Vascular Smooth Muscle Cell Apoptosis Promotes Transplant Arteriosclerosis Through Inducing the Production of SDF‐1α

Transplant arteriosclerosis is a leading cause of late allograft loss. Medial smooth muscle cell (SMC) apoptosis is considered to be an important event in transplant arteriosclerosis. However, the precise contribution of medial SMC apoptosis to transplant arteriosclerosis and the underlying mechanisms remain unclear. We transferred wild‐type p53 to induce apoptosis of cultured SMCs. We found that apoptosis induces the production of SDF‐1α from apoptotic and neighboring viable cells, resulting in increased SDF‐1α in the culture media. Conditioned media from Ltv‐p53‐transferred SMCs activated PI3K/Akt/mTOR and MAPK/Erk signaling in a SDF‐1α‐dependent manner and thereby promoted mesenchymal stem cell (MSC) migration and proliferation. In a rat aorta transplantation model, lentivirus‐mediated BclxL transfer selectively inhibits medial SMC apoptosis in aortic allografts, resulting in a remarkable decrease of SDF‐1α both in allograft media and in blood plasma, associated with diminished recruitment of CD90⁺CD105⁺ double‐positive cells and impaired neointimal formation. Systemic administration of rapamycin or PD98059 also attenuated MSC recruitment and neointimal formation in the aortic allografts. These results suggest that medial SMC apoptosis is critical for the development of transplant arteriosclerosis through inducing SDF‐1α production and that MSC recruitment represents a major component of vascular remodeling, constituting a relevant target and mechanism for therapeutic interventions.     

Kidney‐Induced Cardiac Allograft Tolerance in Miniature Swine is Dependent on MHC‐Matching of Donor Cardiac and Renal Parenchyma

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full‐MHC barrier in miniature swine. However, the renal element(s) responsible for kidney‐induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic‐derived “passenger” cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co‐transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC‐mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC‐matched to each other but MHC‐mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC‐mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC‐mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC‐matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.     

Phospho‐Smad1 modulation by nedd4 e3 ligase in BMP/TGF‐β signaling

A considerable number of studies have focused on the regulation of mothers against decapentaplegic homologue (Smad)–dependent or –independent pathways in the signaling by each transforming growth factor β (TGF‐β) superfamily member in diverse biologic contexts. The sophisticated regulation of the actions of these molecules and the underlying molecular mechanisms still remain elusive. Here we show new mechanisms of ambilateral R (receptor‐regulated)–Smad regulation of bone morphogenetic protein 2 (BMP‐2)/TGF‐β1 signals. In a specific context, both signals regulate the nonclassic Smads pathway reciprocally, BMP‐2 to Smad2/3 and TGF‐β1 to Smad1/5/8, as well as their own classic linear Smad pathway. Interestingly, in this study, we found that C‐terminal phosphorylated forms of each pathway Smad degraded rapidly 3 hours after stimulation of nonclassic signals but are dramatically restored by treatment with via proteasomal inhibition. Furthermore, an E3 ligase, neural precursor cell expressed, developmentally down‐regulated 4 (Nedd4), also was found as one of the important modulators of the p‐Smad1 in both BMP‐2 and TGF‐β1 action. Overexpressed Nedd4 suppressed the BMP‐induced osteoblast transdifferentiation process of premyoblast C2C12 cells or alkaline phosphatase (ALP) level of human osteosarcoma cells and promoted TGF‐β1‐induced degradation of p‐Smad1 via physical interaction and polyubiquitination. Conversely, siNedd4 potentiated BMP signals through upregulation of p‐Smad1 and ALP activity, the effect of which led to an increased the rate of P_i‐induced calcification of human vascular smooth muscle cells. These new insights about proteasomal degradation–mediated phosphorylated nonclassic Smad regulation of BMP‐2/TGF‐β1 could, in part, help to unravel the complex mechanisms of abnormal nonosseous calcification by the aberrant activity of BMP/TGF‐β/Smads. © 2011 American Society for Bone and Mineral Research.     

