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(Stroke. 1996;27:2211-2215.)
© 1996 American Heart Association, Inc.

Articles

-Thrombin Upregulates G_i3 in Human Vascular Endothelial Cells

S. Martin Shreeve, PhD; Marie A. Shatos, PhD Eric Thorin, PhD

the Totman Laboratory for Human Cerebrovascular Research (S.M.S., E.T.) and the Department of Biochemistry (M.A.S.), University of Vermont (Burlington).

Correspondence to S. Martin Shreeve, PhD, Totman Laboratory for Cerebrovascular Research, Department of Pharmacology, College of Medicine, Given Bldg, University of Vermont, Burlington, VT 05405. E-mail sshreeve@moose.uvm.edu.

	Abstract

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Background and Purpose During thrombosis, -thrombin becomes sequestered by fibrin and the subendothelial basement membrane, and it is available to interact with the vasculature over prolonged periods. In this study, we investigated the long-term effect of -thrombin on G_i3 and G_s levels in human vascular endothelial cells (EC). Because obesity is associated with changes in receptor signaling in many animal models, we also explored the influence of this clinical risk factor.

Methods Primary cultures of human EC were exposed to -thrombin for 16 hours, and immunologically detectable G_i3 and G_s levels were measured.

Results -Thrombin (100 nmol/L) increased G_i3 levels in EC derived from the cerebral microvasculature and superficial temporal artery (4.2±1.2-fold and 2.8±0.32-fold, respectively) but had no significant effect on EC derived from omental artery (P>.6) or from the superficial temporal artery of obese (body mass index 28 kg/m²) patients (P>.4). The expression of G_s was unchanged in all cell types (P.1). Two other circulating peptides, vasoactive intestinal peptide and endothelin-1, failed to alter the expression of either G protein in EC from the cerebral microvasculature, further demonstrating the specificity of the -thrombin effect. However, thrombin receptor activating protein-14 mimicked the -thrombin response and increased G_i3 in EC derived from the cerebral microvasculature and superficial temporal artery.

Conclusions We conclude that -thrombin increases G_i3 expression in some EC through activation of its tethered liganded receptor. Obesity appears to suppress this action of -thrombin.

Key Words: endothelium • obesity • G-proteins • thrombin

	Introduction

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Obesity, atherosclerosis, hypertension, diabetes, and elevated levels of procoagulants such as fibrinogen and -thrombin are interrelated risk factors for all forms of stroke and other cerebrovascular events.^{1 2 3 4} During thrombosis, a massive amount of -thrombin is generated, which can overwhelm the protective effects of the plasma protease inhibitors.^{5 6} Consequently, -thrombin circulates downstream, becomes sequestered by the subendothelial extracellular matrix^{7 8} or binds to fibrin in thrombi, and is slowly released on clot degradation.⁹ Bound -thrombin is protected from inactivation by circulating inhibitors, retains its proteolytic activity, and is available to interact with EC, vascular smooth muscle cells, and platelets over prolonged periods. Because of its relevance to stroke and other cardiovascular diseases, we examined the long-term effects of -thrombin on EC.

-Thrombin activates EC via nonproteolytic and proteolytic actions on high- and moderate-affinity receptors, respectively.^{10 11 12 13 14} Most of the cellular activities induced by -thrombin in EC and other cells are mediated by the moderate-affinity receptor that belongs to the seven transmembrane, heterotrimeric G protein–coupled receptor family. This receptor is activated as a consequence of the serine protease activity of -thrombin.^{11 12 13 14 15} -Thrombin binds to an extracellular domain near the amino terminus and proteolyses the receptor protein. This exposes a new amino terminus (NH₂SFLLRN) that acts as a tethered ligand and triggers the receptor. Synthetic peptides, having amino acid residues corresponding to this region, can directly activate the receptor and induce most of the actions of thrombin¹⁴ ; they are consequently called thrombin receptor–activating proteins (TRAPs).

