Transforming growth factor beta3 (TGFB3) gene mutations in patients of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD1) and Loeys-Dietz syndrome-5 (LDS5)/Rienhoff syndrome are associated with cardiomyopathy, cardiac arrhythmia, cardiac fibrosis, cleft palate, aortic aneurysms, and valvular heart disease. Although the developing heart of embryos express Tgfb3, its overarching role remains unclear in cardiovascular development and disease. We used histological, immunohistochemical, and molecular analyses of Tgfb3-/- fetuses and compared them to wildtype littermate controls. The cardiovascular phenotypes were diverse with approximately two thirds of the Tgfb3-/- fetuses having one or more cardiovascular malformations, including abnormal ventricular myocardium (particularly of the right ventricle), outflow tract septal and alignment defects, abnormal aortic and pulmonary trunk walls, and thickening of semilunar and/or atrioventricular valves. Ventricular septal defects (VSD) including the perimembranous VSDs were observed in Tgfb3-/- fetuses with myocardial defects often accompanied by the muscular type VSD. In vitro studies using TGFβ3-deficient fibroblasts in 3-D collagen lattice formation assays indicated that TGFβ3 was required for collagen matrix reorganization. Biochemical studies indicated the 'paradoxically' increased activation of canonical (SMAD-dependent) and noncanonical (MAP kinase-dependent) pathways. TGFβ3 is required for cardiovascular development to maintain a balance of canonical and noncanonical TGFβ signaling pathways.

Embryonic heart valve primordia (cushions) maintain unidirectional blood flow during development despite an increasingly demanding mechanical environment. Recent studies demonstrate that atrioventricular (AV) cushions stiffen over gestation, but the molecular mechanisms of this process are unknown. Transforming growth factor-beta (TGFβ) and serotonin (5-HT) signaling modulate tissue biomechanics of postnatal valves, but less is known of their role in the biomechanical remodeling of embryonic valves. In this study, we demonstrate that exogenous TGFβ3 increases AV cushion biomechanical stiffness and residual stress, but paradoxically reduces matrix compaction. We then show that TGFβ3 induces contractile gene expression (RhoA, aSMA) and extracellular matrix expression (col1α2) in cushion mesenchyme, while simultaneously stimulating a two-fold increase in proliferation. Local compaction increased due to an elevated contractile phenotype, but global compaction appeared reduced due to proliferation and ECM synthesis. Blockade of TGFβ type I receptors via SB431542 inhibited the TGFβ3 effects. We next showed that exogenous 5-HT does not influence cushion stiffness by itself, but synergistically increases cushion stiffness with TGFβ3 co-treatment. 5-HT increased TGFβ3 gene expression and also potentiated TGFβ3 induced gene expression in a dose-dependent manner. Blockade of the 5HT2b receptor, but not 5-HT2a receptor or serotonin transporter (SERT), resulted in complete cessation of TGFβ3 induced mechanical strengthening. Finally, systemic 5-HT administration in ovo induced cushion remodeling related defects, including thinned/atretic AV valves, ventricular septal defects, and outflow rotation defects. Elevated 5-HT in ovo resulted in elevated remodeling gene expression and increased TGFβ signaling activity, supporting our ex-vivo findings. Collectively, these results highlight TGFβ/5-HT signaling as a potent mechanism for control of biomechanical remodeling of AV cushions during development.

Transforming growth factor (TGF)-beta suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells (MECs). During the course of mammary tumorigenesis, genetic and epigenetic changes negate the cytostatic actions of TGF-beta, thus enabling TGF-beta to promote the acquisition and development of metastatic phenotypes. The molecular mechanisms underlying this conversion of TGF-beta function remain poorly understood but may involve signaling inputs from integrins.

