© 1997 American Heart Association, Inc.
Articles |
Inhibition of Proliferation and Stimulation of Migration of Endothelial Cells by Secretoneurin In Vitro
From the Departments of Internal Medicine (C.M.K., R.K., G.K., N.R., C.J.W.) and Pharmacology, (R.F.-C., H.W.), Faculty of Medicine, and the Department of Pharmacology and Toxicology, Faculty of Pharmacology (S.T.K.), University of Innsbruck, Austria; and the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, NY (R.H.-A.).
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Abstract |
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Abstract Vascular cell responses in inflammation are affected by several neuropeptides of perivascular nerve fibers. Secretoneurin is a 33–amino acid peptide that is coreleased from these nerve endings with other proinflammatory neuropeptides, eg, substance P and calcitonin gene–related peptide. Furthermore, secretoneurin has been shown to be chemotactic for human skin fibroblasts and human blood monocytes in vitro and in vivo. An action on cellular components of the vascular wall is not yet reported. We therefore investigated in vitro effects of this novel sensory neuropeptide on endothelial cells. Secretoneurin exerted a potent and reversible inhibitory effect both on endothelial cell growth under low serum conditions (1% fetal calf serum) and endothelial cell growth factor–activated endothelial cell proliferation. We show in the present study that secretoneurin exerts this effect on aortic (rat) and pulmonary artery (bovine) endothelial cells, as well as venous (human umbilical vein) endothelium. Endothelial cell chemotaxis was tested by means of three different migration assays employing nitrocellulose and polycarbonate micropore filters. Secretoneurin consistently exhibited potent chemoattractant activity. The effective concentrations for the observed effects were in the picomolar range. The combination of chemotactic and antiproliferative effects on endothelial cells suggests that secretoneurin may act as a regulatory factor of vascular cell functions.
Key Words: secretoneurin • endothelial cells • proliferation • migration • neuropeptide
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Introduction |
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Endothelial cells are strategically located between the intravascular elements and the parenchyma of organs. They are critical elements in the evolution of all types of inflammation.1 The endothelium, when stimulated by neurotransmitters, hormones, and substances from platelets and the coagulation system, is involved in both acute and chronic inflammatory responses.2 Sequential events are necessary for the process of endothelium repair and neovascularization, including endothelial cell migration and proliferation.2 These processes are mediated by several polypeptide molecules, including FGF, TGF-ß, TNF-
, and platelet-derived growth factor.2 It has been shown that perivascular sensory neuropeptides such as substance P participate in a number of inflammatory responses involving vascular cell biology. Thus, substance P has been reported to act as a growth and chemotactic factor for cultured human fibroblasts3 3 5 and endothelial cells6 7 and to induce differentiation of endothelial cells into capillary-like structures.8 The chromogranins, comprising chromogranin A and B and secretogranin II and 7B29 10 11 represent a family of abundant and acidic proteins with a widespread distribution throughout the nervous and endocrine systems.12 13 14 We have recently shown that proteolytic processing of secretogranin II in the brain leads to the formation of a 33–amino acid peptide, SN.15 16 This novel neuropeptide is found in high concentrations in the brain15 16 17 and afferent C fibers18 and can be determined in human plasma19 ; its distribution in tissue and its biological properties are comparable to those of other neuropeptides, eg, neuropeptide Y, calcitonin gene–related peptide, and substance P.14 15 16 17 18 20 21 22 SN was shown to evoke dopamine release from the rat caudate-putamen with a magnitude of the maximal response higher than that reported for other neuropeptides and comparable to acetylcholine-induced dopamine release.20 It is released from sensory afferent C fibers by capsaicin, indicating a function as sensory neuropeptide.18 In accordance, we demonstrated that SN acts as a potent chemoattractant for human skin fibroblasts and human blood monocytes in vitro and in vivo,21 23 a property also reported for other sensory neuropeptides.24 25 26 27 28 These results are consistent with the hypothesis that SN may serve as a mediator in neurogenic inflammation. Since endothelial cells are critical elements for vascular biology, we were interested in testing effects of SN on endothelial cells in culture.
In this study we demonstrate that the novel neuropeptide SN has a regulatory activity on endothelial cells in vitro, as it exerts an antiproliferative effect on endothelial cell growth and acts as a chemoattractant of endothelial cells in vitro.
