Letrozole ameliorates liver fibrosis through the inhibition of the CTGF pathway and 17ß-hydroxysteroid dehydrogenase 13 expression.

BACKGROUND: To establish a treatment option for liver fibrosis, the possibility of the drug repurposing theory was investigated, with a focus on the off-target effects of active pharmaceutical ingredients. METHODS: First, several active pharmaceutical ingredients were screened for their effects on the gene expression in the hepatocytes using chimeric mice with humanized hepatocytes. As per the gene expression-based screening assay for 36 medications, we assessed the mechanism of the antifibrotic effect of letrozole, a third-generation aromatase inhibitor, in mouse models of liver fibrosis induced by carbon tetrachloride (CCl4) and a methionine choline-deficient (MCD) diet. We assessed liver histology, serum biochemical markers, and fibrosis-related gene and protein expressions in the hepatocytes. RESULTS: A gene expression-based screening assay revealed that letrozole had a modifying effect on fibrosis-related gene expression in the hepatocytes, including YAP, CTGF, TGF-ß, and CYP26A1. Letrozole was administered to mouse models of CCl4- and MCD-induced liver fibrosis and it ameliorated the liver fibrosis. The mechanisms involved the inhibition of the Yap-Ctgf profibrotic pathway following a decrease in retinoic acid levels in the hepatocytes caused by suppression of the hepatic retinol dehydrogenase, Hsd17b13 and activation of the retinoic acid hydrogenase, Cyp26a1. CONCLUSIONS: Letrozole slowed the progression of liver fibrosis by inhibiting the Yap-Ctgf pathway. The mechanisms involved the modification of the Hsd17b13 and Cyp26a1 expressions led to the suppression of retinoic acid in the hepatocytes, which contributed to the activation of Yap-Ctgf pathway. Because of its off-target effect, letrozole could be repurposed for the treatment of liver fibrosis. The third-generation aromatase inhibitor letrozole ameliorated liver fibrosis by suppressing the Yap-Ctgf pathway by partially modifying the Hsd17b13 and Cyp26a1 expressions, which reduced the retinoic acid level in the hepatocytes. The gene expression analysis using chimeric mice with humanized liver revealed that the mechanisms are letrozole specific and, therefore, may be repurposed for the treatment of liver fibrosis.

The use of connective tissue growth factor mimics for flexor tendon repair.

Intrasynovial flexor tendon lacerations of the hand are clinically problematic, typically requiring operative repair and extensive rehabilitation. The small-molecule connective tissue growth factor (CTGF) mimics, oxotremorine M (Oxo-M) and 4-PPBP maleate (4-PPBP), have been shown to improve tendon healing in small animal models by stimulating the expansion and differentiation of perivascular CD146+ cells. To enhance intrasynovial flexor tendon healing, small-molecule CTGF mimics were delivered to repaired canine flexor tendons via porous sutures. In vitro studies demonstrated that Oxo-M and 4-PPBP retained their bioactivity and could be released from porous sutures in a sustained manner. However, in vivo delivery of the CTGF mimics did not improve intrasynovial tendon healing. Histologic analyses and expression of tenogenic, extracellular matrix, inflammation, and remodeling genes showed similar outcomes in treated and untreated repairs across two time points. Although in vitro experiments revealed that CTGF mimics stimulated robust responses in extrasynovial tendon cells, there was no response in intrasynovial tendon cells, explaining the lack of in vivo effects. The results of the current study indicate that therapeutic strategies for tendon repair must carefully consider the environment and cellular makeup of the particular tendon for improving the healing response.

CTGF induces tenogenic differentiation and proliferation of adipose-derived stromal cells.

