Β1 Expression Research Articles

A novel solvent-extruded tacrolimus-eluting suture for attenuated inflammation and scarring in skin repair

This study aimed to design an absorbable suture that locally delivers the immunosuppressive agent, tacrolimus (FK506), to attenuate scar tissue formation by modulating inflammation. As the suture degrades, it releases FK506 directly into the repair site. The poly (l-lactide-co-caprolactone) (PLLA-PCL) polymer and 0.2 % wt FK506 were co-extruded using a solvent extrusion technique to generate the drug-eluting sutures. The properties of the generated sutures were assessed regarding FK506 release in vitro, wound closure in an in vivo murine model, murine skin histology for structural and inflammatory markers, and mechanical strength of both sutures and healed murine skin. The generated suture consistently released FK506 at ≤10 ng/day rates. Following annealing, the sutures retained 60 % of their anticipated tensile strength, exhibiting enhanced ductility. The histological examination and in vivo murine study indicated that wounds treated with FK506-eluting PLLA-PCL sutures produced a thinner epidermal layer, enhanced integrin β4 expression, increased keratinocyte presence, attenuated inflammation, and reduced scarring, demonstrating healed tissue improvements compared with those treated with the placebo, poly (glycolic-co-caprolactone) (PGCL), sutures. Mechanical evaluations of healed tissue indicated that drug-PLLA-PCL sutures did not reduce the mechanical strength of the skin compared to placebo PGCL sutures. The FK506-eluting PLLA-PCL sutures show promising results in advancing wound healing by offering targeted drug delivery. This innovative method can improve wound healing and provide a streamlined approach to scar management. Continued research, optimization, and broad-scale studies are needed to refine the manufacturing process and ensure that the properties of the suture meet clinical requirements.

Innovative 3D-Printed Titanium Specimens Favor a Modulation of Inflammation in Dental Pulp Stem Cells During Liposome-Triggered Mineralization.

developing customized titanium specimens, with innovative surfaces, is a suitable strategy to overcome implant failure. Additionally, a faster and efficient osteogenic commitment assists tissue regeneration. To investigate the interplay between inflammation and differentiation upon implantation, Dental Pulp Stem Cells (DPSCs) were cultured on 3D-printed titanium owning an internal open cell form, administering osteogenic factors by a liposomal formulation (LipoMix) compared to traditional delivery of differentiation medium (DM). osteogenic differentiation was evaluated by western blot, by measuring β1 integrin expression, and by and real-time RT-PCR, by measuring SP7 and Collagen I gene expression; while.angiogenesis was characterized by measuring VEGF secretion levels. Matrix mineralization was assessed by means of Alizarin Red Staining, cell adhesion and inflammation responses through western blot, enzymatic and ELISA assays evaluating Nrf2 expression, catalase activity and Prostaglandin E2 secretion, respectively. LipoMix enhances cell proliferation and adhesion, as revealed by increased integrin β1 expression. Mineralized matrix deposition, SP7 gene expression, Collagen I release and Alkaline Phosphatase activity appear increased in LipoMix condition. Additionally, the redox-sensitive transcription factor Nrf2 is overexpressed at the earliest experimental times, triggering the catalase activity. data reported confirm that internal topography and post-production treatments on titanium surfaces dynamically and positively condition the DPSC progress towards the osteogenic phenotype, moreover, the combination with LipoMix fastens the positive modulation of inflammation under osteogenic conditions. Therefore, the development of customized surfaces along with the administration of differentiating factors enclosed in a liposomal delivery system, could represent a promising and innovative tool in regenerative dentistry.

Expression of interleukin-8 and integrin β3 predicts prognosis of patients with hepatocellular carcinoma after hepatectomy.

As important components in the tumor microenvironment, interleukin-8 (IL-8) and integrin β3 play a key role in the progression and metastasis of hepatocellular carcinoma (HCC). This study aimed to determine the expression of IL-8 and integrin β3 and their prognostic value in patients with HCC after hepatectomy. We investigated the expression of IL-8 and integrin β3, their clinical significance, as well as their correlation in the cancer tissue of 130 patients with HCC using immunohistochemistry. The prognostic value of IL-8 and integrin β3 was investigated through the follow-up of patients with HCC after hepatectomy. In HCC, IL-8 expression had a positive correlation with integrin β3 expression. Increased expressions of IL-8 and integrin β3 were indicators of tumor progression and poor prognosis in patients with HCC after hepatectomy. IL-8 positive specimens exhibited a higher proportion of macrovascular invasion, larger tumor size, poor differentiation, and advanced tumor-node-metastasis (TNM) stage (P < .05, respectively). Integrin β3 positive group exhibited a higher proportion of TNM III-staged tumors (P < .05). The results indicated that macrovascular invasion, advanced TNM stage, and integrin β3 expression were independent unfavorable prognostic factors in HCC after hepatectomy. Integrin β3 expression was proved to be an independent unfavorable prognostic factor in HCC after hepatectomy. Targeting integrin β3 might be a potential therapeutic approach in preventing tumor progression in HCC.

