Wim Martinet

Elevated Levels of Oxidative DNA Damage and DNA Repair Enzymes in Human Atherosclerotic Plaques

Background—The formation of reactive oxygen species is a critical event in atherosclerosis because it promotes cell proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. In the present study, we investigated whether reactive oxygen species-induced oxidative damage to DNA occurs in human atherosclerotic plaques and whether this is accompanied by the upregulation of DNA repair mechanisms. Methods and Results—We observed increased immunoreactivity against the oxidative DNA damage marker 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in plaques of the carotid artery compared with the adjacent inner media and nonatherosclerotic mammary arteries. Strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque including macrophages, smooth muscle cells, and endothelial cells. As shown by competitive ELISA, carotid plaques contained 160±29 8-oxo-dG residues/105 dG versus 3±1 8-oxo-dG residues/105 dG in mammary arteries. Single-cell gel electrophoresis showed elevated levels of DNA strand breaks in the plaque. The overall number of apoptotic nuclei was low (1% to 2%) and did not correlate with the amount of 8-oxo-dG immunoreactive cells (>90%). This suggests that initial damage to DNA occurs at a sublethal level. Several DNA repair systems that are involved in base excision repair (redox factor/AP endonuclease [Ref 1] and poly(ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair pathways (p53, DNA-dependent protein kinase) were upregulated, as shown by Western blotting and immunohistochemistry. Overexpression of DNA repair enzymes was associated with elevated levels of proliferating cell nuclear antigen. Conclusions—Our findings provide evidence that oxidative DNA damage and repair increase significantly in human atherosclerotic plaques.

Phagocytosis of Apoptotic Cells by Macrophages Is Impaired in Atherosclerosis

Objective—Apoptotic cell death has been demonstrated in advanced human atherosclerotic plaques. Apoptotic cells (ACs) should be rapidly removed by macrophages, otherwise secondary necrosis occurs, which in turn elicits inflammatory responses and plaque progression. Therefore, we investigated the efficiency of phagocytosis of ACs by macrophages in atherosclerosis. Methods and Results—Human endarterectomy specimens and human tonsils were costained for CD68 (macrophages) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) (apoptosis). Free and phagocytized ACs were counted in both tissues. The ratio of free versus phagocytized AC was 19-times higher in human atherosclerotic plaques as compared with human tonsils, indicating a severe defect in clearance of AC. Impaired phagocytosis of AC was also detected in plaques from cholesterol-fed rabbits and did not further change with plaque progression. In vitro experiments with J774 or peritoneal mouse macrophages showed that several factors caused impaired phagocytosis of AC including cytoplasmic overload of macrophages with indigestible material (beads), free radical attack, and competitive inhibition among oxidized red blood cells, oxidized low-density lipoprotein and ACs for the same receptor(s) on the macrophage. Conclusion—Our data demonstrate that phagocytosis of ACs is impaired in atherosclerotic plaques, which is at least partly attributed to oxidative stress and cytoplasmic saturation with indigestible material.

Autophagy in Atherosclerosis A Cell Survival and Death Phenomenon With Therapeutic Potential

Autophagy is a reparative, life-sustaining process by which cytoplasmic components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. A growing body of evidence suggests that autophagy is stimulated in advanced atherosclerotic plaques by oxidized lipids, inflammation, and metabolic stress conditions. However, despite the increasing interest in autophagy in various pathophysiological situations such as neurodegeneration, cancer, and cardiac myopathies, the process remains an underestimated and overlooked phenomenon in atherosclerosis. As a consequence, its role in plaque formation and stability is poorly understood. Most likely, autophagy safeguards plaque cells against cellular distress, in particular oxidative injury, by degrading damaged intracellular material. In this way, autophagy is antiapoptotic and contributes to cellular recovery in an adverse environment. An interesting observation is that basal autophagy can be intensified by specific drugs. Excessively stimulated autophagic activity is capable of destroying major proportions of the cytosol, leading finally to type II programmed cell death that lacks several hallmarks of apoptosis or necrosis. Because atherosclerosis is an inflammatory disorder of the arterial intima, pharmacological approaches could be developed to stabilize vulnerable, rupture-prone lesions through selective induction of macrophage autophagic death.

