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promote growth factor-induced proliferation of human vascular smooth muscle cells
1Molecular Cardiology Research Institute and 2Pulmonary, Critical Care, and Sleep Division, Department of Medicine, Tufts-New England Medical Center, and Tufts University School of Medicine, Boston, Massachusetts
Submitted 12 October 2005 ; accepted in final form 22 December 2005
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ABSTRACT |
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(HIF-1), a master regulator of the transcriptional response to hypoxia, is involved in the enhanced mitogen-induced proliferative responses of hypoxic VSMC. Exposure to moderate hypoxia (5% O2) enhanced the proliferative responses of human pulmonary artery SMC (HPASMC) to mitogens including platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF), compared with those in normoxia (20% O2). Moderate hypoxia elicited increased cellular HIF-1 levels, shown by Western blot analysis, and also enhanced PDGF-, FGF-2-, and EGF-induced expression of HIF-1. Knockdown of HIF-1 or HIF-1
levels in HPASMC with specific small interfering RNAs inhibited FGF-2-stimulated proliferation of HPASMC incubated in either 5% or 20% O2 but failed to inhibit the comitogenic effect of hypoxia. Knockdown of HIF-1 similarly inhibited PDGF-stimulated proliferation, whereas HIF-2 knockdown had no effect on HPASMC proliferation. Knockdown of HIF-1 expression also inhibited growth factor-induced expression of cyclin A. We conclude that HIF-1 promotes proliferative responses of human VSMC to FGF-2, PDGF, and EGF by mechanisms that may involve HIF-1-dependent expression of cyclin A, but HIF is apparently not crucial to the enhancement of FGF-2-, PDGF-, and EGF-induced proliferation of VSMC that occurs during hypoxia.
platelet-derived growth factor; fibroblast growth factor; cyclin A; oxygen
One major class of transcriptional regulators potentially involved in hypoxia-induced enhancement of VSMC proliferative responses is the hypoxia-inducible factors (HIFs), a group of heterodimeric transcription factors that consist of an - and -subunit and are thought to be master regulators of the transcriptional response to hypoxia. Targeted deletion of either the - or -subunit of HIF-1 is lethal to the early embryo and is associated with defective vascular development, including a paucity of surrounding SMC in medium-sized blood vessels and an absence of larger blood vessels (15, 18, 19). These findings suggest that HIF-1 may be crucial to VSMC proliferation and/or survival during early vascular development. In addition, partial deficiency of HIF-1 reduces the muscularization of small pulmonary arteries occurring in mice exposed to chronic hypoxia (33), suggesting a role of HIF-1 in hypoxia-induced hyperplasia of VSMC. The hypothesis that HIF-1 may promote VSMC proliferation is further supported by evidence that VSMC express HIF-1 and that growth factors are potent activators of HIF-1 expression in VSMC (25).
Although many studies support a role of HIF-1 in regulating transcriptional responses to severe hypoxia (0%–1% O2), little is known about its role in cellular responses elicited in VSMC by moderate hypoxia (
5% O2). Therefore, in this study we tested whether moderate hypoxia enhances growth factor-induced proliferation in human VSMC and whether such effects are associated with enhanced growth factor-induced HIF-1 expression. Our tests included distinct classes of peptide growth factors, including agonists of tyrosine kinase and G protein-coupled receptors. To selectively reduce expression of HIF-1 subunits and thereby test its role in the proliferative responses of VSMC to mitogens and hypoxia, we used small interfering RNAs (siRNAs) that elicit an endogenous sequence-specific, posttranscriptional gene silencing program when delivered into the cytoplasm (11). The results show that HIF-1 plays an important role in the proliferative responses of human pulmonary artery SMC (HPASMC) to growth factors by mechanisms that may involve stimulatory effects on the expression of cyclin A. Hypoxia, on the other hand, appears to stimulate growth factor-induced proliferation via HIF-independent mechanisms.
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METHODS |
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Hypoxia. HPASMC were exposed to moderate (5% O2) or severe (1% O2) hypoxia in a water-jacketed CO2 incubator that maintains a subambient O2 level by the regulated injection of N2 (Forma Scientific). Control cells were placed in a similar incubator maintained at the ambient O2 level (20%). In all studies, cells remained at the lower O2 level for the entire experimental protocol, without any transient periods of reoxygenation that could affect VSMC proliferation.
