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Am J Physiol Heart Circ Physiol 278: H1815-H1822, 2000;
0363-6135/00 $5.00
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Vol. 278, Issue 6, H1815-H1822, June 2000

Somatostatin receptor subtype expression and function in human vascular tissue

Susan B. Curtis1, Jeff Hewitt2, Svetlana Yakubovitz3, Alexander Anzarut1, York N. Hsiang1, and Alison M. J. Buchan3

Departments of 1 Surgery, 2 Biochemistry and Molecular Biology, and 3 Physiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In animal models the somatostatin analog angiopeptin inhibits intimal hyperplasia by acting primarily through somatostatin receptor 2 (SSTR-2). However, the results of clinical trials using angiopeptin have been disappointing. In this study we showed that human blood vessels express high levels of SSTR-1 with significantly lower levels of SSTR-2 and -4. Samples of normal veins and arteries, as well as atherosclerotic arteries, expressed predominantly SSTR-1. In addition, the levels of SSTR-1 varied between individuals, indicating that the vascular disease process may have affected SSTR gene expression. Immunocytochemical studies demonstrated that SSTR-1 was present in endothelial but not vascular smooth muscle cells. No evidence of SSTR-3 or -5 expression was detected in normal or diseased blood vessels. Two endothelial cell preparations, ECV304 and human umbilical vein endothelial cells, were investigated and shown to express only SSTR-1 and -4. Exposure of these cells to 10 nM somatostatin or 10 nM SSTR-1-specific agonist resulted in alterations to the actin cytoskeleton, as characterized by a loss of actin stress fibers coupled with an increase in lamellipodia formation at the plasma membrane. These results suggest that the lack of effectiveness of angiopeptin in humans may be due to the differential expression of SSTR-1 by human endothelial cells.

endothelial cells; lamellipodia; actin stress fibers; human umbilical vein endothelial cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR REMODELING is an adaptive process that occurs in response to long-term changes in hemodynamic conditions. Any type of interventional treatment, such as angioplasty or bypass surgery, can be damaging to the vessel wall (3). Injuries to the vessel wall lead to a complex cascade of reparative responses starting with mural thrombi formation. Death of medial smooth muscle cells (SMC) from vessel wall injury also initiates the release of growth factors, such as basic fibroblast growth factor (bFGF) and platelet-derived growth factor from platelets, macrophages, and endothelial cells (3, 22). These factors subsequently stimulate the proliferation and migration of medial SMC into the intima. Proliferating SMC synthesize and secrete a wide variety of mitogenic growth factors to promote further SMC proliferation and elaboration of extracellular matrix (13, 29). This reparative process terminates when the damaged endothelium has been restored. However, if cell proliferation and matrix deposition continue, they lead to a pathological condition known as intimal hyperplasia (IH) (14).

Clinically, IH causes renarrowing, or restenosis, of treated arteries in 30-50% of coronary angioplasties within 6 mo and in ~20% of bypass procedures within 2 yr after treatment (4, 25). Apart from intravascular stents and anticoagulation, which appear effective in limiting restenosis in the short term (8), no other interventions have been successful in halting the development of IH.

Recent evidence indicates that a somatostatin (SS) analog, angiopeptin (BIM-23014), is effective in inhibiting IH after arterial injury in animal models (5, 11, 12, 16, 19, 21, 23, 26, 33). Angiopeptin inhibits the release of insulin-like growth factor-1 and bFGF from endothelial cells (15), thus preventing SMC proliferation and migration. Clinical trials using angiopeptin to inhibit IH-causing restenosis, however, have been inconclusive (9, 10, 18).

SS is a neuroendocrine peptide that exerts a wide range of physiological actions that reflect both its widespread distribution and the existence of several receptor subtypes. There are five known SS receptors, SSTR-1 through -5 (6, 17), that can be divided into two subgroups through sequence similarity and affinity for SS analogs (6). The first subgroup of receptors, including SSTR-2, -3, and -5, has a high affinity for SS-14, SS-28, and SS analogs such as angiopeptin. The second subgroup, including SSTR-1 and -4, has a high affinity for SS-14 but a lower affinity for the majority of available SS analogs. All five receptors are coupled to G proteins and affect a number of distinct signal transduction pathways (11, 27).