The tethered liganded receptor couples to phospholipase C, apparently through G_q, although the ß subunits of G_i have also been implicated in platelets.^{11 12 13 14 15 16} -Thrombin can also inhibit adenylyl cyclase through a G_i protein. The identity of the G_i subtype involved and other G protein effector interactions are yet to be determined.

EC responses to -thrombin include the production and release of prostacyclin, nitric oxide, platelet activating factor, plasminogen activators, ET-1, and platelet-derived growth factor. -Thrombin also triggers EC retraction and increases microvascular permeability, induces the expression of adhesion molecules, and is mitogenic for capillary EC and other cells.^{11 17 18}

One of the many ways a cell can respond to prolonged agonist contact is to regulate the levels of various signaling components. This phenomenon has been well documented for certain receptors such as the ß₂-adrenergic receptor, which is downregulated on exposure to agonist.¹⁹ However, receptors are not the only components that can be altered. Downregulation of G protein levels through prolonged contact with agonist-occupied receptors has been shown for a number of G_i- and G_q-linked receptors^{20 21 22} and is used as an approach to determine which G proteins are activated by receptors. In some cases, opposing G proteins are upregulated.^{20 23}

Several pathological states are associated with changes in G protein levels. For example, there is a significant increase in the ratio of G_i to G_s in congestive heart failure (reviewed in Reference 24). G_i1 is decreased in adipocytes from obese human subjects.²⁵ Oxidized low-density lipoprotein downregulates G_i2 in bovine aortic EC,²⁶ and a decrease in either the amount or functionality of G_i in regenerated endothelium may account for some of the injury induced after balloon denudation.²⁷ Excessive glucocorticoid action, hypothyroidism, and insulin deficiency and resistance have also been associated with changes in the levels of certain G proteins.²⁸ Indeed, alterations in G protein function have been implicated in a variety of disorders.²⁹

Because of its possible relevance to the clinical effects of stroke, we examined the long-term effect of -thrombin on levels of G_i3 and its opposing G protein, G_s, in primary cultures of human EC. Since the endothelium is not homogeneous and varies depending on location and disease,^{18 30} we studied EC cultured from three vessels, including the cerebral microvasculature. Some cells came from obese patients, and we were therefore able to explore the effects of this clinical risk factor.

	Materials and Methods

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Human EC
Human EC were cultured from the cerebral microvasculature (HCMEC), omental artery (OEC), and superficial temporal arteries from nonobese (STEC) and obese (O/STEC) patients. Patients with a body mass index 28 kg/m² were considered obese. Donors had no clinical evidence of hypertension, atherosclerosis, or diabetes. The HCMEC used in this study were derived from 9 nonobese donors: 4 men ranging in age from 27 to 65 years and 5 women ranging in age from 29 to 82 years. The OEC were derived from 2 female nonobese patients of 20 and 32 years of age. Clinical data for STEC and O/STEC are shown in the Table. All tissues were collected during surgery and were pathological samples that would have been discarded if not used in these studies. All donors gave written informed consent. Institutional approval was obtained.

View this table: [in this window] [in a new window]   Table 1. Summary of Clinical Data for Donors of Superficial Temporal Artery

HCMEC were isolated as previously described.¹⁸ Briefly, fragments of cerebral cortex, consisting exclusively of gray matter devoid of large blood vessels and assessed to be free of abnormal pathology, were minced and subjected to repeated cycles of collagenase digestion. The supernatant containing the cerebral microvessels was then forced through a 70-µm mesh to separate the cells from large undigested fragments. Cells were pelleted and seeded in supplemented M-199 medium. As cells began to proliferate, HCMEC were further isolated from contaminating cell types by use of conventional cloning technology.

Superficial temporal arterial fragments (ID, 832±66 mm) were obtained from patients undergoing neurosurgery for skull fracture or tumor removal. Omental arterial fragments (ID, 441±67 mm) were collected from patients undergoing gastrointestinal surgery for conditions that did not involve the omentum. Arterial segments were cleaned of connective tissue, cut into small fragments, and placed in a culture dish containing supplemented Dulbecco's modified Eagle's medium. As STEC, O/STEC, and OEC proliferated, they were harvested and cloned using conventional cloning technology.

