Günter Kamp

Software-supported image quantification of angiogenesis in an in vitro culture system: application to studies of biocompatibility

Healing of soft tissue trauma and bone discontinuities following implantation involves acute inflammatory reactions and the formation of blood vessels (angiogenesis). During angiogenesis new capillary vessels arise from the existing vasculature. Endothelial cells (EC) are the major cell type involved in angiogenesis. Corrosion of orthopaedic metallic implant materials (e.g. CoCr alloys) can cause locally high concentrations of heavy metal ions in the peri-implant tissues. Some divalent metal ions (Co2+, Ni2+, Zn2+) lead to the activation of EC in vitro. Upon exposure to these ions. EC release cytokines and chemokines and increase the expression of cell surface adhesion molecules, which represents the pro-inflammatory phenotype. In this study we have examined whether metal ions influence the other endothelial aspect of wound healing, the angiogenic response. Therefore, we utilized an in vitro model of angiogenesis and examined the effects of divalent cobalt ions on the in vitro vessel formation. The quantification of the cobalt/ion-exerted effects on angiogenesis in vitro was performed using a contrast-rich vital staining and analysed by software-supported image quantification.

Changes in Human Endothelial Cell Energy Metabolic Capacities during in vitro Cultivation. The Role of "Aerobic Glycolysis" and Proliferation

Background: In this study the influence of cultivation and proliferation on energy metabolic characteristics of human umbilical vein endothelial cells (HUVEC) has been examined. The energy metabolic capacities of human endothelial cells freshly isolated from the umbilical vein were compared with those after cultivation for three passages and as subconfluent and confluent cultures. Methods: Expression of cell type-specific differentiation markers and proliferative activity were studied in dependency on cultivation characteristics. Furthermore, the energy metabolic characteristics of HUVEC were analyzed by measurement of the maximum catalytic activities of marker enzymes of various metabolic pathways. Results: Examination of a typical marker of proliferation, Ki67, confirmed that HUVEC changed in culture from a non-proliferative to a proliferative state. Compared to other cell types, the enzyme pattern of HUVEC showed a high glycolytic and a high NADPH regenerating capacity. These capacities increased by cultivation nearly to the same degree as marker enzymes of other metabolic pathways (e.g. citric acid cycle). Conclusion: Our data support the theory that metabolism of EC is primarily by “aerobic glycolysis”, i.e. the conversion of glucose to lactate in the presence of oxygen. These characteristics were independent of whether the cells are freshly isolated/non-proliferating or cell culture-adapted/proliferating.

Paradoxical effects of hypoxia-mimicking divalent cobalt ions in human endothelial cells in vitro

Divalent cobalt ions (Co2+) induce the expression of hypoxia responsive genes and are often used in cell biology to mimic hypoxia. In this in vitro study we compared the effects of hypoxia and Co2+ on human endothelial cells and examined processes that are stimulated in hypoxia in vivo (proliferation and angiogenesis). We analyzed the expression of the hypoxia-inducible factor-1α (HIF-1α) under different hypoxic conditions (3% and nearly 0% O2) and Co2+-concentrations (0.01–0.7 mM). As in hypoxia, the amount of HIF-1α protein was enhanced by exposure to Co2+ (did not correlate with mRNA amount). However, contrary to the results of hypoxia, in vitro-angiogenesis was inhibited after exposure to even low Co2+-concentrations (≥0.01 mM). This led to the conclusion that although hypoxia signaling after Co2+-exposure took place, further yet unknown Co2+-induced event(s) must have occurred. (Mol Cell Biochem 270: 157–166, 2005)

Experimental approaches to study vascularization in tissue engineering and biomaterial applications

The success of tissue engineering and biomaterial applications is not only dependent on the growth and functioning of the organ- or tissue-specific cells on the biomaterial but is entirely dependent in most cases on a successful vascularization after implantation. The process of vascularization involves angiogenesis; the formation of new blood vessels which spread into the implant material and supply the existing cells with the nutrients to survive. We have established in vitro methods using human microvascular endothelial cells to evaluate novel biomaterials for endothelial cell attachment, cytotoxicity, growth, angiogenesis and the effects on gene regulation. These in vitro studies can be used to rapidly evaluate the potential success of a new biomaterial and for the development of matrix scaffolds which will promote a physiological vascularization response.

Characteristics of the spermathecal contents of old and young honeybee queens.

Sperm are often stored, for a long time after mating, in females of various animal species. In case of the queen honeybee (Apis mellifera), sperm remain fertile for several years in the spermatheca. Little information is available regarding the effect of long-term storage of sperm on its fertility. To evaluate this, enzymes and/or sperm have been analysed from the spermatheca of 75 queens of various ages (0 year Y0, n=14; one year Y1, n=14; two years Y2, n=7; virgin queen VQ, n=40) and semen samples have been taken from 46 drones. The sperm from the spermatheca of older queens move more slowly (F=11.45, P < 0.0001) and show different movement patterns (Chi2=90.0, P < 0.0001) from those of the other groups. The spermatheca content of differently aged mated queens differ significantly with respect to the activities of lactate dehydrogenase (F=3.37, P < 0.05), citrate synthase (F=6.24, P < 0.005) and arginine kinase (F=9.44, P < 0.0006). Glyceraldehyde 3-phosphate dehydrogenase (F=0.10, P=0.91) does not differ significantly. The results suggest considerable changes in the energy metabolic profile of the spermatheca tissue, of the sperm or of both during sperm storage.

