Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells

PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.     

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.     

Reoxygenation injury in a cultured corneal epithelial cell line protected by the uptake of lactoferrin

PURPOSE: To investigate whether reoxygenation after extended hypoxia causes cellular damage in cultured corneal epithelial cells and to demonstrate the protective effects of lactoferrin. METHODS: Immortalized human corneal epithelial cells (T-HCECs) were cultured to confluence in 96-well culture plates, subjected to stringent hypoxia (1% O2, 5% CO2, 94% N2 at 37 degrees C) for 24 hours, and returned to normoxic conditions (5% CO2, 95% air at 37 degrees C). Cell viability was observed by 1 microM propidium iodide staining 0, 2, 4, and 6 hours after reoxygenation. Inhibition studies were performed after 2 hours' reoxygenation, using 2 mM iron chelator desferrioxamine and 0.2 mg/ml lactoferrin. Confocal immunocytochemistry for human lactoferrin and western blot analysis for lactoferrin-induced ferritin were performed in cultured T-HCECs to demonstrate the internalization of lactoferrin after application. RESULTS: After 2 hours, reoxygenation of T-HCECs after hypoxia produced an increase in cell death that was significantly greater than that observed in normoxic control cells or in cells subjected to hypoxia for the same time span without reoxygenation. The addition of desferrioxamine and lactoferrin at the time of reoxygenation significantly attenuated cellular damage. Confocal immunocytochemistry revealed that lactoferrin is taken into the cytoplasm of T-HCECs as early as 30 minutes after application. This was also demonstrated in western blot analysis by the upregulation of intracellular ferritin at 18 hours by the addition of iron-bound lactoferrin but not by iron-free lactoferrin. CONCLUSION: Reoxygenation is responsible for increased cellular damage after extensive hypoxia, which is attenuated by chelators of free iron in the cytosol, including the major tear protein lactoferrin.     

Carbon monoxide inhibits hypoxic pulmonary vasoconstriction in rats by a cGMP-independent mechanism

Hypoxia activates erythropoietin-producing cells, chemoreceptor cells of the carotid body and pulmonary artery smooth muscle cells (PSMC) with a comparable arterial PO2 threshold of some 70 mmHg. The inhibition by CO of the hypoxic responses in the two former cell types has led to the proposal that a haemoprotein is involved in the detection of the PO2 levels. Here, we report the effect of CO on the hypoxic pulmonary vasoconstriction (HPV). Pulmonary arterial pressure (PAP) was measured in an in situ, blood-perfused lung preparation. PAP in normoxia (20% O2, 5% CO2) was 15.2+/-1.8 mmHg, and hypoxia (2% O2, 5% CO2) produced a DeltaPAP of 6.3+/-0.4 mmHg. Addition of 8% or 15% CO to the hypoxic gas mixture reduced the DeltaPAP by 88.3+/-2.7% and 78.2+/-6.1% respectively. The same levels of CO did not affect normoxic PAP nor reduced the DeltaPAP produced by angiotensin II. The effect of CO was studied after inhibition of the NO-cyclic guanosine monophosphate (cGMP) cascade with N-methyl-l-arginine (5.10(-5) M) or methylene blue (1.4.10(-4) M). It was found that both inhibitors more than doubled the hypoxic DeltaPAP without altering the effectiveness of CO to inhibit the HPV. In in vitro experiments we verified the inhibition of guanylate cyclase by measuring the levels of cGMP in segments of the pulmonary artery. Cyclic GMP levels were 1.4+/-0.2 (normoxia), 2.5+/-0.3 (hypoxia) and 3.3+/-0.5 pmole/mg tissue (hypoxia plus 8% CO); sodium nitroprusside increased normoxic cGMP levels about fourfold. Methylene blue reduced cGMP levels to less than 10% in all cases, and abolished the differences among normoxic, hypoxic and hypoxic plus CO groups. It is concluded that CO inhibits HPV by a NO-cGMP independent mechanism and it is proposed that a haemoprotein could be involved in O2-sensing in PSMC.     