CCN5 Reduces Ligamentum Flavum Hypertrophy by Modulating the TGF‐β Pathway

Ligamentum flavum hypertrophy (LFH) is the most important component of lumbar spinal canal stenosis. Although the pathophysiology of LFH has been extensively studied, no method has been proposed to prevent or treat it. Since the transforming growth factor‐β (TGF‐β) pathway is known to be critical in LFH pathology, we investigated whether LFH could be prevented by blocking or modulating the TGF‐β mechanism. Human LF cells were used for the experiments. First, we created TGF‐β receptor 1 (TGFBR1) knock out (KO) cells with CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 biotechnology and treated them with TGF‐β1 to determine the effects of blocking the TGF‐β pathway. Subsequently, we studied the effect of CCN5, which has recently been proposed to modulate the TGF‐β pathway. To assess the predisposition toward fibrosis, α‐smooth muscle actin (αSMA), fibronectin, collagen‐1, collagen‐3, and CCN2 were evaluated with quantitative real‐time polymerase chain reaction, western blotting, and immunocytochemistry. The TGFBR1 KO LF cells were successfully constructed with high KO efficiency. In wild‐type (WT) cells, treatment with TGF‐β1 resulted in the overexpression of the messenger RNA (mRNA) of fibrosis‐related factors. However, in KO cells, the responses to TGF‐β1 stimulation were significantly lower. In addition, CCN5 and TGF‐β1 co‐treatment caused a notable reduction in mRNA expression levels compared with TGF‐β1 stimulation only. The αSMA protein expression increased with TGF‐β1 but decreased with CCN5 treatment. TGF‐β1 induced LF cell transdifferentiation from fibroblasts to myofibroblasts. However, this cell transition dramatically decreased in the presence of CCN5. In conclusion, CCN5 could prevent LFH by modulating the TGF‐β pathway. © 2019 The Authors. Journal of Orthopaedic Research^® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2634–2644, 2019     

Simvastatin inhibits transforming growth factor‐β1‐induced expression of type I collagen,CTGF, and α‐SMA in keloid fibroblasts

Simvastatin, a 3‐hydroxy‐3‐methylglutaryl coenzyme‐A reductase inhibitor, is used to reduce cholesterol levels. Accumulating evidence has revealed the immunomodulatory and anti‐inflammatory effects of simvastatin that prevent cardiovascular diseases. In addition, the beneficial effects of statins on fibrosis of various organs have been reported. However, the functional effect of statins on dermal fibrosis of keloids has not yet been explored. The objective of this study was to determine whether simvastatin could affect dermal fibrosis associated with keloids. We examined the effect of simvastatin on transforming growth factor (TGF)‐β1‐induced production of type I collagen, connective tissue growth factor (CTGF or CCN2), and α‐smooth muscle actin (α‐SMA). Keloid fibroblasts were cultured and exposed to different concentrations of simvastatin in the presence of TGF‐β1, and the effects of simvastatin on TGF‐β1‐induced collagen and CTGF production in keloid fibroblasts were determined. The type I collagen, CTGF, and α‐SMA expression levels and the Smad2 and Smad3 phosphorylation levels were assessed by Western blotting. The effect of simvastatin on cell viability was evaluated by assessing the colorimetric conversion of 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide. Simvastatin suppressed TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in a concentration‐dependent manner. The TGF‐β1‐induced Smad2 and Smad3 phosphorylation levels were abrogated by simvastatin pretreatment. The inhibition of type I collagen, CTGF, and α‐SMA expression by simvastatin was reversed by geranylgeranyl pyrophosphate, suggesting that the simvastatin‐induced cellular responses were due to inhibition of small GTPase Rho involvement. A RhoA activation assay showed that preincubation with simvastatin significantly blocked TGF‐β1‐induced RhoA activation. The Rho‐associated coiled kinase inhibitor Y27632 abrogated TGF‐β1‐induced production of type I collagen, CTGF, and α‐SMA. However, Y27632 had no significant effect on TGF‐β1‐induced phosphorylation of Smad2 and Smad3. In conclusion, the present study suggests that simvastatin is an effective inhibitor of TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in keloid fibroblasts.     

Genomewide Comprehensive Analysis Reveals Critical Cooperation Between Smad and c‐Fos in RANKL‐Induced Osteoclastogenesis

We have previously reported that transforming growth factor β (TGF‐β) plays an essential role in receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE‐seq and ChIP‐seq) analyses indicated the cooperation of Smad2/3 with c‐Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c‐Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T‐cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF‐β, and c‐Fos binding to open chromatin sites was suppressed by inhibition of TGF‐β signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c‐Fos deficiency. These results suggest that TGF‐β regulates RANKL‐induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c‐Fos. © 2014 American Society for Bone and Mineral Research.     

