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1
* Department of Research and
University Medical Outpatient Department, University Hospital, CH-4031 Basel, Switzerland
1Correspondence: Department of Research and University Medical Outpatient Department, University Hospital, CH-4031 Basel, Switzerland. E-mail: ebattegay{at}uhbs.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: microvessels • smooth muscle • endothelium • embryogenesis • neovascularization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Proliferation of ECs is pivotal in the formation of new microvessels. This process, termed angiogenesis, is important during organ development in embryogenesis (2)
and tumor growth, (3)
, and contributes to diabetic retinopathy, psoriasis, rheumatoid arthritis, and atherosclerosis (3)
.
Hypoxia is an important stimulus of SMC and EC proliferation and is found in atherosclerotic lesions and rapidly growing tumors (4
5
6)
. Responses to hypoxia can be acute, occurring over a period of seconds to minutes, or chronic, with a time course of hours to days. Lasting hypoxia induces the expression of genes encoding transporters, enzymes, and growth factors, which determine molecular and histological modifications to reduce the cellular need and dependence on O2 and increase O2 supply to the tissues by facilitating nonoxidative synthesis of ATP or by increasing the O2-carrying capacity of blood (7)
. Moderate levels of hypoxia (3–5% O2) are easily reversible and enable adaptive physiological responses such as neovascularization (7)
. On the other hand, anoxia or an extremely low pO2 (0–0.5% O2) contributes to the pathophysiology of tumor progression and cell apoptosis rather than to physiological adaptive responses (7)
.
Hypoxia activates hypoxia-inducible transcription factors (HIFs) that induce the expression of vascular endothelial growth factor (VEGF) (8
, 9)
, VEGF receptor flt-1 (10)
, basic fibroblast growth factor (bFGF) (11)
, platelet-derived growth factor (PDGF), nitric oxide synthases, and angiopoietin 2 (12)
. Thus, hypoxia can up-regulate numerous genes that trigger neovascularization (3
, 8
, 9)
, proliferation and remodeling of the vascular wall (7)
.
Little work has been done on the proliferation-promoting signaling pathways that might be induced by hypoxia itself and thereby affect vascular wall cell proliferation directly or influence the response to locally produced growth factors (13)
. The pathways used to achieve cell proliferation in response to hypoxia may be cell specific. Members of the MAP kinase family are activated in different hypoxic tumor cells, the pulmonary arterial wall, and cultured human osteoblastic periodontal ligament cells (13)
. The PI3K-Akt-mTOR pathway has been implicated in the hypoxic response induced by HIF transcription factor in transformed cells (14
, 15)
. Proliferation also increases in vascular wall cells in response to hypoxia, but the intracellular signaling elements mediating this response have not been fully defined (16
, 17)
.
We have investigated the effects of moderate hypoxia (3% O2) and cell-specific, locally produced growth regulatory molecules on proliferation of vascular arterial wall cells and angiogenesis in vitro. We have systematically followed the signaling pathways induced by growth factors and determined elements of signaling that were unaffected, compulsory, or specialized for the response to hypoxia.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actin and Caldesmon staining. RA-SMCs were used between passages 4 and 15. Primary rat aortic endothelial cells (RA-EC) were isolated by outgrowth from intact aortas into a 3-dimensional collagen gel and characterized by endothelial cell markers (CD31, vWF). Primary cultures of human adult aortic SMC (HU-SMC) and mouse NIH3T3 cells were provided by Eleonore Köhler of Basel, Switzerland, and used between passages 4 to 9 and 5 to 20, respectively. Cells were grown in DMEM (Oxoid, Basel, Switzerland) supplemented with 10% fetal calf serum (FCS; Seromed, Berlin, Germany), nonessential amino acids, sodium pyruvate (Oxoid, Basel, Switzerland), and a penicillin-streptomycin-amphotericin formula (Life Technology, Basel, Switzerland). For hypoxia, cultures were incubated in a hypoxia incubator (Ismatec, Basel, Switzerland) with a gas mixture containing 3% O2 and 5% CO2, balanced with nitrogen.
