Intimal hyperplasia is an exaggerated healing response that occurs in the vessel wall after injury. It is a major cause of restenosis, which limits the success of many vascular interventions including bypass grafting, endarterectomy, and balloon angioplasty.1
The development of neointimal hyperplasia is a complex process involving vascular inflammation, re-establishment of the luminal endothelial lining, progenitor cell recruitment, and smooth muscle cell (SMC) proliferation and apoptosis.1,2
It is thought that vascular SMCs undergo a behavioural change in response to injury, transforming from a quiescent and differentiated state to a proliferative and synthetic phenotype.3
Another key characteristic of ‘injured’ SMCs is their resistance to apoptosis. SMCs, isolated from human endarterectomy lesions, were found to be insensitive to apoptotic stimuli.4
As the number of SMCs accumulated in the intimal lesions is determined by the balance between cell proliferation and apoptosis, defining the molecular mechanisms underlying both proliferation and apoptosis is necessary in order to better understand the behavioural changes of SMCs after vascular injury and thus to develop novel therapeutic strategies to inhibit intimal hyperplasia.5
Apoptosis is a multi-step process. In mammalian cells, apoptosis can be initiated through two main pathways. One pathway involves the activation of transmembrane death receptors such as Fas and TNF-α by their specific ligands leading to activation of caspase 8. The other pathway involves mitochondrial depolarization leading to the release of cytochrome c and activation of caspase 9. Both pathways ultimately result in activation of caspase 3, which then leads to apoptotic events including the cleavage of cell proteins, subsequent DNA fragmentation, and cell death.6
Factors that activate death receptors or the mitochondria-mediated apoptotic pathways are found to be abundant at the arterial wall of an injured artery.
Protein kinase C delta (PKCδ) is a novel member of the PKC family, a major group of serine–threonine kinases. All PKC isotypes share a similar structure including an N-terminal regulatory domain and a C-terminal catalytic domain connected via a hinge region. We and others have previously demonstrated in cultured vascular SMCs (VSMCs) that PKCδ is an important mediator of apoptosis in VSMCs.7–9
Inhibition of PKCδ activity or expression diminishes activation of caspase cascade as well as its downstream death events including DNA fragmentation and externalization of phospholipids.7,9
Recently, our group showed that PKCδ undergoes a caspase-3-mediated cleavage in the linker region to produce a catalytic fragment (CF) critical to the execution of cell death.7
Results with PKCδ knockout (KO) mice reveal that mice lacking PKCδ develop normally and are fertile. When subjected to stress or injury, VSMCs of PKCδ null mice showed an anti-apoptotic phenotype.8
In addition, PKCδ null mice are found to exhibit enhanced proliferation of B cells and auto-immunity.10
Using a mouse vein graft bypass model, Leitges and colleagues showed that PKCδ null mice developed an exacerbated graft arteriosclerosis associated with diminished cell apoptosis, suggesting this kinase might be an integral element of vascular injury.8
In cultured VSMCs, PKCδ has been shown to negatively regulate proliferation.11,12
Overexpression of this kinase in VSMC leads to G1 cell cycle arrest12
and impaired activation of mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/211
. However, inhibition of PKCδ either by gene deletion11
or dominant negative mutant also affects ERK activation.13
The precise relationship between PKCδ and ERK remains to be further investigated.
One of the distinctive properties of both human atherosclerosis and restenosis is the locality of lesions, which is related to differences in local hemodynamics as well as the regional differences in vessel wall physiology. Different vascular interventions, namely bypass, endarterectomy, angioplasty, and stenting, produce different kinds of injuries to the vessel which could subsequently elicit distinct injury responses. Clinically, the incidence of intimal hyperplasia and restenosis during long-term follow up varies significantly among different vascular interventions.14,15
Therefore, it is important to study PKCδ and its role in VSMC apoptosis and proliferation in multiple experimental models that mimic various vascular interventions.
In this report, we examined PKCδ function in the arterial wall using two rodent arterial injury models. Through genetic or molecular manipulations, we tested the effect of PKCδ inhibition or overexpression on VSMC apoptosis, proliferation, and intimal hyperplasia. These studies provide an explicit link between PKCδ and the pathogenesis of intimal hyperplasia after arterial injury.