Vascular biology
Abstract
Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease with no effective pharmacological interventions. While single-cell transcriptomics has advanced our understanding of AAA, it lacks spatial context. Here, we employed Seq-Scope, an ultra-high-resolution spatial transcriptomic technology, to decipher the spatial landscape of angiotensin II–induced AAA in Apoe–/– mice. Our analysis revealed the heterogeneity of macrophages, fibroblasts, and smooth muscle cells (SMCs), with specific responses in different layers of the AAA tissue. SMCs in the inner layers showed associations with Mgp-expressing fibroblasts and GPNMB-expressing macrophages, whereas the outer layers had different dominant cell types. Notably, GPNMB-expressing macrophages were concentrated near SMCs in regions of severe elastic lamina damage. Immunofluorescent staining confirmed their colocalization, and scRNA-seq reanalysis independently validated the presence of GPNMB-high macrophages in AAA tissues, highlighting their involvement in inflammation and tissue remodeling. Moreover, we discovered that macrophage-derived soluble GPNMB induces SMC phenotypic switching, reducing contractile markers while increasing cytokines and metalloproteinases. This effect was partly mediated by CD44 signaling. These findings suggest that GPNMB-high macrophages contribute to AAA development by driving SMC dysfunction. This study highlights the importance of high-resolution spatial transcriptomics in complementing single-cell transcriptomics, offering valuable insights into molecular and cellular responses in the AAA microenvironment.
Authors
Guizhen Zhao, Chun-Seok Cho, Hongyu Liu, Yongha Hwang, Yichen Si, Myungjin Kim, Yongjie Deng, Yang Zhao, Chao Xue, Yanhong Guo, Lin Chang, Dogukan Mizrak, Bo Yang, Hyun Min Kang, Jifeng Zhang, Jun Hee Lee, Y. Eugene Chen
Abstract
Pulmonary veno-occlusive disease (PVOD) is a rare and severe subtype of pulmonary arterial hypertension, characterized by progressive remodeling of small pulmonary arteries and veins with no therapies. Using a mitomycin C (MMC)-induced rat model, we previously demonstrated that protein kinase R (PKR)-mediated integrated stress response (ISR) drives endothelial dysfunction and vascular remodeling. To determine if PKR is the primary mediator of ISR and the pathogenesis, we treated control (Ctrl) and PKR knockout (KO) mice with the same dose of MMC. Consistent with rat data, Ctrl mice displayed ISR activation, vascular remodeling, and pulmonary hypertension after MMC treatment, while KO mice showed none of these phenotypes. Proteomic analysis revealed that MMC-mediated ISR activation attenuates protein synthesis in Ctrl but not in KO mice. These findings underscore the critical role of PKR-dependent ISR activation and subsequent perturbation of proteostasis as central mechanisms driving PVOD pathogenesis and identifying PKR as a promising therapeutic target.
Authors
Amit Prabhakar, Rahul Kumar, Meetu Wadhwa, Abhilash Barpanda, Joseph Lyons, Asavari S. Gowda, Simren P. Gupta, Ananyaa Arvind, Prajakta Ghatpande, Arun P. Wiita, Brian B. Graham, Giorgio Lagna, Akiko Hata
Abstract
Bronchopulmonary dysplasia (BPD), a prevalent and chronic lung disease affecting premature newborns, results in vascular rarefaction and alveolar simplification. Although the vasculature has been recognized as a main player in this disease, the recently found capillary heterogeneity and cellular dynamics of endothelial subpopulations in BPD remain unclear. Here, we show Cap2 cells are damaged during neonatal hyperoxic injury, leading to their replacement by Cap1 cells which, in turn, significantly decline. Single cell RNA-seq identifies the activation of numerous p53 target genes in endothelial cells (ECs), including Cdkn1a (p21). While global deletion of p53 results in worsened vasculature, endothelial-specific deletion of p53 reverses the vascular phenotype and improves alveolar simplification during hyperoxia. This recovery is associated with the emergence of a transitional EC state, enriched for oxidative stress response genes and growth factors. Notably, this transitional EC gene signature is conserved in an aberrant capillary population identified in human BPD with pulmonary hypertension, underscoring the biological and clinical relevance of our findings. These results reveal a key role for p53 in maintaining endothelial lineage fidelity during pulmonary capillary repair following hyperoxic injury and highlight the critical contribution of the endothelium to BPD pathogenesis.
