Ken Sabanai

Spontaneous Myocardial Infarction in Mice Lacking All Nitric Oxide Synthase Isoforms

Background— The roles of nitric oxide (NO) in the cardiovascular system have been investigated extensively in pharmacological studies with NO synthase (NOS) inhibitors and in studies with NOS isoform–deficient mice. However, because of the nonspecificity of the NOS inhibitors and the compensatory interactions among NOS isoforms (nNOS, iNOS, and eNOS), the ultimate roles of endogenous NO derived from the entire NOS system are still poorly understood. In this study, we examined this point in mice deficient in all 3 NOS isoforms (triply n/i/eNOS−/− mice) that we have recently developed. Methods and Results— The triply n/i/eNOS−/− mice, but not singly eNOS−/− mice, exhibited markedly reduced survival, possibly due to spontaneous myocardial infarction accompanied by severe coronary arteriosclerotic lesions. Furthermore, the triply n/i/eNOS−/− mice manifested phenotypes that resembled metabolic syndrome in humans, including visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance. Importantly, activation of the renin-angiotensin system was noted in the triply n/i/eNOS−/− mice, and long-term oral treatment with an angiotensin II type 1 receptor blocker significantly suppressed coronary arteriosclerotic lesion formation and the occurrence of spontaneous myocardial infarction and improved the prognosis of those mice, along with ameliorating the metabolic abnormalities. Conclusions— These results provide the first direct evidence that genetic disruption of the whole NOS system causes spontaneous myocardial infarction associated with multiple cardiovascular risk factors of metabolic origin in mice in vivo through the angiotensin II type 1 receptor pathway, demonstrating the critical role of the endogenous NOS system in maintaining cardiovascular and metabolic homeostasis.

Statin Treatment Upregulates Vascular Neuronal Nitric Oxide Synthase Through Akt/NF-κB Pathway

Objective—Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are known to enhance vascular expression of endothelial (eNOS) and inducible nitric oxide synthase (iNOS). In this study, we examined whether statins also upregulate vascular expression of neuronal NOS (nNOS). Methods and Results—In cultured rat aortic smooth muscle cells, treatment with atorvastatin significantly increased nNOS expression, associated with activation of Akt and NF-&kgr;B. Inhibition of Akt by dominant-negative Akt suppressed atorvastatin-induced nNOS expression as well as Akt and NF-&kgr;B activation. Inhibition of NF-&kgr;B by dominant-negative I&kgr;B also attenuated atorvastatin-induced nNOS expression and NF-&kgr;B activation, but not Akt activation. We further examined whether atorvastatin also enhances nNOS expression in isolated mouse aorta, and if so, how much nNOS-derived NO accounts for atorvastatin-induced NOx production. In isolated aortas of wild-type mice, atorvastatin significantly increased all three NOS isoform expression and NOx production. In isolated aortas of doubly i/eNOS−/−, n/eNOS−/−, and n/iNOS−/− mice, which express only nNOS, iNOS, and eNOS, respectively, atorvastatin-induced NOx production was approximately 25%, 25%, and 50% to that of wild-type mice, respectively, suggesting that nNOS accounts for 25% of the atorvastatin-mediated NOx production. Conclusions—These results indicate that atorvastatin upregulates vascular nNOS through Akt/NF-&kgr;B pathway, demonstrating a novel nNOS-mediated vascular effect of the statin.

Genetic Disruption of All NO Synthase Isoforms Enhances BMD and Bone Turnover in Mice In Vivo: Involvement of the Renin-Angiotensin System†

Introduction: NO is synthesized by three different NO synthase (NOS) isoforms, including neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). The roles of NO in bone metabolism have been extensively investigated in pharmacological studies and in studies with NOS isoform–deficient mice. However, because of the nonspecificity of agents and compensation among the NOS isoforms, the ultimate roles of endogenous NO are still poorly understood. To address this point, we successfully generated mice in which all three NOS genes are completely disrupted. In this study, we examined whether bone metabolism is abnormal in those mice.

