Action of Tropoelastin and Synthetic Elastin Sequences on Vascular Tone and on Free Ca²⁺ Level in Human Vascular Endothelial Cells

From the Groupe d'Electrophysiologie Moléculaire (G.F., S.G., J.V.), Laboratoire de Bioénergétique Fondamentale et Appliquée, Université Joseph Fourier, Grenoble, France; the Department of Biochemistry (A.S.W.), University of Sydney, New South Wales, Australia; the Laboratoire de Biochimie Analytique et Synthèse Bioorganique (J.W.), Université Claude Bernard, Villeurbanne, France; the Centre de Recherche en Gérontologie et Gériatrie (T.F.), Hopital d'Youville, Université de Sherbrooke, Québec, Canada; INSERM U460 (M.-P.J.), Paris, France; the Department of Cell Biology and Physiology (R.P.M.), Washington University School of Medicine, St Louis, Mo; and the Laboratoire de Biologie du Vieillissement (L.R.), CHU Pitié et Recherche Ophtalmologique, Hotel-Dieu, Université Paris 6, Paris, France.

Correspondence to Gilles P. Faury, PhD, Department of Cell Biology and Physiology, Washington University, School of Medicine, Campus Box 8228, 660 S Euclid Ave, St Louis, MO 63110. E-mail gfaury{at}cellbio.wustl.edu

	Abstract

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Abstract—The elastic properties of extensible tissues such as arteries and skin are mainly due to the presence of elastic fibers whose major component is the extracellular matrix protein elastin. Pathophysiological degradation of this protein leads to the generation of elastin peptides that have been identified in the circulation in the ng/mL to µg/mL range. Similar concentrations of an elastin peptide preparation (-elastin) were previously demonstrated to induce, among other biological actions, a dose- and endothelium-dependent vasorelaxation mediated by the elastin/laminin receptor and by endothelial NO production. To determine the elastin sequence(s) responsible for vasomotor activity and to learn more about possible signaling pathways, we have compared the action of different concentrations (10^-13 to 10^-7 mol/L) of recombinant human tropoelastin, eight synthetic elastin peptides, and a control peptide (VPVGGA) on both rat aortic ring tension and [Ca²⁺]_i of cultured human umbilical vein endothelial cells. No vasoactivity could be detected for VPVGGA and for the elastin-related sequences VGVGVA, PGVGVA, and GVGVA. Tropoelastin, VGV, PGV, and VGVAPG were found to induce an endothelium- and dose-dependent vasorelaxation and to increase endothelial [Ca²⁺]_i, whereas PVGV and VGVA produced these effects only at low concentration (10^-11 mol/L). A likely candidate for mediating the elastin peptide–related effects is the elastin/laminin receptor, since the presence of lactose strongly inhibited the vasoactivity associated with these compounds. Our results show that although the flanking amino acids modulate its activity, VGV seems to be the core sequence recognized by the elastin receptor.

Key Words: elastin • elastin/laminin receptor • vascular tone • endothelial cell • [Ca²⁺]_i

	Introduction

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Elastin, the extracellular matrix protein responsible for the major part of tissue elasticity in vertebrates, functions as an insoluble component of elastic fibers in many extensible organs, such as skin, lung, and arteries.^{1 2} The alteration of this protein, mediated by elastase-type enzymes, leads to severe pathologies, such as emphysema and arteriosclerosis, and diverse skin diseases.^{3 4 5 6} As a consequence of either physiological (such as aging) or pathological elastin degradation, soluble elastin peptides are released into the surrounding extracellular space and have been detected in the circulation at a concentration range of 10^-6 to 10^-2 mg/mL.^{6 7}