Cell proliferation assays
To determine cell numbers, 103 cells/well were seeded into 96-well plates and after 24 h the normal culture medium was replaced with serum-free medium (FCS substituted with 0.1% BSA). Cultures were incubated for 24 h at 21% O2 (normoxia) and at 3% O2 (hypoxia), then stimulated with growth factors (PDGF-AA, PDGF-BB, bFGF, EGF, VEGF, or insulin; R&D systems, Minneapolis, MN) or diluent. Each condition was performed in octuplicate and cultures were supplied with fresh growth factors every second day. After 96–120 h, cell numbers were assessed using Cell Proliferation Reagent WST-1 (Roche Molecular Biochemicals, Rotkreuz, Switzerland) according to the manufacturer’s specifications. Control experiments comparing cell enumeration by the WST-1 assay or by direct counting after enzymatic dissociation showed an exact proportionality of data.
For DNA synthesis assays, cells were grown to subconfluency in normal culture medium. Medium was changed to serum-free medium and cells were cultured for 24 h under normoxia or hypoxia. Thereafter cultures were exposed for 24 h to growth factors or serum in fresh medium that had been pre-equilibrated to 3% O2 in order to avoid reoxygenation. Cultures were pulsed for the last 8 h with 1 µCi/ml [3H]-thymidine, then washed, and incorporation of [3H]-thymidine was determined as described previously (18)
.
Where used, inhibitors were included at the beginning of, and throughout normoxic and hypoxic incubations. Inhibitors were also added freshly 1 h before growth factor addition. Dose response curves were performed to determine suitable concentrations. The following inhibitors were used: 1) LY 294002, 1–20 µM (Alexis Corp., San Diego, CA; ref 19
), 2) Ro-32–0432, 1 µM (Calbiochem, San Diego, CA; ref 20
), 3) FTase inhibitor III, 200-2000 nM (Calbiochem; ref 21
), 4) FPT inhibitor I, 200-2000 nM (Calbiochem; ref 21
), 5) rapamycin, 0.2-500 nM, (Alexis Corp; ref 22
23
), 6) PD 98059, 3-30 µM (Calbiochem; ref 24
).
Transfection of EC
The complete cDNA of human mTOR tagged with a hemagglutinin extension was obtained from George Thomas (FMI, Basel, Switzerland) and subcloned into a pIRES2-EGFP vector (Clontech, Palo Alto, CA). The vector contained a geneticin resistance gene and an enhanced green fluorescent protein as selection markers. Low passage RA-EC were grown in 15 cm dishes to 60% confluency and transfected with a plasmid preparation of the expression vector by Geneporter I lipofection according to the manufacturer’s instructions (Axonlab, San Diego, CA). Transfection was monitored by green fluorescence of GFP expressing proteins 48 h after transfection. To select for stable transfection, geneticin was added at a titrated concentration (150 µg/ml) for 1 wk. Single colonies of cells were subcloned, assessed for expression of transfected protein by Western blot analysis of protein lysates and used for proliferation assays.
Endothelial sprout formation in 3-dimensional collagen gels (in vitro angiogenesis)
Collagen I gels were prepared by mixing seven parts type I collagen (3 mg/ml), one part 10x DMEM (Oxoid, Basel, Switzerland), and two parts NaHCO3 (11.76 mg/ml) on ice. Fibrin gels were prepared by mixing 3 mg fibrinogen (Sigma-Aldrich, Buchs, Switzerland) per milliliter of serum-free DMEM with 300 µg/ml of thrombin on ice; 100 µl/well of either solution was placed into 48-well plates and allowed to polymerize overnight at 37°C. Gels were overlaid with 300 µl serum-free DMEM and equilibrated overnight. Thoracic aortas were excised from Sprague-Dawley rats (RCC, Füllinsdorf, Switzerland), cut in 1 mm2 squares, placed onto the gels endothelium facing down, and overlaid with three drops of unpolymerized collagen or fibrinogen mixture. After polymerization, gels were overlaid with 300 µl serum-free DMEM and incubated for 24 h under normoxic (21% O2) and hypoxic (3% O2) conditions. Aortic explants were then exposed to growth factors (20 ng/ml PDGF-BB, 20 ng/ml PDGF-AA, 10 ng/ml bFGF, 10 ng/ml VEGF; from R&D systems, Minneapolis, MN), serum (10% FCS; Seromed, Berlin, Germany), or diluent (0.1% HAc, 0.25% BSA) and incubated for 8–14 days under normoxia and hypoxia, with fresh addition of growth factors every third day. Fibrin gels were protected from degradation by adding 300 µg/ml -amino caproic acid (Sigma-Aldrich, Buchs, Switzerland) every third day. Endothelial sprouts were photographed digitally on a light microscope (Zeiss, Feldbach, Switzerland); the extent of sprout formation was determined by comparison with a standardized scale by two independent investigators and averaged.
