Impact of aging on pulmonary cellular responses during mechanical ventilation

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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

An intracellular complement system drives metabolic and proinflammatory reprogramming of vascular fibroblasts in pulmonary hypertension

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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

Short-term disruption of TGFβ signaling in adult mice renders the aorta vulnerable to hypertension-induced dissection

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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

A single-cell atlas of normal and KRASG12D-malformed lymphatic vessels

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Abstract

Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific processes that drive KRAS-mediated CLAs have yet to be fully elucidated. Here, we used single-cell RNA sequencing to construct an atlas of normal and KrasG12D-malformed lymphatic vessels. We identified six subtypes of lymphatic endothelial cells (LECs) in the lungs of adult wild-type mice (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes were specified during development, we integrated our data with data from four stages of development. We found that proliferating and Ptx3 LECs were prevalent during early lymphatic development and that collecting and valve LECs emerged later in development. Additionally, we discovered that the proportion of Ptx3 LECs decreased as the lymphatic network matured but remained high in KrasG12D mice. We also observed that the proportion of collecting and valve LECs was lower in KrasG12D mice than in wild-type mice. Last, we found that immature lymphatic vessels in young mice were more sensitive to the pathologic effects of KrasG12D than mature lymphatic vessels in older mice. Together, our results expand the current model for the development of the lymphatic system and suggest that KRAS mutations impair the maturation of lymphatic vessels.

Authors

Lorenzo M. Fernandes, Danielle Griswold-Wheeler, Jeffrey D. Tresemer, Angelica Vallejo, Neda Vishlaghi, Benjamin Levi, Abigail Shapiro, Joshua P. Scallan, Michael T. Dellinger

Inhibition of vascular smooth muscle cell PERK/ATF4 ER stress signaling protects against abdominal aortic aneurysms

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Abstract

Abdominal aortic aneurysms (AAA) are a life-threatening cardiovascular disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by vascular smooth muscle cell (VSMC) dysfunction and apoptosis, for which the mechanisms regulating loss of VSMCs within the aortic wall remain poorly defined. Using single-cell RNA-Seq of human AAA tissues, we identified increased activation of the endoplasmic reticulum stress response pathway, PERK/eIF2α/ATF4, in aortic VSMCs resulting in upregulation of an apoptotic cellular response. Mechanistically, we reported that aberrant TNF-α activity within the aortic wall induces VSMC ATF4 activation through the PERK endoplasmic reticulum stress response, resulting in progressive apoptosis. In vivo targeted inhibition of the PERK pathway, with VSMC-specific genetic depletion (Eif2ak3fl/fl Myh11-CreERT2) or pharmacological inhibition in the elastase and angiotensin II–induced AAA model preserved VSMC function, decreased elastin fragmentation, attenuated VSMC apoptosis, and markedly reduced AAA expansion. Together, our findings suggest that cell-specific pharmacologic therapy targeting the PERK/eIF2α/ATF4 pathway in VSMCs may be an effective intervention to prevent AAA expansion.

Authors

Brennan Callow, Xiaobing He, Nicholas Juriga, Kevin D. Mangum, Amrita Joshi, Xianying Xing, Andrea Obi, Abhijnan Chattopadhyay, Dianna M. Milewicz, Mary X. O’Riordan, Johann Gudjonsson, Katherine Gallagher, Frank M. Davis

Map2k6 is a potent genetic modifier of arterial rupture in vascular Ehlers-Danlos syndrome mice

