Nox2 regulates endothelial cell cycle arrest and apoptosis via p21 (cip1) and p53

Endothelial cells (EC) express constitutively two major isofonns (Nox2 and Nox4) of the catalytic subunit of NADPH oxidase, which is a major source of endothelial reactive oxygen species. However, the individual roles of these Noxes in endothelial function remain unclear. We have investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC, Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4 similar to 1:13), but was upregulated 24 It after starvation and increased to 8 +/- 3.5-fold at 36 h of starvation. Accompanying the upregulation of Nox2, there was a 2.28 +/- 0.18-fold increase in O-2(-); production, a dramatic induction of p21(cip1) and p53, cell cycle arrest, and the onset of apoptosis (all p < 0.05). All these changes were inhibited significantly by in vitro deletion of Nox2 expression and in coronary microvascular EC isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8 +/- 0.5fold increase in Nox4 mRNA expression after 36 h of starvation (p < 0.01), neither production nor the p21(cip1) or p53 expression was increased significantly and only 0.46% of cells were apoptotic. In conclusion, Nox2-derived O-2(-), through the modulation of p21(cip1) and p53 expression, participates in endothelial cell cycle regulation and apoptosis. (c) 2007 Elsevier Inc. All rights reserved.

The mammalian cell cycle: an overview

In recent years, we have witnessed major advances in our understanding of the mammalian cell cycle and how it is regulated. Normal mammalian cellular proliferation is tightly regulated at each phase of the cell cycle by the activation and deactivation of a series of proteins that constitute the cell cycle machinery. This review article describes the various phases of the mammalian cell cycle and focuses on the cell cycle regulatory molecules that act at each stage to ensure normal cellular progression.

Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.

Can the cardiomyocyte cell cycle be reprogrammed?

Cardiac repair following myocardial injury is restricted due to the limited proliferative potential of adult cardiomyocytes. The ability of mammalian cardiomyocytes to proliferate is lost shortly after birth as cardiomyocytes withdraw from the cell cycle and differentiate. We do not fully understand the molecular and cellular mechanisms that regulate this cell cycle withdrawal, although if we could it might lead to the discovery of novel therapeutic targets for improving cardiac repair following myocardial injury. For the last decade, researchers have investigated cardiomyocyte cell cycle control, commonly using transgenic mouse models or recombinant adenoviruses to manipulate cell cycle regulators in vivo or in vitro. This review discusses cardiomyocyte cell cycle regulation and summarises recent data from studies manipulating the expressions and activities of cell cycle regulators in cardiomyocytes. The validity of therapeutic strategies that aim to reinstate the proliferative potential of cardiomyocytes to improve myocardial repair following injury will be discussed. (c) 2007 Elsevier Inc. All rights reserved.

Reprogramming the cell cycle machinery to treat cardiovascular disease

Coronary artery disease is one of the most common heart pathologies. Restriction of blood flow to the heart by atherosclerotic lesions, leading to angina pectoris and myocardial infarction, damages the heart, resulting in impaired cardiac function. Damaged myocardium is replaced by scar tissue since surviving cardiomyocytes are unable to proliferate to replace lost heart tissue. Although narrowing of the coronary arteries can be treated successfully using coronary revascularisation procedures, re-occlusion of the treated vessels remains a significant clinical problem. Cell cycle control mechanisms are key in both the impaired cardiac repair by surviving cardiomyocytes and re-narrowing of treated vessels by maladaptive proliferation of vascular smooth muscle cells. Strategies targeting the cell cycle machinery in the heart and vasculature offer promise both for the improvement of cardiac repair following MI and the prevention of restenosis and bypass graft failure following revascularisation procedures.

Altered expression of cell cycle proteins and prolonged duration of cardiac myocyte hyperplasia in p27KIP1 knockout mice

The precise role of cell cycle-dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains to be determined. We report that loss of p27(KIP1) in the mouse results in a significant increase in heart size and in the total number of cardiac myocytes. In comparison to p27(KIP1)+/+ myocytes, the percentage of neonatal p27(KIP1)-/- myocytes in S phase was increased significantly, concomitant with a significant decrease in the percentage of G(0)/G(1) cells. The expressions of proliferating cell nuclear antigen, G(1)/S and G(2)/M phase-acting cyclins, and cyclin-dependent kinases (CDKs) were upregulated significantly in ventricular tissue obtained from early neonatal p27(KIP1)-/- mice, concomitant with a substantial decrease in the expressions of G(1) phase-acting cyclins and CDKs. Furthermore, mRNA expressions of the embryonic genes atrial natriuretic factor and alpha-skeletal actin were detectable at significant levels in neonatal and adult p27(KIP1)-/- mouse hearts but were undetectable in p27(KIP1)+/+ hearts. In addition, loss of p27(KIP1) was not compensated for by the upregulation of other CDK inhibitors. Thus, the loss of p27(KIP1) results in prolonged proliferation of the mouse cardiac myocyte and perturbation of myocyte hypertrophy.

Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy

The role of cell cycle dependent molecules in controlling the switch from cardiac myocyte hyperplasia to hypertrophy remains unclear, although in the rat this process occurs between day 3 and 4 after birth. In this study we have determined (1) cell cycle profiles by fluorescence activated cell sorting (FACS); and (2) expressions, co-expressions and activities of a number of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting andin vitrokinase assays in freshly isolated rat cardiac myocytes obtained from 2, 3, 4 and 5-day-old animals. The percentage of myocytes found in the S phase of the cell cycle decreased significantly during the transition from hyperplasia to hypertrophy (5.5, 3.5, 2.3 and 1.9% of cells in 2-, 3-, 4- and 5-day-old myocytes, respectively,P<0.05), concomitant with a significant increase in the percentage of G0/G1phase cells. At the molecular level, the expressions and activities of G1/S and G2/M phase acting cyclins and CDKs were downregulated significantly during the transition from hyperplasia to hypertrophy, whereas the expressions and activities of G1phase acting cyclins and CDKs were upregulated significantly during this transition. In addition, p21CIP1- and p27KIP1- associated CDK kinase activities remained relatively constant when histone H1 was used as a substrate, whereas phosphorylation of the retinoblastoma protein was upregulated significantly during the transition from hyperplasia to hypertrophy. Thus, there is a progressive and significant G0/G1phase blockade during the transition from myocyte hyperplasia to hypertrophy. Whilst CDK2 and cdc2 may be pivotal in the withdrawal of cardiac myocytes from the cell cycle, CDK4 and CDK6 may be critical for maintaining hypertrophic growth of the myocyte during development.

Cell cycle profiles and expression of p21CIP1 and P27KIP1 during myocyte development

The ability of the cardiac myocyte to divide ceases shortly after birth. Thus, following severe injury, e.g., during myocardial infarction, the mature heart is unable to regenerate new tissue to replace the dead or damaged tissue. The identification of the molecules controlling the cessation of myocyte cell division may lead to therapeutic strategies which aim to re-populate the damaged myocardial area. Hence, we have determined the cell cycle profile, expressions and activities of the cyclin-dependent kinase inhibitors (CDKIs), p21CIP1 and p27KIP1, during rat ventricular myocyte development. Fluorescent activated cell sorting (FACS) analyses showed the percentage of S phase myocytes to be decreased significantly throughout development, concomitant with a significant increase in the percentage of G0/G1 and G2/M phase cells. The expression of p21CIP1 and p27KIP1 increased significantly throughout cardiac development and complexed differentially with a number of cyclins and CDKs. Furthermore, an adult myocyte extract reduced neonatal myocyte CDK2 kinase activity significantly (>30%, p<0.05) whereas immunodepletion of p21CIP1 from adult lysates restored CDK2 kinase activity. Thus, p21CIP1 and p27KIP1 may be important for the withdrawal of cardiac myocytes from the cell cycle and for maintaining the G0/G1 and G2/M phase blockades.

The cell cycle and drug discovery: The promise and the hope

In recent years, there have been major developments in the understanding of the cell cycle. It is now known that normal cellular proliferation is tightly regulated by the activation and deactivation of a series of proteins that constitute the cell cycle machinery. The expression and activity of components of the cell cycle can be altered during the development of a variety of diseases where aberrant proliferation contributes to the pathology of the illness. Apart from yielding a new source of untapped therapeutic targets, it is likely that manipulating the activity of such proteins in diseased states will provide an important route for treating proliferative disorders, and the opportunity to develop a novel class of future medicines.

Arresting developments in the cardiac myocyte cell cycle: Role of cyclin-dependent kinase inhibitors

Like most other cells in the body, foetal and neonatal cardiac myocytes are able to divide and proliferate. However, the ability of these cells to undergo cell division decreases progressively during development such that adult myocytes are unable to divide. A major problem arising from this inability of adult cardiac myocytes to proliferate is that the mature heart is unable to regenerate new myocardial tissue following severe injury, e.g. infarction, which can lead to compromised cardiac pump function and even death. Studies in proliferating cells have identified a group of genes and proteins that controls cell division. These proteins include cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs), which interact with each other to form complexes that are essential for controlling normal cell cycle progression. A variety of other proteins, e.g. the retinoblastoma protein (pRb) and members of the E2F family of transcription factors, also can interact with, and modulate the activities of, these complexes. Despite the major role that these proteins play in other cell types, little was known until recently about their existence and activities in immature (proliferating) or mature (non-proliferating) cardiac myocytes. The reason(s) why cardiac myocytes lose their ability to divide during development remains unknown, but if strategies were developed to understand the mechanisms underlying cardiac myocyte growth, it could open up new avenues for the treatment of cardiovascular disease. In this article, we shall review the function of the cell cycle machinery and outline some of our recent findings pertaining to the involvement of the cell cycle in modulating cardiac myocyte growth and hypertrophy.