Intrasynovial tendons are paucicellular and hypovascular, resulting in a poor response to injury. Surgical repair of ruptured or lacerated tendons often lead to complications such as adhesions, repair site gapping, and repair site rupture. Adipose-derived stem cells (ASCs) have shown promise for enhancing tendon repair, as they have the capacity to differentiate into tendon fibroblasts and augment the healing response. Furthermore, connective tissue growth factor (CTGF) has been shown to promote tendon regeneration via the stimulation of endogenous tendon stem cells. Here, we evaluated the potential of CTGF to promote tenogenic differentiation of ASCs in vitro. Gene and protein expression, cell proliferation, and FAK and ERK1/2 signaling were assessed. CTGF increased tenogenic genes in mouse ASCs in a dose- and time-dependent manner. Western blot and immunostaining analyses demonstrated increases in tenogenic protein expression in CTGF-treated ASCs at all timepoints studied. CTGF increased ASC proliferation in a dose-dependent manner. CTGF induced phosphorylation of ERK1/2 within 5 min and FAK within 15 min; both signals persisted for 120 min. Blocking FAK and ERK1/2 pathways by selective inhibitors SCH772984 and PF573228, respectively, attenuated the CTGF-induced tenogenic differentiation and proliferation of ASCs. These results suggest that CTGF induces tenogenic differentiation of ASCs via the FAK and ERK1/2 pathway. Statement of clinical significance: Although prior research has led to advances in tendon operative techniques and rehabilitation methods, clinical outcomes after tendon repair remain variable, with high rates of repair site gapping or rupture. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

NTG-101: A Novel Molecular Therapy that Halts the Progression of Degenerative Disc Disease.

The tremendous cost, pain and disability associated with degenerative disc disease (DDD) makes the development of a biological agent that can mitigate the course of DDD, a critical unmet need. We have identified and reported that a single injection of a combination of recombinant human (rh) Transforming growth factor beta 1 (TGF-ß1) and Connective tissue growth factor (CTGF) proteins into the injured intervertebral disc (IVD) nucleus pulposus (NP) can mediate DDD in a pre-clinical rodent model. In this study, we developed and evaluated the efficacy of a novel molecular therapy (NTG-101) containing rhTGF-ß1 and rhCTGF proteins suspended in an excipient solution using in vivo models of DDD including rat-tail and chondrodystrophic (CD) canines. Needle puncture injury in CD-canine NPs resulted in loss of hydration, disc height and showed radiographic evidence of DDD like humans. However, NTG-101-injected IVDs maintained disc height and demonstrated retention of viscoelastic properties as compared to IVDs injected with phosphate buffer saline (PBS, 1X, pH = 7.2). In addition, a single intra-discal injection of NTG-101 into the injured IVD-NPs resulted in sustained expression of healthy extra-cellular matrix (ECM) proteins (aggrecan, collagen 2A1) and reduced expression of inflammation associated proteins and molecules (IL-1ß, IL-6, IL-8, MMP-13, Cox-2 and PGE2) as compared to vehicle controls. In conclusion, we demonstrated that a single intra-discal injection of the novel formulation, NTG-101 confers a robust anti-inflammatory, anti-catabolic and pro-anabolic effects in pre-clinical models of DDD thereby restoring homeostasis. These findings suggest the therapeutic potential of NTG-101 for clinical use.

The effects of platelet-rich plasma on hypertrophic scars fibroblasts.

We hypothesised that a feedback mechanism of the transforming growth factor (TGF)-ß1 signalling pathway, triggered by high-level TGF-ß1, activates platelet-rich plasma (PRP) release to reduce connective tissue growth factor (CTGF) production and expression of CTGF mRNA in hypertrophic scar dermal fibroblasts. Primary dermal fibroblasts were isolated from cultures of hypertrophic scars. Cells were cultured after addition of serum-free Dulbecco's modified Eagle's medium supplemented with 5% (wt/vol) PRP or platelet-poor plasma (PPP). At 1, 4, 6, 8, 11, and 13 days after addition of PRP or PPP, the TGF-ß1 and CTGF levels in supernatants were determined using solid-phase enzyme-linked immunosorbent assays. Quantitative reverse transcription polymerase chain reactions were performed to quantify TGF-ß1 and CTGF mRNA expression levels. TGF-ß1 mRNA expression in the PRP groups was lower than in the PPP groups from 4 to 13 days of culture, and there was statistically significant difference (P < .01). CTGF level and mRNA expression in the PRP groups was lower than in the PPP groups, and there were statistically significant differences (P < .01). Although further experiments will focus on clarifying the second messenger of the TGF-ß1 negative feedback mechanism, the in vitro data presented show that PRP can potentially reduce CTGF and CTGF gene transcription by triggering the TGF-ß1 signalling negative feedback mechanism.

