Regulatory T cells (Treg cells) play crucial roles in the induction of peripheral tolerance to self- and foreign-antigens. IL-10-producing regulatory T cells (IL-10-producing Treg cells) constitute a Treg cell subset characterized by the production of high amounts of IL-10, cytokine-mediated immunosuppressive capabilities, and independence of Foxp3 expression for their suppressive activity. In the past decade, identifying naturally occurring IL-10-producing Treg cells was difficult due to the lack of suitable surface markers. More recently, lymphocyte activation gene 3 (LAG-3) is a CD4 homologue that has been identified as a marker for IL-10-producing Treg cells. CD4+CD25-LAG3+ T cells produce large amounts of IL-10 and suppress colitis in a mouse model. These CD4+CD25-LAG3+ Treg cells also exhibit suppressive activity in murine models of lupus and humoral immunity in a TGF-β3-dependent manner. Moreover, the combined expression of LAG-3 and CD49b identifies IL-10-producing Treg cells in mice and humans more specifically. Recently, LAG-3 has gained more attention in the context of immune checkpoints because it believed to be related to T cell tolerance and exhausted T cells that infiltrate the tumor microenvironment. Tumors and the tumor microenvironment promote development of IL-10-producing Treg cells and foster tumor growth. This response might interfere with protective immune responses. Understanding LAG-3-expressing IL-10-producing Treg cells may contribute to the development of novel therapeutic strategies in immune-mediated diseases.

BACKGROUND

Recently, much interest has been generated in the use of intense pulsed light (IPL) sources in the treatment of various skin conditions. However, the underlying mechanism for its therapeutic action has not been elucidated.

OBJECTIVE

To investigate the effect of IPL on the in vivo expression of transforming growth factor beta1 (TGF-β1) and on the immunolocalization of Smad3 in biopsies obtained from perilesional skin in patients with mild-to-moderate inflammatory acne vulgaris.

METHODS

Biopsies obtained from 20 patients with inflammatory acne vulgaris at baseline (B1) and post-IPL treatment (B2 = 48 h after first treatment and B3 = 1 week after final treatment) were immunohistochemically analyzed to determine the expression of TGF-β1 and the immunolocalization of Smad3. Digital images were semiquantitatively assessed using image analysis software.

RESULTS

Intense pulsed light elicited a consistent increase in epidermal TGF-β1 expression (B2 vs. B1: P = 0.004 and B3 vs. B1: P = 0.007). Furthermore, it resulted in enhanced nuclear immunolocalization of Smad3 (B2 vs. B1: epidermis, P = 0.000055 and dermis, P = 0.014; B3 vs. B1: epidermis, P = 0.00024 and dermis, P = 0.008).

CONCLUSIONS

Intense pulsed light upregulates TGF-β1/Smad3 signaling in perilesional skin obtained from patients with mild-to-moderate inflammatory acne vulgaris. Further experiments on lesional skin and downstream effects are warranted to determine whether it may play a role in IPL-induced resolution of acne vulgaris.

BACKGROUND

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by recurrent epistaxis, mucocutaneous telangiectasia, and arteriovenous malformations. The efficacy of traditional treatments for HHT is very limited. The aim of this study was to investigate the therapeutic role of thalidomide in HHT patients and the effect in FLI-EGFP transgenic zebrafish model.

METHODS

HHT was diagnosed according to Shovlin criteria. Five HHT patients were treated with thalidomide (100 mg/d). The Epistaxis Severity Score (ESS), telangiectasia spots, and hepatic computed tomography angiography (CTA) were used to assess the clinical efficacy of thalidomide. The Fli-EGFP zebrafish model was investigated for the effect of thalidomide on angiogenesis. Dynamic real-time polymerase chain reaction assay, ELISA and Western blotting from patient's peripheral blood mononuclear cells and plasma were used to detect the expression of transforming growth factor beta 3 (TGF-β3) messenger RNA (mRNA) and vascular endothelial growth factor (VEGF) protein before and after 6 months of thalidomide treatment.

