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Cardiovascular Research 2002 55(1):201-212; doi:10.1016/S0008-6363(02)00326-7
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Effect of hypoxia and endothelial loss on vascular smooth muscle cell responsiveness to VEGF-A: role of flt-1/VEGF-receptor-1

Astrid Parentia, Laura Brogellia, Sandra Filippia, Sandra Donninib and Fabrizio Leddaa,*

aDepartment of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pieraccini 6, 50139 Florence, Italy
bInstitute of Pharmacological Science, University of Siena, Siena, Italy

* Corresponding author. Tel.: +39-55-427-1288; fax: +39-55-427-1280 ledda{at}ds.unifi.it

Received 7 November 2001; accepted 18 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The influence of hypoxia and endothelial loss on the responsiveness of vascular smooth muscle cells (VSMCs) to vascular endothelial growth factor (VEGF-A) was tested. Methods and results: Exposure to hypoxia induced a potentiation of cultured cell proliferation in response to either the agonist for the VEGF receptor 1 (flt-1) placental growth factor (PlGF-1) or to VEGF-A. This effect was mediated by the mitogen activated protein kinase (MAPK) cascade, since it was inhibited by the MAPK kinase inhibitor PD98059 and by the farnesyl transferase inhibitor II. Accordingly, PlGF-1 activated extracellular signal-regulated kinase1/2. In rat aortic rings deprived of endothelium and cultured in three-dimensional fibrin gels, an increased sprouting of tubular structures in response to VEGF-A was observed only under hypoxia. Studies on rat aorta preparations revealed an endothelium-dependent vasorelaxation in response to either VEGF-A or PlGF1, which was reversed to a contractile response in endothelium-deprived preparations exposed to hypoxia. Western blot and immunohistochemistry of endothelium-deprived preparations exposed to hypoxia showed flt-1 receptor expression in all medial cells. Conversely, flt-1 mRNA, of endothelium-deprived aortic preparations and of tubular structures, was unchanged by hypoxia. Conclusion: These findings demonstrate that experimental conditions mimicking pathological vascular injury can make VSMCs responsive to VEGF-A through the induction of functional flt-1 receptors.

KEYWORDS Endothelial factors, hypoxia/anoxia; Remodeling; Smooth muscle; Vasoconstriction/dilation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The vessel wall is a complex structure composed of several types of cells, present in different proportions according to size, type, and localization. Pathologic conditions leading to flow decrease and ischemia activate multistep processes leading to vessel remodeling. Cytokine and growth factors, cell–cell and cell–matrix interactions regulate cell response to biologic events. Proliferation and migration of activated vascular smooth muscle cells (VSMCs), associated with the release of abundant extracellular matrix, are fundamental events involved in neointimal growth during atherosclerotic plaque formation and restenosis. In addition, growing evidence indicates that proliferation of VSMCs represents a major event involved in the growth of collateral vessels following chronic artery occlusion. This remodeling of arterioles into collateral arteries, called arteriogenesis [1], seems the only relevant type of vascular growth potentially able to compensate for flow insufficiency after an artery occlusion. Proliferation of smooth muscle cells occurs following endothelial cell activation in response to shear stress, recruitment of monocytes and release of cytokines and growth factors [1–3]. During vascular remodeling, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A) are secreted by many cell types [1,4] such as VSMCs, macrophages and endothelial cells [5] and their gene expression is upregulated by hypoxia and by many cytokines and growth factors [5,6]. VEGF-A is widely considered a pro-angiogenic molecule specifically active on endothelial cells and provided with endothelium-dependent vasodilatory properties [5,7]; owing to these properties, this factor has been proposed as a therapeutic agent suitable for inducing therapeutic angiogenesis in hypoxic tissues of patients suffering from coronary artery and peripheral vascular diseases [8]. It has also been proposed that locally delivered VEGF-A may act as an endothelium protecting agent, able to prevent vascular wall thickening after stent implantation [9]. The proposals about a therapeutic role of VEGF-A are chiefly based on the concept that receptors for VEGF are restricted to endothelial cells [6,10]. Nevertheless, the expression of VEGF receptor1/fms-like tyrosine kinase-1 (VEGFR-1/flt-1 receptor) for VEGF-A has also been reported in other cells, such as monocytes in which they mediate chemotaxis [11]. It has been shown that functional flt-1 receptors are expressed by VSMCs in the neointima of rat carotid after balloon injury [12]. According to this demonstration, flt-1 receptors, which are normally present on endothelial cells, appear in VSMCs accumulating in the neointima within a week after the injury [12].

