Eisuke Furuya

Implantation of Mesenchymal Stem Cells Overexpressing Endothelial Nitric Oxide Synthase Improves Right Ventricular Impairments Caused by Pulmonary Hypertension

Background— Pulmonary hypertension (PH) is a life-threatening disease. Bone marrow cell transplantation is reported to reduce the development of PH by increasing vascular beds in pulmonary circulation. However, adenoviral overexpression of endothelial nitric oxide synthase (eNOS) in the lung is also known to reduce PH. Because mesenchymal stem cells (MSCs) are potential cell sources for neovascularization, the implantation of MSCs overexpressing eNOS (MSCs/eNOS) may further improve the surgical results. We evaluated the efficacy of MSCs/eNOS implantation in monocrotaline (MCT)-induced PH rats. Methods and Results— MSCs were isolated from rat bone marrow. PH was induced in rats by subcutaneous injection of MCT. One week after MCT administration, the rats received 3 different treatments: MSCs (MSC group), MSCs/eNOS (MSC/eNOS group), or nontreatment (PH group). As the negative control, rats received saline instead of MCT (control group). Right ventricular (RV) hypertrophy and the elevation of RV systolic pressure (RVSP) were evaluated 3 weeks after MCT administration. Moreover, the effects of MSCs/eNOS on survival were investigated in PH induced by MCT 3 weeks earlier. RVSP in both the MSC and MSC/eNOS groups was significantly lower than the PH group. RVSP in the MSC/eNOS group was significantly lower than the MSC group. The RV weight to body weight ratio was significantly lower in the MSC and MSC/eNOS groups than the PH group. The survival time of rats receiving MSCs/eNOS was significantly longer than the nontreatment rats. Conclusion— Intravenous implantation of MSCs/eNOS may offer ameliorating effects on PH-related RV impairment and survival time.

Keloids have continuous high metabolic activity

The adenosine triphosphate (ATP) content of keloids and scars resected from patients was demonstrated by high-performance liquid chromatography. The surface color of hypertrophic scars was red or pink and that of atrophic scars was white. The ATP content of red scars was (in mmol/g protein) 1.06 +/- 0.14, of pink scars 0.12 +/- 0.02, of white atrophic scars 0.19 +/- 0.06, and of keloids 1.06 +/- 0.19. The longer the elapsed time after the trauma, the lower the level of ATP in scar tissues (correlation coefficient = -0.506; p = 0.005 by Spearmans rank correlation). However, ATP levels in keloids were still high 10 years after the injury. Fibroblasts and fibrocytes in keloids and scars were counted in histologic preparations stained with hematoxylin and eosin. The average number of fibroblasts in a definite area (56 x 10(-4) mm at a magnification of x400) was 4.8 in keloids, 5.1 in red scars, 2.4 in pink scars, and 1.3 in white atrophic scars. The number of fibrocytes in the same area was 0.4 in keloids, 0.4 in red scars, 2.3 in pink scars, and 1.3 in white atrophic scars. These results indicate that keloids and red hypertrophic scars have higher ATP levels and contain more fibroblasts than pink or white scars, and they also suggest that the levels of ATP and the number of fibroblasts decrease when red hypertrophic scars change into atrophic scars. In keloids, ATP and fibroblasts seem to remain at high levels for a long time.

Novel Isoforms of Rat Brain Fructose 6‐Phosphate 2‐Kinase/Fructose 2,6‐Bisphosphatase Are Generated by Tissue‐Specific Alternative Splicing

Abstract: Fructose 6‐phosphate 2‐kinase/fructose 2,6‐bisphosphatase catalyzes the synthesis and degradation of fructose 2,6‐bisphosphate, which is the most potent activator of glycolysis. We have shown previously the occurrence of a partial cDNA (RB7) encoding the catalytic core domain of a novel brain‐type isozyme. To elucidate the full‐length sequence of RB7 cDNA, we have carried out cDNA cloning using the 3′‐ and 5′‐rapid amplification cDNA ends method and have isolated eight isoforms from rat brain. The cDNA sequences encoding the 5′‐untranslated region, the amino‐terminal domain, and the catalytic core domain were identical among all the isoforms. However, heterogeneity of the carboxyl‐terminus was found by sequence analysis. This heterogeneity was shown not to have resulted from transcription of multiple genes, as Southern blot and genomic sequence analysis revealed that the gene was a single copy in the rat genome. It is likely that these transcripts represent splice variants of the gene. High‐level expression of the gene was also observed by northern blot analysis in skeletal muscle. However, the pattern of alternative splicing was different from that of brain, and only four isoforms were detected in skeletal muscle.

