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Research ArticleAngiogenesisCardiology
Open Access | 
10.1172/jci.insight.186812
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Yan, L. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Zhou, Z. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Chen, S. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Feng, X. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Mao, J. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Luo, F. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Zhu, J. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Chen, X. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Hu, Y. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Wang, Y. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Wu, B. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Du, L. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Wang, C. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Gong, L. in: JCI | PubMed | Google Scholar
1Department of Pediatrics, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
2Center for Regeneration and Aging and
3Department of Obstetrics and Gynecology, Center for Reproductive Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, China.
4Department of Pediatrics, Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China.
5Liangzhu Laboratory, Zhejiang University, Hangzhou, China.
Address correspondence to: Yanfen Zhu, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, N1, Mall Avenue, Yiwu City, 322000, China. Phone: 0571.87233303; Email: yanfenzhu@zju.edu.cn. Or to: Liang Gong, Liangzhu Laboratory, Zhejiang University, 1369 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311113, China. Phone: 0571.87233350; Email: lianggong@zju.edu.cn. Or to: Chunlin Wang, The First Affiliated Hospital, Zhejiang University School of Medicine, Department of Pediatrics, 1367 Wenyi West Road, Yuhang District, Hangzhou, Zhejiang, 311100, China. Phone: 0571.87233351; Email: hzwangcl@zju.edu.cn.
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Find articles by Zhu, Y. in: JCI | PubMed | Google Scholar
Authorship note: LY, ZZ, and SC contributed equally to this work and have been designated as co–first authors.
Published April 22, 2025 - More info
Published in Volume 10, Issue 8 on April 22, 2025
AbstractSmall for gestational age (SGA), with increased risk of adult-onset cardiovascular diseases and metabolic syndromes, is known to associate with endothelial dysfunction, but the pathogenic mechanisms remain unclear. In this study, the pathological state of human umbilical vein endothelial cells (HUVECs) from SGA individuals was characterized by presenting increased angiogenesis, migration, proliferation, and wound healing ability relative to their normal counterparts. Genome-wide mapping of transcriptomes and open chromatins unveiled global gene expression alterations and chromatin remodeling in SGA-HUVECs. Specifically, we revealed increased chromatin accessibility at active enhancers, along with dysregulation of genes associated with angiogenesis, and further identified CD44 as the key gene driving HUVECs’ dysfunction by regulating pro-angiogenic genes’ expression and activating phosphorylated ERK1/2 and phosphorylated endothelial NOS expression in SGA. In SGA-HUVECs, CD44 was abnormally upregulated by 3 active enhancers that displayed increased chromatin accessibility and interacted with CD44 promoter. Subsequent motif analysis uncovered activating protein-1 (AP-1) as a crucial transcription factor regulating CD44 expression by binding to CD44 promoter and associated enhancers. Enhancers CRISPR interference and AP-1 inhibition restored CD44 expression and alleviated the hyperangiogenesis of SGA-HUVECs. Together, our study provides a foundational understanding of the epigenetic alterations driving pathological angiogenesis and offers potential therapeutic insights into addressing endothelial dysfunction in SGA.
Graphical Abstract
Introduction
Small for gestational age (SGA) describes infants with birth weights below the 10th percentile compared with their appropriate-for-gestational-age (AGA) counterparts (1). Advances in perinatal monitoring and management, along with neonatal resuscitation, have significantly improved survival rates for SGA infants. However, these individuals remain at heightened risk for adverse vascular outcomes, including impaired vasodilatation (2), fetal cardiovascular programming (3), and increased arterial wall thickness (4, 5), ultimately predisposing them to adult-onset cardiovascular and metabolic diseases, particularly in the presence of genetic susceptibilities (6, 7). Endothelial cells, which form the inner lining of blood vessels, serve as a dynamic interface between the circulatory system and surrounding tissues, critically regulating vascular tone and cellular functions (8). Impaired endothelial function is closely related to the pathological vascular processes in cardiovascular diseases and diabetic vascular complications (9). Therefore, preserving endothelial function during early life stages is crucial for maintaining vascular health over the long term. Impairments in early angiogenesis can contribute to lifelong vascular dysfunction, making endothelial cells a compelling therapeutic target for mitigating SGA-associated cardiovascular and metabolic risks in adulthood.