Up‐regulation of fibroblast growth factor (FGF) 9 expression and FGF‐WNT/β‐catenin signaling in laser‐induced wound healing

Fibroblast growth factor (FGF) 9 is secreted by both mesothelial and epithelial cells, and plays important roles in organ development and wound healing via WNT/β‐catenin signaling. The aim of this study was to evaluate FGF9 expression and FGF‐WNT/β‐catenin signaling during wound healing of the skin. We investigated FGF9 expression and FGF‐WNT/β‐catenin signaling after laser ablation of mouse skin and adult human skin, as well as in cultured normal human epidermal keratinocytes (NHEKs) upon stimulation with recombinant human (rh) FGF9 and rh‐transforming growth factor (TGF)‐β1. Our results showed that laser ablation of both mouse skin and human skin leads to marked overexpression of FGF9 and FGF9 mRNA. Control NHEKs constitutively expressed FGF9, WNT7b, WNT2, and β‐catenin, but did not show Snail or FGF receptor (FGFR) 2 expression. We also found that FGFR2 was significantly induced in NHEKs by rhFGF9 stimulation, and observed that FGFR2 expression was slightly up‐regulated on particular days during the wound healing process after ablative laser therapy. Both WNT7b and WNT2 showed up‐regulated protein expression during the laser‐induced wound healing process in mouse skin; moreover, we discerned that the stimulatory effect of rhFGF9 and rhTGF‐β1 activates WNT/β‐catenin signaling via WNT7b in cultured NHEKs. Our data indicated that rhFGF9 and/or rhTGF‐β1 up‐regulate FGFR2, WNT7b, and β‐catenin, but not FGF9 and Snail; pretreatment with rh dickkopf‐1 significantly inhibited the up‐regulation of FGFR2, WNT7b, and β‐catenin. Our results suggested that FGF9 and FGF‐WNT/β‐catenin signaling may play important roles in ablative laser‐induced wound healing processes.     

Osteoblast‐Derived TGF‐β1 Stimulates IL‐8 Release Through AP‐1 and NF‐κB in Human Cancer Cells

Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)‐8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone‐derived growth factors on the IL‐8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast‐derived TGF‐β1 is associated with osteolytic bone diseases. Materials and Methods: IL‐8 mRNA levels were measured using RT‐PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF‐β1, BMP‐2, and IGF‐1. DNA affinity protein‐binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c‐fos, c‐jun, p65, and p50 to the IL‐8 promoter. A transient transfection protocol was used to examine IL‐8, NF‐κB, and activator protein (AP)‐1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL‐8, AP‐1, and NF‐κB promoter in human cancer cells. Osteoblasts were transfected with TGF‐β1, BMP‐2, or IGF‐1 small interfering RNA, and the medium was collected after 48 h. TGF‐β1 but not BMP‐2 or IGF‐1 siRNA inhibited OBCM‐induced IL‐8 release in human cancer cells. In addition, TGF‐β1 also directly induced IL‐8 release in human cancer cells. Activation of AP‐1 and NF‐κB DNA‐protein binding and MAPKs after TGF‐β1 treatment was shown, and TGF‐β1–induced IL‐8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF‐β1, BMP‐2, and IGF‐1. TGF‐β1 is the major contributor to the activation of extracellular signal‐related kinase (ERK), p38, and c‐Jun N‐terminal kinase (JNK), leading to the activation of AP‐1 and NF‐κB on the IL‐8 promoter and initiation of IL‐8 mRNA and protein release, thereby promoting osteoclastogenesis.     