Beneficial effect of fructose-1,6-bisphosphate on mitochondrial function during ischemia-reperfusion of rat liver

BACKGROUND/AIMS Several groups have reported that administration of fructose-1,6-bisphosphate (FBP) reduces ischemic injury. The aim of this study was to determine the protective effect of FBP on the impairment of mitochondrial oxidative phosphorylation by ischemia-reperfusion injury in the rat liver. METHODS The respiratory control ratio (RCR) and the adenine nucleotide content of mitochondria isolated from ischemic and reperfused livers with or without FBP treatment were measured. RESULTS In FBP-treated livers, the cellular adenosine triphosphate level was restored to more than 50% of normal after 120 minutes of reperfusion following 120 minutes of ischemia, whereas that of control livers only reached 15% of normal. The RCR and the adenine nucleotide content of mitochondria isolated from FBP-treated livers were significantly higher than those of mitochondria from control livers after ischemia and reperfusion. FBP strongly suppressed the formation of lipid peroxides during reperfusion. In vitamin E-deficient rats, the RCR decreased markedly during reperfusion, but FBP protected the mitochondria against reperfusion injury. CONCLUSIONS FBP has a protective effect against ischemia-reperfusion injury on the liver and especially preserves the oxidative phosphorylation capacity of hepatic mitochondria.

Inadequate blood supply persists in keloids

We have previously shown that the adenosine triphosphate (ATP) content of keloids remains high for a long time. In keloids, anaerobic glycolysis may be related to the production of ATP. In this study, we counted the vessels in keloids, hypertrophic and atrophic scars in a defined area, and measured the cross-sectional areas of their internal lumens immunohistopathologically and the lactate concentrations. The mean number in a defined area was smallest in keloids as was the mean internal area of vessels. The lactate content of keloids was 39.4 (13.5) mmol/g of protein, of red scars 23.8 (7.5), of pink scars 23.8 (7.6), and of white scars 13.3 (7.3). These results indicate that ATP may be produced by anaerobic glycolysis in keloids, and that the decreased and narrowed vessels may reduce oxygen perfusion. The blood supply to keloids is inadequate, and this persists.

Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.

To identify the proteins involved in 5-fluorouracil (5-FU) resistance, a comparison of the total and phosphorylated proteins between the human colorectal cancer (CRC) cell line DLD-1 and its 5-FU-resistant subclone DLD-1/5-FU was performed. Using 2-DE and MALDI-TOF/TOF-based proteomics, 17 up-regulated and 19 down-regulated protein spots were identified in the 5-FU-resistant DLD-1/5-FU cells compared with the parent cell lines. In DLD-1/5-FU cells, 7 anti-apoptotic proteins (HSPB1, proteasome subunit α-5, transitional endoplasmic reticulum ATPase, 14-3-3 β, 14-3-3 γ, 14-3-3 σ, and phosphoglycerate kinase 1) were up-regulated and 4 proapoptotic proteins (cofilin-1, pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, and nucleophosmin) were down-regulated. The results show that the acquired drug resistance of DLD-1/5-FU cells is caused by the prevention of drug-induced apoptosis, in particular through the enhanced constitutive expression of HSPB1 and its phosphorylated form. Short interfering RNA knockdown of endogenous HSPB1 in DLD-1/5-FU cells restored the sensitivity to 5-FU. Furthermore, MALDI-TOF/TOF and 2-DE Western blot analysis identified the phosphorylated residues of HSPB1 as Ser-15 and Ser-82 in the main (diphosphorylated) form and Ser-15, Ser-78, and Ser-82 in the minor (triphosphorylated) form. The current findings indicate that phosphorylated HSPB1 may play an important role in 5-FU resistance.

Tissue-specific alternative splicing of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase.

We have reported the occurrence of eight splice variants of rat brain fructose 6‐phosphate 2‐kinase/fructose 2,6‐bisphosphatase (RB2K). In the present study, we quantified these splice variants in various tissues using a RNAse protection assay and found a tissue‐specific pattern of alternative splicing of the RB2K gene. Splice variants containing exon F were specifically expressed in brain. Moreover, exons D and E were spliced in brain, skeletal muscle and heart. Consequently, eight, six, four and two splice variants were expressed in brain, skeletal muscle, heart and liver plus testis, respectively. These results suggest that distinct RB2K isoforms could be involved in regulation of glycolysis in a tissue‐specific manner.

Synthetic vascular prosthesis impregnated with mesenchymal stem cells overexpressing endothelial nitric oxide synthase.

