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(Circulation Research. 1995;77:897.)
© 1995 American Heart Association, Inc.

Articles

Expression of cGMP-Dependent Protein Kinase I and Phosphorylation of Its Substrate, Vasodilator-Stimulated Phosphoprotein, in Human Endothelial Cells of Different Origin

Richard Draijer, Arie B. Vaandrager, Christine Nolte, Hugo R. de Jonge, Ulrich Walter, Victor W.M. van Hinsbergh

From the Gaubius Laboratory TNO-PG (R.D., V.W.M. van H.), Leiden, the Netherlands; the Department of Biochemistry (A.B.V., H.R. de J.), Cardiovascular Research Institute COEUR, Erasmus University, Rotterdam, the Netherlands; and the Medical University Clinic (C.N., U.W.), Clinical Biochemistry, and Pathobiochemistry, Würzburg, Germany.

Correspondence to Dr V.W.M. van Hinsbergh, Gaubius Laboratory TNO-PG, PO Box 430, 2300 AK Leiden, the Netherlands.

	Abstract

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Abstract Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca²⁺]_i was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca²⁺ and endothelial permeability. These data indicate that cGMP-PK type I is expressed in various types of human macrovascular and microvascular ECs but is absent or expressed in very low amounts in umbilical vein ECs. cGMP-PK type I expression in ECs may be important in the regulation of endothelial permeability and the release of factors involved in vasoregulation and hemostasis.

Key Words: cGMP • permeability • microvascular endothelial cells • aortic endothelial cells • endothelial contraction

	Introduction

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The endothelium, which lines the blood vessels, actively regulates the extravasation of fluid, nutrients, and hormones into the tissues. In vascular leakage, intercellular exchange of macromolecules and fluid over this barrier is enhanced by EC contraction.^{1 2 3 4} This process can be mimicked in vitro by using tight monolayers of human ECs on porous filters.^{5 6} We and other investigators have demonstrated that the elevation of cGMP in EC counteracts the thrombin- and oxidant-induced increase in endothelial permeability in vitro.^{7 8 9 10} Intracellularly, cGMP acts via cGMP receptor proteins, which can be divided into distinct classes, such as cGMP-regulated ion channels, cGMP-regulated phosphodiesterases, cGMP-PKs, and perhaps even cAMP-dependent protein kinases.^{11 12 13} cGMP-PK I phosphorylates certain cellular proteins, including VASP, a recently cloned proline-rich protein that is associated with actin filaments and focal contacts.^{14 15} Furthermore, cGMP-PK plays an important role in the signal transduction pathway by which endothelium-derived NO and other cGMP-generating agents induce SMC relaxation and inhibition of platelet activation.^{12 16} Activation of cGMP-PK I by cGMP indirectly inhibits agonist-induced activation of phospholipase C and the accumulation of cytoplasmic Ca²⁺ in platelets^{17 18} and causes a reduction of the agonist-induced accumulation of cytoplasmic Ca²⁺ in SMCs.^{19 20} This reduces cell activation and contraction. Although it may be assumed that a similar mechanism underlies the cGMP-dependent reduction of endothelial contraction and vascular leakage, it has been difficult to detect cGMP-PK I in vascular ECs by immunofluorescence studies using intact tissues.^{11 12 21 22} On the other hand, our previous study²³ suggested that the modulating effect of cGMP on agonist-induced endothelial permeability was mediated by cGMP-PK in human aortic ECs in vitro, while another mechanism, probably via phosphodiesterase III, contributed to the cGMP-induced reduction of permeability of umbilical vein EC monolayers.²³ Another isoform of cGMP-PK, type II, which is implicated in the regulation of salt and fluid secretion in the intestine, was recently shown also to be present in tissues outside the intestine.²⁴ Therefore, in the present study, we investigated the presence and possible activation of cGMP-PK I and II in cultures and freshly obtained isolations of various human EC types. Our data demonstrate expression and activation of cGMP-PK I in human aorta and foreskin microvascular ECs but not in umbilical vein ECs. This different expression of cGMP-PK I closely agrees with differences in the effects of cGMP on the cytoplasmic Ca²⁺ concentration and the regulation of permeability in these cells and points to an autoregulatory role of NO-derived cGMP in ECs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Medium 199 supplemented with 20 mmol/L HEPES was obtained from Flow Laboratories; tissue culture plastics, from Corning or Costar; and Transwells (diameter, 0.65 cm; pore size, 3 µm), from Costar. A crude preparation of endothelial cell growth factor was prepared from bovine brain as described by Maciag et al.²⁵ Human serum obtained from a local blood bank was prepared from freshly taken blood of healthy donors. Human sera were pooled and stored at 4°C. Newborn calf serum was obtained from GIBCO and heat-inactivated before use (30 minutes, 56°C). Pyrogen-free human serum albumin (albumin) was purchased from the Central Laboratory of Blood Transfusion Service. HRP (EC 1.11.1.7 type I) was obtained from Sigma Chemical Co; bovine -thrombin, from Leo Pharmaceutical Products; fura 2-AM from Molecular Probes; 8-pCPT-cGMP and Sp-5,6-DCl-cBiMPS, from Biolog Life Science Institute; prostaglandin E,¹ from P-L Biochemicals Inc; ionomycin, from Calbiochem Corp; rhodamine-labeled Ulex europaeus lectin I, from Vector Laboratories Inc; ASM-1, from Sanbio Ltd; and monoclonal antibodies to von Willebrand factor, fluorescein-conjugated swine immunoglobulins to rabbit immunoglobulin, and fluorescein-conjugated rabbit immunoglobulin to mouse immunoglobulin, from Dakopatts. Monoclonal antibody to CD31 was a kind gift from Dr J. van Mourik, CLB, Amsterdam, the Netherlands. Purification of cGMP-PK from bovine lung and the preparation of specific antibodies against it has been described previously.²⁶ Antibodies against VASP and cGMP-PK II were raised as previously described.^{24 27} Rainbow molecular mass markers for SDS-PAGE were purchased from Amersham Life Sciences.

