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1D-adrenoceptors on endothelial cells are potentiated by hypoxiaLaboratory of Vascular Pharmacology, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy
Submitted 29 March 2007 ; accepted in final form 26 July 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-adrenoceptors (1-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O2) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce a trophic effect in human endothelial cells through stimulation of the 1D-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective 1D-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that 1D-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by NG-nitro-L-arginine methyl ester. Conversely, the 1A-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the 1B-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of 1D-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of 1D-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury.
vascular remodeling; low oxygen tension; endothelial cell growth and migration; catecholamines; nitric oxide
1-adrenoceptors (1-ARs) (32, 37). The 1-ARs play key roles in cardiovascular, genitourinary, and central nervous system functions (16). In the cardiovascular system, they are involved in the regulation of cardiac and arteriolar smooth muscle contraction; however, it has also been suggested that they are able to exert trophic actions that may contribute to adaptive growth in the adult artery (11, 13, 29, 38). It has been shown that 1-ARs display growth factor-like activity in arterial smooth muscle cells and adventitial fibroblasts that is mediated by the generation of reactive oxygen species (3) or by the autocrine production of growth factors (29, 36) and that they contribute to hypertrophy and hyperplasia in models of vascular injury. Wall injury strongly increases the sensitivity of cells to adrenergic-mediated growth, leading to enhanced wall thickening and intimal lesion development.
Recent studies (10) have revealed that catecholamines are able to increase angiogenesis in pathological conditions such as hindlimb ischemia, even if the mechanisms and the AR subtypes responsible for the arteriogenic and angiogenic processes are still unclear. Evidence showing that ischemia enhances norepinephrine release from sympathetic nerve terminals (6) reinforces the hypothesis that catecholamines may be involved in the above-mentioned effect. Both the endothelial 2-ARs (34) and the 1-ARs (4, 33) are able to induce nitric oxide (NO)-mediated vasorelaxation in response to catecholamine stimulation. Interestingly, the expression of 1-AR subtypes in human endothelial cells is modulated in pathophysiological conditions such as inflammation, suggesting that this kind of receptor plays an important role in the regulation of vascular tone (23). In a previous study (14), we have shown that 1D-AR is involved in a NO-mediated vasodilatatory response. This study also showed that the 1D-AR-mediated vasorelaxant effect is induced by nanomolar concentrations of phenylephrine (PHE), which are not able to induce any detectable increase in vascular tone. The mechanisms underlying this effect have been elucidated and consist of inositol phosphate stimulation, followed by Ca2+ mobilization from sarcoplasmic reticulum and NO synthase activation (14). Because NO is recognized as a major downstream effector of proangiogenic cytokines (30), and because hypoxia is considered a strong stimulus for neovascularization, we investigated in the present study the possible role of 1D-ARs in inducing a proangiogenic phenotype in human endothelial cells, both in normoxic conditions and in experimental conditions mimicking pathologically low oxygen tension in vascular tissues. We tested the effects of picomolar concentrations of PHE, either in the presence or in the absence of the selective 1D-AR antagonist BMY-7378, in a number of cellular models that are widely considered to predict the trophic/angiogenic potential of molecules.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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To evaluate the functional role of 1D-AR in physiological and pathological conditions, human umbilical vein endothelial cells (HUVECs) were pretreated or not with hypoxia (3% O2) for 12, 24, or 48 h and then were used for the experiments in normoxic conditions. Vessel rings from human umbilical artery were kept in hypoxia or normoxia during the treatment period (15 days) with test substances. The 1-AR responsiveness to PHE stimulation was studied by using the following concentrations, which are devoid of any contractile response (14): 0.005 nM, 0.01 nM, 0.03 nM, 0.1 nM, and 1 nM. The 1-AR characterization was carried out by using selective antagonists: 1 nM (S)-(+)-niguldipine hydrochloride (NIG) (5), 3 µM chloroethylclonidine dihydrochloride (CEC) (12), and 0.3 µM BMY-7378 dihydrochloride, which are 1A-, 1B- (and weak 1D-), and 1D-AR antagonists, respectively. BMY-7378 shows great selectivity for 1D-ARs (pK 
8) compared with the 1A- and 1B-subtypes (pK 6) and at present is the most useful agent (19). The role of NO was also investigated by means of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 100 µM).