Northern blot analysis
Samples of 20 µg total RNA were electrophoresed in a 1% formaldehyde agarose gel, transferred onto nylon, and probed with 32P-labeled human PDGF ß receptor (EcoRI fragment; 2650 bp) and chicken GAPDH (PstI fragment; 1150 bp) at 68°C according to the manufacturer’s instructions (QuickHyb, Stratagene, La Jolla, CA).
Immunoblot analysis
Cells were lysed in 1x SDS buffer (0.28 M Tris-HCl, pH 6.8, 45% glycerol, 0.1 M SDS) supplemented with the inhibitor mixture (Roche Molecular Biochemicals, Rotkreuz, Switzerland) and 1 mM ortho-vanadate. Protein contents were quantified using the BCA Protein Assay Reagent (Pierce, Rockford, Il) and 10 µg protein per lane was subjected to SDS-gel electrophoresis and electroblotting. Polyclonal immunoglobulin (IgG) against PDGF receptor ß subunit, PDGFR receptor subunit, mTOR (sc-431, sc-432, sc-8319; Santa Cruz Biotechnologies, Santa Cruz, CA), monoclonal mouse anti-pY IgG (PY20; Transduction Laboratories, San Diego, CA), and polyclonal anti-PKB-Ser374 (Upstate Biotechnology, Lake Placid, NY) were used for detection. HRPO-conjugated IgGs (Transduction Laboratories, San Diego, CA) were used for visualization of relevant proteins on X-ray films (Kodak, Geroldswil, Switzerland) by a chemiluminescence reaction.
Statistical analysis
All results depicted represent compiled data of experiments repeated at least three times using at least two different cell isolates or aortic explants. Data points are given as the mean values ± SE of three or more individual experiments. After assessing for normal distribution, statistical significance of compiled data from individual experiments was determined by the Bonferroni t test and the Student-Newman-Keuls test using the program Primer of Biostatistics (McGraw-Hill, New York, NY).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A), but not EGF or insulin (data not shown), increased DNA synthesis under conditions of normoxia (Fig. 1A
). Hypoxia further enhanced DNA synthesis above levels seen under normoxia in response to PDGF-BB (1.8±0.1-fold, P<0.001, n=8) or PDGF-AA (1.8±0.1-fold, P<0.005, n=3) but not to FCS (Fig. 1A
). Hypoxia increased DNA synthesis in response to bFGF, but a level of statistical significance was not attained (Fig. 1A
; P=0.08, n=3). There were no differences between hypoxia and normoxia with respect to SMC migration, elongation and apoptosis in response to PDGF (data not shown). This suggests a specific effect of hypoxia on PDGF-mediated cell proliferation.
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The proliferation response to hypoxia and PDGF-BB was further examined in vascular cells from different species by assessing cell numbers. In rat and human aortic SMC, hypoxia increased the cell proliferation response to PDGF-BB significantly (Fig. 1B
; P<0.005, n=3). In rat aortic EC, hypoxia increased the cell proliferation response significantly to PDGF-BB and to bFGF (Fig. 1C
; P<0.005, n=3) but not to VEGF (data not shown). In mouse NIH3T3 fibroblasts, hypoxia doubled cell numbers in response to PDGF-BB (Fig. 1D
; P<0.005, n=3). Hypoxia did not increase cell proliferation to PDGF-BB in a large T antigen-immortalized HMEC-1 (25)
or rat SMC proliferation in response to FCS (data not shown).
Of the growth factors tested in SMC, PDGF-BB elicited the greatest mitogenic response; the amplifying effects of hypoxia were also most marked for PDGF-BB. Therefore we investigated whether the effects of hypoxia could be due to increased PDGF receptor expression or receptor activation (tyrosine phosphorylation). As shown by Northern and Western Blotting, PDGFRß transcript and PDGFRß and - protein levels in RA-SMC after 6–48 h of incubation under hypoxia were similar to those under normoxia (Fig. 2
A, upper and middle panels; data shown for 24 h). Hypoxia did not modulate either PDGF-BB- or PDGF-AA-induced tyrosine phosphorylation of the 180 kDa PDGFR after 5 min of stimulation with PDGF-BB or PDGF-AA in RA-SMC (Fig. 2A
, lower panel). We and others have shown the presence and proangiogenic function of PDGFRß on aortic endothelial cells (26
27
28
29
30)
. In RA-EC, PDGFRß and PDGFR were also expressed but not modulated by hypoxia over a period of 6–48 h of hypoxic incubation (Fig. 2B
; data shown for 24 h). Thus, in RA-SMC and RA-EC, hypoxia does not modulate PDGFRß or PDGFR subunit expression or activation.