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Purification of Peptides and Production of Antisera
Four peptides deriving from the primary amino acid sequence of secretogranin II were synthesized by standard t-butoxycarbonyl chemistry and purified by reversed-phase high-performance liquid chromatography as described previously16 : bovine secretogranin 133-151 (bLF-19), bovine secretogranin 217-286 (bEL-17), rat secretogranin 154-186 (SN), human secretogranin 172-186 (carboxy-terminal fragment of SN), as well as a trypsinized form of SN. Proteolytic cleavage of SN was done by trypsin (350 µg SN in 80 µL of 0.1 mol/L Tris-HCl, pH 7.9, containing 10 µg trypsin for 3 hours at 37°C). Purity of peptides was verified by amino acid sequencing and mass spectrography, and the concentrations were determined by amino acid analysis based on phenylalanine. For antisera production, SN was coupled via an additional N-terminal cysteine to maleimide-activated keyhole limpet hemocyanin and used for generation of antisera in Chinchilla bastard rabbits (Ivanovas, Kislegg, Germany) as already reported in detail.16 Immunoglobulin fractions were prepared from antiserum by affinity chromatography on Protein A Sepharose columns (Pharmacia). Detection of peptide contamination with endotoxin was excluded by using the E-Toxate assay (Limulus amebocyte lysate test for endotoxins, Sigma Chemical Company).
Conditions of Endothelial Cell Cultures
CPAEs
Bovine artery endothelial cells were obtained from the American Type Culture Collection. CPAEs were grown in enriched medium (medium 199 in Earle's salts base with L-glutamine [Biological Industries], 20% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin) in 75-cm2 plastic tissue-culture flasks and kept in a humidified incubator at 37°C in 5% CO2. After reaching confluence, the cells were subcultured and reseeded at a ratio of 1:3. Endothelial cells were used between passages 3 and 12 for all experiments.
HUVECs
HUVECs were isolated and cultured in flasks as described by Jaffe et al.29 Briefly, endothelial cells were isolated from umbilical cords after treatment with 0.5 mg/mL collagenase (Schöller Pharma) in 0.01 mol/L PBS without calcium and magnesium and grown in specific medium (Promo Cell, Biomedica) containing 2% fetal bovine serum, 0.1 mg/mL epidermal growth factor, 1 ng/mL bovine fibroblast growth factor, 1 µg/mL hydrocortisone, and 4 µg/mL ECGS. Cells were cultured in gelatin-coated culture flasks and kept in a humidified incubator at 37°C in 5% CO2. Endothelial cells were used between passages 3 and 5 for all experiments.
rEC
Female Sprague-Dawley rats weighing 230 to 280 g were housed in a light-, temperature-, and humidity-controlled environment and provided with food and water ad libitum. Before being killed by decapitation, 10 rats were anesthetized with diethyl ether, and thoracic aortas were prepared immediately after removal. Aortas were cut into consecutive 2-mm segmental rings, mounted on the plastic surface of 24-well tissue-culture plates coated with a distinct mixture of collagen type I (0.1 µg/mL, Collaborative Biomedical Products), fibronectin (1 µg/mL; Collaborative Biomedical Products), and gelatin (0.5%, Sigma Chemical Company). Cells were cultured in medium 199 with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 100 µg/mL ECGS (Sigma Chemical Company) and kept in a humidified incubator at 37°C in 5% CO2. Endothelial cells were used between passages 3 and 5 for all experiments.
Identification of Endothelial Cells
Identification of rECs and HUVECs was performed by demonstrating specific immunofluorescent staining for factor VIII–related antigen (DAKO Corp) and acetylated LDL labeled with 1,1'-dioctadecyl-3,3-3',3'-tetramethylindocarbocyanine perchlorate (Dil-Ac-LDL; Biomedical Technologies, Inc) and by examining monolayers microscopically for their typical morphology.
Proliferation Assays
Preparation of Endothelial Cells
CPAEs. Replicate confluent endothelial cell cultures were washed and trypsinized, and 100 µL of cell suspension (2.5x103 and 5x103 cells per well in MTT and BrdU assays, respectively) in culture medium containing 10% FCS were plated into 96-well flat-bottomed tissue-culture plates. Twenty-four hours after subculture, the plating medium was aspirated and replaced by medium containing 1% FCS and the substances being tested. Endothelial cell proliferation was compared with that of cells growing in the presence of medium supplemented with 1% FCS (basal conditions) or 20% FCS (optimal growth conditions).