YKL40/Integrin β4 Axis Induced by the Interaction between Cancer Cells and Tumor-Associated Macrophages Is Involved in the Progression of High-Grade Serous Ovarian Carcinoma.

Macrophages in the tumor microenvironment, termed tumor-associated macrophages (TAMs), promote the progression of various cancer types. However, many mechanisms related to tumor-stromal interactions in epithelial ovarian cancer (EOC) progression remain unclear. High-grade serous ovarian carcinoma (HGSOC) is the most malignant EOC subtype. Herein, immunohistochemistry was performed on 65 HGSOC tissue samples, revealing that patients with a higher infiltration of CD68+, CD163+, and CD204+ macrophages had a poorer prognosis. We subsequently established an indirect co-culture system between macrophages and EOC cells, including HGSOC cells. The co-cultured macrophages showed increased expression of the TAM markers CD163 and CD204, and the co-cultured EOC cells exhibited enhanced proliferation, migration, and invasion. Cytokine array analysis revealed higher YKL40 secretion in the indirect co-culture system. The addition of YKL40 increased proliferation, migration, and invasion via extracellular signal-regulated kinase (Erk) signaling in EOC cells. The knockdown of integrin β4, one of the YKL40 receptors, suppressed YKL40-induced proliferation, migration, and invasion, as well as Erk phosphorylation in some EOC cells. Database analysis showed that high-level expression of YKL40 and integrin β4 correlated with a poor prognosis in patients with serous ovarian carcinoma. Therefore, the YKL40/integrin β4 axis may play a role in ovarian cancer progression.

Elevated MMP-9, Survivin, TGB1 and Downregulated Tissue Inhibitor of TIMP-1, Caspase-3 Activities are Independent of the Low Levels miR-183 in Endometriosis.

This study aimed to measure the correlation between miR-183 and gene expression that regulates apoptosis and adhesion mechanism that may be linked to the pathogenesis of endometriosis. Forty-four subjects, including 22 control subjects, participated in this study. We collected ectopic endometriosis and endometrial samples. For the control, the sample was taken from endometrial tissue through pipelle biopsy. RNA was extracted from all tissues using RNA mini kit, and the expression was assessed using quantitative-real time PCR. Relative mRNA and miRNA expression were presented using the formula of the Livak method. The data were statistically analyzed using GraphPad Prism 8. The expression of Caspase-3, Survivin, Integrin β1 (ITGB1), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) (adhesion- and apoptosis-related gene) were calculated using the relative expression method. We found significant differences in Caspase-3, Survivin, ITGB1, MMP-9, and TIMP-1 expression between ectopic endometriosis tissues of women with endometriosis compared to healthy endometrium. MMP-9, Survivin, and ITGB1 was significantly increased in the endometriosis group, while Caspase-3, TIMP-1, and miR-183 were significantly reduced in the endometriosis group. No correlation was found between the expression level of miR-183 and Caspase3, Survivin, ITGB1, and Cadherin in both tissue types. Despite the difference in expression levels of miR-183 and associated adhesion- and apoptosis-related genes, there was no significant association between miR-183 with specific adhesion and apoptosis genes in endometriosis tissue.

Short-Term Exposure to Foodborne Xenoestrogens Affects Breast Cancer Cell Morphology and Motility Relevant for Metastatic Behavior In Vitro.

Breast cancer is highly susceptible to metastasis formation. During the time of disease progression, tumor pathophysiology can be impacted by endogenous factors, like hormonal status, as well as by environmental exposures, such as those related to diet and lifestyle. New lines of evidence point toward a potential role for foodborne endocrine disruptive chemicals in this respect; however, mechanistic understanding remains limited. At the molecular level, crucial steps toward metastasis formation include cell structural changes, alteration of adhesion, and reorganization of cytoskeletal proteins involved in motility. Hence, this study investigates the potential of dietary xenoestrogens to impact selected aspects of breast cancer cell mechanotransduction. Taking the onset of the metastatic cascade as a model, experiments focused on cell-matrix adhesion, single-cell migration, and adaptation of cell morphology. Dietary mycoestrogens alternariol (AOH, 1 μM) and α-zearalenol (α-ZEL, 10 nM), soy isoflavone genistein (GEN, 1 μM), and food packaging plasticizer bisphenol A (BPA, 10 nM) were applied as single compounds or in mixtures. Pursuing the hypothesis that endocrine active molecules could affect cell functions beyond the estrogen receptor-dependent cascade, experiments were performed comparing the MCF-7 cell line to the triple negative breast cancer cells MDA MB-231. Indeed, the four compounds functionally affected the motility and the adhesion of both cell types. These responses were coherent with rearrangements of the actin cytoskeleton and with the modulation of the expression of integrin β1 and cathepsin D. Mechanistically, molecular dynamics simulations confirmed a potential interaction with fragments of the α1 and β1 integrin subunits. In sum, dietary xenoestrogens proved effective in modifying the motility and adhesion of breast cancer cells, as predictive end points for metastatic behavior in vitro. These effects were measurable after short incubation times (1 or 8 h) and contribute to shed novel light on the activity of compounds with hormonal mimicry potential in breast cancer progression.

