Vascular biology
Abstract
The neutrophil-to-lymphocyte ratio (NLR) is associated with unfavorable prognosis and hemorrhagic transformation (HT) in patients with ischemic stroke, yet the underlying mechanisms remain unclear. Using patient samples and a murine stroke model, we identified CD8⁺ regulatory T cells (CD8 Tregs) key regulators of neutrophil homeostasis after ischemic stroke, thereby limiting endothelial disruption and HT. Loss of CD8 Tregs expanded circulating neutrophils by extending their lifespan rather than altering proliferation, bone marrow release, or direct cytotoxicity. Mechanistically, CD8 Tregs shortened neutrophil lifespan by modulating HIF-1α–dependent glycolytic activity and relieving PD-L1–mediated suppression of bone marrow clearance. Finally, co-culture experiments with human CD8 Tregs and neutrophils revealed similar neutrophil-regulatory effects, accompanied by improved endothelial barrier integrity. These findings reveal a previously unrecognized CD8 Treg–neutrophil axis and suggest potential therapeutic strategies for preventing HT after stroke.
Authors
Jianan Lu, Qia Zhang, Jiarui Chen, Huaming Li, An Ping, Ziyang Jin, Xiaotao Zhang, Yichen Gu, Xuejiao Dai, Zihong Chen, Yajun Qian, Guoqiang Zhang, Jun Yu, Jianmin Zhang, Ligen Shi
Abstract
Hereditary Hemorrhagic Telangiectasia type 2 (HHT2), caused by mutations in ACVRL1 ( also known as ALK1), is characterized by brain arteriovenous malformations (bAVMs), abnormal artery–vein connections for which treatment options remain limited. Despite evidence of endothelial cell (EC) heterogeneity, its role in bAVM pathogenesis remains poorly defined. Using endothelial-specific inducible Alk1 knockout mice (Alk1iECKO) and regionally resolved single-cell RNA sequencing, we show that loss of ALK1 signaling induces bAVMs predominantly in the perineural vascular plexus (PNVP). This process is driven by the emergence of a KIT+ angiogenic EC population with human AVM-like transcriptional features, including tip-cell markers and activation of PI3K and KRAS signaling pathways. Cross-species analyses and validation in human samples demonstrate that KIT expression is conserved in endothelial cells from both sporadic and HHT2 brain AVMs. Drug repurposing analysis identified KIT as a top actionable target, and we show that Kit is directly repressed by BMP9–ALK1–SMAD4 signaling. Pharmacological inhibition of KIT reduced angiogenic reprogramming and vascular malformations in vivo without affecting normal vasculature. These findings identify a pathogenic angiogenic EC state and position KIT signaling as a therapeutically actionable pathway in brain AVMs.
Authors
Elise Drapé, Lauranne Carrier, Gael Cagnone, Atik R.M. Fuad, Mathilde Bizou, Damian A. Sanchez, Typhaine Anquetil, Jack Wang, Halima Drissi Touzani Walali, Adnan Gopinadhan, Patrick Piet van Vliet, Joel P. Howard, Mysha Ibnat, Gregor Andelfinger, Ethan Winkler, Bruno Larrivée, Alexandre Dubrac
Abstract
The endocardium is a major source of coronary angiogenesis and arterialization, through coordinated cell fate transition and migration. However, the transcriptional regulatory network synchronizing cell fate determination and movement remains unclear. Here, we identified transcription factor HAND2 as a key candidate for coronary vascular formation. Endocardial deletion of Hand2 in mice disrupted arterial-venous networks and stunted coronary arteries, paralleling a ventricular noncompaction phenotype. Moreover, deletion of Hand2 produced excessive tip cells with defective movement. RNA-seq analysis revealed enhanced hypoxic metabolic activation but declined TGF-β/p38MAPK-dependent endothelial-to-mesenchymal transition (Endo-MT). In consistence, genetic inhibition of the core hypoxic regulators or pharmaceutical administration of TGFβ2 partially recovered the coronary arterial defects in Hand2 mutants. Furthermore, HAND2 was found directly bound to promoters of the target genes, harmonizing cell migration and cell fate transition. These findings pinpoint HAND2 as an essential regulator of the endocardial transcriptional regulatory network for coronary arterialization and provide potential therapeutic targets for coronary artery diseases.
