Parathyroid hormone(PTH), a major bone hormone, inhihits DNA and collagen syntheses in osteohlast-like cells in vitro, but increase the proliferation of osteoblast in vivo as secn in hyperparathyroidism. On the other hand, insulin is known to increase DNA and collagen syntheses and modify the effects of PTH in osteoblast-like cells. We have examined the effects of PTH and insulin in rat osteosarcoma UMR-l06-01 cells and whether PTH plays a role in the insulin-mediated bone formation. When 1 nM PTH and 10 nM insulin were administered to VMR-l06-01 ceils, the rates of DNA synthesis were 124% and 136% of the untreated control, respectively. When the two hormones were administered serially by exposing to 1 nM PTH for 7 days followed by 10 nM insulin lor 24h, the largest increase was observed. The protein synthesis was also increased remarkahly when the two hormones were aclministered serially: the[3H]-leucine incorporation rates, compared to the control group, were 75% and l62% with PTH ancl insulin administration, respectively, but the rate was 297% with the serial administration of the two. The collaeen synthesis, as measured by the (3H)-proline incorporation rates were 60% and l64% with PTH and insulin administration, respectively, but 351% with serial administration, again showing a dramatic effect. These results showed that 1 nM PTH decreased DNA and collagen syntheses in UMR-l06-01 cells after both a 24h and a more prolonged exposure. Similar exposures to insulin tended to increase the syntheses. The comhination of PTH and insulin tended to increase the syntheses. hut not beyond the effect of insulin alone. However, the sequential administration of PTH and insulin markedly increases ihose rales relative to the simultaneous adminstration of these two hormones. Thus, it is possihle that sequential stimulation of PTH and insulin in hone matrix exerts an synergistic effect on hone formation in vivo.
Animals ; Collagen ; DNA ; Hand ; Hyperparathyroidism ; Insulin* ; Osteoblasts ; Osteogenesis ; Osteosarcoma* ; Parathyroid Hormone* ; Rats ; Respiratory Sounds

Cartilage is one of the most commonly manipulated tissue in esthetic and reconstructive surgery. Cartilage has an important role in longitudinal bone growth. Anabolic hormones and locally produced peptide growth factors are known to influence this process Matrix composition changes through proliferation, maturation, and differentiation of chondrocytes, and endochondral ossification thereafter. Defined cartilage matrix is synthesized during the maturation of chondrocytes where the major change is the increment of type II collagen. Variable sulfated mucololysaccharides and hyaluronic acid are also synthesized during this maturation. IGF-I(insulin like growth factor-I), so called somatomedin C, is a prominent growth factor in serum. IGF-I is known to be involved in long growth. IGF-I is affected by pituitary growth hormone. There are few studies done on IGF-I effect in cartilage matrix formation and possible changes of collagen subtypes. This experiment was designed to see the IGF-I effect on the colagen synthesis of cultured chondrocytes. Optimal concentration of IGF-I for the experiment was determined using H3-thymidine incorporation into DNA. The IGF-I effect on collagen synthesis was studied using H3-proline. The IGF-I effect on the synthesis of subtypes of collagen was studied using SDS-PAGE and immunocytochemical staining. Chondrocytes were isolated from the ears of New Zealand white rabbit and cultured in 2 X 10(5) cells/300 microgram density. IGF-I increased DNA synthesis, and optimal concentration of IGF-I was determined by dose-relationship curve as 10ng/ml. Collagen synthesis was increased by IGF-I. Type II collagen was increased on SDS-PAGE with IGF-I and this gel electrophoresis showed type X collagen, also. The increase in type II collagen was confirmed with immunocytochemical staining, the reaction becoming stronger with the addition of IGF-I. Type I collagen was not changed with IGF-I on immunocytochemistry. We conclude that IGE-I is an important modulator influencing not only proliferation and maturation but also terminal different-iation of chondrocytes.
Bone Development ; Cartilage ; Chondrocytes* ; Collagen Type I ; Collagen Type II ; Collagen Type X ; Collagen* ; DNA ; Ear ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Growth Hormone ; Hyaluronic Acid ; Immunohistochemistry ; Insulin-Like Growth Factor I* ; Intercellular Signaling Peptides and Proteins ; New Zealand

