Vascular dysfunction of venous bypass conduits is mediated by reactive oxygen species in diabetes: role of endothelin-1

Diabetes is associated with increased risk for complications following coronary bypass grafting (CABG) surgery. Augmented superoxide (*O2*) production plays an important role in diabetic complications by causing vascular dysfunction. The potent vasoconstrictor endothelin-1 (ET-1) is also elevated in diabetes and following CABG; however, the effect of ET-1 on *O2* generation and/or vascular dysfunction in bypass conduits remain unknown. Accordingly, this study investigated basal and ET-1-stimulated *O2* production in bypass conduits and determined the effect of *O2* on conduit reactivity. Saphenous vein specimens were obtained from nondiabetic (n = 24) and diabetic (n = 24) patients undergoing CABG. Dihydroethidium staining and NAD(P)H oxidase activity assays (5380 +/- 940 versus 16,362 +/- 2550 relative light units/microg) demonstrated increased basal *O2* levels in the diabetes group (p < 0.05). Plasma ET-1 levels were associated with elevated basal *O2* levels, and treatment of conduits with exogenous ET-1 further increased *O2* production and augmented vasoconstriction. Furthermore, vascular relaxation was impaired in the diabetic group (75 versus 40%), which was restored by *O2* scavenger superoxide dismutase. These findings suggest that ET-1 causes bypass conduits dysfunction via stimulation of *O2* production in diabetes. Novel therapies that attenuate *O2* generation in bypass conduits may improve acute and late outcome of CABG in diabetic patients.     

Stepwise genetic changes associated with progression of nontumorigenic HPV-18 immortalized human prostate cancer-derived cell line to a malignant phenotype

Cytogenetics, fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) were used to identify genes that are involved in the development and progression of prostate cancer. For that purpose, we chose a cell line established in vitro from a prostatic adenocarcinoma which was nontumorigenic in nude mice and followed its progression to a tumorigenic cell line. Stepwise changes were observed in the cell line as it became tumorigenic. The composite karyotype at the nontumorigenic stage (CA-HPV-10) was 68 approximately 77,XXY,-(1, 9, 13, 14, 19, 22),+(4, 5, 11, 18, 20, 21),+(del(1) (q23q31)=M1 (two copies), +der(9)t(1;9)(q24 approximately q31;p23)=M5(two copies), der(14)t(14;?)(q10;?)=M17 in the majority of metaphases. These two derivative chromosomes were also observed a previous study. Our CGH analysis clearly showed that this deleted region in M1 is, in fact, translocated with derivative M5 and, in reality, is amplified. The cell line established from nodule (SCID 5019 p11), showed a number of new changes, as described; however, the most significant change was amplification of the 8q23 approximately qter region, harboring c-myc. This region was translocated with chromosomes 2, 4, and 16 as der(2)t(2;8)(q33;q23)=M12, der(4)t(4;8)(q34;q23)=M11, and der(16)t(8;16)(q24;q21)=M9. We deduce from our study that amplification of c-myc and other genes in the 8q23 approximately qter region were important in progression but did not lead to tumorigenicity. The population that became tumorigenic (SCID 5019 II) showed almost all of the same changes in the karyotype as observed in the nodular cell line; the only significant change was the appearance of der(11)t(4;11)(q32;q22)=M7 and the addition of another copy of t(3q;7p)=M2. These new changes lead to loss of chromosomes 3p, 4pter approximately q34, 6, 7q21 approximately qter, 11q22 approximately qter, and 18q, and gain of 3q, 7p, 8q23 approximately qter, and 11pter approximately q22, before the cell line became tumorigenic. The clonal selection of the population is proven by the presence of a number of the same derivative chromosomes in both the nodular and tumorigenic cell line. As it progressed to tumorigenicity, some of the same changes observed in the original study re-appear at different stages of malignancy, although it was absent in the nontumorigenic cell line. These are: der(16)t(8;16)(q24;q21)=M9 in the nodular cell line and der(11)t(4;11)(q32;q22)=M7 in the tumorigenic cell line. In our system, amplification of c-myc and other genes in der(2)t(2;8)(q33;q23)=M12,der(4) t(4;8)(q34;q23)=M11 together with the presence of der(16)t(8;16)(q24;q21)=M9 and der(11)t(4;11)(q32;q22)=M5 makes the cell line tumorigenic. It is either nontumorigenic, with the presence of a marker equivalent to der(16)=M9 and der(11)=M7 observed in the original study, and only nodular (SCID 5019 p11, present study), with the presence of number of markers with c-myc amplification (M9, M11, and M12). There is accumulation of all the above-mentioned changes in the same cell before it becomes tumorigenic.     