Owing to the mentioned observations, the aim of our study was to assess whether VEGF-A can activate VSMC proliferation, differentiation in tubular structures and changes in vascular tone, following hypoxic damage and endothelial loss, thus demonstrating that this growth factor can modulate wall remodeling in addition to promoting angiogenesis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats (220–250 g) were purchased from Harlan (Borchen, Germany).

2.2 Cell culture
VSMCs were isolated from the thoracic aorta of male Wistar rats and cultured as previously described [13]. Briefly, the vessels deprived of adventitia and endothelium were aseptically opened longitudinally, cut into 1-mm2 pieces and incubated with 0.1% collagenase for 20 min at 37 °C 5% CO2. Obtained cells were cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin, and were characterized by immunohistochemical assay with an anti-{alpha}-actin monoclonal antibody (Sigma), showing that >95% of cells were {alpha}-actin positive [13].

2.3 Cell proliferation
VSMC proliferation was quantified by the total cell number as previously reported [13]. Briefly, 5x103 cells were seeded in 48-multiwell plates and allowed to adhere overnight. Cells were kept in starving conditions (0.1% FCS) for 48 h in normoxic (21% O2) or in hypoxic conditions (3% O2, PO2 20 mmHg). Then, media were removed and replaced with 1% FCS medium containing the test substances. Proliferation was evaluated after 96 h in normoxic conditions. The effect of VEGF-A was compared to the control condition in 1% FCS medium and to the effect produced by 10 ng/ml platelet derived growth factor (PDGFbb).

2.4 Ex vivo organs
2.4.1 Cell outgrowth from vessel fragments
Rat aortas without adventitia and endothelium were cut in rings 1 mm long, positioned in 48-multiwell plates and included in a fibrin gel obtained by adding bovine fibrinogen solution and thrombin into each well [14]. After fibrin clotting, 2% FCS medium M199 with test substances were added. The organ culture was kept at 37 °C in 5% CO2 in hypoxic or normoxic conditions for 14 days. The medium with test substances was replaced every 2 days. The extent of fibrin gel occupied by tubular structures was quantified after 5, 10, and 14 days (final evaluation is reported) by an inverted microscope using a squared ocular grid (506 µm2 area at 200x magnification) [14]. Cell populations present in the tubular structures were characterized and quantified at day 14. Vessel rings were removed and fibrin was lysated using plasmin. The cell suspension obtained was centrifuged, spread on a slide, fixed in acetone/chloroform 1:1 and stained for a rat endothelium antigen (mouse anti-rat endothelium, clone MRC OX-43, Biosource Int., Camarillo, CA) or for {alpha}-smooth muscle actin (DakoA/S, Glostrup, Denmark) with an avidin–biotin amplified immunoperoxidase technique.

2.4.2 Studies on vascular tone
Rings (3–4 mm width) of thoracic rat aorta were mounted in a 10-ml organ bath filled with warmed (37 °C) and oxygenated (95% O2, 5% CO2) Tyrode solution for isometric measurement [15]. After 90 min of equilibration, concentration–response curves for NA (0.1–1 µM) were performed and a concentration able to induce 50% of the maximum effect was chosen in order to evaluate the relaxant effect of test substances. The effect of VEGF-A was also tested in rat aorta rings after endothelium deprivation [15], in the absence and in the presence of a 24-h hypoxic pretreatment. Hypoxic treatment was performed by incubating aorta rings in Tyrode solution in a humidified incubator with 5% CO2 and 3% O2 for 24 h. Normoxic treatment was performed by incubating aorta rings in a Tyrode solution continuously bubbled with a 95% O2, 5% CO2 gas mixture during the 24-h incubation. Results are expressed as percent of maximal NA-induced contraction.