Authors
Lisandra Vila Ellis, Jonathan D. Bywaters, Amanda Ceas, Yun Liu, Jennifer M.S. Sucre, Jichao Chen
Abstract
Malignancies increase the risk for thrombosis and metastasis dependent on complex interactions of innate immune cells, platelets, and the coagulation system. Immunosuppressive functions of platelets and macrophage-derived coagulation factors in the tumor microenvironment (TME) drive tumor growth. Here we show that patients with malignancies and tumor-bearing mice have increased levels of coagulation factor (F) X expressing circulating monocytes engaged in platelet aggregate formation. This interaction and resulting thrombin generation on platelets interferes with monocyte differentiation and antigen uptake of antigen-presenting cells (APCs). Myeloid cell-specific deletion of FX or abrogated FXa signaling via protease activated receptor 2 (PAR2) averts the suppressive activity of platelets on tumor cell debris uptake and promotes the immune stimulatory activity of APCs in the TME. Myeloid cell FXa-PAR2 signaling deficiency specifically enhances activation of the cGAS-STING-IFN-I pathway with a resulting expansion of antigen experienced progenitor exhausted CD8+ T cells. Pharmacological blockade of FXa with direct oral anticoagulants expands T cell priming-competent immune cells in the TME and synergizes with the reactivation of exhausted CD8+ T cells by immune checkpoint inhibitors for improved anti-tumor responses. These data provide mechanistic insights into the emerging clinical evidence demonstrating the translational potential of FXa inhibition to synergize with immunotherapy.
Authors
Petra Wilgenbus, Jennifer Pott, Sven Pagel, Claudius Witzler, Jennifer Royce, Federico Marini, Sabine Reyda, Thati Madhusudhan, Thomas Kindler, Anne Hausen, Matthias M. Gaida, Hartmut Weiler, Wolfram Ruf, Claudine Graf
Abstract
Mutations in Cullin-3 (CUL3) cause hypertension (HTN). We examined the role of smooth muscle cell (SMC) CUL3 in the regulation of renin gene expression. Mice with SMC-specific CUL3 deletion (S-CUL3KO) developed severe HTN with paradoxically preserved levels of plasma angiotensin peptides and renal renin expression. Cre-recombinase was active in JG cells resulting in decreased CUL3 expression. We evaluated components of the renin cell baroreceptor and revealed preserved lamin A/C but decreased integrin β1 expression in S-CUL3KO. We hypothesized that Rab proteins are involved in integrin β1 downregulation. Silencing either Rab21 or Rab5 in CUL3-deficient HEK293 cells increased integrin β1 protein. Co-immunoprecipitation revealed a direct interaction between Rab5 and CUL3. CUL3-deficiency increased Rab5 suggesting it is regulated by a CUL3-mediated mechanism and that CUL3-deficiency results in loss of Rab protein turnover leading to enhanced integrin β1 internalization. We conclude that the loss of integrin β1 from juxtaglomerular cells impairs the mechanosensory function of the renin cell baroreceptor, which underlies the persistent renin expression observed in hypertensive S-CUL3KO mice. These findings provide insights into the molecular mechanisms of HTN, revealing that dysregulation of Rab proteins and integrin β1 in the kidney due to CUL3-deficiency contributes to the development of HTN.