The mechanism/s leading to diabetic neuropathy are complex. Transforming growth factor-beta1 (TGF-beta1) has been associated with diabetic nephropathy and retinopathy but not neuropathy. In this study, changes in TGF-beta isoforms were examined in vivo and in vitro. Two groups of animals, streptozotocin diabetic with neuropathy and non-diabetic controls were examined at 4 weeks (n=10/group) and 12 weeks (n=8/group). In diabetic DRG using quantitative real-time PCR (QRT-PCR), TGF-beta1 and TGF-beta2 mRNA, but not TGF-beta3, was increased at 4 and 12 weeks. In sciatic nerve TGF-beta3 mRNA was primarily increased. Immunohistochemistry (DRG) and immunoblotting (sciatic nerve) showed similar differential protein expression. In sciatic nerve TGF-beta formed homo- and hetero-dimers, of which beta(2)/beta(3), beta(1)/beta(1), and beta(1)/beta(3) were significantly increased, while that of the TGF-beta(2)/beta(2) homodimer was decreased, in diabetic compared to non-diabetic rats. In vitro, pretreatment of embryonic DRG with TGF-beta neutralizing antibody prevents the increase in total TGF-beta protein observed with high glucose using immunoblotting. In high glucose conditions, combination with TGF-beta2>beta1 increases the percent of cleaved caspase-3 compared to high glucose alone and TGF-beta neutralizing antibody inhibits this increase. Furthermore, consistent with the findings in diabetic DRG and nerve, TGF-beta isoforms applied directly in vitro reduce neurite outgrowth, and this effect is partially reversed by TGF-beta neutralizing antibody. These findings implicate upregulation of TGF-beta in experimental diabetic peripheral neuropathy and indicate a novel mechanism of cellular injury related to elevated glucose levels. In combination, these findings indicate a potential new target for treatment of diabetic peripheral neuropathy.

Transforming growth factor-beta (TGF-beta), a multifunctional growth factor, represents three mammalian isoforms, TGF-beta1, TGF-beta2, and TGF-beta3. In cutaneous wound healing, combined neutralization of TGF-beta1 and -beta2 or addition of TGF-beta3 reduces scar formation. Here, we investigated whether experimental manipulation of TGF-beta isoforms reduced adhesion formation after injury to the peritoneum. Adhesions were produced in mice by surgical abrasion of adjacent serosa followed by close apposition. In the first part of this study, a detailed analysis of TGF-beta isoform distribution was performed through immunolocalization. TGF-beta isoforms clearly showed a unique temporal and spatial pattern of expression after peritoneal wounding. Based on this pharmacokinetic data, we next administered neutralizing antibodies to TGF-beta1 and -beta2 or exogenous TGF-beta3 peptide by local application and intraperitoneal injection at various times before and after surgery. At day 7 after surgery, addition of neutralizing antibodies to both TGF-beta1 and -beta2 significantly reduced the number and size of adhesions (P < 0.05) compared with the vehicle control. By contrast, exogenous addition of TGF-beta3 either had no effect or increased adhesion formation compared to the vehicle control. In conclusion, these results show that by blocking both TGF-beta1 and TGF-beta2 using neutralizing antibodies, it is possible to prevent abdominal adhesion formation.

Elucidation of pathways involved in mouse strain-dependent variation in the hematopoietic stem cell (HSC) compartment may reveal novel mechanisms relevant in vivo. Here, we demonstrate genetically determined variation in the proliferation of lin-Sca1++kit+ (LSK) primitive hematopoietic progenitor cells in response to transforming growth factor-beta (TGF-beta) 2, the dose response of which was biphasic with a stimulatory effect at low concentrations. In contrast, the dose responses of TGF-beta1 or -beta3 were inhibitory and did not show mouse strain-dependent variation. A quantitative trait locus (QTL) for the effect of TGF-beta2 was identified on chromosome 4 overlapping with a QTL regulating the frequency of LSK cells. These overlapping QTL were corroborated by the observation that the frequency of LSK cells is lower in adult Tgfb2+/- mice than in wild-type littermates, indicating that TGF-beta2 is a genetically determined positive regulator LSK number in vivo. Furthermore, adult Tgfb2+/- mice have a defect in competitive repopulation potential that becomes more pronounced upon serial transplantation. In fetal TGF-beta2-deficient HSCs, a defect only appears after serial reconstitution. These data suggest that TGF-beta2 can act cell autonomously and is important for HSCs that have undergone replicative stress. Thus, TGF-beta2 is a novel, genetically determined positive regulator of adult HSCs.

Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-beta isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.

In this study we investigated whether signalling by TGF-beta3 and Wnt-5a cross-talk during chondrogenic differentiation of chick wing mesenchyme. Using differential display polymerase chain reaction screening, we found the expression of Wnt-5a to be significantly increased during transforming growth factor-beta3 (TGF-beta3)-induced precartilage condensation in mesenchyme micromass cultures. Transfection of cells with a Wnt-5a expression construct promoted precartilage condensation and chondrogenesis in micromass cultures, similar to that observed when chondrogenic-competent cells were exposed to TGF-beta3. Overexpression of Wnt-5a or treatment with TGF-beta3 stimulated the activation of protein kinase C-alpha (PKC-alpha) and p38 mitogen-activated protein kinase (MAPK), both positive regulators of chondrogenic differentiation. Inactivation of PKC-alpha and p38 MAPK by specific inhibitors abrogated chondrogenesis stimulated by both TGF-beta3 and Wnt-5a. Similarly, partial reduction in TGF-beta3-induced Wnt-5a expression by small interfering RNA resulted in decreased activities of PKC-alpha and p38 MAPK, and abolished the chondro-stimulatory effect of TGF-beta3. Collectively, these findings indicate that Wnt-5a, a non-canonical Wnt, can mediate the chondro-stimulatory effect of TGF-beta3 through upregulation of PKC-alpha and p38MAPK signaling.

Epithelial-mesenchymal transformation (EMT), the process by which epithelial cells are converted into motile, invasive mesenchymal cells, is critical to valvulogenesis. Transforming growth factor-beta3 (TGF-beta3), an established mediator of avian atrioventricular (AV) canal EMT, is secreted as a latent complex. In vitro, plasmin-mediated proteolysis has been shown to release active TGF-betas from the latent complex. Annexin II, a co-receptor for tissue plasminogen activator (tPA) and plasminogen, promotes cell-surface generation of the serine protease plasmin. Here, we show that annexin II-mediated plasmin activity regulates release of active TGF-beta3 during chick AV canal EMT. Primary embryonic endocardial-derived cells express annexin II which promotes plasminogen activation in vitro. Incubation of heart explant cultures with either alpha(2)antiplasmin (alpha(2)AP), a major physiological plasmin inhibitor, or anti-annexin II IgG, blocked EMT by approximately 80%, and 50%, respectively. Anti-annexin II IgG-mediated inhibition of EMT was overcome by the addition of recombinant TGF-beta3. Upon treatment with anti-annexin II IgG or alpha(2)AP, conditioned medium from heart explant cultures showed absence of the active fragment of TGF-beta3 by Western blot analysis and a approximately 50% decrease in TGF-beta specific bioactivity. Our results suggest that annexin II-mediated plasmin activity regulates the release of active TGF-beta during cardiac valve development in the avian heart.

