The Vascular Biological Laboratory at the Beijing Institute of Heart, Lung and Blood Vessel Diseases was established in 2008. The laboratory is the foundation for the Key Laboratory of Cardiovascular Re- modeling-related Diseases at the Ministry of Education and Beijing Collaborative Innovation Research Center for Cardiovascular Disease. The Director of the laboratory is Professor Jie Du.

In the Vascular Biological Laboratory, the following research is currently being undertaken: (1) “Studies on the mechanism of interaction between inflammatory micro-environment and smooth muscle cells in aortic dissection” supported by grants from the National Key Research and Development Program; (2) “The role of arachidonic acid metabolites in pathological cardiac remodeling” funded by a key grant from the National Natural Science Foundation of China; (3) Translational Research in Aortic Aneurysm Disease (The Innovation Team of the Ministry of Education); and (4) National Nature China Canada Cooperation Project: Genetic basis of aortic valve calcification and stenosis in European and Asian populations. In addition, research teams have undertaken research projects supported by the National Natural Science Foundation of China, the Beijing Natural Science Foundation, and the Beijing Municipal Science and Technology Commission.

Twenty-five research SCI papers were published by the Vascular Biological Laboratory in 2019, and research group members attended international and domestic academic meetings. There are currently 19 staff members conducting important scientific research in the Laboratory.

Since the establishment of the department, 23 doctoral and 24 masters students have graduated, and 13 doctoral students and 13 masters students are currently under training. The graduates have entered hospitals to undertake clinical and research work in Beijing.

The progress of the subject

Heart failure is the common result of myocardial remodeling caused by a variety of cardiovascular diseases, including hypertension and myocardial infarction. Arachidonic acid (ARA) metabolically ac tive small molecules have regulatory effects on the initiation and development of cardiac remodeling and heart failure. We found that the expression of multiple miRNAs in serum and heart tissue changed in pa tients and mice upon pathological myocardial remodeling. Diseases affected the expression of ARA met abolic enzymes and the pathological myocardial remodeling process. In addition to miRNAs, other small nucleic acid molecules, circRNAs and tRNAs, undergo gene transcription regulation. However, several scientific issues, such as the role of the mutual regulatory network between these small nucleic acid mol ecules and ARA metabolic activity in the pathological myocardial remodeling process, remain unclear. Our project focuses on the regulatory network between ARA metabolically active small molecules and small nucleic acid molecules, as well as the combinations of various small nucleic acid molecules in heart tissue and serum that undergo cardiac remodeling after hypertension or myocardial infarction. Scientific analyses and mass spectrometry data for ARA metabolites have addressed selected research targets and made several important findings as described below.

1. Through screening and re-verification of miRNA expression profiles in sera from more than 200 STEMI patients, it was found that the circulating levels of miR-26a-5p, miR-21-5p, and miR-191-5p were related to the long-term heart rate of STEMI patients and vascular adverse events such 2020 BEIJING INSTITUTE OF HEART,LUNG AND BLOOD VESSEL DISEASES 025 as cardiac death, heart failure, and recurring myocardial infarction. Evaluation of these three miR NAs combined with hs-cTnI as shown to improve the prognostic assessment ability of the GRACE score and TIMI risk score (Can J Cardiol. 2020 Mar 20: S0828-282X(20)30273-7).

2. Further analysis of ARA metabolites in myocardial infarction patients and healthy controls revealed that metabolites of the ARA CYP450 pathway were significantly increased. Continued explora tion of the correlations between metabolites and prognosis identified five fatty acid metabolites that were associated with adverse events in patients with acute myocardial infarction, of which two known protective metabolites (12,13EpOME and 9,10EpOME) were significantly reduced in the poor prognosis group.

3. In a mouse model of cardiac ischemia-reperfusion (I/R), neutrophils recruited from the circula tion into the heart expressed miR-223 at elevated levels. Compared with the control group, miR- 223 knockout mice with I/R had increased cardiac damage and reduced cardiac function. TUNEL staining revealed increased cardiac cell apoptosis. Overexpression of miR-223 by a miR-223 agomir reduced cardiac cell apoptosis after I/R, improved cardiac function, and played a protective role. On GO analysis, the most significantly upregulated genes after miR-223 knockout were relat ed to inflammation and immune signaling pathways. The expression levels of target genes Pla2g7,Cyp4f18, and Ptgs2, which were predicted to be related to ARA metabolism in the heart of miR-223 knockout mice, were significantly increased compared with the levels in the wild-type control group.

