Short-term effects of 7-ketocholesterol on human adipose tissue mesenchymal stem cells in vitro

https://doi.org/10.1016/j.bbrc.2014.01.132Get rights and content

Highlights

  • 7-Ketocholesterol was incubated with mesenchymal stem cells from human adipose tissue.

  • 7-KC promoted dose-dependent hyperpolarization of mitochondria membrane.

  • High concentrations of 7-KC led to increased apoptosis and alterations of F-actin.

Abstract

Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Among them, 7-ketocholesterol (7-KC) is one of the most important. It has potent effects in cell death processes, including cytoxicity and apoptosis induction. Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Very little is known about the effects of oxysterols in MSCs. Here, we describe the short-term cytotoxic effect of 7-ketocholesterol on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from two young, healthy women. After 24 h incubation with 7-KC, mitochondrial hyperpolarization was observed, followed by a slight increase in the level of apoptosis and changes in actin organization. Finally, the IC50 of 7-KC was higher in these cells than has been observed or described in other normal or cancer cell lines.

Introduction

Maintenance of the cellular redox balance is crucial for cell survival. Lipids are very sensitive to oxidative modifications [1]. Lipid oxidation is characterized by complex product patterns, including lipid peroxides, aldehydes, and many others. Moreover, molecules with regulatory functions are generated by the oxidation of lipids and fatty acids [2], [3], [4], [5], [6]. Oxidized phospholipids are recognized as important mediators of cellular signaling [7]. Oxidative stress, antioxidant efficiency, and lipid oxidizability are known to change in different pathophysiological conditions [8].

Cholesterol is an important structural element of cell membranes and an essential substrate for the biosynthesis of several molecules, such as bile acids and steroid hormones [9]. It is transported in plasma mainly by low-density lipoprotein (LDL). The cholesterol molecule is easily oxidized and may be transformed into numerous oxidation products known as oxysterols. They can be considered a way to route the cholesterol molecule for catabolism.

Oxysterols comprise a highly heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation [10]. Among them, 7-ketocholesterol (7-KC) is one of the most important, and is found in relatively large abundance in oxidized low-density lipoprotein (oxLDL) [11], [12].

Oxysterols exhibit several biological activities. They play essential roles in a number of physiological processes, such as cholesterol homeostasis regulation, platelet aggregation, and protein prenylation [13]. They participate in the control of lipid metabolism and regulation of the immune system, and have been associated with several other pathophysiological processes [14].

Oxysterols have potent effects on cell death processes, including apoptosis induction [15], [16]; reactive oxygen species (ROS) are reportedly involved in this effect [15]. In fact, oxysterols have been shown to exhibit cytotoxicity in a number of cell lines, including smooth muscle cells, fibroblasts, and vascular endothelial cells [17].

Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation abilities [18]. In vitro, according to the Society for Cellular Therapy, MSCs are described as cells that are capable of adhering to plastic with fibroblast-like morphology and differentiating into mesenchymal lineages (e.g., chondrogenic, osteogenic, and adipogenic lineages) under appropriate culture conditions. They are also characterized by the expression of CD105, CD73, and CD90, as well as by the lack of expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA class II. The property of self-renewal is essential for the expansion of the stem cell pool during fetal development, as well for maintenance of the stem cell pool throughout the organism’s lifespan [19].

Several oxysterols exert important effects on cell death, proliferation, and differentiation: 7-ketocholesterol is known to induces monocyte differentiation [11]; and 22(R)-hydroxycholesterol and 25-hydroxycholesterol are potent inhibitors of cell growth in thymocytes, lymphocytes, and keratinocytes, as well as inducers of keratinocyte differentiation [20]. However, very little is known about the effects of oxysterols in MSCs. There are few descriptions, and most are related to their osteogenic effect. In fact, some types of oxysterols can behave as anti-adipogenic signals by diverting pluripotent MSCs away from adipogenic and toward osteogenic differentiation [14].

Here we described the short-term cytotoxic effect of 7-ketocholesterol on MSCs derived from human adipose tissue.

Section snippets

Human adipose tissue mesenchymal stem cell isolation and characterization

Adipose tissue was obtained from two young, healthy women (20 and 22 year old) that underwent abdominal plastic surgery for esthetic reasons. The Ethical Committee of the Institution approved the protocol for this study, and patients provided written informed consent. With more than 90% of the volume, adipocytes have been described to represent the major cell type in lipoaspirate material [21]. From each patient, 30 ml of fatty material was collected in a sterile flask. The tissue was dissociated

Results

We used several methods to characterize hAMSCs. First, hAMSCs were isolated from the two samples by their adherence to plastic. Second, hAMSCs expressed the transcription factors Oct-4 and Nanog, as determined by RT-PCR (data not shown). Both markers confirm the undifferentiated state. Third, hAMSCs were cultivated in specific osteogenic and chondrogenic differentiation media to evaluate their plasticity and multipotency. Differentiation was confirmed after 21 days (data not shown). Finally,

Discussion

MSCs were isolated from two human adipose tissue samples. They exhibited all characteristics of MSCs: adherence to plastic, fibroblast-like morphology, expression of the undifferentiated genes Oct-4 and Nanog, membrane markers (CD90+, CD73+, CD49d, and CD34), and osteogenic and chondrogenic differentiation ability.

Oxidative stress is a key pro-apoptotic factor. It results in excessive accumulation of ROS, which directly damage cell membranes, protein, and DNA [26]. There are several reports

Acknowledgments

The authors thank Silvana Cereijido Altram for technical assistance. This work was supported by a grant from Financiadora de Estudos e Projetos (FINEP), Brazil; by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil; and by the Instituto Nacional de Ciencia e Tecnologia – Fluidos Complexos (INCT-FCx), Brazil. It was presented as oral presentation at the 3rd European Network on Oxysterol Research (ENOR) symposium ‘OXYSTEROLS: markers and Pathways’ in Swansea, UK, 19–20

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      The arterial microenvironment is a multicellular dynamic compartment that, among other systemic factors, is continuously stimulated by 7KC. Moreover, 7KC has been shown to induce apoptosis in a variety of cells, including those constituting arterial microenvironment such as endothelial cells, smooth muscle cells, and macrophages at physiological concentrations (Nury et al., 2013; Levy et al., 2014; Luchetti et al., 2015; Silva et al., 2017), besides promoting endothelial dysfunction, including endothelial-dependent arterial relaxation (Deckert et al., 2002; Son et al., 2015; Luchetti et al., 2015). Endothelial cells (ECs) constitute the major cell type in the lining of blood vessels and it is well known that their secreted factors, including proteins, are crucial players in the modulation of vascular biology (Bydlowski et al., 1987a,b; Eele et al., 2015).

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