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Lipid metabolic disorders play critical roles in atherogenesis. Traditionally, it has been suggested that reduced high density lipoprotein (HDL) levels might be an important morbidity indicator for cardiovascular diseases. Therefore, it has been argued that therapeutically raising HDL levels may reduce atherogenesis in patients with dyslipidemia. However, recent clinical trials to elevate serum HDL levels by pharmacologic approaches failed to demonstrate clinical efficacy. Thus, to investigate the functionality of HDL and to explore the possible clinical relevance as well as to define an effective indicator that can represent HDL function may provide another key and reference to disclose the clinical treatment of dyslipidemia. We analyzed the association between the data of dichlorofluorescein assay (assay the functionality of HDL), the effect of HDL on oxidized low density lipoprotein (oxLDL)-stimulated endothelial progenitor cells (EPCs) in vitro, levels of circulating EPCs, and ex vitro EPC colony forming units of each case, we defined the indicator (relative HDL index (RHDL index) = dichlorofluorescein assay result of each subject/dichlorofluorescein assay reading of our young healthy controls) that may represent functionality of HDL. HDL from healthy adults protected oxLDL-treated EPCs by modulating p38 mitogen-activated protein kinase and Rho activation and by promoting nitric oxide production. HDL from subject with RHDL index ≧2 also failed to restore the functionality of oxLDL-treated EPCs via cell-signaling pathways in vitro. The RHDL index significantly correlated with patients' circulating EPC number or EPC colony forming units ex vivo. In conclusions, we explored the RHDL index as a score to predict a patient's EPC functions in vivo and ex vitro. [ABSTRACT FROM AUTHOR]

Abstract: The Cilostazol for RESTenosis (CREST) clinical trial was initiated to evaluate the efficacy of cilostazol, an antiplatelet drug, in inhibiting restenosis after stent implantation in a native coronary artery as evaluated by quantitative coronary angiography. Preliminary results suggest that cilostazol reduces restenosis by 36% over standard therapy alone. Restenosis after coronary stenting is primarily attributed to neointimal formation. Cilostazol decreases the activity of phosphodiesterase type 3, leading to the accumulation of cyclic adenosine monophosphate, which initiates a cascade of events including upregulation of anti-oncogenes p53 and p21 and upregulation of hepatocyte growth factor (HGF). The increase in p53 protein blocks cell cycle progression and induces apoptosis in vascular smooth muscle cells (VSMCs), leading to an antiproliferative effect. Upregulation of local HGF stimulates rapid regeneration of endothelial cells, which inhibits neointimal formation via two mechanisms: inhibition of abnormal VSMC growth and improvement of endothelial function. These mechanisms may be responsible for the improvement in restenosis shown in the CREST trial and a number of other trials in patients who underwent percutaneous transluminal coronary angioplasty. These effects, in addition to antithrombotic and vasodilatory attributes of cilostazol, make it a potentially viable treatment option for preventing restenosis following coronary stenting. [Copyright &y& Elsevier]

Molecular therapy is emerging as a potential strategy for the treatment of various diseases for which few known effective therapies exist. One strategy for combating disease processes has been to target the transcriptional process. Two approaches have been used to accomplish this: the use of antisense complimentary to the mRNA of interest and the use of ribozymes, a unique class of RNA molecules that not only store information but also process catalytic activity. Ribozymes are known to catalytically cleave specific target RNA, leading to its degradation, whereas antisense molecules inhibit translation by binding to mRNA sequences on a stoichiometric basis. More recently, small interfering RNA has been shown to inhibit target gene expression. The application of oligonuclotide technology, such as antisense, to regulate the transcription of disease-related genes in vivo has important therapeutic potential. Transfection of cis-element double-stranded oligodeoxynucleotides has been reported as a powerful tool in a new class of anti-gene strategies for molecular therapy. [Copyright &y& Elsevier]

Potential role of hepatocyte growth factor in the maintenance of renal structure: Anti-apoptotic action of HGF on epithelial cells. Background. Mesangial cells (MC) are known to secrete various vasoactive substances that may control endothelial and epithelial cell growth. Therefore, the cell-cell interactions among these cells may be important in the control of renal function. However, the exact mechanisms of maintaining the cell-cell interactions are not yet understood. We have focused on the role of hepatocyte growth factor (HGF) in the regulation of cell-cell interactions, since HGF has many protective functions in the kidney. To investigate the role of HGF in renal injury, we examined (1 ) the effects of HGF on epithelial injury induced by serum deprivation, and (2 ) the role of local HGF production in the maintenance of renal structure. Methods. Apoptotic changes in epithelial cells were assessed by nuclear morphology and DNA fragmentation assay. Transfection of human HGF vector into epithelial cells was performed by a highly efficient viral-liposome method. The effects of secreted HGF on the growth of renal cells were examined using a co-culture system. Results. The addition of recombinant HGF (rHGF) stimulated the growth of rat and porcine epithelial cells. Moreover, the decrease in number of epithelial cells by serum deprivation was significantly attenuated by rHGF. Interestingly, apoptotic changes in epithelial cells induced by serum deprivation were also significantly attenuated by rHGF (P < 0.01). As a model of gene therapy, the effects of overexpression of human HGF gene in epithelial cells on apoptosis induced by serum deprivation were examined. Transfection of human HGF vector into epithelial cells also attenuated epithelial cell death induced by serum deprivation through the inhibition of apoptosis, accompanied by increased HGF production (P < 0.01). In addition, HGF also prevented endothelial injury induced by tumor necrosis factor-α and... [ABSTRACT FROM AUTHOR]