Integrin profle of circulating tumour cells in breast cancer patients

Background. Integrins, as adhesion molecules, play a key role in the interaction of cells with the basal membrane and intercellular matrix. Numerous studies demonstrate evidence of increased expression of integrins on tumor cells in different types of cancer. Thus, β3 and αV integrins are associated with stem-like features of tumor cells, and β4 integrin as α6β4 heterodimer provides anchorage-independent survival of malignant mammary epithelial cells. However, all the described functions of integrins have been investigated exclusively on primary tumor cells. The functional significance and expression pattern of integrins on circulating tumor cells (CTCs) remains unclear. The aim of the study was to evaluate the β3, β4 and αvβ5 integrin expression on CTCs and its association with molecular subtype, stage and lymph node metastasis in breast cancer patients Material and Methods. The study included 22 patients with T1–4N0–3M0 invasive ductal breast carcinoma. Venous blood was taken from patients without neoadjuvant chemotherapy (group 1) and after neoadjuvant chemotherapy (group 2) in the volume of 12 ml into vacuum tubes with EDTA. The expression of CTC integrins including stemness features CD44/CD24, CD133 and ALDH1, and epithelial-mesenchymal transition (EMT) (N-cadherin) was evaluated by flow cytometry. Results. CTCs with β3+β4-αvβ5- and β3-β4+αvβ5+ phenotypes and stemness properties were associated with larger tumor size (T4) in breast cancer patients. The β3 integrin expression was associated with more aggressive molecular subtypes of breast cancer. Administration of neoadjuvant chemotherapy did not affect the expression pattern of β3, β4 and αvβ5 integrins in CTCs. Conclusion. In breast cancer, most CTCs expressed β3, β4 and αvβ5 integrins despite the lack of attachment to the basal membrane and intercellular matrix. The expression of the above integrins on CTCs was associated with breast cancer molecular subtype, stage and lymph node metastasis, and therefore its evaluation can be considered as one of the objectives of liquid biopsy study.

Abstract 64: Smooth Muscle Cullin-3 Regulates the Renal Baroreceptor Mechanism: a Link Between CUL3 and Integrin β1

Cullin-3 (CUL3) is a critical component of the CUL3-Ring-Ligase (CRL) ubiquitin ligase complex. CUL3 mutants in smooth muscle cells (SMCs) cause vascular dysfunction, arterial stiffness, and severe hypertension (HTN). We hypothesized that conditional deletion of CUL3 in SMCs (S-CUL3KO) led to HTN via 1) a renin-angiotensin system (RAS)-dependent mechanism, and 2) impaired renal baroreceptor mechanism regulating renin expression. CUL3 Flox mice were bred to mice carrying tamoxifen-inducible CRE-recombinase under a smooth muscle promoter (ISM-CRE, control). S-CUL3KO mice exhibited augmented systolic blood pressure (SBP) (ISM-CRE: 124±1 vs. S-CUL3KO: 167±3 mmHg, p&lt;0.05) 3-weeks post tamoxifen. Once HTN was established we used an angiotensin-converting enzyme inhibitor (captopril, 10 mg/kg/day in drinking water) for 1 week to reverse HTN. We observed a greater depressor effect in S-CUL3KO (ΔSBP -49±3 mmHg) compared to ISM-CRE (ΔSBP -33±1 mmHg, p&lt;0.05). A similar effect was identified following angiotensin II type I receptor blocker (ARB, candesartan, 100 mg/kg/day in drinking water) treatment (ΔSBP, ISM-CRE: -27±2 vs. S-CUL3KO: -37±2, p&lt;0.05). We prevented HTN in S-CUL3KO by administering ARB before tamoxifen injection (SBP 120±5 mmHg). Mesenteric arteries exhibited augmented Ang-II induced vasoconstriction, which was prevented with ARB, suggesting that HTN in S-CUL3KO is RAS-dependent and AT 1 R-mediated. Despite severe HTN, S-CUL3KO did not suppress renin mRNA expression in kidneys. We hypothesize that S-CUL3KO exhibits a defective renal baroreceptor mechanism causing impaired suppression of renin expression. We propose that CUL3 participates in the degradation of integrin β1, the key mechanosensory of the renal baroreceptor, via Rab-mediated trafficking and lysosomal fusion. Supporting this, HEK293 cells lacking CUL3 exhibited lower integrin β1 protein expression (0.51±0.08 vs. 0.09±0.01 normalized RFU, p&lt;0.05) concomitant with increased Rab5 and Rab7 expression (3.6±0.3 vs. 1.9±0.1 and 0.95±0.12 vs. 0.37±0.04 normalized RFU). S-CUL3KO demonstrated severely impaired morphological distribution of integrin β1 protein in the juxtaglomerular apparatus. In conclusion, our findings support the hypothesis that 1) CUL3 regulates RAS via the renal baroreceptor, and 2) Rab proteins regulating integrin β1 expression are potential targets for the CRL3 pathway. Understanding CUL3 target proteins might contribute to the development of new strategies to treat HTN.