The data presented in the present study suggest that the underlying reason for the lack of effect of angiopeptin in humans is the SSTR subtypes expressed by human blood vessels. Angiopeptin has a high affinity for SSTR-2, -3, and -5, and although these are expressed in animal blood vessels, SSTR-1 and -4 are the predominant subtypes expressed in human blood vessels.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The transformed human endothelial cell line ECV304 was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in medium 199 (Sigma Chemical, St. Louis, MO) supplemented with 2 mM glutamine, 24 mM sodium bicarbonate, 10 mM HEPES, 10 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated FCS. The primary cell preparations, human umbilical vein endothelial cells (HUVEC) and aortic smooth muscle cells (AoSMC), were obtained from Clonetics (San Diego, CA) and cultured in the appropriate medium from Clonetics, supplemented with 10% FCS. Cells were grown in 75-cm2 Falcon flasks (Becton Dickinson Labware, Franklin Lakes, NJ) and incubated at 37°C in 5% carbon dioxide in humidified air. For immunocytochemical studies, the cell lines were seeded onto 3-aminopropyltriethoxysilane (APES; Sigma)-coated coverslips placed into 24-well Costar tissue culture dishes (Corning, Corning, NY).

Human artery and vein samples (n = 14) of 100-400 mg were collected from the operating room following bypass procedures or amputations or from human donors for organ transplantation in association with the Pacific Organ Retrieval and Transplant Society with ethical permission from the Ethical Committee on Human Experimentation at the University of British Columbia. The nine normal samples included three aortas (2 from females 52 and 53 yr of age and 1 from a male 40 yr of age), three mammary arteries (from males between 60 and 70 yr of age), and three greater saphenous veins (from males between 50 and 75 yr of age). The five arteriosclerotic popliteal arteries were from males 65-75 yr of age. Confirmation that the collected popliteal arteries were atherosclerotic was obtained from the Pathology Department at the University of British Columbia, which received the major portion of the abnormal tissues. After removal, the tissue samples were immediately frozen in liquid nitrogen and stored at -80°C.

RNA isolation and cDNA synthesis. Frozen tissues were weighed and then ground to a powder with the use of a mortar and pestle under liquid nitrogen. TRIzol solution (1 ml/ 100 mg; GIBCO Life Technologies, Grand Island, NY) was added to the ground tissue to lyse the cells. Total RNA was then isolated according to the manufacturer's directions. The RNA was repurified by performing a phenol-chloroform extraction, and the RNA was precipitated. Confluent endothelial cell cultures were treated with trypsin (0.25%; GIBCO) until a cell suspension was obtained; after centrifugation, the cell pellet was lysed in 1 ml of TRIzol solution and total RNA was isolated as described above.

The cDNA synthesis was performed in first-strand buffer [25 mM Tris · HCl (pH 8.3), 37.5 mM KCl, 1.5 mM MgCl2, and 10 mM dithiothreitol containing 1 mM dNTP (Pharmacia Biotech, Uppsala, Sweden), 10 units of RNasin (Pharmacia), and 2 units of DNase (Promega, Madison, WI)] and heated to 37°C for 30 min to degrade any contaminating genomic DNA. Heating the sample to 75°C for 5 min inactivated the DNase. A sample of this reaction was removed and used as template in a subsequent PCR reaction to verify the absence of genomic DNA. SuperScript II reverse transcriptase (100 units, M-MLV; GIBCO), and then 0.5 µg of oligo-dT primer (GIBCO) was added and the samples were incubated at 42°C for 1 h. Heating the samples to 75°C for 15 min inactivated the enzyme. cDNA samples were then stored at -20°C.

Detection of SSTR subtypes expressed in blood vessels and endothelial cell lines by PCR. Oligonucleotide primers were synthesized on an Applied Biosystems model 391 DNA synthesizer (see Table 1 for details). SSTR-1 through -5 primer pairs were designed to hybridize to unique regions of the receptors. The PCR reactions for SSTR-1 through -5 were carried out using 2 µl of cDNA in a 25-µl total volume of PCR buffer [67 mM Tris (pH 9.01), 1.5 mM MgSO4, 166 mM ammonium sulfate, and 10 mM beta -mercaptoethanol] containing 1 mM MgCl2 (5 mM MgCl2 for SSTR-5), 0.2 mM dNTP (Pharmacia), 5% DMSO (SSTR-5 only), and 100 ng of 5' and 3' primer. A negative control (water instead of template) and a positive control (human genomic DNA) were included in each PCR run for each primer pair. Taq polymerase (1.25 units; GIBCO) was added, and the samples were overlaid with mineral oil to prevent evaporation.