RESULTS

The average ESS before and after thalidomide were 6.966 ± 3.093 and 1.799 ± 0.627, respectively (P = 0.009). The "telangiectatic spot" on the tongue almost vanished; CTA examination of case 2 indicated a smaller proximal hepatic artery and decreased or ceased hepatic artery collateral circulation. The Fli-EGFP zebrafish model manifested discontinuous vessel development and vascular occlusion (7 of 10 fishes), and the TGF-β3 mRNA expression of five patients was lower after thalidomide therapy. The plasma VEGF protein expression was down-regulated in HHT patients.

CONCLUSIONS

Thalidomide reverses telangiectasia and controls nosebleeds by down-regulating the expression of TGF-β3 and VEGF in HHT patients. It also leads to vascular remodeling in the zebrafish model.

The present study aimed to investigate the mechanisms underlying microRNA (miRNA)-mediated regulation of chondrogenic differentiation. Mouse embryo-derived stem cells C3H10T1/2 were cultured and chondrogenic differentiation was induced using transforming growth factor-β3 (TGF-β3). In addition, miRNA expression profiles were detected via miRNA array analysis, and quantitative polymerase chain reaction was performed to verify the differentially expressed miRNAs. Furthermore, bioinformatics software was used to predict the putative targets and the prediction was validated by dual-luciferase reporter assays and western blot analysis. In addition, cell proliferation and glycosaminoglycans were measured by a direct cell count method and alcian blue staining, respectively. Compared with the control group, 86 miRNAs were identified as differentially expressed in TGF-β3-induced cells and the expression levels of 28 miRNAs were increased while the remaining 58 miRNAs exhibited a decline in expression. Amongst the differentially expressed miRNAs, miR-30b expression was observed to have significantly decreased during chondrogenic differentiation. SOX9 is a target gene of miR-30b, and miR-30b inhibits SOX9 expression during chondrogenic differentiation. Furthermore, the alcian blue staining results demonstrated that miR-30b inhibited early chondrogenic differentiation. However, the data of the present study indicated that miR-30b had no influence on C3H10T1/2 cell line proliferation. In conclusion, miR-30b is a key negative regulator of TGF-β3-induced C3H10T1/2 cell chondrogenic differentiation, which functions by directly targeting SOX9.

BACKGROUND

Intestinal fibrosis is a serious and often recurrent complication of inflammatory bowel disease despite surgical intervention. The anti-fibrotic potential of prostaglandin E2 (PGE2) and polyenylphosphatidylcholine (PC) was investigated using the murine model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced chronic intestinal inflammation and fibrosis, and murine and human intestinal myofibroblasts.

METHODS

Mice were treated with TNBS enemas weekly for 2 or 6 weeks ± PGE2 (10 mg/kg/day orally) or PC (200 mg/kg/day orally). Inflammation and fibrosis were histologically assessed and scored. Pro-inflammatory cytokines, TLR4, and ECM-related gene expression from the colonic tissue and cultured myofibroblasts were assessed by RT-qPCR. The levels of α-SMA(+) staining and endogenous PGE2 in vivo were also assessed.

RESULTS

Both PGE2 and PC treatment significantly decreased TNBS-induced intestinal inflammation and excess collagen deposition in vivo. This was accompanied by decreased α-SMA(+) staining in the lamina propria and lower collagen type I (COL1α1) expression. Endogenous PGE2 levels demonstrated that PC was not being converted into PGE2, thus mediating its effects primarily via PGE2-independent pathways. Both PGE2 and the PC isoform, 1,2-dilinoleoylphosphatidylcholine (DLPC), regulated primary mouse myofibroblast and CCD-18co COL1α1 production, and induced lower collagen type I to III and TGF-β1 to TGF-β3 ratios, demonstrating their ability to induced normal healing in the presence of phorbol 12-myristate 13-acetate (protein kinase C-dependent inducer of collagen production).

CONCLUSIONS

PGE2 and PC both have potent anti-fibrogenic potentials in their ability to regulate inflammatory cell and myofibroblast accumulation within inflamed tissue, to decrease pro-inflammatory cytokine expression and to maintain normal healing in an inflammatory environment.