Cell proliferation assays. SMC were plated in 48-well plates (5,000/cm2) in growth medium, incubated overnight, and serum-deprived (1% FCS) for 24 h. Replicate wells were then stored at –70°C for baseline (day 0) cell counts, and fresh medium with or without growth factors was added to the remaining wells, which were incubated 72–96 h in 20% or 5% O2. Day 0 and days 3 or 4 cell counts were determined by lysing cells in a buffer containing a fluorescent dye that has minimal fluorescence by itself but fluoresces when bound to DNA or RNA (Cyquant; Molecular Probes). Absolute cell number was calculated by comparing the fluorescence obtained with samples to that of a standard curve similarly prepared using a known number of cells.
Western blot analysis.
To analyze HIF-1 accumulation in the nucleus, nuclear extracts were prepared by standard methods as reported earlier (27). Briefly, cells were placed rapidly on ice in buffer containing 10 mM HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT) supplemented with protease inhibitors (Complete, Boehringer Mannheim), 1 mM NaF, and 1 mM Na orthovanadate and incubated 15 min. NP-40 was added to a final concentration of 0.5%, and nuclei were pelleted by centrifugation (16,000 g for 30 s). The nuclear pellets were lysed on ice for 30 min in buffer containing 20 mM HEPES (pH 7.4), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 0.5 mM DTT, 1 mM NaF, 1 mM Na orthovanadate, and Complete protease inhibitors, cleared by centrifugation and stored in aliquots at –70°C. For analysis of cyclin A, whole cell extracts were prepared by lysis on ice for 15 min in buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5 mM DTT, 5% glycerol, supplemented with protease and phosphatase inhibitors). Whole cell or nuclear proteins were separated on a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane, and the membrane was incubated sequentially with 5% nonfat dry milk, followed by mouse IgG directed against human HIF-1 (BD Transduction Laboratories), HIF-2, or HIF-1 (Novus Biologicals), and peroxidase-conjugated donkey anti-mouse IgG. Cyclin A was detected by using rabbit anti-cyclin A (Santa Cruz), followed by peroxidase-conjugated donkey anti-rabbit IgG. Peroxidase bound to the blot was visualized by chemiluminescence (SuperSignal; Pierce Chemical).
siRNA transfections.
HPASMC were plated and transfected the following day with siRNAs (Dharmacon Research, Lafayette, CO) by using either Oligofectamine (GIBCO) or Lipofectamine 2000 (Invitrogen). The sense and antisense strands of the siRNAs used were ACUUCUGGAUGCUGGUGAUdTdT and AUCACCAGCAUCCAGAAGUdTdT, directed against nucleotides 213–231 downstream of the translation start site of HIF-1; CAAGAUGACAGCCUACAUCdTdT and GAUGUCGGCUGUCAUCUUGdTdT, directed against nucleotides 308–327 downstream of the translation start site of HIF-1; and CUUACGCUGAGUACUUCGAdTdT and UCGAAGUACUCAGCGUAAGdTdT, for nonspecific siRNA. A pool of four HIF-2-specific siRNAs (Dharmacon M-004814–01) were used to inhibit HIF-2 expression, with a pool of four nonspecific siRNAs (Dharmacon D-001206–13) used for control transfections. To determine the effect of siRNA-mediated HIF subunit knockdown on cellular proliferation rates, HPASMC were trypsinized the following day, counted, and transferred to 48-well plates for analysis of cell growth by Cyquant assay, as described above.
Statistical analysis. Significant differences between growth factor-induced proliferation in 20% vs. 5% O2 and between growth factor-induced proliferation in 5% O2 in HPASMC transfected with nonspecific vs. HIF subunit-specific siRNAs were analyzed by two-way ANOVA with paired measurements, followed by Bonferroni posttests with the use of Prism 4.0 software.
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RESULTS |
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1 failed to stimulate proliferation of HPASMC, and moderate hypoxia did not induce or enhance proliferative responses to these agents (Fig. 1D). The results indicate that moderate hypoxia augments proliferation induced by the tyrosine kinase receptor-activating mitogens PDGF-AB, EGF, and FGF-2.