2.5 Receptor expression
2.5.1 Immunoprecipitation and immunoblotting
Rat aortic rings were promptly frozen in liquid N2 and then were homogenized on ice in lysis buffer followed by centrifugation at 14,000xg for 10 min at 4 °C. Aliquots of 600 µg of total proteins were used to immunoprecipitate flt-1 with a polyclonal rabbit IgG antibody (Santa Cruz Biotech., CA). The immunoprecipitates were run on 7% SDS–PAGE gels and proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, and treated with the flt-1 antibody. Immunoreactive proteins were detected by enhanced chemiluminescence (ECL). The intensities of the bands were quantified by densitometric analysis.

2.5.2 Immunohistochemistry
Flt-1 localization was also assessed on endothelium-deprived frozen aortic rings by means of an immunohistochemical technique. Serial sections (4 µm) were incubated with a rabbit polyclonal IgG antibody against VEGFR-1 (1:50, Santa Cruz Biotech) or an anti-{alpha}-actin monoclonal antibody (1:50, Sigma, St. Louis, MO) and then treated with a biotinylated secondary antibody and appropriate reagents using a kit for immunohistochemical analysis (ABC staining System, Santa Cruz Biotech). Sections were counterstained with Mayer's hematoxylin.

2.5.3 Differential RT-PCR analysis

(a) Flt-1 and VEGF-A mRNA expression in aorta. Aortic rings treated or not treated with hypoxia for 24 h were promptly frozen. VSMCs cultured in either normoxic or hypoxic conditions were also used. Total RNA was extracted using the RNAzol method (Ultraspec RNA, Biotecx). Reverse transcription of 1 µg of total RNA was carried out as already described [16] and flt-1 or VEGF-A were amplified with specific primers, the latter designed to detect the four previously described distinct VEGF-A isoforms (VEGF-121, -145, -165, -189), as follows: flt-1 sense 5'-AGG CAG CGG ATT GAC CAA AG-3', antisense 5'-TTC CTG CAC CTG TTG CTT CC-3' [17]; VEGF-A sense 5'-CCA TGA ACT TTC TGC TCT CTT G-3', antisense 5'-GGT GAG AGG TCT AGT TCC CG-3' [18]. The 18S rRNA was chosen as internal standard by using a competimer technology for quantitative RT-PCR (QuantumRNATM Universal 18S, Ambion, Austin, TX). Preliminary experiments established the optimal ratio of 18S Primers:Competimer dilutions (3:7 for flt-1 and 2:8 for VEGF-A). Then a differential RT-PCR was performed in sequential cycles (30) including 30 s denaturation at 94 °C, 30 s annealing conditions at 55 °C for flt-1 and 60 °C for VEGF-A, and 30 s extension at 72 °C. Amplificates were electrophoresed in 3% agarose gel and a quantitative evaluation was obtained as ratios between optical density of the target genes and 18S rRNA amplification products.
(b) flt-1 mRNA expression in tubular structures from aortic rings. Tubular outgrowths were recovered by liquefying fibrin using plasmin [19]. The cells were pelletted and total RNA was isolated using NucleoSpin Nucleic Acid Purification kit (Clontech, Palo Alto, CA). cDNA was synthesized from one-fifth of the total RNA using Sensiscript Reverse Transcriptase (Quiagen, Hilden, Germany) and then was amplified by a differential PCR using sense and antisense primers for flt-1 and for 18S Primers:Competimers. The specific number of cycles was then determined (35 cycles for flt-1 in samples from normoxic conditions, and 40 cycles for flt-1 in samples from hypoxic conditions). Following amplification, amplificates were electrophoresed in 3% agarose gel and a quantitative evaluation was obtained as ratios between optical density of the target genes (flt-1) and 18S rRNA amplification products.

