TGF-β1 Expression Research Articles

Transforming growth factor-β1 and vascular endothelial growth factor levels in senile acute myeloid leukemia and correlation with prognosis.

Acute myeloid leukemia (AML) is a disease in which immature hematopoietic cells accumulate in the bone marrow and continuously expand, inhibiting hematopoiesis. The treatment and prognosis of this disease have always been unsatisfactory. To investigate the correlation between vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGFβ1) expression and prognosis in older adults with AML. This study enrolled 80 patients with AML (AML group), including 36 with complete response (AML-CR), 23 with partial response (AML-PR), and 21 with no response (AML-NR). The expression levels of VEGF and TGFβ1 were detected by reverse transcription polymerase chain reaction in bone marrow mononuclear cells isolated from 56 healthy controls. Kaplan-Meier analysis was performed to assess overall survival (OS) and progression- or disease-free survival (DFS). Prognostic risk factors were analyzed using a Cox proportional hazards model. The AML group showed a VEGF level of 2.68 ± 0.16. VEGF expression was lower in patients with AML-CR than those with AML-PR or AML-NR (P < 0.05). TGFβ1 expression in the AML group was 0.33 ± 0.05. Patients with AML-CR showed a higher TGFβ1 expression than those with AML-PR or AML-NR (P < 0.05). VEGF and TGFβ1 expression in patients with AML was significantly correlated with the counts of leukocytes, platelets, hemoglobin, and peripheral blood immature cells (P < 0.05); Kaplan-Meier survival analysis revealed that patients with high TGFβ1 expression had better OS and DFS than those with low TGFβ1 expression (P < 0.05), whereas patients with low VEGF levels showed better OS and DFS than those with high VEGF levels (P < 0.05). VEGF, TGFβ1, and platelet count were identified by the Cox proportional hazards model as independent risk factors for OS (P < 0.05), while VEGF, TGFβ1, and white blood cell count were independent risk factors for DFS (P < 0.05). Decreased VEGF expression and increased TGFβ1 expression in patients with AML provide valuable references for determining and individualizing clinical treatment strategies.

High Glucose Increases Lactate and Induces the Transforming Growth Factor Beta-Smad 1/5 Atherogenic Pathway in Primary Human Macrophages.

Hundreds of millions of people worldwide are expected to suffer from diabetes mellitus. Diabetes is characterized as a dynamic and heterogeneous disease that requires deeper understanding of the pathophysiology, genetics, and metabolic shaping of this disease and its macro/microvascular complications. Macrophages play an essential role in regulating local immune responses, tissue homeostasis, and disease pathogenesis. Here, we have analyzed transforming growth factor beta 1 (TGFβ1)/Smad signaling in primary human macrophages grown in normal (NG) and high-glucose (HG; +25 mM glucose) conditions. Cell culture lactate concentration and cellular phosphofructokinase (PFK) activity were increased in HG concentrations. High glucose levels in the growth media led to increased macrophage mRNA expression of TGFβ1, and TGFβ-regulated HAMP and PLAUR mRNA levels, while the expression of TGFβ receptor II remained unchanged. Stimulation of cells with TGFβ1 protein lead to Smad2 phosphorylation in both NG and HG conditions, while the phosphorylation of Smad1/5 was detected only in response to TGFβ1 stimulation in HG conditions. The use of the specific Alk1/2 inhibitor dorsomorphin and the Alk5 inhibitor SB431542, respectively, revealed that HG conditions led TGFβ1 to activation of Smad1/5 signaling and its downstream target genes. Thus, high-glucose activates TGFβ1 signaling to the Smad1/5 pathway in primary human macrophages, which may contribute to cellular homeostasis in a harmful manner, priming the tissues for diabetic complications.

The Role of Notch1 Pathway Regulating CD4+CD25+Foxp3+ T Cells in Renovascular Hypertension

