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(Stroke. 1997;28:375-381.)
© 1997 American Heart Association, Inc.

Articles

Human Vascular Endothelium Heterogeneity

A Comparative Study of Cerebral and Peripheral Cultured Vascular Endothelial Cells

Eric Thorin, PhD; Marie A. Shatos, PhD; S. Martin Shreeve, PhD; Carrie L. Walters, MD John A. Bevan, MD

the Departments of Pharmacology (E.T., S.M.S., J.A.B.) and Biochemistry (M.A.S.), Totman Laboratory for Human Cerebrovascular Research, University of Vermont, Burlington, and Neurosurgeons Associates, Phoenix, Ariz (C.L.W.).

Correspondence to E. Thorin, PhD, Institut de Cardiologie de Montreal, Centre de Recherche, 5000 rue Belanger, Montreal (Qc) H1T 1C8, Canada. E-mail thorin@icm.umontreal.ca.

	Abstract

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Background and Purpose Hormones, neurotransmitters, and autacoids play a key role in the regulation of vascular tone as a result of their interaction with the endothelium. The aim of this study was to compare selected properties of three human endothelial cell lines isolated from cerebral pial arteries (PEC) and two peripheral vessels, the superficial temporal (SEC) and omental (OEC) arteries.

Methods Intracellular free calcium concentration ([Ca²⁺]_i) and receptor protein expression were measured in characterized primary cultures of human endothelial cells.

Results All cell lines labeled positively for factor VIII/von Willebrand factor. Growth rate and constitutive release of endothelin-1, expressed as a function of protein, were both significantly lower in cerebral cells (PEC) than in endothelial cells derived from peripheral vessels. Basal [Ca²⁺]_i measured with the fluorescent calcium indicator fura 2-AM (2 µmol/L) did not differ in either of the three cell lines. Although PEC responded to endothelin-1 (0.1 µmol/L) and vasoactive intestinal peptide (1 µmol/L) by a twofold to threefold increase in [Ca²⁺]_i, OEC were unresponsive to these peptides. Moreover, the calcium response to -thrombin (10 nmol/L) was greater in cerebral (PEC) than in peripheral (SEC, OEC) endothelial cells, while bradykinin (100 nmol/L) increased [Ca²⁺]_i to a similar level in all three cell types.

Conclusions This study demonstrates that endothelial cells from different sites of the vasculature exhibit different growth rates and vary in their response to agonists.

Key Words: endothelium • receptors, vasoactive intestinal peptide

	Introduction

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Vascular EC release biologically active agents such as nitric oxide¹ and prostacyclin,² which are powerful vasodilators, and ET-1, a potent endogenous vasoconstrictor.³ The balance between dilators and constrictors released by the endothelium is an important determinant of vascular tone.⁴

Cultured EC, used to study these factors,^{5 6 7} shear stress, and membrane potential,^{1 8 9} have been derived from either human umbilical veins^{1 10 11} or animal vessels.^{12 13} There is increasing evidence that EC differ between vascular beds and between species. For example, microvascular and macrovascular human EC display functional heterogeneity with respect to bradykinin degradation¹⁴ and plasminogen activator inhibitor-1 activity.^{14 15} The spontaneous release of prostanoids is more pronounced in vascular EC than in endocardial EC.¹⁶ Recently, Ohbayashi and colleagues¹⁷ demonstrated that EC isolated from porcine coronary artery released more ET-1 than those from the aorta. Receptors for adenosine, -thrombin, histamine, and acetylcholine appear to be much more abundant in microvascular guinea pig coronary EC than those of bovine aorta.⁹ EC isolated from the cerebral vasculature also have been shown to have distinctive characteristics: membrane composition of cerebral EC from human is different from that of dog.¹⁸ Moreover, -thrombin differentially modulates the fibrinolytic potential of cerebral and umbilical EC.¹⁵

Because of our interest in endothelium-dependent mechanisms involved in the regulation of cerebrovascular tone, we isolated and compared EC from human cerebral and peripheral resistance arteries. Our objectives were to obtain pure cell lines and to characterize the cells with respect to selected biochemical and pharmacological properties.