A human prostatic stromal myofibroblast cell line WPMY-1: a model for stromal-epithelial interactions in prostatic neoplasia.

Here we report the characterization of an SV40 large-T antigen-immortalized stromal cell line, WPMY-1, derived from the same prostate as our previously described epithelial cell lines. The WPMY-1 cells were determined to be myofibroblasts on the basis of co-expression of smooth muscle alpha-actin and vimentin. They also show positive staining for androgen receptor, large-T antigen, and positive but heterogeneous staining for p53 and pRb. Their growth is stimulated by the synthetic androgen mibolerone to 145% of control (100%). Platelet-derived growth factor BB, epidermal growth factor and basic fibroblast growth factor, at 10 ng/ml, stimulated growth to 138, 143 and 146% of control, respectively. Transforming growth factor-beta, at 10 ng/ml, inhibited serum-induced growth to 65% of control in the presence of 1% serum, and bFGF-induced growth to 30% of control. A serum-free medium was developed for optimal growth of WPMY-1 cells. They show anchorage-independent growth in soft agar. Studies on paracrine interactions show that myofibroblast-conditioned medium causes a marked inhibition of growth in WPE1-10 cells, while conditioned medium from WPE1-10 prostatic epithelial cells caused only a small increase in the growth of WPMY-1 cells. WPMY-1 cells secrete very low levels of MMP-9 but high levels of MMP-2, markedly higher than the epithelial cells. These epithelial and myofibroblast cell lines, derived from the same prostate, provide novel and useful models for studies on paracrine stromal-epithelial interactions in carcinogenesis, tumor progression, prevention and treatment of prostate cancer and benign prostatic hyperplasia.     

Characterization of clones of tumorigenic feline cells transformed by a chemical carcinogen

Tumorigenic clonal lines derived from soft agar colonies induced by DMBA-transformed feline embryo cells were isolated and characterized. The morphologically altered clonal cells formed large aggregates, growing in this aggregate form when suspended in liquid growth medium above an agar base and forming colonies in soft agar with high efficiency. When inoculated into athymic nude mice, chemically altered clonal cells produced progressively growing sarcomas. Cells established from the tumors morphologically resembled the DMBA-transformed feline embryo cells and were characterized as cat cells by karyological analysis. The tumorigenic lines were negative for feline oncornavirus-associated cell membrane antigen (FOCMA), and for "gag-X" the transformation-related polyprotein which is encoded by the replication defective feline sarcoma virus.     

