Design, synthesis and evaluation of a pyrazolo[3,4-d]pyrimidine derivative as a novel and potent TGFß1R1 inhibitor.

The transforming growth factor ß1 (TGFß1)/SMAD signaling pathway regulates many vital physiological processes. The development of potent inhibitors targeting activin receptor-like kinase 5 (ALK5) would provide potential treatment reagents for various diseases. A significant number of ALK5 inhibitors have been discovered, and they are currently undergoing clinical evaluation at various stages. However, the clinical demands were far from being met. In this study, we utilized an alternative conformation-similarity-based virtual screening (CSVS) combined with a fragment-based drug designing (FBDD) strategy to efficiently discover a potent and active hit with a novel chemical scaffold. After structural optimization in the principle of group replacement, compound 57 was identified as the most promising ALK5 inhibitor. Compound 57 demonstrated significant inhibitory effects against the TGF-ß1/SMAD signaling pathway. It could markedly attenuate the production of extracellular matrix (ECM) and deposition of collagen. Also, the lead compound showed adequate pharmacokinetic (PK) properties and good in vivo tolerance. Moreover, treatment with compound 57 in two different xerograph models showed significant inhibitory effects on the growth of pancreatic cancer cells. These results suggested that lead compound 57 refers as a promising ALK5 inhibitor both in vitro and in vivo, which merits further validation.

Advances in the discovery of activin receptor-like kinase 5 (ALK5) inhibitors.

Activin receptor­like kinase-5 (ALK5) is an outstanding member of the transforming growth factor-ß (TGF-ß) family. (TGF-ß) signaling pathway integrates pleiotropic proteins that regulate various cellular processes such as growth, proliferation, and differentiation. Dysregulation within the signaling pathway can cause variety of diseases, such as fibrosis, cardiovascular disease, and especially cancer, rendering ALK5 a potential drug target. Hence, various small molecules have been designed and synthesized as potent ALK5 inhibitors. In this review, we shed light on the current ATP-competitive inhibitors of ALK5 through diverse heterocyclic based scaffolds that are in clinical or pre-clinical phases of development. Moreover, we focused on the binding interactions of the compounds to the ATP binding site and the structure-activity relationship (SAR) of each scaffold, revealing new scopes for designing novel candidates with enhanced selectivity and metabolic profiles.

ALK-5 Inhibitors for Efficient Derivation of Mesenchymal Stem Cells from Human Embryonic Stem Cells.

Objectives: Successful tissue regeneration requires a clinically viable source of mesenchymal stem cells (MSCs). We explored activin receptor-like kinase (ALK)-5 inhibitors to rapidly derive an MSC-like phenotype with high cartilage forming capacity from a xeno-free human embryonic cell line. Methods: Embryonic stem cell (ESC) lines (H9 and HADC100) were treated with the ALK-5 inhibitor SB431542; HADC100 cells were additionally treated with ALK-5 inhibitors SB525334 or GW788388. Cells were then seeded upon human fibronectin in the presence of fibroblast growth factor 2 (FGF2) in a serum-free medium. Flow cytometry was used to assess MSC markers (positive for CD73, CD90, and CD105; negative for CD34 and CD45). Differentiation status was assessed through quantitative polymerase chain reaction. Cartilage forming capacity was determined in high-density pellet cultures, in fibrin gels containing extracellular matrix (fibrin-ECM), and after implantation in ex vivo human osteoarthritic cartilage. Gene expression, histology, and immunostaining were used to assess cartilage phenotype, tissue regeneration, and integration. Results: Exposure to all three ALK-5 inhibitors lead to expression of mesodermal gene markers and differentiation into MSC-like cells (embryonic stem cell-derived mesenchymal stem cells [ES-MSCs]) based on surface marker expression. ES-MSC in pellet cultures or in fibrin-ECM gels expressed high levels of chondrogenic genes: COL2A1, ACAN, and COMP; and low levels of COL1A1 and RUNX2. Cell pellets or fibrin constructs implanted into ex vivo human osteoarthritic cartilage defects produced GAG-rich (safranin O positive) and collagen type II-positive neocartilage tissues that integrated well with native diseased tissue. Conclusions: We developed a protocol for rapid differentiation of xeno-free ESC into MSC-like cells with high cartilage forming capacity with potential for clinical applications. Impact statement Osteoarthritis (OA) is a common disease resulting in significant disability and no approved disease modifying treatment (other than total joint replacement). Embryonic stem cell-derived cell therapy has the potential to benefit patients with cartilage lesions leading to OA and may prevent or delay the need for total joint replacement.