Examines the action of hepatocyte growth factor (HGF) as a local modulator in the kidney. Role in pathogenesis of renal disease; Importance of cell-cell interactions among renal cells in the control of renal function; Effects of HGF on the growth of endothelial cells.

Hepatocyte growth factor (HGF) is a heterodimeric protein and shows mitogenic and morphogenic activities toward a variety of epithelial cells. There has been no immunohistochemical evidences for naked DNA mediated transgene expression of HGF into central nervous system. We initially demonstrated a naked plasmid mediated expression of HGF into rat striatum. The immunofluorescence staining revealed that exogenous protein of human HGF was expressed at 7 days after plasmid injection (300 μg). Exogenous HGF was mainly expressed in reactive astrocytes according to dual-labeling staining of HGF and glial fibrillary acidic protein or S100. It is also demonstrated that c-met, specific receptor of HGF, was expressed in the injection site. Intensive expression of c-met was found in the site to which HGF encoded plasmid was injected. These evidences for the exogenous expression of HGF and its receptor c-met may implicate an application of naked plasmid mediated HGF for neuronal disease as well as the other neurotrophic factors. [Copyright &y& Elsevier]

A missense mutation (N1411) in Presenilin-2 (PS-2) gene is associated with early-onset familial Alzheimer''s disease. In this study, SK-N-SH human neuroblastoma cells were transfected with wild-type and mutant PS-2 gene to examine presenilin-2 effects on apoptosis. Serum deprivation resulted in enhanced apoptosis in mutant PS-2 comparing with wild-type PS-2. Similarly, mutant PS-2 induced lactate dehydrogenase release to greater extent than wild-type PS-2. Time course experiment demonstrated that the increase in caspase-3-like activity was more pronounced and accelerated in mutant PS-2, compared to wild-type PS-2. While a significant decrease in bcl-2, an anti-apoptotic molecule, occurred in the cells overexpressing mutant PS-2, no significant change was observed in bax, a pro-apoptotic molecule, as compared with the cells overexpressing wild-type PS-2. Our study demonstrated that mutant PS-2 induces apoptosis accompanied by increased caspase-3-like activity and decreased bcl-2 expression in neuronal cells after serum-deprivation. [Copyright &y& Elsevier]

Abstract: Behavioral and psychological problems are often observed in patients with dementia such as that associated with Alzheimer disease, and these noncognitive symptoms place an extremely heavy burden on the family and caregivers. Although it is well know that these symptoms often are triggered by infection of peripheral organs, the underlying mechanisms for these pathological conditions are still unclear. In this study, using an Alzheimer amyloid precursor protein (APP)-transgenic mouse, we analyzed behavioral changes and brain inflammatory response induced by peripheral administration of lipopolysaccharide. Application of a unique in vivo microdialysis system revealed that the increase in brain inflammatory cytokine (interleukin-6) level was significantly higher in APP-Tg than in wild-type mice after peripheral lipopolysaccharide injection, which was associated with more severe sickness behaviors. The blood–brain barrier became more permeable in APP-Tg mice during peripherally evoked inflammation, suggesting the increased vulnerability of the blood–brain barrier to inflammation in this animal model of Alzheimer''s disease. These findings might provide insight into the pathogenesis of noncognitive symptoms in dementia and a basis to develop new therapeutic treatments for them. [Copyright &y& Elsevier]

Abstract: Based on the potent angiogenic effects of hepatocyte growth factor (HGF), therapeutic angiogenesis using human HGF plasmid DNA increased tissue perfusion and reduced symptoms in patients with critical limb ischemia (CLI) in randomized placebo-controlled clinical trials. To explore further the potent angiogenic activity of HGF, the present study compared the effects of HGF, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) on angiogenesis and vascular inflammation. All of HGF, VEGF and bFGF significantly induced the formation of capillary blood vessel and granulation tissue in the rat paper disc model as an in vivo animal model of angiogenesis. However, although HGF, bFGF and VEGF significantly increased the growth of vascular endothelial cells, bFGF alone, but not HGF or VEGF, significantly increased the growth of vascular smooth muscle cells (VSMCs) in the in vitro proliferation assay. In addition, bFGF, but not HGF or VEGF, significantly activated an essential transcription factor for inflammation, NFκB, and gene expression of its downstream inflammation-related cytokines (IL-8 and MCP-1) in VSMCs, accompanied by an increase in the vascular permeability in the rat paper disc model. Thus, the present results indicated that HGF induced angiogenesis without vascular inflammation, different from bFGF and VEGF. These different properties between HGF, VEGF and bFGF might affect the efficiency of therapeutic angiogenesis. [Copyright &y& Elsevier]

