Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium

Summary Porcine thyroid cells isolated by dispase treatment were cultured in either (a) Matrigel, (b) agarose with the addition of different combinations of basic fibroblast growth factor and laminin, or (c) on agarose-coated dishes. The formation of follicles and the presence of a basal lamina was investigated by routine electron microscopy of Araldite-embedded material and by light and electron microscopical immunocytochemical detection of the basal lamina components, laminin and collagen type IV. After 10 days of culture in Matrigel or agarose, a basal lamina-like structure surrounded most follicles. Follicles of cells growing in agarose and overlaid with a medium containing thyrotropin and fibroblast growth factor showed a fluorescent band at the basal side of the follicles after immunocytochemical staining with anti-laminin and anti-collagen antibodies. Routine electron microscopy showed that a basal lamina-like structure lined the outside of the follicle. This structure could be subdivided into a lamina lucida and a lamina densa. Electron microscopical immunogold labelling revealed that immunologically detectable laminin was confined to the lamina densa. These findings suggest that even in the absence of basal lamina components in the culture medium, thyroid cells are able to form follicles with a regular basal lamina when they are cultured in a three-dimensional environment.     

Phenotypic diversity in cultured cerebral microvascular endothelial cells

Summary Diversity exists in both the structure and function of the endothelial cells (EC) that comprise the microvasculature of different organs. Studies of EC have been aided by our ability to first isolate and subsequently establish cultures from microvascularized tissue. After the isolation of microvessel endothelial cells (MEC) derived from rat cerebrum, we observed morphologic differences in colonies of cells that grew in primary cultures. The morphologies ranged from a cobblestone phenotype considered typical of EC in culture to elongated and stellate cell appearances. Serially passaged cell lines were established based on two parameters: initially by growth and, second, on differences in primary colony morphology using selective weeding techniques. Each culture was examined for the presence of EC-characteristic markers which include Factor-VIII-related antigen, angiotensin-I-converting enzyme activity, collagen type IV synthesis, and PGI₂ production. Variable expression of each of these characteristics among the established EC lines was observed. Growth curves established for each of the EC cultures demonstrated differences in both population doubling rates and cell densities at confluence. The endocytic capacity of each EC line was also evaluated. Our ability to isolate and establish a number of morphologically distinct EC cultures indicates that diversity exists within the EC that comprise the cerebral microvasculature. Diversity in the established cell lines suggests either the EC that line the brain microvasculature exist as a mosaic or that morphologically distinct cultures may originate from different microanatomical origins (arteriolar, true capillary, or venular) or may have resulted from cells at different points in their in vitro life spans at the time of isolation. This research was supported by grants HLO3227 and HLO1514 from the National Institutes of Health, Bethesda, MD.     

Methylmercury-injury effect on tube formation by cultured human vascular endothelial cells

The effect of methylmercury chloride (MeHg) on growth and tube formation by cultured human umbilical vein endothelial cells (HUVECs) was investigated. HUVECs were collected by enzymatic digestion with collagenase. Precultivation of HUVECs with MeHg at concentrations of 1.0–50.0 mol/L exerted negligible effects on the viable cell number, while the viable cell number was slightly reduced at 100 mol/L and fell to zero at concentrations exceeding 500.0 mol/L MeHg. The viable cell number was depressed in a concentration-dependent manner. Tube formation was studied by culturing the cells on gelled basement membrane matrix (Matrigel). Treatment of HUVECs with 0.1–5.0 mol/L MeHg for 24 h inhibited tube formation dose-dependently. Fetal bovine serum (FBS) increased tube formation in a dose-dependent manner, with half-maximum stimulation of tube formation at approximately 3.4% FBS. The length of tube formation decreased time-dependently at concentrations of 0.1 and 1.0 mol/L MeHg. Pretreatment of Matrigel with 1 mol/L MeHg before the cell seeding reduced the tube formation by HUVECs. These results suggest that the growth and tube formation by HUVECs is susceptible to MeHg cytotoxicity, and that MeHg could be injurious to endothelial cell function.Abbreviations MeHg methylmercury chloride - HUVECs human umbilical vein endothelial cells     

Basal lamina secreted by MDCK cells has size- and charge-selective properties

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, respectively, during pressure-driven filtration. Streaming potential measurement indicated the presence of fixed, negative charge in the basal lamina. The sieving coefficient for neutral Ficoll 400 decreased by ～0.0013 for each 1-? increment in solute radius, compared with a decrease of 0.0023 per ? for the anionic Ficoll 400. In this system, molecular charge played a significant role in determining the sieving characteristics of the membrane, pointing to solute charge as a potential contributor to GBM permselectivity.     

Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.     

Stimulation of prostaglandin formation by vasoactive mediators in cultured human endothelial cells

Human endothelial cells in culture synthesize prostaglandins and release these products into the culture medium. The major products of arachidonic acid metabolism were identified by high pressure liquid chromatography or thin layer chromatography, and release of prostaglandins was measured by radioimmunoassays. Addition of histamine or bradykinin enhanced release of prostaglandins in both arterial and venous endothelial cells. Other vasoactive compunds including angiotensin II, vasopressin, substance P, epinephrine, norepinephrine, or isoproterenol were ineffective. Release of prostaglandins by histamine was concentration-related, and involved H₁ receptors, as determined by addition of histamine antagonists. Incubation of endothelial cells with C-arachidonic acid resulted in a time-dependent uptake into cell lipids, where most of the radioactivity was incorporated into phosphatidyl choline and neutral lipids. Endothelian cells released ¹⁴C_arachidonic acid as well as ¹⁴C-prostaglandins in response to either histamine or bradykinin. The enhanced release of ¹⁴C-prostaglandins was inhibited by either indomethacin or mepacrine, but ¹⁴C-arachidonic acid release was inhibited only by mepacrine. We conclude that the vasoactive compounds, histamine and bradykinin, stimulate formation of prostaglandins in endothelial cells by the release of arachidonic acid from phospholipids of the cell membrane.     

Ultracytochemical characteristics of cultured goat brain microvascular endothelial cells [corrected]

This ultrastructural study was undertaken to determine the localization of cytochemically demonstrable blood-brain barrier (BBB)-associated enzymatic activities and of some nonenzymatic constituents in goat [corrected] brain microvascular endothelial cells (ECs) growing in vitro. Positive reactions for alkaline phosphatase (AP), 5'-nucleotidase (5'N), transport ATPase (Na+,K(+)-ATPase), and adenosine diphosphatase (ADPase) were present on both apical and basolateral plasma membranes (PMs) of the ECs. The reaction for calcium-dependent ATPase (Ca(2+)-ATPase) was less intense and was restricted to basolateral PM and associated plasmalemmal pits. These cells also revealed an abundance of anionic sites labeled with cationic colloidal gold (CCG) and Ricinus communis agglutinin 120 (RCA)-binding sites, specific for beta-D-galactosyl residues, on the apical PM. The labeling of the apical PM with Ulex europaeus agglutinin (UEA)-gold complex, specific for alpha-L-fucosyl residues, was negligible. When compared with results of cytochemical examination of the ECs of goat [corrected] brain capillary in vivo, these observations indicate that although cells cultivated in vitro retain at confluence the enzymatic activities typical for BBB-type ECS, they lose their characteristic (polar) localization. This loss is interpreted as a reflection of lost functional polarity of the microvascular endothelium in vitro resulting from deprivation of the normal influence of the components of brain parenchyma.     

Proteomic analysis of the proteins expressed by hydrogen peroxide treated cultured human dermal microvascular endothelial cells

Reactive oxygen species (ROS) have been traditionally regarded as toxic by-products of aerobic metabolism. However, ROS also act as intracellular signaling molecules and can mediate phenotypes in vascular endothelial cells, which may be physiological or pathological in nature. To clarify the molecular mechanisms of ROS signaling, we examined hydrogen peroxide (H(2)O(2))-responsive proteins in cultured human dermal microvascular endothelial cells (HMVEC) using proteomic tools. Protein expression in HMVEC was studied after they had been exposed to low- and high-levels of H(2)O(2) for various times, and intracellular ROS production was examined by flow cytometer and UV spectrophotometer. Proteins obtained from dose- and time-dependent series were separated by two-dimensional gel electrophoresis and tentatively identified by matrix-assisted laser desorption-time of flight mass spectrometry, by matching the tryptic mass maps obtained with entries in the NCBI and Swiss-Prot protein sequence database. At least 163 proteins were changed by H(2)O(2), and 60 proteins were identified. Oxidative stress triggered dramatic change in the expression of proteins in primary microvessel endothelial cells, and their mapping to cellular process provided a view of the ubiquitous cellular changes elicited by H(2)O(2). These results could provide a framework for the understanding of the mechanisms of cellular redox homeostasis and H(2)O(2) metabolism in microendothelium environment in various biological processes as well as pathological conditions.     