	Materials and Methods

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Cell Culture
Human vascular EC were isolated from SEC and OEC fragments and from PEC in cerebral cortex fragments obtained during surgery (Table 1). These vascular fragments would otherwise have been discarded. Donors had no clinical evidence of hypertension, atherosclerosis, or diabetes.

View this table: [in this window] [in a new window]   Table 1. Identification of Donors per Type of Artery

Human PEC and SEC segments were collected from five and seven patients, respectively, undergoing neurosurgery for skull fracture or tumor removal. In the latter cases, segments of arteries used for cell culture were not feeding the tumor. Human OEC segments were collected from three patients undergoing gastrointestinal surgery for conditions that did not involve the omentum.

Each arterial segment was cleaned of connective tissue under sterile conditions, cut in small fragments, and placed in a culture dish containing growth medium (Dulbecco's modified Eagle's medium supplemented with 20% heat-inactivated fetal bovine serum, 1 mmol/L neomycin, 2 mmol/L glutamine, and 20 U/mL nystatin). After 4 to 7 days, cells were seen to proliferate. They were harvested with a rubber policeman, dispersed with a 22-gauge needle, and seeded at a low concentration (100 cells per milliliter) in large culture dishes. Forty-eight hours later, cells were clearly proliferating. We isolated EC under the microscope by scraping away any contaminating cells (mainly pericytes), as described by Ryan and Maxwell.¹⁹ After 48 hours, this operation was repeated if contaminating cells were still present. The cells were harvested with a rubber policeman and seeded in large culture flasks. When confluent (2 to 4 weeks), the cells were trypsinized and seeded (1) in large culture dishes for ET-1 quantification or G protein analysis, (2) in six multiwell plates for growth curve determination, and (3) in glass coverslips for immunocytochemical characterization and [Ca²⁺]_i measurement.

Characterization
Subconfluent EC (passage 3) established on 20x20-mm glass coverslips were fixed with cold methanol for 15 minutes. They were then incubated with anti–factor VIII/von Willebrand factor antibody (1:25 dilution; Jackson Immunoresearch Laboratories) or anti–smooth muscle -actin antibody (1:400 dilution; Sigma) in phosphate buffer solution overnight at 4°C. After they were washed, the coverslips were incubated with fluorescein-labeled goat anti-rabbit IgG (Jackson Immunoresearch Laboratories) for 1 hour at 37°C. Cells were examined with fluorescence microscopy (Zeiss IM35 with fluorescein excitation and emission wavelengths of 340 nm and 510 nm, respectively).

The -subunit of the G_o protein was revealed by Western blotting as previously described.²⁰ Rat brain homogenate was used as a positive control.

Appearance of immunoreactive ET-1 in the culture medium of EC was quantified with the use of anti–ET-1 antibody. Experiments were performed in triplicate. Cells were incubated for 1 hour in 4 mL of serum-free medium. We immobilized 100 µL of the medium on nitrocellulose membrane (0.1 µm pore size) using a slot-blot apparatus (Schleicher & Schuell). The membrane was incubated for 1 hour at room temperature in 5% nonfat dry milk in 1% Tween 20 in Tris-saline buffer (500 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.4) to block nonspecific protein binding. After it was washed with 0.05% Tween 20 in Tris-saline buffer (Tween-Tris-saline buffer), the nitrocellulose paper was incubated for 1 hour at room temperature with rabbit anti–ET-1 primary antiserum (Peninsula Laboratories) diluted 1:1000 in 5% nonfat milk in Tween-Tris-saline buffer. The paper was washed again and incubated for 1 hour with horseradish peroxidase–conjugated secondary antibody (1:2000 dilution in Tween-Tris-saline buffer supplemented with 5% nonfat dry milk). The paper was washed, and detection was accomplished with the use of the enhanced chemiluminescence method (Amersham) and quantified with the use of Optimas Imaging software (Bioscan). We determined protein concentration using a fluorescamine assay.²⁰

Growth Curves
To determine growth curves for the three cell lines (PEC, SEC, and OEC), 10⁴ cells were seeded into six-well plates. The medium was changed every 2 days. Cell counts were repeated every day for 9 days. Doubling time was calculated for each growth curve by logarithmic conversion of the maximum growth rate and expressed as hours. Growth curves of the three cell lines were performed separately on three cell preparations per cell line and repeated for 7 (SEC), 5 (PEC), and 3 (OEC) different patients.