Abstract OBJECTIVE To explore the role and mechanism of the Notch1 pathway in regulating CD4+CD25+Foxp3+ Treg cells in renal vascular hypertension (RVH). METHODS According to the diagnostic criteria of RVH in the 8th edition of Internal Medicine, 220 patients treated in the Department of Nephrology of Sichuan Provincial People’s Hospital from January 2019 to January 2022 were enrolled in RVH group, and 220 patients with essential hypertension (EH) in the same period were selected as EH group; another 220 healthy examinees were selected as control group (NC). The proportion of CD4+CD25+ Treg cells in peripheral blood mononuclear cells (PBMCs) and the expression levels of interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and cell proliferation nuclear antigen-67 (Ki-67) in PBMC of the 3 groups were compared by real-time fluorescence quantitative PCR and western blotting; the relative expression levels of Notch1 receptor and ligand mRNA in PBMC of the 3 groups were compared by real-time fluorescence quantitative PCR; the expression of downstream proteins Hes1 and Hes5 of Notch1 signaling pathway, the proportion of CD4+CD25+Foxp3+ Treg cells, and the effect on TGF-β expression were compared by western blotting after adding Notch1 signaling pathway inhibitor (DAPT). Real-time quantitative PCR and western PCR were used. The proportion of CD4+CD25+ Treg cells in PBMCs and the expression levels of IL-10, TGF-β, and cell proliferating nuclear antigen-67 (Ki-67) in PBMC were compared between the 2 groups. Real-time quantitative PCR was used to compare the mRNA expression levels of the Notch1 receptor and ligand in PBMC between the 2 groups. RESULTS Flow cytometry results showed that compared with the NC group, the RVH group had a significantly lower proportion of CD4+CD25+Foxp3+ Treg cells, TGF-β and Ki-67 expression levels, and higher IL-10 expression levels, with significant differences (P &amp;lt; 0.001). Compared with the RVH group, the EH group had a significantly higher proportion of CD4+CD25+Foxp3+ Treg cells, TGF-β and Ki-67 expression levels, and lower IL-10 expression levels, with significant differences (P &amp;lt; 0.001). Real-time quantitative PCR results showed that the RVH group also had significantly higher relative expression levels of Notch receptor Notch1 mRNA, Notch ligand DLL1, DLL4, Jagged1, and Jagged2 mRNA in PBMC than the NC group and EH group, with significant differences (P &amp;lt; 0.001). Western blot results showed that after adding Notch1 signaling pathway inhibitor (DAPT), Hes1 and Hes5 expression in peripheral CD4+CD25+Foxp3+ Treg cells of patients decreased significantly (P &amp;lt; 0.001), while CD4+CD25+Foxp3+ Treg proportion and TGF-β expression level increased significantly (P &amp;lt; 0.001). CONCLUSIONS The Notch1 signaling pathway plays an immunoregulatory role in RVH regulated by CD4+CD25+Foxp3+ Treg cells. Regulation of the Notch1 signaling pathway can significantly promote the proliferation of CD4+CD25+Foxp3+ Treg cells and the ability to secrete anti-inflammatory factors.

TGFβ signaling pathway is altered by HLA-B27 expression, resulting in pathogenic consequences relevant for spondyloarthritis

BackgroundAssociation of HLA-B27 with spondyloarthritis (SpA) has been known for 50 years, but still remains unexplained. We recently showed that HLA-B27 expressed in wing imaginal disc from HLA-B27/human-β2 microglobulin (hβ2m) transgenic Drosophila deregulated bone morphogenetic protein (BMP) pathway by interacting physically with type I BMP receptor (BMPR1) Saxophone (Sax), leading to crossveinless phenotype.MethodsGenetic interaction was studied between activin/transforming growth factor β (TGFβ) pathway and HLA-B27/hβ2m in transgenic Drosophila wings. The HLA-B27-bound peptidome was characterized in wing imaginal discs. In mesenteric lymph node (mLN) T cells from HLA-B27/hβ2m rat (B27 rat), physical interaction between HLA-B27 and activin receptor-like kinase-2 (ALK2), ALK3 and ALK5 BMPR1s, phosphorylation of small mothers against decapentaplegic (SMADs) and proteins of the non-canonical BMP/TGFβ pathways induced by its ligands, and the transcript level of target genes of the TGFβ pathway, were evaluated.ResultsIn HLA-B27/hβ2m transgenic Drosophila, inappropriate signalling through the activin/TGFβ pathway, involving Baboon (Babo), the type I activin/TGFβ receptor, contributed to the crossveinless phenotype, in addition to deregulated BMP pathway. We identified peptides bound to HLA-B27 with the canonical binding motif in HLA-B27/hβ2m transgenic Drosophila wing imaginal disc. We demonstrated specific physical interaction, between HLA-B27/hβ2m and mammalian orthologs of Sax and Babo, i.e. ALK2 and ALK5 (i.e. TGFβ receptor I), in the mLN cells from B27 rat. The magnitude of phosphorylation of SMAD2/3 in response to TGFβ1 was increased in T cells from B27 rats, showing evidence for deregulated TGFβ pathway. Accordingly, expression of several target genes of the pathway was increased in T cells from B27 rats, in basal conditions and/or after TGFβ exposure, including Foxp3, Rorc, Runx1 and Maf. Interestingly, Tgfb1 expression was reduced in naive T cells from B27 rats, even premorbid, an observation consistent with a pro-inflammatory pattern.ConclusionsThis study shows that HLA-B27 alters the TGFβ pathways in Drosophila and B27 rat. Given the importance of this pathway in CD4 + T cells differentiation and regulation, its disturbance could contribute to the abnormal expansion of pro-inflammatory T helper 17 cells and altered regulatory T cell phenotype observed in B27 rats.