Numerous studies have demonstrated that elastin peptides have diverse biological properties. In particular, elastin peptides have been shown to stimulate fibroblast adhesion to elastin fibers,⁸ to regulate cellular proliferation,⁹ and to be chemotactic for monocytes¹⁰ and (similar to tropoelastin) fibroblasts.^{11 12 13} Moreover, -elastin, a mixture of peptides resulting from the degradation of elastin in KOH, has recently been shown to induce dilation in aortic rings from adult rats but not from younger and older animals.^{14 15 16} These peptides have also been shown to trigger intracellular events, such as an increase in both Ca²⁺ influx and [Ca²⁺]_i in leukocytes^{17 18 19} and in endothelial cells (authors' unpublished data, 1997). The effects of tropoelastin and elastin peptides are mediated by their binding to a multifunctional high-affinity receptor,^{20 21 22 23} which, on most cells, including endothelial cells,^{24 25 26} is a 67- to 69-kD peripheral membrane protein also able to bind laminin.^{21 22 23 24 25 26} One exception, however, is a 59-kD elastin binding protein in Lewis lung carcinoma cells.²⁷ The 67-kD receptor has been shown to also bind lactose-type carbohydrates to a lectin binding site, which has the effect of inducing both the release of elastin from its binding site and the release of the receptor itself from the cell membrane.²¹

Several biologically active sequences in elastin have been identified. In early studies, the repetitive sequence VGVAPG, present in bovine and human tropoelastin, was found to be chemotactic for both monocytes and fibroblasts.²⁸ This same peptide and related sequences GFGVGAGVP and GLGVGAGVP were shown to be chemoattractants for aortic endothelial cells.²⁹ Other repeated elastin sequences were also found to be biologically active, including AGVPGFGVG and GFGVGAGVP, which are chemotactic for fibroblasts,³⁰ and (VPGVG)n, which induces calcification in the rat.³¹ Furthermore, recent results indicate that the elastin peptide VGVAPG and five related shorter or frame-shifted elastin sequences (VGV, VGVA, GVGVA, PGVGVA, and VGVGVA) act in leukocytes on intracellular free Ca²⁺ metabolism, elastase release, released myeloperoxidase activity, or hydrogen peroxide or superoxide anion production.³²

We have previously demonstrated that -elastin induces a dose- and endothelium-dependent vasorelaxation on NA-precontracted rat aortic rings, mediated by the elastin/laminin receptor and involving both NO synthase and cyclooxygenase pathways.^{14 15} To determine the active sequence(s) of elastin on vasomotor activity, we have investigated the action of eight synthetic elastin (VGV, PGV, VGVA, GVGVA, PGVGVA, and VGVAPG) and related (PVGV and VGVGVA) peptides on NA-contracted aortic rings and on [Ca²⁺]_i of cultured HUVECs. The activity of these peptides was also compared with the activity of rTE. The peptide VPVGGA, a rearrangement of the amino acids in the active peptide VGVAPG, served as a control. Our results show that rTE and several elastin-related peptides induce both endothelium-dependent vasorelaxation and an increase in endothelial [Ca²⁺]_i. The core sequence of this activity seems to be VGV, whose effect is modulated by the flanking amino acids.

	Materials and Methods

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Animals
Four- to 6-month-old female Wistar rats were obtained from Iffa-Credo (L'Arbresle, France). All animals were fed with Extralabo rat chow (Pietrement). Twenty animals were used. The housing and surgery procedures were in accordance with institutional guidelines.

Preparation of Aortic Ring Segments and Perfusion Conditions
Rats were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg). The descending thoracic aorta was excised and placed in an oxygenated (O₂ 95%/CO₂ 5%) Krebs' bicarbonate buffer containing (mmol/L) NaCl 118, KCl 5.6, CaCl₂ 2.4, MgCl₂ 1.2, KH₂PO₄ 1.2, NaHCO₃ 20, and D-glucose 11 (pH 7.4). The tissue was cleaned of adhering connective tissue and fat and then cut into 1- to 2-mm transverse segments. Aortic rings were placed horizontally between a stationary stainless steel hook and an isometric force transducer (Biologic) connected to a microcomputer. A peristaltic pump continuously delivered the medium (1.5 mL/min, 37°C) to the organ bath (0.9 mL). A passive tension of 1.5 g was applied for 60 minutes and was then readjusted back to 1.5 g for 15 minutes before starting the experiment. The aortic rings were precontracted and perfused until the end of the experiments with KbNA, which induced 80% of maximal tension developed by NA. The endothelium integrity was tested by adding 10^-6 mol/L acetylcholine to KbNA before recontracting the rings with KbNA alone. In some rings, the endothelium had been removed by gentle rubbing of the intimal surface with a section of polyethylene catheter. Selective removal of the endothelium was assessed by determining the absence of vasorelaxant effect of acetylcholine (10^-6 mol/L) but responsiveness to NA (10^-6 mol/L). The rings were then perfused with KbNA alone for 105 minutes (control) or with increasing concentrations of the test substances (10^-13 to 10^-7 mol/L) for 15 minutes per dose in the absence or presence of lactose (10^-4 mol/L). In this last instance, lactose was applied 15 minutes before and during the application of the elastin-related compounds.

