Vascular biologies
Abstract
Abdominal aortic aneurysm (AAA) is usually asymptomatic until life-threatening complications occur, predominantly involving aortic rupture. Currently, no drug-based treatments are available, primarily due to limited understanding of AAA pathogenesis. Transcriptional regulator PR domain-containing protein 16 (PRDM16) is highly expressed in the aorta, but its functions in the aorta are largely unknown. By RNA-seq analysis, we found that VSMCs-specific Prdm16 knockout mice (Prdm16SMKO) already showed extensive changes in the expression of genes associated with extracellular matrix (ECM) remodeling and inflammation in the abdominal aorta under normal housing conditions without any pathological stimuli. Human AAA lesions displayed lower PRDM16 expression. Periadventitial elastase application to the suprarenal region of the abdominal aorta aggravated AAA formation in Prdm16SMKO. During AAA development, VSMCs undergo apoptosis because of both intrinsic and environmental changes including inflammation and ECM remodeling. Prdm16 deficiency promoted inflammation and apoptosis in VSMCs. A disintegrin and metalloproteinase 12 (ADAM12) is a gelatinase which can degrade various ECM. We found that ADAM12 is a target of transcriptional repression by PRDM16. Adam12 knockdown reversed VSMC apoptosis induced by Prdm16 deficiency. Our study demonstrated that PRDM16 deficiency in VSMCs promoted ADAM12 expression and aggravates AAA formation, which may provide potential targets for AAA treatment.
Authors
Zhenguo Wang, Xiangjie Zhao, Guizhen Zhao, Yanhong Guo, Haocheng Lu, Wenjuan Mu, Juan Zhong, Minerva Garcia-Barrio, Jifeng Zhang, Y. Eugene Chen, Lin Chang
Abstract
To improve our limited understanding of the pathogenesis of thoracic aortic aneurysm (TAA) leading to acute aortic dissection, single-cell RNA sequencing (scRNA-seq) was employed to profile disease-relevant transcriptomic changes of aortic cell populations in a well-characterized mouse model of the most commonly diagnosed form of Marfan syndrome (MFS). As result, two discrete sub-populations of aortic cells (SMC3 and EC4) were identified only in the aorta of Fbn1mgR/mgR mice. SMC3 highly express genes related to extracellular matrix formation and nitric oxide signaling, whereas EC4 transcriptional profile is enriched in SMC, fibroblast, and immune cell-related genes. Trajectory analysis predicted close phenotypic modulation between SMC3 and EC4, which were therefore analyzed together as a discrete MFS-modulated (MFSmod) sub-population. In situ hybridizations of diagnostic transcripts located MFSmod cells to the intima of Fbn1mgR/mgR aortas. Reference-based dataset integration revealed transcriptomic similarity between MFSmod and an SMC-derived cell cluster modulated in human TAA. Consistent with angiotensin II type I receptor (At1r) contribution to TAA development, MFSmod cells were absent in the aorta of Fbn1mgR/mgR mice treated with the At1r antagonist losartan. Altogether, our findings indicate that a discrete dynamic alteration of aortic cell identity is associated with dissecting TAA in MFS mice and increased risk of aortic dissection in MFS patients.
Authors
Yifei Sun, Keiichi Asano, Lauriane Sedes, Anna Cantalupo, Jens Hansen, Ravi Iyengar, Martin J. Walsh, Francesco Ramirez
Abstract
Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1, an inwardly rectifying potassium channel that regulates membrane hyperpolarization, contributes to vasodilation in resistance arteries. First, we show phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data has implied PS may compete with PIP2 binding on Kir2.1. We found 83.33% of Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate PS blocks PIP2 activation of Kir2.1, and addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggests PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and demonstrates the intracellular lipid localization within endothelium is an important determinant of vascular function.
Authors
Claire A. Ruddiman, Richard G. Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson
Abstract
Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler, Smarca4/Brg1, facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein was upregulated in AdvSca1-SM cells after acute vascular injury and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-β1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-β1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-β1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data shed insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide important anti-fibrotic clinical benefit.
Authors
Austin J. Jolly, Sizhao Lu, Allison M. Dubner, Keith A. Strand, Marie F. Mutryn, Aaron Pilotti-Riley, Etienne P. Danis, Raphael A. Nemenoff, Karen S. Moulton, Mark W. Majesky, Mary C.M. Weiser-Evans
Abstract
Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma–/–) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.