HUVECs. For experiments using HUVECs, cells were detached by collagenase treatment and seeded at a concentration of 5x103 cells per well in culture medium containing 10% FCS into 96-well flat-bottomed tissue-culture plates. After 24 hours of resting and allowance for attachment, HUVECs were stimulated with test substances in medium containing 10% FCS and 40 µg/mL ECGS for 72 hours.
rEC. These endothelial cells were harvested by mild trypsinization and seeded in 96-well flat-bottomed tissue- culture plates at a concentration of 5x103 cells per well in culture medium. After 24 hours of incubation, cells were incubated with SN in medium containing 10% FCS and 40 µg/mL ECGS for 72 hours. After this time period, endothelial cell growth was evaluated colorimetrically.
Colorimetric Assay for Cellular Growth and Survival (MTT Assay)
After incubation at 37°C for the indicated time in medium containing assay substances, cellular proliferation was measured using a colorimetric assay for cell growth and chemosensitivity.30 31 This colorimetric assay is based on the tetrazolium salt MTT (Sigma Chemical Company), detects living but not dead cells, and the signal generated is directly proportional to the number of cells.32 As previously described, stock MTT solution was added to all wells of an assay at the indicated time.30 31 After a further period of incubation (6 hours), the medium was aspirated from the adherent cells as completely as possible without disturbing the formazan crystals formed within the cells. Subsequently, DMSO (Merck) was added to each well, the plates were agitated on a plate shaker, and the optical density was read. Optical density measurements were carried out with an enzyme-linked immunosorbent assay reader (model MR 700; Dynatech Labs) at 570 nm.
BrdU Incorporation Assay
After incubation with various drugs, DNA synthesis in endothelial cells was measured using the BrdU-labeling and detection kit (BrdU-Kit III, Boehringer Mannheim). BrdU was added to the culture medium for incorporation into the DNA of replicating cells. After 18 hours of incubation, cells were fixed and nucleases added to partially digest and expose the target haptens. Thereafter, anti–BrdU-peroxidase was added to each well to bind to the BrdU in the DNA, and finally the signal, developed with a water-soluble peroxidase substrate, was measured with the enzyme-linked immunosorbent assay reader at 405 nm.
Migration Assays
A substance was considered a chemotactic agent if it induced cells to migrate from an area of lower test substance concentration to an area of higher concentration. This process is described as chemotaxis or the "directional migration" of cells. Chemokinesis, on the other hand, represents augmented random, nondirectional cell motion.
Preparation of Endothelial Cells
Single-cell suspensions of endothelial cells were prepared from confluent cultures. Cells were first washed and then treated with 0.05% trypsin and 0.02% EDTA at 37°C until they were observed to round up, which typically occurred after 
1 minute. Medium 199 containing 20% FCS was then added to the cultures to stop trypsinization. After centrifugation, endothelial cells were washed and resuspended in RPMI supplemented with 1% BSA (Behring) at 8x105/mL and allowed to recover in suspension for 30 minutes.
Nitrocellulose Assay
Chemotaxis and chemokinesis of bovine endothelial cells in response to the test attractants were measured in a modified multiwell Boyden chamber (Neuro Probe). For each chemotaxis assay, the bottom wells were filled with 28 µL of medium with 1% BSA alone or supplemented with various concentrations of SN, its C-terminal fragment, or other secretogranin II–derived peptides (bLF-19 or bEL-17), and 40 000 cells in 50 µL of RPMI containing 1% BSA were added to the upper wells of the chemotaxis chamber. When both chemotaxis and chemokinesis were studied in an experiment, appropriate concentrations of the test attractant were also added to the upper wells of the Boyden chamber. The two wells were separated by a 5-µm-pore-sized nitrocellulose filter (Sartorius) that had been pretreated with 0.2% bovine dermal gelatin (Sigma Chemical Company). The chemotaxis chambers were then incubated in a humidified atmosphere of 95% air and 5% CO2at 37°C for an optimal period of time (4 hours).33 Cell migration was measured microscopically (leading-front assay). Migration depth of the cells was measured in triplicate after the cells had been fixed in ethanol, dehydrated, and stained with hematoxylin.