2.6 Extracellular signal-regulated kinase1/2 (ERK1/2) activity
VSMCs were starved for 48 h in normoxia or hypoxia, then were stimulated with 10 ng/ml PlGF-1 over a range of times from 15 min to 1 h in normoxic condition. Thirty micrograms of cell lysate [13] were run on 10% SDS–PAGE electrophoresis, proteins were then transferred to a PVDF membrane, and treated with the anti phosphoERK1/2 antibody (Cell Signaling Tech., Beverly, MA). Immunoreactive proteins were detected by ECL.

2.7 Densitometric analysis
PCR bands and immunoblots were analyzed in triplicate by densitometry using NIH Image 1.60B5. Images of the gel or PVDF membranes were scanned with a flat bed scanner UMAX Magic Scanner v 2.3.1.

2.8 Materials
VEGF-A and PDGFbb were purchased from Calbiochem-Novabiochem Int. (San Diego, CA). PlGF-1 was from R&D Systems Inc. (Minneapolis, MN).

2.9 Statistical evaluation
Data are reported as means±S.E.M. Each experiment was run in duplicate. Statistical analysis was performed using Student's t-test for paired data and analysis of variance followed by Scheffe's test. A P value <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of VEGF-A on VSMC growth
VSMCs were serum starved and incubated for 48 h either in normoxic or in hypoxic conditions, and then the test substances were added and left for 4 more days. VEGF-A (10 and 20 ng/ml) slightly increased VSMC growth, while the selective flt-1 receptor ligand placental growth factor (PlGF-1) [20] significantly promoted cell proliferation at the concentration of 10 ng/ml (Fig. 1A). In the cells exposed for 48 h to hypoxia and then stimulated with the two growth factors, a significant potentiation of cell proliferation was observed in response to either VEGF-A (10 ng/ml) or to PlGF-1 (10 and 20 ng/ml). PDGFbb (10 ng/ml) was significantly effective in promoting VSMC growth, but the response was not significantly changed by hypoxic condition (Fig. 1A).


Figure 1
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  		Fig. 1 (A) Effect of two different concentrations of VEGF-A and PlGF-1 on proliferation of VSMCs exposed to normoxia or hypoxia during the 48-h starving incubation. The effect of PDGFbb is shown for comparison. Bars represent means±S.E.M. of three experiments in duplicate. *P<0.05, **P<0.01 vs. basal proliferation. #P<0.05 vs. VEGF-A or PlGF-1 without hypoxic treatment. (B) Effect of the MEK inhibitor PD98059 (30 µM) and FPT inhibitor II (1 µM) on growth of cells stimulated with 10 ng/ml PlGF-1 with or without hypoxic pretreatment. Bars represent means±S.E.M. of three experiments in duplicate. #P<0.05, ##P<0.01 vs. PlGF-1. (C) ERK1/2 activity in response to 10 ng/ml PlGF-1 in cells pretreated or not pretreated with hypoxia for 48 h. Representative time-course experiment. The band of actin, as a control protein, is also shown.

 
The growth-promoting effect of PlGF-1 on VSMCs was mediated by the MAPK-cascade, since it was significantly impaired by the ras activator inhibitor FPT II (1 µM) [21] and by the MEK inhibitor (PD 98059, 30 µM) [22] (Fig. 1B). The two inhibitors did not induce any significant toxic effect on the cells, as shown by Trypan blue exclusion observations (96±3% and 94±5% viability in untreated and treated cells, respectively). PlGF-1 consistently increased ERK1/2 activity in a time-dependent manner, being more potent in cells pretreated with a severe hypoxia, with maximal effect after 1-h stimulation (Fig. 1C). ERK1/2 activity of unstimulated cells were unchanged after hypoxic treatment.

