Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

PURPOSE: To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. METHODS: The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). RESULTS: The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. CONCLUSIONS: The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

Stress responses of human retinal pigment epithelial cells to glyoxal.

PURPOSE: Intracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells. METHODS: Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of Nε-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal. The percentage of apoptotic cells was further quantified by flow cytometry after staining of fixed cells with propidium iodide to determine cells with a subdiploid (fragmented) DNA content. Apoptosis related activation of caspase 3 was determined by Western blotting. Glyoxal induced changes in VEGF-A165a mRNA expression and protein production were determined by real-time PCR and by flow cytometry after immunocytochemical staining. RESULTS: Increasing glyoxal concentrations resulted in enhanced formation of AGEs, such as CML modifications of proteins. This was associated with elevated levels of intracellular reactive oxygen species, a depolarized MMP, and a decreased intracellular pH, resulting in an increased number of apoptotic cells. Apoptosis related caspase 3 activation increased in a dose dependent manner after glyoxal incubation. In consequence, the cells activated compensatory mechanisms and increased the levels of the anti-oxidative and stress-related proteins heme oxygenase-1, osteopontin, heat shock protein 27, copper/zinc superoxide dismutase, manganese superoxide dismutase, and cathepsin D. Furthermore, VEGF-A165a mRNA expression and VEGF-A protein production were significantly increased after incubation with glyoxal in ARPE-19 cells. CONCLUSIONS: The glyoxal-induced oxidative stress and apoptosis in ARPE-19 cells may provide a suitable in vitro model for studying RPE cellular reactions to AGEs that occur in AMD or in DR.

Quality assessment of corneal storage media and their components.

BACKGROUND: To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination. METHODS: A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry). RESULTS: Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches. CONCLUSIONS: The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.

Surfactant metabolism and anti-oxidative capacity in hyperoxic neonatal rat lungs: effects of keratinocyte growth factor on gene expression in vivo.

Development of preterm infant lungs is frequently impaired resulting in bronchopulmoary dysplasia (BPD). BPD results from interruption of physiologic anabolic intrauterine conditions, the inflammatory basis and therapeutic consequences of premature delivery, including increased oxygen supply for air breathing. The latter requires surfactant, produced by alveolar type II (AT II) cells to lower surface tension at the pulmonary air:liquid interface. Its main components are specific phosphatidylcholine (PC) species including dipalmitoyl-PC, anionic phospholipids and surfactant proteins. Local antioxidative enzymes are essential to cope with the pro-inflammatory side effects of normal alveolar oxygen pressures. However, respiratory insufficiency frequently requires increased oxygen supply. To cope with the injurious effects of hyperoxia to epithelia, recombinant human keratinocyte growth factor (rhKGF) was proposed as a surfactant stimulating, non-catabolic and epithelial-protective therapeutic. The aim of the present study was to examine the qualification of rhKGF to improve expression parameters of lung maturity in newborn rats under hyperoxic conditions (85% O(2) for 7 days). In response to rhKGF proliferating cell nuclear antigen mRNA, as a feature of stimulated proliferation, was elevated. Similarly, the expressions of ATP-binding cassette protein A3 gene, a differentiation marker of AT II cells and of peroxiredoxin 6, thioredoxin and thioredoxin reductase, three genes involved in oxygen radical protection were increased. Furthermore, mRNA levels of acyl-coA:lysophosphatidylcholine acyltransferase 1, catalyzing dipalmitoyl-PC synthesis by acyl remodeling, and adipose triglyceride lipase, considered as responsible for fatty acid supply for surfactant PC synthesis, were elevated. These results, together with a considerable body of other confirmative evidence, suggest that rhKGF should be developed into a therapeutic option to treat preterm infants at risk for impaired lung development.

Pulmonary blood flow increases in damaged regions directly after acid aspiration in rats.