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been major difficulties in developing homogenous cultures of human glomerular endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogenous monolayers of human glomerular endothelial cells based on the method of Green DF et al published in 1992. Using the selective media and the sieving method, fibronectin was required as a surface matrix after adequate collagenase treatment, and endothelial cell growth factor and heparin was needed for the continuous growth of endothelial cells. The endothelial cell growth factor was isolated from the bovine hypothalamic extracts. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence and stained homogenously with von Willebrand factor(factor VIII). The cytokeratin and the actin were not stained. This study might be helpful for in vitro study to know the biological characteristics of human glomerular endothelial cells under the predetermined condition.
Actins ; Cell Culture Techniques ; Collagenases ; Endothelial Cells* ; Epithelial Cells ; Fibronectins ; Heparin ; Humans* ; Keratins ; Population Characteristics

BACKGROUND: The expression of adhesion molecules contribute to development of systemic diseases. Vascular cell adhesion molecule-l(VCAM-1) is an endothelial cell membrane glycoprotein that has been implicated in leukocyte/endothelial cell interactions in inflammation. OBJECTIVE: The aim of this study was to characterize the surface expression and regulation of VCAM-1 on two different endothelial cells. METHOD: We examined the effects of the expression of VCAM-1 in two different endothelial cells, isolated from human umbilical cords and human glomerulus. Expression of VCAM-1 was measured by enzyme-linked immunosorbent assay(ELISA) and flow cytometry. RESULTS: In human umbilical cord endothelial cells(HUVECs), both interleukin-l B(IL-lB) and tumor necrosis factor-a (TNF-a) increased VCAM-1 expression. VCAM-1 expression increased by TNF-a was higher than that increased by IL-lB. In human glomerular endothelial cells(HGECs), IL-lB and TNF-a markedly increased VCAM-1 expression. Conclusion. The regulation of VCAM-1 appears to be somewhat different in HGECs compared with HUVECs. These differences between the responsiveness of the two cells may possibly indicate inherent differences in endothelial cell derived from different vascular beds.
Cell Adhesion ; Cell Communication ; Cytokines* ; Endothelial Cells* ; Flow Cytometry ; Humans* ; Inflammation ; Membrane Glycoproteins ; Necrosis ; Umbilical Cord ; Vascular Cell Adhesion Molecule-1*

Objective: : Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. Methods: : Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. Results: : Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. Conclusion : Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

Objective: : Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. Methods: : Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. Results: : Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. Conclusion : Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

The administration of erythropoietin for renal failure patients is frequently associated with a mild-to-marked rise in arterial blood pressure. The erythropoietin-induced hypertension has been variably attributed to the rise in erythrocyte concentration and/or a direct or indirect pressor action of erythropoietin on vascular smooth muscle. Especially erythropoietin-induced hypertension may be mediated by increased production of the potent vasoactive peptide endothelin. This study was designed to determine the effect of erythropoietin to the production of endothelin in human glomerular endothelial cells and human umbilical vein endothelial cells. ELISA method was used to measure the endothelin- 1, after 4 X 10(4) endothelial cells were grown to confluency on 24-well plates, in which erythropoietin 25u/ml was incubated for 24 hours. The results showed that the endothelin production in human glomerular endothelial cells was insignificant (116.0+/-14.0%, P>0.05, mean+/-S.E., n=5, each n means the mean of duplicate experiments), but the endothelin production in human umbilical endothelial cells was increased after 25u/ml erythropoietin treatment(126.5+/-15.2%, P<0.05, mean+/-S.E., n=4). In glomerular endothelial cells, TGF-beta(0.5ng/ml) increased the production of endothelin(218.8+/-57.2%, P<0.01, mean+/-S.E., n=5), also it looks like that TNF-alpha and thrombin might increase the production of endothelin according to the concentration. In conclusion, the responsiveness of endothelial cells to erythropoietin may be different according to the cell type, and glomerular endothelial cells could increase the production of endothelin, if appropriate stimuli were given.
Arterial Pressure ; Endothelial Cells* ; Endothelins* ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; Erythropoietin* ; Human Umbilical Vein Endothelial Cells ; Humans* ; Hypertension ; Muscle, Smooth, Vascular ; Renal Insufficiency ; Thrombin ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha

Cyclosporine(CsA) is a modulator of the immune system used therapeutically to prevent organ transplant rejection. However, it is nephrotoxic and causes thrombotic phenomena after renal and bone marrow transplantation. CsA nephrotoxicity in vivo is associated with elevated levels of von Willebrand factor(vWf), which is a multimeric plasm glycoprotein secreted by endothelial cells and platelets. CsA is not soluble in water and the intravenous form given to patients is dissolved in a vehicle called cremophor EL. The vehicle has been implicated in anaphylatic reactions and associated with the release of histamine in vivo. We hypothesized that CsA or cremophor might affect the release of vWf from human glomerular endothelial cells. vWf was measured in culture supernant using ELISA kit after speed vaccum for 3 hour. The expression of vWf in cultured human glomerular endothelial cells was relatively low compared to human plasm in vivo. CsA alone did not increase vWf release(100%, 99.9+/-0.7%, 99.1+/-2.9%, 106+/-21.5%, CsA 0, 0.1, 1, 10microM mean S.E., n=2), but cremophor EL increased vWf release (100%, 104.0+/-8.6%, 117.6+/-9.5%, 121.3+/-12.2%, mean+/-S.E., n=3). These results were same as the results of experiments under thrombin(1IU/ml) and histamine(10(-4)M). The increased expression of vWf in human glomerular endothelial cells in response to CsA seems to be related to cremophor, the solvent, rather than CsA itself in vitro culture experiments.
Bone Marrow Transplantation ; Cyclosporine ; Endothelial Cells* ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins ; Histamine ; Humans* ; Immune System ; Transplants ; von Willebrand Factor*

PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.
Blotting, Western ; Cell Culture Techniques ; Cell Cycle ; DNA ; Fibroblasts* ; Immunohistochemistry ; Osteoblasts* ; Polymerase Chain Reaction ; RNA, Messenger ; Starvation ; Thymidylate Synthase*

PURPOSE: In the present study, the effects of bFGF on the early responses of proliferation of UMR 106-01 osteoblast cells during cell cycle reentry from the latent(G0/G1) to the proliferative periods(S/M) were investigated. MATERIALS AND METHODS: The synchronized cell culture method using the serum starvation was utilized. After the addition of bFGF, the time courses of protein synthesis, DNA synthesis, thymidylate synthase(TS) activity, TS mRNA level and expression of c-fos were determined. RESULTS: 87% UMR 106-01 cells were synchronized to G0/G1 by serum starvation for seven days in the medium containing 0.1% serum. The protein level began to increase 3 hours after bFGF treatment and reached the maximum at 18 hours. TS activity began to increase 3 hours after the bFGF treatment and reached its peak at 6 hours while its mRNA level, determined by quantitative PCR, reached the maximum at 12 hours. The expression of c-fos protein, determined by western blot analysis and immunocytochemistry, increased 3 hours after bFGF treatment. On the contrary, these prominent changes and responses to bFGF were not observed in the case of using non-synchronized cells cultured in the medium containing 10% serum. CONCLUSION: Based on these data it can be concluded that bFGF-induced DNA synthesis in the early proliferative phase is due to increases in both TS activity and mRNA amount and that the increase in c-fos expression and TS activity occur before the increase in TS mRNA level.
Blotting, Western ; Cell Culture Techniques ; Cell Cycle ; DNA ; Fibroblasts* ; Immunohistochemistry ; Osteoblasts* ; Polymerase Chain Reaction ; RNA, Messenger ; Starvation ; Thymidylate Synthase*