Effects of Hyperglycemia and Metformin on Expression of Adhesion Molecules on Human Aortic Endothelial Cells

Expression of adhesion molecules on the endothelial cell surface as a response to elevated glucose concentration is considered as a main event in the development of atherosclerosis. The influence of high glucose concentration and metformin, a wide used anti-diabetic drug, on the expression of E-selectin, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on human aortic endothelial cells (HAECs) was investigated. HAECs were cultured 4 h in a medium with 5.5, 8.0, 12.0, and 16.5 mmol dm-3 glucose with or without metformin addition. The expression of cell adhesion molecules (CAM) was measured by flow-cytometry. Compared to CAM expression in the medium with 5.5 mmol dm-3 glucose, glucose concentration of 12.0 mmol dm-3 increased expression of E-selectin (62 %), VCAM-1 (four fold) and ICAM-1 (81 %). Metformin administration in the medium with 12.0 mmol dm-3 glucose additionally enhanced E-selectin and VCAM-1 expression compared to effects of corresponding glucose concentration. ICAM-1 expression was only significantly increased in the medium with metformin and 8.0 mmol dm-3 glucose. In conclusion, metformin in condition with elevated glucose concentration additionally increased expression of CAM on HAECs which could contribute to development of macrovascular complications in diabetic patients

Trefoil factor family protein 3 affects cancellous bone formation in the secondary centers of ossification of mouse tibiae

Background and Purpose: Trefoil factor family (TFF) is a small family of peptides, which comprises three peptides, TFF1, TFF2 and TFF3. The primary sites of TFF expression and synthesis are mucous epithelia, with gastrointestinal mucosa being their main localization. They have a role in mucosal restitution and strengthening of the normal mucosal barrier. Although not present in healthy articular cartilage, TFF3 is expressed in osteoarthritis and septic arthritis affected cartilage and promotes cartilage degradation in such conditions. Also, TFF3 is present in endochondral ossification during embryonic development. Since this implies a role for TFF3 in the ossification process, we evaluated the histomorphometric parameters of the trabecular bone in the epiphyses of mouse tibiae from wild type mice and TFF3 knock-out mice. Materials and Methods: Tibiae of wild-type and knock-out mice were isolated, fixed in 4% paraformaldehyde, paraffin embedded and cut into 6μm sections, which were stained using Masson’s trichrome stain. Digital photographs were taken for histomorphometry of the epiphyseal cancellous bone. Results: Trabecular bone volume density, trabecular bone surface density and trabecular number were significantly decreased in TFF3 knock-out mice, when compared to wild-type mice. Trabecular separation was significantly higher in TFF3 knock-out mice, and trabecular thickness did not differ significantly. Conclusions: In addition to its impact on the cartilage degradation, our present study shows that TFF3 might also have a role in the formation of cancellous bone and its properties

Trefoil factor family protein 3 affects cancellous bone formation in the secondary centers of ossification of mouse tibiae

Background and Purpose: Trefoil factor family (TFF) is a small family of peptides, which comprises three peptides, TFF1, TFF2 and TFF3. The primary sites of TFF expression and synthesis are mucous epithelia, with gastrointestinal mucosa being their main localization. They have a role in mucosal restitution and strengthening of the normal mucosal barrier. Although not present in healthy articular cartilage, TFF3 is expressed in osteoarthritis and septic arthritis affected cartilage and promotes cartilage degradation in such conditions. Also, TFF3 is present in endochondral ossification during embryonic development. Since this implies a role for TFF3 in the ossification process, we evaluated the histomorphometric parameters of the trabecular bone in the epiphyses of mouse tibiae from wild type mice and TFF3 knock-out mice. Materials and Methods: Tibiae of wild-type and knock-out mice were isolated, fixed in 4% paraformaldehyde, paraffin embedded and cut into 6μm sections, which were stained using Masson’s trichrome stain. Digital photographs were taken for histomorphometry of the epiphyseal cancellous bone. Results: Trabecular bone volume density, trabecular bone surface density and trabecular number were significantly decreased in TFF3 knock-out mice, when compared to wild-type mice. Trabecular separation was significantly higher in TFF3 knock-out mice, and trabecular thickness did not differ significantly. Conclusions: In addition to its impact on the cartilage degradation, our present study shows that TFF3 might also have a role in the formation of cancellous bone and its properties