The effects of microglia-derived plasminogen (PLGn) on the neurotrophic role of astrocytes were investigated in vitro. The treatment of astrocytes with rat PLGn led to a significant increase in transforming growth factor beta3 (TGFbeta3) in the conditioned medium (CM). This response of astrocytes to PLGn was characteristic and different from that to other stimulators, including lipopolysaccharide, phorbol-12-myristate-13-acetate, interferon-gamma, and ATP. In surveying the signaling molecules that respond to PLGn in astrocytes, we found that Akt/PKB phosphorylation is promoted. The pretreatment of astrocytes with an Akt inhibitor prior to PLGn stimulation resulted in a significant decrease in TGFbeta3 amounts in the CM, suggesting an association of Akt with TGFbeta3 production/secretion. Further survey revealed that phosphatidylinositol 3 kinase (PI3K) is closely associated with TGFbeta3 production/secretion in astrocytes. In fact, PI3K inhibitor clearly depressed the phosphorylation of Akt, indicating that PI3K is localized upstream of Akt. Moreover, the effects of PLGn to increase TGFbeta3 were depressed by pretreatment with a proteinase-activated receptor-1 (PAR-1) inhibitor. Plasmin could mimic the PLGn effects to upregulate TGFbeta3, and the plasmin effects were suppressed by pretreatment with the PAR-1 inhibitor, suggesting the association of PLGn/plasmin effects with PAR-1. In addition, Akt phosphorylation caused by plasmin was inhibited in the presence of PAR-1 inhibitor. We have therefore demonstrated that PLGn/plasmin, probably plasmin, facilitates the production/secretion of TGFbeta3 in astrocytes through both PAR-1 and the subsequent signaling cascade including PI3K and Akt.

Bone marrow mesenchymal stem cells (BMSCs) are considered good candidates for seed cells in bone engineering. The study aim to investigate the synergistic effects of human bone morphogenetic protein 2 (hBMP2) and transforming growth factor beta3 (hTGF-beta3) modified BMSCs on inducing osteogenic differentiation in vitro.

Recent studies have shown that transforming growth factor (TGF)-beta3 regulates blood-testis barrier (BTB) dynamics in vivo, plausibly by determining the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB site via the p38 MAP kinase signaling pathway. Since BTB is composed of coexisting TJs and basal ectoplasmic specializations [ES, a testis-specific adherens junction (AJ) type] in the seminiferous epithelium of the rat testis, we sought to examine if TGF-beta3 would also regulate anchoring junction dynamics. Using an in vivo model in which rats were treated with AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb Sertoli-germ cell AJs without affecting the integrity of TJs at the BTB, it was noted that the event of germ cell loss from the epithelium was associated with a transient surge in TGF-beta3. Furthermore, it was also associated with a surge in the protein levels of Ras, p-ERK, and the intrinsic activity of ERK, illustrating TGF-beta3 apparently regulates Sertoli-germ cell ES function via the Ras/MEK/ERK signaling pathway. Indeed, pretreatment of rats with TbetaRII/Fc chimera, a TGF-beta antagonist, or U0126, a specific MEK inhibitor, could significantly delay and partially block the disruptive effects of AF-2364 in depleting germ cells from the epithelium. While the protein levels of the cadherin/catenin complex were significantly induced during AF-2364-mediated germ cell loss, perhaps being used to retain germ cells in the epithelium, this increase failed to reverse the loss of adhesion function between Sertoli and germ cells because of a loss of protein-protein interactions between cadherins and catenins. Collectively, these results illustrate that the testis has a novel mechanism in place in which an agent that primarily disrupts TJs can induce secondary loss of AJ function, leading to germ cell loss from the seminiferous epithelium. Yet an agent that selectively disrupts AJs (e.g., AF-2364) can limit its effects exclusively at the Sertoli-germ cell adhesive site without perturbing the Sertoli-Sertoli TJs.

Complement activation plays a central role in autoimmune demyelination. To explore the possible effects of C5 on post-inflammatory tissue repair, we investigated the transcriptional profile induced by C5 in chronic experimental allergic encephalomyelitis (EAE) using oligonucleotide arrays. We used C5-deficient (C5-d) and C5-sufficient (C5-s) mice to compare the gene expression profile and we found that 390 genes were differentially regulated in C5-s mice as compared to C5-d mice during chronic EAE. Among them, a group of genes belonging to the family of insulin-like growth factor binding proteins (IGFBP) and transforming growth factor (TGF)-beta3 were found most significantly differentially regulated by C5. The dysregulation of these genes suggests that these proteins might be responsible for the gliosis and lack of remyelination seen in C5-d mice with chronic EAE.

