The mechanism and biomarker function of Cavin-2 in lung ischemia-reperfusion injury

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Abstract

Background

Lung Ischemia Reperfusion injury(LIRI) is one of the most predominant complications of ischemic lung disease. Cavin-2 emerged as a regulator of a variety of cellular processes, including endocytosis, lipid homeostasis, signal transduction and tumorigenesis, but the function of Cavin-2 in LIRI is unknown. The purpose of this study was to determine the predictive potential of Cavin-2 in protecting lung ischemia-reperfusion injury and its corresponding mechanisms.

Methods

We found the strong relationship between Cavin-2 and multiple immune-related genes by deep learning method. To reveal the mechanism of Cavin-2 in LIRI, the LIRI SD rat model was constructed to detect the expression of Cavin-2 in the lung tissue of SD rats after LIRI, and the expression of Cavin-2 in lung cell lines was also detected. The expression of IL-6, IL-10 and MDA in cells after Cavin-2 over-expression or knockdown was examined under hypoxic conditions. The expression levels of p-AKT, p-STAT3 and p-ERK1/2 were measured in over-expressing Cavin-2 cells under hypoxic-ischemia conditions, and then the corresponding blockers of AKT, STAT3 and ERK1/2 were given to verify, whether they play a protective role in LIRI.

Results

After hypoxia, the expression of Cavin-2 in rat lung tissues was significantly increased, and the cellular activity and IL-10 in Cavin-2 over-expressing cells were significantly higher than that of the control group, while IL-6 and MDA were significantly lower than that of the control group, while the above results were reversed in Cavin-2 knockdown cells; Meanwhile, the phosphorylation levels of AKT, STAT3, and ERK1/2 were significantly increased in Cavin-2 over-expression cells after hypoxia. When AKT, STAT3, and ERK1/2 specific blockers were given, they lost their protective effect against LIRI.

Conclusions

Cavin-2 shows biomarker potential in protecting lung from ischemia-reperfusion injury through the survivor activating factor enhancement (SAFE) and reperfusion injury salvage kinase (RISK) pathway.

Introduction

Lung ischemia and reperfusion injury is a common clinical acute event, which can be caused by many reasons, such as lung transplantation, pulmonary embolism, and cardiopulmonary bypass [1,2]. The characteristic of LIRI is that after the first attack of ischemia and hypoxia, than the lung is reperfused with blood, which will cause more damage [[3], [4]]. How to restore the blood perfusion in time and how to restrain the injury after reperfusion are the primary question of LIRI therapy.

Caveolae is a kind of plasma membrane with flask like depression, which is rich in cholesterol, sphingolipids, caveolin and the coat protein cavin [[5], [6], [7], [8]]. In cells, signal molecules aggregate and organize in Caveolae to mediate signal transduction. Recent studies have shown that Caveolae and Caveolae related signaling molecules play an important role in protecting cells from ischemia-reperfusion injury [9,10]. Multiple researches have reported the relationship between caveloae and LIRI [[11], [12]]. For example, cardiac specific Caveolin-3 over-expression has been shown to mimic ischemic pretreatment, while caveolin-3 gene knockout can eliminate ischemic pretreatment [13,14].

Caveolae and its main component Caveolins are involved in many cellular pathophysiological processes, including lipid homeostasis, signal transduction, endocytosis and tumorigenesis. Caveolins is a new membrane protein family, which plays an important role in caveolae formation. The researches show that Cavins can stabilize the structure of caveolae and regulate the function of Caveolins [15]. Up to now, Cavins contain four different proteins, which are Cavin-1, Cavin-2, Cavin-3 and Cavin-4. Cavin-1 and Cavin-2 are the necessary conditions for the formation of Caveolae. Therefore, the researchers speculated that Cavin-1 and Cavin-2 may be related to tumor occurrence [[16], [17], [18]], Cavin-3 may be a risk factor for depression, and Cavin-4 may be related to Caveolin related muscle diseases [[19], [20], [21], [22], [23]].