3.2 Effect of VEGF-A on cell outgrowth
The role of VEGF-A on smooth muscle growth was investigated in an in vitro model of organ culture using rat aorta. Intact and endothelium-deprived aorta fragments were kept in normoxic and hypoxic conditions for up to 2 weeks in M199 with 2% FCS. In normoxic conditions, tubular structures appeared in the whole aorta fragments within 5 days and the maximum response was reached after 2 weeks (mean area of tubules 200±45 mm2). VEGF-A (10 ng/ml) stimulated tube formation (26±9% increase over control), and this effect was significantly increased in hypoxic conditions (45±8% increase over control, P<0.05). In endothelium-deprived aorta fragments, tube formation occurred faster and more abundantly. After 2 weeks the area covered by the new tubules in basal condition was 481±64 mm2. The addition of VEGF-A did not increase the number of tubular structures in normoxia (Fig. 2A and B), while it was significantly effective when vessel fragments were exposed to hypoxia (Fig. 2A and C). PDGFbb strongly promoted tubular structure formation (2.8±0.4-fold), which was unaffected under hypoxic conditions (Fig. 2A). The tubular structures were recovered by enzymatic digestion of the fibrin gel after 2 weeks of culture and the cells were dispersed on a slide. Endothelial and smooth muscle cell characterization was performed by immunohistochemical screening (see Methods). The tubular structures derived from endothelium-deprived artery displayed only ≤2% of endothelium-antigen positive cells either in normoxic or in hypoxic conditions. The addition of VEGF-A to the endothelium-deprived preparation in normoxic conditions did not significantly change the percent of {alpha}-actin positive cells, while significantly reducing them in hypoxia (7±2% fold, n = 3, P<0.01). This finding is in agreement with the knowledge that growing VSMCs appear more spindle-shaped and show less prominent {alpha}-actin staining [23].


Figure 2
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  		Fig. 2 Tube formation from rat aortic fragments deprived of endothelium and included in a 3D fibrin gel in normoxia and hypoxia. (A) Influence of VEGF-A on tubular structure formation expressed as area covered by tubular structures. The effect of PDGFbb is shown for comparison. Bars indicate means±S.E.M. of six experiments. *P<0.05, **P<0.01, vs. serum alone (basal). (B, C) Representative pictures of tubular structures following 14-day incubation with VEGF (10 ng/ml) in normoxia (B) and in hypoxia (C).

 
3.3 Effect of VEGF-A on vascular tone
Since the experiments reported above suggested that hypoxia was able to induce responsiveness to VEGF-A not only in cultured smooth muscle cells but also in ex vivo preparations, we tested the effect of VEGF-A on vascular tone in rat aorta preparations either incubated in physiological conditions or exposed to endothelium deprivation and hypoxic damage. Exposure for 5 min to VEGF-A (10–50 ng/ml) did not modify the resting tone of the preparations. An increase in tension to 536.2±16 mg was induced by exposure to noradrenaline (NA). In NA-precontracted preparations a dose-dependent relaxing effect was induced by VEGF-A (10–50 ng/ml), with a maximal effect observed at 50 ng/ml (Fig. 3A). A concentration-dependent relaxant response was also induced by PlGF1 although to a lesser extent (32±7% relaxation with 50 ng/ml, Fig. 3A and B). In endothelium-deprived preparations the relaxant response to both the growth factors was completely abolished (data not shown). VEGF-A and PlGF-1 did not modify the basal tension when the two growth factors were applied either immediately after endothelium deprivation or after endothelium removal followed by a 24-h exposure to normoxic condition (Fig. 4A and B). However, in endothelium-deprived preparations exposed to hypoxia for 24 h, VEGF-A induced a concentration-dependent contractile effect (Fig. 4A and B) with a maximum response, obtained with 50 ng/ml VEGF-A, amounting to 147.7±3% of the maximum effect of NA. A concentration-dependent contractile response, superimposable on that obtained with VEGF-A, was also induced by PlGF-1 (Fig. 4A).


