Dioscin elevates lncRNA MANTIS in therapeutic angiogenesis for heart diseases

Abstract Dioscin has been widely used in clinics for coronary artery disease (CAD) treatment for years in China. However, the underlying mechanism for Dioscin‐mediated cardioprotective effect has not been elucidated. Here, we showed that Dioscin significantly rescues the cardiac function in mouse model of myocardial infarction (MI), accompanied by the reduction of cardiac fibrosis and apoptosis, resulting from elevated angiogenesis. Mechanistically, Dioscin promotes the proliferation and migration of hypoxic endothelial cells via the up‐regulation of lncRNA MANTIS, which serves as a scaffolding lncRNA within a chromatin remodeling complex. Meanwhile, it enables pol II binding to the transcription start sites, which leads to induced expression of angiogenesis‐related genes, including SOX18, SMAD6, and COUP‐TFII. Conversely, IncRNA MANTIS silencing prevents Dioscin‐induced migration and angiogenesis in hypoxic endothelial cells. Taken together, these data provide new insights that clarifies the cardioprotective effects of Dioscin against myocardial infarcted injury and confirms the effect on angiogenic activity of endothelial cells. This will build a solid theoretical basis for clinical therapeutic strategies.

for those patients, therapeutic angiogenesis has been proposed as an attractive novel strategy for CAD and MI treatment. It is a combined utilization of chemicals and angiogenic factors to enhance neovascularization and growth of collateral blood vessels, which act as endogenous bypass conduits to increase tissue perfusion and provide oxygen and nutrients in the ischemic area. Thus, it is an emerged demand to identify novel and effective angiogenic compound for MI treatment.
Dioscin, a glucoside saponin, is one natural product identified from certain medicinal plants such as Dioscoreazingiberensis Wright and Dioscoreanipponica Makino . Recent studies suggest that Dioscin lowers the lipid levels and represses the inflammatory response, showing the hepatoprotective effects (Yao et al., 2018;Zhang et al., 2017). Additionally, it has been widely used in clinics as CAD treatment for many years in China. However, the mechanism underlying the protective effect of Dioscin on the infarcted hearts needs to be elucidated.
Recently, emerging data indicate that long noncoding RNAs (LncRNA, >200 nucleotides in length), a major population of transcriptome, play critical roles in the physiology and pathophysiology of cardiovascular diseases by distinct mechanisms (Ransohoff et al., 2018). Hypoxia-induced endothelial lncRNA MANTIS (metastasisassociated lung adenocarcinoma transcript 1) identified in the antisense strand of the intron of Annexin A4, a calcium and phospholipid binding protein, is expressed in different cellular types. MANTIS silencing causes the endothelial dysfunction, impairing sprouting and tube formation, and attenuating endothelial migration. MANTIS is required for endothelial cells proliferation, neonatal retina vascularization, and vascular growth in vivo after hindlimb ischemia (Cremer et al., 2019). A key function of lncRNA MANTIS is by interacting and combining with brahma related gene-1 (BRG1), to regulate gene transcription, including SRY (sex determining region Y)-box 18 (SOX18), mothers against decapentaplegic homologue 6 (SMAD6), and nuclear receptor subfamily 2 group F member 2 (COUP-TFII), which are important for angiogenesis in endothelial cells. Although lncRNAs have been proven crucial in ischemic diseases, whether lncRNAs are regulated by Dioscin remains unclear in myocardial infarction.
Here, we demonstrate that a natural product Dioscin alleviates hypoxic-caused cardiac dysfunction in mouse model of myocardial infarction via up-regulating the level of lncRNA MANTIS. We show that Dioscin increases IncRNA MANTIS expression, promoting the complex formation of MANTIS and BRG1, elevating the expression of SOX18, SMAD6, and COUP-TFII, resulting in promoted angiogenesis in myocardial infarction.