HUVEC and human artery vessels were obtained from human term umbilical cords. Italian law does not require any approval by an ethical committee for experimental use of human umbilical cords.
Cell Culture
HUVECs were isolated with collagenase perfusion of term umbilical cord vein (24). HUVECs were grown in Medium 199 (M199) supplemented with 20% FCS, antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), 10 ng/ml basic FGF, 5 U/ml heparin, and 2 mM glutamine on 0.1% gelatin-coated plates, kept in a humidified incubator at 37°C in 5% CO2, and split (1:2) twice a week by using trypsin-EDTA solution. Cells between passages 2 and 5 were used.
The cell type was characterized by a "cobblestone" cell morphology under phase-contrast microscopy and by immunohistochemical assay with a monoclonal anti-human factor VIII-related antigen antibody (Dako). More than 95% of the cells were positive for von Willebrand factor.
Cell Proliferation
HUVEC proliferation was quantified by the total cell number as previously reported (29). Briefly, cells (2.5 x 103/100 µl) suspended in 10% FCS medium were plated on 0.1% gelatin-coated flat-bottom 96-well plates and were allowed to adhere overnight. Cells pretreated or not pretreated with hypoxia (3% O2 for 24 h) were then stimulated with test substances in the presence of 2% FCS and were allowed to grow for 2 days in normoxic conditions. The effect of PHE was compared with control conditions in 2% FCS medium and with the effect produced by 10 ng/ml FGF-2 or 10% FCS. Receptor antagonists and the NO synthase inhibitor were added 30 min before stimulation with tested drugs. After 48 h, cells were fixed with methanol and were stained with Diff-Quik. Cell duplication was assessed by counting the total cell number in 10 random fields of each well at x200 with the aid of a 21-mm2 grid.
Migration Assay
A modified Boyden chamber was used to evaluate HUVEC migration (48-well plates; Neuroprobe) (27). Briefly, polyvinyl-pyrrolidone-free polycarbonate filters (8-µm pore size) were coated with 100 µg/ml collagen type I and 10 µg/ml fibronectin. Cells grew in hypoxic (3% O2) or normoxic (21% O2) conditions for 24 or 48 h; cells were then suspended (50 µl, 1.2 x 104 cells) and were added to the upper wells. The receptor antagonists or NO synthase inhibitor were added to the cell suspension 30 min before seeding, then migration was evaluated by incubating the chamber at 37°C for 4 h. Migrated cells were methanol-fixed, stained with Diff-Quik, and counted by using a microscope (x400 magnification) in 10 random fields per well. Each experimental point was measured in triplicate.
Cell Outgrowth from Human Vessel Fragments
Cell outgrowth from human umbilical artery fragments was performed as previously described (7). Briefly, human umbilical artery was isolated from terminal cords, and the adventitia was mechanically removed, leaving intact endothelium. Rings 1- to 2-mm-long were positioned in 48-well plates and were included in a fibrin gel. After fibrin clotting, 2% FCS-M199 with test substances was added. Receptor antagonists and NO synthase inhibitor were added 30 min before stimulation with PHE and were left for the entire experiment in hypoxic or normoxic conditions at 37°C in 5% CO2. The medium with test substances was replaced every 2 days for 2 wk. The extent of fibrin gel occupied by tubular outgrowth structures was quantified after 5 and 10 days by an inverted microscope at a magnification of x200 and with the use of a squared ocular grid (506-µm2 area at x200 magnification). Data are expressed as total area covered by the new tubular structure formed. Digital images of microvessel outgrowth were obtained by using a Nikon microscope coupled to a video camera (digital sight camera control unit DS-L1; Nikon) on day 5.
Cell populations present in the tubular structures were characterized and quantified at day 10. Vessel rings were removed, and fibrin was lysed by adding plasmin (200 U/ml, 100 µl/well). The cell suspension obtained was centrifuged, spread on a slide, fixed in acetone/chloroform 1:1, and stained for human CD31 endothelial antigen (mouse anti-human clone JC/70A; Dako) or for human -smooth muscle actin (mouse anti-human clone 1A4; Dako) by using an avidin-biotin amplified immunoperoxidase technique.