Figure 3
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  		Fig. 3 (A) Relaxant effect of different concentrations of VEGF-A and PlGF-1 in noradrenaline-preconstricted aortic ring preparations with endothelium. Data expressed as percent relaxation (inhibition of maximal NA-induced contraction). Points represent means±S.E.M. of seven experiments. *P<0.05 vs. 25 ng/ml PlGF-1. (B) Representative tracing showing the effect of two concentrations of PlGF-1 on a preparation with endothelium precontracted by noradrenaline (NA); the response to 3 µM acetylcholine (Ach) is also shown.

 

Figure 4
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  		Fig. 4 (A) Effect of different concentrations of VEGF-A and PlGF-1 on endothelium-deprived aortic ring preparations exposed or not exposed to hypoxia for 24 h. Data are expressed as percent of maximal NA-induced contraction. Points represent means±S.E.M. of 10 experiments. (B) Upper tracing: representative experiment showing the lack of response to VEGF-A in an endothelium-deprived preparation in which the response to noradrenaline (NA) is maintained. Lower tracing: the same as above in an endothelium-deprived preparation exposed to 24-h hypoxia.

 
3.4 Effect of endothelium deprivation and hypoxia on flt-1 expression
Data reported above suggested that the reversal of VEGF-A effect on vascular tone was due to an up-regulation of VEGF-A receptors in VSMCs. The observation that the vasoconstrictor effect of PlGF-1 was superimposable on that elicited by VEGF-A pointed to VEGFR-1/flt-1 receptors as the receptor possibly involved. Flt-1 expression was then measured in endothelium-deprived preparations exposed or not to hypoxia. Immunoblot showed a low expression of flt-1 receptors in lysates of endothelium-deprived preparations. Conversely, in preparations exposed to both endothelial damage and hypoxia, an evident flt-1 up-regulation was detected (Fig. 5A). Immunohistochemical analysis in endothelium-deprived preparations confirmed a weak presence of flt-1 staining confined to medial cells near the lumen (Fig. 5B), whereas all medial cells showed flt-1 immunoreactivity in endothelium-deprived preparations exposed to a 24-h hypoxia (Fig. 5C). Medial cells were also {alpha} actin positive, thus showing that hypoxia-induced flt-1 up-regulation occurred in smooth muscle cells (data not shown). Flt-1 was also detected, as expected, in endothelial cells of intact aortic preparations (data not shown). On the contrary, VEGFR-2/KDR was never revealed in endothelium-deprived preparation either exposed or not exposed to hypoxic treatment (data not shown). Differential RT-PCR analysis revealed the presence of flt-1 mRNA on endothelium-deprived aorta, which was not modified by hypoxic treatment (Fig. 6A). The analysis of flt-1 mRNA from tubular structures showed the presence of flt-1 mRNA on tubules either with or without endothelium (Fig. 6B). The expression of flt-1 mRNA was almost doubled by hypoxic condition in the tubular structures with endothelium while it was unaffected in the endothelium-deprived structures (Fig. 6B). As expected, hypoxia induced an up-regulation of VEGF-A mRNA isoforms in either aortic preparations or in cultured VSMCs (Fig. 6C). The less representative mammalian isoforms, VEGF-145 and -189, were expressed only after hypoxic treatment (Fig. 6C).


Figure 5
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  		Fig. 5 Effect of endothelium removal and hypoxia on flt-1 expression in aortic preparations. (A) Immunoreactive band densitometry (OD, optical density); bars represent means±S.E.M. of five experiments. *P<0.01 vs. normoxia. Figure inset: representative immunoblot in endothelium-deprived aortic rings without (lanes 1 and 2) and with (lanes 3 and 4) 24-h hypoxia. (B, C) Flt-1 immunostaining of VSMCs in the media of endothelium-deprived preparations without (B) and with (C) exposure to hypoxia.