Cavin-2 (serum deprivation protein response, SDPR) maps to chromosome 2q32-33, which shares more than 20% similarities with cavin-1, and is firstly shown in vitro to be a substrate for protein kinase C (PKC) isoforms by Burgener in 1990 [24]. Cavin-2 plays an important role in the formation of Caveolae in lung and fat. Compared with other tissues, the apparent size of Cavin protein complex in lung and fat is smaller. Cavin-2 is responsible for determining the size or stability of Cavin complex. The loss of Cavin-2 increases the apparent size of Cavin complex. In the lung endothelial cells and cultured lung endothelial cells, the residual Caveolae depth detected without Cavin-2 decreased [[25], [26], [27]]. In addition, Cavin-1 and Caveolin 1 were also detected in Cavin-2 −/− cells, in the flat area and shallow cavity of plasma membrane. We believe that the endothelial fovea in different tissues is heterogeneous, and Cavin-2 plays an important role in the formation of this heterogeneity. The role of Cavin-2 in the morphogenesis of fovea is directly related to the shape of the fovea membrane [28]. Therefore,Cavin-2 could be the important factor in ischemia reperfusion(I/R),which is protected by Caveolae.

A large number of experimental studies have shown whether pre-treatment or post-treatment, they all play the protective role of ischemia-reperfusion injury by activating a series of survival promoting signal transduction pathways in the early stage of reperfusion [29]. Among them, it is generally recognized that blocking the opening of mitochondrial permeability transition pore (mPTP) by activating the signal transduction pathway of reperfusion injury salvage kinase (RISK) is a common pathway to alleviate myocardial ischemia reperfusion injury (MIRI) [30]. RISK pathways is composed of activation of the phosphatidylinositol 3-kinase/Akt (PI3K/AKT) and extracellular signal regulated kinase 1/2 (ERK1/2) [31]. The survival activating factor enhancement (SAFE) pathway is composed of tumor necrosis factor-a (TNF-a), signal transducer and activator of transcription-3 (STAT3). STAT3 is another important survival promoting signal transduction pathway to reduce MIRI in the early stage of reperfusion, and STAT3 is the most important downstream target of SAFE pathway [[32], [33]]. In addition, multiple researchers have paid attention to the computational methods. These methods have been widely used in genomics [34], transcriptomics [35], proteomics [[36], [37], [38], [39]], metabolomics [[40], [41], [42], [43]]. These tools and methods fully expand the application of various omics data and the interpretation of disease mechanisms.

In this study, we will investigate whether Cavin-2 can protect lung tissue and reduce LIRI by RISK or SAFE signal transduction pathway.

Section snippets

Predicting the Cavin-2 interaction network by deep learning

Cavin-2 is also called SDPR. As shown in Fig. 1, we searched its gene interaction network in String database and found that 10 genes are reported to interact with it. Due to the complex regulation relationship between genes, we used Graph convolutional network (GCN) as other studies [[44], [45], [46]] to predict genes that can interact with Cavin-2 in large scale.

Since the interaction between genes is a complex network association, we use GCN to extract the hierarchical structure of the

Statistical analysis

All datum were recored as Mean ± standard deviation(SD) and SPSS20.0 software(Chicago,IL,USA) was used for data analysis. The non-parametric Mann-Whitney U test was used to compare the differences between two groups. One-way analysis of variance was performed for among groups comparison of means.DUNNETT’S T3 analysis was used for comparing uneven variance.P < 0.05 was considered statistically significant.

Hypoxia-reoxygenation promotes the expression of Cavin-2

The results showed that the expression of Cavin-2 mRNA and protein was significantly higher than that of sham operated group (P < 0.05) (Fig. 3,A and B, C). At the same time, the expression of Cavin-2 in lung cell lines HPMEC, Beas-2B and HPAEpiC was detected. The results showed that the expression of Cavin-2 was the highest in Beas-2B, and the expression of HPAEpiC was the lowest (Fig. 3, D). The expression of Cavin-2 in Beas-2B cell line was detected after being treated with hypoxia in

Discussion

Lung Ischemia-reperfusion injury is a complex pathophysiological process. The imbalance between organ oxygen supply and metabolic oxygen consumption leads to tissue hypoxia, cell damage or death. At the same time of resuming tissue oxygen supply, reperfusion will aggravate tissue damage due to active oxygen and inflammatory cell recruitment. Because the lung has the double blood supply of pulmonary artery and bronchial artery, and the double oxygen supply of exchange through blood and alveolus

Disclosure of conflict of interest

The authors confirm that there are no conflicts of interest.Address Co-correspondence to: Changsheng Li and Xuefeng Zhou, The Department of thoracic surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071,China. E-mail: [email protected]; [email protected].

Declarations of competing interest

The authors declare that they have no competing interests.

Acknowledgements

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