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expression in HPASMC.
As a first test of whether stimulation of HIF-1 expression might contribute to the mitogenic effects of growth factors in HPASMC, we compared the increases in HIF-1 protein levels induced by these agents, by using Western blot analysis, and determined the extent to which they were correlated with proliferative responses. The HIF-1 levels induced by moderate (5% O2) or severe (1% O2) hypoxia were also measured for comparison. PDGF and FCS, which were strong mitogens (Fig. 1, A and D), likewise strongly induced HIF-1 expression in HPASMC, inducing nuclear accumulation of HIF-1 to levels as high as those elicited by severe hypoxia (Fig. 2, A and B). In contrast, FGF-2 and EGF, which were found to be weaker mitogens (Fig. 1, B and C), induced HIF-1 only weakly (Fig. 2C). ANG II and TGF-1, which were not mitogenic (Fig. 1D), induced the lowest nuclear HIF-1 levels (Fig. 2B). Thrombin was not mitogenic but induced a moderately strong HIF-1 response (Fig. 2B). The strong correlation between the magnitudes of growth factor-induced proliferative responses and the induced levels of nuclear HIF-1 protein supports the hypothesis that HIF-1 plays a role in proliferative responses of HPASMC.
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expression additively.
We tested whether the abilities of FGF-2, EGF, or PDGF to induce HIF-1 expression would be additive with that of moderate hypoxia. Moderate hypoxia alone, in the absence of exogenous growth factors, induced modest increases in nuclear HIF-1 levels (Fig. 2A). Exposure to moderate hypoxia, or to FGF-2, EGF, or PDGF in normoxia, induced modest increases in the cellular HIF-1 levels, as shown by Western blot analysis of whole cell extracts (Fig. 3). (PDGF-induced HIF-1 expression seen in Fig. 3 was not as strong as that seen with HPASMC from a different organ donor in Fig. 2, A and B). In the presence of moderate hypoxia, each of these growth factors induced markedly higher levels of HIF-1 expression compared with those seen in normoxic conditions. Exposure to moderate hypoxia alone, or to PDGF, FGF-2, or EGF under normoxic conditions, also induced modest increases in HIF-2 expression, and like that of HIF-1, the induction of HIF-2 by growth factors was augmented by moderate hypoxia compared with that seen under normoxia (Fig. 3).
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, HIF-1, or HIF-2 in HPASMC, we first determined the transfection efficiency attained by using a lipid transfection reagent and a fluorescent-labeled siRNA and found that Cy3-labeled siRNA was detected in 100% of HPASMC 24 h after transfection (200 of 200 cells identified microscopically by nuclear DNA staining). Transfection of HPASMC with HIF-1-specific siRNA markedly reduced HIF-1 protein levels determined by Western blot analysis (Fig. 4A), whereas nonspecific control siRNA had no effect. Knockdown of HIF-1 protein persisted for at least 6 days after transfection (Fig. 4B). Furthermore, HIF-1-directed siRNA reduced the cellular level of HIF-1 in HPASMC without affecting those of HIF-1 or lamin C (Fig. 4C), demonstrating that its effect was highly specific. In addition, transfection with siRNAs designed to knock down HIF-1 or HIF-2 induced marked and specific reductions in the cellular levels of HIF-1 and HIF-2, respectively, and the effect of each was specific to the targeted HIF subunit (Fig. 4, C and D).
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or HIF-1, but not HIF-2, inhibits FGF-2- and PDGF-induced HPASMC proliferation.
To test the role of HIF-1 in hypoxia-induced augmentation of growth factor-induced proliferation, we determined the ability of hypoxia to enhance FGF-2-stimulated proliferation in HPASMC in which HIF subunit levels were selectively reduced by specific siRNAs. Both basal and FGF-2-induced proliferation rates were similar in HPASMC transfected with nonspecific siRNA to those in untransfected HPASMC (Fig. 5A), indicating that transfection with siRNA does not cause any nonspecific inhibitory effects on cell proliferation. Knockdown of either HIF-1 or HIF-1 inhibited FGF-2-induced proliferation of HPASMC under both hypoxic and normoxic conditions (Fig. 5A). Knockdown of HIF-1 also reduced PDGF-induced proliferation in normoxic HPASMC (Fig. 5B). In contrast, transfection with HIF-2-directed siRNA failed to alter FGF-induced proliferation in either normoxic or hypoxic HPASMC (Fig. 5C). Together these results support a role of HIF-1 in growth factor-induced proliferation. In contrast, hypoxia-induced enhancement of the proliferative effect of FGF-2 was unaffected by transfection with HIF-1-specific siRNA (Fig. 5A). These results indicate that HIF-1 promotes FGF-2- and PDGF-induced proliferation but is not essential for the stimulatory effect of hypoxia on HPASMC proliferation.