HCMEC were identified as endothelial from the following five criteria: positive immunocytochemical staining for factor VIII/von Willebrand factor antigen; binding of Ulex europaeus agglutinin-1 lectin, a specific human EC marker that is particularly sensitive for microvasculature EC; negative staining for glial fibrillary acidic protein; ability to produce prostacyclin, a normal secretory product of the endothelium; and absence of G_o protein, which is present in neurogenic cells.¹⁸ STEC, O/STEC, and OEC stained positively for factor VIII/von Willebrand factor antigen but not for anti-smooth muscle -actin antigen and did not express G_o protein but did secrete ET-1, a normal secretory product of the endothelium.³⁰ STEC, O/STEC, and OEC exhibit a "cobblestone"-like appearance typical of EC cultured from larger vessels or from microvessels of noncerebral origin. HCMEC exhibit an elongated spindle-shape morphology and align longitudinally as they grow.

Drug Treatment and Cell Membrane Preparation
EC at passages one through four were grown in 750-mm² tissue culture flasks and incubated just before confluence with -thrombin (Hematological Technologies), hirudin (Genentech), cycloheximide (Sigma), TRAP-14 (SFLLRNPNDKYEPF; Bachem), ET-1 (Peninsula Laboratories), or VIP (Bachem) for 16 hours. Cells were washed with PBS, scraped off the plates, and pelleted.

Frozen cell pellets were resuspended in 10 mL of ice-cold 10 mmol/L Tris HCl, 0.1 mmol/L EDTA, pH 7.5, and homogenized (20 up-and-down strokes) using a glass Dounce tissue grinder. The homogenate was centrifuged at 500g for 10 minutes and the resultant supernatant at 48 000g for 1 hour. The pellet was resuspended in 100 µL of phosphate buffer (50 mmol/L NaPO₄ and 5 mmol/L MgCl₂, pH 7.4) and 100 µL of boiling 10% SDS and incubated at 80°C for 5 minutes. Protein was measured using a fluorescamine assay.³¹

SDS-Polyacrylamide Gels and Western Immunoblotting
An equal volume of double-strength SDS-reducing sample buffer (10% glycerol, 1% 2-mercaptoethanol, 2% SDS, 0.001% bromophenol blue, 62.5 mmol/L Tris-HCl, pH 6.8) was added to the cell membrane preparation, and the G proteins were analyzed on 1.5-mm-thick 10% or 12% discontinuous SDS-polyacrylamide gels. For each experiment, care was taken to load equal amounts of total protein (10 to 30 µg) on the gel.

Proteins were transferred from the polyacrylamide gel to 0.45-µm nitrocellulose paper (Bio-Rad) at 60 V for 2 hours or 30 V for 90 minutes (minigel) using Towbin buffer.³² The paper was incubated for 1 hour at room temperature in Tris-saline buffer (500 mmol/L NaCl, 20 mmol/L Tris, pH 7.4) containing 5% nonfat dry milk and 0.1% Tween 20 to block nonspecific protein binding. After washing with 0.05% Tween 20 in Tris-saline buffer (Tween-Tris-saline buffer), the nitrocellulose paper was incubated for 1 hour with a 1:1000 dilution of anti–G protein antiserum in Tween-Tris-saline containing 1.5% goat serum. Polyclonal rabbit anti–G protein antisera (Calbiochem) are raised against synthetic peptides that are unique to specific G protein -subunits (C-terminal decapeptides). These antisera are specific for the appropriate G protein subunit.³² The anti-G_i3 antiserum used in this study has been affinity purified (using the C-terminal G_i3 decapeptide) and is specific for G_i3. After being washed, the paper was incubated for 1 hour at room temperature with horseradish peroxidase–conjugated second antibody (1:2000 dilution in Tween-Tris-saline containing 1.5% normal goat serum), and proteins were detected using an enhanced chemiluminescence method and Hyperfilm-ECL according to manufacturer's instructions (Amersham). The relative intensities of bands on film from immunoblots were determined by means of an Optimas imaging program (BioScan).