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PI3K activity is required for the proliferative response of vascular cells and angiogenesis in vitro under normoxia and hypoxia
We then hypothesized that hypoxia might affect signaling pathways involved in proliferative responses to PDGF isoforms. Both PDGF receptor subunits can mediate cellular proliferation of SMC and other cells by activating MAP kinase (MAPK) through p21ras and PKC pathways (31)
. PDGF also activates phosphoinositide-3 kinase (PI3K) (31)
. We therefore investigated the influence of pharmacological inhibitors of the main signaling pathways (PI3K, PKC isoforms, MAPK, p21ras) on DNA synthesis in response to PDGF-BB, PDGF-AA, bFGF, and serum under conditions of normoxia and hypoxia.
Inhibition of p21ras (2 µM FTase III inhibitor) and PKC isoforms , ßI, ßII, 
, and 
(100 µM Ro-32–0432) did not affect DNA synthesis of RA-SMC in response to PDGF-BB (Fig. 3
A). In contrast, the PI3K inhibitor LY294002 dose-dependently (2–20 µM) decreased DNA synthesis in response to PDGF-BB, PDGF-AA, bFGF, and 5% FCS under normoxia and hypoxia (Fig. 3A, B
). At the maximal dose of 20 µM, DNA synthesis of RA-SMC was completely abrogated. We conclude that activity of PI3K is crucial for DNA synthesis in SMC but that it does not contribute to the difference in response between normoxia and hypoxia. PD98059 was used to inhibit MAPK, a downstream target of PI3K and p21ras (31)
. This inhibitor only moderately lowered DNA synthesis induced by PDGF and FCS under normoxic and hypoxic conditions (Fig. 3A
).
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Hypoxia does not modulate phosphorylation of PI3K downstream target protein kinase B (PKB)
The above data suggest that other signal transduction elements downstream of or parallel to PI3K might be regulated by hypoxia. We first analyzed activity of PKB in RA-SMC by assessment of S-473 phosphorylation (32)
. PKB/S-473 phosphorylation induced by PDGF was not different under conditions of hypoxia and normoxia (Fig. 4
). LY294002 dose-dependently inhibited PKB/S-473 phosphorylation under normoxic and hypoxic conditions, whereas rapamycin had no effect (data not shown). Altered activity of PKB thus does not explain the observed hypoxia-mediated potentiation of proliferation. Therefore, we conclude that downstream signaling elements linked to PI3K signaling other than PKB are modulated by hypoxia.
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Rapamycin specifically abrogates the hypoxia-mediated increase in growth factor-mediated vascular cell proliferation
Mammalian TOR (a target of rapamycin) is a PI kinase family-related element of signaling. Rapamycin, a specific mTOR inhibitor, was used to examine the role of mTOR signaling in the hypoxia-induced potentiation of DNA synthesis and proliferation in response to PDGF. Although PDGF-BB- and PDGF-AA-induced DNA synthesis responses of RA-SMC under normoxic conditions were not affected by rapamycin, the mTOR inhibitor caused a dose-dependent abrogation of hypoxia-amplified DNA synthesis responses (Fig. 5
A). Rapamycin also dose-dependently inhibited the hypoxia-potentiated proliferative responses of HA-SMC to PDGF-BB, but was without effect on mitogen-induced proliferation under normoxia (Fig. 5B
). Analogous effects of rapamycin were observed in bFGF- and PDGF-BB-stimulated RA-EC whereby proliferation was inhibited only under hypoxic conditions (Fig. 5C
). We conclude that rapamycin does not affect EC and SMC proliferation under normoxic conditions but specifically blocks the amplifying effects of hypoxia on mitogen-stimulated proliferation.