Vasomotor Data Analysis
Results are expressed as mean±SEM of aortic ring tension (mg) after a 15-minute application of each agent concentration. Initial tensions were normalized to an average value of NA-induced precontraction (1120 mg). Statistical analysis was performed using a two-way ANOVA. In order to perform comparisons between results in the presence and in the absence of lactose, control ring tensions (with or without lactose) were normalized to 1120 mg, and the same normalization factors were then applied to ring tensions in the presence of the tested substances as described previously.¹⁶ The statistical tests were completed by Fisher's least significant difference test for paired value comparisons. Differences were considered significant at P<.05.

HUVEC Isolation and Culture
HUVECs were obtained as previously described³³ and were maintained, at 37°C and humid atmosphere, in fibronectin-coated dishes³⁴ with medium 199 supplemented with Earle's salts and 22% human serum. Trypsinized cells were plated on dishes (containing 13x14-mm glass coverslips) coated with fibronectin so that the cell density was in the range of 10 500 cells/cm.² Only first to fourth passage subconfluent cells were used in the experiments. The purity of the endothelial cell cultures was established using an antibody to von Willebrand factor.

Loading of HUVECs With Fura 2
Fura 2-AM loading was performed generally according to the procedure of Thomas and Delaville.³⁵ The culture dish was washed twice with a Tyrode's solution containing (mmol/L) NaCl 118, KCl 5.6, CaCl₂ 2.4, MgCl₂ 1.2, HEPES 10, and D-glucose 11 (pH 7.4). The dish was then filled with 0.75 mL Tyrode's solution. In the dark, 8 µL of 0.5 mmol/L fura 2-AM in DMSO was added to 250 µL of Tyrode's solution containing 2% BSA and 0.08% pluronic F-127 (wt/vol). This solution (258 µL) was then added to the culture dish (final volume, 1 mL), yielding final concentrations of 4 µmol/L fura 2-AM and <1% DMSO. After a 40-minute incubation (room temperature, in the dark), the cells were washed twice and bathed with Tyrode's solution (1 mL) for 10 minutes.

[Ca²⁺]_i Measurements
Fluorescence measurements were as described previously.^{36 37} Under low light, the HUVEC-containing glass coverslip was removed from the culture dish and fitted vertically and diagonally in a Tyrode's solution–filled quartz cuvette (10x10 mm) with a plastic ring at the bottom that enclosed a small stir bar that mixed the solution. The coverslip was fitted so that the cells faced the dual excitation light (340 and 380 nm), and the emitted fluorescence (510 nm) was read at 90° from behind the coverslip. The cuvette was then placed at 30°C in a Deltascan 4000 spectrofluorometer (Photon Technology International), and measurements were performed after a minimum of 5 minutes and before 60 minutes. The recorded signal resulted from the integrated fluorescence from all the cells facing the excitation path. [Ca²⁺]_i was monitored for 4 minutes after the addition of elastin-related peptides to the cuvette. For investigating the inhibitory effect of lactose, fluorescence was monitored for 2 minutes in the presence of lactose (10^-4 mol/L) alone, before the addition of the test compounds. Control experiments were performed by recording the HUVEC fluorescence alone for 5 minutes. Each measurement was stopped by the addition of 0.002% digitonin (maximum [Ca²⁺]_i), followed by 45 mmol/L EGTA (minimum [Ca²⁺]_i). After the autofluorescence was subtracted, [Ca²⁺]_i was calculated using standard techniques, assuming that the K_d of fura 2 for Ca²⁺ is 224 nmol/L.³⁸

