Blair Henderson

Cadmium Is a Novel and Independent Risk Factor for Early Atherosclerosis Mechanisms and In Vivo Relevance

Objectives—Although cadmium (Cd) is an important and common environmental pollutant and has been linked to cardiovascular diseases, little is known about its effects in initial stages of atherosclerosis. Methods and Results—In the 195 young healthy women of the Atherosclerosis Risk Factors in Female Youngsters (ARFY) study, cadmium (Cd) level was independently associated with early atherosclerotic vessel wall thickening (intima-media thickness exceeding the 90th percentile of the distribution; multivariable OR 1.6[1.1.–2.3], P=0.016). In line, Cd-fed ApoE knockout mice yielded a significantly increased aortic plaque surface compared to controls (9.5 versus 26.0 mm2, P<0.004). In vitro results indicate that physiological doses of Cd increase vascular endothelial permeability up to 6-fold by (1) inhibition of endothelial cell proliferation, and (2) induction of a caspase-independent but Bcl-xL-inhibitable form of cell death more than 72 hours after Cd addition. Both phenomena are preceded by Cd-induced DNA strand breaks and a cellular DNA damage response. Zinc showed a potent protective effect against deleterious effects of Cd both in the in vitro and human studies. Conclusion—Our research suggests Cd has promoting effects on early human and murine atherosclerosis, which were partly offset by high Zn concentrations.

Cigarette smoke metal-catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules

Smoking is a significant risk factor for development of atherosclerosis. However, the pathophysiology of smoking‐mediated vessel wall damage is not understood. With tools ranging from analytical chemistry to cell biology, we show that cigarette smoke contains metals that catalyze the direct oxidation of cellular proteins by smoke oxidants. Oxidation of cellular proteins causes a loss of microtubule function, culminating in microtubule depolymerization and proteasome‐dependent degradation of α‐tubulin. As a consequence of the microtubule collapse, cytoskeletal structures as well as intermediate filaments break down, leading finally to a contraction of vascular endothelial cells. We observed a smoke extract‐induced, calpain‐dependent degradation of the intracellular form of platelet‐endothelial cell adhesion molecule 1/CD31, as well as a release of P‐selectin/CD62P, IL‐6, and IL‐8 from endothelial cells into the supernatant. Increased levels of soluble CD62P and IL‐6 are well known to be associated with smoking in humans. Increased permeability of the vascular endothelium is a crucial event in atherogenesis. This work highlights the compounds and mechanisms by which cigarette smoke induces leakiness of the vascular endothelium. DavidBernhard AdamCsordas BlairHenderson AndreaRossmann MichaelaKind GeorgWick Cigarette smoke metal‐catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules. FASEB J. 19, 1–12 (2005)

T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire

Atherosclerosis is a multifactorial, chronic-inflammatory disease for which the underlying cause remains unknown. It is also well documented that T-cells are among the first cells to migrate into the arterial intimal vessel layer, but their function there is still unexplained. Clinical and experimental data have provided evidence that atherosclerosis starts as an autoimmune reaction based on humoral and cellular immunity against a phylogenetically highly conserved stress protein, heat shock protein 60 (HSP60). In the present study, we phenotypically characterized T-cells from endarterectomized specimens of the carotid artery, and tested their reactivity to human HSP60. In addition, the T-cell receptor repertoire of the T-cell lines was defined by immunoscope analysis. We found a mixed population of CD4(+) and CD8(+) intralesional T-cells, with a slight predominance of CD8(+) cells. IFN-gamma production prevailed over IL-4 production. The T-cell reaction against human HSP60 was significantly increased in intralesional cells compared to peripheral T-cells. The lesion-derived T-cells showed an oligoclonally-restricted repertoire, in contrast to the polyclonal pattern of PBMC. These results clearly show that HSP60 is a major antigenic candidate, and that an oligoclonal T-cell expansion takes place in advanced human atherosclerotic lesions.

Increased serum cadmium and strontium levels in young smokers : Effects on arterial endothelial cell gene transcription

Objective—Metal constituents of tobacco have long been suspected to contribute to cardiovascular diseases. In this study, we determined the serum concentrations of aluminum, cadmium (Cd), cobalt, copper, iron, manganese, nickel, lead, strontium (Sr), and zinc of young nonsmokers, passive smokers, and smokers. Methods and Results—Cd and Sr were found to be significantly increased in smokers compared with nonsmokers. The effects of these metals on primary arterial endothelial cells were then assessed using microarray technology and real-time polymerase chain reaction (RT-PCR). The data showed that Sr does not interfere with endothelial cell transcription. In contrast, the effects of Cd in amounts delivered to the human body by smoking were dramatic. Conclusions—Arterial endothelial cells responded to Cd exposure by massively upregulating metal and oxidant defense genes (metallothioneins) and by downregulating a number of transcription factors. In addition, the mRNA of the intermediate filament protein vimentin, crucial for the maintenance of cellular shape, was reduced. Surprisingly, a number of pro-inflammatory genes were downregulated in response to Cd. The present data suggest that by delivering Cd to the human body, smoking deregulates transcription, exerts stress, and damages the structure of the vascular endothelium; furthermore, in contrast to the effects of cigarette smoke as a whole, Cd seems to possess anti-inflammatory properties.