Transforming growth factor‐β1 suppresses the up‐regulation of matrix metalloproteinase‐2 by lung fibroblasts in response to tumor necrosis factor‐α

Exposed to inflammatory factors or cytokines, fibroblasts appear to play additional roles beyond the deposition of extracellular matrix. It has been reported that tumor necrosis factor‐α (TNF‐α) induces the production of matrix metalloproteinase‐2 (MMP‐2) and transforming growth factor‐β1 (TGF‐β1) in fibroblasts. In this study, we demonstrated that the active MMP‐2 secreted by lung fibroblasts reached the peak level at 12 hours after TNF‐α treatment, whereas, by adding anti‐TGF‐β1 antibody in the culture medium, the MMP‐2 production in response to TNF‐α was maintained at high levels after 24 hours of treatment. We also confirmed that TNF‐α induced up‐regulation of active TGF‐β1 and exogenous TGF‐β1 induced down‐regulation of MMP‐2 synthesis in lung fibroblasts. Moreover, an increased MMP‐2 level was observed in a rat model with pulmonary inflammation and fibrosis induced by bleomycin‐A5. This revealed that MMP‐2 in the lung reached the peak level when TNF‐α reached the peak level at the 7th day, and then MMP‐2 decreased along with an increase in the TGF‐β1 level. Taken together, our results demonstrate that TNF‐α induced an increase of MMP‐2 and TGF‐β1 in lung fibroblasts, and the TGF‐β1 attenuated the up‐regulation of MMP‐2. This suggests that MMP‐2 secreted from fibroblasts modulated by TNF‐α/TGF‐β1 might play an important role in pulmonary inflammation and fibrosis.     

Nedd4 Deficiency in Vascular Smooth Muscle Promotes Vascular Calcification by Stabilizing pSmad1

The nonosseous calcification process such as atherosclerosis is one of the major complications in several types of metabolic diseases. In a previous study, we uncovered that aberrant activity of transforming growth factor β (TGF‐β) signaling pathway could contribute to the vascular smooth muscle cells’ (VSMCs) calcification process. Also, we identified NEDD4 E3 ligase as a key suppressor of bone morphogenetic protein (BMP)/Smad pathway via a polyubiquitination‐dependent selective degradation of C‐terminal phosphorylated Smad1 (pSmad1) activated by TGF‐β. Here, we further validated and confirmed the role of Nedd4 in in vivo vascular calcification progression. First, Nedd4 deletion in SM22α‐positive mouse tissues (Nedd4^fl/fl;SM22α‐Cre) showed deformed aortic structures with disarranged elastin fibers at 24 weeks after birth. Second, vitamin D–induced aorta vascular calcification rate in Nedd4^fl/fl;SM22α‐Cre mice was significantly higher than their wild‐type littermates. Nedd4^fl/fl;SM22α‐Cre mice showed a development of vascular calcification even at very low‐level injection of vitamin D, but this was not exhibited in wild‐type littermates. Third, we confirmed that TGF‐β1–induced pSmad1 levels were elevated in Nedd4‐deficient primary VSMCs isolated from Nedd4^fl/fl;SM22α‐Cre mice. Fourth, we further found that Nedd4^fl/fl;SM22α‐Cre mVSMCs gained mesenchymal cell properties toward osteoblast‐like differentiation by a stable isotope labeling in cell culture (SILAC)‐based proteomics analysis. Finally, epigenetic analysis revealed that methylation levels of human NEDD4 gene promoter were significantly increased in atherosclerosis patients. Collectively, abnormal expression or dysfunction of Nedd4 E3 ligase could be involved in vascular calcification of VSMCs by activating bone‐forming signals during atherosclerosis progression. © 2016 American Society for Bone and Mineral Research.     

T Cells Promote Bronchial Epithelial Cell Secretion of Matrix Metalloproteinase‐9 via a C‐C Chemokine Receptor Type 2 Pathway: Implications for Chronic Lung Allograft Dysfunction

Chronic lung allograft dysfunction (CLAD) is the major limitation of long‐term survival after lung transplantation. CLAD manifests as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Alloimmune reactions and epithelial‐to‐mesenchymal transition have been suggested in BOS. However, little is known regarding the role of allogenicity in epithelial cell differentiation. Primary human bronchial epithelial cells (BECs) were treated with activated T cells in the presence or absence of transforming growth factor (TGF)‐β. The expression of epithelial and mesenchymal markers was investigated. The secretion of inflammatory cytokines and matrix metalloproteinase (MMP)‐9 was measured in culture supernatants and in plasma from lung transplant recipients (LTRs): 49 stable, 29 with BOS, and 16 with RAS. We demonstrated that C‐C motif chemokine 2 secreted by T cells supports TGF‐β–induced MMP‐9 production by BECs after binding to C‐C chemokine receptor type 2. Longitudinal investigation in LTRs revealed a rise in plasma MMP‐9 before CLAD onset. Multivariate analysis showed that plasma MMP‐9 was independently associated with BOS (odds ratio [OR] = 6.19, p = 0.002) or RAS (OR = 3.9, p = 0.024) and predicted the occurrence of CLAD 12 months before the functional diagnosis. Thus, immune cells support airway remodeling through the production of MMP‐9. Plasma MMP‐9 is a potential predictive biomarker of CLAD.     