[Ca²⁺]_i Measurement Data Analysis
For each measurement, initial [Ca²⁺]_i was normalized to an average measured value (50 nmol/L). Statistical analysis was performed using a one-way ANOVA for comparison of simple effects of each elastin-related compound or of lactose. A two-way ANOVA was used to assess statistical analysis of the effect of compounds in the presence or absence of lactose. When necessary, these tests were completed by the nonparametric Mann-Whitney U test for paired value comparisons. Differences were considered significant at P<.05.

Preparation of Synthetic Elastin Peptides and Control Peptide
All peptides were synthesized using 9-fluorenylmethoxycarbonyl chemistry. Purity of the peptides was confirmed by HPLC and by fast bombardment spectrometry. Purity and concentrations of the peptide stock solutions were verified by amino acid analysis.

Preparation of rTE
rTE encoded by a 2210-bp Escherichia coli–optimized synthetic gene was expressed and purified from bacterial extracts as described.³⁹ rTE molecular mass is 63490 kD.

Preparation of -Elastin
Elastin peptides (-elastin) were obtained by hydrolysis of highly purified bovine ligamentum nuchae elastin with 1 mol/L KOH in 80% (vol/vol) aqueous ethanol.⁴⁰ By adjusting the time of hydrolysis, peptides of different molecular mass can be obtained. Peptides used in these experiments had an average molecular mass of 75 kD.

Drugs and Materials
The following drugs and reagents were from Sigma: Krebs' bicarbonate and HEPES-buffered solution components, NA, acetylcholine, lactose, fura 2-AM, DMSO, BSA, pluronic F-127, collagenase 1A, medium 199 with Earle's salts, L-glutamine, and HEPES. Human serum was from the Centre de Transfusion Sanguine de Grenoble. Falcon cell culture dishes were from Becton-Dickinson. The 13x14-mm glass coverslips were from Vitromed.

	Results

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Vasorelaxant Activity of Elastin-Related Compounds
The vasoactivity of elastin-related peptides was found to be compound and concentration dependent on aortic rings with intact endothelium (Table 1, category A). Compared with the control, the peptides VGV, PGV, VGVAPG, and rTE induced dose-dependent vasorelaxation in the NA-precontracted rat aortic rings with maximum values of 32%, 21%, 26%, and 41%, respectively (Fig 1A). Nevertheless, for each concentration for which several peptides were found to be active, no significant difference in activity could be detected between these peptides, except at 10^-9 mol/L, where rTE was more active than VGV (Fig 1A). Moreover, the peptide PGV was slightly active (14% vasorelaxation) at 10^-12 mol/L (Fig 1A). The sequences VGVA and PVGV induced a relaxation of the aortic rings only at a concentration of 10^-11 mol/L, with values of 14% and 19%, respectively, and no activity difference could be detected between these two peptides (Fig 1B). Finally, the peptides PGVGVA, VGVGVA (for which the induced aortic ring tensions presented a slightly higher variance), and GVGVA as well as the control peptide VPVGGA produced tension variations of 12% maximum compared with the control, with all values not statistically significant (P0.1) (Fig 1C).

View larger version (22K): [in this window] [in a new window]   Figure 1. Concentration-response curves showing the effects of elastin-related compounds after a 15-minute exposure on rat aortic ring tone. The rings were precontracted and were then perfused for 105 minutes with 10-6 mol/L NA (control). In some rings, increasing doses of elastin peptides were also added to the perfusion. The peptides added were as follows: rTE, VGV, VGVAPG, and PGV (A); PVGV and VGVA (B); and VPVGGA, GVGVA, VGVGVA, and PGVGVA (C). Each point represents the mean of five to nine experiments. The results are expressed as tension (mg) (mean±SEM). *P<.05 vs control tension by least significant difference test. #P<.05 vs corresponding VGV-induced tension by least significant difference test. P<.065 (least significant difference test) and P<.05 (U test) vs corresponding control tension.