The influence of hypoxia on the cytotoxicity of concomitant KW-2149 and ionizing irradiation in Chinese hamster fibroblasts

7-N-((2-((2-(gamma-L-glutamylamino)ethyl)dithio)ethyl))-mitomycin C (KW-2149) is a newly synthesized water-soluble mitomycin C (MMC) analog. Preclinical testing showed an interesting activity profile and a superior hematological tolerance in murine models. The aim of this study was to investigate the interaction of this compound with ionizing radiation, both under normoxic and hypoxic conditions, in Chinese hamster fibroblasts (V79). V79 cells were irradiated both under normoxic conditions and after a 1 h period of hypoxia. Paired irradiation dose-response curves confirmed the significance of radioresistance under hypoxia with an oxygen enhancement ratio of approximately 3. In contrast to MMC, KW-2149 showed no increased cytotoxic effect on hypoxic V79 cells. The cytotoxic effect of KW-2149 increased with increasing concentration, irrespective of the ambient oxygen pressure. When KW-2149 was combined with irradiation under hypoxic conditions, cytotoxicity was significantly enhanced under these conditions. The difference in survival between normoxic and hypoxic conditions was statistically significant (p < 0.004). These data suggest a radiosensitizing effect of KW-2149, more pronounced under hypoxic conditions. This effect increases with radiation dose. It also corroborates earlier suggestions of a different mode of action of KW-2149 as compared to MMC.     

Direct cytotoxicity of hypoxia-reoxygenation towards sinusoidal endothelial cells in the rat

AIMS/BACKGROUND: Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury. METHODS: Sinusoidal endothelial cells were isolated from rat liver. After 2 3 days of primary culture, the cells were exposed to hypoxia (N2/CO2 95/5) for 120 min and reoxygenation (O2/CO2 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O2 alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondrial dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively. RESULTS: The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type. CONCLUSIONS: The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.     

Hyperthermia and hypoxia increase tolerance of retinal ganglion cells to anoxia and excitotoxicity

PURPOSE: Knowledge of the mechanisms by which retinal ganglion cells are damaged may provide information required to develop novel treatments for diseases that cause retinal ganglion cell death. The authors investigated whether the expression of the 72-kDa heat shock protein in cultured rat retinal ganglion cells increases tolerance to hypoxic and excitotoxic injury. METHODS: Hyperthermia (42 degrees C for 1 hour) and sublethal hypoxia (9% O2 for 6 hours) were used to induce synthesis of the 72-kDa heat shock protein in cultured rat retinal ganglion cells and cultured retinal Müller cells. Induction of the 72-kDa heat shock protein was detected with immunocytochemical and immunoblot techniques. Survival of cultured retinal ganglion cells after exposure to anoxia (< 1% O2 for 6 hours) and glutamate (200 microns for 6 hours) was measured and compared to control cultures stressed previously by hyperthermia or sublethal hypoxia. The effect of quercetin, a blocker of heat shock protein synthesis, was evaluated in parallel experiments. RESULTS: Heat shock protein immunoreactivity was expressed in cultured retinal ganglion cells and Müller cells after hyperthermia and sublethal hypoxia. The mean (+/- standard deviation) retinal ganglion cell survival rates after exposure to anoxia (expressed as a percentage of untreated control cultures) in cells pretreated with sublethal hypoxia (83% +/- 17%) and hyperthermia (82% +/- 19%) were significantly greater than for cells that had no pretreatment (50% +/- 18%, P < 0.001). The mean (+/-standard deviation) retinal ganglion cell survival rate after exposure to glutamate in cells pretreated with sublethal hypoxia (82% +/- 19%) and hyperthermia (86% +/- 17%) were significantly greater than for cells that had no pretreatment (56% +/- 17%, P < 0.001). Inhibition of heat shock protein synthesis with quercetin abolished the protective effects of sublethal hypoxia and hyperthermia on cell survival after anoxia and glutamate exposure. CONCLUSIONS: The neuroprotective effect of hyperthermia and sublethal hypoxia suggests that heat shock proteins confer protection against ischemic and excitotoxic retinal ganglion cell death.     

Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression

Tumor hypoxia and high levels of expression of the urokinase-type plasminogen activator (uPA) receptor (uPAR) represent a poor clinical outcome for patients with various cancers. Here, we examined the effect of hypoxia on in vitro invasion of extracellular matrix and uPAR expression by human carcinoma cells. Compared with culture under 20% O2, culture for up to 24 hr under 1% or 4% O2 resulted in increased cell surface uPAR. However, the highest uPAR levels were observed in cells cultured under 1% O2. Culture of MDA-MB-231 breast carcinoma cells under hypoxia also resulted in increased uPAR mRNA levels. Furthermore, incubation with cobalt chloride or with an iron chelator also resulted in elevated uPAR expression, while presence of 30% carbon monoxide in the hypoxic atmosphere reduced the hypoxia-mediated uPAR mRNA upregulation. Increased uPAR expression was paralleled by higher cell-associated uPA levels and lower levels of secreted uPA as determined by gel zymography performed on cell extracts and culture-conditioned media. In addition, the in vitro invasiveness of MDA-MB-231 breast carcinoma cells was significantly higher when the invasion assay was performed under hypoxic conditions. This effect of hypoxia on invasion was abrogated by including in the assay a monoclonal, function-blocking anti-u PAR antibody or by the presence of 30% carbon monoxide in the hypoxic atmosphere. Our findings indicate that hypoxia stimulates carcinoma cell invasiveness by upregulating uPAR expression on the cell surface through a mechanism that requires a putative heme protein. Through a similar mechanism, hypoxia may stimulate tumor invasion and metastasis in vivo.     

Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension

Vascular endothelial growth factor (VEGF) is a potent mitogenic and permeability factor targeting predominantly endothelial cells. At least two tyrosine kinase receptors, Flk-1 and Flt-1, mediate its action and are mostly expressed by endothelial cells. VEGF and VEGF receptor expression are upregulated by hypoxia in vivo and the role of VEGF in hypoxia-induced angiogenesis has been extensively studied in a variety of disease entities. Although VEGF and its receptors are abundantly expressed in the lung, their role in hypoxic pulmonary hypertension and the accompanying vascular remodeling are incompletely understood. We report in this in vivo study that hypoxia increases mRNA levels for both VEGF and Flk-1 in the rat lung. The kinetics of the hypoxic response differ between receptor and ligand: Flk-1 mRNA showed a biphasic response to hypoxia with a significant, but transient, rise in mRNA levels observed after 9-15 h of hypoxic exposure and the highest levels noted after 3 wk. In contrast, VEGF mRNA levels did not show a significant increase with acute hypoxia, but increased progressively after 1-3 wk of hypoxia. By in situ hybridization, VEGF mRNA was localized predominantly in alveolar epithelial cells with increased signal in the lungs of hypoxic animals compared with controls. Immunohistochemical staining with anti-VEGF antibodies localized VEGF peptide throughout the lung parenchyma and was increased in hypoxic compared with normoxic animals. Furthermore, hypoxic animals had significantly higher circulating VEGF concentrations compared with normoxic controls. Lung vascular permeability as measured by extravasation of Evans Blue dye was not significantly different between normoxic and hypoxic animals, although a tendency for increased permeability was seen in the hypoxic animals. These findings suggest a possible role for VEGF in the pulmonary response to hypoxia.     

Modification of hypoxia-induced injury in cultured rat astrocytes by high levels of glucose

