Bovine Endothelial Cells In Culture Research Articles

Elevation of bovine endothelial cell angiotensin converting enzyme by cationophores and inhibition by ouabain

We recently reported that calcium ionophore A23187 causes a several-fold elevation of angiotensin converting enzyme (ACE) activity of bovine pulmonary artery endothelial cells in culture and that this elevation is dependent upon extracellular calcium. Now we have observed that monensin, a sodium ionophore, also elevates the ACE activity of these cells. This elevation in ACE was not inhibited by 0.2 mM EGTA or the calcium channel inhibitor nifedipine, and monensin did not alter intracellular calcium as measured by fluorimetric assessment of fura-2 AM-loaded cells. When confluent endothelial cells were incubated with monensin or A23187 in the presence of 10–20 nM ouabain, a specific inhibitor of Na + K +- ATPase , the elevation in ACE produced by both of the ionophores was totally eliminated. Concentrations of ouabain greater than 10 nM also inhibited baseline levels of ACE activity. Ca 2+ measurements of fura-2 AM-loaded cells showed that ouabain had no effect on the influx of Ca 2+ produced by A23187. The elevation of ACE seemed to require new protein synthesis, since 0.1 μg/ml cycloheximide inhibited the elevation produced by monensin and A23187. Other sodium transport inhibitors such as amiloride or bumetanide had no effect on ACE elevation caused by monensin. These results suggest that ACE levels of bovine endothelial cells in culture are under cation regulation and may be modulated by ouabain-sensitive Na + K +- ATPase .

Hypoxia increases endothelin release in bovine endothelial cells in culture, but epinephrine, norepinephrine, serotonin, histamine and angiotensin II do not

Calf coronary artery endothelial cells in culture were exposed to normoxic and hypoxic conditions (2% oxygen) for 4hr and 24 hr. After 24 hr of incubation, there was a significant increase in the accumulation of immuno-reactive endothelin (irET) in the media. Incubations with epinephrine, norepinephrine, serotonin, histamine and angiotensin II did not result in an increase in irET production. These studies indicate that hypoxia enhances endothelin release, however, the kinetics of this release suggest that the acute endothelium-dependent vasconstriction occuring after hypoxia does not result from an increase in endothelin release.

Inhibition of Atrial Natriuretic Peptide-induced Cyclic GMP Accumulation in the Bovine Endothelial Cells with Anti-atrial Natriuretic Peptide Receptor Antiserum

Using an antiserum raised against the purified atrial natriuretic peptide (ANP) receptor that has a disulfide-linked homodimeric structure and represents one subtype of the multiple ANP receptors, we showed that the receptor is coupled to the guanylate cyclase activation; formerly, this type of ANP receptor is not considered to be coupled to the cyclase. The specificity of the antiserum was determined by immunoblot analysis and immunoprecipitation. The anti-receptor antiserum did not compete with 125I-ANP for binding to the receptor but it lowered the affinity of the receptor. When added to bovine endothelial cell cultures, the antiserum blocked the cyclic GMP response of the cells triggered by ANP. These results indicate that the subtype of the ANP receptor recognized by the antiserum is responsible for the activation of particulate guanylate cyclase as well as the double function type receptor that has been assumed to contain both the receptor domain and the catalytic domain for cGMP synthesis on the same molecule. The presence of dissociative complexes of ANP receptor and particulate guanylate cyclase was also demonstrated by radiation inactivation analysis.

Characterization of beta-adrenergic receptors in cultured human and bovine endothelial cells

We used radioligand binding methods to characterize beta-adrenergic receptors on endothelial cells cultured from adult human iliac vein (HIVE) and bovine fetal aorta (BFAE). For comparison, we also studied the well-characterized C6 glioma cell line (C6). Both human and bovine endothelial cells showed specific saturable binding of [125I]iodopindolol. There was no difference in the binding affinity (KD) of iodopindolol to membranes from the three cell types. However, the beta-receptor density (Bmax) was greater on HIVE cells and BFAE cells than on C6 cells. Displacement of ligand from HIVE and BFAE cells by zinterol or from BFAE cells by ICI 89,406 was consistent with binding to the beta 2-subtype. In contrast, displacement of ligand from C6 cells by zinterol or ICI 89,406 was consistent with binding to both beta 1- and beta 2-subtypes. Exposing BFAE cells in culture to 10 microM isoproterenol for 6 h resulted in a 55% decrease in Bmax without a change in KD. We conclude that 1) human and bovine endothelial cells in culture contain a substantial population of beta-adrenergic receptors, which are predominantly of the beta 2-subtype, and 2) endothelial beta-receptors exhibit downregulation by beta-agonists in culture.