Abstract: Serotonin (5-hydroxytryptamine, 5-HT) plays a crucial role in peripheral artery disease (PAD) and diabetes mellitus (DM). In these conditions, the balance between the 5-HT2A receptor in smooth muscle cells and the 5-HT1B receptor in endothelial cells (ECs) regulates vascular tonus. In the present study, we focused on the role of 5-HT in endothelial dysfunction using a selective 5-HT2A receptor blocker, sarpogrelate. In human EC, 5-HT markedly stimulated eNOS expression and the phosphorylation of eNOS, Akt and ERK1/2. In addition, a dose-dependent increase in tubule-formation on Matrigel was observed after 5-HT treatment. In contrast, high glucose significantly inhibited tubule formation and eNOS expression through inactivation of Akt, while 5-HT significantly attenuated these actions of high glucose (P <0.01). These results indicate that 5-HT stimulated angiogenesis through activation of Akt in ECs. However, in clinical situations, 5-HT seems to act as the “devil”. To examine the role of 5-HT in diabetic PAD, a hindlimb ischemia model was created in diabetic mice. The blood flow ratio of the ischemic to non-ischemic limb was significantly lower in DM mice than in normal mice, while sarpogrelate significantly attenuated the decrease in the blood flow ratio compared to control (P <0.01). Consistently, the decrease in eNOS expression and Akt activity in DM mice was significantly attenuated by sarpogrelate. Overall, the present study demonstrated that selective inhibition of 5-HT2A by sarpogrelate significantly restored ischemic limb blood perfusion in a severe diabetic mouse model through stimulation of the eNOS/Akt pathway via the endothelial 5-HT1B receptor. Enhancement of vasodilation and angiogenesis by sarpogrelate might provide a unique treatment for PAD and DM patients. [Copyright &y& Elsevier]

Abstract: Chitosan (CS)-modified poly(d,l-lactide-co-glycolide) (PLGA) nanospheres (NS) were developed and evaluated for use with a nuclear factor kappa B (NF-κB) decoy oligonucleotide (ODN) oral delivery system in an experimental model of ulcerative colitis (UC). Decoy ODN-loaded PLGA NS were prepared by an emulsion solvent diffusion method, and the physicochemical properties of NS were investigated. CS-modified PLGA NS (CS-PLGA NS) showed positive zeta potential, while unmodified PLGA NS (plain-PLGA NS) were negatively charged. Decoy ODN uptake studies with Caco-2 cells using confocal laser scanning microscopy (CLSM) indicated that CS-PLGA NS were more effectively taken up by the cells than plain-PLGA NS. Decoy ODN-loaded CS-PLGA NS were able to improve the stability of ODN against DNase I or an acidic medium, such as gastric juice. Daily oral administration of CS-PLGA NS in a rat model significantly improved dextran sulfate sodium-induced diarrhea, bloody feces, shortening of colon length, and myeloperoxidase activity. Furthermore, decoy ODN-loaded CS-PLGA NS were specifically deposited and adsorbed on the inflamed mucosal tissue of the UC model rat. These results suggested that CS-PLGA NS provide an effective means of colon-specific oral decoy ODN delivery in UC. [ABSTRACT FROM AUTHOR]

