Chul Han

Genetic Ablation of the Bmpr2 Gene in Pulmonary Endothelium Is Sufficient to Predispose to Pulmonary Arterial Hypertension

Background— Pulmonary arterial hypertension (PAH) is a rare but fatal lung disease of diverse origins. PAH is now further subclassified as idiopathic PAH, familial PAH, and associated PAH varieties. Heterozygous mutations in BMPR2 can be detected in 50% to 70% of patients with familial PAH and 10% to 40% of patients with idiopathic PAH. Although endothelial cells have been suspected as the cellular origin of PAH pathogenesis, no direct in vivo evidence has been clearly presented. The present study was designed to investigate whether endothelial Bmpr2 deletion can predispose to PAH. Methods and Results— The Bmpr2 gene was deleted in pulmonary endothelial cells using Bmpr2 conditional knockout mice and a novel endothelial Cre transgenic mouse line. Wide ranges of right ventricular systolic pressure were observed in mice with heterozygous (21.7 to 44.1 mm Hg; median, 23.7 mm Hg) and homozygous (20.7 to 56.3 mm Hg; median, 27 mm Hg) conditional deletion of Bmpr2 in pulmonary endothelial cells compared with control mice (19.9 to 26.7 mm Hg; median, 23 mm Hg) at 2 to 7 months of age. A subset of mice with right ventricular systolic pressure >30 mm Hg exhibited right ventricular hypertrophy and an increase in the number and wall thickness of muscularized distal pulmonary arteries. In the lungs of these mice with high right ventricular systolic pressure, the expression of proteins involved in the pathogenesis of PAH such as serotonin transporter and tenascin-C was elevated in distal arteries and had a high incidence of perivascular leukocyte infiltration and in situ thrombosis. Conclusions— Conditional heterozygous or homozygous Bmpr2 deletion in pulmonary endothelial cells predisposes mice to develop PAH.

Elevated plasma osteopontin levels in patients with hepatocellular carcinoma.

OBJECTIVES:Osteopontin (OPN) is a secreted glycoprotein, frequently associated with various tumors. We investigated the usefulness of plasma OPN level as a biomarker for hepatocellular carcinoma (HCC).METHODS:We determined plasma levels of OPN, α-fetoprotein (AFP), and prothrombin induced by vitamin K absence II (PIVKA II) in a group of 62 HCC patients, in 60 patients with chronic liver diseases, and in 60 healthy control individuals using a standardized ELISA kit. To determine the source of elevated plasma level of OPN, immunohistochemical analysis of 285 HCC samples on tissue microarray was performed.RESULTS:Plasma OPN levels in the HCC patients (median 954 ng/mL, range 168–5,742) were significantly higher (p-value < 0.001) than those patients with chronic liver diseases (381 ng/mL, 29–1,688) or of a healthy control group (155 ng/mL, 10–766). Within the HCC patient group, plasma OPN level increased significantly with advancing degree of Child-Pugh class and of tumor stage. Diagnostic sensitivity and specificity of OPN for HCC was 87% and 82%, respectively (cut-off value: 617.6 ng/mL). OPN had a greater area under curve value (0.898) than AFP (0.745) or PIVKA II (0.578), suggesting superior diagnostic accuracy of OPN. Immunohistochemistry of 285 samples of HCC showed that OPN was expressed in 92 of 285 tumors (32.3%). OPN expression was found in the malignant hepatocytes and cancer-infiltrating macrophages, not in the noncancerous hepatocytes or Kupffer cells.CONCLUSIONS:These data propose elevated plasma OPN levels as a potential biomarker for HCC.

