Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression.

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

Selective Blockade of Periostin Exon 17 Preserves Cardiac Performance in Acute Myocardial Infarction.

We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon 17) and Pn-4 (lacking exons 17 and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon 17 in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming growth factor-ß1. Importantly, the number of α-smooth muscle actin-positive myofibroblasts was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited fibroblast and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-17, via a neutralizing antibody or drug, without suppressing other periostin variants might offer a new class of medication for the treatment of acute myocardial infarction patients.

miR-24 limits aortic vascular inflammation and murine abdominal aneurysm development.

Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.

Endothelial progenitor cells in clinical settings.

Senescence of cells is associated with shortened or damaged telomeres and is characterized by permanent exit from the cell cycle and altered function. Cellular senescence is caused by repeated cell division, and also conditions of stress including inflammation and reactive oxygen species can lead to the development of premature senescence. At the cellular level, proliferative and oxidative-stress induced cell senescence related to a pro-inflammatory state might strongly contribute to age-associated impaired tissue and organ functions. Vascular cells (endothelial cells, vascular smooth muscle cells) and bone marrow-derived endothelial progenitor cells have been repeatedly shown to have pivotal role in the maintenance and regeneration of cardiovascular tissue. Therefore, the molecular mechanisms of vascular cell senescence have been extensively studied. However, therapeutic approaches to prevent cellular senescence in cardiovascular disease (CVD) are still limited. Hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) are all potent angiogenic growth factors in animal models of ischemia, but their therapeutic effects are not the same in animal experiments and clinical trials. A multicenter, double-blind, placebo-controlled phase III clinical trial in Japan and a US phase II clinical trial of HGF gene therapy for critical limb ischemia (CLI) demonstrated a significant improvement in primary end points and an increase in transcutaneous partial pressure of oxygen even after one year compared with placebo, whereas effectiveness of VEGF and FGF treatment for CLI has not yet been shown. Moreover, our recent publication and another researcher demonstrated that HGF acts as an anti-inflammatory cytokine, while VEGF and FGF act as pro-inflammatory cytokine. This review overviews the outcomes of clinical trials using angiogenic growth factors, which have shown a dramatic effect in several animal studies. Additionally, interventions with HGF aimed at improving the regenerative capacity of stem/progenitor cells and vascular cells by preventing cellular senescence are discussed.

MicroRNA-29b regulation of abdominal aortic aneurysm development.

Tremendous efforts have been initiated to elucidate the molecular and pathophysiological characteristics of abdominal aortic aneurysm (AAA) disease, which is a significant contributor to morbidity and mortality in the Western world. Recently, a novel class of small noncoding RNAs, called microRNAs, was identified as important transcriptional and posttranscriptional inhibitors of gene expression thought to simultaneously "fine tune" the translational output of multiple target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. Several research groups were able to identify the miR-29 family, and miR-29b in particular, as crucial regulators of-not only vascular fibrosis-but also cardiac-, kidney-, liver-, and skin-fibrosis. The current review briefly points out data indicating a causal role for miR-29 in various diseases, while focusing on its potential benefit during AAA initiation and propagation.

Therapeutic Angiogenesis by Gene Therapy for Critical Limb Ischemia: Choice of Biological Agent.

Peripheral artery disease (PAD) is caused by atherosclerosis, hardening and narrowing arteries over time due to buildup of fatty deposit in vascular bed called plaque. Severe blockage of an artery of the lower extremity markedly reduce blood flow, resulting in critical limb ischemia (CLI) manifested by a variety of clinical syndromes including rest pain in the feet or toes, ulcer and gangrene with infection. Despite significant advances in clinical care and interventions for revascularization, patients with CLI remain at high risk for amputation and cardiovascular death. To overcome this unmet need, therapeutic angiogenesis using angiogenic growth factors has evolved in an attempt to increase blood flow in ischemic limb. Initial animal studies and phase I clinical trials with vascular endothelial growth factor (VEGF) or fibroblast growth factor (FGF) demonstrated promising results, inspiring scientists to progress forward. However, more rigorous phase II and III clinical trials have failed to demonstrate beneficial effects of these angiogenic growth factors to date. Recently, two multicenter, double-blind, placebo-controlled clinical trials in Japan (phase III) and US (phase II) demonstrated that hepatocyte growth factor (HGF) gene therapy for CLI significant improved primary end points and tissue oxygenation up to two years in comparison to placebo. These clinical results implicate a distinct action of HGF on cellular processes involved in vascular remodeling under pathological condition. This review presents data from phase I-III clinical trials of therapeutic angiogenesis by gene therapy in patients with PAD. Further, we discuss the potential explanation for the success or failure of clinical trials in the context of the biological mechanisms underlying angiogenesis and vascular remodeling, including cellular senescence, inflammation, and tissue fibrosis.