Enhancement of Anterior cruciate ligament injury repairing using connective tissue growth factor in a rabbit model.

The study was to evaluate the contribution of connective tissue growth factor (CTGF) to the regeneration of the torn anterior cruciate ligament (ACL) in a rabbit. ACL transection surgeries were performed on both knees of male New Zealand rabbits. Then injury reparation was done as follows: 0.5ml fibrin glue (FG) alone (FG-treated group, n=24 knees) and 0.5ml FG dissolve with 15ng CTGF (CTGF/FG-treated group, n=24 knees). At 2 or 6 weeks after surgery, the ACLs were characterized histologically (n=6 knees) and biomechanically (n=6 knees). The healing effect of the CTGF/FG-treated group was obviously better than that of the FG-treated group, with an increased amount of collagen fibers and fibroblasts in the ligament tissue. After 2 or 6 weeks of healing, CTGF/FG-treated group exhibited significantly higher maximum loads of 8.50±0.58N and 16.35±1.16N, compared with the control group (7.52±0.80N and 13.60±1.35N). And the stiffness of CTGF/FG-treated group at 2 or 6 weeks post-intervention (5.59±1.24N/mm and 11.64±2.21N/mm) was remarkably higher than that the control group (3.74±0.89N/mm and 6.83±2.51N/mm). CTGF could serve as a potentially attractive tool for improving ACL injury treatment by promoting the regeneration of related cells.

CTGF/CCN2 Postconditioning Increases Tolerance of Murine Hearts towards Ischemia-Reperfusion Injury.

BACKGROUND AND PURPOSE: Previous studies of ischemia-reperfusion injury (IRI) in hearts from mice with cardiac-restricted overexpression of CCN2 have shown that CCN2 increases tolerance towards IRI. The objectives of this study were to investigate to what extent post-ischemic administration of recombinant human CCN2 (rhCCN2) would limit infarct size and improve functional recovery and what signaling pathways are involved. EXPERIMENTAL APPROACH: Isolated mice hearts were perfused ad modum Langendorff, subjected to no-flow, global ischemia, and subsequently, exposed to mammalian cell derived, full-length (38-40kDa) rhCCN2 (250 nM) or vehicle during the first 15 min of a 60 min reperfusion period. KEY RESULTS: Post-ischemic administration of rhCCN2 resulted in attenuation of infarct size from 58 ± 4% to 34 ± 2% (p < 0.001) which was abrogated by concomitant administration of the PI3 kinase inhibitor LY294002 (45 ± 3% vs. 50 ± 3%, ns). In congruence with reduction of infarct size rhCCN2 also improved recovery of left ventricular developed pressure (p < 0.05). Western blot analyses of extracts of ex vivo-perfused murine hearts also revealed that rhCCN2 evoked concentration-dependent increase of cardiac phospho-GSK3ß (serine-9) contents. CONCLUSIONS AND IMPLICATIONS: We demonstrate that post-ischemic administration of rhCCN2 increases the tolerance of ex vivo-perfused murine hearts to IRI. Mechanistically, this postconditioning effect of rhCCN2 appeared to be mediated by activation of the reperfusion injury salvage kinase pathway as demonstrated by sensitivity to PI3 kinase inhibition and increased CCN2-induced phosphorylation of GSK3ß (Ser-9). Thus, the rationale for testing rhCCN2-mediated post-ischemic conditioning of the heart in more complex models is established.

