Bone morphogenetic protein 2 (BMP-2) and transforming growth factor β (TGF-β) isoforms are important in advancing bone regeneration. The aim of the present study was to investigate the positive and reciprocal effect of TGF‑β3, one of the three TGF‑β isoforms, on BMP‑2 in promoting osteogenic differentiation. Exogenous BMP‑2 and TGF‑β3 genes were separately, and in combination, overexpressed in rabbit bone marrow‑derived mesenchymal stem cells (rBMSCs). Expression levels of BMP‑2 and TGF‑β3 were evaluated using reverse‑transcription‑polymerase chain reaction (RT‑PCR) and Western blotting assays. Furthermore, the osteogenic differentiation capacities of BMSCs were assessed by measuring Alizarin Red S staining, an alkaline phosphatase activity assay, and quantification of the osteogenic‑specific genes, Runt‑related transcription factor 2 (Runx2) and Osterix (Osx). Using lentiviral‑mediated transfection, robust co‑transfection efficiency of >90% was achieved. RT‑PCR and immunoblotting results indicated a marked elevated expression of BMP‑2 and TGF‑β3 in rBMSCs undergoing co‑transfection, compared with transfection with BMP‑2 or TGF‑β3 alone, indicating that BMP‑2 and TGF‑β3 are synergistically expressed in rBMSCs. Furthermore, enhanced osteogenic differentiation was observed in rBMSCs co‑transfected with BMP‑2/TGF‑β3. The present study successfully delivered BMP‑2 together with TGF‑β3 into rBMSCs with high efficiency for the first time. Furthermore, TGF‑β3 overexpression was demonstrated to enhance the osteogenic efficacy of BMP‑2 in rBMSCs, and vice versa. This provides a potential clinical therapeutic approach for regenerating the function of osseous tissue, and may present a promising strategy for bone defect healing.

BACKGROUND

The epithelial-mesenchymal transition (EMT) is a distinguishing characteristic of chronic rhinosinusitis with nasal polyps (CRSwNP). The underlying mechanism remains largely unknown. Arachidonate 15-lipoxygenase (ALOX15), an enzyme involved in arachidonic acid metabolism, has been reported to cause airway epithelial injury and thus may further promote the EMT. The aim of this study was to evaluate the role of ALOX15 during the EMT process in CRSwNP.

METHODS

A total of 54 samples were obtained, including 10 from healthy control, 16 from non-eosinophilic CRSwNP, and 28 from eosinophilic CRSwNP. Hematoxylin and eosin staining was performed to determine the basement membrane (BM) thickness. The concentration of molecules mediating remodeling was assayed by Luminex. The messenger RNA (mRNA) and protein levels of target genes were measured by quantitative real-time polymerase chain reaction (PCR) and Western blotting.

RESULTS

EMT was enhanced in eosinophilic CRSwNP compared with the healthy controls and non-eosinophilic CRSwNP infiltrated with lymphocytes and/or plasma cells. The expression pattern of molecules related to remodeling, including matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs), and transforming growth factor β (TGF-β) family members, differed between the subtypes of CRSwNP. The mRNA level of ALOX15 was correlated with the BM thickness and MMP-1 and TGF-β3 expression. The inhibition of ALOX15 by PD146176 could induce claudin-1, claudin-4, claudin-7, zonula occludens (ZO)-1, ZO-2, E-Cadherin, TIMP-1, and TIMP-3 expressions and reduce the levels of MMP-1 and N-Cadherin in epithelial cells acquired from eosinophilic CRSwNP patients.

CONCLUSIONS

The specific inhibition of ALOX15 could attenuate the EMT, which may provide an alternative method for the treatment of CRSwNP.