Fluocinolone Acetonide Is a Potent Synergistic Factor of TGF‐β3–Associated Chondrogenesis of Bone Marrow–Derived Mesenchymal Stem Cells for Articular Surface Regeneration

Articular cartilage repair remains a challenging problem. Based on a high‐throughput screening and functional analysis, we found that fluocinolone acetonide (FA) in combination with transforming growth factor beta 3 (TGF‐β3) strongly potentiated chondrogenic differentiation of human bone marrow–derived mesenchymal stem cells (hBMSCs). In an in vivo cartilage defect model in knee joints of immunocompromised mice, transplantation of FA/TGF‐β3–treated hBMSCs could completely repair the articular surface. Analysis of the intracellular pathways revealed that FA enhanced TGF‐β3–induced phosphorylation of Smad2 and Smad3. Additionally, we performed a pathway array and found that FA activates the mTORC1/AKT pathway. Chemical inhibition of mTORC1 with rapamycin substantially suppressed FA effect, and inhibition of AKT completely repressed chondrogenesis of hBMSCs. Inhibition of glucocorticoid receptor with mifepristone also suppressed FA effect, suggesting that FA involves binding to the glucocorticoid receptor. Comparative analysis with other glucocorticoids (triamcinolone acetonide [TA] and dexamethasone [DEX]) revealed the unique ability of FA to repair articular cartilage surgical defects. Analysis of intracellular pathways showed that the mTORC1/AKT pathway and the glucocorticoid receptor was highly activated with FA and TA, but to a lesser extent with DEX. Collectively, these results show a unique ability of FA to enhance TGF‐β3–associated chondrogenesis, and suggest that the FA/TGF‐β3 combination may be used as major inducer of chondrogenesis in vitro. Additionally, FA/TGF‐β3 could be potentially applied in a clinical setting to increase the efficiency of regenerative approaches based on chondrogenic differentiation of stem cells. © 2015 American Society for Bone and Mineral Research.     

Regulation of the friction coefficient of articular cartilage by TGF‐β1 and IL‐1β

Articular cartilage functions to provide a low‐friction surface for joint movement for many decades of life. Superficial zone protein (SZP) is a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage that contributes to effective boundary lubrication. In both cell and explant cultures, TGF‐β1 and IL‐1β have been demonstrated to, respectively, upregulate and downregulate SZP protein levels. It was hypothesized that the friction coefficient of articular cartilage could also be modulated by these cytokines through SZP regulation. The friction coefficient between cartilage explants (both untreated and treated with TGF‐β1 or IL‐1β) and a smooth glass surface due to sliding in the boundary lubrication regime was measured with a pin‐on‐disk tribometer. SZP was quantified using an enzyme‐linked immunosorbant assay and localized by immunohistochemistry. Both TGF‐β1 and IL‐1β treatments resulted in the decrease of the friction coefficient of articular cartilage in a location‐ and time‐dependent manner. Changes in the friction coefficient due to the TGF‐β1 treatment corresponded to increased depth of SZP staining within the superficial zone, while friction coefficient changes due to the IL‐1β treatment were independent of SZP depth of staining. However, the changes induced by the IL‐1β treatment corresponded to changes in surface roughness, determined from the analysis of surface images obtained with an atomic force microscope. These findings demonstrate that the low friction of articular cartilage can be modified by TGF‐β1 and IL‐1β treatment and that the friction coefficient depends on multiple factors, including SZP localization and surface roughness. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:249–256, 2009     

Induced pluripotent stem cells attenuate chronic allogeneic vasculopathy in an integrin beta‐1‐dependent manner