Compensatory growth is mediated by multiple cell types that interact during organ repair. To elucidate the relationship between stem/progenitor cells that proliferate or differentiate and somatic cells of the lung, we used a novel organotypic ex vivo pneumoexplant system. Applying this technique, we identified a sustained culture of repopulating adult progenitors in the form of free-floating anchorage-independent cells (AICs). AICs did not express integrin proteins α5, β3 and β7, and constituted 37% of the total culture at day 14, yielding a mixed yet conservative population that recapitulated RNA expression patterns of the healthy lung. AICs exhibited rapid proliferation manifested by a marked 60-fold increase in cell numbers by day 21. More than 50% of the AIC population was c-KIT(+) or double-positive for CD45(+) and CD11b(+) antigenic determinants, consistent with cells of hematopoietic origin. The latter subset was found to be enriched with prosurfactant protein-C and SCGB1A1 expressing putative stem cells and with aquaporin-5 producing cells, characteristic of terminally differentiated alveolar epithelial type-1 pneumocytes. At the air/gel interface, AICs undergo remodeling to form a cellular lining, whereas TGF(β)1 treatment modifies protein expression properties to further imply a robust effect of the microenvironment on AIC phenotypic changes. These data confirm the active participation of clonogenic hematopoietic stem cells in a mammalian model of lung repair and validate mixed stem/somatic cell cultures, which license sustained cell viability, proliferation and differentiation, for use in studies of compensatory pulmonary growth.

NK4 may be a promising agent to inhibit tumor invasion and metastasis. To observe the effects of NK4 on the cardiovascular system with pathological injury and to discuss the mechanism, we established an experimental model of viral myocarditis (VCM) by coxsackievirus B3 infection in Balb/c mice on Day 0 and administered NK4 twice daily to the VCM and control mice from Day 20 to Day 45. We then evaluated the cardiac function by means of ultrasonic inspection. Hepatocyte growth factor, TNF (tumor necrosis factor)-alpha, and angiotensin II levels in the myocardial tissue were measured with enzyme-linked immunosorbent assay. Myocardium histopathology was examined with hematoxylin and eosin stain. Collagen deposition of the myocardium was detected through Masson staining. Microvessel staining with the RECA antibody and apoptosis detection with terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling were performed in the myocardium. The changes in MMP3 (matrix metalloproteinase 3), MMP9, TIMP1 (tissue inhibitor of metalloproteinase 1), and TGF (transforming growth factor)-beta1 expression in the myocardium were measured by reverse-transcriptase polymerase chain reaction. We found that NK4 intervention increased TGF-beta and angiotensin II expression, suppressed MMPs, improved the activities of TIMPs, and then promoted collagen deposition in the myocardium. NK4 intervention also decreased the microvessels' density and increased the apoptotic cell count in the myocardia of VCM mice. However, we did not observe the obvious changes in the myocardia of control mice after NK4 intervention. These data suggest that NK4 made negative impacts on the restoration of cardiac function and the recovery from VCM in the experimental mice.

This study employed transforming growth factor beta 3 (TGF-β3) induction of the C3H10T1/2 mesenchymal stem cell (MSC) line to construct an in vitro chondrogenic differentiation model for MSCs. A C3H10T1/2 MSC cell line was cultured, amplified, and the seventh generation of cells was centrifuged to construct pellets, which were divided into a non-induced group and an induced group (treated with TGF-β3, vitamin C, dexamethasone, and ITS). Specimens were taken after 7, 14, 21, and 28 days under non-induced and induced culture to compare these two groups by Alcian Blue staining, collagen type II immunohistochemical staining, and transmission election microscopy (TEM) on days 21 and 42. Cell pellets in the non-induced group were smaller than those in the induced group on days 7, 14, 21, and 28 with the pellet morphology of the induced group being more regular and nearly spherical. Alcian blue staining in the induced group was consistently stronger than that in non-induced group across all time points, and type II collagen immunohistochemical IOD values were significantly higher in the induced group over the non-induced group across all time points. On days 21 and 42, TEM revealed that the induced group displayed greater karyokinesis and a higher euchromatin ratio compared to the non-induced group. This specially constructed pellet model treated with TGF-β3-containing chondrogenic medium can effectively promote chondrogenic differentiation of C3H10T1/2 MSC cells in vitro. This in vitro pellet model should be of value in providing a preliminary cell model reference for further studies of the mechanism of chondroblast differentiation of stem cells.

