The dataset describes: Phenotypic changes induced by cholesterol loading in smooth muscle cells isolated from the aortae of C57BL/6 mice

The data presented in this article is related to the research article entitled “ABCA1 and HDL3 are Required to Modulate Smooth Muscle Cells Phenotypic Switch after Cholesterol Loading” (Castiglioni et al., 2017) [1]. This data describes the characterization of the phenotypic changes induced by cholesterol loading in smooth muscle cells (SMCs) isolated from the aortae of C57BL/6 mice. Upon cholesterol loading, there is a significant and concentration-dependent decrease in the expression of Acta2 and a parallel increase in Mac-2, and ATP binding cassette (ABC) transporters Abca1 and Abcg1. Cholesterol incubation causes the transformation of SMCs into foam cells with a 3-fold increase in cellular total cholesterol content and a 2.5-fold stimulation of the activity of the esterifying enzyme Acyl-CoA:cholesterol acyltransferase (ACAT). The addition of the same amount of cholesterol, either dissolved in ethanol or as lipoprotein cholesterol (AcLDL or native LDL) only slightly induces the activity of the enzyme ACAT, and does not cause the accumulation of lipid droplets into SMCs. We describe also the knock down of ABCA1 expression by siRNA treatment in mouse smooth muscle cells.


a b s t r a c t
The data presented in this article is related to the research article entitled "ABCA1 and HDL 3 are Required to Modulate Smooth Muscle Cells Phenotypic Switch after Cholesterol Loading" (Castiglioni et al., 2017) [1]. This data describes the characterization of the phenotypic changes induced by cholesterol loading in smooth muscle cells (SMCs) isolated from the aortae of C57BL/6 mice. Upon cholesterol loading, there is a significant and concentration-dependent decrease in the expression of Acta2 and a parallel increase in Mac-2, and ATP binding cassette (ABC) transporters Abca1 and Abcg1. Cholesterol incubation causes the transformation of SMCs into foam cells with a 3-fold increase in cellular total cholesterol content and a 2.5-fold stimulation of the activity of the esterifying enzyme Acyl-CoA:cholesterol acyltransferase (ACAT). The addition of the same amount of cholesterol, either dissolved in ethanol or as lipoprotein cholesterol (AcLDL or native LDL) only slightly induces the activity of the enzyme ACAT, and does not cause the accumulation of lipid droplets into SMCs. We describe also the knock down of ABCA1 expression by siRNA treatment in mouse smooth muscle cells. Real time PCR and western blot analyses were performed on mouse smooth muscle cells previously incubated with cholesterol complexed with methyl-betacyclodextrin Data source location

Milan, Italy
Data accessibility Data is within this article.

Value of the data
This data describes the cholesterol-induced phenotypic changes in murine smooth muscle cells. Cholesterol loading downregulates the expression of smooth muscle cell markers, and increases the expression of inflammation-related genes. In addition, cholesterol transforms smooth muscle cells into foam cells and affects lipid metabolism.
The present data gives insights on how deliver cholesterol to metabolically active intracellular lipid pools, independently of lipoprotein receptor pathway.

Data
This dataset extends and completes the data presented in the recently published article [1]. Cholesterol loading of smooth muscle cells (SMCs) isolated from the aortae of C57BL/6 mice causes a significant and concentration-dependent decrease in Acta2 mRNA levels ( Fig. 1A) and an increase in Mac-2, Abca1 and Abcg1 mRNA (up to 10-fold, 16-fold and 160-fold, respectively). The effects on mRNAs are confirmed by western blot analysis (Fig. 1B). Cholesterol incubation causes the transformation of SMCs into foam cells, with intracellular accumulation of lipid droplets (Oil Red O staining; Fig. 2A). There is a 3-fold increase in cellular total cholesterol content, due to the accumulation of both free and esterified cholesterol (Fig. 2B). The increased cellular esterified cholesterol content is caused by a 2.5-fold stimulation of the activity of the esterifying enzyme ACAT that is completely blocked by the addition of Sandoz 58-035, a specific ACAT inhibitor (Fig. 3A). The addition of the same amount of cholesterol, either dissolved in ethanol or as lipoprotein cholesterol (native low-density lipoprotein (LDL) or Acetylated LDL) slightly induces the activity of the ACAT enzyme (Fig. 3A), and does not cause the accumulation of lipid droplets into SMCs (data not shown). The cholesterol delivered is available for downloading in the presence of HDL 3 (Fig. 3B).
As shown in Fig. 4, the incubation of WT SMC with Abca1 siRNA reduces Abca1 expression to the levels observed in Abca1 KO cells, as measured by either qRT-PCR or western blot analysis (Fig. 4).

Cell cultures
SMCs were isolated from the intimal-medial layer of aortae of littermate Abca1 WT and KO mice of both sexes (The Jackson Lab, Bar Harbor, ME, USA). The mice, originally on a DBA background, have been backcrossed into C57BL/6 mice for at least nine times. All mice were kept in accordance with guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and the Italian Ministry of Health and the local University of Milan ethics committee approved the protocol. Mice were anesthetized with 2% isoflurane and killed by cervical dislocation. Aorta was rapidly dissected from the aortic root to the iliac bifurcation and SMCs prepared according to the procedure described by Ross [2].
Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% FCS. The medium was changed every three days. SMC lineage was confirmed by the presence of immunoreactivity for Acta2 in 4 99% of the cells. The experiments have been performed using 8 cell lines isolated from different mice of both genotypes. Cells were used between the 4th and 10th passage.

HDL 3 isolation
HDL 3 were isolated from the plasma of healthy normolipidemic volunteers by sequential preparative ultracentrifugation [3].

Oil Red O staining
Cholesterol-loaded SMCs were fixed for 30 min with 4% paraformaldehyde solution in PBS, stained with Oil Red O for 4 h and counterstained with hematoxylin for other 5 min [6]. Cells were examined with a light microscope (X80) and pictures of representative fields were taken.

Cholesterol esterification
After cholesterol loading, 14 C-oleic acid was added and cholesterol esterification measured following the incorporation of labelled oleate into cellular cholesteryl esters [7].

Extraction and analysis of cellular cholesterol
Cell lipids were extracted in hexane/isopropyl alcohol (3:2) containing BHT 0.01%. Free-and esterified cholesterol were separated by HPTLC plates and quantified on a DANI 1000 gas liquid chromatographer [8]. Results were normalized by protein content.

mRNA and miRNA levels measurement
Total RNA was extracted by using the TRIZOL solution (Invitrogen) according to manufacturer's protocol and reverse transcribed (Thermo Scientific). SYBR Green-based real time PCR was used to measure mRNA or miRNA levels [9]. The primers used for specific mouse genes are listed in Table 1.

Table 1
Sequences of mouse primers. α-tubulin (1.5000; Sigma-Aldrich). Quantification was performed by densitometric analysis using the Image Studio Lite software from Li-Cor Bioscience.

Statistical analysis
Data were expressed as means 7 SD. All experiments were repeated at least four-five times in triplicates.