The management of mucositis is the subject of many controversies, and the optimal treatment is still not known. Several evaluation scoring systems have been described, but no one of these is appropriate to all clinical situations: a simple scale such as that devised by the WHO can be used routinely, and more sophisticated ones can be implemented by trained experimenters working in research. We have considered the impact of each of the treatments currently available on each stage of mucositis. In attempts at prevention, self-care, in the sense of oral hygiene, must remain atraumatic. It is probably advisable to differentiate patients with good previous oral care, in whom tooth brushing is beneficial, from others, in whom the risk of hemorrhage and infection excludes any brushing. Before the dosage of chemotherapy is reduced, the curative or palliative intent of the strategy must be carefully evaluated. In the vascular phase protection of the proliferating cells is attempted by means of vasoconstriction (cryotherapy), cytoprotection (prostaglandin E2 and other antioxidants) or epithelial cell-inhibiting factors such as TGF-B3. Treatments applied in the epithelial phase are directed at increasing the cell proliferation to accelerate epithelial restoration by sucralfate and several growth factors: hematopoietic GF, which has demonstrated a direct effect on the mucosa (GM-CSF), or epithelial growth factors such as keratinocyte GF. In the ulcerative and bacteriological phase attempts are made to attenuate sepsis by means of antiseptics (chlorhexidine), amphotericin B and antiviral agents or antibiotic lozenges. In the healing phase application of the low-energy helium-neon laser has demonstrably been followed by a later time of onset, less pronounced peak severity and shorter duration of oral mucositis. After cancer treatment, oral hygiene, inhibition of oral flora, and pain relief are the main goals. Physiopathogen-specific treatment is the next step, with the emphasis on the inhibition of epithelial cell proliferation during drug exposure and facilitation of epithelial maturation and healing.

The objective of the study was to explore the effects of galectin-9 on myeloid suppressor cells in Coxsackievirus B3 (CVB3)-induced myocarditis and the possible mechanisms involved. For this purpose, BALB/c male mice were infected with CVB3 on day 0 and then received intraperitoneal (IP) administration of recombinant galectin-9 or phosphate-buffered saline (PBS) daily from day 3 to day 7. The phenotypes and functions of myeloid suppressor cells were evaluated. The role and mechanism of myeloid suppressor cells and subsets in CVB3-induced myocarditis in vitro were explored. We found that galectin-9 remarkably increased the frequencies of CD11b+Gr-1+ cells in the cardiac tissue and spleen with myocarditis. Ly-6G+ cells were decreased and Ly-6C+ cells were increased in galectin-9-treated mice. In addition, CD11b+Gr-1+ cells were highly effective in suppressing CD4+ T cells. Moreover, our data demonstrate that CD11b+Gr-1+ cells are capable of expanding regulatory T cells (Tregs) from a preexisting population of natural Tregs, which depends on IL-10 but not TGF-β. Our results indicate that galectin-9 therapy may represent a useful approach to ameliorate CVB3-induced myocarditis.

Transforming growth factor-β (TGF-β) and heat shock protein 70 (HSP70) are important for the hair follicle (HF) cycle, but it is unclear whether they participate in HF regression in yak skin. In this study, we investigated the role of TGF-β, TGF-βRII, and HSP70 in the transition from anagen to catagen of HFs. The results showed that TGF-β2 transcription was significantly higher than that of TGF-β1 and TGF-β3 in the same periods. Meanwhile, the expressions of TGF-β2, TGF-βRII, and caspase-3 were higher in the catagen phase than that in mid-anagen, and some TGF-βRII-positive HF cells were terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive. Moreover, the HSP70 protein levels in mid-anagen were higher than those in late-anagen and catagen. These results suggested that TGF-β2 plays a major role in catagen induction in yak HFs, which might be achieved via TGF-βRII-mediated apoptosis in HF epithelial cells. In contrast, HSP70 might protect epithelial cells from apoptosis and ultimately inhibit HF regression. In conclusion, TGF-β2 has positive effects, whereas HSP70 has negative effects, on catagen induction.

UNASSIGNED

To determine whether (1) the in vitro expression of epithelial basement membrane components nidogen-1, nidogen-2, and perlecan by keratocytes, corneal fibroblasts, and myofibroblasts is modulated by cytokines/growth factors, and (2) perlecan protein is produced by stromal cells after photorefractive keratectomy.

UNASSIGNED

Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-β1, IL-1α, IL-1β, TGF-β3, platelet-derived growth factor (PDGF)-AA, or PDGF-AB. Real-time quantitative RT-PCR was used to detect expression of nidogen-1, nidogen-2, and perlecan mRNAs. Western blotting evaluated changes in protein expression. Immunohistochemistry was performed on rabbit corneas for perlecan, alpha-smooth muscle actin, keratocan, vimentin, and CD45 at time points from 1 day to 1 month after photorefractive keratectomy (PRK).