Measurement of [Ca²⁺]_i
The concentration of [Ca²⁺]_i was measured in EC monolayer as previously described.²¹ Briefly, confluent EC established on 9x22-mm glass coverslips were incubated with the penta-acetoxymethylester form of fura 2 (fura 2-AM, 2 µmol/L, Molecular Probes). A Hitachi f-2000 fluorescence spectrofluorimeter was used for [Ca²⁺]_i recording; the excitation monochromator alternated between 340 nm and 380 nm every 2 seconds, and fluorescence emission was collected at 509 nm. Data were analyzed as previously described.²¹

SDS–Polyacrylamide Gel Electrophoresis and Autoradiography
Cells were scraped from a 1500-mm² tissue culture flask in PBS containing the following protease inhibitor mix: soybean trypsin inhibitor (100 U/mL), leupeptin (10 µmol/L), bacitracin (5 U/mL), pepstatin A (0.14 µmol/L), benzamide (1 mmol/L), aprotonin (14 U/mL), sodium azide (3 µmol/L), and PMSF (100 µmol/L). After centrifugation, the cells were resuspended in ice-cold Tris-EDTA buffer (0.1 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.4) containing protease inhibitors with a 25-gauge needle and homogenized with a Dounce homogenizer. After centrifugation at 500g for 10 minutes, the supernatant was recentrifuged at 48 000g for 30 minutes. The resultant pellet was washed and resuspended in Tris-EDTA buffer containing protease inhibitors at a protein concentration of 4 mg/mL.

Next, 250 µg membrane protein was incubated for 30 minutes at room temperature (20°C) with 250 pmol/L ¹²⁵I-VIP in Tris-EDTA buffer containing protease inhibitors. Membranes were collected by centrifugation and resuspended in phosphate-magnesium buffer (50 mmol/L NaPO₄, 1 mmol/L MgCl₂, pH 7.4). ¹²⁵I-VIP was then cross-linked to membrane proteins by incubating with 5 mmol/L disuccinimidyl suberate for 30 minutes at room temperature, as previously described.^{20 22} After cell membranes were quenched and washed, they were resuspended in 200 µmol/L of 10% glycerol, 1% 2-mercaptoethanol, 2% SDS, 0.001% bromophenol blue, 62.5 mmol/L Tris-HCl, pH 6.8 (SDS-sample buffer) and incubated for 20 minutes at room temperature before either immediate electrophoresis or storage at -80°C.

VIP receptors in SDS-sample buffer were analyzed in 1.5-mm-thick discontinuous SDS-polyacrylamide gels. These gels were subjected to autoradiography (for 2 to 14 days) with Kodak x-ray film (XAR-5), as previously reported.^{20 22}

Statistical Analysis
Results were expressed as mean±SEM of n=7 (SEC), n=5 (PEC), and n=3 (OEC) cell lines; n indicates the number of cell lines (donors) tested per concentration of agonist. Each experiment was performed in triplicate for each cell line. Comparisons were made with a Student's t test or one-way ANOVA, followed by Fisher's correction for multiple comparisons when appropriate. Differences between means were considered significant at P<.05.

Materials
Anti–smooth muscle -actin antibody, bradykinin triacetate, ethylene glycol-bis(ß-amino-ethyl ether)N,N,N',N'-tetraacetic acid, ionomycin, soybean trypsin inhibitor, and bacitracin were obtained from Sigma. VIP and ET-1 were obtained from Peninsula Laboratories. Anti-G_o antibody and ¹²⁵I-VIP (2200 Ci/mmol) were purchased from New England Nuclear. Aprotinin, leupeptin, and pepstatin A were obtained from Boehringer Mannheim. The homobifunctional cross-linker disuccinimidyl suberate was from Pierce. PMSF, acrylamide, and SDS were obtained from Serva. Human -thrombin with a specific activity of 3433 NIH units/mg was obtained from Hematologic Technologic. All media and regents from cell culture were obtained from Gibco BRL. Other materials were of the highest purity available.

