Cultures Of Aortic Endothelial Cells Research Articles

Angiogenic oligosaccharides of hyaluronan enhance the production of collagens by endothelial cells

The present study demonstrates a relationship between angiogenic oligosaccharides of hyaluronan (HA) and the production of collagens during the process of angiogenesis in vivo and in vitro. The addition of angiogenic oligosaccharides of HA to the chorioallantoic membrane of the chick embryo induced a deposition of collagen fibrils. The treatment of sub-confluent cultures of bovine aortic endothelial cells with the same oligosaccharides (1 microgram/ml) increased the uptake of [3H]proline by approximately 60%. SDS-polyacrylamide gel electrophoresis of treated cultures demonstrated the enhanced synthesis of type I and type VIII collagens. The production of type VIII collagen was confirmed by western blotting and immunocytochemistry using antibodies to sheep and bovine type VIII collagen. Type VIII collagen is a short chain collagen that has a high degree of homology to cartilage-specific type X collagen. The biological functions of type VIII and type X collagens are unknown. We have suggested that the two collagens play a role in the process of angiogenesis.

Disintegration of cytoskeletal structure of actin filaments in energy-depleted endothelial cells.

In a previous study [Watanabe, H., W. Kuhne, R. Spahr, P. Schwartz, and H. M. Piper. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H1344-H1352, 1991] metabolic inhibition (5 mM KCN + 5 mM 2-deoxy-D-glucose, for 2 h) was found to cause disintegration of F-actin filaments, cell retraction, and augmented paracellular macromolecule permeability in monolayer cultures of porcine aortic endothelial cells after a rapid depletion of ATP stores (90% in 5 min). These changes were reversible. In the present study, the nature of this cytoskeletal disintegration was investigated. 1) Disintegration of F-actin filaments within 2-h incubation under metabolic inhibition was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. 2) Cytosolic Ca2+ levels rapidly rose in metabolically inhibited cells; after 2 h a 10-fold increase was observed. 3) Presence of the Ca2+ ionophore A23187 (10 microM) mimicked the reversible effect of metabolic inhibition on F-actin filaments and monolayer permeability but not the extensive depletion of ATP stores. 4) Existence of the Ca(2+)-activatable actin-severing protein gelsolin in endothelial cells was demonstrated. The results show that during the reversible phase of endothelial energy depletion disintegration of F-actin filaments is only partial, since it is based on their fragmentation and not depolymerization. Increase in cytosolic Ca2+ levels seems to be the primary cause for the fragmentation, possibly through the activation of gelsolin.

NADPH-diaphorase histochemistry identifies isolated endothelial cells at sites of traumatic injury in the adult rat brain

In addition to labelling endothelium, some ependymal cells (including tanycytes), and a subpopulation of neurons, nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry of stab lesion sites in the neocortex revealed a large population of cells concentrated within several hundred micrometres of the lesion site. To determine the identity of these cells, NADPH-diaphorase reactivity was compared to binding with either the I-B 4 isolectin from Bandeiraea simplicifolia (which has previously been shown to identify endothelial cells and activated mononuclear phagocytes), or a monoclonal antibody (OX-42) that recognizes activated mononuclear phagocytes. Many I-B 4 lectin-labelled cells were also NADPH-diaphorase reactive, and other I-B 4 lectin-labelled cells were also OX-42 immunoreactive, but co-existence of OX-42 immunoreactivity and NADPH-diaphorase reactivity was not observed. Only a small minority of NADPH-diaphorase-reactive cells did not exhibit I-B 4 lectin binding. In contrast to the simple somatic morphology of the majority of NADPH-diaphorase-reactive cells, the I-B 4 lectin-negative cells had a ramified appearance, and while readily observed at two days postlesion, they were only rarely seen at three days postlesion. Primary cultures of bovine aortic endothelial cells also exhibited NADPH-diaphorase reactivity which occupied most of the cytoplasm in a filamentous web pattern. Endothelial cells possess a constitutive form of nitric oxide synthase which, as demonstrated in NADPH-diaphorase-reactive neurons, may be the basis of their NADPH-diaphorase reactivity. These findings indicate that NADPH-diaphorase-reactive cells observed at lesion sites are probably angiogenic endothelial cells not associated with extant blood vessels. Thus, NADPH-diaphorase histochemistry offers an effective method of visualizing neovascularization in the brain and other tissues.

Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts

SPARC (secreted protein, acidic and rich in cysteine), also known as osteonectin, is an extracellular Ca+2-binding glycoprotein that inhibits the incorporation of [3H]-thymidine and delays the onset of S-phase in synchronized cultures of bovine aortic endothelial (BAE) cells. This effect appears not to be dependent on the functional properties of SPARC associated with changes in cell shape or inhibition of cell spreading. In this study we investigate the conditions under which cell cycle modulation occurs in different types of cells. Human umbilical vein endothelial cells, a transformed fetal BAE cell line, and bovine capillary endothelial cells exhibited a sensitivity to SPARC and a cationic peptide from a non-Ca+2-binding region of SPARC (peptide 2.1, 0.2-0.8 mM) similar to that observed in BAE cells. In contrast, human foreskin fibroblasts and fetal bovine ligament fibroblasts exhibited an increase in the incorporation of [3H]-thymidine in the presence of 25 microM-0.2 mM peptide 2.1; inhibition was observed at concentrations in excess of 0.4 mM. This biphasic modulation could be further localized to a sequence of 10 amino acids comprising the N-terminal half of peptide 2.1. A synthetic peptide from another cationic region of SPARC (peptide 2.3) increased [3H]-thymidine incorporation by BAE cells and fibroblasts in a dose-dependent manner. In endothelial cells, a stimulation of 50% was observed at a concentration of 0.01 mM; fibroblasts required approximately 100-fold more peptide 2.3 for levels of stimulation comparable to those obtained in endothelial cells. The observation that SPARC and unique SPARC peptides can differentially influence the growth of fibroblasts and endothelial cells in a concentration-dependent manner suggests that SPARC might regulate proliferation of specific cells during wound repair and remodeling.

PMA-activated neutrophils decrease ectoenzyme activities in rabbit aortic endothelial cells in culture.

We have studied the effects of phorbol 12-myristate 13-acetate (PMA)-activated neutrophils [polymorphonuclear leukocytes (PMN)] on endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] activities in cultured rabbit aortic endothelial cells (EC) with the use of [3H]benzoyl-Phe-Ala-Pro and 14C-labeled AMP as substrates, respectively, under first-order reaction conditions. PMA (1-1,000 ng/ml) or PMN alone had no effect on ACE activity. When PMA was incubated together with PMN (PMN/EC = 1.25:1 or 2.5 x 10(5) neutrophils/ml) for 4 h in Earle's salts, a PMA dose-dependent decrease in ACE activity was observed. Threshold PMA concentration was 2 ng/ml. At 8 ng PMA/ml, ACE activity was totally abolished, without any evidence of cytotoxicity, as inferred from release of 51Cr from prelabeled EC. The decrease in ACE activity was also dependent on PMN concentration and was detectable at PMN/EC values as low as 1.25:10 (0.25 x 10(5) PMN/ml). Inhibition of ACE occurred as early as 1 h after incubation (PMA 10 ng/ml, PMN/EC = 1.25:1). PMA alone caused a small but significant increase in NCT activity, whereas PMA coincubation with PMN produced a significant decrease in NCT activity (20-29%), which was PMA and PMN concentration independent. PMA increased PMN adherence to endothelial monolayers in a concentration-dependent manner. Pretreating PMN with monoclonal antibody 60.3 (raised against the adhesion glycoprotein CD18) or placing a 2-microns filter between PMN and EC, protected the decrease in ACE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro

In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin ( M r ~ 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.

The acute effect of FK506 and cyclosporine on endothelial cell function and renal vascular resistance.