Unique remodeling processes after vascular injury in intracranial arteries: analysis using a novel mouse model.

The effectiveness of angioplasty and stenting in intracranial atherosclerotic diseases is controversial due to high rates of delayed restenosis and hemorrhage compared with extracranial arteries. However, the mechanisms underlying these differences are still unclear, because their pathophysiology is yet to be examined. To address this issue, we established a novel vascular injury model in the intracranial internal carotid arteries (IICAs) in mice, and analyzed the remodeling process in comparison to that of the femoral arteries (FAs). In IICAs, neointimal hyperplasia was observed from day 14 and grew until day 56. Although smooth muscle cells (SMCs) emerged in the neointima from day 28, SMCs in the injured media were continuously lost with eventual extinction of the media. Re-endothelialization was started from day 7 and completed on day 28. Accumulation of macrophages was continued in the adventitia until day 56. Compared with FAs, the following points are unique in IICAs: (1) delayed continuous formation of neointima; (2) accumulation of macrophages in the media on day 14; (3) continuous loss of SMCs in the media followed by extinction of the media itself; and (4) continuously growing adventitia. These pathophysiologic differences might be associated with unfavorable outcomes in percutaneous transluminal angioplasty and stenting in intracranial arteries.

Therapeutic option of plasmid-DNA based gene transfer.

Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common method in clinical cases because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid deoxyribonucleic acid (DNA)-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a phase III clinical trial did not show sufficient efficiency. In this situation, more efficient plasmid DNA gene transfer is needed all over the world. This review focuses on plasmid DNA gene transfer and its enhancement, including ultrasound with microbubbles, electroporation, hydrodynamic method, gene gun, jet injection, cationic lipids and cationic polymers.

Hepatocyte growth factor inhibits lipopolysaccharide-induced oxidative stress via epithelial growth factor receptor degradation.

OBJECTIVE: Lipopolysaccharide (LPS) triggers sepsis and systemic inflammatory response syndrome, which results in multiple organ failure. Our recent reports demonstrated that hepatocyte growth factor (HGF) attenuated angiotensin II-induced oxidative stress via epithelial growth factor receptor (EGFR) degradation in vascular smooth muscle cells. Here, we examined whether HGF can protect against systemic inflammatory response syndrome induced by LPS and investigated the mechanism. METHODS AND RESULTS: HGF inhibited the increase in the expression of vascular cell adhesion molecule-1 and EGFR by LPS in vitro. HGF inhibited colocalization of EGFR and Src homology domain 2-containing inositol 5'-phosphatase 2. Furthermore, HGF inhibited reactive oxygen species production. We also injected LPS into HGF transgenic mice with increased HGF serum concentration and their littermates. HGF transgenic mice reduced LPS-induced vascular cell adhesion molecule-1 and reactive oxygen species compared with control, accompanied by significant EGFR degradation. Furthermore, HGF transgenic mice significantly improved survival in the LPS injection model. CONCLUSIONS: The present study revealed inhibition of LPS-induced vascular cell adhesion molecule-1 expression by HGF via the degradation of EGFR. We demonstrated that HGF regulated Src homology domain 2-containing inositol 5'-phosphatase 2 recruitment to EGFR and inhibited LPS-induced inflammation via EGFR degradation. This effect of HGF may be useful for the treatment of inflammatory disease.

Long-term follow-up evaluation of results from clinical trial using hepatocyte growth factor gene to treat severe peripheral arterial disease.

