Comparison of cell-type-specific vs transmural aortic gene expression in experimental aneurysms.
OBJECTIVE
Abdominal aortic aneurysm (AAA) progression and disease resistance are related to mural cellularity; adventitial macrophages and neocapillaries predominate in larger, advanced aneurysms, whereas smaller AAAs have fewer macrophages and retain more medial smooth muscle cells (SMCs). Expression analysis of mRNA derived from the entire aorta may mask the role that specific cell types play in modulating disease progression. We used laser capture microdissection (LCM) to isolate SMC and macrophage-predominant mural cell populations for gene expression analysis in variable-flow AAA.


METHODS
Rat AAAs were created via porcine pancreatic elastase (PPE) infusion. Aortic flow was increased via femoral arteriovenous fistula creation (HF-AAA) or reduced via unilateral iliac ligation (LF-AAA) in selected cohorts. SMC and macrophage-predominant cell populations were isolated via LCM and analyzed for expression of pro-inflammatory transcription factors and chemokines, cytokines, and proteolytic enzymes via real-time polymerase chain reaction.


RESULTS
Aortic PPE infusion precipitated endothelial cell (EC) denudation, SMC apoptosis, and elastic lamellar degeneration. Increased aortic flow (HF > NF > LF) stimulated restorative EC and SMC proliferation (45.8 +/- 6.6 > 30.5 +/- 2.1 > 21 +/- 3.6 and 212.2 +/- 9.8 > 136.5 +/- 8.9 > 110 +/- 13.5, respectively, for both cell types; P < .05) at 5 days after PPE infusion, while simultaneously reducing medial SMC apoptosis and transmural macrophage infiltration. Expression of nuclear factor kappa B (NF-kappab), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage migration inhibitory (MIF), heparin-binding EGF-like factor (HB-EGF) and inducible nitric oxide synthase (iNOS) varied between cell types and flow conditions at all time points examined. Gelatinolytic protease expression varied by cell type in response to flow loading (eg, increased in SMCs, decreased in macrophages), consistent with observed patterns of elastolysis and SMC proliferation reported in prior experiments.


CONCLUSIONS
Flow differentially regulates cell-specific AAA gene expression. Whole-organ analysis of AAA tissue lysates obscures important cellular responses to inflammation and flow, and may explain previous seemingly contradictory observations regarding proteolysis and cell proliferation. Cell-type specific expression and functional analyses may substantially clarify the pathophysiology of AAA disease.


CONCLUSIONS
Understanding aneurysmal aortic degeneration at the most fundamental level is a critical precursor to the development of next-generation therapies such as drug-eluting endografts and/or medical therapies to limit expansion of preclinical AAA in high-risk or elderly patients. Although animal modeling is necessary to gain insight into the early initiating events of AAA disease, the methods used in such analyses have critical bearing on the conclusions drawn regarding pathogenesis and potential therapeutic derivations. By analyzing cell-type-specific gene expression rather than whole-organ tissue lysates, the precise roles of important mediators such as metalloproteinases can be placed in the appropriate context. Further refinement of these techniques may allow cell-specific therapies to be applied at defined time points in disease progression with improved patient outcome and reduced procedural morbidity.
Sho
Eiketsu
E
Division of Vascular Surgery, Stanford University, USA.
Sho
Mien
M
Nanjo
Hiroshi
H
Kawamura
Koichi
K
Masuda
Hirotake
H
Dalman
Ronald L
RL
D000818:Animals; D017544:Aortic Aneurysm, Abdominal / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology; D017209:Apoptosis / Q000235:genetics; D002452:Cell Count; D002455:Cell Division; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D005819:Genetic Markers; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007261:Infusions, Intra-Arterial; D036341:Intercellular Signaling Peptides and Proteins; D008263:Macrophage Migration-Inhibitory Factors / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism / Q000648:ultrastructure; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010196:Pancreatic Elastase / Q000008:administration & dosage / Q000633:toxicity; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005819:Genetic Markers; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008263:Macrophage Migration-Inhibitory Factors; D016328:NF-kappa B; D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D010196:Pancreatic Elastase; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9