By contrast, on aortic rings without endothelium, the 10 compounds (rTE, VGV, PGV, VGVA, PVGV, GVGVA, VGVAPG, VGVGVA, PGVGVA, and VPVGGA) had no effect on the tone of NA-precontracted aortic rings for the two tested concentration ranges: 10^-11 mol/L and 10^-8 to 10^-7 mol/L, the active concentrations determined above (Table 2). All the tested compound-induced tension values were not different from the corresponding control tension values (Table 1, category B).

The effect of the elastin/laminin receptor antagonist "lactose" was then tested on the endothelium-dependent vaso-relaxant action of the active elastin-related sequences (VGV, PGV, VGVA, PVGV, VGVAPG, and rTE) for concentrations demonstrated to induce a submaximal vasorelaxation. VGV, VGVAPG, and rTE were used at 10^-8 mol/L. Since only one concentration was found to be active in the range of the maximal effect for PGV (10^-7 mol/L), VGVA, and PVGV (10^-11 mol/L), this concentration was used for these compounds. The direct effect of lactose (10^-4 mol/L) was first studied on the NA-precontracted rat aortic rings with endothelium. Lactose (10^-4 mol/L)–induced tension (mg) was identical to the control tension after 15 minutes but was lower than the control tension after 30 minutes and lower than the control tension and the initial lactose-induced tension after 45 minutes. At these times, the slight vasorelaxation induced by lactose was in the range of 5% compared with the control value (Fig 2 and Table 1, category C). After assessment of a second normalization of the tension values to balance the effect of lactose itself, the effect of lactose on the vasorelaxant effect induced by the active compounds (rTE, PGV, VGV, PVGV, VGVA, and VGVAPG) was investigated. As shown in Tables 1 (category D) and 3, the presence of lactose had a significant inhibitory effect on the elastin peptide–induced vasorelaxation, and the inhibition was independent of the active peptide used. These results indicate that lactose strongly inhibited the vaso-relaxant action of all the active elastin-related compounds.

View larger version (28K): [in this window] [in a new window]   Figure 2. Action of lactose (Lac, 10-4 mol/L) on aortic ring tone. The rings were precontracted with NA (10-6 mol/L), and then the tension was recorded in the presence or absence of Lac for 45 minutes. Each point represents the mean of 9 (absence of Lac) to 17 (presence of Lac) experiments. The results are expressed as tension (mg) (mean±SEM). *P<.05 vs corresponding control tension without Lac by least significant difference test. P<.05 vs Lac-induced tension after 15 minutes by least significant difference test.

Action of Elastin-Related Compounds on HUVEC [Ca²⁺]_i
To investigate the intracellular mechanisms explaining the vasorelaxant action of rTE and certain elastin-related sequences, we studied the effect of these compounds on [Ca²⁺]_i in adherent HUVECs loaded with the fluorescent dye fura 2. We first verified that the -elastin–induced [Ca²⁺]_i increase previously demonstrated on pooled suspended HUVECs loaded with indo 1 or on individual adherent HUVECs loaded with fluo 3 (authors' unpublished data, 1997) was also occurring in the fura 2–loaded adherent HUVECs stimulated with 10^-3 mg/mL (10^-8 mol/L) -elastin (Fig 3A). In these conditions, the -elastin–induced [Ca²⁺]_i increase exhibited slow kinetics and an amplitude (average raw increase, 2.06-fold, or 1.73 times the control value) similar to those previously observed in pooled suspended HUVECs loaded with indo 1 (average raw increase, 2-fold). The control peptide VPVGGA (10^-7 mol/L) had no effect on HUVEC [Ca²⁺]_i compared with cells with no peptide added (Fig 3B and 3C and Table 4). The effects of the elastin-related compounds on HUVEC [Ca²⁺]_i were then investigated at concentrations in the range of 10^-8 to 10^-7 mol/L for the peptides found to have no activity on the vascular tone and at active concentrations for the peptides found to induce a vasorelaxation. Only rTE (10^-9 to 10^-7 mol/L), VGV (10^-8 to 10^-7 mol/L), PGV (10^-8 to 10^-7 mol/L), VGVA (10^-11 mol/L), PVGV (10^-11 mol/L), and VGVAPG (10^-8 to 10^-7 mol/L), ie, all the peptides found active on the vascular tone, induced an increase in HUVEC [Ca²⁺]_i compared with the control cells (Fig 4A and 4C and Tables 1 [category E] and 4). Nevertheless, no statistically significant difference (U test) between the [Ca²⁺]_i increases induced by the different active compounds could be detected. The compounds found to have no activity on the vascular tone (VPVGGA, PGVGVA, VGVGVA, and GVGVA) also exhibited no significant activity on HUVEC [Ca²⁺]_i (Fig 3B and Tables 1 [category E] and 4).