OBJECTIVE: As angiogenic growth factors can stimulate the development of collateral arteries, a concept called therapeutic angiogenesis, we performed a phase I/IIa open-label clinical trial using intramuscular injection of naked plasmid DNA encoding hepatocyte growth factor (HGF). We reported long-term evaluation of 2 years after HGF gene therapy in 22 patients with severe peripheral arterial disease. METHODS AND RESULTS: Twenty-two patients with peripheral arterial disease or Buerger disease staged by Fontaine IIb (n=7), III (n=4), and IV (n=11) were treated with HGF plasmid, either 2 mg or 4 mg ×2. Increase in ankle-branchial pressure index >0.1 was observed in 11 of 14 patients (79 %) at 2 years after gene therapy and in 11 of the 17 patients (65%) at 2 months. Reduction in rest pain (>2 cm in visual analog scale) was observed in 9 of 9 patients (100%) at 2 years and in 8 of 13 (62%) patients at 2 months. At 2 years, 9 of 10 (90%) ischemic ulcers reduced by >25%, accompanied by a reduction in the size of ulcer. Severe complications and adverse effects caused by gene transfer were not detected in any patient throughout the period up to 2 years. CONCLUSIONS: Overall, the present study demonstrated long-term efficacy of HGF gene therapy up to 2 years. These findings may be cautiously interpreted to indicate that intramuscular injection of naked HGF plasmid is safe, feasible, and can achieve successful improvement of ischemic limbs as sole therapy.

Inhibition of Lp(a)-induced functional impairment of endothelial cells and endothelial progenitor cells by hepatocyte growth factor.

BACKGROUND: Lipoprotein (a) (Lp(a)) is one of the risk factors for peripheral artery disease (PAD). Our previous report demonstrated that hepatocyte growth factor (HGF) gene therapy attenuated the impairment of collateral formation in Lp(a) transgenic mice. Since risk factors for atherosclerosis accelerate endothelial senescence and impair angiogenesis, we examined the role of Lp(a) in dysfunction and senescence of endothelial progenitor cells (EPC) and endothelial cells. METHODS: In vitro and in vivo incorporation assays were performed using ex-vivo expanded DiI-labeled human EPC. Senescence of cultured endothelial cells, production of oxidative stress and angiogenesis function were evaluated by SA-ß-galactosidase staining, dihydroethidium (DHE) staining and Matrigel assay, respectively. RESULTS: EPC transplantation significantly stimulated recovery of ischemic limb perfusion, while EPC pre-treated with Lp(a) did not increase ischemic limb perfusion. Impairment of angiogenesis by EPC with Lp(a) was associated with a significant decrease in CD31-positive capillaries and DiI-labeled EPC. Importantly, Lp(a) significantly accelerated the onset of senescence and production of reactive oxygen species (ROS) in human aortic endothelial cells, accompanied by a significant increase in the protein expression of p53 and p21. On the other hand, HGF significantly attenuated EPC dysfunction, senescence, ROS production, and p53 and p21 expression induced by Lp(a). CONCLUSION: Lp(a) might affect atherosclerosis via acceleration of senescence, ROS production, and functional impairment of the endothelial cell lineage. HGF might have inhibitory effects on these atherogenic actions of Lp(a).

Hepatocyte growth factor reduces cardiac fibrosis by inhibiting endothelial-mesenchymal transition.

The purpose of this study was to investigate the effect of hepatocyte growth factor (HGF) on the pathogenesis of cardiac fibrosis induced by pressure overload in mice. Although cardiac fibrosis is attributed to excess pathological deposition of extracellular matrix components, the mechanism remains unclear. Recent reports revealed that α-smooth muscle actin-expressing myofibroblasts are primarily responsible for fibrosis. It is believed that myofibroblasts are differentiated from resident fibroblasts, whereas the transformation of vascular endothelial cells into myofibroblasts, known as endothelial-mesenchymal transition, has been suggested to be intimately associated with perivascular fibrosis. Thus, we hypothesized that HGF prevents cardiac fibrosis by blocking these pathways. We analyzed the pressure-overloaded HGF-transgenic mouse model made by transverse aortic constriction. Human coronary artery endothelial cells and human cardiac fibroblasts were examined in vitro after being treated with transforming growth factor-ß1 or angiotensin II with or without HGF. The amount of cardiac fibrosis significantly decreased in pressure-overloaded HGF-transgenic mice compared with pressure-overloaded nontransgenic controls, particularly in the perivascular region. This was accompanied by a reduction in the expression levels of fibrosis-related genes and by significant preservation of echocardiographic measurements of cardiac function in the HGF-transgenic mice (P<0.05). The survival rate 2 months after transverse aortic constriction was higher by 45% (P<0.05). HGF inhibited the differentiation of human coronary artery endothelial cells into myofibroblasts induced by transforming growth factor-ß1 and the phenotypic conversion of human cardiac fibroblasts into myofibroblasts. We conclude that HGF reduced cardiac fibrosis by inhibiting endothelial-mesenchymal transition and the transformation of fibroblasts into myofibroblasts.