For each gel, results were compared with control. The data are expressed as mean±SE relative to control. One-way repeated measures ANOVA was performed to determine whether significant changes in the expression of each G protein occurred in HCMEC or STEC in response to several agonists. If P<.05, then this was followed by Dunnett's t test for multiple comparisons versus control. A paired t test was used to determine whether exposure to -thrombin significantly altered the expression of each G protein in OEC or O/STEC. Differences were considered significant when P<.05.

Cross-Linking of VIP and ET-1 to Their Receptors and Autoradiography
HCMEC membranes (100 µg protein) were incubated for 30 minutes at 23°C with 250 pmol/L ¹²⁵I-VIP or ¹²⁵I–ET-1 (New England Nuclear) in the presence or absence of 10 nmol/L nonradioactive peptide in phosphate buffer. Free peptide was removed by centrifugation and resuspension of the membranes in phosphate buffer. Covalent cross-linking of the peptide to its receptors was accomplished by incubation (30 minutes at 23°C) with 5 mmol/L of the homobifunctional cross-linker ethylene glycol-bis(succinimidylsuccinate) (Pierce Chemical Co).³² Samples were analyzed on 7.5% or 8% SDS-polyacrylamide gels, and receptor complexes were visualized using autoradiography and Kodak X-OMAT film.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Treatment of HCMEC with -thrombin induced a dose-dependent upregulation in membrane G_i3 (M_r=41 000) (Fig 1A). -Thrombin (100 nmol/L [10 NIH U/mL]) increased G_i3 by 4.2±1.2-fold (n=5). However, the expression of both long (M_r=52 000) and short (M_r=45 000) splice variants of G_s was not significantly (P.1, n=3) changed by -thrombin (Fig 1B).

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of agonists on the expression of Gi3 and Gs in HCMEC membranes. Cells were treated with agonists for 16 hours. Cont indicates control (no drug treatment); ET, 100 nmol/L ET-1; VIP, 100 nmol/L VIP; and thr, 10 or 100 nmol/L -thrombin. Membranes derived from harvested cells were analyzed with SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with antiserum against Gi3 (A) or Gs (B) as indicated. Arrows indicate G protein; lines denote the position of Mr markers (shown in kilodaltons). The upper panels display immunoblot data from a typical study; the histograms in the lower panels show the quantification of each G protein band relative to control (mean±SE). *Significantly different from control at P<.05.

The selectivity of this effect was further demonstrated by the finding that two other circulating factors, ET-1 and VIP, had no significant effect on either G_i3 or G_s expression (Fig 1). To show that this was not due to the absence of receptors for these peptides, VIP and ET receptors in HCMEC were analyzed using SDS-polyacrylamide gel electrophoresis and autoradiography.

Covalent cross-linking of ¹²⁵I-VIP to HCMEC membranes revealed two radiolabeled protein bands centered at M_r=58 000 and M_r=71 000. Labeling to both bands was of high affinity and specific because it was completely blocked by 10 nmol/L nonradioactive VIP and the VIP antagonist [4-Cl-D-Phe⁶,Leu¹⁷]VIP (IC₅₀, 300 nmol/L). Similar M_r values for the VIP receptor have been reported in other tissues.^{33 34 35} The two bands may reflect the existence of distinct subtypes, although both human VIP receptors cloned to date have a similar predicted M_r of approximately 50 000 (excluding glycosylation).^{36 37} In this case, it seems more probable that the two bands are a consequence of major differences in glycosylation.³⁵

¹²⁵I-ET-1 labeled two protein bands at approximately M_r=50 000 and a third at M_r=35 000. The labeling was specific (IC₅₀ of nonradioactive ET-1 was 30 nmol/L for all bands) and of high affinity. Cross-linking in other tissues has revealed a similar range of specifically labeled proteins.³⁸ Again, the multiple bands may reflect major differences in glycosylation, the expression of distinct subtypes (although cloned ET_A, ET_B, and ET_C receptors have a similar predicted M_r of approximately 50 000^{39 40} ), and proteolysis (the endothelin receptor is highly susceptible to proteolytic enzymes, despite the presence of inhibitors). We conclude that HCMEC express receptors for VIP and ET-1 but that these peptides, unlike -thrombin, do not induce changes in G_i3 levels.