UNASSIGNED

IL-1α or -1β significantly upregulated perlecan mRNA expression in keratocytes. TGF-β1 or -β3 markedly downregulated nidogen-1 or -2 mRNA expression in keratocytes. None of these cytokines had significant effects on nidogen-1, -2, or perlecan mRNA expression in corneal fibroblasts or myofibroblasts. IL-1α significantly upregulated, while TGF-β1 significantly downregulated, perlecan protein expression in keratocytes. Perlecan protein expression was upregulated in anterior stromal cells at 1 and 2 days after -4.5 or -9 diopters (D) PRK, but the subepithelial localization of perlecan became disrupted at 7 days and later time points in -9-D PRK corneas when myofibroblasts populated the anterior stroma.

UNASSIGNED

IL-1 and TGF-β1 have opposing effects on perlecan and nidogen expression by keratocytes in vitro. Proximate participation of keratocytes is likely needed to regenerate normal epithelial basement membrane after corneal injury.

Acne vulgaris is a universal skin disease and it may leave a scar when the original skin lesion disappears. These scars can cause cosmetic problems and psychological burden, leading to poor quality of life of patients. Acne scars are classified into atrophic scars and hypertrophic scars. As most of the acne scars are atrophic, many studies have been conducted focusing on the treatment of atrophic lesions. This study was conducted to investigate the underlying pathogenesis of acne hypertrophic scars by identifying roles of fibrogenetic and inflammatory markers. Skin biopsy samples were obtained from hypertrophic scars of face and back and from adjacent normal tissues as control group. Some samples from back were immature hypertrophic scars and the other samples were in mature stages. Immunohistochemistry staining and quantitative PCR were performed for fibrogenetic and inflammatory markers. Both in mature and immature hypertrophic scars, vimentin and α-SMA were increased. Production of TGF-β3 protein as well as transcription of TGF-β3 was also significantly elevated. In contrast, expression of TGF-β1 showed no increase. Instead, expression levels of SMAD2 and SMAD4 were increased. Elevations of CD45RO, TNF-α and IL-4 and reduction of IL-10 were observed. In immature hypertrophic scars, IGF-1R and insulin-degrading enzyme expression were increased. Increased apoptosis was observed in immature stages of hypertrophic scars but not in mature stages. Elevations of TGF-β3, SMAD2 and SMAD4 in hypertrophic scars and increase of IGF-1R in immature stages may give some clues for acne hypertrophic scar formation.

OBJECTIVE

To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization.

METHODS

Experimental.

METHODS

Laboratory.

METHODS

Women with symptomatic leiomyomas.

METHODS

Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness.

METHODS

Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF).

RESULTS

Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression.

CONCLUSIONS

Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma.

Thymoma represents the most common anterior mediastinal compartment neoplasm, originating from the epithelial cell population in the thymus. Various histological types of thymoma feature different clinical characteristics. Furthermore, thymoma is frequently associated with autoimmune disorders, esp. myasthenia gravis (MG). However, the underlying molecular tumourigenesis of thymoma remains largely unknown. The goal of our current study is to demonstrate the underlying genetic abberations in thymoma, so as to understand the possible cause of MG in thymoma patients. By using CapitalBio mRNA microarray analysis, we analyzed 31 cases of thymoma including 5 cases of type AB thymoma, 6 B1-type cases, 12 B2-type cases, 5 B2B3-type cases and 3 type-B3 cases. 6 cases of thymoma were not associated with myasthenia gravis, while 25 cases were with myasthenia gravis. By comparisons between thymoma and the paratumoral tissues, differentially expressed genes were identified preliminarily. Among them, 292 genes increased more than 2-fold, 2 genes more than 5-fold. On the other hand, 596 genes were decreased more than 2-fold, 6 genes more than 20-fold. Interestingly, among these genes upregulated more than 2-fold, 6 driver genes (FANCI, NCAPD3, NCAPG, OXCT1, EPHA1 and MCM2) were formerly reported as driver oncogenes. This microarray results were further confirmed through real-time PCR. 8 most dysregulated genes were verified: E2F2, EPHA1, CCL25 and MCM2 were upregulated; and IL6, FABP4, CD36 and MYOC were downregulated. Supervised clustering heat map analysis of 2-fold upregulated and 2-fold downregulated genes revealed 6 distinct clusters. Strikingly, we found that cluster 1 was composed of two type-B2 thymoma; and cluster 6 was three type-B2/B3 thymoma. KEGG database analysis revealed possible genetic mechanisms of thymoma and functional process. We further compared gene expression pattern between thymoma with and without MG, and found 5 genes were upregulated more than 2-fold, more than 30 genes were downregulated more than 2-fold. KEGG analysis revealed 2 important signaling pathways with more than 2-fold upregulated genes (TGF- beta signaling pathway and HTLV-I signaling pathway) as differially functioning between MG positive and negative thymomas. Real-time PCR analysis confirmed that CCL25 was upregulated; and MYC, GADD45B, TNFRSF12 downregulated in thymoma with MG. Our study thus provided important genetic information on thymoma. It shed light on the molecular bases for analyzing the functional process of thymoma and finding potential biomarkers for pathological categorizing and treatment. Our work may provide important clues in understanding possible causes of MG in thymoma patients.