Isolation and Culture of ECs
Human umbilical vein ECs were isolated by the method of Jaffe et al²⁸ and characterized as described previously.²⁹ Isolation and characterization of human ECs from the aorta, iliac vein and artery, and foreskin microvasculature were performed as earlier described.^{30 31} The blood vessels of human origin were obtained according to the guidelines of the Institutional Review Board of the University Hospital Leiden. Cells were cultured on fibronectin-coated dishes in medium 199 supplemented with 10% human serum, 10% newborn calf serum, 150 µg/mL crude endothelial cell growth factor, 5 U/mL heparin, 100 U/mL penicillin, and 0.1 mg/mL streptomycin at 37°C under 5% CO₂/95% air. Medium was renewed every other day. Confluent EC monolayers were released with trypsin-EDTA and subcultured on fibronectin-coated dishes or filters. For the evaluation of the presence of cGMP-PK, the phosphorylation of VASP, [Ca²⁺]_i measurements, and the barrier function, human aortic ECs (after 2 to 9 passages), iliac vein ECs (after 8 passages), umbilical vein ECs (after 1 and 2 passages), and foreskin microvascular ECs (after 6 to 10 passages) were used. ECs of aorta, iliac artery, and umbilical vein were also used immediately after isolation to determine the presence of cGMP-PK.

Evaluation of the Barrier Function
ECs cultured on filters were used between 4 and 6 days after seeding. Exchange of macromolecules through the endothelial monolayers was investigated by assay of the transfer of HRP. Passage of HRP through human EC monolayers was performed as described previously.^{5 32} Briefly, EC monolayers were cultured on porous membranes (fibronectin-coated filters of the Transwell system, 0.33 cm², 3-µm pore size) to form a tight monolayer. Before the start of the experiment, cells were incubated for 1 hour in medium 199 with albumin. In pretreatment, the cells were incubated for 15 minutes with 8-pCPT-cGMP (100 µmol/L) in the upper and lower compartments. At the start of the experiment, 5 µg/mL HRP in medium 199 with albumin was added to the upper compartment of the Transwell system in the presence or absence of thrombin (1 U/mL). Samples were taken from the lower compartment (at the other side of the endothelial monolayer) at various time intervals, and an equal volume of medium 199 containing albumin was added to this lower compartment. Cells were kept at 37°C under 5% CO₂/95% air. All passage experiments were performed in triplicate. The concentration of HRP was derived from the HRP activity in each sample with peroxide and tetramethyl benzidine as substrate and expressed as nanograms passed per square centimeter in a certain time interval.

