Although murine models of coronary atherosclerotic disease have been used extensively to determine mechanisms, limited new therapeutic options have emerged. Pigs with familial hypercholesterolemia (FH pigs) develop complex coronary atheromas that are almost identical to human lesions. We reported previously that insulin-like growth factor 1 (IGF-1) reduced aortic atherosclerosis and promoted features of stable plaque in a murine model. We administered human recombinant IGF-1 or saline (control) in atherosclerotic FH pigs for 6 months. IGF-1 decreased relative coronary atheroma in vivo (intravascular ultrasound) and reduced lesion cross-sectional area (postmortem histology). IGF-1 increased plaque’s fibrous cap thickness, and reduced necrotic core, macrophage content, and cell apoptosis consistent with promotion of a stable plaque phenotype. IGF-1 reduced circulating triglycerides, markers of systemic oxidative stress and CXCL12 chemokine levels. We used spatial transcriptomics (ST) to identify global transcriptome changes in advanced plaque compartments and to obtain mechanistic insights into IGF-1 effects. ST analysis shows that IGF-1 suppressed FOS/FOSB factors and gene expression of MMP9 and CXCL14 in plaque macrophages, suggesting possible involvement of these molecules in IGF-1’s effect on atherosclerosis. Thus, IGF-1 reduced coronary plaque burden and promoted features of stable plaque in a pig model, providing support for consideration of clinical trials.
Sergiy Sukhanov, Yusuke Higashi, Tadashi Yoshida, Svitlana Danchuk, Mitzi Alfortish, Traci Goodchild, Amy Scarborough, Thomas E. Sharp III, James S. Jenkins, Daniel Garcia, Jan Ivey, Darla L. Tharp, Jeffrey D. Schumacher, Zach Rozenbaum, Jay K Kolls, Douglas K. Bowles, David Lefer, Patrice Delafontaine
Ciprofloxacin use may be associated with adverse aortic events. However, the mechanism underlying the effect of ciprofloxacin on the progression of thoracic aortic aneurysm (TAA) is not well understood. Using an in vitro microphysiological model, we treated human aortic smooth muscle cells (HASMCs) derived from patients with bicuspid aortic valve (BAV)- or tricuspid aortic valve (TAV)-associated TAAs with ciprofloxacin. TAA C57/BL6 mouse models were utilized to verify the effects of ciprofloxacin exposure. In the microphysiological model, real-time polymerase chain reaction, western blotting, and RNA sequencing showed that ciprofloxacin exposure was associated with a downregulated contractile phenotype, an upregulated inflammatory reaction, and extracellular matrix (ECM) degradation in the normal HASMCs derived from the non-diseased aorta. Ciprofloxacin induced mitochondrial dysfunction in the HASMCs and further increased apoptosis by activating the ERK1/2 and P38 mitogen-activated protein kinase pathways. These adverse effects appeared to be more severe in the HASMCs derived from BAV- and TAV-associated TAAs than in the normal HASMCs when the ciprofloxacin concentration exceeded 100 µg/mL. In the aortic walls of the TAA-induced mice, ECM degradation and apoptosis were aggravated after ciprofloxacin exposure. Therefore, ciprofloxacin should be used with caution in patients with BAV- or TAV-associated TAAs.
Bitao Xiang, Mieradilijiang Abudupataer, Gang Liu, Xiaonan Zhou, Dingqian Liu, Shichao Zhu, Yang Ming, Xiujie Yin, Shiqiang Yan, Yongxin Sun, Hao Lai, Chunsheng Wang, Jun Li, Kai Zhu
Vascular smooth muscle cells (vSMC) exert a critical role in sensing and maintaining vascular integrity. These cells abundantly express the low-density lipoprotein receptor-related protein 1 (LRP1), a large endocytic and signaling receptor that recognizes numerous ligands including ApoE-rich lipoproteins, proteases, and protease-inhibitor complexes. We observed the spontaneous formation of aneurysms in the superior mesenteric artery (SMA) of both male and female mice in which LRP1 was genetically deleted in v SMC (smLRP1-/- mice). Quantitative proteomics revealed elevated abundance of several proteins in smLRP1-/- mice that are known to be induced by angiotensin II (AngII)-mediated signaling, suggesting that this pathway is dysregulated. Administration of losartan, an AngII type I receptor antagonist, or an angiotensinogen antisense oligonucleotide to reduce plasma angiotensinogen concentrations restored the normal SMA phenotype in smLRP1-/- mice and prevented aneurysm formation. Additionally, employing a vascular injury model, we noted excessive vascular remodeling and neointima formation in smLRP1-/- mice that was restored by losartan administration. Together, these findings reveal that LRP1 regulates vascular integrity and remodeling of the SMA by attenuating excessive AngII-mediated signaling.
