BACKGROUND: We have developed a novel system for expansion of gene-modified hematopoietic stem/progenitor cells to overcome the low efficiency of current gene transfer methodology. This system involves 'selective amplifier genes', that encode fusion proteins between the granulocyte colony-stimulating factor receptor (GCR) and the hormone-binding domain of estrogen receptor (ER). Hematopoietic progenitors expressing the chimeras showed estrogen-responsive growth in a controllable manner. However, endogenous estrogen may activate the fusion proteins in vivo, depending on the hormonal status of the subjects. METHODS: We replaced ER with a mutant receptor (TmR) which specifically binds to 4-hydroxytamoxifen (Tm), to overcome limitations with wild-type ER. Interleukin-3 (IL-3)-dependent Ba/F3 cells and hematopoietic progenitor cells transduced with the resultant fusion proteins (GCRTmR and delta GCRTmR) were examined for ligand-inducible growth. RESULTS: GCRTmR- and delta GCRTmR-expressing Ba/F3 showed IL-3-independent growth in response to Tm, while the cells were unresponsive to estrogen at concentrations up to 10(-7)-10(-6) M. Furthermore, murine bone marrow cells transduced with GCRTmR and delta GCRTmR formed colonies in methyl-cellulose medium in response to Tm, while virtually no colonies appeared with 10(-7) M estrogen or without cytokines. CONCLUSIONS: These results suggest that influences of the endogenous estrogen can be almost eliminated by using the GCRTmR/Tm or delta GCRTmR/Tm system to expand gene-modified hematopoietic stem/progenitor cells.

Animals↗

PubMedDate not supplied

Alzheimer's disease and estrogen.

The preventive effect of estrogen on Alzheimer's disease (AD) has become clear with epidemiological data. Therapeutic effects of estrogen have not yet been established. In this presentation, we report our new basic and clinical data. The estrogen receptor, (ER)alpha, and ERbeta mRNA were investigated in rat brain. Estradiol-17beta (E(2)) treatment following OVX reduced the levels of ERalpha mRNA in the hypothalamus. In the substantia innominata (SI), the number of choline acetyltransferase immunoreacive cells increased significantly in the estrogen treatment rat. The neurons in SI projecting to the forebrain cortex contained ERalpha. Increasing amounts of intracellular calcium, peroxidation, and apoptosis with amyloid beta were suppressed in neuronal cells from rat pheochromocytoma (PC12) cells with E(2). ERalpha cDNA transfected PC 12 cells elaborated more neurite-like processes with E(2). In clinics, we are currently preparing vaginal progesterone tablets, which essentially may concentrate in the endometrium to prevent endometrial cancer, with few general circulation of progesterone inviting less depression. The therapeutic effects of cyclic estrogen, such as its preventive effect, are suggested in these studies, at least on mild AD.

Alzheimer Disease↗

PubMedDate not supplied

Atheroprotective effect of estriol and estrone sulfate on human vascular smooth muscle cells.

In patients with atherosclerosis, fibrosclerotic focuses are induced by multiplication of vascular smooth muscle cells (VSMC), and they are regulated by cytokines and regulators. There have been few reports about the atheroprotective effect of estriol (E(3)). Estrone sulfate (E(1)-S) is the predominant estrogen of conjugated equiline estrogens, which is commonly used in hormone replacement therapy, but it should be hydrolyzed by steroid sulfatase (STS) to enter the cells of target tissues. The purpose of this study was to detect STS in VSMC and to investigate whether E(3) and E(1)-S have atheroprotective effects like E(2). First, we detected the presence of STS mRNA in VSMC by in situ hybridization. We then examined the changes in the expression of mRNAs of cytokines, namely, PDGF-A chain, IL-1, IL-6 and TGF-beta, in VSMC, in the presence and absence of E(3) and estrogens. As a result, the expression of PDGF-A chain, IL-1 and IL-6 mRNAs was suppressed by E(3) (P<0.05 vs control) significantly like E(1)-S and E(2), but that of TGF-beta mRNA was not significantly affected by any estrogen. These results indicate that E(1)-S can be hydrolyzed by STS in VSMC, and that E(3) may regulate the cytokines by suppressing the production of mRNAs. It is suggested that there is a possibility of E(1)-S and E(3) having a direct effect on vessels in atherogenesis.

Arteriosclerosis↗

PubMedDate not supplied

Gene transfer into rat renal cells using adeno-associated virus vectors.

Adeno-associated virus (AAV) vectors have a number of attractive features, including lack of cytotoxicity, ability to transduce nondividing cells, and long-term transgene expression. We investigated whether rat renal cells could be efficiently transduced with AAV vectors. Rat glomerular mesangial cells were transduced with AAV-lacZ vector containing beta-galactosidase gene in vitro, and the expression of beta-galactosidase was evaluated by X-gal staining and ELISA. For ex vivo experiments, sections of rat kidneys were incubated with AAV-lacZ, and then evaluated by X-gal histochemical staining. The level of beta-galactosidase expression in cultured rat mesangial cells increased in a dose-dependent manner (ranging from 1 x 10(5) to 5 x 10(6) particles/cell). When transduced with 5 x 10(6) vector particles/cell of AAV-lacZ, about 50% of mesangial cells were stained positively with X-gal, and the level of beta-galactosidase expression reached 9.9 +/- 1.5 ng/mg protein. Expression was detectable during the culture period for at least 7 days. X-gal histochemical examination of the ex vivo transduced renal tissue revealed tubular cell and interstitial tissue staining. However, gene transfer was not clearly observed in glomeruli. These findings suggest that AAV vectors have the potential for gene therapy of renal diseases.

Animals↗

PubMedDate not supplied

Menopause and hyperlipidemia: pravastatin lowers lipid levels without decreasing endogenous estrogens.

In study 1, serum lipid and estrogen levels were determined in 30 women (aged 40 to > 60 years). Total cholesterol (TC) levels increased significantly with age, but no significant association was found between TC levels and menopausal status. Hypercholesterolemia (TC > 220 mg/dl) was identified in 10 women and hypertriglyceridemia (> 150 mg/dl) in 5 women. Among women not receiving estrogen replacement therapy, a significant negative correlation was found between TC levels and estradiol-17 beta levels. In study 2, serum lipid and estrogen levels were determined in 74 women; 12 of the 74 were receiving conjugated estrogen for the treatment of the menopausal syndrome and 21 hyperlipidemic women were receiving pravastatin (2.5 to 30 mg daily). Among postmenopausal women, high-density lipoprotein cholesterol levels were significantly higher and low-density lipoprotein (LDL) cholesterol levels significantly lower in the estrogen-treated than untreated women. Serum TC and LDL cholesterol levels were significantly reduced during pravastatin treatment. Levels of endogenous estrogens (estradiol-17 beta, estrone, and estrone sulfate) were not significantly affected by pravastatin treatment. The results indicate that pravastatin can be used to reduce hyperlipidemia in menopausal women without affecting endogenous estrogen levels.

Adult↗