Current Pharmaceutical Biotechnology - Volume 7, Issue 2, 2006

Reactive oxygen species (ROS) are produced by many cell types of cardiovascular system. In healthy subjects, intrinsic antioxidative substances including alpha-tocopherol (vitamin E) and vitamin C reduces ROS and protect cardiovascular cells from injury. However, excessive production of ROS and/or decreased antioxidative activity in cardiovascular cells induces oxidative stress and causes endothelial dysfunction and a variety of cardiovascular diseases, which is one of the main topic of cardiovascular medicine. In this issue, review by top researchers are organized as follows: a). What is the present and real questions to be solved for reducing cardiovascular diseases caused by oxidative stress from the viewpoints of cardiologists? Professor Ueda summarizes the applications of vitamin E antioxidative properties. b). For the evaluation of oxidative stress in cardiovascular cells, experimental animals and whole human body by the biochemical and biotechnological methods, Dr. Yasunari exemplifies the measurement of oxidative stress of leukocytes by fluorescence compounds and flow cytometry. c). Modification of oxidative stress by the natural and pharmaceutical compounds in cellular and whole body. Drs. Suzuki and Eguchi review the role of angiotensin II on oxidative stress in cardiovascular system. Dr. Ozono describes new biotechological methods to reduce oxidative stress in the cardiovascular system, focusing on the Bach1/heme oxygenase-1 pathway. d). New biotechnological methods to reduce oxidative stress in cardiovascular medicine include bone marrow cell transplantation and gene therapy. Dr. Nakagami presents a gene therapy approach using by NF-kB decoy oligonucleotides. Dr. Yao and Fukuda review the oxidative stress on progenitor and stem cells in cardiovascular diseases and Dr. Higashi in bone bone marrow cell transplantation. This special issue should provide the reader with a better understanding of some of the clinical applications of pharmaceutical biotechnology and should also help researchers in the field of biotechnological pharmacology.

There is no doubt that oxidative stress is pivotally involved in the process of atherosclerosis. Thus antioxidants, particularly vitamin E, have been expected to retard the development of atherosclerosis. In fact, several cohort studies suggested reduced cardiovascular risk in persons taking vitamin E supplements. However, randomized clinical trials of vitamin E did not show any benefit of vitamin E supplementation in terms of prevention of coronary heart disease and death. Discrepancy between cohort studies and randomized clinical trials may be partly explained by difference in coronary risk in study participant. However, use of vitamin E supplementation in low risk population has not been justified yet.

To better identify patients at high risk for cardiovascular events, several markers of risk have been proposed for use in screening. Recently, oxidative stress and inflammation have been evaluated as potential tools for prediction of the risk of cardiovascular events. Among them, we have measured reactive oxygen species (ROS) formation by polymorphonuclear cells (PMNs) and mononuclear cells (MNCs), since they may be a possible link between inflammation and oxidative stress. ROS formation by PMNs and MNCs was measured by a gated flow cytometric assay. Such biotechnological method of measuring ROS formation by PMNs and MNCs will make it possible that we measure vascular oxidative stress and vascular inflammation at the same time from only small amount of blood. We will state in this review that ROS formation by PMNs and MNCs are regulated by different mechanisms, although PMNs and MNCs are circulating in the same blood. Moreover, we will state that ROS formation by PMNs are regulated by blood pressure, Hb A 1C and oxidided LDL. ROS formation by MNCs are regulated by vascular inflammation, and that ROS formation by MNCs are also related to various cardiovascular risks such as LV mass, norepinephrine, IMT, and nocturnal blood pressure.

Reactive oxygen species (ROS) are proposed to induce cardiovascular diseases, such as atherosclerosis and hypertension, through several mechanisms. One such mechanism involves ROS acting as intracellular second messengers, which lead to induction of unique signal transductions. Angiotensin II (AngII), a potent cardiovascular pathogen, stimulates ROS production through vascular NADPH oxidases. The ROS production induced by AngII activates downstream ROS-sensitive kinases that are critical in mediating cardiovascular remodeling. Recent advances in gene transfer/knockout techniques have lead to numerous in vitro and in vivo studies that identify the potential components and mechanisms of ROS signal transduction by AngII which promote cardiovascular remodeling. In this review, we will focus our discussion on the signal transduction research elucidating ROS production and function induced by AngII using currently available molecular biotechnologies.

