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1.  Multifactorial, Site-Specific Recurrence Models after Radical Cystectomy for Urothelial Carcinoma: External Validation in a Cohort of Korean Patients 
PLoS ONE  2014;9(6):e100491.
Purpose
The aim of this study was to evaluate the accuracy of site-specific recurrence models after radical cystectomy in the Korean population.
Materials and Methods
We conducted a review of an electronic medical record of 572 patients who underwent radical cystectomy for urothelial carcinoma of the bladder. Primary end point was the site-specific recurrence after radical cystectomy.
Results
The median follow-up in the validation cohort was 42.3 months (interquartile range: 23.0–89.3 months). During the follow-up period, there were 165 patients (28.8%), 85 (14.9%), 31 (5.4%), and 78 (13.6%) who recurred in abdomen/pelvis, thoracic region, upper urinary tract, and bone, respectively. The c-indices of abdomen/pelvis, thoracic region, upper urinary tract, and bone models 3 years after radical cystectomy were 0.69 (95% confidence interval [CI], 0.65–0.73), 0.69 (95% CI, 0.64–0.75), 0.61 (95% CI, 0.52–0.69), and 0.65 (95% CI, 0.59–0.71), respectively. Kaplan-Meier curves demonstrated that models discriminated well and log-rank test were all highly significant (all p<0.001), except upper urinary tract model (p = 0.366). Decision curve analysis revealed that the use of prediction models for abdomen/pelvis, thoracic region, and bone recurrence was associated with net benefit gains relative to the treat-all strategy, but not the model for upper urinary tract recurrence.
Conclusions
Abdomen/pelvis, thoracic region, and bone models demonstrate moderate discrimination, adequate calibration, and meaningful net benefit gains, whereas upper urinary tract model does not seem applicable to patients from Asia because it has suboptimal accuracy.
doi:10.1371/journal.pone.0100491
PMCID: PMC4061079  PMID: 24937260
2.  Heredity and cardiometabolic risk: naturally occurring polymorphisms in the human neuropeptide Y2 receptor promoter disrupt multiple transcriptional response motifs 
Journal of hypertension  2013;31(1):123-133.
Objectives
The neuropeptide Y2 G-protein-coupled receptor (NPY2R) relays signals from PYY or neuropeptide Y toward satiety and control of body mass. Targeted ablation of the NPY2R locus in mice yields obesity, and studies of NPY2R promoter genetic variation in more than 10 000 human participants indicate its involvement in control of obesity and BMI. Here we searched for genetic variation across the human NPY2R locus and probed its functional effects, especially in the proximal promoter.
Methods and results
Twin pair studies indicated substantial heritability for multiple cardiometabolic traits, including BMI, SBP, DBP, and PYY, an endogenous agonist at NPY2R. Systematic polymorphism discovery by resequencing across NPY2R uncovered 21 genetic variants, 10 of which were common [minor allele frequency (MAF) >5%], creating one to two linkage disequilibrium blocks in multiple biogeographic ancestries. In vivo, NPY2R haplotypes were associated with both BMI (P =3.75E–04) and PYY (P =4.01E–06). Computational approaches revealed that proximal promoter variants G-1606A, C-599T, and A-224G disrupt predicted IRF1 (A>G), FOXI1 (T>C), and SNAI1 (A>G) response elements. In neuroendocrine cells transfected with NPY2R promoter/luciferase reporter plasmids, all three variants and their resulting haplotypes influenced transcription (G-1606A, P <2.97E–06; C-599T, P <1.17E–06; A-224G, P <2.04E–06), and transcription was differentially augmented or impaired by coexpression of either the cognate full-length transcription factors or their specific siRNAs at each site. Endogenous expression of transcripts for NPY2R, IRF1, and SNAI1 was documented in neuroendocrine cells, and the NPY2R mRNA was differentially expressed in two neuroendocrine tissues (adrenal gland, brainstem) of a rodent model of hypertension and the metabolic syndrome, the spontaneously hypertensive rat.
Conclusion
We conclude that common genetic variation in the proximal NPY2R promoter influences transcription factor binding so as to alter gene expression in neuroendocrine cells, and consequently cardiometabolic traits in humans. These results unveil a novel control point, whereby cis-acting genetic variation contributes to control of complex cardiometabolic traits, and point to new transcriptional strategies for intervention into neuropeptide actions and their cardiometabolic consequences.
doi:10.1097/HJH.0b013e32835b053d
PMCID: PMC3615981  PMID: 23149563
autonomic; genetics; hypertension; nervous system; obesity
3.  Genes and environment: novel, functional polymorphism in the human cathepsin L (CTSL1) promoter disrupts a xenobiotic response element (XRE) to alter transcription and blood pressure 
Journal of hypertension  2012;30(10):1961-1969.
Background
Cathepsin L (CTSL1) catalyzes the formation of peptides that influence blood pressure (BP). Naturally occurring genetic variation or targeted ablation of the Ctsl1 locus in mice yield cardiovascular pathology. Here, we searched for genetic variation across the human CTSL1 locus and probed its functional effects, especially in the proximal promoter.
