Explore the vast range of titles available.

Hepatocyte growth factor (HGF) is a potent angiogenic factor. The efficacy and safety of intramuscular injection of a naked plasmid encoding human HGF gene (beperminogene perplasmid, Collategene) was investigated in patients with critical limb ischemia (CLI) in a multicenter, randomized, double-blind, placebo-controlled trial. The randomization ratio for plasmid to placebo was 2:1. Injection sites were selected in each patient limb based on angiographic findings. Placebo or plasmid was injected on days 0 and 28. Evaluation of efficacy was carried out after 12 weeks. The primary end point was the improvement of rest pain in patients without ulcers (Rutherford 4) or the reduction of ulcer size in patients with ulcer(s) (Rutherford 5). Secondary end points were ankle-brachial pressure index, amputation, and quality of life (QOL). Forty-four patients were treated, and we performed interim analysis of efficacy in 40 patients. The overall improvement rate of the primary end point was 70.4% (19/27) in HGF group and 30.8% (4/13) in placebo group, showing a significant difference ( P =0.014). In Rutherford 5 patients, HGF achieved a significantly higher improvement rate (100% [11/11]) than placebo (40% [2/5]; P =0.018). HGF plasmid also improved QOL. There were no major safety problems. HGF gene therapy is safe and effective for CLI.

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.

Background: Bacteria are implicated in certain forms of model chronic colitis but the identity and role of bacteria in human ulcerative colitis (UC) are uncertain. Aims: To isolate pathogenic bacteria from inflamed mucosa of patients with UC, to examine whether the bacteria have a toxin to Vero cells, and to determine whether the toxin induces UC-like lesions in animals. Methods: Bacteria were isolated from UC patients and supernatants from cultures were filtered and tested for cytotoxicity to Vero cells. Bacterial cells producing the cytotoxic supernatants were examined by polymerase chain reaction for verotoxin genes. Culture supernatants of cytotoxic strains were examined by high performance liquid chromatography for organic acid concentrations. Mice were given enemas containing organic acid at the mean concentration in the supernatants of cytotoxic strains to ascertain whether colonic lesions appear in UC. Results: Only supernatants from cultures of Fusobacterium varium killed Vero cells. Bacterial cells lacked verotoxin genes. Bacterial culture supernatants contained high concentrations of n-butyric acid and the mean concentration (32 mmol/l) was cytotoxic to Vero cells. Twenty four hours after mice were given enemas containing either butyric acid or F varium culture supernatants, colonic ulcers with crypt abscesses, inflammatory cell infiltration, and apoptotic changes were observed. Conclusions: Butyric acid in culture supernatants from cultures of F varium caused UC-like lesions in mice. This study indicates that F varium may be one of the elusive pathogenic factors in UC.

Aims/hypothesis In populations of East Asian descent, we performed a replication study of loci previously identified in populations of European descent as being associated with obesity measures such as BMI and type 2 diabetes. Methods We genotyped 14 single nucleotide polymorphisms (SNPs) from 13 candidate loci that had previously been identified by genome-wide association meta-analyses for obesity measures in Europeans. Genotyping was done in 18,264 participants from two general Japanese populations. For SNPs showing an obesity association in Japanese individuals, we further examined diabetes associations in up to 6,781 cases and 7,307 controls from a subset of the original, as well as from additional populations. Results Significant obesity associations ( p  < 0.1 two-tailed, concordant direction with previous reports) were replicated for 11 SNPs from the following ten loci in Japanese participants: SEC16B , TMEM18 , GNPDA2 , BDNF , MTCH2 , BCDIN3D – FAIM2 , SH2B1 – ATP2A1 , FTO , MC4R and KCTD15 . The strongest effect was observed at TMEM18 rs4854344 ( p  = 7.1 × 10 −7 for BMI). Among the 11 SNPs showing significant obesity association, six were also associated with diabetes (OR 1.05−1.17; p  = 0.04−2.4 × 10 −7 ) after adjustment for BMI in the Japanese. When meta-analysed with data from the previous reports, the BMI-adjusted diabetes association was found to be highly significant for the FTO locus in East Asians (OR 1.13; 95% CI 1.09−1.18; p  = 7.8 × 10 −10 ) with substantial inter-ethnic heterogeneity ( p  = 0.003). Conclusions/interpretation We confirmed that ten candidate loci are associated with obesity measures in the general Japanese populations. Six (of ten) loci exert diabetogenic effects in the Japanese, although relatively modest in size, and independently of increased adiposity.

