Factor Messenger Ribonucleic Acid Levels Research Articles

Decreased platelet derived growth factor expression during fracture healing in diabetic animals.

Animal model experiments have suggested that diabetes inhibits cell proliferation during fracture healing. Immunohistochemical analysis of proliferating cell nuclear antigen revealed significant reductions in cellular proliferation rates in the fracture callus of spontaneously diabetic BB Wistar rats as compared with healthy BB Wistar rats. Because platelet derived growth factor is associated with the early stage of fracture healing, it was hypothesized that diabetes causes decreased platelet derived growth factor expression during the early phase of fracture healing with a concomitant decrease in cell proliferation. Midshaft femur fractures were created in healthy and spontaneously diabetic BB Wistar rats and analyzed at Days 2, 4, and 7 after fracture for expression of platelet derived growth factor. Immunohistochemistry showed decreased localization of platelet derived growth factor in early diabetic fracture callus compared with healthy controls. Platelet derived growth factor messenger ribonucleic acid levels, as determined by reverse transcription and polymerase chain reaction, also were decreased in early diabetic fractures compared with healthy controls. Therefore the decreased cell proliferation rates associated with diabetic fracture healing are consistent with decreased platelet derived growth factor levels and suggest a causal relationship. These results suggest that diabetes is affecting the early phase of fracture healing by inhibiting cell proliferation through decreasing expression of platelet derived growth factor.

Developmental regulation of vascular endothelial growth/permeability factor messenger ribonucleic acid levels in and vascularization of the villous placenta during baboon pregnancy.

Vascular endothelial growth/permeability factor (VEG/PF) has an important role in angiogenesis; however, very little is known about the developmental regulation of VEG/PF and the vascular system within the placenta during human pregnancy. In the present study, therefore, a developmental approach was used in the baboon to determine the placental source of VEG/PF and its fms-like tyrosine kinase (flt-1) and kinase-insert domain containing (KDR/flk-1) receptors, and whether the rise in estrogen with advancing pregnancy was associated with a corresponding increase in placental VEG/PF expression and vascularization. VEG/PF messenger RNA (mRNA) levels were determined by competitive RT-PCR in villous cell fractions isolated by Percoll gradient centrifugation from placentas obtained on days 45 and 54 (very early), 60 (early), 100 (mid), and 165-170 (late) of baboon pregnancy (term = 184 days). Maternal peripheral serum estradiol increased from very low concentrations early in gestation (0.15-0.20 ng/ml) to an early surge of over 2.5 ng/ml on days 60-85, and peak levels of 4-6 ng/ml late in baboon pregnancy. VEG/PF mRNA was expressed in low level in the syncytiotrophoblast (<2,000 attomol/microgram total RNA), and values in this fraction did not change significantly with advancing gestation. VEG/PF mRNA expression was slightly greater in the inner villous core cell fraction; however, levels decreased (P < 0.05) between early and late gestation. Cytotrophoblasts were a major source of VEG/PF mRNA and levels increased (P < 0.01) from 3,631 +/- 844 attomol/microgram total RNA on day 45 to 25,807 +/- 5,873 attomol/microgram total RNA on day 170. VEG/PF protein expression determined by immunocytochemistry was abundant in cytotrophoblasts and lower in the syncytiotrophoblast and inner villous core cells. The flt-1 and KDR/flk-1 receptors were expressed in the vascular endothelial cells of the baboon villous placenta. The percentage of villous placenta occupied by blood vessels and the number of vessels/mm(2) villous tissue, determined by image analysis, progressively increased (P < 0.001; r = 0.97) from 3.4 +/- 0.2% and 447 +/- 29, respectively, on day 54 to 15.9 +/- 0.9% and 1,375 +/- 71, respectively, on day 170. In summary, the present study shows that villous cytotrophoblasts were a major source of VEG/PF mRNA and protein in the baboon villous placenta, and that cytotrophoblast VEG/PF mRNA levels and vascularization of the villous placenta closely paralleled the increase in estradiol concentrations of advancing pregnancy. These results are consistent with the concept that estrogen has an important role in establishing the new vascular system within the developing placenta during primate pregnancy and that VEG/PF mediates this process.