Methods and results
Systematic polymorphism discovery by re-sequencing across CTSL1 in 81 patients uncovered 38 genetic variants, five of which were relatively common (MAF >5%), creating a single linkage disequilibrium block in multiple biogeographic ancestries. One of these five common variants lay in a functional domain of the gene: promoter C-171A (rs3118869), which disrupts a predicted xenobiotic response element (XRE; match C>A). In transfected CTSL1 promoter/luciferase reporter plasmids, C-171A allele influenced transcription (C>A, P = 3.36E-6), and transcription was also augmented by co-exposure to the aryl hydrocarbon receptor (AHR) complex (AHR:ARNT) in the presence of their ligand dioxin (P = 6.81E-8); allele (C vs. A) and AHR:ARNT/dioxin stimulus interacted to control gene expression (interaction P = 0.033). Endogenous Ctsl1, Ahr, and Arnt transcripts were present in chromaffin cells. Promoter functional C-171A genotype also predicted hypertension (P = 1.0E–3), SBP (P = 4.0E–4), and DBP (P = 3.0E–3), in an additive pattern for diploid genotypes (A/A > C/A > C/C) in 868 patients, and the results were extended by validation analysis into an independent population sample of 986 patients.
Conclusion
We conclude that common genetic variation in the proximal CTSL1 promoter, especially at position C-171A, is functional in cells, and alters transcription so as to explain the association of CTSL1 with BP in vivo. At the XRE, endogenous genetic variation plus exogenous aryl hydrocarbon stimulation interact to control CTSL1 gene expression. These results unveil a novel control point whereby heredity and environment can intersect to control a complex trait, and point to new transcriptional strategies for intervention into transmitter biosynthesis and its cardiovascular consequences.
doi:10.1097/HJH.0b013e328356b86a
PMCID: PMC3478326  PMID: 22871890
autonomic; genetics; hypertension; nervous system
4.  Connective Tissue Growth Factor (CTGF) Expression Modulates Response to High Glucose 
PLoS ONE  2013;8(8):e70441.
Connective tissue growth factor (CTGF) is an important mediator of fibrosis; emerging evidence link changes in plasma and urinary CTGF levels to diabetic kidney disease. To further ascertain the role of CTGF in responses to high glucose, we assessed the consequence of 4 months of streptozotocin-induced diabetes in wild type (+/+) and CTGF heterozygous (+/−) mice. Subsequently, we studied the influence of glucose on gene expression and protein in mice embryonic fibroblasts (MEF) cells derived from wildtype and heterozygous mice. At study initiation, plasma glucose, creatinine, triglyceride and cholesterol levels were similar between non-diabetic CTGF+/+ and CTGF+/− mice. In the diabetic state, plasma glucose levels were increased in CTGF+/+ and CTGF+/− mice (28.2 3.3 mmol/L vs 27.0 3.1 mmol/L), plasma triglyceride levels were lower in CTGF+/− mice than in CTGF+/+ (0.7 0.2 mmol/L vs 0.5 0.1 mmol/L, p<0.05), but cholesterol was essentially unchanged in both groups. Plasma creatinine was higher in diabetic CTGF+/+ group (11.7±1.2 vs 7.9±0.6 µmol/L p<0.01), while urinary albumin excretion and mesangial expansion were reduced in diabetic CTGF+/− animals. Cortices from diabetic mice (both CTGF +/+ and CTGF +/−) manifested higher expression of CTGF and thrombospondin 1 (TSP1). Expression of nephrin was reduced in CTGF +/+ animals; this reduction was attenuated in CTGF+/− group. In cultured MEF from CTGF+/+ mice, glucose (25 mM) increased expression of pro-collagens 1, IV and XVIII as well as fibronectin and thrombospondin 1 (TSP1). In contrast, activation of these genes by high glucose was attenuated in CTGF+/− MEF. We conclude that induction of Ctgf mediates expression of extracellular matrix proteins in diabetic kidney. Thus, genetic variability in CTGF expression directly modulates the severity of diabetic nephropathy.
doi:10.1371/journal.pone.0070441
PMCID: PMC3741286  PMID: 23950936
5.  Mefloquine Exposure Induces Cell Cycle Delay and Reveals Stage-Specific Expression of the pfmdr1 Gene 
Drug-resistant Plasmodium falciparum malaria is a major public health problem. An elevated pfmdr1 gene copy number (CN) is known to decrease parasite sensitivity to the commonly used antimalarial mefloquine (MFQ). To understand the relationship between pfmdr1 CN and mefloquine resistance, we evaluated pfmdr1 transcript levels in three P. falciparum strains with different CNs in the presence and absence of MFQ. Parasite strains with multiple pfmdr1 gene copies exhibited higher relative transcript levels than single-copy parasites, and MFQ induced pfmdr1 expression above the levels without treatment in all three strains evaluated. Concomitant morphology analyses of the sampled cultures revealed that MFQ treatment of synchronized ring-stage parasites induced a delay in parasite maturation through the intraerythrocytic cycle. pfmdr1 expression peaks in the ring stage, and MFQ could be causing increased transcription by delaying parasite maturation. However, pretreatment with mefloquine did not affect the artemisinin in vitro half-maximal inhibitory concentration (IC50). These results suggest that MFQ-induced increases in pfmdr1 expression are the direct result of the maturation delay at the ring stage but that this change in expression does not affect the antimalarial activity of artemisinin.
doi:10.1128/AAC.01006-12
PMCID: PMC3553685  PMID: 23208721
6.  Reduced Alpha-Lipoic Acid Synthase Gene Expression Exacerbates Atherosclerosis in Diabetic Apolipoprotein E–Deficient Mice 
Atherosclerosis  2012;223(1):137-143.