Measurement of [Ca²⁺]_i
ECs were cultured on 1.5-cm² glass coverslips and loaded with fura 2 by incubation with 2 µmol/L fura 2-AM for 45 minutes at 37°C in medium 199 containing 1% albumin with or without 8-pCPT-cGMP (100 µmol/L, 15-minute preincubation). Then the cells were washed with Tyrode’s buffer. The coverslips were mounted in a holder and placed in a quartz cuvette containing 1.2 mL Tyrode’s buffer. Fura 2 fluorescence was continuously measured, before and after the addition of thrombin (1 U/mL), with a luminescence spectrometer (model LS 50B, Perkin Elmer Ltd). The mean [Ca²⁺]_i was determined from a cell area of 0.6 cm² and was calculated by the following equation (nmol/L):

where R represents the ratio of the fluorescence values at 340 nm and 380 nm; R_max and R_min are the maximal and minimal ratio values, respectively, being determined after each experiment by the addition of 1 µmol/L ionomycin and 10 mmol/L EGTA, respectively; ß represents the ratio of the fluorescence at 380 nm of free fura 2 and fura 2 completely saturated with Ca²⁺ (the fluorescence values were corrected for autofluorescence of unloaded cells); and K_d, the dissociation constant of the fura 2-Ca²⁺ complex, was assumed to be 224 nmol/L at 37°C, according to Grynkiewicz et al.³³

Immunocytochemistry
Glass coverslips were coated for 45 minutes with 1% gelatin, which was cross-linked by an additional incubation of 15 minutes with 0.5% glutaraldehyde. The glass coverslips were washed five times with medium 199. ECs were seeded on the glass coverslips and, at confluence, washed with PBS, fixed for 10 minutes with methanol (-20°C), and incubated 30 minutes with PBS/20% human serum. The endothelial monolayers were then stained with anti-cGMP-PK I, anti-CD31, anti-von Willebrand factor, and/or Ulex europaeus lectin I for 30 minutes, washed three times for 5 minutes each with PBS, and incubated with a second fluorescent-conjugated antibody. After 30 minutes, the cells were washed three times for 5 minutes each with PBS and embedded in p-phenylenediamine. When indicated, immunocytochemistry was also carried out as described by Reinhard et al.¹⁴

Detection of cGMP-PK and VASP by Immunoblotting
Confluent monolayers of ECs were washed three times with PBS and suspended in reducing SDS-PAGE sample buffer and boiled for 5 minutes. Samples (6 µg of protein) were separated on SDS-PAGE using a 10% gel and transferred to nitrocellulose.³⁴ Blots were blocked overnight at 4°C in 20 mmol/L Tris-HCl, pH 7.5, 500 mmol/L NaCl plus 0.1% Tween 20 and incubated for 1 hour at room temperature with antibodies against cGMP-PK I (1:500), VASP (1:3000), and cGMP-PK II (1:3000) in the same buffer. The immunoreactive proteins were detected by the enhanced chemiluminescence method as described by the manufacturer (Amersham Life Sciences).

RNA Isolation and Northern Blotting
Total RNA was isolated according to the method of Chomczynski and Sacchi.³⁵ Formaldehyde-agarose gel electrophoresis, Northern blotting, and hybridization were performed as previously indicated.³⁶ An EcoRI fragment from the expression vector pMM9, containing the complete coding sequence of the human cGMP-PK Iß, was used as a probe.³⁷