Oxidative stress is involved in the mechanism of atherosclerotic lesion formation and in the mechanisms underlying the development of other pathogenic conditions of the cardiovascular system, including endothelial dysfunction, hypertension, and heart failure. Reducing oxidative stress may be a reasonable therapeutic approach to treat cardiovascular diseases. HO-1 is a cytoprotective enzyme that is induced in response to oxidative stress and degrades heme into carbon monoxide (CO) and bilirubin, both of which have cytoprotective effects. A substantial body of evidence suggests that introduction of HO-1, either pharmacologically or by a gene delivery technique, confers cytoprotection in ischemic heart disease and atherosclerosis in animals. Recent studies have revealed that CO has anti-inflammatory properties and that administration of CO provides protection against atherosclerosis and ischemic heart disease. Discovery of Bach1, a transcriptional repressor of HO-1, has greatly contributed to the understanding of the regulation of HO-1 expression, providing a clue to a development of alternative method to enhance HO activity. Bach1 normally represses HO-1 expression. However, upon exposure to oxidative stress, Bach1 loses its repressive activity and is exported out of the nucleus, which in turn results in the upregulation of HO-1. Bach1 knockout mice, expressing an increased amount of HO-1, are resistant to pro-atherosclerotic and ischemic stresses. These findings indicate that inhibition of Bach1 may be a novel approach to enhance protection against stress. In summary, the Bach1-HO-1 system is an important defense mechanism against oxidative stress. Development of a safe and effective method to enhance this pathway, such as Bach1 inhibitor, may be of great clinical relevance.

Oxidative stress to cardiovascular cells induced by an interaction of multiple cytokines and adhesion molecules has been postulated to be responsible for cardiovascular disease. Since nuclear factor-kB (NFkB) also plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes, we utilized oligodeoxynucleotides (ODNs) as "decoy" cis-elements that block the binding of nuclear factors to promoter regions of targeted genes, resulting in the inhibition of gene transactivation. Indeed, transfection of NFkB decoy ODNs into coronary artery effectively prevented transactivation of essential cytokine and adhesion molecule protein expression, and thereby protected the myocardium from infarction. Transfection of NFkB decoy ODNs into balloon-injured carotid artery or porcine coronary artery markedly reduced neointimal formation. Thus, a clinical trial using NFkB decoy ODNs to treat restenosis was started in 2002. Recently, the therapeutic target utilizing NFkB decoy has been expanded to glomerulonephritis, rheumatoid arthritis, atopic dermatitis and osteoporosis. Moreover, the clinical trials to treat RA patients were initiated in 2003 and a Phase I/IIa human clinical trial using NFkB decoy ODNs to treat atopic dermatitis was initiated in December 2001. Topical application of NFkB decoy ODNs exhibited marked therapeutic effects on the facial skin condition of patients with atopic dermatitis. The covalently modified ODNs were developed by enzymatically ligating two identical molecules, thereby preventing their degradation by exonucleases. Indeed, the modified decoy ODNs possess increased nuclease resistance and are transported more efficiently into cells. Although there are still unresolved issues, decoy ODN drugs should become a reality. Type 2 diabetes is associated with a two to four-fold increased risk of both coronary heart disease and stroke. Dysfunction of endothelial cells (EC) is known to promote abnormal vascular growth such as that in atherosclerosis and arteriosclerosis, and has been postulated as an initial trigger of the progression of atherosclerosis in patients with diabetes mellitus. Moreover, hyperglycemia is an independent risk factor for the development of cardiovascular disease. We and others have previously demonstrated high D-glucose induced apoptosis through the activation of the bax-caspase proteases pathway in human EC, and the potential contribution of hepatocyte growth factor as an anti-apoptotic factor for the pathogenesis of endothelial dysfunction. The anti-apoptotic action of HGF was due to bcl-2 upregulation and the phosphatidylinositol 3- kinase pathway, which is involved in Akt activation. Although it has been known for years that cardiovascular tissues can release a large amount ROS, including superoxide, hydrogen peroxide, and nitric oxide, the role of oxidative stress in atherogenesis has received increasing attention in recent years. Recent works strongly suggest that NADPH oxidase is a major source of superoxide in cardiovascular cells, and oxidative stress can be involved in the process of endothelial dysfunction. NADPH oxidase can be activated in hyperglycemia through the protein kinase C pathway. From a viewpoint of these molecular mechanisms, HMG-CoA reductase inhibitors (statins) might inhibit the high glucose-induced NADPH oxidase activation through inhibition of Rac activity and finally prevent the increase in ROS production in diabetes. Actually, recent clinical trial suggested that statins prevent several vascular events in patients with type 2 diabetes without a high concentration of LDL-cholesterol. These pleiotropic effects of statins can be expected to improve endothelial dysfunction through nitric oxide production and/or an anti-oxidant effect on diabetic patients.

There is accumulating evidence that reactive oxygen species (ROS) play major roles in the initiation and progression of cardiovascular dysfunction associated with diseases such as hyperlipidemia, diabetes mellitus, hypertension, ischemic heart disease, and chronic heart failure. ROS produced by migrating inflammatory cells as well as vascular cells (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) have distinct functional effects on each cell type. These effects include cell growth, apoptosis, migration, inflammatory gene expression and matrix regulation. ROS, through regulating vascular cell function, can play a central role in normal vascular physiology, and contribute substantially to the development of cardiovascular diseases. Excessive production of ROS is an essential mechanism underlying the pathogenesis of endothelial dysfunction and cardiovascular disease. Stem cells hold great promise for tissue repair and regenerative medicine, and endothelial progenitor cells (EPC) play a significant role in neovascularization of ischemic tissue. Recent studies have shown that cardiovascular risk factors such as hypertension, hypercholesterolemia, diabetes and cigarette smoking are inversely correlated with EPC number and function. Understanding the mechanisms, that regulate EPC function may provide new insights into the pathogenesis of vasculogenesis and may promote development of specific therapies to prevent ROS production and ultimately correct EPC dysfunction. We have demonstrated the angiotensin II receptor blockers improve EPC dysfunction through antioxidative mechanisms. In the present review, we describe our current understanding of the contributions of oxidative stress to progenitor and stem cell dysfunction in cardiovascular disease and focus on the potential mechanisms that underlie oxidative stress-induced damage of progenitor and stem cells.