Objectives
To study the effects of reduced lipoic acid gene expression on diabetic atherosclerosis in apolipoprotein E null mice (Apoe−/−).
Methods and Results
Heterozygous lipoic acid synthase gene knockout mice (Lias+/−) crossed with Apoe−/− mice were used to evaluate the diabetic effect induced by streptozotocin on atherosclerosis in the aortic sinus of the heart. While diabetes markedly increased atherosclerotic plaque size in Apoe−/− mice, a small but significant effect of reduced expression of lipoic acid gene was observed in diabetic Lias+/−Apoe−/− mice. In the aortic lesion area, the Lias+/−Apoe−/− mice exhibited significantly increased macrophage accumulation and cellular apoptosis than diabetic Lias+/+Apoe−/− littermates. Plasma glucose, cholesterol, and interleukin-6 were also higher. These abnormalities were accompanied with increased oxidative stress including a decreased ratio of reduced glutathione/oxidized glutathione in erythrocytes, increased systemic lipid peroxidation, and increased Gpx1 and MCP1 gene expression in the aorta.
Conclusions
Decreased endogenous lipoic acid gene expression plays a role in development of diabetic atherosclerosis. These findings extend our understanding of the role of antioxidant in diabetic atherosclerosis.
doi:10.1016/j.atherosclerosis.2012.04.025
PMCID: PMC3389304  PMID: 22658261
lipoic acid; Lias mouse model; atherosclerosis; diabetes; and apolipoprotein E null mice
7.  Pro- and anti-atherogenic effects of a dominant negative P465L mutation of PPARγ in apolipoprotein E-null mice 
Objective
The dominant-negative mutation, P467L, in Peroxisome Proliferator Activated Receptor gamma (PPARγ) affects adipose tissue distribution, insulin sensitivity and blood pressure in heterozygous humans. We hypothesized that the equivalent mutation, PPARγ-P465L, in mice will worsen atherosclerosis.
Methods and Results
ApolipoproteinE-null mice with and without PPARγ-P465L mutation were bred in 129S6 inbred genetic background. Mild hypertension and lipodystrophy of PPARγ-P465L persisted in the apoE-null background. Glucose homeostasis was normal, but plasma adiponectin was significantly lower and resistin was higher in PPARγ-P465L mice. Plasma cholesterol and lipoprotein distribution were not different, but plasma triglycerides tended to be reduced. Surprisingly, there were no overall changes in the atherosclerotic plaque size or composition. PPARγ-P465L macrophages had a small decrease in CD-36 mRNA and a small yet significant reduction in VLDL uptake in culture. In unloaded apoE-null macrophages with PPARγ-P465L, cholesterol uptake was reduced while apoAI-mediated efflux was increased. However, when cells were cholesterol loaded in presence of acetylated LDL, no genotype difference in uptake or efflux was apparent. A reduction of VCAM1 expression in aorta suggests a relatively anti-atherogenic vascular environment in mice with PPARγ-P465L.
Conclusions
Small, competing pro- and anti-atherogenic effects of PPARγ-P465L mutation result in unchanged plaque development in apoE-deficient mice.
doi:10.1161/ATVBAHA.112.248682
PMCID: PMC3389794  PMID: 22539598
mouse model; macrophage; bone marrow transfer; PPAR gamma; atherosclerosis
8.  Aldosterone deficiency and mineralocorticoid receptor antagonism prevent angiotensin II-induced cardiac, renal, and vascular injury 
Kidney international  2012;82(6):643-651.
Angiotensin II causes cardiovascular injury in part by aldosterone-induced mineralocorticoid receptor activation, and it can also activate the mineralocorticoid receptor in the absence of aldosterone in vitro. Here we tested whether endogenous aldosterone contributes to angiotensin II/salt-induced cardiac, vascular, and renal injury by the mineralocorticoid receptor. Aldosterone synthase knockout mice and wild type littermates were treated with angiotensin II or vehicle plus the mineralocorticoid receptor antagonist spironolactone or regular diet while drinking 0.9- saline. Angiotensin II/salt caused hypertension in both the knockout and wild type mice; an effect significantly blunted in the knockout mice. Either genetic aldosterone deficiency or mineralocorticoid receptor antagonism reduced cardiac hypertrophy, aortic remodeling, and albuminuria, as well as cardiac, aortic, and renal plasminogen activator inhibitor-1 mRNA expression during angiotensin II treatment. Mineralocorticoid receptor antagonism reduced angiotensin II/salt-induced glomerular hypertrophy, but aldosterone deficiency did not. Combined mineralocorticoid receptor antagonism and aldosterone deficiency reduced blood urea nitrogen and restored nephrin immunoreactivity. Angiotensin II/salt also promoted glomerular injury through the mineralocorticoid receptor in the absence of aldosterone. Thus, mineralocorticoid antagonism may have protective effects in the kidney beyond aldosterone synthase inhibition.
doi:10.1038/ki.2012.170
PMCID: PMC3434275  PMID: 22622494
9.  Human Thrombomodulin Knockin Mice Reveal Differential Effects of Human Thrombomodulin on Thrombosis and Atherosclerosis 
Objective
We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo.
Methods and Results
Knockin mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knockin mice exhibited decreased protein C activation in the aorta (P < 0.01) and lung (P < 0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knockin mice compared with wild-type mice (P < 0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knockin mice (12 ± 3 minutes) than wild-type mice (31 ± 6 minutes; P < 0.05). No differences in serum cytokine levels were detected between knockin and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knockin mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis.
