Insulin Resistance as a Risk Factor for Gallbladder Stone Formation in Korean Postmenopausal Women

BACKGROUND/AIMS: The objective of this study was to determine whether insulin resistance is associated with gallbladder stone formation in Korean women based on menopausal status. METHODS: The study included 4,125 consecutive Korean subjects (30-79 years of age). Subjects who had a medical history of diabetes, hypertension, dyslipidemia, other cardiovascular disorders, or hormone replacement therapy were excluded. The women were subdivided into two groups according to their menopausal status. RESULTS: Analysis of premenopausal women showed no significant differences in the homeostasis model of assessment-insulin resistance (HOMA-IR) index between the two groups in terms of gallstone disease. The associations between the occurrence of gallbladder stones and age, obesity, abdominal obesity, hyperinsulinemia, and high HOMA-IR index were statistically significant in the analysis with postmenopausal women. In a multiple logistic regression analysis, low high density lipoprotein-cholesterol was an independent predictor of gallbladder stone formation in premenopausal women. However, the multiple logistic regression analysis also showed that age and HOMA-IR were significantly associated with gallbladder stone formation in postmenopausal women. In an additional analysis stratified by obesity, insulin resistance was a significant risk factor for gallbladder stone formation only in the abdominally obese premenopausal group. CONCLUSIONS: Insulin resistance may be associated with gallbladder stone formation in Korean postmenopausal women with abdominal obesity.

Characteristics of gene transfer and VEGF expression using naked plasmid vectors in the rat heart / 대한내과학회지

BACKGROUND: The purpose of this study was to compare gene expression among newly designed eukaryotic expression vectors, and to characterize the pattern of vascular endothelial growth factor(VEGF) expression using the most potent plasmids DNA vector. METHODS: After exposure of a beating rat heart (Sprague-Dawley, 250-300g), 5 different types of plasmid DNA was injected directly into the myocardium. Reporter protein was analyzed by ELISA in the extracted heart. RESULTS: The vector harboring cytomegalovirus (CMV) promoter and enhancer induced the strongest expression of reporter gene (chloramphenicol acetyl transferase; CAT) compared to those of pC3.1, pEF1a, RSV, pActin in the rat heart via direct injection of plasmid DNA into the apex (p<0.001). Using pCN-CAT, gene expression showed a dose-dependent response over a range of 0.3-10 (mu)g. CAT expression could be detected up to 30 days after 10 (mu)g of pCN-CAT injection with the maximal expression on day 5. In X-gal staining of injected pCN-lacZ gene, beta-galactosidase was found only around the needle track in the apex. The expressed hVEGF121 had biologic activity with vascular permeability assay (Miles assay) in guinea pigs. After injection of pCN-hVEGF121 into the apex of the rat heart, the expression of VEGF protein was dose-dependent over the range of 25 and 500 (mu)g. VEGF expression was detected up to 14 days with its peak on day 2 after injection of 250 (mu)g of pCN-hVEGF121. When plasmid was injected into the apex of the rat heart, the expression of VEGF in the heart showed concentration gradient from the apex to the base. However, the expression of CAT was detected only in the apex. CONCLUSION: Plasmids vector with hCMV IE promoter/enhancer will provide clear advantages over other previously developed plasmids and the information regarding the behaviors of VEGF expression may be useful in angiogenic gene therapy of the heart.

Probing the Utility of Vascular Smooth Muscle Cells as a Target Cell for ex vivo Cardiovascular Gene Therapy

BACKGROUND AND OBJECTIVES: Compared to other target cells examined for gene therapy, vascular smooth muscle cells (VSMCs) have the unique advantages including proximity to blood stream and relative abundance in vasculature. With an ultimate goal of developing VSMC-based therapies for cardiovascular disorders, we explored the utility of VSMC as a target cell for ex vivo gene therapy using a set of retroviral vectors. MATERIALS AND METHODS: Cultured VSMCs were transduced with replication-defective recombinant retroviruses harboring LacZ, nlsLacZ, mVEGF, mGM-CSF or bacterial CAT reporter. The VSMCs were examined for G418-selection, transduction efficiency, the level of transgene expression, and longevity of gene expression. ResultsVSMCs were readily transduced with different kinds of retroviral vectors. The bacterial neo r gene-transduced VSMCs were successfully selected with G418. The G418-selected VSMCs could express the transduced genes at a level comparable to NIH3T3. The level of transgene expression did not appear to be affected by the increasing number of passages. CONCLUSION: The results demonstrate an efficient transduction of VSMCs by retroviral vectors in vitro and an sustained expression of retrovirally transduced genes in VSMCs. VSMCs could be one of the ideal target cells for ex vivo cardiovascular gene therapy employing retroviral vector.

