(Circulation. 1995;92:11-14.)
© 1995 American Heart Association, Inc.
Articles |
Basic Fibroblast Growth Factor Upregulates the Expression of Vascular Endothelial Growth Factor in Vascular Smooth Muscle Cells
Synergistic Interaction With Hypoxia
From the Departments of Medicine and Surgery, King's College School of Medicine and Dentistry, London, SE5 9PJ, UK.
Correspondence to Dr Jorge D. Erusalimsky, Department of Medicine, KCSMD, Besssemer Rd, London SE5 9PJ, UK.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Vascular endothelial growth factor (VEGF) is a hypoxia-inducible direct angiogenic factor. Upregulation of VEGF is thought to mediate many of the angiogenic effects of growth factors that are not direct endothelial cell mitogens. Like VEGF, basic fibroblast growth factor (bFGF) is considered to induce angiogenesis by a direct effect on endothelial cells. This study investigated the possibility that bFGF may also act indirectly by regulating VEGF expression in vascular smooth muscle cells (VSMCs).
Methods and Results Incubation of confluent and quiescent cultures of rabbit VSMCs with bFGF caused a time- and concentration-dependent increase in steady-state levels of VEGF mRNA, as analyzed by Northern blot hybridization. Exposure of VSMCs to a threshold hypoxic stimulus (2.5% O2) caused a modest increase in VEGF mRNA levels. However, the combination of 2.5% O2 with bFGF had a marked synergistic effect. This effect was specific for VEGF as hypoxia did not enhance bFGF-induced expression of the proto-oncogene c-myc. Synergistic upregulation of VEGF mRNA expression also was observed between hypoxia and TGF-ß1.
Conclusions These results suggest that bFGF may promote angiogenesis both by a direct effect on endothelial cells and also indirectly by the upregulation of VEGF in VSMCs. The synergy demonstrated between hypoxia and either bFGF or TGF-ß1 suggests that multiple diverse stimuli may interact via the upregulation of VEGF expression in VSMCs to amplify the angiogenic response.
Key Words: growth substances • atherosclerosis • oncogenes • hypoxia • angiogenesis
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Angiogenesis, the formation of new blood vessels, involves the activation, migration, and proliferation of endothelial cells1 and is regulated by several peptide and nonpeptide molecules.2 Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are endothelial cell mitogens in vitro and have potent angiogenic activity in vivo.3 4 These molecules are referred to as "direct" angiogenic growth factors and are considered key regulators of this process. In contrast, "indirect" angiogenic growth factors stimulate angiogenesis in vivo but do not demonstrate mitogenic activity for endothelial cells in vitro. They include platelet-derived growth factor5 (PDGF) and transforming growth factor-ß6 (TGF-ß), which may promote angiogenesis by modulating the expression of the direct angiogenic growth factors.7
VEGF is an endothelial cell–specific mitogen8 that also induces increased vascular permeability9 and monocyte migration through endothelial layers.10 Four polypeptide species of VEGF have been identified.11 These are encoded by a single gene and formed by alternative splicing. Two isoforms, VEGF121 and VEGF165, are secreted and freely soluble, whereas the other two, VEGF189 and VEGF206, are found bound to the cell surface or the extracellular matrix via heparin-like molecules. VEGF is expressed in the ischemic areas of solid tumors where it is believed to contribute to tumor neovascularization.12 Recently, it was shown to be induced in an experimental animal model of myocardial ischemia.13 In vitro, it is strongly induced by hypoxia in cultured cells, including tumor cell lines,14 cardiac myocytes,15 and vascular smooth muscle cells7 16 (VSMCs). Like VEGF, bFGF stimulates endothelial cell proliferation in vitro17 and angiogenesis in vivo.2 In addition, it has mitogenic activity in other cells, including VSMCs and fibroblasts.4 In the arterial wall, bFGF is synthesized by endothelial cells and VSMCs,18 and although it lacks a secretory signal peptide,4 it is found sequestered within the basement membrane19 or the extracellular matrix.20 There is evidence that bFGF may contribute to the development of vascular disease following its release from dying cells or as a result of extracellular matrix proteolysis.21
Hypoxia of the arterial wall22 and the release of PDGF-BB following "endothelial cell injury"23 have both been implicated in the pathogenesis of atherosclerosis. We recently reported that these two diverse stimuli synergistically upregulate VEGF mRNA expression in cultured rabbit VSMCs.16 In this study, we investigated the possibility that bFGF might also modulate VEGF mRNA expression. We demonstrate that bFGF causes a marked increase of VEGF mRNA levels. Furthermore, we show that in a similar way to PDGF-BB, bFGF and TGF-ß1 interact with a threshold level of hypoxia to produce a synergistic upregulation of VEGF expression. These results suggests that bFGF promotes the proliferation of new blood vessels by acting as a direct angiogenic factor and by indirectly modulating the expression of VEGF.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Reagents
Tissue culture media and additives were from Gibco Life Techologies and fetal calf serum (FCS) from Sigma Chemical Co. Recombinant human bFGF, TGF-ß1, and PDGF-BB were purchased from R&D Systems. Radiolabeled nucleotides and Megaprime DNA labeling system were from Amersham Intl. Other reagents were from standard suppliers or as listed in the text.