Apoptotic versus autophagic cell death in heart failure

OBJECTIVE Progressive loss of cardiomyocytes is one of the most important pathogenic characteristics of heart failure. Apoptosis may be an important mode of cell death in heart failure but it must be demonstrated by multiple criteria and not just TUNEL staining alone. Previously, we and others have demonstrated that besides apoptosis other phenomena like active gene transcription can result in TUNEL positivity. Moreover, other types of cell death that are caspase-independent could be important in heart failure. This study examined the hypothesis whether TUNEL labeling parallels caspase activation. METHODS Cardiac tissue of patients in the terminal stage of heart failure as a consequence of ischaemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) were studied. Embryonic mice hearts were used for positive control for detection of the classical apoptosis. RESULTS In mice embryonic hearts we could clearly find apoptotic cell death detected by TUNEL labeling and immunohistochemistry for activated caspase-3. In heart failure, TUNEL-positive cardiomyocytes were negative for active caspase-3 but showed signs of active gene transcription (SC-35). However, autophagic cell death could be found in 0.3% of the cardiomyocytes. Autophagic cell death was demonstrated by granular cytoplasmic ubiquitin inclusions, an established marker of autophagocytosis in neurons. Interestingly, these autophagic cardiomyocytes were TUNEL and activated caspase-3 negative but were also negative for C9, a marker for necrosis. Western blot analysis confirmed that in cardiomyopathies no cleavage of caspase-3 and caspase-7 occurred. CONCLUSION The present study demonstrates two fundamentally different situations of cell death in cardiac tissue. In embryonic mice, cardiomyocytes undergo caspase-dependent cell death. However, cardiomyocytes in heart failure show caspase-independent autophagic cell death rather than apoptotic cell death.

Molecular and cellular mechanisms of skeletal muscle atrophy: an update

Skeletal muscle atrophy is defined as a decrease in muscle mass and it occurs when protein degradation exceeds protein synthesis. Potential triggers of muscle wasting are long-term immobilization, malnutrition, severe burns, aging as well as various serious and often chronic diseases, such as chronic heart failure, obstructive lung disease, renal failure, AIDS, sepsis, immune disorders, cancer, and dystrophies. Interestingly, a cooperation between several pathophysiological factors, including inappropriately adapted anabolic (e.g., growth hormone, insulin-like growth factor 1) and catabolic proteins (e.g., tumor necrosis factor alpha, myostatin), may tip the balance towards muscle-specific protein degradation through activation of the proteasomal and autophagic systems or the apoptotic pathway. Based on the current literature, we present an overview of the molecular and cellular mechanisms that contribute to muscle wasting. We also focus on the multifacetted therapeutic approach that is currently employed to prevent the development of muscle wasting and to counteract its progression. This approach includes adequate nutritional support, implementation of exercise training, and possible pharmacological compounds.

Autophagy in the cardiovascular system

Autophagy is a catabolic pathway for bulk turnover of long-lived proteins and organelles via lysosomal degradation. Growing evidence reveals that autophagy is involved in the progression or prevention of many human diseases. Here we discuss the role of autophagy in the normal heart, in heart disease and atherosclerosis. In the heart, autophagy functions predominantly as a pro-survival pathway during cellular stress by removing protein aggregates and damaged organelles, protecting the heart against famine, excessive beta-adrenergic stimulation and ischemia. However, when severely triggered, e.g. during reperfusion, the autophagic machinery may lead to cell death. Furthermore, autophagy modulates cardiac hypertrophy and the transition from hypertrophy to heart failure. During aging, lipofuscin is formed via autophagy in the heart and impairs autophagy. Basal autophagy in atherosclerotic plaques is a survival mechanism safeguarding plaque cells against cellular distress, in particular oxidative injury, metabolic stress and inflammation, by removing harmful oxidatively modified proteins and damaged components. Hence, autophagy is anti-apoptotic and contributes to cellular recovery in an adverse environment. However, excessively stimulated autophagy causes autophagic death in plaque cells and is detrimental. Ceroid that is formed via autophagy in atherosclerotic arteries impairs autophagy and induces apoptosis. Basal autophagy can be intensified by appropriate drugs and pharmacological approaches have been developed to stabilize rupture-prone plaques through selective induction of macrophage autophagic death, without affecting the plaque stabilizing smooth muscle cells.

Therapeutic potential of helminth soluble proteins in TNBS‐induced colitis in mice

Background: The hygiene hypothesis suggests an inverse relationship between the incidence of parasitic infections and chronic inflammatory bowel diseases (IBD). We investigated the therapeutic potential of Schistosoma mansoni and Ancylostoma caninum soluble proteins on experimental colitis in mice. Methods: Colitis was induced by intrarectal administration of 10 mg trinitrobenzene sulfonic acid (TNBS) in 30% ethanol. Six hours after TNBS injection, mice were treated intraperitoneally with helminth proteins. Three days later, colonic inflammation was scored based on 5 inflammatory parameters: clinical disease activity, macroscopic and microscopic inflammation score, extent of inflammation, and myeloperoxidase (MPO) activity. To determine immunological pathways induced by S. mansoni proteins we measured cytokine profiles of T‐lymphocytes from colon, mesenteric lymph nodes (MLN), and spleen by real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR). Results: Control mice showed no signs of inflammation, whereas all inflammatory parameters were significantly increased in mice with colitis. Treatment of mice with colitis with S. mansoni or A. caninum proteins decreased the macroscopic inflammation score, extent of inflammation, and MPO activity. Immunologically, induction of colitis significantly increased expression of IFN‐γ mRNA in the inflamed colon. Treatment with S. mansoni proteins caused a decrease of proinflammatory cytokines (IFN‐γ, IL‐17) in colon and MLN, whereas the production of regulatory cytokines (IL‐10, TGF‐β) increased significantly in colon tissue. Conclusions: Treatment with proteins of S. mansoni and A. caninum ameliorated TNBS‐induced colitis in mice. S. mansoni proteins increased mRNA expression of regulatory cytokines while suppressing expression of proinflammatory cytokines. Therefore, we suggest a therapeutic potential for helminth proteins in the treatment of IBD. (Inflamm Bowel Dis 2009)

Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death

Although reactive oxygen species (ROS) at physiological concentrations are required for normal cell function, excessive production of ROS is detrimental to cells. Neuroglobin and cytoglobin are two globins, whose functions are still a matter of debate. A potential role in the detoxification of ROS is suggested. The influence of neuroglobin and cytoglobin on cell death after oxidative stress in human neuroblastoma SH-SY5Y cells was evaluated. Exposure of SH-SY5Y cells to paraquat or H(2)O(2) resulted in a concentration- and time-dependent induction of apoptotic and necrotic cell death. H(2)O(2) was 16 times more potent to induce cell death as compared to paraquat. SH-SY5Y cells transfected with plasmid DNA containing the neuroglobin or cytoglobin sequence showed enhanced survival after exposure to 300 microM H(2)O(2) for 24h as compared to untransfected controls. This finding suggests that neuroglobin and cytoglobin protect SH-SY5Y cells against oxidative stress-induced cell death.

Apoptosis in atherosclerosis: focus on oxidized lipids and inflammation

An increasing body of evidence from both animal models and human specimens suggests that apoptosis or programmed cell death is a major event in the pathophysiology of atherosclerosis. Although the significance of apoptosis in atherosclerosis remains unclear, it has been proposed that apoptotic cell death contributes to plaque instability, rupture and thrombus formation. Biochemical and genetic analyses of apoptosis provide an increasingly detailed picture of the intracellular signaling pathways involved. Nevertheless, it remains to be determined whether apoptosis can become a clinically important approach to modulate plaque progression. In this review, we have outlined some of the most recent results concerning apoptosis in atherosclerosis with a special focus on oxidized lipids, inflammation and therapeutic regulation of the apoptotic cell death process.

Autophagy in Atherosclerosis: A Potential Drug Target for Plaque Stabilization

Evidence is accumulating that autophagy occurs in advanced atherosclerotic plaques. Although there is an almost relentless discovery of molecules that are involved in autophagy, studies of selective autophagy induction or inhibition using knockout mice are just now beginning to reveal its biological significance. Most likely, autophagy safeguards plaque cells against cellular distress, in particular oxidative injury, by degrading the damaged intracellular material. In this way, autophagy is protective and contributes to cellular recovery in an unfavorable environment. Pharmacological approaches have recently been developed to stabilize vulnerable, rupture-prone lesions through induction of autophagy. This approach has proven to be successful in short-term studies. However, how autophagy induction affects processes such as inflammation remains to be elucidated and is currently under investigation. This review highlights the possibilities for exploiting autophagy as a drug target for plaque stabilization.