| Dioscin attenuates cardiac dysfunction in mouse model of myocardial infarction
To explore the effects of Dioscin in response to MI, we applied Dioscin as a therapeutic treatment in mouse MI model. Echocardiography showed that Dioscin significantly improved cardiac functions after MI (Figure 1a). Ejection fraction (EF), a vital parameter for cardiac function, was improved after Dioscin treatment in infarcted hearts, compared to vehicle treatment (Figure 1b). Similarly, significant improvement was observed for fraction shortening (FS) with Dioscin administration (Figure 1b). Additionally, declination of the left ventricular end-diastolic diameters (LVEDD) and left ventricular end-systolic diameters (LVESD) in response to MI was dramatically rescued by Dioscin in mice suffered with MI, indicating Dioscin could help to maintain cardiac structure in infarcted hearts (Figure 1c).
Dioscin repressed the increase of heart size caused by myocardial infarction ( Figure 1d). As shown in Figure 1e,f, MI caused the increase of the ratios of heart weight (HW) to body weight (BW) or tibia length (TL). However, this hypertrophic tendency was alleviated by Dioscin administration. Additionally, the mRNA levels of biomarkers for heart failure, including Anp, Bnp, and β-Mhc, were declined after MI with Dioscin treatment (Figure 1g). These findings suggest that Dioscin can rescue cardiac function and delay the progress of heart failure caused by myocardial infarction.

| Dioscin promotes angiogenesis in mice after myocardial infarction
To further confirm whether Dioscin attenuates the cardiac dysfunction after MI, we explored the therapeutical effect of Dioscin in the infarcted hearts. Administration of Dioscin significantly reduced the size of cardiac fibrosis in response to MI, compared to vehicle  Figure 2b). What is more, the ratio for Bcl2/Bax mRNAs was declined in infarcted hearts, and this reduction was blocked by Dioscin ( Figure 2c). These data indicate that Doscin enhances angiogenesis and alleviates cardiac apoptosis and fibrosis in response to infarction.

| Increase for lncRNA MANTIS is associated with Dioscin treatment
As lncRNAs are involved in the development of cardiovascular diseases, we examined whether angiogenesis is involved in regulating the expression of lncRNAs that were reported in endothelial cells. We profiled 50 lncRNAs, reported which play a vital role in endothelial cells, in hypoxic HUVECs with/without Dioscin treatment using real-time PCR assays ( Figure 4a). As depicted in (e,f) Ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were shown, respectively. (g) Real-time PCR analysis of the messenger RNA (mRNA) expression of Anp, Bnp, and β-Mhc was performed using heart tissues from Sham group and MI group treated with Vehicle or Dioscin. Data were presented as mean ± SEM. *p < 0.05 relative to Sham; #p < 0.05 relative to MI; &p < 0.05 relative to MI with 40 μg/g Dioscin

| DISCUSS ION
In this study, we demonstrated that Dioscin efficiently rescues cardiac dysfunction in response to myocardial infarction. Interestingly, cardioprotective Dioscin promotes the increase of lncRNA MANTIS, formatting the complex of MANTIS and BRG1, resulting in tube formation and angiogenesis, alleviating cardiac fibrosis and apoptosis.
Our results suggest that Dioscin is a novel candidate for therapeutic strategy for MI.
Dioscin has a potential effect of angiogenesis and anti-apoptosis on infarcted hearts. Currently, United States Food and Drug Administration (FDA) has not approved any therapeutic angiogenic strategies for CAD/MI treatment (Bergers & Hanahan, 2008;Fischer et al., 2008). However, new angiogenic factors and chemicals are urgent to be identified and applied in clinics. In our study, Dioscin is considered as a potential and effective therapeutic nature product for infarcted hearts, rescuing the cardiac function in mouse model of MI via promoting of angiogenesis. Additionally, the cardiac fibrosis and apoptosis were alleviated by Dioscin in infarcted hearts. F I G U R E 2 Dioscin promotes angiogenesis in MI mouse model. (a) Representative Masson trichrome staining from heart tissues (scale bar: 1mm) and their quantification. (b) Representative immunofluorescent images were staining with CD31 (red) using heart tissues from mice (scale bar: 100 μm), and the quantification was showed at the right. (c) Real-time PCR assays for the ratio of Bax/Bcl 2 mRNA levels were performed. Date were expressed as mean ± SEM. *p < 0.05 relative to Sham; #p < 0.05 relative to MI; &p < 0.05 relative to MI with 40 μg/g Dioscin

