Vascular biology
Abstract
Genetic factors are fundamental in the etiology of thoracic aortic aneurysm and dissection (TAAD), but the genetic cause is detected in only about 30% of cases. To define unreported TAAD-associated sequence variants, exome and gene panel sequencing was performed in 323 patients. We identified heterozygous CDKL1 variants [c.427T>C p.(Cys143Arg), c.617C>T p.(Ser206Leu), and c.404C>T p.(Thr135Met)] in 6 patients from 3 families with TAAD-spectrum disorders. CDKL1 encodes a protein kinase involved in ciliary biology. Amino acid substitutions were predicted to affect CDKL1 catalytic activity or protein binding properties. CDKL1 was expressed in vascular smooth muscle cells in normal and diseased human aortic wall tissue. Cdkl1 knockdown and transient knockout in zebrafish resulted in intersomitic vessel (ISV) malformations and aortic dilation. Co-injection of human CDKL1wildtype, but not CDKL1Cys143Arg and CDKL1Ser206Leu RNA, rescued ISV malformations. All variants affected CDKL1 kinase function and profiling data, and altered protein-protein binding properties, particularily with ciliary transport molecules. Expression of CDKL1 variants in heterologeous cells interfered with cilia formation and length, CDKL1 localization, and p38-MAPK and Vegf signaling. Our data suggest a role of CDKL1 variants in the pathogenesis of TAAD-spectrum disorders. The association between primary cilia dysregulation and TAAD expands our knowledge of the underlying molecular pathophysiology.
Authors
Theresa Nauth, Melanie Philipp, Sina Renner, Martin D. Burkhalter, Helke Schüler, Ceren Saygi, Kristian Händler, Bente Siebels, Alice Busch, Thomas Mair, Verena Rickassel, Sophia Deden, Konstantin Hoffer, Jakob Olfe, Thomas S. Mir, Yskert von Kodolitsch, Evaldas Girdauskas, Meike Rybczynski, Malte Kriegs, Hannah Voß, Thomas Sauvigny, Malte Spielmann, Malik Alawi, Susanne Krasemann, Christian Kubisch, Till J. Demal, Georg Rosenberger
Abstract
Adams-Oliver Syndrome (AOS) is a rare congenital disorder characterized by scalp, limb, and cardiovascular defects. While variants in the NOTCH1 receptor, DLL4 ligand, and RBPJ transcription factor have been implicated in AOS, the driving tissue types and molecular mechanisms by which these variants cause pathogenesis are unknown. Here, we used quantitative binding assays to show that AOS-associated RBPJ missense variants compromise DNA binding but not cofactor binding. These findings suggest that AOS-associated RBPJ variants do not function as loss-of-function alleles but instead act as dominant-negative proteins that sequester cofactors from DNA. Consistent with this idea, mice carrying an AOS-associated Rbpj allele develop dominant phenotypes that include increased lethality and cardiovascular defects in a Notch1 heterozygous background, whereas Notch1 and Rbpj compound heterozygous null alleles are well-tolerated. To facilitate studies into the tissues driving AOS pathogenesis, we employed conditional genetics to isolate the contribution of the vascular endothelium to the development of AOS-like phenotypes. Importantly, our studies show that expression of the Rbpj AOS allele in endothelial cells is both necessary and sufficient to cause lethality and cardiovascular defects. These data establish that reduced Notch1 signaling in the vasculature is a key driver of pathogenesis in this AOS mouse model.
Authors
Alyssa F. Solano, Kristina Preusse, Brittany Cain, Rebecca Hotz, Parthav Gavini, Zhenyu Yuan, Benjamin Bowen, Gabrielle Maco, Hope Neal, Ellen K. Gagliani, Christopher Ahn, Hee-Woong Lim, Laura Southgate, Rhett A. Kovall, Raphael Kopan, Brian Gebelein
Abstract
In peripheral tissues, an endothelial cell (EC) protein, GPIHBP1, captures lipoprotein lipase (LPL) from the interstitial spaces and transports it to the capillary lumen. LPL mediates the margination of triglyceride-rich (TG-rich) lipoproteins (TRLs) along capillaries, allowing the lipolytic processing of TRLs to proceed. TRL-derived fatty acids are used for fuel in oxidative tissues or stored in adipose tissue. In mice, GPIHBP1 is absent from capillary ECs of the brain (which uses glucose for fuel); consequently, LPL and TRL margination are absent in mouse brain capillaries. However, because fatty acids were reported to play signaling roles in the brain, we hypothesized that LPL-mediated TRL processing might occur within specialized vascular beds within the central nervous system. Here, we show that GPIHBP1 is expressed in capillary ECs of human and mouse choroid plexus (ChP) and that GPIHBP1 transports LPL (produced by adjacent ChP cells) to the capillary lumen. The LPL in ChP capillaries mediates both TRL margination and processing. Intracapillary LPL and TRL margination are absent in the ChP of Gpihbp1–/– mice. GPIHBP1 expression, intracapillary LPL, and TRL margination were also observed in the median eminence and subfornical organ, circumventricular organs implicated in the regulation of food intake.