BACKGROUND: After gastric aspiration events, patients are at risk of pulmonary dysfunction and the development of severe acute lung injury and acute respiratory distress syndrome, which may contribute to the development of an inflammatory reaction. The authors' aim in the current study was to investigate the role of the spatial distribution of pulmonary blood flow in the pathogenesis of pulmonary dysfunction during the early stages after acid aspiration. METHODS: The authors analyzed the pulmonary distribution of radiolabeled microspheres in normal (n = 6) and injured (n = 12) anesthetized rat lungs using positron emission tomography, computed tomography, and histological examination. RESULTS: Injured regions demonstrate increased pulmonary blood flow in association with reduced arterial pressure and the deterioration of arterial oxygenation. After acid aspiration, computed tomography scans revealed that lung density had increased in the injured regions and that these regions colocalized with areas of increased blood flow. The acid was instilled into the middle and basal regions of the lungs. The blood flow was significantly increased to these regions compared with the blood flow to uninjured lungs in the control animals (middle region: 1.23 [1.1; 1.4] (median [25%; 75%]) vs. 1.04 [1.0; 1.1] and basal region: 1.25 [1.2; 1.3] vs. 1.02 [1.0; 1.05], respectively). The increase in blood flow did not seem to be due to vascular leakage into these injured areas. CONCLUSIONS: The data suggest that 10 min after acid aspiration, damaged areas are characterized by increased pulmonary blood flow. The results may impact further treatment strategies, such as drug targeting.

Myocardial accumulation of bupivacaine and ropivacaine is associated with reversible effects on mitochondria and reduced myocardial function.

BACKGROUND: Mechanisms of local anesthetic cardiac toxicity are still not completely understood. In this study, we analyzed whether concentrations of local anesthetics found in clinical toxicity affect myocardial mitochondrial structure and oxygen consumption. METHODS: Guinea pig isolated heart Langendorff preparations were exposed to bupivacaine (3.0 and 7.5 µg/mL) and ropivacaine (3.6 and 9.0 µg/mL) for 10 minutes. Heart rate, systolic blood pressure, the first derivative of left ventricular pressure (+dP/dt), electrocardiogram, and coronary flow were recorded. The local anesthetic tissue concentration was measured either immediately after local anesthetic exposure, or after 20- and 60-minute washout periods. In addition, electron microscopy of myocardial mitochondria was performed using a scoring system for structural damage of mitochondria. Cardiomyocyte cell culture was incubated with bupivacaine, and oxygen consumption ratio, extracellular acidification, and relative amounts of PGC-1α mRNA, a regulator of cellular energy metabolism, were determined. RESULTS: Bupivacaine and ropivacaine induced reversible PR interval and QRS prolongation, and left ventricular pressure and +dP/dt reduction. Myocardial tissue concentration of local anesthetics was 3-fold the arterial concentration. Mitochondria showed a significant concentration-dependent morphological swelling after local anesthetic application. These changes were reversed by a 20-minute washout period for ropivacaine and by a 60-minute washout for bupivacaine. Bupivacaine reduced mitochondrial oxygen consumption and increased PGC-1α expression in neonatal cardiomyocyte cell cultures, whereas fatty acid metabolism remained unaffected. CONCLUSIONS: Bupivacaine and ropivacaine accumulate in the myocardium. Reversible local anesthetic-induced mitochondrial swelling occurs at concentrations that induce a negative inotropic effect. Bupivacaine reduces cellular metabolism, whereas this reduction is reversible by fatty acids. Interaction with mitochondria may contribute to the negative inotropic effect of local anesthetics.

Blue light stress in retinal neuronal (R28) cells is dependent on wavelength range and irradiance.