RT-PCR Analysis
HUVECs were seeded at 80% confluence in 6-cm-diameter Petri dishes in M199 plus 10% FCS and were allowed to grow in normoxic or hypoxic conditions (12, 24, 48 h) for the treatment period.
Total HUVEC RNA was isolated according to the manufacturer's protocol (Purescript; Gentra Systems, Minneapolis, MN) and was reverse transcribed by using random primers. The RNA purity was validated by PCR and gel electrophoresis by using primers for the GAPDH gene. A typical PCR reaction (HotStarTaq; Qiagen) was prepared for amplification of 1D-AR mRNA, and calibration was performed by amplification of the same cDNA sample with primers for GAPDH mRNA. Primer sequences were as follows: 1D-AR, 5'-CTA TTT CAT CGT GAA CCT GG-3' (sense, bases 400–419) and 5'-TCG GYG ATA CCG CAG AAG CG-3' (antisense, annealing to bases 734–753) (16); GAPDH, 5'-CCA TGG AGA AGG CTG GGG-3' (sense) and 5'-CAA AGT TGT CAT GGA TGA CC-3' (antisense). Amplification was performed in sequential cycles including 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 2 min of extension at 72°C for 1D-AR and 45 s of denaturation at 94°C, 45 s of annealing at 56°C, and 1 min of extension at 72°C for GAPDH. The predicted product sizes were 354 bp and 194 bp for 1D-AR and GAPDH, respectively. Amplification products were highlighted with ethidium bromide on 1% agarose gel. The intensities of the bands corresponding to the amplificates were quantified by densitometric analysis.
Immunoprecipitation and Western Blot Analysis
HUVECs were seeded at 80% confluence in 10-cm-diameter Petri dishes in M199 plus 10% FCS and were allowed to grow in normoxic or hypoxic conditions (24 h) for the treatment period.
Immunoprecipitation.
Total cell proteins were extracted with 200 µl of lysis buffer (50 mM Tris·HCl, 1% Triton X-100, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 25 µg/ml leupeptin, 10 µg/ml aprotinin, 10 mM NaF, 150 mM NaCl, 10 mM 
-glycerophosphate, and 5 mM pyrophosphate, pH 7.4) at 4°C, preserved for 30 min, and finally centrifuged at 12,000 g for 15 min. The supernatants containing 500 µg of cell proteins, measured by the BCA method (Pierce Chemical), were immunoprecipitated overnight with 1 µg anti-human 1D-AR antibody (rabbit polyclonal; Santa Cruz Biotechnology) in 0.5 ml immunoprecipitation buffer (50 mM Tris·HCl, 0.5% Triton X-100, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 25 µg/ml leupeptin, 10 µg/ml aprotinin, 50 mM NaF, 100 mM NaCl, 20 mM -glycerophosphate, and 10 mM pyrophosphate, pH 7.4) at 4°C. Protein G-agarose was added for 2 h at 4°C, and the tubes were then centrifuged at 1,000 g for a few seconds. The pellets were washed three times with ice-cold PBS (pH 7.4) and were suspended in reducing Laemmli buffer. Samples were resolved by 8% SDS-PAGE and were blotted onto PVDF membranes (Millipore). Preblocked membranes were probed overnight at 4°C with rabbit polyclonal anti-human 1D-AR antibody (1:500; Santa Cruz Biotechnology) and then with horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. The results were recorded by using X-ray film after visualization with an ECL luminescence-detection kit (Amersham).
Western blot.
Cells were lysed as previously described (35), with slight modifications. The samples were boiled, and 30 µl of total cell extract was run on 8% SDS-PAGE, blotted onto PVDF membranes, and immunostained with anti-human 1D-AR antibodies (rabbit polyclonal, 1:500; Santa Cruz Biotechnology) or anti-human -tubulin (1:2,000; Sigma). The antigen-antibody complexes were visualized by using appropriate secondary antibodies and an ECL detection system.