Long-term effects of the trehalase inhibitor trehazolin on trehalase activity in locust flight muscle.

SUMMARY Trehalase (EC 3.2.1.28) hydrolyzes the main haemolymph sugar of insects, trehalose, into the essential cellular substrate glucose. Trehalase in locust flight muscle is bound to membranes that appear in the microsomal fraction upon tissue fractionation, but the exact location in vivo has remained elusive. Trehalase has been proposed to be regulated by a novel type of activity control that is based on the reversible transformation of a latent (inactive) form into an overt (active) form. Most trehalase activity from saline-injected controls was membrane-bound (95%) and comprised an overt form (∼25%) and a latent form (75%). Latent trehalase could be assayed only after the integrity of membranes had been destroyed. Trehazolin, a potent tight-binding inhibitor of trehalase, is confined to the extracellular space and has been used as a tool to gather information on the relationship between latent and overt trehalase. Trehazolin was injected into the haemolymph of locusts, and the trehalase activity of the flight muscle was determined at different times over a 30-day period. Total trehalase activity in locust flight muscle was markedly inhibited during the first half of the interval, but reappeared during the second half. Inhibition of the overt form preceded inhibition of the latent form, and the time course suggested a reversible precursor–product relation (cycling) between the two forms. The results support the working hypothesis that trehalase functions as an ectoenzyme, the activity of which is regulated by reversible transformation of latent into overt trehalase.

Expression and compartmentalisation of the glycolytic enzymes GAPDH and pyruvate kinase in boar spermatogenesis

Boar spermatozoa contain isoforms of both glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) and pyruvate kinase (PK, EC 2.7.1.40). The sperm-specific forms, GAPDH-S and PK-S, are tightly bound to cell structures. By immunofluorescence microscopy GAPDH-S and PK-S were localised in the principal piece of the boar sperm flagellum as well as in the acrosomal region of the sperm head and at the head-midpiece junction. The midpiece of the flagellum, however, contains isoforms of GAPDH and PK that were only recognised by antibodies against somatic GAPDH and PK, respectively, but not by the antibodies against GAPDH-S and PK-S. In sections of boar testis, GAPDH-S and PK-S were first detected in elongating spermatids when both the developing flagellum and the head were labelled with antibodies against GAPDH-S and PK-S. In contrast, antibodies against rabbit muscle GAPDH and PK labelled all developmental stages of germ cells and also neighbouring contractile cells. Thus, the structure-bound sperm-specific enzymes, GAPDH-S and PK-S, appeared only late in spermatogenesis simultaneously with the development of the structures to which they are bound. Anchoring glycolytic enzymes to structures in these mitochondria-free regions may secure ATP-production for both motility and acrosome function.

Responses and adaptations of collembolan communities (Hexapoda: Collembola) to flooding and hypoxic conditions

Standard ecological methods (pitfall traps, trunk eclectors and soil cores) were used to evaluate collembolan community responses to different flooding intensities. Three sites of a floodplain habitat near Mainz, Germany, with different flooding regimes were investigated. The structures of collembolan communities are markedly different depending on flooding intensity. Sites more affected by flooding are dominated by hygrophilic and hygrotolerant species, whereas the hardwood floodplain is dominated by mesophilic species. The survival strategies of the hygrophilic and hygrotolerant species include egg diapause and passive drifting. The physiological adaptations to hypoxic conditions of several collembolan species were analyzed using a microcalorimeter. The activities were tested under normoxic and hypoxic/anoxic conditions as well as during post-hypoxic recovery. Lactate was increased after hypoxic intervals in the species studied, suggesting that, in addition to a massive decrease in metabolic rate, a modest glycolytic activity may be involved in the tolerance to hypoxia.

In vivo validation of in vitro quality tests for cryopreserved honey bee semen.

Development of cryopreservation protocols for honey bee semen is hampered by the lack of validated laboratory tests that allow the prediction of in vivo performance of frozen-thawed semen. Here we analyzed correlations between seven in vitro tests and indicators of semen performance after insemination. These tests included measures of motility, cell conformation, and membrane permeability before and after exposure to physiochemical stress. We show that the proposed protocol for motility measurement yields results that correlate well with the number of sperm reaching the storage organ of queens (correlation coefficient ρ=0.67) and the proportion of viable eggs in inseminated queens (ρ=0.48). The conventional live/dead assay of membrane permeability by dual fluorescent staining and a new test based on the leakage of the glycolytic enzyme glucose-phosphate-isomerase (GPI) from damaged cells were also correlated to the number of sperm reaching the spermatheca (ρ=0.54 and -0.61, respectively). We conclude that motility, live/dead-staining and the assay for GPI-leakage are valuable tools for the improvement of cryopreservation of honey bee semen.

Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria).

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 microg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.