The aim of our study was to elucidate the role of wavelength and irradiance in blue light retinal damage. We investigated the impact of blue light emitted from light-emitting diode (LED) modules with peaks at either 411nm (half bandwidth 17nm) or 470nm (half bandwidth 25nm) at defined irradiances of 0.6, 1.5 and 4.5W/m(2) for 411nm and 4.5W/m(2) for 470nm on retinal neuronal (R28) cells in vitro. We observed a reduction in metabolic activity and transmembrane potential of mitochondria when cells were irradiated at 411nm at higher irradiances. Furthermore, production of mitochondrial superoxide radicals increased significantly when cells were irradiated with 411nm light at 4.5W/m(2) . In addition, such irradiation caused an activation of the antioxidative glutathion system. Using vital staining, flow cytometry and western blotting, we were able to show that apoptosis only took place when cells were exposed to 411nm blue light at higher irradiances; necrosis was not observed. Enhanced caspase-3 cleavage product levels confirmed that this effect was dependent on light irradiance. Significant alterations of the above-mentioned parameters were not observed when cells were irradiated with 471nm light despite a high irradiance of 4.5W/m(2) , indicating that the cytotoxic effect of blue light is highly dependent on wavelength. The observed phenomena in R28 cells at 411nm (4.5W/m(2) ) point to an apoptosis pathway elicited by direct mitochondrial damage and increased oxidative stress. Thus, light of 411nm should act via impairment of mitochondrial function by compromising the metabolic situation of these retinal neuronal cells.

Influence of blue light on photoreceptors in a live retinal explant system.

PURPOSE: The present study was performed to investigate the early effects of blue light irradiation of photoreceptors in retinal explant cultures. METHODS: Murine retinal explant cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm2. Dihydroethidium was used to determine the production of reactive oxygen species. Morphological alterations of photoreceptor outer segments were determined by live imaging microscopy with mitochondrial dye JC-1. Transmission and scanning electron microscopy were used for ultrastructural evaluations. Cell death in the retina was assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay method. RESULTS: Live retinal explants displayed an increase in reactive oxygen species production, as revealed by fluorescent dihydroethidium products in photoreceptor cells after 30 min of blue light exposure. After 3 h of exposure, blue light caused disorganization of the normally neatly stacked outer segments of living photoreceptors. Ultrastructural analysis revealed breaks in the cell membrane surrounding the outer segments, especially in the middle section. The outer segments appeared tortuous, and the lamellar structures had been disrupted. TUNEL-staining revealed that long-term blue light exposure induced photoreceptor cell death. CONCLUSIONS: In vitro blue light irradiation of retinal explants is a suitable model system for investigating early ultrastructural changes, as well as damage that leads to cell death in photoreceptor cells.

The influence of sublethal blue light exposure on human RPE cells.

PURPOSE: To evaluate the in vitro response of retinal pigment epithelial (RPE) cells to a nonlethal dose of blue light. METHODS: The human RPE cell line ARPE-19 was irradiated with blue light (405 nm) at an output power of 1 mW/cm(2) or 0.3 mW/cm(2). The following parameters were studied: metabolic activity; apoptosis; reactive oxygen species (ROS) production; mitochondrial membrane potential (MMP); ultrastructural changes of mitochondria; production of advanced glycation endproducts (AGEs); and stress-related cellular proteins. RESULTS: Nonlethal doses of blue light irradiation significantly reduced ARPE-19 metabolic activity and MMP while increasing intracellular ROS levels and expression of stress-related proteins heme oxygenase-1 (HO-1), osteopontin, heat shock protein 27 (Hsp-27), manganese superoxide dismutase (SOD-Mn), and cathepsin D. Blue light irradiation also induced ultrastructural conformation changes in mitochondria, resulting in the appearance of giant mitochondria after 72 h. We further found enhanced formation of AGEs, particularly N(epsilon)-(carboxymethyl) lysine (CML) modifications, and a delay in the cell cycle. CONCLUSIONS: ARPE-19 cells avoid cell death and recover from blue light irradiation by activating a host of defense mechanisms while simultaneously triggering cellular stress responses that may be involved in RPE disease development. Continuous light exposure can therefore detrimentally affect metabolically stressed RPE cells. This may have implications for pathogenesis of age-related macular degeneration.

Effects of advanced glycation end products-inductor glyoxal and hydrogen peroxide as oxidative stress factors on rat retinal organ cultures and neuroprotection by UK-14,304.