Iron-laden macrophage-mediated paracrine profibrotic signaling induces lung fibroblast activation.

Idiopathic pulmonary fibrosis (IPF) is a devastating condition characterized by progressive lung scarring and uncontrolled fibroblast proliferation, inevitably leading to organ dysfunction and mortality. Although elevated iron levels have been observed in patients and animal models of lung fibrosis, the mechanisms linking iron dysregulation to lung fibrosis pathogenesis, particularly the role of macrophages in orchestrating this process, remain poorly elucidated. Here we evaluate iron metabolism in macrophages during pulmonary fibrosis using both in vivo and in vitro approaches. In murine bleomycin- and amiodarone-induced pulmonary fibrosis models, we observed significant iron deposition and lipid peroxidation in pulmonary macrophages. Intriguingly, the ferroptosis regulator glutathione peroxidase 4 (GPX4) was upregulated in pulmonary macrophages following bleomycin instillation, a finding corroborated by single-cell RNA sequencing analysis. Moreover, macrophages isolated from fibrotic mouse lungs exhibited increased transforming growth factor (TGF)-β1 expression that correlated with lipid peroxidation. In vitro, iron overload in bone marrow-derived macrophages triggered lipid peroxidation and TGF-β1 upregulation, which was effectively suppressed by ferroptosis inhibitors. When cocultured with iron-overloaded macrophages, lung fibroblasts exhibited heightened activation, evidenced by increased α-smooth muscle actin and fibronectin expression. Importantly, this profibrotic effect was attenuated by treating macrophages with a ferroptosis inhibitor or blocking TGF-β receptor signaling in fibroblasts. Collectively, our study elucidates a novel mechanistic paradigm in which the accumulation of iron within macrophages initiates lipid peroxidation, thereby amplifying TGF-β1 production, subsequently instigating fibroblast activation through paracrine signaling. Thus, inhibiting iron overload and lipid peroxidation warrants further exploration as a strategy to suppress fibrotic stimulation by disease-associated macrophages. NEW & NOTEWORTHY This study investigates the role of iron in pulmonary fibrosis, specifically focusing on macrophage-mediated mechanisms. Iron accumulation in fibrotic lung macrophages triggers lipid peroxidation and an upregulation of transforming growth factor (TGF)-β1 expression. Coculturing iron-laden macrophages activates lung fibroblasts in a TGF-β1-dependent manner, which can be mitigated by ferroptosis inhibitors. These findings underscore the potential of targeting iron overload and lipid peroxidation as a promising strategy to alleviate fibrotic stimulation provoked by disease-associated macrophages.

Effects of Bushen Huoxue on integrin β3 and integrin β5 in the placental tissue of mice with repeated implantation failure caused by phospholipid antibodies.

This study aimed to investigate the effects of the Kidney-Tonifying and Blood-Activating Formula on combating the downregulation of integrin β3 and integrin β5 in mouse placental tissue induced by phospholipid antibodies. This study aimed to investigate the effects of kidney-tonifying and blood-activating formulations on combating the downregulation of integrin β3 and integrin β5 in mouse placental tissue induced by phospholipid antibodies. Mice in the phospholipid antibody group and phospholipid antibody + kidney-tonifying and blood-activating formula group underwent repeated implantation experiments, and the expression of integrin β3 and integrin β5 in placental tissue was observed. The effects of the kidney-tonifying and blood-activating formula on mouse placental tissue were evaluated through biochemical index tests, histopathological observations, and immunohistochemical staining. After intervention with the kidney-tonifying and blood-activating formula, the expression of integrin β3 and integrin β5 in placental tissue was notably upregulated, and placental structure was restored. Kidney-Tonifying and Blood-Activating Formula significantly improved abnormal biochemical indices and exerted a remarkable protective effect on placental tissue pathology. The kidney-tonifying and blood-activating formula effectively counteracted the downregulation of integrin β3 and integrin β5 in mouse placental tissue induced by phospholipid antibodies and alleviated placental tissue pathology through structural improvement and protection. Therefore, kidney-tonifying and blood-activating formulas may serve as potential therapeutic agents for recurrent implantation failure, offering new insights and approaches for clinical treatment.