Therapeutic Effect of Melatonin on CCl4-Induced Fibrotic Liver Model by Modulating Oxidative Stress, Inflammation, and TGF-β1 Signaling Pathway in Pinealectomized Rats.

The study aimed to determine the CCl4-induced liver fibrosis model in pinealectomized rats and biochemically, immunohistochemically, and histopathologically investigate the therapeutic effect of melatonin on liver fibrosis. The surgical procedure for pinealectomy was performed at the beginning of the study, and the sham and pinealectomized rats were administered CCl4 dissolved in corn oil (1:1) alone every other day to induce liver fibrosis or together with melatonin (10 mg/kg) therapy for 15 days. Melatonin is an essential therapeutic agent and offers an alternative therapeutic strategy in CCl4-induced liver fibrosis by suppressing inflammation, oxidative stress, and the TGF-β1 signaling pathway. Treatment with melatonin ameliorated CCl4-induced liver fibrosis by restoring hepatocellular damage and reducing plasma AST, ALT, and ALP values. Melatonin increases the activity of SOD and CAT, which are important enzymes for antioxidant defence, and raises GSH levels, which further enhances antioxidant function. Also, melatonin reduced hepatic inflammation (IL-6 and IL-1β) and oxidative stress indices. Moreover, histopathological changes and immunohistochemical expression of TGF-β1 were restored following melatonin supplementation in the CCl4-induced liver fibrosis model in pinealectomized rats. Our study shows that melatonin supplementation has a beneficial effect in protecting the liver fibrosis induced by CCl4 in pinealectomized rats.

EGFR mutations induce the suppression of CD8+ T cell and anti-PD-1 resistance via ERK1/2-p90RSK-TGF-β axis in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) patients with EGFR mutations exhibit an unfavorable response to immune checkpoint inhibitor (ICI) monotherapy, and their tumor microenvironment (TME) is usually immunosuppressed. TGF-β plays an important role in immunosuppression; however, the effects of TGF-β on the TME and the efficacy of anti-PD-1 immunotherapy against EGFR-mutated tumors remain unclear.MethodsCorresponding in vitro studies used the TCGA database, clinical specimens, and self-constructed mouse cell lines with EGFR mutations. We utilized C57BL/6N and humanized M-NSG mouse models bearing EGFR-mutated NSCLC to investigate the effects of TGF-β on the TME and the combined efficacy of TGF-β blockade and anti-PD-1 therapy. The changes in immune cells were monitored by flow cytometry. The correlation between TGF-β and immunotherapy outcomes of EGFR-mutated NSCLC was verified by clinical samples.ResultsWe identified that TGF-β was upregulated in EGFR-mutated NSCLC by EGFR activation and subsequent ERK1/2-p90RSK phosphorylation. TGF-β directly inhibited CD8+ T cell infiltration, proliferation, and cytotoxicity both in vitro and in vivo, but blocking TGF-β did not suppress the growth of EGFR-mutated tumors in vivo. Anti-TGF-β antibody combined with anti-PD-1 antibody significantly inhibited the proliferation of recombinant EGFR-mutated tumors in C57BL/6N mice, which was superior to their monotherapy. Mechanistically, the combination of anti-TGF-β and anti-PD-1 antibodies significantly increased the infiltration of CD8+ T cells and enhanced the anti-tumor function of CD8+ T cells. Moreover, we found that the expression of TGF-β1 in EGFR-TKI resistant cell lines was significantly higher than that in parental cell lines. The combination of anti-TGF-β and nivolumab significantly inhibited the proliferation of EGFR-TKI resistant tumors in humanized M-NSG mice and prolonged their survival.ConclusionsOur results reveal that TGF-β expression is upregulated in NSCLC with EGFR mutations through the EGFR-ERK1/2-p90RSK signaling pathway. High TGF-β expression inhibits the infiltration and anti-tumor function of CD8+ T cells, contributing to the “cold” TME of EGFR-mutated tumors. Blocking TGF-β can reshape the TME and enhance the therapeutic efficacy of anti-PD-1 in EGFR-mutated tumors, which provides a potential combination immunotherapy strategy for advanced NSCLC patients with EGFR mutations.Graphical

Therapeutic Effect of Lecigel, Cetiol®CC, Activonol-6, Activonol-M, 1,3-Propanediol, Soline, and Fucocert® (LCAA-PSF) Treatment on Imiquimod-Induced Psoriasis-like Skin in Mice.