Retinal ganglion cell degeneration is supposed to be mediated by reactive oxygen species (ROS) and advanced glycation end products (AGEs). The alpha2-adrenergic agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (brimonidine; UK-14,304), is said to exert a neuroprotective effect. To investigate these mechanisms in detail, we exposed rat whole mounts to glyoxal or H(2)O(2) and treated them with either UK-14,304 alone or additionally with the phosphatidylinositide 3 kinase (PI3) kinase inhibitor, 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly 294002). The accumulation of Nepsilon-[carboxymethyl] lysine (CML) was assessed immunohistochemically and changes in intracellular pH (pHi), mitochondrial transmembrane potential (MTMP) and ROS production in cell bodies of multipolar ganglion cell layer were studied by intravital fluorescence microscopy and confocal laser scanning microscopy. Ultrastructural changes in mitochondria of multipolar ganglion cell layer cell bodies were determined by transmission electron microscopy. We found that glyoxal and H(2)O(2) increased accumulation of CML-modified proteins and ROS production and decreased pHi and MTMP in cell bodies of multipolar ganglion cell layer. UK-14,304 could prevent production of ROS, accumulation of CML-modified proteins, ameliorate acidification, preserve MTMP and attenuate ultrastructural damages of ganglion cell mitochondria. Ly 294002 reversed the UK-14,304-mediated attenuation of CML and ROS production. We conclude that the protective effects of UK-14,304 seem partly to be mediated by PI3 kinase-dependent pathways.

Effects of IGM-enriched solution on polymorphonuclear neutrophil function, bacterial clearance, and lung histology in endotoxemia.

Immunological interventions in endotoxemia and sepsis have been tested in experimental and clinical studies. Our group evaluated the effects of an immunoglobulin (Ig)M-enriched solution in an established model of Gram-negative bacteraemia. Ten New Zealand White rabbits (2-3 kg) were randomized to a treatment or control group. In both groups, LPS was infused at a rate of 40 mg kg(-1) h(-1). Immunoglobulin M-enriched solution (Pentaglobin; 2 mL kg(-1) h(-1)) was applied in the intervention group 15 min after beginning LPS infusion. 1 x 10(8) colony forming units of Escherichia coli were injected 30 min after LPS infusion was commenced. Baseline hemodynamic and respiratory parameters, blood E. coli concentration (30 min before and 1, 15, 30, 60, 90, 120, and 180 min after E. coli injection), polymorphonuclear neutrophil oxidative burst activity, and phagocytosis dead space (both 30 min before and 1, 15, 60, 120, and 180 min postinjection) were measured. Ex vivo phagocytosis activity was measured in a separate experiment and evaluated by electron microscopy. Diffuse alveolar damage (DAD) was measured. Organ colonization (kidney, lung, liver, spleen) was assessed in aseptic organ samples. Hemodynamic parameters did not differ between the two groups. Bacterial blood clearance was not influenced by application of IgM-enriched solution. Liver and spleen colonization was significantly reduced in the IgM group. Immunoglobulin M-enriched solution reduced in vitro residual phagocytosis capacity at 30, 90, and 180 min and improved respiratory burst at 180 min. Correspondingly, ex vivo phagocytosis activity as documented by electron microscopy was increased in the IgM group. The sum of all weighted DAD scores (except overdistension) was significantly better in the IgM group (23+/-5 vs. 30+/-8). Immunoglobulin M-enriched solution significantly improved six of seven DAD score parameters and reduced liver and spleen E. coli count. Residual phagocytosis capacity was significantly decreased in the IgM group, whereas burst activity was increased, pointing to an increased in vivo phagocytosis efficiency. Short-term IgM-enriched solution intervention had an especially beneficial effect on LPS-induced pulmonary histological changes.

Effects of vaporized perfluorohexane and partial liquid ventilation on regional distribution of alveolar damage in experimental lung injury.

