Targeting TGF-beta Research Articles

A-118 Targeting TGF-beta activation in cutaneous T-cell lymphomas

A-118 Targeting TGF-beta activation in cutaneous T-cell lymphomas

Elevated secretion of pro-collagen I-alpha and vascular endothelial growth factor as biomarkers of acetabular labrum degeneration and calcification in hip osteoarthritis: An explant study

BackgroundHip osteoarthritis (OA) involves structural degeneration of different joint compartments, including femoral head cartilage, periarticular ligaments and the acetabular labrum. However, the molecular mechanisms underlying labrum degeneration in hip OA remain poorly understood. AimTo assess secretion of putative biomarkers for OA from explanted human labrum tissues under basal and inflammatory conditions and to determine whether these could differentiate between OA and calcification status compared to fracture controls. MethodsIntact labrum specimens were collected from patients undergoing joint arthroplasty for primary hip OA (n ​= ​15, mean age 70) or non-OA femoral neck fracture (n ​= ​5, mean age 64). Tissues were dissected in equal-sized samples and explanted for one week. To mimic activation of inflammatory signaling by endogenous damage-associated molecular patterns (DAMP) tissue were stimulated with a toll-like receptor 4 (TLR4) agonist (1 ​μg/mL LPS). The involvement of transforming growth factor-beta (TGF-beta) signaling was evaluated by treatment with a TGF-beta type 1 receptor inhibitor (10 ​μM SB-505124). Secretion of aggrecan (ACAN), pro-collagen-I alpha (Pro-Col-Iα), cartilage oligomeric matrix protein (COMP), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay (ELISA). Labrum calcification was evaluated by 3D whole mount fluorescent microscopy of ethyl cinnamate-based optically cleared tissues stained with Alcian blue/Alizarin red. ResultsWhole mount microscopy revealed non-OA fracture controls were non-calcified, whereas six OA labra (40%) were partially calcified or ossified. Basal secretion of Pro-Col-Iα and VEGF was increased four-fold in OA versus non-OA labra. Pro-Col-Iα levels were correlated with those of VEGF (r ​= ​0.65) and COMP (r ​= ​0.54). Stimulation of DAMP signaling through TLR4 affected secretion of IL-6, VEGF, COMP and Pro-Col-Iα, with distinct responses between non-OA and OA tissues. Inhibition of TGF-beta signaling specifically reduced elevated secretion of Pro-Col- Iα and VEGF in calcified OA labrum. ConclusionsSecretion of the putative OA biomarkers Pro-Col-Iα and VEGF is elevated in degenerated human acetabular labrum and may serve as indicators of OA and calcification status. Secretion of both factors was partially regulated by TGF-beta signaling in calcified OA labrum tissues.The Translational potential of this article:Our findings suggest that a biomarker panel consisting of Pro-Col-Iα/VEGF/COMP may be valuable for assessing subradiographic labrum degeneration and calcification in hip OA. Targeting TGF-beta signaling may offer a means to reduce vascular invasion and fibrosis in acetabular labrum tissue.

Pancreatic Adenocarcinoma Therapeutics Targeting RTK and TGF Beta Receptor.

Despite the improved overall survival rates in most cancers, pancreatic cancer remains one of the deadliest cancers in this decade. The rigid microenvironment, which majorly comprises cancer-associated fibroblasts (CAFs), plays an important role in the obstruction of pancreatic cancer therapy. To overcome this predicament, the signaling of receptor tyrosine kinases (RTKs) and TGF beta receptor (TGFβR) in both pancreatic cancer cell and supporting CAF should be considered as the therapeutic target. The activation of receptors has been reported to be aberrant to cell cycle regulation, and signal transduction pathways, such as growth-factor induced proliferation, and can also influence the apoptotic sensitivity of tumor cells. In this article, the regulation of RTKs/TGFβR between pancreatic ductal adenocarcinoma (PDAC) and CAFs, as well as the RTKs/TGFβR inhibitor-based clinical trials on pancreatic cancer are reviewed.

