Aika Yamawaki-Ogata

Novel Small-Caliber Vascular Grafts With Trimeric Peptide for Acceleration of Endothelialization

BACKGROUND Both rapid endothelialization and the prevention of intimal hyperplasia are essential to improve the patency of small-caliber vascular grafts (SCVGs). Using the peptide array based screening system, we identified the peptide CAG (cysteine-alanine-glycine), which has a high affinity for endothelial cells and a low adhesive property for smooth muscle cells (SMCs). In this article, we report an in vivo analysis of novel vascular grafts that were constructed with a biodegradable polymer (poly-ε-caprolactone [PCL]) containing CAG peptide. METHODS The novel SCVG, which measured 0.7 mm in diameter and 7 mm in length, was fabricated using the electrospinning technique. Carotid arterial replacement was performed on Sprague-Dawley rats using SCVGs with (group CAG) or without CAG (group C). Histologic and biochemical assessments were performed at 1, 2, and 6 weeks after implantation. RESULTS The ratio of endothelialization was significantly higher in group CAG compared with group C (CAG versus C, 64.4±20.0% versus 42.1±8.9% at 1 week; p=0.017; 98.2±2.3% versus 72.7±12.9% at 2 weeks; p=0.001; and 97.4±4.6% versus 76.7±5.4% at 6 weeks; p<0.001). Additionally, Western blot analysis showed that the level of endothelial nitric oxide synthase (eNOS) at 1 week in group CAG was significantly higher than that in group C (CAG versus C, 1.20±0.37 versus 0.34±0.16; p=0.013), and that α-smooth muscle actin (ASMA) at 6 weeks in group CAG was significantly lower than that in group C (CAG versus C, 0.89±0.06 versus 1.25±0.22; p=0.04). CONCLUSIONS The graft with CAG promoted rapid endothelialization and the potential for inhibition of intimal hyperplasia.

Intravenous administration of mesenchymal stem cells prevents angiotensin II-induced aortic aneurysm formation in apolipoprotein E-deficient mouse

BackgroundMesenchymal stem cells (MSCs) are known to be capable of suppressing inflammatory responses. We previously reported that intra-abdominal implantation of bone marrow-derived MSCs (BM-MSCs) sheet by laparotomy attenuated angiotensin II (AngII)-induced aortic aneurysm (AA) growth in apolipoprotein E-deficient (apoE−/−) mice through anti-inflammation effects. However, cell delivery by laparotomy is invasive; we here demonstrated the effects of multiple intravenous administrations of BM-MSCs on AngII-induced AA formation.MethodsBM-MSCs were isolated from femurs and tibiae of male apoE−/− mice. Experimental AA was induced by AngII infusion for 28 days in apoE−/− mice. Mice received weekly intravenous administration of BM-MSCs (n=12) or saline (n=10). After 4 weeks, AA formation incidence, aortic diameter, macrophage accumulation, matrix metalloproteinase (MMP)’ activity, elastin content, and cytokines were evaluated.ResultsAngII induced AA formation in 100% of the mice in the saline group and 50% in the BM-MSCs treatment group (P < 0.05). A significant decrease of aortic diameter was observed in the BM-MSCs treatment group at ascending and infrarenal levels, which was associated with decreased macrophage infiltration and suppressed activities of MMP-2 and MMP-9 in aortic tissues, as well as a preservation of elastin content of aortic tissues. In addition, interleukin (IL)-1β, IL-6, and monocyte chemotactic protein-1 significantly decreased while insulin-like growth factor-1 and tissue inhibitor of metalloproteinases-2 increased in the aortic tissues of BM-MSCs treatment group.ConclusionsMultiple intravenous administrations of BM-MSCs attenuated the development of AngII-induced AA in apoE−/− mice and may become a promising alternative therapeutic strategy for AA progression.

A doxycycline loaded, controlled-release, biodegradable fiber for the treatment of aortic aneurysms.