OBJECTIVE: To determine whether the patterns of distribution of histological effects of vaporized perfluorohexane (PFH) and partial liquid ventilation (PLV) differ significantly in acute lung injury. DESIGN AND SETTING: Experimental study in an animal research laboratory. SUBJECTS: Eighteen pigs. INTERVENTIONS: After induction of acute lung injury by means of infusion of oleic acid animals were randomly assigned to PFH, PLV, or gas ventilation (GV) groups. Six hours thereafter animals were killed, and lung tissue samples were taken for analysis. MEASUREMENTS AND RESULTS: Histopathological analysis revealed less damage with PFH than with GV or PLV in the nondependent and central regions. PFH and PLV showed less injury in the dependent regions than GV. GV and PFH were associated with less histological damage in the nondependent than the dependent regions, whereas PLV presented the opposite pattern. Morphometric analysis showed increased aeration in nondependent than dependent regions with PFH and GV. PLV led to more aeration in the periphery than in central areas. CONCLUSIONS: PFH was associated with a more homogeneous attenuation of alveolar damage across the lungs, although this therapy had more pronounced effects in nondependent zones. PLV showed the opposite pattern, with more important reduction in alveolar damage in dependent lung regions. Interestingly, reduction in alveolar damage with PFH was as effective as with PLV in dependent zones. Our findings suggest that vaporized perfluorocarbon could be advantageous as adjunctive therapy in the treatment of acute lung injury.

Distribution of endothelin receptor subtypes ETA and ETB in the rat kidney.

The endothelin (ET) receptor system is markedly involved in the regulation of renal function under both physiological and pathophysiological conditions. The present study determined the detailed cellular localization of both ET receptor subtypes, ET(A) and ET(B), in the vascular and tubular system of the rat kidney by immunofluorescence microscopy. In the vascular system we observed both ET(A) and ET(B) receptors in the media of interlobular arteries and afferent and efferent arterioles. In interlobar and arcuate arteries, only ET(A) receptors were present on vascular smooth muscle cells. ET(B) receptor immunoreactivity was sparse on endothelial cells of renal arteries, whereas there was strong labeling of peritubular and glomerular capillaries as well as vasa recta endothelium. ET(A) receptors were evident on glomerular mesangial cells and pericytes of descending vasa recta bundles. In the renal tubular system, ET(B) receptors were located in epithelial cells of proximal tubules and inner medullary collecting ducts, whereas ET(A) receptors were found in distal tubules and cortical collecting ducts. Distribution of ET(A) and ET(B) receptors in the vascular and tubular system of the rat kidney reported in the present study supports the concept that both ET receptor subtypes cooperate in mediating renal cortical vasoconstriction but exert differential and partially antagonistic effects on renal medullary function.

Inhibition of apoptosis and reduction of intracellular pH decrease in retinal neural cell cultures by a blocker of carbonic anhydrase.

PURPOSE: Methylglyoxal and glyoxal are intermediates of advanced glycation end products (AGEs). These substances, as well as hydrogen peroxide, induce retinal neurons to reduce their intracellular pH and augment their production of reactive oxygen species, leading to apoptosis. Because these processes may play a role in diabetic retinopathy, the authors undertook this study to investigate the protective action of dorzolamide, an inhibitor of carbonic anhydrase, on retinal neural cells. METHODS: E1A-NR3 cells were incubated with varying concentrations of glyoxal, methylglyoxal, and H2O2 for different periods of time in the presence or absence of dorzolamide. Apoptotic changes were determined by cytofluorometry after the cells were incubated with appropriate dyes and antibodies. The parameters studied were DNA strand breaks (TUNEL assay), subdiploid DNA content (sub-G1 assay), annexin V binding, reactive oxygen species intermediates production, active caspase-3, N(epsilon)-(carboxymethyl)lysine (a glycation product), and intracellular pH. RESULTS: Optimal conditions for detection of the cell-protecting effect of dorzolamide were incubation with 0.6 to 0.8 mM glyoxal or methylglyoxal for 5 hours or with 0.1 mM H2O2 for 30 minutes, respectively, followed by 20-hour incubation with fresh medium. All apoptotic changes were reduced in the assays in which dorzolamide was included. CONCLUSIONS: Dorzolamide reduced the damage inflicted on retinal neural cells by agents that induced apoptosis and, therefore, can be considered a neuroprotectant.

