Objective: To explore the effect of microRNA-21 (miR-21) on myocardial fibrosis in mice with chronic viral myocarditis (CVMC) and related mechanisms. Methods: Forty 4-week-old Balb/c male mice were randomly divided into 4 groups (n=10 each): phosphate buffer saline (PBS) group, CVMC group, CVMC+miR-21 inhibitor group, CVMC+isotype control group. The first injection of Coxsackie virus B3 (CVB3) or PBS was performed on day 0, and the total study time was 42 days. Each mouse in CVMC group, CVMC+miR-21 inhibitor group and CVMC+isotype control group was intraperitoneally (i.p) injected with 100TCID50 CVB3 0.1, 0.15, and 0.2 ml on day 0, 14, and 28, respectively. The mice in PBS group were i.p injected with the same dose of PBS at the same time point. After the initial infection, each mouse in CVMC+miR-21 inhibitor group and CVMC+isotype control group was intravenously injected with 0.1 ml miR-21 inhibitor or 0.1 ml isotype control, on day 14 and 28. Cardiac function was measured on surviving mice of 4 groups by echocardiography on day 42. Then, the hearts were removed aseptically to observe the expressions of green fluorescence protein (GFP). The myocardial pathological changes were examined with HE, Masson staining and the myocardial pathological scores (PS), the collagen volume fraction (CVF) were calculated respectively. The levels of miR-21, collagen typeⅠ-A1 (COL1-A1) and collagen type Ⅲ-A1 (COL3-A1) mRNA in heart were detected by quantitative real-time polymerase chain reaction (RT-qPCR). Furthermore, the expressions of transforming growth factor-β1 (TGF-β1) and mothers against decapentaplegic homolog 7(Smad7) in heart were determined with Western blot assay. Results: (1) Cardiac function in 4 groups: Compared with PBS group, left ventricular end systolic diameter (LVESD) and left ventricular end diastolic diameter (LVEDD) were markedly increased in CVMC group and CVMC+isotype control group (all P<0.05), whereas the left ventricular ejection fraction (LVEF) was decreased (P<0.05). LVESD and LVEDD were significantly decreased, and LVEF was increased in CVMC+miR-21 inhibitor group compared with those in CVMC group and CVMC+isotype control group (all P<0.05). (2) Myocardial pathological changes: The expressions of GFP in CVMC+miR-21 inhibitor group and CVMC+isotype control group were visible in heart tissues frozen sections. The hearts in CVMC group and CVMC+isotype control group were enlarged and stiff, inflammatory cells were visible and significantly increased myocardial fibrosis was evidenced in mice of these two groups. Higher PS and CVF were evidenced in CVMC group (PS: 1.14±0.69 vs. 0, CVF: (17.86±2.61)% vs. (5.70±1.42)%, all P<0.05) and CVMC+isotype control group(PS: 1.00±0.63 vs. 0, CVF: (16.78±2.58)% vs. (5.70±1.42)%, all P<0.05) compared to PBS group. Compared with CVMC group and CVMC+isotype control group, degree of cardiac fibrosis was reduced in mice of CVMC+miR-21 inhibitor group (CVF: (11.01±2.55)% vs. (17.86±2.61)%, (11.01±2.55)% vs. (16.78±2.58)%, all P<0.05), whereas PS were similar between them (PS: 0.89±0.60 vs. 1.14±0.69, 0.89±0.60 vs. 1.00±0.63, all P>0.05). (3) Cardiac expressions of miR-21, COL1-A1 and COL3-A1 mRNA: The cardiac expressions of miR-21, COL1-A1 mRNA, COL3-A1mRNA in CVMC group and CVMC+isotype control group were markedly higher than those in PBS group (all P<0.05), which were significantly downregulated in CVMC+miR-21 inhibitor group (all P<0.05 vs. CVMC group and CVMC+isotype control group). (4) The cardiac expressions of TGF-β1 and Smad7 protein: The cardiac expressions of TGF-β1 protein in CVMC group and CVMC+isotype control group were markedly higher, whereas the cardiac Smad7 protein expressions were significantly lower (all P<0.05) than those in PBS group (all P<0.05), these changes could be reversed in CVMC+miR-21 inhibitor group (P<0.05 vs. CVMC group and CVMC+isotype control group). Conclusions: Our results suggest that miR-21 contributes to the myocardial fibrosis in CVMC mice through modulating TGF-β1/Smad7 signaling pathway.