Authors
Huijuan Wang, Haosheng Zhang, Leiyin Zheng, Peihan Zhang, Yuqian Wang, Sijia Ding, Wenping Liu, Yuanming Cheng, Zhongzhou Yang, Wen Luo
Abstract
Orphan GPCRs of the GPRC5 family regulate macrophage activity and vascular contractility by dimerizing with other GPCRs, but pharmacological modulation of this process has not been explored. We previously identified the dimerization interface of receptor GPRC5B and show here that both its mutation and inhibition by a decoy peptide disturbed the interaction with the prostaglandin E2 receptor EP2 in macrophages, resulting in reduced EP2 signaling, enhanced migration and phagocytosis, and protection from bacterial peritonitis in mice. Furthermore, we show that a similar interface exists in related receptor GPRC5C, and, the same as in GPRC5B, mutation or inhibition by decoy peptide improved host defense. Through a virtual docking screen, we identified a small molecule inhibitor of both GPRC5B and GPRC5C dimerization, K303MP20, and showed that it reduced EP2 signaling, enhanced macrophage activity, and improved host defense in bacterial peritonitis and influenza A infection. Interestingly, K303MP20 not only blocked dimerization between GPRC5B/C and EP2, but also with prostacyclin receptor IP and angiotensin II receptor AT1, resulting in reduced AT1-dependent contraction and enhanced IP-dependent relaxation in human and murine smooth muscle cells. In vivo, K303MP20 did not affect basal blood pressure, but protected mice from angiotensin II–induced hypertension. Taken together, inhibition of orphan GPCR dimerization by small molecules is feasible and improves infection control and arterial hypertension.
Authors
Jeonghyeon Kwon, Margherita Persechino, Jingchen Shao, Jamal Shamsara, Birgit Spitznagel, Isabelle Salwig, Miloslav Sanda, Stefan Offermanns, Peter Kolb, Nina Wettschureck
Abstract
Neddylation is highly activated in many human cancers and may serve as a therapeutic target for clinical treatment. However, it remains unclear regarding the role of neddylation in tumor angiogenesis. Here, we demonstrate that the neddylation E2 enzyme UBE2M is upregulated in tip cells and is essential for tumor vascular sprouting. We show that UBE2M-mediated neddylation of STAT1 enhances its phosphorylation and promotes the transcription of DLL4. This elevated DLL4 expression in tip cells activates Notch signaling in adjacent stalk cells, thereby maintaining the tip-stalk cell balance and ensuring organized vascular patterning. Consequently, endothelial-specific deletion of UBE2M reduces DLL4 expression, leading to excessive but non-productive sprouting due to uncontrolled tip cell formation and lack of stalk cell support, which ultimately suppresses tumor growth. Importantly, targeting endothelial neddylation potently sensitizes various tumors to anti-VEGF therapy. Together, our findings unveil UBE2M as a key regulator of angiogenic signaling and identify it as a promising anti-angiogenic target in cancer.
Authors
Xinyi Jiang, Jie Zhang, Li Zhou, Zonglin Li, Ningcong Sun, Xian Xu, Jisong Zhang, Yizhou Huang, Xue Zhang, Enguo Chen, Hongqiang Cheng, Yuehai Ke
Abstract
Efferocytosis, the clearance of apoptotic cells by macrophages, promotes tissue resolution. Efficient resolution requires efferocytosis-induced macrophage proliferation (EIMP) to expand pro-resolving macrophages. Here, we show that efferocytosis activates base excision repair (BER) to remove 8-OHdG from DNA, enabling EIMP. Mechanistically, efferocytosis promotes poly(ADP-ribose) polymerase-1 (PARP1) chromatin binding and PARylation to facilitate DNA repair complex assembly, and increases nuclear MTH1/NUDT1, which hydrolyzes 8-OHdG. Both processes require DNA-methyltransferase-3A (DNMT3A), which is activated during efferocytosis. Using a model where dexamethasone-induced thymocyte apoptosis triggers efferocytosis-mediated thymic repair, we showed that DNMT3A is required for increases in nuclear PARP1/MTH1, oxidized DNA suppression, EIMP in thymic macrophages, and thymic repair. We next studied a human-relevant model of atherosclerosis regression, where efferocytosis drives protective lesional fibrous cap thickening. We compared WT mice with a model of DNMT3A-clonal hematopoiesis (CH), in which loss-of-function DNMT3A mutations promote atherosclerotic disease. Atherosclerosis regression in WT mice led to decreased nuclear 8-OHdG and increases in nuclear PARP1/MTH1 and EIMP in lesional macrophages and fibrous cap thickening, all of which were impaired in DNMT3A-CH regression. These findings reveal that efferocytosis initiates a BER pathway to allow macrophage proliferation for tissue resolution, with possible therapeutic relevance to atherosclerosis regression and DNMT3A-CH.