Currently, the mechanism governing the regeneration of the soft tissue-to-bone interface, such as the transition between the anterior cruciate ligament (ACL) and bone, is not known. Focusing on the ACL-to-bone insertion, this study tests the novel hypothesis that interactions between cells from the ligament (fibroblasts) and bone (osteoblasts) initiate interface regeneration. Specifically, these heterotypic cell interactions direct the fibrochondrogenic differentiation of interface-relevant cell populations, defined here as ligament fibroblasts and bone marrow stromal cells (BMSC). The objective of this study is to examine the effects of heterotypic cellular interactions on BMSC or fibroblast growth and biosynthesis, as well as expression of fibrocartilage-relevant markers in tri-culture. The effects of cell-cell physical contact and paracrine interactions between fibroblasts and osteoblasts were also determined. It was found that, in tri-culture with fibroblasts and osteoblasts, BMSC exhibited greater fibrochondrogenic potential than ligament fibroblasts. The growth of BMSC decreased while proteoglycan production and TGF-β3 expression increased. Moreover, tri-culture regulated BMSC response via paracrine factors, and interestingly, fibroblast-osteoblast contact further promoted proteoglycan and TGF-β1 synthesis as well as induced SOX9 expression in BMSC. Collectively, the findings of this study suggest that fibroblast-osteoblast interactions play an important role in regulating the stem cell niche for fibrocartilage regeneration, and the mechanisms of these interactions are directed by paracrine factors and augmented with direct cell-cell contact.

Objective: To explore the effect of kartogenin (KGN) on proliferation and chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSC) in vitro, and the synergistic effects of KGN and transforming growth factor (TGF)-β3 on hUCMSC.

Methods: Human umbilical cord mesenchymal stem cells were isolated and cultured. Then the differentiation properties were identified by flow cytometry analysis. HUCMSC were divided into four groups: control group, KGN group, TGF-β3 group, and TK group (with TGF-β3 and KGN added into the medium simultaneously). Cells in all groups were induced for 21 days using the suspension ball culture method. Hematoxylin and eosin, immunofluorescence, and Alcian blue staining were used to analyze chondrogenic differentiation. Real-time reverse transcriptase polymerase chain reaction was performed to investigate genes associated with chondrogenic differentiation.

Result: Hematoxylin and eosin staining showed that cells in the TGF-β3 group and the TK group had formed cartilage-like tissue after 21 days of culture. The results of immunofluorescence and Alcian blue staining showed that compared with the control group, cells in the KGN and TGF-β3 groups demonstrated increased secretion of aggrecan after 21 days of culture. In addition, cells in the group combining KGN with TGF-β3 (5.587 ± 0.27, P < 0.01) had more collagen II secretion than cells in the TGF-β3 alone group (2.86 ± 0.141, P < 0.01) or the KGN group (1.203 ± 0.215, P < 0.01). The expression of aggrecan (2.468 ± 0.097, P < 0.05) and SRY-Box 9 (4.08 ± 0.13, P < 0.05) in cells in the group combining KGN with TGF-β3 was significantly higher than those in the TGF-β3 group (2.216 ± 0.09, 3.02 ± 0.132, P < 0.05).'