Cigarette smoke is an endothelial stressor and leads to cell cycle arrest

The molecular mechanisms underlying the atherogenic activity of cigarette smoke have yet to be fully elucidated. In the present study, genome-wide microarray analysis was performed on endothelial cells exposed to an aqueous cigarette smoke extract (CSE) for 3, 7, and 24 h, to obtain a better insight into how smoking may lead to endothelial damage. Microarray analysis showed the transcriptional response to CSE was dominated by heat shock, stress responsive, and inflammatory genes, along with genes encoding for anti-oxidant and metal detoxification proteins. The heat shock response was shown to be a result of short lived reactive species of CSE, with the abrogation of the effect by the addition of old CSE, the anti-oxidant N-acetyl cysteine, or the removal of metals from CSE implying that reactive oxygen species are the main culprit. This was further supported by a strong decline in the level of intracellular protein oxidation levels seen under these conditions compared to freshly prepared CSE. Mitochondrial integrity was also found to be significantly compromised after CSE treatment, resulting in a threefold increase in depolarised mitochondria after 6 h. Finally, cell cycle analysis showed the induction of G1 cell cycle arrest. An increased stress and inflammation response indicates that endothelial damage from smoking could contribute to immune cell infiltration, while decreased growth rates reduce endothelial layer repair, promoting atherogenesis.

Gene expression profiling of human endothelial cells exposed to 50-Hz magnetic fields fails to produce regulated candidate genes.

Abstract To address the question of a possible effect of magnetic fields (MF) at 50 Hz on living systems, gene expression analyses were performed on human primary vascular endothelial cells exposed to MF of various intensities compared to control cells. Exposure protocols included continuous exposure at a single intensity (10 and 700 μT), intermittent exposure at a single intensity (700 μT), and continuous exposure to a variable-intensity field (10–30 μT). The transcriptional response of the cells was investigated using oligonucleotide microarrays containing up to 30 000 unique features. Although in individual experiments genes were identified where the expression appeared to be affected by exposure to MF, none of these genes were regulated in the same manner in subsequent repetition experiments. This is the first report of a transcriptome-wide analysis of the effects of MF exposure on human cells. The lack of a reproducible effect of MF on the expression of any genes in our investigation adds further weight to the evidence that 50-Hz MF are not capable of interacting with biological systems and thus do not represent an endothelial stress factor.

Expression levels of heat shock protein 60 in human endothelial cells in vitro are unaffected by exposure to 50 Hz magnetic fields

Abstract Magnetic fields (MFs) from domestic power sources have been implicated as being a potential risk to human health. A number of epidemiological studies have found a significant link between exposure to MFs and increased rates of cancers. There have also been a number of in vivo and in vitro studies reporting effects of MFs in animal disease models and on the expression or activity of a range of proteins. In the past decade, our group proposed that atherosclerosis may have an autoimmune component, with heat shock protein 60 (Hsp60) expressed in endothelial cells as the dominant autoantigen. A number of stressors have been shown to induce the expression of Hsp60, including the classical risk factors for atherosclerosis. We were interested to see if the exposure of endothelial cells to an MF elicited increased expression of Hsp60, as has been reported previously for Hsp70. The present work describes the exposure of endothelial cells to domestic power supply (50 Hz) MFs at an intensity of 700 μT. The results from our system indicate that cultured endothelial cells exposed to a high intensity of MF either alone or in combination with classical heat stress show no effects on the expression of Hsp60 at either the messenger ribonucleic acid or the protein level. As such, there is no evidence that exposure to extremely low–frequency MF would be expected to increase the expression of Hsp60 and therefore the initiation or progression of atherosclerosis.

Dynamics of heat shock protein 60 in endothelial cells exposed to cigarette smoke extract

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the “HSP60 pathway.” It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the “auto-immune hypothesis of atherosclerosis.”

Progression of arteriovenous bypass restenosis in mice exposed to a 50 Hz magnetic field.

Abstract The controversy over whether magnetic fields (MF) produced by electrical wiring and appliances contribute to diseases such as cancer has been debated in the literature for more than 2 decades. These extremely low frequency fields at 50 or 60 Hz are omnipresent in the industrialized world and have been linked to various forms of cancer by epidemiological studies. Little has been published investigating any possible role of MF and cardiovascular disease, and this is the first study looking specifically at the effect of exposure to high-intensity MF on the development and progression of restenosis. A mouse arteriovenous bypass model was used, and mice were exposed to MF for periods of 1, 2, or 3 weeks. Neointima formation, infiltration of mononuclear cells, and heat shock protein 60 expression were all studied at the conclusion of the exposure regimen. Animals exposed to the MF for 1 week showed significantly smaller neointima formation compared with control mice exposed to a null field, although this difference was not observed in mice exposed for 2 or 3 weeks. No difference was found between mice exposed to MF and controls in any of the other parameters investigated.

CHAPTER 64 – Atherosclerosis: Autoimmunity to Heat-Shock Proteins

Publisher Summary This chapter focuses on atherosclerosis and autoimmune hypothesis of atherogenesis. Heat shock proteins (HSPs) are expressed by prokaryotic and eukaryotic cells as a response to various stress factors. HSP60 is a special case since it is not only an important and highly antigenic component of bacteria and parasites, but also plays a major role in several autoimmune diseases. The autoimmune hypothesis of atherogenesis postulates that pre-existing adaptive and innate immunity to HSP60 exposed on the surface of arterial endothelial cells (ECs) leads to the development of an initial inflammatory stage of this disease. Immunity against HSP60 seems to be an important facet of the defense system, e.g., as reflected by the fact that a considerable proportion of lymphocytes within the vascular-associated lymphoid tissue (VALT) show reactivity against HSP60. Also, every healthy animal and human possesses humoral antibodies against HSP60. It is noted that cells of the innate immune system recognize HSP60 as a danger signal, be it of microbial or autologous origin. The development of atherosclerosis therefore depends on the expression and accessibility of atherogenic HSP60 epitopes on the surface of ECs.