Conclusion
Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This “humanized” animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.
doi:10.1161/ATVBAHA.111.236828
PMCID: PMC3202707  PMID: 21885846
thrombomodulin; protein C; inflammation; atherosclerosis; thrombosis
10.  Apolipoprotein E4 Exaggerates Diabetic Dyslipidemia and Atherosclerosis in Mice Lacking the LDL Receptor 
Diabetes  2011;60(9):2285-2294.
OBJECTIVE
We investigated the differential roles of apolipoprotein E (apoE) isoforms in modulating diabetic dyslipidemia—a potential cause of the increased cardiovascular disease risk of patients with diabetes.
RESEARCH DESIGN AND METHODS
Diabetes was induced using streptozotocin (STZ) in human apoE3 (E3) or human apoE4 (E4) mice deficient in the LDL receptor (LDLR−/−).
RESULTS
Diabetic E3LDLR−/− and E4LDLR−/− mice have indistinguishable levels of plasma glucose and insulin. Despite this, diabetes increased VLDL triglycerides and LDL cholesterol in E4LDLR−/− mice twice as much as in E3LDLR−/− mice. Diabetic E4LDLR−/− mice had similar lipoprotein fractional catabolic rates compared with diabetic E3LDLR−/− mice but had larger hepatic fat stores and increased VLDL secretion. Diabetic E4LDLR−/− mice demonstrated a decreased reliance on lipid as an energy source based on indirect calorimetry. Lower phosphorylated acetyl-CoA carboxylase content and higher gene expression of fatty acid synthase in the liver indicated reduced fatty acid oxidation and increased fatty acid synthesis. E4LDLR−/− primary hepatocytes cultured in high glucose accumulated more intracellular lipid than E3LDLR−/− hepatocytes concomitant with a 60% reduction in fatty acid oxidation. Finally, the exaggerated dyslipidemia in diabetic E4LDLR−/− mice was accompanied by a dramatic increase in atherosclerosis.
CONCLUSIONS
ApoE4 causes severe dyslipidemia and atherosclerosis independent of its interaction with LDLR in a model of STZ-induced diabetes. ApoE4-expressing livers have reduced fatty acid oxidation, which contributes to the accumulation of tissue and plasma lipids.
doi:10.2337/db11-0466
PMCID: PMC3161311  PMID: 21810592
11.  Percutaneous Radiofrequency Thermocoagulation Under Fluoroscopic Image-Guidance for Idiopathic Trigeminal Neuralgia 
Objective
We retrospectively investigated the long-term results of percutaneous radiofrequency thermocoagulation (RFT) using fluoroscopic image-guidance for treatment of trigeminal neuralgia.
Methods
A total of 38 patients diagnosed and treated with RFT as an idiopathic trigeminal neuralgia were investigated. To minimize the risks related to conventional technique based on cutaneous landmarks, and to eliminate the need to frequent reposition of cannula, we adopted a technique of image-guided fluoroscopic cannulation of the foramen ovale. To minimize sensory complication following thermal lesion, our target response was a generation of a lesion with mild to moderate hypalgesia rather than dense hypalgesia.
Results
The immediate pain-relief was achieved in all patients underwent RFT. With mean duration of follow-up of 38.2 months (range,12-72), 11 (28.9%) experienced recurrence of pain. The mean timing of recurrence was 26.1 months (range,12-46). A 42.7% recurrence rate was estimated by Kaplan-Meier analysis for the 38 patients at 46 months; 20.2% within 2 years, 29.1% within 3 years. In the long-term, 27 patients (71%) and 6 patients (15.8%) showed Barrow Neurological Institute (BNI) score I and BNI score II responses. 3 (7.9%) patients was assessed as BNI score III, 2 patients (5.3%) showed BNI score IV response. As a complication, troublesome dysesthesia occurred in 3 of 38 patients (7.9%), however, there was no permanent cranial nerve palsy or morbidity.
Conclusion
These results indicates that RFT under fluoroscopic image-guided cannulation of foramen ovale is a safe, effective, and reliable means of treating trigeminal neuralgia.
doi:10.3340/jkns.2011.50.5.446
PMCID: PMC3259465  PMID: 22259692
Fluoroscopy; Foramen ovale; Radiofrequency; Thermocoagulation; Trigeminal neuralgia
12.  Role of Reactive Oxygen Species in Hyper-Adrenergic Hypertension: Biochemical, Physiological, and Pharmacological Evidence from Targeted Ablation of the Chromogranin A (Chga) Gene 
Background
Oxidative stress, an excessive production of reactive oxygen species (ROS) outstripping antioxidant defense mechanisms, occurs in cardiovascular pathologies including hypertension. Here, we used biochemical, physiological, and pharmacological approaches to explore the role of derangements of catecholamines, ROS, and NO• in the development of a hyper-adrenergic model of hereditary hypertension: targeted ablation (knockout, KO) of chromogranin A (Chga) in the mouse.
Methods and Results
Homozygous (−/−) Chga gene knockout (KO) mice were compared to wild-type (WT, +/+) controls. In the KO mouse, elevations of systolic and diastolic BP were accompanied by not only elevated catecholamine (norepinephrine and epinephrine) concentrations, but also increased ROS (H2O2) and isoprostane (an index of lipid peroxidation), as well as depletion of NO•. Renal transcript analyses implicated changes in Nox1/2, Xo/Xdh, and Sod1,2 mRNAs in ROS elevation by the KO state. KO alterations in BP, catecholamines, H2O2, isoprostane, and NO• could be abrogated or even normalized (rescued) by either sympathetic outflow inhibition (with clonidine) or NADPH oxidase inhibition (with apocynin). In cultured renal podocytes, H2O2 production was substantially augmented by epinephrine (likely through b2-adrenergic receptors) and modestly diminished by norepinephrine (likely through a1-adrenergic receptors).
