Toshiaki Ishizuka

PHOTOCROSSLINKABLE CHITOSAN HYDROGEL CONTAINING FIBROBLAST GROWTH FACTOR-2 STIMULATES WOUND HEALING IN HEALING-IMPAIRED DB/DB MICE

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) resulted within 30s in an insoluble, flexible hydrogel. About 20% of the FGF-2molecules were released from the FGF-2-incorporated chitosan hydrogel into phosphate buffered saline (PBS) within 1 day, after which no further significant release occurred under in vitro non-degradation conditions of the hydrogel. The FGF-2molecules retained in the chitosan hydrogel remained biologically active, and were released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In order to evaluate its accelerating effect on wound healing, full thickness skin incisions were made on the back of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure in both db/db and db/+ mice. However, the addition of FGF-2 in the chitosan hydrogel further accelerated wound closure in db/db mice, although not in db/+ mice. Histological examination also has demonstrated an advanced granulation tissue formation, capillary formation and epithelialization in wounds treated with FGF-2-incorporated chitosan hydrogels in db/db mice.

Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source.

Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivowas examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs.

Vascularization in vivo caused by the controlled release of fibroblast growth factor-2 from an injectable chitosan/non-anticoagulant heparin hydrogel

Addition of various heparinoids to the lactose-introduced, water-soluble chitosan (CH-LA) aqueous solution produces an injectable chitosan/heparinoid hydrogel. In the present work, we examined the capability of the chitosan/non-anticoagulant heparin (periodate-oxidized (IO(4)-) heparin) hydrogel to immobilize fibroblast growth factor (FGF)-2, as well as the controlled release of FGF-2 molecules from the hydrogel in vitro and in vivo. The hydrogel was biodegraded in about 20 days after subcutaneous injection into the back of a mouse. When the FGF-2-incorporated hydrogel was subcutaneously injected into the back of both mice and rats, a significant neovascularization and fibrous tissue formation were induced near the injected site. These results indicate that the controlled release of biologically active FGF-2 molecules is caused by biodegradation of the hydrogel, and that subsequent induction of the vascularization occurs.

Increased endothelin-1 production in fibroblasts derived from patients with systemic sclerosis.

OBJECTIVES--To determine whether fibroblasts from patients with systemic sclerosis (SSc) produce excessive amounts of endothelin-1 (ET-1), which is recognised as having vasoconstrictive properties and as having a potent mitogenic effect on fibroblasts. METHODS--Dermal fibroblasts were removed from 11 patients with SSc and from five normal controls (NC). The assay of ET-1 protein was measured by an ELISA that used two anti-ET-1 antibodies. The gene expression of prepro ET-1 mRNA was evaluated by a reverse-transcriptase polymerase chain reaction (RT-PCR) method. RESULTS--Levels of ET-1 protein were significantly higher in SSc fibroblast cultures than in those of normal fibroblasts (p < 0.01). The expression of prepro ET-1 mRNA was also higher in SSc fibroblasts than in normal fibroblasts. The addition of interleukin-1 beta (IL-1 beta) increased the production of ET-1 by fibroblasts. CONCLUSION--The findings indicate that the overproduction of ET-1 is a novel abnormal function in SSc fibroblasts, and that ET-1 induced by fibroblasts may play a role in the fibrosis and Raynauds phenomenon of SSc.

Endothelin-1 enhances vascular cell adhesion molecule-1 expression in tumor necrosis factor α-stimulated vascular endothelial cells

Vascular cell adhesion molecule-1 (VCAM-1) is a mononuclear leukocyte-selective adhesion molecule that is expressed in human vascular endothelial cells at sites of local inflammation. It participates in local endothelial-monocyte interactions during the initiation of atherosclerosis. In the present study, endothelin alone did not induce the surface expression and mRNA accumulation of VCAM-1 in human vascular endothelial cells, but inhibition of endogenous nitric oxide (NO) by N(G)-monomethyl-L-arginine enhanced the surface expression and mRNA accumulation of VCAM-1 stimulated by endothelin-1. It is conceivable that in human vascular endothelial cells, stimulation of an endothelin receptor results in the production of nitric oxide (NO), suppressing the expression of VCAM-1. Endothelin-1 enhanced the surface expression and mRNA accumulation of VCAM-1 in cells treated with tumor necrosis factor alpha (TNF-alpha). The enhancement by endothelin-1 may be explained by the inhibitory effect of TNF-alpha on endothelin-induced NO production. Pretreatment with BQ788 (an endothelin ET(B) receptor antagonist) or inhibitors of nuclear factor kappa B (NF-kappaB) activation completely diminished the synergistic enhancement of VCAM-1 expression by endothelin-1 in TNF-alpha-stimulated vascular endothelial cells, both at the protein and mRNA levels. These findings suggest that the synergistic enhancement of VCAM-1 expression by TNF-alpha and endothelin ET(B) receptor stimulation may be augmented by the induction of NF-kappaB binding activity in human vascular endothelial cells.