Authors
Kleopatra Avrampou, Santosh R. Sukka, David Ngai, Patrick Ampomah, Xiaobo Wang, George Kuriakose, Jacob Glass, Bernhard Dorweiler, Hanna Winter, Lars Maegdefessel, Hanrui Zhang, Aaron Viny, Ira Tabas
Abstract
Cerebral malaria (CM) from Plasmodium falciparum is a major cause of death in African children. Since bradykinin (BK) is a mediator of vasogenic edema, we hypothesized that it contributes to the pathogenesis of CM in Kenyan children and Plasmodium berghei ANKA (PbA) infected C57BL/6J mice in experimental cerebral malaria (ECM). Cleaved plasma high molecular weight kininogen (cHK) is a marker for BK release. 40% of children with central nervous system malaria had plasma cHK versus 18% of children with uncomplicated malaria. Wild-type PbA-infected mice with ECM had circulating cHK, elevated BK levels, and reduced HK and prekallikrein activity/antigen levels. HK null (Kng1–/–), combined BK B1 and B2 receptor null (Bdkrb1–/–/Bdkrb2–/–), BK B2 (Bdkrb2–/–) or BK B1 (Bdkrb1–/–) receptor null mice were protected significantly from neurologic deterioration and brain edema compared to wild-type mice. F12–/– mice were not protected from neurological deterioration. Prekallikrein null (Klkb1–/–), prolylcarboxypeptidase hypomorphs (Prcpgt/gt), and brain endothelial cell conditional knockout of PRCP (Prcpfl/fl Cre) mice with ECM had reduced neurologic deterioration and brain edema. Adjuvant plasma kallikrein inhibition combined with artesunate treatment in PbA-infected mice reversed neurologic deterioration and brain edema and significantly prolonged survival over artesunate alone. BK-induced vasogenic edema contributes to human and murine CM.
Authors
Alessandro S. de Sa Pinheiro, Douglas E. Teixeira, Rodrigo P. Silva-Aguiar, Young Jun Shim, Alona A. Merkulova, Sadiq Silbak, Yelenna Skomorovska-Prokvolit, David Midem, Sidney Ogolla, Bjoern B. Burckhardt, Tanja Gangnus, Julio Scharfstein, Celso Caruso-Neves, Owen J.T. McCarty, David Gailani, Michael Bader, Philip J. Rosenthal, Arlene E. Dent, Chris J. Janse, Keith R. McCrae, Ana Acacia de Sa Pinheiro, James W. Kazura, Alvin H. Schmaier
Abstract
Lymphatic valves are essential for maintaining tissue fluid homeostasis, and their dysfunction leads to lymphedema, a morbid and disfiguring disease without a cure. Mechanical forces due to lymph flow are required for proper lymphatic valve development, yet it remains unclear how lymphatic endothelial cells (LECs) sense and decode mechanical signals. In this study, we identify the cell guidance semaphorin receptor plexin D1 (PLXND1) as a lymphatic mechanosensor required for lymphatic valve morphogenesis. Conditional genetic ablation of Plxnd1 in LECs caused major defects in lymphatic valve development in 2 different lymphatic vascular beds. Mechanistically, PLXND1 acted as a mechanosensor within a lymphatic mechanocomplex, initiating distinct mechanical signals and activating the lymphatic valve transcriptional program through an unconventional pathway. Screening of patients with primary lymphedema identified PLXND1 missense variants, and functional analysis established 2 pathogenic variants that selectively disrupt the ligand versus mechanosensing functions of this receptor. Variants associated with lymphedema in members of the mechanocomplex disrupted its formation, underscoring the central role of this complex in lymphatic valve biology. Our work uncovers a mechanosensing mechanism guiding lymphatic valve development, and has profound implications for the understanding and treatment of primary lymphedema in humans.