	Results

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Isolation and Characterization of EC
The internal diameters of the PEC and OEC samples were similar (464±51 and 441±67 µm, respectively) and less than those of the SEC segments (832±66 µm, P<.05). In all three arteries, EC were isolated following the same protocol. An enzymatic digestion with the use of collagenase would have led to complete digestion of the tissue or collection of an insufficient number of cells. We therefore used a vessel explant that favored the proliferation of EC. Because pericytes/smooth muscle cells could potentially contaminate the culture, a subsequent cloning of each preparation was necessary. As shown in Fig 1A, 1B, and 1C, the general morphological characteristics ("cobblestone-like" appearance) were similar to those previously described for vascular endothelium.^{11 14 23} All the cells stained positively for factor VIII/von Willebrand–associated antigen (Fig 1D) but not for anti–smooth muscle -actin antigen (1:400 dilution) (data not shown).

View larger version (105K): [in this window] [in a new window]   Figure 1. Cultured human vascular EC. A, B, C, Contrast-phase micrograph (x100) of a confluent monolayer of PEC, SEC, and OEC, respectively. D, Positive staining for the factor VIII/von Willebrand factor antigen specific for EC. The EC did not stain for anti–smooth muscle -actin antigen, demonstrating the absence of smooth muscle cells and pericytes (data not shown). E, Absence of expression of the -subunit of the Go protein normally expressed in cells of neuroepithelial (ectodermal) origin such as neurons, glial cells, and astrocytes.

Glial cells or astrocytes could contaminate the pial EC preparations. However, tissues were always carefully cleaned of connective tissue, gray matter, and cerebral cortex tissues. Furthermore, we showed that the cell culture did not express the -subunit of the G_o protein (Fig 1E), which is present in neurogenic cells.²⁴

Peripheral EC (OEC and SEC) exhibited a greater rate of cell proliferation than PEC (P<.05; Fig 2). During the logarithmic growth phase, doubling times of SEC and OEC were shorter and the final cell count was lower in PEC (Table 2, Fig 2). The amount of protein present in each well on day 9 of the growth curve, which was standardized for all cell lines, suggested a lower cell density in PEC (PEC, 0.37±0.02 mg, n=48; SEC, 0.41±0.02 mg, n=59; OEC, 0.38±0.03, n=27).

View larger version (25K): [in this window] [in a new window]   Figure 2. Growth curves of human SEC, OEC, and PEC. Each point represents cumulative data from human SEC (n=4), OEC (n=3), and PEC (n=6) segments. Cell growth and final cell density were greater for peripheral (OEC and SEC) than for central (PEC) EC.

View this table: [in this window] [in a new window]   Table 2. Heterogeneity of EC Lines Isolated From Human Small Arteries

All three cell types constitutively secreted significant amounts of ET-1. A lower amount of ET-1 per milligram of cell protein was released from PEC compared with SEC, whereas the amount of ET-1 per milligram of cell protein secreted by OEC was not significantly different from the two other cell lines (Table 2).

Measurement of [Ca²⁺]_i
Although resting [Ca²⁺]_i levels were similar in all three types of EC, cells differed in their responses to a variety of agonists (Table 2). Although -thrombin (10 nmol/L) triggered an increase in [Ca²⁺]_i in all three cell lines, the peak increase in [Ca²⁺]_i induced by -thrombin (0.1 to 100 nmol/L) in PEC was significantly greater than that in OEC and SEC (Table 2, Fig 3). However, the response to bradykinin (0.1 µmol/L) was similar in the three human EC lines. Cerebral and peripheral EC also differed in their responsiveness to VIP and ET-1. Both peptides induced a significant increase in [Ca²⁺]_i in cerebral PEC but had no effect on peripheral OEC (Table 2).

View larger version (23K): [in this window] [in a new window]   Figure 3. Thrombin-induced concentration-dependent increase in [Ca2+]i in cultured human OEC and PEC. *P<.05 compared with SEC (each point represents 4 to 6 independent series of experiments; ANOVA, Scheffe's F test).