Advantages of Modified Osteosynthesis in Treatment of Osteoporotic Long Bones Fractures – Experimental Model

In surgery of fractured long bones, a patient suffering from osteoporosis represents constant challenge to a surgeon and applied material and instruments that need to destroy as little as possible of an already damaged bone. One potential way of increasing the contact surface between the implants and osteoporotic bone is injection of bone cement (methyl-metacrilat, Palakos) into a prepared screw bed. This method of osteosynthesis was therefore subjected to experimental research to prove that application of modified osteosynthesis using bone cement in treatment of fractures in osteoporotic patients has advantage over the standard method of osteosynthesis because this modified method enables significantly greater firmness and stability of the osteosynthesis, which is the essential precondition of a successful fracture healing. The research was carried out on six macerated cadaveric preparations of a shin bone from the osteological collection from Institute for Anatomy, School of Medicine, University »J. J. Strossmayer«. All samples of long bones were artificially broken in the middle part of the diaphysis and then standard osteosynthesis and modified osteosynthesis with screws filled with bone cement were performed on the samples. Results show that under identical static action of the moment of torsion in the modified osteosynthesis torsion angle deviation is lower than in the standard osteosynthesis. In modified osteosynthesis with bone cement the first results for angle of torsion deviation greater than 0.2 degrees were noticed after 120 minutes, while in the standard method of osteosynthesis they were noticed already in the first minute

Localization of trefoil factor family peptide 3 (TFF3) in epithelial tissues originating from the three germ layers of developing mouse embryo

Trefoil factor family (TFF) peptides are involved in the maintenance of epithelial integrity and epithelial restitution. Mature epithelial tissues originate from different embryonic germ layers. The objective of this research was to explore the presence and localization of TFF3 peptide in mouse embryonic epithelia and to examine if the occurrence of TFF3 peptide is germ layer-dependent. Mouse embryos (14-18 days old) were fixed in 4% paraformaldehyde and embedded in paraffin. Immunohistochemistry was performed with affinity purified rabbit anti-TFF3 antibody, goat anti-rabbit biotinylated secondary antibody and streptavidin-horseradish peroxidase, followed by 3, 3’-diaminobenzidine. TFF3 peptide was present in the gastric and intestinal mucosa, respiratory mucosa in the upper and lower airways, pancreas, kidney tubules, epidermis, and oral cavity. The presence and localization of TFF3 peptide was associated with the embryonic stage and tissue differentiation. TFF3 peptide distribution specific to the germ layers was not observed. The role of TFF3 peptide in cell migration and differentiation, immune response, and apoptosis might be associated with specific embryonic epithelial cells. TFF3 peptide may also be considered as a marker for mucosal maturation

Analysis of Fibroblast Growth Factor Influence on Growth and Developmental Potential of Rat Foetuses in the In Vitro Culture Model

The fibroblast growth factor’s (FGF) influence on the growth and differentiation of 8- and 9- day-old rat foetus has been studied, whereas foetuses were grown in an in vitro culture model. Proliferation was analysed by the expression of proliferating cell nuclear antigen (PCNA). It was established that the usage of FGF in the first period of the culture lowers the growth no matter the foetus age at the moment of culturing and no matter whether it is a medium with or without a serum. If FGF is applied in the second culture period, it also lowers the growth, however younger foetuses in the in vitro culture model are more sensible to FGF negative influence.When FGF was applied in a lower concentration the growth of whole foetuses was improved in the in vitro culture model, which shows that the FGF influence on growth depends on the concentration. Stereological analyses have been done and showed that, in the in vitro culture model, FGF has no influence on proliferating cartilage tissue, but it stimulates the survival of nervel tissue cells. It has been shown that the quantitative research of growth processes in cultivated foetuses can precisely be done by combining classic methods of measuring whole foetus diameters and analysing the expression of proliferating antigen

