Effects of High Glucose and Advanced Glycosylation Endproducts(AGE) on the in vitro Permeability Model

PURPOSE: We describe the changes of rat glomerular epithelial cells when exposed to high levels of glucose and advanced glycosylation endproducts(AGE) in the in vitro diabetic condition. We expect morphological alteration of glomerular epithelial cells and permeability changes experimentally and we may correlate the results with a mechanism of proteinuria in DM. METHODS: We made 0.2 M glucose-6-phsphate solution mixed with PBS(pH 7.4) containing 50 mg/mL BSA and protease inhibitor for preparation of AGE. As control, we used BSA. We manufactured and symbolized five culture dishes as follows; B5 - normal glucose(5 mM) + BSA, B30 - high glucose(30 mM) + BSA, A5 - normal glucose(5 mM) + AGE, A30 - high glucose(30 mM) + AGE, A/B 25 - normal glucose(5 mM) + 25 mM of mannitol(osmotic control). After the incubation period of both two days and seven days, we measured the amount of heparan sulfate proteoglycan(HSPG) in each dish by ELISA and compared them with the B5 dish at 2nd and 7th incubation days. We observed the morphological changes of epithelial cells in each culture dish using scanning electron microscopy(SEM). We tried the permeability assay of glomerular epithelial cells using cellulose semi-permeable membrane measuring the amount of filtered BSA through the apical chamber for 2 hours by sandwich ELISA. RESULTS: On the 2nd incubation day, there was no significant difference in the amount of HSPG between the 5 culture dishes. But on the 7th incubation day, the amount of HSPG increased by 10% compared with the B5 dish on the 2nd day except the A30 dish(P0.05). In the osmotic control group (A/B 25) no significant correlation was observed. On the SEM, we could see the separated intercellular junction and fused microvilli of glomerular epithelial cells in the culture dishes where AGE was added. The permeability of BSA increased by 19% only in the A30 dish on the 7th day compared with B5 dish on the 7th day in the permeability assay(P<0.05). CONCLUSION: We observed not only the role of a high level of glucose and AGE in decreasing the production of HSPG of glomerular epithelial cells in vitro, but also their additive effect. However, the role of AGE is greater than that of glucose. These results seems to correlate with the defects in charge selective barrier. Morphological changes of the disruption of intercellular junction and fused microvilli of glomerular epithelial cells seem to correlate with the defects in size-selective barrier. Therefore, we can explain the increased permeability of glomerular epithelial units in the in vitro diabetic condition.

Studies on Differentiation of Cardiac Myoblast Induced by Co-culture with Isolated Neonatal Rat Cardiac Myocytes / 대한해부학회지

Cellular cardiomyoplasty has recently emerged as a potential new treatment of ischemic heart disease. Combining cellular cardiomyoplasty with gene therapy using myogenic transcription factor might facilitate myocardial regeneration. In this study, we engineered H9c2, L6 using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. Each cell overexpressing MEF2 and Nkx2.5 was generated by gene transfection and clonal selection. CO-culture was performed that each cell line added over cultured cardiomyocyte. H9c2-MEF2c and H9c2-Nkx2.5 became long, spindle shape like cardiomyocyte. Troponin T, cardiac specific marker, was found spot-like pattern in H9c2-Nkx2.5. However, co-culture with cardiomyocyte did not induce differentiation all kinds of cells into cardiomyocyte. Connexin43, which is gap junction marker was increased in H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c and L6-Nkx2.5. Especially, co-culture with cardiomyocyte resulted in elevation of connexin43 levels more than monoculture. Ultrastructurally, formations of gap junction and desmosome were found apparently in L6-Nkx2.5. Long-standing, strong, regular and more frequent contraction were observed in cardiomyocyte co-cultured with H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c, L6-Nkx2.5, respectively. Neverthless, any cell did not have active contraction itself, but passive movement except cardiomyocyte. H9c2-MEF2c, L6-MEF2c and L6-Nkx2.5 had resistance to hypoxia compared with other groups. These results suggested that co-culture and overexpressions of MEF2c and Nkx2.5 induced differentiation into cardiomyocyte and played an important role on intercellular junction formation and hypoxic resistance. This would be a promising source of cellular cardiomyoplasty. Therefore, much more research would be essential for clinical application of cellular cardiomyoplasty and this study would be a basic source for further study of MEF2c and Nkx2.5 in cellular cardiomyoplasty.

