Objective-To examine the impact of low-density lipoprotein (LDL), an established mediator of atherosclerosis, on the transcription factor cAMP-response element-binding protein (CREB), which is a regulator of vascular smooth muscle cell (VSMC) quiescence. Methods and Results-VSMC CREB content is diminished in rodent models of diabetes and pulmonary hypertension. We examined aortic CREB content in rodent models of aging, hypertension, and insulin resistance, and we determined nuclear CREB protein in the medial VSMC of high-fat-fed LDL receptor-null mice. There was significant loss of CREB protein in all models. In vitro, primary culture rat aortic VSMC exposed to LDL and oxidized LDL exhibited a rapid, transient increase in CREB phosphorylation and transient phosphorylation/activation of Akt, ERK, JNK, ans p38 MAPK. Exposure to oxidized LDL, but not to LDL, for 24 to 48 hours decreased CREB protein in a dose-dependent fashion and led to nuclear exclusion of CREB. Pharmacological reactive oxygen species scavengers and inhibition of ERK activation blocked oxidized LDL-mediated CREB downregulation. Conclusion-These data support a model wherein loss of VSMC CREB protein, which renders these cells more susceptible to activation and apoptosis, is a common pathological response to vascular injury and potentially contributes to plaque progression. The end result is a proinflammatory microenvironment that promotes a vicious cycle of plaque progression. Interruption of this cycle is necessary to slow atherosclerosis progression and stabilize existing plaque. Medications that lower low-density lipoprotein (LDL) cholesterol have revolutionized therapy for cardiovascular disease but do not fully normalize cardiovascular risk. 3 Our laboratory has identified the transcription factor cAMP-response element-binding protein (CREB) as a modulator of smooth muscle cell phenotype. CREB blunts mitogen-stimulated VSMC proliferation, migration, and matrix protein expression, and it protects smooth muscle cells from apoptosis. 4 -6 In previous reports, we observed decreased levels of CREB protein and the active form of CREB (phosphoserine 133 CREB) in medial VSMC in rodent models of insulin-resistant and insulin-deficient diabetes. 6 Loss of VSMC CREB protein in this model was, in part, secondary to oxidant stress and could be modeled in vitro by exposure of aortic VSMC to H 2 O 2 or glucose oxidase. 6 Similarly, in a model of pulmonary vascular injury (hypoxia-induced pulmonary hypertension), we reported loss of CREB protein concurrent with pulmonary artery hypertrophy. 5 This could be modeled in vitro by exposing pulmonary VSMC to platelet-derived growth factor, which induced CREB nuclear export and degradation by a pathway downstream of Akt and casein kinase 2 (CK2).In this article we report on the response of VSMC in vivo to rodent models of traditional cardiovascular risk factors, such as hyperlipidemia, hypertension, and obesity, and in vitro in the context of hyperlipidemia. We present data demonstrating loss of aortic CREB conten...

Aims/hypothesis Transplantation of islets is a viable option for the treatment of diabetes. A significant proportion of islets is lost during isolation, storage and after transplantation as a result of apoptosis. cAMP response element binding protein (CREB) is an important cell survival factor. The aim of the present study was to determine whether preservation of CREB function is needed for survival of human islets. Materials and methods To determine the effects of downregulation of CREB activity on beta cell apoptosis in a transplantation setting, adenoviral vectors were used to express two dominant negative mutant forms of CREB in human islets isolated from cadaveric donors. Markers of apoptosis were determined in these transduced islets under basal conditions and following treatment with growth factor. Results Expression of CREB mutants in human islets resulted in significant (p<0.001) activation of caspase-9, a key regulatory enzyme in the mitochondrial pathway of apoptosis, when compared with islets transduced with adenoviral beta galactosidase. Immunocytochemical analysis showed the activation of caspase-9 to be predominantly in beta cells. Other definitive markers of apoptosis such as parallel activation of caspase-3, accumulation of cleaved poly-(ADP-ribose) polymerase and nuclear condensation were also observed. Furthermore, the anti-apoptotic action of growth factors exendin-4 and betacellulin in human islets exposed to cytokines was partially lost when CREB function was impaired. Conclusions/interpretation Our findings suggest that impairment of CREB-mediated transcription could lead to loss of islets by apoptosis with potential implications in islet transplantation as well as in the mechanism of beta cell loss leading to diabetes.

Hypertension, dyslipidemia, inflammation, impaired fibrinolysis, and hyperglycemia all play a role in the damage done to the vasculature by type 2 diabetes and insulin resistance. In health, vascular smooth muscle cells (SMCs) are quiescent and contractile and have low proliferative and migratory activities. However, in the case of diabetes and metabolic stress, vascular smooth muscle cells undergo a change in phenotype to an increased proliferative and migratory state. This is known as phenotypic modulation and in excess it is the hallmark of atherosclerosis. The transcription factor CREB (cAMP response element binding protein) has been shown to be important for maintenance of the highly differentiated proliferation-resistant phenotype of vascular SMCs by our lab. We have observed early injury-induced activation of CREB in rodent models of atherosclerosis, a presumed early cytoprotective response to injury. The mechanism of CREB activation in this setting is unknown. Recently a novel signaling mechanism mediated by protein kinase N (PKN or PRK1/PRK2) was identified and shown to increase the transcription of SMC contractile proteins in a p38 MAPK dependent fashion. We hypothesized that PKN would increase in response to early vascular injury and activate CREB via phosphorylation. To address this hypothesis we measured protein content by Western blot analysis of PKN in aortic vessel wall lysates from young (4 wk) and old (14 wk) Zucker rats as compared with age-matched control rats. In the 4-week animals, there was a high level of PKN, which was higher in the Zucker animal (OD = 1.61 total PKN) than the control animals (OD = 1.54 total PKN). Overall PKN content decreased with age but remained higher in the Zucker cohort (OD = 1.29 for Zucker vs 0.82 in control animals). Preliminary studies assessing PKN activity (phosphoPKN) indicated than activation paralleled content. These values correlated positively with values of active CREB previously found in the same animals. In summary, expression of PKN decreases with age in the vasculature and its expression is higher in a model of insulin resistance. This is the first in vivo observation of disease-related induction of PKN in response to insulin resistance. Future studies will determine whether PKN directly activates CREB. In conclusion, PKN may be a part of a cytoprotective vascular response to injury that loses effectiveness with age.