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Hypoxia increases sprout formation in rat and mouse 3-dimensional models of angiogenesis via a rapamycin-sensitive mechanism
Endothelial proliferation is a key step in angiogenesis, so we examined the effects of hypoxia and rapamycin using an in vitro model for this process. Mouse (Fig. 6
A–G) and rat (Fig. 6H
) aortas were embedded in fibrin or collagen gels and endothelial sprout formation quantified after 10 days culture under conditions of normoxia and hypoxia without or with PDGF-BB or bFGF. Hypoxia promoted in vitro angiogenesis of mouse and rat aortas in the absence of growth factors (Fig. 6A, B, I
) and massively amplified sprout formation in the presence of PDGF-BB (Fig. 6C, D, I
) or bFGF (data not shown).
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We studied the effects of rapamycin (10 nM: Fig. 6E-G
; 50 nM: Fig. 6H
) in this in vitro angiogenesis model. Rapamycin potently inhibited PDGF- and bFGF-induced sprout formation from rat and mouse aortas under conditions of hypoxia (Fig. 6F-H
), whereas the effect under normoxia was insignificant (Fig. 6E, G, H
).
mTOR overexpression in EC further enhances proliferation under hypoxia
To further assess the role of mTOR in hypoxia-induced endothelial sprout formation, we investigated the effects of mTOR overexpression in proliferation of EC that had been derived from rat aortic endothelial sprouts. We subcloned the human mTOR wild-type cDNA into a mammalian expression vector (pIRES-EGFP) driven by a CMV promotor and transfected low-passage rat aortic EC by lipofection. Geneticin was used to select stably transfected EC and transfection was monitored microscopically by visualization of GFP. Expression of transfected mTOR was confirmed by immunoblot detection of the hemagglutinin tag (HA tag), fused with the mTOR-cDNA (Fig. 7
A, upper panel). Immunoblotting using anti-mTOR (mTOR) antibody confirmed increased expression (1.4-fold) in transfected EC (Fig. 7A
, left lower panel). We also assessed whether mTOR protein levels in wild-type (wt) EC were increased under conditions of hypoxia. However, mTOR protein levels from cells cultured for 30 h under conditions of hypoxia were unchanged when compared with normoxia (Fig. 7A
, right lower panel). Cell proliferation of mTOR transfected and wt EC was assessed under conditions of normoxia and hypoxia in the presence of 20 ng/ml PDGF-BB and increasing concentrations of rapamycin (0.2–200 nM). mTOR overexpression in EC further enhanced proliferation under conditions of hypoxia compared with untransfected EC by 
1.6-fold (Fig. 7B
). The proliferation of mTOR transfected EC under normoxic conditions was increased compared with untransfected EC, albeit to a much lesser extent (Fig. 7B
). Rapamycin inhibited the proliferation under hypoxia in mTOR transfected and untransfected EC (Fig. 7B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, hypoxia altered neither the levels of PDGF receptor and ß subunits nor the extent of their phosphorylation.
Inhibition of the main signaling pathways induced by PDGF and bFGF clearly showed that neither Ro-32–0432-sensitive PKC isoforms nor p21ras were involved in mediating the mitogenic response in SMC. Hypoxia can enhance the activity of various MAP kinases in various tumor cells (13)
. In our SMC, MAPK (ERK1/2) was responsible for one-fifth of the overall proliferation rate under hypoxia and normoxia (Fig. 3A
). Thus, activity of ERK1/2 was not specific for the increase in proliferation observed under hypoxia in vascular cells. Complete abrogation of proliferation was observed when PI3K was blocked with a high dose of LY294002. Increasing concentrations of LY294002 dose-dependently inhibited proliferation under normoxia and hypoxia (Fig. 3B
), suggesting that PI3K is required for growth factor-induced mitogenesis but not differentially regulated by hypoxia. Accordingly, activity of the PI3K downstream target PKB was not enhanced when comparing PDGF-induced S473 phosphorylation under normoxia vs. hypoxia (Fig. 4)
. However, rapamycin specifically blocked the increase in proliferation observed under hypoxia in rat and human aortic SMC and in rat aortic EC (Fig. 5)
. This specific inhibition of the hypoxia-amplified response by rapamycin was also observed in PDGF and bFGF-induced angiogenesis of mouse and rat aortas in vitro (Fig. 6)
. These observations with rapamycin allow us to conclude that hypoxia amplifies vascular proliferation and angiogenesis in vitro via the target of rapamycin, mTOR. This hypothesis is further strengthened by our observation that proliferation of mTOR overexpressing RAEC was 1.6-fold higher under conditions of hypoxia than with untransfected cells. Thus, this is a novel, additional mechanism at the postreceptor level of growth factors by which hypoxia can enhance cell proliferation.