Viral myocarditis (VMC) is an inflammation of heart muscle in infants and young adolescents. This study explored the function of halofuginone (HF) in Coxsackievirus B3 (CVB3) -treated suckling mice. HF-treated animal exhibited higher survival rate, lower heart/body weight, and more decreased blood sugar concentration than CVB3 group. HF also reduced the expressions of interleukin(IL)-17 and IL-23 and the numbers of Th17 cells. Moreover, HF downregulated pro-inflammatory cytokine levels and increased anti-inflammatory cytokine levels. The expressions of transforming growth factor(TGF-β1) and nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) p65/ tumor necrosis factor-α (TNF-α) proteins were decreased by HF as well. Finally, the overexpression of TGF-β1 counteracted the protection effect of HF in CVB3-treated suckling mice. In summary, our study suggests HF increases the survival of CVB3 suckling mice, reduces the Th17 cells and pro-inflammatory cytokine levels, and may through downregulation of the TGF-β1-mediated expression of NF-κB p65/TNF-α pathway proteins. These results offer a potential therapeutic strategy for the treatment of VMC.

UNASSIGNED

To study the transforming growth factor beta (TGF-β) signaling pathway in interactions with estrogen receptor alpha (ERα) signaling pathway mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs).

UNASSIGNED

The subcultured UL cells were used to determine the validation of TGF-β3 for the viability of human UL cells using CCK-8 assay, mRNA expressions of ERα, and c-fos by quantitative reverse transcription polymerase chain reaction method, and expressions of p-Smad3, SnoN, and c-fos proteins by Western blot assay in each treatment group.

UNASSIGNED

Compared with each of EEs or TGF-β3 treatment, slightly decrease in the proliferation rate of UL was detected in the coexistence of each EE with TGF-β3. Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs. Somehow, the expression of p-Smad3 and c-fos proteins significantly decreased in each of E2, bisphenol A (BPA), nonylphenol (NP), and octylphenol (OP) group, as well as the expression of SnoN protein significantly reduced only in BPA and NP groups, followed by TGF-β3 treatment. With the overlaid action of ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group, but slightly increased in E2, BPA, NP, and OP groups. However, compared with the control group, the expression of SnoN and c-fos proteins significantly decreased in the same setting.

UNASSIGNED

Both ERα signaling pathway and TGF-β signaling pathway have different roles in governing UL cell proliferation. The phenolic EEs can be a promoter to the proliferation of UL cells, which is mediated by ERα signaling pathway and cross-talked with TGF-β signaling pathway.

UNASSIGNED

Transforming growth factor-β (TGF-β) may inhibit the development of atherosclerosis. We evaluated serum levels of TGF-β isoforms concurrently with serum levels of endotoxin and various inflammatory markers. In addition, we determined if any association exists between polymorphisms in the TGF-β1 gene and atherosclerosis in South African CKD patients.

UNASSIGNED

We studied 120 CKD patients and 40 healthy controls. Serum TGF-β1, TGF-β2, TGF-β3, endotoxin, and inflammatory markers were measured. Functional polymorphisms in the TGF-β1 genes were genotyped using a polymerase chain reaction-sequence specific primer method and carotid intima media thickness (CIMT) was assessed by B-mode ultrasonography.

UNASSIGNED

TGF-β isoforms levels were significantly lower in the patients with atherosclerosis compared to patients without atherosclerosis (p<0.001). Overall, TGF-β isoforms had inverse relationships with CIMT. TGF-β1 and TGF-β2 levels were significantly lower in patients with carotid plaque compared to those without carotid plaque [TGF-β1: 31.9 (17.2 - 42.2) versus 45.9 (35.4 - 58.1) ng/ml, p=0.016; and TGF-β2: 1.46 (1.30 - 1.57) versus 1.70 (1.50 - 1.87) ng/ml, p=0.013]. In multiple logistic regression, age, TGF-β2, and TGF-β3 were the only independent predictors of subclinical atherosclerosis in CKD patients [age: odds ratio (OR), 1.054; 95% confidence interval (CI): 1.003 - 1.109, p=0.039; TGF-β2: OR, 0.996; 95% CI: 0.994-0.999, p=0.018; TGF-β3: OR, 0.992; 95% CI: 0.985-0.999, p=0.029). TGF-β1 genotypes did not influence serum levels of TGF-β1 and no association was found between the TGF-β1 gene polymorphisms and atherosclerosis risk.

UNASSIGNED

TGF-β isoforms seem to offer protection against the development of atherosclerosis among South African CKD patients.