Conclusion: The combination of KGN and TGF-β3 had synergistic effects and induced hUCMSC chondrogenesis. This could represent a new approach for clinical application and studies on cartilage repair and regeneration.

Keywords: Chondrogenesis; Kartogenin; Mesenchymal stem cells; Transforming growth factor-β3.

Posterior capsule opacification (PCO) is the most common complication that causes visual decrease after extracapsular cataract surgery. The primary cause of PCO formation is the proliferation of the residual lens epithelial cells (LECs). The mammalian target of rapamycin (mTOR) plays an important role in the growth and migration of LECs. In the current study, we used small interfering RNA (siRNA) to specifically attenuate mTOR in human lens epithelial B3 cells (HLE B3). We aimed to examine the effect of mTOR-siRNA on the proliferation, migration and epithelial-to-mesenchymal transition (EMT) of HLE B3 cells and explore the underlying mechanisms. The mTOR-siRNA was transfected into HLE B3 cells using lipofectamine 2000. The mRNA and protein levels of mTOR were examined to confirm the efficiency of mTOR-siRNA. The levels of mRNA and protein as well as the activity of mTOR down-stream effectors p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB, AKT) were examined using real-time PCR or Western blot, respectively. The cell proliferation was determined using cell counting kit (CCK) 8 and cell growth curve assay. The cell migration was examined using Transwell system and Scratch assay. MTOR-siRNA effectively eliminated mTOR mRNA and protein. The proliferation and migration were significantly suppressed by mTOR-siRNA transfection. mTOR-siRNA reduced the mRNA of p70S6K and AKT in a time-dependent manner. Furthermore, the phosphorylation of p70S6K and AKT was decreased by mTOR-siRNA. MTOR-siRNA also eliminated the formation of mTORC1 and mTORC2 protein complex and blocked the transforming growth factor (TGF)-β-induced EMT. Our results suggested that mTOR-siRNA could effectively inhibit the proliferation, migration and EMT of HLE B3 cells through the inhibition of p70S6K and AKT. These results indicated that mTOR-siRNA might be an effective agent inhibiting HLE cells growth and EMT following cataract surgery and provide an alternative therapy for preventing PCO.

Mesenchymal cell proliferation is one of the processes in shelf outgrowth. Both all-trans retinoic acid (atRA) and transforming growth factor-β3 (TGF-β3) play an important role in mouse embryonic palate mesenchymal (MEPM) cell proliferation. The cellular effects of TGF-β are mediated by Smad-dependent or Smad-independent pathways. In the present study, we demonstrate that atRA promotes TGF-β3 promoter demethylation and protein expression, but can cause depression of mesenchymal cell proliferation, especially at embryonic day 14 (E14). Moreover, the inhibition of MEPM cell proliferation by atRA results in the downregulation of Smad signaling mediated by transforming growth interacting factor (TGIF). We speculate that the effects of atRA on MEPM cell proliferation may be mediated by Smad pathways, which are regulated by TGIF but are not related to TGF-β3 expression. Finally, the cellular effects of TGF-β3 on MEPM cell proliferation may be mediated by Smad-independent pathways.

Gene silencing using small interfering RNA has been proposed as a therapy for cancer, viral infections and other diseases. This study aimed to investigate whether layer-by-layer polymer surface modification could deliver small interfering RNA to decrease fibrotic processes associated with medical device implantation. Anti-green fluorescent protein labelled small interfering RNA was applied to tissue culture plates and polyurethane using a layer-by-layer technique with small interfering RNA and poly-L-lysine. In vitro studies showed that the level of down-regulation of green fluorescent protein was directly related to the number of coatings applied. This layer-by-layer coating technique was then used to generate Rhodamine-Flii small interfering RNA-coated implants for in vivo studies of small interfering RNA delivery via subcutaneous implantation in mice. After two days, Rh-positive cells were observed on the implants' surface indicating cellular uptake of the Rhodamine-Flii small interfering RNA. Decreased Flii gene expression was observed in tissue surrounding the Rhodamine-Flii small interfering RNA coated implants for up to seven days post implantation, returning to baseline by day 21. Genes downstream from Flii, including TGF-β1 and TGF-β3, showed significantly altered expression confirming a functional effect of the Rhodamine-Flii small interfering RNA on gene expression. This research demonstrates proof-of-principle that small interfering RNA can be delivered via layer-by-layer coatings on biomaterials and thereby can alter the fibrotic process.