Authors
Wenxin Song, Madison Hung, Ellen Kozlov, Megan Hung, Anh P. Tran, James Carroll, Le Phoung Nguyen, Troy L. Lowe, Paul Kim, Hyesoo Jung, Yiping Tu, Joonyoung Kim, Ashley M. Presnell, Julia Scheithauer, Jenna P. Koerner, Ye Yang, Shino D. Magaki, Christopher K. Williams, Michael Ploug, Haibo Jiang, Christer Betsholtz, Maarja Andaloussi Mäe, Liqun He, Anne P. Beigneux, Loren G. Fong, Stephen G. Young
Abstract
Plasminogen activator inhibitor-1 (PAI-1), encoded by SERPINE1, contributes to age-related cardiovascular diseases (CVD) and other aging-related pathologies. Humans with a heterozygous loss-of-function SERPINE1 variant exhibit protection against aging and cardiometabolic dysfunction. We engineered a mouse model mimicking the human mutation (Serpine1TA700/+) and compared cardiovascular responses with wild-type littermates. Serpine1TA700/+ mice lived 20% longer than littermate controls. Under L-NG-Nitro-arginine methyl ester (L-NAME)-induced vascular stress, Serpine1TA700/+ mice exhibited diminished pulse wave velocity (PWV), lower systolic hypertension (SBP), and preserved left ventricular diastolic function compared to controls. Conversely, PAI-1-overexpressing mice exhibited measurements indicating accelerated cardiovascular aging. Single cell transcriptomics of Serpine1TA700/+ aortas revealed a vascular-protective mechanism with downregulation of extracellular matrix regulators Ccn1 and Itgb1. Serpine1TA700/+ aortas were also enriched in a cluster of smooth muscle cells that exhibited plasticity. Finally, PAI-1 pharmacological inhibition normalized SBP and reversed L-NAME-induced PWV elevation. These findings demonstrate that PAI-1 reduction protects against cardiovascular aging-related phenotypes, while PAI-1 excess promotes vascular pathological changes. Taken together, PAI-1 inhibition represents a promising strategy to mitigate age-related CVD.
Authors
Alireza Khoddam, Anthony Kalousdian, Mesut Eren, Saul Soberanes, Andrew Decker, Elizabeth J. Lux, Benjamin W. Zywicki, Brian Dinh, Bedirhan Boztepe, Baljash S. Cheema, Carla M. Cuda, Hiam Abdala-Valencia, Arun Sivakumar, Toshio Miyata, Lisa D. Wilsbacher, Douglas E. Vaughan
Abstract
Severe systemic inflammatory reactions, including sepsis, often lead to shock, organ failure and death, in part through an acute release of cytokines that promote vascular dysfunction. However, little is known about the vascular endothelial signaling pathways regulating the transcriptional profile in failing organs. This work focuses on signaling downstream of IL-6, due to its clinical importance as a biomarker for disease severity and predictor of mortality. Here, we show that loss of endothelial expression of the IL-6 pathway inhibitor, SOCS3, promoted a type I interferon (IFNI)-like gene signature in response to endotoxemia in mouse kidneys and brains. In cultured primary human endothelial cells, IL-6 induced a transient IFNI-like gene expression in a non-canonical, interferon-independent fashion. We further show that STAT3, which we had previously shown to control IL-6-driven endothelial barrier function, was dispensable for this activity. Instead, IL-6 promoted a transient increase in cytosolic mitochondrial DNA and required STAT1, cGAS, STING, and the IRFs 1, 3, and 4. Inhibition of this pathway in endothelial-specific STING knockout mice or global STAT1 knockout mice led to reduced severity of an acute endotoxemic challenge and prevented the endotoxin-induced IFNI-like gene signature. These results suggest that permeability and DNA sensing responses are driven by parallel pathways downstream of this cytokine, provide new insights into the complex response to acute inflammatory responses, and offer the possibility of potential novel therapeutic strategies for independently controlling the intracellular responses to IL-6 in order to tailor the inflammatory response.
