Poldip2 deficiency protects against lung edema and vascular inflammation in a model of acute respiratory distress syndrome

Forrester, Steven J.; Xu, Qian; Kikuchi, Daniel S.; Okwan-Duodu, Derick; Campos, Ana Carolina Pinheiro; Faidley, Elizabeth A.; Zhang, Guogang; Lassègue, Bernard; Sadikot, Ruxana T.; Griendling, Kathy K.; Hernandes, Marina S.

doi:10.1042/cs20180944

Cited by 20 publications

(31 citation statements)

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“…Furthermore, the pathways identified here are consistent with previous studies from our laboratory performed in cultured cells [10, 31, 32], which revealed the importance of POLDIP2 in cytoskeletal dynamics. Our results are also consistent with studies carried out in Poldip2 gene trap mice and cells derived from them [1, 4, 7, 12, 25, 26, 30, 36, 37, 40, 51], which provided insights into the functions of Poldip2 in the vasculature, in inflammation and mitochondrial function. Therefore, with the exception of homozygous lethality, data provided by the gene trap model are in agreement with the present results.…”

Section: Discussionsupporting

confidence: 92%

“…The integrity of the blood brain barrier was also preserved by intracerebral administration of Poldip2 siRNA in a mouse meningitis model [42]. Similarly, Poldip2 +/- mice are protected against lung edema and inflammation after LPS [1]. These studies suggest that POLDIP2 is a promising therapeutic target for pathologies involving breakdown of a barrier, such as stroke and sepsis, for which new treatments are urgently needed.…”

Section: Introductionmentioning

confidence: 99%

“…POLDIP2 is a protein with a surprisingly large variety of roles, affecting apparently unrelated cellular processes and physiological functions. This pleiotropism of POLDIP2 results in part from its expression in most tissues [1][2][3][4][5][6][7][8][9][10][11][12] and subcellular compartments, including the nucleus, cytosol and mitochondria [13][14][15]. In addition, POLDIP2 can potentially bind to numerous other proteins, currently nearly 200, according to its NCBI human gene record.…”

Section: Introductionmentioning

confidence: 99%

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Characterization ofPoldip2knockout mice: avoiding incorrect gene targeting

Lassègue

Kumar

Mandavilli

et al. 2021

Preprint

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POLDIP2 is a multifunctional protein whose roles are only partially understood. Our laboratory previously reported physiological studies performed using a mouse gene trap model, which suffered from two limitations: perinatal lethality in homozygotes and constitutive Poldip2 inactivation. To overcome these limitations, we developed a new conditional floxed Poldip2 model. The first part of the present study shows that our initial floxed mice were affected by an unexpected mutation, which was not readily detected by Southern blotting and traditional PCR. It consisted of a 305 kb duplication around Poldip2 with retention of the wild type allele and could be traced back to the original targeted ES cell clone. We offer simple suggestions to rapidly detect similar accidents, which may affect genome editing using both traditional and CRISPR-based methods. In the second part of the present study, correctly targeted floxed Poldip2 mice were generated and used to produce a new constitutive knockout line by crossing with a Cre deleter. In contrast to the gene trap model, many homozygous knockout mice were viable, in spite of having no POLDIP2 expression. To further characterize the effects of Poldip2 ablation in the vasculature, an RNA-seq experiment was performed in constitutive knockout carotid arteries. Results support the involvement of POLDIP2 in multiple cellular processes and provide new opportunities for future in-depth study of its functions.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization ofPoldip2knockout mice: avoiding incorrect gene targeting

Lassègue

Kumar

Mandavilli

et al. 2021

Preprint

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“…Evans blue extravasation into whole brains was measured in Poldip2 +/+ and Poldip2 +/− mice after 18 h of LPS treatment (Fig. 1a) since we previously reported that there is a 57% mortality rate among Poldip2 +/+ animals after 20 h of LPS treatment (18 mg/kg) [32]. The large increase in Evans blue extravasation in Poldip2 +/+ mice was significantly reduced in Poldip2 +/− mice (Fig.…”

Section: Resultsmentioning

confidence: 80%

Poldip2 mediates blood-brain barrier disruption in a model of sepsis-associated encephalopathy