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reduces growth factor-induced cyclin A expression in HPASMC.
We have identified potential HIF-1-binding sites in the promoter region of the cyclin A gene promoter (D. Beasley, unpublished results), raising the possibility that HIF-1 promotes growth factor-induced proliferation via direct effects on cyclin A gene expression. We therefore tested the effects of HIF-1 knockdown on growth factor-induced expression of cyclin A. Both PDGF-AB- and FGF-2-induced cyclin A expression were significantly reduced in HPASMC transfected with HIF-1-directed siRNA compared with those in HPASMC transfected with nonspecific siRNA (Fig. 6), suggesting that HIF-1 may promote growth factor-induced cyclin A expression in HPASMC.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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contributes to growth factor-induced proliferation, even though HIFs do not seem to be crucial for hypoxia-induced enhancement of HPASMC proliferation. The results may be relevant to the pathophysiology of atherosclerosis or pulmonary hypertension.
Moderate hypoxia enhanced the mitogenic responses of HPASMC to three different tyrosine kinase receptor-activating growth factors, including PDGF, FGF-2, and EGF, in agreement with earlier reports that the stimulatory effect of hypoxia on VSMC proliferation requires costimulation with growth factors in both human and nonhuman VSMC. For example, serum-induced, but not basal, proliferation was enhanced by exposure to moderate hypoxia (3%–5% O2) in VSMC derived from human saphenous vein explants (6, 29) and bovine pulmonary artery (9, 10, 12). In addition, presently we found that the effect of hypoxia is dependent on the nature of the comitogenic stimulus. PDGF- and FGF-2-induced proliferation was increased in HPASMC exposed to moderate hypoxia, whereas serum-induced proliferation was not affected, similar to previous findings with rat aortic SMC (14). Hypoxia also did not induce or enhance responsiveness to other putative mitogens known to signal via G protein-coupled receptors (thrombin and ANG II) or via a serine/threonine kinase receptor (TGF-). Because the latter agents failed to stimulate HPASMC proliferation in either hypoxic or normoxic conditions, the inability of hypoxia to stimulate proliferation in response to these factors could simply be attributable to inadequate expression of the cognate receptors or downstream signaling molecules in HPASMC, rather than to a selective influence of hypoxia on the actions of certain classes of mitogens. Nevertheless, considered together with the other available evidence, the present results indicate that the nature of the primary mitogen used to stimulate proliferation is an important determinant of the proliferative response to hypoxia in VSMC. Whether the stimulatory effect of hypoxia on HPASMC proliferation is specific to tyrosine kinase receptor-activating growth factors will be an interesting question for future research.
The present studies also revealed the novel finding that HIF-1 contributes to the proliferative responses of HPASMC to FGF-2 and PDGF, as each was found to induce HIF-1 expression, and knockdown of either HIF-1 or HIF-1 expression markedly reduced their proliferative effects. Growth factors induced higher levels of HIF-1 expression in HPASMC incubated in moderate hypoxia (5% O2) compared with those in normoxia (20% O2), which suggested a potential role of HIF-1 in the promitogenic effect of hypoxia. However, our finding that hypoxia produced similar (30%) enhancement of FGF-induced proliferation irrespective of whether nuclear HIF-1 protein levels were depleted by RNA interference indicates that HIF-1 may not play a primary role in the enhanced VSMC proliferation seen in 5% O2. The reason for the failure of HIF-1 knockdown to reduce hypoxia-induced proliferation was not that it was insufficient to inhibit functional responses, because it did reduce proliferative responses to FGF-2. Therefore, the stimulatory effect of moderate hypoxia on proliferation in HPASMC appears to involve HIF-1-independent mechanisms.
