Hideyuki Miyauchi

p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload

Cardiac hypertrophy occurs as an adaptive response to increased workload to maintain cardiac function. However, prolonged cardiac hypertrophy causes heart failure, and its mechanisms are largely unknown. Here we show that cardiac angiogenesis is crucially involved in the adaptive mechanism of cardiac hypertrophy and that p53 accumulation is essential for the transition from cardiac hypertrophy to heart failure. Pressure overload initially promoted vascular growth in the heart by hypoxia-inducible factor-1 (Hif-1)-dependent induction of angiogenic factors, and inhibition of angiogenesis prevented the development of cardiac hypertrophy and induced systolic dysfunction. Sustained pressure overload induced an accumulation of p53 that inhibited Hif-1 activity and thereby impaired cardiac angiogenesis and systolic function. Conversely, promoting cardiac angiogenesis by introducing angiogenic factors or by inhibiting p53 accumulation developed hypertrophy further and restored cardiac dysfunction under chronic pressure overload. These results indicate that the anti-angiogenic property of p53 may have a crucial function in the transition from cardiac hypertrophy to heart failure.

Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway.

The signaling pathway of insulin/insulin‐like growth factor‐1/phosphatidylinositol‐3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF‐16, a forkhead transcription factor. Although reduction‐of‐function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Here we show that Akt activity increases along with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence‐like arrest of cell growth via a p53/p21‐dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt‐induced senescence may be involved in vascular pathophysiology.

Angiotensin II Induces Premature Senescence of Vascular Smooth Muscle Cells and Accelerates the Development of Atherosclerosis via a p21-Dependent Pathway

Background— Angiotensin II (Ang II) has been reported to contribute to the pathogenesis of various human diseases including atherosclerosis, and inhibition of Ang II activity has been shown to reduce the morbidity and mortality of cardiovascular diseases. We have previously demonstrated that vascular cell senescence contributes to the pathogenesis of atherosclerosis; however, the effects of Ang II on vascular cell senescence have not been examined. Methods and Results— Ang II significantly induced premature senescence of human vascular smooth muscle cells (VSMCs) via the p53/p21-dependent pathway in vitro. Inhibition of this pathway effectively suppressed induction of proinflammatory cytokines and premature senescence of VSMCs by Ang II. Ang II also significantly increased the number of senescent VSMCs and induced the expression of proinflammatory molecules and of p21 in a mouse model of atherosclerosis. Loss of p21 markedly ameliorated the induction of proinflammatory molecules by Ang II, thereby preventing the development of atherosclerosis. Replacement of p21-deficient bone marrow cells with wild-type cells had little influence on the protective effect of p21 deficiency against the progression of atherogenesis induced by Ang II. Conclusions— We demonstrated that Ang II promotes vascular inflammation by inducing premature senescence of VSMCs both in vitro and in vivo. Our results suggest a critical role of p21-dependent premature senescence of VSMCs in the pathogenesis of atherosclerosis.

Critical Roles of Muscle-Secreted Angiogenic Factors in Therapeutic Neovascularization

The discovery of bone marrow–derived endothelial progenitors in the peripheral blood has promoted intensive studies on the potential of cell therapy for various human diseases. Accumulating evidence has suggested that implantation of bone marrow mononuclear cells effectively promotes neovascularization in ischemic tissues. It has also been reported that the implanted cells are incorporated not only into the newly formed vessels but also secrete angiogenic factors. However, the mechanism by which cell therapy improves tissue ischemia remains obscure. We enrolled 29 “no-option” patients with critical limb ischemia and treated ischemic limbs by implantation of peripheral mononuclear cells. Cell therapy using peripheral mononuclear cells was very effective for the treatment of limb ischemia, and its efficacy was associated with increases in the plasma levels of angiogenic factors, in particular interleukin-1&bgr; (IL-1&bgr;). We then examined an experimental model of limb ischemia using IL-1&bgr;–deficient mice. Implantation of IL-1&bgr;–deficient mononuclear cells improved tissue ischemia as efficiently as that of wild-type cells. Both wild-type and IL-1&bgr;–deficient mononuclear cells increased expression of IL-1&bgr; and thus induced angiogenic factors in muscle cells of ischemic limbs to a similar extent. In contrast, inability of muscle cells to secrete IL-1&bgr; markedly reduces induction of angiogenic factors and impairs neovascularization by cell implantation. Implanted cells do not secret angiogenic factors sufficient for neovascularization but, instead, stimulate muscle cells to produce angiogenic factors, thereby promoting neovascularization in ischemic tissues. Further studies will allow us to develop more effective treatments for ischemic vascular disease.