BACKGROUND AND PURPOSE: Preexisting hyperglycemia exacerbates central nervous system injury after transient global and focal cerebral ischemia. Increased anaerobic metabolism with resultant lactic acidosis has been shown to cause the hyperglycemic, neuronal injury. The contribution of astrocytes in producing lactic acidosis under hyperglycemic/ischemic conditions is unclear, whereas the protective role of astrocytes in ischemic-induced neuronal injury has been documented. The ability of astrocytes to maintain energy status and ion homeostasis under hyperglycemic conditions could ultimately reduce neuronal injury. Therefore, we determined the effects of increased glucose concentrations on glucose utilization, lactate production, extracellular pH, and adenosine triphosphate concentrations in hypoxia-treated astrocyte cultures. METHODS: Primary astrocytes were prepared from neonatal rat cerebral cortices. After 35 days in vitro, cultures were incubated with 0-60 mmol/L glucose and subjected to hypoxic conditions at 95% N2/5% CO2 for 24 hours. In addition, under high-glucose conditions (30 mmol/L), astrocytes were exposed to up to 72 hours of hypoxia. Determination of lactate dehydrogenase efflux, adenosine triphosphate concentrations, and extracellular lactate concentrations defined astrocyte status. Equiosmolar levels of mannitol were added in place of high glucose concentrations to distinguish hyperosmotic effect. RESULTS: When physiological concentrations of glucose (7.5 mmol/L) or lower concentrations were used, significant cell damage occurred with 24 hours of hypoxia, as determined by increased efflux of lactate dehydrogenase and loss of cell protein. When higher glucose concentrations (15-60 mmol/L) were used, efflux of lactate dehydrogenase was similar to that observed in normoxic cultures, despite an increased utilization of glucose. Lactate concentrations in the media at low or normal glucose concentrations exceeded normoxic levels, but higher glucose concentrations (15-30 mmol/L) failed to increase lactate levels further. Values of adenosine triphosphate for hypoxic astrocytes treated with high glucose concentrations were significantly higher than those of astrocytes with zero or low glucose levels. In cultures exposed to hypoxia and high glucose levels (30 mmol/L), no cellular injury was observed before 48 hours of hypoxia. Lactate concentrations in the media increased during the first 24 hours of hypoxia and reached steady state. The pH of the media decreased to 6.4 after 24 hours and 5.5 at 48 hours. The latter pH was concomitant with a marked increase in extracellular lactate dehydrogenase activity. Hyperosmotic mannitol failed to protect cultured astrocytes against hypoxia. CONCLUSIONS: Hypoxic injury to mature astrocytes was reduced by the presence of 15-60 mmol/L glucose in the medium during 24-30 hours of hypoxia. Injury occurred when the pH of the medium was < 5.5. This protection was not afforded by the hyperosmotic effect of high glucose concentrations, nor was the hypoxic injury at later time periods with 30 mmol/L glucose mediated solely by lactate accumulation.     

Alterations in alpha 1-adrenergic receptor-mediated phosphatidylinositol turnover in hypoxic cardiac myocytes

The purpose of these studies was to examine the effects of hypoxia on alpha 1-adrenergic receptor (alpha 1AR) mediated phosphatidylinositol (PI) turnover in cultured neonatal rat cardiac myocytes. Cells were pre-labeled with [3H]-inositol and incubated for 1 h in either normoxia or hypoxia. Phenylephrine, an alpha 1AR agonist, was added at various time intervals (0-60 min) before termination of the incubation. There was a time-dependent release of radioactivity from the lipid fraction to the aqueous fraction with alpha 1AR stimulation. alpha 1AR-mediated PI turnover was biphasic in normoxic cells and monophasic in hypoxic cells. Using ion-exchange chromatography, radioactivity in the inositol trisphosphate (IP3) peak was increased with acute phenylephrine stimulation (5 min) in the normoxic cells, while inositol phosphate (IP) and inositol bisphosphate (IP2) were increased with chronic stimulation (60 min). After 5 min of alpha 1AR stimulation, hypoxia did not alter total aqueous radioactivity when compared to normoxia, but there was a significant increase in IP2. However, there was decreased PI turnover in chronically stimulated (30-60 min) hypoxic cells when compared to normoxic cells. Hypoxia had no effect on radioactivity in the IP3 fraction with either 0, 5, or 60 min of alpha 1AR stimulation, but there was a significant increase in [1,4,5]-IP3 in hypoxic cells with 30 s alpha 1AR stimulation. With hypoxia, there was no difference in radioactivity in the phosphatidylinositols with either 0 or 5 min stimulation when compared to normoxia. However, after 60 min of alpha 1AR stimulation, hypoxia resulted in increased PI and PIP, when compared to normoxic cells, but PIP2 radioactivity was unchanged. There was no effect of pertussis toxin on either the acute or chronic phase of PI turnover, negating involvement of Gi or G(o). These data suggest that alpha 1AR stimulation in neonatal rat cardiac myocytes is biphasic, and that hypoxia produces a slower monophasic response during extended alpha 1-agonist exposure as would be found with ischemia.     

