Flow-induced modulation of the permeability of endothelial cells cultured on microcarrier beads

The maintenance of endothelial barrier function is important in the regulation of fluid and solute balance between the vascular space and the surrounding tissue. Since fluid flow across endothelial cells stimulates a wide variety of endothelial responses, the effect of shear stress on barrier function was investigated. Bovine pulmonary artery endothelial cells were cultured on permeable microcarrier beads, placed in a chromatography column, and perfused. Indicator-dilution techniques were used to estimate the permeability of the cell-covered beads to low molecular weight tracers (sodium fluorescein—NaFlsc; cyanocobalamin—B12) as a function of flow rate through the column. Permeability values for both tracers were significantly increased (9.3 ± 0.6 to 19.3 ± 1.7 for NaFlsc; 8.2 ± 0.5 to 20.4 ± 3.1 for B12; mean ± SEM, × 10⁻⁵ cm/s, P < .05) when the flow rate was increased from 0.9 ml/min to 3.2 ml/min (corresponding to average shear stresses of 4.7 and 16.8 dynes/cm²). The permeability increase occurred within minutes of the flow increase, and was reversed by decreasing the flow rate to 0.9 ml/min. In the presence of cytochalasin D, the flow-induced permeability increase was not reversible. Neither inhibition of nitric oxide synthase (with N^G-monomethyl-L -arginine) nor inhibition of cyclooxygenase (with indomethacin) was capable of blocking the flow-induced permeability increase. These results indicate that the rapid modulation of endothelial barrier by flow in vitro is probably not due to prostacyclin or nitric oxide. © 1996 Wiley-Liss, Inc.     

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.     

Effects of serum concentration on some growth characteristics of cultured murine parietal yolk sac carcinoma cells

Murine parietal yolk sac carcinoma cells were examined by scanning and transmission electron microscopy to determine the ultrastructural changes resulting from growth, in vitro, in media containing different serum concentrations. Cells grown in medium supplemented with 10% fetal bovine serum (FBS) formed spherical bodies, were generally oval with numerous surface microvilli, well-organized microtubules, abundant free polysomes and a well-developed Golgi apparatus. By contrast, cells grown in 1% FBS failed to form multicellular spheres, were generally flattened over the growth surface and lacked the surface and intracellular features demonstrated when cells were grown in 10% serum. These differences could explain the alterations in the glycosylation of secreted glycoprotein associated with culture in the presence of low serum.     

Density dependent growth of corneal endothelial cells cultured in vitro

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.     

Apical and basolateral CO2-HCO-3 permeability in cultured bovine corneal endothelial cells

Corneal endothelial function is dependent onHCO₃ transport. However, the relativeHCO₃ permeabilities of the apical andbasolateral membranes are unknown. Using changes in intracellular pHsecondary to removingCO₂-HCO₃ (at constant pH) or removing HCO₃alone (at constant CO₂) fromapical or basolateral compartments, we determined the relative apicaland basolateral HCO₃ permeabilities and their dependencies on Na⁺ andCl. Removal ofCO₂-HCO₃from the apical side caused a steady-state alkalinization (+0.08 pHunits), and removal from the basolateral side caused an acidification(0.05 pH units). Removal ofHCO₃ at constantCO₂ indicated that the basolateralHCO₃ fluxes were about three to fourtimes the apical fluxes. Reducing perfusateNa⁺ concentration to 10 mM had noeffect on apical flux but slowed basolateralHCO₃ flux by one-half. In the absence of Cl, there was anapparent increase in apical HCO₃ fluxunder constant-pH conditions; however, no net change could be measuredunder constant-CO₂ conditions.Basolateral flux was slowed ~30% in the absence ofCl, but the net flux wasunchanged. The steady-state alkalinization after removal ofCO₂-HCO₃apically suggests that CO₂diffusion may contribute to apicalHCO₃ flux through the action of amembrane-associated carbonic anhydrase. Indeed, apicalCO₂ fluxes were inhibited by theextracellular carbonic anhydrase inhibitor benzolamide and partiallyrestored by exogenous carbonic anhydrase. The presence ofmembrane-bound carbonic anhydrase (CAIV) was confirmed byimmunoblotting. We conclude that theNa⁺-dependent basolateralHCO₃ permeability is consistent withNa⁺-nHCO₃cotransport. Changes inHCO₃ flux in the absence ofCl are most likely due toNa⁺-nHCO₃cotransport-induced membrane potential changes that cannot bedissipated. Apical HCO₃ permeabilityis relatively low, but may be augmented byCO₂ diffusion in conjunction witha CAIV.