View larger version (21K): [in this window] [in a new window]   Figure 3. Action of 10-3 mg/mL -elastin (A) and the control peptide VPVGGA at 10-7 mol/L (B) on [Ca2+]i in cultured HUVECs compared with the control experiment (C). Each trace is representative of six, seven, and three experiments, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 4. A and B, Action of the elastin sequence VGV at 10-7 mol/L on [Ca2+]i in HUVECs in the absence (A) or presence (B) of 10-4 mol/L lactose. Each trace is representative of five and three experiments, respectively. C and D, Action of 10-9 mol/L rTE on [Ca2+]i in HUVECs in the absence (C) or presence (D) of 10-4 mol/L lactose. Each trace is representative of five experiments.

Furthermore, compared with the control condition, application of lactose (10^-4 mol/L) to HUVECs was not found to induce a significant change in [Ca²⁺]_i over a 2-minute period (Table 1, category F). However, the addition of 10^-4 mol/L lactose inhibited the [Ca²⁺]_i increase induced by the active elastin-related peptides (Fig 4B and 4D and Table 4). The presence of lactose had a significant inhibitory effect for all the active elastin-related compounds tested, since the inhibition was independent of the active peptide type (Table 1, category G).

	Discussion

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Elastin or elastin-related compounds, such as the precursor molecule "tropoelastin" or its degradation products (elastin peptides), have been demonstrated to induce several biological effects.¹ The results of the present study demonstrate that the whole tropoelastin molecule (rTE) as well as some elastin-related sequences induce a strong vasorelaxation on NA-precontracted adult rat aortic rings (Fig 1) and an increase in the endothelial [Ca²⁺]_i (Table 4). These results are consistent with previous findings indicating that -elastin produced the same effects (References 14¹⁴ to 16 and authors' unpublished data, 1997). Similar to the results obtained using -elastin, the absence of endothelium totally abolished the vasorelaxant action of both rTE and active synthetic elastin-related peptides (Table 2). The -elastin–induced endothelium- and NO-dependent vasorelaxation was shown to be mediated by the elastin/laminin receptor.¹⁵ Inhibition of elastin binding to the 67-kD subunit of this receptor by lactose was used to confirm the mediation of the receptor on the vasorelaxant action of -elastin. Lactose was shown to strongly inhibit the -elastin–induced vasorelaxation.^{14 15 16} In the present study, using this same approach, the actions of rTE and other vasoactive elastin-related peptides (VGV, PGV, VGVAPG, PVGV, and VGVA, at active concentrations) were also strongly inhibited by the presence of 10^-4 mol/L lactose (Table 3), indicating that their action is most likely mediated by the 67-kD subunit of the elastin/laminin receptor.