In adult mammals such as rats, the blood-testis barrier (BTB) conferred by adjacent Sertoli cells in the seminiferous epithelium segregates post-meiotic germ cell development from the systemic circulation and is one of the tightest blood-tissue barriers. Yet it must "open" transiently at stages VIII to IX of the epithelial cycle to accommodate the migration of preleptotene/leptotene spermatocytes. While this is a vital event of spermatogenesis, the mechanism(s) that regulates BTB dynamics is virtually unknown. Recent studies have suggested that transforming growth factor-beta3 (TGF-beta3) and tumor necrosis factor alpha (TNFalpha) secreted by Sertoli and germ cells into the microenvironment of the BTB are capable of inducing reversible BTB disruption in vivo, apparently by reducing the steady-state levels of occludin and zonula occludens-1 (ZO-1) at the BTB via the p38 mitogen activated protein (MAP) kinase signaling pathway. In this study, local administration of TGF-beta3 (200 ng/testis) to the testis was shown to reversibly perturb the BTB integrity in vivo. We next sought to delineate the mechanism by which these cytokines maintain the steady-state level of integral membrane proteins: occludin, junctional adhesion molecule-A (JAM-A) and N-cadherin at the BTB. Primary Sertoli cells cultured in vitro were shown to establish intact tight junctions and functional BTB within two days when assessed by transepithelial electrical resistance (TER) measurement across the cell epithelium. Sertoli cell integral membrane protein internalization at the BTB was assessed by biotinylation of cell surface proteins, to be followed by tracking the endocytosed/biotinylated proteins by using specific antibodies. Both TGF-beta3 (3 ng/ml) and TNFalpha (10 ng/ml) were shown to significantly accelerate the kinetics of internalization of JAM-A, N-cadherin, and occludin versus controls. Treatment of cells with phenylarsine oxide (PAO) at 10 microM that blocks clathrin-mediated endocytosis was shown to inhibit the TGF-beta3-induced protein internalization. This inhibition of TGF-beta3-mediated protein endocytosis was further validated by silencing of clathrin. The specific effect of TGF-beta3 on protein internalization was further confirmed by RNAi using specific TGF-beta receptor I (TbetaR1) siRNA duplexes. When TbetaR1 was knocked down, the TGF-beta3-induced increase in the kinetics of JAM-A and occludin endocytosis was abolished, making them indistinguishable from controls, illustrating the specificity of the TGF-beta3 effects on protein endocytosis. In summary, this report demonstrates for the first time that BTB dynamics are regulated by TGF-beta3 and TNFalpha via an enhancement of protein endocytosis at the BTB.

There are a wide variety of extracellular matrices that can be used for regenerative purposes. Placental tissue-based matrices are quickly becoming an attractive option given the availability of the tissue source and the wide variety of bioactive molecules knows to exist in unprocessed placental tissues. As fresh placental tissues are seldom an option at the point of care, we examined both the composition and bioactivity of a commercially packaged flowable placental connective tissue matrix (FPTM) (BioECM® , Skye Biologics, Inc.) that was preserved by the proprietary HydraTek® process. The FPTM contained significant amounts of collagen and various growth factors such as bFGF, EGF, PDGF, KGF, and PIGF. In addition, it contained high levels of tissue inhibitors of metalloproteinases (TIMP-1 and 2) and molecules known to modulate the immune response including TGF-β and IL-4. In terms of its bioactivity, the FPTM displayed the ability (1) to suppress INF-γ secretion in activated T-cells nearly fourfold over control media, (2) to inhibit methicillin resistant Staphylococcus aureus (MRSA) and Staphylococcus saprophyticus proliferation, (3) to increase the migration of adipose-derived stem cells (ASCs) nearly threefold over control media and (4) to adhere to ASCs in culture. When ASCs were exposed to FPTM in culture, the cells maintained healthy morphology and showed no significant changes in the expression of five genes involved in tissue growth and repair as compared to culture in standard growth media. © 2018 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2731-2740, 2018.