Actions of nitric oxide on the release of prostacyclin from bovine endothelial cells in culture

Endothelial cells release the potent vasodilator prostacyclin, as well as the highly labile endothelium-derived relaxing factor (EDRF) which mediates vascular relaxation induced by some vasodilators including acetylcholine and bradykinin. EDRF has recently been characterised as nitric oxide (NO). The effects of NO on prostacyclin release, measured as 6-keto-PGF 1α, from endothelial cells obtained from bovine thoracic aorta, have now been investigated. Incubation of endothelial cells in culture with bradykinin (10–100 nM) stimulated the release of 6-keto-PGF 1α. Pre-incubation (0.5–2 min) with NO (13–130 μM) caused a significant dose-dependent inhibition of 6-keto-PGF 1α release, reaching a maximum of 29 ± 4% inhibition. Pre-incubation with superoxide dismutase (30 units ml −1) which prevents the breakdown of NO, significantly augmented the degree of inhibition, as did the selective inhibitor of cyclic GMP phosphodiesterase, M & B 22948 (5 μM), reaching 51 ± 2% inhibition. The potentiation by M & B 22948 suggests that this inhibitory effect of high concentrations of NO is brought about by elevation of intracellular cyclic GMP levels following activation of guanylate cyclase. Whether endogenous NO is produced by endothelial cells under physiological conditions in sufficient quantities to modulate prostacyclin release remains to be established.

Anti-endothelial cell antibodies: detection and characterization in sera from patients with autoimmune hypoparathyroidism.

In a previous report, we described antibodies in autoimmune hypoparathyroidism (AHP) that are cytotoxic for cultured bovine parathyroid cells. In the present study, we show that sera from six AHP patients, but not from 26 patients with other autoimmune diseases or from 7 healthy subjects, react with bovine endothelial cells in culture (by flow cytometry and fluorescence microscopy) and in tissue sections (by immunohistology). We found uniformly that the immunoglobulin class reacting is IgM. Adsorption experiments showed that the antigenic determinants reacting with AHP sera were similar on bovine cultured endothelial cell membranes and in tissue sections of bovine parathyroid glands. The AHP sera also reacted with endothelial cells cultured from bovine adrenal medulla and pulmonary artery. Immunoblot analysis showed antibody binding to two major bands of 200 and 130 kDa solubilized from the membrane fraction of bovine parathyroid endothelial cells. Only one AHP serum consistently recognized endothelium-related structures on frozen sections of three different human parathyroid adenomas; two other sera reacted with one adenoma each; and three did not react with human adenomas. This indicates that human material is less suitable than bovine in detecting endothelium-related immune phenomena in AHP sera. The anti-endothelium IgM antibodies appear to be disease-specific but are not organ- or species-specific. The identification of endothelial cells as the target for antibodies in AHP raises the possibility that the endothelium subserves an important local function for endocrine epithelium.

ENDOGENOUS NITRIC OXIDE INHIBITS HUMAN PLATELET ADHESION TO VASCULAR ENDOTHELIUM

The adhesion of human platelets to monolayers of bovine endothelial cells in culture was studied to determine the role of endothelium-derived nitric oxide in the regulation of platelet adhesion. The adhesion of unstimulated and thrombin-stimulated platelets, washed and labelled with indium-111, was lower in the presence than in the absence of bradykinin or exogenous nitric oxide. The inhibitory action of both bradykinin and nitric oxide was abolished by haemoglobin, but not by aspirin, and was potentiated by superoxide dismutase to a similar degree. It is suggested that the effect of bradykinin is mediated by the release of nitric oxide from the endothelial cells, and that nitric oxide release contributes to the non-adhesive properties of vascular endothelium.