Conclusions
ROS seem to play a necessary role in the development of hyper-adrenergic hypertension in this model, in a process mechanistically linking elevated BP with catecholamine excess, renal transcriptional responses, ROS elevation, lipid peroxidation, and NO• depletion. Some of the changes seem to be dependent on transcription, while others are immediate. The cycle could be disrupted by inhibition of either sympathetic outflow or NADPH oxidase. Since common genetic variation at the human CHGA locus alters BP, the results have implications for antihypertensive treatment as well as prevention of target-organ consequences of the disease. The results document novel pathophysiological links between the adrenergic system andc oxidative stress, and suggest new strategies to probe the role and actions of ROS within this setting.
doi:10.1161/CIRCGENETICS.109.924050
PMCID: PMC3052689  PMID: 20729505
Chromogranin A; hypertension; reactive oxygen species; nitric oxide
13.  Genetic reduction of lipoic acid synthase expression modestly increases atherosclerosis in male, but not in female, apolipoprotein E deficient mice 
Atherosclerosis  2010;211(2):424-430.
Objectives
To evaluate the effects of a genetic reduction of Lias gene expression on atherosclerosis development.
Methods and Results
Heterozygous knockout mice for the lipoid acid synthase gene (Lias+/−) were crossed with apolipoprotein E–deficient (ApoE−/−) mice, and the plaque size in aortic sinuses of Lias+/− ApoE−/− mice was evaluated at 6 months of age. Lesions at the aortic sinus in Lias+/− ApoE−/− males were significantly larger (1.5X) than those in Lias+/+ ApoE−/− littermate males. The lesion size was inversely correlated with an increased erythrocyte reduced glutathione/ oxidized glutathione (GSH/GSSH) ratio, an systemic index of body redox balance. Lias+/− ApoE−/− males also had significantly increased plasma cholesterol and reduced pyruvate dehydrogenase complex activity in the liver. Significant reductions in the expression of genes for antioxidant enzymes, including superoxide dismutase 1 (SOD1) and SOD2, were observed in aortas of Lias+/− ApoE−/− males. Female Lias+/− ApoE−/− also exhibited changes in these parameters, parallel to those observed in males. However, the Lias gene effects for the majority of these factors, including atherosclerotic lesion size, were not significant in females.
Conclusions
Our data provide evidence that Lias deficiency enhances atherosclerosis in male mice, at least in part due to reduce antioxidant capacity. The notable absence of such effects in females leaves open the possibility of a gender-specific protection mechanism.
doi:10.1016/j.atherosclerosis.2010.03.009
PMCID: PMC2914155  PMID: 20347443
antioxidant; lipoic acid; Lias mouse model; atherosclerosis; apolipoprotein E null mice
14.  A Mouse Strain Where Basal Connective Tissue Growth Factor Gene Expression Can Be Switched from Low to High 
PLoS ONE  2010;5(9):e12909.
Connective tissue growth factor (CTGF) is a signaling molecule that primarily functions in extracellular matrix maintenance and repair. Increased Ctgf expression is associated with fibrosis in chronic organ injury. Studying the role of CTGF in fibrotic disease in vivo, however, has been hampered by perinatal lethality of the Ctgf null mice as well as the limited scope of previous mouse models of Ctgf overproduction. Here, we devised a new approach and engineered a single mutant mouse strain where the endogenous Ctgf-3′ untranslated region (3′UTR) was replaced with a cassette containing two 3′UTR sequences arranged in tandem. The modified Ctgf allele uses a 3′UTR from the mouse FBJ osteosarcoma oncogene (c-Fos) and produces an unstable mRNA, resulting in 60% of normal Ctgf expression (Lo allele). Upon Cre-expression, excision of the c-Fos-3′UTR creates a transcript utilizing the more stable bovine growth hormone (bGH) 3′UTR, resulting in increased Ctgf expression (Hi allele). Using the Ctgf Lo and Hi mutants, and crosses to a Ctgf knockout or Cre-expressing mice, we have generated a series of strains with a 30-fold range of Ctgf expression. Mice with the lowest Ctgf expression, 30% of normal, appear healthy, while a global nine-fold overexpression of Ctgf causes abnormalities, including developmental delay and craniofacial defects, and embryonic death at E10-12. Overexpression of Ctgf by tamoxifen-inducible Cre in the postnatal life, on the other hand, is compatible with life. The Ctgf Lo-Hi mutant mice should prove useful in further understanding the function of CTGF in fibrotic diseases. Additionally, this method can be used for the production of mouse lines with quantitative variations in other genes, particularly with genes that are broadly expressed, have distinct functions in different tissues, or where altered gene expression is not compatible with normal development.
doi:10.1371/journal.pone.0012909
PMCID: PMC2943916  PMID: 20877562
15.  The Efficacy and Safety of Photoselective Vaporization of the Prostate with a Potassium-titanyl-phosphate Laser for Symptomatic Benign Prostatic Hyperplasia according to Prostate Size: 2-Year Surgical Outcomes 
Korean Journal of Urology  2010;51(5):330-336.