Neuronal protein 3.1 (P311), a conserved RNA-binding protein, represents the first documented protein known to stimulate transforming growth factor (TGF)-β1 to -β3 translation in vitro and in vivo. Because TGF-βs play critical roles in fibrogenesis, we initiated efforts to define the role of P311 in skin scar formation. Here, we show that P311 is up-regulated in skin wounds and in normal and hypertrophic scars. Genetic ablation of p311 resulted in a significant decrease in skin scar collagen deposition. Lentiviral transfer of P311 corrected the deficits, whereas down-regulation of P311 levels by lentiviral RNA interference reproduced the deficits seen in P311-/- mice. The decrease in collagen deposition resulted in scars with reduced stiffness but also reduced scar tensile strength. In vitro studies using murine and human dermal fibroblasts showed that P311 stimulated TGF-β1 to -β3 translation, a process that involved eukaryotic translation initiation factor 3 subunit b as a P311 binding partner. This resulted in increased TGF-β levels/activity and increased collagen production. In addition, P311 induced dermal fibroblast activation and proliferation. Finally, exogenous TGF-β1 to -β3, each restituted the normal scar phenotype. These studies demonstrate that P311 is required for the production of normal cutaneous scars and place P311 immediately up-stream of TGF-βs in the process of fibrogenesis. Conditions that decrease P311 levels could result in less tensile scars, which could potentially lead to higher incidence of dehiscence after surgery.

Skin injury in adult mammals brings about a series of events and inflammation in the wounded area is initiated first and provides lots of inflammatory factors, which is critical for the final scar formation. While the postinjured skin of fetus and nude mice heals scarlessly owing to the absence of inflammation or immunodeficient, we designed a feasible acid-responsive ibuprofen-loaded poly(L-lactide) (PLLA) fibrous scaffolds via doping sodium bicarbonate to prevent excessive inflammation and achieve scarless healing finally. The morphological results of in vivo experiments revealed that animals treated with acid-responsive ibuprofen-loaded PLLA fibrous scaffolds exhibited alleviative inflammation, accelerated healing process, and regulated collagen deposition via interference in the collagen distribution, the α-smooth muscle actin (α-SMA), and the basic fibroblast growth factor (bFGF) expression. The lower ratios of collagen I/collagen III and TGF-β1/TGF-β3 and higher ratio of matrix metalloproteinase-1 (MMP-1)/tissue inhibitor of metalloproteinase-1 (TIMP-1) in acid-responsive ibuprofen-loaded PLLA fibrous scaffolds group were confirmed by real-time qPCR as well. These results suggest that inhibiting the excessive inflammation will result in regular collagen distribution and appropriate ratio between the factors, which promote or suppress the scar formation, then decrease the scar area, and finally achieve the scarless healing.

Background. The purpose of this study was to evaluate the effects of ASCs on full-thickness skin grafts. Specifically, we investigated the anti-inflammatory effects of ASCs that are mediated via regulation of the phenotypes of activated macrophages. Methods. ASCs were isolated, cultured, and injected under full-thickness skin grafts in 15 rats (ASC group). An additional 15 rats served as controls (PBS group). Skin graft survival assessment and vascularization detection were assessed with H&E staining and laser Doppler blood flowmetry (LDF). The effects of ASCs on angiogenesis, anti-inflammation, collagen accumulation-promoting, and antiscarring were assessed. Results. We found that the skin graft survival rate was significantly increased in the ASC group. The neovascularization, collagen deposition, collagen type I to type III ratio, and levels of VEGF and TGF-β3 in the ASC group were markedly higher than those in the PBS group at day 14. Additionally, in the ASC group, the levels of iNOS, IL-1β, and TNF-α were remarkably decreased, whereas the levels of IL-10 and Arg-1 were substantially increased. Conclusions. Our results confirm that ASCs transplantation can effectively improve full-thickness skin graft survival. Additionally, the anti-inflammatory role of ASCs may indirectly contribute to skin graft survival via its effect on macrophage polarization.

