ere determined using specific EIA kits, according to the manufacturer’s instructions. Each measurement was performed in duplicate. 3 / 13 LPA Effects on Liver Sinusoidal Endothelial Cells Q-RT-PCR Q-RT-PCR was performed as in a previous study. Briefly, total RNA was MedChemExpress IMR 1 isolated from liver sinusoidal endothelial cells using RNAzol B reagent. Then, cDNA was prepared from 2 g of total RNA using random hexamer primers. LPAR1-6, Cyr61, TIMP-1, C5/C5a, M-CSF, MCP-5, SDF-1, gp130, CCL28, and CXCL16 mRNA levels were determined with a quantitative real-time PCR detection system. The primers used are shown in Statistical analysis Results are given as means SD’s. Two-tailed t-tests were used to compare the results between the indicated groups in Results. A p-value of < 0.05 was considered significant. Results LPAR1, LPAR3, and LPAR6 are expressed in liver sinusoidal endothelial cells Because lysophosphatidic acid receptor expression has been shown to be involved during liver regeneration, we focused on the effects of LPA on liver sinusoidal endothelial cells. We isolated liver sinusoidal endothelial cells by using Dynabeads to positively isolate CD31positive endothelial cells from mouse liver tissue. The purity of isolated liver sinusoidal endothelial cells was verified by determining the CD31 positive rate by flow cytometry and by expression of the hematopoietic cell marker CD45 that has also been reported to be expressed on liver sinusoidal endothelial cells. The purities of liver sinusoidal endothelial cells during five serial passages were determined, which showed that from the first to the fifth passage, 4 / 13 LPA Effects on Liver Sinusoidal Endothelial Cells Fig 1. Liver sinusoidal endothelial cell isolation and LPA receptor expression patterns. Liver sinusoidal endothelial cells were isolated from mouse liver tissue and their purity was determined by CD45 and CD31 positives rate by flow cytometry analysis. Liver sinusoidal endothelial cell purity during five serial passages. LPA receptors' mRNA expression determined by qRT-PCR. LPAR1 and LPAR6 mRNA expression was normalized to that of GAPDH mRNA expression. Strategy for investigating LPA effects on liver sinusoidal endothelial cells using proteome profile arrays. doi:10.1371/journal.pone.0122060.g001 the CD31 positive rates were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761586 title=View Abstract(s)”>PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761601 from 94.4 2.3% to 80.3 4% and the CD45 positive rates were from 82.5 4.7% to 74.3 3.9%. For this study, liver sinusoidal endothelial cells were only used after the fifth passage. We also determined the mRNA expressions of LPAR’s by qRT-PCR. This showed that LPAR1 and LPAR3 mRNA’s were strongly expressed and that LPAR6 mRNA was weakly expressed in liver sinusoidal endothelial cells. To determine the effects of LPA on liver sinusoidal endothelial cells, we screened the conditioned medium derived 
from LPA-treated liver sinusoidal endothelial cells for specific biological functions based on the results of proteome profile arrays. LPA treatment enhances angiogenesis related factors Cyr61 and TIMP1 expression in liver sinusoidal endothelial cells Conditioned media derived from liver sinusoidal endothelial cells after vehicle treatment or after LPA treatment were used to determine angiogenesis-related factors’ expression. When using an angiogenesis related protein array, the spots for Cyr61 and TIMP-1 had different intensities between conditioned media after vehicle treatment and after LPA treatment. Quantitative results showed that Cyr61 and TIMP-1 expressions were 3.61 0.2