Polycarbonate Assay
Forty thousand cells in 28 µL of RPMI containing 1% BSA were placed in each well of the lower compartment of the chemotaxis chamber. Wells were then overlaid with a 5-µm porous polyvinyl-pyrrolidone–free polycarbonate membrane (thickness of <10 µm; Nuclepore) that had been coated with 0.2% gelatin. After this, chemotaxis chambers were inverted and incubated for 90 minutes at 37°C (5% CO2 and fully humidified), which allowed the cells to attach to the membrane. Chambers were then placed upright and 50 µL of the test solutions was added to the upper well and incubated for another 4 hours.33 After this period, membranes were recovered and cells on the attachment side scraped off, leaving only those cells that had migrated through the pores to the other side of the filter membrane. Membranes were submerged in methanol to fix cells, which were then stained with Diff-Quick (Dade) and mounted onto glass slides. For each well, the number of cells that had migrated through the filter was calculated by taking the average of the number of cells counted in three separate x400 microscopic fields.
Transwell Assay
This migration assay was performed as previously described34 using modified Boyden chambers with a 6.5-mm, 5-µm-pore-size, 10-µm-thickness polystyrene membrane separating the two chambers (Transwells, Costar). Assay substances dissolved in medium containing 1% BSA were placed into the lower chamber of a 24-well plate containing Transwells and allowed to stand at 37°C for 30 minutes before placement of the cells into the insert (106 cells per milliliter). Transwell inserts were pretreated with 0.2% gelatin. Cell migration was measured after 4 hours of incubation at 37°C. All nonmigrant cells were removed from the upper face of the Transwell membrane with a cotton swab, and migrant cells (those attached to the lower face) were fixed and stained with 0.1% crystal violet in 0.1 mol/L borate, pH 9.0, and 2% ethanol. Stained cells were subsequently extracted with 10% acetic acid, absorbance at 600 nm was determined, and migration was calculated from a standard calibration curve.
In all experiments we used EGF (10 ng/mL) as positive chemotactic control33 and medium supplemented with 1% BSA as negative control to determine random cell migration.
Calculations
The data are expressed as mean±SEM. The nonparametrical analysis was performed for independent samples after Kruskal-Wallis. Differences were compared using the Mann-Whitney U test or the two-tailed Student's t test for paired or unpaired samples.
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SN Inhibits Endothelial Cell Proliferation In Vitro
Studies on proliferation of bovine endothelial cells were carried out in medium containing 1% FCS to minimize possible interactions with serum-associated proteins, such as platelet-derived growth factor. CPAEs, seeded at 2.5x106/0.32 cm2, undergo a nearly threefold increase in cell number under basal conditions (medium 199 supplemented with 1% FCS) over a period of 3 days and a fivefold to sixfold increase in the presence of 4 µg/mL ECGS, which is a bovine acidic fibroblast growth factor preparation. SN and two other secretogranin II–derived peptides, bEL-17 and bLF-19, were tested for their effect on basal (Figs 1
and 2) and ECGS-stimulated (Fig 3) proliferation of bovine endothelial cells over a broad concentration range. SN inhibits both basal and ECGS-induced proliferation of CPAEs not only in the MTT assay (Fig 1) but also when assayed alternatively by BrdU labeling, measuring DNA synthesis (Fig 2). The antiproliferative effect of SN was concentration dependent, with maximal inhibition occurring at 10-7 mol/L, the highest concentration tested. The growth-inhibitory effect was consistently observed in early- as well as late-passage endothelial cells. On the other hand, bEL-17 and bLF-19 did not affect endothelial cell proliferation at any concentrations tested. Proteolytic cleavage of SN by trypsin abolished the antiproliferative activity observed by the complete neuropeptide (concentration tested was 10-7 mol/L; Fig 4). Inhibition of endothelial cell growth was further observed by a fragment of SN consisting of its 15 C-terminal amino acids at a concentration of 10-7 mol/L to a significant extent (Fig 4). Furthermore, the observed antiproliferative effect of SN on CPAE growth was comparable to the growth-inhibitory potency of TNF- at a concentration of 10 ng/mL, as shown in Fig 4.
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By microscopic inspection, the morphology of the endothelial cell cultures was not altered after incubation with SN. To exclude a toxic effect, we additionally re-stimulated SN-treated (10-8 mol/L) endothelial cells with a mitogenic concentration of ECGS (4 µg/mL). CPAE proliferation did resume within 3 days after removal of SN from the culture medium (data not shown). Endotoxin contamination of peptides used was excluded by a sensitive Limulus amebocyte lysate assay (data not shown). Furthermore, the antiproliferative effect of SN on both basal proliferation and ECGS-induced endothelial cell growth could not be overcome by the simultaneous addition of polymyxin B (10 µg/mL) to the culture medium, whereas the effect of lipopolysaccharide (10 µg/mL) could be neutralized (Fig 5). These data demonstrate that the neuropeptide SN is a potent inhibitor of basal and ECGS-induced endothelial cell growth and that the process is reversible.
