Loren E. Wold

Endothelin-1 enhances oxidative stress, cell proliferation and reduces apoptosis in human umbilical vein endothelial cells: role of ETB receptor, NADPH oxidase and caveolin-1.

1 Endothelin‐1 (ET‐1), an endothelium‐derived vasoactive peptide, participates in the regulation of endothelial function through mechanisms that are not fully elucidated. This study examined the impact of ET‐1 on oxidative stress, apoptosis and cell proliferation in human umbilical vein endothelial cells (HUVEC). HUVECs were challenged for 24u2003h with ET‐1 (10u2003pM–10u2003nM) in the absence or presence of the ETB receptor antagonist BQ788 (1u2003μM) or the NADPH oxidase inhibitor apocynin (1u2003μM). Reactive oxygen species (ROS) were detected using chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate. Apoptosis was evaluated with 4′,6′‐diamidino‐2′‐phenylindoladihydrochloride staining and by the caspase‐3 assay. Cell proliferation was measured by a colorimetric assay. Expression of NADPH oxidase, Akt, pAkt, Bcl‐2, Bax, IκB, caveolin‐1 and eNOS was evaluated by Western blot analysis. 2 ET‐1 significantly enhanced ROS generation and cell proliferation following 24‐h incubation, both of which were prevented by BQ788 or apocynin, consistent with the ability of ET‐1 to directly upregulate NADPH oxidase. ET‐1 itself did not affect apoptosis but attenuated homocysteine‐induced apoptosis through an ETB receptor‐mediated mechanism. Western blot analysis indicated that ET‐1 alleviated homocysteine (Hcy)‐induced apoptosis, likely acting by antagonizing the Hcy‐induced decreases in Akt, pAkt, pAkt‐to‐Akt, Bcl‐2‐to‐Bax ratios and increases in Bax and caveolin‐1 expression. Furthermore, ET‐1 downregulated expression of caveolin‐1 and eNOS, which was attenuated by BQ788 or apocynin. 3 In summary, our results suggest that ET‐1 affects oxidative stress, proliferation and apoptosis possibly through ETB, NADPH oxidase, Akt, Bax and caveolin‐1‐mediated mechanisms.

Oxidative stress and stress signaling: menace of diabetic cardiomyopathy

AbstractCardiovascular disease is the most common cause of death in the diabetic population and is currently one of the leading causes of death in the United States and other industrialized countries. The health care expenses associated with cardiovascular disease are staggering, reaching more than US

Measurement of Cardiac Mechanical Function in Isolated Ventricular Myocytes from Rats and Mice by Computerized Video-Based Imaging.

350 billion in 2003. The risk factors for cardiovascular disease include high fat/cholesterol levels, alcoholism, smoking, genetics, environmental factors and hypertension, which are commonly used to gauge an individuals risk of cardiovascular disease and to track their progress during therapy. Most recently, these factors have become important in the early prevention of cardiovascular diseases. Oxidative stress, the imbalance between reactive oxygen species production and breakdown by endogenous antioxidants, has been implicated in the onset and progression of cardiovascular diseases such as congestive heart failure and diabetes-associated heart dysfunction (diabetic cardiomyopathy). Antioxidant therapy has shown promise in preventing the development of diabetic heart complications. This review focuses on recent advances in oxidative stress theory and antioxidant therapy in diabetic cardiomyopathy, with an emphasis on the stress signaling pathways hypothesized to be involved. Many of these stress signaling pathways lead to activation of reactive oxygen species, major players in the development and progression of diabetic cardiomyopathy.

Increases in insulin-like growth factor-1 level and peroxidative damage after gestational ethanol exposure in rats

Isolated adult cardiac ventricular myocytes have been a useful model for cardiovascular research for more than 20 years. With the recent advances in cellular physiology and transgenic techniques, direct measurement of isolated ventricular myocyte mechanics is becoming an increasingly important technique in cardiac physiology that provides fundamental information on excitation-contraction coupling of the heart, either in drug intervention or pathological states. The goal of this article is to describe the isolation of ventricular myocytes from both rats and mice, and the use of real-time beat-to-beat simultaneous recording of both myocyte contraction and intracellular Ca2+ transients.