Organoids from human tooth showing epithelial stemness phenotype and differentiation potential.

Insight into human tooth epithelial stem cells and their biology is sparse. Tissue-derived organoid models typically replicate the tissue's epithelial stem cell compartment. Here, we developed a first-in-time epithelial organoid model starting from human tooth. Dental follicle (DF) tissue, isolated from unerupted wisdom teeth, efficiently generated epithelial organoids that were long-term expandable. The organoids displayed a tooth epithelial stemness phenotype similar to the DF's epithelial cell rests of Malassez (ERM), a compartment containing dental epithelial stem cells. Single-cell transcriptomics reinforced this organoid-ERM congruence, and uncovered novel, mouse-mirroring stem cell features. Exposure of the organoids to epidermal growth factor induced transient proliferation and eventual epithelial-mesenchymal transition, highly mimicking events taking place in the ERM in vivo. Moreover, the ERM stemness organoids were able to unfold an ameloblast differentiation process, further enhanced by transforming growth factor-ß (TGFß) and abrogated by TGFß receptor inhibition, thereby reproducing TGFß's known key position in amelogenesis. Interestingly, by creating a mesenchymal-epithelial composite organoid (assembloid) model, we demonstrated that the presence of dental mesenchymal cells (i.e. pulp stem cells) triggered ameloblast differentiation in the epithelial stem cells, thus replicating the known importance of mesenchyme-epithelium interaction in tooth development and amelogenesis. Also here, differentiation was abrogated by TGFß receptor inhibition. Together, we developed novel organoid models empowering the exploration of human tooth epithelial stem cell biology and function as well as their interplay with dental mesenchyme, all at present only poorly defined in humans. Moreover, the new models may pave the way to future tooth-regenerative perspectives.

Lung-restricted ALK5 inhibition avoids systemic toxicities associated with TGFß pathway inhibition.

Systemic therapies targeting transforming growth factor beta (TGFß) or TGFßR1 kinase (ALK5) have been plagued by toxicities including cardiac valvulopathy and bone physeal dysplasia in animals, posing a significant challenge for clinical development in pulmonary indications. The current work aims to demonstrate that systemic ALK5-associated toxicities can be mitigated through localized lung delivery. Lung-selective (THRX-144644) and systemically bioavailable (galunisertib) ALK5 inhibitors were compared to determine whether lung selectivity is sufficient to maintain local tissue concentrations while mitigating systemic exposure and consequent pathway-related findings. Both molecules demonstrated potent ALK5 activity in rat precision cut lung slices (PCLS; p-SMAD3 half-maximal inhibitory concentration [IC50], 141 nM and 1070 nM for THRX-144644 and galunisertib, respectively). In 14-day repeat-dose studies in rats, dose-related cardiac valvulopathy was recapitulated with oral galunisertib at doses ≥150 mg/kg/day. In contrast, inhaled nebulized THRX-144644 did not cause similar systemic findings up to the maximally tolerated doses in rats or dogs (10 and 1.5 mg/kg/day, respectively). THRX-144644 lung-to-plasma ratios ranged from 100- to 1200-fold in rats and dogs across dose levels. THRX-144644 lung trough (24 h) concentrations in rats and dogs ranged from 3- to 17-fold above the PCLS IC50 across tolerated doses. At a dose level exceeding tolerability (60 mg/kg/day; 76-fold above PCLS IC50) minimal heart and bone changes were observed when systemic drug concentrations reached pharmacologic levels. In conclusion, the current preclinical work demonstrates that localized pulmonary delivery of an ALK5 inhibitor leads to favorable TGFß pathway pharmacodynamic inhibition in lung while minimizing key systemic toxicities.

Synthesis and biological evaluation of 4-(pyridine-4-oxy)-3-(tetrahydro-2H-pyran-4-yl)-pyrazole derivatives as novel, potent of ALK5 receptor inhibitors.

