Release Of TGF-β1 Research Articles

Nanoparticle-Mediated TGF-β Release from Microribbon-Based Hydrogels Accelerates Stem Cell-Based Cartilage Formation In Vivo.

Conventional nanoporous hydrogels often lead to slow cartilage deposition by MSCs in 3D due to physical constraints and requirement for degradation. Our group has recently reported macroporous gelatin microribbon (μRB) hydrogels, which substantially accelerate MSC-based cartilage formation in vitro compared to conventional gelatin hydrogels. To facilitate translating the use of μRB-based scaffolds for supporting stem cell-based cartilage regeneration in vivo, there remains a need to develop a customize-designed drug delivery system that can be incorporated into μRB-based scaffolds. Towards this goal, here we report polydopamine-coated mesoporous silica nanoparticles (MSNs) that can be stably incorporated within the macroporous μRB scaffolds,and allow tunable release of transforming growth factor (TGF)-β3. We hypothesize that increasing concentration of polydopamine coating on MSNs will slow down TGF- β3 release, and TGF-β3 release from polydopamine-coated MSNs can enhance MSC-based cartilage formation in vitro and in vivo. We demonstrate that TGF-β3 released from MSNs enhance MSC-based cartilage regeneration in vitro to levels comparable to freshly added TGF-β3 in the medium, as shown bybiochemical assays, mechanical testing, and histology. Furthermore, when implanted in vivo in a mouse subcutaneous model, onlythe group containing MSN-mediated TGF-β3 release supported continuous cartilage formation, whereas control group without MSN showed loss of cartilage matrix and undesirable endochondral ossification. The modular design of MSN-mediated drug delivery can be customized for delivering multiple drugs with individually optimized release kinetics, and may be applicable to enhance regeneration of other tissue types.

Targeting GARP-TGFβ axis enhances anti-tumor efficacy of immune checkpoint blockade therapy

Abstract Although immune checkpoint blockade (ICB) has emerged as a promising cancer therapy, a majority of cancers, such as pancreatic cancer and triple negative breast cancer, fail to respond to ICB. A potential mechanism is the accumulation of transforming growth factor-β1 (TGFβ1) in tumor microenvironment (TME) which drives immune dysfunction by inducing regulatory T cells (Treg), excluding and inhibiting the function of effector CD8+ T cells. Glycoprotein-A repetitions predominant protein (GARP) is a cell surface docking and activating receptor for latent TGFβ1 and highly expressed in breast cancer cells, Tregs and platelets. Previous work from our lab has demonstrated a critical role for GARP in dampening anti-tumor immunity via TGFβ1 pathway. To validate if GARP is a therapeutic target, we generated an anti-human GARP antibody (4D3). We found that 4D3 blocked GARP-latent TGFβ1 interaction and limited active TGFβ1 release. We expressed human GARP in murine 4T1 breast cancer cells. In this orthotopic syngeneic mouse tumor model, combination therapy with 4D3 and anti-PD1 antibody suppressed tumor growth and metastasis more effectively than treatment with either agent alone, resulting in significantly enhanced survival. Mechanistically, the combination therapy reduced p-SMAD3 expression as well as collagen deposition in the TME. In addition, 4D3 treatment reduced tumor-associated macrophages and their PD-L1 expression. Collectively, our preclinical data demonstrated that simultaneous blockade of the PD1 and GARP-TGFβ1 axis elicits potent and superior anti-tumor efficacy relative to monotherapies, suggesting a potentially more effective strategy against cancers that are resistant to current immune checkpoint blockade.

Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration.

Inflammation plays an essential role in the development of lumbar disc degeneration (LDD), although the exact effects of macrophage subtypes on LDD remain unclear. Based on previous studies, we hypothesized that M2-polarization of local macrophages and simultaneous suppression of their production of fibrotic transforming growth factor beta 1 (TGFβ1) could inhibit progression of LDD. Thus, we applied an orthotopic injection of adeno-associated virus (AAV) carrying shRNA for DNA Methyltransferase 1 (DNMT1) and/or shRNA for TGFβ1 under a macrophage-specific CD68 promoter to specifically target local macrophages in a mouse model for LDD. We found that shDNMT1 significantly reduced levels of the pro-inflammatory cytokines TNFα, IL-1β and IL-6, significantly increased levels of the anti-inflammatory cytokines IL-4 and IL-10, significantly increased M2 macrophage polarization, significantly reduced cell apoptosis in the disc degeneration zone and significantly reduced LDD-associated pain. The anti-apoptotic and anti-pain effects were further strengthened by co-application of shTGFβ1. Together, these data suggest that M2 polarization of macrophages induced by both epigenetic modulation and suppressed production and release of TGFβ1 from polarized M2 macrophages, may have a demonstrable therapeutic effect on LDD.

