Hmvecs d

Human PAECs and human SMCs (PASMCs) were purchased from PromoCell. Human microvascular ECs of the dermis and the lings (MVECs-D and MVECs-L) and human coronary artery ECs (CAEC) were purchased from Clonetics. PAECs and CAECs were cultured in HuMedia-EG2 (Kurabo), and hMVECs-L and hMVECs-D were cultured in EGM-MV2 (Lonza). When the cells became subconfluent (~90%), RNA was extracted from cultured ECs using TRIzol (Invitrogen). RNAs for 5-donor pooled human lungs, heart, kidneys, and liver were obtained from BioChain Institute. Labeled cRNA was prepared using 1 vial of the mixed RNAs for each sample according to the One-Color Microarray-Based Gene Expression Analysis ver. 6.5 protocol, and 600 ng of cRNAs was used for hybridization with microarray chips (SurePrint G3 Human GE; G4851B) for 17 h. After washing, signals were scanned using an Agilent DNA microarray scanner, and the data were analyzed using Feature Extraction ver. 10.7.1.1 (Agilent Technologies).

Human umbilical vein endothelial cells (HUVECs, ATCC), the murine endothelial cell line EOMA (ATCC, Manassas, VA), which exhibits properties characteristic of microvascular endothelial cells, and human dermal microvascular endothelial cells (HMVECs-d) (Lonza, Walkersville, MD) were grown as monolayers in EBM2 medium (Clonetics, Walkersville, MD) supplemented with EGM2 (Clonetics) [14] (link), [17] (link). For in vitro flow chamber adhesion experiments, ECs were cultured until they reached confluence on clear glass slides pre-coated with 2% gelatin.

Human dermal MVECs (HMVECs-d, Lonza, Walkersville, MD) were grown in the manufacturer's EGM-2MV media for up to 5 passages and seeded into 8-well LabTak CC2-coated chamber slides (Nalge Nunc International, Rochester, NY) to achieve ∼30% confluency. Trophozoite-infected RBCs were magnetically purified and adjusted to 3–5% parasitemia (with equal parasitemias for samples compared on the same slide) and 0.5% hematocrit using uninfected RBCs in binding medium (BM; RPMI 1640, 0.5% BSA). Parasitized RBC suspensions were added to MVEC-coated chamber slides (200 μl/well) and incubated on an orbital shaker (100 rpm) for 1 h at room temperature. Whenever possible, each parasite isolate was tested for binding in duplicate wells on the same slide. After the parasitized RBC suspensions were removed from each well, the gasket was detached from the slide. The slide was then washed by dipping horizontally in BM four times, fixed in 2% glutaraldehyde overnight at room temperature, and stained in 10% Giemsa for 30 min. The number of parasitized RBCs bound to ∼350 MVECs in each well was counted, and data expressed as number of parasitized RBCs per MVEC. Duplicate comparisons were made whenever sufficient numbers of parasites were available. Seventy-six percent (25/33) of comparisons were performed in duplicate, 50% of which had a ratio of replicate 1∶2 between 0.80 and 1.3 (range 0.5–2.5).

Human PAECs and human SMCs (PASMCs) were purchased from PromoCell. hMVECs-L and hMVECs-D were purchased from Clonetics. PAECs were cultured in HuMedia-EG2 (Kurabo), and PASMCs were cultured in Smooth Muscle Cell Growth Medium 2 (PromoCell). hMVECs-L and hMVECs-D were cultured in EGM-MV2 (Lonza).