We further conclude that PI3K-dependent signaling mediates the mitogenic response of vascular wall cells but is not increased by hypoxia per se (Fig. 8
). Rather, hypoxic mTOR signaling converges with mitogenic signaling further downstream and thereby increases proliferation in vascular cells up to 2-fold (Fig. 5)
and, as seen in the angiogenesis assay in vitro, up to 10-fold (Fig. 6I
). Under normoxia and hypoxia, growth factor (PDGF, bFGF) -induced signaling is partially transmitted via ERK1/2 (Fig. 3A
and Fig. 8
), possibly via PI3K-dependent activation of MAPK (Fig. 8)
(33)
. In contrast, inhibition of p21ras signaling upstream of MAPK did not affect vascular wall cell proliferation.
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The preferred mTOR downstream target that mediates the increase in vascular cell proliferation and angiogenesis under hypoxia remains to be determined. One downstream target of mTOR, the serine/threonine kinase p70s6k, increases activation of the ribosomal machinery and induces G1 phase-promoting genes such as cyclin D (34
35
36)
. mTOR may further transmit hypoxia-induced signals via 4E-BP1 (37)
, the p70s6k homologue p54S6kinase2 (S6K2) (38)
, or other downstream targets (Fig. 8)
.
mTOR (also known as FRAP, RAFT, and RAPT) was the initially identified member of a novel family of PI kinase-related kinases that function in surveillance pathways (39)
. mTOR plays a central role in controlling cellular growth (39
40
41)
. The molecular mechanisms that regulate mTOR activity are still unclear. The availability of nutrients (amino acid levels) regulates mTOR function (42
43
44
45
46)
. mTOR signaling is also modulated by growth factors via the PI3K/PKB pathway (44)
. Intracellular concentrations of ATP have been shown to regulate mTOR (42)
. In a recent study, phosphatidic acid (PA) was shown to directly bind to the domain in mTOR targeted by rapamycin and thereby increase mTOR’s ability to activate downstream effectors (47)
. Previous reports have indicated that hypoxia increased PA levels via activation of diacylglycerol kinase (48)
. Here we show that low oxygen levels increase mTOR function (Fig. 8)
. It remains to be determined whether hypoxia increases mTOR signaling via PA. The mTOR pathway has been implicated in the regulation of HIF-1a, the master control transcription factor of the cellular response to hypoxia (14)
.
The importance of our findings is underscored by recent clinical studies with rapamycin. Rapamycin has been efficacious in treating solid tumors in patients with metastatic renal cell carcinoma and non-small cell lung, prostate, and breast cancers (49)
. The anti-tumor efficacy of rapamycin has supported the development of compounds targeting the mTOR signal transduction pathways (49)
even though it is not clear whether tumor inhibition is due to inhibition of tumor vascularization and/or direct inhibition of tumor cell growth. Our results suggest that rapamycin may inhibit angiogenesis and vascular proliferation and thus inhibit tumor vascularization. In the field of cardiovascular disease, rapamycin-coated stents have been extremely effective in inhibiting excessive SMC proliferation (restenosis) in humans (50
, 51)
. Hypoxia occurs in tumors, vascular medial areas, and atherosclerotic lesions (4
, 52)
. Similarly, growth factor receptors are up-regulated in tumors (53)
and during all phases of atherogenesis (54)
. Therefore, our findings demonstrating mTOR involvement in hypoxia-facilitated growth responses to mitogens (PDGF, bFGF) may provide a molecular basis for the efficacy of rapamycin in tumors and restenosis.
In conclusion, our data suggest that in addition to energy levels (42)
, nutrients (46)
, and lipids (47)
, oxygen levels regulate mTOR (Fig. 8)
. This is a new role for the mTOR pathway that may contribute to control morphogenic, repair, and disease processes in which vascular cell proliferation and angiogenesis are involved.
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ACKNOWLEDGMENTS |
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Received for publication November 15, 2001.
Revision received February 19, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1.4-fold greater than in wt EC (lower left blot). Expression of mTOR protein in wt EC is not altered by culture under hypoxia (30 h, lower right blot). B) Cell numbers in cultures of mTOR-transfected and wt EC were determined after culture under normoxia (open circles) and hypoxia (closed circles) in the presence of PDGF-BB (20 ng/ml) and without or with the inclusion of the indicated concentrations of rapamycin. Data are given as mean ± SE, n = 3 individual experiments.