The individual ingredients of 1,3-Propanediol, Soline, and Fucocert® (PSF) are often used as cosmetic formulations in skin care. In addition, the mixture of Lecigel, Cetiol®CC, Activonol-6, and Activonol-M (LCAA) is often used as a cosmetic base. However, whether the combination of LCAA with PSF (LCAA-PSF) exerts a therapeutic effect on psoriasis remains unclear. In this study, mice induced with imiquimod (IMQ) were divided into three groups and administered 100 mg/day of LCAA, 100 mg/day of LCAA-PSF, or Vaseline on the dorsal skin of each mouse. Weight-matched mice treated with Vaseline alone were used as controls. Hematoxylin and eosin (H&E) staining and enzyme-linked immunosorbent assay(ELISA) were used to assess tissue morphology and inflammatory cytokines. RNA sequencing analysis was used to predict the mechanism underlying the action of LCAA-PSF against psoriasis, while immunohistochemical analysis validation was used to identify pertinent molecular pathways. The results demonstrated that LCAA-PSF alleviated IMQ-induced keratinocyte differentiation/ proliferation bydecreasingthe serum levels of inflammatory cytokines such as IL-6, TNF-α, IL-23, and IL-17A and the epidermisof TGFβ, Ki67, CK5/6, and VEGF expression, which is associated with angiogenesis and keratinocyte differentiation/ proliferation. These findings highlight the antipsoriatic activity of LCAA-PSF in a psoriasis-like mouse model and suggest this may occurvia the inhibition of inflammatory factor secretionand the TGFβ-related signal pathway.

The integrin receptor beta7 subunit mediates airway remodeling and hyperresponsiveness in allergen exposed mice

BackgroundFibroblast differentiation to a myofibroblast phenotype is a feature of airway remodeling in asthma. Lung fibroblasts express the integrin receptor α4β7 and fibronectin induces myofibroblast differentiation via this receptor.ObjectivesTo investigate the role of the β7 integrin receptor subunit and α4β7 integrin complex in airway remodeling and airway hyperresponsiveness (AHR) in a murine model of chronic allergen exposure.MethodsC57BL/6 wild type (WT) and β7 integrin null mice (β7 -/-) were sensitized (days 1,10) and challenged with ovalbumin (OVA) three times a week for one or 4 weeks. Similar experiments were performed with WT mice in the presence or absence of α4β7 blocking antibodies. Bronchoalveolar (BAL) cell counts, AHR, histological evaluation, soluble collagen content, Transforming growth factor-β (TGFβ) and Interleukin-13 (IL13) were measured. Phenotype of fibroblasts cultured from WT and β7 -/- saline (SAL) and OVA treated mice was evaluated.ResultsEosinophil numbers were similar in WT vs β7-/- mice. Prolonged OVA exposure in β7-/- mice was associated with reduced AHR, lung collagen content, peribronchial smooth muscle, lung tissue TGFβ and IL13 expression as compared to WT. Similar findings were observed in WT mice treated with α4β7 blocking antibodies. Fibroblast migration was enhanced in response to OVA in WT but not β7 -/- fibroblasts. α-SMA and fibronectin expression were reduced in β7-/- fibroblasts relative to WT.ConclusionsThe β7 integrin subunit and the α4β7 integrin complex modulate AHR and airway remodeling in a murine model of allergen exposure. This effect is, at least in part, explained by inhibition of fibroblast activation and is independent of eosinophilic inflammation.

Extracts from Artemisia annua Alleviates Myocardial Remodeling through TGF-β1/Smad2/3 Pathway and NLRP3 Inflammasome