     

Effects of myostatin and other growth factors on cultured human cells

A dedicated cell-based biological test system was used for studying specific effects of myostatin and other human growth factors on the proliferation of cultured myoblasts and fibroblasts. Myostatin inhibited myoblast growth without affecting human fibroblasts. In this test system, human growth hormone and insulin-like growth factor I acted as antagonists of myostatin, which indicates that these agents have a potential for blocking its effects in vivo.     

Effects of hyperoxia on DNA synthesis in cultured porcine aortic endothelial cells

The mode of action of hyperoxia on the inhibition of DNA synthesis from thymidine (dThd) was studied in primary cultures of porcine aortic endothelial cells (EC) at confluence. A significant effect of hyperoxia on dThd uptake was detected only after a 48-h exposure to 95% O2. On the other hand, decrease in dThd kinase activity was already observed after a 12-h exposure, and the time course of its reduction followed closely that of the inhibition of dThd incorporation into DNA. The incorporation of dThd triphosphate into DNA in permeabilized EC was unaffected by hyperoxia. Determination of DNA alpha- and beta-polymerase activities showed that hyperoxia reduced the activity of the alpha-polymerase and increased that of the beta-polymerase. We conclude that most of the O2 effects on DNA synthesis from dThd can be attributed to dThd kinase inhibition. The increased activity of DNA beta-polymerase, an enzyme involved in DNA repair, also supports the view that hyperoxia could damage DNA.     

Hyperoxia causes increased albumin permeability of cultured endothelial monolayers

The effect of phasic eye movement activity on ventilation during rapid-eye-movement (REM) sleep was studied in seven healthy young adults by use of the respiratory inductive plethysmograph. Mean ventilation (VE) and ventilatory components during REM sleep were not significantly different from that seen in either stages 1-2 or 3-4 sleep. The percent of rib cage contribution to ventilation in REM sleep, 29.3 +/- 5.1%, was reduced compared with 54.4 +/- 5.8% in stage 1-2 and 52.2 +/- 4.3% in stage 3-4 sleep (P less than 0.005). When one separated breaths by the degree of associated phasic eye movement activity, it became apparent that breathing during REM sleep is very heterogeneous. Increasing eye movement activity was associated with inhibition of ventilation with a reduction in VE from 5.1 +/- 0.3 to 3.8 +/- 0.3 l/min. Tidal volume and frequency both fell, whereas inspiratory duration was unchanged. Compartmental ventilation was also affected, with the fall in the rib cage contribution from 37.8 +/- 6.4 to 15.3 +/- 5.6%. Chest wall and abdominal movement became more asynchronous as phasic-eye-movement activity increased and frank paradoxical breathing was seen.     

Disassembly of F-actin filaments in human endothelial cells cultured on type V collagen.

We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and beta 1 integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and beta 1 integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and beta 1 integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete F-actin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen.     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Effects of adipokines on expression of adrenomedullin and endothelin-1 in cultured vascular endothelial cells

Obesity is a major risk factor for the development of hypertension. Adipokines may cause hypertension by acting both centrally and directly on the vascular vessels. We wished to clarify whether three adipokines, leptin, resistin and tumor necrosis factor-alpha, affect expression of adrenomedullin and endothelin-1 in vascular endothelial cells. Human umbilical vein endothelial cells were cultured for 24 h with leptin (1-10 nmol/l), resistin (1-10 nmol/l) or tumor necrosis factor-alpha (1-10 ng/ml). Expression of adrenomedullin and endothelin-1 was examined by radioimmunoassay and northern blot analysis. Immunoreactive-adrenomedullin in the medium and adrenomedullin mRNA expression levels were decreased by treatment of tumor necrosis factor-alpha time- and dose-dependently, whereas endothelin-1 secretion was not significantly changed by it. Leptin or resistin had no significant effects on expression of adrenomedullin or endothelin-1 in human umbilical vein endothelial cells. Under hypoxic conditions (1% O2), expression of both adrenomedullin and endothelin-1 was induced in these cells. Immunoreactive-adrenomedullin levels in the medium were decreased by treatment of tumor necrosis factor-alpha under hypoxia. Leptin or resistin had no significant effects on adrenomedullin or endothelin-1 expression also in hypoxia. These findings have raised the possibility that decreased expression of adrenomedullin by tumor necrosis factor-alpha may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects.     