Currently, there is no effective clinical treatment to prevent abdominal aortic aneurysm (AAA). To develop a novel therapeutic approach, we modified decoy oligodeoxynucleotide (ODN) against nuclear factor κB (NFκB) and ets, to a ribbon-shaped circular structure without chemical modification, to increase its resistance to endonuclease for systemic administration. Intraperitoneal administration of ribbon-type decoy ODNs (R-ODNs) was performed in an elastase-induced rat AAA model. Fluorescent isothiocyanate (FITC)-labeled R-ODNs could be detected in macrophages migrating into the aneurysm wall, and NFκB and ets activity were simultaneously inhibited by chimeric R-ODN. Treatment with chimeric R-ODN significantly inhibited aortic dilatation, whereas conventional phosphorothioate decoy ODN failed to prevent aneurysm formation. Significant preservation of elastic fibers was observed with chimeric R-ODN, accompanied by a reduction of secretion of several proteases from macrophages. Activation of matrix metalloproteinase (MMP)-9 and MMP-12, but not MMP-2, was suppressed in the aneurysm wall by chimeric R-ODN, whereas recruitment of macrophages was not inhibited. Treatment with chimeric R-ODN also inhibited the secretion of cathepsin B and K from macrophages. Overall, the present study demonstrated that systemic administration of chimeric R-ODNs prevented aneurysm formation in a rat model. Further modification of the decoy strategy would provide a means of less invasive molecular therapy for human AAA. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra. The ultimate treatment of PD is to protect and repair dopaminergic neurons from degenerative process. As neurotrophic factors have been shown to support the survival and enhance the function of dopaminergic neurons, gene therapy using neurotrophic factors such as glial cell line-derived neurotrophic factor becames center of interests. In the present study, we focused on hepatocyte growth factor (HGF) as a novel neurotrophic and angiogenic growth factor that is a well-known potent pleiotrophic cytokine exhibiting mitogenic, motogenic, and morphogenic activities in a variety of cells.Prevention study: we have extended the preclinical exploration to primate model of PD. Seven days after stereotaxic transfection of human HGF plasmid or lacZ plasmid into the unilateral striatum, infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the right internal carotid artery of the monkey produces toxin- induced injury to the right nigro-striatal pathway. After MPTP infusion for 4 weeks, control animals exhibited stable moderate parkinsonian features. However, primates transfecterd with HGF plasmid showed normal states. PD model primates transfected with lacZ plasmid demonstrated the apomorphine and amphetamine- induced rotational asymmetry. On the other hand, transfection of human HGF plasmid resulted in a significant inhibition of abnormal rotation for 6 months. Microdialysis demonstrated that concentrations of dopamine in striatum and substantia nigra were higher level compared with PD model and lacZ plasmid transfected animals. PET imaging demonstrated remarkable and persistent increase of [11C]-L-dopa uptake and CFT binding in HGF plasmid injected striatum, while [11C]-raclopride did not change at the HGF plasmid terated site. Overall, the present study demonstrated that over-expression of human HGF prevented neuronal death in PDmodel.Repair study: Primate models which infused MPTP into the right internal carotid artery ,exhibited stable moderate parkinsonian features. PD primates were stereotaxic transfection of human HGF plasmid or lacZ plasmid was performed into the unilateral striatum. Primates transfecterd with HGF plasmid into striatum showed to improved normal states gradually. PD model primates transfected with lacZ plasmid demonstrated the apomorphine and amphetamine- induced rotational asymmetry. On the other hand, transfection of human HGF plasmid resulted in a almost inhibition of abnormal rotation for over 6 months. Doparminergic neurons of PD model primates transfected with human HGF plasmid into striatum were repaired significantly as assessed by immunohistchemistry. The present study demonstrated that over-expression of human HGF repaired neuronal degeneration and improved of symptoms in PD model. These two studies results indicate that HGF gene therapy may represent a promising candidate treatment for PD.Molecular Therapy (2006) 13, S160–S160; doi: 10.1016/j.ymthe.2006.08.480 [ABSTRACT FROM AUTHOR]

Epidemiological studies suggest that statins (hydroxymethylglutaryl-CoA reductase inhibitors) could reduce the risk of Alzheimer disease. Although one possible explanation is through an effect on β-amyloid (Aβ) metabolism, its effect remains to be elucidated. Here, we explored the molecular mechanisms of how statins influence Aβ metabolism. Fluvastatin at clinical doses significantly reduced Aβ and amyloid precursor protein C-terminal fragment (APP-CTF) levels among APP metabolites in the brain of C57BL/6 mice. Chronic intracerebroventricular infusion of lysosomal inhibitors blocked these effects, indicating that up-regulation of the lysosomal degradation of endogenous APP-CTFs is involved in reduced Aβ production. Biochemical analysis suggested that this was mediated by enhanced trafficking of APP-CTFs from endosomes to lysosomes, associated with marked changes of Rab proteins, which regulate endosomal function. In primary neurons, fluvastatin enhanced the degradation of APP-CTFs through an isoprenoid-dependent mechanism. Because our previous study suggests additive effects of fluvastatin on Aβ metabolism, we examined Aβ clearance rates by using the brain efflux index method and found its increased rates at high Aβ levels from brain. As LRP1 in brain microvessels was increased, up-regulation of LRP1-mediated Aβ clearance at the blood-brain barrier might be involved. In cultured brain microvessel endothelial cells, fluvastatin increased LRP1 and the uptake of Aβ, which was blocked by LRP1 antagonists, through an isoprenoid-dependent mechanism. Overall, the present study demonstrated that fluvastatin reduced Aβ level by an isoprenoid-dependent mechanism. These results have important implications for the development of disease-modifying therapy for Alzheimer disease as well as understanding of Aβ metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Although it is well known that Lipoprotein(a) (Lp(a)) is an atherogenic lipoprotein and an independent risk factor for cardiovascular disease, there is no confirmed therapy to decrease Lp(a) or prevent atherosclerosis induced by Lp(a). Thus, it is mandatory to develop novel therapy to prevent atherosclerosis in high Lp(a) concentration. Here, we focused on the effect of estrogen on Lp(a) level and Lp(a)-induced vascular remodeling. Methods: We employed Lp(a) transgenic mice (human apo(a) yeast artificial chromosome (YAC) and human apoB double transgenic mice). Vascular remodeling was induced by ligation of the common carotid artery and the effect of estrogen was evaluated in female mice after ovariectomy with or without estrogen replacement. Results: Estrogen deficiency caused by ovariectomy increased serum Lp(a), and continuous replacement of 17β-estradiol (20μg/kg/day) reversed the change. In the vascular remodeling model induced by carotid artery occlusion, neointima formation was significantly increased in ovariectomized female Lp(a) transgenic mice, but few in male Lp(a) transgenic mice, as compared to wild FVB mice. Importantly, continuous replacement of estrogen in ovariectomized mice significantly attenuated it. In cultured endothelial cells and macrophages, addition of Lp(a) increased mRNA of ICAM-1, VCAM-1, E-selectin and MCP-1 in endothelial cells and TNF-α, IL-1β and MCP-1 in macrophages in a dose-dependent manner. Importantly, pre-treatment with estrogen attenuated these changes in a dose-dependent manner. Conclusion: Estrogen negatively regulates both plasma Lp(a) level and Lp(a)-induced vascular remodeling, suggesting that estrogen might be a strong candidate to reduce serum Lp(a) concentration. [Copyright &y& Elsevier]