The transforming growth factor type ß receptor I (TGF-ß R1, also known as activin-like kinase 5 or ALK5) plays a significant role in the pathogenesis of multiple diseases such as malignant tumors and tissue fibrosis. Specific inhibition of ALK5 provides a novel method for controlling the development of cancers and fibrotic diseases. Herein, a novel series of 4-(pyridine-4-oxy)-3-(tetrahydro-2H-pyran-4-yl)-pyrazole derivatives was synthesized and identified as ALK5 inhibitors. Among them, compound 8h inhibited ALK5 autophosphorylation and NIH3T3 cell activity with IC50 values of 25 nM and 74.6 nM, respectively. Compound 8h also showed favorable pharmacokinetic profile and ameliorated hERG inhibition. More importantly, 30 mg/kg oral administration of 8h could significantly induce tumour growth inhibition in CT26 xenograft model without obvious toxicity.

Disease modeling of pulmonary fibrosis using human pluripotent stem cell-derived alveolar organoids.

Although alveolar epithelial cells play a critical role in the pathogenesis of pulmonary fibrosis, few practical in vitro models exist to study them. Here, we established a novel in vitro pulmonary fibrosis model using alveolar organoids consisting of human pluripotent stem cell-derived alveolar epithelial cells and primary human lung fibroblasts. In this human model, bleomycin treatment induced phenotypes such as epithelial cell-mediated fibroblast activation, cellular senescence, and presence of alveolar epithelial cells in abnormal differentiation states. Chemical screening performed to target these abnormalities showed that inhibition of ALK5 or blocking of integrin αVß6 ameliorated the fibrogenic changes in the alveolar organoids. Furthermore, organoid contraction and extracellular matrix accumulation in the model recapitulated the pathological changes observed in pulmonary fibrosis. This human model may therefore accelerate the development of highly effective therapeutic agents for otherwise incurable pulmonary fibrosis by targeting alveolar epithelial cells and epithelial-mesenchymal interactions.

The local wound environment is a key determinant of the outcome of TGFß signaling on the fibrotic response of CD44+ leader cells in an ex vivo post-cataract-surgery model.

The cytokine transforming growth factor beta (TGFß) has a role in regulating the normal and pathological response to wound healing, yet how it shifts from a pro-repair to a pro-fibrotic function within the wound environment is still unclear. Using a clinically relevant ex vivo post-cataract surgery model that mimics the lens fibrotic disease posterior capsule opacification (PCO), we investigated the influence of two distinct wound environments on shaping the TGFß-mediated injury response of CD44+ vimentin-rich leader cells. The substantial fibrotic response of this cell population occurred within a rigid wound environment under the control of endogenous TGFß. However, TGFß was dispensable for the role of leader cells in wound healing on the endogenous basement membrane wound environment, where repair occurs in the absence of a major fibrotic outcome. A difference between leader cell function in these distinct environments was their cell surface expression of the latent TGFß activator, αvß3 integrin. This receptor is exclusively found on this CD44+ cell population when they localize to the leading edge of the rigid wound environment. Providing exogenous TGFß to bypass any differences in the ability of the leader cells to sustain activation of TGFß in different environments revealed their inherent ability to induce pro-fibrotic reactions on the basement membrane wound environment. Furthermore, exposure of the leader cells in the rigid wound environment to TGFß led to an accelerated fibrotic response including the earlier appearance of pro-collagen + cells, alpha smooth muscle actin (αSMA)+ myofibroblasts, and increased fibrotic matrix production. Collectively, these findings show the influence of the local wound environment on the extent and severity of TGFß-induced fibrotic responses. These findings have important implications for understanding the development of the lens fibrotic disease PCO in response to cataract surgery wounding.

Long-Acting Tumor-Activated Prodrug of a TGFßR Inhibitor.

Inhibition of TGFß signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFß receptor (TGFßR) inhibitors in cancer therapy. To restrict the activity of TGFßR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFßR1 inhibitor 1 were prepared by appending 1 to a serine protease substrate and a half-life extension fatty acid carbon chain. The prodrugs were shown to be selectively metabolized in tumor tissues relative to the heart and blood and demonstrated a prolonged favorable increase in the tumor-to-heart ratio of the active drug in tissue distribution studies. Once-weekly administration of the most tissue-selective compound 10 provided anti-tumor efficacy comparable to the parent compound and reduced systemic exposure of the active drug.