Statistical Analysis
Data are presented as mean±SEM. Statistical analysis as indicated in the text was performed with the Wilcoxon rank sum test or the Mann-Whitney two-sample test. Statistical significance was assumed at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of ECs in Culture
ECs were identified by the characteristic morphology and specific EC markers: von Willebrand factor, CD31 (PECAM-1), uptake of DiI-acetylated low-density lipoprotein, and the binding of Ulex europaeus lectin I. Discrimination between ECs and possibly contaminating SMCs was made by their different morphology and an antibody against SMC -actin. Only pure EC cultures, entirely free of SMC contamination, have been used in the present study. Contamination of cell types other than SMCs in EC cultures from large vessels is not likely, because of the mild method used for isolation of the ECs (enzymatic separation of ECs from the matrix without scraping). Cultures of microvascular ECs from foreskin were SMC -actin negative, both in immunofluorescence cell preparations and cell lysates on Western blot (not shown); they were free of elongated or spindle-shaped cells. The EC cultures had a cobblestone appearance and formed a barrier to solutes and macromolecules, indicated by the transendothelial electrical resistance and the permeability of tracer molecules.

cGMP-PK Identification in Human ECs
The presence of cGMP-PK in human ECs was evaluated by immunofluorescence localization studies and by Western blot analysis of cGMP-PK immunoreactivity in EC lysates. To ascertain the EC nature of the cells, immunofluorescence localization of cGMP-PK I in ECs (Fig 1A, FITC labeling) was combined with staining with rhodamine-labeled Ulex europaeus lectin I (Fig 1C), which recognizes ECs but not SMCs. The EC nature of the cells was further demonstrated by the presence of CD31 at the cell-cell contacts (Fig 1D). The virtual absence of SMCs was verified by establishing the absence of SMC -actin in the cultures (not shown). cGMP-PK I was diffusively present in ECs in culture derived from aorta, iliac artery and vein, and foreskin microvessels. Freshly isolated ECs from three different aortas and an iliac artery were also positive for cGMP-PK I (Fig 1E). In contrast, ECs that were cultured or freshly isolated from umbilical vein were negative for cGMP-PK I. SMCs derived from the same umbilical veins, after additional trypsin digestion, stained positive for cGMP-PK I and SMC -actin (not shown).

View larger version (105K): [in this window] [in a new window]   Figure 1. Immunofluorescence analysis of cGMP-PK and EC markers in aortic ECs in culture and 1 hour after isolation. Isolation, culture, and immunocytochemical staining of ECs on glass coverslips were performed as described in "Materials and Methods.‘ Panels A through D show aortic ECs in culture. A, cGMP-PK. B, Negative control (aspecific staining of the nuclei with an FITC-labeled second antibody). C, Ulex europaeus lectin I (EC marker, double staining with cGMP-PK in panel A). D, EC marker CD31 (PECAM-1). Panels E and F show aortic ECs 1 hour after isolation. E, cGMP-PK. Some of the cells are already spreading; because most of the cells are still rounded 1 hour after isolation, the aggregate of ECs makes an unfocused appearance. F, Negative control.

The nature of cGMP-PK I immunoreactivity in EC lysates was further studied by Western blot analysis (Fig 2). Purified cGMP-PK I from bovine lung and the lysate of a bovine aortic SMC culture were used as a reference (Fig 2, lanes 1 to 3 and 12). Pure EC cultures from aorta (four different isolations), iliac vein (one isolation), and foreskin microvessels contained detectable amounts of cGMP-PK I, usually estimated to be 0.15 to 0.5 µg cGMP-PK per milligram EC protein (Fig 2, lanes 4 to 10). This amount is comparable to the cGMP-PK content of isolated rat aortic and bovine tracheal SMCs.^{19 20} cGMP-PK I immunoreactivity in aortic ECs remained constant in early-passage EC cultures but decreased during serial propagation of aortic ECs, usually after six to eight passages. This decrease resembles the loss of cGMP-PK in SMCs during prolonged culture.²⁰ In contrast to most tissues,¹¹ but similar to human platelets and cultured bovine aortic ECs,^{38 39} cGMP-PK I was found predominantly in particulate fractions of human aortic EC extracts, and this localization did not change after activation of cGMP-PK with 8-pCPT-cGMP (not shown). However, cGMP-PK I immunoreactivity could not be detected in five freshly isolated EC preparations and in subcultures of ECs from 15 different umbilical veins (Fig 2, lane 11). The 35- to 40-kD band on the Western blot appeared to be nonspecific and not related to any form of cGMP-PK, as was previously observed in certain cell extracts with preimmune and antisera prepared against cGMP-PK.³⁸ By Northern hybridization assay, cGMP-PK I mRNA was detected in RNA preparations of aortic and microvascular ECs but was absent in preparations of umbilical vein ECs (data not shown).

