The role of endothelin-1 in vascular remodeling in vivo

See article by Dao et al.[4](pages 61–68) in this issue.

Vascular smooth muscle cells (VSMCs) are a major component of the arterial wall and play a critical role in the development of occlusive vascular lesions. In normal vessels, VSMCs are quiescent, differentiated, and contractile and function to maintain vascular tone and blood pressure. In pathological processes such as the response to vascular injury, VSMCs undergo a phenotypic transition whereby they proliferate, migrate from the medial to the intimal layer, and lead to neointimal formation and subsequent vascular remodeling. These resultant vascular changes contribute to the pathological basis of atherosclerosis and restenosis that occur after revascularization procedures such as angioplasty, stenting, and bypass grafting.

Endothelin-1 (ET-1) is a potent 21-amino acid vasoconstrictor peptide. ET-1 is mainly generated from the endothelium of blood vessels and functions in a paracrine or autocrine manner on ET_A and ET_B receptors. These receptors present on VSMCs have been shown to induce contraction and stimulate cell hypertrophy and/or hyperplasia, while endothelial ET_B receptors stimulate the production of nitric oxide and prostacyclin and accordingly elicit vasorelaxation. Accumulating evidence indicates that endogenous ET-1 is involved in the vascular remodeling in cardiovascular diseases. For example, local ET-1 production has been shown to be increased in vascular lesions, in neointimal formation after vascular injury, and in atherosclerosis, hypercholesterolemia, and hypertension. Indeed, the administration of ET receptor antagonists reduced the neointimal formation after vascular injury and prevented vascular remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats; this suggested the possibility that ET-1 induces VSMC hyperplasia in this process in vivo. However, several studies using cultured VSMCs showed that ET-1 stimulates VSMC hypertrophy but not hyperplasia in vitro [1–3]. Therefore, it is necessary to develop an understanding of the effect of ET-1 on VSMC hypertrophy and/or hyperplasia in vivo.

In the current issue of Cardiovascular Research, Dao et al. [4] have examined the mitogenic or trophic effect of ET-1 in rat small mesenteric arteries in vivo and demonstrated that administration of low doses of ET-1 stimulated protein synthesis (hypertrophy) in the arteries, whereas high doses stimulated both protein and DNA synthesis (hyperplasia). The EC₅₀ of ET-1 for protein synthesis was 4 times lower than that for DNA synthesis, suggesting that VSMC hypertrophy and/or hyperplasia in response to ET-1 stimulation might be regulated in a concentration-dependent manner. These findings provide a new insight into the mechanisms underlying the regulation of vascular hypertrophy and hyperplasia in small arteries in vivo.

Regulation by cell cycle-regulatory proteins determines whether VSMCs undergo hypertrophy or hyperplasia in response to external stimuli. The activation of cyclin-dependent kinases (CDKs) followed by the phosphorylation of the retinoblastoma gene product (Rb) and the activation of transcriptional factor E2F are required for the cell cycle progression of VSMCs [5,6]. The kinase activity of CDKs is negatively regulated by several molecules such as p16^Ink4, p21^Waf1/Cip1, and p27^Kip1[7]; these are referred to as CDK inhibitors. In particular, although p27^Kip1 and p21^Waf1/Cip1 have been implicated in VSMC hypertrophy and hyperplasia [8–10], the effect of ET-1 on hypertrophy and hyperplasia remains unclear.

In contrast to ET-1, the mitogenic or trophic effect of angiotensin II (AII) in VSMCs has been intensively investigated. Braun-Dullaeus et al. [8] investigated the induction of expression of cell cycle-regulatory proteins by AII and serum, which induce hypertrophy and hyperplasia, respectively, in VSMCs. Both AII and serum clearly induced the upregulation of cyclinD1, CDK2, and CDK1. However, in the AII-stimulated cells, the p27^Kip1 levels remained high, and the CDK2 activation was inhibited. In the serum-stimulated cells, p27^Kip1 was markedly downregulated, and the CDK2 activation was induced. Moreover, the ablation of p27^Kip1 expression using an antisense oligonucleotide blocked hypertrophy and promoted hyperplasia in the AII-stimulated VSMCs, suggesting that p27^Kip1 functions as a molecular switch in determining whether VSMCs undergo hypertrophy or hyperplasia in response to AII stimulation. Similarly, Rao [11] showed that in cultured VSMCs, AII had no significant effect on the steady-state levels of p27^Kip1 expression and on the activities of CDK2 and CDK4, whereas platelet-derived growth factor (PDGF) induced the depletion of p27^Kip1 and increased the level of cyclin D1 and the activities of CDK2 and CDK4. In fact, p27^Kip1 has been implicated in the termination of VSMC proliferation in neointimal formation after vascular injury in vivo [12]. Regarding p21^Waf1/Cip1, Okamoto et al. [10] recently reported that AII produced its sustained induction in VSMCs and that the adenovirus-mediated overexpression of p21^Waf1/Cip1 caused VSMC hypertrophy, suggesting a role for p21^Waf1/Cip1 in VSMC hypertrophy in response to AII stimulation.

In the present study, Dao et al. [4] identified that CDK2 binding by p27^Kip1 but not by p21^Waf1/Cip1 functions as a molecular switch to prevent or promote VSMC hyperplasia in response to ET-1 stimulation in mesenteric arteries. This finding was supported by Yang et al. [3], who reported that ET-1 failed to downregulate p27^Kip1, activate CDK2, and stimulate hyperplasia in human cultured VSMCs. Taken together, these studies show that p27^Kip1 plays a critical role in determining whether VSMCs undergo hypertrophy or hyperplasia in response to ET-1 stimulation in vivo and in vitro.

Although the observations of Dao et al. [4] significantly extend our understanding of VSMC hypertrophy and hyperplasia in response to ET-1 stimulation, several questions remain. First, does a high ET-1 concentration produce any additional effects due to other factors? Interestingly, ET-1 markedly potentiates VSMC hyperplasia in response to several growth factors such as PDGF, basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) [3,13]. In addition, ET-1 upregulates PDGF receptor expression in VSMCs [14]; therefore, it is possible that ET-1 might interact with the growth factors produced locally and influence the determination of whether VSMCs undergo hyperplasia or hypertrophy in vivo. Second, does ET-1 exert similar effects on other vessels such as coronary or cerebral arteries? Since the expression pattern of ET_A and ET_B receptors might differ between the sites of vessels, the effects of ET-1 on other vessels need to be elucidated. Third, which signalling pathways are responsible for determining whether VSMCs undergo hypertrophy or hyperplasia in response to ET-1 stimulation? Dao et al. [4] showed that the PI-3K–Akt pathway was not involved in the ET-1-induced hypertrophy of VSMCs. Hence, further investigations are required to understand the detailed mechanisms by which ET-1 regulates VSMC hypertrophy and hyperplasia in vivo.