Atherosclerosis is a complex process in which long-term exposure of the vessel wall to vascular insults induces the vicious cycle of vascular pathology (reviewed in 1, 2
). Regardless of the etiological factor or factors, most atherosclerotic lesions share overlapping features including endothelial dysfunction, neointimal proliferation, lipid accumulation, inflammation and necrosis 1, 2
. Risk factors such as dyslipidemia, hypertension, diabetes, smoking and age likely have both common and unique effects on the cellular and extracellular components of the vessel wall. We previously have reported that oxidant stress, mitogens and cytokines lead to CREB downregulation in pulmonary and aortic VSMC as well as neurons and beta cells 4–6, 8, 9
. Here, we present evidence that diminished aortic medial CREB expression is a common deleterious response to a variety of atherosclerotic risk factors. This series of experiments demonstrates significant age, hypertension, insulin resistance and obesity -induced aortic VSMC CREB downregulation. Importantly, we directly demonstrate decreased aortic nuclear CREB expression in response to high fat feeding in both C57BL/6 and LDLR −/− mice. Furthermore, we demonstrate that in vitro,
exposure of rat primary VSMC to oxLDL leads to decreased CREB protein expression specifically via a ROS/ERK-dependent pathway. These data support a model wherein loss of VSMC CREB expression is a common pathological response to vascular injury, likely due to oxidant stress, which renders the vessel wall more susceptible to proliferation, migration, inflammation and apoptosis and potentially contributes to plaque progression.
Others and we have reported that CREB is critical for oxidant defense and survival of VSMC, cardiac myocytes, neurons and adipocytes 4, 14–17,18
. Cardiac selective expression of dominant negative CREB leads to accelerated heart failure and blocks the adaptive cardiac response to exercise training 14, 19
. In this context cardiac CREB appears to regulate not only myocardial apoptosis but also metabolic adaptation by affecting mitochondrial biogenesis and efficiency 17
. We have further reported that overexpression of CREB in cultured neurons and beta cells can protect these cell types from oxidant and cytokine mediated apoptosis 8, 9
. It seems likely that CREB plays a similar protective role in the vessel wall where inflammation and oxidant stress accelerate atherosclerosis.
CREB protects cells from oxidant injury through transcriptional regulation of numerous targets including genes involved in anti-apoptotic mechanisms, upregulation of antioxidant defense, and augmentation of growth factor signaling. First, CREB inhibits apoptosis by stabilizing mitochondria through induction of bcl-2 and by augmenting mitochondrial biogenesis via PGC-1. In addition, oxidative stress leads to the induction of CREB-mediated antioxidant defenses on two levels: by inducing expression of antioxidant enzymes such as manganese superoxide dismutase (MnSOD), thioredoxin (Trx) and heme-oxygenase-1 (HO-1)20,21, 22
and by increasing mitochondrial biogenesis via upregulation of PGC-1. Finally, CREB enhances appropriate growth factor signaling through regulation of BDNF and IRS-2 23, 24
. Thus, loss of VSMC CREB function may contribute to disruption of vascular growth regulation, antioxidant and anti-apoptotic defenses.
In this report we demonstrate dramatic upregulation at the protein level of one CREB target anti-oxidant, HO-1, in response to oxLDL treatment. We hypothesize that this upregulation of HO-1 is at least in part a response to CREB activation and is one beneficial effect of CREB activation that could be lost with CREB downregulation. The persistence of HO-1 protein after CREB downregulation is not inconsistent with a CREB role in HO-1 activation, as the half-life of HO-1 mRNA is increased to up to 11 hours under certain types of oxidant stress25
. However, HO-1 regulation is complex and multifactorial, involving many signaling pathways and transcription factors other than CREB, in addition to non-transcriptional regulatory mechanisms26, 27
. Overall, the HO-1 activation we demonstrate is a marker of severe oxidant stress imposed by oxLDL treatment, and our studies do not rule out a role for other regulatory pathways. In fact, the oxLDL resistance inferred by ERK inhibition with UO126 may be the result of early HO-1 activation and represent a preconditioning that allows cells to cope with the oxidant stress imposed by oxLDL.