View larger version (52K): [in this window] [in a new window]   Figure 2. Autoradiogram of cGMP-PK immunoreactivity in EC lysates. Western blots of various human EC lysates (6 µg per lane) were prepared as described in "Materials and Methods." Molecular mass markers (in kilodaltons) are indicated on the left side of the figure. Lanes are as follows: lanes 1 to 3, purified cGMP-PK standard at 4, 2, and 1 ng, respectively; lanes 4 and 5, cultures of aortic ECs (donor 1) after 6 and 8 passages, respectively; lanes 6 to 8, cultures of aortic ECs (donor 2) after 2, 5, and 9 passages, respectively; lane 9, culture of foreskin microvascular ECs after 10 passages; lane 10, culture of iliac vein ECs after 8 passages; lane 11, culture of umbilical vein ECs after 1 passage; and lane 12, culture of bovine aortic SMCs after 2 passages. From these and other blots we could estimate that the various EC preparations contained 1 to 3 ng cGMP-PK per 6 µg EC protein.

In addition to the cGMP-PK I, the presence of the exclusively membrane-bound cGMP-PK II, which has recently been cloned from rat intestine,²⁴ was evaluated in the EC types. Neither ECs from human aortic and foreskin microvessels nor ECs from the umbilical vein showed a positive immunoreactivity with a cGMP-PK II-specific antibody (not shown).

cGMP-PK Phosphorylates VASP in Response to 8-pCPT-cGMP
To determine whether the cGMP-PK I in ECs is active, cultures of various EC types were incubated with the cell membrane-permanent cGMP analogue 8-pCPT-cGMP, which is a selective cGMP-PK activator.^{40 41} cGMP-PK activation was measured by determination of the phosphorylation of VASP, which is an established substrate for both cGMP-PK and cAMP-dependent protein kinase.^{14 42} In confluent human umbilical vein ECs, VASP was localized at cell-cell contacts (Fig 3, left). In subconfluent human ECs, VASP was mainly found to be associated with focal contact areas and microfilaments (Fig 3, right), similar to previous observations made with other cell types.¹⁴ In all untreated EC types, VASP was predominantly present as a 46-kD form (Fig 4). After cGMP-PK activation, the 50-kD form of VASP (phosphorylated at serine 157) was generated in intact ECs from aorta, iliac vein, and foreskin microvessels (Figs 4 and 5). The diminished cGMP-PK expression during subculturing of aortic ECs (Fig 2) was reflected in the cGMP-PK activity: VASP phosphorylation after the addition of 8-pCPT-cGMP to the cells disappeared after prolonged culture (Fig 4). VASP phosphorylation induced by 8-pCPT-cGMP was not observed in umbilical vein ECs, in accordance with the absence of cGMP-PK immunoreactivity in these cells. However, VASP phosphorylation can occur in umbilical vein ECs in response to cAMP-dependent protein kinase activation by prostaglandin E₁ and Sp-5,6-DCl-cBiMPS (Fig 6).

View larger version (106K): [in this window] [in a new window]   Figure 3. Immunocytochemical staining of human umbilical vein ECs in culture by a monospecific VASP antiserum. Left, Arrows indicate the localization of VASP at cell-cell contacts of confluent primary cultures. Right, In subconfluent cells, strong immunoreactivity is found to be associated with stress fibers and focal attachment sites. Experimental procedures and antibodies used were essentially as described by Reinhard et al.14 Bar=10 µm.

