Single-Cell Transcriptomics Reveals the Relationship Between Cardiac Fibroblasts and Angiogenesis: The Key Role of ANGPTL4 in HFpEF

Background Introduction

Heart failure is one of the major global health challenges today. Heart failure is categorized into heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF). HFpEF accounts for 50% of all heart failure cases, affecting approximately 32 million people worldwide. Despite its high morbidity and mortality, effective treatments for HFpEF are lacking due to the incomplete understanding of its pathophysiological mechanisms. Therefore, in-depth research into the cellular heterogeneity and potential mechanisms of HFpEF, particularly the role of angiogenesis impairment, has become a focus of current studies.

The development of single-cell RNA sequencing (scRNA-seq) technology has provided unprecedented opportunities for investigating complex cellular heterogeneity and intercellular communication. At single-cell resolution, researchers can more precisely dissect the roles of different cell types in diseases and their regulatory interactions. In this study, the authors utilized scRNA-seq to comprehensively analyze the transcriptome of non-myocardial cells in HFpEF hearts, revealing potential links between cardiac fibroblasts (CFs) and angiogenesis, and identifying ANGPTL4 as a key molecule regulating this process.

Source of the Paper

This paper was co-authored by Guoxing Li, Huilin Zhao, Zhe Cheng, Junjin Liu, Gang Li, and Yongzheng Guo, from Chongqing Medical University and its affiliated hospitals in China. It was published in 2025 in the Journal of Advanced Research with the title “Single-cell transcriptomic profiling of heart reveals ANGPTL4 linking fibroblasts and angiogenesis in heart failure with preserved ejection fraction.” The doi is 10.1016/j.jare.2024.02.006.

Research Process and Results

1. Construction and Validation of the HFpEF Mouse Model

The research team first constructed an HFpEF mouse model using a high-fat diet combined with the NOS inhibitor L-NAME. Eight-week-old male C57BL/6N mice were divided into HFpEF and control groups. The HFpEF group received a high-fat diet and L-NAME treatment, while the control group was fed a normal diet. Echocardiography and Doppler imaging were used to assess ejection fraction and diastolic function. The results showed that the HFpEF group exhibited impaired diastolic function and reduced exercise tolerance despite normal ejection fraction, successfully mimicking the pathological features of HFpEF.

2. Preparation and Analysis of Single-Cell RNA Sequencing Samples

From both the HFpEF and control groups, three mice were selected for heart tissue isolation and enzymatic digestion to obtain single-cell suspensions. Non-myocardial cells were retained for further analysis. The Chromium Single Cell 50 v2 kit (10x Genomics) was used to prepare single-cell RNA libraries, and CellRanger was employed for data alignment and expression matrix generation. The Seurat package was used for data integration, quality control, dimensionality reduction, clustering, and identification of differentially expressed genes. Monocle 2 was used to construct pseudotime trajectories, further analyzing cell differentiation and transcriptional dynamics.

3. Single-Cell Data Analysis and Cell Subpopulation Identification

Through scRNA-seq analysis of six heart samples, the research team obtained 53,040 cells. Based on specific marker genes for each cell lineage, 10 cell types were identified, including cardiac fibroblasts (CFs), macrophages (Macs), smooth muscle cells (SMCs), endothelial cells (ECs), and others. In the HFpEF group, the proportions of vascular-related cell lineages (such as arterial ECs, venous ECs) were significantly downregulated, while the proportions of smooth muscle cells and immune-related cell lineages increased, indicating significant angiogenesis impairment and inflammatory responses in HFpEF mice.

4. Differentiation and Functional Analysis of Cardiac Fibroblasts

The research team further analyzed subpopulations of cardiac fibroblasts, dividing them into seven subgroups, including anti-fibrotic WIF1+ CFs, highly fibrotic CILP+ CFs, and highly metabolic TXNIP+ CFs. In the HFpEF group, the proportions of CILP+ CFs and TXNIP+ CFs significantly increased, indicating that these fibroblasts exhibited higher fibrosis and metabolic activity in HFpEF. Through pseudotime trajectory analysis, the team found that CFs gradually transitioned toward highly fibrotic and metabolic phenotypes during differentiation, further validating the critical role of CFs in HFpEF pathological progression.

5. Validation of Angiogenesis Impairment and Molecular Mechanisms

Through bulk RNA sequencing and immunohistochemical analysis, the research team found that the expression of multiple pro-angiogenic factors was significantly downregulated in the HFpEF group, accompanied by reduced vascular density. Further research revealed that CFs interacted with ECs by secreting ANGPTL4, which exerted an anti-angiogenic function by inhibiting the RAF/MEK/ERK signaling pathway. In the HFpEF group, the expression of ANGPTL4 was significantly upregulated, suggesting its role in exacerbating pathological progression by inhibiting angiogenesis.

Research Conclusions

This study is the first to reveal the transcriptomic characteristics of non-myocardial cells in HFpEF hearts using single-cell RNA sequencing technology, elucidating the critical role of cardiac fibroblasts in HFpEF. The study found that fibroblasts in HFpEF hearts interact with endothelial cells by secreting ANGPTL4, inhibiting angiogenesis and thereby leading to cardiac microvascular rarefaction. Additionally, the study highlighted that high-fat diet-induced metabolic reprogramming may be a significant driver of cellular differentiation and functional changes in HFpEF. These findings provide new insights into the pathological mechanisms of HFpEF and suggest ANGPTL4 as a potential therapeutic target.

Research Highlights

1. First Comprehensive Single-Cell Transcriptomic Analysis of HFpEF Hearts: Using scRNA-seq technology, the research team conducted a systematic analysis of non-myocardial cells in HFpEF hearts, revealing cellular heterogeneity and its pathological roles in HFpEF.
2. Key Role of Cardiac Fibroblasts in HFpEF: The study found that fibroblasts in HFpEF hearts interact with endothelial cells by secreting ANGPTL4, inhibiting angiogenesis, providing a new perspective on the pathological mechanisms of HFpEF.
3. ANGPTL4 as a Potential Therapeutic Target: Using the machine learning tool DrugnomeAI, the study predicted the druggability of ANGPTL4, indicating its potential as a novel therapeutic target for HFpEF.

Research Significance

This study not only provides new theoretical foundations for the pathological mechanisms of HFpEF but also lays the groundwork for developing therapeutic strategies targeting ANGPTL4. Through in-depth analysis of single-cell data, the research team uncovered the multifunctional roles of cardiac fibroblasts in HFpEF, offering important references for future precision medicine in HFpEF treatment.