Transcriptional Remodeling of Ion Channel Subunits by Flow Adaptation in Human Coronary Artery Endothelial Cells

Endothelial cells (ECs) are constantly exposed to blood flowinduced shear forces in the vessels and this is a major determinant of endothelial function. Ion channels have a major role in endothelial function and in the control of vascular tone. We hypothesized that shear force is a general regulator of ion channel expression, which will have profound effects on endothelial function. We examined this hypothesis using large-scale quantitative real-time RT-PCR. Human coronary artery ECs were exposed to two levels of flow-induced shear stress for 24 h, while control cells were grown under static conditions. The expression of ion channel subunits was compared between control and flow-adapted cells. We used primers against 55 ion channel and exchanger subunits and were able to detect 54 subunits. Five dyn/cm 2 of shear induced downregulation of 1 (NCX1) and upregulation of 18 subunits, including K Ca 2.2, K Ca 2.3, CX37, K v 1.5 and HCN2. Fifteen dyn/cm 2 of shear stress induced the expression of 30 ion channel subunits, including K Ca 2.3, K Ca 2.2, CX37, K ir 2.3 and K Ca 3.1. Our data demonstrate that substantial remodeling of endothelial ion channel subunit expression occurs Received: July 30, 2010 Accepted after revision: November 23, 2010 Published online: March 9, 2011 Dr. William A. Coetzee Pediatric Cardiology, New York University School of Medicine 560 First Avenue, TCH-521 New York, NY 10016 (USA) Tel. +1 212 263 8518, E-Mail william.coetzee @ med.nyu.edu © 2011 S. Karger AG, Basel Accessible online at: www.karger.com/jvr D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /5 /2 01 7 7: 07 :0 1 P M Kefaloyianni /Coetzee J Vasc Res 2011;48:357–367 358 changes in flow [5] . Moreover, abnormalities in flow-mediated responses of ECs, for example during endothelial dysfunction, are associated with vascular pathology. Accumulating data suggest that disturbed or oscillatory flow is proatherogenic, through the induction of proinflammatory, procoagulant, proliferative and proapoptotic molecules, and sustained laminar flow provides protection from atherosclerosis through the induction of antithrombotic and antiproliferative factors [3, 5, 6] . Among the first cellular components identified to mediate the endothelial response to shear were ion channels. In particular, it was shown that shear stress induces rapid activation of inward-rectifying K + currents that result in membrane hyperpolarization and Ca 2+ entry. Subsequently, various types of Ca 2+ -permeable cation channels, K + channels, and Cl – channels have also been implicated in endothelial mechanosensing [7–11] . The longterm effects that flow has on ion channel expression and function are poorly investigated. It has been reported that flow mediates changes in the expression of specific ion channel subunits in the endothelium. For example, the intermediate-conductance Ca 2+ channel subunit K Ca 3.1 [12] , ATP-sensitive K + (K ATP ) channel subunit K ir 6.2 [13] and the T-type voltage-gated Ca 2+ channel subunit Ca v 3.1 [14] are reported to be upregulated by flow. Based on these findings, we hypothesized that shear force is a general regulator of ion channel expression, which will have profound effects on endothelial function. In order to test our hypothesis, we used a large-scale quantitative RTPCR assay (qRT-PCR) to examine the transcriptional levels of about 60 ion channel and exchanger subunits in ECs that have been cultured under static or flow-adapted conditions. We found that a remarkable transcriptional upregulation of ion channels occurs with flow adaptation that is also dependent on the levels of flow. Materials and Methods Flow Adaptation of ECs Human coronary artery ECs (HCAECs; Lonza, Walkersville, Md., USA) were grown in EGM2-MV (Lonza) and were used between the 4th and 6th passage. HCAECs were exposed to defined amounts of shear stress using an artificial capillary system (FiberCell TM 4300-C2025) as previously described [15] . Artificial capillaries were activated according to the manufacturer’s instructions and precoated with 0.2% gelatin. Cells were allowed to attach for 18 h and constant laminar flow sufficient to generate 5 or 15 dyn/ cm 2 of shear force was applied for 24 h. The two levels of shear stress were selected based on a large body of