 

Figure 6
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  		Fig. 6 (A) RT-PCR for mRNA expression of VEGFR-1/flt-1 in endothelium-deprived preparations. Densitometrical analysis (OD) of PCR bands, expressed as OD ratio of flt-1 receptor with the 18S rRNA; means±S.E.M. of three experiments. Figure inset: representative RT-PCR: preparation not exposed (lane 2) and exposed (lane 3) to hypoxia; lane 1: marker, 100 bp DNA ladder. (B) Flt-1 mRNA expression in tubular structures with and without endothelium, exposed or not exposed to hypoxia. Densitometrical analysis (OD) of PCR bands, expressed as OD ratio (flt-1/18S); means±S.E.M. of three experiments. Figure inset: representative RT-PCR: tubular structures with (lanes 1 and 2) and without (lanes 4 and 5) endothelium following hypoxic treatment (lanes 2 and 5); marker, 100 bp DNA ladder (lane 3); *P<0.05 vs. normoxia. (C) Representative experiment showing VEGF-A mRNA isoforms in endothelium-denuded aortic preparations (lanes 2, 3) and in cultured VSMCs (lanes 4, 5) under normoxia (lanes 2 and 4) and hypoxia (lanes 3 and 5).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study produces evidence, for the first time, that experimental conditions mimicking events possibly occurring in vascular pathological conditions, such as a severe hypoxia associated with endothelial damage, can make VSMCs responsive to VEGF-A. Cultured VSMCs responded to VEGF-A by proliferating, and endothelium-deprived aorta preparations produced tubular structures in a 3D fibrin gel in response to VEGF-A when exposed to hypoxia. Moreover, the relaxant response to VEGF-A was reversed to a contractile effect in endothelium-deprived preparations exposed to hypoxia. Severe hypoxic treatment of cultured VSMCs was able to induce mitogenic activity of VEGF-A. The mitogenic response was likely linked to activation of VEGFR-1/flt-1 receptor, since it was elicited also by the selective receptor flt-1 ligand PlGF-1, which is even more potent than VEGF-A. Moreover, this proliferation increase was linked to ras/MAPK kinase activation, since it was prevented by a farnesyl transferase inhibitor and by a MEK inhibitor. It is noteworthy that activation of MAPK in response to PlGF-1 has been previously demonstrated only in cultured endothelial cells, and that it has been considered involved in their proliferative response to the growth factor [24]. Thus, the present observations suggest a possible role in vascular pathology for PlGF, whose biological role is still uncertain and submitted to investigation [25,26].

The growth of VSMCs represents an early event in vascular wall remodeling, such as that which occurs in arteriogenesis after prolonged arterial occlusion. During this process, the collateral vessels increase their diameter through an increased mitogenesis of vascular cell populations [27]. Arteriogenesis, even more than angiogenesis, is necessary to limit the size of the area submitted to hypoxic damage and to restore the blood flow after the occlusion of a major artery. Hypoxia is a potent regulator of a variety of biological processes, including angiogenesis and vascular contractility [28,29]; however, less is known about its role in arteriogenesis. Experimental data demonstrated that VEGF-A and VEGFR-1/flt-1 (and, to a lesser extent, VEGFR-2/KDR) are up-regulated by hypoxia in endothelial cells [5,6,30]. The present observation that VSMCs can become responsive to VEGF-A through an up-regulation of flt-1 receptors induced by hypoxia, suggests that VEGF-A may have a role in vascular wall remodeling in the presence of hypoxia. Our experimental findings, indeed, showed that hypoxia is able to disclose an activity of VEGF-A on VSMCs, while the effect of PDGFbb was independent of O2 tension. This hypoxia-induced growth effect of medial cells was observed not only in cultured cells but also in an organ culture model. The observation that VEGF-A promoted pseudo-capillary sprouting from aortic rings with endothelium is in agreement with previous findings [31], demonstrating the role of VEGF-A on angiogenesis. However, we showed a sprouting of tubular structures in the absence of endothelium, demonstrating that the mitogenic response of medial/VSMC cells to VEGF-A is not simply dependent on a loss of differentiation of cultured cells, but is strictly dependent on exposure to hypoxia.