We have previously documented that cyclosporine exerts a direct cytotoxic effect on endothelial cells and causes an increase in renal vascular resistance (RVR) in the rat. In the present study we investigated whether FK506, a novel immunosuppressive agent thought to be less nephrotoxic than CsA, impairs endothelial cell function in vitro and affects RVR in vivo. In vitro eicosanoid release and endothelin release were measured in bovine aortic endothelial cells in culture exposed for 1, 6, and 24 hr to increasing concentrations of FK506 (1 nM to 10 microM) or CsA (0.5, 10 microM). No significant changes in TxB2 and 6-keto-PGF1 alpha (the stable breakdown products of TxA2 and PGI2, respectively) and endothelin release were found after 1 and 6 hr of incubation with all the concentrations of FK506 and CsA considered. FK506 did not affect endothelin release even after 24 hr of incubation. In contrast, cell exposure to CsA was associated with a dose-dependent increase in TxB2, 6-keto-PGF1 alpha, and endothelin release that reached statistical significance after incubation with 10 microM CsA. FK506 did not induce cell detachment or lysis at any concentration and time considered, while 10 microM CsA induced a significant reduction in cell count accompanied by cell lysis. In vivo studies showed that a single i.v. injection of FK506 to rats within a broad range of doses (28 ng/kg to 2.8 micrograms/kg) did not modify RVR. This was true even for a dose as high as 20 mg/kg, while 20 mg/kg CsA caused a dramatic increase in RVR. We conclude that FK506, unlike CsA, does not induce endothelial cell injury in vitro. Whether this explains the differences in renovascular resistance observed in vivo after acute injection of FK506 and CsA is an attractive possibility that needs to be further explored.

Development and persistence of Cowdria ruminantium specific antibodies following experimental infection of cattle, as detected by the indirect fluorescent antibody test

Different breeds of cattle were experimentally infected with Palm River, a Zimbabwean isolate, or Ball-3, a South African isolate of Cowdria ruminantium, derived from tissue culture or tick or blood stabilates. C. ruminantium specific antibody responses were detected by an indirect fluorescent antibody test (IFAT) using C. ruminantium-infected bovine aortic endothelial (BAE) cell cultures as antigen. The first detection of antibodies to C. ruminantium generally coincided with the peak of the febrile reaction and the antibodies remained detectable for a period of 8–30 weeks in the Palm River infected group and 18–30 weeks in the Ball-3 infected group. Peak reciprocal antibody titres in both groups ranged from 64 to 2048 between 3 and 6 weeks post-infection. No apparent serological differences were observed among the various C. ruminantium isolates when tested in homologous and heterologous IFATs. Post-infection sera to Anaplasma marginale, Theileria parva parva, Babesia bigemina and Rickettsia conorii did not exhibit reactivity with the C. ruminantium antigen. These results indicate the possible use of C. ruminantium-infected cultures as antigen in IFATs to detect similar C. ruminantium-specific antibody responses in the field in clinically sick, recovered and carrier animals.

Prothrombotic phenotype diversity of human aortic endothelial cells in culture

We have previously demonstrated that human aortic endothelium exhibits morphologic heterogeneity in situ, and this heterogeneity can be reproduced in culture. In this study, we have compared prothrombotic properties of cultured endothelial cells (EC) from areas of human aorta at high risk for atherosclerosis (HP-EC) with EC from areas at low risk (LP-EC). Using paired cultures from the same donors, we have found that the expression of cell surface thrombomodulin (TM) — as measured by the ability to generate activated protein C (APC) from protein C in the presence of thrombin — is relatively reduced on HP-EC compared to LP-EC (respectively, 4.98 ± 4.43 vs. 5.83 ± 4.37 pM APC/min/cm 2; p=.03, n=12). Furthermore, HP-EC more efficiently assemble the prothrombinase complex on their cellular surface, resulting in an increased rate of thrombin generation from prothrombin (9.81 ± 3.10 (HP-EC) vs. 7.96 ± 3.20 nM thrombin/min/cm2 (LP-EC); p <.03, n =7). The combination of reduced TM expression and increased prothrombinase complex assembly on HP-EC suggests a prothrombotic phenotype in these cells. These findings may be important in the pathogenesis of thrombosis associated with atherosclerotic plaques.

Cell density dependent effects of TGF-beta demonstrated by a plasminogen activator-based assay for TGF-beta.