Cell Culture
Primary cultures of VSMCs were grown by the
explant technique
from the thoracic aorta of New Zealand White rabbits, as previously
described.16 Cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) containing 20% FCS, 0.1 mmol/L nonessential
amino acids, 1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin at 37°C under 5%
CO2/95% air in a humidified incubator. For
experiments, semiconfluent explant cultures were trypsinized and the
cells subcultured in 100-mm-diameter tissue culture dishes (Costar
Corp) at a density of 5x105 cells/dish. When these first
passage cells became confluent (usually 4 to 5 days after plating), the
medium was replaced with DMEM containing 0.5% FCS, and the cells were
incubated for a further 48 hours to render them quiescent before the
initiation of each experiment.
Hypoxic Conditions
Experiments at low oxygen tensions were
performed in a
custom-made, air-tight, humidified environmental chamber (Wellcome
Research Laboratories) maintained at 37°C and flushed with a mixture
of 5% CO2 and O2 in the range of 0 to 5%, the
balance made up with N2. The desired oxygen concentration
was adjusted and monitored by an electronic gas controller, as
previously described.16
Northern Blot Analysis
Total cellular RNA was extracted
according to the acid
guanidinium thiocyanate–phenol–chloroform method, as previously
described.16 RNA (20 µg per lane) was electrophoresed on
1% agarose/6% formaldehyde gels and transferred to Duralon-UV
membranes (Strategene). Hybridizations were performed at 64°C for 1
hour in QuickHyb Solution (Stratagene) containing 0.1 mg/mL
denatured salmon sperm DNA and 1.5 to 2x106 cpm/mL
32P-labeled cDNA probes (specific activity of 0.5 to
1x109 cpm/µg). The following DNA probes were used: a
540-bp BamHI-HindIII fragment of human
VEGF121 cDNA (kindly provided by Dr Werner Risau, Max
Planck Institute) and a 850-bp Pst I-HindIII
fragment (nucleotides 540 to 1390) of human
c-myc cDNA (kindly provided by Dr H. Land, Imperial Cancer
Research Laboratories). Following hybridization, filters were washed in
1xSSC/0.1% SDS for 15 minutes at room temperature and then for 15
minutes at 55°C. Filters were autoradiographed and the resulting
bands quantified by densitometry by a UVP Gel Documentation System
model GDS2000 and UVP SW2000 software (Ultra-Violet Products Ltd).
To verify the relative amount of total RNA, filters were hybridized
with a 32P-labeled 28S rRNA anti-sense
oligonucleotide probe (Clontech Laboratories)
(0.2x106 cpm/mL). Each experiment was performed at
least two times, and results from one representative
experiment are shown in each case.
Statistical Methods
Statistical significance of densitometric
data was determined by
ANOVA (SAS).
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Basic FGF Upregulates VEGF mRNA Expression in VSMCs
Incubation of confluent and quiescent cultures of rabbit VSMCs with 10 ng/mL bFGF resulted in a marked time-dependent increase in the steady-state levels of a major 3.7-kb VEGF mRNA species (Fig 1A
). The rise in the expression of this transcript was
clearly evident within 1 hour of incubation with bFGF, reached a
maximum (25-fold increase) by 12 hours, and persisted for up to 24
hours. A previously described minor 1.5-kb VEGF-related
transcript16 was also upregulated by bFGF.
Analysis of mRNA expression for the proto-oncogene
c-myc showed that the same concentration of bFGF caused a
more rapid increase in the level of this transcript, which reached a
maximum within 1 hour and was sustained for up to 24 hours.
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Hypoxia Synergises With bFGF in Upregulation of VEGF
mRNA Expression
We have previously shown that exposure of rabbit VSMCs
to an
atmosphere of 2.5% O2 acts as a threshold hypoxic stimulus
for upregulation of VEGF expression.16 Fig 1B
shows that
incubation of VSMCs with bFGF for 4 hours at 21% O2 caused
a concentration-dependent increase in VEGF mRNA expression with a
maximal effect seen above 3 ng/mL bFGF. Incubation with the same
concentrations of bFGF at 2.5% O2 resulted in a more
marked increase in the levels of VEGF mRNA (Fig 1B). As judged
by
scanning densitometry, this was a significant enhancement
(P=.001), approximately two times greater than the additive
effect of the two respective stimuli given alone, and was observed at
all the concentrations of bFGF tested (Fig 1C). To investigate
whether
the observed synergy between bFGF and hypoxia was selective for
VEGF mRNA expression, we examined the effect of these stimuli on
c-myc expression. Basic FGF increased c-myc mRNA
expression in quiescent rabbit VSMCs in a concentration-dependent
manner and in a similar fashion to the upregulation of VEGF mRNA (Fig
1B). However, in contrast to the enhancement of VEGF mRNA,
c-myc expression was not significantly enhanced by
incubation at 2.5% O2 (P=.331) (Fig
1B and 1D).
