Cardiac fibrosis is a common occurrence in patients with chronic hypertension and advanced heart failure. Cardiac fibrosis is characterized by an increased number of fibroblasts in the myocardium and excessive deposition of the extracellular matrix (ECM) (1, 2). Myocardial fibrosis can lead to decreased ventricular compliance that contributes to diastolic and systolic dysfunction, ultimately leading to heart failure and possible premature death (1, 3).
The development of fibrosis in the heart was reported to follow a pathway similar to that of other organs, such as the liver, lung, kidney and solid tumors (4, 5). It is well known that myofibroblasts in fibrotic tissues are derived from the expansion and activation of resident tissue fibroblasts, tissue migration of bone marrow derived circulating fibrocytes, and the transition of epithelial cells into mesenchymal cells (4). Recently, endothelial to mesenchymal transition (EndMT) has emerged as a possible source of tissue myofibroblasts, and has been proven to contribute to cardiac fibrosis (6, 7). However, the molecular mechanism underlying the role of EndMT in cardiac fibrosis remain unclear.
The transcription factor ETS-1 (E26 avian leukemia oncogene 1, 5′ domain) is composed of 450 amino acids and contains the DNA-binding ETS domain which binds to the ETS-binding motif GGAA/T cis-acting element in target genes. It has been identified as a critical regulator of embryogenesis, angiogenesis and inflammation. Previous studies have demonstrated that ETS-1 is rapidly induced in endothelial cells and smooth muscle cells of mouse aorta that are subjected to chronic Ang II infusion, leading to vascular inflammation and remodeling (8). Furthermore, Ang II infusion results in an increase in heart size and ventricular wall thickness in mice, but these effects are significantly diminished in global
Previous studies have reported that ETS-1 participates in EMT in tumor initiation and metastasis, and renal fibrosis (11–13). In this study we hypothesize that ETS-1 regulates EndMT in the murine heart in response to chronic hypertension.
ETS-1 is expressed in endothelial cells, smooth muscle cells and fibroblasts in the heart. To assess the contribution of endothelial-specific ETS-1 in cardiac fibrosis, We crossed Tie2-Cre+ mice with
Blood pressure of the mice was measured 3 days before and 7 and 14 days after Ang II infusion. No significant difference in systolic blood pressure was observed in Tie2-Cre+;
To investigate the effect of endothelial deletion of
Echocardiography analysis indicated that the left ventricular wall thickness in mice was remarkably increased, as shown by the increase in IVSd and LVPWd. Tie2-Cre+;
We then assessed whether endothelial deletion of
Myocardial fibrosis is an early manifestation of hypertrophic cardiomyopathy (3). To investigate the effect of endothelial deletion of
Because EndMT plays an important role in cardiac fibrosis (6), we investigated whether endothelial deletion of
To further elucidate the effect of endothelial deletion of
To further confirm the role of ETS-1 in EndMT, we knocked down
We further investigated the mRNA levels of endothelial and mesenchymal markers by using qPCR in TGF-β1-treated H5V cells. The expression of α-SMA, FSP-1 and N-cadherin were increased and expression levels of CD31 and E-cadherin were decreased in H5V cells after treatment with TGF-β1 for 2 days. Reduced mRNA expression of mesenchymal markers and an increase in the mRNA expression of endothelial markers showed that knockdown of
We also examined the changes in expression levels of transcription factors that play an important role in EndMT, such as Snail, Slug, Twist1 and ZEB1. Consistent with our
In this study, we found that deletion of
Endothelial cells are critical for maintaining cardiovascular system function, and endothelial dysfunction result in cardiovascular disease. Organ fibrosis is characterized by EndMT, a process in which endothelial cells in small vessels undergo a transition to form fibroblasts that show active synthesis and secretion of numerous ECM factors (6). A recent study found that cardiac endothelial cells are converted to myofibroblasts via EndMT during cardiac fibrosis induced by pressure overload in mice, and cardiac function can be substantially improved by inhibiting the EndMT process. This indicates that EndMT plays an important role in cardiac fibrosis and in the development of heart failure (6). Thus, further exploration of EndMT and identification of the key factors that regulate EndMT may offer potential therapeutic targets for clinical treatment.