Depressed contractile function and adrenergic responsiveness of cardiac myocytes in an experimental model of Parkinson disease, the MPTP-treated mouse

Ethanol exposure during pregnancy elicits profound detrimental developmental and behavioral effects such as reduced levels of insulin-like growth factor-1 (IGF-1) in the fetus. However, few reports have addressed its impact on postpartum dams. This study was designed to examine the influence of gestational ethanol exposure on postpartum maternal organ oxidative damage and IGF-1 level. Pregnant female rats were pair-fed from Day 2 of gestation until labor with control or ethanol (6.36% (v/v)) liquid diets and were sacrificed 6 weeks after parturition (ethanol withdrawn after parturition). There was no difference in body weight during or after the gestational period between the control and ethanol groups. Litter size was significantly less for ethanol-fed dams. One-week postnatal pup survival was significantly lower in the ethanol-fed (57.1%) than the control (97.8%) group. Liver and kidney tissue IGF-1 levels and mRNA were elevated in the ethanol-fed mothers, accompanied by hepatic but not renal oxidative damage, indicated by profound lipid peroxidation (measured by malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)) and protein carbonyl formation. The levels of glutathione (GSH), glutathione disulfide (GSSG) and GSH/GSSG ratios in liver and kidney were not different between the ethanol-fed and control dams. Collectively, these data suggest that gestational ethanol exposure may lead to postpartum oxidative organ damage and a possible compensatory increase in organ IGF-1 levels.

Insulin-like growth factor I (IGF-1) supplementation prevents diabetes-induced alterations in coenzymes Q9 and Q10

Radiotracer and biochemical studies have shown that patients with Parkinson disease lack functional sympathetic innervation to the heart. The same observation was made in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson disease. This study examined the mechanical properties, adrenergic receptor level and intracellular Ca2+ handling in cardiac myocytes isolated from C57/BL6 mice that received either MPTP (30 mg/kg, i.p., twice in 24 h) or vehicle. Mechanical properties were evaluated using an IonOptix MyoCam system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocities of shortening and relengthen (+/-dL/dt). Intracellular Ca2+ handling was evaluated with fura 2. Myocytes from MPTP-treated mice exhibited a depressed PS (85% of normal), normal TPS, prolonged TR90 (147% of normal), and reduced +/-dL/dt (both 79% of normal). These results were correlated with a 67% reduction of beta-adrenergic receptor expression in myocardial membranes from MPTP-treated mice when compared to normal. Myocytes from MPTP-treated mice also exhibited a reduced peak of intracellular Ca2+ sequestration and sarcoplasmic reticulum (SR) Ca2+ load (55 and 38% of normal, respectively). The resting intracellular Ca2+ and Ca2+-transient decay were comparable to the values seen in myocytes from untreated mice. Myocytes from MPTP-treated and untreated mice were equally responsive over a range of stimulation frequencies (0.1, 0.5, 1, 3 and 5 z). Response to norepinephrine (1 microM) and isoproterenol (1 microM) was reduced in myocytes from MPTP-treated mice. These results demonstrate substantial cardiac dysfunctions in this model of experimental Parkinson disease, probably due to reduced adrenergic responsiveness and SR Ca2+ load.

Prenatal ethanol exposure alters ventricular myocyte contractile function in the offspring of rats