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Abstract

Aortic dissection or rupture is a major cause of mortality in vascular Ehlers-Danlos Syndrome (vEDS), a connective tissue disorder caused by heterozygous mutations in the COL3A1 gene. C57BL6/J (BL6) mice carrying the Col3a1 G938D/+ mutation recapitulate the vEDS vascular phenotype and die suddenly of aortic rupture/dissection. However, 129S6/SvEvTac (129) mice expressing the same Col3a1 G938D/+ mutation show near-complete life-long protection from vascular rupture. To identify genetic modifiers of vascular risk in vEDS, we performed genome-wide genotyping of intercrossed BL6/129 vEDS mice stratified by survival and identified a significant protective locus encompassing a variant in Map2k6, encoding Mitogen-Activated Protein Kinase Kinase 6 (M2K6), a p38-activating kinase. Genetic ablation of Map2k6 rendered previously protected 129 vEDS mice susceptible to aortic rupture, in association with reduced protein phosphatase 1 activity and increased PKC and ERK phosphorylation. Accelerated vascular rupture in vEDS mice treated with a pharmacological inhibitor of p38 was rescued by concomitant ERK antagonism, supporting an opposing role for ERK and p38 in the modification of aortic rupture risk in vEDS. These results suggest that pharmacologic strategies aimed at mimicking the effect of this natural protective pathway may improve prevention of aortic rupture risk in vEDS.

Authors

Caitlin J. Bowen, Rebecca Sorber, Juan F. Calderon Giadrosic, Jefferson J. Doyle, Graham Rykiel, Zachary Burger, Xiaoyan Zhang, Wendy A. Espinoza Camejo, Nicole K. Anderson, Simone Sabnis, Chiara Bellini, Elena MacFarlane, Harry C. Dietz

Casz1 and Znf101/Zfp961 differentially regulate apolipoproteins A1 and B, alter plasma lipoproteins, and reduce atherosclerosis

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Abstract

High apolipoprotein B–containing (apoB-containing) low-density lipoproteins (LDLs) and low apoA1–containing high-density lipoproteins (HDLs) are associated with atherosclerotic cardiovascular diseases. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both significantly decreases apoB and increases apoA1 expression by inducing mRNA degradation of 2 different transcription factors, Znf101 and Casz1. We found that Znf101 enhances apoB expression, while Casz1 represses apoA1 expression. The hepatic knockdown of Casz1 in mice increased plasma apoA1, HDL, and cholesterol efflux capacity. The hepatic knockdown of Zfp961, an ortholog of Znf101, reduced lipogenesis and production of triglyceride-rich lipoproteins and atherosclerosis, without causing hepatic lipid accumulation. This study identifies hepatic Znf101/Zfp961 and Casz1 as potential therapeutic targets to alter plasma lipoproteins and reduce atherosclerosis without causing liver steatosis.

Authors

Abulaish Ansari, Pradeep Kumar Yadav, Liye Zhou, Binu Prakash, Laraib Ijaz, Amanda Christiano, Sameer Ahmad, Antoine Rimbert, M. Mahmood Hussain

Endothelial Response to Blood-Brain Barrier Disruption in the Human Brain

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Abstract

Cerebral endothelial cell (EC) injury and blood-brain barrier (BBB) permeability contribute to neuronal injury in acute neurological disease states. Preclinical experiments have used animal models to study this phenomenon, yet the response of human cerebral ECs to BBB disruption remains unclear. In our Phase 1 clinical trial (NCT04528680), we used low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce transient BBB disruption of peri-tumoral brain in patients with recurrent glioblastoma. We found radiographic evidence that BBB integrity was mostly restored within 1-hour of this procedure. Using single-cell RNA sequencing and transmission electron microscopy, we analyzed the acute response of human brain ECs to ultrasound-mediated BBB disruption. Our analysis revealed distinct EC gene expression changes after LIPU/MB, particularly in genes related to neurovascular barrier function and structure, including changes to genes involved in the basement membrane, EC cytoskeleton, and junction complexes, as well as caveolar transcytosis and various solute transporters. Ultrastructural analysis showed that LIPU/MB led to a decrease in luminal caveolae, the emergence of cytoplasmic vacuoles, and the disruption of the basement membrane and tight junctions, among other things. These findings suggested that acute BBB disruption by LIPU/MB led to specific transcriptional and ultrastructural changes and could represent a conserved mechanism of BBB repair after neurovascular injury in humans.