Severe dyslipidaemia, atherosclerosis, and sudden cardiac death in mice lacking all NO synthases fed a high-fat diet

AIMS The precise role of the nitric oxide synthase (NOS) system in lipid metabolism remains to be elucidated. We addressed this point in mice that we have recently developed and that lack all three NOS isoforms. METHODS AND RESULTS Wild-type (WT), singly, doubly, and triply NOS(-/-) mice were fed either a regular or high-cholesterol diet for 3-5 months. The high-cholesterol diet significantly increased serum low-density lipoprotein (LDL) cholesterol levels in all the genotypes when compared with the regular diet. Importantly, when compared with the WT genotype, the serum LDL cholesterol levels in the high-cholesterol diet were significantly and markedly elevated only in the triply NOS(-/-) genotype, but not in any singly or doubly NOS(-/-) genotypes, and this was associated with remarkable atherosclerosis and sudden cardiac death, which occurred mainly in the 4-5 months after the high-cholesterol diet. Finally, hepatic LDL receptor expression was markedly reduced only in the triply NOS(-/-) genotype, accounting for the diet-induced dyslipidaemia in the genotype. CONCLUSION These results provide the first direct evidence that complete disruption of all NOS genes causes severe dyslipidaemia, atherosclerosis, and sudden cardiac death in response to a high-fat diet in mice in vivo through the down-regulation of the hepatic LDL receptor, demonstrating the critical role of the whole endogenous NOS system in maintaining lipid homeostasis.

Spontaneous Differentiation of Human Mesenchymal Stem Cells on Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold

Introduction Mesenchymal stem cells (MSCs) have immunosuppressive activity and can differentiate into bone and cartilage; and thus seem ideal for treatment of rheumatoid arthritis (RA). Here, we investigated the osteogenesis and chondrogenesis potentials of MSCs seeded onto nano-fiber scaffolds (NFs) in vitro and possible use for the repair of RA-affected joints. Methods MSCs derived from healthy donors and patients with RA or osteoarthritis (OA) were seeded on poly-lactic-glycolic acid (PLGA) electrospun NFs and cultured in vitro. Results Healthy donor-derived MSCs seeded onto NFs stained positive with von Kossa at Day 14 post-stimulation for osteoblast differentiation. Similarly, MSCs stained positive with Safranin O at Day 14 post-stimulation for chondrocyte differentiation. Surprisingly, even cultured without any stimulation, MSCs expressed RUNX2 and SOX9 (master regulators of bone and cartilage differentiation) at Day 7. Moreover, MSCs stained positive for osteocalcin, a bone marker, and simultaneously also with Safranin O at Day 14. On Day 28, the cell morphology changed from a spindle-like to an osteocyte-like appearance with processes, along with the expression of dentin matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), suggesting possible differentiation of MSCs into osteocytes. Calcification was observed on Day 56. Expression of osteoblast and chondrocyte differentiation markers was also noted in MSCs derived from RA or OA patients seeded on NFs. Lactic acid present in NFs potentially induced MSC differentiation into osteoblasts. Conclusions Our PLGA scaffold NFs induced MSC differentiation into bone and cartilage. NFs induction process resembled the procedure of endochondral ossification. This finding indicates that the combination of MSCs and NFs is a promising therapeutic technique for the repair of RA or OA joints affected by bone and cartilage destruction.

Fluorescent Visualisation of Oxytocin in the Hypothalamo-neurohypophysial/-spinal Pathways After Chronic Inflammation in Oxytocin-Monomeric Red Fluorescent Protein 1 Transgenic Rats

Oxytocin (OXT) is a well‐known neurohypophysial hormone that is synthesised in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. The projection of magnocellular neurosecretory cells, which synthesise OXT and arginine vasopressin in the PVN and SON, to the posterior pituitary plays an essential role in mammalian labour and lactation through its peripheral action. However, previous studies have shown that parvocellular OXTergic cells in the PVN, which project to the medulla and spinal cord, are involved in various physiological functions (e.g. sensory modulation and autonomic). In the present study, we examined OXT expression in the PVN, SON and spinal cord after chronic inflammation from adjuvant arthritis (AA). We used transgenic rats that express OXT and the monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualise both the magnocellular and parvocellular OXTergic pathways. OXT‐mRFP1 fluorescence intensity was significantly increased in the PVN, SON, dorsal horn of the spinal cord and posterior pituitary in AA rats. The levels of OXT‐mRFP1 mRNA were significantly increased in the PVN and SON of AA rats. These results suggested that OXT was up‐regulated in both hypothalamic magnocellular neurosecretory cells and parvocellular cells by chronic inflammation, and also that OXT in the PVN‐spinal pathway may be involved in sensory modulation. OXT‐mRFP1 transgenic rats are a very useful model for visualising the OXTergic pathways from vesicles in a single cell to terminals in in vitro preparations.

