Transplantation of erythropoietin gene-transfected umbilical cord mesenchymal stem cells as a treatment for limb ischemia in rats.

The aim of this study was to investigate the mechanisms of erythropoietin (EPO)-transfected umbilical cord mesenchymal stem cells (UC-MSCs) in the treatment of rats with ischemic limb and provide a theoretical basis for optimization of UC-MSC transplantation. Sixty SD rats were randomly divided into four groups: ischemia control group, EPO treatment group, UC-MSCs treatment group, EPO gene transfected UC-MSC treatment groups (15 rats in each group). The left femoral hind artery and its branches were ligated to develop hind limb ischemia in male SD rats. Five points were injected in the adductor and gastrocnemius muscles with medium, cDNA3-EPO gene DNA-liposome complex solution or UC-MSCs in control groups and EPO-transfected-UC-MSCs in the experimental group. Western blot confirmed in vitro EPO expression in EPO gene-transfected human UC-MSCs. Arterial angiography at 4 weeks post-transplantation showed no development of blood vessels in the control group and moderate angiogenesis in the EPO- and UC-MSC-treated groups. However, a large number of freshmen angiogenesis and abundant collateral circulation was observed in the EPO-transfected-UC-MSC-treated experimental group. Rat capillary density measurement results confirmed the angiographs quantitatively and showed no statistically significant difference between EPO- and UC-MSC-treated groups (P > 0.05). CM-Dil-positive cell numbers were (0 ± 0.00), (0 ± 0.00), (32.46 ± 6.68), (59.64 ± 10.38)/HP (P < 0.05). RT-PCR detected that the in vivo mRNA expression of the EPO gene was relatively higher in the EPO-transfected-UC-MSC-treated group than the EPO-treated group (0.79 ± 0.06 vs 0.19 ± 0.04, P < 0.05). Thus, this study revealed that using UC-MSCs as vector in gene therapy for limb ischemia facilitates stable and effective expression of EPO compared to direct gene injection.

Directional laser emission from a wavelength-scale chaotic microcavity.

We demonstrate directional output from a deformed disk laser of dimensions comparable to the emission wavelength. Unlike larger deformed cavity lasers, which exhibit universal output directionality determined by chaotic ray dynamics, the far-field patterns differ between lasing modes. The directional emission results from weak coupling of isotropic high-quality modes to anisotropic low-quality modes, combined with chiral symmetry breaking of clockwise and counterclockwise propagating waves. This mechanism makes it possible to control the output properties of wavelength-scale lasers.

Improving optical confinement in nanostructures via external mode coupling.

We demonstrate the formation of long-lived resonances via external coupling in open nanostructures. In the examples of three dielectric nanorods or strips, external coupling of modes is induced by tuning of the rod spacing and increases the lifetime over an order of magnitude. Such an enhancement results from the destructive interference of fields that minimizes light leakage. Our results illustrate an effective way of storing light in nanostructures, and have potential applications to nanoscale photonic devices.

Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway.

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72h. Peak expression occurred at 12h (3- to 4-fold, p<0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs' proliferation and migration. Over-expression of Bcl-2 increased HAECs' tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation. Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.

Related transcriptional enhancer factor-1 induces fibroblast growth factor receptor-1 expression in endothelial cells.