Transforming growth factor betas (TGF-βs) belong to three isoforms (TGF-β1, TGF-β2 and TGF-β3) members of a large pleiotropic superfamily of around 100 distinct proteins participating in the regulation of key events of development and disease, and tissue repair. In the central nervous system (CNS), all the three isoforms are produced by both glial and neuronal cells and are involved in essential tissue functions such as cell-cycle control, regulation of early development and differentiation, neuronal survival and astrocytes differentiation. Recent findings have shown abnormally increase of the levels of TGF-β1 in the brain of patients suffering Alzheimer's disease (AD), an elderly pathology reaching individuals over 65-years-old which present well-known hallmarks, including cerebrovascular deficiency, abnormal deposition of amyloid beta (Aβ), cholinergic denervation, neuroinflammation, neurofibrillary tangles and progressive loss of memory. However, related to the pathological features of AD, the brain overexpression of TGF-β1 was associated with neuroinflammation, accumulation of extracellular matrix compounds and cerebrovascular stiffness, neuronal apoptosis along with the development of vascular hypertrophy. Consistent with these observations, transgenic mice model (TGF mice) overexpressing constitutively TGF-β1 fully mimicked AD-like cerebrovascular pathology. Taken altogether, these data suggest the involvement of TGF-β1in the pathogenesis of AD, particularly in the cerebrovascular pathology which is of interest in the present review that will discuss the contribution of TGF-β1 in the cerebrovascular physiopathology of AD.

Recurrent mutational activation of the MAP kinase pathway in plasma cell myeloma implicates growth factor-like signaling responses in the biology of Ab-secreting cells (ASCs). Physiological ASCs survive in niche microenvironments, but how niche signals are propagated and integrated is poorly understood. In this study, we dissect such a response in human ASCs using an in vitro model. Applying time course expression data and parsimonious gene correlation network analysis (PGCNA), a new approach established by our group, we map expression changes that occur during the maturation of proliferating plasmablast to quiescent plasma cell under survival conditions including the potential niche signal TGF-β3. This analysis demonstrates a convergent pattern of differentiation, linking unfolded protein response/endoplasmic reticulum stress to secretory optimization, coordinated with cell cycle exit. TGF-β3 supports ASC survival while having a limited effect on gene expression including upregulation of CXCR4. This is associated with a significant shift in response to SDF1 in ASCs with amplified ERK1/2 activation, growth factor-like immediate early gene regulation and EGR1 protein expression. Similarly, ASCs responding to survival conditions initially induce partially overlapping sets of immediate early genes without sustaining the response. Thus, in human ASCs growth factor-like gene regulation is transiently imposed by niche signals but is not sustained during subsequent survival and maturation.

Fat grafting can reduce radiation-induced fibrosis. Improved outcomes are found when fat grafts are enriched with adipose-derived stromal cells (ASCs), implicating ASCs as key drivers of soft tissue regeneration. We have identified a subpopulation of ASCs positive for CD74 with enhanced antifibrotic effects. Compared to CD74- and unsorted (US) ASCs, CD74+ ASCs have increased expression of hepatocyte growth factor, fibroblast growth factor 2, and transforming growth factor β3 (TGF-β3) and decreased levels of TGF-β1. Dermal fibroblasts incubated with conditioned media from CD74+ ASCs produced less collagen upon stimulation, compared to fibroblasts incubated with media from CD74- or US ASCs. Upon transplantation, fat grafts enriched with CD74+ ASCs reduced the stiffness, dermal thickness, and collagen content of overlying skin, and decreased the relative proportions of more fibrotic dermal fibroblasts. Improvements in several extracellular matrix components were also appreciated on immunofluorescent staining. Together these findings indicate CD74+ ASCs have antifibrotic qualities and may play an important role in future strategies to address fibrotic remodeling following radiation-induced fibrosis.

Keywords: FACS; adipose; adipose stem cells; stem cells; transplantation.