Artemisinin and its derivatives, the well-known anti-malarial drugs extracted from traditional Chinese medicine, Artemisia annua, have been implicated in treating fibrotic diseases. However, whether artemisinin affects cardiac fibrosis in the pathogenesis of heart failure is still unknown. This study aimed to evaluate the possible effects of artemisinin on cardiac function and myocardial fibrosis in the heart failure model and to explore the underlying mechanisms. Isoproterenol was injected subcutaneously for induction of the cardiac fibrosis model. Proteomic analysis was performed after 4 four weeks of artemisinin treatment. Echocardiography was used to evaluate cardiac function and structure. Hematoxylin and eosin (H&E) staining, as well as Masson trichrome staining, were performed for histopathology. The α-SMA, collagen I, and III expression in the myocardium was detected by immunohistochemical staining. The ratio of heart weight to body weight (HW/BW, mg/kg) and the ratio of heart weight to tibia length (HW/TL, mg/mm) were calculated as indicators for cardiac remodeling. Brain natriuretic peptide (BNP) levels were quantified in rat plasma using enzymelinked immunosorbent assay (ELISA). In contrast, the protein levels of TGF-β1, p-Smad2/3, and Smad2/3 were assessed in the myocardium and fibroblasts via western blot analysis. RT-qPCR was performed to analysis the expression of Col-I, Col-III, α-SMA, NLRP3, Caspase-1, IL-1β, and IL-18. Proteomic analysis identified 227 differentially expressed proteins (DEPs), including 119 upregulated and 108 downregulated proteins. These proteins were identified as the core proteins targeted by artemisinin for improving myocardial remodeling. GO annotation of the DEPs indicated that the DEPs were mainly associated with biological processes such as inflammation regulation. In the in vivo study of an isoproterenol-induced rat cardiac remodeling model, we found that artemisinin administration significantly ameliorated cardiac dysfunction and reduced collagen production by suppressing TGFβ-1/Smads signaling and inhibiting NLRP3 inflammasome activation. As manifested by downregulating the expression of α-SMA, Col-I, and Col-III, NLRP3, IL-1β, IL-18, Caspase-1 mRNA, and TGF-β1, p-SMAD 2/3 protein in the myocardium. Similar beneficial effects of artemisinin were consistently observed in TGF-β1 treated primary cardiac fibroblasts. Extracts from Artemisia annua relieves myocardial remodeling through TGF-β1/Smad2/3 pathway and NLRP3 inflammasome

Cellular In Vitro Responses Induced by Human Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Obtained from Suspension Culture.

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-β and IL-1β was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

The Effect of Photobiomodulation and Akkermansia muciniphila on THP-1 Derived Macrophage Polarization Treated with Gliadin Peptide.

Introduction: Photobiomodulation (PBM) and Akkermansia muciniphila have been shown to be effective in improving inflammatory conditions with positive effects on increasing the population of anti-inflammatory M2 macrophages (MQs). In this study, gliadin-stimulated THP-1 derived MQs were treated with A. muciniphila and PBM to evaluate their effects on promoting the polarization of M2 MQs. Methods: The human monocyte cell line (THP-1) was differentiated to MQs. MQs were stimulated with 200 μg/mL gliadin for 24 hours and then treated with PBM 810 nm alone and in combination with A. muciniphila for the following 24 hours to evaluate their effects on MQs polarization. THP-1 derived MQs were also treated with PBM and A. muciniphila to evaluate their effects on non-stimulated MQs. CD11b, CD80, and CD206 levels were evaluated by using the flow cytometry technique. Moreover, the expression of some M1 and M2-related cytokines was determined. Results: PBM therapy of gliadin-stimulated MQs decreased IL-6 and increased TGF-β, IL-10 and TNF-α expression compared with gliadin exposed MQs. PBM along with A. muciniphila treatment induced IL-6, TNF-α, and IL-10 expression in MQs in comparison to the untreated group. It also elevated TGF-β, IL-10 and TNF-α levels in gliadin-triggered MQs in comparison to gliadin-stimulated MQ cells. Conclusion: The result of this study showed the potential of PBMT and A. muciniphila for modulating inflammatory responses and MQs polarization. This may open new perspectives to find possible therapeutic targets for celiac diseases.

Pro-fibrotic effect of the susceptible gene PCSK5 in vascular fibrosis of Takayasu arteritis via TGF-β and SMAD3 signaling pathway activation

BackgroundVascular fibrosis directly causes vascular thickening in Takayasu arteritis (TAK), in which sustained transforming growth factor beta (TGF-β) activation is critical. Understanding TGF-β activation regulation and blocking it might yield a therapeutic effect in TAK. Proprotein convertase subtilisin/kexin type 5 (PCSK5) rs6560480 (T/C) is associated with TAK development. In this study, we assessed the association between the PCSK5 rs6560480 genotype and PCSK5 expression in TAK and explored its molecular role in TGF-β activation and vascular fibrosis development. MethodsIn TAK patients, PCSK5 and TGF-β expression in plasma and aortic tissue was examined by ELISA and immunohistochemical staining, and PCSK5 rs6560480 was genotyped. The correlation between PCSK5 and extracellular matrix (ECM) expression was examined by Western blotting (WB) and immunohistochemistry staining. Detection by co-immunoprecipitation was performed to detect the interaction between PCSK5 and TGF-β in adventitial fibroblasts (AAFs). Downstream signaling pathways were detected by WB and validated with appropriate inhibitors. Potential immunosuppressive agents to inhibit the effects of PCSK5 were explored in cell culture and TAK patients. ResultsPatients with PCSK5 rs6560480 TT patients had significantly higher PCSK5 levels and more thickened vascular lesions than patients with PCSK5 rs6560480 CT. PCSK5 expression was significantly increased in alpha smooth muscle actin (α-SMA)-positive myofibroblasts in TAK vascular lesions. Overexpressing PCSK5 facilitated TGF-β and downstream SMAD2/3 activation and ECM expression in AAFs and aorta in in-vitro culture. The mechanistic study supported that PCSK5 activated precursor TGF-β (pro-TGF-β) to the mature form by binding the pro-TGF-β cleavage site. Leflunomide inhibited PCSK5 and pro-TGF-β binding, decreasing TGF-β activation and ECM expression, which was also partially validated in leflunomide-treated patients. ConclusionThe findings revealed a novel pro-fibrotic mechanism of PCSK5 in TAK vascular fibrosis via TGF-β and downstream SMAD2/3 pathway activation. Leflunomide might be anti-fibrotic by disrupting PCSK5 and pro-TGF-β binding, presenting a new TAK treatment approach.