Fibroblast growth factor receptor-1 (FGFR-1) has been implicated in the process of cardiogenesis, although the underlying molecular mechanisms are poorly understood. In this study, we report the regulation of FGFR-1 expression by related transcriptional enhancer factor-1 (RTEF-1) in vitro (endothelial cells) and in vivo (RTEF-1 transgenic mice). FGFR-1 promoter activity, FGFR-1 mRNA and protein level were measured in bovine aortic endothelial cells (BAEC) in response to RTEF-1 and in endothelial cells isolated from livers in RTEF-1 transgenic mice. RTEF-1 stimulated FGFR-1 promoter activity in a dose-dependent manner. RTEF-1 enhanced FGFR-1 mRNA (4-fold) and protein expression (3.5-fold) whereas RTEF-1 siRNA decreased FGFR-1 protein expression (4-fold). FGFR-1 mRNA and protein expression were also increased in endothelial cells isolated from livers of RTEF-1 transgenic mice. Furthermore, RTEF-1 enhanced tubule formation whereas this was decreased by RTEF-1 knockdown. Moreover, increased relaxation of microvessels was found in RTEF-1 transgenic mice compared to wild-type mice. Our results indicate that RTEF-1 acts as a transcriptional stimulator of FGFR-1 in endothelial cells through its activation of the FGFR-1 promoter. RTEF-1 thus plays an important role in the regulation of FGFR-1 expression. These findings help further understand FGFR activity in angiogenesis and may lead to new therapeutic targets in ischemic vascular disorders.

Hypoxia-induced expression of RTEF-1 (related transcriptional enhancer factor-1) in endothelial cells is independent of HIF-1 (hypoxia-inducible factor-1).

Related transcriptional enhancer factor-1 (RTEF-1) plays an important role in transcriptional regulation of angiogenic genes in hypoxic endothelial cells. The mechanisms involved in the induction of RTEF-1 expression in hypoxia are poorly understood. In bovine aortic endothelial cells (BAEC) subjected to hypoxia, Western blot and quantitative PCR analysis revealed that RTEF-1 protein and mRNA levels were significantly increased by hypoxia. To address the potential role of the hypoxia-inducible factor-1 (HIF-1) in RTEF-1 induction, a hepatoma cell line deficient in HIF-1 (c4) and a control HIF-1 positive cell line (vT{2}) were exposed to hypoxia. We report that RTEF-1 protein expression assessed by either Western blotting or immunofluorescence was increased in both cell lines. This demonstrates that HIF-1 is not required for RTEF-1 upregulation by hypoxia. Conversely, RTEF-1 appeared to regulate the expression of HIF-1: HIF-1alpha promoter activity was increased (3.6-fold) by RTEF-1 overexpression in BAEC. Furthermore, RTEF-1 enhanced BAEC proliferation and tubule formation; these were inhibited by RTEF-1 knockdown with siRNA. We propose that RTEF-1, acting via HIF-1, is a key regulator of angiogenesis in response to hypoxia.

VEGF regulates FGF-2 and TGF-beta1 expression in injury endothelial cells and mediates smooth muscle cells proliferation and migration.

BACKGROUND: Vascular endothelial growth factor (VEGF) is implicated in the development of restenosis after percutaneous transluminal coronary angioplasty (PTCA) as well as atherosclerosis. The purpose of our study was: 1) to evaluate the expression of endothelial cell (EC) fibroblast growth factor 2 (FGF-2) and transforming growth factor beta1 (TGF-beta1) mRNA expression following vascular injury and VEGF modulation and 2) to assess whether VEGF indirectly stimulates smooth muscle cell (SMC) migration and proliferation via growth factors released by injured EC. METHODS: Bovine aortic endothelial cells (BAEC) were cultured to near confluency and were serum starved. Linear wounds were made in medium with and without VEGF. FGF-2 and TGF-beta1 mRNA expression were evaluated. Bovine aortic organ culture experiments were also carried out and growth factor expression was assessed. SMC proliferation and migration was assessed in response to EC injury medium with/without VEGF. RESULTS: EC injury in the presence of VEGF increased FGF-2 mRNA. EC injury also induced TGF-beta1 mRNA expression; however VEGF inhibited TGF-beta1 mRNA expression in both injured and noninjured ECs. VEGF increased FGF-2 mRNA stability and did not alter TGF-beta1 mRNA stability. SMC proliferation and migration was found to be induced by injured EC media and injury EC medium with VEGF, respectively CONCLUSIONS: The results demonstrate that 1) VEGF indirectly stimulates SMC proliferation and migration through stimulation of the expression of FGF-2 and 2) VEGF inhibits the expression of TGF-beta1 released by EC. Theses data further suggest an integral role for FGF-2 and TGF-beta1 in wound repair.