Cytochemical study of the effect of aluminium on cultured brain microvascular endothelial cells

Summary The cytotoxic effect of aluminium was studied on cultured goat brain microvascular endothelial cells used as an in vitro model of the blood—brain barrier. Confluent monolayers of these cells were exposed for 4 days to aluminium maltol and, for control purposes, to maltol alone, and also to cadmium chloride as a known cytotoxic substance. The localization of plasmalemma-bound enzymatic activities of 5-nucleotidase and Ca²⁺-ATPase and the distribution of sialic acid residues were studied at the ultrastructural level.It was observed that the reaction for 5-nucleotidase activity was only insignificantly affected, indicating its resistance to the cytotoxic action of both substances used. On the contrary, the activity of Ca²⁺-ATPase was evidently suppressed, especially in the interendothelial clefts where junctional complexes are presumably to be formed. Aluminium also affects the density of sialic acid residues, as shown by their redistribution, leading to the appearance of relatively long segments of unlabelled apical cell surface.The data obtained suggest that observed changes in the localization of Ca²⁺-ATPase and sialic acid residues can lead ultimately to impairment of the formation and maintenance of intercellular junctions and to disturbances in the negatively charged domains of the endothelial cell surface. Whether these alterations, induced in vitro, contribute to in vivo disturbances of blood—brain barrier function requires further experimental study.     

C-reactive protein induced expression of adhesion molecules in cultured cerebral microvascular endothelial cells

Ischemic stroke can trigger an acute phase response resulting in a rise of plasma concentration of C-reactive protein (CRP). Clinical data about the relationship between CRP and prognosis suggest that CRP might be involved in the pathogenesis of cerebral ischemia. In the present work, a significant increase of circulating level of CRP was observed in an vivo rat brain ischemia model of middle cerebral artery occlusion. To determine the possible effects of CRP on brain microvessel endothelium, we performed a dose-dependent experiment in mouse brain microvascular endothelial cells (bEnd.3 cells) with emphasis on its relation to cell adhesions molecules. Incubation with CRP (1-75 mg/L) for 24 h significantly increased Lactate dehydrogenase (LDH) leakage from bEnd.3 cells (P<0.01) in a dose-dependent manner, and induced significant up-regulations of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions analyzed by Western blotting (P<0.01). In contrast to earlier report, CRP also induced significant increase in ICAM-1 expression in the absence of serum (P<0.01). In conclusion, the present results suggest that CRP may be involved directly in the development of inflammation in response to cerebral ischemia.     

Role of JNK in network formation of human lung microvascular endothelial cells

The signaling mechanisms in vasculogenesis and/or angiogenesis remain poorly understood, limiting the ability to regulate growth of new blood vessels in vitro and in vivo. Cultured human lung microvascular endothelial cells align into tubular networks in the three-dimensional matrix, Matrigel. Overexpression of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates the ERK, JNK, and p38 pathways, inhibited network formation of these cells. Adenoviral-mediated overexpression of recombinant MKP-3 (a dual specificity phosphatase that specifically inactivates the ERK pathway) and dominant negative or constitutively active MEK did not attenuate network formation in Matrigel compared with negative controls. This result suggested that the ERK pathway may not be essential for tube assembly, a conclusion which was supported by the action of specific MEK inhibitor PD 184352, which also did not alter network formation. Inhibition of the JNK pathway using SP-600125 or l-stereoisomer (l-JNKI-1) blocked network formation, whereas the p38 MAPK blocker SB-203580 slightly enhanced it. Inhibition of JNK also attenuated the number of small vessel branches in the developing chick chorioallantoic membrane. Our results demonstrate a specific role for the JNK pathway in network formation of human lung endothelial cells in vitro while confirming that it is essential for the formation of new vessels in vivo.     