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To further investigate the specificity of the observed effect of our SN preparation, we studied the effect of SN in the presence of specific anti-SN immunoglobulins. Cells were preincubated with an immunoglobulin fraction for 10 minutes before addition of a submaximal concentration of the neuropeptide (10-8 mol/L). After an incubation period of 3 days, cellular proliferation was measured. In the presence of an optimal concentration of the specific antibody, the antiproliferative effect of SN was completely abolished (Fig 6). Reversal of growth inhibition by specific antibodies was concentration dependent, with a maximum at a dilution of 1 to 1000. Anti-SN immunoglobulins themselves did not affect cellular growth.
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To estimate whether the observed effect is peculiar to a certain endothelial cell type, we investigated the effect of SN on endothelial cells deriving from two other tissues (rat arterial and human venous endothelium). As described above, SN reduced the proliferative response of CPAEs significantly and inhibited the growth response of all other endothelial cell types tested (HUVECs, rECs). As shown in Figs 7 and 8, the effect of ECGS (40 µg/mL) was completely inhibited. HUVECs as well as rECs were also growth inhibited significantly to an extent similar to experiments employing TNF- at a concentration of 10 ng/mL.
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SN-Induced Stimulation of Endothelial Cell Migration
A further purpose of this study was to determine whether SN can function as a chemoattractant for endothelial cells. The migratory response of endothelial cells to the neuropeptide was evaluated over a wide concentration range (from 10-5 to 10-14 mol/L) using three different assay systems, ie, a modified Boyden chamber technique using nitrocellulose or polycarbonate filters, and a Transwell assay. We observed a significant chemotactic response of bovine endothelial cells toward the neuropeptide (Figs 9 and 10) in all migration assays. The chemotactic effect was concentration dependent and showed a maximum at 10-6 mol/L in the nitrocellulose (Fig 9) and polycarbonate (Fig 10a) assays and a maximum at 10-8 mol/L in the Transwell assay (Fig 10b), whereas it decreased at higher concentrations. In all experiments we used medium 199 supplemented with 1% BSA as negative control and medium 199 containing 10 ng/mL EGF as positive chemotactic control. The peak migratory response toward SN was significantly greater than that toward the positive control. To elucidate whether this effect is mediated by the C terminus of the peptide, we tested a human carboxyterminal 15–amino acid–long SN fragment for its ability to elicit a migratory response (Fig 9). The C-terminal fragment of SN caused a significant migratory response, with a maximal effect at 10-6 mol/L, similar to that obtained from the complete peptide in the nitrocellulose assay (Fig 9). These findings suggest that the N terminus of SN is not required for the chemotactic response elicited from endothelial cells.
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To further ascertain whether the SN-induced migratory response of endothelial cells is specific, we investigated the effect of SN on endothelial cell chemotaxis in the presence of specific anti-SN immunoglobulins. The addition of antibodies to SN-containing medium significantly abolished the neuropeptide-induced migratory response (Fig 11).
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Additionally, a checkerboard assay was performed to determine whether the migratory response evoked by SN represents chemotaxis and/or chemokinesis (Table). The results showed a chemokinetic response of endothelial cells to SN (from 10-6 to 10-10 mol/L). However, the chemokinetic response did not equal the chemotactic response toward the neuropeptide. Nevertheless, SN was shown to stimulate the speed and/or number of cells migrating at random as well as the cells' directed migration.
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Discussion |
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We investigated the effect of SN on arterial and venous endothelial cells in vitro. Endothelial cells in culture form ordered monolayers, and the cells express the characteristics of in vivo endothelium. We demonstrate here that SN inhibits both basal and ECGS-stimulated proliferation of CPAE. This effect was not peculiar to this endothelial cell line, as SN also inhibited and/or reduced growth of endothelial cells from other sources (rECs and HUVECs). The growth-inhibitory potency of this novel neuropeptide was similar to the effect of TNF-
at a concentration of 10 ng/mL, as observed in correlating experiments.