Kaempferol 3-O-gentiobioside, an ALK5 inhibitor, affects the proliferation, migration, and invasion of tumor cells via blockade of the TGF-ß/ALK5/Smad signaling pathway.

Overactivation of TGF-ß/ALK5/Smad signaling pathway has been observed in the advanced stage of various human malignancies. As a key component of TGF-ß/ALK5/Smad signaling pathway transduction, TGF-ß type I receptor (also known as ALK5) has emerged as a promising therapeutic target for cancer treatment. In this study, to discover a novel ALK5 inhibitor, a commercial natural products library was screened using docking-based virtual screening, followed by luciferase reporter assay. A flavonoid glycoside kaempferol 3-O-gentiobioside (KPF 3-O-G) was identified as a potent ALK5 inhibitor through directly bound to the ATP-site of ALK5, resulting in the inhibitory effects on phosphorylation and translocation of Smad2 and expression of Smad4. Additionally, we found that KPF 3-O-G reduced cell proliferation and inhibited TGF-ß-induced cell migration and invasion. Moreover, western blotting and immunofluorescent analysis showed that KPF 3-O-G significantly reversed the TGF-ß-induced EMT biomarkers, including upregulation of E-cadherin and downregulation of N-cadherin, vimentin, and snail. In vivo study showed that KPF 3-O-G administration reduced tumor growth in human ovarian cancer xenograft mouse model, without obvious toxic effect. This study provided novel insight into the anticancer effects of KPF-3-O-G and indicated that KPF-3-O-G might be developed as potential therapeutics for cancer treatment after further validation.

Tofacitinib Ameliorates Lupus Through Suppression of T Cell Activation Mediated by TGF-Beta Type I Receptor.

Autoreactive T cells play a crucial role in the pathogenesis of systemic lupus erythematosus (SLE). TGF-ß type I receptor (TGFßRI) is pivotal in determining T cell activation. Here, we showed that TGFßRI expression in naïve CD4+ T cells was decreased in SLE patients, especially in those with high disease activity. Moreover, IL-6 was found to downregulate TGFßRI expression through JAK/STAT3 pathway in SLE patients. In vitro, the JAK inhibitor tofacitinib inhibited SLE T cell activating by upregulating TGFßRI expression in a dose-dependent manner. In MRL/lpr mice, tofacitinib treatment ameliorated the clinical indicators and lupus nephritis, as evidenced by reduced plasma anti-dsDNA antibody levels, decreased proteinuria, and lower renal histopathological score. Consistently, tofacitinib enhanced TGFßRI expression and inhibited T cell activation in vivo. TGFßRI inhibitor SB431542 reversed the effects of tofacitinib on T cell activation. Thus, our results have indicated that tofacitinib can suppress T cell activation by upregulating TGFßRI expression, which provides a possible molecular mechanism underlying clinical efficacy of tofacitinib in treating SLE patients.

T Cell-Mediated Antitumor Immunity Cooperatively Induced By TGFßR1 Antagonism and Gemcitabine Counteracts Reformation of the Stromal Barrier in Pancreatic Cancer.

The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFß antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFß inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFß-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFß inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFß inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFß inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFß inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery.

Discovery of 4-aminoquinolines as highly selective TGFßR1 inhibitors with an attenuated MAP4K4 profile for potential applications in immuno-oncology.

The tumor microenvironment contains high concentrations of TGFß, a crucial immunosuppressive cytokine. TGFß stimulates immune escape by promoting peripheral immune tolerance to avoid tumoricidal attack. Small-molecule inhibitors of TGFßR1 are a prospective method for next-generation immunotherapies. In the present study, we identified selective 4-aminoquinoline-based inhibitors of TGFßR1 through structural and rational-based design strategies. This led to the identification of compound 4i, which was found to be selective for TGFßR1 with the exception of MAP4K4 in the kinase profiling assay. The compound was then further optimized to remove MAP4K4 activity, since MAP4K4 is vital for proper T-cell function and its inhibition could exacerbate tumor immunosuppression. Optimization efforts led to compound 4s that inhibited TGFßR1 at an IC50 of 0.79 ± 0.19 nM with 2000-fold selectivity against MAP4K4. Compound 4s represents a highly selective TGFßR1 inhibitor that has potential applications in immuno-oncology.