Prostate cancer is the leading type of cancer diagnosed, and the most frequent cause of worldwide male cancer-related deaths annually. The limitations of current prostate cancer screening tests demand the identification of novel biomarkers for the early diagnosis of prostate cancer bone metastasis. In the present study, we performed a proteomic analysis of secreted proteins from the prostate cancer bone metastasis cell line, PC-3, and the normal prostate cell line, RWPE-1. We thus quantified 917 proteins, of which 68 were found to be secreted at higher levels by PC-3 than by RWPE-1 cells via LC-MS/MS. To characterize the highly secreted proteins in the PC-3 cell line and thereby identify biomarker proteins, we divided the quantifiable proteins into four quantitative categories (Q1-Q4). The KEGG lysine degradation and osteoclast differentiation pathways were demonstrated to be enriched in the highly secreted Q4 protein group. Transforming growth factor (TGF) beta family proteins related to osteoclast differentiation were identified as key regulators of PC-3 cell proliferation. Immunoblotting was used to confirm the observed high level of pentraxin, follistatin, TGF-beta family members, and serpin B3 secretion by PC-3 cells. From the collective results of the present study, we suggest that serpin B3 is a promising novel biomarker candidate for the diagnosis of prostate cancer bone metastasis.

Fibrocartilage is typically found in regions subject to complex, multi-axial loads and plays a critical role in musculoskeletal function. Mesenchymal stem cell (MSC)-mediated fibrocartilage regeneration may be guided by administration of appropriate chemical and/or physical cues, such as by culturing cells on polymer nanofibers in the presence of the chondrogenic growth factor TGF-β3. However, targeted delivery and maintenance of effective local factor concentrations remain challenges for implementation of growth factor-based regeneration strategies in clinical settings. Thus, the objective of this study was to develop and optimize the bioactivity of a biomimetic nanofiber scaffold system that enables localized delivery of TGF-β3. To this end, we fabricated TGF-β3-releasing nanofiber meshes that provide sustained growth factor delivery and demonstrated their potential for guiding synovium-derived stem cell (SDSC)-mediated fibrocartilage regeneration. TGF-β3 delivery enhanced cell proliferation and synthesis of relevant fibrocartilaginous matrix in a dose-dependent manner. By designing a scaffold that eliminates the need for exogenous or systemic growth factor administration and demonstrating that fibrochondrogenesis requires a lower growth factor dose compared to previously reported, this study represents a critical step towards developing a clinical solution for regeneration of fibrocartilaginous tissues. STATEMENT OF SIGNIFICANCE: Fibrocartilage is a tissue that plays a critical role throughout the musculoskeletal system. However, due to its limited self-healing capacity, there is a significant unmet clinical need for more effective approaches for fibrocartilage regeneration. We have developed a nanofiber-based scaffold that provides both the biomimetic physical cues, as well as localized delivery of the chemical factors needed to guide stem cell-mediated fibrocartilage formation. Specifically, methods for fabricating TGF-β3-releasing nanofibers were optimized, and scaffold-mediated TGF-β3 delivery enhanced cell proliferation and synthesis of fibrocartilaginous matrix, demonstrating for the first time, the potential for nanofiber-based TGF-β3 delivery to guide stem cell-mediated fibrocartilage regeneration. This nanoscale delivery platform represents an exciting new strategy for fibrocartilage regeneration.

OBJECTIVE

To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration.

METHODS

Proliferation was assessed by PrestoBlue™, morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay.

RESULTS

Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-β1) and 3 (TGF-β3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression.