Targeting TGF-beta pathway with COVID-19 Drug Candidate ARTIVeda/PulmoHeal Accelerates Recovery from Mild-Moderate COVID-19

Our COVID-19 drug candidate ARTIVedaTM/PulmoHeal is a novel gelatin capsule formulation of the Artemisia extract Ayurveda for oral delivery of TGF-β targeting anti-malaria phytomedicine Artemisinin with documented anti-inflammatory and anti-SARS-CoV-2 activity. Here we report the safety and efficacy of ARTIVedaTM in adult COVID-19 patients with symptomatic mild-moderate COVID-19, who were treated in a randomized, open-label Phase 4 study in Bangalore, Karnataka, India (Clinical Trials Registry India identifier: CTRI/2020/09/028044). ARTIVeda showed a very favorable safety profile, and the only ARTIVeda-related adverse events were transient mild rash and mild hypertension. Notably, ARTIVeda, when added to the SOC, accelerated the recovery of patients with mild-moderate COVID-19. While all patients were symptomatic at baseline (WHO score=2-4), 31 of 39 (79.5%) of patients treated with ARTIVeda plus SOC became asymptomatic (WHO score=1) by the end of the 5-day therapy, including 10 of 10 patients with severe dry cough 7 of 7 patients with severe fever. By comparison, 12 of 21 control patients (57.1%) treated with SOC alone became asymptomatic on day 5 (p=0.028, Fisher’s exact test). This clinical benefit was particularly evident when the treatment outcomes of hospitalized COVID-19 patients (WHO score=4) treated with SOC alone versus SOC plus ARTIVeda were compared. The median time to becoming asymptomatic was only 5 days for the SOC plus ARTIVeda group (N=18) but 14 days for the SOC alone group (N=10) (p=0.004, Log-rank test). These data provide clinical proof of concept that targeting the TGF-β pathway with ARTIVeda may contribute to a faster recovery of patients with mild-moderate COVID-19 when administered early in the course of their disease.

The potential regulatory role of hsa_circ_0004104 in the persistency of atrial fibrillation by promoting cardiac fibrosis via TGF-\u03b2 pathway

IntroductionThe progression of paroxysmal AF (PAF) to persistent AF (PsAF) worsens the prognosis of AF, but its underlying mechanisms remain elusive. Recently, circular RNAs (circRNAs) were reported to be associated with cardiac fibrosis. In case of the vital role of cardiac fibrosis in AF persistency, we hypothesis that circRNAs may be potential regulators in the process of AF progression.Materials and methods6 persistent and 6 paroxysmal AF patients were enrolled as derivation cohort. Plasma circRNAs expressions were determined by microarray and validated by RT-PCR. Fibrosis level, manifested by serum TGF-β, was determined by ELISA. Pathways and related non-coding RNAs involving in the progression of AF regulated were predicted by in silico analysis.ResultsPsAF patients showed a distinct circRNAs expression profile with 92 circRNAs significantly dysregulated (fold change ≥ 2, p < 0.05), compared with PAF patients. The validity of the expression patterns was subsequently validated by RT-PCR in another 60 AF patients (30 PsAF and PAF, respectively). In addition, all the 5 up and down regulated circRNAs were clustered in MAPK and TGF-beta signaling pathway by KEGG pathway analysis. Among the 5 circRNAs, hsa_circ_0004104 was consistently downregulated in PsAF group (0.6 ± 0.33 vs 1.46 ± 0.41, p < 0.001) and predicted to target several AF and/or cardiac fibrosis related miRNAs reported by previous studies. In addition, TGF-β1 level was significantly higher in the PsAF group (5560.23 ± 1833.64 vs 2236.66 ± 914.89, p < 0.001), and hsa_circ_0004104 showed a significant negative correlation with TGF-β1 level (r = − 0.797, p < 0.001).ConclusionCircRNAs dysregulation plays vital roles in AF persistency. hsa_circ_0004104 could be a potential regulator and biomarker in AF persistency by promoting cardiac fibrosis via targeting MAPK and TGF-beta pathways.

P0321 : Increased anti-tumor effect of vitamin D after CYP24A1 inhibition on HCC cell lines

P0320 TARGETING TGF-BETA I WITH THE TRANSFORMING GROWTH FACTOR RECEPTOR TYPE I KINASE INHIBITOR, LY2157299, MODULATES STEMNESS-RELATED BIOMARKERS IN HEPATOCELLULAR CARCINOMA B. Rani, F. Dituri, Y. Cao, U. Engstrom, L. Lupo, S. Dooley, A. Moustakas, G. Giannelli. Department of Biomedical Sciences and Human Oncology, University of Bari, Bari, Italy; Biomedical Center, Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala, Sweden; Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy; Molecular Hepatology-Alcohol associated diseases, Department of medicine II, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; Department of Medical Biochemistry and Microbiology, and Ludwig Institute for Cancer Research, Science for Life Laboratory, Box 581 Biomedical Center, Uppsala, Sweden E-mail: bhavna.rani@uniba.it