Ginseng saponins induce store-operated calcium entry in Xenopus oocytes

1 We investigated the effect of the active ingredients of Panax ginseng, ginsenosides, on store-operated Ca2+ entry (SOCE) using a two-electrode voltage clamp technique in Xenopus oocytes in which SOCE is monitored through Ca(2+)-activated Cl- currents. 2 Under hyperpolarizing voltage clamp conditions, treatment with ginsenosides produced a biphasic Ca(2+)-activated Cl- current consisting of a rapid transient inward current and a slowly developing secondary sustained inward current. The transient inward current was inactivated rapidly, whereas the sustained inward current persisted for nearly 10 min. The effect of ginsenosides on the biphasic current was dose-dependent and reversible. The EC50 was 42.8+/-11.6 and 46.6+/-7.1 microg ml(-1) for the transient and sustained inward current, respectively. 3 In the absence of extracellular Ca2+ ginsenosides induced only a transient inward current but in the presence of extracellular Ca2+ ginsenosides induced the biphasic current. Magnitudes of the sustained currents were dependent on extracellular Ca2+ concentration. Sustained inward current induced by ginsenosides, but not transient inward current, and ginsenoside-induced store-operated Ca2+ (SOC) currents (ISOC) were blocked by La3+, a Ca2+ channel blocker, suggesting that the sustained inward current and ISOC was derived from an influx of extracellular Ca2+. 4 Treatment with 2-APB and heparin, which are IP3 receptor antagonists, inhibited the ginsenoside-induced biphasic current. Treatment with the PLC inhibitor, U73122, also inhibited the ginsenoside-induced biphasic current. Intraoocyte injection of ATP-gammaS, but not adenylyl AMP-PCP, induced a persistent activation of ginsenoside-induced sustained current but did not affect the transient current. 5 In rat hippocampal neurons, ginsenosides inhibited both carbachol-stimulated intracellular Ca2+ release and intracellular Ca2+ depletion-activated SOCE. 6 These results indicate that ginsenoside might act as a differential regulator of intracellular Ca2+ levels in neurons and Xenopus oocytes.     

HYP-1, a novel diamide compound, relieves inflammatory and neuropathic pain in rats

In the present study, we investigated whether a novel compound, 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)-2-oxoethylamino)-N-(3,4,5-trimethoxybenzyl)acetamide (HYP-1), is capable of binding to voltage-gated sodium channels (VGSCs) and evaluated both its inhibitory effect on Na⁺ currents of the rat dorsal root ganglia (DRG) sensory neuron and its in vivo analgesic activity using rat models of inflammatory and neuropathic pain. HYP-1 showed not only high affinity for rat sodium channel (site 2), but also potent inhibitory activity against the TTX-R Na⁺ currents of the rat DRG sensory neuron. HYP-1 co-injected with formalin (5%, 50 μl) under the plantar surface of rat hind paw dose-dependently reduced spontaneous pain behaviors during both the early and late phases. This result was confirmed by c-Fos immunofluorescence in the L4-5 spinal segments. A large number of c-Fos-positive neurons were observed in rat injected with a mixture of formalin and vehicle, but not in rat treated with a mixture of formalin and HYP-1. In addition, the effectiveness of HYP-1 (6 and 60 mg/kg, i.p.) in suppression of neuropathic pain, such as mechanical, cold and warm allodynia, induced by rat tail nerve injury was investigated. HYP-1 showed limited selectivity over hERG, N-type and T-type channels. Our present results indicate that HYP-1, as a VGSC blocker, has potential analgesic activities against nociceptive, inflammatory and neuropathic pain.     

Roles of 7-ketocholesterol on the Homeostasis of Intracellular Cholesterol Level

ABSTRACT:: Atherosclerotic plaque contains materials, such as cholesterol, oxysterols, cell debris, modified fatty acids, and infiltrated cells. Among them, cholesterol is the major component in plaque. Cholesterol is known to originate from the influx of extracellular materials, but this explanation is not enough for the cholesterol accumulation observed in atherosclerotic plaque. This study examined the origins of cholesterols in plaques. The main focus was to determine if the intracellular cholesterol levels are affected by oxysterols in human vascular smooth muscle cells. The results showed that the cholesterol levels increased in response to a 7-ketocholesterol (7K)-treatment in a dose-dependent manner. Eight enzymes involved in cholesterol biosynthesis were examined. Among them, squalene epoxidase (SQLE) was increased by 7K but not by 7α-hydroxycholesterol, 27-hydroxycholesterol (27OH-chol), or cholesterol. The 7K-induced SQLE expression was suppressed in the presence of the enzyme inhibitor SB203580 but not by UO126 and SP600125. The SQLE immunoreactivity was detected in the atherosclerotic plaque of the aortic roots from apoE mice. In addition, 7K increased the cholesterol level and SQLE expression in murine bone marrow-derived macrophages. This suggests that 7K increases the intracellular cholesterol level through an elevation of SQLE expression, which might affect the progress of cholesterol accumulation in the atherosclerotic lipid core.