Authors
Kar-Lai Pang, Vedanta Mehta, Claire Aitken, Sara E. Dobbins, Jing Yu, Gabriele Bonetti, Adam N. Keen, Feiran Zhang, Amélie Sabine, Tatiana V. Petrova, Paul R. Riley, E. Yvonne Jones, Sandro Michelini, Matteo Bertelli, John S. Reader, Pia Ostergaard, Ellie Tzima
Abstract
The blood and lymphatic vascular systems are regulated by angiopoietin (ANGPT) growth factors, which signal via endothelial TIE receptor tyrosine kinases and integrins. However, mechanistic understanding of how these receptors crosstalk is limited. Here, we show how β1-integrin inactivation regulates endothelial ANGPT/TIE2 signaling. By integrating biophysical analyses, X-ray crystallography, size-exclusion chromatography–small-angle X-ray scattering and atomistic molecular dynamics simulations, we show that ANGPT2 binds through its asymmetrically positioned C-terminal fibrinogen-like domains to both TIE2 and α5β1-integrin, forming a trimeric complex compatible with the inactive α5β1-integrin conformation. Inactive β1-integrin colocalizes with ANGPT-induced TIE2 in cell-cell junctions and stabilizing β1-integrin in its inactive state enhances junctional TIE2 accumulation and promotes nuclear exclusion of the TIE2 transcriptional effector FOXO1 in cultured endothelial cells. Endothelial-specific β1-integrin deletion in adult mice reduces venous TIE2 phosphorylation, whereas endotoxemia diminishes junctional β1-integrin along with decreased phosphorylated TIE2. In contrast, without TIE2, ANGPT2 uniquely engages active β1-integrin, via its N-terminal superclustering domain. Altogether, our results provide structural and mechanistic evidence of ANGPT signaling via α5β1-integrin and support a model in which inactive α5β1-integrin acts as a junctional scaffold for ANGPT/TIE2/FOXO1 signaling, explaining how integrin conformational switching spatially organizes growth factor signaling in the endothelium.
Authors
Tuomas Sipilä, Srinivas Kumar Ponna, Abhinandan Venkatesha Murthy, Anne Pink, Giray Enkavi, Shraman Kumar Bohra, Klaudia Lewna, Keerthana Ganesh, Qina Liu, Mirka Korhonen, Tommi Kajander, Michael Potente, Johanna Ivaska, Ilpo Vattulainen, Veli-Matti Leppänen, Pipsa Saharinen
Abstract
Enhanced TGFβ signaling caused by mutations in Fibrillin-1 (FBN1) in patients with Marfan syndrome (MFS) leads to myxomatous degeneration of the mitral valve (MDMV). MDMV can result in mitral valve prolapse, severe regurgitation, and sudden cardiac death. However, it remains unknown whether lymphatic vessel (LV) dysfunction contributes to MDMV development in MFS. Here, we show that lymphangiogenesis in murine mitral valves (MVs) begins postnatally. However, this process is inhibited in a mouse MFS model, Fbn1 mutant (Fbn1C1039G/+) mice, accompanied by disrupted lymphatic cell-cell junctions, impaired lymphatic drainage, and an abnormally widespread distribution of MHCII+ infiltrating macrophages. Treatment of Fbn1 mutant mice with VEGF-C156S, a selective VEGFR3 agonist, stimulates the ERK and Akt pathways, increases LV density in MVs, and ameliorates MDMV. Fbn1 mutant MVs display disorganized valvular endothelial cells (VECs) and decreased expression of the anti-inflammatory modulator Zfp36 (zinc finger protein 36) in VECs and immune cells. Treatment with FTY720 (Fingolimod), a ZFP36 activator and S1P antagonist, rescues MDMV phenotypes in Fbn1 mutant mice by reducing immune cell infiltration and restoring lymphatic cell junctions and drainage. These findings suggest that the Fbn1 mutation causes LV hypoplasia and defective lymphatic drainage in MVs, driven in part by pro-inflammatory VECs, leading to MFS-related MDMV.
Authors
Can Tan, Ziyou Ren, Shreya Kurup, Xianpeng Liu, Zhi-Dong Ge, Shodai Suzuki, Pritika Jakka, Cheryl Tang, M. Luisa Iruela-Arispe, Tsutomu Kume
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)