C1q Deficiency Promotes Pulmonary Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury

Shah, Dilip; Romero, Freddy; Zhu, Ying; Duong, Michelle; Sun, Jianxin; Walsh, Kenneth; Summer, Ross

doi:10.1074/jbc.m115.690784

Cited by 21 publications

(23 citation statements)

References 42 publications

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“…Studying endothelial function: previous studies have shown that cultured MLECs can be used for multiple assays for assessing endothelial function, such as angiogenesis 28 , vascular tone 29 , and inflammation 30 . Here, we used LPS induced inflammatory response and monocyte adhesion as a model to test the responsiveness of cultured cells.Validation of endothelium-specific knockout or transgenic mice as well as performing phenotypic, genetic, or proteomic studies.…”

Section: Discussionmentioning

confidence: 99%

A simple protocol for isolating mouse lung endothelial cells

Wang

Niu

et al. 2019

Sci Rep

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Endothelial dysfunction is the common molecular basis of multiple human diseases, such as atherosclerosis, diabetes, hypertension, and acute lung injury. Therefore, primary isolation of high-purity endothelial cells (ECs) is crucial to study the mechanisms of endothelial function and disease pathogenesis. Mouse lung ECs (MLECs) are widely used in vascular biology and lung cell biology studies such as pulmonary inflammation, angiogenesis, vessel permeability, leukocyte/EC interaction, nitric oxide production, and mechanotransduction. Thus, in this paper, we describe a simple, and reproducible protocol for the isolation and culture of MLECs from adult mice using collagenase I-based enzymatic digestion, followed by sequential sorting with PECAM1 (also known as CD31)- and ICAM2 (also known as CD102)-coated microbeads. The morphology of isolated MLECs were observed with phase contrast microscope. MLECs were authenticated by CD31 immunoblotting, and immunofluorescent staining of established EC markers VE-cadherin and von Willebrand factor (vWF). Cultured MLECs also showed functional characteristics of ECs, evidenced by DiI-oxLDL uptake assay and THP-1 monocyte adhesion assay. Finally, we used MLECs from endothelium-specific enhancer of zeste homolog 2 (EZH2) knockout mice to show the general applicability of our protocol. To conclude, we describe here a simple and reproducible protocol to isolate highly pure and functional ECs from adult mouse lungs. Isolation of ECs from genetically engineered mice is important for downstream phenotypic, genetic, or proteomic studies.

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Section: Discussionmentioning

confidence: 99%

A simple protocol for isolating mouse lung endothelial cells

Wang

Niu

et al. 2019

Sci Rep

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“…6D. A recent study demonstrated that targeted deletion of C1q in mice alters pulmonary vascular homeostasis by augmenting the expression of endothelial adhesion markers associated with enhanced susceptibility of the lung endothelium to injury (49). To examine the potential mechanism by which fibrosis was still observed in C1qA Ϫ/Ϫ mice, we measured mRNA levels of two adhesion molecules involved in endothelial cell injury, ICAM-1 and VCAM-1.…”

Section: F521mentioning

confidence: 99%

Pericytes and immune cells contribute to complement activation in tubulointerstitial fibrosis

Xavier

Sahu

Landes

et al. 2017

American Journal of Physiology-Renal Physiology

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We have examined the pathogenic role of increased complement expression and activation during kidney fibrosis. Here, we show that PDGFRβ-positive pericytes isolated from mice subjected to obstructive or folic acid injury secrete C1q. This was associated with increased production of proinflammatory cytokines, extracellular matrix components, collagens, and increased Wnt3a-mediated activation of Wnt/β-catenin signaling, which are hallmarks of myofibroblast activation. Real-time PCR, immunoblots, immunohistochemistry, and flow cytometry analysis performed in whole kidney tissue confirmed increased expression of C1q, C1r, and C1s as well as complement activation, which is measured as increased synthesis of C3 fragments predominantly in the interstitial compartment. Flow studies localized increased C1q expression to PDGFRβ-positive pericytes as well as to CD45-positive cells. Although deletion of C1qA did not prevent kidney fibrosis, global deletion of C3 reduced macrophage infiltration, reduced synthesis of C3 fragments, and reduced fibrosis. Clodronate mediated depletion of CD11bF4/80 high macrophages in UUO mice also reduced complement gene expression and reduced fibrosis. Our studies demonstrate local synthesis of complement by both PDGFRβ-positive pericytes and CD45-positive cells in kidney fibrosis. Inhibition of complement activation represents a novel therapeutic target to ameliorate fibrosis and progression of chronic kidney disease.

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“…Lung tissue was dissociated into a single-cell suspension using a mouse lung dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) with the gentle gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described (19). The purity of the endothelial cell population was confirmed by flow cytometry, as previously described (17).…”

Section: Lung Endothelial Cell Isolationmentioning

confidence: 99%

Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury

Shah¹,

Romero²,

Guo³

et al. 2017

Am J Respir Cell Mol Biol

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Obesity is a significant risk factor for acute respiratory distress syndrome. The mechanisms underlying this association are unknown. We recently showed that diet-induced obese mice exhibit pulmonary vascular endothelial dysfunction, which is associated with enhanced susceptibility to LPS-induced acute lung injury. Here, we demonstrate that lung endothelial dysfunction in diet-induced obese mice coincides with increased endoplasmic reticulum (ER) stress. Specifically, we observed enhanced expression of the major sensors of misfolded proteins, including protein kinase R-like ER kinase, inositol-requiring enzyme α, and activating transcription factor 6, in whole lung and in primary lung endothelial cells isolated from diet-induced obese mice. Furthermore, we found that primary lung endothelial cells exposed to serum from obese mice, or to saturated fatty acids that mimic obese serum, resulted in enhanced expression of markers of ER stress and the induction of other biological responses that typify the lung endothelium of diet-induced obese mice, including an increase in expression of endothelial adhesion molecules and a decrease in expression of endothelial cell-cell junctional proteins. Similar changes were observed in lung endothelial cells and in whole-lung tissue after exposure to tunicamycin, a compound that causes ER stress by blocking N-linked glycosylation, indicating that ER stress causes endothelial dysfunction in the lung. Treatment with 4-phenylbutyric acid, a chemical protein chaperone that reduces ER stress, restored vascular endothelial cell expression of adhesion molecules and protected against LPS-induced acute lung injury in diet-induced obese mice. Our work indicates that fatty acids in obese serum induce ER stress in the pulmonary endothelium, leading to pulmonary endothelial cell dysfunction. Our work suggests that reducing protein load in the ER of pulmonary endothelial cells might protect against acute respiratory distress syndrome in obese individuals.

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C1q Deficiency Promotes Pulmonary Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury

Cited by 21 publications

References 42 publications

A simple protocol for isolating mouse lung endothelial cells

A simple protocol for isolating mouse lung endothelial cells

Pericytes and immune cells contribute to complement activation in tubulointerstitial fibrosis

Obesity-Induced Endoplasmic Reticulum Stress Causes Lung Endothelial Dysfunction and Promotes Acute Lung Injury

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