A pyrrole derivative from Paeonia suffruticosa.

A new pyrrole derivative, 2-methyl-5-(2'-O-alpha-D-glucopyranosyl-1'-oxygen)-propylpyrrole, named paesuffrioside (1), was isolated from the water-soluble extract of the root cortex of Paeonia suffruticosa, together with 11 known compounds. Their structures were elucidated by spectroscopic analysis and chemical transformation. Compounds 2 and 3 were isolated from this plant for the first time.

Role of epidermal growth factor and epidermal growth factor receptor on hemidesmosome complex formation and integrin subunit beta4.

Epidermal growth factor (EGF) stimulates integrin beta4 expression and synthesis in corneal epithelium through ligand binding to the EGF receptor, receptor dimerization and activation of the intracellular domain. We hypothesized that inhibition of EGF receptor messenger RNA (mRNA) would block integrin beta4 expression, which is induced by EGF. We also tested the hypothesis that EGF would cause the degradation of hemidesmosomes in control and injured corneal organ cultures. Primary rabbit corneal epithelial cell cultures or corneas were cultured in keratinocyte medium in the presence or absence of an antisense 20-mer phosphorothioate oligonucleotide complementary to the initiation codon region of EGF receptor mRNA. Cells were also cultured in the presence or absence of EGF. Sense and scrambled oligonucleotides similarly modified were used as controls. The concentration of EGF receptor mRNA was semiquantitatively determined by reverse transcriptase/polymerase chain reaction (RT-PCR). We found that transfection did inhibit EGFR expression and migration of epithelial cells and also demonstrated that EGFR mediated expression of integrin beta4 mRNA. Injury induced a decrease in hemidesmosomes that was enhanced with EGF but was not caused by the presence of growth factor in unwounded tissue. These results indicate that injury causes the activation of EGFR but that EGF alone is not responsible for the degradation of hemidesmosomes and that other growth factors play a role in the complex repair of wounds in an avascular tissue.

TGF-beta1 regulates TGF-beta1 and FGF-2 mRNA expression during fibroblast wound healing.

AIMS: To evaluate the expression of transforming growth factor beta1 (TGF-beta1) and fibroblast growth factor 2 (FGF-2) mRNA in stromal cells in response to injury in the presence of either TGF-beta1 or FGF-2. It has been shown previously that heparan sulfate proteoglycans and FGF-2 are present transiently during wound repair in vivo and that an increase in TGF-beta1 mRNA is detected rapidly after injury. METHODS: Primary corneal fibroblasts were cultured to confluency, serum starved, and linear wound(s) were made in medium containing TGF-beta1 or FGF-2. TGF-beta1 and FGF-2 mRNA expression were evaluated using both northern blot analysis and in situ hybridisation. Both dose dependent and time course experiments were performed. Whole eye organ culture experiments were also carried out and growth factor expression was assessed. RESULTS: Injury and exogenous TGF-beta1 increased TGF-beta1 mRNA values. The increase in expression of FGF-2 mRNA was not detected until wound closure. In contrast, FGF-2 inhibited the expression of TGF-beta1. TGF-beta1 increased TGF-beta1 mRNA stability but did not alter that of FGF-2. Migration assay data demonstrated that unstimulated stromal cells could be activated to migrate to specific growth factors. CONCLUSIONS: TGF-beta1 specifically enhances cellular responsiveness, as shown by increased stability after injury and the acquisition of a migratory phenotype. These data suggest that there is an integral relation during wound repair between TGF-beta1 and FGF-2.

Long term effects of Toki-shakuyaku-san on brain dopamine and nerve growth factor in olfactory-bulb-lesioned mice.