Thioredoxin inhibits microvascular endothelial capillary tubule formation

Thioredoxin (Trx) inhibited human HMEC-1 dermal microvascular endothelial cell capillary tubule forming capacity in a Matrigel based assay in vitro. Inhibition of capillary tubule formation was Trx catalytic site and thioredoxin reductase (TrxR) dependent, mediated at the Matrigel matrix level, and associated with a shift from morphological differentiation to continuous proliferation, with enhanced cell spreading resulting in eventual monolayer formation. Soluble complex carbohydrates, which inhibited capillary tubule formation on Matrigel without induction of cell spreading or monolayer formation, failed to impair Trx promotion of cell spreading and mono-layer formation, suggesting a shift away from carbohydrate-mediated cell/matrix adhesive interactions. Laminin peptides YIGRS and SIKVAV, which impaired tubule formation on Matrigel without inducing cell spreading or monolayer formation, partially impaired cell spreading upon Trx-treated Matrigel without restoring tubule formation, consistent with a potential role for laminin in Trx-mediated effects. Trx reduced laminin and destabilised laminin/galectin-3 complexes within Matrigel. Native purified EHS Laminin (also containing galectin-3), but not recombinant galectin-3, restored HMEC-1 capillary tubule formation on Trx-treated Matrigel. These data highlight a novel deregulatory effect of extracellular Trx upon morphological capillary differentiation that appears to depend upon the reduction of laminin and destabilisation of its interaction with galectin-3, possibly leading to galectin-3 neutralisation that shifts cell/matrix adhesive interactions away from being carbohydrate mediated and results in loss of proliferation-inhibiting and differentiation promoting cues from this tumor basement membrane matrix.     

Basal lamina formation on thyroid epithelia in separated follicles in suspension culture

When thyroid follicles are isolated by collagenase treatment of minced thyroid lobes, the basal lamina around each follicle is removed. The basal lamina does not reform when follicles are cultured in suspension in Coon's modified Ham's F-12 medium containing, in addition, 0.5% calf serum, insulin, transferrin, and thyrotropin. We have added acid soluble collagen and/or laminin to see if they would result in the formation of a basal lamina. An extended basal lamina did not form when follicles were embedded in a gel formed from acid-soluble rat tendon collagen or from calf skin collagen when added at a concentration of 100 micrograms collagen/ml. However, laminin at a concentration of 5.1 micrograms/ml gave rise to short segments of a basal lamina within 30 min. At longer time intervals, the segments lengthened and covered the base of many cells, and were continuous across the gap between cells and across the mouth of a coated pit. Not all basal surfaces were covered, and no exposed apical surfaces with microvilli had a basal lamina. There was no obvious difference in the appearance of the basal lamina if collagen was added in addition to laminin, but collagen, in contact with the plasma membrane when added alone, was lifted off the membrane in the presence of the basal lamina. The basal lamina appeared denser if formed in the presence of 5% serum instead of 0.5%.     

Retinoids upregulate phagocytosis by human dermal microvascular endothelial cells

Animals fed a diet deficient in vitamin A show severe physiological changes that often result in death. At the cellular level, retinoids have been shown to induce differentiation of cells dervied from a wide spectrum of tissues, including the vasculature. To understand further the mechanisms for these events, we studied the effects of 13-cis-retinoic acid, all trans-retinoic acid, all-trans-retinol, and all-trans-retinol acetate on human dermal microvascular endothelial cells (HDMEC). Concentrations of retinoids in the physiological range from 0 to 1 μM were used in our experiments. These concentrations were nontoxic to HDMEC. Here we report that in addition to the known effect of retinoids on keratinocytes and sebacytes, retinoids induced morphological and functional changes in HDMEC that gave these cells macrophage like characteristics. 13-Cis-retinoic acid and all-trans-RA induced HDMEC to phagocytize and to increase the production of hydrogen peroxide and superoxide anion. These two retinoids also changed the morphology of endothelial cells from typical small compact cuboidal epithelioid cells to cells with larger cytoplasm and indistinct cell membranes. The retinoidstimulated HDMEC deposited increased amounts of extracellular matrix. All-trans-retinol and all-trans-retinol acetate did not significantly affect HDMEC in all parameters tested. The induction of these properties provides a new model with which to study how retinoids regulate gene expression using a normal, nontransformed cell line. © 1994 wiley-Liss, Inc.     

