Macrophage-derived IL-6 trans-signaling as a novel target in the pathogenesis of bronchopulmonary dysplasia

RationalePremature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD.Methods and ResultsFirst, transcriptomic analysis with in-silico cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines (Ccl2, Ccl7, Cxcl5) and markers (Il6, Il17A, Mmp12). Second, hyperoxia-activated IL-6/STAT3 signaling was measured in vivo and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. Il6 null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fiber assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signaling and IL-6 trans-signaling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly via Klf4; hyperoxia-conditioned medium of macrophages and IL-6 impaired ATII proliferation. Finally, clinical data demonstrate elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived IL6 and active STAT3 were related to loss of epithelial cells in BPD lungs.ConclusionWe present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis, and disrupts elastic fiber formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signaling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease.

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Lung matrix and vascular remodeling in mechanically ventilated elastin haploinsufficient newborn mice

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00278.2014 ◽

2015 ◽

Vol 308 (5) ◽

pp. L464-L478 ◽

Cited By ~ 19

Author(s):

Anne Hilgendorff ◽

Kakoli Parai ◽

Robert Ertsey ◽

Edwin Navarro ◽

Noopur Jain ◽

...

Keyword(s):

Lysyl Oxidase ◽

Elastic Fiber ◽

Growth Arrest ◽

Subsequent Development ◽

Fiber Formation ◽

Lung Growth ◽

Elastase Activity ◽

Newborn Mice ◽

Fibrillin 1 ◽

Lung Capillaries

Elastin plays a pivotal role in lung development. We therefore queried if elastin haploinsufficient newborn mice ( Eln+/−) would exhibit abnormal lung structure and function related to modified extracellular matrix (ECM) composition. Because mechanical ventilation (MV) has been linked to dysregulated elastic fiber formation in the newborn lung, we also asked if elastin haploinsufficiency would accentuate lung growth arrest seen after prolonged MV of neonatal mice. We studied 5-day-old wild-type ( Eln+/+) and Eln+/− littermates at baseline and after MV with air for 8–24 h. Lungs of unventilated Eln+/− mice contained ∼50% less elastin and ∼100% more collagen-1 and lysyl oxidase compared with Eln+/+ pups. Eln+/− lungs contained fewer capillaries than Eln+/+ lungs, without discernible differences in alveolar structure. In response to MV, lung tropoelastin and elastase activity increased in Eln+/+ neonates, whereas tropoelastin decreased and elastase activity was unchanged in Eln+/− mice. Fibrillin-1 protein increased in lungs of both groups during MV, more in Eln+/− than in Eln+/+ pups. In both groups, MV caused capillary loss, with larger and fewer alveoli compared with unventilated controls. Respiratory system elastance, which was less in unventilated Eln+/− compared with Eln+/+ mice, was similar in both groups after MV. These results suggest that elastin haploinsufficiency adversely impacts pulmonary angiogenesis and that MV dysregulates elastic fiber integrity, with further loss of lung capillaries, lung growth arrest, and impaired respiratory function in both Eln+/+ and Eln+/− mice. Paucity of lung capillaries in Eln+/− newborns might help explain subsequent development of pulmonary hypertension previously reported in adult Eln+/− mice.

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Recent updates on the molecular network of elastic fiber formation

Essays in Biochemistry ◽

10.1042/ebc20180052 ◽

2019 ◽

Vol 63 (3) ◽

pp. 365-376 ◽

Cited By ~ 4

Author(s):

Seung Jae Shin ◽

Hiromi Yanagisawa

Keyword(s):