Abstract. Diabetes, which causes enhanced oxidative stress, is a multifactorial disease that leads to deleterious effects in many organ systems within the body. Ubiquinones (coenzyme Q9 and Q10) are amphipathic molecular components of the electron transport chain that function also as endogenous antioxidants and attenuate the diabetes-induced decreases in antioxidant defense mechanisms. Insulin-like growth factor 1 (IGF-1) is considered to be an “essential surviving factor”, the level and function of which are compromised in diabetes. This study investigated the impact of IGF-1 supplementation on ubiquinone levels in a rat model of type I diabetes. Adult male Sprague-Dawley rats were divided into four groups: control, control plus IGF-1, diabetic and diabetic plus IGF-1. Diabetic animals received a single intravenous injection of streptozotocin (STZ, 55 mg/kg). IGF-1 supplementation groups received a daily intraperitoneal dose of 3 mg IGF-1 per kilogram body weight for 7 weeks. Coenzyme Q9 and Q10 levels were assessed by ultraviolet detection on high pressure liquid chromatography. STZ caused a significant reduction in body weight and an elevation in blood glucose level, which were not prevented by IGF-1 supplementation. In addition Q9 and Q10 levels in diabetic liver were significantly elevated. IGF-1 supplementation prevented liver alterations in Q10 but not Q9 levels. Q9 and Q10 levels in diabetic kidney were significantly depressed, and these deleterious effects were abolished by IGF-1 treatment. These data suggest that IGF-1 antagonizes the diabetes-induced alterations in endogenous antioxidants including coenzyme Q10, and hence may have a therapeutic role in diabetes.

Influence of ovariectomy on ventricular myocyte contraction in simulated diabetes.

Fetal alcohol syndrome (FAS) is often associated with cardiac hypertrophy and impaired ventricular function in a manner similar to postnatal chronic alcohol ingestion. Chronic alcoholism has been shown to lead to hypomagnesemia, and dietary Mg2+ supplementation was shown to ameliorate ethanol-induced cardiovascular dysfunction such as hypertension. However, the role of gestational Mg2+ supplementation on FAS-related cardiac dysfunction is unknown. This study was conducted to examine the influence of gestational dietary Mg2+ supplementation on prenatal ethanol exposure-induced cardiac contractile response at the ventricular myocyte level. Timed-pregnancy female rats were fed from gestation day 2 with liquid diets containing 0.13 g/L Mg2+ supplemented with ethanol (36%) or additional Mg2+ (0.52 g/L), or both. The pups were maintained on standard rat chow through adulthood, and ventricular myocytes were isolated and stimulated to contract at 0.5 Hz. Mechanical properties were evaluated using an IonOptix™ soft-edge system, and intracellular Ca2+ transients were measured as changes in fura-2 fluorescence intensity (ΔFFI). Offspring from all groups displayed similar growth curves. Myocytes from the ethanol group exhibited reduced cell length, enhanced peak shortening (PS), and shortened time to 90% relengthening (TR90) associated with a normal ΔFFI and time to PS (TPS). Mg2+ reverted the prenatal ethanol-induced alteration in PS and maximal velocity of relengthening. However, it shortened TPS and TR90, and altered the ΔFFI, as well as Ca2+ decay rate by itself. Additionally, myocytes from the ethanol group exhibited impaired responsiveness to increased extracellular Ca2+ or stimulating frequency, which were restored by gestational Mg2+ supplementation. These data suggest that although gestational Mg2+ supplementation may be beneficial to certain cardiac contractile dysfunctions in offspring of alcoholic mothers, caution must be taken, as Mg2+ supplementation affects cell mechanics itself.

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats

We studied the effect of ovariectomy (OVX) on cardiac contraction in myocytes maintained under a diabetes-simulated high-glucose environment. Female rats were ovariectomized or sham operated (SHAM) and kept for 6 weeks. Isolated myocytes were maintained in a diabetes-simulated high [glucose] medium (HG; 25.5 mM) for 24 h before mechanical properties were measured. Contractile indices analyzed included peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca2+ fura-2 fluorescence intensity and decay rate (tau). Nitric oxide synthase (NOS) activity was also evaluated. OVX myocytes displayed a longer TR(90), slower +/- dL/dt, lower fluorescence intensity and higher tau (slower decay rate) when compared to SHAM myocytes. In the SHAM group, HG exerted diabetes-like contractile dysfunctions, including depressed PS, prolonged TR90, reduced fluorescence intensity, higher tau and enhanced NOS activity when compared to myocytes maintained in low [glucose] medium (5.5 mM). Interestingly, the HG- induced mechanical alterations were significantly exaggerated (TPS, TR90 and tau), reversed (PS and NOS) or lost (+/- dL/dt and fluorescence intensity) in the OVX group. These data suggest that ovarian hormones play a role in the regulation of cardiac contractile function, and may have potentially protective effects against diabetes-associated cardiac dysfunction.