Jackie M. Zhang, Dianaly T. Au, Hisashi Sawada, Michael K. Franklin, Jessica J. Moorleghen, Deborah A. Howatt, Pengjun Wang, Brittany O. Aicher, Brian Hampton, Mary Migliorini, Fenge Ni, Adam E. Mullick, Mashhood M. Wani, Areck A. Ucuzian, Hong S. Lu, Selen C. Muratoglu, Alan Daugherty, Dudley K. Strickland
Recent studies have shown that cellular metabolism is tightly linked to the regulation of immune cells. Here, we show that activation of cholesterol metabolism, involving cholesterol uptake, synthesis, and autophagy/lipophagy, is integral to innate immune responses in macrophages. In particular, cholesterol accumulation within endosomes and lysosomes is a hallmark of the cellular cholesterol dynamics elicited by Toll-like receptor 4 activation and is required for amplification of myeloid differentiation primary response 88 (Myd88) signaling. Mechanistically, Myd88 binds cholesterol via its CLR recognition/interaction amino acid consensus domain, which promotes the protein’s self-oligomerization. Moreover, a novel supramolecular compound, polyrotaxane (PRX), inhibited Myd88‑dependent inflammatory macrophage activation by decreasing endolysosomal cholesterol via promotion of cholesterol trafficking and efflux. PRX activated liver X receptor, which led to upregulation of ATP binding cassette transporter A1, thereby promoting cholesterol efflux. PRX also inhibited atherogenesis in Ldlr–/– mice. In humans, cholesterol levels in circulating monocytes correlated positively with the severity of atherosclerosis. These findings demonstrate that dynamic changes in cholesterol metabolism are mechanistically linked to Myd88‑dependent inflammatory programs in macrophages and support the notion that cellular cholesterol metabolism is integral to innate activation of macrophages and is a potential therapeutic and diagnostic target for inflammatory diseases.
Sumio Hayakawa, Atsushi Tamura, Nikita Nikiforov, Hiroyuki Koike, Fujimi Kudo, Yinglan Cheng, Takuro Miyazaki, Marina Kubekina, Tatiana V. Kirichenko, Alexander N. Orekhov, Nobuhiko Yui, Ichiro Manabe, Yumiko Oishi
Vascular smooth muscle cells (SMCs) are heterogeneous, and their differential responses to vascular injury are not well understood. To address this question, we performed single-cell analysis of vascular cells to a ligation injury in mouse carotid arteries after 3 days. While endothelial cells had a homogeneous activation of mesenchymal genes, less than 30% of SMCs responded to the injury and generated 2 distinct clusters — i.e., proinflammatory SMCs and stress-responsive SMCs. Proinflammatory SMCs were enriched with high levels of inflammatory markers such as vascular cell adhesion molecule-1 while stress-responsive SMCs overexpressed heat shock proteins. Trajectory analysis suggested that proinflammatory SMCs were potentially derived from a specific subpopulation of SMCs. Ligand-receptor pair analysis showed that the interaction between macrophages and proinflammatory SMCs was the major cell-cell communication among all cell types in the injured arteries. In vitro coculture demonstrated that VCAM1+ SMCs had a stronger chemotactic effect on macrophage recruitment than VCAM1– SMCs. Consistently, the number of VCAM1+ SMCs significantly increased in injured arteries and atherosclerotic lesions of ApoE–/– mice and human arteries. These findings provide insights at the single-cell level on the distinct patterns of endothelial cells and SMC responses to vascular injury.
Xili Ding, Qin An, Weikang Zhao, Yang Song, Xiaokai Tang, Jing Wang, Chih-Chiang Chang, Gexin Zhao, Tzung Hsiai, Guoping Fan, Yubo Fan, Song Li
Endothelial mitochondria play a pivotal role in maintaining endothelial cell (EC) homeostasis through constantly altering their size, shape, and intracellular localization. Studies show that the disruption of the basal mitochondrial network in EC, forming excess fragmented mitochondria, implicates cardiovascular disease. However, cellular consequences underlying the morphological changes in the endothelial mitochondria under distinctively different, but physiologically occurring, flow patterns (i.e., unidirectional flow [UF] versus disturbed flow [DF]) are largely unknown. The purpose of this study was to investigate the effect of different flow patterns on mitochondrial morphology and its implications in EC phenotypes. We show that mitochondrial fragmentation is increased at DF-exposed vessel regions, where elongated mitochondria are predominant in the endothelium of UF-exposed regions. DF increased dynamin-related protein 1 (Drp1), mitochondrial reactive oxygen species (mtROS), hypoxia-inducible factor 1, glycolysis, and EC activation. Inhibition of Drp1 significantly attenuated these phenotypes. Carotid artery ligation and microfluidics experiments further validate that the significant induction of mitochondrial fragmentation was associated with EC activation in a Drp1-dependent manner. Contrarily, UF in vitro or voluntary exercise in vivo significantly decreased mitochondrial fragmentation and enhanced fatty acid uptake and OXPHOS. Our data suggest that flow patterns profoundly change mitochondrial fusion/fission events, and this change contributes to the determination of proinflammatory and metabolic states of ECs.