Hypoxia and TFG-ß Synergistically Upregulate VEGF
mRNA Expression
TGF-ß has been reported to induce the expression
of VEGF in
fibroblasts,24 adenocarcinoma cells,24 and
VSMCs.7 This prompted us to investigate whether
hypoxia could also synergize with this growth factor in the
induction of VEGF. Exposure of VSMCs to 2.5% O2 for 4
hours caused a 1.5- to 2-fold increase in the steady-state levels of
VEGF mRNA. A similar increase in expression was observed when the cells
were incubated for the same length of time with 0.3 ng/mL
TGF-ß1 at 21% O2. The combination of
treatment with TGF-ß1 and incubation at 2.5%
O2 caused an 
7-fold increase in the level of VEGF mRNA,
an extent that was greater than the additive effect of the two stimuli
given alone. The magnitude of this interaction was similar to that
observed between hypoxia and bFGF or between hypoxia
and PDGF-BB (Fig 2).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The results reported in this study demonstrate that bFGF leads to a marked time- and concentration-dependent increase in VEGF mRNA levels in rabbit VSMCs. This finding raises the possibility that in addition to its direct angiogenic properties, bFGF may also regulate neovascularization indirectly by modulating VEGF expression. It was noted that the bFGF-induced upregulation of VEGF mRNA expression was slower than the observed effect of bFGF on the expression of c-myc mRNA. This kinetic difference suggests that the mechanisms for upregulation of these two transcripts are different.
We recently reported that PDGF-BB synergizes with a threshold level of hypoxia in the upregulation of VEGF mRNA transcripts corresponding to the soluble isoforms VEGF121 and VEGF165.16 In this study we examined the possibility that hypoxia might interact with other direct or indirect angiogenic factors. We demonstrate that hypoxia also synergizes with bFGF or TGF-ß1 in the upregulation of VEGF mRNA expression. This synergistic effect between hypoxia and angiogenic growth factors was selective for VEGF as hypoxia did not modify the induction of c-myc expression. The human VEGF gene contains two hypoxia-sensitive enhancer elements25 and several consensus binding sites for growth factor–regulated transcription factors.26 The presence of these gene regulatory sequences suggest that the synergy between hypoxia and growth factors results from interactions taking place at the level of transcription. However, other mechanisms, such as hypoxic induction of increased mRNA stability27 or interactions between signaling pathways, could also explain the observed results.
In the arterial wall, bFGF is sequestered inside cells4 and is also bound to the basement membrane19 and the extracellular matrix.20 Because it lacks a secretory signal peptide, it remains unclear how this molecule is made biologically available. One possible mechanism is that it is released in injured tissue following cell death and extracellular matrix degradation. Impaired oxygenation of the arterial wall may lead to hypoxia within the tunica media,28 causing cell death and release of bFGF. Thus, bFGF may interact concurrently with reduced oxygen tension in the hypoxic arterial wall to upregulate VEGF expression and result in amplification of the angiogenic process. However, it should be emphasized that in cultured VSMCs, under the experimental conditions used in this work, hypoxic treatment is unlikely to cause release of bFGF, at least to any great extent. Otherwise, this factor would have had no effect when added exogenously under these conditions.
TGF-ß has no mitogenic activity for endothelial cells in vitro. However, in vivo it does stimulate the proliferation of new blood vessels.6 In cultured VSMCs, TGF-ß induces both bFGF and VEGF expression.7 This upregulation of direct angiogenic molecules may explain at least in part the observed in vivo angiogenic properties of TGF-ß. Our results demonstrate that hypoxia synergizes with TGF-ß1 as well as with PDGF-BB in the upregulation of VEGF mRNA expression. Thus, the amplification of the angiogenic process by the combination of reduced oxygen tension and growth factors also operates with indirect angiogenic molecules.
Pericytes that are associated with the abluminal surface of capillary endothelial cells29 are phenotypically related to VSMCs. Recent studies have shown that the conditioned medium of pericytes incubated under hypoxic conditions contains angiogenic activity.30 It is therefore possible that our findings in VSMCs may also apply to pericytes and thus have important implications for the neovascularization of tumors.