Abstract We122: Suppression of defender against cell death 1 (Dad1) induces cardiomyocyte death by regulating cell adhesion proteins.

Introduction: Suppression of cardiomyocyte loss is considered a promising strategy for the prevention of the onset of heart failure (HF). Previously, defender against cell death 1 ( Dad1 ) was identified as a cytoprotective gene. Dad1 forms a complex with staurosporine and temperature sensitive 3A (Stt3A), a catalytic subunit of oligosaccharyltransferase (OST) complex which is responsible for N-glycosylation. However, the function of Dad1 in cardiomyocytes remains unknown. Hypothesis: The disruption of cell-extracellular matrix interactions results in cell death, known as anoikis. Because various cell adhesion proteins are N-glycosylated, we hypothesized that Dad1 suppressed anoikis by regulating N-glycosylation of cell adhesion proteins in cardiomyocytes. Methods: Neonatal rat cardiomyocytes were cultured and the expression of Dad1 or Stt3A was knocked down using siRNA. The protein expression was assessed with immunoblot analysis, and cell viability was evaluated using a CellTiter-Blue assay kit. Results: The suppression of Dad1 using siRNAs reduced cardiomyocyte viability ( p &lt;0.01, n=4). Interestingly, suppression of Dad1 impaired the N-glycosylation of integrin β1 and decreased the expression of mature integrin β1 ( p &lt;0.01, n=3). These changes inactivated integrin-mediated signal transduction through dephosphorylation of focal adhesion kinase ( p &lt;0.01, n=5). In addition, enhanced cell-extracellular matrix interactions by using adhesamine rescued the cardiomyocyte death by Dad1 knockdown ( p &lt;0.01, n=4). Next, we pursued the relationship between Dad1 and Stt3A. Dad1 knockdown decreased the expression of Stt3A ( p &lt;0.01, n=6). Suppression of Stt3A induced cardiomyocyte death accompanied by the changes of cell adhesion proteins ( p &lt;0.01, n=4). Finally, double knockdown of Dad1 and Stt3A did not induce a further reduction in cell viability compared with the knockdown of only Dad1 ( p &lt;0.05, n=4), suggesting that cardiomyocyte death by Dad1 knockdown was dependent on Stt3A. Conclusion: Dad1 is required for the stabilization of Stt3A and suppresses cardiomyocyte anoikis by regulating N-glycosylation of cell adhesion proteins in an OST-dependent manner. Dad1-mediated cardiomyocyte survival could be a therapeutic target against HF.

Postoperative radiotherapy with docetaxel versus cisplatin for high-risk oral squamous cell carcinoma: a randomized phase II trial with exploratory analysis of ITGB1 as a potential predictive biomarker

BackgroundOral squamous cell carcinoma (OSCC) causes significant mortality and morbidity worldwide. Surgical resection with adjuvant radiotherapy remains the standard treatment for locally advanced resectable OSCC. Results from landmark trials have established postoperative concurrent cisplatin-radiotherapy (Cis-RT) as the standard treatment for OSCC patients with high-risk pathologic features. However, cisplatin-related toxicity limits usage in clinical practice. Given the need for effective but less toxic alternatives, we previously conducted a single-arm trial showing favorable safety profiles and promising efficacy of concurrent docetaxel-radiotherapy (Doc-RT).MethodsIn this randomized phase 2 trial, we aimed to compare Doc-RT with the standard Cis-RT in postoperative OSCC patients. Eligible patients had AJCC stage III–IV resectable OSCC with high-risk pathologic features. Two hundred twenty-four patients were enrolled and randomly assigned to receive concurrent Doc-RT or Cis-RT. The primary endpoint was 2-year disease-free survival (DFS). Secondary endpoints included overall survival (OS), locoregional-free survival (LRFS), distant metastasis-free survival (DMFS), and adverse events (AEs). Integrin β1 (ITGB1) expression was analyzed as a biomarker for efficacy.ResultsAfter a median 28.8-month follow-up, 2-year DFS rates were 63.7% for Doc-RT arm and 56.1% for Cis-RT arm (p = 0.55). Meanwhile, Doc-RT demonstrated comparable efficacy to Cis-RT in OS, LRFS, and DMFS. Doc-RT resulted in fewer grade 3 or 4 hematological AEs. Low ITGB1 was associated with improved Doc-RT efficacy versus Cis-RT.ConclusionsThis randomized trial directly compared Doc-RT with Cis-RT for high-risk postoperative OSCC patients, with comparable efficacy and less toxicity. ITGB1 merits further validation as a predictive biomarker to identify OSCC patients most likely to benefit from Doc-RT. Findings indicate docetaxel may be considered as a concurrent chemoradiation option in this setting.Trial registrationwww.ClinicalTrials.gov. NCT02923258 (date of registration: October 4, 2016)

