Distinct regulation of mitogen-activated protein kinases and p27Kip1 in smooth muscle cells from different vascular beds: A potential role in establishing regional phenotypic variance.

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Atherosclerotic cardiovascular disease is the leading cause of mortality and morbidity in developed countries. Although percutaneous transluminal angioplasty has become a well-established technique for revascularization of patients with arterial occlusive disease, the occurrence of restenosis at the site of angioplasty remains the major limitation despite a successful procedure. The molecular basis of atherosclerosis and restenosis involves dedifferentiation of vascular SMCs to a socalled “synthetic state” characterized by abundant production of matrix components and excessive proliferative and migratory activities (1-3). Therefore, a better understanding of the molecular mechanisms underlying these processes should help develop novel therapeutic approaches for the treatment of cardiovascular disease.

Cellular proliferation is regulated by the balance between multiple cyclin-dependent kinase (CDK)/cyclin holoenzymes and members of the Cip/Kip and INK4 families of CDK inhibitors (CKIs) (4,5). Active CDK/cyclin complexes promote cell cycle progression by phosphorylating the retinoblastoma gene product (pRb) and the related pocket proteins p107 and p130 from mid G1 to mitosis. CKIs associate with and inhibit the activity of CDK/cyclin holoenzymes. Studies arguing for a role of the Cip/Kip protein p27 in the pathophysiology of the cardiovascular system include the following: 1) p27 may contribute to the reestablishment of the quiescent phenotype after the initial proliferative response to balloon angioplasty in rat and porcine arteries, and adenovirus-mediated overexpression of p27 inhibited neointimal growth in these experimental models (6-8); 2) p27 may function as a molecular switch that regulates the phenotypic response of vascular SMCs to both hyperplastic and hypertrophic stimuli (9,10); 3) p27 is a negative regulator of endothelial cell proliferation and migration in vitro, and adenovirus-mediated overexpression of p27 inhibited angiogenesis in vivo (11,12); 4) p27 may contribute to integrin-mediated control of vascular SMC proliferation (13); 5) p27 may limit cardiomyocyte proliferation during early postnatal development and after injury in adult mice (14,15); 6) changes in p27 expression might regulate human vascular cell proliferation within atherosclerotic lesions (7,16), and a causal link between reduced p27 expression and atherosclerosis has been established in apolipoprotein E-deficient mice (17). It has been established that expression of p27 is regulated mainly at the level of translation and protein turnover (18).

Multiple growth factors and cytokines interact with specific receptors located in the cytoplasmic membrane of vascular cells in response to a variety of pathological stimuli, thus triggering a complex signal transduction cascade which culminates in changes in gene expression that execute a proliferative and migratory response (2,3). Activation of the MAPK signal transduction pathway is thought to play an important role during cardiovascular disease (19-23).

It has been well established that different segments of the arterial tree display significant differences in their susceptibility to atherosclerosis, both in animal models and humans. In this regard, it is notable that vascular SMCs display regional phenotypic variance, both when comparing cells obtained from different compartments of the same vessel or cells isolated from vessels from different vascular beds (24-30). The findings of the present study demonstrate that p27 and MAPKs are critical regulators of vascular SMC proliferation and migration. Our results suggest that intrinsic differences in the regulation of p27 and MAPKs may contribute to the establishment of regional variance in the proliferative and migratory capacity of SMCs from distinct regions of the vascular system.

 

Vascular SMCs undergo dedifferentiation and excessive proliferation and migration during atherosclerosis and restenosis post-angioplasty (1-3). Upregulation of the growth suppressor p27 in the arterial wall might limit SMC proliferation at late time points after balloon angioplasty in rat and porcine arteries (6,7), and adenovirus-mediated overexpression of p27 inhibited neointimal thickening in these animal models (8,41). Regarding the role of p27 on atherosclerosis, genetic disruption of p27 increased arterial cell proliferation and accelerated atheroma formation in hypercholesterolemic apolipoprotein E-deficient mice (17). Moreover, p27 might mediate TGF-b-dependent inhibition of cell growth in human atheromas (16), and proliferating cells within human coronary atheromas appear to express low level of p27 (7). Consistent with the observation that p27 overexpression attenuated human vascular endothelial cell migration in vitro (12), and that p27 inactivation reduced rapamycin-dependent inhibition of vascular SMC migration (42), we found that retrovirus-mediated overexpression of p27 inhibited vascular SMC migration. Thus, p27 might control neointimal thickening via regulation of both cell proliferation and migration.