Stimulation with thromboxane A2 (TXA2) receptor agonist enhances ICAM-1, VCAM-1 or ELAM-1 expression by human vascular endothelial cells

A previous study reported that intercellular adhesion molecule‐1 (ICAM‐1) expression by human vascular endothelial cells (HUVEC) is augmented by intracellular signal transmission mainly through the protein kinase C (PKC) system stimulated by TXA2 receptors. In the present study, we show that a TXA2 receptor agonist, U46619, augments the expression of not only ICAM‐1, but also vascular cell adhesion molecule‐1 (VCAM‐1) or endothelial leucocyte adhesion molecule‐1 (ELAM‐1) in HUVEC both at protein and mRNA levels. Pretreatment with SQ29,548 (a TXA2 receptor antagonist) or PKC inhibitors greatly diminished the extent of U46619‐induced mRNA accumulation and surface expression of the adhesion molecules. An inhibitor of nuclear factor κB (NF‐κB) activation, PDTC, diminishes U46619‐induced VCAM‐1 mRNA accumulation. NAC, which inhibits NF‐κB and activation protein 1 (AP‐1) binding activity, inhibits the expression of ICAM‐1 or ELAM‐1 at protein and mRNA levels. These findings suggest that ICAM‐1 or ELAM‐1 expression of HUVEC stimulated via TXA2 receptors is augmented by induction of NF‐κB and AP‐1 binding activity through the PKC system, and that VCAM‐1 expression is augmented by induction of NF‐κB binding activity.

Filtration leukocytapheresis therapy in rheumatoid arthritis: A randomized, double‐blind, placebo‐controlled trial

OBJECTIVE To determine the efficacy and safety of filtration leukocytapheresis (LCP) for the treatment of rheumatoid arthritis (RA). METHODS Twenty-five patients with drug-resistant RA were randomly assigned to undergo filtration LCP and 7 to undergo sham apheresis (control group) in a randomized, double-blind, placebo-controlled study. Three apheresis procedures were performed, with 1-week intervals between procedures. The efficacy of filtration LCP was evaluated according to the American College of Rheumatology definition of improvement in RA. Medications for each patient were unchanged for at least 6 months prior to enrollment and throughout the study. RESULTS Tender joint counts, swollen joint counts, patient assessment of pain and global severity, physician assessment of global severity, and Health Assessment Questionnaire Disability Index were significantly improved in the LCP group compared with the control group (P < 0.05 for patient assessment of pain; P < 0.01 for all others). Seventy-nine percent of the patients in the LCP group exhibited significant overall improvement, while none of the patients in the control group were improved (P < 0.001). CONCLUSION The results indicate that filtration LCP is an effective and well-tolerated treatment for patients with drug-resistant RA.

Thromboxane A2 receptor blockade suppresses intercellular adhesion molecule-1 expression by stimulated vascular endothelial cells

Inhibition of the thromboxane A2-synthesizing enzyme (DP-1904: [+/-]-6-[1-imidazolylmethyl]-5,6,7,8-tetrahydronaphthalene-2-carbo xylic acid hydrochloride hemihydrate) reportedly suppresses intercellular adhesion molecule-1 (ICAM-1) expression on the surface of stimulated vascular endothelial cells (Ishizuka et al., 1994, Eur. J. Pharmacol 262, 113). In the present study, thromboxane A2 receptor antagonists suppressed the expression of ICAM-1 on the surface of human vascular endothelial cells that were stimulated by tumor necrosis factor alpha (TNF alpha), platelet activating factor (PAF), or U46619 (9,11-dideoxy-9 alpha, 11 alpha-epoxymethanoprostaglandin F2 alpha). Augmentation of ICAM-1 expression on human vascular endothelial cells stimulated by U46619 was suppressed by protein kinase C inhibitors. Thromboxane A2 receptor antagonist suppressed U46619 stimulation of protein kinase C activity of a cell membrane fraction. These results indicate that in human vascular endothelial cells, thromboxane A2, the production and secretion of which is stimulated by TNF alpha or PAF, binds to the thromboxane A2 receptors on cell membranes and augments ICAM-1 expression on the cell surfaces mainly through protein kinase C.

A novel method to prevent secondary exposure of medical and rescue personnel to toxic materials under biochemical hazard conditions using microwave radar and infrared thermography

In order to prevent secondary exposure of medical personnel to toxic materials under biochemical hazard conditions, we performed a noncontact determination of exposure to toxic conditions via 1215-MHz microwave radar and thermography. A toxic condition was induced by intravenous administration of lipopolysaccharide (LPS) in rabbits. The exposure to LPS was determined by linear discriminant analysis using noncontact derived variables.

Hyaluronan production in human rheumatoid fibroblastic synovial lining cells is increased by interleukin 1β but inhibited by transforming growth factor β1

OBJECTIVES To investigate the regulatory roles of interleukin 1β (IL1β), tumour necrosis factor α (TNFα), interferon γ (IFNγ) or transforming growth factor β1 (TGFβ1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells. METHODS Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1β, TNFα, IFNγ or TGFβ1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography. RESULTS HA synthesis by the cells was not significantly augmented by TNFα or by IFNγ. It was significantly stimulated by IL1β but inhibited by TGFβ1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNFα. They were remarkably increased by stimulation with IL1β and IFNγ, but reduced with TGFβ1. CONCLUSION IL1β is an up regulator of HA synthesis, while TGFβ1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.