Possible Involvement of the Rat Hypothalamo-Neurohypophysial/-Spinal Oxytocinergic Pathways in Acute Nociceptive Responses.

Oxytocin (OXT)‐containing neurosecretory cells in the parvocellular divisions of the paraventricular nucleus (PVN), which project to the medulla and spinal cord, are involved in various physiological functions, such as sensory modulation and autonomic processes. In the present study, we examined OXT expression in the hypothalamo‐spinal pathway, as well as the hypothalamo‐neurohypophysial system, which includes the magnocellular neurosecretory cells in the PVN and the supraoptic nucleus (SON), after s.c. injection of saline or formalin into the hindpaws of transgenic rats that express the OXT and monomeric red fluorescent protein 1 (mRFP1) fusion gene. (i) The numbers of OXT‐mRFP1 neurones that expressed Fos‐like immunoreactivity (‐IR) and OXT‐mRFP1 intensity were increased significantly in the magnocellular/parvocellular PVN and SON after s.c. injection of formalin. (ii) OXT‐mRFP1 neurones in the anterior parvocellular PVN, which may project to the dorsal horn of the spinal cord, were activated by s.c. injection of formalin, as indicated by a significant increases of Fos‐IR and mRFP1 intensity intensity. (iii) Formalin injection caused a significant transient increase in plasma OXT. (iv) OXT, mRFP1 and corticotrophin‐releasing hormone mRNAs in the PVN were significantly increased after s.c. injection of formalin. (v) An intrathecal injection of OXT‐saporin induced hypersensitivity in conscious rats. Taken together, these results suggest that the hypothalamo‐neurohypophysial/‐spinal OXTergic pathways may be involved in acute nociceptive responses in rats.

Effects of central administration of oxytocin-saporin cytotoxin on chronic inflammation and feeding/drinking behaviors in adjuvant arthritic rats

An increase in the arthritis index as a marker of chronic inflammation and suppression of food intake are observed in adjuvant arthritic (AA) rats. Our previous study demonstrated that central oxytocin (OXT)-ergic pathways were activated potently in AA rats. In the present study, OXT-saporin (SAP) cytotoxin, which chemically disrupts OXT signaling was administered centrally to determine whether central OXT may be involved in the developments of chronic inflammation and alteration of feeding/drinking behavior in AA rats. The arthritis index was significantly enhanced in AA rats pretreated with OXT-SAP administered intrathecally (i.t.) but not intracerebroventricularly (i.c.v.). Suppression of food intake was significantly attenuated transiently in AA rats pretreated with OXT-SAP administered i.c.v. but not i.t. Suppression of drinking behavior was not affected by i.t. or i.c.v. administration of OXT-SAP in AA rats. In addition, intraperitoneal administration of an OXT receptor antagonist did not change the arthritis index or feeding/drinking behavior in AA rats. These results suggest that central OXT-ergic pathways may be involved in anti-inflammation at the spinal level and suppression of feeding behavior at the forebrain-brainstem level in AA rats.

Systemic bone loss, impaired osteogenic activity and type I muscle fiber atrophy in mice with elastase-induced pulmonary emphysema: Establishment of a COPD-related osteoporosis mouse model

Although it is suggested that chronic obstructive pulmonary disease (COPD) and bone are related, almost all of the pathological mechanisms of COPD-related osteoporosis remain unknown. There is a mouse model showing a deterioration of bone quality after cigarette smoke exposure; however, in smoking exposure models, various factors exist that affect bone metabolism, such as smoking and body weight loss (muscle and fat mass loss). We considered it appropriate to use an elastase-induced emphysema model to exclude factors influencing bone metabolism and to investigate the influence of pulmonary emphysema on bone metabolism. The purpose of this study was to establish a COPD/emphysema-related osteoporosis mouse model by using the elastase-induced emphysema model. The lumbar vertebrae and femurs/tibiae exhibited trabecular bone loss and impaired osteogenic activity in 24-week-old male elastase-induced emphysema model mice. In addition, the model mice showed atrophy of type I muscle fibers without atrophy of type II muscle fibers. We believe that the mice described in this experimental protocol will be accepted as a COPD/emphysema-related osteoporosis mouse model and contribute to further investigations.