Protective Role of SIRT1 in Diabetic Vascular Dysfunction

Objective—Calorie restriction (CR) prolongs the lifespan of various species, ranging from yeasts to mice. In yeast, CR extends the lifespan by increasing the activity of silencing information regulator 2 (Sir2), an NAD+-dependent deacetylase. SIRT1, a mammalian homolog of Sir2, has been reported to downregulate p53 activity and thereby prolong the lifespan of cells. Although recent evidence suggests a link between SIRT1 activity and metabolic homeostasis during CR, its pathological role in human disease is not yet fully understood. Methods and Results—Treatment of human endothelial cells with high glucose decreases SIRT1 expression and thus activates p53 by increasing its acetylation. This in turn accelerates endothelial senescence and induces functional abnormalities. Introduction of SIRT1 or disruption of p53 inhibits high glucose–induced endothelial senescence and dysfunction. Likewise, activation of Sirt1 prevents the hyperglycemia-induced vascular cell senescence and thereby protects against vascular dysfunction in mice with diabetes. Conclusions—These findings represent a novel mechanism of vascular cell senescence induced by hyperglycemia and suggest a protective role of SIRT1 in the pathogenesis of diabetic vasculopathy.

Ras Induces Vascular Smooth Muscle Cell Senescence and Inflammation in Human Atherosclerosis

Background—Vascular cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related vascular disorders. Ras, an important signaling molecule involved in atherogenic stimuli, is known to promote aging in yeast and cellular senescence in primary human fibroblasts. The aim of this study was to investigate the role of Ras-induced vascular smooth muscle cell (VSMC) senescence in atherogenesis. Methods and Results—We introduced an activated ras allele (H-ras V12) into human VSMCs using retroviral infection. Introduction of H-ras V12 induced a growth arrest with phenotypic characteristics of cellular senescence, such as enlarged cell shapes and increases in expression of cyclin-dependent kinase inhibitors and senescence-associated &bgr;-galactosidase (SA-&bgr;-gal) activity. Activation of Ras drastically increased expression of proinflammatory cytokines, in part through extracellular signal-regulated kinase activation. To determine whether Ras activation induces cellular senescence in vivo, we transduced the adenoviral vector encoding H-ras V12 into rat carotid arteries injured by a balloon catheter. Introduction of Ras into the arteries enhanced vascular inflammation and senescence compared with mock-infected injured arteries. Moreover, SA-&bgr;-gal–positive VSMCs were detected in the intima of advanced human atherosclerotic lesions and exhibited increased levels of extracellular signal-regulated kinase activity and proinflammatory cytokine expression. Conclusions—Our results suggest that atherogenic stimuli mediated by Ras induce VSMC senescence and vascular inflammation, thereby contributing to atherogenesis. This novel mechanism of atherogenesis may provide insights into a new antisenescence treatment for atherosclerosis.

Cellular senescence impairs circadian expression of clock genes in vitro and in vivo.