To determine whether -thrombin regulates G_i3 in EC derived from other vessels, we examined the effects of this enzyme on STEC. Again, G_i3 was upregulated (by 2.8±0.32-fold, n=6), whereas both forms of G_s were not significantly changed (P.4, n=3) (Fig 2).

View larger version (44K): [in this window] [in a new window]   Figure 2. Effect of -thrombin and TRAP-14 on the expression of G proteins in STEC membranes. Cells were treated for 16 hours with 100 nmol/L -thrombin without or with either 0.5 µmol/L hirudin or 5 µg/mL cycloheximide, or 10 µmol/L TRAP-14. Membranes derived from harvested cells were analyzed with SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with antiserum against Gi3 (A left and B) or Gs (A right ) as indicated. The histogram (B) shows quantification of Gi3 (mean±SE), and the inset displays immunoblot data from a typical study. *Significantly different from control at P<.05.

The effective dose range, while physiological, suggests that -thrombin is producing its effect on G_i3 expression via the moderate-affinity G protein tethered liganded receptor. This is supported by the finding that the thrombin receptor agonist TRAP-14 (a synthetic 14–amino acid peptide corresponding to residues Ser⁴² through Phe⁵⁵ of the thrombin receptor that forms the new NH₂ terminus after receptor cleavage) also induces G_i3 upregulation (by 2.6±0.4-fold, n=2) (Fig 2). TRAP-14 also significantly upregulated G_i3 in HCMEC (by 4.0±1.9-fold, n=2). Hirudin inhibited the effect of -thrombin (Fig 2), demonstrating that the upregulation of G_i3 is the result of its proteolytic action. Again, this finding is consistent with -thrombin acting through its tethered liganded receptor.

However, not all EC are regulated by -thrombin in this way. -Thrombin treatment of OEC did not significantly (P>.6, n=7) alter the expression of G_i3 (Fig 3); both forms of G_s were also unchanged (P>.4, n=3). Moreover, expression of G_i3 and G_s in O/STEC, which unlike STEC are derived from obese patients (Table), was also unaffected (P>.4, n=3) (Fig 3). This difference in EC was not due to variations in the level of basal G_i3, since this did not significantly differ (P>.1) among HCMEC, STEC, O/STEC, and OEC.

View larger version (51K): [in this window] [in a new window]   Figure 3. Effect of -thrombin on the expression of Gi3 and Gs in OEC and O/STEC. Cells were treated with 100 nmol/L -thrombin for 16 hours. Membranes derived from harvested cells were analyzed with SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with antiserum against Gi3 (top) or Gs (bottom) as indicated. Immunoblot data from a typical study are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study reveals a novel long-term effect of -thrombin: in HCMEC and STEC -thrombin upregulates G_i3 (Figs 1 and 2). Three observations lead us to conclude that this response is mediated by the tethered liganded receptor. First, hirudin (which prevents both the binding of -thrombin to its receptor, as well as its proteolytic action⁴¹ ) inhibits the response. Second, the effective -thrombin concentration is in the nanomolar range, which is characteristic of the tethered liganded receptor but not of the higher affinity thrombomodulin receptor.^{11 12 13 14 15} Third, TRAP-14, which binds to an as yet undefined region of the tethered liganded receptor and so mimics most of the responses of thrombin,¹⁴ also upregulates G_i3 in HCMEC and STEC. Although we have not examined the mechanism of -thrombin–induced G_i3 upregulation, the inhibitory action of cycloheximide (Fig 2) suggests that -thrombin induces de novo synthesis of G_i3.

