Role And Mechanism Of Exosomes Derived From Cardiac Fibroblasts In Ang â…¡-induced Cardiac Hypertrophy

Background:Currently the prevention strategy of chronic heart failure has shifted from treatment of cardiotonic to the regulation of myocardial remodeling and inhibiting excessive activation of Ang Ⅱ and RAS. Myocardial remodeling is characterized by cardiomyocyte hypertrophy, cardiomyocyte apoptosis, increased cardiac fibroblasts proliferation and extracellular matrix secretion.Although early cardiac hypertrophy is thought to be a compensatory response under the stress state, but continuous cardiac hypertrophy tends to result in increasing incidence of heart failure or sudden cardiac death. Reduced SERCA2a play an important role in the process of myocardial remodeling and heart failure.Many signal pathways could induce pathological myocardial hypertrophy, of which MAPKs pathway plays a crucial role. MAPKs family contains three main members:extracellular signal regulated kinase (ERK), stress activated protein kinase (SAPK, also called JNK) and p38 kinase.Renin-angiotensin system (RAS) is an important hormone and humoral regulation system in the human body. Myocardial RAS is more important than circulating RAS in the regulation of myocardial remodeling. Ang Ⅱ typel receptor (AT1R) play a leading role in the process of myocardial remodeling, and the function of Ang Ⅱ type2 receptor(AT2R) is still controversial.60%-70% cells in normal heart are cardiac fibroblasts which account for about a third of the heart volume. Cardiac fibroblasts produce extracellular matrix, provide support for myocardial cells, participate in cardiac repair after injury.In addtion, they also secrete a variety of growth factors which mediate crosstalk with cardiomyocytes, and influence the function of the latter. Exosomes are a kind of extracellular vesicles with a cup-like shape and diameter of 30-100 nm.Aims1. To set up the protocol to isolation of cardiac fibroblasts-exosomes;2. To explore the role and mechanism of cardiac fibroblasts-exosomes in cardiac hypertrophy.Methods1. Cell cultureNeonatal rat cardiac myocytes and fibroblasts were isolated from hearts of 1-to 3-day old Sprague Dawley (SD) rats were finely minced and digested with type II collagenase2. Exosome isolationExosomes were isolated and purified using a multi-step centrifugation protocol with slight modifications.3. Measure of cardiomyocyte hypertrophyCell area measure and 3H-leucine uptake assay were used to test cardiomyocyte hypertrophy.4. Real-time PCR, Western blot and ICCDeterminating mRNA was used 2-△△CT. Western blot and ICC were operatesd with standard protocol.5. Angiotensin II MeasurementThe concentrations of angiotensin Ⅱ (Ang Ⅱ) in exosomes or culture medium of rat neonatal cardiomyocytes were measured by an Ang Ⅱ enzyme immunoassay (EIA) Kit6. Statistical AnalysisData are presented as means ± SD. Statistical analyses were performed by one-way ANOVA by use of SPSS (SPSS Inc, Chicago, IL, USA). Probability (P) values of< 0.05 were considered statistically significant.Results1. Conditioned medium of cardiac fibroblasts (CF CM) induced pathological cardiomyocyte hypertrophy.2 Telmisartan blocked CF CM-induced cardiomyocyte hypertrophy, and PD123319 inhibited partly CF CM-induced cardiomyocyte hypertrophy.3 Cardiac fibroblasts (CF) exosomes were isolated with a multi-step centrifugation protocol with slight modifications.4 CF exosomes could be uptaked by cardiomyocytes.5 CF exosomes induced pathological cardiomyocyte hypertrophy and downregulated SERCA2a mRNA in cardiornyocytes.6 Losing exosome decreased pro-hypertrophic effect of CF CM.7 CF exosomes induced upregulated mRNA level of angioteninogen, AT1R, AT2R in cardiomyocytes. And cardiac fibroblasts exosomes also induced enhanced Ang Ⅱ secretion from cardiomyocytes.8 EIA data suggested CF exosomes contained no Ang Ⅱ.9 Telmisartan and PD123319 inhibited cardiac fibroblasts exosomes-induce cardiomyocyte hypertrophy.10 Inhibitors of ERK, JNK and p38 blocked CF exosomes-induce RAS and Ang Ⅱ secretion.11 Inhibitors of ERK, JNK and p38 inhibited CF exosomes-induce cardiomyocyte hypertrophy.Conclusions1. CF exosomes induced cardiomyocyte hypertrophy and Ang Ⅱ secretion via activating RAS.2 CF exosomes activated RAS via MAPKs pathway.BackgroundHeart failure is the end-stage phase of various kinds of heart diseases. Currently the prevention strategy of chronic heart failure has shifted from the treatment of cardiotonic to the regulation of myocardial