Transforming growth factor-beta 1 (TGF-beta 1) induces a decrease in plasminogen activator (PA) expression in confluent cultures of bovine aortic endothelial (BAE) cells. We describe an assay using the suppression of PA expression in confluent BAE cells by TGF-beta 1 which detects concentrations of the growth factor ranging from 5 to 200 pg/ml and has an ED50 of 15-20 pg/ml. The assay can be performed in 96-well plates and requires a minimum of 35 ul of solution per sample, thereby limiting the amount of reagents required and allowing many samples to be tested in a single assay. Here we demonstrate that the effect of TGF-beta 1 on PA expression in BAE cells depends on the length of time the cells are exposed to the growth factor and the density at which the cells are plated. In cells plated at a high density (3.5 x 10(5) cells/cm2), both 4 h and 24 h exposures to TGF-beta 1 suppress PA expression. However, with cells plated sparsely (3.5 x 10(4) cells/cm2), a 4 h exposure to TGF-beta 1 increases PA expression 2-fold, whereas a 24 h exposure results in an 85% inhibition of basal PA expression. The paradoxical stimulation of PA expression in cells at a sparse density upon 4 h exposure to TGF-beta 1 occurs in a dose-dependent manner with an ED50 of 15-20 pg/ml. This bifunctional response of PA production in cells exposed to TGF-beta 1 may have implications with regard to the role of TGF-beta 1 in angiogenesis.

Biphasic regulation by dexamethasone of IL-1- and LPS-stimulated endothelial prostacyclin production.

Body reaction to injury comprises two major pathways: the immune response, predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in an increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied. Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10(-6) M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 micrograms/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI2 in a concentration- and time-dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24 + 2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below the baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase A2 (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA2-modulating protein, possibly lipocortin, that accumulate during exposure to Dex.

Endothelium-derived kinins account for the immediate response of endothelial cells to bacterial lipopolysaccharide.

We have recently shown that cultured endothelial cells produce kinins that can stimulate endothelial nitric oxide (NO) production in an autocrine manner. Because both the kallikrein-kinin system and the L-arginine/NO pathway have been implicated in the pathogenesis of septic shock, we investigated the possible involvement of endothelium-derived kinins in the response of cultured endothelial cells to bacterial lipopolysaccharide (LPS). In primary cultures of human umbilical vein and porcine aortic endothelial cells, LPS (0.3 to 3 micrograms/ml) induced significant concentration-dependent increases in cyclic GMP and 6-keto-PGF1 alpha, both of which were abolished in the presence of the selective bradykinin B2-receptor antagonist HOE 140 (0.1 microM). These LPS-induced increases in cyclic GMP and 6-keto-PGF1 alpha were short lived, being maximal after 5 min but were not apparent after 60 min. In parallel with these effects, LPS (30 micrograms/ml) induced a distinct, HOE 140-sensitive increase in the intracellular calcium concentration of human endothelial cells loaded with indo-1. In summary, these data suggest that the release of endothelium-derived kinin and subsequent stimulation of endothelial cells, followed by the enhanced production of NO and prostacyclin (PGI2), are implicated in the immediate hypotension induced by LPS in vivo.

Biphasic regulation by dexamethasone of IL-1-and LPS-stimulated endothelial prostacyclin production

Body reaction to injury comprises two major pathways: the immune response is predominantly mediated by IL-1 and other cytokines, and neuroendocrine mechanisms, resulting in increased glucocorticoid production. Each has distinct effects on prostaglandin (PG) production, which may in turn mediate both systemic and local inflammatory responses. The interactions, if any, between the two systems on PG synthesis have not been studied. Bovine aortic endothelial cell cultures were used and prostacyclin (PGI2) production was monitored. Cells were treated with dexamethasone (Dex) 10(-6) M and IL-1 10-30 U/ml in one experiment, and lipopolysaccharide (LPS, 0.1-1.0 microgram/ml) in another experiment, separately or in combination, for either 2 or 24 + 2 h. While Dex was without effect, IL-1 and LPS stimulated PGI2 in a concentration and time dependent manner. Short exposure to Dex (2 h) enhanced the stimulation by IL-1 and LPS. On the contrary, more prolonged exposure (24 + 2 h) reversed the effects of IL-1 and LPS, resulting in PGI2 levels below baseline. A biphasic regulation by Dex was also observed with increasing concentrations of LPS. Dex was actually ineffective by itself, but it enhanced PGI2 production in combination with lower concentrations of LPS, while abolishing the influence of higher concentrations of this agonist. The data suggest that Dex may initially stimulate phospholipase A2 (PLA2) activity, while inhibiting it later. This biphasic behavior may be attributed to different concentrations of a PLA2-modulating protein, possibly lipocortin, that accumulate during exposure to Dex.