Transplantation of bioengineered dermal derived matrix-scaffold in combination with hyperbaric oxygen therapy improves wound healing in diabetic rats

Successful treatment of diabetic wounds requires multifactorial approaches. Herein we investigated the effects of a bioengineered three-dimensional dermal derived matrix-scaffold (DMS) in combination with hyperbaric oxygen (HBO) in repairing of wound model in diabetic rats. Thirty days after induction of diabetes, a circular wound was created and treatments were performed for 21 days. Animals were randomly allocated into the untreated group, DMS group, HBO group, and DMS+HBO group. On days 7, 14, and 21, tissue samples were obtained for stereological, molecular, and tensiometrical assessments. Our results showed that the wound closure rate, volume of new dermis and epidermis, numerical density fibroblasts and blood vessels, collagen density, and biomechanical characterize were significantly higher in the treatment groups than in the untreated group, and these changes were more obvious in the DMS+HBO ones. Moreover, the expression of TGF-β, bFGF, miRNA-21, miRNA-146a, and VEGF genes were meaningfully upregulated in treatment groups compared to the untreated group and were greater in the DMS+HBO group. This is while expression of TNF-α and IL-1β, as well as the numerical density of neutrophil and macrophage decreased more considerably in the DMS+HBO group than in the other groups. Overall, using both DMS engraftment and HBO treatment has more effects on diabetic wound healing.

PACAP binds conserved receptors and modulates cytokine gene expression and protein secretion in trout cell lines

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1β and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1β protein from previously accumulated transcripts or the cleavage of accumulated IL-1β precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.

Assessment of Probability of Pulmonary Fibrosis Progression in Patients with Interstitial Lung Diseases to Determine Further Treatment Tactics

The objective: to develop a method for assessing the probability of progression of pulmonary fibrosis in patients with interstitial lung diseases in order to determine further treatment tactics.Subjects and Methods. 80 patients with interstitial lung diseases underwent lung biopsies with consequent morphological and immunohistochemical tests. The group with progressive fibrosis consisted of 42 patients. The prognostic model was built using the binary logistic regression method.Results. A statistical model was developed in the form of a regression equation consisting of 3 parameters: the presence of a morphological pattern of interstitial fibrosis; intensity of CTGF expression; and intensity of TGF-β expression. This mathematical model makes it possible to predict the progression of pulmonary fibrosis in patients with interstitial lung diseases with 83.3% sensitivity and 84.2% specificity.

Local vascular Klotho mediates diabetes-induced atherosclerosis via ERK1/2 and PI3-kinase-dependent signaling pathways

Background and aimsDiabetes is one of the major causes of cardiovascular disease (CVD). As high as 29 % of patients with diabetes develop atherosclerosis. Vascular Smooth Muscle Cells (VSMCs) are a key mediator in the pathogenesis of atherosclerosis, generating pro-inflammatory and proliferative characteristics in atherosclerotic lesions. MethodsWe used human atherosclerotic samples, developed diabetes-induced atherosclerotic mice, and generated loss of function and gain of function in Klotho human aortic smooth muscle cells to investigate the function of Klotho in atherosclerosis. ResultsWe found that Klotho expression is decreased in smooth muscle actin-positive cells in patients with diabetes and atherosclerosis. Consistent with human data, we found that Apoe knockout mice with streptozotocin-induced diabetes fed on a high-fat diet showed decreased expression of Klotho in SMCs. Additionally, these mice showed increased expression of TGF-β, MMP9, phosphorylation of ERK and Akt. Further, we utilized primary Human Aortic Smooth Muscle Cells (HASMCs) with d-glucose under dose-response and in time-dependent conditions to study the role of Klotho in these cells. Klotho gain of function and loss of function studies showed that Klotho inversely regulated the expression of atherosclerotic markers TGF-β, MMP2, MMP9, and Fractalkine.Further, High Glucose (HG) induced Akt, and ERK1/2 phosphorylation were enhanced or mitigated by endogenous Klotho deficiency or its overexpression respectively. PI3K/Akt and MAPK/ERK inhibition partially abolished the HG-induced upregulation of TGF-β, MMP2, MMP9, and Fractalkine. Additionally, Klotho knockdown increased the proliferation of HASMCs and enhanced α-SMA and TGF-β expression. ConclusionsTaken together, these results indicate that local vascular Klotho is involved in diabetes-induced atherosclerosis, which is via PI3K/Akt and ERK1/2-dependent signaling pathways.