Use of Complementary and Alternative Medicine among Korean Cancer Patients

Background : Complementary and alternative medicine (CAM) is now being increasingly used among cancer patients. The objectives of our study were to assess the prevalence, types, cost, subjective effects, and side effects of CAM use, reasons for CAM use, characteristics of CAM users compared to those of nonusers, and patients’ expectations of doctors regarding their CAM use among Korean cancer patients at a single cancer center. Methods : From April to August, 2003, we interviewed 186 cancer patients hospitalized in the Korea Cancer Center Hospital using a structured questionnaire, and analyzed the data. Results : 78.5% of experimental subjects (146 patients) had been treated with at least one type of CAM, in addition to conventional Western treatment, with a mean monthly cost of 1,380,000 Won/person (approximately, 1,100 U.S. dollars on July, 2004). The most prevalent types of CAM used by these patients included medicinal mushrooms (67.1%), herbs (54.1%), vegetable diets (50.6%), and ginseng (46.5%). The main reported reasons for the use of CAM in addition to conventional medicine were nutritional support (19.1%) and physical strengthening (17.8%). 5% of CAM users experienced side effects. The younger and more educated the patients were, the more likely they were to employ CAM. 66% of CAM users wanted to discuss CAM techniques with their doctors. Conclusion : More than two-thirds of cancer patients used various kinds of CAM, incurring considerable costs. Therefore, in order to help patients make informed decisions, medical society should be open to communication with patients. Not only the scientific aspects, but also the economic aspects of CAM usage should be examined more thoroughly, in order to ensure proper distribution of medical resources.

VEGF neutralization can prevent and normalize arteriovenous malformations in an animal model for hereditary hemorrhagic telangiectasia 2

Arteriovenous malformation (AVM) refers to a vascular anomaly where arteries and veins are directly connected through a complex, tangled web of abnormal AV fistulae without a normal capillary network. Hereditary hemorrhagic telangiectasia (HHT) types 1 and 2 arise from heterozygous mutations in endoglin (ENG) and activin receptor-like kinase 1 (ALK1), respectively. HHT patients possess AVMs in various organs, and telangiectases (small AVMs) along the mucocutaneous surface. Understanding why and how AVMs develop is crucial for developing therapies to inhibit the formation, growth, or maintenance of AVMs in HHT patients. Previously, we have shown that secondary factors such as wounding are required for Alk1-deficient vessels to develop skin AVMs. Here, we present evidences that AVMs establish from nascent arteries and veins rather than from remodeling of a preexistent capillary network in the wound-induced skin AVM model. We also show that VEGF can mimic the wound effect on skin AVM formation, and VEGF-neutralizing antibody can prevent skin AVM formation and ameliorate internal bleeding in Alk1-deficient adult mice. With topical applications at different stages of AVM development, we demonstrate that the VEGF blockade can prevent the formation of AVM and cease the progression of AVM development. Taken together, the presented experimental model is an invaluable system for precise molecular mechanism of action of VEGF blockades as well as for preclinical screening of drug candidates for epistaxis and gastrointestinal bleedings.

Addition of exogenous NAD+ prevents mefloquine-induced neuroaxonal and hair cell degeneration through reduction of caspase-3-mediated apoptosis in cochlear organotypic cultures.

Background Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects. Principal Findings In this study, we show that the coenzyme NAD+, known to play a critical role in maintaining the appropriate cellular redox environment, protects cochlear axons and sensory hair cells from mefloquine-induced degeneration in cultured rat cochleae. Mefloquine alone destroyed hair cells and nerve fiber axons in rat cochlear organotypics cultures in a dose-dependent manner, while treatment with NAD+ protected axons and hair cells from mefloquine-induced degeneration. Furthermore, cochlear organs treated with mefloquine showed increased oxidative stress marker levels, including superoxide and protein carbonyl, and increased apoptosis marker levels, including TUNEL-positive nuclei and caspases-3. Treatment with NAD+ reduced the levels of these oxidative stress and apoptosis markers. Conclusions/Significance Taken together, our findings suggest that that mefloquine disrupts the cellular redox environment and induces oxidative stress in cochlear hair cells and nerve fibers leading to caspases-3-mediated apoptosis of these structures. Exogenous NAD+ suppresses mefloquine-induced oxidative stress and prevents the degeneration of cochlear axons and sensory hair cells caused by mefloquine treatment.

