Vascular biology
Abstract
The induced membrane technique (IMT) is a 2-stage surgical intervention for critical-sized bone defects (CSBD), yet the metabolic mechanisms driving neovascularization within the induced membrane remain unclear. Here, we combined a rat IMT model, metabolomic profiling, and endothelial assays to delineate the role of purine metabolism in neovascularization of type H vessels. Using a rat IMT model and metabolomic profiling, we identified purine metabolism as the most substantial pathway during the formation of induced membranes, with consistent trends of adenosine, inosine, hypoxanthine, and xanthosine found in both serum and induced membranes. Histological analysis revealed abundant CD31hiEMCNhi type H vessels, critical for osteogenesis, within the induced membrane. Inhibition of purine metabolism suppressed the CD31hiEMCNhi type H phenotype in human umbilical vein endothelial cells, whereas treatment with inosine, hypoxanthine, or xanthosine promoted endothelial activation and the type H phenotype. Notably, inosine and hypoxanthine displayed parallel changes across consistent systemic (serum) and local alterations (induced membranes), highlighting their potential as serum indicators of induced membrane formation. Collectively, these findings uncover a previously unrecognized metabolic mechanism driving neovascularization of type H vessels in induced membranes and suggest purine metabolites as promising indicators and therapeutic targets for improving IMT outcomes as well as CBSD treatment.
Authors
Yung-Heng Hsu, Guan-Lin Lee, Yu-Chih Lin, Mei-Feng Chen, Yuhan Chang, Ying-Yu Wu, Chih-Chien Hu
Abstract
A single-nucleotide missense polymorphism (rs1800449, R158Q) in the propeptide domain of lysyl oxidase (LOX-PP) is associated with increased risk of coronary artery disease (CAD) independent of changes in plasma lipid levels. Although the enzymatic function of LOX has an essential role for the cross-linking of extracellular matrix proteins in connective tissues, whether and how LOX-PP R158Q contributes to the development of atherosclerosis has not been clearly established. Here, hypercholesterolemia was induced in mice that were WT or homozygous for the LOX-PP R158Q polymorphism by adeno-associated virus-8–mediated overexpression of Pcsk9 followed by high-fat diet feeding for 16 weeks. We found that the R158Q polymorphism promoted atherosclerosis and induced proliferation of macrophages and vascular smooth muscle cells without altering LOX enzymatic activity. Using single-cell RNA sequencing, we found the transcriptional program of atherosclerotic plaques from mice harboring R158Q was strongly enriched for proliferation- and calcification-related genes in a regionally distinct manner. Together, these results establish an enzymatically independent proatherogenic role for the LOX-PP and suggest its potential as a novel therapeutic target.
Authors
In-Hyuk Jung, Junedh M. Amrute, Sofia E. Luna, Ryan E. Wagoner, Arturo Alisio, Paul C. Lee, Kendall H. Burks, Joohee Oh, Hannah C. Plunkett Paletta, Chul Joo Kang, Nathan O. Stitziel
Abstract
Renin cells are essential for survival and serve as key regulators of blood pressure and fluid-electrolyte homeostasis. Their function and identity are dependent on signals from their local microenvironment afforded by neighboring cells and nerves. Whether and how renin cells contribute to the development and maintenance of this microenvironment remains unclear. Because renin cells are rare -0.01 % of kidney cells- conventional histological approaches cannot capture their interaction with nerve fibers and surrounding cells within the nephron and its vasculature. Using high-resolution 3D imaging, cell-specific multicolor reporter mice, single-cell RNA-Seq, and conditional gene deletions, we mapped how renin cells assemble within arterioles and communicate with axon fibers to organize the growth and orientation of the kidney arterioles during development and disease. This co-inductive process is mediated by Ngf produced by renin cell precursors and is necessary for renin cell survival and innervation. Interestingly, renin enzymatic insufficiency elevates Ngf and drives arteriolar hypertrophy with aberrant axon sprouting and hyperinnervation. These findings indicate that renin cells regulate kidney neurovascular development revealing them as active organizers of their local neuroregulatory microenvironment in health and disease.