Purpose
We investigated 2-year follow-up outcomes of patients who underwent potassium-titanyl-phosphate (KTP)-photoselective vaporization of the prostate (PVP) laser therapy for symptomatic benign prostatic hyperplasia (BPH).
Materials and Methods
Of a total of 169 patients who underwent 80 W KTP-PVP by a single surgeon, we retrospectively analyzed the clinical data of 74 patients who completed 2 years of follow-up. The efficacy of the PVP was assessed at 1, 3, 6, 12, and 24 months postoperatively by use of the International Prostate Symptom Score (IPSS) and uroflowmetry with postvoid residual urine volume (PVR). Safety, including complications, was evaluated at each visit.
Results
Mean preoperative total prostate and transitional zone volumes were 42.3 ml (range, 34.0-59.0 ml) and 18.6 ml (range, 10.1-28.6 ml) respectively. According to both IPSS and uroflowmetry, compared with baseline, the improvement in each parameter was sustained significantly at both 1 and 2 years postoperatively (p<0.05). There were no serious intraoperative complications, such as massive hemorrhage requiring transfusion or transurethral resection syndrome. Transient gross hematuria occurred in 16 (21.6%) cases, urgency incontinence in 6 (8.1%) cases, bladder neck contracture (BNC) in 3 (4.1%) cases, and urethral stricture in 1 (2.7%) case. The cases of urethral stricture and BNC developed only in the group with a prostate size of less than 45 ml. No cases required reoperation due to re-growing prostatic tissue.
Conclusions
PVP seems to be a safe and effective procedure for the surgical treatment of symptomatic BPH. After PVP, the subjective and objective improvements in the micturition parameters were sustainable up to 2 years, with minimal complications.
doi:10.4111/kju.2010.51.5.330
PMCID: PMC2873887  PMID: 20495696
Laser therapy; Potassium titanylphosphate; Prostatic hyperplasia
16.  Glomerular type 1 angiotensin receptors augment kidney injury and inflammation in murine autoimmune nephritis 
Studies in humans and animal models indicate a key contribution of angiotensin II to the pathogenesis of glomerular diseases. To examine the role of type 1 angiotensin (AT1) receptors in glomerular inflammation associated with autoimmune disease, we generated MRL-Faslpr/lpr (lpr) mice lacking the major murine type 1 angiotensin receptor (AT1A); lpr mice develop a generalized autoimmune disease with glomerulonephritis that resembles SLE. Surprisingly, AT1A deficiency was not protective against disease but instead substantially accelerated mortality, proteinuria, and kidney pathology. Increased disease severity was not a direct effect of immune cells, since transplantation of AT1A-deficient bone marrow did not affect survival. Moreover, autoimmune injury in extrarenal tissues, including skin, heart, and joints, was unaffected by AT1A deficiency. In murine systems, there is a second type 1 angiotensin receptor isoform, AT1B, and its expression is especially prominent in the renal glomerulus within podocytes. Further, expression of renin was enhanced in kidneys of AT1A-deficient lpr mice, and they showed evidence of exaggerated AT1B receptor activation, including substantially increased podocyte injury and expression of inflammatory mediators. Administration of losartan, which blocks all type 1 angiotensin receptors, reduced markers of kidney disease, including proteinuria, glomerular pathology, and cytokine mRNA expression. Since AT1A-deficient lpr mice had low blood pressure, these findings suggest that activation of type 1 angiotensin receptors in the glomerulus is sufficient to accelerate renal injury and inflammation in the absence of hypertension.
doi:10.1172/JCI34862
PMCID: PMC2662542  PMID: 19287096
17.  Increased energy expenditure, dietary fat wasting and resistance to diet-induced obesity in mice lacking renin 
Cell metabolism  2007;6(6):506-512.
SUMMARY
An overactive renin angiotensin system is associated with obesity and the metabolic syndrome. However, mechanisms behind it are unclear. Cleaving angiotensinogen to angiotensin I by renin is a rate-limiting step of angiotensin II production, but renin is suggested to have angiotensin-independent effects. We generated mice lacking renin (Ren1c) using embryonic stem cells from C57BL/6 mouse, a strain prone to diet-induced obesity. Ren1c-/- mice are lean, insulin sensitive, and resistant to diet-induced obesity without changes in food intake and physical activity. The lean phenotype is likely due to a higher metabolic rate, and gastrointestinal loss of dietary fat. Most of the metabolic changes in Ren1c-/- mice were reversed by angiotensin II administration. These results support a role for angiotensin II in the pathogenesis of diet-induced obesity and insulin resistance.
doi:10.1016/j.cmet.2007.10.011
PMCID: PMC2174204  PMID: 18054319
18.  Intermittent pressure overload triggers hypertrophy-independent cardiac dysfunction and vascular rarefaction 
Journal of Clinical Investigation  2006;116(6):1547-1560.