Concordance of End-Tidal Carbon Dioxide and Arterial Carbon Dioxide in Severe Traumatic Brain injury

OBJECTIVE It is important for preventing hyperventilation so as to improve the outcomes of patients with severe traumatic brain injuries. End-tidal CO2 (Petco2) reflects arterial CO2 (Paco2), noninvasively and continuously. The aim of this study is to examine the concordance between Paco2 and Petco2 among adults presenting to the emergency department with severe traumatic brain injury and to elucidate the patient groups who had discordance. METHODS From July 1, 2006 to December 31, 2007, 77 patients who presented in the emergency department of the hospital after severe brain injury were enrolled. Glasgow Coma Scale, blood pressure, heart rate, respiration rate, body temperature, arterial blood gas variables, and serum lactate levels were measured under the mechanical ventilation support. Finally, Injury Severity Score and Abbreviated Injury Scale of head and chest were recorded. The concordance between Petco2 and Paco2 was analyzed using Bland-Altman plot. We defined that normal gap of Paco2 and Petco2 [P(a-et)co2] is -5 mm Hg to 5 mm Hg, and compared the normal and high P(a-et)co2 groups to find the factors affecting the P(a-et)co2. RESULTS Sixty-six patients after exception of 11 patients with nontraumatic brain injury were analyzed. Ten patients (15.2%) were below 30 mm Hg of Paco2. Pearsons correlation coefficient between Paco2 and Petco2 was 0.666 (p < 0.001) and the concordance between Paco2 and Petco2 was 77.3%. The patients with high P(a-et)co2 showed significantly higher Injury Severity Score, lower systolic blood pressure, lower arterial pH, lower base deficit, and higher serum lactate levels than patients with normal P(a-et)co2. Patients with severe chest trauma, hypotension, and metabolic acidosis increased the risk of the discordance between Paco2 and Petco2. Twenty-eight patients who had no hypotension, no metabolic acidosis, and no severe chest trauma showed concordance rate of 100.0% between Paco2 and Petco2. CONCLUSIONS Severe chest trauma and hemodynamic and tissue perfusion state should be considered when attempting to monitor the hyperventilation in severe brain injury patients using Petco2.

Health Effects of Long-Term Rapamycin Treatment: The Impact on Mouse Health of Enteric Rapamycin Treatment from Four Months of Age throughout Life

Rapamycin, an mTOR inhibitor, has been shown to extend lifespan in a range of model organisms. It has been reported to extend lifespan in multiple strains of mice, administered chronically or acutely early or late in life. The ability of rapamycin to extend health (healthspan) as opposed to life is less well documented. To assess the effects chronic rapamycin treatment on healthspan, enteric rapamycin was given to male and female C57BL/6J mice starting at 4 months of age and continued throughout life. Repeated, longitudinal assessments of health in individual animals were made starting at 16 months of age (=12 months of treatment) until death. A number of health parameters were improved (female grip strength, female body mass and reduced sleep fragmentation in both sexes), others showed no significant difference, while at least one (male rotarod performance) was negatively affected. Rapamycin treatment affected many measures of health in a highly sex-specific manner. While sex-specific phenotypic effects of rapamycin treatment have been widely reported, in this study we document sex differences in the direction of phenotypic change. Rapamycin-fed males and females were both significantly different from controls; however the differences were in the opposite direction in measures of body mass, percent fat and resting metabolic rate, a pattern not previously reported.