Adipose-derived stem cells (ADSCs) are promising for cartilage repair due to their easy accessibility and chondrogenic potential. Although chondrogenesis of transforming growth factor-β (TGF-β) mediated mesenchymal stem cells (MSCs) is well established in vitro, clinical tissue engineering requires effective and controlled delivery of TGF-β in vivo. In this work, a self-assembled peptide scaffold was employed to construct cartilages in vivo through the chondrogenesis from ADSCs controlled by recombinant fusion protein LAP-MMP-mTGF-β3 that was transfected by lentiviral vectors. During this course, the addition of matrix metalloproteinases (MMPs) can trigger the release of mTGF-β3 from the recombinant fusion protein of LAP-MMP-mTGF-β3 in the combined scaffolds, thus stimulating the differentiation of ADSCs into chondrogenesis. The specific expression of cartilage genes was analyzed by real-time polymerase chain reaction and Western blot. The expression of chondrocytic markers was obviously upregulated to a higher level compared to the one by commonly used TGF-β3 alone. After 3 weeks of in vitro culturing, the hybrids with differentiated chondrogenesis were then injected subcutaneously into nude mice and retrieved after 4 weeks of culturing in vivo. Histological analysis also confirmed that the recombinant fusion protein was more effective for the formation of cartilage matrix than the cases either with TGF-β3 alone or without LAP-MMP-mTGF-β3 (P<0.05). This study demonstrates that controlled local delivery of the LAP-MMP-mTGF-β3 constructs can accelerate differentiation of ADSCs into the cartilage in vivo, which indicates the great potential of this hybrid in rapid therapy of osteoarthritis.

Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-β1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine.

The aim of this study was to evaluate how low-intensity pulsed ultrasound (LIPUS) modulates the effect of transforming growth factor-β3 (TGF-β3) on the differentiation of scaffold-free dedifferentiated bovine articular chondrocyte tissues toward a cartilage-like phenotype. Specifically, the effect of these stimuli on the expression of hypertrophic markers collagen type I, collagen type X, and cartilage-degrading collagenase gene expression for a scaffold-free model was analyzed. A bioreactor that applied LIPUS directly from the transducer through a silicone gel to a six-well plate containing the tissues allowed simple, sterile, and large-scale experiments. Tissues were subjected to LIPUS of 55 mW/cm(2) in a 200 μs burst sine wave of 1 MHz over a 10-day period with or without TGF-β3 (10 ng/mL). Tissues exposed to TGF-β3 had significantly increased glycosaminoglycan and total collagen protein production along with upregulated cartilage-specific gene expression, resulting in tissues with a higher Young's Modulus. However, these tissues had also upregulated gene expression for hypertrophic markers collagen type I, collagen type X, MMP-1, MMP-13, MMP-2, and also an increase in the phosphorylation of p38. The expression of these matrix-degrading enzymes was remediated by hypertrophic development and differentiate dedifferentiated bovine articular chondrocytes towards a chondrogenic lineage allowing it to be a valuable tool in cartilage tissue engineering.

Melanoma is an aggressive type of cutaneous malignancy. Transforming growth factor (TGF)‑β has been demonstrated to be an important mediator of tumor progression. However, to the best of our knowledge, the systemic roles of plasma TGF‑β and TGF‑β in situ have not been investigated in Han Chinese melanoma patients. The results of the present study demonstrated that the in situ and plasma levels of TGF‑β1, TGF‑β2 and TGF‑β3 protein and messenger RNA were significantly elevated in tumor tissues compared with those of normal tissues. The survival rates of the patients which were triple‑positive (TGF‑β1+, TGF‑β2+ and TGF‑β3+) were found to be markedly decreased compared to those which were single‑ (TGF‑β1+, TGF‑β2+ or TGF‑β3+) or double‑positive (TGF‑β1+, TGF‑β2+; TGF‑β2+, TGF‑β3+; or TGF‑β1+, TGF‑β3+). These results may therefore contribute to the use of TGF‑β as a prognostic biomarker, and to the development of novel therapies for melanoma treatment.

BACKGROUND

The problem for transmyocardial revascularization is the occlusion of transmural channels. We have developed a novel heparinized basic fibroblast growth factor (bFGF)-incorporating tubular stent by polymer materials of poly D, L-lactic/glycolic acid (PLGA) and polycaprolactone (PCL) with preferable biocompatibility and mechanical elasticity. This study investigated the effect of this new stent on transmyocardial perfusion.