Circadian rhythms are regulated by a set of clock genes that form transcriptional feedback loops and generate circadian oscillation with a 24-hour cycle. Aging alters a broad spectrum of physiological, endocrine, and behavioral rhythms. Although recent evidence suggests that cellular aging contributes to various age-associated diseases, its effects on the circadian rhythms have not been examined. We report here that cellular senescence impairs circadian rhythmicity both in vitro and in vivo. Circadian expression of clock genes in serum-stimulated senescent cells was significantly weaker compared with that in young cells. Introduction of telomerase completely prevented this reduction of clock gene expression associated with senescence. Stimulation by serum activated the cAMP response element-binding protein, but the activation of this signaling pathway was significantly weaker in senescent cells. Treatment with activators of this pathway effectively restored the impaired clock gene expression of senescent cells. When young cells were implanted into young mice or old mice, the implanted cells were effectively entrained by the circadian rhythm of the recipients. In contrast, the entrainment of implanted senescent cells was markedly impaired. These results suggest that senescence decreases the ability of cells to transmit circadian signals to their clocks and that regulation of clock gene expression may be a novel strategy for the treatment of age-associated impairment of circadian rhythmicity.

Reduced Nitric Oxide Causes Age-Associated Impairment of Circadian Rhythmicity

Impairment of circadian rhythmicity in the elderly has been suggested to cause age-associated diseases such as atherosclerosis and hypertension. Endothelium-derived nitric oxide (NO) is a critical regulator of cardiovascular homeostasis, but its production declines with aging, thereby inducing vascular dysfunction. We show here that impaired circadian rhythmicity is related to a decrease of NO production with aging. Treatment with an NO donor significantly upregulated the promoter activity of the clock gene Period via the cAMP response element–dependent and the E-box enhancer element–dependent pathways. Both phosphorylation and S-nitrosylation by NO are involved in this upregulation. In aged animals, endothelial NO synthase activity was markedly decreased during the daytime, along with impairment of clock gene expression and the circadian variation in blood pressure. Treatment of aged animals with an NO donor significantly improved the impairments. Inhibition of NO synthase activity also led to impairment of clock gene expression and blood pressure rhythm. These results suggest that NO is a key regulator of the circadian clock in the cardiovascular system and may be a novel target for the treatment of age-associated alteration of circadian rhythms.

Vascular Endothelial Growth Factor Receptor-1 Regulates Postnatal Angiogenesis Through Inhibition of the Excessive Activation of Akt

Vascular endothelial growth factor (VEGF) binds both VEGF receptor-1 (VEGFR-1) and VEGF receptor-2 (VEGFR-2). Activation of VEGFR-2 is thought to play a major role in the regulation of endothelial function by VEGF. Recently, specific ligands for VEGFR-1 have been reported to have beneficial effects when used to treat ischemic diseases. However, the role of VEGFR-1 in angiogenesis is not fully understood. In this study, we showed that VEGFR-1 performs “fine tuning” of VEGF signaling to induce neovascularization. We examined the effects of retroviral vectors expressing a small interference RNA that targeted either the VEGFR-1 gene or the VEGFR-2 gene. Deletion of either VEGFR-1 or VEGFR-2 reduced the ability of endothelial cells to form capillaries. Deletion of VEGFR-1 markedly reduced endothelial cell proliferation and induced premature senescence of endothelial cells. In contrast, deletion of VEGFR-2 significantly impaired endothelial cell survival. When VEGFR-1 expression was blocked, VEGF constitutively activated Akt signals and thus induced endothelial cell senescence via a p53-dependent pathway. VEGFR-1+/− mice exhibited an increase of endothelial Akt activity and showed an impaired neovascularization in response to ischemia, and this impairment was ameliorated in VEGFR-1+/− Akt1+/− mice. These results suggest that VEGFR-1 plays a critical role in the maintenance of endothelial integrity by modulating the VEGF/Akt signaling pathway.

Akt-Induced Cellular Senescence: Implication for Human Disease

Reduction-of-function mutations in components of the insulin/insulin-like growth factor-1/Akt pathway have been shown to extend the lifespan in organisms ranging from yeast to mice. It has also been reported that activation of Akt induces proliferation and survival of mammalian cells, thereby promoting tumorigenesis. We have recently shown that Akt activity increases with cellular senescence and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, leading to endothelial dysfunction. This novel role of Akt in regulating the cellular lifespan may contribute to various human diseases including atherosclerosis and diabetes mellitus.