Effect of extracellular matrix stiffness on efficacy of Dapagliflozin for diabetic cardiomyopathy

BackgroundExtracellular matrix (ECM) stiffness is closely related to the progress of diabetic cardiomyopathy (DCM) and the response of treatment of DCM to anti-diabetic drugs. Dapagliflozin (Dapa) has been proven to have cardio-protective efficacy for diabetes and listed as the first-line drug to treat heart failure. But the regulatory relationship between ECM stiffness and treatment efficacy of Dapa remains elusive.Materials and methodsThis work investigated the effect of ECM stiffness on DCM progression and Dapa efficacy using both in vivo DCM rat model and in vitro myocardial cell model with high glucose injury. First, through DCM rat models with various levels of myocardial injury and administration with Dapa treatment for four weeks, the levels of myocardial injury, myocardial oxidative stress, expressions of AT1R (a mechanical signal protein) and the stiffness of myocardial tissues were obtained. Then for mimicking the stiffness of myocardial tissues at early and late stages of DCM, we constructed cell models through culturing H9c2 myocardial cells on the polyacrylamide gels with two stiffness and exposed to a high glucose level and without/with Dapa intervention. The cell viability, reactive oxygen species (ROS) levels and expressions of mechanical signal sensitive proteins were obtained.ResultsThe DCM progression is accompanied by the increased myocardial tissue stiffness, which can synergistically exacerbate myocardial cell injury with high glucose. Dapa can improve the ECM stiffness-induced DCM progression and its efficacy on DCM is more pronounced on the soft ECM, which is related to the regulation pathway of AT1R-FAK-NOX2. Besides, Dapa can inhibit the expression of the ECM-induced integrin β1, but without significant impact on piezo 1.ConclusionsOur study found the regulation and effect of biomechanics in the DCM progression and on the Dapa efficacy on DCM, providing the new insights for the DCM treatment. Additionally, our work showed the better clinical prognosis of DCM under early Dapa intervention.

Pec 1 of Pseudomonas aeruginosa Inhibits Bacterial Clearance of Host by Blocking Autophagy in Macrophages.

Pseudomonas aeruginosa (P. aeruginosa), a common opportunistic pathogen, is highly prone to chronic infection and is almost impossible to eradicate, especially attributed to virulence factors and adaptive mutations. In the present study, pseudomonas effector candidate 1 (Pec 1), a novel virulence factor of P. aeruginosa, was investigated, which inhibited bacterial clearance by the host and aggravated lung injury. Further, it demonstrated that Pec 1 inhibited miR-155 via suppressing integrin β3 expression, thereby activating PI3K-AKT-mTOR and inhibiting autophagy in macrophages. Additionally, the identification of Pec 1 in sputum was related to the bacterial load and assisted in rapid diagnosis of P. aeruginosa infection. This finding underlined the importance of Pec 1 in the pathogenesis of P. aeruginosa infection and indicated that Pec 1 could be a vital independent virulence factor during chronic infection with P. aeruginosa, providing new insights in rapid diagnosis, therapeutic targets, and vaccine antigens of P. aeruginosa infection.

Thymosin β4 and β10 Expression in Human Organs during Development: A Review.

This review summarizes the results of a series of studies performed by our group with the aim to define the expression levels of thymosin β4 and thymosin β10 over time, starting from fetal development to different ages after birth, in different human organs and tissues. The first section describes the proteomics investigations performed on whole saliva from preterm newborns and gingival crevicular fluid, which revealed to us the importance of these acidic peptides and their multiple functions. These findings inspired us to start an in-depth investigation mainly based on immunochemistry to establish the distribution of thymosin β4 and thymosin β10 in different organs from adults and fetuses at different ages (after autopsy), and therefore to obtain suggestions on the functions of β-thymosins in health and disease. The functions of β-thymosins emerging from these studies, for instance, those performed during carcinogenesis, add significant details that could help to resolve the nowadays so-called "β-thymosin enigma", i.e., the potential molecular role played by these two pleiotropic peptides during human development.

Downregulation of TGF-β1 in fibro-adipogenic progenitors initiates muscle ectopic mineralization.