Fibroblast growth factor modulates synthesis of collagen in cultured vascular endothelial cells

Vascular endothelial cells derived from adult bovine aortic arch can be grown in two ways, either in the presence or absence of fibroblast growth factor. The types of collagen produced by cultures under these two conditions have been compared. In the presence of fibroblast growth factor, cells grow in an orderly fashion, express their normal phenotype and synthesize primarily type III collagen plus collagens types IV and V at a ratio of 10:1:3. Cultures grown in the absence of the factor lose their orderly pattern of growth, lose polarity and normal phenotypic expression. They devote twice the proportion of total protein-synthesizing capacity to collagen, and now synthesize type I in addition to the other collagen types. The ratio of collagen types I:III:IV:V is approximately 30:70:1:13. The kinds of type V collagen chains expressed are also altered. Fibroblast growth factor appears to modulate collagen synthesis, the major component of the extracellular matrix, and indirectly modulates the phenotypic expression of cultured vascular endothelial cells. In atherosclerosis, type I collagen is found in association with the intimal layer. The disorderly growth and the abnormal production of type I collagen by these vascular endothelial cells cultured in the absence of fibroblast growth factor is a model for a number of pathological situations including atherosclerotic plaque formation.     

Nuclear localization of endogenous basic fibroblast growth factor in cultured endothelial cells

Indirect immunofluorescence using anti-human placental bFGF antibodies demonstrates the presence of bFGF-like reactivity in the cytoplasm and in the nucleus of adult bovine aortic endothelial cells and of normal and transformed fetal bovine aortic endothelial AG 7680 and GM 7372 cells. Biologically active immunoreactive Mr 18,000 bFGF can be isolated by heparin-Sepharose affinity chromatography from the extract of GM 7372 cell nuclei. Quantitation of bFGF content by biological and immunological methods indicates that 100,000 bFGF molecules per nucleus are present in GM 7372 cells, with nuclear bFGF corresponding to 25-30% of total cellular bFGF. Immunoprecipitation experiments demonstrate that the nuclear localization of newly synthesized bFGF occurs when GM 7372 cells are biosynthetically labeled both in the absence and in the presence of suramin, a molecule that inhibits the binding of bFGF to its plasma membrane receptor. Thus the data indicate that endogenous bFGF undergoes an intracellular sorting to the nucleus of the endothelial cell.     

Anti-angiogenic effects of homocysteine on cultured endothelial cells

High levels of homocysteine induce a sustained injury on arterial endothelial cells which accelerates the development of thrombosis and atherosclerosis. Some of the described effects of homocysteine on endothelial cells are features shared with an anti-angiogenic response. Therefore, we studied the effects of homocysteine on key steps of angiogenesis using bovine aorta endothelial cells as a model. Homocysteine decreased proliferation and induced differentiation. Furthermore, 5 mM homocysteine produced strong inhibitions of matrix metalloproteinase-2 and urokinase, two proteolytic activities that play a key role in extracellular matrix re-modeling, and decreased migration and invasion, other two key steps of angiogenesis. This study demonstrates that homocysteine can inhibit several steps of the angiogenic process.     

Effects of hypoxia on phosphoinositide turnover and adenylate cyclase system in cultured endothelial cells]

By studying the effects of oxygen deficiency upon signal-transducing system it has been shown that in long hypobaric hypoxia activates PI-turnover in cultured human endothelial cells. The sensitivity of cells to histamine was decreased as well as the adenylate cyclase activity in membranes of this cells. The amount of beta-adrenoreceptors was not influenced significantly. Incubation of endothelial cells with histamine (10(-5) M) and phorbol ester (10(-9) M) = activator of protein kinase C within 1-2 h resulted in desensitization of cellular responses which can be seen not only as a disappearance of histamine-induced activation of PI-turnover but also as a decrease of beta-adrenoreceptor amount and adenylate cyclase activity. It seems that hypoxia may change the action of Ca-mobilizing hormones on PI-turnover and suppress adenylate cyclase in human endothelial cells. However the effects of hypoxia on signal-transducing systems in this cells are developed slower than those of Ca-mobilizing hormones.