                              
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  		Table 1.   Human SSTR primers

The amplification reaction was carried out in a RoboCycler Gradient 96 (Stratagene, La Jolla, CA) for 35 cycles. Each cycle consisted of denaturation for 45 s at 94°C, annealing for 45 s at the relevant temperature (see Table 1), and an extension for 45 s at 72°C. A final extension step at 72°C for 5 min terminated the amplification. The PCR products were separated by electrophoresis through a 1% agarose gel. The DNA was visualized and photographed using the Eagle Eye II Video System (Stratagene). The DNA fragments obtained with the use of primers for SSTR-1, -2, and -5 were isolated from the gels and ligated into pGEM-T (Stratagene). DNA sequencing of the subclone was performed using the dideoxynucloetide chain-termination procedure with T7 sequenase (Pharmacia). The DNA fragments obtained with the use of primers for SSTR-3 and -4 were electroluted from the agarose gel, and diagnostic restriction digest analysis performed to confirm that the PCR products were SSTR-3 and -4.

Detection of von Willebrand factor in endothelial cells. Oligonucleotide primers with the sequence 5'-CCCACCCTTTGATGAACACA-3' for the forward primer and 5'-CCTCACTTGCTGCACTTCCT-3' for the reverse primer were used in a PCR reaction to detect von Willebrand factor (vWF) cDNA in human artery samples. The PCR reaction was performed in PCR buffer [20 mM Tris · HCl (pH 8.4) and 50 mM KCl] containing 2.0 mM MgCl2, 0.2 mM dNTP (Pharmacia), 5% DMSO, and 100 ng of 5' and 3' primer. A negative control (water instead of template) and a positive control (vWF PCR product) were included in each PCR run. Taq polymerase (1.25 U; GIBCO) was added, and the samples were overlaid with mineral oil to prevent evaporation. The 35 PCR cycles were performed as described in Detection of SSTR subtypes expressed in blood vessels and endothelial cell lines by PCR, with an annealing temperature of 60°C. The PCR product was separated and visualized as described earlier. The DNA fragment was isolated from the gel, and sequence analysis confirmed that the PCR product was vWF.

Immunocytochemistry. A small section of blood vessel from each sample was fixed in 4% paraformaldehyde, placed on a piece of cork, covered with optimum cutting temperature compound (Fisher Scientific, Fair Lawn, NJ), and frozen in liquid nitrogen-chilled isopentane (approximately -60°C). Cryosections (10 µm) were mounted on glass slides and stored overnight at -20°C. Cryosections were warmed to 37°C for a few hours and then incubated in PBS with 50 mM ammonium chloride and 50 mM glycine for 1 h to neutralize excess aldehydes. Cultured cells were fixed in 4% paraformaldehyde for 5 min at room temperature and then washed in PBS. All antisera were diluted in PBS containing 5% heat-inactivated horse serum. When whole cells were to be immunostained, 0.1% Triton X-100 (Sigma) was added to the diluent. A rabbit antiserum to vWF (1:1,000 dilution; Sigma) was overlaid on sections or whole cells on coverslips and incubated at 4°C overnight. Rabbit antisera to human SSTR-1 and SSTR-2 (a kind gift from Dr. J. H. Walsh, CURE/Gastroenteric Biology Center Antibody/RIA Core, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41301) were diluted to 1:100, and sections or cells were incubated with the antibody for 48 h at 4°C. The sections (or cells) were washed in PBS and the bound antibodies localized using Cy3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:1,000 for 1 h at room temperature. Slides were screened using a Zeiss Axiophot microscope equipped with epifluorescence. Representative sections were digitized using a Bio-Rad MRC 600 confocal laser scanning microscope equipped with a krypton-argon laser. The resultant images were converted using NIH Image software and processed using Adobe Photoshop software.