This difference between cells derived from cerebral and peripheral regions was further investigated by analysis of VIP receptors with the use of SDS–polyacrylamide gel electrophoresis (Fig 4). Covalent cross-linking of ¹²⁵I-VIP to membranes from OEC revealed a single specifically labeled protein centered at an M_r of 58 kDa. The electrophoresis profile of membrane from PEC was different, revealing an additional M_r of 71 kDa. The broad bands presumably resulted from the proteins having a heterogeneous carbohydrate content.

View larger version (29K): [in this window] [in a new window]   Figure 4. Comparison of membrane-bound VIP receptors with detergent-solubilized receptors using a cross-linking agent and analysis by SDS–polyacrylamide gel electrophoresis in OEC and PEC. Note cross-linking of 125I-VIP to its receptor on PEC but not OEC. The arrow indicates the specifically labeled receptor protein characteristic of the human VIP receptor (Mr71 000). The lower band might represent a second type of VIP receptor expressed in both cell lines. EC membranes were incubated for 30 minutes at room temperature with 250 pmol/L 125I-VIP in the absence (-) or presence (+) of 100 nmol/L nonradioactive VIP.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
This study demonstrates for the first time that cultured human EC exhibit distinct differences in growth rate, protein synthesis, ET-1 release, and responsiveness to endogenous factors such as ET-1, -thrombin, and VIP.

The general morphological features of cultured EC derived from human pial, superficial temporal, and omental beds were not different. Phase-contrast microscopy demonstrated the classic morphological characteristics of a confluent monolayer with cobblestone-like appearance with little or no cellular overlaps. Overlaps and interdigitations between cells were visible in cultured human endocardial EC.¹⁶ We occasionally saw similar overlaps in what appeared to be pure EC with no evidence of nonendothelial cells or cell transformation. Similarly, cobblestone-like appearance is only one of the five phenotypes reported as representative of EC in culture.²⁵ With the use of electron microscopy, it has been demonstrated that cerebral EC in vitro are capable of forming tight junctions reminiscent of the tight junctions found in vivo that are postulated to play a role in maintaining the integrity of the blood-brain barrier.²⁶

All cells isolated from human small arteries were positively identified as EC by specific, well-documented, EC markers. All EC stained positively for the factor VIII/von Willebrand factor–related antigen, the most reliable marker for cells of endothelial origin.²³ Although a variety of EC from large and small blood vessels take up acetylated LDL in vitro and in vivo, brain capillary EC²⁷ and heart EC²⁸ reportedly lack the receptor for modified LDL. Therefore, we did not use acetylated LDL uptake as a marker for EC identification in this study. Moreover, the absence of the -subunit of the G_o protein in EC lines isolated from small pial arteries demonstrated that our cultures did not include contaminating neurogenic cells.²⁴

Although EC growth characteristics were similar to those previously reported,^{16 29} PEC were slower growing and achieved a lower final cell density (Fig 2). Because there was no difference in protein content per well at day 9 in any of the cell lines studied, this suggests that PEC (Fig 1A) are larger than SEC and OEC (Fig 1B and 1C, respectively). Differences in size have been previously reported; for example, porcine coronary EC are larger than aortic EC.¹⁷

SEC released more ET-1 per milligram total protein into the culture medium than PEC, whereas the quantity of ET-1 released by OEC was intermediate. Such differences in peptide formation are not unique. For example, cultured pig coronary EC release more ET-1 than pig aortic EC.¹⁷ It is possible that in our experiments, the size of the vessel rather than its anatomic origin influenced the formation of the peptide. Since the SEC segment diameter was greater than that of the other two vessels, it is possible that the role of ET-1 is different between large and small blood vessels.³⁰

Although VIP and ET-1 triggered an increase in [Ca²⁺]_i in cerebral PEC, these peptides had no effect on peripherally derived OEC. ET-1 receptors are members of the superfamily of receptors linked with guanine nucleotide binding (G) proteins. In EC, their stimulation leads to the activation of phospholipase C and formation of inositol 1,4,5-trisphosphate and diacylglycerol, with the subsequent release of [Ca²⁺]_i and activation of protein kinase C. The former induces nitric oxide release, leading to vasodilation.³¹ There is considerable evidence that VIP exerts many of its effects through G_s and stimulation of adenylyl cyclase (Reference 20 and references therein). Nevertheless, there are also data suggesting that the VIP receptor may couple to other G proteins and signaling mechanisms, including phospholipase C and Ca²⁺ signaling.^{32 33} Because all cells responded similarly to bradykinin, the absence of a Ca²⁺ response to ET-1 and VIP in OEC suggested either the absence of receptors for these peptides or activation of a different intracellular pathway.