Basic fibroblast growth factor messenger ribonucleic acid levels in human placentas from normal and pathological pregnancies

This study was designed to determine whether basic fibroblast growth factor (bFGF) gene expression in human placenta varies as a function of gestational age and to evaluate whether bFGF synthesis might be altered in pathological pregnancies. Moreover, we also investigated whether human placental cells express the bFGF receptor gene. The presence of mRNA for bFGF and its receptor was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on total RNA derived from human placental cells at different times of culture. Levels of bFGF mRNA were determined by competitive RT-PCR in human placental tissues collected at the beginning and at the end of pregnancy. Competitive RT-PCR was also employed to evaluate bFGF synthesis in term placentas derived from pregnancies complicated by diabetes. mRNA for both bFGF and its receptor were demonstrated in human placenta starting as early as 8 weeks of pregnancy. Values of bFGF mRNA were significantly higher in first trimester compared with term placentas. Term placentas derived from pregnancies associated with type I diabetes expressed levels of bFGF mRNA higher than those present in normal term placentas. These data demonstrate that bFGF and its receptor are synthesized in human placental cells throughout gestation. Moreover, bFGF gene expression is developmentally regulated. Finally, bFGF might also be partially responsible for the placental alterations observed in pregnancies complicated by diabetes.

Modulation of leukemia inhibitory factor gene expression and protein biosynthesis in the human fallopian tube

OBJECTIVE: The fallopian tube is the site of fertilization and early embryonic growth and a common site of ectopic implantation. Although the factors responsible for early embryogenesis and implantation are incompletely understood, leukemia inhibitory factor may have an important role in early embryonic development and implantation. We set out to evaluate the production and modulation of leukemia inhibitory factor in the fallopian tube. STUDY DESIGN: We first investigated leukemia inhibitory factor messenger ribonucleic acid levels in fallopian tubes. We then investigated leukemia inhibitory factor messenger ribonucleic acid and protein production in tubal epithelial and stromal cell cultures. RESULTS: Leukemia inhibitory factor messenger ribonucleic acid is expressed in the fallopian tube with only slight variation during the menstrual cycle; however, it is markedly elevated in association with ectopic pregnancy. The level is higher in the tubal mucosa than in the remaining layers and is higher in the more distal segments of the fallopian tube. Estradiol and progesterone did not modulate leukemia inhibitory factor expression in epithelial or stromal cell cultures. Interleukin-1α, tumor necrosis factor-α, and transforming growth factor-β enhanced leukemia inhibitory factor expression in epithelial and stromal cells, with transforming growth factor-β1 enhancing expression by fourfold in stromal cells. Epithelial cells secreted high levels of leukemia inhibitory factor compared with stromal cells (332 ± 89 vs 25 ± 42 pg/mg total protein). Yet stromal cells treated with transforming growth factor-β alone or in combination with epidermal growth factor and platelet-derived growth factor, as well as TNF-α alone or in combination with interleukin-1α enhanced secretion of leukemia inhibitory factor at or above the levels found with epithelial cells. CONCLUSIONS: We speculate that the high constitutive levels of leukemia inhibitory factor expressed in the ampullary portion of the fallopian tube may play a role in early embryonic development. Additionally, elevated expression with ectopic implantation and the marked induction of secretion in the tubal stroma by growth factors and cytokines suggest a link between inflammation, leukemia inhibitory factor, and tubal ectopic pregnancies. (Am J Obstet Gynecol 1996;175:1611-9.)

Basic fibroblast growth factor messenger ribonucleic acid levels in eutopic and ectopic human endometrial stromal cells as assessed by competitive polymerase chain reaction amplification

Previous studies have localized basic fibroblast growth factor (bFGF) and its mRNA in normal and neoplastic endometrium but the expression of bGFG mRNA in endometriosis cells is virtually unknown. Our purpose was to investigate the presence of bFGF and its receptor mRNAs in highly purified primary cultures of stromal cells isolated from six eutopic endometrial samples obtained from patients without evidence of endometriosis and five endometriosis cyst linings. Using reverse transcriptase-polymerase chain reaction (RT-PCR), single major DNA bands of the expected sizes for bFGF and its receptor (354 and 661 bp, respectively) were detected in both endometrial and endometriosis samples. A competitive RT-PCR, that uses coprimer extension and amplification of a bovine RNA as an internal standard, was developed for semiquantitative estimation of bFGF gene expression. The target to standard mean ratios ± SEM in six normal endometrial samples and in five endometriosis cultures were 26.7 ± 10.7 and 9.2 ± 3.0, respectively. Furthermore, when data were analyzed according to the cyst diameter, levels of bFGF mRNA resulted statistically higher ( P < 0.05) in bigger cysts when compared to those detected in smaller ones (16 ± 2.7 and 4.7 ± 1.8, respectively). These results demonstrate that the genes coding for bFGF and its receptor are expressed in endometriosis cells, but levels of bFGF mRNA are generally lower than those detected in their eutopic counterpart. Moreover, they indicate that endometriosis cells derived from large cysts have increased bFGF mRNA levels. Thus, bFGF could be one of the factors responsible for a more or less active behavior of the endometnotic lesion.