Developmental Potential of Mouse Embryos without Extraembryonic Membranes in Modified Organ Culture

The long term stationary culture of postimplanatation embryos without extraembryonic membranes is a method to assess their developmental potential in vitro. The method was almost exclusively used on rat embryos, while mouse embryos were considered unsuitable due to their poor differentiation. In present study the postimplantation mouse embryos were used to verify potential of this method in mice. In addition, the course of in vitro differentiation was compared to embryo development in situ. Embryos were cultivated for maximum of 14 days and morphology and differentiation was analysed on serial semithin sections. Although anatomical relationships were lost from the beginning of the cultivation, the differentiation was only delayed, and the developmental potential after long term culture was comparable to those observed in rats. Therefore the advantages of long term cultivation could be utilized to analyse the differentiation of numerous lines of genetically modified mice with impaired postimplantation development

Experimental models in the research of the early embriogenesis

Razvojna biologija u Hrvatskoj ima 50-godišnju tradiciju zahvaljujući njenom osnivaču prof. dr. Nikoli Škrebu, te velikom broju njegovih suradnika i učenika. Istraživanja su bila usmjerena na kritična razdoblja razvoja u ranoj embriogenezi sisavaca (miš, štakor). Razrađenim postupcima izolacije zametnih listića te, potom, transplantacije istih na ektopična mjesta domaćina (korioalantoisna membrana, prednja očna komora, bubrežna čahura) dobiveni su teratomi, koji su omogućili posredno proučavanje razvojnih sposobnosti nediferenciranog presatka. Postupci kulture zametaka in vitro i transplantacije in vivo pokazali su stabilnost diferencijacije. U potrazi za genima koji upravljaju embrionalnim razvojem, primijenio se postupak genske zamke, kojim se mogu otkriti novi geni s ograničenim uzorkom ekspresije.The period of continuous and internationally recognized research in developmental biology began in Croatia in the early fifties. It was initiated by Nikola Škreb and continued by his coworkers. His research was focused to the analysis of the early postimplantation rodent embryo with special attention on the crucial period of gastrulation. The next step was the analysis of growth and histological differentiation after transfer of embryos and separated germ lyers to ectopic sites (kidney capsule, anterior chamber of the eye, chorioalantois membrane). The potential of early rat embryo to grow and differentiate was shown using a method of organ culture in vitro. After establishing molecular methods and identification of genes involved in the development, the same developmental problems are investigated in a new way. One of them is the gene trap procedure, which utilises the possibility to select trapped genes active during embryogenesis

Pojavnost intramiokardijalnih masnih stanica u stijenci desne pretklijetke i desne klijetke – postmortalna humana analiza