Myocyte adaptation to chronic hypoxia and development of tolerance to subsequent acute severe hypoxia

Studies in animal models and humans suggest that myocardium may adapt to chronic or intermittent prolonged episodes of reduced coronary perfusion. Stable maintenance of partial flow reduction is difficult to achieve in experimental models; thus, in vitro cellular models may be useful for establishing the mechanisms of adaptation. Since moderate hypoxia is likely to be an important component of the low-flow state, isolated adult rat cardiac myocytes were exposed to 1% O2 for 48 hours to study chronic hypoxic adaptation. Hypoxic culture did not reduce cell viability relative to normoxic controls but did enhance glucose utilization and lactate production, which is consistent with an anaerobic pattern of metabolism. Lactate production remained transiently increased after restoration of normal O2 tension. Myocyte contractility was reduced (video-edge analysis), as was the amplitude of the intracellular Ca2+ transient (indo 1 fluorescence) in hypoxic cells. Relaxation was slowed and was accompanied by a slowed decay of the Ca2+ transient. These changes were not due to alterations in the action potential. Tolerance to subsequent acute severe hypoxia occurred in cells cultured in 1% O2 and was manifested as a delay in the time to full ATP-depletion rigor contracture during severe hypoxia and enhanced morphological recovery of myocytes at reoxygenation. The latter was still seen after normalization of the data for the prolonged time to rigor, suggesting a multifactorial basis for tolerance. An intervening period of normoxic exposure before subsequent acute severe hypoxia did not result in loss of tolerance but rather increased the delay to subsequent ATP depletion rigor. Cellular glycogen was preserved during chronic hypoxic exposure and increased after the restoration of normal O2 tension. As mitochondrial cytochromes should be fully oxygenated at levels well below 1% O2, hypoxic adaptation may be mediated by a low-affinity O2-sensing process. Thus, adaptations that occur during prolonged periods of moderate hypoxia are proposed to poise the myocyte in a better position to tolerate impending episodes of severe O2 deprivation.     

The effect of selenium supplementation during the early post-mating period on embryonic survival in sheep

The functioning of the guinea-pig isolated portal vein was monitored by measuring spontaneous mechanical activity, responses to electrical stimulation and administered noradrenaline in normoxic conditions. The effect of hypoxia, induced by bubbling the physiological bathing solution with a 95% N(2)/5% CO(2) gas mixture, on the mechanical performance of the vein was then assessed. Spontaneous activity declined in hypoxia, with mean contraction tension reduced by 55 + or - 8.8%. The responses to electrical field stimulation (2-32 Hz, 0.7 msec. 70 V) were lowered by 14 + or - 4.6% but contractions produced by a range of noradrenaline concentrations (0.01-160 mu M) were unaffected by hypoxia. Substitution of glucose in the bathing solution with sucrose, a substrate unavailable to the cells for energy generation, produced a marked enhancement of the effect of hypoxia. Spontaneous activity was reduced by 76 + or - 8.3%, electrically-induced activity by 80 + or - 14.4% and noradrenaline-induced responses by 85 + or - 6.8%. Although in normoxia the activity and responses of the portal vein were unaffected by the presence of alpha-tocopherol, it significantly protected the functioning of the vein in hypoxic conditions. This effect was concentration-dependent within the range 10-160 mu M and was most marked when glucose was replaced by sucrose in the bathing solution.     