End-to-end anastomosis in the treatment for bile duct injury during laparoscopic cholecystectomy has been associated with stricture formation. The aim of this study was to experimentally investigate the effect of oral tamoxifen (tmx) treatment on fibrosis, collagen content and transforming growth factor-β1, -β2 and -β3 expression in common bile duct anastomosis of pigs. Twenty-six pigs were divided into three groups [sham (n = 8), control (n = 9) and tmx (n = 9)]. The common bile ducts were transected and anastomosed in the control and tmx groups. Tmx (40 mg/day) was administered orally to the tmx group, and the animals were euthanized after 60 days. Fibrosis was analysed by Masson's trichrome staining. Picrosirius red was used to quantify the total collagen content and collagen type I/III ratio. mRNA expression of transforming growth factor (TGF)-β1, -β2 and -β3 was quantified using real-time polymerase chain reaction (qRT-PCR). The control and study groups exhibited higher fibrosis than the sham group, and the study group showed lower fibrosis than the control group (P = 0.011). The control and tmx groups had higher total collagen content than the sham group (P = 0.003). The collagen type I/III ratio was higher in the control group than in the sham and tmx groups (P = 0.015). There were no significant differences in the mRNA expression of TGF-β1, -β2 and -β3 among the groups (P>> 0.05). Tmx decreased fibrosis and prevented the change in collagen type I/III ratio caused by the procedure.

We investigated enhancement of articular cartilage regeneration using a combination of human adipose derived stem cells (hADSCs) and TGF-β3 microspheres (MS) in vivo. Poly-lactic-co-glycolic acid (PLGA)MS were prepared using a solid/oil/water emulsion solvent evaporation-extraction method. The morphology of the MS was evaluated by scanning electron microscopy (SEM). The release characteristic of the TGF-β3 MS was evaluated. A New Zealand rabbit model for experimental osteoarthritis (OA) was established using the anterior medial meniscus excision method. Thirty OA rabbits were divided randomly into three groups according to different treatments of the right knee joints on day 7 after surgery: hADSCs/MS group received injection of both hADSCs and TGF-β3 MS; hADSCs group was injected with hADSCs; control group was injected with normal saline. Gross observation, histological staining and RT-PCR for collagen II and aggrecan) were used to assess the severity of OA and for evaluating the effect of combined use of hADSCs and TGF-β3 MS on articular cartilage regeneration in vivo. The MS were spherical with a smooth surface and the average diameter was 28 ± 2.3 µm. The encapsulation efficiency test showed that 73.8 ± 2.9% of TGF-β3 were encapsulated in the MS. The release of TGF- β3 lasted for at least 30 days. At both 6 and 12 weeks after injection, three groups exhibited different degrees of OA. Histological analysis showed that the hADSCs/MS group exhibited less OA than the hADSCs group, and the control group exhibited the most severe OA. Real-time RT-PCR showed that the gene expression of both collagen II and aggrecan were significantly up-regulated in the hADSCs/MS group. At 12 weeks after injection, the hADSCs/MS group also exhibited less OA than the other two groups. Combined use of hADSCs and TGF-β3 MS promoted articular cartilage regeneration in rabbit OA models.

Chronic fatigue syndrome (CFS) is a prevalent and disabling condition among adolescent. The disease mechanisms are unknown. Previous studies have suggested elevated plasma levels of several cytokines, but a recent meta-analysis of 38 articles found that of 77 different cytokines measured in plasma, transforming growth factor beta (TGF-β) was the only one that was elevated in patients compared to controls in a sufficient number of articles. In the present study we therefore compared the plasma levels of the three TGF-β isoforms in adolescent CFS patients and healthy controls. In addition, the study explored associations between TGF-β levels, neuroendocrine markers, clinical markers and differentially expressed genes within the CFS group.

CFS patients aged 12-18 years (n = 120) were recruited nation-wide to a single referral center as part of the NorCAPITAL project (ClinicalTrials ID: NCT01040429). A broad case definition of CFS was applied, requiring 3 months of unexplained, disabling chronic/relapsing fatigue of new onset, whereas no accompanying symptoms were necessary. Healthy controls (n = 68) were recruited from local schools. The three isoforms of TGF-β (TGF-β1, TGF-β2, TGF-β3) were assayed using multiplex technology. Neuroendocrine markers encompassed plasma and urine levels of catecholamines and cortisol, as well as heart rate variability indices. Clinical markers consisted of questionnaire scores for symptoms of post-exertional malaise, inflammation, fatigue, depression and trait anxiety, as well as activity recordings. Whole blood gene expression was assessed by RNA sequencing in a subgroup of patients (n = 29) and controls (n = 18).