P0320 : Targeting TGF-beta I with the transforming growth factor receptor type I kinase inhibitor, LY2157299, modulates stemness-related biomarkers in hepatocellular carcinoma

Targeting Tgf-Beta I With The Transforming Growth Factor Receptor Type I Kinase Inhibitor, Ly2157299, Modulates Stemness-Related Biomarkers In Hepatocellular Carcinoma

Immunotherapy of pancreatic carcinoma with siRNA targeting TGF-beta

Increasing evidence suggests that immune responses are important in the control of tumors. Pancreatic cancers are often infiltrated with lymphocytes, which correlates with a better prognosis. Continued tumor growth in the presence of a lymphocytic infiltration indicates a failure of immune control. TGF-beta is a highly active immunosuppressive cytokine and high serum levels correlate with a poor prognosis in pancreatic cancer, making it an interesting target for immunotherapy. The ubiquitously expressed cytosolic helicase retinoic acid-inducible protein I (RIG-I) is an immunoreceptor that signals the presence of viral RNA and activates the immune system via type I IFN. RIG-I detects RNA with a triphosphate group at the 5' end. Modification of siRNA as 5'-triphosphat-RNA (3p-siRNA) allows combining gene silencing and RIG-I activation. Here, we describe immunotherapy of murine pancreatic cancer with 3p-siRNA targeting TGF-beta. The 3p-siRNA reduced TGF-beta production by pancreatic carcinoma cells. In addition, RIG-I activation in tumor cells resulted in up-regulation of MHC-I expression, secretion of type I IFN and IP-10. As a consequence, tumor cells were sensitized to T cell-mediated killing. Moreover, 3p-siRNA induced tumor cell apoptosis via the intrinsic pathway. In vivo, 3p-siRNA against TGF-beta induced a Th1-type cytokine profile, with high levels of IL-12 and type I IFN. Systemic therapy slowed down tumor growth and prolonged survival in mice with orthotopic pancreatic tumors. Bi-functional RNA molecules that combine siRNA activity and activation of the innate immune system are promising candidates for the treatment of pancreatic cancer.

Tranilast, an antifibrogenic agent, ameliorates a dietary rat model of nonalcoholic steatohepatitis†

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease and is one of the most common liver diseases in the developed world. The histological findings of NASH are characterized by hepatic steatosis, inflammation, and fibrosis. However, an optimal treatment for NASH has not been established. Tranilast, N-(3',4'-dimethoxycinnamoyl)-anthranilic acid, is an antifibrogenic agent that inhibits the action of transforming growth factor beta (TGF-beta). This drug is used clinically for fibrogenesis-associated skin disorders including hypertrophic scars and scleroderma. TGF-beta plays a central role in the development of hepatic fibrosis, and tranilast may thus ameliorate the pathogenesis of NASH. We investigated the effects of tranilast using an established dietary animal model of NASH, obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and nondiabetic control Long-Evans Tokushima Otsuka (LETO) rats fed a methionine-deficient and choline-deficient diet. Treatment with 2% tranilast (420 mg/kg/day) for 8 weeks prevented the development of hepatic fibrosis and the activation of stellate cells, and down-regulated the expression of genes for TGF-beta and TGF-beta-target molecules, including alpha1 procollagen and plasminogen activator-1. In addition, tranilast attenuated hepatic inflammation and Kupffer cell recruitment, and down-regulated the expression of tumor necrosis factor alpha. Unexpectedly, tranilast ameliorated hepatic steatosis and up-regulated the expression of genes involved in beta-oxidation, such as peroxisome proliferator-activated receptor alpha and carnitine O-palmitoyltransferase-1. Most of these effects were observed in LETO rats and OLETF rats, which suggest that the action of tranilast is mediated through the insulin resistance-independent pathway. Our findings suggest that targeting TGF-beta with tranilast represents a new mode of therapy for NASH.