In previous studies, we have demonstrated that stress response-induced high glucocorticoid levels could be the underlying cause of traumatic heterotopic ossification (HO), and we have developed a glucocorticoid-induced ectopic mineralization (EM) mouse model by systemic administration of a high dose of dexamethasone (DEX) to animals with muscle injury induced by cardiotoxin injection. In this model, dystrophic calcification (DC) developed into HO in a cell autonomous manner. However, it is not clear how DC is formed after DEX treatment. Therefore, in this study, we aimed to explore how glucocorticoids initiate muscle EM at a cellular and molecular level. We showed that DEX treatment inhibited inflammatory cell infiltration into injured muscle but inflammatory cytokine production in the muscle was significantly increased, suggesting that other non-inflammatory muscle cell types may regulate the inflammatory response and the muscle repair process. Accompanying this phenotype, transforming growth factor β1 (TGF-β1) expression in fibro-adipogenic progenitors (FAPs) was greatly downregulated. Since TGF-β1 is a strong immune suppressor and FAP's regulatory role has a large impact on muscle repair, we hypothesized that downregulation of TGF-β1 in FAPs after DEX treatment resulted in this hyperinflammatory state and subsequent failed muscle repair and EM formation. To test our hypothesis, we utilized a transgenic mouse model to specifically knockout Tgfb1 gene in PDGFRα-positive FAPs to investigate if the transgenic mice could recapitulate the phenotype that was induced by DEX treatment. Our results showed that the transgenic mice completely phenocopied this hyperinflammatory state and spontaneously developed EM following muscle injury. On the contrary, therapeutics that enhanced TGF-β1 signaling in FAPs inhibited the inflammatory response and attenuated muscle EM. In summary, these results indicate that FAPs-derived TGF-β1 is a key molecule in regulating muscle inflammatory response and subsequent EM, and that glucocorticoids exert their effect via downregulating TGF-β1 in FAPs.

β2-adrenergic receptor activation decreases the mechanical sensitivity of rat masticatory muscle afferent fibres.

Activation of β2 adrenergic receptors reduces cutaneous mechanical pain thresholds in rats. While β2 adrenergic receptor activation may contribute to mechanisms that underlie temporomandibular joint pain, its effect on masticatory muscle pain sensitivity is uncertain. The current study sought to determine the extent to which β adrenergic receptors are expressed by masticatory muscle afferent fibres, and to assess the effect of local activation of these receptors on the mechanical sensitivity of masticatory muscle afferent fibres in rats. Trigeminal ganglion neurons that innervate the rat (n = 12) masseter muscle and lower lip were identified by tissue injection of fluorescent dyes and were then stained with antibodies against β1 or β2 adrenergic receptors. Extracellular recordings from 60 trigeminal ganglion neurons that innervate the masticatory muscle were undertaken in a second group of anaesthetised rats of both sexes (n = 37) to assess afferent mechanical activation thresholds. Thresholds were assessed before and after injection of the β adrenergic receptor agonists into masticatory muscle. β1 and β2 adrenergic receptor expression was greater in labial skin than in masticatory muscle ganglion neurons (p < .05, one-way ANOVA, Holm-Sidak test). There was a higher expression of β2 adrenergic receptors in masticatory muscle ganglion neurons in males than in females. The mixed β agonist isoproterenol increased afferent mechanical activation threshold in male but not female rats (p < .05, Mann-Whitney test). In male rats, salbutamol, a β2 selective agonist, also increased afferent mechanical activation threshold but hydralazine, a vasodilator, did not (p < .05, Mann-Whitney test). Activation of β2 adrenergic receptors decreases the mechanical sensitivity of masticatory muscle afferent fibres in a sex-related manner.

Region and layer-specific expression of GABAA receptor isoforms and KCC2 in developing cortex.

γ-Aminobutyric acid (GABA) type A receptors (GABAARs) are ligand-gated Cl-channels that mediate the bulk of inhibitory neurotransmission in the mature CNS and are targets of many drugs. During cortical development, GABAAR-mediated signals are significantly modulated by changing subunit composition and expression of Cl-transporters as part of developmental processes and early network activity. To date, this developmental evolution has remained understudied, particularly at the level of cortical layer-specific changes. In this study, we characterized the expression of nine major GABAAR subunits and K-Cl transporter 2 (KCC2) in mouse somatosensory cortex from embryonic development to postweaning maturity. We evaluated expression of α1-5, β2-3, γ2, and δ GABAAR subunits using immunohistochemistry and Western blot techniques, and expression of KCC2 using immunohistochemistry in cortices from E13.5 to P25 mice. We found that embryonic cortex expresses mainly α3, α5, β3, and γ2, while expression of α1, α2, α4, β2, δ, and KCC2 begins at later points in development; however, many patterns of nuanced expression can be found in specific lamina, cortical regions, and cells and structures. While the general pattern of expression of each subunit and KCC2 is similar to previous studies, we found a number of unique temporal, regional, and laminar patterns that were previously unknown. These findings provide much needed knowledge of the intricate developmental evolution in GABAAR composition and KCC2 expression to accommodate developmental signals that transition to mature neurotransmission.