Recently, clinical studies have shown that novel therapies, including cell implantation and transfer of gene encoding for angiogenic growth factors, are effective in patients with critical limb ischemia who have no other treatment option. This concept is called therapeutic angiogenesis. Cell therapy involves implantation of bone-marrow or peripheral mononuclear cells and endothelial progenitor cells (CD34+ cells) in the gastrocnemius of the ischemic leg. Gene therapy involves delivery of vascular endothelial growth factor and fibroblast growth factor using a plasmid or adenoviral vector. Critical limb ischemia is associated with endothelial dysfunction as well as excess oxidative stress. A balance of oxidative stress and nitric oxide play an important role in the development of atherosclerosis in patients with peripheral arterial diseases. It has been reported that both cell therapy and gene therapy improve endothelial function in the resistant artery of an ischemic limb. Cell therapy or gene therapy in combination with pharmacological therapy as an antioxidant could be useful for restoration of endothelial function and prevention of development of atherosclerosis in patients with critical limb ischemia. In this review, we discuss the relationships between oxidative stress, endothelial function, and angiogenesis and the mechanism by which therapeutic angiogenesis improves endothelial function.

The respiratory tract as the main entrance for various inhalative substances has great potential to generate reactive species directly or indirectly in excess. Thus, heavy smokers are at high risk for development, impairment and failed response to treatment of chronic obstructive pulmonary disease (COPD). The article is an update regarding the influence of reactive oxygen (ROS) and nitrogen (RNS) species on COPD; however, we do not intend to describe ROS and RNS actions on the entire lung tissue. Here, we focus on the airways, because in human most of the described effects of ROS and RNS species are measured on respiratory epithelial cells obtained by bronchoscopy. ROS and RNS species are physiological compounds in cells and risk factors for several respiratory diseases. In general, both kinds of species are thermodynamically stabile, but their reaction behaviors in cellular environments are very different. For example, the life times of the superoxide anion radical range from micro/milliseconds up to minutes and even hours in in-vitro model systems. Oxidative stress by cigarette smoke was investigated in detail by the authors of this article. In addition, original studies by the authors on the amount of fine particulate matter and trace elements in lung biopsies after defined inhalation indicate a distortion of the equilibrium between oxidants and antioxidants. We also try to present some modern views with respect to genomic medicine for future therapeutic perspectives, although this is an upcoming sector of COPD therapy.

Several factors are known to be involved in the regulation of vitamin D and sunlight and diet are the two sources in humans, but the relative importance of each of them is not well defined. Vitamin D, parathyroid hormone and serum insulin-like growth factor-I (IGF-I) were found to be independent predictors of total bone density. Thus, the growth hormone (GH)/IGF-I is thought to play an important role in the regulation of bone mineral density and the skeleton is second only to the liver as a source of circulating levels of IGF-I. The mechanisms by which IGF-I may influence bone metabolism is not fully understood but they are a predictor of bone mass density and are positively associated with vitamin D concentrations. There is a physiological decline of the GH/IGF axis with ageing. The high affinity IGFbinding proteins (IGFBP-1 to 6) have also been involved in IGF-I regulation, and it is important to include the IGFindependent properties, particularly those of IGFBP3 that may be involved in the osteoblastic differentiation observed in human bone marrow stromal cell cultures. These hormones have been shown to up regulate each other. 1,25-(OH) D3 has been shown to promote the action of IGF-I by increasing IGF-I receptors and IGF-I can also elevate 1,25-(OH) D3 concentrations by stimulating the hydroxylation of 25-(OH) D3 in the active 1,25-(OH) D3 hormone. Both GH and IGF-I significantly increased renal 1a-hydroxylase expression and serum 1, 25-(OH) D3 concentrations. In prostate cells, 1,25-(OH) D3 is growth inhibitory for many established cell lines and the role of IGFBPs, especially IGFBP-3, can be growth inhibitory or stimulatory and IGFBP-3 expression increases in response to 1,25-(OH) D3, or its analogs, in established prostate cancer cell lines. Body fat is inversely associated with 25-(OH) D3 in relation to with anthropometric measures, indicating a specific role of adipose tissue. IGF-I may be involved in both normal and abnormal fetal growth and stimulation of IGF-I synthesis during normal pregnancy may be associated with an increase in GH production by the placenta. Thus, maternal and umbilical cord serum IGF-I and 1,25-(OH) D3 concentrations are lower in preeclampsia and umbilical cord serum IGF-I, IGFBP-1 and IGFBP-3 concentrations are associated with low newborn birth weights.