Authors
Daria Golosova, Gaurav Kumar, Ko-Ting Lu, Patricia C. Muskus Veitia, Ana Hantke Guixa, Kelsey K. Wackman, Eva M. Fekete, Daniel T. Brozoski, Justin L. Grobe, Maria Luisa S. Sequeira-Lopez, R. Ariel Gomez, Pablo Nakagawa, Curt D. Sigmund
Abstract
The pathobiology of pulmonary hypertension (PH) is complex and multiple cell types contribute to disease pathogenesis. We sought to characterize the molecular crosstalk between endothelial and mesenchymal cells that promote PH in the tumor necrosis factor alpha transgenic (TNF-Tg) model of PH. Pulmonary endothelial and mesenchymal cells were isolated from WT and TNF-Tg mice underwent single-cell RNA sequencing. Data were analyzed using clustering, differential gene expression and pathway analysis, ligand-receptor interaction, transcription factor binding, and RNA velocity assessments. Significantly altered ligand-receptor interactions were confirmed with immunofluorescent staining. TNF-Tg mice had increases in smooth muscle cells and Col14+ fibroblasts, and reductions in general capillary (gCAP) endothelial cells, Col13+ fibroblasts, pericytes, and myofibroblasts. Pathway analysis demonstrated NF-kB, JAK/STAT, and interferon mediated inflammation, endothelial apoptosis, loss of vasodilatory pathways, increased TGF-beta signaling, and smooth muscle cell proliferation. Ligand-receptor analysis demonstrated a loss of BMPR2 signaling in TNF lungs and establishment of a maladaptive BMP signaling cascade which functional studies revealed stems from endothelial NFkB activation and subsequent endothelial SMAD2/3 signaling. This system highlights a complex set of changes in cellular composition, cell communication, and cell fate driven by TNF signaling which lead to aberrant BMP signaling which is critical for development of PH.
Authors
ML Garcia-Hernandez, Javier Rangel-Moreno, Qingfu Xu, Ye Jin Jeong, Soumyaroop Bhattacharya, Ravi Misra, Stacey Duemmel, Ke Yuan, Benjamin D. Korman
Abstract
BACKGROUND Obesity, a growing health concern, often leads to metabolic disturbances, systemic inflammation, and vascular dysfunction. Emerging evidence suggests that adipose tissue-derived extracellular vesicles (adiposomes) may propagate obesity-related complications. However, their lipid composition and effect on cardiometabolic state remain unclear.METHODS This study examined the lipid composition of adiposomes in 122 participants (75 in obesity group, 47 in lean group) and its connection to cardiometabolic risk. Adiposomes were isolated via ultracentrifugation and characterized using nanoparticle tracking and comprehensive lipidomic analysis by mass spectrometry. Cardiometabolic assessments included anthropometry, body composition, glucose-insulin homeostasis, lipid profiles, inflammatory markers, and vascular function.RESULTS Compared with lean controls, individuals with obesity exhibited elevated adiposome release and shifts in lipid composition, including higher ceramides, free fatty acids, and acylcarnitines, along with reduced levels of phospholipids and sphingomyelins. These alterations strongly correlated with increased BMI, insulin resistance, systemic inflammation, and impaired vascular function. Pathway enrichment analyses highlight dysregulation in glycerophospholipid and sphingolipid metabolism, bile secretion, proinflammatory pathways, and vascular contractility. Machine-learning models utilizing adiposome lipid data accurately classified obesity and predicted cardiometabolic conditions, such as diabetes, hypertension, dyslipidemia, and liver steatosis, achieving accuracy above 85%.CONCLUSION Obesity profoundly remodels the adiposome lipid landscape, linking lipid changes to inflammation, metabolic dysfunction, and vascular impairment. These findings underscore adiposome lipids as biomarkers for obesity and related cardiometabolic disorders, supporting personalized interventions and offering therapeutic value in risk stratification and treatment.FUNDING This project was supported by NIH grants R01HL161386, R00HL140049, P30DK020595 (PI: AMM), R01DK104927, and P30DK020595 as well as by a VA Merit Award (1I01BX003382, PI: BTL).
Authors
Abeer M. Mahmoud, Imaduddin Mirza, Elsayed Metwally, Mohammed H. Morsy, Giorgia Scichilone, Monica C. Asada, Amro Mostafa, Francesco M. Bianco, Mohamed M. Ali, Mario A. Masrur, Chandra Hassan, Brian T. Layden
Abstract
Systemic arterial stenosis, including moyamoya disease (MMD) and middle aortic syndrome (MAS), is a rare condition of unclear etiology. MMD is a cerebral angiopathy, and MAS affects the abdominal and thoracic aorta. Although some genetic associations with MAS have been identified, the causes remain elusive. In this study, de novo heterozygous missense variants of RING finger protein 213 (RNF213) (p.His4058Pro and p.Thr4155Pro) in 2 unrelated families with MAS and MMD were studied by whole-exome sequencing. To elucidate the significance of these variants, we produced knockin mice carrying the Rnf213 p.His4058Pro variant. Homozygous knockin mice exhibited perinatal lethality because of respiratory failure and lung dysplasia, suggesting that this variant is pathogenic. Lung dysplasia in homozygous knockin mice was associated with upregulated innate immunity and inflammatory responses and downregulated cell proliferation. These findings suggested that in mice, the RNF213 p.His4058Pro variant plays critical roles in regulation of innate immunity and inflammation that affect lung development, revealing the complexity of RNF213 function in various tissues and species. In conclusion, this study provides insights into the genetic basis of MAS and MMD, highlights the potential involvement of RNF213 variants in systemic vasculopathy, and identifies unexpected associations with lung development and immune processes.