Role of Cardiac Transcription Factor Nkx2.5 on Cardiomyoplasty Model in vitro / 대한해부학회지

Despite therapeutic advance, the prevalence of ischemic heart disease continues to increase. Recently, cell transplantation of stem cell has been proposed as a strategy for cardiac repair following myocardial damage. However, low differentiation efficiency into cardiomyocyte and poor cell viability associated with transplantation have limited the reparative capacity of these cell. In this study, we engineered P19 embryonal carcinoma cells using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction of P19 in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. An P19 embryonal carcinoma cell line expressing GFP, MEF2c, Nkx2.5 was generated by gene transfection and clonal selection. Nkx2.5 overexpression induced connexin43 expression level decrease. Electron microscopy revealed myofibril organization and immunostaining with cTnT showed positive staining in P19-Nkx2.5, consistent with early stage cardiomyocyte. Connexin43 and N-cadherin was expressed between P19-MEF2c and cardiomyocyte, P19- Nkx2.5 and cardiomyocyte in co-culture. And beating rate of cardiomyocyte co-cultured with P19-Nkx2.5 increased much more than other group, even if P19-Nkx2.5 did not have synchronous contraction with cardiomyocyte. Additionally, P19-Nkx2.5 had a resistance against hypoxia. These result suggest that overexpression of Nkx2.5 induced differentiation of P19 into cardiomyocyte and would be electro-mechanical coupling with cardiomyocyte after transplantation. Futhermore, Nkx2.5 overexpression had protection potential to hypoxic injury. Therefore, P19 cell overexpressed Nkx2.5 would be promising cell source for further study of new therapy of myocardial disease and building up in vitro model.

Formation of Intercellular Junction between Cardiomyocyte and H9c2 Cell Line in Co-Culture / 체질인류학회지

Recently, new treatments for human heart disease such as ischemia, infarction, cardiomyopathy, coronary heart disease have been developed. transplantation various kinds of cells from skeletal muscle, endothelium, mesenchyme, hemopoietic tissue to injured area after infarction were challenged. It's so called 'Cell Transplantation'. This therapeutic strategy already adopted and got a good result in clinical trial. But several limitations are still remained, including ethics, donor cell numbers, side effects, therapeutic efficiency. In this research, we investigated the formation of intercellular junction and synchronous contraction between cardiomyocyte and H9c2 cell line in co-culture to establish experimental model in vitro for cell transplantation. For this purpose, two kinds of cells, primary cultured cardiomyocyte and H9c2 (cardiomyoblast cell line) were used. Cultured cardiomyocytes had repetitive contraction-relaxation pattern along longitudinal axis both in single and coculture. But their contractions were slower, less regular, less strong in co-culture than in cardiomyocyte culture only. H9c2 cells did not contracted actively themselves, but moved toward cardiomyocyte passively coincided with contraction. In contact region between two kinds of cells, there was no signal after immunocytochemical staining labeled with connexin43 (gap junction), desmoplakin (desmosome), N-cadherin (adherent junction) even though they had membrane contact. Moreover, F-actin and striation were less developed. These results suggested that co-culture system interfere with remodelling of contractile apparatus, intercellular junction formation as well as contraction-relaxation. Furthermore cardiomyocyte could not induce H9c2 cells differentiation into cardiomyocyte. Therefore, much more research would be essential for clinical application of cell transplantation and this study would be the basic source for further study of new therapy of myocardial disease and building up in vitro model.