Effect of SS-14 on endothelial cell structure. To determine whether SS or a peptide agonist of SSTR-1 affected endothelial cell function, we investigated the effect of the peptide on the actin cytoskeleton in the two cell preparations, HUVEC and ECV304. The cells were cultured on APES-coated coverslips for 48 h as previously described. Before peptides were added, the cells were washed to remove growth medium, fresh medium (lacking serum) was then added (1 ml/well), and the cells were cooled to 4°C for 30 min. After cells were cooled, 25 µl of medium containing either 400 nM SS-14 (Peninsula Laboratories, Belmont, CA) or 400 nM SSTR-1-specific agonist (a kind gift from Dr. J. Rivier, Salk Institute; Ref. 20), or 25 µl of control medium were added to the wells, and the cells were returned to the 37°C incubator for 30 min. Fixation was then carried out in 4% paraformaldehyde for 5 min, followed by three PBS washes. The actin cytoskeleton was visualized by incubating the cells with ALEXA-488-conjugated phalloidin (1:50; Molecular Probes) for 15 min at room temperature. Cells were screened using a Zeiss Axiophot microscope equipped with epifluorescence, and the final images were collected using the confocal microscope as previously described.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SSTR expression in normal vessels. Normal aorta (n = 3) expressed high levels of SSTR-1 with lower levels of SSTR-2 and -4 (Fig. 1A). However, there was no evidence of mRNA for SSTR-3 or -5, although PCR products were obtained with the use of human genomic DNA as a control (Fig. 1A). Samples from the internal mammary artery (n = 3) and greater saphenous vein (n = 3) showed a similar pattern of receptor expression, with SSTR-1, -2, and -4 but not SSTR-3 and -5 being detected (Fig. 1B).


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  		Fig. 1.   PCR analysis of somatostatin receptor (SSTR) expression in human arteries. A: gel shows results of PCR reactions to establish SSTR subtype expression in human aorta. Note that although SSTR-3 and -5 products were amplified from genomic DNA, no product was detected in vascular samples. Ladder is 100 bp. Ao, aorta; G, genomic DNA; vWF, von Willebrand factor. B: gel shows PCR products obtained after amplification of mRNA isolated from internal mammary artery (MA) or greater saphenous vein (GSV). Note presence of SSTR-1, -2, and -4 and absence of SSTR-3 and -5. C: comparison of levels of SSTR-1 and -2 expressed in internal mammary arteries from different patients (MA1-MA3). Internal control was vWF mRNA. Note variability of SSTR-1 levels but not vWF among samples. Also note that SSTR-2 mRNA was absent from MA3 sample.

A comparison of the levels of expression of SSTR-1 and -2 compared with vWF in three internal mammary artery specimens demonstrated constant levels of vWF, whereas the levels of the two receptors varied between samples (Fig. 1C). In all cases, the levels of SSTR-1 were greater than those of SSTR-2, and in one preparation SSTR-2 was absent even though both SSTR-1 and vWF were detected.

SSTR expression in diseased vessels. In arteriosclerotic popliteal arteries (n = 5), SSTR-1, -2, and -4 were expressed with no evidence of SSTR-3 or -5 (Fig. 2A). A comparison of SSTR levels among samples from the five patients available indicated changes in receptor expression but not in that of the internal control, vWF. The presence of vWF mRNA also confirmed the continued presence of endothelial cells in the injured arteries (Fig. 2B). The level of SSTR-1 was consistently higher than that of SSTR-2 or -4 in these samples.


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  		Fig. 2.   A: PCR analysis of SSTRs expressed by an atherosclerotic artery. SSTR-1 and -2 were predominant receptors, with a lower level of SSTR-4. Scl, sclerotic artery sample. B: comparison of PCR products obtained for SSTR-1, -2, -4 and vWF in 2 atherosclerotic artery samples. Note that level of expression of vWF did not vary, whereas that of SSTRs differed significantly.

In all samples, PCR reactions were performed on the same day using the same batch of cDNA from the respective vessels. Thus differences in SSTR expression were not due to differences in cDNA or PCR efficiency. vWF was used as the internal control rather than actin because the former is present only on endothelial cells.

SSTR expression in cultured cells. The ECV304 cells and primary HUVEC were analyzed to determine SSTR subtype expression of endothelial cells. Both ECV304 and early passages (passage 3) of HUVEC expressed high levels of SSTR-1 with low levels of SSTR-4 (Fig. 3, A and B). The remaining SSTR subtypes were undetectable. Interestingly, in later passages (passage 15) of HUVEC, low levels of SSTR-2 were detected (data not shown).