Asbestos fiber analysis in seven asbestosis cases

OBJECTIVE: Smoking is a risk factor for the development of thyroid-associated ophthalmopathy, an inflammatory process primarily affecting the fibroblasts in extraocular muscles. We wished to determine whether the extraocular muscle fibroblasts are more sensitive than dermal fibroblasts to T-cell derived cytokines, as a reason for this anatomical localization, and whether hypoxia alters fibroblast function, as one explanation for the susceptibility conferred by smoking. DESIGN: Fibroblasts derived from the skin or extraocular muscles of healthy subjects were cultured with cytokines under normal (5% CO2:95% air) and hypoxic (5% CO2:95% N2) conditions. MEASUREMENTS: Glycosaminoglycan, protein and DNA synthesis were measured by assessing incorporation of D-6-3H-glucosamine, 3H-amino acids, and 3H-thymidine respectively. RESULTS: alpha-interferon and interleukin-6 had no effect on fibroblasts. gamma-interferon, tumour necrosis factor and interleukin-1 stimulated glycosaminoglycan synthesis; this effect was greater in orbital than in dermal fibroblasts with gamma-interferon and interleukin-1 (P < 0.05). The same cytokines stimulated total protein with a greater response in orbital fibroblasts with gamma-interferon. Interleukin-1 inhibited DNA synthesis in orbital fibroblasts but stimulated DNA synthesis in dermal fibroblasts (P < 0.01); tumour necrosis factor also displayed a differential effect (P < 0.01). Hypoxia caused a significant increase in glycosaminoglycan, protein and DNA synthesis in both types of fibroblasts, under both basal and cytokine-treated conditions (P < 0.05). CONCLUSIONS: Extraocular muscle fibroblasts respond differently from dermal fibroblasts following cytokine stimulation, which may explain in part the anatomical localization of ophthalmopathy. Hypoxia stimulates fibroblasts and this could contribute, as an enhancing factor, to the adverse effects of smoking on thyroid eye disease.     

Hypoxia inhibits gastric emptying and gastric acid secretion in conscious rats

This study examines the effects of hypoxia in the gastric function in conscious rats which adapted to a meal-feeding schedule, that allowed free access to a high protein (HP) diet (550 g casein/kg diet, Exp.1,2 and 4), a normal protein (NP) diet (200 g casein/kg diet, Exp.3) or a nonpurified rat (NPR) diet (Exp. 5 and 6) for 4 h every day for 2 wk. In Exp. 1, after 4 h of consuming the HP diet, rats were exposed to 7.6 or 10.5% O2 normobaric hypoxia. Hypoxia delayed the excretion of urinary urea for 12 h. In Exp.2 and 3, when rats were exposed to 7.6%O2 after 4 h of consuming the HP diet and exposed to 10.5% O2 after 4 h of consuming the NP diet, respectively, a significant delay in gastric emptying was found in the hypoxic rats. In Exp. 4, when rats were exposed to 7.6 O2 hypoxia after 4 hr of eating the HP diet, the plasma gastrin concentration in the 7.6% O2 hypoxic rats was 2.3-fold that of the normoxic rats after 6 h of hypoxia. Furthermore, when rats that did not consume any HP diet on the day of the experiment were exposed to 7.6 or 10.5% O2 hypoxia, the plasma gastrin concentration was higher in both hypoxic groups than in the normoxic group after 3 and 6 of hypoxia. In Exp. 5, rats that were not fed the NPR diet on the day of study were exposed to 7.6 or 10.5% O2 hypoxia for 3 h after pylorus ligation. Hypoxia inhibited the secretion of gastric acid and elevated the plasma gastrin concentration. In Exp. 6, unfed rats that had been consuming the NPR diet were exposed to 7.6% O2 hypoxia for 3 h after pylorus ligation and were orally administered HCl. The rise of the gastrin concentration due to hypoxia was completely inhibited by oral HCl. These results demonstrate that hypoxia inhibits gastric emptying and gastric acid secretion and that the inhibitory effect of hypoxia on gastric acid secretion stimulates gastrin release through positive feedback regulation.     

Effects of in vivo hypoxia on acetylcholine synthesis by rat brain synaptosomes

Synaptosomes from normoxic and hypoxic rats were incubated aerobically in the presence and absence of veratridine. In the absence of veratridine, no significant difference was observed between the two types of preparation regarding either ATP/ADP ratio or 14CO2 or [14C]acetylcholine synthesis from D-[U-14C]glucose. However, in the presence of veratridine, significant reductions in the output of 14CO2 and [14C]acetylcholine by synaptosomes from hypoxic rats were apparent. It was concluded that irreversible metabolic lesions occur at the synapse as a result of hypoxia, which are apparent only when the metabolism of the preparation is accelerated to a level comparable with the maximal rate occurring in vivo. The presence of such lesions is further evidenced by the significant reductions in ATP/ADP ratio, 14CO2 output, and [14C]acetylcholine synthesis that occur in synaptosomes from hypoxic rats made anoxic in vitro and permitted to recover. Such decreases are not seen when synaptosomes from normoxic rats are similarly treated.