In vivo transcriptional response of cardiac endothelium to lipopolysaccharide.
OBJECTIVE
Vascular endothelial cells must integrate stimuli from multiple sources, including plasma, leukocytes, and neighboring components of the vessel. These stimuli are difficult to recapitulate in vitro. We have developed a method to examine the in vivo regulation of gene expression in endothelial cells and have applied it to a model of sepsis.


RESULTS
We used fluorescent-activated cell sorting to isolate highly purified endothelial cells from the hearts of transgenic mice that express green fluorescent protein driven by the endothelial-specific promoter Tie2. We treated these mice with intraperitoneal lipopolysaccharide and identified those genes within cardiac endothelium that were >3-fold dysregulated 4 and 24 hours later by microarray analysis. These findings were confirmed by real-time polymerase chain reaction and compared with in vitro regulation in a murine endothelial cell line.


CONCLUSIONS
The in vivo regulation was distinct and, in general, more robust than that seen in vitro. We identified endothelial-expressed genes not previously recognized to be regulated in response to lipopolysaccharide. This approach provides insight into the cardiac-specific responses of the endothelium that contribute to the specific responses of the heart to sepsis, and can be generalized to the exploration of endothelial responses in any organ.
Maresh
J Gregory
JG
Internal Medicine-Cardiology, University of Texas Southwestern Medical Center, Dallas, Tex 75390-8573, USA.
Xu
Huaxia
H
Jiang
Nan
N
Shohet
Ralph V
RV
D000209:Acute-Phase Proteins / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D016000:Cluster Analysis; D003199:Computer Systems; D042783:Endothelial Cells / Q000737:chemistry / Q000378:metabolism; D004730:Endothelium, Vascular / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods / Q000706:statistics & numerical data; D006321:Heart / Q000502:physiology*; D017403:In Situ Hybridization / Q000379:methods; D000071068:Lipocalin-2; D054834:Lipocalins; D008070:Lipopolysaccharides / Q000097:blood / Q000276:immunology*; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000706:statistics & numerical data; D015513:Oncogene Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret; D012313:RNA / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*
D000209:Acute-Phase Proteins; D000071068:Lipocalin-2; D054834:Lipocalins; D008070:Lipopolysaccharides; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; C071332:Lcn2 protein, mouse; D012313:RNA; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases