Department of Physiology and Biophysics, University of Alabama Birmingham, Birmingham Alabama 35294-0005
Submitted 25 November 2003 ; accepted in final form 29 November 2003
Aberrant vascular smooth muscle cell (VSMC) growth and migration are associated with many vascular occlusive diseases, including atherosclerosis, transplant vasculopathy, and restenosis after percutaneous transluminal angioplasty (PTCA). Upon injury of the arterial wall, VSMC dedifferentiate into a synthetic, proliferative phenotype. These initial growth events are then followed by the directional migration of VSMC from the media to the intima and subsequent neointima formation. A myriad of vasoactive agents induce VSMC growth and migration, including growth factors, vasoconstrictors, and oxidative stress.
The mammalian target of rapamycin (mTOR), also referred to as FKBP12 and rapamycin-associated protein (FRAP), plays a key role in the regulation of VSMC proliferation and migration. Pioneering studies by Marx and colleagues (6) demonstrated that rapamycin inhibited VSMC proliferation by blocking cell cycle progression at the G1/S transition. Rapamycin also inhibited rat and human VSMC migration in response to PDGF (9). Subsequent in vivo studies demonstrated that systemic administration of rapamycin reduced neointima formation after balloon angioplasty in porcine coronary arteries (3).
These compelling preliminary data provided the impetus for developing the use of rapamycin to prevent stent restenosis after percutaneous transluminal coronary angioplasty (PTCA). Attempts to reduce restenosis with the use of systemic and local delivery of pharmacological agents up to this point had been disappointing. A pilot study with 32 patients established that implantation of sirolimus-coated stents significantly reduced neointimal proliferation after PTCA (11). This promising study was followed by a larger trial (the randomized double blind study with the sirolimus-eluting BX velocity balloon expandable stent in patients with de novo native coronary lesions or RAVEL study) that showed no evidence of restenosis in the rapamycin-coated stent group compared to 26% restenosis in the uncoated stent group (7). A larger study of patients with more complex coronary lesions (the multicenter randomized double blind study of sirolimus-coated BX velocity balloon expandable stents in the treatment of patients with de novo coronary artery lesions or SIRIUS) demonstrated that the rate of restenosis of the target vessel was reduced from 21% with a standard stent to 8.6% in a sirolimus-coated stent, 270 days after PTCA (8).
The encouraging results of these clinical trials have been attributed to the antiproliferative and antimigratory effects of rapamycin on VSMC. It is well recognized that synchronized cell cycle control and coordinated regulation of protein synthesis are essential for VSMC proliferation. The regulation of G1 progression and G1/S transition of the cell cycle is governed by a delicate interplay of cyclins, cyclin-dependent kinases (cdks) and their inhibitors (cdkI), such as p21cip and p27kip (4). p21cip and p27kip1 are important negative regulators of cyclin/cdk interactions. The retinoblastoma protein (Rb) is a major target of cdk4/6 and upon phosphorylation, Rb dissociates from the E2F transcription factor, enabling E2F to initiate gene transcription. Inhibition of mTOR by rapamycin results in the upregulation of p27kip1 and p21cip, leading to growth arrest of cultured VSMC. In vivo studies of balloon injury also associated rapamycin treatment with decreased cyclin D and cdk2 expression (1), increased p27kip1 expression, and reduced pRb phosphorylation (3) within the vessel wall.
The mechanisms by which rapamycin reduces VSMC migration are less clear. Sun et al. (12) showed that the sensitivity of VSMCs to the antimigratory activity of rapamycin depends on the presence of p27kip1. More recently, Díez-Juan and Andrés (2) provide provocative data that establish p27kip1-cdk-pRb as a link between VSMC migration and proliferation. In general, they found that a low level of p27kip1 expression was correlated with high proliferative and migratory capacity, whereas nuclear accumulation of p27kip1 was associated with a quiescent and static phenotype. Thus, it is possible that G1/S-dependent gene expression of cytoskeletal proteins, matrix regulating enzymes, or components of focal adhesions is necessary for efficient cell motility.
In the current article in focus (Ref. 5, see p. C507 in this issue), Martin et al. provide compelling data for a new role for rapamycin as a promoter of VSMC differentiation. Their major finding is that mTOR inhibition promotes the coordinated regulation of not only cell cycle progression but also the expression of contractile proteins to induce the differentiated phenotype. Rapamycin treatment of primary human, porcine, or rat VSMC cultured in low (2.5%) serum caused a marked increase in the expression of smooth muscle (SM)-myosin heavy chain, SM-actin, and calponin. Interestingly, overexpression of the mTOR target p70 S6 kinase (S6K1) reversed the effects of rapamycin on contractile protein and p21cip expression. Although abrogation of PI3-K/Akt (upstream activators of mTOR) signaling has been shown to change PDGF-induced proliferative response of VSMC toward enhanced contractile protein expression (10), the study by Martin and coworkers (5) provides the first evidence that S6K1 actively opposes VSMC differentiation. Moreover, because VSMC dedifferentiation, (characterized by decreases contractile protein expression) is a prerequisite for the transformation of VSMC into a migratory, proliferative phenotype, these novel results add new mechanistic insight into the efficacy of rapamycin-coated stents for the prevention of restenosis. It is possible that rapamycin may prove to be a superior choice for coating stents because it may promote the maintenance of functional, quiescent VSMC at the site of injury. However, further studies are necessary to determine the requirement for p27kip1 and p21cip upregulation in rapamycin-induced VSMC differentiation and whether or not rapamycin can promote the expression of contractile proteins in vivo after PTCA.
Address for reprint requests and other correspondence: P. A. Lucchesi, Dept. of Physiology and Biophysics, Univ. of Alabama Birmingham, 1918 Univ. Blvd, MCLM-986, Birmingham, AL 35294-000 (E-mail: plucche{at}uab.edu).
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