-β/SMAD pathway.

Afatinib plus PEM and CBDCA overcome osimertinib resistance in EGFR-mutated NSCLC with high thrombospondin-1 expression.

Osimertinib induces a marked response in non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) gene mutations. However, acquired resistance to osimertinib remains an inevitable problem. In this study, we aimed to investigate osimertinib-resistant mechanisms and evaluate the combination therapy of afatinib and chemotherapy. We established osimertinib-resistant cell lines (PC-9-OR and H1975-OR) from EGFR-mutant lung adenocarcinoma cell lines PC-9 and H1975 by high exposure and stepwise method. Combination therapy of afatinib plus carboplatin (CBDCA) and pemetrexed (PEM) was effective in both parental and osimertinib-resistant cells. We found that expression of thrombospondin-1 (TSP-1) was upregulated in resistant cells using cDNA microarray analysis. We demonstrated that TSP-1 increases the expression of matrix metalloproteinases through integrin signaling and promotes tumor invasion in both PC-9-OR and H1975-OR, and that epithelial-to-mesenchymal transition (EMT) was involved in H1975-OR. Afatinib plus CBDCA and PEM reversed TSP-1-induced invasion ability and EMT changes in resistant cells. In PC-9-OR xenograft mouse models (five female Balb/c-Nude mice in each group), combination therapy strongly inhibited tumor growth compared with afatinib monotherapy (5 mg/kg, orally, five times per week) or CBDCA (75 mg/kg, intraperitoneally, one time per week) + PEM (100 mg/kg, intraperitoneally, one time per week) over a 28-day period. These results suggest that the combination of afatinib plus CBDCA and PEM, which effectively suppresses TSP-1 expression, may be a promising option in EGFR-mutated NSCLC patients after the acquisition of osimertinib resistance.

Myeloid-Specific Thrombospondin-1 Deficiency Exacerbates Aortic Rupture via Broad Suppression of Extracellular Matrix Proteins.

Rupture of abdominal aortic aneurysms (AAA) is associated with high mortality. However, the precise molecular and cellular drivers of AAA rupture remain elusive. Our prior study showed that global and myeloid-specific deletion of matricellular protein thrombospondin-1 (TSP1) protects mice from aneurysm formation primarily by inhibiting vascular inflammation. To investigate the cellular and molecular mechanisms that drive AAA rupture by testing how TSP1 deficiency in different cell populations affects the rupture event. We deleted TSP1 in endothelial cells and macrophages --- the major TSP1-expressing cells in aneurysmal tissues ---- by crossbreeding Thbs1 flox/flox mice with VE-cadherin Cre and Lyz2-cre mice, respectively. Aortic aneurysm and rupture were induced by angiotensin II in mice with hypercholesterolemia. Myeloid-specific Thbs1 knockout, but not endothelial-specific knockout, increased the rate of lethal aortic rupture by more than 2 folds. Combined analyses of single-cell RNA sequencing and histology showed a unique cellular and molecular signature of the rupture-prone aorta that was characterized by a broad suppression in inflammation and extracellular matrix production. Visium spatial transcriptomic analysis on human AAA tissues showed a correlation between low TSP1 expression and aortic dissection. TSP1 expression by myeloid cells negatively regulates aneurysm rupture, likely through promoting the matrix repair phenotypes of vascular smooth muscle cells thereby increasing the strength of the vascular wall.

Critical role of thrombospondin-1 in promoting intestinal mucosal wound repair.

Thrombospondin-1 (TSP1) is a matricellular protein associated with the regulation of cell migration through direct binding interactions with integrin proteins and by associating with other receptors known to regulate integrin function, including CD47 and CD36. We previously demonstrated that deletion of an epithelial TSP1 receptor, CD47, attenuates epithelial wound repair following intestinal mucosal injury. However, the mechanisms by which TSP1 contributes to intestinal mucosal repair remain poorly understood. Our results show upregulated TSP1 expression in colonic mucosal wounds and impaired intestinal mucosal wound healing in vivo upon intestinal epithelium-specific loss of TSP1 (VillinCre/+ Thbs1fl/fl or Thbs1ΔIEC mice). We report that exposure to exogenous TSP1 enhanced migration of intestinal epithelial cells in a CD47- and TGF-β1-dependent manner and that deficiency of TSP1 in primary murine colonic epithelial cells resulted in impaired wound healing. Mechanistically, TSP1 modulated epithelial actin cytoskeletal dynamics through suppression of RhoA activity, activation of Rho family small GTPase (Rac1), and changes in filamentous-actin bundling. Overall, TSP1 was found to regulate intestinal mucosal wound healing via CD47 and TGF-β1, coordinate integrin-containing cell-matrix adhesion dynamics, and remodel the actin cytoskeleton in migrating epithelial cells to enhance cell motility and promote wound repair.

Role of TSP-1 and its receptor ITGB3 in the renal tubulointerstitial injury of focal segmental glomerulosclerosis

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 β3 (ITGB3) in FSGS. Previously the renal interstitial chip analysis of FSGS patients with tubular interstitial injury showed that the expressions of TSP-1 and ITGB3 were up-regulated. We found that the level of TSP-1 and ITGB3 increased in the tubular cells of FSGS patients. The serum level of TSP-1 increased and was correlated to the degree of tubulointerstitial lesions in FSGS patients. THBS1/ITGB3 signaling induced renal tubular injury in HK-2 cells exposure to BSA and the ADR-induced nephropathy model. THBS1 knockout 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 BSA, 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.

Proteomic analysis of plasma at the preterminal stage of rhesus nonhuman primates exposed to a lethal total-body dose of gamma-radiation

The identification and validation of radiation biomarkers is critical for assessing the radiation dose received in exposed individuals and for developing radiation medical countermeasures that can be used to treat acute radiation syndrome (ARS). Additionally, a fundamental understanding of the effects of radiation injury could further aid in the identification and development of therapeutic targets for mitigating radiation damage. In this study, blood samples were collected from fourteen male nonhuman primates (NHPs) that were exposed to 7.2 Gy ionizing radiation at various time points (seven days prior to irradiation; 1, 13, and 25 days post-irradiation; and immediately prior to the euthanasia of moribund (preterminal) animals). Plasma was isolated from these samples and was analyzed using a liquid chromatography tandem mass spectrometry approach in an effort to determine the effects of radiation on plasma proteomic profiles. The primary objective was to determine if the radiation-induced expression of specific proteins could serve as an early predictor for health decline leading to a preterminal phenotype. Our results suggest that radiation induced a complex temporal response in which some features exhibit upregulation while others trend downward. These statistically significantly altered features varied from pre-irradiation levels by as much as tenfold. Specifically, we found the expression of integrin alpha and thrombospondin correlated in peripheral blood with the preterminal stage. The differential expression of these proteins implicates dysregulation of biological processes such as hemostasis, inflammation, and immune response that could be leveraged for mitigating radiation-induced adverse effects.

#2144 Expression profile induced by TGF-β1 in senescent human peritoneal mesothelial cells is less pronounced than in young cells

Abstract Background and Aims Transforming growth factor β1 (TGF-β1) is a recognized driver of both mesothelial-to-mesenchymal transition (MMT) and cellular senescence observed in vitro in human peritoneal mesothelial cells (HPMCs), and have a significant role in adverse peritoneal remodelling of patients treated with peritoneal dialysis (PD). We have previously shown that senescent HPMCs spontaneously acquire some phenotypic features of MMT which in young HPMCs are induced by TGF-β1. At the same time, senescent HPMCs appeared to be less responsive to TGF-β. It is not clear what determines which pathway is activated by TGF-β1. Here, we aimed to extend previous observations and used electron microscopy, transcriptomics, and proteomics to identify differences between young and senescent cells in their responsiveness to TGF-β. Method Replicative senescence of omentum-derived HPMCs isolated from 4 separate donors was induced by serial passages until cells ceased to grow and expressed senescence-associated β-galactosidase. Serum-starved young and senescent cells were treated in parallel with 1 ng/mL of TGF-β1 for 72 hours. Fixed cells were analyzed with a Jeol transmission electron microscope at 80 kV (Jeol, Tokyo, Japan). Cell lysates were analysed for global gene and protein expression using an integrated transcriptomic (Array-based) and proteomic (LC/MS-based) approach. Results Exposure to TGF-β1 led to phenotypic changes consistent with MMT in young, but not in senescent HPMCs (Fig. 1A). The omics analyses revealed that the response to TGF-β1 was associated with 89 genes being differentially expressed (55 down- and 34 up-upregulated) in young HPMCs and with 13 genes (5 down- and 8 up-regulated) in senescent HPMCs (Fig. 1B). In addition, TGF-β1 changed the expression of 131 proteins in young HPMCs (72 down- and 59 up-regulated) and 232 proteins in senescent HPMCs (127 down- and 105 up-regulated) (Fig. 1C). The responses to TGF-β identified at both the transcriptome and protein levels (Fig. 1D) and present in both young and senescent HPMCs included TGFBI (TGF-β-induced) and ITGB3 (β3 integrin), which were upregulated, and UPK1B (uroplakin 1B) and SEMA3B (semaphorin 3B), which were downregulated. Changes in response to TGF-β seen at the RNA and protein level included also VCAN (versican), ITGA11 (integrin α11), FN1 (fibronectin 1), CCNYL1 (Cyclin Y-like 1) and MYOZ2 (myozenin 2), however, these were observed only in young cells. A decrease in KLK7 (Kallikrein 7) gene expression after exposure to TGF-β was seen both in young and senescent HPMCs, with a decrease in KLK7 protein detected only in senescent cells. Finally, exposure to TGF-β led to an increase in THBS1 (thrombospondin 1) expression in young cells and at the protein level in senescent cells. Conclusion In conclusion, the profound morphological changes seen in senescent HPMCs are not significantly exacerbated by TGFβ. Likewise, changes in global gene and protein profiles in response to TGF-β appear to be less pronounced in senescent cells. Among the genes and proteins affected by TGF-β, those involved in angiogenesis and fibrosis deserve further attention as they may contribute to adverse peritoneal remodelling in PD patients.

Abstract 3057: Vascular Smooth Muscle Cell Cd47 Contributes To Atherosclerosis Development

Aims: Thrombospondin-1 (TSP1), a secretory protein, has been associated with various cardiovascular diseases. TSP1 mainly functions via its cognate receptors CD47 and CD36. Recently, we observed elevated TSP1 expression in human and murine atherosclerotic arteries. However, the role of vascular smooth muscle cell (VSMC) TSP1-CD47 signaling in regulating VSMC phenotype and atherogenesis is unclear. Methods and Results: Human arterial SMCs were treated with human TSP1 and analyzed for SMC markers and proliferation. The data revealed decreased expression of SMC markers, ACTA, CNN1, and SM22α in TSP1-exposed VSMCs compared with vehicle-treated control cells. Further, TSP1 treatment increased VSMC proliferation in vitro , suggesting TSP1 induces VSMC hyperproliferative phenotype. Additional experiments showed CD47 as the major TSP1’s receptor in VSMCs. To investigate the in vivo role of VSMC Cd47 in atherosclerosis, we generated tamoxifen-inducible SMC-specific Cd47 knockout mice ( Cd47 f/f Myh11 Cre +/– , Cd47 ΔVSMC ) by crossing female Cd47 f/f mice with male Myh11 Cre +/- mice. Male tamoxifen-injected Cd47 ΔVSMC and corn oil-administered Cd47 WT mice were given a single injection of AAV8- hPCSK9 (1х10 11 viral genomes/mouse, IP) and fed a Western diet for 12 weeks. En face oil red O (ORO) staining of whole aortas exhibited reduced atherosclerosis in Cd47 ΔVSMC mice compared with control Cd47 WT mice. Further histochemical staining performed on aortic root sections revealed attenuated neointimal lesion area, lipid accumulation and necrotic area in Cd47 ΔVSMC mice. However, there were no significant differences in weight gain, body composition (fat and lean mass), plasma total cholesterol, and fasting blood glucose between Cd47 ΔVSMC and Cd47 WT mice. Conclusions: Taken together, these findings suggest that TSP1 promotes VSMC phenotype switch and VSMC-specific Cd47 deletion in hypercholesterolemic mice suppresses atherosclerosis progression.

Skin-derived precursor conditioned medium alleviated photoaging via early activation of TGF-β/Smad signaling pathway by thrombospondin1: In vitro and in vivo studies

Photoaging is one major exogenous factor of skin aging. Efficacy and safety of current anti-photoaging therapies remained to be improved. Our previous studies indicated that skin-derived precursors (SKPs) alleviated photodamage by early activation of TGF-β/Smad signaling pathway via thrombospondin1 (TSP1). However, the research concerning SKP conditioned medium (SKP-CM) has never been reported. In the current study, we aimed to explore the anti-photoaging effects of SKP-CM both in vitro and in vivo, and to elucidate the possible mechanisms. Mouse SKP-CM (mSKP-CM) collection was optimized by a comparative method. The concentration of protein and growth factors in mSKP-CM was detected using BCA protein assay kit and growth factor protein chip. The anti-photoaging effects of mSKP-CM and its regulation of key factors in the TGF-β/Smad signaling pathway were explored using UVA + UVB photoaged mouse fibroblasts (mFBs) and nude mice dorsal skin. The research revealed that mSKP-CM contained significantly higher-concentration of protein and growth factors than mouse mesenchymal stem cell conditioned medium (mDMSC-CM). mSKP-CM alleviated mFBs photoaging by restoring cell viability and relieving senescence and death. ELISA, qRT-PCR, and western blot results implied the potential mechanisms were associated with the early activation of TGF-β/Smad signaling pathway by TSP1. In vivo experiments demonstrated that compared with the topical intradermal mDMSC-CM injection and retinoic acid cream application, the photodamaged mice dorsal skin intradermally injected with mSKP-CM showed significantly better improvement. Consistent with the in vitro results, both western blot and immunohistochemistry results confirmed that protein expression of TSP1, smad2/3, p-smad2/3, TGF-β1, and collagen I increased, and matrix metalloproteinases decreased. In summary, both in vitro and in vivo experiments demonstrated that mSKP-CM alleviated photoaging through an early activation of TGF-β/Smad signaling pathway via TSP1. SKP-CM may serve as a novel and promising cell-free therapeutical approach for anti-photoaging treatment and regenerative medicine.