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  		Fig. 3.   PCR analysis of SSTR and vWF expression in ECV304 cells and human umbilical vein endothelial cells (HUVEC). A: ECV304 cells expressed SSTR-1 and -4 as well as vWF, but not the other SSTRs. B: HUVEC showed same expression pattern as ECV304 cells. C: aortic smooth muscle cells (AoSMC) expressed SSTR-1 and -2 but also VWF, indicating that this was a mixed cell population. EC, ECV304; Hv, HUVEC; Ao, AoSMC.

These data suggested that SSTR-1 and -4 mRNA detected in the samples of arteries and veins originated, at least in part, from the endothelial cells lining the vessels. The origin of the low levels of SSTR-2 mRNA was considered to be nonendothelial. To determine whether arterial smooth muscle cells express SSTR-2, a primary AoSMC preparation was investigated. These cells expressed SSTR-1 with low levels of SSTR-2 (Fig. 3C). None of the other SSTR subtypes were detected. Screening these cells with primers to vWF demonstrated low levels of mRNA, indicating that the AoSMC preparation was not pure and suggesting that the SSTR-1 mRNA may have been endothelial in origin.

Immunocytochemistry. Endothelial cells in blood vessels from normal and arteriosclerotic tissues were positive for SSTR-1 immunoreactivity (IR; Fig. 4, A and B). However, the vascular smooth muscle cells from both normal and arteriosclerotic arteries showed no IR. In addition, no immunostaining of vascular tissue was obtained with an SSTR-2-specific antibody, although the antibody detected SSTR-2 in sections of human antrum fixed using identical conditions (Fig. 4C), indicating that this was not due to the inability of the antibody to detect the protein. vWF-IR was limited to endothelial cells in normal and arteriosclerotic vessels, as expected (Fig. 4D).


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  		Fig. 4.   Immunocytochemical staining of a cross section of a human artery. A: SSTR-1 immunoreactivity (IR) in a normal artery. Staining is restricted to cell surface of endothelial cells (arrows). B: SSTR-1-IR in a sclerotic artery. Only endothelial cells are positive for SSTR-1-IR (arrow). C: SSTR-2-IR gastrin cells in a section of gastric antrum showing that the antibody was capable of detecting native protein (arrows). D: vWF-IR endothelial cells in an atherosclerotic artery confirming presence of endothelial cells (arrow). Bar, 10 µm.

ECV304 cells and HUVEC were immunostained for both SSTR-1 and vWF. The ECV304 cells showed SSTR-1-IR in both the cytoplasm and on the plasma membrane (Fig. 5A). The SSTR-1-IR at the periphery was clustered into regions resembling lamellipodia. Both the ECV304 and HUVEC showed vWF-IR; however, the percentage of positive cells varied among the different cell samples. All ECV304 cells (100%) were immunoreactive for vWF (Fig. 5B). However, the earlier passage of HUVEC contained 95% vWF-IR cells compared with ~50% vWF-IR in a later passage (Fig. 5C). A small number (<10%) of cells in the AoSMC culture were vWF-IR, again indicating that this was not a homogeneous cell preparation.


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  		Fig. 5.   Immunocytochemical staining of ECV304 and HUVEC. A: SSTR-1-IR in ECV304 cells was located in perinuclear region (small arrows) and on the plasma membrane (large arrows). B: vWF-IR in ECV304 cells stained typical Weibel-Palade bodies (arrows), indicating presence of mature endothelial cells. C: vWF-IR in a later passage of HUVEC. Note that not all cells are positively stained; arrows indicate cells that do not contain vWF-IR.

Effect of SS on endothelial cell function. In control HUVEC and ECV304 cells, abundant actin stress fibers stretching the entire length of the cell with low levels of cortical actin and few lamellipodia were seen (Fig. 6A). In both cell preparations, SS-14 and SSTR-1 analog-treated cells consistently showed a significant decrease in long stress fibers, and the remaining fibers were short and lacked a directional organization (Fig. 6, B and C). In addition to the changes to the stress fibers, cells exposed to both peptides demonstrated a significant increase in cortical actin and in the number and size of lamellipodia at the plasma membrane (Fig. 6, B and C).