Authors
Nina Martino, Erin K. Sanders, Ramon Bossardi Ramos, Iria Di John Portela, Fatma Awadalla, Shuhan Lu, Dareen Chuy, Neil Poddar, Mei Xing G Zuo, Uma Balasubramanian, Peter A. Vincent, Pilar Alcaide, Alejandro P. Adam
Abstract
The role of endothelial dysfunction in tubulointerstitial fibrosis associated with chronic kidney disease (CKD) is not well understood. In this study, we demonstrate that the activation of the endothelial tyrosine kinase TIE2 alleviates renal pathology in experimental CKD in mice. TIE2 activation was achieved using a human angiopoietin-2 (ANGPT2)-binding and TIE2-activating antibody (ABTAA), or through adult-induced endothelial-specific knockout of the vascular endothelial protein tyrosine phosphatase gene (Veptp). Both methods significantly protected CKD mice from endothelial dysfunction, peritubular capillary loss, tubular epithelial injury, and tubulointerstitial fibrosis. Conversely, silencing TIE2 through adult-induced endothelial-specific knockout of the Tie2 gene exacerbated CKD pathology. Additionally, we found that endothelial dysfunction promotes renal fibrosis not through endothelial-to-mesenchymal transition as previously expected, but by inducing the expression of pro-fibrotic PDGFB in tubular epithelial cells, a process that is inhibited by TIE2 activation. Our findings suggest that TIE2 activation via ABTAA warrants investigation as a therapy in human CKD, where there is a substantial unmet medical need.
Authors
Riikka Pietilä, Amanda M. Marks-Hultström, Liqun He, Sami Nanavazadeh, Susan E. Quaggin, Christer Betsholtz, Marie Jeansson
Abstract
Peripheral artery disease (PAD) often advances to chronic limb-threatening ischemia (CLTI), resulting in severe complications such as limb amputation. Despite the potential of therapeutic angiogenesis, the mechanisms of cell-cell communication and transcriptional changes driving PAD are not fully understood. Profiling long non-coding RNAs (lncRNAs) from gastrocnemius muscles of human subjects with or without CLTI revealed that a vascular smooth muscle cell (SMC)-enriched lncRNA CARMN, was reduced with CLTI. This study explored how a SMC lncRNA-miRNA signaling axis regulates angiogenesis in limb ischemia. CARMN knockout (KO) mice exhibited reduced capillary density and impaired blood flow recovery and tissue necrosis following limb ischemia. We found that CARMN KO SMC supernatants inhibited endothelial cell (EC) proliferation, spheroid sprouting, and network formation. RNA-sequencing identified downregulation of the Hedgehog signaling pathway in CARMN KO models and revealed that CARMN regulates this pathway through its downstream miRNA, miR-143-3p, which targets Hedgehog-interacting protein (HHIP), an antagonist of Hedgehog signaling. Delivery of HHIP-specific siRNA or miR-143-3p mimics rescued EC angiogenic defects and improved blood flow recovery in both CARMN KO and WT mice. These findings underscore the critical role of CARMN in modulating angiogenesis through the miR-143-3p-HHIP-Hedgehog signaling axis, providing insights into SMC-EC interactions and potential therapeutic strategies for CLTI.
Authors
Ming Zhai, Anurag Jamaiyar, Jun Qian, Winona W. Wu, Emre Bektik, Vinay Randhawa, Camila De Oliveira Vaz, Arvind K. Pandey, Akm Khyrul Wara, Madhur Sachan, Yi Hu, Jéssica L. Garcia, Claire E. Alford, Terence E. Ryan, Wenhui Peng, Mark W. Feinberg
Abstract
Direct interaction of RAS with the PI3K p110α subunit mediates RAS-driven tumor development: however, it is not clear how p110α/RAS-dependant signaling mediates interactions between tumors and host tissues. Here, using a murine tumor cell transfer model, we demonstrated that disruption of the interaction between RAS and p110α within host tissue reduced tumor growth and tumor-induced angiogenesis, leading to improved survival of tumor-bearing mice, even when this interaction was intact in the transferred tumor. Furthermore, functional interaction of RAS with p110α in host tissue was required for efficient establishment and growth of metastatic tumors. Inhibition of RAS and p110α interaction prevented proper VEGF-A and FGF-2 signaling, which are required for efficient angiogenesis. Additionally, disruption of the RAS and p110α interaction altered the nature of tumor-associated macrophages, inducing expression of markers typical for macrophage populations with reduced tumor-promoting capacity. Together, these results indicate that a functional RAS interaction with PI3K p110α in host tissue is required for the establishment of a growth-permissive environment for the tumor, particularly for tumor-induced angiogenesis. Targeting the interaction of RAS with PI3K has the potential to impair tumor formation by altering the tumor-host relationship, in addition to previously described tumor cell–autonomous effects.