Aortic endothelial cells in culture: factors which modulate their morphological and biosynthetic phenotypes.

New capillaries arise from the pre-existing vasculature (arterioles, venules and capillaries) through a series of steps that involve the endothelium and the surrounding extracellular matrix (ECM). The principal morphological events involved in angiogenesis were described in the 1940s. Electron microscopic studies performed over 20 years later have confirmed these initial observations and added further detailed information [1–3].

The direct effect of light therapy on endothelial cell proliferation in vitro.

During repair, new blood vessels are formed by the process of angiogenesis. The prerequisite to blood vessel formation is the proliferation of endothelial cells. The purpose of this study was to find out if light therapy is capable of affecting endothelial cell proliferation in vitro. The direct effect of light at wavelengths of either 660 or 820 nm was studied on primary cultures of bovine aortic endothelial cells using varying energy densities, of either 1, 2, 4, or 8 J/cm2. The proliferation of the endothelial cells was assessed over a period of five days after a single irradiation.

Identification of structural domains in protein C involved in its interaction with thrombin-thrombomodulin on the surface of endothelial cells.

The structural domains of protein C involved in its interaction with thrombin-thrombomodulin on the endothelial cell surface have been investigated using isolated intact domains of bovine protein C produced from controlled proteolytic digests of the protein. The fragments investigated include the gamma-carboxyglutamic acid (Gla)-rich module, the two epidermal growth factor (EGF)-like modules, and a fragment consisting of the Gla and the two EGF-like modules. The effects of these fragments on the catalytic efficiency (Km and Vmax) of activation of protein C by the endothelial cell surface thrombin-thrombomodulin complex (IIa-TM) have been evaluated in vitro using a stirred microcarrier cell culture of bovine aortic endothelial cells and purified proteins. Neither the Gla nor the two EGF-like modules alone had any discernible effect on protein C activation. The intact Gla-EGF fragment, however, inhibited protein C activation. The results are consistent with a rapid equilibrium competitive inhibition model, in which the Gla-EGF fragment competes with protein C for binding to IIa-TM, and indicate that the Gla-EGF fragment alone accounts for most of the binding energy of intact protein C for IIa-TM. In addition, a requirement for the Gla residues of protein C for binding is implied by the observation that heat-decarboxylated Gla-EGF fragment was not an inhibitor of protein C activation. In addition, chloromethyl ketone-inactivated activated protein C was found to bind to IIa-TM with the same affinity as protein C, suggesting that the changes which occur in protein C upon activation do not affect that part of the protein responsible for binding to IIa-TM, that is the Gla-EGF region. The Gla-EGF region from factor X also weakly inhibited the IIa-TM activation of protein C.

Platelet Stimulation for Prostacyclin Production in Aortic Endothelial Cell Cultures: Alteration in Diabetes Mellitus

We evaluated the effect of platelets on prostacyclin (PGI2) production in bovine aortic endothelial cell cultures. Human platelet extract significantly stimulated PGI2 production by cultured aortic endothelial cells in a time- and dose-dependent manner, suggesting that platelets contain PGI2-stimulatory activity (PSA). Supernatant fluid separated from platelets activated by collagen also exhibited PSA. The factor(s) causing the PSA of platelets was non-dialysable and heat-stable (56 degrees C for 30 min or 100 degrees C for 3 min), was completely inhibited by trypsin pretreatment, and exhibited an affinity to heparin agarose. Furthermore, gel filtration chromatography showed that the factor(s) responsible for the platelet PSA was eluted at three different peaks with approximate molecular weights of 50,000, 25,000 and 11,000. The PSA of platelet extract from patients with non-insulin-dependent diabetes mellitus (NIDDM) (n = 10) was compared to that from age-matched control subjects (n = 10). Platelet extract from patients with NIDDM stimulated cultured aortic endothelial cells to produce greater amounts of PGI2 than did that from control subjects. These data suggest that the increased PSA of platelets isolated from diabetic patients may contribute to the abnormal interaction between platelets and the vascular wall in diabetic patients.