The hypothesis argued above, that CREB downregulation contributes to vascular pathology in atherosclerosis, implies that CREB activity is important for maintaining normal vascular physiology. CREB is a differentiation factor in VSMC, but CREB’s function in normal physiology is not as clearly delineated in the vasculature as it is in the nervous system 28, 29
. Acute CREB activation by a broad array of toxins may be a cytoprotective response to injury 18
. Recently, a number of observations have been published demonstrating acute CREB activation by established mediators of vascular injury such as fatty acids, LDL cholesterol, VLDL cholesterol, thrombin, endothelin 1, angiotensin II and tumor necrosis factor-α in endothelial cells and VSMC, both in vitro
and in vivo 11, 30–34,35
. We postulate that acute CREB activation by vascular toxins is a healthy response that is intended to decrease oxidant injury and inflammation and maintain VSMC survival and function. However, we acknowledge that there are conflicting data on the role of CREB in acute vascular injury. CREB activity was found to be increased in carotid arteries following wire injury 36
. Introduction of a dominant negative CREB adenovirus at the time of wire injury blunted neointimal lesion formation and led to increased VSMC apoptosis and decreased proliferation 36
. The explanation for these differences from our studies on CREB and VSMC phenotype remains unclear. They are likely dependent on intracellular pathway activation profiles and co-adaptor/co-repressor recruitment to CREB target genes which are unique depending upon the external stimulus and cell context 37, 38,39, 40
The mechanism of CREB down-regulation in the vasculature also remains unclear. Numerous mechanisms have been shown to contribute to the CREB dysfunction observed in Alzheimer’s disease, HIV, cocaine addiction and pulmonary hypertension 4, 34, 41–47
. It is likely that decreased vascular CREB content across the different rodent models presented in this manuscript is also the consequence of more than one mechanism. First, CREB is a self-regulating protein, such that loss of upstream signaling to CREB can result in decreased CREB expression. For example, depletion of cyclic nucleotides and decreased PKA/PKG activation decreases CREB expression 48, 49
. The loss of eNOS stimulated cyclic nucleotide accumulation in endothelial dysfunction results could contribute to decreased vascular CREB expression. Expression of PDGF and its receptor are increased in models of atherosclerosis 50, 2, 51
, as such PDGR stimulation of AKT/CK2 and CREB ubiquination could also occur in the systemic vasculature 4
. It is probable that generation of ROS is the major underlying contributor to CREB downregulation across vascular disease models. ROS have been shown to disrupt signaling to CREB 9
and to promote CREB degradation in Alzheimer’s disease 47
. We have reported downregulation of CREB in aortic VSMC, neurons, and beta cells in response to direct oxidant stress 4, 6, 8, 9
. In further support of this hypothesis we now report that the potent CV risk factor, oxLDL, directly induces CREB downregulation in VSMC in vitro
. In cell culture, simple screening with pharmacological inhibitors indicates that generation of ROS and activation of ERK, but not PKA or Akt, are critical for oxLDL-mediated loss of CREB expression. Another recent report has also suggested that induction of oxidant defense can block effects of LDL on VSMC 10
. A more exhaustive series of studies will be needed to define fully the oxidant-induced signaling pathways important for CREB downregulation.
Loss of vascular medial CREB expression appears to be an early response of the vessel wall to cardiovascular risk exposure; as such, interventions aimed at restoring or maintaining vascular CREB are an appealing goal. Lifestyle interventions demonstrated to improve longevity and decrease cardiovascular risk also increase CREB function 52, 53
. In the CNS, exercise training enhances nNOS expression leading to induction of CREB 54, 18
. Furthermore, exercise increases active CREB and contributes to improved recovery from ischemia, stroke, and memory tasks post-seizure 52, 55
. Preliminary observations indicate that age-related decrease in aortic CREB protein content can be abrogated by caloric restriction (unpublished 2009 JEBR, PAW, JG). Watson et al also recently reported increased cardiac CREB content with modest exercise training in a rodent model of heart failure 17
. Unfortunately, the compensation for VSMC CREB deficiency is not as easy as adding CREB to the vessel wall because inflammatory cell differentiation also employs CREB (see the lumen –). Thus, interventions aimed at restoring CREB will likely need to be directed at the underlying mechanism(s) of CREB loss and will require a better understanding of these mechanism(s). Interestingly, thiazolidinediones, agents which increase insulin sensitivity, have also been found to increase CREB content in the heart and blood vessels of the insulin resistant ob/ob mouse model (6
and unpublished observations). In an Alzheimer’s disease model, blocking CREB degradation with an inhibitor of ubiquitin 1 ligase led to increased CREB expression and improved cognition 43
. Overall, clarification of the mechanism(s) of CREB loss in vascular disease may suggest novel therapeutic target(s) for vascular intervention and augmentation of CREB dependent vascular defenses.
In summary, we report loss of CREB expression across a spectrum of rodent models of vascular risk including insulin resistance, aging, and high fat diet. The relevance of oxLDL to the in vivo vascular CREB depletion model is borne out by the loss of CREB upon direct challenge of VSMC with oxLDL in vitro. Simple analysis of critical pathways reveals that blockade of ROS generation or ERK activation protects cells from CREB depletion and from oxLDL toxicity. This in vitro model will allow further analysis of the mechanism of oxLDL-mediated CREB loss in VSMC. These experiments provide new information on a common response to vascular injury that may be an early event in atherosclerosis and is a potential new target for CVD intervention.