Glucocorticoid induction of angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) converts angiotensin I (Angio I) to angiotensin II (Angio II) and inactivates bradykinin (BK). Glucocorticoids in the physiological range increase ACE in rabbit alveolar macrophages and bovine endothelial cells in culture. Since Angio I and BK are cleaved by ACE catalysis during passage through the pulmonary vasculature we have studied the steroid modulation of ACE in the rat lung. The conversion of Angio I to Angio II by isolated lungs from normal or adrenalectomized male Wistar rats has been evaluated. The initial conversion of Angio I to Angio II in lungs from normal rats was about 60%. In contrast the initial converting activity in lungs from adrenalectomized rats was about 30%. In both groups the converting activity progressively decreased. After 3 h it was about 30% in normal lungs and virtually undetectable in lungs from adrenalectomized rats. Dexamethasone infusion (1 microgram/ml) prevented the decrease in ACE activity observed in normal lungs and induced a gradual enhancement of converting activity in lungs from adrenalectomized animals up to the control level. The effect of dexamethasone was abolished by simultaneous infusion of cycloheximide (1 microgram/ml). These results demonstrate that glucocorticoids induce ACE synthesis in the rat lung. By this induction glucocorticoids promote the increase of both Angio II formation and BK degradation. Thus ACE induction may represent a possible mechanism whereby glucocorticoids might control vascular tone and permeability according to the general mode of action of steroid hormones.

Synergistic stimulation of alkaline phosphatase activity in bovine aortic endothelial cells grown in the presence of retinoids and glucocorticoids.

Low concentrations of retinol (10 nM-10 microM) and dexamethasone (0.1 nM-1.0 microM) elevated activity of alkaline phosphatase (E.C. 3.1.3.1) in bovine endothelial cells in culture. The effect was apparent after 48 hr of growth in the presence of either of the two compounds, prior to any growth stimulatory effects. A synergistic stimulation of alkaline phosphatase was achieved in the presence of both retinol and dexamethasone implying different mechanisms of induction. Retinoic acid and retinyl acetate also elevated alkaline phosphatase but the retinol analogue reduced in the side chain (perhydroretinol) was inactive. The ability of steroids to elevate alkaline phosphatase activity was associated with the structure commonly required for glucocorticoid activity; however, competitors for the steroid receptor binding failed to prevent the elevation by dexamethasone or the synergism in the presence of retinol and dexamethasone.

Vascular Smooth Muscle Cells Inhibit the Plasminogen Activators Secreted by Endothelial Cells

Pure cultures of bovine endothelial cells (EC) produce and secrete large amounts of plasminogen activators (PA). Cocultivation of EC with vascular smooth muscle cells (SMC) resulted in a significant decrease of PA activities secreted by the EC, whereas the cellular PA activities remained unaffected. Secreted PA activities were absent in the growth medium as long as the SMC to EC ratio was 2:1 or higher. The PA inhibitory activity of the SMC was rapid and cell-to-cell contact was not necessary. The PA inhibitory activity was present in homogenates of SMC as well as in the medium conditioned by them but not in the extracellular matrix elaborated by these cells. Serum free medium conditioned by SMC neutralized both tissue type (t-PA) and urokinase like (u-PA) plasminogen activators. Gel electrophoretic analysis of SMC conditioned medium followed by reverse fibrin autography demonstrated PA inhibitory activities in the molecular weight (Mr) range of 50,000 to 52,000 similar to those present in media conditioned by bovine endothelial cells or fibroblasts. Regular fibrin zymography of SMC conditioned medium incubated with u-PA or t-PA revealed the presence of a component with a calculated approximate Mr of 45,000 to 50,000 which formed SDS resistant complexes with both types of PA. These data demonstrate that vascular SMC produce and secrete (a) inhibitor(s) of PAs which may influence the fibrinolytic potential of EC.

Hormonal influence on endothelial cell angiotensin-converting enzyme activity.