Blockade of extracellular high-mobility group box 1 attenuates inflammation-mediated damage and haze grade in mice with corneal wounds

PurposeTo investigate the expression of extracellular high mobility group box 1 (HMGB1) and the effect of its inhibitor glycyrrhizin (GL) in corneal wound healing. MethodsWe treated C57BL/6J mice with GL or PBS before and after establishing a corneal injury model. Fluorescein staining, Ki-67 expression, haze grade, and haematoxylin/eosin (H&E) staining were used to assess treatment efficacy. The expression of HMGB1, NF-κB-p65, the NLRP3 inflammasome, IL-1β, CCL2, CXCL2, TGF-β1, α-SMA, fibronectin, and collagen III and neutrophil influx were examined by immunohistochemical staining, western blot, and RT-qPCR at various time points after corneal injury. ResultsAfter corneal injury, HMGB1 transferred from the nucleus to the cytoplasm and was passively released or actively secreted into the corneal stroma from epithelial cells and inflammatory cells; however, this increase was attenuated by GL treatment. Furthermore, GL indirectly attenuated the expression of IL-1β by directly inhibiting extracellular HMGB1 functions, which activated the NF-κB-p65/NLRP3/IL-1β signalling pathway. Moreover, application of GL alleviated the neutrophil infiltration that delays wound healing, accompanied by the downregulation of expression of the chemokines CCL2 and CXCL2. More interestingly, application of GL reduced the degree of haze grade through inactivating extracellular HMGB1 functions that induced TGF-β1 release and myofibroblast differentiation. In addition, fluorescein and H&E staining and Ki-67 levels suggest that GL promotes regeneration of corneal epithelium. ConclusionsAfter corneal injury, extracellular HMGB1 can be an essential driver to trigger a neutrophil- and cytokine-mediated inflammatory injury amplification loop. The application of GL promotes the cornea to restore transparency and integrity, which may be related to the attenuation of extracellular HMGB1 levels and function.

Transforming growth factor beta variation with physical activity in knee osteoarthritis

