Opposing Actions of AKT (Protein Kinase B) Isoforms in Vascular Smooth Muscle Injury and Therapeutic Response.

OBJECTIVE: Drug-eluting stent delivery of mTORC1 (mechanistic target of rapamycin complex 1) inhibitors is highly effective in preventing intimal hyperplasia after coronary revascularization, but adverse effects limit their use for systemic vascular disease. Understanding the mechanism of action may lead to new treatment strategies. We have shown that rapamycin promotes vascular smooth muscle cell differentiation in an AKT2-dependent manner in vitro. Here, we investigate the roles of AKT (protein kinase B) isoforms in intimal hyperplasia. APPROACH AND RESULTS: We found that germ-line-specific or smooth muscle-specific deletion of Akt2 resulted in more severe intimal hyperplasia compared with control mice after arterial denudation injury. Conversely, smooth muscle-specific Akt1 knockout prevented intimal hyperplasia, whereas germ-line Akt1 deletion caused severe thrombosis. Notably, rapamycin prevented intimal hyperplasia in wild-type mice but had no therapeutic benefit in Akt2 knockouts. We identified opposing roles for AKT1 and AKT2 isoforms in smooth muscle cell proliferation, migration, differentiation, and rapamycin response in vitro. Mechanistically, rapamycin induced MYOCD (myocardin) mRNA expression. This was mediated by AKT2 phosphorylation and nuclear exclusion of FOXO4 (forkhead box O4), inhibiting its binding to the MYOCD promoter. CONCLUSIONS: Our data reveal opposing roles for AKT isoforms in smooth muscle cell remodeling. AKT2 is required for rapamycin's therapeutic inhibition of intimal hyperplasia, likely mediated in part through AKT2-specific regulation of MYOCD via FOXO4. Because AKT2 signaling is impaired in diabetes mellitus, this work has important implications for rapamycin therapy, particularly in diabetic patients.

Chronic miR-29 antagonism promotes favorable plaque remodeling in atherosclerotic mice.

Abnormal remodeling of atherosclerotic plaques can lead to rupture, acute myocardial infarction, and death. Enhancement of plaque extracellular matrix (ECM) may improve plaque morphology and stabilize lesions. Here, we demonstrate that chronic administration of LNA-miR-29 into an atherosclerotic mouse model improves indices of plaque morphology. This occurs due to upregulation of miR-29 target genes of the ECM (col1A and col3A) resulting in reduced lesion size, enhanced fibrous cap thickness, and reduced necrotic zones. Sustained LNA-miR-29 treatment did not affect circulating lipids, blood chemistry, or ECM of solid organs including liver, lung, kidney, spleen, or heart. Collectively, these data support the idea that antagonizing miR-29 may promote beneficial plaque remodeling as an independent approach to stabilize vulnerable atherosclerotic lesions.

Inducing mitophagy in diabetic platelets protects against severe oxidative stress.

Diabetes mellitus (DM) is a growing international concern. Considerable mortality and morbidity associated with diabetes mellitus arise predominantly from thrombotic cardiovascular events. Oxidative stress-mediated mitochondrial damage contributes significantly to enhanced thrombosis in DM A basal autophagy process has recently been described as playing an important role in normal platelet activation. We now report a substantial mitophagy induction (above basal autophagy levels) in diabetic platelets, suggesting alternative roles for autophagy in platelet pathology. Using a combination of molecular, biochemical, and imaging studies on human DM platelets, we report that platelet mitophagy induction serves as a platelet protective mechanism that responds to oxidative stress through JNK activation. By removing damaged mitochondria (mitophagy), phosphorylated p53 is reduced, preventing progression to apoptosis, and preserving platelet function. The absence of mitophagy in DM platelets results in failure to protect against oxidative stress, leading to increased thrombosis. Surprisingly, this removal of damaged mitochondria does not require contributions from transcription, as platelets lack a nucleus. The considerable energy and resources expended in "prepackaging" the complex mitophagy machinery in a short-lived normal platelet support a critical role, in anticipation of exposure to oxidative stress.

Phosphorylation of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) by Akt promotes stability and mitogenic function of S-phase kinase-associated protein-2 (Skp2).

The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.

Epigenetic regulation of smooth muscle cell plasticity.