We investigated this further and analyzed VIP receptor expression in cerebral PEC and peripheral OEC membranes using ¹²⁵I-VIP labeling followed by SDS–polyacrylamide gel electrophoresis and autoradiography. A single radiolabeled protein band of M_r 58 000 was identified in OEC membranes (Fig 4). The labeling of this band was of high affinity and specific. We conclude that this protein represents a ¹²⁵I-VIP receptor complex. If we assume that VIP binds to its receptor in a 1:1 stoichiometry, the size of the VIP receptor itself is estimated to have an M_r of 55 000. Although a wide range of M_r values have been reported for the VIP receptor, this value is in excellent agreement with the M_r value obtained for the receptor in bovine aorta, rat liver, lung, brain,³² peritoneal³⁴ and alveolar³⁵ macrophages and with cloned human VIP₁ and VIP₂ receptors^{34 37} (predicted M_r, 52 000 and 50 000, respectively, excluding glycosylation). PEC were also found to express the M_r 55 000 receptor, together with a specifically labeled M_r 68 000 protein (Fig 4). A receptor protein of approximately this size has also been reported in human liver³⁷ and a human neuroblastoma cell line.³⁸ Possible explanations for this difference in the M_r of the VIP receptor complex include heterogeneity of receptor proteins and variation in glycosylation.

We conclude that both PEC and OEC express VIP receptors and speculate that cerebral PEC may express a distinct VIP receptor that is not only different in size but that may also signal through Ca²⁺. Although previous studies have shown that VIP can regulate peripheral vascular tone by acting on EC,^{39 40} in cerebral arteries VIP induces endothelium-independent relaxation.^{41 42 43 44} Therefore, the role of VIP, acting through the cerebral endothelium, remains to be elucidated.

Stimulation of thrombin receptors induces an array of intracellular events. -Thrombin modulates the large-vessel–derived EC expression of numerous components, including the fibrinolytic protein tissue-type plasminogen activator, urokinase-type plasminogen activator, and plasminogen activator inhibitor-1.¹⁵ It causes various cells in culture to migrate and proliferate.⁴⁵ -Thrombin induces rapid acidification and subsequent gradual alkalinization that is dependent on the Na⁺-H⁺ exchanger.⁴⁶ This peptide also inhibits forskolin-stimulated adenylate cyclase activity, with a subsequent decrease in intracellular cAMP levels in human smooth muscle cells.⁴⁷ Within seconds, -thrombin also activates PLC and induces the hydrolysis of phosphoinositides into inositol-3 and inositol-2 phosphates and subsequently the production of prostaglandin I₂.⁴⁸ The increase in cytosolic free calcium induced by thrombin in human EC was therefore expected. The high sensitivity of cerebral EC to the peptide compared with peripheral EC reflects another differential feature related to the vascular bed of origin of the EC.⁴⁹ The endothelium derived from the cerebral microvasculature is distinctly different in its interactions with -thrombin compared with other large- and small-vessel endothelium, suggesting that urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the major fibrinolytic enzyme in the microvasculature of the brain.¹⁵ The physiological significance of the increased responsiveness of human cerebrovascular EC to -thrombin remains to be established, but our findings strengthen previous data highlighting the uniqueness of endothelium located within the cerebral vasculature.¹⁵