View larger version (33K): [in this window] [in a new window]   Figure 4. Autoradiogram of VASP immunoreactivity in aortic EC lysates analyzed on Western blot. cGMP-PK activation by 8-pCPT-cGMP induces phosphorylation of VASP. EC lysates were prepared for Western blot as described in "Materials and Methods." The apparent molecular masses of the unphosphorylated (46-kD) and the phosphorylated (50-kD) VASP forms are indicated. A, Aortic ECs (donor 2) (lanes 1, 3, and 5, untreated cells; lanes 2, 4, and 6, cells incubated for 30 minutes with 100 µmol/L 8-pCPT-cGMP). The preparations were made for confluent cells after 5 passages (lanes 1 and 2), after 7 passages (lanes 3 and 4), and after 9 passages (lanes 5 and 6). B, Aortic ECs (donor 1) (lanes 1 and 3, untreated cells; lanes 2 and 4, cells incubated for 30 minutes with 100 µmol/L 8-pCPT-cGMP). Lanes 1 and 2 were after 6 passages; lanes 3 and 4, after 8 passages.

View larger version (35K): [in this window] [in a new window]   Figure 5. Autoradiogram of VASP immunoreactivity in iliac vein, foreskin microvascular, and umbilical vein EC lysates analyzed on Western blot. cGMP-PK activation by 8-pCPT-cGMP induces phosphorylation of VASP. EC lysates were prepared for Western blot as described in "Materials and Methods." The molecular masses of the unphosphorylated (46-kD) and the phosphorylated (50-kD) VASP forms are indicated. Lanes are as follows: lanes 1, 3, and 5, untreated cells; lanes 2, 4, and 6, cells incubated for 30 minutes with 100 µmol/L 8-pCPT-cGMP. Lanes 1 and 2 represent iliac vein ECs after 8 passages; lanes 3 and 4, foreskin microvascular ECs after 10 passages; and lanes 5 and 6, umbilical vein ECs after 1 passage.

View larger version (24K): [in this window] [in a new window]   Figure 6. Top, Autoradiographs demonstrating the effects of prostaglandin E1 (PG-E1) and sodium nitroprusside (SNP) on VASP phosphorylation in cultured human umbilical vein ECs. Cells (passage 2) were incubated for 5 minutes with various concentrations of PG-E1 or SNP as indicated. VASP phosphorylation was analyzed by Western blots as previously described27 and is indicated by the shift of VASP from the 46- to the 50-kD form. Bottom, Quantitative analysis of VASP phosphorylation in cultured human umbilical vein ECs incubated with 500 µmol/L Sp-5,6-DCl-cBiMPS or 200 µmol/L 8-pCPT-cGMP for the time periods indicated. VASP phosphorylation was analyzed by Western blots and is expressed as the appearance of 50-kD VASP as percentage of total VASP (46-kD plus 50-kD VASP as percentage of total VASP (46-kD plus 50-kD forms). Data represent the mean±SD of three separate experiments.

Cellular Consequences of cGMP-PK Activation
Elevation of intracellular cGMP attenuates thrombin-induced increase in endothelial permeability.^{7 10} We used 8-pCPT-cGMP to selectively activate cGMP-PK, without affecting cGMP-regulated phosphodiesterases. Thrombin increased endothelial permeability (passage of HRP) in aortic, umbilical vein, and foreskin microvascular EC monolayers (Fig 7A through 7C), which was associated with a transient increase of [Ca²⁺]_i (Fig 7D through 7F). Preincubation with 8-pCPT-cGMP reduced the thrombin-stimulated permeability in aortic ECs to 59±6% (mean±SEM of 6 experiments performed in triplicate, P<.05, Fig 7A) and in foreskin microvascular ECs to 45±4% (3 experiments, Fig 7B). However, the cGMP-PK activator had no effect on the permeability through umbilical vein EC monolayers: 112±16% (4 experiments, Fig 7C) and 89±18% (8 experiments) with 100 µmol/L and 1 mmol/L 8-pCPT-cGMP, respectively. Similarly, the thrombin-induced [Ca²⁺]_i elevation was inhibited by 8-pCPT-cGMP in aortic and foreskin microvascular ECs: the [Ca²⁺]_i peak value decreased from 754±161 to 264±50 nmol/L in aortic ECs (P=.002, 10 experiments, Fig 7D) and from 791±106 to 410±103 nmol/L in foreskin microvascular ECs (P=.05, 5 experiments, Fig 7E). However, thrombin-induced [Ca²⁺]_i elevation was not significantly affected in umbilical vein ECs (from 796±83 to 649±67 nmol/L, 16 experiments, Fig 7F).

