We demonstrated that alcohol (EtOH) inhibits VSMC proliferation in vitro by inhibiting Notch signaling in these cells. Moreover, daily alcohol feeding markedly reduced intima-media thickening concomitant with inhibition of induced Notch 1 and HRT expression after carotid ligation injury in the mouse. These findings provide new mechanistic data that may be relevant to how moderate alcohol consumption affects SMC proliferation and vascular remodeling and, consequently, cardiovascular mortality.
In the adult, SMCs proliferate at an extremely low rate and their principal function is contraction and regulation of blood vessel diameter. Unlike either skeletal or cardiac muscle, which is terminally differentiated, SMCs within adult animals retain plasticity and can undergo reversible changes in phenotype in response to a variety of stimuli, including growth factors and hemodynamic forces.24,25
An example of this plasticity is seen in response to vascular injury when SMCs dramatically increase their rate of cell growth and synthetic capacity (including the production of extracellular matrix components). VSMC proliferation plays a critical role in atherosclerosis and during restenosis after angioplasty and its regulation; therefore, it is of considerable clinical interest.
Previous studies have investigated the effect of alcohol on SMC growth and on injury-induced vascular remodeling. Alcohol inhibited SMC proliferation and migration in vitro,15,16,26
inhibited neointimal hyperplasia after balloon injury in animals,18–20
reduced restenosis after percutaneous transluminal coronary angioplasty and stent implantation in patients,27
and inhibited the progression and initiation of atherosclerotic lesions in hyperlipidemic mice.28
Herein, we demonstrate that alcohol treatment inhibits HCASMC proliferation in vitro and that daily feeding of alcohol, in amounts considered equivalent to those found in moderate drinkers, inhibited carotid artery vessel remodeling (ie, intimal-medial thickening) in response to flow reduction. Because SMCs are the predominant cell type responsible for intimal-medial thickening in this model23,29
and given our in vitro findings, our data are supportive of the concept that moderate alcohol consumption inhibits SMC proliferation and, thus, vascular remodeling. EtOH feeding also strongly inhibited the increase in adventitial volume seen in this model. The adventitia is predominately composed of fibroblasts and connective tissue cells; thus, the inhibitory effect of alcohol on the adventitia cannot be ascribed to inhibition of VSMC growth and warrants separate investigation.
Notch signaling is an important regulator of cell fate decisions during embryogenesis and also in adults in certain circumstances.30
SMCs preferentially express the receptors Notch 1 and Notch 3. Engagement of Notch by ligand results in 2 cleavage events that allow release of the IC of Notch (also referred to as notch intracellular domain [NICD]), thereby allowing translocation to the nucleus.31
Therefore, enforced expression of Notch IC provides a constitutively active signaling form of the receptor. The major signaling pathway evoked by Notch IC translocation into the nucleus is relayed by the transcriptional repressor, RBP-Jk, which is also (CBF1/(Su[H]Lag-1)-type transcription factor (CSL). Notch IC/CSL signaling leads to cell type–dependent activation of Notch target genes, including the HES
gene and HES
-related transcription factors (HRTs).32,33
Researchers have previously shown that Notch receptors and downstream target genes (HES
) are crucial in controlling adult SMC growth, migration, and apoptosis in vitro and in vivo.8–10
Herein, overexpression of HRT-1 increased SMC proliferation and inhibited the EtOH antiproliferative effect, supporting a role for Notch 1–regulated gene expression in the regulation of SMC growth. It was also demonstrated that hemodynamic forces, such as cyclic strain, can modulate Notch receptor signaling and proliferation in VSMCs.34
Moreover, previous studies suggest that the expression of several Notch components, including receptors (Notch 1 and 3), ligands (Jagged 1 and Jagged 2), and target genes (HRT-1
) are altered after experimentally induced vascular injury (balloon catheter denudation)9,11,14,35
in human atherosclerotic lesions36
and after flow-induced injury.37
Intimal hyperplasia after vascular injury (wire injury) was significantly decreased in HRT-2−/−
and SMCs from HRT-2−/−
mice revealed that these mutant cells proliferate at a reduced rate compared with wild-type cells. The overexpression of HRT-1 or HRT-2 in VSMCs led to increased VSMC proliferation associated with reduced levels of the cyclin-dependent kinase inhibitors p21waf1/cip1 (Cdkn1a) or p27kip1 (Cdkn1b).39
However, despite the evidence supporting an important role for Notch in regulating SMC proliferation/vascular remodeling, and the growth inhibitory effects of EtOH on SMCs, to our knowledge, there have been no previous studies investigating the effect of alcohol on the Notch pathway in SMCs. Therefore, our study represents the first evidence of an alcohol-induced modulation of Notch signaling in SMCs. EtOH, at the dose studied, inhibited Notch 1 in HCASMCs, in the absence of any effect on Notch 3. Moreover, Notch 1 mRNA expression was exclusively induced in the carotid artery after ligation injury, an induction that was inhibited by alcohol feeding. Our data support the important role of Notch 1 IC (but not Notch 3 IC) in contributing to the intimal hyperplasia typical of this animal model. This fits well with the recent report by Li et al,14
indicating a role for Notch 1, but not Notch 3, in SMCs in mediating neointimal formation in a carotid ligation vascular injury model. Notch signaling is a tightly controlled pathway with several potential regulatory points. Studies investigating precisely how EtOH inhibits Notch signaling in SMCs are under way in our laboratory. Interestingly, it was recently reported that EtOH promotes Notch activity and proliferation in endothelial cells,22
the opposite of the effect seen herein in SMCs. Taken together, our studies point to an intriguing vascular cell-specific effect of EtOH with respect to Notch that merits further investigation.
In conclusion, alcohol, at levels consistent with moderate consumption, inhibits Notch signaling and, thus, proliferation in VSMCs in vitro and during vessel remodeling in response to flow reduction in vivo. Modulation of Notch by alcohol in SMCs may be an important mechanism involved in alcohol’s purported cardiovascular protective effects.