Plasma levels of all three isoforms of TGF-β were equal in the CFS patients and the healthy controls. Subgrouping according to the Fukuda and Canada 2003 criteria of CFS did not reveal differential results. Within the CFS group, all isoforms of TGF-β were associated with plasma cortisol, urine norepinephrine and urine epinephrine, and this association pattern was related to fatigue score. Also, TGF-β3 was related to expression of the B cell annotated genes TNFRSF13C and CXCR5.

Plasma levels of all TGF-β isoforms were not altered in adolescent CFS. However, the TGF-β isoforms were associated with neuroendocrine markers, an association related to fatigue score. Furthermore, TGF-β3 might partly mediate an association between plasma cortisol and B cell gene expression. Trial registration Clinical Trials NCT01040429.

Personal exposure to fungal bioaerosols derived from contaminated building materials or agricultural commodities may induce or exacerbate a variety of adverse health effects. The genomic mechanisms that underlie pulmonary immune responses to fungal bioaerosols have remained unclear.

The impact of fungal viability on the pulmonary microRNA and messenger RNA profiles that regulate murine immune responses was evaluated following subchronic inhalation exposure to Aspergillus fumigatus conidia.

Three groups of naïve B6C3F1/N mice were exposed via nose-only inhalation to A. fumigatus viable conidia, heat-inactivated conidia (HIC), or HEPA-filtered air twice a week for 13 weeks. Total RNA was isolated from whole lung 24 and 48 h postfinal exposure and was further processed for gene expression and microRNA array analysis. The molecular network pathways between viable and HIC groups were evaluated.

Comparison of data sets revealed increased Il4, Il13 and Il33 expression in mice exposed to viable vs. HIC. Of 415 microRNAs detected, approximately 50% were altered in mice exposed to viable vs. HIC 48 h postexposure. Significantly down-regulated (P ≤ 0.05) miR-29a-3p was predicted to regulate TGF-β3 and Clec7a, genes involved in innate responses to viable A. fumigatus. Also significantly down-regulated (P ≤ 0.05), miR-23b-3p regulates genes involved in pulmonary IL-13 and IL-33 responses and SMAD2, downstream of TGF-β signalling. Using Ingenuity Pathway Analysis, a novel interaction was identified between viable conidia and SMAD2/3.

Examination of the pulmonary genetic profiles revealed differentially expressed genes and microRNAs following subchronic inhalation exposure to A. fumigatus. MicroRNAs regulating genes involved in the pulmonary immune responses were those with the greatest fold change. Specifically, germinating A. fumigatus conidia were associated with Clec7a and were predicted to interact with Il13 and Il33. Furthermore, altered microRNAs may serve as potential biomarkers to evaluate fungal exposure.

Wnt-signalling cascade is one of the crucial pathways involved in the development and homeostasis of cartilage. Influencing this pathway can potentially contribute to improved cartilage repair or regeneration. One key molecular regulator of the Wnt pathway is the glycogen synthase kinase-3 enzyme, the inhibition of which allows initiation of the signalling pathway. This study aims to utilise a binary SiO2-Li2O sol-gel derived glass for controlled delivery of lithium, a known glycogen synthase kinase-3 antagonist. The effect of the dissolution products of the glass on chondrogenic differentiation in an in vitro 3D pellet culture model is reported. Dissolution products that contained 5 mM lithium and 3.5 mM silicon were capable of inducing chondrogenic differentiation and hyaline cartilaginous matrix formation without the presence of growth factors such as TGF-β3. The results suggest that sol-gel derived glass has the potential to be used as a delivery vehicle for therapeutic lithium ions in cartilage regeneration applications.

Scar formation in severe burn injury is a major health concern. Herein, we developed a hybrid collagen scaffold with an incorporated ZnO-curcumin nanocomposite, which facilitates scarless wound healing. Biocompatibility and hemocompatibility studies unveiled that the hybrid scaffold is apt for in vivo wound healing studies. Histological and immunohistochemical analyses demonstrate that the hybrid scaffold accelerated scarless burn wound healing in albino rats owing to the ZnO-curcumin nanocomposite induced up-regulation of angiogenesis and TGF-β3 expression. The semi-quantitatively measured scar elevation index of the hybrid scaffold-treated animals is on a par with that of the unwounded or normal skin. The studies suggest that the prepared hybrid biomaterial could be a potential candidate for scarless healing in severe burn injuries.