The findings are similar to results of proliferation studies obtained with TGF-ß and TNF-. Both of these substances were shown to inhibit endothelial cell proliferation in vitro.35 36 37 38 39 40 However, the precise mechanisms of their antiproliferative properties are unknown. Thus, some effects of TGF-ß on cellular proliferation appear to be entirely mediated through intracellular mechanisms that alter the cell's ability to progress through the proliferative cycle. Other effects, however, are indirect in that they result from changes in extracellular matrix or autocrine cytokine production by TGF-ß.41 Furthermore, TGF-ß opposes the proliferative effect of mitogens by interfering at a level distal from mitogen receptors and their early signals in the pathway of mitogenic stimulation.41 Recent observations suggest that TGF-ß lengthens or arrests the cell cycle in G-1 phase.42 43 44 Since we observed reduced proliferation in cultures treated with SN compared with cultures growing under basal conditions or treated with ECGS, and as the antiproliferative effect could be overcome by a known mitogen, SN could possibly affect cell cycle progression in a manner similar to TGF-ß.
In a second set of experiments, we demonstrated that SN affects endothelial cell migration in a way similar to TNF-. Dose-dependent stimulation of chemokinesis and chemotaxis was induced by SN and its C-terminal fragment. The migratory response toward SN could be antagonized by the use of specific anti-SN antibodies. The test systems used for measuring endothelial cell migration employed different chamber types, which means that the concentration gradients that are formed between the two wells of the chemotaxis chambers probably differ. Therefore, differences in dose-response curves may be obtained in the different assay systems. The observed migratory responses are consistent with our previous observations regarding the migration of human blood monocytes toward this novel neuropeptide.21
Several polypeptides have been identified as regulatory factors of endothelial cell functions on the basis of in vivo and in vitro activity. Most notable are acidic and basic FGFs, TGF- and TGF-ß, angiogenin, TNF-, and to a lesser extent EGF.2 All of these factors are markedly active in a number of in vitro and in vivo assays. FGF, for example, is mitogenic and chemotactic for endothelial cells in vitro45 46 47 48 and stimulates endothelial cell production of collagenase and plasminogen activator proteases.49 Additionally, acidic and basic FGF are angiogenic factors in the cornea bioassay.50 The neuropeptide substance P is important in a number of inflammatory processes in which the endothelium plays a critical role. In addition to the participation of substance P in vascular leak,51 migration, and activation of white blood cells,24 25 26 more recent data have also revealed an angiogenic activity.6 7 8 In marked contrast to this, TGF-ß is a potent inhibitor of both large-vessel35 36 37 and microvessel endothelial cell proliferation.38 It inhibits both baseline and growth factor (eg, FGF)–stimulated growth and blocks endothelial cell motility.35 TNF- inhibits endothelial cell proliferation and promotes the migration of cultured microvascular endothelial cells, although not of large-vessel endothelial cells.39 40 TNF- is angiogenic in a variety of in vivo assays.2 Thus, it is similar to TGF-ß, because both molecules promote angiogenesis in vivo and capillary tube formation in vitro but inhibit endothelial cell proliferation in vitro. Recently, Pandey and coworkers52 described a role for B61, a cytokine-inducible endothelial gene product that is the ligand for the Eck receptor protein tyrosine kinase in angiogenesis evoked by TNF-. In contrast, angiogenesis induced by fibroblast growth factor was not altered by an antibody to B61. As the effect of the neuropeptide SN on endothelial cell function is similar to TNF-, its potency to affect endothelial cell functions could also be related to this ligand of the Eck receptor protein tyrosine kinase. However, the precise mechanisms of SN on the endothelium remain to be elucidated.
The properties of SN affecting endothelial cells in vitro seem to be similar to those of TNF- and in part similar to the properties of TGF-ß, since SN inhibits endothelial cell proliferation while promoting endothelial cell migration in vitro. As the precise in vivo role of TNF- in endothelial cells (in particular during neovascularization and in causing endothelial dysfunction in the pathogenesis of atherosclerosis) is unknown, it will be interesting to more extensively compare activities of SN, TNF-, and TGF-ß, as was previously reported for the interacting effects of SP, EGF, and FGF.32 Although more information is needed to exactly define the role of SN in modulating endothelial functions, including angiogenesis, this study provides evidence of a new biological activity of this neuropeptide in vitro that remains to be tested in vivo.
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Reprint requests to Univ Doz Dr Christian J. Wiedermann, Department of Internal Medicine, Faculty of Medicine, University of Innsbruck, Anichstraße 35, Austria.
Received June 27, 1996; accepted July 23, 1996.
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References |
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