| Mouse model
Male C57BL/6 mice were unrestricted access to food and water and were housed in the controlled environment, with regulation of temperature (22 ± 1°C) and humidity (55%), a 12: 12-h dark-light cycle.
All animal experiments were carried out in accordance with the principles provided by the National Institute of Health Guideline and were approved by the Animal Care and Use Committee of Nanjing Medical University (IACUC-1912034).
Mouse model of MI was generated by ligation of the left anterior descending (LAD) as described previously with male mice at the age of 8-10 weeks (Lu et al., 2016). Briefly, the mice were anesthetized with isoflurane and then intubated with a fine polyethylene cannula connected to a small animal ventilator. A thoracotomy incision was performed in the second intercostal space, and the heart was exteriorized out of the chest. The LAD coronary artery was ligated permanently with a 7-0 nonabsorbable surgical suture, and the heart was then returned inside the chest. The chest wall was closed in layers, and skin incision was closed by sutures. Mice with sham-operation were subjected to the same surgical treatment, but the LAD was not ligated.
Dioscin (Di'ao group, Chengdu, China) was intragastric administration 1 day after ligation and continued until the mice were sacrificed 2 weeks. Mice in the control group received an equal volume of the solvent orally.

| Transfections
Primary human umbilical vein endothelial cells (HUVECs) were isolated from the human umbilical vein, as previously described (Cai et al., 2015). The cells were cultured in the EGM medium containing 5% (v/v) fetal bovine serum (FBS) and EGM-2 Single Quots     Migration was quantified as the ratio of the area covered with cells to the cell-free area.

| Matrigel assays (tube formation)
Human umbilical vein endothelial cells in EBM were seeded onto Matrigel (Corning, USA) with Dioscin or negative control. After 8 h of incubation, 2 μg/ml of Calcein AM (Invitrogen, USA) was added directly to the well and incubated for 15 min. The images were visualized and captured. The number of vessel tubes formed was analyzed as described previously (Lu et al., 2016).

| Real-time RT-PCR assays
Total RNA was isolated from mouse hearts or cultured cells using TRIzol (Invitrogen, USA) according to the manufacturer's instruction . A total of 0.5 μg of RNA samples were reversetranscribed using M-MLV Reverse Transcriptase, following the manufacturer's protocol (Promega, USA). Real-time PCR assay was then performed using the FastStart Universal SYBR Green Master (Roche, Switzerland) and Real-time PCR Detection System (Bio-Rad, USA) as described previously. Experiments were performed in triplicate, and the relative expression levels of the genes were calculated using the 2 −ΔΔCT method. The primers used were listed in Table 1 and Table 2.

| Histological analysis
The fixed hearts were sectioned, and cardiac fibrosis was assessed by staining with Masson's trichrome. Immunohistochemical staining was performed on paraffin-embedded sections with a primary antibody against CD31 (AF3628, R&D, USA) and BRG1 (SC-17796, Santa, USA) which was followed by incubation with a biotinylated secondary antibody as described previously (Wu et al., 2017).

| Statistics
All the data were expressed as means ± standard error of the mean (SEM) using GraphPad Prism 8. Two-group comparisons were analyzed by a Student's t test. For comparisons of more than two groups, one-way ANOVA was employed. Statistical significance is indicated by *p < 0.05.

ACK N OWLED G EM ENTS
We want to thank Dr. Changhan Ouyang for excellent technical assistance, and Dr. Shan Lu for manuscript revise.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R CO NTR I B UTI O N
QL designed the study. CK, DL, CH, and RL conducted searches, extracted, and analyzed the data. CK and DL wrote the manuscript.
All authors contributed to the article and approved the submitted version.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.