The influence of various hormones on angiotensin-converting enzyme (ACE) production and release by bovine endothelial cells in culture was studied. Dexamethasone, thyroxine (T4), and triiodothyronine (T3) stimulated ACE activity in cells and their culture supernatants without affecting cell number or protein content. The stimulating effects of dexamethasone and thyroid hormones were additive, suggesting that these hormones may have different sites of action. In addition, their stimulating effects were blocked by cycloheximide, indicating that increased enzymatic activity occurred through new protein synthesis. The exposure of cells to insulin reduced ACE activity of cells and their culture supernatants without influencing cell counts or protein content; insulin also partially inhibited the stimulation of ACE activity by dexamethasone or T3. Our studies suggest that the production of ACE by endothelial cells is under hormonal regulation. Release of ACE activity into culture supernatants parallels changes in cellular ACE activity. The results supplement previous observations made in vitro and in vivo.

Processing of insulin by bovine endothelial cells in culture. Internalization without degradation.

Insulin binding and processing was studied in monolayer cultures of bovine aortic endothelial cells. Specific 125I-insulin binding was both time and temperature dependent. Maximum binding at 37 degrees C occurred at 90 min, and was 3.8%/mg protein and, at 15 degrees C, 7%/mg protein at 4 h. 125I-insulin was crosslinked to its receptor using disuccinimidyl suberate (DSS), and the structure of the receptor complex was identified by SDS-polyacrylamide gel electrophoresis and autoradiography; a major band with Mr = 145,000 was identified, which corresponds to the alpha-subunit of the insulin receptor reported in other tissues. Receptor-bound insulin was internalized, and both the rate and the amount of internalization were temperature dependent. The rate of internalization was slowest at 4 degrees C, and fastest at 37 degrees C, and the maximum amount of 125I-insulin internalized in 120 min was 16% at 4 degrees C, 45% at 15 degrees C, and 81% at 37 degrees C. Despite the high rate of internalization, endothelial cells do not appear to degrade insulin significantly, as determined by gel chromatography and TCA solubility (7% at 4 h) of media-associated radioactivity. In addition, the majority of internalized insulin (75%) was released by 60 min, largely as intact insulin. Chloroquine treatment at high concentration did not exert any major effect on insulin binding or degradation within the first 60 min, but thereafter produced a marked increase in cell-associated radioactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Processing of insulin by bovine endothelial cells in culture. Internalization without degradation

Processing of insulin by bovine endothelial cells in culture. Internalization without degradation

Induction by glucocorticoids of angiotensin converting enzyme production from bovine endothelial cells in culture and rat lung in vivo.

The effect of corticosteroids on angiotensin converting enzyme was investigated in endothelial cell cultures and intact rat lung. Cultured endothelial cells from bovine aorta showed net production of angiotensin converting enzyme (ACE) over 2 d culture in serum-free medium. Dexamethasone (DM) increased cell ACE activity six- to sevenfold at 100 nM with a threshold effect at 0.3 nM. The effect of DM on ACE production was completely inhibited by actinomycin D or cycloheximide. Deoxycorticosterone (DOC) and aldosterone were markedly less active, with a threshold near 100 nM and significant (two to threefold) stimulation of ACE activity at 1 muM. In cells incubated in the presence of 10 nM DM, DOC (10 muM) significantly inhibited ACE production compared with 10 nM DM alone, suggesting that DOC is a partial agonist/partial antagonist in this enzyme system. Protein content of cells or medium was unchanged by steroids at all doses used. In vivo, adrenalectomized rats showed lower pulmonary ACE compared with intact controls, and when injected with DM (40 mug/d for 4 d) showed a significant (twofold, P < 0.002) increase in lung ACE over oil-injected, adrenalectomized controls; serum ACE did not change. Injection with DOC (40 mug/d) or aldosterone (10 mug/d) had no effect on lung or serum ACE. Over a range (0.6 to 2,000 mug) of concentrations of DM administered daily for 7 d, the dose-response curve of DM for induction of pulmonary ACE mirrored that for thymolysis; for both, half-maximal effects were seen at approximately 6 mug DM/d, and plateau levels at 60 mug/d. We conclude that glucocorticoids are potent inducers of ACE activity in endothelial cells in culture and in rat lung in vivo, and that the action of aldosterone and DOC reflects occupancy of glucocorticoid receptors. This effect may be of (patho)physiological relevance in regulating levels of ACE in local vascular beds, and thereby modulating local levels of the vasoactive peptides angiotensin II and bradykinin.