We used olfactory-bulb-lesioned mice induced by intranasal irrigation with zinc sulfate as a model of dementia, to investigate the effects of Toki-shakuyaku-san (TSS) on monoamines and nerve growth factor (NGF) in brain regions. TSS was given daily through the drinking water for either 1, 2, 3, 4 or 8 weeks from the day after olfactory lesion. The administration of TSS significantly suppressed the decrease of 3,4-dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) in olfactory bulb of olfactory-lesioned mice at 1 week, and tended to suppress the decrease of DOPAC and HVA during the experimental session. However, the administration of TSS had no influence on dopamine contents. NGF contents in the olfactory bulb were increased after the irrigation, and the value returned to the same level as the control at 8 weeks after. Although the NGF contents in the olfactory bulb of TSS-treated mice were immediately increased at 1 and 2 weeks, the value returned to normal level within 3 weeks. These findings indicate that oral administration of TSS prevents the reduction of dopamine metabolites, DOPAC and HVA, and immediately increased NGF contents in the olfactory bulb. This suggested that TSS treatment promotes the NGF contents in olfactory nerves and rescue the neurons from damage.

Injury and EGF mediate the expression of alpha6beta4 integrin subunits in corneal epithelium.

Our goal was to evaluate the role of epidermal growth factor and injury on the expression of integrin subunits alpha6(alpha6) and beta4(beta4). An in vitro wound model was used to evaluate corneal wound repair and cellular migration. Primary rabbit corneal epithelial cell cultures were serum-starved and injured in the presence or absence of EGF or tyrphostin AG1478, an inhibitor of EGF receptor kinase activity. Repair was monitored morphologically and expression was analyzed using in situ hybridization and immunohistochemistry accompanied by confocal microscopy. The addition of EGF to cell cultures induced a dose-dependent increase in beta4 mRNA expression but the constitutive expression of alpha6 was several fold greater. In the wounded cultures there was a rapid change in expression at the edge of the wound that was enhanced with EGF. In our model there was an increase in beta4 and alpha6 protein in migrating cells. Changes in integrin expression were accompanied by a transient increase in activation of the EGF receptor. The addition of tyrphostin inhibited migration of cells and wound repair, the activation of the EGF receptor and phosphorylation of beta4 in the cytoplasm. These data indicate that the activation of the EGF receptor plays a critical role in the regulation of integrin receptors and the mediation of cellular migration.

Effects of hokoei-to (pugongying-tang), a Kampo formula, on monoamine content in brain regions and mitogenic activity of splenic lymphocytes in ovariectomized mice.

Hokoei-to (pugongying-tang) is one of the Kampo formulae clinically used for gynecological disturbances such as lack of lactation and mammary swelling. We investigated the effect of hokoei-to on the nervous and immune systems in ovariectomized mice as a climacteric disorder model. Hokoei-to suppressed the decrease of monoamines in the ventral hippocampus and dorsal hippocampus of ovariectomized mice. It was shown that the hokoei-to could improve the metabolic turnover of dopamine. The mitogenic activity of lymphocytes in the spleen was reduced after ovariectomy; a suppression of this reduced activity was observed in the group given hokoei-to.

Pancreatic islet regeneration by ephedrine in mice with streptozotocin-induced diabetes.

In this experiment, we investigated the effects of crude Ephedrae herba, alkaloid extract of Ephedrae herba and 1-ephedrine, a major alkaloid component, on diabetic mice induced by streptozotocin (STZ). The alkaloid extract and 1-ephedrine showed suppression on the hyperglycemia. The suppression by Ephedrae herba of hyperglycemia may therefore be due to 1-ephedrine. Furthermore, we found that Ephedrae herba, alkaloid and 1-ephedrine promoted the regeneration of pancreas islets following atrophy induced by STZ. It is therefore suggested that Ephedrae herba may regenerate atrophied pancreatic islets, restore the secretion of insulin, and thus correct hyperglycemia.