TGF-β/SMAD Pathway Mediates Cadmium Poisoning-Induced Chicken Liver Fibrosis and Epithelial-Mesenchymal Transition.

Cadmium(Cd) is a toxic heavy metal widely present in the environment, capable of accumulating in the liver and causing liver damage. In this study, the mechanism of cadmium-induced liver fibrosis in chickens was investigated from the perspective of hepatocyte epithelial-mesenchymal transition (EMT) based on the establishment of a model of chicken cadmium toxicity and a model of cadmium-stained cells in a chicken hepatocellular carcinoma cell line (LMH). The 7-day-old chickens were randomly divided into the regular group (C group) and cadmium poisoning group (Cd group), and the entire test cycle was 60days. Three sampling time points of 20days, 40days, and 60days were established. By testing the liver coefficient, histopathological and ultrastructural changes in chicken livers were observed. The enzyme activities of liver function and the expression changes of fibrosis markers (COL1A1, Fibronectin), epithelial-mesenchymal transition markers (E-cadherin, Vimentin, and α-SMA), and the critical factors of the TGF-β/SMAD signaling pathway (TGF-β1, SMAD 2, and SMAD 3) were detected in the liver expression changes. The results showed that at the same sampling time point, the chicken liver coefficient in group Cd was significantly higher than that in control group (P < 0.01); the activities of the liver function enzymes ALT and AST in chickens in the Cd group were significantly higher than those in the C group (P < 0.01); liver hepatocytes degenerated and necrotic, the number of erythrocytes in the blood vessels was increased, and inflammatory cells infiltrated in the sinusoidal gap; the perisinusoidal gap of the liver was enlarged, and there was an apparent aggregation of collagen fibers in the intervening period as seen by transmission electron microscopy. The results of Masson staining showed that the percentage of fiber area was significantly higher in the chickens' livers of the Cd group. The fiber area percentage was significantly higher. The results of real-time fluorescence quantitative PCR and Western Blot showed that the expression of E-cadherin in the livers of chickens in the Cd group was significantly lower than that in the C group (P < 0.01). The expression of α-SMA, Vimentin, COL1A1, Fibronectin, TGF-β1, SMAD 2, and SMAD 3 was significantly higher than that in the C group (P < 0.01). The results of in vitro assays showed that in the LMH cell model established by adding trimethylamine N-oxide, an activator of the TGF-β/SMAD signaling pathway, and oxidized picric acid, an inhibitor of the TGF-β/SMAD signaling pathway, the expression of E-cadherin was significantly reduced in cadmium-stained LMH cells (P < 0.01). The expression of α-SMA, Vimentin, COL1A1, Fibronectin, TGF-β, SMAD 2, and SMAD 3 was significantly elevated (P < 0.01). Cadmium and Trimethylamine N-oxide, an activator of the TGF-β/SMAD signaling pathway, promoted the expression of these factors. In contrast, the inhibitor of the TGF-β/SMAD signaling pathway, Oxymatrine, a TGF-β/SMAD signaling pathway inhibitor, significantly slowed down these changes. These results suggest that cadmium induces hepatic epithelial-mesenchymal transition by activating the TGF-β/SMAD signaling pathway in chicken hepatocytes, promoting hepatic fibrosis.

The methanol extract of Centaurea depressa M. Bieb. -Containing nanoemulsion formulation: In vitro characterization and scratch assay