Bovine endothelial cells in culture produce thromboxane as well as prostacyclin.

Bovine aortic endothelial and smooth muscle cells in culture were incubated with arachidonic acid or prostaglandin H2. The amount of prostacyclin nd thromboxane A2 synthesized ws then determined by specific radioimmunoassay for 6-keto-prostaglandin F1 alpha and thromboxane B2. Although smooth muscle cells produced only 6-keto-prostaglandin F1 alpha and thromboxane B2 in a ratio of 5:1 to 10:1. The same ratio of these metabolites of arachidonic acid ws also found when prostaglandin production from endogenous arachidonic acid was stimulated in endothelial cells by the ionophore A23187. Cyclooxygenase inhibitors inhibited the production of both metabolites equally, whereas thromboxane synthetase inhibitors selectively inhibited the production of thromboxane B2. Cells in culture were also incubated with [1-14C]arachidonic acid and the extracted products were identified by two-dimensional thin-layer chromatography. 6-Keto-prostaglandin F1 alpha was the only metabolite produced by smooth muscle cells, but endothelial cells synthesized 6-keto prostaglandin F1 alpha, thromboxane B2, prostaglandin E2, and prostaglandin F2 alpha.

Characterisation of an endothelial cell proliferation factor from cultured 3T3 cells

A high molecular weight polypeptide from cultured 3T3 cells enhanced the rate of proliferation of both vascular and non-vascular bovine endothelial cells in culture, and induced DNA synthesis in resting confluent cultures of these cells. This proliferation factor showed no activity towards bovine aortal smooth muscle cells, dog kidney cells, bovine corneal stromal fibroblasts or BALB/c 3T3 cells. Activity was resistant to 6 M guanidine hydrochloride, β-mercaptoethanol, periodate oxidation or RNase, but was inactivated by heating at 100 °C or by pronase. The isoelectric point was found to be 6.5 and its molecular weight, determined by gel filtration and sedimentation velocity, was 120000 D. These properties clearly distinguish this growth factor from other polypeptide factors known to stimulate endothelial cell proliferation.

Inhibition of endothelial cell growth by a factor isolated from cartilage

An endothelial cell growth inhibitor (EGI), which affects proliferation of vascular endothelial cells, has been isolated from a 1 M guanidine hydrochloride extract of bovine cartilage by ion exchange chromatography on CM-Sephadex C-25. Two peaks of EGI activity were observed, both of which eluted in positions separate from a peak of trypsin inhibitory activity. Using bovine endothelial cells in culture, we have demonstrated that EGI is not cytotoxic, but rather affects the cell cycle. The average population doubling time increased from 24 h for cells grown in control media to 40 h when cells were grown in the presence of 2 μg EGI/ml of culture medium. The trypsin inhibitor isolated from cartilage had no effect on endothelial cell proliferation. As predicted by segmented regression analysis of the growth curves, we have shown that EGI remains active in the media for a period of 3 days. An appendix of the statistical treatment of the data by segmented regression analysis is presented.

Uptake and degradation of low density lipoproteins (LDL) by confluent, contact-inhibited bovine and human endothelial cells exposed to physiological concentrations of LDL

Metabolism of low density lipoproteins (LDL) was studied in cultures of endothelial cells derived from bovine aorta or heart and from human umbilical veins. At low LDL concentrations nonconfluent cultures of bovine endothelial cells catabolized more LDL protein than contact-inhibited confluent cultures but this difference was reduced at high LDL concentrations. Nonconfluent human endothelial cells displayed also a higher rate of LDL degradation than their contact-inhibited counterparts, but this difference was less pronounced than in the bovine cells. Bovine endothelial cells grown in the presence of fibroblast growth factor metabolized less LDL than those cultured without fibroblast growth factor (FGF), but this difference was not consistent in the human endothelial cells. The data presented provide evidence that contact-inhibited confluent human endothelial cells are capable of catabolizing LDL when exposed to physiological concentrations of this lipoprotein.