Purpose: Transforming growth factor beta (TGF-β) is a multifunctional cytokine with three different mammalian isoforms. All three isoforms of TGF-β are secreted as inactive latent complexes. Bound to latency-associated peptide, TGF-β is unable to bind to membrane receptors. Therefore, even though TGF-β is constitutively expressed throughout the body, only the free ligand is biologically active and able to modulate the metabolic activity of cartilage. An existing theory likens the latent TGF-β complex to a molecular sensor that monitors the extracellular matrix and responds to perturbations by releasing free TGF-β. In the cartilage, TGF-β1 plays an essential role in regulating cartilage matrix metabolism. The goal of this study was to assess the effects of physical activity on free and total TGF-β isoforms in plasma from individuals with knee osteoarthritis (OA). Methods: To assess the impact of physical activity on biomarker variation, participants (n=20; 65% female; 80% white; mean age 70 years; mean BMI 31.72 kg/m2) were recruited on the basis of symptomatic OA in at least one knee. Knee OA severity was assessed from a postero-anterior semi-flexed radiograph with a SynaFlexer positioning device and quantified by Kellgren Lawrence (K-L) score for each knee. Blood was obtained at 4 different time points (Figure 1). The interval from T0 to T1A was devoted to physical activity including activities of daily living and non-brisk walking for 1 hour, with no more than 10 minutes rest at any one time. All procedures were approved by the Institutional Review Board. Using commercially available ELISA assays, we quantified free and total TGF-β1 (BioLegend) in plasma samples (n=80) and available matched serum (n=79) samples. Total TGF-β2 and TGF-β3 (MSD U-PLEX ELISA) were also quantified in plasma (n=80). The data for TGF-β3 should be interpreted with caution, as 47.5% of samples had concentrations below the LLOD so were imputed as 1/2 LLOD for analyses. To assess variations in TGF-β over this time period, biomarker concentrations for the four time points were normalized to the mean across all four time points for each individual. Significance was assessed by non-parametric Friedman test and Dunn’s post-hoc test. Spearman correlation was used to evaluate associations between non-normalized: free and total plasma TGF-β1; serum and plasma free and total TGF-β1; all total TGF-β isoforms; and free and total TGF-β1 (T1A and change from T0 to T1A) with sum K-L grade. Results: Median plasma concentrations (range pg/ml) were as follows: free TGF-β1: 8.53 (0-40.71); total TGF-β1: 27343.00 (6760.00-81815.00); total TGF-β2: 24.99 (0-170.30); and total TGF-β3: 1.03 (0-3.10). Free plasma TGF-β1 represented 0.032% of total plasma TGF-β1. Free and total TGF-β1, and total TGF-β2, increased significantly after one hour of activity (Figure 2; time point T0 to T1A). The mean fold-change increase with activity was 1.7 for free TGF-β1, 1.4 for total TGF-β1, and 1.5 for total TGF-β2. After food consumption (T1B), TGF-β3 concentrations decreased an average of 62% from T1A, but no significant changes occurred in the other TGF-β isoforms at this time point. In plasma, total TGF-β1 strongly correlated with free TGF-β1 (rs=0.36, p=0.008), total TGF-β2 (rs=0.69, p<0.0001), and total TGF-β3 (rs=0.36, p=0.001). Total TGF-β2 also correlated with Total TGF-β3 (rs=0.29, p=0.01). Serum TGF-β1 concentrations were consistently higher than plasma concentrations (a mean 17.5-fold for free TGF-β1 and a mean 1.6-fold for total TGF-β1). Plasma and matched sera did not correlate for free TGF-β1 (rs=0.09, p=0.43) or total TGF-β1 (rs=0.19, p=0.09). Neither free nor total TGF-β1 were associated with sum K-L grade. Conclusions: This study demonstrates that plasma concentrations of both free and total TGF-β1, and total TGF-β2, all significantly increased with physical activity, suggesting release from a mechanically sensitive reservoir. These results are consistent with known release of TGF-β1 from extracellular matrix of myofibroblasts upon mechanical contraction in vitro. We observed an effective 1.7-fold increase of circulating free (active) TGF-β1 during an hour of activity (from a mean 8.45 to a mean 13.08 pg/ml). Based upon previous published studies, these concentrations may represent a physiologically relevant modulation. These results suggest the need for standardization of activity prior to biofluid sampling intended for TGF-β analyses to assess its impact on musculoskeletal health and disease.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

CCL11 increases the proportion of CD4+CD25+Foxp3+ Treg cells and the production of IL‑2 and TGF‑β by CD4+ Tcells via the STAT5 signaling pathway.

CD4+ regulatory T (Treg) cells are associated with immune tolerance and antitumor immunosuppression. The aim of the present study was to investigate the role and molecular mechanism of C-C motif chemokine ligand 11 (CCL11) in the regulation of Treg cells from patients with breast cancer (BC) and healthy individuals in vitro, and from tumor-bearing mice in vivo. CD4+ T cells isolated from patients with BC or healthy individuals were incubated with anti-CCL11 neutralizing antibodies or recombinant human CCL11 protein, in the presence or absence of a STAT5 inhibitor. The serum CCL11 level and proportion of Treg cells characterized as CD4+CD25+forkhead box P3+ (Foxp3) among the CD4+ T cells in patients with BC and healthy individuals were analyzed by ELISA and flow cytometry, respectively. CCL11, C-C motif chemokine receptor 3 (CCR3), Foxp3, phosphorylated-STAT5 and STAT5 expression levels were determined by western blotting. The serum CCL11 level and the proportion of CD4+CD25+Foxp3+ Treg cells were significantly increased in patients with BC compared with healthy individuals. CCL11 blockade reduced the proportion of CD4+CD25+Foxp3+ Treg cells, the expression of CCR3 and Foxp3, and the level of STAT5 activation in tumor-associated CD4+ T cells, in a dose-dependent manner. CCL11 blockade also reduced the proportion of CD4+CD25+Foxp3+ Treg cells and the serum levels of interleukin (IL)-2 and transforming growth factor (TGF)-β1 in tumor-bearing mice. The recombinant human CCL11 protein increased the proportion of CD4+CD25+Foxp3+ Treg cells, the expression of CCR3 and Foxp3, and the release of IL-2 and TGF-β1 in non-tumor-associated CD4+ T cells via the STAT5 signaling pathway. The results of the present study may aid in identifying therapeutics that could further modulate the immune system during BC.