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  		Fig. 6.   Phalloidin-labeled ECV304 cells. A: control ECV304 cells with abundant stress fibers stretching entire length of cell (arrows). B: ECV304 cells treated with 10 nM SS-14 for 30 min at 37°C. Note loss of long stress fibers. Remaining fibers are short and disorganized (small arrows), and there was an increase in number and size of lamellipodia at plasma membrane (large arrows). C: ECV304 cells treated with 10 nM SSTR-1 agonist. Note loss of stress fibers (small arrows) and presence of lamellipodia (large arrows).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiopeptin, an SS analog that preferentially binds to SSTR-2, -3, and -5, has been used to prevent the development of IH in animal models (5, 12, 16, 19, 23, 33). In a previous study from this laboratory, Chen et al. (2) showed that rat arteries express SSTR-2 and -3 subtypes. The data indicated that the ability of angiopeptin to inhibit the development of IH was due to activation of one or both of these receptors (2). However, other studies comparing the effect of SS on vascular reactivity demonstrated species-specific responses (8). This suggests that the lack of clinical efficacy of angiopeptin to inhibit restenosis could be due to the expression of different SSTR subtypes. To investigate this possibility, we designed experiments to determine the expression and localization of SSTRs in human vascular tissues.

These studies demonstrated differential expression of SSTR subtypes in human blood vessels. In normal and arteriosclerotic vessels, mRNA encoding SSTR-1 was invariably detected, whereas the presence of SSTR-2 and -4 mRNA was more variable. There was a complete absence of mRNA encoding SSTR-3 or -5. These data indicate that of the three receptor subtypes that bind angiopeptin with a high affinity, only one, SSTR-2, was expressed in the normal samples. The normal blood vessels were collected from females and males ranging in age from 40 to 75 yr, and therefore this pattern of expression appears constant in adults.

A comparison of expression levels of SSTR-1 and -2 from normal mammary arteries and saphenous veins consistently showed that SSTR-1 was the more abundant SSTR, although the amount detected varied among individuals. The intensity of the SSTR-1 bands was compared with those of the internal control vWF. We chose to use vWF rather than a housekeeping gene such as 18S ribosomal RNA or actin because we have shown that SSTR-1 expression is limited to the endothelial cells in the blood vessels. The general cell markers would reflect all the nonendothelial cells present in the tissue samples that would be expected to vary more between individuals.

These data indicate that the predominant SSTR subtypes expressed by human vessels belong to the second group of SSTRs, i.e., SSTR-1 and -4. Less information is available concerning the function of SSTR-1 and -4 compared with SSTR-2, -3, and -5 because of the lack of subtype-specific analogs for these receptors. The majority of available SS analogs interact with SSTR-2, -3, and -5 (7, 27). Clinically, the most important of these analogs (such as octreotide) are used to treat a variety of endocrine tumors. Angiopeptin, a second analog activating the SSTR-2 group of receptors, has produced disappointing results in clinical trials that evaluated the ability of the analog to prevent development of IH (9, 10, 18). Our data suggest that this failure may be due to the expression of SSTR-1 and not SSTR-2 on human endothelial cells.

A comparison of the ligand selectivity of human SSTRs expressed in cell lines showed that whereas the half-maximal inhibitory concentration of SS-14 at SSTR-1, -2, and -4 was comparable at 1 nM, angiopeptin selectively activates SSTR-2 (1 nM) and SSTR-5 (5 nM) but has a low affinity for both SSTR-1 and -4 (1 µM) (28). These results indicate that 1,000-fold higher concentrations of angiopeptin would be required to activate SSTR-1 or -4 on the endothelial cells compared with the native agonist. The use of high levels of angiopeptin would not be advisable in view of the known side effects including diarrhea and abdominal pain.

The pattern of SSTR subtype expression in arteriosclerotic vessels did not differ significantly from that found in normal vascular tissue. Because the arteriosclerotic samples were obtained from individuals with chronic disease, we were unable to determine, in this study, whether SSTR expression would be altered during the acute phase of vascular injury, such as following angioplasty. In a rodent model of vascular injury, Chen et al. (2) showed that SSTR-2 was upregulated. However, by 2 mo after the initial injury, the levels of SSTR-2 were declining (S. B. Curtis and A. M. J. Buchan, unpublished data). It is probable that SSTR expression in human blood vessels will respond to intimal injury, but to demonstrate such a response would require availability of acutely injured samples.