Imaging of the three-dimensional alveolar structure and the alveolar mechanics of a ventilated and perfused isolated rabbit lung with Fourier domain optical coherence tomography.

In this feasibility study, Fourier domain optical coherence tomography (FDOCT) is used for visualizing the 3-D structure of fixated lung parenchyma and to capture real-time cross sectional images of the subpleural alveolar mechanics in a ventilated and perfused isolated rabbit lung. The compact and modular setup of the FDOCT system allows us to image the first 500 microm of subpleural lung parenchyma with a 3-D resolution of 16 x 16 x 8 microm (in air). During mechanical ventilation, real-time cross sectional FDOCT images visualize the inflation and deflation of alveoli and alveolar sacks (acini) in successive images of end-inspiratory and end-expiratory phase. The FDOCT imaging shows the relation of local alveolar mechanics to the setting of tidal volume (VT), peak airway pressure, and positive end-expiratory pressure (PEEP). Application of PEEP leads to persistent recruitment of alveoli and acini in the end-expiratory phase, compared to ventilation without PEEP where alveolar collapse and reinflation are observed. The imaging of alveolar mechanics by FDOCT will help to determine the amount of mechanical stress put on the alveolar walls during tidal ventilation, which is a key factor in understanding the development of ventilator induced lung injury (VILI).

Upregulation of endothelin receptor B in human endothelial cells by low-density lipoproteins.

Low-density lipoproteins (LDLs) represent the most important treatable risk factors for coronary artery disease. Although it has been previously shown that hypercholesterolemia stimulates the endothelin system, the effects of increased levels of LDL on endothelial endothelin receptors have not been previously studied. In particular, the influence of native and oxidatively modified LDLs (nLDLs and oxLDLs) and the regulatory mechanisms in endothelial cells are currently unknown. Human endothelial cells almost exclusively express the endothelin receptor type B (ET(B)). Therefore, the effect of nLDL and oxLDL on the expression of ET(B) was studied in primary cultures of human umbilical vein endothelial cells (HUVEC). HUVEC were stimulated by nLDL and oxLDL in a time-dependent (1-12 hrs) and dose-dependent (25-100 microg/ml) manner. To analyze signal transduction pathways involved in the regulation of ET(B), protein kinase C (PKC) was inhibited using 100 nM Ro-31-8220. The mRNA expression of ET(B) was determined by quantitative reverse transcription-polymerase chain reaction and ET(B) protein expression by Western blot. Native LDL induced ET(B) mRNA after 1 hr (100 microg/ml, 199 +/- 35%, n = 15, P < 0.05 vs. control). Stimulation of HUVEC with oxLDL increased ET(B) mRNA expression (1 hr, 100 microg/ml oxLDL: 308 +/- 48%, n = 15, P < 0.05 vs. control) as well. Induction of ET(B) was also found on the protein level. nLDL was even more potent than oxLDL in inducing ET(B) protein expression. Induction of ET(B) expression by oxLDL is mediated by PKC. These data demonstrate that low-density lipoproteins even independent of oxidative modification are potent inducers of ET(B) receptors at the mRNA and protein level in HUVEC. Given the nitric oxide-releasing capacity of endothelial ET(B) receptors, this effect may represent a possible vasoprotective mechanism.

Effects of Narrow-band IR-A and of Water-Filtered Infrared A on Fibroblasts.