Shear Stress-Induced miR-143-3p in Collateral Arteries Contributes to Outward Vessel Growth by Targeting Collagen V-α2.

Arteriogenesis, describing the process of collateral artery growth, is activated by fluid shear stress (FSS). Since this vascular mechanotransduction may involve microRNAs (miRNAs), we investigated the FSS-induced expression of vascular cell miRNAs and their functional impact on collateral artery growth during arteriogenesis. Approach and Results: To this end, rats underwent femoral artery ligation and arteriovenous anastomosis to increase collateral blood flow to maximize FSS and trigger collateral vessel remodeling. Five days after surgery, a miRNA expression profile was obtained from collateral tissue, and upregulation of 4 miRNAs (miR-24-3p, miR-143-3p, miR-146a-5p, and miR-195-5p) was verified by quantitative polymerase chain reaction. Knockdown of miRNAs at the same time of the surgery in an in vivo mouse ligation and recovery model demonstrated that inhibition of miR-143-3p only severely impaired blood flow recovery due to decreased arteriogenesis. In situ hybridization revealed distinct localization of miR-143-3p in the vessel wall of growing collateral arteries predominantly in smooth muscle cells. To investigate the mechanotransduction of FSS leading to the increased miR-143-3p expression, cultured endothelial cells were exposed to FSS. This provoked the expression and release of TGF-β (transforming growth factor-β), which increased the expression of miR-143-3p in smooth muscle cells in the presence of SRF (serum response factor) and myocardin. COL5A2 (collagen type V-α2)-a target gene of miR-143-3p predicted by in silico analysis-was found to be downregulated in growing collaterals. These results indicate that the increased miR-143-3p expression in response to FSS might contribute to the reorganization of the extracellular matrix, which is important for vascular remodeling processes, by inhibiting collagen V-α2 biosynthesis.

A cancer invasion model of cancer-associated fibroblasts aggregates combined with TGF-β1 release system.

IntroductionThe objective of this study is to design a cancer invasion model where the cancer invasion rate can be regulated in vitro.MethodsCancer-associated fibroblasts (CAF) aggregates incorporating gelatin hydrogel microspheres (GM) containing various concentrations of transforming growth factor-β1 (TGF-β1) (CAF-GM-TGF-β1) were prepared. Alpha-smooth muscle actin (α-SMA) for the CAF aggregates was measured to investigate the CAF activation level by changing the concentration of TGF-β1. An invasion assay was performed to evaluate the cancer invasion rate by co-cultured of cancer cells with various CAF-GM-TGF-β1.ResultsThe expression level of α-SMA for CAF increased with an increased in the TGF-β1 concentration. When co-cultured with various types of CAF-GM-TGF-β1, the cancer invasion rate was well correlated with the α-SMA level. It is conceivable that the TGF-β1 concentration could modify the level of CAF activation, leading to the invasion rate of cancer cells. In addition, at the high concentrations of TGF-β1, the effect of a matrix metalloproteinase (MMP) inhibitor on the cancer invasion rate was observed. The higher invasion rate would be achieved through the higher MMP production.ConclusionsThe present model is promising to realize the cancer invasion whose rate can be modified by changing the TGF-β1 concentration.

Fibrocytes, Wound Healing, and Corneal Fibrosis.