Objectives: Technetium-99m–labeled matrix metalloproteinase inhibitor (MPI) was used for the noninvasive assessment of matrix metalloproteinase (MMP) activity in atherosclerotic plaques after minocycline (MC) intervention. Background: MMP activity in atherosclerosis contributes to plaque instability. Some antimicrobial agents may attenuate MMP activity. Methods: Atherosclerotic lesions were produced in 38 rabbits with a high cholesterol diet for 4 months; 5 groups of rabbits, in the fourth month, received fluvastatin (FS) (n = 6), low-dose MC (n = 7), high-dose MC (n = 7), a combination of low-dose MC and FS (n = 6), or no intervention (n = 12); 8 unmanipulated rabbits were used as disease controls. Micro–single-photon emission computed tomography imaging was performed in all animals after intravenous MPI administration, followed by pathologic characterization of the aorta. A cell culture study evaluated the effect of MC on MMP production by activated human monocytes. Results: MPI uptake was visualized best in untreated atherosclerotic animals (percent injected dose per gram MPI uptake, 0.11 ± 0.04%). MPI uptake was reduced in the FS (0.06 ± 0.01%; p < 0.0001), high-dose MC (0.05 ± 0.01%; p < 0.0001), and MC-FS (0.05 ± 0.005%; p < 0.0001) groups. Low-dose MC did not resolve MPI uptake significantly (0.08 ± 0.02; p = 0.167). There was no incremental benefit of the combination of MC and FS. MPI uptake showed a significant correlation with plaque MMP-2, and MMP-9 activity. MMP-9 release from tumor necrosis factor-α–activated macrophages was abrogated by incubation with MC. Conclusions: Molecular imaging of MMP activity in atherosclerotic plaque allows for the study of the efficacy of therapeutic interventions. MC administration resulted in substantial reduction in plaque MMP activity and histologically verified plaque stabilization. MC was found to be equally effective as FS. [Copyright &y& Elsevier]

Objectives: The purpose of this study was to investigate the efficiency of small interfering ribonucleic acid (siRNA) in murine arteries. We transfected it using a nonviral ultrasound-microbubble-mediated in vivo gene delivery system. Background: siRNA is an effective methodology to suppress gene function. The siRNA can be synthesized easily; however, a major obstacle in the use of siRNA as therapeutics is the difficulty involved in effective in vivo delivery. Methods: To investigate the efficiency of nonviral ultrasound-microbubble-mediated in vivo siRNA delivery, we used a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA in murine arteries. Murine femoral arteries were injured using flexible wires to establish arterial injury. Results: The fluorescein-labeled siRNA and GFP siRNA showed that this nonviral approach could deliver siRNA into target arteries effectively without any tissue damage and systemic adverse effects. ICAM-1 siRNA transfection into murine injured arteries significantly suppressed the development of neointimal formation in comparison to those in the control group. Immunohistochemistry revealed that accumulation of T cells and adhesion molecule positive cells was observed in nontreated injured arteries, whereas siRNA suppressed accumulation. Conclusions: The nonviral ultrasound-microbubble delivery of siRNA ensures effective transfection into target arteries. ICAM-1 siRNA has the potential to suppress arterial neointimal formation. Transfection of siRNA can be beneficial for the clinical treatment of cardiovascular and other inflammatory diseases. [ABSTRACT FROM AUTHOR]

Abstract: Parameters affecting the particle sizes of poly(DL-lactide-co-glycolide) (PLGA) nanospheres produced by the Emulsion Solvent Diffusion (ESD) method were evaluated in this study, so that suitable PLGA nanospheres could be prepared to pass through a membrane filter with 0.2μm pore size and used as a sterile product. Experimental results demonstrated that the particle sizes of PLGA nanospheres could be reduced by the following efforts. [(1)] Increase stirring rate of poor solvent. [(2)] Decrease feed rate of good solvent. [(3)] Increase poor solvent ratio. [(4)] Increase the temperature of poor solvent. [(5)] Decrease polyvinyl alcohol concentration in poor solvent. [(6)] Increase ethanol concentration in good solvent. [(7)] Decrease PLGA concentration in good solvent. After optimization, PLGA nanospheres with a mean particle size of 102–163nm and the 100–98% of filtration fraction could be produced and passed the bacteria challenge tests. This study found PLGA nanospheres can be efficiently prepared as a sterile product. [Copyright &y& Elsevier]