This study aimed to determine the mechanism of isogeneic‐induced pluripotent stem cells (iPSCs) homing to vascular transplants and their therapeutic effect on chronic allogeneic vasculopathy. We found that integrin β1 (Intgβ1) was the dominant integrin β unit in iPSCs that mediates the adhesion of circulatory and endothelial cells (ECs). Intgβ1 knockout or Intgβ1‐siRNAs inhibit iPSC adhesion and migration across activated endothelial monolayers. The therapeutic effects of the following were examined: iPSCs, Intgβ1‐knockout iPSCs, iPSCs transfected with Intgβ1‐siRNAs or nontargeting siRNAs, iPSC‐derived ECs, iPSC‐derived ECs simultaneously overexpressing Intgα4 and Intgβ1, iPSCs precultured in endothelial medium for 3 days (endothelial‐prone stem cells), primary aortic ECs, mouse embryonic fibroblasts, and phosphate‐buffered saline (control). The cells were administered every 3 days for a period of 8 weeks. iPSCs, iPSCs transfected with nontargeting siRNAs, and endothelial‐prone stem cells selectively homed on the luminal surface of the allografts, differentiated into ECs, and decreased neointimal proliferation. Through a single administration, we found that iPSCs trafficked to allograft lesions, differentiated into ECs within 1 week, and survived for 4‐8 weeks. The therapeutic effect of a single administration was moderate. Thus, Intgβ1 and pluripotency are essential for iPSCs to treat allogeneic vasculopathy.     

Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

Allogeneic demineralized bone is used extensively as a clinical graft material because it has osteo/chondroinductive and osteoconductive properties. Demineralized bone powder (DBP) induces chondrogenic differentiation of human dermal fibroblasts (hDFs) in three‐dimensional collagen cultures, but the initiating mechanisms have not been fully characterized nor has it been shown that bone morphogenetic proteins (BMPs) recapitulate DBP's effects on target cells. Among the many signaling pathways regulated in hDFs by DBP prior to in vitro chondrogenesis, there are changes in Wnts and their receptors that may contribute to DBP actions. This study tests the hypothesis that DBP modulation of Wnt signaling entails both BMP and TGF‐β pathways. We compared the effects of DBP, TGF‐β1, or BMP‐2 on Wnt signaling components in hDFs by Wnt signaling macroarray, RT‐PCR, in situ hybridization, and Western immunoblot analyses. Many effects of DBP on Wnt signaling components were not shared by BMP‐2, and likewise DBP effects on Wnt genes and β‐catenin only partially required the TGF‐β pathway, as shown by selective inhibition of TGF‐β/activin receptor‐like kinase. The analyses revealed that 64% (16/25) of the Wnt signaling components regulated by DBP were regulated similarly by the sum of effects by BMP‐2 and by TGF‐β1. In conclusion, signaling mechanisms of inductive DBP in human dermal fibroblasts involve the modulation of multiple Wnt signals through both BMP and TGF‐β pathways. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 554–560, 2013     

Insulin‐like growth factor‐I improves chondrogenesis of predifferentiated human umbilical cord mesenchymal stromal cells

Human umbilical cord mesenchymal stromal cells (hUCMSCs) are an attractive cell source for tissue engineering with numerous advantages over other adult stem cell sources, such as great expansion ability in vitro and extensive availability. The objective of this 6‐week study was to test the hypothesis that switching from chondrogenic transforming growth factor‐beta3 (TGF‐β3) to anabolic insulin‐like growth factor‐I (IGF‐I) at the 3‐week time point would produce more cartilage‐like matrix than TGF‐β3 alone. hUCMSCs were seeded into polyglycolic acid (PGA) scaffolds and then cultured in chondrogenic medium containing TGF‐β3 for 3 weeks. The TGF‐β3‐treated hUCMSCs were then exposed for 3 more weeks to one of four different conditions: (1) continued in chondrogenic medium, (2) control medium (no TGF‐β3), (3) control medium with 10 ng/ml IGF‐I, or (4) control medium with 100 ng/ml IGF‐I. Compared to continuing with TGF‐β3, switching to IGF‐I increased collagen production, and furthermore increased both collagen type II gene expression and immunostaining. In conclusion, the shift from TGF‐β3 to IGF‐I at week 3 resulted in a significant increase of cartilage‐like extracellular matrix, confirming our hypothesis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1109–1115, 2009