Human umbilical vein endothelial cells (HUVECs), which bridge the maternal and fetal circulation, play a pivotal role in fetal growth and development by regulating the formation and function of the fetal vascular system (10). Emerging evidence highlights significant differences in the angiogenic capacity of HUVECs derived from SGA neonates compared with their AGA counterparts (11, 12). These disparities are accompanied by distinct protein expression profiles and DNA methylation patterns (9, 10, 13). Furthermore, epigenetic modifications have been implicated in the transgenerational inheritance of impaired vascular function, as observed in studies on SGA rats (14, 15). Despite these insights, the precise contributions of epigenetic factors to HUVEC dysfunction in SGA and the underlying mechanisms remain largely unresolved. Given the critical role of HUVECs in fetal vascular biology and their accessibility from umbilical cords, primary cultured HUVECs offer a robust model for investigating fetal endothelial dysfunction and the developmental origins of adult cardiovascular and metabolic diseases in SGA (10, 16–18).
CD44, a cell surface glycoprotein widely expressed on mammalian cell surfaces, including endothelial cells, epithelial cells, fibroblasts, and white blood cells (19), plays essential roles in cell-cell interactions, cell adhesion, cell proliferation, and migration (20). Evidence from Cd44-knockout mice further supports its potential role in both physiological vascular development and pathological vascular dysfunction (21, 22). Here, we characterized the dysfunction of HUVECs derived from SGA status and examined the landscape of chromatin openness by performing RNA-Seq and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq). We identified CD44 as the major causal gene driving the expression of pro-angiogenic genes and activating phosphorylated ERK1/2 and phosphorylated endothelial NOS (p-ERK1/2 and p-eNOS) expression in SGA and further elucidated aberrant accessible enhancers targeted by activating protein-1 (AP-1) transcription factors, resulting in upregulated CD44 expression and enhanced angiogenic potential in SGA-HUVECs. These findings suggest that the epigenetic regulation of CD44 may be crucial in determining endothelial function and angiogenesis in SGA individuals and could influence the development and progression of fetal-origin, adult-onset diseases.
ResultsPromoted proliferation, migration, and angiogenesis of HUVECs in SGA. To examine the epigenetic changes associated with SGA status, we conducted comprehensive analysis as shown in Figure 1A. We collected umbilical cord samples from 12 SGA neonates diagnosed by birth weight below 10th percentile for gestational age as well as 9 AGA neonates with birth weight ranging from 10th to 90th percentiles as a control group following the criteria outlined in China (23). To minimize the impact of maternal factors, all specimens were obtained exclusively from full-term individuals without medical or obstetrical complications, except for 1 SGA mother who experienced preeclampsia during pregnancy. Compared with AGA, the SGA group exhibited significant reductions in birth weight (SGA: 2.15 ± 0.16 kg; AGA: 3.47 ± 0.12 kg; P < 0.0001), birth length (SGA: 46.08 ± 0.76 cm; AGA: 49.67 ± 0.47 cm; P = 0.0020), and maternal BMI at delivery (P = 0.0130) but nonsignificant changes in maternal age, gestational age, and pre-pregnancy BMI, as detailed in Supplemental Tables 1 and 2; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.186812DS1 In addition, a notably smaller diameter of both umbilical cord and umbilical vein in the SGA group was observed (Figure 1B), suggesting potential abnormalities in HUVECs’ functions in SGA neonates.
Figure 1Distinctive phenotypes of HUVECs derived from SGA and AGA individuals. (A) Schematic overview of the study design. CRISPRi, CRISPR interference; Hi-C, high-throughput chromosome conformation capture; OE, overexpression; TF, transcription factor. (B) Representative images of the cross section of umbilical cord tissues stained by H&E. The enlarged parts represent umbilical veins. Scale bars: 1 mm (left) and 200 μm (right); n = 4 per group. (C) Representative immunofluorescence staining of primary HUVECs’ identity; scale bar: 20 μm; n = 4 per group. (D) Representative images of angiogenesis; scale bar: 100 μm. (E) Analysis of the total tube length, total tube branching length, and total segment length for samples shown in Supplemental Figure 1A (n = 9 AGA and 12 SGA). (F) Representative images of scratches at 0 hours and 12 hours; scale bar: 200 μm. (G) Analysis of the percentage of migration area; n = 4 per group. (H) Representative images of HUVECs that migrated through the pores; scale bar: 100 μm. (I) Analysis of percentage of migrating cell number; n = 4 per group. (J) Analysis of EdU assay; n = 4 per group. (K) Analysis of CCK8 assay results at 0 hours, 24 hours, 48 hours, and 72 hours; n = 4 per group. Two-way ANOVA with Holm-Šídák multiple comparisons test was used for comparing cell proliferation at different times. (L) Analysis of glucose consumption; n = 4 per group. Data presented as mean ± SEM. Statistical analysis was performed using 2-tailed unpaired Student’s t test.