METHODS

Miniswine were grouped into bare stent group (BS), heparinized stent group (HS), and bFGF- and heparin-incorporated stent group (HBS). Two stents were implanted into the ischemic region. At 6 wk postoperatively, von Willebrand Factor (vWF) and Ki-67 antigen were immunohistologically stained. The expression of transforming growth factor-beta3 (TGF-β3), vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1β), and vWF mRNA from the cardiac tissue around the stent were performed by reverse transcription polymerase chain reaction (RT-PCR). Left ventricular function and myocardial perfusion by echocardiography and nuclear scanning were also documented.

RESULTS

Ki-67 positively-staining proliferating cells, expressions of proangiogenic factors of TGF-β3, VEGF, vWF, IL-1β, and vascular density in HBS group were significantly different from those in HS and BS groups (P < 0.05). Neovascularization with endothelization, improvement in perfusion, and fractional shortening in BHS and HS groups were significantly better than those in BS group (P < 0.001).

CONCLUSIONS

Heparinized bFGF-incorporating stent developed in the present study may keep the myocardial channel open with full luminal endothelization. By inducing cellular migration and expression of angiogenic factors, this novel stent may further enhance neovascular formation, increase myocardial perfusion, and improve cardiac function.

Objective: To investigate the effect of transforming growth factor-β3 (TGF-β3) and dental pulp stem cells (DPSC) in promoting the implant's osteointegration. Methods: Thirty-three New Zealand white rabbits were randomly divided into phosphate buffer saline (PBS) group, DPSC group and TGF-β3 + DPSC group (12 rabbits/group). Two teeth from the rabbits's mandibular incisors or molars were pulled out randomly, then implant were placed in the tooth extraction site immediately. In PBS group, the implant area was filled with Bio-Oss powder 0.30 g mixed by PBS 20 μl only; while the implant area was filled with Bio-oss powder 0.30 g and 1×10(8)/L DPSC 20 μl in DPSC group; in the the TGF-β3+DPSC group the implant area was filled with Bio-Oss powder 0.30 g mixed with 1×10(8)/L DPSC 20 μl and 80 μg/L TGF-β3 20 μl. Eighteen New Zealand rabbits were executed in the 4 weeks and 8 weeks respectively. The treated alveolar bone tissue and implant were collected for plastic section. Alizarin red staining (ARS), immunohistochemical detection (IHC) of bone sialoprotein (BSP), osteocalcin (OC) and type Ⅰ collagen (COL-Ⅰ) were performed after 4 weeks and 8 weeks. Combined bone lamelta width (CBLW) and implant bone contact rate (IBCR), trabecular width (TW) and trabecular area percentage (TA) were observed by histomorphometric measurement. Results: ARS staining: 4 weeks after the operation, the TGF-β3+ DPSC group showed more red calcified nodules than the other two groups; 8 weeks after operation, the red calcified nodule was further increased. 4 weeks after the operation, the expression of BSP, OC and COL-Ⅰ was (0.35± 0.04), (0.36 ± 0.03) and (0.39 ± 0.01) respectively in TGF-β3+ DPSC group, (0.27 ± 0.02), (0.24 ± 0.01) and (0.28±0.03) respectively in DPSC group, and (0.13±0.03), (0.15±0.02) and (0.16±0.02) respectively in PBS group. Eight weeks after operation, the expression of BSP, OC and COL-Ⅰ was (0.51±0.02), (0.49±0.03) and (0.53±0.02) respectively in TGF-β3+DPSC group, (0.35±0.02), (0.37±0.01) and (0.38±0.01) respectively in DPSC group, and (0.21±0.03), (0.19±0.01) and (0.22±0.02) respectively in PBS group. After 4 weeks and 8 weeks, the expression of BSP, OC and COL-Ⅰ in TGF-β3+DPSC group were significantly higher than the other groups (P<0.05), there was no significant difference between DPSC group and PBS group (P>0.05). Eight weeks after operation, the CBLW, IBCR, TW and TA around implant in TGF-β3+ DPSC group were significantly higher than that in the other groups (P<0.05), there was no significant difference between DPSC group and PBS group (P>0.05). Conclusions: The DPSC has the potential osteogenic differentiation ability; TGF-β3 can accelerate the osteogenic differentiation of DPSC to some extent; TGF-β3 combined with DPSC can effectively promote the implant's osseointegration.