The pathogenesis of aortic aneurysm (AA) is characterized by degradation of extracellular matrix with increased matrix metalloproteinases (MMPs) and inflammatory reaction. Doxycycline (DOXY) has been reported to control the extension of AA by regulation of MMP. However, systemic administration may cause adverse side effects. In this study, we demonstrated the possibility of local administration of DOXY controlled-release biodegradable fiber (DCRBF) for AA in mice. DCRBF was fabricated by biodegradable polymer (polylactic acid; PLA) mixed with DOXY using an electrospinning technique. DCRBF was cocultured with SMCs, macrophages and aortic tissue, and placed on an abdominal aortic aneurysm which induced apolipoprotein E-deficient mice. We evaluated gene and protein expression of proteases, elastin and inflammatory markers. In the presence of DCRBF, MMP-12 was significantly decreased, TGF-β1 and Lox were significantly increased in SMC gene expression, MMP-9 and -12 significantly decreased gene expression of macrophages. The DCRBF preserved elastin content and decreased MMP-2 and -9 in aortic tissue. In addition, IGF-1 and TIMP-1 were significantly increased and IL-6 and TNF-α were significantly decreased with DCRBF in vivo. In conclusion, our results suggested that local administration of DCRBF may become a promising alternative therapeutic strategy for AA.

Therapeutic potential of bone marrow-derived mesenchymal stem cells in formed aortic aneurysms of a mouse model

OBJECTIVES An aortic aneurysm (AA) is caused by atherosclerosis with chronic inflammation. Mesenchymal stem cells (MSCs) have potential anti-inflammatory properties. In this study, we examined whether an already-formed AA can be treated by intravenous injection of bone marrow-derived (BM)-MSCs in a mouse model. METHODS AA was induced in apolipoprotein E-deficient mice by angiotensin II-infusion for 28 days through sub-cutaneous osmotic mini-pumps. After that, 1 × 10(6) BM-MSCs (in 0.2 ml saline) or 0.2 ml saline as a control was injected via the tail vein. Mice were sacrificed at 2 (saline group n = 10, BM-MSC group n = 10), 4 (saline group n = 6, BM-MSC group n = 7) or 8 weeks (saline group n = 5, BM-MSC group n = 6) after injection. The aortic tissues of each group were dissected. Aortic diameter, elastin content, matrix metalloproteinase (MMP)-2 and -9 enzymatic activity and cytokine concentrations were measured, as was macrophage infiltration, which was also evaluated histologically. RESULTS The incidence of AA in the BM-MSC group was reduced at 2 weeks (BM-MSC 40% vs saline 100%, P < 0.05), and aortic diameter was reduced at 2 and 4 weeks (2 weeks: 1.40 vs 2.29 mm, P < 0.001; 4 weeks: 1.73 vs 2.32 mm, P < 0.05). The enzymatic activities of MMP-2 and -9 were reduced in the BM-MSC group at 2 weeks (active-MMP-2: 0.28 vs 0.45 unit/ml, P < 0.05; active-MMP-9: 0.16 vs 0.34 unit/ml, P < 0.05). Inflammatory cytokines were down-regulated in the BM-MSC group (interleukin-6: 2 weeks: 1475.6 vs 3399.5 pg/ml, P < 0.05; 4 weeks: 2184.7 vs 3712.8 pg/ml, P < 0.05 and monocyte chemotactic protein-1: 2 weeks: 208.0 vs 352.7 pg/ml, P < 0.05) and insulin-like growth factor (IGF)-1 and tissue inhibitor of metalloproteinase (TIMP)-2 were up-regulated in the BM-MSC group at 2 weeks (IGF-1: 4.7 vs 2.0 ng/ml, P < 0.05; TIMP-2: 9.5 vs 4.0 ng/ml, P < 0.001). BM-MSC injection inhibited infiltration of M1 macrophages and preserved the construction of elastin. CONCLUSIONS Our results suggest that BM-MSCs might be an effective treatment for AA. Further investigation is necessary to optimize the injected dosage and the frequency of BM-MSCs to prevent a transient effect.

Effect of GDF‐5 and BMP‐2 on the expression of tendo/ligamentogenesis‐related markers in human PDL‐derived cells

OBJECTIVES The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament-derived cells was investigated with special reference to tendo/ligamentogenesis-related markers. MATERIALS AND METHODS Effects of each factor were analyzed by quantitative PCR for scleraxis and tenomodulin and by western blotting for scleraxis. After exposure to those factors, STRO-1-positive and STRO-1-negative fractions of human periodontal ligament tissues were isolated with an immunomagnetic cell sorting system, and the expression of scleraxis in each fraction was analyzed by western blotting. Non-separated crude cells were used as a control. RESULTS Growth differentiation factor 5 and bone morphogenetic protein 2 did not increase alkaline phosphatase activity in crude periodontal ligament-derived cells. Growth differentiation factor 5, but not bone morphogenetic protein 2, increased the expression of scleraxis in crude, STRO-1-positive and STRO-1-negative periodontal ligament-derived cells. The expression of scleraxis in STRO-1-positive periodontal ligament-derived cells was significantly less compared to that in crude P2 and STRO-1-negative periodontal ligament-derived cells. CONCLUSION Growth differentiation factor 5 induced the expression of scleraxis and may enhance tendo/ligamentogenesis in human periodontal ligament-derived cells. The expression of scleraxis was higher in STRO-1-negative fraction, suggesting more differentiated state of the cells.