Statistical Analysis
Data are reported as means ± SE. The experiments were in triplicate. Statistical analysis was performed by using Student's t-test for unpaired data. A P value <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1D-AR Expression in Human Endothelial Cells
RT-PCR analysis revealed that, following 24 h of hypoxia, 1D-AR mRNA levels in HUVEC were higher than those detected in cells exposed to normoxic conditions (Fig. 1A). A longer exposure (48 h) to hypoxia did not induce any further increase in 1D-AR mRNA expression. Immunoblot and immunoprecipitation experiments showed a single protein band with an apparent mass of 80 kDa in HUVEC, thus confirming the presence of 1D-AR protein. Cells exposed to hypoxia for 24 h had increased 1D-AR expression (Fig. 1B). No bands were detected when primary antibody was omitted (data not shown).
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Because the experiments reported above demonstrated that 1D-AR mRNA expression in human endothelium is increased by exposure to hypoxic conditions, the effect of the selective 1-AR agonist PHE on cell outgrowth from vascular fragments was tested to investigate whether 1-AR stimulation was able to promote tissue architectural changes in the vessel wall.
In normoxic conditions, tubular structures appeared in the whole vessel fragments within 3 days and the maximum response was reached after 10 days (mean area of tubules, 2.290 ± 0.48 mm2; Fig. 2B). A significant stimulatory effect of PHE on tubular growth (2-fold increase over control) was evident after 5 days of culture only at the 0.1 nM concentration of the agonist (Fig. 2A). In preparations kept in hypoxia, the sprouting of pseudocapillaries from vascular fragments occurred faster and more abundantly. A glaring tubular outgrowth was observed within 3 days in response to the lowest PHE concentration tested (0.03 nM). After 5 days of culture of vessel fragments in the hypoxic environment, a significant potentiation of sprouting was already measured at the 0.03 nM concentration (Fig. 2A). The increase in the number of tubular structures was twofold greater than that observed with the same agonist concentration in normoxic conditions (P < 0.05 vs. normoxia). The maximal PHE effect was observed at 0.1 nM concentration, which induced a threefold increase in pseudocapillary formation, an effect that is also statistically different from that promoted in normoxic conditions (P < 0.05 vs. normoxia). However, the number of tubular structures from both normoxic and hypoxic preparations was identical after 10 days of culture (Fig. 2B).
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90% of the cells obtained from vessel fragments, either in normoxic or in hypoxic conditions, were positive for human CD31 endothelial antigen (Fig. 2C), whereas only 
2% of cells were positive for -smooth muscle actin.
Effect of 1-AR Antagonists and of the NO Synthase Inhibitor L-NAME on Tubular Outgrowth
The pseudocapillary formation in response to PHE (0.03 and 0.1 nM for 5 days) in hypoxic conditions was almost completely prevented in the presence of the 1D-receptor-selective antagonist BMY-7378 (0.3 µM) and of the NO synthase inhibitor L-NAME (100 µM) (Fig. 3). Conversely, the 1A- and 1B-AR antagonists NIG (3 µM) and CEC (1 nM) were devoid of any inhibitory effect on pseudocapillary growth induced by the agonist (Fig. 3A). It is noteworthy that the concentrations of the antagonists used in these experiments were devoid of any effect on tubular structure formation in basal conditions (data not shown).
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The findings of the above-mentioned experiments prompted us to further investigate the effect of picomolar PHE concentrations on HUVEC migration and proliferation, because both these events are crucial steps during the angiogenetic response. Cells grown either in hypoxic or in normoxic conditions for 24 and 48 h were used for these experiments. Figure 4A shows that PHE (0.03–1 nM) slightly stimulated migration of HUVECs exposed to normoxic conditions, inducing a significant increase in cellular movement (25% over control) only at the concentration of 0.1 nM. Similar results were obtained when the effect of the agonist on cell proliferation was tested (Fig. 4B). However, the exposure of cells to hypoxic conditions strongly potentiated cell migration induced by PHE (0.03–1 nM) in a concentration-dependent manner (Fig. 4A). The maximum stimulatory effect was observed in cells exposed to 24 h hypoxia: 1 nM PHE induced a significant cell-migration response (67% over control) whose degree was comparable with that induced by 10 ng/ml FGF-2 (Fig. 4A). It is noteworthy that the stimulatory effect of the growth factor was not influenced by the previous exposure to hypoxia. Proliferation of HUVECs was also potentiated by exposure to hypoxia, which significantly increased sensitivity and responsiveness of the cells to the agonist. In fact the proliferative effect of PHE was already significant at the lowest concentration used (0.005 nM) and was significantly greater than that observed with the same agonist concentrations in cells exposed to normoxic conditions (###P < 0.001, ##P < 0.01, and #P < 0.05 vs. same PHE concentration in normoxia; Fig. 4B). The maximal effect obtained by 0.01 nM PHE amounted to 60% of the effect obtained with 10 ng/ml FGF-2 and to 30% of that obtained with 10% FCS. Proliferation of cells previously exposed to hypoxia for 24 h was also significantly enhanced by noradrenaline (0.01 nM; 33 ± 3% increase over unstimulated cells; P < 0.001 vs. unstimulated cells and P < 0.001 vs. 0.1 nM noradrenaline in normoxia; n = 3; Fig. 4B).