View larger version (32K): [in this window] [in a new window]   Figure 7. Thrombin-induced increase of endothelial permeability and [Ca2+]i in the absence or presence of 8-pCPT-cGMP. The endothelial permeability was measured by the passage of HRP through EC monolayers, and the [Ca2+]i was determined in fura 2-loaded ECs, as described in "Materials and Methods." A, Passage of HRP through aortic EC monolayers under control conditions (), after addition of 1 U/mL thrombin without (•) and with () a preincubation of 100 µmol/L 8-pCPT-cGMP. The arrow indicates the time point of thrombin addition. B and C, Conditions similar to those described in panel A for foreskin microvascular ECs and umbilical vein ECs, respectively. D, [Ca2+]i in aortic ECs after addition of thrombin (indicated by an arrow on the x axis) without (•) and with () a preincubation of 8-pCPT-cGMP. E and F, Conditions similar to those described in panel D for foreskin microvascular ECs and umbilical vein ECs, respectively.

Whereas activation of cGMP-PK had no effect on the permeability of umbilical vein ECs, activation of cAMP-PK by prostaglandin E₁ reduced both basal and thrombin-induced endothelial permeability in vitro (Fig 8). This agrees with previous studies with the stable prostacyclin-analogue iloprost.^{6 43} The effect of cAMP-PK activation did not depend on the [Ca²⁺]_i, because elevation of the cellular cAMP concentration had no effect on the [Ca²⁺]_i in these cells (not shown).

View larger version (16K): [in this window] [in a new window]   Figure 8. Effect of prostaglandin E1 (PGE1) on the basal and thrombin-stimulated passage of HRP through human umbilical vein EC monolayers evaluated over a 2-hour incubation period. A, Passage of HRP under basal conditions. B, Passage of HRP under thrombin (1 U/mL)-stimulated conditions. Values are mean±SEM of triplicate determinations. Similar results were obtained with another tracer molecule (FITC-labeled 38.9-kD dextran).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Results reported in the present study demonstrate that ECs from adult human arteries and vein and from foreskin microvessels, but not those from human umbilical vein, contain active cGMP-PK I. The selective cGMP-PK activator 8-pCPT-cGMP caused VASP phosphorylation, reduced thrombin-stimulated [Ca²⁺]_i accumulation, and reduced thrombin-induced increase in permeability. These effects were restricted to cGMP-PK-containing ECs.

cGMP-PK was originally not found in cultured bovine pulmonary arterial and aortic ECs,⁴⁴ but MacMillan-Crow et al³⁹ and Diwan et al⁴⁵ recently reported the presence of cGMP-PK I in extracts of bovine aortic and rat pulmonary microvascular ECs, respectively. The different results in these studies may be due to a culture phenomenon, because the cGMP-PK contents decrease below detection level during serial propagation of human aortic ECs (Fig 2). We found cGMP-PK in a range of 0.15 to 0.5 µg/mg cellular protein in adult artery and vein ECs and in microvascular ECs, which is somewhat less than is found in isolated human platelets⁴⁶ but similar to the level detected in SMCs in vitro.^{19 20} Interestingly, human umbilical vein ECs, which have many properties in common with postcapillary venule ECs, did not contain cGMP-PK either in culture or immediately after isolation from the native blood vessel.

The differences in cGMP-PK content of various types of ECs may largely determine the effect of cGMP-enhancing agents on the endothelium of a particular type of blood vessel. This suggestion is supported by the observation that a thrombin-induced increase in [Ca²⁺]_i and endothelial permeability was reduced by 8-pCPT-cGMP in cGMP-PK-containing aortic and microvascular ECs, whereas this cGMP analogue was ineffective in cGMP-PK-negative umbilical vein ECs (Fig 7). Elevation of [Ca²⁺]_i has been associated with an increase in endothelial contraction and permeability.^{2 3 5 23 47 48 49} The observed cGMP-PK-mediated reduction of endothelial permeability may therefore be a direct consequence of the reduction of the thrombin-induced [Ca²⁺]_i rise. In this respect, regulation of EC contraction, which underlies the increase in intercellular permeability, appears to be rather similar to the regulation of SMC contraction and platelet activation.^{16 50} However, SMC contraction may also be modulated by mechanisms other than [Ca²⁺]_i regulation.⁵¹ Therefore, we cannot exclude the possibility that cGMP-dependent phosphorylation also contributes by additional Ca²⁺-independent mechanisms to a change in the interaction of actin filaments with myosin or cell-cell contact areas and hence influences endothelial permeability. One possible additional mechanism could be cGMP-PK-mediated VASP phosphorylation.