Exposures of the skin with electromagnetic radiation of wavelengths between 670 nm and 1400 nm are often used as a general treatment to improve wound healing and reduce pain, for example, in chronic diabetic skin lesions. We investigated the effects of water-filtered infrared A (wIRA) and of narrow-band IR-A provided by a light-emitting diode LED (LED-IR-A) irradiation in vitro on 3T3 fibroblast cultures under defined conditions with and without glyoxal administration. Glyoxal triggers the formation of advanced glycation end products, thereby mimicking a diabetic metabolic state. Cell viability and apoptotic changes were determined by flow cytometry after vital staining with Annexin V, YO-PRO-1 and propidium iodide (PI), and by SubG1 assay. Mitochondrial function and oxidative stress were examined by vital staining for radical production, mitochondrial membrane potential (MMP) and the ratio of reduced-to-oxidized glutathione (GSH/GSSG). The metabolic state was monitored by a resazurin conversion assay. The numbers of apoptotic cells were reduced in cultures irradiated with wIRA or LED-IR-A. More mitochondria showed a well-polarized MMP after wIRA irradiation in glyoxal damaged cells. LED-IR-A treatment specifically restored the GSH/GSSG ratio. The immediate positive effects of wIRA and LED-IR-A observed in living cells, particularly on mitochondria, reflect the therapeutic benefits of wIRA and LED-IR-A.

Muscular ETB receptors develop postnatally and are differentially distributed in specific segments of the rat vasculature.

The endothelin/endothelin-receptor system is a key player in the regulation of vascular tone in mammals. We raised and characterized an antiserum against rat ETB receptor and investigated the distribution of ETB receptors in different vascular beds during postnatal development (day 0 through day 28) and in the adult rat. We report the tissue-specific and age-dependent presence of vasoconstrictor ETB receptors. At the time of birth, vascular smooth muscle cells from all tissues examined did not exhibit ETB receptor immunoreactivity. The occurrence of ETB receptor immunoreactivity in the postnatal development was time dependent and started in small coronary and meningeal arteries at day 5, followed by small mesenteric arteries as well as brachial artery and vein at day 14. At day 21, ETB receptors were present in the media of muscular segments of pulmonary artery, large coronary arteries, and intracerebral arterioles. At day 28, ETB receptor immunoreactivity was evident in interlobular renal arteries, vas afferens, and efferens. Large renal arteries, mesenteric artery, and elastic segments of pulmonary arteries, as well as coronary and mesenteric veins, did not exhibit ETB receptor immunoreactivity. These data demonstrate the age-dependent and tissue-specific presence of ETB receptors, mainly on arterial smooth muscle cells in the vascular system of the rat.

Serum-free medium and hydroxyethyl starch supports cell survival better than Minimal Essential Medium and dextran in organ-cultured mouse corneas.

BACKGROUND/AIMS: In a previous study, we observed a deleterious effect of serum-supplemented Minimal Essential Medium (MEM) on human corneal endothelial cell survival in a cell culture model. Consequently, here we studied the effects of conventional, serum-supplemented MEM and a serum-free medium in combination with two different deswelling substances on cell survival in whole corneas in a mouse model. METHODS: Murine corneas were cultured for 4 days in MEM+2% fetal calf serum (FCS) or serum-free Human Endothelial-SFM (SFM), both supplemented with either 6% dextran T500 or 7.5% hydroxyethyl starch (HES) 130/0.4. Cells were examined by differential interference contrast microscopy, H&E staining, immunocytochemistry for cleaved caspase-3, Bcl-2, haem oxygenase-1 and immunoblotting for cleaved caspase-3. RESULTS: In MEM, endothelial cells were almost completely lost after 4 days and the number of epithelial cells was markedly reduced. The remaining cells showed fragmented nuclei and were positive for cleaved caspase-3 and strongly positive for Bcl-2. Corneas cultured in SFM retained an almost closed layer of endothelial cells. Fewer cells were positive for Bcl-2, and only a few cells were positive for cleaved caspase-3 even under staurosporine administration. HES supplementation was well tolerated by corneal cells over 4 days, while a 4-day supplementation with dextran resulted in the loss of endothelial and epithelial cells. CONCLUSIONS: Serum-free medium, Human Endothelial-SFM, promoted cell survival during corneal organ culture better than MEM+2% FCS. HES 130/0.4 appeared to be tolerated better by the cells than dextran T500.