Our studies with fat-fed rabbits showed that aortic arch tissue displays increased cell proliferation and atherogenicity as compared to femoral artery. We found that primary cultures of ASMCs and FSMCs maintained marked differences in their growth and migratory potential, which might be related, at least in part, to their distinct primary embryonic lineage (neural crest and mesoderm, respectively) (1,27,38). Indeed, ASMCs and CSMCs, which are thought to derive from neural crest ectoderm, behaved similarly in our proliferation and migration assays. We chose to examine ASMCs and FSMCs as an in vitro model to elucidate molecular mechanisms involved in the establishment of dissimilar atherogenicity in distinct vessel segments. Greater ASMC proliferation and migration correlated with lower expression of p27 when compared to FSMCs, and retrovirus-mediated overexpression of p27 attenuated the growth and migratory potential of ASMCs. Previous studies also support the notion that distinct regulation of p27 expression plays an important role in establishing differences in the phenotypic response of vascular SMCs toward a variety of stimuli. First, Yang et al. (29) reported reduced proliferation of human internal mammary artery (IMA) compared with saphenous vein (SV) SMCs. Importantly, PDGF-BB markedly downregulated p27 protein level in SV, but this response was much less pronounced in IMA. Thus, sustained p27 expression in spite of growth stimuli may contribute to the resistance to growth of SMCs from IMA, and to the longer patency of arterial versus venous grafts. Second, p27 may regulate the proliferative response of vascular SMCs toward fibroblast growth factor 2 (FGF2 or basic FGF). While FGF2 plays a critical role in the induction of medial SMC proliferation after balloon angioplasty (30,43,44), neutralizing antibodies to FGF2 failed to inhibit neointimal SMC proliferation in balloon-injured arteries (45). Moreover, only a small increase in growth was observed when arteries with existing neointimal lesions were esposed to FGF2 (30,43). Attenuated FGF2-dependent proliferation of neointimal SMCs occurred despite a robust induction of positive cell cycle regulators (30). Interestingly, neointimal SMCs expressed high levels of p27 compared with medial SMCs, and FGF2 infusion did not reduce the level of this inhibitor in arteries with established neointimal lesions.

Protein turnover is thought to play a major role in the regulation of p27 expression. Phosphorylation of p27 on Thr187 triggers its ubiquitination and rapid turnover in the proteasome (18). Our Western blot assays demonstrate that the majority (90 %) of p27 in ASMCs corresponds to a slow migrating form that undergoes phosphorylation on Thr187 and ubiquitination. In marked contrast, approximately 96% of p27 in FSMCs corresponded to a faster migrating p27 band that was not recognized by the phospho-specific antibody and did not contain ubiquininated protein. Thus, the relative amount of p27 phosphorylated on Thr187 and ubiquitinated appears higher in ASMCs compared to FSMCs, which might account for the lower level of p27 detected in ASMCs. Of note, ubiquitinated p27 in the faster migrating band that does not contain phosphorylated Thr187 was also detected in ASMCs (cf. Fig. 4E). This finding is in agreement with recent studies demonstrating an additional pathway for p27 ubiquitination and proteolysis independent of phosphorylation of p27 on Thr187 (46,47).

We investigated additional regulatory networks involved in the establishment of vascular SMC phenotypic variance. A wealth of evidence implicates the rapid activation of the MAPK signal transduction pathway during the pathogenesis of cardiovascular disease (19,21). For example, it has been suggested that persistent activation and hyperexpression of ERK1/2 is a critical element to initiate and perpetuate cell proliferation during diet-induced atherogenesis in the rabbit (48). Moreover, ERK1/2 activation occurs rapidly after angioplasty of porcine and rat arteries, (20,22), and all three MAPKs are activated in human failing hearts (49). Our results indicate that ERK1/2 contribute to establishing phenotypic differences between ASMCs and FSMCs. First, mitogen-dependent activation of ERK1/2 was more robust in ASMCs than in FSMCs. Second, reduced ERK1/2 activation by exposure of ASMCs to PD98059 impaired their growth and migratory capacity. By contrast, forced activation of ERK1/2 greatly increased FSMC proliferation and migration. We observed increased p27 expression upon ERK1/2 blockade in ASMCs, and diminished p27 expression upon forced ERK1/2 activation in FSMCs. Thus, in agreement with previous studies in NIH 3T3 fibroblasts and cancer cells (50-53), our findings suggest an important role for the MAPK pathway in the control of p27 expression in ASMCs and FSMCs. Solid ERK1/2 activation in mitogen-stimulated ASMC cultures might facilitate p27 degradation, thus favoring proliferation and migration of these cells. In contrast, weaker ERK1/2 activation might contribute to comparably higher expression of p27 in FSMCs, thus hindering their proliferative and migratory responses. In consideration of this model, it is noteworthy that PDGF-BB induced similar MAPK activation in cultures of SV and IMA in spite of distinct regulation of p27 in these cells (29), suggesting that MAPK-independent mechanisms of p27 regulation might operate in SMCs of different vascular beds.

In conclusion, we propose that intrinsic differences in MAPK-dependent signaling and p27 expression in rabbit ASMCs and FSMCs contribute to establishing variance in their proliferative and migratory potential. These dissimilarities might be attributable, at least in part, to their distinct primary embryonic origin. Further clarification of the molecular networks underlying vascular SMC phenotypic variance should shed significant insight into the mechanisms leading to regional variability in the susceptibility to intimal lesion development.