Spiking of intracellular calcium ion concentration in single cultured pig aortic endothelial cells stimulated with ATP or bradykinin

Single pig aortic endothelial cells in culture loaded with the Ca(2+)-sensitive fluorescent dye Indo-1 were stimulated with ATP (0.1-100 microM) or bradykinin (0.1-5.0 nM). Spiking or oscillations of [Ca2+]i were seen in approx. 50% of cells stimulated with either agonist. Non-spiking or transient responses in which [Ca2+]i returned to pre-stimulation levels rapidly 9120-250 s), or sustained responses in which [Ca2+]i remained elevated for many minutes, were seen in a further 20% of cells in each case, stimulated with either agonist. There was a marked variation between individual cells in the latency, magnitude, frequency and overall pattern of oscillations induced by ATP and bradykinin, although the patterns of response to bradykinin were less variable. In cells where repetitive spikes were seen, a relation between concentration of ATP and the latency of the response and the frequency of spiking was evident. Effects of removal of extracellular Ca2+, elevation of extracellular K+ concentration (35 or 70 mM) or exposure to phorbol 12,13-dibutyrate or 1,2-dioctanoyl-sn-glycerol were tested on the spiking Ca2+ responses. Each of these procedures reversibly slowed or prevented Ca2+ spiking evoked by ATP or bradykinin. In contrast, the inactive phorbol ester 4 alpha-phorbol didecanoate had no effect on Ca2+ spiking evoked by these hormones. Our results thus indicate that the responses of single cells to ATP or bradykinin exhibit marked heterogeneity, and suggest that secretory events driven by extracellular Ca2+ may be regulated by repetitive spikes or oscillations of Ca2+.

Rabbit Aortic Endothelial Cell Hypoxia Induces Secretion of Transforming Growth Factor Beta and Augments Macrophage Adhesion In Vitro

Hypoxic injury of vascular endothelial cells is hypothesized to be the initial cellular event in the formation of an atherosclerotic lesion. We studied the effect of various oxygen tensions on rabbit aortic endothelial cells in culture to determine macrophage adhesion and analyzed endothelial cell-conditioned media for fibroblast mitogenesis and transforming growth factor beta production. Fibroblast mitogenesis assay of endothelial cell-conditioned media revealed decreased activity at lower oxygen tensions. Further study revealed an inverse relationship between oxygen tension and aortic endothelial cell production of transforming growth factor beta despite lower total numbers of viable aortic endothelial cells at lower oxygen tensions. When rabbit aortic endothelial cells grown at various oxygen tensions were incubated with five day old bone marrow macrophages, an increase in macrophage adherence to aortic endothelial cells was noted at low oxygen tensions. Our observations suggest that aortic endothelial cell hypoxia leads to the production of transforming growth factor beta, a known monocyte chemoattractant. Monocytes may marginate and then adhere to endothelial cells, their adherence being augmented by endothelial cell hypoxia. This may contribute to the initial cellular events in the formation of an atherosclerotic lesion.

Stimulation of heparan sulphate release from cultured endothelial cells by plasmin

We investigated the effect of plasmin on the release of glycosaminoglycans (GAG) from bovine aortic endothelial cells in culture. Plasmin significantly increased the release of [35S]sulphate-labelled GAG (35S-GAG) from the cell layer, although plasminogen did not show such a stimulatory effect. The percentage of heparan sulphate in the released 35S-GAG (about 80%) was not changed by plasmin. Plasmin enhanced the detachment of endothelial cells from the monolayer only slightly. From these results, it was suggested that the local anticoagulant activity on endothelium mediated by the anticoagulant heparan sulphate will be increased by plasmin; the conversion of plasminogen to plasmin may be mediated by plasminogen activators released from thrombin-stimulated endothelial cells.