4. Coronary heart disease and hypertension lead to cardiac remodeling. In patients with coronary heart disease and hypertension, coronary artery calcification and increased vascular stiffness of ten occur. Transcriptomics analysis showed that the mRNA expression of ARA metabolism-re lated enzymes was significantly altered in calcified blood vessels. We detected nearly 100 metab olites of ARA, DHA, and EPA in the aortic tissue and plasma of a mouse aortic calcification model and found that the contents of metabolites in the LOX and COX pathways were increased in the calcified aorta, while the CYP450 pathway products did not change significantly. Among them, the COX pathway product PGD2 and LOX pathway product 12-HETE showed the greatest increases.Knockout of the PGD2 receptor CRTH2 significantly increased the vascular calcification, and the use of 12/15-Alox enzyme (12-HETE) inhibitors also significantly increased the vascular calcifica tion. These findings suggest that ARA metabolites may have a protective effect on vascular calci fication. In our follow-up study, we will further explore the molecular mechanism of this protective effect.

5. RNA modification by m6A methylation affects pathological myocardial remodeling by regulating the metabolism of ARA. The m6A methyltransferase METTL3 was specifically knocked out in cardio myocytes in mice, leading to spontaneous pathological myocardial remodeling such as myocardial hypertrophy and myocardial fibrosis, which in turn led to heart failure. Transcriptome sequencing of the myocardial tissue from myocardium-specific METTL3 knockout mice revealed that the genes downregulated upon METTL3 knockout were mainly enriched in the fatty acid metabolism pathway.In vitro experiments confirmed that the expression of PPARA protein in METTL3 knockout cardio-2020 myocytes was significantly down-regulated, and its fatty acid oxidation ability was significantly re duced. In our follow-up study, we will further clarify the specific mechanism for the myocardial cell METTL3 regulation of fatty acid metabolism.

6. The nanoparticle delivery system mediated by CHO-PGEA efficiently delivers miRNAs into car diomyocytes. The CHO-PGEA/miR-182 complex efficiently increases the expression of heart miR-182 and reduces the protein level of its target gene Foxo3. Previous studies showed that miR-182 aggravates myocardial hypertrophy after TAC. Thus, we delivered a miR-182 inhibitor into the heart using the CHO-PGEA system, and observed significant inhibition of hypertrophy and improvement of cardiac function. After testing, the CHO-PGEA/miRNA complex had no significant effects on liver and kidney tissue structures or on liver and kidney function, thereby confirming the safety of CHO-PGEA treatment. Therefore, CHO-PGEA nanoparticles were identified as potential miRNA carriers that can efficiently target and safely deliver miRNAs for the treatment of heart dis eases (Adv Sci (Weinh). 2019 Apr 6;6(11):1900023).

7. Use of heparin polysaccharide nanoparticles (HepNPs) to deliver vascular endothelial growth fac- tors VEGF-C and VEGF-A (Hep@VEGF-C/Hep@VEGF-A = 3:1) in vivo effectively promoted angiogenesis after myocardial infarction and reduced cardiac edema, thereby significantly improv- ing cardiac function after myocardial infarction. Therefore, the HepNP in vivo delivery system can be used to deliver protein drugs safely and effectively and has a wide range of transformational applications in cardiovascular diseases and other diseases (Small. 2020 Jan;16(4):e1905925.).

Thoracic aortic aneurysms (TAAs) cause substantial mortality worldwide. Familial and syndromic TAAs are highly correlated with genetics. However, the incidence of sporadic isolated TAAs (iTAAs)  is much higher, and the genetic contribution remains unclear. To examine the genetic characteristics  of sporadic iTAAs, we performed a genetic screen of 551 sporadic iTAA cases and 1071 controls by  0262020 BEIJING INSTITUTE OF HEART,  LUNG AND BLOOD VESSEL DISEASES  027 whole-exome sequencing. The prevalence of pathogenic mutations in known causal genes was 5.08%  in the iTAA cohort. We selected 100 novel candidate genes using a strict strategy and found that the  suspected functional variants of these genes were significantly enriched in cases compared with con-trols and carried by 60.43% of patients. We found more severe phenotypes and a lower proportion of  hypertension in cases with pathogenic mutations or suspected functional variants. Among the candidate  genes, TESTIN (TES), which encodes a focal adhesion (FA) scaffold protein, was identified as a potential  TAA causal gene, accounting for four patients with two missense variants in the LIM1 domain (c.751T>C  encoding p.Y251H; c.838T>C encoding p.Y280H), and highly expressed in the aorta. The two variants  led to a decrease in TES expression. The thoracic aorta was spontaneously dilated in TesY249H knockin and Tes knockout mice. Mechanistically, the p.Y249H variant or knockdown of TES led to repression  of vascular smooth muscle cell (VSMC) contraction genes and disturbed the VSMC contractile phenotype. Interestingly, suspected functional variants of other FA scaffold genes, including TLN1 and ZYX,  were also significantly enriched in patients with iTAA; moreover, their knockdown resulted in decreased  contractility of VSMCs. For the first time, this study revealed the genetic landscape across iTAAs and  showed that FA scaffold genes are critical for the pathogenesis of iTAAs.