4R-cembranoid suppresses glial cells inflammatory phenotypes and prevents hippocampal neuronal loss in LPS-treated mice.

Chronic neuroinflammation has been implicated in neurodegenerative disease pathogenesis. A key feature of neuroinflammation is neuronal loss and glial activation, including microglia and astrocytes. 4R-cembranoid (4R) is a natural compound that inhibits hippocampal pro-inflammatory cytokines and increases memory function in mice. We used the lipopolysaccharide (LPS) injection model to study the effect of 4R on neuronal density and microglia and astrocyte activation. C57BL/6J wild-type mice were injected with LPS (5 mg/kg) and 2 h later received either 4R (6 mg/kg) or vehicle. Mice were sacrificed after 72 h for analysis of brain pathology. Confocal images of brain sections immunostained for microglial, astrocyte, and neuronal markers were used to quantify cellular hippocampal phenotypes and neurons. Hippocampal lysates were used to measure the expression levels of neuronal nuclear protein (NeuN), inducible nitrous oxide synthase (iNOS), arginase-1, thrombospondin-1 (THBS1), glial cell-derived neurotrophic factor (GDNF), and orosomucoid-2 (ORM2) by western blot. iNOS and arginase-1 are widely used protein markers of pro- and anti-inflammatory microglia, respectively. GDNF promotes neuronal survival, and ORM2 and THBS1 are astrocytic proteins that regulate synaptic plasticity and inhibit microglial activation. 4R administration significantly reduced neuronal loss and the number of pro-inflammatory microglia 72 h after LPS injection. It also decreased the expression of the pro-inflammatory protein iNOS while increasing arginase-1 expression, supporting its anti-inflammatory role. The protein expression of THBS1, GDNF, and ORM2 was increased by 4R. Our data show that 4R preserves the integrity of hippocampal neurons against LPS-induced neuroinflammation in mice.

Abstract 572: Identifying potential therapeutic targets through gene expression analysis of peritoneal cavity cells in gastric cancer with peritoneal dissemination

Abstract Background: Peritoneal dissemination (PD) is the primary mode of metastasis in gastric cancer (GC) and is strongly associated with a poor prognosis. However, the mechanisms underlying PD formation in GC remain largely unknown.Methods and Results: In this study, we collected peritoneal washings from three cytology-positive (CY1) and five cytology-negative (CY0) gastric cancer patients undergoing laparotomy. We performed RNA sequencing on cell components in the peritoneal washings and identified 13 genes that were differentially expressed and upregulated in the CY1 group. Among these genes, we focused on thrombospondin 1 (THBS1) and 2 (THBS2). We evaluated their expression levels in 317 patients with gastric or gastroesophageal junction cancer who underwent primary curative resection at the Kanagawa Cancer Center between January 2002 and December 2012. Patients were stratified into two groups based on the median expression values of these genes, and Kaplan-Meier analysis was conducted. THBS2 expression was found to be inversely associated with disease-free survival (DFS) (p = 0.041), peritoneal dissemination-specific DFS (p = 0.041), and overall survival (OS) (p = 0.041) in the study cohort. In contrast, THBS1 expression did not exhibit a significant association with any of the aforementioned outcomes.Conclusion: Our results suggest that THBS2 expression in gastric cancer tissues may serve as a useful independent prognostic factor for patients, potentially playing a crucial role in the formation of PD. Citation Format: Yukihiko Hiroshima, Wataru Kawase, Itaru Hashimoto, Shizune Onuma, Hayato Watanabe, Mitsuhiro Furuta, Takashi Oshima, Yohei Miyagi. Identifying potential therapeutic targets through gene expression analysis of peritoneal cavity cells in gastric cancer with peritoneal dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 572.