The control peptide (VPVGGA) and the elastin-related peptides VGVGVA, PGVGVA, and GVGVA were found to have no significant effect on the vascular tone. The vasorelaxant effect induced by rTE and by the peptides VGV, PGV, and VGVAPG appeared to be dose dependent (Fig 1). The 25% to 40% maximum relaxation of NA-contracted aortic rings induced by these elastin peptides is important, since its amplitude approximately corresponds to one third to half of the 70% to 80% maximum relaxation induced by the highly potent agent acetylcholine (10^-6 to 10^-5 mol/L) on NA-contracted aortic rings.⁴¹ Nevertheless, whereas activity of the peptides VGV, PGV, and VGVAPG directly increased with their concentration, rTE activity increased first with concentration before exhibiting a clear maximum activity at 10^-9 mol/L and then decreasing when the concentration increased (Fig 1). This rTE activity peak at 10^-9 mol/L, in the range of the K_d of tropoelastin binding to the elastin/laminin receptor,²⁰ is consistent with previous findings indicating a maximum chemotactic activity of tropoelastin toward fibroblasts at the same concentration.^{12 13} In contrast, the effects produced by the peptides PVGV and VGVA are different from the effects of the other active peptides, since they induced a significant vaso-relaxant effect only at 10^-11 mol/L. Matching this result, another elastin-related peptide (VGVGVA) has already been demonstrated to act on superoxide anion production at only one concentration in a dose-effect experiment on leukocytes.³² Nevertheless, such activity at only one concentration is uncommon for endothelium-dependent vasoactive agents. However, some agents have already been demonstrated to exhibit a vasodilation maximum at one particular concentration, eg, acetylcholine (10^-6 mol/L) in the rabbit aorta and in the bovine coronary arteries^{42 43} and endothelin 3 (10^-8 mol/L) in the mesenteric artery.⁴⁴ If the maximum effect induced by a vasoactive molecule is slight, a statistically significant activity could then be observed only at a single concentration, potentially explaining our results concerning PVGV or VGVA.

In addition, the experiments performed on cultured adherent HUVECs confirmed that only the vasoactive synthetic elastin peptides and rTE produced an increase in HUVEC [Ca²⁺]_i with no observed significant difference in maximal amplitude (Table 4). Furthermore, enhancement of HUVEC [Ca²⁺]_i by the above-mentioned agonists was inhibited by lactose (10^-4 mol/L), suggesting the involvement of the 67-kD subunit of the endothelial elastin/laminin receptor. The long-lasting pattern of the elastin peptide–induced [Ca²⁺]_i increase (Figs 3 and 4) is similar to the -elastin–induced [Ca²⁺]_i increase in suspended HUVECs and probably also results mainly from membrane Ca²⁺ channel activation (authors' unpublished data, 1997). This slow kinetic, different from the fast [Ca²⁺]_i increase (peak), involving both extracellular Ca²⁺ influx and mobilization of intracellular Ca²⁺ stores, induced by -elastin on leukocytes,^{19 45} matches the slow and long-lasting extracellular Ca²⁺ influx produced by -elastin in fibroblasts and smooth muscle cells.¹⁸ Furthermore, the slow elastin peptide–induced [Ca²⁺]_i increase in HUVECs resembles the 1.3-fold [Ca²⁺]_i increase induced within a few minutes by laminin in neurons, which is also mainly mediated by extracellular Ca²⁺ influx and which is involved in neuron growth.⁴⁶

It has previously been demonstrated that activation of the endothelial 67-kD elastin/laminin receptor by -elastin results in a dose-dependent vasorelaxation via endothelial production of NO.^{14 15} In the present study, it has been shown that the different active elastin peptides are probably acting through the same 67-kD receptor pathway, since both their dose-dependent vasorelaxant effect and the observed increase in endothelial [Ca²⁺]_i that they induce at 10^-7 to 10^-9 mol/L are inhibited by lactose, an inhibitor of elastin binding to its receptor. Moreover, it has already been demonstrated that in the vascular endothelial cells there is a close correlation between [Ca²⁺]_i and the amount of released NO, as well as with the resulting vasorelaxation.^{47 48} Therefore, it is likely that in the endothelial cells stimulated by -elastin or the different active elastin peptides, the observed NO- and dose-dependent vasorelaxation occurs concomitantly with a dose-dependent endothelial [Ca²⁺]_i increase. Concerning the elastin peptides exhibiting an activity only at one concentration, it is likely that the same relation between elastin peptide–induced [Ca²⁺]_i increase and vasorelaxation is applicable. At the active concentration (10^-11 mol/L), PVGV induces an increase in endothelial [Ca²⁺]_i and vasorelaxation, whereas at higher concentrations (10^-8 mol/L), where it is inactive on vascular tone (Fig 1B), this peptide has no effect on endothelial [Ca²⁺]_i (data not shown).