The Effects of Aldosterone and Cytokines IL-1beta, TNF-alpha on the Expression of Angiotensin Converting Enzyme Gene in Vascular Smooth Muscle Cells

BACKGROUND: It has been suggested that all components of the renin-angiotensin-aldosterone system (RAAS) are present in the vascular wall and that the vascular RAAS modulates vascular tone and vascular hypertrophy. One of the catalytic step in the RAAS cascade is the local conversion of angiotensin I to angiotensin II (Ang II) by angiotensin converting enzyme (ACE). One of the major sources of ACE in the vasculature is vascular smooth muscle cells (VSMC). Here, we provide insight into the intrinsic mechanisms by which the components of RAAS regulate gene expression of ACE in cultured smooth muscle cells of the rat and we also investigated the effects of cytokines on ACE mRNA. METHODS: RNA was extracted from the primary cultured VSMCs. We analyzed the expression levels of ACE by competitive reverse transcription-PCR using recombinant RNA as an internal standard. RESULTS: 1) ACE mRNA level was increased markedly by aldosterone in a dose- and time-dependent manner, indicating that there exists positive feedback mechanism within RAAS. 2) The induction of ACE mRNA by aldosterone was inhibited by spironolactone. 3) Aldosterone-stimulated expression of ACE was also inhibited by Ang II, which shows that Ang II acts as a negative regulator of the expression of ACE in RAAS cascade. 4) Interleukin-1beta or TNF-alpha did not induce ACE mRNA expression. 5) However, mixture of interleukin-1betaand TNF-alpha(CytoMix) significantly increased the expression of ACE. It was also shown that CytoMix increased aldosterone-stimulated ACE mRNA expression in an additative manner. CONCLUSION: These results indicate that the expression of ACE in smooth muscle cells is modulated by the components of RAAS and cytokines. The intrinsic positive and negative feedback controls of RAAS would play an important role in the pathogenesis of vascular diseases.

Gene Transfer into Cultured Cardiac Myocytes Mediated by Retrovirus

BACKGROUND: Transplantation of cardiac myocytes (CMs) into the injured heart emerges as a potential alternative for the treatment of heart failure. Genetic modification of CMs could enhance and/or modify its therapeutic effects. The characteristics of retroviral gene delivery, which is most commonly used in human trial, has been minimally studied in CMs due to its low efficiency in non-dividing cells. In this study, using newly developed high-titer retrovirus, we evaluated 1) the efficiency of gene transfer into CMs, 2) whether S phase during infection is necessary for the transduction, and 3) characteristics of gene delivery to mononucleated vs binucleated CMs. METHODS: Enriched CMs were cultured from the ventricles of 1 day-old rat hearts. The cells were transduced by MFG-nls-LacZ retroviruses (5x107 IU/ml) in the presence or absence of polybrene. 3H-thymidine was added to label cells in S phase. The cells were stained for b-galactosidase activity and then immunostained using MF20Ab to identify CMs. The cells were subsequently processed for in vitro autoradiography. RESULTS: 1)With 3 rounds of infection, 5.9% of total cultured cells were LacZ-positive. The efficiency of transduction reached upto 7.4% in the presence of polybrene 8 microgram/ml. 2)Nuclear morphology of LacZ-positive CMs was pleomorphic from mononucleated to multinucleated, mostly binucleated. 3)Among mononucleated CMs, 17% of cells were labelled with thymidine. Transduction efficiency (TDE) of thymidine-positive and -negative mononucleated CMs were 37.9% and 3.1%, respectively. Among binucleated cells, 28.9% of cells were labelled with thymidine. TDE of thymidine-positive and -negative binucleated CMs were 75.4% and 13.6%, respectively. 4)In total, TDE of binucleated cells were 3.5 times compared to the one of mononucleated cells (31.5% vs 9.0%). CONCLUSION: TDE of CMs using high-titer retrovirus is relatively low. S phase of cells during retroviral infection is not mandatory for the retroviral transduction. Binucleated CMs are susceptible to retroviral gene delivery and their TDE is higher than the one of mononucleated CMs.

Effect of Local Administration of Lovastatin on Preventing Neointimal Hyperplasia in the Rat Carotid Artery Injury Model

BACKGROUND AND OBJECTIVES: ovastatin, a HMG-CoA reductase inhibitor, is known to show antiproliferative effects on VSMC after vessel injury, but a large amount of the drug is needed orally for this purpose. This study investigated the effects of lovastatin given locally to injured carotid arteries of rats on reducing neointimal hyperplasia. MATERIALS AND METHOD: Lovastatin was given perivascularly to balloon-injured carotid arteries of 21 rats in 1 microM to the low-dose group, and 30 microM to the high-dose group. The control group was treated with pluronic gel only. Two weeks later, the lumen area, neointimal areas and the number of actively proliferating cells were obtained and compared. RESULTS: eointimal area was 0.113+/-0.032 mm2, 0.065+/-0.017 mm2, 0.072+/-0.017 mm2 in the control, low-dose and high-dose groups respectively. The area was significantly smaller in the treatment groups (p<0.05), but no significant difference was observed between the treatment groups. The number of actively proliferating cells per mm2 of neointimal area were 714.5+/-227.4, 688.4+/-333.7, and 1526.3+/-744.0 in the groups respectively, and the number was significantly high in the high-dose group (p<0.05). CONCLUSION: Local administration of lovastatin is effective in reducing neointimal hyperplasia after vascular injury, but extremely high doses are not needed locally for this purpose.