Our data show that the cGMP-PK-I in ECs is active and phosphorylates VASP. Very recent data suggest that VASP phosphorylation does not directly regulate phospholipase C activity or Ca²⁺ mobilization but appears to be involved in regulating the actin filament system, possibly via profilin and proteins associated with cell-matrix (focal adhesions) and cell-cell contacts.^{15 37 52} In this context, our finding that VASP is localized at cell-cell contact areas of confluent ECs, whereas it is predominantly present in focal adhesion sites in subconfluent ECs, is of interest. This spatial distribution has much similarity with the distribution of cadherins in ECs⁵³ and suggests that VASP may participate in the organization of cell-cell contacts of the adherens type. Further studies are clearly necessary to elucidate whether VASP is involved in the maintenance of the endothelial barrier function and whether the phosphorylation of VASP contributes to the regulation of endothelial permeability. If this is the case, VASP may act as a convergence point for cGMP and cAMP, which both induce a phosphorylation of VASP at three distinct sites (Reference 42⁴² and the present study), and both can inhibit an agonist-induced increase in permeability.^{10 54}

In addition to endothelial permeability, the synthesis and/or release of a number of EC products involved in vasoregulation and hemostasis is enhanced by vasoactive substances, at least in part via an increase in [Ca²⁺]_i. They involve the release of NO⁵⁵ and prostacyclin,⁵⁶ the acute release of tissue-type plasminogen activator and von Willebrand factor,⁵⁷ and the intracellular production of platelet-activating factor.⁵⁸ Elevation of cGMP by the administration of atrial natriuretic peptide or 8-bromo-cGMP reduced the acute release of tissue-type plasminogen activator induced by vasoactive substances in the perfused rat hind leg.⁵⁹ The same mediators inhibited thrombin-induced release of endothelin 1 by rat aortic ECs.⁶⁰ Furthermore, the production of NO has been reported to counterregulate its own production,⁶¹ probably because it reduces the accumulation of [Ca²⁺]_i via its activation of guanylyl cyclase.

The present data suggest that the expression of cGMP-PK I, VASP, and probably additional substrates may be important in the regulation of various EC functions. The presence of cGMP-PK in combination with NO synthase and soluble and natriuretic peptide receptor-linked guanylyl cyclase equips the EC with a potent (counter)regulatory mechanism for maintaining hemostasis.

	Selected Abbreviations and Acronyms

 

8-pCPT-cGMP = 8-(4-chlorophenylthio)-cGMP ASM-1 = monoclonal antibody to SMC actin cGMP-PK = cGMP-dependent protein kinase cGMP-PK I and cGMP-PK II = cGMP-PK types I and II, respectively EC = endothelial cell FITC = fluorescein isothiocyanate HRP = horseradish peroxidase NO = nitric oxide PECAM-1 = platelet/endothelial cell adhesion molecule-1 SMC = smooth muscle cell Sp-5,6-DCl-cBiMPS = 5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole-3',5'-cyclic monophosphorothioate, Sp-isomer VASP = vasodilator-stimulated phosphoprotein

	Acknowledgments

 
This study was supported by the Netherlands Heart Foundation (grant 90.085), by the Netherlands Organisation for Scientific Research (NWO), and by the Deutsche Forschungsgemeinschaft (SFB 355). The authors also thank Susanne Stumpf for excellent technical assistance and Matthias Reinhard for his help with the immunofluorescence analysis of VASP.

	Footnotes

 
Previously presented as preliminary results in abstract form (Endothelium. 1993;1:S31; and the Seventh International Symposium on the Biology of Vascular Cells, Heidelberg, Germany, 1994).

© 1995 American Heart Association, Inc.

Received March 31, 1995; accepted July 10, 1995.

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