Authors

Andrew Gould, Yu Luan, Ye Hou, Farida V. Korobova, Li Chen, Victor A. Arrieta, Christina Amidei, Rachel Ward, Cristal Gomez, Brandyn Castro, Karl Habashy, Daniel Zhang, Mark Youngblood, Crismita Dmello, John Bebawy, Guillaume Bouchoux, Roger Stupp, Michael Canney, Feng Yue, M. Luisa Iruela-Arispe, Adam M. Sonabend

Talin1 dysfunction is genetically linked to systemic capillary leak syndrome

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Abstract

Systemic capillary leak syndrome (SCLS) is a rare life-threatening disorder due to profound vascular leak. The trigger and the cause of the disease are currently unknown and there is no specific treatment. Here, we identified a rare heterozygous splice-site variant in the TLN1 gene in a familial SCLS case, suggestive of autosomal dominant inheritance with incomplete penetrance. Talin1 has a key role in cell adhesion by activating and linking integrins to the actin cytoskeleton. This variant causes in-frame skipping of exon 54 and is predicted to affect talin’s C-terminal actin-binding site (ABS3). Modeling the SCLS-TLN1 variant in TLN1-heterozygous endothelial cells (ECs) disturbed the endothelial barrier function. Similarly, mimicking the predicted actin-binding disruption in TLN1-heterozygous ECs resulted in disorganized endothelial adherens junctions. Mechanistically, we established that the SCLS-TLN1 variant, through the disruption of talin’s ABS3, sequestrates talin’s interacting partner, vinculin, at cell–extracellular matrix adhesions, leading to destabilization of the endothelial barrier. We propose that pathogenic variants in TLN1 underlie SCLS, providing insight into the molecular mechanism of the disease that can be explored for future therapeutic interventions.

Authors

Naama Elefant, Georgia Rouni, Christina Arapatzi, Danit Oz-Levi, Racheli Sion-Sarid, William J.S. Edwards, Neil J. Ball, Shira Yanovsky-Dagan, Alana R. Cowell, Vardiella Meiner, Vladimir Vainstein, Sofia Grammenoudi, Doron Lancet, Benjamin T. Goult, Tamar Harel, Vassiliki Kostourou

Sphingosine-1-phosphate (S1P) receptor type 1 signaling in macrophages reduces atherosclerosis in LDL receptor-deficient mice

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Abstract

Sphingosine 1-phosphate (S1P) is a lysosphingolipid with anti-atherogenic properties, but mechanisms underlying its effects remain unclear. We here investigated atherosclerosis development in cholesterol-rich diet-fed LDL receptor-deficient mice with high or low overexpression levels of S1P receptor type 1 (S1P1) in macrophages. S1P1-overexpressing macrophages showed increased activity of transcription factors PU.1, IRF8, and LXR and were skewed towards a M2-distinct phenotype characterized by enhanced production of IL-10, IL-1RA, and IL-5, increased ATP-binding cassette transporter A1- and G1-dependent cholesterol efflux, increased expression of MerTK and efferocytosis, and reduced apoptosis due to elevated Bcl6 and MafB. A similar macrophage phenotype was observed in mice administered S1P1-selective agonist KRP203. Mechanistically, the enhanced PU.1, IRF8, and LXR activity in S1P1-overexpressing macrophages led to down-regulation of the cAMP-dependent protein kinase A and activation of the signaling cascade encompassing protein kinases Akt and mTOR complex 1 (mTORC1) as well as the late endosomal/lysosomal adaptor MAPK and mTOR activator 1 (Lamtor-1). Atherosclerotic lesions in aortic roots and brachiocephalic arteries were profoundly or moderately reduced in mice with high and low S1P1 overexpression in macrophages, respectively. We conclude that S1P1 signaling polarizes macrophages towards an anti-atherogenic functional phenotype and countervails the development of atherosclerosis in mice.

Authors

Francesco Potì, Enrica Scalera, Renata Feuerborn, Josephine Fischer, Lilli Arndt, Georg Varga, Evangelia Pardali, Matthias D. Seidl, Manfred Fobker, Gerhard Liebisch, Bettina Hesse, Alexander H. Lukasz, Jan Rossaint, Beate E. Kehrel, Frank Rosenbauer, Thomas Renné, Christina Christoffersen, Manuela Simoni, Ralph Burkhardt, Jerzy-Roch Nofer