To further investigate the influence of SGA status on HUVECs’ function, we successfully isolated and cultured the primary HUVECs from both SGA and AGA groups in vitro and confirmed their identity by the characteristic cobblestone cell shapes and high expression of endothelial markers vWF and CD31 (Figure 1C). Interestingly, relative to AGA-HUVECs, SGA-HUVECs exhibited increased in vitro angiogenic capacity with significantly increased total length (P = 0.0002), total branching length (P < 0.0001), and total segment length (P < 0.0001) of tubes in the tube formation assay (Figure 1, D and E, and Supplemental Figure 1A). Meanwhile, promoted migration and invasive ability were observed in SGA-HUVECs, as evidenced, respectively, by a significantly larger migration area 12 hours after injury in the scratch wound healing assay (Figure 1, F and G, P = 0.0014) and by a significantly increased number of cells migrating through the pores in the Transwell assay (Figure 1, H and I, P = 0.0003) relative to AGA-HUVECs. Assessment of proliferation ability by 5-ethynyl-2′-deoxyuridine (EdU) nucleic acid labeling technique and cell counting kit-8 (CCK8) assays revealed a significantly higher proliferation rate (P = 0.0309 in EdU assay and P = 0.0017 at 24 hours in CCK8 assay) in SGA-HUVECs compared with AGA-HUVECs (Figure 1, J and K). Consistent with their increased cellular activity, SGA-HUVECs displayed a higher consumption of glucose compared with the AGA group after 24 hours of culture, indicating an elevated energy requirement to support their improved survival (Figure 1L, P = 0.0185). There was also an elevated reactive oxygen species (ROS) generation in SGA-HUVECs compared with AGA (Supplemental Figure 1B).
Taken together, our results suggest that the SGA condition may hinder umbilical cord development, particularly affecting the formation of the umbilical vein and the function of HUVECs. Specifically, the SGA state exerts strong stimulatory effects on the angiogenesis, migration, proliferation, and wound healing capacity of HUVECs.
Dysregulation of angiogenic genes in SGA-HUVECs. To further explore the mechanisms underlying the impaired SGA-HUVEC function, we performed RNA-Seq on 4 SGA-HUVECs and 4 AGA-HUVECs with 3 technical replicates. Both hierarchical clustering and principal component analysis (PCA) separated the SGA and AGA groups well (Figure 2, A and B), indicating a distinct gene regulation pattern between them. A total of 399 differentially expressed genes (DEGs) were identified in SGA relative to AGA with fold-change ≥ 1.50 and adjusted P value < 0.05, 223 of which were upregulated and 176 downregulated (Figure 2C and Supplemental Figure 2A). Real-time quantitative PCR (RT-qPCR) assay verified the upregulation of CXCL8, IL1A, TNFSF18, CD44, and FOXF1 and the downregulation of COL3A1, IGFBP3, SFRP1, CDH11, and SULF1 in SGA-HUVECs (Figure 2D). Among them, CD44 is a major cell surface receptor of hyaluronic acid that plays an essential role in physiological activities, including cell proliferation, adhesion, angiogenesis, and migration (24). CXCL8, also known as IL-8, promotes angiogenesis during wound healing, tissue repair, and cancer progression (25). Reduction of CXCL8 expression attenuated tumor-associated angiogenesis (26). SULF1 is a known antiangiogenic gene in multiple processes (27). To understand the functional association of the 399 DEGs, we performed gene ontology (GO) enrichment analysis (Figure 2E). We observed the enrichment of processes associated with development (embryo development, circulatory system development, and anatomical structure formation involved in morphogenesis), indicating low birth weight in SGA may disturb the impaired HUVECs’ function. Corresponding to the pro-angiogenic state of SGA-HUVECs, GO terms related to angiogenesis (blood vessel morphogenesis, blood vessel development, and tube morphogenesis), and extracellular matrix (cell-substrate adhesion and extracellular matrix organization), a structure playing significant roles in blood vessel formation and signaling (28), were enriched. Moreover, Kyoto Encyclopedia of Genes and Genomes analysis also identified ECM-receptor interaction as the top significant term (data not shown), indicating ECM-associated receptor and ligand proteins play a role here. These analyses indicated that genes associated with angiogenesis were dysregulated in SGA-HUVECs, which may result in impaired function of umbilical veins.
