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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) . 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.
Value of the data
This dataset extends and completes the data presented in the recently published article . 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 HDL3 (Fig. 3B).
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 .
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 > 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.
HDL3 were isolated from the plasma of healthy normolipidemic volunteers by sequential preparative ultracentrifugation .
Cholesterol was delivered to cells by using a Chol:MβCD complex as ‘‘water-soluble cholesterol’’ containing ≈ 50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MβCD, Sigma-Aldrich) , . All treatment concentrations involving Chol:MβCD were based on cholesterol weight. SMCs were incubated with DMEM supplemented with 0.2% Essential Fatty Acid Free albumin alone or containing Chol:MβCD (50 µg/ml) or HDL3 (100 µg/ml) or apoAI (35 µg/ml) for 48 h.
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 . Cells were examined with a light microscope (X80) and pictures of representative fields were taken.
After cholesterol loading, 14C-oleic acid was added and cholesterol esterification measured following the incorporation of labelled oleate into cellular cholesteryl esters .
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 . Results were normalized by protein content.
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 . The primers used for specific mouse genes are listed in Table 1.
Wild type SMCs were transfected for 24 h with Abca1 siRNA (ON-TARGETplus SMARTpool Mouse Abca1, Dharmacon) or a nonsense strand of siRNA (scramble, ON-TARGETplus Non-Targeting pool, Dharmacon), using INTERFERin siRNA transfection reagent (Polyplus). Then, cells were treated for 48 hours with Chol:MβCD (50 μg/mL) in presence or absence of HDL3 (100 μg/mL). Total mRNA was extracted and subjected to qRT-PCR analysis.
Mouse SMCs were processed for Western blotting as described . The different proteins were detected using the following primary antibodies: Abca1 (1:1000; Abcam), Abcg1 (1:500; Novus Biologicals), Acta2 (1:10,000; Abcam), Mac-2 (1:1000: Abcam), β-actin (1:5000; Sigma-Aldrich), α-tubulin (1.5000; Sigma-Aldrich). Quantification was performed by densitometric analysis using the Image Studio Lite software from Li-Cor Bioscience.
Data were expressed as means ± SD. All experiments were repeated at least four-five times in triplicates.
Transparency documentTransparency document associated with this article can be found in the online version at https://doi.org/10.1016/j.dib.2017.11.050