Vascular biology
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
Abstract
Single dose radiotherapy (SDRT) is a highly-curative modality that may transform radiotherapy practice. Unfortunately, only ~50% of oligometastatic lesions are SDRT treatable due to adjacent radiosensitive normal organs at risk. Here we address extent to which an anti-angiogenic drug, VEGFR2-antagonist DC101, radiosensitizes SDRT using murine MCA/129 fibrosarcomas and Lewis Lung Carcinomas, which display a dose range for SDRT lesional eradication virtually identical to that employed clinically (10-30Gy). SDRT induces unique tumor cure, stimulating rapid endothelial acid sphingomyelinase (ASMase)/ceramide signaling that yields marked vasoconstriction and perfusion defects in tumor xenografts and human oligometastases. Ensuing tumor parenchymal oxidative damage initiates a SUMO Stress Response (SSR), which inactivates multiple homologous recombination repair enzymes, radiosensitizing all tumor types. While VEGF inhibits neo-angiogenic ASMase, optimal radiosensitization occurs only upon anti-angiogenic drug delivery at ~1h preceding SDRT. Obeying these principles, we find DC101 radiosensitizes SSR, DNA double strand break unrepair and tumor cure by 4-8Gy at all clinically-relevant doses. Critically, DC101 fails to sensitize small intestinal endothelial injury or lethality from the gastrointestinal-acute radiation syndrome.
Authors
Jin Cheng, Liyang Zhao, Sahra Bodo, Prashanth K. B. Nagesh, Rajvir Singh, Adam O. Michel, Regina Feldman, Zhigang Zhang, Simon N. Powell, Zvi Fuks, Richard Kolesnick
Abstract
There is an emerging role for Stimulator of interferon genes (STING) signaling in pulmonary hypertension (PH) development. Related, prior resesarch has demonstrated the relevance of the immune checkpoint protein Programmed death ligand 1 (PD-L1) expression by immunoregulatory myeloid cells in PH. However, there remains a need to elucidate the cell-specific role of STING expression, and the STING/PD-L1 signaling axis in PH, before readily available disease-modifying therapies can be applied to patients with disease. Here, through generation of bone marrow chimeric mice, we show that STING-/- mice receiving wild-type (WT) bone marrow are protected against PH secondary to chronic hypoxia. We further demonstrate a cellular dichotomous role for STING in PH development with STING expression by smooth muscle cells contributing to PH, and its activation on myeloid cells being pivotal in severe disease prevention. Finally, we provide evidence that a STING-PD-L1 axis modulates disease severity, suggesting future potential therapeutic applications. Overall, these data provide concrete evidence of STING involvement in PH in a cell-specific manner, establishing biologic plausibility for cell-targeted STING-related therapies in PH treatment.
Authors
Ann T. Pham, Shiza Virk, Aline C. Oliveira, Matthew D. Alves, Chunhua Fu, Yutao Zhang, Jimena Alvarez-Castanon, Brian B. Lee, Keira L. Lee, Radwan Mashina, Katherine E. Ray, Patrick Donabedian, Elnaz Ebrahimi, Harsh Patel, Reeha Patel, Duncan Lewis, Zhiguang Huo, Harry Karmouty-Quintana, Li Chen, Lei Jin, Andrew J. Bryant
Abstract
Pathologic implications of dysregulated pulmonary vascular metabolism to pulmonary arterial hypertension (PAH) are increasingly recognized, but their clinical applications have been limited. We hypothesized that metabolite quantification across the pulmonary vascular bed in connective tissue disease–associated (CTD-associated) PAH would identify transpulmonary gradients of pathobiologically relevant metabolites, in an exercise stage–specific manner. Sixty-three CTD patients with established or suspected PAH underwent exercise right heart catheterization. Using mass spectrometry–based metabolomics, metabolites were quantified in plasma samples simultaneously collected from the pulmonary and radial arteries at baseline and during resistance-free wheeling, peak exercise, and recovery. We identified uptake and excretion of metabolites across the pulmonary vascular bed, unique and distinct from single vascular site analysis. We demonstrated the physiological relevance of metabolites previously shown to promote disease in animal models and end-stage human lung tissues, including acylcarnitines, glycolytic intermediates, and tryptophan catabolites. Notably, pulmonary vascular metabolite handling was exercise stage specific. Transpulmonary metabolite gradients correlated with hemodynamic endpoints largely during free-wheeling. Glycolytic intermediates demonstrated physiologic significance at peak exercise, including net uptake of lactate in those with more advanced disease. Contribution of pulmonary vascular metabolism to CTD-PAH pathogenesis and therapeutic candidacy of metabolism modulation must be considered in the context of physiologic stress.