Figure 2Dysregulation of angiogenic genes in SGA-HUVECs. (A) Clustering of RNA-Seq data by correlation of fragments per kilobase million (FPKM) between samples. (B) PCA of RNA-Seq data based on FPKM values. (C) Heatmap of the normalized expression of the 399 DEGs in SGA relative to AGA. Selected genes are labeled. Red color indicates upregulation and blue color indicates downregulation. (D) Validation of RNA-Seq data by RT-qPCR. Relative expression of 10 selected genes in 4 biological replicates were displayed. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (E) The top 20 significantly enriched GO terms of biological process in DEGs with the FDR < 0.05. (F) PPI subnetwork with CD44 as hub nodes. First neighbors of hub nodes were shown. The thicker the edge, the higher the combined score between 2 nodes. (G) Representative IHC images of paraffin-embedded umbilical cord samples using CD44 antibody. Arrows indicate HUVECs with high CD44 expression; scale bars: 200 μm (upper) and 25 μm (lower). (H) Analysis of percentage of CD44-positive cells in intima of umbilical veins from AGA and SGA; n = 4 per group. (I and J) Analysis of Western blot showing the high CD44 protein levels in HUVECs from SGA relative to AGA; n = 6 per group. The data in D, H, and I are presented as mean ± SEM and were analyzed using 2-tailed unpaired Student’s t test.
As 17.20% of protein-coding DEGs encoded receptor or ligand proteins (i.e., CD44, CXCL8, SULF1, LAMB2, IGFBP3, ANGPT2, and ITGB4), twice the proportion (8.00%) found in genome-wide protein-coding genes (29), we thus performed Protein-Protein Interaction (PPI) analysis of DEGs using the STRING database (30). A PPI network consisted of 105 nodes and 108 edges, with confidence scores ≥ 0.7 detected (Supplemental Figure 2B). CD44 was the top hub node with 10 first-level connected proteins, involving proteins functioning in growth (i.e., IGFBP3) (31, 32), angiogenesis, and vessel development (i.e., VCAM1 and LYVE1) (33, 34) (Figure 2F). Next, we verified the substantial increase of CD44 in the endothelial cells of umbilical cord tissues by immunohistochemistry (IHC) (Figure 2, G and H, and Supplemental Figure 3A). ELISA for CD44 in umbilical cord blood serum revealed a slight elevation of CD44 levels though without statistical significance in SGA compared with the AGA group (Supplemental Figure 3B). We further verified the elevated CD44 protein levels in SGA-HUVECs by Western blot compared with AGA-HUVECs (Figure 2, I and J). Inspired by the highly differential CD44 expression and the key role of CD44 in ECM signaling and angiogenesis, we speculated that CD44 may function as the primary molecular determinant causing SGA-HUVECs’ dysfunction.
CD44 as a key regulator strengthening angiogenesis of SGA-HUVECs. To validate our hypothesis, we employed small interfering RNA (siRNA) to specifically decrease CD44 expression in the SGA condition. Following transfection with CD44 siRNA (Si-CD44), CD44 mRNA and protein levels were reduced by approximately 76.00% and 80.00%, respectively (Supplemental Figure 4, A–C). In SGA-HUVECs, diminished CD44 expression led to reduced tube-forming ability (Figure 3A), marked by decreased total branching length (P = 0.0011), total segment length (P = 0.0060), and total length (P = 0.0011) of tubes (Figure 3C and Supplemental Figure 4, D and E), which is accordant with the previously reported results in Cd44-deficient mice that endothelial Cd44 led to reduced vascularization and angiogenesis together with impaired vascular integrity (22, 35). Furthermore, reduced migration (Figure 3, B and D, P = 0.0058), wound healing capacity (Figure 3E and Supplemental Figure 4F, P = 0.0476), proliferation rate (Figure 3F and Supplemental Figure 4G, P < 0.0001 in CCK8 assay and P = 0.0026 in EdU assay), and glucose consumption (
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