Transcriptomic screening of microvascular endothelial cells implicates novel molecular regulators of vascular dysfunction after spinal cord injury.
Microvascular dysfunction is a critical pathology that underlies the evolution of secondary injury mechanisms after traumatic spinal cord injury (SCI). However, little is known of the molecular regulation of endothelial cell (EC) plasticity observed acutely after injury. One reason for this is the relative lack of methods to quickly and efficiently obtain highly enriched spinal microvascular ECs for high-throughput molecular and biochemical analyses. Adult C57Bl/6 mice received an intravenous injection of fluorescein isothiocyanate (FITC)-conjugated Lycopersicon esculentum lectin, and FITC-lectin-bound spinal microvessels were greatly enriched by fluorescence-activated cell sorter (FACS) purification. This technique allows for rapid (<1.5 h postmortem) isolation of spinal cord microvascular ECs (smvECs). The results from cell counting, reverse-transcription polymerase chain reaction (RT-PCR), and western blot analyses show a high degree of EC enrichment at mRNA and protein levels. Furthermore, a focused EC biology microarray analysis identified multiple mRNAs dramatically increased in the EC compartment 24 h after SCI, which is a time point associated with the pathologic loss of spinal vasculature. These included thrombospondin-1, CCL5/RANTES, and urokinase plasminogen activator, suggesting they may represent targets for therapeutic intervention. Furthermore, these novel methodologic approaches will likely facilitate the discovery of molecular regulators of endothelial dysfunction in a variety of central nervous system (CNS) disorders including stroke and other neurodegenerative diseases having a vascular component.
Benton
Richard L
RL
Kentucky Spinal Cord Injury Research Center, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA. rlbent01@louisville.edu
Maddie
Melissa A
MA
Worth
Christopher A
CA
Mahoney
Edward T
ET
Hagg
Theo
T
Whittemore
Scott R
SR
D000818:Animals; D017302:Annexins / Q000235:genetics; D004730:Endothelium, Vascular / Q000473:pathology / Q000503:physiopathology*; D005260:Female; D005341:Fibrinolysin / Q000235:genetics; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008833:Microcirculation / Q000473:pathology / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D037121:Plant Lectins; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000098:blood supply*; D013119:Spinal Cord Injuries / Q000235:genetics* / Q000503:physiopathology*; D019699:Thrombospondins / Q000235:genetics; D014158:Transcription, Genetic; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D017302:Annexins; D037121:Plant Lectins; D012333:RNA, Messenger; D019699:Thrombospondins; C034981:tomato lectin; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator