Trevor D. Littlewood

The role of c-myc in cell growth

Recent experiments have established that the c-myc oncogene encodes a sequence-specific DNA-binding protein that interacts with a specific intracellular partner, Max, and probably manifests its effects through transcriptional modulation. In addition, the range of biological functions attributed to expression of c-myc has grown to include not only transformation and mitogenesis but also cell death.

Chronic Apoptosis of Vascular Smooth Muscle Cells Accelerates Atherosclerosis and Promotes Calcification and Medial Degeneration

Vascular smooth muscle cell (VSMC) accumulation is implicated in plaque development. In contrast, VSMC apoptosis is implicated in plaque rupture, coagulation, vessel remodeling, medial atrophy, aneurysm formation, and calcification. Although VSMC apoptosis accompanies multiple pathologies, there is little proof of direct causality, particularly with the low levels of VSMC apoptosis seen in vivo. Using a mouse model of inducible VSMC–specific apoptosis, we demonstrate that low-level VSMC apoptosis during either atherogenesis or within established plaques of apolipoprotein (Apo)E−/− mice accelerates plaque growth by two-fold, associated with features of plaque vulnerability including a thin fibrous cap and expanded necrotic core. Chronic VSMC apoptosis induced development of calcified plaques in younger animals and promoted calcification within established plaques. In addition, VSMC apoptosis induced medial expansion, associated with increased elastic lamina breaks, and abnormal matrix deposition reminiscent of cystic medial necrosis in humans. VSMC apoptosis prevented outward remodeling associated with atherosclerosis resulting in marked vessel stenosis. We conclude that VSMC apoptosis is sufficient to accelerate atherosclerosis, promote plaque calcification and medial degeneration, prevent expansive remodeling, and promote stenosis in atherosclerosis.

c-Myc functionally cooperates with Bax to induce apoptosis.

ABSTRACT c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.

Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-κB activation

Normal fibroblasts are resistant to the cytotoxic action of tumor necrosis factor (TNF), but are rendered TNF‐sensitive upon deregulation of c‐Myc. To assess if oncoproteins induce the cytotoxic TNF activity by modulating TNF signaling, we investigated the TNF‐elicited signaling responses in fibroblasts containing a conditionally active c‐Myc protein. In association with cell death, c‐Myc impaired TNF‐induced activation of phospholipase A2, JNK protein kinase and cell survival‐signaling‐associated NF‐κB transcription factor complex. The TNF‐induced death of mouse primary fibroblasts expressing deregulated c‐Myc was inhibited by transient overexpression of the p65 subunit of NF‐κB, which increased NF‐κB activity in the cells. Unlike other TNF‐induced signals, TNF‐induced accumulation of the wild‐type p53 mRNA and protein was not inhibited by c‐Myc. TNF, with c‐Myc, induced apoptosis in mouse primary fibroblasts but only weakly in p53‐deficient primary fibroblasts. The C‐terminal domain of p53, which is a transacting dominant inhibitor of wild‐type p53, failed to inhibit apoptosis by c–Myc and TNF, suggesting that the cell death was not dependent on the transcription‐activating function of p53. Taken together, the present findings show that the cytotoxic activity of TNF towards oncoprotein‐expressing cells involves p53 and an impaired signaling for survival in such cells.

Statins Use a Novel Nijmegen Breakage Syndrome-1–Dependent Pathway to Accelerate DNA Repair in Vascular Smooth Muscle Cells

Although the hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used in atherosclerosis to reduce serum cholesterol, statins have multiple other effects, including direct effects on cells of the vessel wall. Recently, DNA damage, including telomere shortening, has been identified in vascular smooth muscle cells (VSMCs) in human atherosclerosis. Although statins reduce DNA damage in vitro, the mechanisms by which they might protect DNA integrity in VSMCs are unknown. We show that human atherosclerotic plaque VSMCs exhibit increased levels of double-stranded DNA breaks and basal activation of DNA repair pathways involving ataxia telangiectasia–mutated (ATM) and the histone H2AX in vivo and in vitro. Oxidant stress induced DNA damage and activated DNA repair pathways in VSMCs. Statin treatment did not reduce oxidant stress or DNA damage but markedly accelerated DNA repair. Accelerated DNA repair required both the Nijmegen breakage syndrome (NBS)-1 protein and the human double minute protein Hdm2, accompanied by phosphorylation of Hdm2, dissociation of NBS-1 and Hdm2, inhibition of NBS-1 degradation, and accelerated phosphorylation of ATM. Statin treatment reduced VSMC senescence and telomere attrition in culture, accelerated DNA repair and reduced apoptosis in vivo after irradiation, and reduced ATM/ATR (ATM and Rad3-related) activity in atherosclerosis. We conclude that statins activate a novel mechanism of accelerating DNA repair, dependent on NBS-1 stabilization and Hdm2. Statin treatment may delay cell senescence and promote DNA repair in atherosclerosis.

Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3

Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis.

Cell death in the cardiovascular system

Cell death is important for both development and tissue homeostasis in the adult. As such, it is tightly controlled and deregulation is associated with diverse pathologies; for example, regulated cell death is involved in vessel remodelling during development or following injury, but deregulated death is implicated in pathologies such as atherosclerosis, aneurysm formation, ischaemic and dilated cardiomyopathies and infarction. We describe the mechanisms of cell death and its role in the normal physiology and various pathologies of the cardiovascular system.

Bone Marrow–Derived Smooth Muscle–Like Cells Are Infrequent in Advanced Primary Atherosclerotic Plaques but Promote Atherosclerosis

Objective—Although vascular smooth muscle cells (VSMCs) provide the major structural integrity of atherosclerotic plaques, their origin has been questioned. In particular, although some studies identified plaque VSMCs originating from bone marrow or peripheral blood, their frequency is controversial and their function unknown. We used genetic tracking of cell fate through smooth muscle cell (SMC)–specific LacZ reporter activity and VSMC-selective apoptosis to investigate the frequency, distribution, and role of marrow-derived VSMCs in atherogenesis. Methods and Results—Cultured mouse bone marrow–derived smooth muscle–like cells expressed SMC markers and functional SMC promoter-driven transgenes over time. Transplantation of apolipoprotein E (ApoE)−/− mice with smooth muscle myosin heavy chain–Cre/ROSA26R/ApoE−/− marrow showed that 0.7±0.14% cells expressed LacZ in atherosclerotic plaques, located superficially in early plaques, and in necrotic cores but not fibrous caps of advanced lesions. Cells expressing both progenitor and SMC markers showed a similar distribution and frequency. Apoptosis of marrow-derived SMC-like cells transplanted from SM22&agr;–human diphtheria toxin receptor/ApoE−/− mice retarded atherogenesis, with reduced plaque macrophage content. Cultured marrow-derived SMC-like cells secreted proinflammatory cytokines and promoted macrophage migration, VSMC proliferation, and collagen synthesis. Conclusion—Bone marrow–derived SMC-like cells are infrequent in advanced primary atherosclerotic plaques and absent in fibrous caps. However, these cells secrete proinflammatory cytokines and mitogens and promote atherosclerosis.

Smooth Muscle Cell Apoptosis Promotes Vessel Remodeling and Repair via Activation of Cell Migration, Proliferation, and Collagen Synthesis

Objective—Although vascular smooth muscle cell (VSMC) apoptosis occurs after vessel injury and during remodeling, the direct role of VSMC death in determining final vessel structure is unclear. We sought to determine the role of VSMC apoptosis in vessel remodeling, medial repair, and neointima formation and to identify the mediators involved. Methods and Results—The left common carotid artery was ligated in SM22&agr;-human diphtheria toxin receptor mice, in which diphtheria toxin treatment selectively induces VSMC apoptosis. Apoptosis induced from day 7 to day 14 after ligation significantly increased neointimal and medial areas, cell proliferation, migration, and vessel size. Neointima formation depended on VSMCs, as VSMC depletion before ligation significantly reduced neointimal area and cellularity. In culture, conditioned media from apoptotic VSMCs promoted VSMC migration, proliferation, and collagen synthesis. Interleukin-6 (IL-6) secretion increased 5-fold and IL-1&agr; 1.5-fold after apoptosis, whereas IL-6 inhibition negated the effect of apoptotic VSMC supernatants on VSMC migration, proliferation, and matrix synthesis. Conclusion—Signaling from apoptotic VSMCs directly promotes vessel remodeling, medial repair, and neointima formation after flow reduction. Although lumen size appears to depend on flow, VSMC apoptosis is an important determinant of vessel, medial, and neointimal size after flow reduction.

Oxidative stress regulates IGF1R expression in vascular smooth-muscle cells via p53 and HDAC recruitment

Apoptosis of VSMCs (vascular smooth-muscle cells) leads to features of atherosclerotic plaque instability. We have demonstrated previously that plaque-derived VSMCs have reduced IGF1 (insulin-like growth factor 1) signalling, resulting from a decrease in the expression of IGF1R (IGF1 receptor) compared with normal aortic VSMCs [Patel, Zhang, Siddle, Soos, Goddard, Weissberg and Bennett (2001) Circ. Res. 88, 895-902]. In the present study, we show that apoptosis induced by oxidative stress is inhibited by ectopic expression of IGF1R. Oxidative stress repressed IGF1R expression at multiple levels, and this was also blocked by mutant p53. Oxidative stress also induced p53 phosphorylation and apoptosis in VSMCs. p53 negatively regulated IGF1R promoter activity and expression and, consistent with this, p53-/- VSMCs demonstrated increased IGF1R expression, both in vitro and in advanced atherosclerotic plaques in vivo. Oxidative-stress-induced interaction of endogenous p53 with TBP (TATA-box-binding protein) was dependent on p53 phosphorylation. Oxidative stress also increased the association of p53 with HDAC1 (histone deacetylase 1). Trichostatin A, a specific HDAC inhibitor, or p300 overexpression relieved the repression of IGF1R following oxidative stress. Furthermore, acetylated histone-4 association with the IGF1R promoter was reduced in cells subjected to oxidative stress. These results suggest that oxidative-stress-induced repression of IGF1R is mediated by the association of phosphorylated p53 with the IGF1R promoter via TBP, and by the subsequent recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF1R promoter-TBP-p53 complex.