Background— Endothelial dysfunction is known to exaggerate coronary artery disease, sometimes leading to irreversible myocardial damage. In such cases, repetitive coronary revascularization including coronary artery bypass grafting is needed, which may cause a shortage of graft conduits. On the other hand, endothelial nitric oxide synthase (eNOS) is an attractive target of cardiovascular gene therapy. The vascular prostheses, of which the inner surfaces are covered with mesenchymal stem cells (MSCs) overexpressing eNOS, are expected to offer feasible effects of NO and angiogenic effects of MSCs on the native coronary arterial beds, as well as improvement of self-patency. Herein, we attempted to develop small caliber vascular prostheses generating the bioactive proteins. Also, we attempted to transduce eNOS cDNA into MSCs. Methods and Results— The MSCs were isolated from rat bone marrow and transduced with each adenovirus harboring rat eNOS cDNA and &bgr;-galactosidase (&bgr;-gal) (eNOS/MSCs and &bgr;-gal/MSCs). The &bgr;-gal/MSCs were impregnated into vascular prostheses, then the expressions of &bgr;-gal on the inner surfaces of them were evaluated by 5-bromo-4-chloro-3-indolyl &bgr;-d-galactoside staining. The NOS activity of eNOS/MSCs was assayed by monitoring the conversion of 3H-arginine to 3H-citrulline. The inner surfaces of the vascular prostheses were covered with MSCs expressing &bgr;-gal. The amount of the 3H-citrulline increased, and eNOS/MSCs were determined to generate enzymatic activity of eNOS. This activity was completely inhibited by NG-nitro-l-arginine methyl ester. Conclusions— The inner surface of expanded polytetrafluoroethylene vascular prostheses seeded with lacZ gene-transduced MSCs exhibited recombinant proteins. Development of eNOS/MSC-seeded vascular prostheses would promise much longer graft patency and vasculoprotective effects.

A new method of quantitating serum and urinary levels of 1,5-anhydroglucitol in insulin-dependent diabetes mellitus

A new method was developed for quantitating the serum and urinary levels of 1,5-anhydroglucitol (AG), a sensitive and informative marker of glycemic control. This method utilized a combination of ODS and pyranose oxidase-immobilized columns for HPLC, and monitored hydrogen peroxide production with an electrochemical detector. We applied this method to determine the serum and urinary AG levels in 15 patients with insulin-dependent diabetes mellitus (IDDM) as well as in control subjects. Baseline separation of AG from other sugars such as glucose and myoinositol was achieved. Quantitation of AG was achieved over the range from 0.2 ng to 0.3 micrograms based upon peak heights. The serum and urinary AG levels in the IDDM patients were 4.4 +/- 8.3 mg/l and 5.1 +/- 4.3 mg/day, respectively. We found that the urinary AG to serum AG ratio showed a linear correlation with the urinary glucose level in the IDDM patients (urinary glucose (y) vs. urinary AG to serum AG ratio (x): y = 9.071x-0.991; r = 0.968, P < 0.001). This method proved efficient and reliable for quantitating urinary AG. Since determination of both the AG and glucose levels in urine gives equivalent clinical information to the serum AG level, urinary monitoring could provide a valuable addition to the available methods for assessing the glycemic status of IDDM patients.

Activation of β2-adrenergic receptor plays a pivotal role in generating the protective effect of ischemic preconditioning in rat hearts

Background. Ischemic preconditioning (IPC) protects hearts against ischemia by reducing infarct size. However, IPC does not preserve cardiac function, such as left ventricular peak developed pressure (LVPDP). Moreover, IPC fails to protect the post-myocardial infarct (MI) heart. Design. Rat hearts were transfected with β2-adrenergic receptor (B2AR) cDNA by the hemagglutinating virus of Japan–liposome method. After the gene transfer, the hearts were perfused in a Langendorff mode and preconditioned with two cycles of 5 min of ischemia and reperfusion. After 20 min of global ischemia, the hearts were reperfused under aerobic conditions for 90 min. LVPDP was measured as an indicator of the cardiac function. Results. LVPDP of ischemic hearts was well preserved by the combination treatment of IPC and gene transfer of B2AR, but not IPC or gene transfer of B2AR alone. Moreover, the treatment was beneficial to even the post-MI heart. On the contrary, gene transfer of β-adrenergic receptor kinase 1 (BARK1) reduced the protective effect of IPC. We also found that the mRNA ratio of B2AR and BARK1 was well correlated with the preservation of the LVPDP. Conclusions. The combination treatment of IPC and gene transfer of B2AR protects cardiac function against ischemia and it shows the beneficial effect also in post-MI hearts.