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Exposure to the NO synthase inhibitor L-NAME (100 µM) and to the 1D-AR antagonist BMY-7378 (0.3 µM) significantly prevented the stimulatory effect of hypoxia on cell migration (Fig. 5) and proliferation (Fig. 6) induced by PHE stimulation. On the contrary, the effect of PHE on endothelial cells was not affected by pretreatment with NIG (1 nM) or CEC (3 µM), 1A-AR and 1B-AR antagonists, respectively (Fig. 5B and Fig. 6B). Moreover, receptor antagonists and the NO synthase inhibitor, at the above-mentioned concentrations, were devoid of any effect on endothelial proliferation and migration under basal conditions (Figs. 5 and 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-adrenoceptors in producing vasorelaxation through NO production has been largely recognized. Conversely, 1-ARs have been widely considered as vasoconstrictor receptors because their stimulation induces contraction of resistance vessels in most vascular beds (31). However, the role of this kind of receptor in the endothelial response during noradrenaline-induced vascular constriction has been only recently highlighted (4, 33). Moreover, the presence of different 1-AR subtypes on human endothelial cells has been detected in recent years, although the functional implications of their heterogeneous presence are still poorly understood (23, 26).
In a previous study, we have produced evidence for the involvement of an 1D-AR subtype in the endothelium-dependent vasodilatory response in the rat mesenteric vascular bed (14). This vasorelaxant response was induced by nanomolar concentrations of PHE (which were devoid of any contractile vascular effect), was mediated by NO synthase activation, and was antagonized by the selective 1D-AR antagonist BMY-7378. This observation about the sensitivity of 1D-ARs to such small concentrations of PHE is in full agreement with the observation (25) that this kind of receptor displays a much higher binding affinity for agonists compared with those of the other 1-AR subtypes. Thus 1D-ARs seem to be characterized by high intrinsic activity with respect to the formation of inositol phosphate and the activation of extracellular signal-regulated kinase (15, 25). Therefore, because selective 1D-AR agonists are not available, an experimental design in which very low 1-adrenergic agonist concentrations are used may help disclose the role of catecholamines at the vascular level, most likely through the stimulation of 1D-ARs. It is known that catecholamines, besides the regulation of vascular tone, exert trophic effects on vascular smooth muscle cells (VSMCs). Evidence of this effect comes from both in vitro experiments (29) and in vivo studies showing that a chronic local increase in noradrenaline concentration induces neointimal growth and lumen loss after balloon injury in the carotid artery of the rat (32, 37). The recent findings of a trophic activity of catecholamines during collateral vessel growth and angiogenesis in hindlimb ischemia (10), together with that showing the expression of different endothelial 1-AR mRNA in pathological conditions such as inflammation (23), clearly suggest that endothelial 1-ARs may have an additional biological role beyond the mere regulation of vascular tone and VSMC hyperplasia.