Application of ultrasound elastography for monitoring the effects of TßR1 shRNA therapy on hepatic fibrosis in a rat model.

BACKGROUND: To investigate the application of ultrasound elastography in monitoring the effects of the transforming growth factor (TGF)-ß1 signaling pathway-targeted combination therapy for hepatic fibrosis. METHODS: 1. Short hairpin RNA (shRNA) constructs targeted towards TßR1 were designed, synthesized, and packaged using an adeno-associated virus (AAV), and the effective target shRNA was selected based on transfection results. 2. Fifty rats were randomly allocated (n = 10 per group) to the (A) control group, (B) model group, (C) 0-week therapy group, (D) 4-week therapy group, and (E) combination therapy group. At weeks 2, 4, 6, 8, 10, and 12, acoustic radiation force impulse (ARFI) elastography was used to measure the liver stiffness, inner diameter of the portal vein diameter, and blood velocity; radio frequency ultrasound imaging was used to measure the abdominal aortic elasticity parameter and pulse wave velocity (PWV) of the rats. 3. At week 12, portal vein puncture was performed to measure the portal venous pressure, and rat liver specimens were obtained for the pathological measurement of the degree of hepatic fibrosis. RESULTS: 1. An shRNA interference sequence targeted towards TßR1 was successfully designed, screened, and packaged using an AAV, and small-animal imaging results indicated expression of the specific shRNA in the liver. 2. At week 12, the ultrasound elastography results were significantly different between the experimental groups and the control group (p < 0.01); among the experimental groups, differences were significant between the therapy groups and the model group (p < 0.01). For groups C and E, the therapeutic effects on hepatic fibrosis in rats were significant, with the pathological results indicating a significant reduction in the degree of hepatic fibrosis (p < 0.01). The therapeutic effectiveness of group D was less than that of group C (p < 0.05). Significant differences existed between the portal venous pressure of the experimental groups and of the control group (p < 0.01). For the abdominal aortic elasticity parameter measured by radio frequency ultrasound imaging, differences existed between the values obtained from the experimental groups and from that of the control group (p < 0.05), while statistically significant differences were not found among the various experimental groups. 3. Continuous ultrasound examination results indicated that the elasticity value of group A was significantly different from those of the other groups after 2 weeks of model establishment (p < 0.01); after 6 weeks, the elasticity values of groups C and E were significantly different compared with those of groups B and D (p < 0.01). For the abdominal aortic elasticity parameter and pulse wave velocity (PWV), there were no significant differences among the various groups (p > 0.05). CONCLUSION: CCl4-induced hepatic fibrosis can be treated through shRNA silencing of TßR1. Ultrasound ARFI elastography is superior to external force-assisted elastography as it can reflect the degree of fibrosis in moderate to severe hepatic fibrosis and the variations in the degree of fibrosis after treatment. Portal venous pressure was positively correlated with the degree of fibrosis; with early combination therapy, both the degree of fibrosis and portal venous pressure could be effectively reduced.

N-cadherin mediates the migration of bone marrow-derived mesenchymal stem cells toward breast tumor cells.