Functional studies also showed that the well-known vasorelaxant effect of VEGF-A [32] was reversed into a vasoconstrictor response of an intensity comparable to that elicited by noradrenaline in endothelium-deprived preparations only when hypoxia was applied. PlGF-1 similarly induced an endothelium-dependent vasorelaxant response in intact preparations, which was never described before. However, this response was changed to a vasoconstriction in endothelium-deprived preparations exposed to hypoxia. It has been recently shown that microvascular constriction is present during acute severe myocardial ischemic damage [33]. This paradoxical vasoconstrictor response probably allows redistribution of collateral circulation and might represent a mechanism able to maintain correct pressure in the ipo-perfused vascular units [33]. Extrapolation of the results of our study to in vivo pathological conditions is obviously difficult, since our findings have been obtained in cultured cells and in preparations obtained from a vessel (aorta) which is not involved in arteriogenesis. Nevertheless, one may speculate that the vasoconstrictor response observed in the present study could represent one of the events, aimed at rescue from severe hypoxia, induced by VEGF-A following flow insufficiency.

Altogether the above-mentioned data suggest that a phenotypic change occurs in VSMCs in response to hypoxia, which likely consists of the expression of specific VEGF receptors in VSMCs. This hypothesis is supported by results obtained using Western blot and immunohistochemistry. VSMCs of the aortic tunica media did not express flt-1 receptors in the presence of endothelium. After endothelium-deprivation, only some cells near the lumen were positive for this receptor; however, in endothelium-deprived preparations exposed to hypoxia, almost all medial VSMCs expressed flt-1 receptors. Receptor expression by internal VSMCs was evidently insufficient to cause an increase in tone in response to VEGF-A, while the response became functionally significant when receptor expression was induced in the majority of cells. Moreover, the observation that expression of flt-l receptor mRNA was not influenced by hypoxia either in aortic preparations (24 h hypoxia) or in tubular structures (14 days hypoxia), suggests that the mechanism activated by acute and chronic hypoxia is similar and that the regulation of this receptor occurs at the translational level, regulation which is transcriptional for other cell types such as for endothelial cells [5,6].

An important role of PlGF/flt-1 in vascular pathology is also suggested by this manuscript. It has been proposed that PlGF stimulates angiogenesis by displacing VEGF from flt-1, which may function as an inert ‘decoy’ by binding VEGF, thus regulating the availability of VEGF for activation of VEGFR-2 [25]. Thus, PlGF potentiates VEGF effects on the endothelium, but might also directly stimulate endothelial cells independently of VEGF. Despite these interesting findings, the role of PlGF/flt-1 on the activation of VSMCs in pathological condition is still unknown. Present data demonstrate that, in a pathological setting of hypoxia and endothelial loss, an involvement of flt-1 on VSMC activation occurs and that it is probably not mediated by VEGF-2, since this receptor was never present and/or upregulated in the medial cells and in cultured VSMCs.

Regulation of VSMC function is potentially important in the understanding of events responsible for the activation of VSMCs in pathological conditions. Since it is known that VSMCs are able to express VEGF-A, the possibility that VEGF-A itself [4] and VEGF receptors (present data) can be upregulated by hypoxia, suggests that autocrine regulation of VSMC functions can be involved in pathological processes characterized by vascular remodeling [13]. Activation and proliferation of VSMCs are processes common to different types of wall remodeling and to atherosclerotic plaque formation. It is important to identify the effect of a single factor on a single cell population in the context of the vessel wall, in order to understand mechanisms leading to either beneficial or detrimental conditions. VEGF-A stimulates new capillary formation and thus is necessary to restoration of the blood flow in ischemic tissue. Nevertheless, the acquisition of a mitogenic phenotype by vascular smooth muscle cells in response to VEGF-A (present data) prompts speculation of a possible involvement of VEGF-A in vascular pathology.

Time for primary review 35 days.


   Acknowledgements
 
Supported by grants from the Italian Ministry of University and Scientific and Technological Research to FL, and from University of Florence to AP.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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