Authors
Ayako Kashimada, Tomoko Mizuno, Eriko Tanaka, Susumu Hosokawa, Tomohiro Udagawa, Yuichi Hiraoka, Keisuke Uchida, Tomohiro Morio, Kenjiro Kosaki, Masatoshi Takagi
Abstract
Vascular smooth muscle cells (VSMCs) exhibit significant heterogeneity and plasticity, enabling them to switch between contractile and synthetic states, which is crucial for vascular remodeling. NEXN has been identified as a high confidence gene associated with dilated cardiomyopathy (DCM). Existing evidence indicate NEXN is involved in phenotypic switching of VSMCs. However, a comprehensive understanding of the cell-specific roles and precise mechanisms of NEXN in vascular remodeling remains elusive. Using integrative transcriptomics analysis and smooth muscle specific lineage tracing mice, we demonstrate NEXN is highly expressed in VSMCs, and the expression of NEXN is significantly reduced during the phenotypic transformation of VSMCs and intimal hyperplasia induced by vascular injury. VSMC-specific NEXN deficiency promoted the phenotypic transition of VSMCs and exacerbated neointimal hyperplasia in mice following vascular injury. Mechanistically, we found NEXN primarily mediated VSMCs proliferation and phenotypic transition through endoplasmic reticulum (ER) stress and KLF4 signaling. Inhibiting ER stress ameliorated VSMCs phenotypic transition by reducing cell cycle activity and proliferation caused by NEXN deficiency. These findings indicate targeting NEXN could be explored as a promising therapeutic approach for proliferative arterial diseases.
Authors
Zexuan Lin, Chaojie Wang, Zhuohua Wen, Zhaohui Cai, Wenjie Guo, Xin Feng, Zengyan Huang, Rongjun Zou, Xiaoping Fan, Canzhao Liu, Hanyan Yang
Abstract
Microvascular rarefaction substantially contributes to renal dysfunction following ischemia-reperfusion injury (IRI). We characterized the microRNA signature of extracellular vesicles (EVs) released during endothelial apoptosis to identify biomarkers and regulators of microvascular rarefaction and renal dysfunction. Using in vitro models and RNA-Seq, we found miR-423-5p, let-7b-5p, and let-7c-5p enriched in small EVs from apoptotic endothelial cells. In mouse models of renal IRI and a cohort of 51 patients who have undergone renal transplant with delayed graft function, serum miR-423-5p correlated with circulating EVs, while let-7b-5p and let-7c-5p were also present in free form. Early acute kidney injury saw increased serum miR-423-5p levels linked to small EVs with endothelial markers. Over time, higher serum miR-423-5p levels were associated with large EVs and correlated with greater renal microvascular density and reduced fibrosis. Microvascular density and fibrosis predicted renal function 3 years after transplantation. We explored miR-423-5p’s role in renal homeostasis, finding that its injection during renal IRI preserved microvascular density and inhibited fibrosis. Endothelial cells transfected with miR-423-5p showed enhanced resistance to apoptosis, increased migration, and angiogenesis. Localized miR-423-5p injection in hindlimb ischemia model accelerated revascularization. These findings position miR-423-5p as a predictor of renal microvascular rarefaction and fibrosis, highlighting potential strategies for preserving renal function.
Authors
Francis Migneault, Hyunyun Kim, Alice Doreille, Shanshan Lan, Alexis Gendron, Marie-Hélène Normand, Annie Karakeussian Rimbaud, Martin Dupont, Isabelle Bourdeau, Éric Bonneil, Julie Turgeon, Sylvie Dussault, Pierre Thibault, Mélanie Dieudé, Éric Boilard, Alain Rivard, Héloïse Cardinal, Marie-Josée Hébert
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