PurposeThis review highlights the roles of fibrocytes—their origin, markers, regulation and functions—including contributions to corneal wound healing and fibrosis.MethodsLiterature review.ResultsPeripheral blood fibroblast-like cells, called fibrocytes, are primarily generated as mature collagen-producing cells in the bone marrow. They are likely derived from the myeloid lineage, although the exact precursor remains unknown. Fibrocytes are identified by a combination of expressed markers, such as simultaneous expression of CD34 or CD45 or CD11b and collagen type I or collagen type III. Fibrocytes migrate into the wound from the blood where they participate in pathogen clearance, tissue regeneration, wound closure and angiogenesis. Transforming growth factor beta 1 (TGF-β1) and adiponectin induce expression of α-smooth muscle actin and extracellular matrix proteins through activation of Smad3 and adenosine monophosphate-activated protein kinase pathways, respectively. Fibrocytes are important contributors to the cornea wound healing response and there are several mechanisms through which fibrocytes contribute to fibrosis in the cornea and other organs, such as their differentiation into myofibroblasts, production of matrix metalloproteinase, secretion of tissue inhibitor of metalloproteinase, and release of TGF-β1. In some tissues, fibrocytes may also contribute to the basement membrane regeneration and to the resolution of fibrosis.ConclusionsNew methods that block fibrocyte generation, fibrocyte migration, and their differentiation into myofibroblasts, as well as their production of matrix metalloproteinases, tissue inhibitor of metalloproteinase, and TGF-β1, have therapeutic potential to reduce the accumulation of collagens, maintain tissue integrity and retard or prevent the development of fibrosis.

Ginsenoside Rb1/TGF-β1 loaded biodegradable silk fibroin-gelatin porous scaffolds for inflammation inhibition and cartilage regeneration.

Creating a microenvironment with low inflammation and favorable for the chondrogenic differentiation of endogenous stem cells plays an essential role in cartilage repairing. In the present study, we design a novel ginsenoside Rb1/TGF-β1 loaded silk fibroin-gelatin porous scaffold (GSTR) with the function of attenuating inflammation and promoting chondrogenesis. The scaffold has porous microstructure, proper mechanical strength, degradation rate and sustained release of Rb1 and TGF-β1. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) seeded into GSTR scaffolds are homogeneously distributed and display a higher proliferation rate than non-loaded scaffolds (GS). GSTR scaffolds promote the chondrogenic differentiation of rBMSCs and suppress the expression of inflammation genes. Under the stimulation of IL-1β, the inflammation level of the chondrocytes seeded in GSTR scaffolds is also significantly down-regulated. Moreover, GSTR scaffolds implanted into the osteochondral defects in rats effectively promote the regeneration of hyaline cartilage 12weeks after surgery when compared with other groups. It is demonstrated that this scaffold loaded with Rb1 and TGF-β1 can synergistically create a microenvironment favorable for cartilage regeneration by promoting the chondrogenesis and suppressing the inflammation levels in vivo. These results prove it has a great potential to develop this Rb1/TGF-β1 releasing scaffold into a novel and promising therapeutic for cartilage repair.

Effect of mesenchymal stem cell-derived exosomes on the induction of mouse tolerogenic dendritic cells.

Dendritic cells (DCs) orchestrate innate inflammatory responses and adaptive immunity through T‐cell activation via direct cell–cell interactions and/or cytokine production. Tolerogenic DCs (tolDCs) help maintain immunological tolerance through the induction of T‐cell unresponsiveness or apoptosis, and generation of regulatory T cells. Mesenchymal stromal cells (MSCs) are adult multipotent cells located within the stroma of bone marrow (BM), but they can be isolated from virtually all organs. Extracellular vesicles and exosomes are released from inflammatory cells and act as messengers enabling communication between cells. To investigate the effects of MSC‐derived exosomes on the induction of mouse tolDCs, murine adipose‐derived MSCs were isolated from C57BL/6 mice and exosomes isolated by ExoQuick‐TC kits. BM‐derived DCs (BMDCs) were prepared and cocultured with MSCs‐derived exosomes (100 μg/ml) for 72 hr. Mature BMDCs were derived by adding lipopolysaccharide (LPS; 0.1μg/ml) at Day 8 for 24 hr. The study groups were divided into (a) immature DC (iDC, Ctrl), (b) iDC + exosome (Exo), (c) iDC + LPS (LPS), and (d) iDC + exosome + LPS (EXO + LPS). Expression of CD11c, CD83, CD86, CD40, and MHCII on DCs was analyzed at Day 9. DC proliferation was assessed by coculture with carboxyfluorescein succinimidyl ester‐labeled BALB/C‐derived splenocytes p. Interleukin‐6 (IL‐6), IL‐10, and transforming growth factor‐β (TGF‐β) release were measured by enzyme‐linked immunosorbent assay. MSC‐derived exosomes decrease DC surface marker expression in cells treated with LPS, compared with control cells ( ≤ .05). MSC‐derived exosomes decrease IL‐6 release but augment IL‐10 and TGF‐β release (p ≤ .05). Lymphocyte proliferation was decreased (p ≤ .05) in the presence of DCs treated with MSC‐derived exosomes. CMSC‐derived exosomes suppress the maturation of BMDCs, suggesting that they may be important modulators of DC‐induced immune responses.