SMAD1 Deficiency in Either Endothelial or Smooth Muscle Cells Can Predispose Mice to Pulmonary Hypertension

A deficiency in bone morphogenetic protein receptor type 2 (BMPR2) signaling is a central contributor in the pathogenesis of pulmonary arterial hypertension (PAH). We have recently shown that endothelial-specific Bmpr2 deletion by a novel L1Cre line resulted in pulmonary hypertension. SMAD1 is one of the canonical signal transducers of the BMPR2 pathway, and its reduced activity has been shown to be associated with PAH. To determine whether SMAD1 is an important downstream mediator of BMPR2 signaling in the pathogenesis of PAH, we analyzed pulmonary hypertension phenotypes in Smad1-conditional knockout mice by deleting the Smad1 gene either in endothelial cells or in smooth muscle cells using L1Cre or Tagln-Cre mouse lines, respectively. A significant number of the L1Cre(+);Smad1 (14/35) and Tagln-Cre(+);Smad1 (4/33) mutant mice showed elevated pulmonary pressure, right ventricular hypertrophy, and a thickening of pulmonary arterioles. A pulmonary endothelial cell line in which the Bmpr2 gene deletion can be induced by 4-hydroxy tamoxifen was established. SMAD1 phosphorylation in Bmpr2-deficient cells was markedly reduced by BMP4 but unaffected by BMP7. The sensitivity of SMAD2 phosphorylation by transforming growth factor-&bgr;1 was enhanced in the Bmpr2-deficient cells, and the inhibitory effect of transforming growth factor-&bgr;1–mediated SMAD2 phosphorylation by BMP4 was impaired in the Bmpr2-deficient cells. Furthermore, transcript levels of several known transforming growth factor-&bgr; downstream genes implicated in pulmonary hypertension were elevated in the Bmpr2-deficient cells. Taken together, these data suggest that SMAD1 is a critical mediator of BMPR2 signaling pertinent to PAH, and that an impaired balance between BMP4 and transforming growth factor-&bgr;1 may account for the pathogenesis of PAH.

Maintaining good hearing: Calorie restriction, Sirt3, and glutathione

Reducing calorie intake extends the lifespan of a variety of experimental models and delays progression of age-related hearing loss (AHL). AHL is a common feature of aging and is characterized by age-related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. Sirtuins are a family of NAD(+)-dependent enzymes that regulate lifespan in lower organisms and have emerged as broad regulators of cellular fate. Our recent study indicated that mitochondrial Sirt3, a member of the sirtuin family, mediates the anti-aging effects of calorie restriction (CR) on AHL in mice. Interestingly, we also found that weight loss alone may not be sufficient for maintaining normal hearing. How does CR slow the progression of AHL through regulation of Sirt3? Here we review the evidence that during CR, Sirt3 slows the progression of AHL by promoting the glutathione-mediated mitochondrial antioxidant defense system in mice. A significant reduction in food consumption in ones daily life may not be a desirable and realistic option for most people. Therefore, identification/discovery of compounds that induce the activation of SIRT3 or glutathione reductase, or that increase mitochondrial glutathione levels has potential for maintaining good hearing through mimicking the anti-aging effects of CR in human inner ear cells.

The serine protease HtrA2/Omi cleaves Parkin and irreversibly inactivates its E3 ubiquitin ligase activity

The serine protease HtrA2 is important in regulating not only apoptosis but also cellular homeostasis. Recently, several lines of evidence suggest that HtrA2 may be intimately associated with Parkin; however, little is known about the functional relationships between HtrA2 and Parkin. Here we have shown that HtrA2 is co-localized with Parkin in the cytosol through the release of HtrA2 from the mitochondria upon cellular stresses. Moreover, endogenous levels of Parkin were significantly decreased in wild-type (HtrA2(+/+)) mouse embryonic fibroblasts (MEF) compared with those in HtrA2-knockout (HtrA2(-/-)) MEF under the same stress conditions. Using cleavage and binding assays, we have demonstrated that HtrA2 specifically binds to and directly cleaves the E3 ubiquitin (Ub) ligase Parkin. Interestingly, the HtrA2-mediated Parkin cleavage irreversibly disrupts Parkin-mediated synphilin-1 ubiquitination and autoubiquitination, indicating that HtrA2 may play a critical role in the Parkin-related pathway involved in the ubiquitin proteasome system.