Smooth muscle cells (SMC) are the major cell type in blood vessels. Their principal function in the body is to regulate blood flow and pressure through vessel wall contraction and relaxation. Unlike many other mature cell types in the adult body, SMC do not terminally differentiate but retain a remarkable plasticity. They have the unique ability to toggle between a differentiated and quiescent "contractile" state and a highly proliferative and migratory "synthetic" phenotype in response to environmental stresses. While there have been major advances in our understanding of SMC plasticity through the identification of growth factors and signals that can influence the SMC phenotype, how these regulate SMC plasticity remains unknown. To date, several key transcription factors and regulatory cis elements have been identified that play a role in modulating SMC state. The frontier in understanding the molecular mechanisms underlying SMC plasticity has now advanced to the level of epigenetics. This review will summarize the epigenetic regulation of SMC, highlighting the role of histone modification, DNA methylation, and our most recent identification of a DNA demethylation pathway in SMC that is pivotal in the regulation of the SMC phenotypic state. Many disorders are associated with smooth muscle dysfunction, including atherosclerosis, the major underlying cause of stroke and coronary heart disease, as well as transplant vasculopathy, aneurysm, asthma, hypertension, and cancer. An increased understanding of the major regulators of SMC plasticity will lead to the identification of novel target molecules that may, in turn, lead to novel drug discoveries for the treatment of these diseases. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) and nuclear factor-κB (NF-κB): a feed-forward loop for systemic and vascular inflammation.

The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation. However, the mechanisms underlying these effects are not fully understood. Because of the fundamental role that inflammation plays in vascular diseases, we hypothesized that EBP50 mediates macrophage activation and the response of vessels to inflammation. Indeed, EBP50 expression increased in primary macrophages and VSMC, and in the aorta of mice, upon treatment with LPS or TNFα. This increase was nuclear factor-κB (NF-κB)-dependent. Conversely, activation of NF-κB was impaired in EBP50-null VSMC and macrophages. We found that inflammatory stimuli promote the formation of an EBP50-PKCζ complex at the cell membrane that induces NF-κB signaling. Macrophage activation and vascular inflammation after acute LPS treatment were reduced in EBP50-null cells and mice as compared with WT. Furthermore, macrophage recruitment to vascular lesions was significantly reduced in EBP50 knock-out mice. Thus, EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation.

EBP50 promotes focal adhesion turnover and vascular smooth muscle cells migration.

The ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ-containing scaffolding protein that regulates a variety of physiological functions. In the vasculature, EBP50 promotes neointima formation following arterial injury. In this study the role of EBP50 on vascular smooth muscle cell (VSMC) migration was characterized. The spreading and motility of primary VSMC isolated from EBP50 knockout (KO) mice were significantly reduced compared to wild-type (WT) cells. EBP50-null VSMC had fewer and larger focal adhesions than wild-type cells. Assembly and disassembly of focal adhesion-assessed by live-cell total internal reflection fluorescence imaging-in response to epidermal growth factor (EGF) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with EGF receptor via the PDZ2 domain and with focal adhesion kinase (FAK) via the C-terminal ERM domain. EBP50 promoted the formation of a complex containing both EGF receptor and FAK. Phosphorylation of Tyr-925 of FAK in response to EGF was significantly reduced in KO cell compared to WT cells. The residence time of FAK in focal adhesions-determined by fluorescence recovery after photobleaching-was increased in WT cells. Collectively, these studies indicate that EBP50, by scaffolding EGF receptor and FAK, facilitates activation of FAK, focal adhesion turnover, and migration of VSMC.

The scaffolding protein EBP50 promotes vascular smooth muscle cell proliferation and neointima formation by regulating Skp2 and p21(cip1).

OBJECTIVE: The Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffolding protein known to regulate ion homeostasis in the kidney and intestine. Previous work showed that EBP50 expression increases after balloon injury in rat carotids. This study was designed to determine the role of EBP50 on vascular smooth muscle cells (VSMC) proliferation and the development of neointimal hyperplasia. METHODS AND RESULTS: Wire injury was performed in wild type (WT) and EBP50 knockout (KO) mice. Two weeks after injury, neointima formation was 80% lower in KO than in WT mice. Proliferation of KO VSMC was significantly lower than WT cells and overexpression of EBP50 increased VSMC proliferation. Akt activity and expression of S-phase kinase protein2 decreased in KO cells resulting in the stabilization of the cyclin-dependent kinase inhibitor, p21(cip1). Consequently, KO cells were arrested in G(0)/G(1) phase. Consistent with these observations, p21(cip1) was detected in injured femoral arteries of KO but not WT mice. No differences in apoptosis between WT and KO were observed. CONCLUSIONS: EBP50 is critical for neointima formation and induces VSMC proliferation by decreasing S-phase kinase protein2 stability, thereby accelerating the degradation of the cell cycle inhibitor p21(cip1).

Characterization and detection of cellular and proteomic alterations in stable stathmin-overexpressing, taxol-resistant BT549 breast cancer cells using offgel IEF/PAGE difference gel electrophoresis.

Stathmin/oncoprotein 18, a protein that regulates microtubule dynamics, is highly expressed in a number of tumors including leukemia, lymphoma, neuroblastoma, breast, ovarian, and prostate cancers. High stathmin levels have been associated with the development of resistance to the widely used anti-cancer drug taxol ((®)Taxol, paclitaxel). The mechanisms of stathmin-mediated taxol resistance are not well-understood at the molecular level. To better understand the role of stathmin in taxol resistance, we stably overexpressed stathmin twofold in BT549 human breast cancer cells and characterized several cell processes involved in the mechanism of action of taxol. After stable overexpression of stathmin, neither the cell doubling time nor the mitotic index was altered and the microtubule polymer mass was reduced only modestly (by 18%). Unexpectedly, microtubule dynamicity was reduced by 29% after stathmin overexpression, resulting primarily from reduction in the catastrophe frequency. Sensitivity to taxol was reduced significantly (by 44%) in a clonogenic assay, and stathmin appeared to protect the cells from the spindle-damaging effects of taxol. The results suggest that in the stably stathmin-overexpressing clones, compensatory gene expression occurred that resulted in normal rates of cell proliferation and prevented the increase in catastrophe frequency expected in response to stathmin. Stathmin overexpression protected the cells from taxol-induced abnormal mitoses, and thus induced taxol resistance. Using offgel IEF/PAGE difference gel electrophoresis, we identified a number of proteins whose expression is reduced in the taxol-resistant stathmin-overexpressing cell lines, including proteins involved in the cytoskeleton and cell structure, the stress response, protein folding, glycolysis, and catalysis.

EBP50 inhibits the anti-mitogenic action of the parathyroid hormone type 1 receptor in vascular smooth muscle cells.

Parathyroid hormone-related protein (PTHrP) and the parathyroid hormone type 1 receptor (PTH1R) are important regulators of vascular remodeling. PTHrP expression is associated to increased proliferation of vascular smooth muscle cells (VSMC). In contrast, signaling via the PTH1R inhibits cell growth. The mechanisms regulating the dual effect of PTHrP and PTH1R on VSMC proliferation are only partially understood. In this study we examined the role of the adaptor protein ezrin-radixin-moesin-binding phosphoprotein (EBP50) on PTH1R expression, trafficking, signaling and control of A10 cell proliferation. In normal rat vascular tissues, EBP50 was restricted to the endothelium with little expression in VSMC. EBP50 expression significantly increased in VSMC following angioplasty in parallel with PTHrP. Interestingly, PTHrP was able to induce EBP50 expression. In the clonal rat aortic smooth muscle cell line A10, EBP50 increased the recruitment of PTH1R to the cell membrane and delayed its internalization in response to PTHrP(1-36). This effect required an intact C-terminal motif in the PTH1R. In naïve A10 cells, PTHrP(1-36) stimulated cAMP production but not intracellular calcium release. In contrast, PTHrP(1-36) induced both cAMP and calcium signaling in A10 cells over-expressing EBP50. Finally, EBP50 attenuated the induction of p27(kip1) and the anti-proliferative effect of PTHrP(1-36). In summary, this study demonstrates the dynamic expression of EBP50 in vessels following injury and the effects of EBP50 on PTH1R function in VSMC. These findings highlight one of the mechanisms leading to increased VSMC proliferation and have important implication in the understanding of the molecular events leading to restenosis.