Authors
Manako Yamaguchi, Georgina Gyarmati, Liam McLaughlin, Hiroki Yamaguchi, Jason P. Smith, Lucas Ferreira de Almeida, Daisuke Matsuoka, Alexandre G. Martini, Sara M. Wilmsen, Sijie Hao, Kazuki Tainaka, Silvia Medrano, Sanjay Jain, Janos Peti-Peterdi, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez
Abstract
Authors
Lun Li, Rhonda Lightle, Bader Ali, Georgeio Sader, Robert Shenkar, Sean P. Polster, Douglas A. Marchuk, Jan-Karl Burkhardt, Issam A. Awad, Mark L. Kahn
Abstract
Pulmonary Arterial Hypertension (PAH) is a rare vascular disorder characterized by elevated pressure in pulmonary arteries, eventually leading to right ventricular failure. Approximately 50% of pediatric disease and 20% of adult disease can be linked to a genetic mutation, with nearly 70% of these cases involving mutations in the bone morphogenetic protein receptor type 2 (BMPR2) locus. Investigations using rodent models have made substantial advances in our understanding of BMPR2 signaling; however, limited data exist regarding the onset and course of PAH, and etiologies for phenotypic expression in these patients remain unknown. In this work, we describe the development of an ovine model of heritable PAH. Because homozygous disruption of BMPR2 is embryonic lethal, we developed heterozygous BMPR2 sheep by using a PAM-disrupting synonymous single stranded oligodeoxyribonucleotide alongside a single guide RNA and Cas9 mediated gene editing strategy. The resulting BMPR2(+/-) lambs demonstrated cardiac and pulmonary vascular pathology that are consistent with BMPR2 mutation-driven PAH observed in humans. Given the genetic and physiological similarities of BMPR2(+/-) sheep to humans with heritable PAH, this large animal model will serve as a vital platform for mechanistic molecular studies and will provide a much-needed pre-clinical model for extensive treatment evaluations.
Authors
Sanjeev A. Datar, Nicholas Werry, Austin R. Brown, Devon S. Fitzpatrick, Oluwafemi Falade, Josephine F. Trott, Rachel Hutchings, Elena K. Amin, Jessica M. Morgan, Hythem Nawaytou, Gail H. Deutsch, Eric G. Johnson, Omar A. Gonzales Viera, Thomas F. Bishop, Tara Urbano Beach, Bret R. McNabb, Eric D. Austin, Jeffrey R. Fineman, Alison L. Van Eenennaam
Abstract
Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) leading to renal fibrosis. Here, we investigate the kinetics of autophagy, apoptosis, and necroptosis activation in tubular epithelial cells (TECs) and peritubular capillaries (PTCs) after renal IRI, and their relative contributions to renal fibrogenesis. IRI with renal artery clamping in GFP-LC3 transgenic mice induced a predominant and sustained necroptotic response in TECs, while apoptosis and autophagy played minor roles. PTCs showed early and persistent activation of apoptosis, brief necroptosis induction, and increased autophagy at a distance from IRI. Disruption of the autophagic process with chloroquine (CHQ) injections in association with renal IRI did not modulate tubular death but enhanced PTC apoptosis and increased microvascular rarefaction and fibrosis. Apoptosis-deficient GFP-LC3/Caspase-3–/– mice exposed to renal IRI showed enhanced PTC autophagy, reduced PTC rarefaction, and inhibition of renal fibrosis, in spite of increased necroptosis in TECs. Inhibition of both autophagy with CHQ and apoptosis in GFP-LC3/Caspase-3–/– mice led to a marked switch toward necroptosis in PTCs. This was associated with aggravated microvascular rarefaction, increased leukocyte infiltration, and enhanced renal fibrosis. These findings establish a predominant role for PTC autophagy and caspase-3–dependent apoptosis in the development of renal fibrosis after IRI.
Authors
Hyunyun Kim, Francis Migneault, Shanshan Lan, Imane Kaci, Julie Turgeon, Annie Karakeussian Rimbaud, Martin Dupont, Shijie Qi, Mélanie Dieudé, Marie-Josée Hébert
Abstract
Pulmonary arterial hypertension (PAH) is a progressive vascular syndrome characterized by aberrant signaling, severe pulmonary artery remodeling, and right ventricular (RV) failure, a major driver of morbidity and mortality. Dysregulation of the apelinergic pathway has been implicated in pulmonary vascular remodeling in PAH. Using a sugen-hypoxia rat model of PAH, we assessed the ability of a novel apelin analog, resistant to native peptidase degradation, to reverse the pathological hallmarks of PAH and RV dysfunction. Apelin analog therapy corrected the vascular lesions in the lungs and nearly normalized pulmonary arterial pressures. Early cardiorenal syndrome, RV dilation and dysfunction as well as RV cardiomyocyte and fibroblast activation induced by pressure overload, were also reversed by apelin analog treatment. Single-nucleus RNA sequencing of the lungs and RV revealed apelin-analog treatment activated several protective pathways, including rebalancing protective BMPR2 (bone morphogenetic protein receptor type 2) signaling to counteract excessive pathogenic TGFBR2 (transforming growth factor β receptor 2) activity in PAH. These findings highlight the therapeutic potential of exogenous apelin in reversing pulmonary vascular and cardiac pathologies in PAH and support further investigation to evaluate the clinical benefits of apelin analog treatment in patients with PAH and RV failure.