previously published data. Control cells (same passage and confluence) were grown in plates under static culturing conditions. Large-Scale qRT-PCR We used qRT-PCR to quantify the mRNA expression of a large number of ion channel transcripts, as described previously [16] . We used primers against 56 ion channel and exchanger subunits, designed with the Primer3 software [17] . The selection was made based on expression profiling data obtained from microarray data with ECs and other literature sources. Primers were designed to span two exons to avoid PCR amplification of genomic sequences. Other design considerations included a melting temperature of 60 ° C and a PCR amplicon of 90–120 base pairs. Total RNA was extracted from control and flow-adapted cells by the acid phenol guanidinium method (TriReagent, Sigma-Aldrich, St. Louis, Mo., USA). Total RNA was reverse transcribed using the Superscript III enzyme (Invitrogen) with random hexamer primers according to the manufacturer’s guidelines. PCRs were performed with an ABI Prism 7900HT sequence detection system (Perkin-Elmer Applied Biosystems) with a SYBR green master mix (Perkin-Elmer Applied Biosystems). The thermal cycling conditions comprised an initial denaturing step at 95 ° C for 10 min and 40 cycles at 95 ° C for 5 s, 60 ° C for 15 s, and 72 ° C for 15 s, followed by a melting curve analysis. Data Analysis The PCR threshold cycle (the fractional cycle number at which the fluorescence level is distinguishable from the background, Ct) values were measured within the exponential phase of the PCR using SDS software (version 2.2, Applied Biosystems). Reactions with any evidence of nonspecificity (low melting temperatures or multiple peaks in melting-point analysis) were excluded from the analysis. We performed an initial characterization of the qRTPCR assay in terms of its reproducibility and sensitivity. There was a high degree of reproducibility within plates [16] . The median value of the coefficient of variation of Ct values for triplicate reactions for the data presented in this study was 1.58% (range: 0.01–18.68%). It is increasingly common with qRT-PCR to use a set of reference genes to estimate gene expression levels (e.g. [16, 18] ). We have used this technique in the present study and calculated expression values of each gene relative to the geometric mean of the three reference genes (‘housekeeping’ genes). For a given experiment, the expression of each gene was measured in triplicate (technical replicates), which was averaged to obtain a single expression value for that gene. The values plotted in figure 1 represent the average from 6 independent experiments (biological replicates). The subset of reference genes used included histone H3, hydroxymethylbilane synthase and dihydrofolate reductase, as previously described [16] . The selection of stable reference genes (i.e. that did not change their expression with flow adaptation) was made using the Normfinder algorithm [19] . The relative expression of ion channel subunits calculated for the flow-adapted cells was compared with the relative expression of the subunits calculated for the control cells (cultured under static conditions) to obtain the fold induction or suppression of the subunit expression. Statistical analysis was performed using a nonparametric test, the Significance Analysis of Microarrays (SAM) [20] . For this analysis, we used the Multi Experiment Viewer software (MEV, version 4.5.1); the parameters of the analysis included: use of all unique permutations, selection of S0 using the method of Tusher et al. [20] and cutoff -value at false discovery rate zero. Data in figures 1 and 4 are presented as means 8 SEM. Heat maps were produced using the MEV software. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /5 /2 01 7 7: 07 :0 1 P M Endothelial Flow Adaptation and Ion Channel Remodeling J Vasc Res 2011;48:357–367 359 Analysis of Microarray Data Microarray data produced by different investigators are publicly available in Gene Expression Omnibus provided by the National Center for Biotechnology Information. We examined studies that investigated changes in endothelial gene expression levels due to changes in shear forces. We then looked for changes in ion channel and transporter gene expression. The data used were from the GSE1518 data set record. Raw data were analyzed using the Genespring GX10 software.