Translational Research in Aortic Aneurysm Disease (The Innovation Team of the Ministry of  Education) Aortic aneurysms cause weakness in the wall of the aorta and increase the risk of aortic rupture.  When rupture occurs, massive internal bleeding results and, unless treated immediately, shock and  death can occur. Aortic aneurysms usually cause no symptoms until they rupture. It is usually difficult  to diagnose an aortic aneurysm by physical examination and it is very difficult to predict how fast an  abdominal aortic aneurysm may grow. Current treatment options consist of surgical reconstruction or  minimally invasive intravascular procedures. However, both options have significant limitations that do  not address the underlying pathways driving this devastating disease.  For these reasons, the development of novel imaging techniques, biomarker panels, regionspecific gene therapies, and targeted pharmacologic treatments for the prediction and treatment of aortic  aneurysms, as well as translation of the discoveries into potential therapeutics remain critical goals of the research program. This proposal will help to recruit and retain outstanding faculty members, enhance  education and training, and positively impact human life.

Calcified aortic valve stenosis (CAVS) is the most common valvular heart disease in elderly people,  and its prevalence continues to rise. There are no effective drug treatments that can prevent or stop the  progression of AVS. Strengthening knowledge on the etiology of AVS and elucidating its pathogenesis  are major issues that need to be solved urgently. AVS has a high degree of genetic susceptibility. The  implementation of this project will deepen understanding of the etiology of AVS, promote the molecular  typing of AVS, and provide support for studies on AVS pathogenic mechanisms, drug screening, and  accurate diagnosis and treatment. The current progress for the clinical part and basic research part of  the project is as follows. The clinical part has two elements: (a) GWAS analysis and (b) polygenic risk  score. For the GWAS analysis, the first batch of case group and control group sample chip (Illumina  ASA) testing has been completed, and some SNPs that were significantly associated with onset of CAVS  have been found, including 8 SNPs with P<1×10-5 and 263 SNPs with P<5×10-4. For the polygenic risk  score, the design and implementation of the CAVS polygenic risk score project have been completed, and we have established multiple PRSs predicting the onset of CAVS through pruning and thresholding,  as well as LDpred methods, obtained good model discrimination and internal cross-validation results,  and found that PRS is related to disease severity. For the basic research part, previous literature reported that PALMD is a susceptibility gene for CAVS. However, the expression and location of PALMD in  different tissues and how it affects the occurrence and development of CAVS remain unclear. We performed quantitative mRNA analyses on different tissues and organs from mice and found that PALMD  was abundantly expressed in the valves, aorta, adipose tissue, lung, and other tissues, but showed low  expression in the spleen, skeletal muscle, and kidney. Single-cell sequencing analysis of the aorta revealed that PALMD was specifically expressed in the endothelial cell population. Immunofluorescence  and histochemical staining analyses confirmed that endothelial cells and PALMD were co-localized in  human and mouse valve and aorta tissues. Among primary human valve interstitial cells and valve endothelial cells (VECs), PALMD was found to be more highly expressed in VECs. To clarify the causal  relationship and molecular mechanism for CAVS susceptibility gene PALMD and CAVS, we successfully  constructed PALMD knockout mice using CRISPR/CAS9 technology. By using this mouse valve disease  model for further in vivo experiments such as endothelial cell-specific overexpression of PALMD and  vascular function detection, combined with primary valve endothelial cell experiments, we will clarify how  PALMD regulates the initiation and development of CAVS by affecting endothelial function.