Authors
Meaghan J. Griffiths, Sarah A. Marshall, Fiona L. Cousins, Lauren R. Alesi, Jordan Higgins, Saranya Giridharan, Urooza C. Sarma, Ellen Menkhorst, Wei Zhou, Alison S. Care, Jacqueline F. Donoghue, Sarah J. Holdsworth-Carson, Peter A.W. Rogers, Evdokia Dimitriadis, Caroline E. Gargett, Sarah A. Robertson, Amy L. Winship, Karla J. Hutt
A miRNA-CXCR4 signaling axis impairs monopoiesis and angiogenesis in diabetic critical limb ischemia
Abstract
Patients with peripheral artery disease (PAD) and diabetes have the highest risk of critical limb ischemia (CLI) and amputation, yet the underlying mechanisms remain incompletely understood. MicroRNA (miRNA)-sequencing of plasma from diabetic patients with or without CLI was compared to diabetic mice with acute or subacute limb ischemia to identify conserved miRNAs. miRNA knockout mice on high fat diet were generated to explore impact on CLI. Comparison of dysregulated miRNAs from diabetic human subjects with PAD and diabetic mice with limb ischemia revealed conserved miR-181 family members. High fat-fed, diabetic Mir181a2b2 knockout (KO) mice had impaired revascularization in limbs due to abrogation of circulating Ly6Chi monocytes with reduced accumulation in ischemic skeletal muscles. M2-like KO macrophages under diabetic conditions failed to produce pro-angiogenic cytokines. Single cell transcriptomics of the bone marrow niche revealed that the reduced monocytosis in diabetic KO mice is a result of impaired hematopoiesis with increased CXCR4 signaling in bone marrow Lineage-Sca1+Kit+ (LSK) cells. Exogenous Ly6Chi monocytes from non-diabetic KO mice rescued the impaired revascularization in ischemic limbs of diabetic KO mice. Increased Cxcr4 expression is mediated by the novel miR-181 target, Plac8. Taken together, MiR-181a/b is a putative mediator of diabetic CLI and contributes to alterations in hematopoiesis, monocytosis, and macrophage polarization.
Authors
Henry S. Cheng, Rulin Zhuang, Daniel Pérez-Cremades, Jingshu Chen, Anurag Jamaiyar, Winona Wu, Grasiele Sausen, Aspasia Tzani, Jorge Plutzky, Jorge Henao-Mejia, Philip P. Goodney, Mark A. Creager, Marc S. Sabatine, Marc P. Bonaca, Mark W. Feinberg
Abstract
Based upon our demonstration that the smooth muscle (SMC)-selective putative methyltransferase, Prdm6, interacted with myocardin-related transcription factor A, we examined Prdm6’s role in SMCs in vivo using cell-type specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well-tolerated, Prdm6 depletion in Wnt1 expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2Prdm6flox/floxROSA26LacZ mice revealed normal neural crest-derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile-associated proteins that was down-regulated by Prdm6 depletion. Chromatin immunoprecipitation (ChIP)-seq experiments in outflow tract SMCs demonstrated that 50% of the genes altered by Prdm6 depletion contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified a SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.
Authors
Meng Zou, Kevin D. Mangum, Justin C. Magin, Heidi H. Cao, Michael T. Yarboro, Elaine L. Shelton, Joan M. Taylor, Jeff Reese, Terrence S. Furey, Christopher P. Mack
Abstract
The main estrogen, estradiol (E2), exerts several beneficial vascular actions through estrogen receptor (ER)α in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we reported the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, that preserves smooth muscle cells (SMCs), E2, E3 and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4 action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its action independently of ERα membrane initiated signaling by contrast to E2. Consistently, RNAseq analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differ from those of E2. Thus, whereas acceleration of endothelial healing by estrogens was viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.
