The role of vascular smooth muscle cells and adventitial fibroblasts in flow-mediated mechanisms of intimal hyperplasia and arterialization
Open Access
- Author:
- Garanich, Jeffrey Stock
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 09, 2005
- Committee Members:
- Peter J Butler, Committee Chair/Co-Chair
John Michael Tarbell, Committee Chair/Co-Chair
Herbert Herling Lipowsky, Committee Member
David A Antonetti, Committee Member
Michael J Eppihimer, Committee Member - Keywords:
- vascular smooth muscle cells
shear stress
migration
interstitial flow
intimal hyperplasia
nitric oxide
matrix metalloproteinase-2
platelet-derived growth factor
adventitial fibroblasts
myofibroblasts
phenotype
contraction
arterialization - Abstract:
- Vascular smooth muscle cells (SMCs) reside in the medial layer of blood vessels and under physiological conditions, maintain a quiescent phenotype through which they carry out their primary function: regulation of the systemic blood pressure via control of vessel dilation and contraction. In regions of vascular injury, such as at the site of vessel repair by angioplasty and at the anastomoses of a vascular graft with the native artery, SMCs transform to a proliferative phenotype, resulting in abnormal proliferation in the media and subsequent migration to the intima. The contribution of hemodynamics to the regulation of this intimal hyperplasia (IH; accumulation of cells and matrix in the intimal layer) has received considerable attention. The conventional view of mechanical forces on SMCs is that due to the presence of the endothelium, SMCs are shielded from direct blood flow and its subsequent shear stress (SS) and are primarily exposed to circumferential stress and strain associated with the pulsatility of blood flow. However, recent modeling studies have shown that medial SMCs in an intact vessel are subjected to physiologically relevant shear stresses through their exposure to transmural interstitial flow driven by the transmural pressure gradient (arterial pressure – tissue pressure) that are near the order of magnitude of those experienced by the endothelium due to luminal blood flow. In fact, due to the funneling of transmural flow through the pores of the internal elastic lamina (IEL), the most superficial layer of SMCs is exposed to SS much greater in magnitude than the underlying layers. This, accompanied by the fact that SMCs are directly subjected to blood flow SS in cases of vascular injury (endothelial denudation and IEL damage), necessitates an understanding of the role of SS in the control of SMC migration and proliferation. There is considerable in vivo evidence suggesting that IH progression is attenuated in regions of elevated blood flow and, conversely, that IH is enhanced in low flow areas. In vitro studies have supported this notion by reporting inhibition of SMC proliferation in response to SS, but similar studies to evaluate the role of SS on SMC migration have yet to be conducted. We therefore seeded rat aortic SMCs on porous cell culture inserts coated with Matrigel (a representative basement membrane matrix) and quantified their migratory activity toward platelet-derived growth factor (PDGF)-BB when exposed to 1, 10, or 20 dyn/cm2 SS for 1-4 h with a rotating disk apparatus. Four h of either 10 or 20 dyn/cm2 SS significantly inhibited SMC migration through the Matrigel layer to the underside of the insert. This inhibition was associated with downregulation of SMC matrix metalloproteinase (MMP)-2 activation in response to SS. Four h of 10 dyn/cm2 markedly increased the SMC production of nitric oxide (NO). Addition of a NO synthase inhibitor (NG-nitro-L-arginine methyl ester; 100 microM) to cells abolished the shear-mediated increase in NO production as well as the inhibition of SMC migration and MMP-2 activity. Through the use of a NO donor (S-nitroso-N-acetylpenicillamine; 500 microM), it was shown that NO acts to suppress SMC migration via the reduction of both total (proenzyme + active) and active MMP-2 levels. Addition of 50 ng/ml exogenous active MMP-2 to inserts significantly elevated SMC migration levels whereas addition of 10 microM MMP-2 Inhibitor I to inserts significantly reduced SMC migration levels, lending further support to the role of MMP-2 in SMC migration. Western blots showed no effect of 4 h of 20 dyn/cm2 SS on SMC production of PDGF-AA, another chemical known to suppress SMC migration. Thus, it appears that SS inhibits SMC migration by upregulating the cellular production of NO which subsequently downregulates MMP-2 activity. Vascular fibroblasts (FBs) are primarily contained in the outer adventitial layer of blood vessels, which has historically been considered a supporting tissue. Recent in vivo work, however, suggests that, along with SMCs, FBs also contribute to intimal lesion formation and that this involvement is at least in part mediated by their acquisition of the smooth muscle (SM)-specific phenotypic marker, SM alpha-actin, and differentiation to smooth muscle-like cells, the myofibroblasts (myoFBs). FBs are sparsely populated in the adventitia and come into closer contact with other FBs and medial SMCs as they migrate to the intima following injury. We hypothesize that the proximity of FBs to neighboring cells plays a role in modulating their conversion to myoFBs through the expression of SM alpha-actin. As described above for SMCs, FBs are exposed to transmural interstitial flow (SS) driven by the transmural pressure gradient. Other studies conducted on the microcirculatory level have shown that interstitial FBs are involved in the arterialization of capillaries that connect two high pressure arteriolar trees, allowing for improved regulation of local blood flow to tissues during processes such as exercise hypertrophy and wound healing. The expression of SM alpha-actin by interstitial FBs as they approach the arterializing vessel and come into closer contact with neighboring cells suggests that the degree of confluence of FBs may affect their expression of SM-specific phenotypic markers. The recruitment of FBs to arterializing vessels has been suggested to be regulated in part by local hemodynamic conditions, namely elevated circumferential stresses in the arterializing vessel due to elevated luminal pressure compared to surrounding capillaries. We suggest that the elevated luminal pressure in the arterializing vessel drives enhanced interstitial flow that subjects nearby FBs to enhanced levels of fluid SS, and that this force directly recruits these cells to the vicinity of the vessel. The involvement of FBs and myoFBs in physiologic and pathologic processes on both the macrocirculatory and microcirculatory levels accompanied by the lack of existing studies of the effects of mechanical and chemical stimulation on their function motivated the present study. Rat adventitial FBs were isolated and maintained at either subconfluent (30-50% confluence in culture) or confluent (100% confluence in culture) culture conditions. Both cell populations were analyzed for their expression of two SM-specific phenotype markers: SM alpha-actin and SM myosin heavy chain (MHC), a marker of mature, fully differentiated SMCs. Cells maintained at subconfluent culture conditions expressed a modest amount of SM alpha-actin and virtually no SM-MHC whereas confluent cells expressed a large amount of SM alpha-actin and modest SM-MHC. Based on these results, subconfluent cells were labeled “fibroblasts” and confluent cells were termed “myofibroblasts” according to literature convention. Each cell population was seeded on Matrigel-coated porous cell culture inserts in the presence of PDGF-BB and exposed to 4 h of 20 dyn/cm2 SS in experiments similar to those conducted using SMCs above. The contractile responses of serum-starved FBs and myoFBs to an interstitial fluid solute (serum) were also determined. In stark contrast to the SMC migratory response to SS and contractile response to serum, FBs enhanced their migration upon exposure to shear and did not contract when re-exposed to serum. MyoFBs, on the other hand, displayed SM-like responses by inhibiting their migratory activity when exposed to SS and significantly contracting within 2 min of re-exposure to serum. These data suggest that modulation of FB phenotype appears to be one way to regulate their involvement in both physiologic and pathologic processes.