Effects of progesterone on uterine leiomyoma growth and apoptosis

Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. Recently we have found that the use of levonorgestrel-releasing intrauterine system (IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. These clinical experiences prompted us to characterize the effects of progestin on the proliferation and apoptosis of leiomyoma cells cultured in vitro. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either E(2) (10 ng/ml) or P(4) (100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells; whereas in cultures of normal myometrial cells, the addition of E(2) augmented PCNA expression in the cells, but P(4) did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P(4) treatment resulted in an increase in EGF expression in the cells. In contrast, E(2) treatment augmented EGF-R expression in cultured leiomyoma cells, but P(4) did not. These results indicate that P(4) up-regulates the expression of PCNA and EGF in leiomyoma cells, whereas E(2) up-regulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P(4) and E(2) act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium, suggesting that the abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Furthermore, Bcl-2 protein expression in leiomyoma cells was up-regulated by P(4), but down-regulated by E(2). Therefore, it seems likely that P(4) may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells.     

Treatment of mouse limb ischemia with an integrative hypoxia-responsive vector expressing the vascular endothelial growth factor gene

Constitutive vascular endothelial growth factor (VEGF) gene expression systems have been extensively used to treat peripheral arterial diseases, but most of the results have not been satisfactory. In this study, we designed a plasmid vector with a hypoxia-responsive element sequence incorporated into it with the phiC31 integrative system (pVHAVI) to allow long-term VEGF gene expression and to be activated under hypoxia. Repeated activations of VEGF gene expression under hypoxia were confirmed in HEK293 and C2C12 cells transfected with pVHAVI. In limb ischemic mice, the local administration of pVHAVI promoted gastrocnemius mass and force recovery and ameliorated limb necrosis much better than the group treated with hypoxia-insensitive vector, even this last group had produced more VEGF in muscle. Histological analyses carried out after four weeks of gene therapy showed increased capillary density and matured vessels, and reduced number of necrotic cells and fibrosis in pVHAVI treated group. By our study, we demonstrate that the presence of high concentration of VEGF in ischemic tissue is not beneficial or is less beneficial than maintaining a lower but sufficient and long-term concentration of VEGF locally.     

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior

Background

Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity.

Methods

MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses.

Results

After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months.

Conclusions

These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.     

A rapid PCR-aided selection of a rice line containing the Rf-1 gene which is involved in restoration of the cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is widely known in higher plants, the mechanism of which is believed to involve incompatibility between nuclei and cytoplasms. In rice lines with the CMS trait, fertility is restored by the aid of a nuclear-encoded gene, Rf-1, whose locus has been determined in chromosome 10. We found a particular PCR-amplified fragment, designated fL601, that specifically amplified using the DNAs from Rf-1 lines tested as templates. RFLP mapping of the fL601 locus revealed that there are two loci for the fL601, and that both are tightly linked to the Rf-1 locus. Progeny analysis also showed high frequency of their co-segregation. Southern analysis of the genomic DNA demonstrated that the Rf-1 lines shared a unique sequence in the fL601 region. These results enabled us to construct a system for specific detection of the corresponding regions. Utilizing this detection system, we established a simple PCR-mediated selection method for the Rf-1 lines, which may facilitate the breeding for hybrid rice.