Authors
Miguel Manuel Murillo, Santiago Zelenay, Emma Nye, Esther Castellano, Francois Lassailly, Gordon Stamp, Julian Downward
Abstract
Brain metastasis is a major cause of breast cancer (BC) mortality, but the cellular and molecular mechanisms have not been fully elucidated. BC cells must breach the blood-brain barrier in order to colonize the brain. Here, we determined that integrin β3 (ITGB3) expression mediated by hypoxia-inducible factor 1 (HIF-1) plays a critical role in metastasis of BC cells to the brain. Hypoxia stimulated BC cell migration and invasion ex vivo and brain colonization in vivo. Knockdown of either HIF-1α or ITGB3 expression impaired brain colonization by human or mouse BC cells injected into the cardiac left ventricle. Exposure of BC cells to hypoxia increased expression of ITGB3 and its incorporation into small extracellular vesicles (EVs). EVs harvested from the conditioned medium of hypoxic BC cells showed increased retention in the brain after intracardiac injection that was HIF-1α and ITGB3 dependent. EVs from hypoxic BC cells showed binding to brain endothelial cells (ECs), leading to increased EC–BC cell interaction, increased vascular endothelial growth factor receptor 2 signaling, increased EC permeability, and increased transendothelial migration of BC cells. Taken together, our studies implicate HIF-1–stimulated production of ITGB3+ EVs as a key mechanism by which hypoxia promotes BC brain metastasis.
Authors
Yongkang Yang, Chelsey Chen, Yajing Lyu, Olesia Gololobova, Xin Guo, Tina Yi-Ting Huang, Vijay Ramu, Varen Talwar, Elizabeth E. Wicks, Shaima Salman, Daiana Drehmer, Dominic Dordai, Qiaozhu Zuo, Kenneth W. Witwer, Kathleen L. Gabrielson, Gregg L. Semenza
Abstract
Lymphatic vessels maintain tissue fluid homeostasis and modulate inflammation, yet their spatial organisation and molecular identity in the healthy human kidney, and how these change during chronic transplant rejection, remain poorly defined. Here, we show that lymphatic capillaries initiate adjacent to cortical kidney tubules and lack smooth muscle coverage. These vessels exhibit an organ-specific molecular signature, enriched for CCL14, DNASE1L3, and MDK, with limited expression of canonical immune-trafficking markers found in other organ lymphatics, such as LYVE1 and CXCL8. In allografts with chronic mixed rejection, lymphatics become disorganised and infiltrate the medulla, with their endothelial junctions remodelling from a button-like to a continuous, zipper-like architecture. Lymphatics in rejecting kidneys localise around and interconnect tertiary lymphoid structures at different maturation stages, with altered intra- and peri-lymphatic CD4⁺ T cell distribution. The infiltrating T cells express IFNγ, which upregulates co-inhibitory ligands in lymphatic endothelial cells, including PVR and LGALS9. Simultaneously, lymphatics acquire HLA class II expression and exhibit C4d deposition, consistent with alloantibody binding and complement activation. Together, these findings define the spatial and molecular features of human kidney lymphatics, revealing tolerogenic reprogramming, accompanied by structural perturbations, during chronic transplant rejection.
Authors
Daniyal J. Jafree, Benjamin J Stewart, Karen L. Price, Maria Kolatsi-Joannou, Camille Laroche, Barian Mohidin, Benjamin Davis, Hannah Mitchell, Lauren G. Russell, Lucía Marinas del Rey, Chun Jing Wang, William J. Mason, Byung Il Lee, Lauren Heptinstall, Ayshwarya Subramanian, Gideon Pomeranz, Dale Moulding, Laura Wilson, Tahmina Wickenden, Saif N. Malik, Natalie Holroyd, Claire L. Walsh, Jennifer C. Chandler, Kevin X. Cao, Paul J.D. Winyard, Adrian S. Woolf, Marc Aurel Busche, Simon Walker-Samuel, Lucy S.K. Walker, Tessa Crompton, Peter J. Scambler, Reza Motallebzadeh, Menna R. Clatworthy, David A. Long
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)