Rationale: Bone marrow-derived mesenchymal stem cells (BM-MSCs) recruited into breast tumors regulate the behavior of tumor cells via various mechanisms and affect clinical outcomes. Although signaling molecules, such as transforming growth factor ß (TGF-ß), are known to transmit signals between BM-MSCs and breast tumor cells for recruiting BM-MSCs, it is unclear which specific intrinsic molecules involved in cell motility mediate the migration of BM-MSCs into breast tumor. It is also unclear as to how specific intrinsic molecules contribute to the migration. Methods: Conditioned medium (CM) from breast tumor cells (MCF-7 and MDA-MB-231) that simulates breast tumor secreting TGF-ß was used to examine the migration of BM-MSCs into breast tumors. A three-dimensional migration assay was performed to investigate the collective migration of BM-MSCs, maintaining cell-cell adhesion, toward breast tumor cells. Results: N-cadherin formed adherens junction-like structures on the intercellular borders of BM-MSCs, and TGF-ß increased the expression of N-cadherin on these borders. Knockdown of Smad4 impaired the TGF-ß-mediated increase in N-cadherin expression in BM-MSCs, but inhibitors of non-canonical TGF-ß pathways, such as extracellular signal-regulated kinases, Akt, and p38, did not affect it. siRNA-mediated knockdown of N-cadherin and Smad4 impaired the migration of BM-MSCs in response to TGF-ß. Conditioned medium from breast tumor cells also enhanced the expression of N-cadherin in BM-MSCs, but inactivation of TGF-ß type 1 receptor (TGFBR1) with SB505124 and TGFBR1 knockdown abolished the increase in N-cadherin expression. BM-MSCs collectively migrated toward CM from MDA-MB-231 in vitro while maintaining cell-cell adhesion through N-cadherin. Knockdown of N-cadherin abolished the migration of BM-MSCs toward the CM from breast tumor cells. Conclusion: In the present study, we identified N-cadherin, an intrinsic transmembrane molecule in adherens junction-like structures, on BM-MSCs as a mediator for the migration of these cells toward breast tumor. The expression of N-cadherin increases on the intercellular borders of BM-MSCs through the TGF-ß canonical signaling and they collectively migrate in response to breast tumor cells expressing TGF-ß via N-cadherin-dependent cell-cell adhesion. We, herein, introduce a novel promising strategy for controlling and re-engineering the breast tumor microenvironment.

Effects of TGF-ß1 Receptor Inhibitor GW788388 on the Epithelial to Mesenchymal Transition of Peritoneal Mesothelial Cells.

We investigated the effectiveness of the transforming growth factor beta-1 (TGF-ß) receptor inhibitor GW788388 on the epithelial to mesenchymal transition (EMT) using human peritoneal mesothelial cells (HPMCs) and examined the effectiveness of GW788388 on the peritoneal membrane using a peritoneal fibrosis mouse model. HPMCs were treated with TGF-ß with or without GW788388. Animal experiments were conducted on male C57/BL6 mice. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate. GW788388 was administered by once-daily oral gavage. The morphological change, cell migration, and invasion resulted from TGF-ß treatment, but these changes were attenuated by cotreatment with GW788388. TGF-ß-treated HPMCs decreased the level of the epithelial cell marker and increased the levels of the mesenchymal cell markers. Cotreatment with GW788388 reversed these changes. Phosphorylated Smad2 and Smad3 protein levels were stimulated with TGF-ß and the change was attenuated by cotreatment with GW788388. For the peritoneal fibrosis mice, thickness and collagen deposition of parietal peritoneum was increased, but this change was attenuated by cotreatment with GW788388. GW788388, an orally available potent TGF-ß receptor type 1 inhibitor, effectively attenuated TGF-ß-induced EMT in HPMCs. Cotreatment with GW788388 improved peritoneal thickness and fibrosis, and recovered peritoneal membrane function in a peritoneal fibrosis mouse model.

The culture microenvironment of juvenile idiopathic arthritis synovial fibroblasts is favorable for endochondral bone formation through BMP4 and repressed by chondrocytes.

BACKGROUND: We examined influences of conditioned media from chondrocytes (Ch) on juvenile idiopathic arthritis synovial fibroblasts (JFLS) and potential for JFLS to undergo endochondral bone formation (EBF). METHODS: Primary cells from three control fibroblast-like synoviocytes (CFLS) and three JFLS were cultured in Ch-conditioned media and compared with untreated fibroblast-like synoviocytes (FLS). RNA was analyzed by ClariomS microarray. FLS cells cultured in conditioned media were exposed to either TGFBR1 inhibitor LY3200882 or exogenous BMP4 and compared with FLS cultured in conditioned media from Ch (JFLS-Ch). Media supernatants were analyzed by ELISA. RESULTS: In culture, JFLS downregulate BMP2 and its receptor BMPR1a while upregulating BMP antagonists (NOG and CHRD) and express genes (MMP9, PCNA, MMP12) and proteins (COL2, COLX, COMP) associated with chondrocytes. Important TGFß superfamily member gene expression (TGFBI, MMP9, COL1A1, SOX6, and MMP2) is downregulated when JFLS are cultured in Ch-conditioned media. COL2, COLX and COMP protein expression decreases in JFLS-Ch. BMP antagonist protein (NOG, CHRD, GREM, and FST) secretion is significantly increased in JFLS-Ch. Protein phosphorylation increases in JFLS-Ch exposed to exogenous BMP4, and chondrocyte-like phenotype is restored in BMP4 presence, evidenced by increased secretion of COL2 and COLX. Inhibition of TGFBR1 in JFLS-Ch results in overexpression of COL2. CONCLUSIONS: JFLS are chondrocyte-like, and Ch-conditioned media can abrogate this phenotype. The addition of exogenous BMP4 causes JFLS-Ch to restore this chondrocyte-like phenotype, suggesting that JFLS create a microenvironment favorable for endochondral bone formation, thereby contributing to joint growth disturbances in juvenile idiopathic arthritis.