Abstract: One of the major risk factors for ischemic disease is hyperlipidemia, which is mainly regulated by endogenous cholesterol synthesis in the liver and dietary absorption in the small intestine. In this study, we evaluated the vascular protective effects of a potent cholesterol absorption inhibitor, ezetimibe. ApoE-deficient mice were fed a chow or high-fat diet with or without ezetimibe (5mg/(kgday)) for 3 months. Co-treatment with ezetimibe significantly reduced plasma cholesterol (by 76%; from 1592 to 381mg/dL) and LDL cholesterol (by 78%; from 1515 to 319mg/dL), and increased HDL cholesterol (by 187%; from 16 to 46mg/dL) in high-fat diet mice. Consistently, a marked inhibitory effect of ezetimibe on the development of lipid-rich plaque was observed, as assessed by oil red O staining. Of importance, treatment with ezetimibe significantly improved endothelial dysfunction as assessed by the vasodilator response to acetylcholine, accompanied by inhibition of interleukin-6 mRNA and an increase in endothelial nitric oxide synthase (eNOS) mRNA in the aorta. Ezetimibe also suppressed oxidative stress and the ubiquitination–proteasome system in the aorta. Although changes in body weight and several tissue weights were similar in the groups with and without ezetimibe administration, only liver weight was significantly decreased in the ezetimibe-treated group. Interestingly, ezetimibe markedly inhibited lipid accumulation in the liver. Furthermore, ezetimibe increased the mRNA expression of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase as a counteraction in the liver, but not in the aorta. Overall, ezetimibe significantly prevented atherosclerosis through not only lipid-lowering effects, but also other direct and/or indirect vascular protective actions in ApoE-deficient mice. [Copyright &y& Elsevier]

Abstract: Recently, atherosclerosis has been considered to be the result of inflammation. Interestingly, hydroxymethylglutaryl-coenzyme (HMG-Co) A inhibitors (statins), which are clinically used as lipid-lowering agents, have been reported to have various anti-inflammatory effects. As abdominal aortic aneurysm (AAA) is a common degenerative condition associated with atherosclerosis, this study was designed to investigate the inhibitory effect of a statin, atorvastatin, on aneurysm formation apart from its lipid-lowering effect. We employed an elastase-induced rat AAA model, as statins do not lower cholesterol in rats. Mean aneurysm diameter was significantly smaller in the atorvastatin treatment group as compared to control at 4 weeks after surgery (P <0.05). Interestingly, atorvastatin inhibited the expression of ICAM and MCP-1, followed by the suppression of macrophage recruitment into the aortic wall at 1 week after operation. A significant reduction in MMP-12, but not MMP-2, -3 and -9, expression was also observed by treatment with atorvastatin at 1 week after surgery. In addition, synthesis of collagen and elastin in the vascular wall were significantly increased by atorvastatin. Here, the present study demonstrated a direct effect of atorvastatin to inhibit the progression of aortic aneurysm, independent of its lipid-lowering effect. This study suggests new therapeutic aspects of statins to inhibit the progression of aneurysms. [Copyright &y& Elsevier]

Peripheral nerve injury occasionally causes chronic neuropathic pain with hyperalgesia and allodynia. However, its treatment is difficult. Here, we used a chronic constriction injury (CCI) model in rats to investigate the effects on experimental neuropathic pain of the human hepatocyte growth factor (HGF) gene delivered into the nervous system by retrograde axonal transport following its repeated intramuscular transfer, using liposomes containing the hemagglutinating virus of Japan (HVJ). CCI (control) rats exhibited marked mechanical allodynia and thermal hyperalgesia, and decreased blood flow in sciatic nerve and hind paw. All these changes were significantly reversed by HGF gene transfer. In the sciatic nerve in HGF-treated rats, the size-frequency distributions for myelinated and unmyelinated axons each showed a rightward shift, the number of myelinated axons >5 µm in diameter was significantly increased, and the mean diameter of unmyelinated axons was significantly increased (versus CCI rats). Levels of P2X3, P2X4, and P2Y1 receptor mRNAs, and of interleukin-6 (IL-6) and activating transcription factor 3 (ATF3) mRNAs, were elevated in the ipsilateral dorsal root ganglia and/or sciatic nerve by CCI, and these levels were decreased by HGF gene transfer. These results may point toward a potential new treatment strategy for chronic neuropathic pain in this model.Molecular Therapy (2008) 17 1, 42–50 doi:10.1038/mt.2008.214 [ABSTRACT FROM AUTHOR]