Abstract 7520: NRT-YHD_001, targeting CD47 for liver cancer immunotherapy

Abstract Previously, we demonstrated that recovery of histone deacetylase 6 (HDAC6) suppressed let-7i-5p expression 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. In addition, N-acetylgalactosamine was attached to NRT-YHD_001 for specific delivery to the liver. In vitro phagocytosis assay showed better macrophage phagocytosis activity than non-modified antisense let-7i-5p. To investigate the in vivo efficacy of NRT-YHD_001, spontaneous (Ras-transgenic) mouse liver cancer model was used and compared with that of sorafenib treatment. This mouse liver cancer model usually develops spontaneous liver cancer mass after 15 ~ 20 weeks of birth. We confirmed that both weekly i.v. injection of 1mg/kg and biweekly i.v. injection of 2mg/kg NRT-YHD_001 showed more significant therapeutic efficacy than the sorafenib-treated group. Furthermore, xenograft assay with Hep3B human liver cancer cells in athymic-nude mouse also showed that weekly i.v. injection of 1mg/kg NRT-YHD_001 exhibited better survival and tumor growth inhibition compared to non-treated group. In mouse and cynomolgus monkey maximum tolerated dose (MTD) studies, no toxicity of NRT-YHD_001 was found at doses up to 500 mg/kg intravenously. These results showed that NRT-YHD_001 had a robust 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, CD47 Citation Format: Suk Woo Nam, Sangyean Kim, Minjeong Na, Eunbi Shin, Jin Woong Ha, Soyoung Jeon, Sungpil Yoon. NRT-YHD_001, targeting CD47 for liver cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7520.

Abstract 1393: Tumor-infiltrating monocyte-like cells create metastasis-promoting immunosuppressive microenvironment via THBS1 production in aggressive colorectal cancer

Abstract Mesenchymal subtype of colorectal cancer (CRC) is characterized by high metastatic potential, poor prognosis and an immunosuppressive tumor microenvironment (TME). Although tumor-infiltrating myeloid cells constitute the primary population of immune components in the TME, targeting myeloid cells remains highly challenging owing to the developmental and functional heterogeneity of their subsets. Thus, a deeper understanding of how tumor-infiltrating myeloid cells contribute to the creation of a tumor-promoting TME is needed to identify rational strategies against CRCs with aggressive phenotypes. Recent studies have revealed that multiple inflammatory signatures are elevated in mesenchymal CRCs, suggesting a strong association between inflammation and stromal activation. In this study, we focus on thrombospondin-1 (THBS1), which is a matricellular protein, typically expressed in inflammatory processes. Immunostaining and RNA in situ hybridization on human CRC samples revealed a clear localization of THBS1 expression in tumor stroma. Analyses of the cancer genome atlas (TCGA) and tissue microarray of human CRCs show a positive correlation between increased expression of THBS1 and the mesenchymal phenotype, immunosuppression, and poor prognosis in human CRCs. Utilizing immunocompetent orthotopic implantation systems with aggressive mouse tumor organoids (MTO) on wild-type and Thbs1 knockout (Thbs1−/−) mice, we demonstrated that loss of THBS1 in the TME suppressed metastatic formation accompanied by an increased infiltration of tumor-infiltrating CD8+ T cells. Depletion of CD8+ T cells by neutralizing antibody restored metastases in MTO-bearing Thbs1−/− mice, indicating that THBS1 promotes metastatic formation by suppressing CD8+ T cell-mediated anti-tumor immunity. Analyses of single cell RNA sequencing data of human CRCs and orthotopic MTO tumors revealed that bone marrow (BM)-derived monocyte-like cells are the primary source of THBS1. This notion was further supported by analyzing the orthotopic MTO tumors in chimeric mice generated by bone marrow transplantation. Finally, we identified stroma-derived CXCL12 as a key chemokine for the recruitment of THBS1-producing monocyte-like cells by analyzing orthotopic MTO tumors treated with CXCL12 inhibitor. Our study contributes to a better understanding of the processes underlying THBS1 production by monocyte-like cells that mediates immune evasion that confer aggressive features to CRCs. These findings provide insights for the treatment of this poor-prognosis malignancy. Citation Format: Mayuki Omatsu, Yuki Nakanishi, Kosuke Iwane, Naoki Aoyama, Takahisa Maruno, Hiroaki Kasashima, Masakazu Yashiro, Akihisa Fukuda, Hiroshi Seno. Tumor-infiltrating monocyte-like cells create metastasis-promoting immunosuppressive microenvironment via THBS1 production in aggressive colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1393.