Authors
Morgane Davezac, Rana Zahreddine, Melissa Buscato, Natalia F. Smirnova, Chanaelle Febrissy, Henrik Laurell, Silveric Gilardi-Bresson, Marine Adlanmerini, Philippe Liere, Gilles Flouriot, Rachida Guennoun, Muriel Laffargue, Jean-Michel Foidart, Françoise Lenfant, Jean-François Arnal, Raphaël Métivier, Coralie Fontaine
Abstract
Vascular calcification (VC) is concomitant with atherosclerosis, yet it remains uncertain why rupture-prone high-risk plaques do not typically show extensive calcification. Intraplaque hemorrhage (IPH) deposits erythrocyte-derived cholesterol enlarging the necrotic core and promoting the high-risk plaque development. Pro-atherogenic CD163+ alternative macrophages engulf hemoglobin-haptoglobin (HH) complexes at IPH sites. However, their role in VC has never been examined. Here we show, in human arteries, the distribution of CD163+ macrophages correlates inversely with VC. In vitro experiments using vascular smooth muscle cells (VSMC) cultured with HH-exposed human macrophages supernatant (M(Hb)) reduced calcification, while arteries from ApoE-/-CD163-/- mice showed greater VC. M(Hb) supernatant-exposed VSMC showed activated NFκB, while blocking NFκB attenuated the anti-calcific effect of M(Hb) on VSMCs. CD163+ macrophages altered VC through NFκB-induced transcription of hyaluronan synthase (HAS), an enzyme which catalyzes the formation of the extracellular matrix glycosaminoglycan, hyaluronan, within VSMCs. M(Hb) supernatants enhanced HAS production in VSMC, while knocking-down HAS attenuated its anti-calcific effect. NFκB blockade in ApoE-/- mice reduced hyaluronan and increased VC. In human arteries, hyaluronan/HAS were increased in areas of CD163+ macrophage presence. Our findings highlight an important mechanism by which CD163+ macrophages inhibit VC through NFκB-induced HAS augmentation and thus promote the high-risk plaques development.
Authors
Atsushi Sakamoto, Rika Kawakami, Masayuki Mori, Liang Guo, Ka Hyun Paek, Jose Verdezoto Mosquera, Anne Cornelissen, Saikat Kumar B. Ghosh, Kenji Kawai, Takao Konishi, Raquel Fernandez, Daniela T. Fuller, Weili Xu, Aimee E. Vozenilek, Yu Sato, Hiroyuki Jinnouchi, Sho Torii, Adam W. Turner, Hirokuni Akahori, Salome Kuntz, Craig C. Weinkauf, Parker J. Lee, Robert Kutys, Kathryn Harris, Alfred Lawrence Killey, Christina M. Mayhew, Matthew Ellis, Leah M. Weinstein, Neel V. Gadhoke, Roma Dhingra, Jeremy Ullman, Endale Armelle Dikongue Emene, Maria E. Romero, Frank D. Kolodgie, Clint L. Miller, Renu Virmani, Aloke V. Finn
Abstract
Most circulating endothelial cells are apoptotic, but rare circulating endothelial colony forming cells (C-ECFCs, also known as blood outgrowth endothelial cells (BOECs)) with proliferative and vasculogenic activity can be cultured; the origin and naïve function of these C-ECFCs remains obscure. Herein, detailed lineage tracing reveals murine C-ECFCs emerge in the early postnatal period, display high vasculogenic potential, with enriched frequency of clonal proliferative cells compared to tissue-resident ECFCs, and are not committed to or derived from the bone marrow hematopoietic system but from tissue-resident ECFCs. In human subjects, C-ECFCs are present in the CD34bright cord blood mononuclear subset, possess proliferative potential and in vivo vasculogenic function in a naïve or cultured state, and display a single cell transcriptome sharing some umbilical venous endothelial cell features like, higher Protein C Receptor and extracellular matrix gene expression. This study provides an advance for the field by identifying the origin, naïve function, and antigens to prospectively isolate C-ECFCs for translational studies.
Authors
Yang Lin, Kimihiko Banno, Chang-Hyun Gil, Jered Myslinski, Takashi Hato, W. Christopher Shelley, Hongyu Gao, Xiaoling Xuei, Yunlong Liu, David Basile, Momoko Yoshimoto, Nutan Prasain, Stefan P Tarnawsky, Ralf H. Adams, Katsuhiko Naruse, Junko Yoshida, Michael P. Murphy, Kyoji Horie, Mervin C. Yoder
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