Histologic and radiologic studies describe intramyocardial fat tissue as a normal finding or as part of cardiac pathology. The role of fat cells within the myocardium is not fully understood. The aim of this study was to assess fat tissue distribution in the myocardium of right atrium (RA) and right ventricle (RV) and age differences in subjects free from cardiac disease. The study included 10 males without cardiac disease divided into two groups according to age (below/above 50 years). Three cross sections were performed (RV free wall and apex and RA free wall) with histomorphological analysis on digital photographs. The shares of total myocardial fat (TMF), perivascular fat (PVF) and non-perivascular (nPVF) fat were calculated. Samples from the older group had larger amounts of fat in the epicardium and myocardium, without statistically significant difference (TMF p=0.847, PVF p=0.4 and nPVF p=0.4). The largest quantities of fat tissue were found in the RV apex samples (14.9%), followed by RV free wall (7.5%) and RA (4.5%), where total apical RV fat share was significantly larger than in RA sample (p=0.044). Intramyocardial fat cells were present within the non-diseased RA and RV in all samples, mostly in the apex. Further investigations on age difference, effect of visceral obesity and sex differences are needed.Dosadašnja histološka i radiološka istraživanja opisuju pojavnost intramiokardijalnog masnog tkiva kao dio normalnog nalaza ili kao dio patološkog nalaza srca. Uloga intramiokardijalnih masnih stanica nije još razjašnjena. Cilj ovoga istraživanja bio je utvrditi raspodjelu masnog tkiva u miokardu desne pretklijetke (DP) i desne klijetke (DK) ovisno o dobi i lokalizaciji kod ispitanika bez kardijalnog morbiditeta. Patohistološka postmortalna studija uključila je 10 muškaraca bez kardijalnih bolesti podijeljenih u dvije skupine prema dobi (ispod/iznad 50 godina). Uzorci su uzeti u tri presjeka (projekcija slobodnog zida DK, slobodni zid vrha DK te stijenka DP) s histomorfološkom analizom na digitalnim fotografijama. Izračunati su udjeli ukupnog masnog tkiva unutar srčanog mišića (UMT), perivaskularnog masnog tkiva (PMT) i ne-perivaskularnog masnog tkiva (nPMT). U uzorcima starijih ispitanika prisutna je veća količina masnog tkiva u epikardu i miokardu, bez statistički značajne razlike (UMT p=0,847, PMT p=0,4, nPMT p=0,4). Najveće količine masnog tkiva pronađene su u uzorcima vrha DK (14,9%), nakon čega slijedi stijenka slobodnog zida DK (7,5%) i DP (4,5%), gdje je ukupni udio masnog tkiva DK bio znatno veći nego kod uzorka DP (p=0,044). Intramiokardijalne masne stanice sastavni su dio histološkog nalaza DP i DK u svim uzorcima bez prisutnih kardijalnih bolesti, dominantno u vrhu DK. Neophodna su daljnja istraživanja utjecaja dobi, spola i visceralne pretilosti na pojavnost intramiokardijalnih masnih stanica unutar stijenke desnog srca

Experimental models in the research of the early embriogenesis

Razvojna biologija u Hrvatskoj ima 50-godišnju tradiciju zahvaljujući njenom osnivaču prof. dr. Nikoli Škrebu, te velikom broju njegovih suradnika i učenika. Istraživanja su bila usmjerena na kritična razdoblja razvoja u ranoj embriogenezi sisavaca (miš, štakor). Razrađenim postupcima izolacije zametnih listića te, potom, transplantacije istih na ektopična mjesta domaćina (korioalantoisna membrana, prednja očna komora, bubrežna čahura) dobiveni su teratomi, koji su omogućili posredno proučavanje razvojnih sposobnosti nediferenciranog presatka. Postupci kulture zametaka in vitro i transplantacije in vivo pokazali su stabilnost diferencijacije. U potrazi za genima koji upravljaju embrionalnim razvojem, primijenio se postupak genske zamke, kojim se mogu otkriti novi geni s ograničenim uzorkom ekspresije.The period of continuous and internationally recognized research in developmental biology began in Croatia in the early fifties. It was initiated by Nikola Škreb and continued by his coworkers. His research was focused to the analysis of the early postimplantation rodent embryo with special attention on the crucial period of gastrulation. The next step was the analysis of growth and histological differentiation after transfer of embryos and separated germ lyers to ectopic sites (kidney capsule, anterior chamber of the eye, chorioalantois membrane). The potential of early rat embryo to grow and differentiate was shown using a method of organ culture in vitro. After establishing molecular methods and identification of genes involved in the development, the same developmental problems are investigated in a new way. One of them is the gene trap procedure, which utilises the possibility to select trapped genes active during embryogenesis