Evidence for synergy between sarcomeres and fibroblasts in an in vitro model of myocardial reverse remodeling.

We have created a novel in-vitro platform to study reverse remodeling of engineered heart tissue (EHT) after mechanical unloading. EHTs were created by seeding decellularized porcine myocardial sections with a mixture of primary neonatal rat ventricular myocytes and cardiac fibroblasts. Each end of the ribbon-like constructs was fixed to a plastic clip, allowing the tissues to be statically stretched or slackened. Inelastic deformation was introduced by stretching tissues by 20% of their original length. EHTs were subsequently unloaded by returning tissues to their original, shorter length. Mechanical characterization of EHTs immediately after unloading and at subsequent time points confirmed the presence of a reverse-remodeling process, through which stress-free tissue length was increased after chronic stretch but gradually decreased back to its original value within 9 days. When a cardiac myosin inhibitor was applied to tissues after unloading, EHTs failed to completely recover their passive and active mechanical properties, suggesting a role for actomyosin contraction in reverse remodeling. Selectively inhibiting cardiomyocyte contraction or fibroblast activity after mechanical unloading showed that contractile activity of both cell types was required to achieve full remodeling. Similar tests with EHTs formed from human induced pluripotent stem cell-derived cardiomyocytes also showed reverse remodeling that was enhanced when treated with omecamtiv mecarbil, a myosin activator. These experiments suggest essential roles for active sarcomeric contraction and fibroblast activity in reverse remodeling of myocardium after mechanical unloading. Our findings provide a mechanistic rationale for designing potential therapies to encourage reverse remodeling in patient hearts.

COX-2 promotes the osteogenic potential of BMP9 through TGF-ß1/p38 signaling in mesenchymal stem cells.

This study investigated the effects of transforming growth factor-ß1 (TGF-ß1) and cyclooxygenase-2 (COX-2) on bone morphogenetic protein 9 (BMP9) in mesenchymal stem cells (MSCs). We found that BMP9 increased mRNA levels of TGF-ß1 and COX-2 in C3H10T1/2 cells. BMP9-induced osteogenic markers were enhanced by TGF-ß1 and reduced by TGF-ßRI-specific inhibitor LY364947. BMP9 increased level of p-Smad2/3, which were either enhanced or reduced by COX-2 and its inhibitor NS398. BMP9-induced osteogenic markers were decreased by NS398 and it was partially reversed by TGF-ß1. COX-2 increased BMP9-induced osteogenic marker levels, which almost abolished by LY364947. BMP9-induced bone formation was enhanced by TGF-ß1 but reduced by silencing TGF-ß1 or COX-2. BMP9's osteogenic ability was inhibited by silencing COX-2 but partially reversed by TGF-ß1. TGF-ß1 and COX-2 enhanced activation of p38 signaling, which was induced by BMP9 and reduced by LY364947. The ability of TGF-ß1 to increase the BMP9-induced osteogenic markers was reduced by p38-specific inhibitor, while BMP9-induced TGF-ß1 expression was reduced by NS398, but enhanced by COX-2. Furthermore, CREB interacted with Smad1/5/8 to regulate TGF-ß1 expression in MSCs. These findings suggest that COX-2 overexpression leads to increase BMP9's osteogenic ability, resulting from TGF-ß1 upregulation which then activates p38 signaling in MSCs.