HIF-la is originally identified as a transcription factor that activates gene expression in response to hypoxia. In metazoans, HIF-la functions as a master regulator of oxygen homeostasis and regulates adaptive responses to change in oxygen tension during embryogenesis, tissue ischemia, and tumorigenesis. Because H~tL1a~deficient mice exhibit a number of develop- mental defects, the precise role of HIF-la in early cardiac morphogenesis has been uncertain. Therefore, to clarify the role of HIF-la in heart development, we investigated the effect of knockdown of HIF-la in Xenopus embryos using antisense morpholino oligonucleotide microinjection techniques. Knock- down of HIF-la resulted in defects of cardiogenesis. Whole mount in situ hybridization for cardiac troponin I (cTnI) showed the two separated populations of cardiomyocytes, which is indicative of cardia bifida, in HIF-la-depleted embryos. Fur- thermore, the depletion of HIF-la led to the reduction in cTnI expression, suggesting the correlation between HIF-la and car- diac differentiation. We further examined the expression of several heart markers, nkx2.5, gata4, tbx5, bmp4, handi, and hand2 in HIF-la-depleted embryos. Among them, the expression of nkx2.5 was significantly reduced. Luciferase reporter assay using the Nkx2.5 promoter showed that knockdown of HIF-lix decreased its promoter activity. The cardiac abnormality in the HIF-la-depleted embryo was restored with co-injection of nkx2.5 mRNA. Collectively, these findings reveal that HIF-la- regulated nkx2.5 expression is required for heart development in Xenopus. [ABSTRACT FROM AUTHOR]

Abstract: Hepatocyte growth factor (HGF) is well known to be involved in many biological functions, such as organ regeneration and angiogenesis, and to exert neurotrophic effects on motor, sensory, and parasympathetic neurons. In this study, we gave repeated intramuscular injections of the human HGF gene, using nonviral HVJ (hemagglutinating virus of Japan) liposome method, to examine whether transfection of the rat nervous system with this gene is able to exert neurotrophic effects facilitating recovery of a crushed nerve. The expression of HGF protein and HGF mRNA indicated that gene transfer into the nervous system did occur via retrograde axonal transport. At 4 weeks after crush, electrophysiological examination of the crushed nerve showed a significantly shorter mean latency and a significantly greater mean maximum M-wave amplitude with repeated injections of HGF gene. Furthermore, histological findings showed that the mean diameter of the axons, the axon number and the axon population were significantly larger in the group with repeated injections of HGF gene. The above results show that repeated human HGF gene transfer into the rat nervous system is able to promote crushed-nerve recovery, both electrophysiologically and histologically, and suggest that HGF gene transfer has potential for the treatment of crushed nerve. [Copyright &y& Elsevier]

Although viral vector systems are efficient to transfect foreign genes into a variety of tissues, safety issues remain in relation to human gene therapy. In this study, we examined the feasibility of a novel nonviral vector system by using high-frequency, low-intensity ultrasound irradiation for transfection into vascular cells, kidneys and the central nerve systems of fetal mice. As a result, expression of the reporter gene, Venus, was readily detected in the central nervous system. The transfected cells were mainly detected in meningeal cells with intracisternal injection. Overall, the present study demonstrated the feasibility of efficient plasmid DNA transfer into several organs, especially the central nervous system, providing a new option for treating various diseases. [Copyright &y& Elsevier]

Atherosclerosis has a close relationship to inflammation, particularly T helper type 1 lymphocyte (Th1) response. Interleukin-10 (IL-10), is thought to suppress Th1 response. To target therapeutic strategy for atherosclerosis, we tested whether IL-10 gene transfer suppresses atherosclerosis in apolipoprotein E-knockout (apoE-KO) mice. Four-week-old apoE-KO mice were divided into two groups and either murine IL-10 cDNA plasmid or empty control vector was transferred to the femoral muscle with the use of Hemagglutinating virus of Japan (HVJ)-liposome. At 1 week after transfection, high cholesterol diet was started and continued for 8 weeks. After euthanasia, histological studies of atherosclerotic lesions and quantitative RT-PCR for Th1 cytokines (IL-12 and IFN-γ) in spleens were performed. IL-10 cDNA gene transfer to the muscle increased plasma IL-10 levels and depressed expression of Th1 cytokines without changing plasma cholesterol levels. IL-10 gene transfer significantly reduced the atherosclerotic plaque area and the macrophage infiltrated area. IL-12 and IFN-γ mRNA expressions in spleens and plasma IFN-γ levels were decreased by IL-10 gene transfer. Therefore, IL-10 gene transfer changed the Th1 response and suppressed atherosclerotic lesion formation in apoE-KO mice. IL-10 could be a new target as a therapeutic tool for the treatment of atherosclerosis. [Copyright &y& Elsevier]