On the basis of our recent identification of multiple potential HIF-1 binding sites in the promoter region of the cyclin A2 gene (D. Beasley, unpublished results), we considered whether HIF-mediated enhancement of the proliferative responses of HPASMC to growth factors may involve a role of cyclin A expression. The results indicate that HIF-1 knockdown leads to decreased growth factor-induced expression ofcyclin A, an effect that would inhibit cell proliferation. Therefore, these findings raise the possibility that HIF-1 promotes growth factor-induced proliferation by stimulating cyclin A gene transcription.
Pulmonary hypertension is often caused by chronic alveolar hypoxia (13) and is characterized by pathogenic VSMC hypertrophy and hyperplasia in distal pulmonary arteries (21, 24). HIF-1 may contribute to the pulmonary vascular remodeling caused by chronic hypoxia, a hypothesis supported by evidence that hypoxia-induced development of pulmonary hypertension and muscularization of small pulmonary arteries are delayed in heterozygous HIF-1 +/– mice that express reduced levels of HIF-1 compared with wild-type HIF-1 +/+ mice (33). Chronic alveolar hypoxia leads to reduced O2 tension in multiple pulmonary cell types, including endothelial cells, VSMC, and adventitial fibroblasts, and HIF-1 expression in each cell type could contribute to vascular remodeling. For example, hypoxia-induced release of growth stimulants by rabbit pulmonary artery fibroblasts is dependent on the transcriptional effects of HIF, as recently shown by using HIF decoy oligonucleotides (26). Also, HIF-1 expression in VSMC may promote their proliferative responses to endothelium-, fibroblast-, or VSMC-derived growth factors, as supported by the present findings that reducing HIF-1 expression inhibits human VSMC proliferation. However, HIF-independent mechanisms are also likely to contribute to pulmonary vascular remodeling, as supported by our finding that hypoxia promotes VSMC proliferation in the presence of HIF-1 knockdown.
In addition to its relevance to classically hypoxic pathological states, the promitogenic effects of hypoxia on VSMC may potentially be relevant to the progression of atherosclerotic lesions. Several converging lines of evidence indicate that both systemic and local hypoxia may promote atherogenesis (7), a process that occurs over decades and is characterized by increased VSMC proliferation in its earliest stages. Cigarette smoking (28), carbon monoxide exposure (1), and chronic sleep apnea (8) lead to systemic hypoxia and are associated with increased incidence of atherosclerosis. Also, experimental chronic arterial hypoxia enhances the development of atherosclerotic lesions (17), whereas arterial hyperoxia inhibits lesion formation in cholesterol-fed rabbits (16). It is also likely that O2 tension is further reduced in atherosclerotic lesions, due to factors including intimal thickening, which impairs the diffusion of oxygen from the vessel lumen, and the high rates of oxygen consumption by intimal foam cells, which continuously hydrolyze and reesterify cholesteryl esters (4, 5, 32). Furthermore, experimental induction of local hypoxia by thrombus formation within the vasa vasorum results in intimal hyperplasia (2, 3, 20, 23). Presumably, systemic hypoxia could affect both endothelium and VSMC function, whereas local hypoxia within the arterial wall or developing lesion may primarily influence the function of VSMC.
The results show that HIF-1 plays an important role in the proliferative responses of HPASMC to growth factors, by mechanisms that may involve direct stimulatory effects on the expression of cyclin A. An important question raised by the present findings is the following: What are the HIF-independent mechanisms involved in hypoxia-induced enhancement of growth factor-induced proliferation in human VSMC? Elucidating the HIF-independent pathway and the roles of cyclin A or other HIF-regulated cell cycle modulators in promoting proliferative responses of VSMC to growth factors will be important goals of future research.
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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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B kinases- and - in serum and IL-1-induced human VSMC proliferation. Am J Physiol Heart Circ Physiol 278: H1823–H1831, 2000.. J Clin Invest 103: 691–696, 1999.[Web of Science][Medline]This article has been cited by other articles:
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K. Schultz, V. Murthy, J. B. Tatro, and D. Beasley Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1{alpha}-dependent mechanisms Am J Physiol Lung Cell Mol Physiol, June 1, 2009; 296(6): L921 - L927. [Abstract] [Full Text] [PDF]
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