Modified Hongteng Baijiang decoction enema improves sequelae of pelvic inflammatory disease by regulating the LIF/JAK2/STAT3 pathway and gut microbiota.

The sequelae of pelvic inflammatory disease (SPID) are major causes of secondary infertility. Modified Hongteng Baijiang decoction (MHTBD) has produced positive results in the treatment of patients with chronic pelvic inflammatory disease; however, its role in SPID remains elusive. Therefore, this study clarified the role of MHTBD in SPID pathogenesis. The main components in MHTBD were analyzed by using liquid chromatography‒mass spectrometry (LC/MS). An SPID rat model was established, and the rats were treated with different doses of MHTBD (0.504 g of raw drug/kg, 1.008 g of raw drug/kg, and 2.016 g of raw drug/kg). Endometrial pinopodes were observed via scanning electron microscopy, endometrial thickness and inflammatory cell infiltration were assessed via HE staining, and the expression of estrogen receptor (ER), progesterone receptor (PR), integrin β3 (ITGB3), and CD31 in the endometrium was detected by using immunohistochemistry. Western blot analysis was used to detect the protein expression of LIF, JAK2, p-JAK2, STAT3, and p-STAT3 in the endometrium. Moreover, the changes in the gut microbiota were analyzed via 16S rRNA sequencing. MHTBD improved endometrial receptivity, attenuated endometrial pathologic damage, reduced inflammatory cell infiltration, decreased ER and PR expression in the endometrium, and promoted the expression of LIF, p-JAK2, and p-STAT3 in the endometrium (p < .05) in SPID rats. Additionally, MHTBD treatment affected the composition of the gut microbiota in SPID rats. Furthermore, MHTBD attenuated endometrial receptivity and pathological damage in SPID rats by promoting the LIF/JAK2/STAT3 pathway. MHTBD attenuates SPID in rats by promoting the LIF/JAK2/STAT3 pathway and improving the composition of the gut microbiota. MHTBD may be a valuable drug for SPID therapy.

#2376 Tubular exosomes in renal ECM microenvironment promotes fibrosis progression by activating latent TGF-β1

Abstract Background and Aims Chronic kidney disease (CKD) is characterized with progressive fibrosis caused by excessive extracellular matrix (ECM) deposition. Tubular epithelial cells secreted increasing exosomes into extracellular space under pathological conditions with integrin as the prominent constituent membrane cargo. Due to the critical role of integrin in activation of TGF-β1 signalling, this study aims to investigate the performance of tubular-derived exosomes in the ECM microenvironment, exploring the potential mechanism of exosomal integrin mediated activation of latent TGF-β1. Method A fibrosis mouse model was established with unilateral ureteral obstruction (UUO) and an ITGβ6 overexpression cell line were established to observe the effect of ITGβ6+exosomes in vitro. Elastic or stress-relaxing hydrogels based on Polymethyl Methacrylate (PMMA) and acrylic acid-WGG(KA)3-heparin were constructed to mimic the in situ ECM under normal and fibrotic conditions with different stiffness (2kPa, 50kPa). Exosomes in hydrogels were tracked by single particle tracking to investigate their mobility and retention capability. Purified exosomal integrin β6 and inhibitor of exosome secretion, GW4869 was applied to clarify the role of exosomal integrin in latent TGF-β1 activation. Results In UUO model, immunofluorescence staining showed that latent TGF-β1 localized to ECM space and stored in the renal tubular interstitial which was activated with fibrotic progression. Additionally, we observed integrin β6 expression in tubule or interstitial increased with fibrotic progression. Isolated exosomes derived from cultured tubule cells under serum-free condition or renal tissues of UUO mice showed higher proportion of integrin β6+ subpopulation, as demonstrated via single exosome analysis by Nanoimaging (Oxford) . Interestingly, these exosomes diffused through hydrogels, while maintaining their original morphology and particle size. It turned out that stress-relaxing hydrogels with lower stiffness exhibited better retention of exosomes. Then, transwell experiment and single-particle tracking both revealed increased exosome diffusion rate in ECM with higher stiffness. Due to the critical role of integrin in the activation of latent TGFβ, we propose integrin β6+ exosome may exert such effect in ECM microenvironment. Impressively, purified ITGB6+ exosomes significantly increased TGF-β1 activation in TGF-β1 overexpressed HK-2 cells, while inhibiting exosome secretion via GW4869 treatment reversed such activation remarkably. Conclusion We demonstrated that tubular epithelial cell derived exosomal integrin β6 diffused in ECM space and promoted the activation of latent TGF-β1. Higher ECM stiffness correlates with accelerated movement of exosome which enhanced TGFβ1 activation. Exosomal integrin β6 may represent a novel manner of TGF-β1 activation with signal-spreading potential which may become a new therapeutic target for suppression of TGF-β1 activation in CKD treatment.