For over a century, there has been intense debate as to the reason why some cardiac stresses are pathological and others are physiological. One long-standing theory is that physiological overloads such as exercise are intermittent, while pathological overloads such as hypertension are chronic. In this study, we hypothesized that the nature of the stress on the heart, rather than its duration, is the key determinant of the maladaptive phenotype. To test this, we applied intermittent pressure overload on the hearts of mice and tested the roles of duration and nature of the stress on the development of cardiac failure. Despite a mild hypertrophic response, preserved systolic function, and a favorable fetal gene expression profile, hearts exposed to intermittent pressure overload displayed pathological features. Importantly, intermittent pressure overload caused diastolic dysfunction, altered β-adrenergic receptor (βAR) function, and vascular rarefaction before the development of cardiac hypertrophy, which were largely normalized by preventing the recruitment of PI3K by βAR kinase 1 to ligand-activated receptors. Thus stress-induced activation of pathogenic signaling pathways, not the duration of stress or the hypertrophic growth per se, is the molecular trigger of cardiac dysfunction.
doi:10.1172/JCI25397
PMCID: PMC1464895  PMID: 16741575
19.  Senescence-associated phenotypes in Akita diabetic mice are enhanced by absence of bradykinin B2 receptors 
The Journal of Clinical Investigation  2006;116(5):1302-1309.
We have previously reported that genetically increased angiotensin-converting enzyme levels, or absence of the bradykinin B2 receptor, increase kidney damage in diabetic mice. We demonstrate here that this is part of a more general phenomenon — diabetes and, to a lesser degree, absence of the B2 receptor, independently but also largely additively when combined, enhance senescence-associated phenotypes in multiple tissues. Thus, at 12 months of age, indicators of senescence (alopecia, skin atrophy, kyphosis, osteoporosis, testicular atrophy, lipofuscin accumulation in renal proximal tubule and testicular Leydig cells, and apoptosis in the testis and intestine) are virtually absent in WT mice, detectable in B2 receptor–null mice, clearly apparent in mice diabetic because of a dominant mutation (Akita) in the Ins2 gene, and most obvious in Akita diabetic plus B2 receptor–null mice. Renal expression of several genes that encode proteins associated with senescence and/or apoptosis (TGF-β1, connective tissue growth factor, p53, α-synuclein, and forkhead box O1) increases in the same progression. Concomitant increases occur in 8-hydroxy-2′-deoxyguanosine, point mutations and deletions in kidney mitochondrial DNA, and thiobarbituric acid–reactive substances in plasma, together with decreases in the reduced form of glutathione in erythrocytes. Thus, absence of the bradykinin B2 receptor increases the oxidative stress, mitochondrial DNA damage, and many senescence-associated phenotypes already present in untreated Akita diabetic mice.
doi:10.1172/JCI26958
PMCID: PMC1430357  PMID: 16604193
20.  Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system 
Journal of Clinical Investigation  2005;115(4):1092-1099.
Angiotensin II, acting through type 1 angiotensin (AT1) receptors, has potent effects that alter renal excretory mechanisms. Control of sodium excretion by the kidney has been suggested to be the critical mechanism for blood pressure regulation by the renin-angiotensin system (RAS). However, since AT1 receptors are ubiquitously expressed, precisely dissecting their physiological actions in individual tissue compartments including the kidney with conventional pharmacological or gene targeting experiments has been difficult. Here, we used a cross-transplantation strategy and AT1A receptor–deficient mice to demonstrate distinct and virtually equivalent contributions of AT1 receptor actions in the kidney and in extrarenal tissues to determining the level of blood pressure. We demonstrate that regulation of blood pressure by extrarenal AT1A receptors cannot be explained by altered aldosterone generation, which suggests that AT1 receptor actions in systemic tissues such as the vascular and/or the central nervous systems make nonredundant contributions to blood pressure regulation. We also show that interruption of the AT1 receptor–mediated short-loop feedback in the kidney is not sufficient to explain the marked stimulation of renin production induced by global AT1 receptor deficiency or by receptor blockade. Instead, the renin response seems to be primarily determined by renal baroreceptor mechanisms triggered by reduced blood pressure. Thus, the regulation of blood pressure by the RAS is mediated by AT1 receptors both within and outside the kidney.
doi:10.1172/JCI200523378
PMCID: PMC1070417  PMID: 15841186
21.  Hypertension and abnormal fat distribution but not insulin resistance in mice with P465L PPARγ 
Journal of Clinical Investigation  2004;114(2):240-249.
Peroxisome proliferator–activated receptor γ (PPARγ), the molecular target of a class of insulin sensitizers, regulates adipocyte differentiation and lipid metabolism. A dominant negative P467L mutation in the ligand-binding domain of PPARγ in humans is associated with severe insulin resistance and hypertension. Homozygous mice with the equivalent P465L mutation die in utero. Heterozygous mice grow normally and have normal total adipose tissue weight. However, they have reduced interscapular brown adipose tissue and intra-abdominal fat mass, and increased extra-abdominal subcutaneous fat, compared with wild-type mice. They have normal plasma glucose levels and insulin sensitivity, and increased glucose tolerance. However, during high-fat feeding, their plasma insulin levels are mildly elevated in association with a significant increase in pancreatic islet mass. They are hypertensive, and expression of the angiotensinogen gene is increased in their subcutaneous adipose tissues. The effects of P465L on blood pressure, fat distribution, and insulin sensitivity are the same in both male and female mice regardless of diet and age. Thus the P465L mutation alone is sufficient to cause abnormal fat distribution and hypertension but not insulin resistance in mice. These results provide genetic evidence for a critical role for PPARγ in blood pressure regulation that is not dependent on altered insulin sensitivity.