Elastic Fiber ◽

Building Blocks ◽

Fiber Formation ◽

Elastic Fibers ◽

Fiber Network ◽

Associated Proteins ◽

Fibrillin 1 ◽

A Cell ◽

Self Aggregation

Abstract Elastic fibers confer elasticity and recoiling to tissues and organs and play an essential role in induction of biochemical responses in a cell against mechanical forces derived from the microenvironment. The core component of elastic fibers is elastin (ELN), which is secreted as the monomer tropoelastin from elastogenic cells, and undergoes self-aggregation, cross-linking and deposition on to microfibrils, and assemble into insoluble ELN polymers. For elastic fibers to form, a microfibril scaffold (primarily formed by fibrillin-1 (FBN1)) is required. Numerous elastic fiber-associated proteins are involved in each step of elastogenesis and they instruct and/or facilitate the elastogenesis processes. In this review, we designated five proteins as key molecules in elastic fiber formation, including ELN, FBN1, fibulin-4 (FBLN4), fibulin-5 (FBLN5), and latent TGFβ-binding protein-4 (LTBP4). ELN and FBN1 serve as building blocks for elastic fibers. FBLN5, FBLN4 and LTBP4 have been demonstrated to play crucial roles in elastogenesis through knockout studies in mice. Using these molecules as a platform and expanding the elastic fiber network through the generation of an interactome map, we provide a concise review of elastogenesis with a recent update as well as discuss various biological functions of elastic fiber-associated proteins beyond elastogenesis in vivo.

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Cell motion-coordinated fibrillar assembly of soluble collagen I to promote MDCK cell branching formation

10.1101/869438 ◽

2019 ◽

Author(s):

Jiajia Wang ◽

Jia Guo ◽

Bo Che ◽

Mingxing Ouyang ◽

Linhong Deng

Keyword(s):

Mdck Cells ◽

Fiber Strength ◽

Genetic Diseases ◽

Myosin Ii ◽

Branching Morphogenesis ◽

Fiber Formation ◽

Collagen I ◽

Fiber Assembly ◽

Cell Motion

AbstractExtracellular Matrix (ECM) assembly and remodeling are critical physiological events in vivo, and abnormal ECM assembly or remodeling is related to pathological conditions such as osteoarthritis, fibrosis, cancers, and genetic diseases. ECM assembly/remodeling driven by cells represents more physiological processes. Collagen I (COL) is very abundant in tissues, which assembly/remodeling is mediated by biochemical and mechanical factors. How cells regulate COL assembly biomechanically still remains to be well understood. Here we used fluorescent COL in the medium to study how cells assembled ECM which represents more physiological structures. The results showed that MDCK cells actively recruited COL from the medium and helped assemble the fibers, which in turn facilitated cell branching morphogenesis, both displaying high spatial associations and mutual dependency. Inhibition of cellular contraction force by ROCK and Myosin II inhibitors attenuated but did not block the COL fiber formation, while cell motion showed high consistency with the fiber assembly. Under ROCK or Myosin II inhibition, further analysis indicated high correlation between local cell movement and COL fiber strength as quantified from different regions of the same groups. Blocking cell motion by actin cytoskeleton disruption completely inhibited the fiber formation. These suggest that cell motion coordinated COL fiber assembly from the medium, possibly through generated strain on deposited COL to facilitate the fiber growth.

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Involvement of Hdac3-mediated inhibition of microRNA cluster 17-92 in bronchopulmonary dysplasia development

Molecular Medicine ◽

10.1186/s10020-020-00237-4 ◽

2020 ◽

Vol 26 (1) ◽

Author(s):

Di Wang ◽

Hui Hong ◽

Xiao-Xia Li ◽

Jing Li ◽

Zhi-Qun Zhang

Keyword(s):