Synovial chondromatosis (SC) of temporomandibular joint is rare proliferative disorder featured by the formation of cartilaginous nodules in synovium and joint space. Transforming growth factor beta 3 (TGF-β3) is closely related to chondrogenic differentiation, and might participate in pathogenesis of SC. We discovered that increased quantity of synoviocytes and blood vessels were observed in SC synovium. The vessel wall and sublining fibroblasts were stained positively by the antibodies against TGF-β3, fibroblast growth factor 2 (FGF-2), and CD34. In loose bodies (LBs), TGF-β3 was mainly expressed in chondrocytes and FGF-2 was expressed in chondrocytes, fibroblasts, and vessel walls. Expressions of TGF-β1, TGF-β3, FGF-2, Sox9, Wnt-4, Foxc2, and VEGF-A mRNA were significantly higher in SC synovium. Stimulation of TGF-β3 on synoviocytes increased alkaline phosphatase (ALP) activity and expressions of chondrogenic genes (Sox9, Col2α1, Aggrecan, Wnt-4, and Wnt-11), osteogenic genes (Runx2, Foxc2, osteocalcin, and Col1α1), and VEGF-A, but failed to influence FGF-2 expression. However, the addition of FGF-2 increased TGF-β3 expression. In conclusion, TGF-β3 existed in synovium and LBs of SC, and was responsible for the pathogenesis of SC.

Heart failure with preserved ejection fraction (HFpEF) represents an important cardiac condition because of its increasing prevalence, resistance to treatment and high associated morbidity and mortality. Two of the major mechanisms responsible for HFpEF are impaired cardiomyocyte sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a), which is responsible for calcium reuptake into the SR, and cardiac fibroblasts/myofibroblasts that produce collagen or myocardial fibrosis. Phospholamban (PLB), in the SR and endoplasmic reticulum, is the primary regulator of SERCA2a in the heart and acts as a reversible inhibitor of SERCA2a. Glucagon-like peptide-1, a 30 amino acid peptide, improves diastolic function through increasing SERCA2a expression and activity as well as by decreasing phosphorylation of Ryanodine receptors. It also enhances collagen production through enhanced procollagen IalphaI/IIIalphaI, connective tissue growth factor, fibronectin, TGF-β3 as well as Interleukin -10, -1beta, and -6 gene expression. Relaxin-2, a two chain, 53 amino acid peptide, increases Ser16- and Thr17-phosphorylation levels of PLB, thereby relieving SERCA2a of its inhibition. H3 Relaxin inhibits TGF-β1-stimulated collagen deposition through H3 relaxin-induced increases in pSmad2. Neuregulin-1, an epidermal growth factor, induces nitric oxide and PI-3 kinase activation that enhance SERCA2 activity. Neuregulin-1 was associated with less myocardial macrophage infiltration and cytokine expression reducing collagen deposition. Ghrelin, a 28 amino acid peptide, improves SERCA2a function by inducing PLB phosphorylation. Ghrelin also reduces cardiac fibrosis. In summary, Glucagon-like peptide-1, Relaxin-2, Neuregulin-1, and Ghrelin each modify calcium dynamics, collagen expression, and myocardial fibrosis through attenuation of deleterious signaling cascades, and induction of adaptive pathways, representing potential therapeutic targets for HFpEF.

BACKGROUND

Dermatomyositis (DM) and systemic lupus erythematosus (SLE) have common skin features, including dermal mucin deposition and interferon signature, although their roles are unknown.

OBJECTIVE

To identify common or specific molecular changes in DM and SLE skin.

METHODS

Proteomic analysis was performed using DM and healthy skin. Glycosaminoglycans were analysed by high-performance liquid chromatography.

RESULTS

The expression of 60 proteins was upregulated or downregulated in DM skin compared with healthy skin in the proteomic analysis. Among those proteins, PSMB9, an immunoproteasome subunit, was upregulated in the epidermis of DM and SLE, but not in other skin diseases. Furthermore, versican V1, a core protein for glycosaminoglycans, was upregulated, while type I collagen was downregulated in the dermis of DM and SLE skin. Interferon stimulated PSMB9 expression in cultured keratinocytes and reduced collagen expression in dermal fibroblasts, but did not affect versican expression. The PSMB9 knock-down in keratinocytes led to significant suppression of transforming growth factor (TGF)-β2 and TGF-β3, inducers of versican synthesis. TGF-β3 expression was upregulated in both DM and SLE, while TGF-β2 expression was increased only in the DM epidermis. ΔDiHS-diS1, a component of heparan sulfate, was significantly increased only in DM. TGF-β2 expression significantly increased the ΔDiHS-diS1 expression in dermal fibroblasts in vitro.