remodeling and the inhibiting of excessive activation of Ang II.Myocardial remodeling is characterized by cardiomyocyte hypertrophy, cardiomyocyte apoptosis, cardiac fibroblasts proliferation and increased extracellular matrix secretion.Although the early cardiac hypertrophy is thought to be a compensatory response under the stress state, but continuous cardiac hypertrophy tends to result in increasing incidence of heart failure or sudden cardiac death. Transgenic overexpression of SERCA2a can strengthen the pump function of the heart, improve hemodynamics, and benefit the patients with long-term.Various factors can cause myocardial remodeling, such as mechanical stretch, pressure load and various neurohumoral factors such as catecholamine, angiotensin II (Ang II), endothelin 1 (ET-1), atrial natriuretic peptide (ANP).Some growth factors can also induce myocardial remodeling such as the epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF). Mechanical stretch and pressure load both stimulate signal into the cell through the transmembrane extracellular matrix receptor, and also activate the myocyte hypertrophy and fibroblast proliferation related pathways by activating catecholamine, Ang II and ET-1.So Ang II is the important participant of myocardial remodeling.Aims1. To explore effect of Ang II on cardiac fibroblasts exosomes release;2. To explore role of cardiac fibroblasts exosomes in Ang II-induced myocardial remodelling.Methods1 LDH AssayLDH assay from Roche company was used to test cytotoxin of GW4869 and DMA to cardiomyocytes and cardiac fibroblasts.2 Transwell cocultureCo-culture of neonatal rat cardiac fibroblasts and myocytes was performed using transwell 24-well plates by cardiac fibroblasts (CF) in the top wells and cardiomyocytes in the bottom wells, and a 0.4-μm porous membrane between the wells.3. AnimalsMale C57BL/6N mice were purchased from Charles River Laboratories. Fifty two mice at age of 8 weeks were randomly divided into six groups which were treated with vehicle, angiotension Ⅱ (Ang Ⅱ), GW4869, and dimethyl amiloride (DMA) as follows:(a) Control group (n=10) was treated with vehicle saline alone. (b) Ang Ⅱ group (n=10) was treated with subcutaneous delivery of Ang Ⅱ dissolved in saline (0.9% NaCl) via Alzet osmotic minipumps at a dose of 2.4 mg/kg body weight/day with an infusion rate of 0.5μ/h as described elsewhere. (c) Ang Ⅱ plus GW4869 group (n=10) was treated with the subcutaneous delivery of Ang Ⅱ and intraperitoneal (i.p.) injection of GW4869 (2.5 mg/kg body weight) daily in 0.9% saline. (d) Ang Ⅱ plus DMA group (n=10) was treated with the subcutaneous delivery of Ang Ⅱ and i.p. injection of DMA (1 μmol/kg body weight) daily in 0.9% saline. (e) GW4869 (n=6) and (f) DMA (n=6) groups were treated with the same amount of GW4869 and DMA alone as described above.. The mice were euthanized 2 weeks after the treatment. After clearing blood with saline, the hearts were harvested.4. Blood pressure and heart rateBlood pressure and heart rate were measured noninvasively by the tail-cuff instrument (IITC Inc., Woodland Hills, California, USA) as previously described 2’35. HistologParaffin sections (5 μm) of the hearts were prepared using a rotary microtome and stored at room temperature until staining. Briefly, cardiomyocyte cross-sectional area was measured via wheat germ agglutinin (WGA) staining. For myocardial fibrosis, tissue sections were stained for collagen with a Masson’s Trichrome Kit.6. Statistical AnalysisData are presented as means ± SD. Statistical analyses were performed by one-way ANOVA by use of SPSS (SPSS Inc, Chicago, IL, USA). Probability (P) values of< 0.05 were considered statistically significant.Results1. Ang II enhanced exosomes release from cardiac fibroblasts.2. Telmisartan and PD123319 inhibited Ang Ⅱμ enhanced exosomes release from cardiac fibroblasts.3. GW4869 and DMA decreasd Ang μ and CF co-culture induced cardiomyocyte hypertrophy.4 Intraperitoneal injection of GW4869 and DMA decreasd Ang Ⅱ infusion-induced cardiac hypertrophy.5 Intraperitoneal injection of GW4869 and DMA decreasd Ang Ⅱ infusion-induced cardiac fibrosis.6 Intraperitoneal injection of GW4869 and DMA increased Ang Ⅱinfusion-decreasd LV SERCA2a.Conclusions1. Ang Ⅱ enhanced exosomes release from cardiac fibroblasts via AT1R and AT2R.2 Intraperitoneal injection of GW4869 and DMA decreasd Ang Ⅱ infusion-induced cardiac hypertrophy and fibrosis.3. Intraperitoneal injection of GW4869 and DMA increased Ang Ⅱ infusion-decreasd LV SERCA2a.