A-Kinase Anchoring Proteins Diminish TGF-β1/Cigarette Smoke-Induced Epithelial-To-Mesenchymal Transition.

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in the lung EMT process are lacking. The human bronchial epithelial cell line (BEAS-2B) and primary human airway epithelial (pHAE) cells were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers (collagen Ӏ, α-SMA, fibronectin) were analyzed (mRNA, protein). ELISA measured TGF-β1 release. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced while using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, and forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin, but increased collagen Ӏ and cell migration, a process that was reversed by the inhibitor of δ/epsilon casein kinase I, PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95, and Yotiao. St-Ht31, the AKAP antagonist, decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration, and reduced E-cadherin expression, a process that was blocked by TGF-β1 neutralizing antibody. The silencing of Ezrin, AKAP95, and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells, pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95, and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members might define the ability of fenoterol, rolipram, and cilostamide to modulate the EMT process, and they might represent potential relevant targets in the treatment of COPD.

The endocannabinoid receptors CB1 and CB2 affect the regenerative potential of adipose tissue MSCs

Human adipose tissue includes large quantities of mesenchymal stromal cells (atMSCs), which represent an abundant cell source for therapeutic applications in the field of regenerative medicine. Adipose tissue secrets various soluble factors including endocannabinoids, and atMSCs express the cannabinoid receptors CB1 and CB2. This indicates that adipose tissue possesses an endocannabinoid system (ECS). The ECS is also ascribed great significance for wound repair, e.g. by modulating inflammation. However, the exact effects of CB1/CB2 activation in human atMSCs have not been investigated, yet. In the present study, we stimulated human atMSCs with increasing concentrations (1–30 μM) of the unspecific cannabinoid receptor ligand WIN55,212–2 and the specific CB2 agonist JWH-133, either alone or co-applied with the receptor antagonist Rimonabant (CB1) or AM 630 (CB2). We investigated the effects on metabolic activity, cell number, differentiation and cytokine release, which are important processes during tissue regeneration. WIN decreased metabolic activity and cell number, which was reversed by Rimonabant. This suggests a CB1 dependent mechanism, whereas the number of atMSCs was increased after CB2 ligation. WIN and JWH increased the release of VEGF, TGF-β1 and HGF. Adipogenesis was enhanced by WIN, which could be reversed by blocking CB1. There was no effect on osteogenesis, and only WIN increased chondrogenic differentiation. Our results indicate that definite activation of the cannabinoid receptors exerted different effects in atMSCs, which could be of specific value in cell-based therapy for wound regeneration.

Placental Hofbauer Cell Polarization Resists Inflammatory Cues In Vitro

Feto-placental Hofbauer cells (HBCs) are macrophages residing in placental stroma. They are generally described as anti-inflammatory M2 polarized cells, promoting tolerance and tissue remodeling. In certain pathologies, however, a possible phenotypical switch towards pro-inflammatory M1 macrophages has been proposed. The study aimed to determine if HBCs can acquire an M1 phenotype under pro-inflammatory conditions in vitro. HBCs were isolated from healthy human term placentas. Cells were cultivated upon addition of LPS and INF-γ or IL-4 and IL-13 to induce the M1 and M2 phenotype, respectively. Specific cell polarization markers and cytokines, associated with respective phenotypes, were investigated by flow cytometry and ELISA. THP-1 macrophages served as positive control. Pro-inflammatory stimuli reduced M2 markers CD163 and DC-SIGN, but did not induce M1 markers. TNF-α release was increased, but at the same time TGF-β and IL-10 release was upregulated, resembling in part the M2b sub-phenotype. Anti-inflammatory stimuli had no effect on HBC polarization. HBCs maintain their M2 phenotype in vitro despite inflammatory stimuli, which might represent a state of adaption and tolerance to avoid rejection of the semiallogeneic feto-placental unit.