CONCLUSIONS

The interferon signature in DM and SLE skin reduces collagen in dermal fibroblasts, whereas overexpression of PSMB9 induced by interferon stimulates versican inducers in epidermal keratinocytes. In addition, the TGF-β2-ΔDiHS-diS1 pathway may be responsible for the specific molecular change in DM skin.

A challenge in using stromal cells for intervertebral disc (IVD) regeneration is their limited differentiation capacity in vivo without exogenous growth factor (GF) supplementation. Priming of stromal cells prior to transplantation may offer a feasible strategy to overcome this limitation. Furthermore, the ability to cryopreserve cells could help alleviate logistical issues associated with storage and transport. With these critical translational challenges in mind, we aimed to develop a strategy involving priming and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs). In phase one, we utilised the electrohydrodynamic atomisation process to fabricate BMSC-encapsulated microcapsules that were primed with TGF-β3 for 14 d after which they were cultured for a further 21 d under basal or GF supplemented media conditions. Results showed that priming induced differentiation of BMSC microcapsules such that they synthesised significant amounts of sGAG (61.9 ± 2.0 μg and 55.3 ± 6.1 μg for low and high cell densities) and collagen (24.4 ± 1.9 μg and 55.3 ± 4.6 μg for low and high cell densities) in continued culture without GF supplementation compared to Unprimed microcapsules. Phase two of this work assessed the extracellular matrix forming capacity of Primed BMSC microcapsules over 21 d after cryopreservation. Notably, primed and cryopreserved BMSCs successfully retained the ability to synthesise both sGAG (24.8 ± 2.7 μg and 75.1 ± 11.6 μg for low and high cell densities) and collagen (26.4 ± 7.8 μg and 93.1 ± 10.2 μg for low and high cell densities) post-cryopreservation. These findings demonstrate the significant potential of priming and cryopreservation approaches for IVD repair and could possibly open new horizons for pre-designed, 'off-the-shelf' injectable therapeutics.

BACKGROUND

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive condition with right ventricular myocardium being replaced by fibro-fatty tissue. The spectrum of the expression may range from benign palpitations to the most malignant sudden death. Most of the mutations identified for the condition are localized in desmosomal proteins although three other nondesmosomal genes (cardiac ryanodine receptor-2, TGF-β3, and TMEM43) have also been implicated in ARVC. Both desmosomal and nondesmosomal genes were screened in a set of patients from local population.

METHODS

A set of 34 patients from local population were included in this study. Diagnosis was based on the criteria proposed by task force of European Society of Cardiology/International Society and Federation of Cardiology. Polymerase chain reaction-based single-strand conformation polymorphism analysis was carried out, and samples with abnormal band pattern were commercially sequenced.

RESULTS

Screening of cardiac ryanodine receptor revealed an insertion of a base in the intronic region of exon-28 in a patient, leading to a creation of a cryptic splice site. Screening of plakohilin-2 for mutations revealed an abnormal band pattern in three patients. Two of them had similar abnormal band pattern for exon-3.1. Sequencing revealed a novel 2 base pair deletion (433_434 delCT), which would lead to premature truncation of the protein (L145EfsX8). Another patient showed abnormal band pattern for exon-3.2 and sequencing revealed a missense mutation C792T leading to amino acid change P244L, in N-terminal, and this substitution may cause disturbances in the various protein-protein interactions.

CONCLUSIONS

This study reports novel cardiac ryanodine receptor (RyR-2) mutations and Pkp-2 for the first time from Indian population.

Transforming growth factor beta (TGF-β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF-β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF-β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF-β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF-β1 in the absence of the latency-associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP-TGF-β1, we were able to show that processing of the latent complex by a furin-like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP-TGF-β1, and co-expression of human furin enabled the proteolytic processing of latent TGF-β1. Engineering the plant post-translational machinery by co-expressing human furin also enhanced the accumulation of biologically active TGF-β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.