Connective Tissue Growth Factor (CTGF/CCN2): at the heart of cardiopulmonary and renal fibrosis.
1.Purpose: Fibroblasts in heart, kidneys and lungs play a key role in regulating normal tissue function and in the adverse tissue remodeling that occurs with cardiac, pulmonary and renal fibrosis. Fibroblasts respond to various bioactive molecules including cytokines, vasoactive peptides and growth factors. Connective tissue growth factor (CTGF/CCN2), a matricellular protein is known to be involved in tissue fibrosis. Our data suggests that the CTGF C-terminal domains 3 and 4 are necessary for its signaling activity. To study the role of CCN2 in cardiac, pulmonary and renal tissue remodeling, we established a transgenic mouse line (LSLd34CTGF/d34CTGF-loxP). Using Cre mice with a cell specific promoter facilitates cell Cre recombinase activity leading to specific overexpression of the transgene.
Here, we investigate the autocrine/paracrine effect of the transgenic overexpression of the active CTGF in fibroblasts. We will cross our floxed LSLd34CTGF/Tg-d34CTGF-loxP mice (housed at KPM-RH, FOTS 19482; hereafter referred as Tg-d34CTGF-loxP) with the well-established Cre model with B-Tcf21-iCreERT2 mice (Strain: C57BL/6-Tcf21tm1(iCre/ERT2)/Bcgen), purchased from Biocytogen Jiangsu Co., Ltd. B-Tcf21-iCreERT2 transgene contains the Transcription factor 21 (Tcf21) gene promoter directing expression of a tamoxifen-inducible Cre recombinase (MerCreMer) in fibroblast. Breeding floxed Tg-d34CTGF-loxP mice with B-Tcf21-iCreERT2 mice will result in generation of a hybrid line with Tamoxifen inducible over-expression of d34CTGF in fibroblasts.
Subsequently we will evaluate the direct role of d34CTGF, as a modulator of cardiac, pulmonary and renal fibrosis in mice. Cardiac function will be evaluated by cardiac echocardiography, whereas molecular mechanisms involved in CCN2 mediated tissue fibrosis will be investigated by histological and biochemical analysis. Understanding of the basic mechanisms regulating the tissue fibrosis is crucial for the development of intervention strategies to inhibit the fibrosis and promote injury repair in adverse tissue remodeling.
2. Distress: For induction of transgene, 10week upto 7 month old hybrid mice will be treated with Tamoxifen by intraperitoneal injection at a dosage of 40 mg/kg (40ug/g body weight) once with endpoint 28 days post Tamoxifen. Cardiac function will be evaluated using echocardiography and tissue will be harvested at termination of study. None of the procedures are expected to be distress inflicting to animals.
3. Expected benefit: The adverse tissue remodeling that occurs with fibrosis leads to organ dysfunction. Therefore, it is necessary to understand the details of the events that lead either to physiological or to pathological tissue fibrosis in order to design new therapeutic targets or strategies. Several scientific reports established that CTGF/CCN2 is a profibrotic molecule. The purpose of this study is to understand the molecular mechanisms of active CTGF/CCN2 (d34CTGF) mediated tissue fibrosis and enable the design of new, safe and effective treatment strategies.
4. Number of animals, and what kind: 490 mice
5. How to adhere to 3R: Our previous experience with echocardiographic analysis and physiologic assessment of cardiac function and tissue harvesting, facilitate adhering to 3R with lowest number of animals. Well-trained personnel and experienced handling using refined methods will reduce the suffering of the animals
Here, we investigate the autocrine/paracrine effect of the transgenic overexpression of the active CTGF in fibroblasts. We will cross our floxed LSLd34CTGF/Tg-d34CTGF-loxP mice (housed at KPM-RH, FOTS 19482; hereafter referred as Tg-d34CTGF-loxP) with the well-established Cre model with B-Tcf21-iCreERT2 mice (Strain: C57BL/6-Tcf21tm1(iCre/ERT2)/Bcgen), purchased from Biocytogen Jiangsu Co., Ltd. B-Tcf21-iCreERT2 transgene contains the Transcription factor 21 (Tcf21) gene promoter directing expression of a tamoxifen-inducible Cre recombinase (MerCreMer) in fibroblast. Breeding floxed Tg-d34CTGF-loxP mice with B-Tcf21-iCreERT2 mice will result in generation of a hybrid line with Tamoxifen inducible over-expression of d34CTGF in fibroblasts.
Subsequently we will evaluate the direct role of d34CTGF, as a modulator of cardiac, pulmonary and renal fibrosis in mice. Cardiac function will be evaluated by cardiac echocardiography, whereas molecular mechanisms involved in CCN2 mediated tissue fibrosis will be investigated by histological and biochemical analysis. Understanding of the basic mechanisms regulating the tissue fibrosis is crucial for the development of intervention strategies to inhibit the fibrosis and promote injury repair in adverse tissue remodeling.
2. Distress: For induction of transgene, 10week upto 7 month old hybrid mice will be treated with Tamoxifen by intraperitoneal injection at a dosage of 40 mg/kg (40ug/g body weight) once with endpoint 28 days post Tamoxifen. Cardiac function will be evaluated using echocardiography and tissue will be harvested at termination of study. None of the procedures are expected to be distress inflicting to animals.
3. Expected benefit: The adverse tissue remodeling that occurs with fibrosis leads to organ dysfunction. Therefore, it is necessary to understand the details of the events that lead either to physiological or to pathological tissue fibrosis in order to design new therapeutic targets or strategies. Several scientific reports established that CTGF/CCN2 is a profibrotic molecule. The purpose of this study is to understand the molecular mechanisms of active CTGF/CCN2 (d34CTGF) mediated tissue fibrosis and enable the design of new, safe and effective treatment strategies.
4. Number of animals, and what kind: 490 mice
5. How to adhere to 3R: Our previous experience with echocardiographic analysis and physiologic assessment of cardiac function and tissue harvesting, facilitate adhering to 3R with lowest number of animals. Well-trained personnel and experienced handling using refined methods will reduce the suffering of the animals