Transcriptomic profiling of ZMPSTE24 knockout endothelial cells: implication of endothelial dysfunction in HGPS

Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare and fatal disease with features of premature aging and cardiovascular diseases (atherosclerosis, myocardial infarction, and stroke). The molecular basis underlying premature aging driven cardiovascular diseases remains incompletely understood. Since endothelial dysfunction is involved in the initiation and progression of cardiovascular diseases. We hypothesized that HGPS-causing progerin (a mutant form of lamin A) and farnesylated prelamin A drives endothelial dysfunction and cardiovascular disease. In our study, we performed transcriptomic profiling of ZMPSTE24-/- mouse endothelial cells, hoping to find the missing link between progeria and atherosclerosis. We observed that ZMPSTE24-/- endothelial cells have increased farnesylated prelamin A accumulation, and show nuclear structure abnormality. Analysis of RNA-seq data revealed that multiple endothelial homeostasis-related genes and pathways are altered by ZMPSTE24 deletion. Further studies are needed to characterize the biological functions of ZMPSTE24 in the pathogenesis of progeria-associated atherosclerosis. Overall design: Mouse lung endothelial cells (MLECs, primary culture, no passage) were isolated from ZMPSTE24 wild type (wt) and knockout (ko) mice (gifted by Prof. Loren Fong, UCLA) using CD31+ and ICAM2+-dynabeads double sorting. Cells were phenotypically validated by CD31, VE-cadherin, and vWF immunostaining and efficient DiI-oxLDL uptake. Cell purity was >99% endothelial cells. MLECs were seeded in 0.2% gelatin-coated dishes till confluence. Total RNA was isolated using the RNA-Easy Mini Plus kit (QIAGEN). High quality RNA samples (pre-assessed by Nanodrop measurements) were further processed in the Genome Research Center of the University of Rochester Medical Center. The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer's protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3' adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt are generated for each sample and aligned to the organism specific reference genome. Sequenced reads were cleaned using Trimmomatic-0.32 before mapping some of to the mouse reference genome (GRCm38.p6) with STAR-2.4.2a. Raw read counts were obtained using HTSeq and gencode M17 mouse gene annotations. DESeq2-1.10.1 was used to perform data normalization and differential expression analysis with an adjusted p-value threshold of 0.05. Then, Cufflinks-2.0.2 Software was used with the gencode 23 human gene annotations to perform differential expression analysis with an FDR cutoff of 0.05.