The peptide VGVAPG, chemotactic for fibroblasts and monocytes,²⁸ and the peptides VGV and VGVA have been found to be active in both leukocytes³² and endothelial cells (present study). Nevertheless, the peptides GVGVA, PGVGVA, and VGVGVA, previously found to be active in leukocytes,³² were not found to be active in endothelial cells in our experiments. These differences could suggest that slight changes in the elastin/laminin receptor and, thus, in the sequence specificity might occur as a function of the cell type. Nevertheless, these modifications of the receptor sequence specificity could produce only minor differences, since despite numerous interspecies sequence differences, rTE is active in both human and rat cells. Moreover, the number of active sequence occurrences in the rTE molecule does not seem to be a major factor related to sequence activity: PGV (26 copies in rTE) is no more active than VGV (13 copies in rTE). A more likely explanation is provided by the results obtained in leukocytes, showing that the different elastin peptides are acting via different pathways.³² For example, in leukocytes, VGVAPG and GVGVA have been demonstrated to trigger hydrogen peroxide production and to release myeloperoxidase activity, whereas only VGVAPG, and not GVGVA, was able to induce elastase release, superoxide anion production, and [Ca²⁺]_i increase.³² The possibility of different peptides triggering different pathways could explain why we did not observe activity in our model for three peptides active in some pathways in leukocytes (PGVGVA, VGVGVA, and GVGVA): these peptides could be active in pathways and cellular events that we were not investigating.

Finally, although the flanking amino acids modulate the activity of the active sequences, VGV seems to be the core sequence interacting with the elastin receptor. The activity of this core sequence seems to decrease or at least to be strongly modified (at matching concentrations) by the presence of proline residues at the N-terminus but not at the C-terminus of the peptide. However, the presence of only hydrophobic amino acids (V or A) on either side of the sequence VGV seems to decrease its activity.

An elastin peptide–induced decrease in the vascular tone is likely to induce, in vivo, an increase in the diameter of the aorta or of other elastic arteries and therefore a decrease in the resistance of these vessels. This phenomenon could be of major importance in the regulation of arterial tone or in counterbalancing the effect of hypertensive physiopathological situations involving elastin degradation, such as aging or atherosclerosis. Further in vivo studies should investigate the potential of elastin sequence–based vasorelaxant treatments.

	Selected Abbreviations and Acronyms

 

DMSO = dimethyl sulfoxide HUVEC = human umbilical venous endothelial cell KbNA = Krebs' bicarbonate buffer containing 10-6 mol/L NA NA = noradrenaline rTE = recombinant human tropoelastin

	Acknowledgments

 
The authors thank Dr Guy Lallement with the Centre de Recherche du Service de Santé des Armées (Grenoble, France) for having provided access to the spectrofluorometer used in these experiments. Thanks are also extended to Jacques Bernillon (Laboratoire de Biochimie Analytique et Synthèse Bioorganique, Université Lyon I, France), Francine Cand (Groupe d'Electrophysiologie Moléculaire, Laboratoire de Bioénergétique Fondamentale et Appliquée, Université Grenoble I, France), and Bernadette Vrhovski and Suzanne Mithieux (Department of Biochemistry, University of Sydney, Australia) for their technical assistance.

Received October 6, 1997; accepted November 20, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Robert L, Hornebeck W. Elastin and Elastases. Vols 1 and 2. Boca Raton, Fla: CRC Press; 1989.

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