Authors
Jennie Vu, Pavel Zhabyeyev, Kemar J. Brown, Joshua M. Gorham, Daniel M. DeLaughter, Huachen Chen, Thilina U. Jayawardena, Ander Vergara, Maria Alexiou, Anjalee Wijewardane, Conrad Fischer, Charlotte Avet, Abby Ewasiuk, Faqi Wang, Mark C. Chappell, Yuri Kim, Michel Bouvier, John C. Vederas, Christine E. Seidman, Jonathan G. Seidman, Gavin Y. Oudit
Abstract
Vascular tortuosity (VT) is a critical biomarker of disease progression and decision to treat ischemic retinal disorders, particularly retinopathy of prematurity (ROP). The murine oxygen-induced retinopathy model is the most widely-used model of ischemic retinopathy. Although VT has been described in OIR, its temporal dynamics have not been systematically defined. In this study, a semi-automated artificial intelligence (AI)-based pipeline was used to quantify VT throughout OIR. Retinal flat mounts from age-matched normoxic and OIR mice (postnatal days [P]10-P56) underwent vessel segmentation using a generative adversarial network (GAN), and VT was quantified as a cumulative tortuosity index (CTI) with the iROP-Assist algorithm. Concurrently, standard OIR outcomes of neovascularization (NV) and vaso-obliteration (VO) were quantified using OIRseg.org. NV peaked at P17 and resolved by P23, while VO regressed over a similar interval. VT peaked with NV at P17 but remained elevated through P56. These temporal changes mirror both the development of VT and its persistence after NV regression observed clinically in ROP. Collectively, these findings establish VT as a durable, quantifiable phenotype in OIR and expand the model’s utility beyond neovascular endpoints, providing a translational platform for investigating VT pathogenesis and evaluating the effects of therapeutic agents on vascular tortuosity.
Authors
Kyle V. Marra, Tomoya Murakami, Jimmy S. Chen, Edith Aguilar, Jacob I. Robinson, Maxwell Prenner, Richard Daneman, Martin Friedlander, Eric Nudleman
Abstract
Spreading depolarizations (SDs) are propagating waves of near-complete breakdown of transmembrane ion gradients that occur during acute ischemic stroke and worsen outcome by driving calcium overload and glutamate release in neurons and astrocytes. The plasmalemmal sodium-calcium exchanger (NCX) plays a key role in such changes, in that the complex ionic disequilibrium during ischemia induces reverse-mode activity of NCX, leading to cellular calcium overload in exchange for sodium. However, the cell type-specific roles of NCX in neurons and astrocytes during SDs remain unclear. Here, we used ion and glutamate reporters in an in vivo stroke model in mice carrying inducible, cell-specific deletions of NCX isoform-1. Neuronal NCX1 deletion reduced neuronal and astrocytic calcium transients, increased neuronal sodium transients, decreased extracellular glutamate levels, and raised SD initiation threshold. In contrast, astrocytic NCX1 deletion increased sodium transients in both neurons and astrocytes, and increased neuronal calcium as well as extracellular glutamate levels. A computational model of ischemia confirmed that these effects are consistent with reverse-mode NCX1 activity. Together, these findings indicate opposing roles of reverse-mode NCX1 during ischemia. Neuronal NCX1 promotes SD susceptibility, calcium overload and glutamate release, whereas astrocytic NCX1 exerts protective effects by attenuating glutamate elevation and neuronal calcium accumulation.
Authors
Somayyeh Hamzei Taj, Pawan Kumar Thapaliya, Cordula Rakers, Niklas J. Gerkau, Christine R. Rose, Ghanim Ullah, Gabor C. Petzold
Abstract
Idiopathic Pulmonary Fibrosis (IPF) is a fatal, aging-related disease characterized by persistent lung fibroblast activation, progressive lung scarring and several vascular abnormalities. We have previously demonstrated that aging-associated vascular dysfunction drives maladaptive endothelial responses to injury and exacerbates lung fibrosis via secretion of pro-fibrotic endothelial-derived factors. However, regulatory mechanisms governing endothelial dysfunction during progressive lung fibrosis remain poorly understood. Here, using preclinical mouse models of progressive lung fibrosis as well as human IPF lungs, we demonstrate that miR-205-5p is overexpressed in lung ECs from fibrotic lungs, and coordinates gene expression programs implicated in endothelial dysfunction and progressive fibrosis. Mechanistically, miR-205-5p induces senescence in lung ECs, mirroring the senescent phenotype of IPF lung ECs. Consistently, conditioned medium derived from lung ECs overexpressing miR-205-5p promotes lung fibroblast activation. Importantly, miR-205-5p inhibition in IPF lung ECs attenuates endothelial senescence and limits paracrine fibroblast activation. Finally, inhibition of miR-205-5p in vivo preserves the pulmonary vascular network and attenuates lung fibrosis progression in aged mice challenged with bleomycin. Collectively, our findings support a novel connection between lung endothelial miR-205-5p, endothelial senescence and pro-fibrotic alteration of the endothelial secretome, and highlight miR-205-5p inhibition as a potential therapeutic intervention for pulmonary fibrosis.
Authors
Giuseppe Muscato, Benjamin B. Roos, Sharonda Harris, Xiaoyu Tracy Cai, Gina Civettini, Enrico Sciacca, Ahmed Raslan, Alessandra Castaldi, Sharon Elliot, Marilyn K. Glassberg, Carlo Vancheri, Daniel J. Tschumperlin, Giovanni Ligresti, Nunzia Caporarello
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