Patients aged 60 years and older with essential hypertension were treated with an angiotensin-converting enzyme inhibitor (ACE-I), delapril (Adecut) or a long-acting calcium (Ca)-antagonist, manidipine (Calslot) for 3 years. The incidences of cardiovascular events as well as drug-related side effects were compared between the two groups to investigate whether both classes of antihypertensive drugs are beneficial in elderly hypertensive patients. There were no significant differences in characteristics of patients between the two intervention groups, except for slightly lower blood pressure (P = .08) in the Ca-antagonist group at the initiation of the study. There were no significant differences in total death between the two groups. Cardiovascular events (both fatal and nonfatal) were noted in 34 of 699 patients (22.5/1000 patient-years) in the ACE-I group and 50 of 1049 patients (19.7/1000 patient-years) in the Ca-antagonist group, with no significant difference found between the two groups. The correlation between cardiovascular incidence and the blood pressure attained during treatment showed a J-shaped phenomenon and suggests that an excessive reduction less than 120 mm Hg in systolic blood pressure (SBP) is unnecessary and may be harmful in certain cases. Side effects were more frequent in the ACE-I group than in the Ca-antagonist group (P = .01). Cough was the major adverse event, occurring in 5.0% of patients in the ACE-I group. In conclusion, the study indicates that both ACE-I (delapril) and Ca-antagonist (manidipine) were equally beneficial for reducing cardiovascular morbidity and mortality in elderly hypertensive patients. However, tolerability of ACE-I was lower due to the adverse event of coughing.

Aims/hypothesis Endothelial cells are considered to be essential for normal pancreatic beta cell function. However, there have been no reports showing their importance for beta cell function. Materials and methods Using mice with disrupted vascular endothelial growth factor-A gene specifically in beta cells, we investigated the relation between islet vascular structure and beta cell function. Results Mice with disrupted vascular endothelial growth factor-A gene specifically in beta cells had reduced islet vascular density with impaired formation of endothelial fenestration. While their fasting glucose and body weight were comparable with control mice, their glucose- and tolbutamide-induced rapid insulin release were impaired, thus resulting in glucose intolerance. On the other hand, glucose and KCl enhanced the levels of insulin secreted from islets isolated from these mice. In addition, the production of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in the islets was increased. Insulin content and expression of insulin I and pancreas duodenum homeobox 1 mRNA in the islets were also increased. Conclusions/interpretation Our results indicate that an abnormal quality and quantity of blood vessels due to reduced expression of vascular endothelial growth factor-A in beta cells could be a cause of impaired insulin secretion without impairment of beta cell function.

Aims/hypothesis To test fasting glucose association at four loci recently identified or verified by genome-wide association (GWA) studies of European populations, we performed a replication study in two Asian populations. Methods We genotyped five common variants previously reported in Europeans: rs1799884 (GCK), rs780094 (GCKR), rs560887 (G6PC2-ABCB11) and both rs1387153 and rs10830963 (MTNR1B) in the general Japanese (n = 4,813) and Sri Lankan (n = 2,319) populations. To identify novel variants, we further examined genetic associations near each locus by using GWA scan data on 776 non-diabetic Japanese samples. Results Fasting glucose association was replicated for the five single nucleotide polymorphisms (SNPs) at p < 0.05 (one-tailed test) in South Asians (Sri Lankan) as well as in East Asians (Japanese). In fine-mapping by GWA scan data, we identified in the G6PC2-ABCB11 region a novel SNP, rs3755157, with significant association in Japanese (p = 2.6 × 10⁻⁸) and Sri Lankan (p = 0.001) populations. The strength of association was more prominent at rs3755157 than that of the original SNP rs560887, with allelic heterogeneity detected between the SNPs. On analysing the cumulative effect of associated SNPs, we found the per-allele gradients (β = 0.055 and 0.069 mmol/l in Japanese and Sri Lankans, respectively) to be almost equivalent to those reported in Europeans. Conclusions/interpretation Fasting glucose association at four tested loci was proven to be replicable across ethnic groups. Despite this overall consistency, ethnic diversity in the pattern and strength of linkage disequilibrium certainly exists and can help to appreciably reduce potential causal variants after GWA studies.

Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells, and acts as a survival factor against endothelial cell death. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. We have previously reported that intra-arterial administration of recombinant HGF induced angiogenesis in a rabbit hindlimb ischemia model. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy, due to its disadvantages. Initially, we examined the transfection of 'naked' human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imaging, accompanied by the detection of human HGF protein. A significant increase in capillary density was found in rats transfected with human HGF as compared with control vector, in a dose-dependent manner (P < 0.01). Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid once on day 10 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (P < 0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-transfected animals. In addition, a significant increase in blood flow was assessed by a Doppler flow wire and the ratio in blood pressure of the ischemic limb to the normal limb was observed in rabbits transfected with HGF plasmid as compared with rabbits transfected with control vector (P < 0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hindlimb models, as potential therapy for peripheral arterial disease.

Impairment of cardiac function in ischemic cardiomyopathy has been postulated to be due to the decrease in blood flow and increase in collagen synthesis. Therefore, an approach to alter them directly by means of a growth factor may open up a new therapeutic concept in ischemic cardiomyopathy. From this viewpoint, hepatocyte growth factor (HGF) is a unique growth factor with angiogenic and antifibrotic effects. Thus, we examined the feasibility of gene therapy using HGF plasmid DNA for ischemic cardiomyopathy. Human HGF plasmid DNA at a dose of 0.4 or 4 mg was injected into ischemic myocardium of pigs induced by ameroid constrictor with the NOGA system. At 1 month after injection, the ischemic area was significantly reduced in the HGF group, accompanied by a significant increase in capillary density and regional myocardial perfusion in the ischemic area ( P <0.01). In contrast, a significant decrease in fibrotic area was observed in the HGF group, associated with a significant decrease in collagen I, III and TGF- β synthesis as compared to the control group ( P <0.01). Consistently, cardiac function was significantly improved in the 4 mg HGF group as compared to the control group ( P <0.05). Overall, the present in vivo experiments demonstrated that intramyocardial injection of human HGF plasmid DNA in ischemic cardiomyopathy resulted in a significant improvement in cardiac function through an increase in blood flow and decrease in fibrosis. These favorable outcomes suggest potential utility to treat patients with ischemic heart disease using HGF gene transfer. Currently, a phase I study using human HGF plasmid DNA is ongoing to test the validity of this concept.

The feasibility of a novel therapeutic strategy using angiogenic growth factors to expedite and/or augment collateral artery development has recently entered the realm of treatment of ischemic diseases. Hepatocyte growth factor (HGF) is a novel member of endothelium-specific growth factors whose mitogenic activity on endothelial cells is very potent. Although it has been demonstrated that HGF is a potential angiogenic growth factor in in vitro culture systems, there is no direct in vivo evidence for the angiogenic activity of HGF in physiological conditions. In this study, we hypothesized that transfection of HGF gene into infarcted myocardium could induce angiogenesis, potentially resulting in a beneficial response to hypoxia. Human HGF gene or control vector driven by the SRalpha promoter was transfected into rat myocardium by the HVJ-liposome method. Four days after in vivo transfection of human HGF gene, there was a marked increase in human immunoreactive HGF as compared with control vector (P < 0.01). In myocardium transfected with HGF vector, a significant increase in PCNA-positive endothelial cells was observed, while few PCNA-positive endothelial cells were detected in both control-vector-transfected and untreated myocardium. The number of vessels around the HGF injection sites was significantly increased as compared with control vector or vehicle (P < 0.01). Angiogenic activity induced by the transfection of HGF vector was also confirmed by the activation of a transcription factor, ets, which is essential for angiogenesis. Furthermore, we studied the pathophysiological role of HGF in a myocardial infarction model. The concentration of endogenous HGF was significantly decreased in infarcted myocardium. Therefore, we hypothesized that transfection of HGF gene into infarcted myocardium could induce a beneficial response to the decreased endogenous HGF. Indeed, transfection of human HGF into infarcted myocardium also resulted in a significant increase in the number of vessels (P < 0. 01), accompanied by marked induction of ets binding activity and a significant increase in blood flow. Overall, the present results provide direct in vivo evidence for the induction of angiogenesis by transfection of the human HGF gene in rat non-infarcted and infarcted myocardium. The constant production of local HGF resulting from the transgene may be considered as an innovative therapeutic angiogenesis strategy for ischemic diseases such as myocardial infarction. Gene Therapy (2000) 7, 417-427.