Angiotensin II stimulates proliferation of adventitial fibroblasts cultured from rat aortic explants

It has been proposed that the local renin-angiotensin system is activated in the adventitia after vascular injury. However, the physiological role of Angiotensin II (Ang II) in the adventitia has not been studied at a cellular level. This study was designed to assess the role of Ang II in the growth response of cultured adventitial fibroblasts (AFs). Adventitial explants of the rat thoracic aorta showed outgrowth of AFs within 5-7 days. Ang II caused hyperplastic response of AF cultures. The Ang II-induced mitogenic response of AFs was mediated primarily by the AT1 receptor. Ang II caused a rapid induction of immediate early genes (c-fos, c-myc and jun B). Induction of c-fos expression was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. Epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) induced DNA synthesis in AFs. Co-stimulation of AFs with the growth factors and Ang II potentiated the incorporation of 3H-thymidine into DNA. Results from this study indicate that Ang II causes mitogenesis of AFs via AT1 receptor stimulation and potentiates the responses to other mitogens. These data suggest that the Ang II may play an important role in regulating AF function during vascular remodeling following arterial injury.

Antiproliferative Effect of Lovastatin on Vascular Smooth Muscle Cell

BACKGROUND AND OBJECTIVES: This study was performed to investigate the antiproliferative effect of lovastatin on vascular smooth muscle cell, especially to determine whether lovastatin induces apoptosis in vascular smooth muscle cell and the products of mevalonate pathway can reverse the antiproliferative effect of lovastatin. METHODS AND MATERIALS: Lovastatin only and lovastatin with one of the products of mevalonate pathway such as isopentenyl adenine, farnesol, mevalonate, cholesterol were added respectively in cultured rat vascular smooth muscle cells stimulated with 10% fetal calf serum. DNA synthesis was measured by tritiated-thymidine incorporation. Cell number was determined by hemocytometric counting. Cells were Giemsa-stained to evaluate morphological changes of apoptosis. Extracted DNA from the cells treated with lovastatin was assessed by gel electrophoresis. RESULTS: 1)Lovastatin inhibited DNA synthesis and cell proliferation in a dose-dependent manner. 2)The inhibitory effects of lovastatin could be reversed almost completely by mevalonate, partially by farnesol. 3)Lovastatin-treated vascular smooth muscle cells showed typical morphological changes of apoptosis. 4)A distinct ladder of DNA bands was visualized by gel electrophoresis of the DNA from the cells treated with lovastatin. CONCLUSION: Mevalonate metabolism is essential for vascular smooth muscle cell proliferation. The antiproliferative effect of lovastatin may result from the induction of apoptosis in vascular smooth muscle cells.

Development of Quantitative Reverse Transcription-Polymerase Chain Reaction for the Mesurement of Angiotensin Converting Enzyme mRNA

BACKGROUND: The analysis of ACE gene expression in vital to study the role of angiotensin conveting enzyme(ACE) in the pathogenesis of cardiovascular disease. Traditionally, levels of individual mRNA expression have been analyzed by semiquantitative Northern blotting, which requires a large quantity of tissue. Therefore, gene expression of a little biopsy specimen from the human heart or atherectomy specimen from the blood vessel cannot be measured easily. Reverse transcription-polymerase chain reaction(RT-PCR) is very effective, sensitive and rapid method of detecting the method of quantitative RT-PCR(QRT-PCR) using recombinant RNA template as internal standard to measure the expression of ACE. METHOD: Recombinant RNA(rcRNA) was designed to yield PCR product which differs in size by about 200bp from that of the target RNA. Initially, spacer gene, which was composed of ACE sense primer, antisense primer, T7 promotor and poly(dT) tail with glutathione transferase(GSTM) gene of 180bp in the middle, was constructed. Then, standard rcRNA was obtained by in vitro transcription. Target RNA was mixed with rcRNA and amplified by PCR, togather with P-dCTP. PCR products were analyzed by gel electrophoresis. For quantitation, either gel was cut and radioactivity was counted or gel was dried and exposed to X-ray film and density was measured using image densitometer. We carried out semiquantitative RT-PCR to study the modulation of ACE expression in vascular smooth muscle cell(VSMC) by dexamethasone and basis FGF(bFGF). RESULT: The size difference of PCR products from the standard RNA and the extracted target RNA was matched as designed. By using QRT-PCR, there was 1.7*10(8) ACE mRNA molecules in 1 ng of rat lung total RNA. bFGF and dexamethasone upregulated ACE mRNA expression in cultured VSMC. CONCLUSION: These results suggest that RT-PCR using rcRNA as internal standard is a very useful method for quantitation or semiquantitation of ACE mRNA from a small amount of tissue or cultured cells. Expression of ACE in VSMC can be modulated by various stimuli such as basic FGF and dexamethasone. QRT-PCR could be widely used in the studies of expression of specific human genes.