602: Expression and function of ATP-binding cassette and solute carrier family transporters in isolated human placental microvascular endothelial cells

Abstract

To identify and characterize the expression and function of ABC and SLC transporter proteins in isolated human placental microvascular endothelial cells (HPMVEC). This study was performed at a single center utilizing placentas from non-laboring cesarean delivery patients. Isolation of HPMVEC was accomplished through collagenase digestion of the placenta followed by positive selection with CD31 microbeads. Cell purity was determined by immunofluorescence staining for UEA-1 lectin, a robust endothelial marker. Relative mRNA abundance of ABC and SLC transporters in HPMVEC (n=4) was measured by qrtPCR using validated primers present on RT2 Profiler PCR array. The mean delta cycle threshold (ΔCq) was calculated by the difference in threshold cycle between the target gene and reference gene (RPLP0) in each sample. Transporter protein expression in HPMVEC was confirmed by Western blot analysis using commercially available antibodies. The ABC efflux transporters P-gp and BCRP are active components of the placental barrier and play an important role in fetal protection. The functional activity of P-gp and BCRP was assessed by measuring the intracellular accumulation of calcein AM and Hoescht 33342, in the presence or absence of transporter specific inhibitors. We obtained a proportion of 90% lectin-positive HPMVEC which was considered to be of sufficient purity (Fig 1). Transcripts from multiple ABC and SLC transporters were present in HPMVEC (Fig 2). BCRP was expressed in placental syncytium and vascular endothelium, while P-gp was predominately localized to the syncytiotrophoblasts. However, P-gp functional activity was present in HPMVEC, which showed a 64% increase in Calcein AM accumulation after 60-minute incubation in the presence of a P-gp inhibitor Elacridar (p<0.05). There was no calculated difference in Hoechst 33342 accumulation in the presence of the BCRP selective blocker Ko143. HPMVEC line blood vessels of the villous tree, where they form the last line of defense to the fetal circulation. The expression and function of uptake and efflux transporter proteins in HPMVEC may be as important as their expression in syncytiotrophoblasts in regulating transplacental transport of drugs and xenobiotics.View Large Image Figure ViewerDownload Hi-res image Download (PPT)