This effect of -thrombin was selective. Although upregulating G_i3, -thrombin had no effect on what can be considered the opposing protein, G_s (Figs 1 and 2). Moreover, two other circulating peptides that act on the endothelium had no effect on G_i3 levels. VIP is released from neurons innervating some blood vessels and induces vasorelaxation by activating receptors in smooth muscle. However, it has been demonstrated that in vessels such as the human saphenous vein, VIP interacts with the endothelium, which mediates a significant part of its vasorelaxant effect.⁴² ET-1 is synthesized by EC and is released both abluminally and into the blood. ET-1 binds to receptors on smooth muscle and acts as an endothelium-derived contracting factor, while circulating ET-1 can induce vasodilation as a consequence of activating endothelin receptors on EC.⁴³ It is generally accepted that ET_B subtypes are expressed in the endothelium, but there is also evidence for the existence of ET_A and possibly ET_C receptors in some endothelial cells.⁴⁴ Although receptors for both VIP and ET-1 were identified on HCMEC, these peptides, unlike -thrombin, had no effect on G_i3 expression (Fig 1).

-Thrombin did not increase G_i3 in all EC (Fig 3). Other studies have also shown that EC of diverse origin respond differently to -thrombin. For example, -thrombin differentially modulates the fibrinolytic potential of cerebral and umbilical EC.¹⁸ We have also found differences among human EC derived from pial, superficial temporal, and omental arteries in that the increases in intracellular calcium triggered by ET-1, VIP, and -thrombin were greater in pial artery EC (unpublished observations, 1996). The lower agonist sensitivity of OEC may reflect differences in receptors and other signaling components.

In other receptor systems, long-term exposure to agonists has been shown to induce changes in G protein levels. Typically, these studies reveal downregulation of G_i or G_q, although there are a few reports of agonist-induced increases in G proteins, including G_s.^{20 21 22 23} Alteration of receptor or G protein levels is associated with changes in signaling efficiency and specificity. Thus, by increasing G_i3, -thrombin may alter the responsivity of the EC to circulating agonists. G_i3 has recently been shown to couple receptors to inhibition of adenylyl cyclase and stimulation of both phospholipase C and phospholipase A₂,⁴⁵ indicating that -thrombin can potentially modulate the activity of various receptor/effector systems. In addition, recent observations that G_i3 is located in the Golgi apparatus of some cells⁴⁶ raise the intriguing possibility that -thrombin may be regulating protein trafficking.

Finally, in STEC (Fig 3), the observation that obesity suppresses the effect of -thrombin is of clinical interest. Previous studies of obesity and G protein levels have typically demonstrated G protein downregulation in nonvascular tissues. For example, compared with findings in lean controls, there is a decrease in the amount of G_s and some G_i proteins in adipocytes⁴⁷ and liver⁴⁸ of ob/ob mice and a decrease in G_i1 protein in adipocytes of obese humans.²⁵ In contrast, we find that there is no difference in the basal levels of G_i3 and G_s in STEC from obese and lean subjects. We make the novel observation that it is the response of the EC to -thrombin that is affected by obesity.

	Selected Abbreviations and Acronyms

 

EC = endothelial cells ET = endothelin HCMEC = human cerebral microvascular endothelial cells Mr = molecular weight O/STEC = human superficial temporal artery endothelial cells from obese patients OEC = human omental artery endothelial cells STEC = human superficial temporal artery endothelial cells from nonobese patients TRAP = thrombin receptor activating protein VIP = vasoactive intestinal peptide

	Acknowledgments

 
This work was supported by The Totman Medical Research Fund and the Collen Foundation. The authors gratefully acknowledge Carrie L. Walters, MD, from Neurological Surgeons (Phoenix, Ariz) and Paul L. Penar, MD, Michael Ricci, MD, and Neil Hyman, MD, from the Department of Surgery, University of Vermont (Burlington) for providing us with blood vessels. We also thank Jacqueline M. Doherty for assistance with various aspects of this study.

Received June 28, 1996; revision received August 29, 1996; accepted August 29, 1996.

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