Background: For regenerative therapies in the orthopedic field, one prerequisite for therapeutic success in the treatment of cartilage defects is the potential of body's own cells to migrate, proliferate and differentiate into functional cells. While this has been demonstrated for mesenchymal stem and progenitor cells (MPC) from healthy tissue sources, the potential of cells from degenerative conditions is unclear. In this study the regenerative potential of MPC derived from subchondral cancellous bone with diagnosed osteoarthritis is evaluated in vitro. Methods: OaMPC isolated from bone chips of three individual patients with Kellgren grade 3 osteoarthritis were characterized by analysis of cell surface antigen pattern. Cell proliferation was evaluated by doubling time and population doubling rate. Cell migration was assessed using a multi-well migration assay. Multi-lineage potential was evaluated by histological staining of adipogenic, osteogenic and chondrogenic markers. In addition, chondrogenic differentiation was verified by qPCR. Results: OaMPC showed a stable proliferation and a typical surface antigen pattern known from mesenchymal stem cells. Cell migration of oaMPC can be induced by human blood serum. OaMPC were capable of adipogenic, osteogenic and chondrogenic differentiation comparable to MPC derived from healthy conditions. Conclusion: OaMPC derived from knee joints affected by osteoarthritic conditions showed regeneration potential regarding migration, proliferation and chondrogenic differentiation. This suggests that oaMPC are able to contribute to cartilage repair tissue formation.

The present study was undertaken to investigate whether transforming growth factor-beta (TGF-beta) isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) and SMADs (SMAD2 and SMAD3) are involved in herpes simplex virus type 1 (HSV-1) corneal infection.

The three mammalian isoforms of transforming growth factor-beta(TGF-beta1, beta2, beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin differentiation, we investigated the expression of TGF-beta isoforms on cell growth and differentiation induction of the human keratinocyte cell line, HaCaT by elevating the Ca(2+) concentration. An ELISA and RT-PCR assay revealed secreted TGF-beta 1 protein and TGF-beta 1 mRNA were increased during calcium-induced differentiation. In contrast, major differences were seen for TGF-beta 2 and TGF-beta 3 mRNA which were decreased during differentiation, but TGF-beta 2 and TGF-beta3 protein were not evident on an ELISA. These results suggest different functions for each TGF-beta isoforms in epidermal differentiation, such that TGF-beta 1 is associated with the more differentiated state, and TGF-beta 2 and TGF-beta 3 may be associated the more proliferated state.

Extracorporeal shockwave therapy (ESWT) has emerged as the important choice for the treatment of many orthopedic disorders. Our previous mechanistic studies suggest that ESWT promoted osteogenesis of human mesenchymal stem cells (hMSCs) through mechanisms that involve adenosine 5'-triphosphate (ATP) release. In this study, we investigated the effect of ESWT on chondrogenesis of hMSCs. We demonstrate that ESWT treatment caused a significant release of adenosine from hMSCs; ESWT treatment increased the levels of A2B receptor (A2BR) in hMSCs under 3-D culture conditions. ESWT, exogenous adenosine and specialized A2BR agonist suppressed hMSC chondrogenic differentiation through downregulating the expressions of aggrecan (ACAN), Collagen Type I alpha 2(COL1A2), Collagen Type II alpha 1(COL2A1), Sex-Determining Region YBox 9 (SOX9) and Sex-Determining Region YBox 6 (SOX6). Selective A2BR antagonists induced chondrogenic differentiation of hMSCs. This study indicated that shockwave therapy inhibits hMSC chondrogenic differentiation through or partially through regulation of adenosine release and activation of A2B receptor under 3-D culture conditions.