As cartilage tissue lacks the innate ability to mount an adequate regeneration response, damage to it is detrimental to the quality of life of the subject. The emergence of three-dimensional bioprinting (3DBP) technology presents an opportunity to repair articular cartilage defects. However, widespread adoption of this technique has been impeded by difficulty in preparing a suitable bioink and the toxicity inherent in the chemical crosslinking process of most bioinks. Our objective was to develop a crosslinker-free bioink with the same biological activity as the original cartilage extracellular matrix (ECM) and good mechanical strength. We prepared bioinks containing different concentrations of silk fibroin and decellularized extracellular matrix (SF-dECM bioinks) mixed with bone marrow mesenchymal stem cells (BMSCs) for 3D bioprinting. SF and dECM interconnect with each other through physical crosslinking and entanglement. A porous structure was formed by removing the polyethylene glycol from the SF-dECM bioink. The results showed the SF-dECM construct had a suitable mechanical strength and degradation rate, and the expression of chondrogenesis-specific genes was found to be higher than that of the SF control construct group. Finally, we confirmed that a SF-dECM construct that was designed to release TGF-β3 had the ability to promote chondrogenic differentiation of BMSCs and provided a good cartilage repair environment, suggesting it is an ideal scaffold for cartilage tissue engineering.

Keywords: 3D bioprinting; Articular cartilage; Bioink; Bone marrow mesenchymal cells; Chondrogenesis; Extracellular matrix; Silk fibroin.

OBJECTIVE

We investigated the role of human cytomegalovirus (HCMV) and its mechanism in extravillous cytotrophoblast (EVT) proliferation and invasion in vitro.

METHODS

Differential enzymatic digestion combined with gradient centrifugation, was used to isolate primary EVT from human chorionic villi collected from early placentae of healthy pregnant women. HCMV infection was determined by immunofluorescence staining of HCMVpp65 antigen expression. An MTT assay was used to examine the role of HCMV in the proliferation of EVT. Quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemical staining and Western blots were carried out in a control group (EVT) and a virus group (EVT+HCMV) to examine the expression of major genes and protein in TGF-β/Smad signaling pathways in EVT 48 h after inoculation with HCMV. An in vitro cell invasion assay was performed to analyze the influence of HCMV on EVT invasion.

RESULTS

HCMV significantly inhibited the proliferation of EVT 48 h after viral infection (P < 0.05). The expression of TGF-β1, Smad1, Smad2, Smad3, Smad4, and Smad5 genes was significantly increased (P < 0.05), but that of TGF-β2, TGF-β3, TGFβRI, TGFβRII, Smad7, MMP2, and MMP9 was significantly decreased in the virus group 48 h after HCMV infection (P < 0.05). Smad7, MMP-2 and MMP-9 protein levels were significantly decreased and the TGF-β1 protein level was significantly increased in infected EVT (all P < 0.05).

CONCLUSIONS

HCMV may act on multiple steps of the TGF-β/Smad signaling pathway to impede EVT proliferation and invasion.

Human induced pluripotent stem cells (hiPSCs) offer promise in regenerative medicine, however more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte‑like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC‑402‑05a cells or supplemented with transforming growth factor β3 (TGF‑β3), and to assess the relative utility of the most commonly used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from the mesoderm and thus share a wide range of properties with chondrocytes, which also originate from the mesenchyme. Thus, the exclusion of dedifferentiation instead of chondrogenic differentiation is crucial. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in a chondrogenic medium supplemented with TGF‑β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC‑402‑05a cells. Based on immunofluorescence and reverse transcription‑quantiative polymerase chain reaction analysis, the results indicated that hiPSCs have the capacity for effective chondrogenic differentiation, in particular cells differentiated in the HC‑402‑05a‑conditioned medium, which present morphological features and markers that are characteristic of mature human chondrocytes. By contrast, cells differentiated in the presence of TGF‑β3 may demonstrate hypertrophic characteristics. Several genes [paired box 9, sex determining region Y-box (SOX) 5, SOX6, SOX9 and cartilage oligomeric matrix protein] were demonstrated to be good markers of early hiPSC chondrogenic differentiation: Insulin‑like growth factor 1, Tenascin‑C, and β‑catenin were less valuable. These observations provide valuable data on the use of hiPSCs in cartilage tissue regeneration.