A new murine model for atherosclerosis with inflammation in the periodontal tissue induced by immunization with heat shock protein 60.

It has recently become apparent that the anti-heat shock protein (HSP) antibody plays an important role in the pathogenesis of atherosclerosis. We studied whether immunization with human HSP60 could lead to atherosclerosis in mice. We attempted to induce atherosclerosis in C57BL/6NJcl mice by immunization with human HSP60 under a high-cholesterol diet. The size of fatty streak lesions was significantly enhanced in mice immunized with human HSP60 under a high-cholesterol diet relative to the number in control mice receiving a high-cholesterol diet alone. In addition, these new atherosclerotic model mice showed lesions of inflammation in the periodontal tissue. This new model may thus provide theoretical support for the clinical observation that patients suffering from periodontitis are frequently found to have atherosclerosis. The cytokine ratio of interferon-gamma/interleukin-4 in the high-cholesterol diet group was significantly higher than that in the standard chow group (p<0.05). This suggests the presence of a predominantly Th1-type immune response in atherosclerosis.

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice.

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.

Hepatoprotective effects of Inula britannica on hepatic injury in mice.

Inula britannica, a Kampo medicine, is prepared from the heads of Compositae plants such as Inula britannica L., which has been used clinically as a remedy for nausea, hiccup and excessive sputum. Here it is shown that administration of Inula britannica improves the survival rate of mice with hepatic injury induced by LPS/PA. It is also suggested that administration of Inula britannica significantly reduces the fluctuation in the amount of cytokine in the spleen of mice with hepatic injuries, and that the Th1/Th2 control effect is related to the inhibitory action of Inula britannica against hepatic injury. In vitro testing suggests that Inula britannica suppresses Th1 differentiation and induces Th2 differentiation by inhibiting the production of macrophage IL-12 and promoting the production of IL-10, thus showing the immunological effect of hepatic injury inhibition by affecting the balance between Th1 and Th2.

Transforming growth factor-beta1 expression in cultured corneal fibroblasts in response to injury.

The mechanisms underlying TGF-beta regulation in response to injury are not fully understood. We have developed an in vitro wound model to evaluate the expression and localization of transforming growth factor-beta1 in rabbit corneal fibroblasts in response to injury. Experiments were conducted in the presence or absence of serum so that the effect of the injury could be distinguished from exogenous wound mediators. Cultures were wounded and evaluations conducted over a number of time points. Expression of TGF-beta1 RNA was determined using Northern blot analysis and in situ hybridization, while the TGF-beta receptors were identified by affinity cross-linking. Injury increased the expression of TGF-beta1 mRNA in cells at the wound edge after 30 min; this response was amplified by the addition of serum. TGF-beta1 mRNA expression was observed in a number of cells distal from the wound. After wound closure, TGF-beta1 mRNA was negligible and resembled unwounded cultures. The half-life of TGF-beta1 mRNA was two times greater in the wounded cultures, indicating that the injury itself maintained the expression, while cell migration was present. Analogous to these findings, we found that binding of TGF-beta to its receptors was maximal at the wound edge, decreasing with time and distance from the wound. These results indicate that injury increases the level of expression of TGF-beta1 mRNA and maintains a higher level of receptor binding during events in wound repair and that these might facilitate the migratory and synthetic response of stromal fibroblasts.

Laser-induced modification of DNA and Poly[A,G] at guanine moiety using acetone as photosensitizer.

The interactions of triplet acetone with polyadenylic acid (Poly[A]), polyguanylic acid (Poly[G]), polyadenylic-guanylic acid (Poly[A,G]) and single-stranded DNA (ssDNA) were investigated in neutral aqueous solution using KrF (248 nm) laser flash photolysis. The transient absorption spectra and kinetics of DNA and polynucleotides obtained under acetone sensitization demonstrated that the predominant transient species was guanine radical. These novel findings have offered time-resolved evidence for photochemical modification of DNA and Poly[A,G] at guanine moiety.