Immunocytochemistry was used to localize cells expressing SSTR-1 and -2 in the vessel wall. The results indicated that SSTR-1, but not SSTR-2, was predominantly expressed on the surface of endothelial cells, a finding similar to previous observations (2) in rat arteries that SSTR receptor expression was limited to the endothelial cells. The localization of SSTR-1 to endothelial cells is consistent with the finding that removal of endothelial cells abolished the vascular effects of SS (24, 32). In the case of SSTR-2 it is probable that, given the low levels of mRNA amplified by RT-PCR, the resultant protein was below the level of detection of immunocytochemistry. The antibody used, however, was capable of detecting the native protein in other cell types such as the gastrin cells of the gastric antrum.

To determine whether human endothelial cells expressed SSTR-2, we examined SSTR expression by both a spontaneously transformed endothelial cell line (ECV304) and a primary cell preparation derived from human umbilical veins (HUVEC). In both cell preparations SSTR-1 and -4 mRNA were identified, but not SSTR-2, -3 and -5 mRNA. Interestingly, late passages of HUVEC (passage 15) did express low levels of SSTR-2; however, these late passage cells cannot be considered a model for normal endothelial cell type. It is unclear whether expression of SSTR-2 in the late passage cells represented a culture artifact, dedifferentiation of the endothelial cells, or the presence of a low level of contaminating cells.

Immunostaining of the ECV304 and HUVEC with an antibody to vWF demonstrated that 100% of ECV304 and 95% of HUVEC in early passages were positively stained. The pattern of intracellular vWF-IR was characteristic of Weibel-Palade bodies, indicating that the ECV304 cells represent a mature endothelial cell population. In later passages of the HUVEC only 50-60% of the cells were positive. These data support the suggestion that either the HUVEC preparation contains nonendothelial cells or that the cells dedifferentiate with time in culture.

The consistent finding of SSTR-1 expression on the endothelial cells led to experiments designed to determine whether there was a functional alteration in endothelial cells after SS treatment. These experiments used the well-established methodology of phalloidin-labeled actin filaments to monitor changes in the cytoskeleton of the ECV304 and HUVEC. The rationale underlying these experiments was that in cell lines transfected with SSTR-1, activation of the receptors caused both a decrease in intracellular cAMP and an inhibition of the sodium-hydrogen exchanger 1 (NHE-1), leading to intracellular acidification (1). Furthermore, because NHE-1 activity modulates the actin cytoskeleton (31), we predicted that activation of SSTR-1 on the ECV304 cells would result in changes to the actin cytoskeleton. We could not determine the result of selective activation of SSTR-4 on endothelial cells because of the lack of a specific agonist of this receptor subtype.

The results of the phalloidin-labeling experiments indicated that SS-14 and an SSTR-1 agonist caused disassembly of actin stress fibers and the production of lamellipodia. These effects would be consistent with the activation of the small GTPase Rac, previously shown to increase cortical actin and stimulate the formation of lamellipodia in endothelial cells (34). Interestingly, activation of SSTR-1 resulted in a reduction in the number of stress fibers, indicating that although Rac was activated, the downstream kinase Rho was inhibited. Inhibition of Rho in HUVEC has previously been shown to result in the loss of stress fibers and an increase in cortical actin (34). The precise mechanism underlying this action of SS has yet to be determined.

In conclusion, our data indicate that SSTR-1 is the predominant SS receptor subtype expressed in normal and atherosclerotic human blood vessels and cultured human endothelial cells. Endothelial cells and arteries expressed low levels of SSTR-4 mRNA, whereas low levels of SSTR-2 mRNA were expressed in intact blood vessels but not in endothelial cells. Activation of SSTR-1 receptors on ECV304 and HUVEC resulted in significant changes to the actin cytoskeleton. These data indicate that analogs capable of activating either SSTR-1 or -4 should be investigated in the treatment of vascular diseases such as IH in humans.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Heart and Stroke Foundation of British Columbia and the Yukon.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. M. J. Buchan, Dept. of Physiology, Univ. of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail: buchan{at}cs.ubc.ca).

Received 8 September 1999; accepted in final form 7 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 278(6):H1815-H1822
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