Comment on "Topically Applied Connective Tissue Growth Factor/CCN2 Improves Diabetic Preclinical Cutaneous Wound Healing: Potential Role for CTGF in Human Diabetic Foot Ulcer Healing".

A recent paper in this journal, presented a novel method by topical application of growth factors in stimulating diabetic cutaneous wound healing that caught our attention. We believe that the experimental method in the article is efficient and creative, but it also has some controversies and shortcomings to be discussed. We noted that the authors used "Tegaderm" as a semiocclusive dressing film and stated that it exerted a "splinting effect" on the wound margins and controlled contraction. Indeed, the "Tegaderm" itself can serve as a dressing film to isolate the wound bed with outside environments while the "splinting effect" is mainly achieved by adding silicone splints around the wound. Considering the unique properties of silicone splints and "Tegaderm," our experimental group propose an alternative method named "combined-suturing" technique that is not only suturing the silicone splints but also securing the "Tegaderm" around the wound. The specific reasons and operative procedures are explained in detail in this letter.

Topically applied connective tissue growth factor/CCN2 improves diabetic preclinical cutaneous wound healing: potential role for CTGF in human diabetic foot ulcer healing.

AIMS/HYPOTHESIS: Topical application of CTGF/CCN2 to rodent diabetic and control wounds was examined. In parallel research, correlation of CTGF wound fluid levels with healing rate in human diabetic foot ulcers was undertaken. METHODS: Full thickness cutaneous wounds in diabetic and nondiabetic control rats were treated topically with 1 µg rhCTGF or vehicle alone, on 2 consecutive days. Wound healing rate was observed on day 14 and wound sites were examined for breaking strength and granulation tissue. In the human study across 32 subjects, serial CTGF regulation was analyzed longitudinally in postdebridement diabetic wound fluid. RESULTS: CTGF treated diabetic wounds had an accelerated closure rate compared with vehicle treated diabetic wounds. Healed skin withstood more strain before breaking in CTGF treated rat wounds. Granulation tissue from CTGF treatment in diabetic wounds showed collagen IV accumulation compared with nondiabetic animals. Wound α-smooth muscle actin was increased in CTGF treated diabetic wounds compared with untreated diabetic wounds, as was macrophage infiltration. Endogenous wound fluid CTGF protein rate of increase in human diabetic foot ulcers correlated positively with foot ulcer healing rate (r = 0.406; P < 0.001). CONCLUSIONS/INTERPRETATION: These data collectively increasingly substantiate a functional role for CTGF in human diabetic foot ulcers.

Inhibition of connective tissue growth factor suppresses hepatic stellate cell activation in vitro and prevents liver fibrosis in vivo.

Activation of hepatic stellate cells (HSC) represents a critical event in fibrosis, and connective tissue growth factor (CTGF) plays a profibrotic activity and a key factor in the pathogenesis of tissue fibrosis. The current study aimed to determine whether lentivirus-mediated short hairpin RNA (shRNA)-targeted CTGF downregulates the CTGF expression and furthermore whether it suppresses the activation and proliferation of HSC in vitro and prevents liver fibrosis in vivo. HSC-T6 cells were treated with recombinant lentivirus carrying CTGF siRNA. Real-time PCR, Western blotting, MTT, and flow cytometry were performed to investigate the activation and proliferation of HSC-T6 cells in response to CTGF silence. CCl4-induced rats were received lentivirus containing CTGF siRNA by intraportal vein injection. Levels of liver fibrosis were assessed by biochemical and histopathologic examinations. Recombinant lentivirus containing CTGF siRNA could effectively and specifically downregulate the expression of CTGF in both HSC-T6 cells and CCl4-induced rats with liver fibrosis. Blockade of CTGF resulted in significant inhibition of HSC activation and proliferation with decrease in TIMPs, MMP2, MMP9, and collagen I, as well as increase in cells in S phase. Silencing CTGF expression with siRNA prevented liver fibrosis in CCl4-induced rat model. These findings indicated that CTGF plays a key role in the pathogenesis of liver fibrosis and lentiviral-mediated CTGF siRNA has the potential to be an effective treatment for liver fibrosis.

[Enhancement of meniscal tearing damage repairing in the avascular zone using connective tissue growth factor (CTGF) in the rabbit model].

OBJECTIVE: To investigate effect of connective tissue growth factors (CTGF) on secretion of extracellular matrix synthesis of meniscal fibrochondrocytes, expression of vascular endothelial growth factors (VEGF), and angiogenesis during the repair of meniscal tearing damage. METHODS: Meniscal fibrochondrocytes were isolated from the inner--1/2 of rabbits' meniscus by collagenase enzymatic digestion, centrifugal separation, and treated with 100 ng/ml CTGF in vitro. Characterization of fibrochondrocytes was identified by flow cytometry analysising CD31, CD44, CD45 and CD105, and was further tested by type II collagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. In vitro, the sections of the 3 mm of the longitudinal teared in the middle of the rabbit's meniscus, and then the defects were dealed with simple suture, suture and implanting with PBS-fibrin glue, sutured and implanting with 1.5 microg CTGF respectively. Expression and distribution of type I and II collagen and VEGF, the tearing healing were observed by fluorescence-immunohistochemisty analysis on the 1st week, the 4th week and the 10th week. RESULTS: Quantitative RT-PCR assays showed that type I and type II collagen,and VEGF mRNA expression in the 100 ng/ml CTGF group had been remarkably enhanced than in the PBS group on the 14th day. Consistent with these effects in vitro, fluorescence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue, type I collagen, type I collagen and capillaries completely filled the defect on the 10th week postoperatively. In contrast, only soft tissue repair occurred after the PBS-fibrin glue was implanted. CONCLUSION: CTGF can significantly promote extracellular matrix (I collagen, II collagen) of the meniscal avascular zone synthesis, and CTGF can greatly heighten the expression of VEGF activity at the same time in vitro, so that it can further enhance the repair of meniscal tearing damage in the avascular zone.

Connective tissue growth factor promotes interleukin-1ß-mediated synovial inflammation in knee osteoarthritis.

Connective tissue growth factor (CTGF), also known as CCN2, is a key proinflammatory mediator. In the present study, the involvement of the CTGF signaling pathway in human knee osteoarthritis (OA) fibroblast-like synoviocytes (FLSs) was investigated. FLSs were isolated from human OA synovium and incubated with CTGF in the absence or presence of interleukin­1ß (IL­1ß). The expression of relevant genes and proteins was analyzed by qPCR, western blotting and enzyme-linked immunosorbent assay (ELISA). Matrix metalloproteinase (MMP) activity and nuclear factor (NF)-κB activation were also evaluated. CTGF stimulation resulted in the significant production of IL-6, IL-8, C-C motif ligand 2 (CCL2), CCL20, MMP-1 and MMP-3 in FLSs in the presence, but not in the absence, of IL-1ß. CTGF also enhanced the levels of phosphorylated extracellular signal-related kinase 1/2 (ERK1/2) and p38. In addition, CTGF at 25 ng/ml, in the presence of IL­1ß, significantly potentiated NF-κB activation. The results indicated that CTGF interacted with IL­1ß in FLSs to promote the inflammatory response in the synovium, leading to the initiation of the inflammatory cascade. These results support the proinflammatory role of CTGF in synovitis and joint destruction in OA.

Targeting TGFß superfamily ligand accessory proteins as novel therapeutics for chronic lung disorders.