Evaluation of the Bactericidal and Fungicidal Activities of Poly([2-(methacryloyloxy)ethyl]trimethyl Ammonium Chloride)(Poly (METAC))-Based Materials

Poly([2-(methacryloyloxy)ethyl]trimethyl ammonium chloride) (METAC) and the gels were prepared and evaluated for their bactericidal and fungicidal activities. The antimicrobial properties of poly(METAC) were tested against Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Saccharomyces cerevisiae (Sa. cerevisiae), methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), and Candida albicans (C. albicans). Moreover, the structural forms of the linear and cross-linked poly(METAC) were investigated for their influences on bacterial aggregation, precipitation, and cell-death. To our knowledge, this is the first report on the comparison of the antimicrobial properties of poly(METAC) and poly(METAC)-gels. The bactericidal and fungicidal activities were evaluated by determining minimum inhibitory concentrations (MICs), UV–Vis spectroscopy, and fluorescence and confocal microscopies. The MICs were found to be 123 (MSSA), 123 (MRSA), 123 (P. aeruginosa), 370 (E. coli), 123 (B. subtilis), 370 (C. albicans), and 370 μg/mL (Sa. cerevisiae), as determined by broth dilution, and 370 (MSSA), 370 (MRSA), 370 (P. aeruginosa), 3300 (E. coli), 370 (B. subtilis), 1100 (C. albicans), and >10,000 μg/mL (Sa. cerevisiae), as determined by paper disc diffusion (on solid medium). The poly(METAC)-gels achieved rapid adsorption/precipitation of bacteria via the cationic surface charge. Thus, these poly(METAC)-based polymers can potentially be used as antibacterial materials.

The oral administration of clarithromycin prevents the progression and rupture of aortic aneurysm

Objective: The pathogenesis of aortic aneurysm (AA) is associated with chronic inflammation in the aortic wall with increased levels of matrix metalloproteinases (MMPs). Clarithromycin (CAM) has been reported to suppresses MMP activity. In this study, we investigated whether CAM could prevent the formation and rupture of AA. Methods: Male apolipoprotein E‐deficient mice (28–30 weeks of age) were infused with angiotensin II for 28 days. CAM (100 mg/kg/d) or saline (as a control) was administered orally to the mice every day (CAM group, n = 13; control group, n = 13). After the administration period, the aortic diameter, elastin content, macrophage infiltration, MMP levels, and levels of inflammatory cytokines, including nuclear factor &kgr;B (NF‐&kgr;B), were measured. Results: The aortic diameter was significantly suppressed in the CAM group (P < .001). No rupture death was observed in the CAM group in contrast to five deaths (38%) in the control group (P < .01). CAM significantly suppressed the degradation of aortic elastin (56.3% vs 16.5%; P < .001) and decreased the infiltration of inflammatory macrophages (0.05 vs 0.16; P < .01). Compared with the controls, the enzymatic activity of MMP‐2 and MMP‐9 was significantly reduced in the CAM group (MMP‐2, 0.15 vs 0.56 [P < .01]; MMP‐9, 0.12 vs 0.60 [P < .01]), and the levels of interleukin 1&bgr; (346.6 vs 1066.0; P < .05), interleukin 6 (128.4 vs 346.2; P < .05), and phosphorylation of NF‐&kgr;B were also decreased (0.3 vs 2.0; P < .01). Conclusions: CAM suppressed the progression and rupture of AA through the suppression of inflammatory macrophage infiltration, a reduction in MMP‐2 and MMP‐9 activity, and the inhibition of elastin degradation associated with the suppression of NF‐&kgr;B phosphorylation. Clinical Relevance: This article shows that high‐dose clarithromycin (CAM) suppressed aortic aneurysm formation and rupture. Fortunately, no mice had adverse effects of CAM in the laboratory test. For clinical relevance, we will try a low dose of CAM for the same model of mice, such as 10 mg/kg/d or less. Although we think we need to try bigger animals, such as a pig, we have no study design of an aortic aneurysm model for big animals.

Bone marrow–derived mesenchymal stromal cells regress aortic aneurysm via the NF-kB, Smad3 and Akt signaling pathways

BACKGROUND AIMS We have confirmed that aortic aneurysm (AA) can be regressed by the administration of bone marrow-derived mesenchymal stromal cells (BM-MSCs). We investigated the kinetics of signaling pathways in AA following treatment with BM-MSCs. METHODS Angiotensin II-infused apolipoprotein E-deficient mice were treated by intravenous injection of 1 × 106 BM-MSCs in 0.2 mL saline (BM-MSCs group, n = 5) or 0.2 mL saline (saline group, n = 5). Mice were sacrificed 2 weeks after injection and subjected to measurements of the incidence of AA and levels of phosphorylated proteins. Levels of proteins in conditioned media of BM-MSCs were also measured. RESULTS The incidence of AA in the BM-MSCs group was reduced (BM-MSC 40% versus saline 100%, P <0.05). Levels of pNF-kB and pSTAT1 were reduced (pNF-kB: 0.28 versus 0.45 unit/mL, P <0.05, pSTAT1: 0.16 versus 0.34, P <0.05), whereas levels of pAkt and pSmad3 were elevated (pAkt: 0.13 versus 0.07, P <0.01, pSmad3: 1.07 versus 0.47, P <0.05) in the BM-MSCs group. The levels of pNF-kB, pAkt, and pSmad3 were correlated with aortic diameters. Trophic factors including IGFPB-3, NRF, Activin A and PDGF-AA were secreted from BM-MSCs (IGFBP-3: 35.2 pg/mL, NRF: 3.1 pg/mL, Activin A: 3.1 pg/mL, PDGF-AA: 0.45 pg/mL). CONCLUSIONS Our findings suggested that the therapeutic mechanism of BM-MSC-mediated AA regression could contribute to regulation of the NF-kB, Smad3 and Akt signaling pathways. In addition, paracrine actions by factors including NRF, IGFBP-3, Activin A and PDGF-AA might have affected these signaling pathways.

Prevention of arterial graft spasm in rats using a vasodilator-eluting biodegradable nano-scaled fibre

OBJECTIVES Arterial graft spasm occasionally causes circulatory collapse immediately following coronary artery bypass graft. The aim of this study is to evaluate the efficacy of our developed materials, which were composed of milrinone (phosphodiesterase III inhibitor) or diltiazem (calcium-channel blocker), with nano-scaled fibre made of biodegradable polymer to prevent arterial spasm. METHODS Milrinone- or diltiazem-releasing biodegradable nano-scaled fibres were fabricated by an electrospinning procedure. In vivo milrinone- or diltiazem-releasing tests were performed to confirm the sustained release of the drugs. An in vivo arterial spasm model was established by subcutaneous injection of noradrenalin around the rat femoral artery. Rats were randomly divided into four groups as follows: those that received 5 mg of milrinone-releasing biodegradable nano-scaled fibre (group M, n = 14); 5 mg of diltiazem-releasing biodegradable nano-scaled fibre (group D, n = 12); or those that received fibre without drugs (as a control; group C, n = 14) implanted into the rat femoral artery. In the fourth group, sham operation was performed (group S, n = 10). One day after the implantation, noradrenalin was injected in all groups. The femoral arterial blood flow was measured continuously before and after noradrenalin injection. The maximum blood flow before noradrenalin injection and minimum blood flow after noradrenalin injection were measured. RESULTS In vivo drug-releasing test revealed that milrinone-releasing biodegradable nano-scaled fibre released 78% of milrinone and diltiazem-releasing biodegradable nano-scaled fibre released 50% diltiazem on the first day. The ratios of rat femoral artery blood flow after/before noradrenalin injection in groups M (0.74 ± 0.16) and D (0.72 ± 0.05) were significantly higher than those of groups C (0.54 ± 0.09) and S (0.55 ± 0.16) (P < 0.05). CONCLUSION Noradrenalin-induced rat femoral artery spasm was inhibited by the implantation of milrinone-releasing biodegradable nano-scaled fibre or diltiazem-releasing biodegradable nano-scaled fibre. These results suggested that our materials might be effective for the prevention of arterial graft spasm after coronary artery bypass graft.