α-Lipoic acid prevents the ionizing radiation-induced epithelial-mesenchymal transition and enhances the radiosensitivity in breast cancer cells

Radiotherapy is routinely used in the treatment of breast cancer. However, its efficiency is often limited by the development of radioresistance and metastasis. The cancer cells surviving irradiation show epithelial-mesenchymal transition (EMT) along with increased migration, invasion and metastasis. In this study, we have evaluated the role of α-lipoic acid in preventing the radiation-induced EMT and in sensitizing the breast cancer cells to radiation. The breast cancer cell lines, MCF-7 and MDA-MB-231 were pretreated with lipoic acid, irradiated and the changes associated with cell growth, clonogenicity, migration, matrix metalloproteinases (MMPs), EMT and TGFβ signaling were measured. Our data showed that lipoic acid pretreatment sensitized the breast cancer cells to the ionizing radiation and inhibited the radiation-induced migration and the release of MMP2 and MMP9. Lipoic acid also prevented the TGFβ1 release and inhibited the radiation-induced EMT in breast cancer cells. The inhibition of TGFβ signaling by lipoic acid is associated with the inhibition of radiation-induced activation and translocation of NF-κB. These results suggest that α-lipoic acid inhibits the radiation-induced TGFβ signaling and nuclear translocation of NF-κB, thereby inhibiting the radiation-induced EMT and sensitizing the breast cancer cells to ionizing radiation.

Cryo-EM Reveals Integrin-Mediated TGF-β Activation without Release from Latent TGF-β

Integrin αvβ8 binds with exquisite specificity to latenttransforming growth factor-β (L-TGF-β). This binding is essential for activating L-TGF-β presented by a variety of cell types. Inhibiting αvβ8-mediated TGF-β activation blocks immunosuppressive regulatory Tcell differentiation, which is a potential therapeuticstrategy in cancer. Using cryo-electron microscopy, structure-guided mutagenesis, and cell-based assays, we reveal the binding interactions between the entire αvβ8 ectodomain and its intact natural ligand, L-TGF-β, as well as two different inhibitory antibody fragments to understand the structural underpinnings of αvβ8 binding specificity and TGF-β activation. Our studies reveal a mechanism of TGF-β activation where mature TGF-β signals within the confines of L-TGF-β and the release and diffusion of TGF-β are not required. The structural details of this mechanism provide a rational basis for therapeutic strategies to inhibit αvβ8-mediated L-TGF-β activation.

Investigating the effect of antiseptic solution on the release of interleukin-6 and transforming growth factor beta 1 from human gingival fibroblasts using wound healing assays.

This in vitro study evaluated the effect of different antiseptics and different concentrations thereof in a model of wound healing using human gingival fibroblasts. The fibroblasts were rinsed with four different antiseptic solutions: sodium hypochlorite (HYP), hydrogen peroxide (H2O2), chlorhexidine digluconate (CHX), and benzalkonium chloride (BC). The effect on the release of interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF-β1) was investigated using enzyme-linked immunosorbent assays (ELISAs). In addition, the effects of the antiseptics on wound healing at 1, 12, 24, and 48 h were assessed through a wound healing assay. The viability of the fibroblasts rinsed with antiseptics was investigated with respect to the concentrations inhibiting cell growth by 50% (IC50), 25% (IC25), and ≤2% (IC2). A statistically significant increased release of IL-6 was obtained with BC IC25 and IC2 after 12, 24, and 48 h (P < 0.01). For TGF-β1, no significant release was found for CHX IC2 after 24 and 48 h or for IC50 and IC25 after 12 h. There was no significant effect on wound healing capacity for CHX or for BC IC25 and IC2. This study demonstrated that antiseptic rinses of human gingival fibroblasts alter the release of IL-6 and TGF-β1 and impact wound healing capacity, with both BC and CHX conferring neutral effects.

Dual growth factor delivery using PLGA nanoparticles in silk fibroin/PEGDMA hydrogels for articular cartilage tissue engineering.