CONCLUSIONS

Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

Current implantable materials are limited in terms of function as native tissue, and there is still no effective clinical treatment to restore articular impairments. Hereby, a functionalized polyacrylamide (PAAm)-alginate (Alg) Double Network (DN) hydrogel acting as an articular-like tissue is developed. These hydrogels sustain their mechanical stability under different temperature (+4 °C, 25 °C, 40 °C) and humidity conditions (60% and 75%) over 3 months. As for the functionalization, transforming growth factor beta-3 (TGF-β3) encapsulated (NP_TGF-β3) and empty poly(lactide-co-glycolide) (PLGA) nanoparticles (PLGA NPs) are synthesized by using microfluidic platform, wherein the mean particle sizes are determined as 81.44 ± 9.2 nm and 126 ± 4.52 nm with very low polydispersity indexes (PDI) of 0.194 and 0.137, respectively. Functionalization process of PAAm-Alg hydrogels with ester-end PLGA NPs is confirmed by FTIR analysis, and higher viscoelasticity is obtained for functionalized hydrogels. Moreover, cartilage regeneration capability of these hydrogels is evaluated with in vitro and in vivo experiments. Compared with the PAAm-Alg hydrogels, functionalized formulations exhibit a better cell viability. Histological staining, and score distribution confirmed that proposed hydrogels significantly enhance regeneration of cartilage in rats due to stable hydrogel matrix and controlled release of TGF-β3. These findings demonstrated that PAAm-Alg hydrogels showed potential for cartilage repair and clinical application.

Keywords: Alginate; Articular cartilage; Double network hydrogels; Growth factor; Nanoparticles.

The objective of the present study was to 1) analyze the ascending aortic proteome within a mouse model of Marfan syndrome (MFS; Fbn1C1041G/+) at early and late stages of aneurysm and 2) subsequently test a novel hypothesis formulated on the basis of this unbiased proteomic screen that links changes in integrin composition to transforming growth factor (TGF)-β-dependent activation of the rapamycin-independent component of mammalian target of rapamycin (Rictor) signaling pathway. Ingenuity Pathway Analysis of over 1,000 proteins quantified from the in vivo MFS mouse aorta by data-independent acquisition mass spectrometry revealed a predicted upstream regulator, Rictor, that was selectively activated in aged MFS mice. We validated this pattern of Rictor activation in vivo by Western blot analysis for phosphorylation on Thr1135 in a separate cohort of mice and showed in vitro that TGF-β activates Rictor in an integrin-linked kinase-dependent manner in cultured aortic vascular smooth muscle cells. Expression of β3-integrin was upregulated in the aged MFS aorta relative to young MFS mice and wild-type mice. We showed that β3-integrin expression and activation modulated TGF-β-induced Rictor phosphorylation in vitro, and this signaling effect was associated with an altered vascular smooth muscle cell proliferative-migratory and metabolic in vitro phenotype that parallels the in vivo aneurysm phenotype in MFS. These results reveal that Rictor is a novel, context-dependent, noncanonical TGF-β signaling effector with potential pathogenic implications in aortic aneurysm. NEW & NOTEWORTHY We present the most comprehensive quantitative analysis of the ascending aortic aneurysm proteome in Marfan syndrome to date resulting in novel and potentially wide-reaching findings that expression and signaling by β3-integrin constitute a modulator of transforming growth factor-β-induced rapamycin-independent component of mammalian target of rapamycin (Rictor) signaling and physiology in aortic vascular smooth muscle cells.

A silk scaffold exhibits high potential for the human anterior cruciate ligament (ACL) reconstruction due to its exceptional mechanics as well as biocompatibility. Inefficient ACL interface restoration is thought to be a major hurdle for the common implementation of a silk-based ligament graft. By integrating a stratified approach and gene immobilization, here we developed a gene-immobilized triphasic silk scaffold to enhance ACL osseointegration. Isotropic silk was divided into three regions (respectively corresponding to a ligament, fibrocartilage and the bone region of the native ACL interface) using a custom-made divider, and the lentiviral vector-encoded transforming growth factor beta-3 (TGF-β3) and bone morphogenetic protein-2 (BMP2) was further, respectively, immobilized to phosphatidylserine (PS)-coated fibrocartilage and the bone region of the triphasic silk scaffold. The in vitro assessments displayed that this gene-immobilized triphasic silk scaffold significantly promotes bone marrow mesenchymal stem cell (BMSC) proliferation and differentiation into corresponding cell lineage. Moreover, the gene-modified triphasic silk scaffold combined with BMSCs alone, which was rolled into a compact shaft to be implanted onto rabbit ACL-defect models, revealed roughly complete osseointegration restoration as a result of apparent three-layered tissue formation and robust mechanical ability as early as 12 weeks postoperatively. These outcomes demonstrated that employing the stratified approach and gene immobilization efficiently expedites silk-mediated ACL interface formation, expanding the therapeutic potential of the silk-based ligament graft for ACL reconstruction.