doi:10.1172/JCI200420964
PMCID: PMC449746  PMID: 15254591
22.  Interactions between endothelial nitric oxide synthase and sex hormones in vascular protection in mice 
The vasculoprotective effects of sex hormones, particularly estrogens, have been attributed to their ability to increase the bioavailability of nitric oxide through activation of endothelial nitric oxide synthase (eNOS). To dissect the relative contribution in vivo of eNOS, sex hormones, and their interaction in two complex vascular phenotypes, hypertension and atherosclerosis, we used mice doubly deficient in eNOS and apoE (nnee) or lacking only apoE (NNee). Females and males were gonadectomized at 1 month of age and implanted either with control pellets or pellets releasing 17β-estradiol (E2). Hormonally intact nnee mice have elevated blood pressure (BP) and increased atherosclerosis compared with NNee mice, but on removal of gonads, BP and atherosclerosis decreased significantly in nnee mice but not in NNee mice. Three months of treatment with exogenous E2 dramatically reduced atherosclerosis and significantly lowered BP in both NNee and nnee mice compared with animals treated with control pellets. Thus exogenous E2 has strong BP-lowering and atheroprotective effects in apoE-deficient mice, but eNOS is not essential for either effect. Endogenous sex hormones, on the other hand, cause significant damage to the vasculature in the absence of eNOS, but these effects are overridden by interactions between eNOS and sex hormones.
doi:10.1172/JCI14066
PMCID: PMC150875  PMID: 11854327
23.  The prostaglandin E2 EP1 receptor mediates pain perception and regulates blood pressure 
Journal of Clinical Investigation  2001;107(3):325-331.
The lipid mediator prostaglandin E2 (PGE2) has diverse biological activity in a variety of tissues. Four different receptor subtypes (EP1–4) mediate these wide-ranging effects. The EP-receptor subtypes differ in tissue distribution, ligand-binding affinity, and coupling to intracellular signaling pathways. To identify the physiological roles for one of these receptors, the EP1 receptor, we generated EP1-deficient (EP1–/–) mice using homologous recombination in embryonic stem cells derived from the DBA/1lacJ strain of mice. The EP1–/– mice are healthy and fertile, without any overt physical defects. However, their pain-sensitivity responses, tested in two acute prostaglandin-dependent models, were reduced by approximately 50%. This reduction in the perception of pain was virtually identical to that achieved through pharmacological inhibition of prostaglandin synthesis in wild-type mice using a cyclooxygenase inhibitor. In addition, systolic blood pressure is significantly reduced in EP1 receptor–deficient mice and accompanied by increased renin-angiotensin activity, especially in males, suggesting a role for this receptor in cardiovascular homeostasis. Thus, the EP1 receptor for PGE2 plays a direct role in mediating algesia and in regulation of blood pressure.
PMCID: PMC199184  PMID: 11160156
24.  Reproductive failure and reduced blood pressure in mice lacking the EP2 prostaglandin E2 receptor 
Journal of Clinical Investigation  1999;103(11):1539-1545.
Prostaglandins (PGs) are bioactive lipids that modulate a broad spectrum of biologic processes including reproduction and circulatory homeostasis. Although reproductive functions of mammals are influenced by PGs at numerous levels, including ovulation, fertilization, implantation, and decidualization, it is not clear which PGs are involved and whether a single mechanism affects all reproductive functions. Using mice deficient in 1 of 4 prostaglandin E2 (PGE2) receptors — specifically, the EP2 receptor — we show that Ep2–/– females are infertile secondary to failure of the released ovum to become fertilized in vivo. Ep2–/– ova could be fertilized in vitro, suggesting that in addition to previously defined roles, PGs may contribute to the microenvironment in which fertilization takes place. In addition to its effects on reproduction, PGE2 regulates regional blood flow in various vascular beds. However, its role in systemic blood pressure homeostasis is not clear. Mice deficient in the EP2 PGE2 receptor displayed resting systolic blood pressure that was significantly lower than in wild-type controls. Blood pressure increased in these animals when they were placed on a high-salt diet, suggesting that the EP2 receptor may be involved in sodium handling by the kidney. These studies demonstrate that PGE2, acting through the EP2 receptor, exerts potent regulatory effects on two major physiologic processes: blood pressure homeostasis and in vivo fertilization of the ovum.
PMCID: PMC408376  PMID: 10359563
25.  Reduced angiotensinogen expression attenuates renal interstitial fibrosis in obstructive nephropathy in mice 
A novel approach was employed to assess the contribution of the renin-angiotensin system (RAS) to obstructive nephropathy in neonatal mice having zero to four functional copies of the angiotensinogen gene (Agt). Two-day-old mice underwent unilateral ureteral obstruction (UUO) or sham operation; 28 days later, renal interstitial fibrosis and tubular atrophy were quantitated. In all Agt genotypes, UUO reduced ipsilateral renal mass and increased that of the opposite kidney. Renal interstitial collagen increased after UUO linearly with Agt expression, from a fractional area of 25% in zero-copy mice to 54% in two-copy mice. Renal expression of transforming growth factor-β1 was increased by ipsilateral UUO in mice expressing Agt, but not in zero-copy mice. However, the prevalence of atrophic tubules due to UUO did not vary with Agt expression. Blood pressure was not different in all groups, except for a reduction in sham zero-copy mice. We conclude that a functional RAS is not necessary for compensatory renal growth. This study demonstrates conclusively that angiotensin regulates at least 50% of the renal interstitial fibrotic response in obstructive nephropathy, an effect independent of systemic hemodynamic changes. Angiotensin-induced fibrosis likely is a mechanism common to the progression of many forms of renal disease.
PMCID: PMC407859  PMID: 9884332

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