Accelerated endothelialization and suppressed thrombus formation of acellular vascular grafts by modifying with neointima-inducing peptide: A time-dependent analysis of graft patency in rat-abdominal transplantation model.

Acellular blood vessels have clinical potential as tissue-engineered vascular grafts. However, neointima is hard to form on their luminal surface. We recently reported the integrin α4ß1 ligand peptide (Arg-Glu-Asp-Val) conjugated with a repetitive Pro-Hyp-Gly sequence as luminal surface modifier. By using this peptide, excellent patency of tissue-engineered small-caliber long-bypass grafts in minipig transplantation model was achieved. Here, the time-dependent change of the graft patency is investigated by using rat abdominal transplantation model. In vitro test showed that 86% of the endothelial cells were adhered to the peptide-modified graft surface, while cells were scarcely adhered on the unmodified and random peptide-modified surfaces. After transplantation in the abdominal aorta, the patency of unmodified and random peptide-modified grafts gradually decreased during two to three weeks and reached 20-40% in four weeks. In contrast, 80% of the modified grafts were patent without any thrombus formation at four weeks. These results suggest that the luminal surface modifier was bound to acellular surface through (Pro-Hyp-Gly)7 sequence and improved the in vivo graft patency by endothelialization and thrombus formation suppression.

Micro-CT evaluation of high pressure-decellularized cardiovascular tissues transplanted in rat subcutaneous accelerated-calcification model.

We have succeeded in reducing the calcification of acellular aortas or valves in porcine allogeneic system by removing the DNA and phospholipids, but its further reduction is desirable. Here, the calcification of the acellular tissue was evaluated in rat subcutaneous transplantation model which is known as calcification model. Acellular samples prepared by high-hydrostatic pressure (HHP) protocols with different washing media were implanted and the calcification was monitored under micro-computed tomography for 1 and 3 months. The amount of the calcium deposition was quantitatively evaluated by atomic absorption spectroscopy. A cell culture medium showed very good cell removal ability but led to severe calcification at 1 month, and surprisingly the calcium deposition increased as the washing period increased. This calcification was suppressed by removing the DNA fraction with high DNase concentration. On the other hand, the calcification was greatly reduced when washed with saline even at low DNase concentration after 2 weeks washing. These results suggest that the ion species in the washing medium and the residual DNase cooperatively affect the tendency of in vivo calcification, which led us to the possibility of reduced calcification of acellular cardiac tissues.

Three-layer microfibrous peripheral nerve guide conduit composed of elastin-laminin mimetic artificial protein and poly(L-lactic acid).

We developed a microfibrous poly(L-lactic acid) (PLLA) nerve conduit with a three-layered structure to simultaneously enhance nerve regeneration and prevent adhesion of surrounding tissue. The inner layer was composed of PLLA microfiber containing 25% elastin-laminin mimetic protein (AG73-(VPGIG)30) that promotes neurite outgrowth. The thickest middle layer was constructed of pure PLLA microfibers that impart the large mechanical strength to the conduit. A 10% poly(ethylene glycol) was added to the outer layer to prevent the adhesion with the surrounding tissue. The AG73-(VPGIG)30 compositing of an elastin-like repetitive sequence (VPGIG)30 and a laminin-derived sequence (RKRLQVQLSIRT: AG73) was biosynthesized using Escherichia coli. The PLLA microfibrous conduits were fabricated using an electrospinning procedure. AG73-(VPGIG)30 was successfully mixed in the PLLA microfibers, and the PLLA/AG73-(VPGIG)30 microfibers were stable under physiological conditions. The PLLA/AG73-(VPGIG)30 microfibers enhanced adhesion and neurite outgrowth of PC12 cells. The electrospun microfibrous conduit with a three-layered structure was implanted for bridging a 2.0-cm gap in the tibial nerve of a rabbit. Two months after implantation, no adhesion of surrounding tissue was observed, and the action potential was slightly improved in the nerve conduit with the PLLA/AG73-(VPGIG)30 inner layer.

Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.

Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous V(H) and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in V(H) genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the V(H) gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.