Kikuchi

Campos

et al. 2019

J Neuroinflammation

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BackgroundSepsis-associated encephalopathy (SAE), a diffuse cerebral dysfunction in the absence of direct CNS infection, is associated with increased rates of mortality and morbidity in patients with sepsis. Increased cytokine production and disruption of the blood-brain barrier (BBB) are implicated in the pathogenesis of SAE. The induction of pro-inflammatory mediators is driven, in part, by activation of NF-κΒ. Lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, potently activates NF-κΒ and its downstream targets, including cyclooxygenase-2 (Cox-2). Cox-2 catalyzes prostaglandin synthesis and in the brain prostaglandin, E2 is capable of inducing endothelial permeability. Depletion of polymerase δ-interacting protein 2 (Poldip2) has previously been reported to attenuate BBB disruption, possibly via regulation of NF-κΒ, in response to ischemic stroke. Here we investigated Poldip2 as a novel regulator of NF-κΒ/cyclooxygenase-2 signaling in an LPS model of SAE.MethodsIntraperitoneal injections of LPS (18 mg/kg) were used to induce BBB disruption in Poldip2+/+ and Poldip2+/− mice. Changes in cerebral vascular permeability and the effect of meloxicam, a selective Cox-2 inhibitor, were assessed by Evans blue dye extravasation. Cerebral cortices of Poldip2+/+ and Poldip2+/− mice were further evaluated by immunoblotting and ELISA. To investigate the role of endothelial Poldip2, immunofluorescence microscopy and immunoblotting were performed to study the effect of siPoldip2 on LPS-mediated NF-κΒ subunit p65 translocation and Cox-2 induction in rat brain microvascular endothelial cells. Finally, FITC-dextran transwell assay was used to assess the effect of siPoldip2 on LPS-induced endothelial permeability.ResultsHeterozygous deletion of Poldip2 conferred protection against LPS-induced BBB permeability. Alterations in Poldip2+/+ BBB integrity were preceded by induction of Poldip2, p65, and Cox-2, which was not observed in Poldip2+/− mice. Consistent with these findings, prostaglandin E2 levels were significantly elevated in Poldip2+/+ cerebral cortices compared to Poldip2+/− cortices. Treatment with meloxicam attenuated LPS-induced BBB permeability in Poldip2+/+ mice, while having no significant effect in Poldip2+/− mice. Moreover, silencing of Poldip2 in vitro blocked LPS-induced p65 nuclear translocation, Cox-2 expression, and endothelial permeability.ConclusionsThese data suggest Poldip2 mediates LPS-induced BBB disruption by regulating NF-κΒ subunit p65 activation and Cox-2 and prostaglandin E2 induction. Consequently, targeted inhibition of Poldip2 may provide clinical benefit in the prevention of sepsis-induced BBB disruption.Electronic supplementary materialThe online version of this article (10.1186/s12974-019-1575-4) contains supplementary material, which is available to authorized users.

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“…Sepsis is a common condition that can lead to ICU hospitalization and cause damage to multiple organs, among which the lung is the most vulnerable (Kuebler, 2019;Liu et al, 2020b). Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common complications of sepsis that are often lifethreatening (Van Wessem and Leenen, 2018;Forrester et al, 2019). Although great progress has been made for the treatment of ALI, the death rate is still as high as 40% (Villar et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Knockdown of lncRNA MALAT1 Alleviates LPS-Induced Acute Lung Injury via Inhibiting Apoptosis Through the miR-194-5p/FOXP2 Axis

Nan

Zhang

Cheung

et al. 2020

Front. Cell Dev. Biol.

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Purpose: We aimed to identify and verify the key genes and lncRNAs associated with acute lung injury (ALI) and explore the pathogenesis of ALI. Research showed that lower expression of the lncRNA metastasis-associated lung carcinoma transcript 1 (MALAT1) alleviates lung injury induced by lipopolysaccharide (LPS). Nevertheless, the mechanisms of MALAT1 on cellular apoptosis remain unclear in LPS-stimulated ALI. We investigated the mechanism of MALAT1 in modulating the apoptosis of LPS-induced human pulmonary alveolar epithelial cells (HPAEpiC). Methods: Differentially expressed lncRNAs between the ALI samples and normal controls were identified using gene expression profiles. ALI-related genes were determined by the overlap of differentially expressed genes (DEGs), genes correlated with lung, genes correlated with key lncRNAs, and genes sharing significantly high proportions of microRNA targets with MALAT1. Quantitative real-time PCR (qPCR) was applied to detect the expression of MALAT1, microRNA (miR)-194-5p, and forkhead box P2 (FOXP2) mRNA in 1 µg/ml LPS-treated HPAEpiC. MALAT1 knockdown vectors, miR-194-5p inhibitors, and ov-FOXP2 were constructed and used to transfect HPAEpiC. The influence of MALAT1 knockdown on LPS-induced HPAEpiC proliferation and apoptosis via the miR-194-5p/FOXP2 axis was determined using Cell counting kit-8 (CCK-8) assay, flow cytometry, and Western blotting analysis, respectively. The interactions between MALAT1, miR-194-5p, and FOXP2 were verified using dualluciferase reporter gene assay. Results: We identified a key lncRNA (MALAT1) and three key genes (EYA1, WNT5A, and FOXP2) that are closely correlated with the pathogenesis of ALI. LPS stimulation promoted MALAT1 expression and apoptosis and also inhibited HPAEpiC viability. MALAT1 knockdown significantly improved viability and suppressed the apoptosis of LPS-stimulated HPAEpiC. Moreover, MALAT1 directly targeted miR-194-5p, a downregulated miRNA in LPS-stimulated HPAEpiC, when FOXP2 was overexpressed.

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Poldip2 deficiency protects against lung edema and vascular inflammation in a model of acute respiratory distress syndrome

Cited by 20 publications

References 40 publications

Characterization ofPoldip2knockout mice: avoiding incorrect gene targeting

Characterization ofPoldip2knockout mice: avoiding incorrect gene targeting

Poldip2 mediates blood-brain barrier disruption in a model of sepsis-associated encephalopathy

Knockdown of lncRNA MALAT1 Alleviates LPS-Induced Acute Lung Injury via Inhibiting Apoptosis Through the miR-194-5p/FOXP2 Axis

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