In conclusion, although EC throughout the vasculature share basic structural and functional characteristics, marked differences exist in their morphology and growth characteristics, ET-1 release, and responsiveness to a variety of mediators, all of which are related to their bed of origin. Our findings demonstrate that it is not possible to generalize on the basis of observations made on a limited number of tissues. On the contrary, our findings emphasize the necessity of performing studies on cells derived from a large number of vascular beds. It must also be noted that EC variations in some instances have also been related to embryologic origin,^{50 51} artery size,⁴⁹ sex,⁵² age,⁵³ and disease state.¹³

	Selected Abbreviations and Acronyms

 

EC = endothelial cells ET-1 = endothelin-1 OEC = omental artery endothelial cells PEC = pial artery endothelial cells PMSF = phenylmethylsulfonyl fluoride SDS = sodium dodecyl sulfate SEC = superficial temporal artery endothelial cells VIP = vasoactive intestinal peptide

	Acknowledgments

 
This study was supported by the Totman Medical Research Trust Fund.

	Footnotes

 
Presented in part at the Experimental Biology Meeting, Atlanta, Ga, April 9-13, 1995, and published in abstract form (FASEB J. 1995;9:A618).

Received July 23, 1996; revision received September 16, 1996; accepted October 8, 1996.

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Editorial Comment

A Comparative Study of Cerebral and Peripheral Cultured Vascular Endothelial Cells

William G. Mayhan, PhD, Guest Editor

Department of Physiology and BiophysicsUniversity of Nebraska Medical CenterOmaha, Neb

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Many vasoactive factors (ie, hormones, neurotransmitters, and autacoids) contribute to the regulation of vascular tone by interactions with the endothelium. It appears that the function of the endothelium in the control of vascular tone varies between vascular beds.^{1R 2R 3R} The purpose of the present study was to characterize the properties of three human EC lines (one derived from cerebral and two derived from peripheral arteries) in response to vasoactive agonists.

Using endothelium isolated from human pial arteries, the superficial temporal artery, and the omental artery, the authors examined cell proliferation, the secretion of ET-1, and basal and stimulated increases in [Ca²⁺]_i (fura 2). The authors report that the proliferation and release of endothelin were lower from endothelium derived from pial arteries than that derived from endothelium of peripheral arteries. In addition, although basal [Ca²⁺]_i was similar in all cell lines, [Ca²⁺]_i in response to stimulation with ET-1, -thrombin, and VIP was greater in cerebral endothelium than peripheral endothelium. In contrast, bradykinin tended to increase [Ca²⁺]_i to a similar level in all cell types.

Thus, the findings of the present study suggest that EC derived from various sites of the vasculature exhibit different rates of growth and responsiveness to several vasoactive agonists. Because of this heterogeneity, it may not be appropriate to generalize the functional role of the endothelium based on observation made on a limited number of tissues.

	Selected Abbreviations and Acronyms

 

EC = endothelial cells ET-1 = endothelin-1 OEC = omental artery endothelial cells PEC = pial artery endothelial cells PMSF = phenylmethylsulfonyl fluoride SDS = sodium dodecyl sulfate SEC = superficial temporal artery endothelial cells VIP = vasoactive intestinal peptide

NT indicates not tested. PEC, SEC, and OEC lines were obtained from 5, 7, and 3 patients, respectively. Growth curves and ET-1 determination were performed in duplicate. Individual concentrations of agonists were tested in triplicate for each cell line (donor).

*Statistically significant difference at P<.05 compared with other groups (ANOVA, Scheffe's F test).

Statistically significant difference at P<.05 compared with SEC (ANOVA, Scheffe's F test).

Statistically significant difference at P<.05 compared with OEC (Student's t test).

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1R. Mehrke G, Pohl U, Daut J. Effects of vasoactive agonists on the membrane potential of cultured bovine aortic and guinea-pig coronary endothelial cells. J Physiol (Lond).. 1990;437:277-299.

2R. Mebazaa A, Wetzel R, Cherian M, Abraham M. Comparison between endocardial and great vessel endothelial cells: morphology, growth, and prostaglandin release. Am J Physiol.. 1995;268:H250-H259.

3R. Ohbayashi A, Hiraga T, Okubo M, Murase T, Matsushita H, Hara M. Characteristics of porcine coronary artery endothelial cells in culture: comparison with aortic endothelium. Biochem Biophys Res Commun.. 1994;202:504-511.

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