Estrogen control of uterine tissue factor messenger ribonucleic acid levels.

One of the early responses of the immature rat uterus to stimulation by estradiol (E2) is an increase in the specific activity and synthesis of a discrete set of proteins including tissue factor (TF). Increases in TF are associated with stimulation of cell growth in mouse and human fibroblasts and endothelial cells. The increase in TF activity following E2 stimulation of the uterus is due to an increase in TF messenger RNA (mRNA). A 2- to 4-fold increase in mRNA is observed 1 h after injection, reaches a maximum at 3 h, and is reduced at 6 h. The increase is hormone specific and occurs at low levels of E2 (0.66 micrograms/kg). The E2 effect is abolished by actinomycin but not by cycloheximide. The changes in TF mRNA occur in a similar time scale and at similar E2 doses as the increases in the uterine proto-oncogenes c-jun and c-fos.

Estrogen control of uterine tissue factor messenger ribonucleic acid levels

One of the early responses of the immature rat uterus to stimulation by estradiol (E2) is an increase in the specific activity and synthesis of a discrete set of proteins including tissue factor (TF). Increases in TF are associated with stimulation of cell growth in mouse and human fibroblasts and endothelial cells. The increase in TF activity following E2 stimulation of the uterus is due to an increase in TF messenger RNA (mRNA). A 2- to 4-fold increase in mRNA is observed 1 h after injection, reaches a maximum at 3 h, and is reduced at 6 h. The increase is hormone specific and occurs at low levels of E2 (0.66 micrograms/kg). The E2 effect is abolished by actinomycin but not by cycloheximide. The changes in TF mRNA occur in a similar time scale and at similar E2 doses as the increases in the uterine proto-oncogenes c-jun and c-fos.

Effect of Hypophysectomy and Growth Hormone Replacement on Hypothalamic Growth Hormone‐Releasing Factor Messenger Ribonucleic Acid Levels

Abstract The mechanisms by which the pituitary gland, and growth hormone (GH) in particular, affect growth hormone-releasing factor (GRF) gene expression have been addressed using the technique of in situ hybridization. Anatomically matched sections through the mediobasal hypothalamus of control and hypophysectomized male rats, with or without GH hormone replacement, were analysed to obtain information on GRF mRNA levels within the arcuate nucleus and around the ventromedial hypothalamus. Hypophysectomy resulted in a 70% increase in the amount of GRF mRNA per cell (P<0.001), within neurons in the arcuate nucleus. GH replacement and T4 replacement separately partially attenuated this increase (GH replacement P< 0.001 versus hypophysectomy, T4 replacement P<0.05 versus hypophysectomy). Additionally, after hypophysectomy there was an 80% increase in the number of cells expressing the GRF gene in neurons around the ventromedial hypothalamus, when compared to shamoperated controls (P<0.01). Both GH and T4 replacement separately partially attenuated this phenomenon (P<0.01 versus hypophysectomized animals). Hypothyroidism alone did not affect GRF mRNA levels in either the arcuate nucleus or in the area surrounding the ventromedial hypothalamus. These results show that hypophysectomy increases GRF mRNA levels in two separate ways: by increasing the amount of mRNA produced per cell within the arcuate nucleus, and by increasing the number of cells expressing the gene in the area surrounding the ventromedial hypothalamus. This increase in the number of GRF mRNA-containing cells after hypophysectomy could result from the recruitment of neurons which previously did not express the GRF gene, and may reflect the plasticity of the adult central nervous system in response to a changing endocrine environment. This could represent part of a sensor mechanism to drive the production of GRF in the arcuate nucleus in response to extreme disruption of the GRF/ GH feedback loop.