The purpose of this study is to examine if aqueous autotaxin (ATX) and TGF-β levels could be used for differentiating glaucoma subtypes. This prospective observational study was performed using aqueous humor samples obtained from 281 consecutive patients. Open angle glaucoma patients were classified into three groups: primary open-angle glaucoma (POAG), secondary open-angle glaucoma (SOAG), and exfoliation glaucoma (XFG). Aqueous levels of ATX and TGF-βs were quantified. The AUC as well as sensitivity and specificity for the classification into normal and glaucoma subtypes using four indicators-ATX, TGF-β1, TGF-β2, and TGF-β3, upon the application of three machine learning methods. ATX, TGF-β1, and TGF-β3 were positively correlated with IOP, and ATX was significantly and negatively correlated with the mean deviation. From least absolute shrinkage and selection operator regression analysis, the AUC values to distinguish each subgroup [normal, POAG, SOAG, and XFG] ranged between 0.675 (POAG vs. normal) and 0.966 (XFG vs. normal), when four variables were used. High AUC values were obtained with ATX for discriminating XFG from normal eyes and with TGF-β3 for discriminating XFG from normal eyes, POAG, or SOAG. Aqueous TGF-β and ATX exhibited high diagnostic performance in detecting glaucoma subtypes, and could be promising biomarkers for glaucoma.

Expression of indoleamine 2,3-dioxygenase (IDO) in mesenchymal stem cells (MSC) is thought to contribute to MSC-mediated immunosuppression. A lentiviral-based transgenic system was used to generate bone marrow stem cells (BMSC) which stably expressed IDO (IDO-BMSCs). Coculture of IDO-BMSCs with dendritic cells (DC) or T cells was used to evaluate the immunomodulatory effect of IDO-BMSCs. A heterotopic heart transplant model in rats was used to evaluate allograft rejection after IDO-BMSC treatment. Mechanisms of IDO-BMSC-mediated immunosuppression were investigated by evaluating levels of proinflammatory and anti-inflammatory cytokines, and production of Tregs. A significant decrease in DC marker-positive cells and a significant increase in Tregs were observed in IDO-BMSC cocultured. Treatment of transplanted rats with IDO-BMSCs was associated with significantly prolonged graft survival. Compared with the control groups, transplanted animals treated with IDO-BMSCs had a (1) significantly higher ejection fraction and fractional shortening, (2) significantly lower expression of CD86, CD80, and MHCII, and significantly higher expression in CD274, and Tregs, and (3) significantly higher levels of interleukin-10 (IL-10), transforming growth factor beta-1 (TGF-β1), TGF-β2, and TGF-β3, and significantly lower levels of IL-2 and interferon gamma. Our results expand our understanding of the molecular mechanisms underlying suppression of heart allograft rejection via IDO-expressing BMSCs.

Electrospinning is a valuable technology for cartilage tissue engineering (CTE) due to its ability to produce fibrous scaffolds mimicking the nanoscale and alignment of collagen fibers present within the superficial zone of articular cartilage. Coaxial electrospinning allows the fabrication of core-shell fibers able to incorporate and release bioactive molecules (e.g., drugs or growth factors) in a controlled manner. Herein, we used coaxial electrospinning to produce coaxial poly(glycerol sebacate) (PGS)/poly(caprolactone) (PCL) aligned nanofibers (core:PGS/shell:PCL). The obtained scaffolds were characterized in terms of their structure, chemical composition, thermal properties, mechanical performance and in vitro degradation kinetics, in comparison to monoaxial PCL aligned fibers and respective non-aligned controls. All the electrospun scaffolds produced presented average fiber diameters within the nanometer-scale and the core-shell structure of the composite fibers was clearly confirmed by TEM. Additionally, fiber alignment significantly increased (>2-fold) the elastic modulus of both coaxial and monoxial scaffolds. Kartogenin (KGN), a small molecule known to promote mesenchymal stem/stromal cells (MSC) chondrogenesis, was loaded into the core PGS solution to generate coaxial PGS-KGN/PCL nanofibers. The KGN release kinetics and scaffold biological performance were evaluated in comparison to KGN-loaded monoaxial fibers and respective non-loaded controls. Coaxial PGS-KGN/PCL nanofibers showed a more controlled and sustained KGN release over 21 days than monoaxial PCL-KGN nanofibers. When cultured with human bone marrow MSC in incomplete chondrogenic medium (without TGF-β3), KGN-loaded scaffolds enhanced significantly cell proliferation and chondrogenic differentiation, as suggested by the increased sGAG amounts and chondrogenic markers gene expression levels. Overall, these findings highlight the potential of using coaxial PGS-KGN/PCL aligned nanofibers as a bioactive scaffold for CTE applications.