We have developed a simple method for converting the lipid envelope of an inactivated virus to a gene transfer vector. Hemagglutinating virus of Japan (HVJ; Sendai virus) envelope vector was constructed by incorporating plasmid DNA into inactivated HVJ particles. This HVJ envelope vector introduced plasmid DNA efficiently and rapidly into various cell lines, including cancer cells and several types of primary cell culture. Efficiency of gene transfer was greatly enhanced by protamine sulfate and centrifugation. Fluorescein isothiocyanate-labeled oligodeoxynucleotides (FITC-ODN) were also delivered to cells at > 95% efficiency. When HVJ envelope vector was injected into organs directly, reporter gene expression was observed in organs including liver, brain, skin, uterus, tumor masses, lung, and eye. When HVJ envelope vector containing luciferase gene was injected into mouse tail vein, luciferase gene expression was detected primarily in spleen. FITC-ODN were also delivered to spleen cells by intravenous injection of HVJ envelope. These results suggest that HVJ envelope vector will be useful for both ex vivo and in vivo gene therapy experiments. [Copyright &y& Elsevier]

Intrauterine injection of naked DNA expressing luciferase, green fluorescent protein (GFP), or β-galactosidase (β-gal) and fluorescein isothiocyanate–labeled oligodeoxynucleotide (FITC-ODN), in combination with microbubble-enhanced ultrasound (US), referred to as the “shotgun method” (SGM), produced high-level protein expression in fetal mice. With the SGM, luciferase expression increased ∼103-fold in comparison with expression after injection of naked DNA alone. Electron microscopic analysis demonstrated transient formation of pores on the skin surface after intraamniotic (i.a.) injection with the SGM. Widespread expression of GFP and β-gal and delivery of FITC-ODN were observed in multiple fetal tissues adjacent to the injection points. PCR analysis indicated that germline transfection was only transient following intraperitoneal (i.p) injection, and there was no evidence of transfer of the reporter gene to the offspring. Thus, SGM might provide a useful means to clarify the molecular mechanisms of genetic diseases in utero, as well as a tool to develop gene therapies in utero. [Copyright &y& Elsevier]

An abstract of the article "Development of a Novel Ribbon-type NF-κB Decoy Oligodeoxynucleotides to Treat Cardiovascular Diseases," by Mariana K. Osako, Naruya Tomita, Yasuo Kunugiza, Keishi Sekiguchi, Keiko Izawa, and Ryuichi Morishita is presented.

Isolation of effective therapeutic genes to control angiogenesis is critical for the advancement of gene therapy for various diseases, including cardiovascular diseases and cancer. The goal of the present study is to screen a human cDNA library using Hemagglutinating Virus of Japan envelope (HVJ-E) vector to isolate candidate genes to regulate endothelial cell growth with potent therapeutic potential. cDNA library were randomly infused into HVJ-E vector, which were then transferred to Human Aortic Endothelial Cells (HAEC) that were seeded in 96-well plates. Then, the MTS assay revealed variable HAEC proliferation among individual wells. To characterize the candidate genes responsible for inducing or reducing endothelial cell proliferation, DNA was extracted from the cell population showing the highest or lowest degree of proliferation and was then transformed directly into E. coli. Use of this system allowed isolation of candidate genes rapidly, because HVJ-E vector does not require preparation of a viral library or packaging cell constructions. In addition, use of this vector allows easy cloning of candidate genes by transformation of E. Coli (12–16 hours). These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes.We further evaluated the candidate genes by c-fos promoter assay, and finally got three pro-angiogenic and two anti-angiogenic candidate genes. Interestingly, one of the identified growth suppressive gene is also known as CHMP1B or CHMP1.5, a human homologue of yeast SNF7p (sucrose non-fermenting). The CHMP family of proteins participates in intracellular membrane traffic event, of which the endocytic pathway is an important component. Further, a recent study reported that CHMP 1B binds to spastin, a protein that is abnormal in the most common form of pure hereditary spastic paraplegia. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.Molecular Therapy (2006) 13, S336–S336; doi: 10.1016/j.ymthe.2006.08.961 [ABSTRACT FROM AUTHOR]

In this study, we focused on NFkB decoy oligodeoxynucleotides (NFkB decoy) as a new therapeutic strategy to attenuate osteoporosis. Tartrate resistant acid phosphatase (TRAP) positive multinucleated cells (osteoclasts) formed from neonatal rabbit bone marrow culture by 1,25-vitamin D3 stimulation were decreased by NFkB decoy treatment in dose dependent manner and pits formed on dentine slices by mature osteoclasts were reduced. In M-CSF and RANKL stimulated osteoclast culture system, NFkB decoy also inhibited the diffentiation and activation of osteoclast as well. In rat ovariectomized model of estrogen deficiency, continuous administration of NFkB decoy using osmotic pump attenuated the increase of TRAP activity, accompanied by a singificant increase in calcium concentration in femur and tibia and decreased in urinary deoxypyridinoline. In additional osteoporosis model of vitamin C deficient rat, inhibiton of NFkB by decoy dramatically improved the bone length, weight and mineral density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targetting of NFkB might be potential therapy in various bone metabolic diseases.Molecular Therapy (2006) 13, S275–S275; doi: 10.1016/j.ymthe.2006.08.792 [ABSTRACT FROM AUTHOR]