Bronchopulmonary Dysplasia ◽

Promoter Region ◽

Placental Growth Factor ◽

Lung Fibroblasts ◽

Newborn Mice ◽

Histone Deacetylase 3 ◽

Adult Mice ◽

Medical Advances

Abstract Background The incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, has been paradoxically rising despite medical advances. Histone deacetylase 3 (Hdac3) has been reported to be a crucial regulator in alveologenesis. Hence, this study aims to investigate the mechanism of Hdac3 in the abnormal pulmonary angiogenesis and alveolarization of BPD. Methods A hyperoxia-induced BPD model of was developed in newborn mice, and primary lung fibroblasts were isolated from adult mice. Hdac3 was knocked out in vivo and knocked down in vitro, while microRNA (miR)-17 was downregulated in vivo and in vitro to clarify their roles in abnormal pulmonary angiogenesis and alveolarization. Mechanistic investigations were performed on the interplay of Hdac3, miR-17-92 cluster, enhancer of zeste homolog 1 (EZH1), p65 and placental growth factor (Pgf). Results Hdac3 was involved in abnormal alveolarization and angiogenesis in BPD mice. Further, the expression of the miR-17-92 cluster in BPD mice was downregulated by Hdac3. miR-17 was found to target EZH1, and Hdac3 rescued the inhibited EZH1 expression by miR-17 in lung fibroblasts. Additionally, EZH1 augmented Pgf expression by recruiting p65 thus enhancing the progression of BPD. Hdac3 augmented the recruitment of p65 in the Pgf promoter region through the miR-17/EZH1 axis, thus enhancing the transcription and expression of Pgf, which elicited abnormal angiogenesis and alveolarization of BPD mice. Conclusions Altogether, the present study revealed that Hdac3 activated the EZH1-p65-Pgf axis through inhibiting miR-17 in the miR-17-92 cluster, leading to accelerated abnormal pulmonary angiogenesis and alveolarization of BPD mice.

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Vitamin D treatment improves survival and infant lung structure after intra-amniotic endotoxin exposure in rats: potential role for the prevention of bronchopulmonary dysplasia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00344.2013 ◽

2014 ◽

Vol 306 (5) ◽

pp. L420-L428 ◽

Cited By ~ 34

Author(s):

Erica Mandell ◽

Gregory Seedorf ◽

Jason Gien ◽

Steven H. Abman

Keyword(s):

Vitamin D ◽

Bronchopulmonary Dysplasia ◽

Tube Formation ◽

Saline Control ◽

Alveolar Type ◽

Lung Growth ◽

Fetal Sheep ◽

Rat Pups ◽

Lung Structure

Vitamin D (vit D) has anti-inflammatory properties and modulates lung growth, but whether vit D can prevent lung injury after exposure to antenatal inflammation is unknown. We hypothesized that early and sustained vit D treatment could improve survival and preserve lung growth in an experimental model of bronchopulmonary dysplasia induced by antenatal exposure to endotoxin (ETX). Fetal rats (E20) were exposed to ETX (10 μg), ETX + Vit D (1 ng/ml), or saline (control) via intra-amniotic (IA) injections and delivered 2 days later. Newborn pups exposed to IA ETX received daily intraperitoneal injections of vit D (1 ng/g) or saline for 14 days. Vit D treatment improved oxygen saturations (78 vs. 87%; P < 0.001) and postnatal survival (84% vs. 57%; P < 0.001) after exposure to IA ETX compared with IA ETX alone. Postnatal vit D treatment improved alveolar and vascular growth at 14 days by 45% and 25%, respectively ( P < 0.05). Vit D increased fetal sheep pulmonary artery endothelial cell (PAEC) growth and tube formation by 64% and 44%, respectively ( P < 0.001), and prevented ETX-induced reductions of PAEC growth and tube formation. Vit D directly increased fetal alveolar type II cell (ATIIC) growth by 26% ( P < 0.001) and enhanced ATIIC growth in the presence of ETX-induced growth suppression by 73% ( P < 0.001). We conclude that antenatal vit D therapy improved oxygenation and survival in newborn rat pups and enhanced late lung structure after exposure to IA ETX in vivo, which may partly be due to direct effects on vascular and alveolar growth.

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Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice.

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00362.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. L3-L14 ◽

Cited By ~ 76

Author(s):

Richard D. Bland ◽

Robert Ertsey ◽

Lucia M. Mokres ◽

Liwen Xu ◽

Berit E. Jacobson ◽

...

Keyword(s):

Mechanical Ventilation ◽

Lysyl Oxidase ◽

Prolonged Mechanical Ventilation ◽

Elastic Fiber ◽

Elastase Activity ◽

Fiber Density ◽

Newborn Mice ◽

Fiber Assembly ◽

Expression Of Genes ◽

Fibrillin 1

Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.

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Molecular Analysis of Fibulin-5 Function during De Novo Synthesis of Elastic Fibers

Molecular and Cellular Biology ◽

10.1128/mcb.01330-06 ◽

2007 ◽

Vol 27 (3) ◽

pp. 1083-1095 ◽

Cited By ~ 41

Author(s):

Qian Zheng ◽

Elaine C. Davis ◽

James A. Richardson ◽

Barry C. Starcher ◽

Tiansen Li ◽

...