MicroRNA-21 blocks abdominal aortic aneurysm development and nicotine-augmented expansion.

Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid-modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.

Telmisartan exerts renoprotective actions via peroxisome proliferator-activated receptor-γ/hepatocyte growth factor pathway independent of angiotensin II type 1 receptor blockade.

Angiotensin (Ang) II type 1 receptor blockers have demonstrated beneficial effects beyond blood pressure control in the treatment of chronic kidney disease. There is clinical evidence that telmisartan is more effective than losartan in reducing proteinuria in hypertensive patients with diabetic nephropathy, because it is a partial agonist of peroxisome-proliferator activated receptor-γ (PPARγ), as well as an Ang II type 1 receptor blocker (AMADEO Study [A comparison of telMisartan versus losArtan in hypertensive type 2 DiabEtic patients with Overt nephropathy]). In this study, we examined the role of PPARγ activation in the renal protective actions of telmisartan using Ang II type 1 receptor-deficient mice. Renal injury was induced in Ang II type 1 receptor-deficient mice by producing unilateral ureteral obstruction, which exhibited severe renal interstitial fibrosis and inflammation. In these mice, telmisartan prevented hydronephrosis induced by unilateral ureteral obstruction more strongly than did losartan. Importantly, the prevention of renal atrophy and fibrosis by telmisartan was significantly attenuated by GW9662, a PPARγ antagonist. Interestingly, the downstream effector of PPARγ activation by telmisartan is hepatocyte growth factor (HGF), a well-known antifibrotic factor, because renal HGF expression was significantly increased by telmisartan, and a neutralizing antibody against HGF diminished the renal protective action of telmisartan. These beneficial changes by telmisartan were associated with a decrease in the expression of transforming growth factor-ß1 and other proinflammatory and profibrotic cytokine genes through PPARγ/HGF activation. Our findings provide evidence of organ protective actions of telmisartan through the PPARγ/HGF pathway, independent of Ang II type 1 receptor blockade. Further development of the next generation of Ang II type 1 receptor blockers with added organ protective actions, such as PPARγ activation, might provide new beneficial drugs to treat renal and cardiovascular diseases.

Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development.

MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.

Three-dimensional microstructural changes in murine abdominal aortic aneurysms quantified using immunofluorescent array tomography.

This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell (SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.

Role of serotonin in angiogenesis: induction of angiogenesis by sarpogrelate via endothelial 5-HT1B/Akt/eNOS pathway in diabetic mice.

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.

Plasmid DNA-based gene transfer with ultrasound and microbubbles.

Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

Selective Glucocorticoid Receptor (GR-II) Antagonist Reduces Body Weight Gain in Mice.

Previous research has shown that mifepristone can prevent and reverse weight gain in animals and human subjects taking antipsychotic medications. This proof-of-concept study tested whether a more potent and selective glucocorticoid receptor antagonist could block dietary-induced weight gain and increase insulin sensitivity in mice. Ten-week-old, male, C57BL/6J mice were fed a diet containing 60% fat calories and water supplemented with 11% sucrose for 4 weeks. Groups (n = 8) received one of the following: CORT 108297 (80 mg/kg QD), CORT 108297 (40 mg/kg BID), mifepristone (30 mg/kg BID), rosiglitazone (10 mg/kg QD), or vehicle. Compared to mice receiving a high-fat, high-sugar diet plus vehicle, mice receiving a high-fat, high-sugar diet plus either mifepristone or CORT 108297 gained significantly less weight. At the end of the four week treatment period, mice receiving CORT 108297 40 mg/kg BID or CORT 108297 80 mg/kg QD also had significantly lower steady plasma glucose than mice receiving vehicle. However, steady state plasma glucose after treatment was not highly correlated with reduced weight gain, suggesting that the effect of the glucocorticoid receptor antagonist on insulin sensitivity may be independent of its mitigating effect on weight gain.