Targeting TGFbeta Bioavailability to Regulate Vascular Stability and Leakage

Significance of ECM as a regulator of vascular function has been hypothesized but not fully investigated. We assessed vessel stability and response to acute tissue stress in vivo in mice where type I collagen metabolism is altered due to presence of a knock-in mutation in the Col1a1 gene, e.g., Colα1(I)r/r mice. Our data revealed that plasma protein extravasation and vessel leakiness following acute stimulation of skin by topical exposure to mustard oil (MO) or intradermal injection of serotonin, histamine or VEGF-A, in Colα1(I)r/r mice was attenuated by 50% indicating that vessels resist acute responses due to maintenance of stability. We found that maintenance of vessel stability in Colα1(I)r/r mice was due to chronic activation of a metalloprotease (MP) in skin since treatment of Colα1(I)r/r mice with the MP inhibitor GM6001 restored acute vascular responses to mutant animals. We assessed vascular leakage in MMP-2, -8, -13 and -14-deficient mice and found that only in MMP14-deficient mice was vessel stability compromised as evidenced by their increased steady state level of plasma protein leakage. To reveal the molecular pathway being regulated by MMP14, we treated Colα1(I)r/r mice with neutralizing antibodies to TGFβ or an ALK5 inhibitor and found that vascular responsiveness in Colα1(I)r/r mice was normalized to characteristic levels as observed in wt mice. The implications of these findings are that pharmacologic manipulation of type I collagen metabolism, MMP14 activity, or TGFβ-induced signaling represents efficacious targets for regulating vascular stability and/or leakiness in vivo.

Inhibition of TGF‐β1 expression in human peritoneal mesothelial cells by pcDU6 vector‐mediated TGF‐β1 shRNA

Transforming growth factor-beta(1) (TGF-beta(1)) is an important mediator of the fibrosis process. High expression of TGF-beta(1) is closely related to peritoneal fibrosis. RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. The aim of this study was to assess the effect of shRNA targeting TGF-beta(1) on the expression of TGF-beta(1) in human peritoneal mesothelial cells (HPMC). TGF-beta(1) specific shRNA expression vectors were constructed and introduced to HPMC stimulated with 4.25% D-glucose (Gs) and 10 microg/mL of lipopolysaccharide (LPS). Expression of TGF-beta(1) mRNA was assessed by semiquantification reverse transcription polymerase chain reaction (RT-PCR). The TGF-beta(1) protein level in the culture supernatant was determined by sandwich enzyme-linked immunosorbent assay. The expression of TGF-beta(1) was upregulated significantly in HPMC stimulated with 4.25% D-Gs and 10 microg/mL LPS P < 0.01. TGF-beta(1) expression in pcDU6 plasmid vector-mediated TGF-beta(1) shRNA groups were obviously downregulated when compared to the 4.25% D-Gs and 10 microg/mL LPS group (P < 0.01) and the pcDU6 void vector group (P < 0.05), with no significant difference among pcDU6 plasmid vector -mediated TGF-beta(1) shRNA groups (P > 0.05). No significant difference was found between teh pcDNA3.1(-) vector plasmid-mediated TGF-beta(1) antisense RNA group and pcDU6 void vector group (P > 0.05). The expression of TGF-beta(1) in pcDU6 plasmid vector-mediated TGF-beta(1) shRNA groups were obviously downregulated when compared to the pcDNA3.1(-) plasmid vector-mediated TGF-beta(1) antisense RNA group (P < 0.05). TGF-beta(1)-specific shRNA can significantly inhibit the expression of TGF-beta(1) in HPMC stimulated with 4.25% D-Gs and 10 microg/mL LPS in HPMC. These results suggest the possible application of TGF-beta(1)-specific shRNA in preventing peritoneal fibrosis in patients receiving peritoneal dialysis.

Targeting TGF-beta in human keratinocytes and its potential role in wound healing.

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix (required for cell migration) and growth factors and their receptors. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated expression of angiogenic bFGF and VEGF, and collagenases MMP-2 and MMP-9 in tissue samples from chronic dermal wounds by immunohistochemistry. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of these factors was analysed by ELISA in human keratinocytes. Immunohistochemical investigation demonstrated a decreased expression of bFGF and VEGF protein, and an increased expression of MMP-2 and MMP-9 in tissue samples from chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment down-regulated collagenase secretion activity and up-regulated VEGF secretion in vitro. Therefore, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction, and stimulation of angiogenesis in chronic wounds. Improving basic knowledge and pharmaceutical intervention in this area ultimately may help clinicians to identify and proactively intervene in an effort to prevent normal wounds from becoming chronic.