In the present study, we have attempted to identify the possible pathophysiological role played by endothelial 1D-ARs. The study has demonstrated that human endothelial cells possess 1D-ARs whose presence is significantly upregulated in an experimental model of hypoxia (3% O2) aimed to mimic a condition of pathological low oxygen tension. It is well known that hypoxia is able to induce profound effects on blood vessel tone and cell growth and that it represents the main stimulus able to trigger an angiogenic response (9). Evidence obtained in this study suggests that the stimulation of 1D-ARs induced by picomolar concentrations of PHE may exert trophic effects on vascular endothelium. In fact, PHE, at a concentration lower than those able to induce a vasoconstrictor response (0.03 nM), was able to promote the outgrowth of tubular structures from fragments of mature human vessels. The finding that only the selective 1D-AR antagonist BMY-7378 completely antagonized this kind of effect strongly supports the hypothesis that the tubular sprouting was due to the activation of 1D-AR. In agreement with our previous results, in which endothelial NO synthase activation is also demonstrated in cultured endothelium in response to nanomolar PHE concentrations (14), it was found that the activation of this endothelial receptor was associated with NO synthase stimulation, because tube outgrowth was impaired by exposure to L-NAME. Although human artery rings cultured in three-dimensional fibrin gel represent a valid experimental tool for disclosing proangiogenic properties (28), we decided to obtain further confirmation of the trophic effect induced by small concentrations of PHE by testing the effect of the agonist on human endothelial cell proliferation and migration. Even if microvascular endothelial cells are the most suitable cellular model for studying the angiogenic process in vitro, we chose HUVECs to investigate the cellular mechanisms of pseudocapillary sprouting from human umbilical artery rings. However, HUVECs have played a major role as a model system for study of the regulation of human endothelial cell function as well as the role of the endothelium in the blood vessel wall response to stretch, hypoxia, and the development of atherosclerotic plaques and angiogenesis.
HUVEC proliferation and migration were induced in a concentration-dependent manner by picomolar concentrations of the agonist and were highly potentiated by exposure of the cells to a hypoxic environment. Again, this effect on endothelial cells was impaired by the 1D-AR selective antagonist BMY-7378 and NO synthase inhibitor L-NAME, thus confirming a role of 1D-AR acting through the involvement of NO synthase activation.
The 1D-ARs are functionally expressed in arteries, such as the aorta, iliac, carotid, mesenteric, and femoral arteries (2, 20), where their constitutive activity is responsible for maintenance of blood pressure in the conductance vessels (17). The main role of these receptors seems to be modulation of sudden changes in vessel diameter, without affecting the small resistance arteries (39). The observations of a constitutive activity of the 1D-ARs with a high degree of basal inositol phosphate production (25, 18), together with those of high agonist affinity and ability to induce NO production (14), suggest that endothelial 1D-ARs are involved in the modulation of vascular tone. However, the functional significance of these receptors may be more prominent. It has been recently shown that catecholamines contribute to arteriogenesis and angiogenesis in ischemic tissue (10) and that they contribute to angiogenesis in the wound-healing process (8). In support of these findings, there is also evidence that injury and ischemia augment norepinephrine release from nerves (6, 21). Taking into account these observations, it may be suggested that 1D-ARs represent an important transduction mechanism for the effects of catecholamines on vascular endothelium. This hypothesis is supported by results of the present study, which show, for the first time, that picomolar concentrations of PHE induce endothelial cell proliferation and migration through direct stimulation of 1D-ARs acting through the involvement of the NO synthase pathway. The demonstration that this trophic effect is significantly potentiated by exposure to low oxygen tension suggests that the role of 1D-ARs may become more prominent in the adaptive responses to hypoxic injury of the vasculature.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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1- and 2-adrenoceptors. Eur J Pharmacol 361: 1–15, 1998.[CrossRef][Web of Science][Medline]-adrenoceptor subtypes by smooth muscle cells and adventitial fibroblasts in rat aorta and in cell culture. J Pharmacol Exp Ther 298: 441–452, 2001.1D-Adrenoceptors cause endothelium-dependent vasodilatation in the rat mesenteric vascular bed. J Pharmacol Exp Ther 296: 869–875, 2001.1-adrenergic receptor mRNA expression in human monocytic cells and endothelial cells. J Neuroimmunol 125: 66–72, 2002.[CrossRef][Web of Science][Medline]1A-adrenoceptor antagonist inhibits neointimal growth after balloon injury of rat carotid artery. Am J Physiol Heart Circ Physiol 284: H385–H392, 2003.1-adrenoceptor-mediated constriction of arterioles. Am J Physiol Heart Circ Physiol 281: H873–H881, 2001.1-adrenoceptor subtypes. Circ Res 89: 815–822, 2001.-adrenoceptors mediate migration of smooth muscle cells and adventitial fibroblast in vitro. Am J Physiol Heart Circ Physiol 282: H2364–H2370, 2002.
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