Hemodynamics and Progenitor Cells in AAA Disease

Ronald L. Dalman MD
Stanford University School of Medicine

Abdominal aortic aneurysm (AAA) disease is a common and highly lethal condition. The impact of AAA disease on public health stimulated the National Heart, Lung and Blood Institute to announce a disease-specific special interest program in December, 1998. This program has succeeded in generating considerable new knowledge regarding aneurysm pathogenesis, as well as increasing public awareness about AAA disease. Despite these accomplishments, however, the pathophysiology of AAA disease and specifically the role that adverse hemodynamic consequences play in initiating and promoting aneurysm progression remain incompletely understood.

In addition to systemic risk factors such as cigarette smoking, “regional” pathogenic influences are increasingly being recognized as significant determinants of AAA risk, localization and progression. The hemodynamic environment of the infrarenal aorta is one such regional influence. Compared with the suprarenal aorta, the resting infrarenal environment is characterized by increased peripheral resistance, increased oscillatory wall shear stress (WSS) and reduced resting flow1,2. These hemodynamic conditions increase pro-inflammatory and pro-apoptotic gene expression3,4, predisposing arteries to degenerative diseases. Major lower limb amputation, chronic spinal cord injury (SCI), and severe peripheral vascular disease have all recently been recognized as potential new risk factors for AAA disease, associations that highlight the pathogenic significance of resistive hemodynamic conditions.

Medial SMC depletion is an important component of human AAA disease profoundly influenced by luminal flow conditions. Medial SMC density is decreased in human AAA tissue, increased production of p53, a potential mediator of cell cycle arrest and programmed cell death, is present within SMCs harvested from AAA tissue, and SMCs from human AAAs express markers of apoptosis. Exactly how aortic smooth muscle cell density influences structural integrity remains poorly understood. In experimental aneurysms, luminal seeding of syngenic SMCs attenuates elastin degradation, decreases monocyte and macrophage infiltration and limits AAA progression, despite ongoing proteolytic enzyme expression and extracellular activation from seeded cells5. The fact that luminal seeding reduces transmural inflammation and proteolysis suggests that diffusible anti-inflammatory paracrine mediators are released in response to flow effects on SMCs. Although the identity of these mediators remains uncertain, they may include at least in part growth or chemotactic factors. Delivery of basic fibroblast growth factor in expression plasmids to aortic SMCs via electroporation increases SMC proliferation, reduces AAA diameter and downregulates proteolytic enzyme expression6. Maintaining medial cellularity either by reducing apoptosis or increasing cellular proliferation may significantly modify the pathogenesis and natural history of human abdominal aortic aneurysm disease.

We have recently investigated the morphologic and cellular consequences of variable flow conditions in experimental AAAs7,8. High flow (HF) increases AAA endothelial cells (ECs) and SMCs compared to low flow (LF) AAAs. HF AAAs have more medial SMC proliferation (as indicated by BrdU + staining) and less apoptosis (TUNEL + cells). Increased EC and SMC growth factor expression (VEGF-D, KDR and PDGF-b) is present in HF AAA at 5 days, consistent with evidence of increased EC coverage and SMC proliferation with BrdU staining. We employed laser capture microdissection (LCM) to isolate homogeneous collections of medial or adventitial AAA mural cells to gain insight into the cell-specific proliferation response to variable hemodynamic conditions. We amplified message using quantitative RT-PCR for comparison of expression of genes of interest between medial SMCs and adventitial macrophages at specific time points following flow modification. Using LCM we identified strong proliferative signals elicited in SMCs in response to flow loading. In particular, expression of c-myc is dramatically increased in HF AAA. c-myc is a proto-oncogene that is quickly induced in response to injury and similar proliferative stimuli that facilitates cell-cycle transition from G1 to S phase. Relatively more anti-apoptotic message was also present in HF vs. LF medial SMCs at early time points, and in HF medial vs. HF adventitial cells.

Further investigation into the phenomenon of flow-mediated mural cellular modulation demonstrates that many vascular cells present in HF or regenerating AAA arise from vascular progenitor cells (CD34+)(Sho E and Dalman RL, data on file). These adult peripheral blood cells may transdifferentiate into cardiomyocytes, endothelial or smooth muscles cells, have previously been identified in human AAAs9 and may play a critical role in modulating arterial disease resistance and pathogenesis. Using our variable flow murine AAA model we analyzed mural CD34+ cell localization, growth factor expression and adventitial neovascularization as functions of luminal flow conditions. Consistent with findings in models of vein graft hyperplasia and arterial injury10, significant numbers of CD34+ cells were present in the regenerating media and adventitia. The absolute percentage of CD34+ vs. CD31+ and the ultimate differentiation fate of these cells is currently being examined in our laboratory.

Our data suggests that circulating vascular progenitor cells localize and differentiate into distinct cell types within experimental AAA as functions of luminal flow conditions. One important corollary to these findings is the observation that flow may influence inflammation at least in part via modulation of neocapillary formation in addition to effects on macrophage infiltration. We are currently uncertain as to what degree either proliferation of existing medial cells or localization and/or proliferation of circulating vascular progenitor cells account for increased smooth muscle cellularity and preserved aortic architecture in HF AAA. It is clear, however, from our own experience and that of others that AAA cellularity is highly regulated by luminal flow conditions. The evolving field of cellular imaging promises to provide detailed information on the kinetics and stimuli for progenitor cell migration, localization and differentiation.

References

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6. Hoshina K, Koyama H, Miyata T, Shigematsu H, Takato T, Dalman RL, Nagawa H. Aortic wall cell proliferation via bFGF gene transfer limits progression of experimental abdominal aortic aneurysm.  J Vasc Surg 2004 submitted, pending revision.

7. Hoshina K, Nakahashi TK, Sho E, Sho M, Tsao P, and Dalman RL. Aortic wall shear stress modulates aneurysm cellularity and structure. J Vasc Surg 2003;37:1067-74.

8. Sho E, Sho M, Hoshina K, Kimura H, Nakahashi TK and Dalman RL. Hemodynamic forces regulate mural macrophage infiltration in experimental aortic aneurysms. Exp Mol Path, 1/14/2004 epub ahead of print.

9. Kobayashi M, Matsubara J, Matsushita M, Nishikimi N, Sakurai T, Nimura Y. Expression of angiogenesis and angiogenic factors in human aortic vascular disease. J Surg Res 2002;106:239-45

10. Zhang L, Freedman NJ, Brian L, Peppel K. Graft-extrinsic cells predominate in vein graft arterialization. Arteriosclero Thromb Vasc Biol 2004;Jan 22 epub ahead of print.