Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf.

RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.

Yin/Yang expression of CCN family members: Transforming growth factor beta 1, via ALK5/FAK/MEK, induces CCN1 and CCN2, yet suppresses CCN3, expression in human dermal fibroblasts.

The role of the microenvironment in driving connective tissue disease is being increasingly appreciated. Matricellular proteins of the CCN family are signaling modifiers that are secreted by cells into the extracellular matrix microenvironment where they have profound, context-dependent effects on organ development, homeostasis and disease. Indeed, CCN proteins are emergent targets for therapeutic intervention. Recent evidence suggests that, in vivo, CCN3 has effects opposing CCN2. Moreover, when CCN3 expression is high, CCN2 expression is low. That is, they appear to be regulated in a yin/yang fashion, leading to the hypothesis that the CCN2:CCN3 ratio is important to control tissue homeostasis. To begin to test the hypothesis that alterations in CCN2:CCN3 expression might be important in skin biology in vivo, we evaluated the relative ex vivo effects of the profibrotic protein TGFbeta1 on dermal fibroblasts on protein and RNA expression of CCN3 and CCN2, as well as the related protein CCN1. We also used signal transduction inhibitors to begin to identify the signal transduction pathways controlling the ability of fibroblasts to respond to TGFbeta1. As anticipated, CCN1 and CCN2 protein and mRNA were induced by TGFbeta1 in human dermal fibroblasts. This induction was blocked by TAK1, FAK, YAP1 and MEK inhibition. Conversely, TGFbeta1 suppressed CCN3 mRNA expression in a fashion insensitive to FAK, MEK, TAK1 or YAP1 inhibition. Unexpectedly, CCN3 protein was not detected in human dermal fibroblasts basally. These data suggest that, in dermal fibroblasts, the profibrotic protein TGFbeta1 has a divergent effect on CCN3 relative to CCN2 and CCN1, both at the mRNA and protein level. Given that the major source in skin in vivo of CCN proteins are fibroblasts, our data are consistent that alterations in CCN2/CCN1: CCN3 ratios in response to profibrotic agents such as TGFbeta1 may play a role in connective tissue pathologies including fibrosis.

Evaluation of genistein ability to modulate CTGF mRNA/protein expression, genes expression of TGFß isoforms and expression of selected genes regulating cell cycle in keloid fibroblasts in vitro.

Keloids are characterized by overgrowth of connective tissue in the skin that arises as a consequence of abnormal wound healing. Normal wound healing is regulated by a complex set of interactions within a network of profibrotic and antifibrotic cytokines that regulate new extracellular matrix (ECM) synthesis and remodeling. These proteins include transforming growth factor ß (TGFß) isoforms and connective tissue growth factor (CTGF). TGFß1 stimulates fibroblasts to synthesize and contract ECM and acts as a central mediator of profibrotic response. CTGF is induced by TGFß1 and is considered a downstream mediator of TGFß1action in fibroblasts. CTGF plays a crucial role in keloid pathogenesis by promoting prolonged collagen synthesis and deposition and as a consequence sustained fibrotic response. During keloids formation, besides imbalanced ECM synthesis and degradation, fibroblast proliferation and it's resistance to apoptosis is observed. Key genes that may play a role in keloid formation and growth involve: suppressor gene p53.,cyclin-depend- ent kinase inhibitor CDKN1A (p21) and BCL2 family genes: antiapoptotic BCL-2 and proapoptotic BAX. Genistein (4',5,7-trihydroxyisoflavone) exhibits multidirectional biological action. The concentration of genistein is relatively high in soybean. Genistein has been shown as effective antioxidant and chemopreventive agent. Genistein can bind to estrogen receptors (ERs) and modulate estrogen action due to its structure similarity to human estrogens. Genistein also inhibits transcription factors NFκB. Akt and AP-l signaling pathways, that are important for cytokines expression and cell proliferation, differentiation, survival and apoptosis. The aim of the study was to investigate genistein as a potential inhibitor of CTGF and TGFß1, ß2 and ß3 isoforms expression and a potential regulator of p53. CDKN1A(p21), BAX and BCL-2 expression in normal fibroblasts and fibroblasts derived from keloids cultured in vitro. Real time RT-QPCR was used to estimate transcription level of selected genes in normal and keloid fibroblasts treated with genistein. Secreted/cell-associated CTGF protein was evaluated in cell growth's medium by ELISA. Total protein quantification was evaluated by fluorimetric assay in cells llsates (Quant-iT TM Protein Assay Kit). It was found that TGFß1, ß2 and ß3 genes expression are decreased by genistein. Genistein suppresses the expression of CTGF mRNA and CTGF protein in a concentration dependent manner, p53 and p21 genes expression are modulated by genistein in concentration dependent manner. The agent also modulates BAX/BCL-2 ratio in examined cells in vitro.

Optimum scratch assay condition to evaluate connective tissue growth factor expression for anti-scar therapy.

To evaluate a potential anti-scar therapy, we first need to have a reliable in vitro wound model to understand dermal fibroblast response upon cell injury and how cytokine levels are changed upon different wound heal phases. An in vitro wound model with different scratch assay conditions on primary human foreskin fibroblast monolayer cultures was prepared and cytokine levels and growth properties were evaluated with the aim of determining optimum injury conditions and observation time. Morphological characteristics of differently scratched fibroblasts from 0 to 36 h post injury (1 line, 2 lines and 3 lines) were investigated. The expression of connective tissue growth factor, CTGF, which is a key mediator in hyper-tropic scarring, and relative intensity of CTGF as a function of time were determined by western blot and gelatin Zymography. After injury (1 line), CTGF level was increased more than 2-fold within 1 h and continuously increased up to 3-fold at 6 h and was leveled down to reach normal value at 36 h, at which cell migration was complete. In more serious injury (2 lines), higher expression of CTGF was observed. The down regulation of CTGF expression after CTGF siRNA/lipofectamine transfection in control, 1 line and 2 lines scratch conditions were 40%, 75% and 55%, respectively. As a model anti-CTGF based therapy, CTGF siRNA with different ratios of linear polyethyleneimine (PEI) complexes (1:1, 1:5, 1:10, 1:20 and 1:30) were prepared and down-regulation efficacy of CTGF was evaluated with our optimized scratch assay, which is 1 line injury at 6 h post injury observation time. As the cationic linear PEI ratio increased, the down regulation efficacy was increased from 20% (1:20) to 55% (1:30). As CTGF level was increased to the highest at 6 h and leveled down afterwards, CTGF level at 6 h could provide the most sensitive response upon CTGF siRNA transfection. The scratch assay in the present study can be employed as a useful experimental tool to differentiate between anti-scar therapies for their down regulation efficacy of CTGF.

Cannabinoids inhibit fibrogenesis in diffuse systemic sclerosis fibroblasts.

OBJECTIVE: It has been demonstrated that the endocannabinoid system is up-regulated in pathologic fibrosis and that modulation of the cannabinoid receptors might limit the progression of uncontrolled fibrogenesis. The aim of this study was to investigate whether the synthetic cannabinoid receptor agonist WIN55,212-2 could modulate fibrogenesis in an in vitro model of dcSSc. METHODS: The expression of cannabinoid receptors CB1 and CB2 was assessed in dcSSc fibroblasts and healthy control fibroblasts. To investigate the effect of WIN55,212-2 on dcSSc fibrogenesis, we studied type I collagen, profibrotic cytokines, fibroblast transdifferentiation into myofibroblasts, apoptotic processes and activation of the extracellular signal-related kinase 1/2 pathway prior to and after the treatment with the synthetic cannabinoid at increasing concentrations. RESULTS: Both CB1 and CB2 receptors were over-expressed in dcSSc fibroblasts compared with healthy controls. WIN55,212-2 caused a reduction in extracellular matrix deposition and counteracted several behavioural abnormalities of scleroderma fibroblasts including transdifferentiation into myofibroblasts and resistance to apoptosis. The anti-fibrogenic effect of WIN55,212-2 was not reverted by selective cannabinoid antagonists. CONCLUSIONS: Our preliminary findings suggest that cannabinoids are provided with an anti-fibrotic activity, thereby possibly representing a new class of agents targeting fibrosis diseases.