Centaurea depressa M. Bieb. is used in traditional medicine as wound healing, antidiabetic, antibacterial, antimalarial, antirheumatic, diuretic, antipyretic, antibacterial, tonic, astringent, choleretic. It is popularly used as poultice, pounded, infusion, decoction, external, and internal. This study aimed to develop and in vitro characterize the methanol extract of Centaurea depressa (MCD)-containing nanoemulsion formulation (MCD-NEF) to heal the wound effectively and analyze the phenolic compounds. MCD-NEF was prepared and its droplet size, PDI, and ZP, and viscosity were determined. The effect of MCD and MCD-NEF on human dermal fibroblast cell viability was examined by MTT assay. MCD's phenolic compound profiles were analyzed by LC-MS/MS. Also, MCD and MCD-NEF's wound healing potential was evaluated using the scratch assay. The droplet size, PDI, ZP, pH and viscosity values for the MCD-NEF were found to be 152.42 ± 3.50 nm, 0.187 ± 0.020, (−)27.67 ± 1.68 mV, 5.57 ± 0.03, and 1.891 ± 0.010 cP, respectively. In the cell viability assay, the highest percentage of living cells was obtained at concentration of 16 μg/mL for the MCD and MCD-NEF. In the scratch assay, MCD-NEF showed significantly ameliorated wound closure % rates at 24 and 30 h than the control, MCD and blank-NEF (without MCD) groups (p < 0.001). In this study, immunofluorescence staining was carried out. TGF-β1 and FGFR-2 expressions in MCD-NEF-treated fibroblast cells increased significantly at 24 and 30 h compared to the control, MCD, and B-NEF groups (p < 0.001). MCD's. Twenty-seven phenolic compounds, including quinic acid, chlorogenic acid, cynaroside, cosmosiin, protocatechuic acid and isoquercitrin were determined by LC-MS/MS to be major phytochemical compounds in MCD. Our findings show that MCD-NEF may be promising for better wound healing.

Resolvin D2-induced reparative dentin and pulp stem cells after pulpotomy in a rat model

IntroductionVital pulp therapy (VPT) is performed to preserve dental pulp. However, the biocompatibility of the existing materials is of concern. Therefore, novel materials that can induce pulp healing without adverse effects need to be developed. Resolvin D2 (RvD2), one of specialized pro-resolving mediators, can resolve inflammation and promote the healing of periapical lesions. Therefore, RvD2 may be suitable for use in VPT. In the present study, we evaluated the efficacy of RvD2 against VPT using in vivo and in vitro models. MethodsFirst molars of eight-week-old male Sprague–Dawley rats were used for pulpotomy. They were then divided into three treatment groups: RvD2, phosphate-buffered saline, and calcium hydroxide groups. Treatment results were assessed using radiological, histological, and immunohistochemical (GPR18, TNF-α, Ki67, VEGF, TGF-β, CD44, CD90, and TRPA1) analyses. Dental pulp-derived cells were treated with RvD2 in vitro and analyzed using cell-proliferation and cell-migration assays, real-time PCR (Gpr18, Tnf-α, Il-1β, Tgf-β, Vegf, Nanog, and Trpa1), ELISA (VEGF and TGF-β), immunocytochemistry (TRPA1), and flow cytometry (dental pulp stem cells: DPSCs). ResultsThe formation of calcified tissue in the pulp was observed in the RvD2 and calcium hydroxide groups. RvD2 inhibited inflammation in dental pulp cells. RvD2 promoted cell proliferation and migration and the expression of TGF-β and VEGF in vitro and in vivo. RvD2 increased the number of DPSCs. In addition, RvD2 suppressed TRPA1 expression as a pain receptor. ConclusionRvD2 induced the formation of reparative dentin, anti-inflammatory effects, and decreased pain, along with the proliferation of DPSCs via the expression of VEGF and TGF-β, on the pulp surface in pulpotomy models.

Core needle biopsy changes the expression of TGFβ1 and TGFβRII at protein level, and the distribution of CD4 and CD8 positive T cells in primary breast cancer

Core needle biopsy (CNB) has become a paradigm in preoperative breast cancer (BC) diagnosis. Although considered safe, it is an invasive procedure, which changes the tumor microenvironment. It facilitates a tumor supportive immune response, induces epithelial-mesenchymal transition (EMT), and enables the release of circulating tumor cells. The cytokine Transforming Growth Factor β (TGFβ) with its pleiotropic immunologic functions has an important role in this process. The aim of this study was to clarify the specific impact of CNB on the activity of the TGFβ pathway in early BC. We compared formalin fixed paraffin embedded samples from CNBs to the corresponding surgical resection specimens (SRSs) of 49 patients with BC. We found that the expression of TGFβ1 at protein level was significantly higher in both tumor epithelial and benign stromal cells in the SRSs (p=0.001), whereas the expression of TGFβRII in tumor cells was lower (p=0.001). The frequency of intra tumoral CD8 and CD4 positive T lymphocytes was lower in SRSs (p=0081 and p=0001, respectively), while in the peripheral stroma their prevalence was increased (p=0001 and p=0012, respectively). Our results show that CNB changes the hallmarks of the TGFβ path way in early BC. These CNB-induced changes in the tumor and in its microenvironment suggest that the procedure may change the immunological anti-tumor response of the host.