Degeneration of articular cartilage due to damages, diseases, or age-related factors can significantly decrease the mobility of the patients. Various tissue engineering approaches which take advantage of stem cells and growth factors in a three-dimensional constructs have been used for reconstructing articular tissue. Proliferative impact of basic fibroblast growth factor (bFGF) and chondrogenic differentiation effect of transforming growth factor-beta 1 (TGF-β1) over mesenchymal stem cells have previously been verified. In this study, silk fibroin (SF) and of poly(ethylene glycol) dimethacrylate (PEGDMA) were used to provide a versatile platform for preparing hydrogels with tunable mechanical, swelling and degradation properties through physical and chemical crosslinking as a microenvironment for chondrogenic differentiation in the presence of bFGF and TGF-β1 releasing nanoparticles (NPs) for the first time. Scaffolds with compressive moduli ranging from 95.70 ± 17.82 to 338.05 ± 38.24 kPa were obtained by changing both concentration PEGDMA and volume ratio of PEGDMA with 8% SF. Highest cell viability was observed in PEGDMA 10%-SF 8% (1:1) [PEG10-SF8(1:1)] hydrogel group. Release of bFGF and TGF-β1 within PEG10-SF8(1:1) hydrogels resulted in higher DNA and glycosaminoglycans amounts indicating synergistic effect of dual release over proliferation and chondrogenic differentiation of dental pulp stem cells in hydrogels, respectively. Our results suggested that simultaneous delivery of bFGF and TGF-β1 through utilization of PLGA NPs within PEG10-SF8(1:1) hydrogel provided a novel and versatile means for articular cartilage regeneration as they allow for dosage- and site-specific multiple growth factor delivery.

Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-β1 from ECM stores

In healing tissue, fibroblasts differentiate to α-smooth muscle actin (SMA)-expressing contractile-myofibroblasts, which pull the wound edges together ensuring proper tissue repair. Uncontrolled expansion of the myofibroblast population may, however, lead to excessive tissue scarring and finally to organ dysfunction. Here, we demonstrate that the loss of low-density lipoprotein receptor-related protein (LRP) 1 overactivates the JNK1/2-c-Jun-Fra-2 signaling pathway leading to the induction of α-SMA and periostin expression in human lung fibroblasts (hLF). These changes are accompanied by increased contractility of the cells and the integrin- and protease-dependent release of active transforming growth factor (TGF)-β1 from the extracellular matrix (ECM) stores. Liberation of active TGF-β1 from the ECM further enhances α-SMA and periostin expression thus accelerating the phenotypic switch of hLF. Global gene expression profiling of LRP1-depleted hLF revealed that the loss of LRP1 affects cytoskeleton reorganization, cell-ECM contacts, and ECM production. In line with these findings, fibrotic changes in the skin and lung of Fra-2 transgenic mice were associated with LRP1 depletion and c-Jun overexpression. Altogether, our results suggest that dysregulation of LRP1 expression in fibroblasts in healing tissue may lead to the unrestrained expansion of contractile myofibroblasts and thereby to fibrosis development. Further studies identifying molecules, which regulate LRP1 expression, may provide new therapeutic options for largely untreatable human fibrotic diseases.

Mesangial cells are key contributors to the fibrotic damage seen in the lupus nephritis glomerulus

BackgroundLupus nephritis (LN) affects up to 80% of juvenile-onset systemic lupus erythematosus patients. Mesangial cells (MCs) comprise a third of the glomerular cells and are key contributors to fibrotic changes within the kidney. This project aims to identify the roles of MCs in an in vitro model of LN.MethodsConditionally immortalised MCs were treated with pro-inflammatory cytokines or with patient sera in an in vitro model of LN and assessed for their roles in inflammation and fibrosis.ResultsMCs were shown to produce pro-inflammatory cytokines in response to a model of the inflammatory environment in LN. Further the cells expressed increased levels of mRNA for extracellular matrix (ECM) proteins (COL1A1, COL1A2, COL4A1 and LAMB1), matrix metalloproteinase enzymes (MMP9) and tissue inhibitors of matrix metalloproteinases (TIMP1). Treatment of MCs with serum from patients with active LN was able to induce a similar, albeit milder phenotype. Treatment of MCs with cytokines or patient sera was able to induce secretion of TGF-β1, a known inducer of fibrotic changes. Inhibition of TGF-β1 actions through SB-431542 (an activin A receptor type II-like kinase (ALK5) inhibitor) was able to reduce these responses suggesting that the release of TGF-β1 plays a role in these changes.ConclusionsMCs contribute to the inflammatory environment in LN by producing cytokines involved in leukocyte recruitment, activation and maturation. Further the cells remodel the ECM via protein deposition and enzymatic degradation. This occurs through the actions of TGF-β1 on its receptor, ALK5. This may represent a potential therapeutic target for treatment of LN-associated fibrosis.