Dysregulation of the transforming growth factor ß (TGFß) pathway has been implicated to underlie a number of disease indications including chronic lung disorders such as asthma, chronic obstructive pulmonary disease (COPD), interstitial pneumonias, and pulmonary arterial hypertension (PAH). Consequently, the pharmaceutical industry has devoted significant resources in the pursuit of TGFß pathway inhibitors that target the cognate type I and II receptors and respective ligands. The progress of these approaches has been painfully slow, due in part to dose-limiting safety issues that result from the antagonism of a pathway that is responsible for regulating many fundamental biological processes including immune surveillance and cardiovascular responses. These disappointments have led many in the field to conclude that modulating the TGFß pathway for chronic indications with a sufficient safety window using conventional approaches may be extremely difficult to achieve. Here we review the rationale and limitations of the use of TGFß pathway inhibitors in chronic lung disorders and the possibility of targeting TGFß superfamily ligand accessory proteins to allow rheostatic regulation of signaling to achieve efficacy while maintaining a sufficient therapeutic index.

Connective tissue growth factor acts as a therapeutic agent and predictor for peritoneal carcinomatosis of colorectal cancer.

PURPOSE: Here, we aimed to investigate the role of connective tissue growth factor (CTGF) in peritoneal carcinomatosis (PC) associated with colorectal cancer (CRC) and to characterize the underlying mechanism of CTGF mediating adhesion. EXPERIMENTAL DESIGN: A cohort of 136 CRC patient specimens was analyzed in this study. CRC cell lines were used for in vitro adhesion assay and in vivo peritoneal dissemination experiment. Recombinant CTGF protein treatment, transfection of CTGF expression plasmids, and knockdown of CTGF expression in CRC cells were utilized to evaluate the integrin α5, which served as a target of CTGF in inhibiting peritoneal seeding. RESULTS: The analysis of CRC tissues revealed an inverse correlation between CTGF expression and prevalence of PC. Lower CTGF level in CRC patients was associated with higher peritoneal recurrence rate after surgery. Inducing CTGF expression in cancer cells resulted in decreased incidence of PC and increased rate of mice survival. The mice received intraperitoneal injection of recombinant CTGF protein simultaneously with cancer cells or following tumor formation; in both cases, peritoneal tumor dissemination was found to be effectively inhibited in the mouse model. Functional assay revealed that CTGF significantly decreased the CRC cell adhesion ability, and integrin α5 was confirmed by reverse transcriptase PCR and functional blocking assay as a downstream effector in the CTGF-mediated inhibition of CRC cell adhesion. CONCLUSIONS: CTGF acts as a molecular predictor of PC and could be a potential therapeutic target for the chemoprevention and treatment of PC in CRC patients.

Potential applications for cell regulatory factors in liver progenitor cell therapy.

Orthotopic liver transplant represent the state of the art treatment for terminal liver pathologies such as cirrhosis in adults and hemochromatosis in neonates. A limited supply of transplantable organs in relationship to the demand means that many patients will succumb to disease before an organ becomes available. One promising alternative to liver transplant is therapy based on the transplant of liver progenitor cells. These cells may be derived from the patient, expanded in vitro, and transplanted back to the diseased liver. Inborn metabolic disorders represent the most attractive target for liver progenitor cell therapy, as many of these disorders may be corrected by repopulation of only a portion of the liver by healthy cells. Another potential application for liver progenitor cell therapy is the seeding of bio-artificial liver matrix. These ex vivo bioreactors may someday be used to bridge critically ill patients to other treatments. Conferring a selective growth advantage to the progenitor cell population remains an obstacle to therapy development. Understanding the molecular signaling mechanisms and micro-environmental cues that govern liver progenitor cell phenotype may someday lead to strategies for providing this selective growth advantage. The discovery of a population of cells within the bone marrow possessing the ability to differentiate into hepatocytes may provide an easily accessible source of cells for liver therapies.

Regenerative medicine: a surgeon's perspective.

More than 200 million incisions are made in the world each year on children and adults. They all end up with a scar unless there is an unusual situation where we are operating on an early gestation fetus. The question is, "why do we not regenerate?" and "why do we always heal with either a 'normal amount of scarring' or, approximately 15% of the time, with a pathologic amount of scarring (hypertrophic scar or keloid)?"