Tetrahydrobiopterin Inhibits PDGF-stimulated Migration and Proliferation in Rat Aortic Smooth Muscle Cells via the Nitric Oxide Synthase-independent Pathway

Tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS) activity, is known to play important roles in modulating both NO and superoxide production during vascular diseases such as atherosclerosis. However, the role of BH4 in functions of vascular smooth muscle cells is not fully known. In this study, we tested the effects of BH4 and dihydrobiopterin (BH2), a BH4 precursor, on migration and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) in rat aortic smooth muscle cells (RASMCs). Cell migration and proliferation were measured using a Boyden chamber and a 5-bromo-2'-deoxyuridine incorporation assay, respectively, and these results were confirmed with an ex vivo aortic sprout assay. Cell viability was examined by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assays. BH4 and BH2 decreased PDGF-BB-induced cell migration and proliferation in a dose-dependent manner. The inhibition of cell migration and proliferation by BH4 and BH2 was not affected by pretreatment with N G-nitro-L-arginine methyl ester, a NOS inhibitor. Moreover, the sprout outgrowth formation of aortic rings induced by PDGF-BB was inhibited by BH4 and BH2. Cell viability was not inhibited by BH4 and BH2 treatment. The present results suggest that BH4 and BH2 may inhibit PDGF-stimulated RASMC migration and proliferation via the NOS-independent pathway. Therefore, BH4 and its derivative could be useful for the development of a candidate molecule with an NO-independent anti-atherosclerotic function.

Proteomic Analysis of Penile Protein Alterations in a Rat Model of Cavernous Nerve Injury

PURPOSE: Cavernous nerve resection (CNR) in rats is a standard model of animal experiments on erectile dysfunction (ED) that occurs after radical prostatectomy (RP). Injured cavernous nerves after surgery can cause fibrosis and apoptosis that lead to penile structural changes that may be accompanied by alterations of protein expression. This study aimed to analyze the changes in protein after CNR in Wistar Kyoto rats. MATERIALS AND METHODS: Using 8-week-old male Wistar Kyoto rats, sham and CNR operation under a microscope were performed. Two and 8 weeks after surgery, we applied 2-DE and MALDI-TOF/TOF (AB 4700) to identify differently expressed penile proteins after CNR. 2-DE gels were stained with silver nitrate and were analyzed with PDQuest. After in-gel digestion, peptide mass spectra were obtained by MALDI-TOF/TOF mass spectrometry in the positive ion reflector mode. The obtained data were screened with a rat database from both the NCBI and the Swiss-Prot/TrFMBL home page. RESULTS: The proteins that were changed more than 1.5-fold compared with the sham group were annexin A4 and pyruvate kinase (PK). Annexin A4 was increased by 1.75-fold after 2 weeks, whereas PK was decreased by 4.16 after 8 weeks. These results were confirmed by immunohistochemistry. CONCLUSIONS: Annexin A4 in the CNR group was increased, which may be related to emiocytosis during apoptosis. The decrease in PK of the CNR group is assumed to be related to a decrease in efficacy during glycolysis. Further study will be needed to elucidate the molecular pathophysiology of ED after cavernous nerve injury.

Olibanum Extract Inhibits Vascular Smooth Muscle Cell Migration and Proliferation in Response to Platelet-Derived Growth Factor

Olibanum (Boswellia serrata) has been shown to have anti-inflammatory, anti-arthritic and anti- cancer effects. This study determined the role of a water extract of olibanum in platelet-derived growth factor (PDGF)-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). PDGF-BB induced the migration and proliferation of RASMCs that were inhibited by olibanum extract in a dose-dependent manner. The PDGF-BB-increased phosphorylation of p38 mitogen-activated protein kinase (MAPK); the heat shock protein (Hsp) 27 was significantly inhibited by the olibanum extract. The effects of PDGF-BB-induced extracellular signal-regulated kinase1/2 was not altered by the olibanum extract. Treatment with olibanum extract inhibited PDGF-BB-stimulated sprout out growth of aortic rings. These results suggest that the water extract of olibanum inhibits PDGF-BB-stimulated migration and proliferation in RASMCs as well as sprout out growth, which may be mediated by the inhibition of the p38 MAPK and Hsp27 pathways.

p38 MAPK Participates in Muscle-Specific RING Finger 1-Mediated Atrophy in Cast-Immobilized Rat Gastrocnemius Muscle

Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.

Propofol Inhibits Platelet-derived Growth Factor-stimulated Migration in Rat Aortic Smooth Muscle Cells / 대한마취과학회지

BACKGROUND: Propofol is the extensively used general anesthetic-sedative agent.Although propofol is known to be involved in migration of various cells, migration response to it in vascular smooth muscle cells is not investigated. This study was carried out to determine the role of propofol in migration of rat aortic smooth muscle cells (RASMCs). METHODS: A7r5 RASMCs were used.Cell migration was examined by the analysis of 5 ng/ml of platelet-derived growth factor (PDGF)-induced RASMC response after treatment of cells with propofol (1-100micrometer) in the Boyden chamber.The activity of cofilin by propofol in RASMCs was measured by the Western blot analysis for the change of cofilin dephosphorylaton in cells treated with 10micrometer propofol for 5, 10, 15 and 20 min, for the effect of propofol (1, 10 and 100micrometer) on cofilin phosphorylation, and for the effects of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (2 mM; EGTA), Na3VO4 (200micrometer), and calyculin A (10 nM) on 10micrometer propofol-induced cofilin dephosphorylation. RESULTS: PDGF increased RASMC migration and this response was dose-dependently inhibited by treatment with propofol. Propofol attenuated the cofilin phosphorylation in RASMCs in a dose- and time-dependent manner.Propofol-induced dephosphorylation of cofilin in RASMCs was abolished by calyculin A, a protein phosphatase 2A inhibitor, but not by EGTA, a Ca2+ chelating agent, or Na3VO4, a protein tyrosine phosphatase inhibitor. CONCLUSIONS: The present results suggest that propofol induces the diminution of PDGF-stimulated RASMC migration and this response may be associated with dephosphorylation of cofilin mediated by the protein phosphatase 2A-dependent pathway.

Anti-tumor immunostimulatory effect of heat-killed tumor cells

As a part of our ongoing search for a safe and efficient anti-tumor vaccine, we attempted to determine whether the molecular nature of certain tumor antigens would influence immune responses against tumor cells. As compared with freeze-thawed or formaldehyde-fixed tumor antigens, heat-denatured tumor antigens elicited profound anti-tumor immune responses and greatly inhibited the growth of live tumor cells. The heat-denatured tumor antigens induced a substantial increase in the anti-tumor CTL response in the absence of any adjuvant material. This response appears to be initiated by strong activation of the antigen-presenting cells, which may recognize heat-denatured protein antigens. Upon recognition of the heat-denatured tumor antigens, macrophages and dendritic cells were found to acutely upregulate the expression of co-stimulatory molecules such as B7.2, as well as the secretion of inflammatory cytokines such as IL-12 and TNF-alpha. The results of this study indicate that heat-denatured tumor extracts might elicit protective anti-tumor adaptive immune responses and also raise the possibility that a safe and efficient adjuvant-free tumor vaccine might be developed in conjunction with a dendritic cell-based tumor vaccine.