Transforming growth factor beta-3 (TGF-β3) has been shown to decrease scar formation after scheduled topical applications to the cutaneous wounds. This study aimed to continuously deliver TGF-β3, during the early phase of wound healing, by engineering a dermal equivalent (DE) using TGF-β3 expressing bone marrow stromal cells (BM-SCs) and human dehydrated amniotic membrane (hDAM). To engineer a DE, rat BM-SCs were seeded on the hDAM and TGF-β3 was transiently transfected into the BM-SCs using a plasmid vector. Pieces of the dermal equivalent were transplanted onto the full-thickness excisional skin wounds in rats. The process of wound healing was assessed by image analysis, Manchester Scar Scale (MSS), and histopathological studies 7, 14, 21, and 85 days after the excision. The results confirmed accurate construction of recombinant pcDNA3.1-TGF-β3 expression system and showed that the transfected BM-SCs seeded on hDAM expressed TGF-β3 mRNA and protein from day 3 through day 7 after transfection. After implantation of the DE, contraction of the wounds was measured from day 7 through 21 and analyzed by linear regression, which revealed that the rate of wound contraction in all experimental groups was similar. Histologic evaluation demonstrated that transfected BM-SCs decreased retention and recruitment of the cells during the early stage of wound healing, decreased the formation of vascular structures and led to formation of uniformly parallel collagen bundles. MSS scores showed that TGF-β3 secreting cells significantly improved the cosmetic appearance of the healed skin and decreased the scar formation. From these results, it could be concluded that transient secretion of TGF-β3, during the early phase of healing, by BM-SCs seeded on hDAM can improve the cosmetic appearance of the scar in cutaneous wounds without negatively affecting the process of wound repair.

Leucine-rich α-2-glycoprotein-1 (LRG1) is involved in several pathophysiological processes, including angiogenesis, cutaneous wound repair and cancer metastasis. In this study, we investigated the potential role and mechanism of LRG1 in corneal re-epithelialisation and nerve regeneration in streptozotocin-induced diabetic mice. We found decreased levels of LRG1 in the corneal epithelium after wounding in diabetic mice compared to normal controls. Hyperglycaemia downregulated the LRG1 expression in the corneal epithelium in vivo, as well as in vitro in a cultured mouse corneal epithelial stem/progenitor cell line (TKE2 cells) exposed to high glucose (HG; 30 mM) in the culture medium. Exogenous application of LRG1 accelerated corneal re-epithelialisation and nerve regeneration in normal mice and diabetic mice. LRG1 also overcame the suppression of wound healing in TKE2 cells by HG conditions, and it activated repair-related signalling by JAK2/STAT3, AKT, epidermal growth factor receptor (EGFR) and transforming growth factor (TGF)-β3. We also found that LRG1 treatment overcame the hyperglycaemia-suppressed expression of matrix metalloproteinase 3 (MMP3) and metalloproteinase 13 (MMP13) in the regenerated corneal epithelium. The promoted effects of LRG1 on corneal re-epithelialisation and nerve regeneration were blocked by inhibitors of MMP3 and MMP13. Subconjunctival injection of 0.5 μg MMP inhibitors did not cause any obvious toxic damage in corneal epithelial cells. Immunoprecipitation and proximity ligation assay experiments confirmed that endogenous LRG1 coprecipitated with MMP3 and MMP13 in TKE2 cells. These results indicate that LRG1 promoted wound repair and nerve regeneration in the diabetic corneal epithelium by regulation of MMPs. Our findings reveal a new function and mechanism for LRG1 in the cornea, and they provide new insights for a better understanding of diabetic keratopathy.

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of transforming growth factor beta 3 (TGF-β3). Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGF-β signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 breast cancer patients. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are promising for regenerating degenerated intervertebral discs (IVDs), but the low efficiency of nucleus pulposus (NP)-specific differentiation limits their clinical applications. The Sonic hedgehog (Shh) signaling pathway is important in NP-specific differentiation of ADSCs, and Smoothened Agonist (SAG) is a highly specific and effective agonist of Shh signaling. In this study, we proposed a new differentiation strategy with the use of the small molecule SAG. The NP-specific differentiation and extracellular matrix (ECM) synthesis of ADSCs were measured in vitro , and the regenerative effects of SAG pretreated ADSCs in degenerated IVDs were verified in vivo . The results showed that the combination of SAG and transforming growth factor-β3 (TGF-β3) is able to increase the ECM synthesis of ADSCs. In addition, the gene and protein expression levels of NP-specific markers were increased by treatment with SAG and TGF-β3. Furthermore, SAG pretreated ADSCs can also improve the disc height, water content, ECM content, and structure of degenerated IVDs in vivo . Our new differentiation scheme has high efficiency in inducing NP-specific differentiation of ADSCs and is promising for stem cell-based treatment of degenerated IVDs.