Keyword(s):

Calcium Binding ◽

De Novo ◽

Elastic Fiber ◽

Elastic Fibers ◽

Cellular Behavior ◽

Fiber Assembly ◽

Structural Support ◽

The Matrix ◽

Epidermal Growth

ABSTRACT Elastic fibers contribute to the structural support of tissues and to the regulation of cellular behavior. Mice deficient for the fibulin-5 gene (fbln5 − / −) were used to further elucidate the molecular mechanism of elastic fiber assembly. Major elastic fiber components were present in the skin of fbln5 − / − mice despite a dramatic reduction of mature elastic fibers. We found that fibulin-5 preferentially bound the monomeric form of elastin through N-terminal and C-terminal elastin-binding regions and to a preexisting matrix scaffold through calcium-binding epidermal growth factor (EGF)-like (CB-EGF) domains. We further showed that adenovirus-mediated gene transfer of fbln5 was sufficient to regenerate elastic fibers and increase elastic fiber-cell connections in vivo. A mutant fibulin-5 lacking the first 28 amino acids of the first CB-EGF domain, however, was unable to rescue elastic fiber defects. Fibulin-5 thus serves as an adaptor molecule between monomeric elastin and the matrix scaffold to aid in elastic fiber assembly. These results also support the potential use of fibulin-5 as a therapeutic agent for the treatment of elastinopathies.

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Fibulin-2 Is Dispensable for Mouse Development and Elastic Fiber Formation

Molecular and Cellular Biology ◽

10.1128/mcb.01876-07 ◽

2007 ◽

Vol 28 (3) ◽

pp. 1061-1067 ◽

Cited By ~ 44

Author(s):

Francois-Xavier Sicot ◽

Takeshi Tsuda ◽

Dessislava Markova ◽

John F. Klement ◽

Machiko Arita ◽

...

Keyword(s):

Matrix Protein ◽

Elastic Fiber ◽

Embryonic Stem ◽

Fiber Formation ◽

Extracellular Matrix Protein ◽

Elastic Fibers ◽

Mouse Development ◽

Fibrillin 1

ABSTRACT Fibulin-2 is an extracellular matrix protein belonging to the five-member fibulin family, of which two members have been shown to play essential roles in elastic fiber formation during development. Fibulin-2 interacts with two major constituents of elastic fibers, tropoelastin and fibrillin-1, in vitro and localizes to elastic fibers in many tissues in vivo. The protein is prominently expressed during morphogenesis of the heart and aortic arch vessels and at early stages of cartilage development. To examine its role in vivo, we generated mice that do not express the fibulin-2 gene (Fbln2) through homologous recombination of embryonic stem cells. Unexpectedly, the fibulin-2-null mice were viable and fertile and did not display gross and anatomical abnormalities. Histological and ultrastructural analyses revealed that elastic fibers assembled normally in the absence of fibulin-2. No compensatory up-regulation of mRNAs for other fibulin members was detected in the aorta and skin tissue. However, in the fibulin-2 null aortae, fibulin-1 immunostaining was increased in the inner elastic lamina, where fibulin-2 preferentially localizes. The results demonstrate that fibulin-2 is not required for mouse development and elastic fiber formation and suggest possible functional redundancy between fibulin-1 and fibulin-2.

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Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200611026 ◽

2007 ◽

Vol 176 (7) ◽

pp. 1061-1071 ◽

Cited By ~ 110

Author(s):

Maretoshi Hirai ◽

Tetsuya Ohbayashi ◽

Masahito Horiguchi ◽

Katsuya Okawa ◽

Akari Hagiwara ◽

...

Keyword(s):

Lysyl Oxidase ◽

Elastic Fiber ◽

Elastic Fibers ◽

Cross Linking ◽

Fiber Assembly ◽

Organizer Activity ◽

Serum Dependent ◽

Serum Free Conditions ◽

Deficient Mice

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat–containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5–deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171–175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168–171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.

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