Vascular endothelial sampling and analysis of gene transcripts: a new quantitative approach to monitor vascular inflammation.
BACKGROUND
Limited access to endothelial tissue is a major constraint when investigating the cellular mechanisms of vascular inflammation in patients with cardiovascular and metabolic diseases. We introduce venous endothelial sampling coupled to quantitative analysis of gene transcripts by real-time PCR, as a novel approach to study endothelial gene expression in human subjects.


METHODS
Endothelial cells were collected from a superficial forearm vein using five guide wires sequentially inserted through a 20-gauge angiocatheter in seven patients with history of cardiovascular events related to advanced vascular disease and in 17 healthy subjects. Endothelial cells were purified using magnetic beads coated with endothelial specific antibodies. Endothelial mRNA was amplified using RiboAmp HS RNA Amplification kit (Molecular Devices, Sunnyvale, CA). Amplified RNA was analyzed by real-time PCR.


RESULTS
Linearity of RNA amplification was validated by real-time PCR using RNA from 1,000 human umbilical endothelial cells (HUVECs) before and after amplification. In human subjects, vascular disease was associated with significant induction of proatherosclerotic genes: early growth response gene product (Egr-1) and monocyte chemoattractant protein-1 (MCP-1).


CONCLUSIONS
Venous endothelial sampling coupled to real-time PCR analysis is a minimally invasive, safe, and reliable technique to monitor vascular inflammation in human subjects. Expression of genes implicated in the atherosclerotic process is increased in the venous endothelium of patients with arterial vascular disease. Venous endothelial sampling and quantitative analysis of gene expression may help develop new vascular-targeted biomarkers to identify and track the impact of disease states and therapeutic interventions in vascular diseases.
Onat
Duygu
D
Department of Medicine, Columbia Univ., New York, NY, USA.
Jelic
Sanja
S
Schmidt
Ann Marie
AM
Pile-Spellman
John
J
Homma
Shunichi
S
Padeletti
Margherita
M
Jin
Zhezhen
Z
Le Jemtel
Thierry H
TH
Colombo
Paolo C
PC
Feng
Lei
L
D000368:Aged; D002318:Cardiovascular Diseases / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D002469:Cell Separation; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000032:analysis / Q000235:genetics; D051766:Early Growth Response Protein 1 / Q000032:analysis / Q000235:genetics; D004730:Endothelium, Vascular / Q000737:chemistry*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007249:Inflammation / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D014158:Transcription, Genetic
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D051766:Early Growth Response Protein 1; D012333:RNA, Messenger