Authors
Michael H. Lee, Thaís C. F. Menezes, Julie A. Reisz, Francesca I. Cendali, Eloara V. M. Ferreira, Jaquelina S. Ota-Arakaki, Priscila A. Sperandio, Rahul Kumar, Claudia Mickael, Martin M. Ieong, Juliana Lucena Santos, Ana Carolina B. Duarte, Dara C. Fonseca Balladares, Kevin Nolan, Rubin M. Tuder, Paul M. Hassoun, Angelo D’Alessandro, Rudolf K. F. Oliveira, Brian B. Graham
Abstract
Vascular smooth muscle cells (VSMCs) possess significant phenotypic plasticity, shifting between a contractile phenotype and a synthetic state for vascular repair/remodelling. Dysregulated VSMC transformation, marked by excessive proliferation and migration, primarily drives intimal hyperplasia. N6-methyladenosine (m6A), the most prevalent RNA modification in eukaryotes, plays a critical role in gene expression regulation; however, its impact on VSMC plasticity is not fully understood. This research investigates the alterations in m6A modification and its regulatory factors during VSMC phenotypic shifts and their influence on intimal hyperplasia. We demonstrate that METTL14, crucial for m6A deposition, significantly promotes VSMC dedifferentiation. METTL14 expression, initially negligible, is elevated in synthetic VSMC cultures, post-injury neointimal VSMCs, and human restenotic arteries. Reducing METTL14 in mouse primary VSMCs decreases pro-synthetic genes, suppressing their proliferation and migration. m6A-RIP-seq profiling shows key VSMC gene networks undergo altered m6A regulation in Mettl14-deficient cells. METTL14 enhances KLF4 and SERPINE1 expression through increased m6A deposition. Local METTL14 knockdown significantly curbs neointimal formation post-arterial injury, and reducing METTL14 in hyperplastic arteries halts further neointimal development. We found that METTL14 is a pivotal regulator of VSMC dedifferentiation, influencing KLF4- and SERPINE1-mediated phenotypic conversion. Inhibiting METTL14 is a viable strategy for preventing restenosis and halting restenotic occlusions.
Authors
Grace Chensee, Bob S.L. Lee, Immanuel D. Green, Jessica Tieng, Renhua Song, Natalia Pinello, Quintin Lee, Majid Mehravar, David A. Robinson, Mian Wang, Mary M. Kavurma, Jun Yu, Justin Jong Leong Wong, Renjing Liu
Abstract
Despite the accumulation of cisplatin in proximal tubules, direct visualization of the surrounding peritubular microcirculation, including its alteration in cisplatin-induced acute kidney injury (AKI), is lacking. Here, using fluorescence and cellular angiography through video-rate high-resolution intravital microscopy, progressive disturbance of peritubular microcirculation in cisplatin-induced AKI in mice was demonstrated. Fluorescence angiography revealed increasing perfusion defects, with a stepwise rise in time to peak (TTP), originating from capillaries surrounding S1 segments. Cellular angiography demonstrated a progressive decrease in the velocity and track length of individual erythrocytes during AKI progression, accompanied by a sequential decrease in the functional capillary ratio (FCR). Alterations in the perfusion area, TTP, and FCR preceded significant changes in blood urea nitrogen and cystatin C, suggesting the potential for early diagnosis. Although neutrophil infiltration near proximal tubules increased throughout the progression, it did not cause obstruction of the peritubular microcirculation. Depletion of neutrophils increased mortality due to systemic side effects, whereas functional inactivation of neutrophils using an anti-CD11b antibody improved peritubular microcirculation in cisplatin-induced AKI. This approach enables direct visualization and quantification of peritubular microcirculation and immune cell dynamics, providing insights into renal pathophysiology and potential therapeutic strategies.
Authors
Inwon Park, Seonghye Kim, Young Woo Um, Hee Eun Kim, Jae Hyuk Lee, Sejoong Kim, PILHAN KIM, You Hwan Jo
Abstract
The notion of clonal cell populations in human atherosclerosis has been suggested but not demonstrated. Somatic mutations are used to define cellular clones in tumors. Here, we characterized the mutational landscape of human carotid plaques through whole-exome sequencing to explore the presence of clonal cell populations. Somatic mutations were identified in 12 of 13 investigated plaques, while no mutations were detected in 11 non-atherosclerotic arteries. Mutated clones often constituted over 10% of the sample cell population, with genes related to the contractile apparatus enriched for mutations. In CHIP (clonal hematopoiesis of indeterminate potential) carriers, hematopoietic clones had infiltrated the plaque tissue and constituted substantial fractions of the plaque cell population alongside locally expanded clones. Our findings establish somatic mutations as a common feature of human atherosclerosis and demonstrate the existence of mutated clones expanding locally, as well as CHIP clones invading from the circulation. While our data do not support plaque monoclonality, we observe a pattern suggesting the coexistence of multiple mutated clones of considerable size spanning different regions of plaques. Mutated clones are likely to be relevant to disease development, and somatic mutations will serve as a convenient tool to uncover novel pathological processes of atherosclerosis in future studies.
Authors
Lasse Bach Steffensen, Stephanie Kavan, Pia Søndergaard Jensen, Matilde Kvist Pedersen, Steffen Møller Bøttger, Martin J. Larsen, Maja Dembic, Otto Bergman, Ljubica Matic, Ulf Hedin, Lars vB Andersen, Jes Sanddal Lindholt, Kim Christian Houlind, Lars P. Riber, Mads Thomassen, Lars Melholt Rasmussen
Abstract
Acute respiratory distress syndrome (ARDS) results in significant morbidity and mortality, especially in the elderly. Mechanical ventilation, a common supportive treatment for ARDS, is necessary for maintaining gas exchange, but can also propagate injury. We hypothesized that aging leads to alterations in surfactant function, inflammatory signaling, and microvascular permeability within the lung during mechanical ventilation. Young and aged male mice were mechanically ventilated, and surfactant function, inflammation, and vascular permeability were assessed. Additionally, single-cell RNA sequencing was used to delineate cell-specific transcriptional changes. The results showed that in aged mice, surfactant dysfunction and vascular permeability were significantly augmented, while inflammation was less pronounced. Differential gene expression and pathway analyses revealed that alveolar macrophages in aged mice showed a blunted inflammatory response, while aged endothelial cells exhibited altered cell-cell junction formation. In vitro functional analysis revealed that aged endothelial cells had an impaired ability to form a barrier. These results highlight the complex interplay between aging and mechanical ventilation, including an age-related predisposition to endothelial barrier dysfunction, due to altered cell-cell junction formation, and decreased inflammation, potentially due to immune exhaustion. It is concluded that age-related vascular changes may underlie the increased susceptibility to injury during mechanical ventilation in elderly patients.
Authors
Aminmohamed Manji, Lefeng Wang, Cynthia M. Pape, Lynda A. McCaig, Alexandra Troitskaya, Onon Batnyam, Leah J.J. McDonald, C. Thomas Appleton, Ruud A.W. Veldhuizen, Sean E. Gill
Abstract
The complement system is central to the innate immune response, playing a critical role in pro-inflammatory and autoimmune diseases such as pulmonary hypertension (PH). Recent discoveries highlight the emerging role of intracellular complement, or the "complosome," in regulating cellular processes like glycolysis, mitochondrial dynamics, and inflammatory gene expression. This study investigates the hypothesis that intracellular complement proteins C3, CFB, and CFD are upregulated in PH fibroblasts (PH-Fibs) and drive their metabolic and inflammatory states, contributing to PH progression. Our results reveal a pronounced upregulation of CFD, CFB, and C3 in PH-Fibs from human and bovine models, both in vivo and in vitro. Elevated levels of C3 activation fragments, including C3b, C3d, and C3a, emphasize enhanced C3 activity. PH-Fibs exhibit notable metabolic reprogramming and increased pro-inflammatory mediators such as MCP1, SDF1, IL6, IL13, and IL33. Silencing CFD via shRNA reduced CFB activation and C3a production while normalizing glycolysis, tricarboxylic acid (TCA) cycle activity, and fatty acid metabolism. Metabolomic and gene expression analyses of CFD knockdown PH-Fibs revealed restored metabolic and inflammatory profiles, underscoring CFD’s crucial role in these changes. This study emphasizes the crucial role of intracellular complement in PH pathogenesis, highlighting the potential for complement-targeted therapies in PH.
Authors
Ram Raj Prasad, Sushil Kumar, Hui Zhang, Min Li, Cheng-Jun Hu, Suzette Riddle, Brittany A. McKeon, M.G. Frid, Konrad Hoetzenecker, Slaven Crnkovic, Grazyna Kwapiszewska, Rubin M. Tuder, Kurt R. Stenmark
Abstract
Hypertension and transient increases in blood pressure from extreme exertion are risk factors for aortic dissection in patients with age-related vascular degeneration or inherited connective tissue disorders. Yet, a common experimental model of angiotensin II-induced aortopathy in mice appears independent of high blood pressure as lesions do not occur in response to an alternative vasoconstrictor, norepinephrine, and are not prevented by co-treatment with a vasodilator, hydralazine. We investigated vasoconstrictor administration to adult mice following 1 week of disrupted TGFβ signaling in smooth muscle cells (SMCs). Norepinephrine increased blood pressure and induced aortic dissection by 7 days and even within 30 minutes (as did angiotensin II) that was prevented by hydralazine. Initial medial injury manifested as blood extravasation among SMCs and fibrillar matrix, progressive delamination from accumulation of blood, and stretched or ruptured SMCs with persistent attachments to elastic fibers. Altered regulatory contractile molecule expression was not of pathological importance. Rather, reduced synthesis of extracellular matrix yielded a vulnerable aortic phenotype by decreasing medial collagen, most dynamically basement membrane-associated multiplexin collagen, and impairing cell-matrix adhesion. We conclude that transient and sustained increases in blood pressure can cause dissection in aortas rendered vulnerable by inhibition of TGFβ-driven extracellular matrix production by SMCs.
Authors
Bo Jiang, Pengwei Ren, Changshun He, Mo Wang, Sae-Il Murtada, María Jesús Ruiz-Rodríguez, Yu Chen, Abhay B. Ramachandra, Guangxin Li, Lingfeng Qin, Roland Assi, Martin A. Schwartz, Jay D. Humphrey, George Tellides
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