Synovial fluid-derived mesenchymal stem cells (SF-MSCs) represent a superior source of stem cells and have great potential for autologous transplantation for cartilage regeneration. Transforming growth factor-β3 (TGF-β3) has been demonstrated to stimulate the chondrogenic differentiation of MSCs. Recently, the small molecule kartogenin (KGN) was reported to enhance chondrogenic differentiation and cartilage regeneration. The effects of KGN and TGF-β3 on the in vitro chondrogenic differentiation of rabbit SF-MSCs were studied. The monolayer and pellet cultures of rabbit SF-MSCs were stimulated in vitro using either KGN or TGF-β3 alone or in combination for 21 days. The in vivo therapeutic effects of KGN combined with TGF-β3 were studied using an intra-articular delivery of autologous rabbit SF-MSCs to cartilage defects in a rabbit model. Compared to a single treatment, the in vitro results demonstrated that the combination of KGN and TGF-β3 resulted in significantly increased protein expression levels of type II collagen (COL II) and SRY-box 9 (SOX9) and decreased the expression level of type X collagen (COL X). Compared with the regenerated cartilage in the single treatment groups, the intra-articular injection of rabbit SF-MSCs mixed with TGF-β3 and KGN exhibited substantial amounts of regenerated cartilage in the defective areas in the medial femoral condyles. We noted that the thicker, hyaline-like cartilaginous tissue contained abundant levels of extracellular matrix, which is characteristic of cartilage. This study demonstrated that TGF-β3 and KGN exhibit synergistic effects for the promotion of the chondrogenesis of rabbit SF-MSCs and can effectively repair cartilage defects through the regeneration of hyaline cartilage.

Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimuli-responsive delivery systems can facilitate 'on-demand' release of therapeutics in response to a variety of physiologic triggering mechanisms (e.g. pH, temperature). However, few systems to date have taken advantage of mechanical inputs from the microenvironment to initiate drug release. Here, we developed mechanically-activated microcapsules (MAMCs) that are designed to deliver therapeutics in an on-demand fashion in response to the mechanically loaded environment of regenerating musculoskeletal tissues, with the ultimate goal of furthering tissue repair. To establish a suite of microcapsules with different thresholds for mechano-activation, we first manipulated MAMC physical dimensions and composition, and evaluated their mechano-response under both direct 2D compression and in 3D matrices mimicking the extracellular matrix properties and dynamic loading environment of regenerating tissue. To demonstrate the feasibility of this delivery system, we used an engineered cartilage model to test the efficacy of mechanically-instigated release of TGF-β3 on the chondrogenesis of mesenchymal stem cells. These data establish a novel platform by which to tune the release of therapeutics and/or regenerative factors based on the physiologic dynamic mechanical loading environment, and will find widespread application in the repair and regeneration of numerous musculoskeletal tissues.

Keywords: biomaterials; drug delivery; microcapsules; stimuli-responsive; tissue engineering.

The blood-brain barrier (BBB) is a functional structure which regulates and restricts the transfer of circulating molecules and immune cells into the central nervous system. The barrier is formed by the presence of tight junctions (TJ) between the specialized brain endothelial cells. The volatile anesthetic isoflurane may affect the permeability of the BBB. Previous studies have proven that isoflurane alters hypoxia-inducible factor-1α (HIF-1α) expression, which may affect the TJ proteins; however, the mechanism of how TJ proteins are affected by isoflurane is still unclear. Primary human brain vascular endothelial cells (HBVEC) were exposed to isoflurane at various concentrations (0-2.5%) and different time periods (0-6 h). The cell viability, occludin expression, paracellular permeability, VEGF expression, TGF-β3 expression and occludin protein endocytosis were quantified. Isoflurane treatment induced a time- and concentration-dependent decrease in occludin mRNA and protein levels in HBVEC. This effect was partially abrogated by silencing the HIF-1α expression. Isoflurane could activate HIF-1α, and the overexpression HIF-1α up-regulated the level of VEGF and TGF-β3, VEGF decreased the expression of occludin and TGF-β3 accelerated the endocytosis of occludin. RNA interference targeting HIF-1α reduced both VEGF and TGF-β3 expression after isoflurane treatment.

CONCLUSIONS

This study provides direct evidence in vitro that exposing isoflurane to HBVECs can trigger HIF-1α activation, leading to lower protein levels of occludin, and increased permeability of the BBB.