Soon-Gook Hong, Junchul Shin, Soo Young Choi, Jeffery C. Powers, Benjamin M. Meister, Jacqueline Sayoc, Jun Seok Son, Ryan Tierney, Fabio A. Recchia, Michael D. Brown, Xiaofeng Yang, Joon-Young Park
Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch‑dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch‑dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.
Michael O’Hare, Gema Esquiva, Mary K. McGahon, Jose Manuel Romero Hombrebueno, Josy Augustine, Paul Canning, Kevin S. Edgar, Peter Barabas, Thomas Friedel, Patrizia Cincolà, Jennifer Henry, Katie Mayne, Hannah Ferrin, Alan W. Stitt, Timothy J. Lyons, Derek P. Brazil, David J. Grieve, J. Graham McGeown, Tim M. Curtis
A central feature of progressive vascular remodeling is altered smooth muscle cell (SMC) homeostasis; however, the understanding of how different cell populations contribute to this process is limited. Here, we utilized single cell RNA sequencing to provide insight into cellular composition changes within isolated pulmonary arteries (PA) from pulmonary arterial hypertension (PAH) and donor lungs. Our results revealed that remodeling skewed the balanced communication network between immune and structural cells, in particular SMC. Comparative analysis with murine PA showed that human PA harbor heterogeneous SMC populations with an abundant intermediary cluster displaying a gradient transition between SMC and adventitial fibroblasts. Transcriptionally distinct SMC populations were enriched in specific biological processes and could be distinguished into four major clusters: oxygen sensing (enriched in pericytes), contractile, synthetic and fibroblast-like. End-stage remodeling was associated with phenotypic shift of pre-existing SMC populations and accumulation of synthetic SMC in neointima. Distinctly regulated genes in clusters built non-redundant regulatory hubs encompassing stress response and differentiation regulators. The current study provides a blueprint of cellular and molecular changes on a single cell level that are defining pathological vascular remodeling process.
Slaven Crnkovic, Francesco Valzano, Elisabeth Fließer, Juergen Gindlhuber, Helene Thekkekara Puthenparampil, Maria C. Basil, Michael P. Morley, Jeremy Katzen, Elisabeth Gschwandtner, Walter Klepetko, Edward Cantu, Heimo Wolinski, Horst Olschewski, Jorg Lindenmann, You-Yang Zhao, Edward E. Morrisey, Leigh M. Marsh, Grazyna Kwapiszewska
Lymphangiectasia, an anomalous dilation of lymphatic vessels first described in the 17th century, is frequently associated with chylous effusion, respiratory failure, and high mortality in young patients, yet the underlying molecular pathogenesis and effective treatments remain elusive. Here, we identify an unexpected causal link between MAPK activation and defective development of the lymphatic basement membrane that drives lymphangiectasia. Human pathological tissue samples from patients diagnosed with lymphangiectasia revealed sustained MAPK activation within lymphatic endothelial cells. Endothelial KRASG12D–mediated sustained MAPK activation in newborn mice caused severe pulmonary and intercostal lymphangiectasia, accumulation of chyle in the pleural space, and complete lethality. Pathological activation of MAPK in murine vasculature inhibited the Nfatc1-dependent genetic program required for laminin interactions, collagen crosslinking, and anchoring fibril formation, driving defective development of the lymphatic basement membrane. Treatment with ravoxertinib, a pharmacological inhibitor of MAPK, reverses nuclear-to-cytoplasmic localization of Nfatc1, basement membrane development defects, lymphangiectasia, and chyle accumulation, ultimately improving survival of endothelial KRAS mutant neonatal mice. These results reveal defective lymphatic basement membrane assembly and composition as major causes of thoracic lymphangiectasia and provide a potential treatment.
Harish P. Janardhan, Karen Dresser, Lloyd Hutchinson, Chinmay M. Trivedi
The (Pro)renin receptor ((P)RR), also known as ATP6AP2, is a single-transmembrane protein that is implicated in a multitude of biological processes. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell (EC)-specific Atp6ap2 knockout mouse model to investigate the role of ATP6AP2 during both physiological and pathological angiogenesis in vivo. We observed that postnatal deletion of Atp6ap2 in ECs results in cell migration defects, loss of tip cell polarity and subsequent impairment of retinal angiogenesis. In vitro, Atp6ap2 deficient ECs similarly displayed reduced cell migration, impaired sprouting, and defective cell polarity. Transcriptional profiling of ECs isolated from Atp6ap2 mutant mice further indicated regulatory roles in angiogenesis, cell migration and extracellular matrix composition. Mechanistically, we provided evidence that expression of various extracellular matrix components is controlled by ATP6AP2 via the extracellular-signal-regulated kinase (ERK) pathway. Furthermore, Atp6ap2 deficient retinas exhibited reduced revascularization in an oxygen induced retinopathy model. Collectively, our results demonstrated a critical role of ATP6AP2 as a regulator of developmental and pathological angiogenesis.
Nehal R. Patel, Rajan K C, Avery E. Blanks, Yisu Li, Minolfa C. Prieto, Stryder M. Meadows
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