Preparation of poly(vinyl alcohol)/DNA hydrogels via hydrogen bonds formed on ultra-high pressurization and controlled release of DNA from the hydrogels for gene delivery.

Poly(vinyl alcohol) (PVA) hydrogels interacting with DNA mediated by hydrogen bonds (PVA/DNA hydrogel) were developed using ultra-high pressure (UHP) technology. The goal was to create a new method of gene delivery by controlled release of DNA. Mixed solutions of DNA and PVA at various concentrations were pressurized at 10,000 atmospheres at 37 degrees C for 10 min. PVA/DNA hydrogels with good formability were produced at PVA concentrations of more than 5% w/v. The presence of DNA in the obtained hydrogels was confirmed by spectroscopic analysis and nucleic acid dye staining. DNA release from the hydrogels was investigated using PVA/DNA hydrogel samples of 5% and 10% w/v formed by UHP treatment or by conventional freeze-thaw methods. The DNA release curves from both types of samples showed a rapid phase in the initial 15 h followed by a sustained release phase. However, there was a difference in the amount of DNA released. Less DNA was released by the pressurized hydrogels than by the freeze-thaw hydrogels. Also, the cumulative amount of DNA released decreased as the PVA content in the hydrogels increased. These results indicate that DNA release from the hydrogels can be modulated by changing the preparation method and the PVA content. Furthermore, it was demonstrated that DNA release could be controlled by varying the amount and duration of pressurizing used to form the hydrogels. Intact fractions of plasmid DNA released from the hydrogels were separated by agarose gel electrophoretic analysis. These results suggest that, using controlled release, DNA from PVA/DNA hydrogels formed by UHP treatment can be transfected into cells.

Myocardial glucose utilisation in type II diabetes mellitus patients treated with sulphonylurea drugs.

PURPOSE: Chronic sulphonylurea treatment maintains improved glycaemic control through mechanisms other than enhancement of insulin secretion and may act on various organs. The aim of this study was to investigate whether the chronic use of sulphonylurea drugs influences PET measurement of myocardial glucose utilisation (MGU) in type II diabetes mellitus. METHODS: Forty-two patients with type II diabetes mellitus and 17 control subjects underwent dynamic (18)F-FDG PET to measure MGU during hyperinsulinaemic euglycaemic clamping. Twenty-one patients had been taking sulphonylurea drugs for more than 1 year (SU group), and the other 21 patients were drug naive (non-SU group). The haemoglobin A1c levels in the two patient groups were similar. Glucose disposal rate (GDR) was also determined as a marker of whole-body insulin resistance. RESULTS: GDR in the SU group (9.01+/-2.53 mg min(-1) kg(-1)) was significantly higher than that in the non-SU group (4.10+/-2.47, p<0.01) and was similar to that in the controls (9.76+/-2.97). MGU in the SU group (7.66+/-3.02 mg min(-1) 100 g(-1)) was significantly higher than that in the non-SU group (5.53+/-2.05, p<0.01) and was similar to that in the controls (7.49+/-2.74). CONCLUSION: Chronic sulphonylurea treatment influences MGU independent of the degree of glycaemic control. The effect of medication should be kept in mind when measuring and interpreting MGU in patients with type II diabetes mellitus.

Measurement of skeletal muscle glucose utilization by dynamic 18F-FDG PET without arterial blood sampling.

OBJECTIVE: Skeletal muscle glucose utilization (SMGU) can be measured by 18F-FDG PET to characterize insulin resistance. The aim of this study was to determine whether femoral muscle SMGU can be measured without arterial blood sampling by sequential PET imaging of the thoracic and femoral regions. METHODS: Ten patients with possible insulin resistance underwent dynamic 18F-FDG PET of the femoral region during hyperinsulinaemic euglycaemic clamping (group A), and femoral muscle SMGU was calculated using PET data of various time periods and measured arterial input. SMGU was also calculated using venous plasma activity, instead of arterial activity, as input during the late phase. Another five patients underwent sequential PET of the thoracic and femoral regions after single tracer injection (group B). The input function was estimated from aorta activity on thoracic images during the early phase and from venous activity during the late phase, and SMGU with this estimated input was compared with that with measured arterial input. RESULTS: In group A, exclusion of early dynamic PET data from analysis had essentially no effect on the calculated SMGU, and partial substitution of venous activity for arterial activity only marginally changed the estimates. The difference between SMGUs with measured and estimated inputs was minimal in group B. CONCLUSION: Femoral muscle SMGU can be calculated without femoral imaging early after tracer injection, and the input function can be assessed using data of thoracic imaging and venous blood samples. These results support the validity of measuring femoral muscle SMGU without arterial sampling, simultaneously with measurement of myocardial glucose utilization.

Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis.

NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.

Impaired myocardial vasodilatation during hyperaemic stress is improved by simvastatin but not by pravastatin in patients with hypercholesterolaemia.

AIMS: Impaired myocardial vasodilatation during hyperaemic stress with dipyridamole has been documented in hypercholesterolaemics without evidence of ischaemia. This study investigated whether two commonly used hydroxymethylglutaryl coenzyme A reductase inhibitors, simvastatin and pravastatin, are equally effective in restoring myocardial vasodilatation. METHODS AND RESULTS: Forty-four hypercholesterolaemics with a low probability of coronary artery disease and 22 controls were studied. Before and after lipid-lowering therapy with simvastatin (n = 22) or pravastatin (n = 22), myocardial blood flow at rest and during dipyridamole loading was measured using positron emission tomography with [(13)N]ammonia, and myocardial vasodilatation was assessed. Treatments with simvastatin and pravastatin similarly reduced plasma total cholesterol and plasma low-density lipoprotein cholesterol. Resting myocardial blood flow was comparable in the controls, simvastatin group, and pravastatin group and unchanged after therapy. Myocardial blood flow during dipyridamole loading and myocardial vasodilatation was lower in the two therapy groups before treatment than in the controls. These parameters improved significantly after therapy with simvastatin, whereas no improvement was observed after pravastatin therapy. The per cent change in myocardial vasodilatation after simvastatin therapy was significantly and inversely correlated with per cent changes in plasma lipid fractions. CONCLUSION: Diminished myocardial vasodilatation in hypercholesterolaemics is improved by simvastatin but not by pravastatin, suggesting differences in vascular effects among statins.

Simple quantification of skeletal muscle glucose utilization by static 18F-FDG PET.

UNLABELLED: Skeletal muscle glucose utilization (SMGU) can be measured by dynamic PET imaging with (18)F-FDG to characterize insulin resistance. The aim of this study was to determine the validity of simple methods to quantify SMGU by static PET imaging. METHODS: Ten patients underwent dynamic (18)F-FDG PET of the femoral region during hyperinsulinemic euglycemic clamping. SMGU was determined by Patlak graphical analysis using data from dynamic imaging with frequent arterial blood sampling. Standardized uptake values (SUVs) were calculated at 45 and 55 min after tracer injection. Skeletal muscle-to-background ratio (SM/B ratio), tissue count divided by venous plasma activity, was also computed at 45 and 55 min. These simple indices were compared by linear regression with the SMGU measured as above, and an estimated SMGU was obtained using the regression equation thus generated, together with a simple index. RESULTS: SMGU was highly correlated with SUVs (r = 0.941 at 45 min, r = 0.951 at 55 min) and SM/B ratios (r = 0.968 at 45 min, r = 0.984 at 55 min). Although SMGU was almost proportional to SM/B ratios, the y-intercepts of the regression lines for SUVs significantly differed from zero. The residual in estimating SMGU using the regression equation was marginally smaller for SM/B ratios than for SUVs and for indices at 55 min than at 45 min, but these differences did not reach statistical significance. Correction for plasma glucose level slightly elevated the correlation coefficients between SMGU and these simple indices. CONCLUSION: It is proposed that the simple quantitative indices, SUV and the SM/B ratio, are reliable indicators of SMGU during hyperinsulinemic euglycemic clamping. Static imaging with or without a single venous blood sampling may therefore be able to replace dynamic imaging with frequent arterial blood sampling, offering substantially greater convenience in evaluating insulin resistance.