ETS-1 is a transcription factor that is critical for embryogenesis, angiogenesis and inflammation. Previous studies have reported that ETS-1 participates in the EMT process during tumor formation, and also regulates the EMT process of renal tubular epithelial cells under pathological conditions (11, 12). In this study, we found that EndMT was significantly decreased in Tie2-Cre+;
The TGF-β1/Smads signaling pathways along with some downstream transcription factors, such as Snail, Slug, Twist and ZEB family of proteins, play an important regulatory role in cardiac EndMT (14). Previous studies have demonstrated that ETS-1 regulates TGF-β1/Smads pathways, is an agonist of the profibrotic effects of TGF-β1, and facilitates Smad2/3 binding to the promoters of genes involved in these pathways (15, 16). Additional studies have reported that ETS-1 transactivates
We generated endothelial-specific
Three-month-old male Tie2-Cre+;
Heart ventricle tissue was minced into 1 mm3 pieces, and then digested using 2 mg/ml collagenase II (LS004174, Worthington, Bio Corp, USA). Tissue debris was removed by centrifugation to produce the single cell suspension. Single cells were magnetically labeled with anti-CD31 super paramagnetic polystyrene beads (11155D, Thermo Fisher Scientific Life Sciences, USA) at 4°C for 20 min and separated using a magnet (12303D, Thermo Fisher Scientific Life Sciences). CD31+ cells bound to the beads were washed out, collected and used for western blotting analysis.
Echocardiography was performed using a Vevo 2100 instrument (Fuji Film Visual Sonics) equipped with an MS-400 imaging transducer (18–38 MHz) as previously described (20). Mice were anaesthetized using 1–2% isoflurane that was administered via inhalation. Interventricular septal thickness at diastole (IVSd), left ventricular internal diastolic diameter (LVIDd); left ventricular posterior wall thickness at diastole (LVPWd) were measured. Percent left ventricular ejection fraction (EF) was calculated using M-mode measurements.
Systolic blood pressure was measured via a tail-cuff method using a non-invasive blood pressure instrument (BP-98A, Softron, China) at 3 days before and at 7 and 14 days after Ang II infusion as previously described (21).
Mice were anesthetized by isoflurane inhalation and were sacrificed by CO2 asphyxiation. The hearts were dissected and fixed in 4% paraformaldehyde overnight at 4°C, and then embedded in paraffin. Longitudinal sections or transverse sections were cut at 10 μm thickness from paraffin blocks and stained with hematoxylin and eosin. Masson’s Trichrome stain and Sirius Red stain were performed as described previously (22).
Immunofluorescence staining was performed on the cryosections as described previously (10). Briefly, tissues were fixed in 4% paraformaldehyde overnight at 4°C and then cryo-embedded in optimum cutting temperature compound (OCT) (Tissue Tek, USA). Transverse heart sections were cut at 10 μm thickness, and the cryosections were incubated with primary antibodies overnight at 4°C. After washing with 0.5% TritonX-100 in phosphate buffer saline, sections were incubated with secondary antibodies for 2 hours at room temperature. The following antibodies were used: CD31 (SC-1506, Santa Cruz Biotechnology, USA), α-SMA (ab5694, Abcam, USA), FSP-1 (ab27957, Abcam) and ETS-1 (SC-350; Santa Cruz Biotechnology).
Western blot analysis was performed as previously described (22). The blots were incubated overnight with the primary antibodies against ETS-1 (SC-350; Santa Cruz Biotechnology), CD31 (SC-1506, Santa Cruz Biotechnology), α-SMA (ab5694, Abcam), and FSP-1 (ab27957, Abcam) at 4°C overnight. Then, blots were washed and incubated with secondary antibodies.
The mouse heart endothelium cells (H5V) were a gift from Dr. Xin Ma (23). The cells were cultured in 90% Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum as described previously (23).
For TGF-β1 treatment, H5V cells were starved in a serum-free culture medium for 48 hours, and then administrated TGF-β1 (10 ng/ml) (14–8342, eBioscience, USA) for two consecutive days.
Total RNA was extracted from mouse left ventricle or cultured H5V cells using an RNeasy kit (9747, Takararr, Japan) according to the manufacturer’s recommendations. 500 μg of RNA was retro-transcribed to cDNA using cDNA reverse transcription kit (037A, Takararr). qRT-PCR was performed using in a QuantStudio DS Real-time fluorescence quantitative PCR System with SYBR Green Master Mix (420A, Takararr) and gene-specific primers. Primer sequences are listed in Supplemental Table S1. Standard and melting curves were measured for every plate and for every gene to ensure efficiency and specificity of the reaction. Ct value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control and the 2–ΔΔCT method was used to analyze the relative expression levels of various genes.
Graphpad prism 6.01 was used for statistical analysis. All data were presented as mean ± standard deviation (SD). Shapiro-Wilk normality test was first applied for checking normality distribution of a variable. Student’s t-test was used for two-group comparisons. Multiple comparisons were tested using one-way ANOVA followed by the post hoc Dunnett’s test (Levene’s tests for equal variance). Differences were considered as statistically significant at P < 0.05.
This work was supported by the Natural Science Foundation of Shanghai (17ZR1423800) and the Science and Technology Commission of Shanghai Municipality (18140903402).
The authors have no conflicting interests.
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