Basal lamina and other extracellular matrix produced by bovine granulosa cells in anchorage-independent culture

Bovine granulosa cells from 3–7 mm follicles were cultured without anchorage in soft agar/methylcellulose solution for 14 days, with or without 50 ng/ml basic fibroblast growth factor. The granulosa cells divided to form colonies of cells. These were analysed by light and electron microscopy, immunohistochemistry and Western immunoblotting. In approximately 20% of the colonies extracellular matrix was clearly visible at the light-microscope level. Ultrastructurally the matrix resembled a basal lamina 30–100 nm thick and was composed of tangled fibres or cords. Unidentified spherical structures of less than 50 nm diameter were sometimes present and attached to this basal lamina. The basal lamina of follicles had similar features, except that the basal lamina produced in vitro was a large aggregate of many convoluted layers. The cells produced collagen type IV and the cellular form of fibronectin. Intercellular areas not associated with basal lamina were identified. Ruthenium red staining revealed these areas to be rich in proteoglycan granules. Free granules were clustered near the cell surface, and the lumina of these areas were rich in fibres decorated with ruthenium red. This material did not resemble follicular fluid of antral follicles. Thus, granulosa cells in anchorage-independent cultures have a follicular cell morphology and secrete two distinct extracellular matrices, one similar to the follicular basal lamina.This study was funded by the Flinders Medical Centre Research Foundation, Flinders University, and the National Health and Medical Research Council of Australia     

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

Basal lamina formation by epithelial cell lines correlates with laminin A chain synthesis and secretion.

Laminin is a major component of the basal lamina upon which all epithelial cells reside in vivo. The synthesis of basal lamina components and their subsequent assembly into a morphologically distinct basal lamina is a differentiated function of epithelial cells in vivo. Ultrastructural studies in our laboratory show that some epithelial cell lines (P-MDCK) form a basal lamina when cultured on membrane-permeable substrate (Millipore Millicells or type I collagen gels). Under the same conditions other epithelial cell lines (MDCK-AA7, M-mTAL-1P, and T84) do not form a basal lamina. When metabolically labeled with [35S]methionine, laminin A and B chains can be immunoprecipitated from the culture medium and culture lysates of P-MDCK cells. In contrast, labeled laminin chains cannot be immunoprecipitated from the culture medium of MDCK-AA7, M-mTAL-1P, and T84 cells. Immunoprecipitates of MDCK-AA7, M-mTAL-1P, and T84 culture lysates demonstrate the presence of one or both B chains but not A chains. These results suggest that laminin B chain synthesis is constitutive in MDCK-AA7, M-mTAL-1P, and T84 cells and that B chains, in the absence of A chains, are not secreted. Furthermore, laminin secretion and basal lamina formation are not required to maintain structural and functional polarity in these cell lines.     

Basal lamina: Schwann cells wrap to the rhythm of space-time

Schwann cells form myelin in the peripheral nervous system. All Schwann cells are surrounded by a basal lamina. Extracellular matrix molecules in the basal lamina, such as laminin, regulate key aspects of Schwann cell development including the formation, architecture and function of myelin. Recent genetic and cell biological experiments suggest that Schwann cells regulate the basal lamina and its receptors in both time and space, resulting in differential functions. These findings have important implications for diseases resulting from laminin dysfunction, such as congenital muscular dystrophy 1A.     

Secretory response of endothelin-1 in cultured human glomerular microvascular endothelial cells to shear stress

The shear-induced secretory response of endothelin-1 (ET-1) by human microvascular endothelial cells was studied using paired human glomerular microvascular endothelial cell (HGMEC) cultured monolayers exposed to steady-state laminar shear stress for up to 10 hours. The first cell monolayer was subjected to a shear stress of 0.65 N m-2 and the second, 1.3 N m-2. ET-1 secretion was determined by radioimmunoassay. Over 10 hours of shear, the total cumulative secretion of ET-1 was 237.4 pg/cm2 for the monolayer exposed to 1.3 N m-2 and 143.6 pg/cm2 for the monolayer exposed to 0.65 N m-2. The average ET-1 secretion rate was 20.90 +/- 2.15 and 12.45 +/- 1.05 pg/cm2.h at 0.65 N m-2 and 1.3 N m-2, respectively. The results showed that ET-1 secretion varied with the time of shear in a nonlinear fashion. Although the level of shear stress affected the absolute value of ET-1 cumulative secretion and secretion rate, the major secretion period for both monolayers occurred between 2.0 and 8.0 hours, with the peak secretion rate occurring at approximately 5 hours. Thus, the response of cultured human microvascular endothelial cells to shear stress differed from that of large vessel endothelial cell cultures in terms of ET-1 secretion. In addition to the level of shear stress, the time of shear was also an important determinant of ET-1 secretion. Consequently, the heterogeneity of vascular endothelial cells and the time of shear should both be considered in future research on the secretion of vascular endothelial cell cultures.