Hypoxia of the arterial wall22 and growth factors released following endothelial cell injury, such as PDGF-BB, TGF-ß, and bFGF,23 have been implicated in the pathogenesis of atherosclerosis. Our findings raise the possibility that these growth factors may act in concert with reduced oxygen tension to further enhance VEGF expression in the arterial wall. This upregulation of VEGF could be involved in promoting the previously observed neovascularization of the atherosclerotic plaque by proliferating vasa vasorum.31 In addition to its well-documented angiogenic properties, VEGF also increases vascular permeability9 and promotes monocyte migration through endothelial layers.10 These events are important in early atherogenesis. It is therefore possible that VEGF may have a more complex role in pathological conditions of the arterial wall and hence be involved in both the early and advanced stages of atherosclerosis.
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Acknowledgments |
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Mr G. Stavri was supported by a junior research fellowship from the British Heart Foundation. We are grateful to Dr Kay Southgate, Department of Cardiac Surgery, Bristol University, UK, for her help in the establishment of VSMC cultures. We are also indebted to Ying Hong for her expert technical assistance and to Ian Cameron, Tony Reeves, and Kevin Hobbs from the Technology Group, Wellcome Research Laboratories, Beckenham, UK, for their dedication in the design and construction of the hypoxic chambers.
Received April 4, 1995; revision received May 11, 1995; accepted May 15, 1995.
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P. Bausero, M.-H. Ben-Mahdi, J.-P. Mazucatelli, C. Bloy, and M. Perrot-Applanat Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2033 - H2042. [Abstract] [Full Text] [PDF]
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 R. M. Fleming and L. B. Boyd The Effect of High-Protein Diets on Coronary Blood Flow Angiology, October 1, 2000; 51(10): 817 - 826. [Abstract] [PDF]
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 E. K. Rofstad and E. F. Halsør Vascular Endothelial Growth Factor, Interleukin 8, Platelet-derived Endothelial Cell Growth Factor, and Basic Fibroblast Growth Factor Promote Angiogenesis and Metastasis in Human Melanoma Xenografts Cancer Res., September 1, 2000; 60(17): 4932 - 4938. [Abstract] [Full Text]
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W. Philipp, L. Speicher, and C. Humpel Expression of Vascular Endothelial Growth Factor and Its Receptors in Inflamed and Vascularized Human Corneas Invest. Ophthalmol. Vis. Sci., August 1, 2000; 41(9): 2514 - 2522. [Abstract] [Full Text]
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 S. Boussat, S. Eddahibi, A. Coste, V. Fataccioli, M. Gouge, B. Housset, S. Adnot, and B. Maitre Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells Am J Physiol Lung Cell Mol Physiol, August 1, 2000; 279(2): L371 - L378. [Abstract] [Full Text] [PDF]
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 L. D. Ke, Y.-X. Shi, S.-A. Im, X. Chen, and W. K. A. Yung The Relevance of Cell Proliferation, Vascular Endothelial Growth Factor, and Basic Fibroblast Growth Factor Production to Angiogenesis and Tumorigenicity in Human Glioma Cell Lines Clin. Cancer Res., June 1, 2000; 6(6): 2562 - 2572. [Abstract] [Full Text]
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J. Dulak, A. Jozkowicz, A. Dembinska-Kiec, I. Guevara, A. Zdzienicka, D. Zmudzinska-Grochot, I. Florek, A. Wojtowicz, A. Szuba, and J. P. Cooke Nitric Oxide Induces the Synthesis of Vascular Endothelial Growth Factor by Rat Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 659 - 666. [Abstract] [Full Text] [PDF]
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 J. Dulak, A. Jozkowicz, A. Ratajska, A. Szuba, J. P Cooke, and A. Dembinska-Kiec Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells Vascular Medicine, February 1, 2000; 5(1): 33 - 40. [Abstract] [PDF]
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 M. F. Flanagan, T. Aoyagi, L. W. Arnold, C. Maute, A. M. Fujii, J. Currier, D. Bergau, H. B. Warren, and K. Rakusan Effects of Chronic Heparin Administration on Coronary Vascular Adaptation to Hypertension and Ventricular Hypertrophy in Sheep Circulation, August 31, 1999; 100(9): 981 - 987. [Abstract] [Full Text] [PDF]
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J. R Kersten, P. S Pagel, W. M Chilian, and D. C Warltier Multifactorial basis for coronary collateralization: a complex adaptive response to ischemia Cardiovasc Res, July 1, 1999; 43(1): 44 - 57. [Abstract] [Full Text] [PDF]
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 C. J. Corral, A. Siddiqui, L. Wu, C. L. Farrell, D. Lyons, and T. A. Mustoe Vascular Endothelial Growth Factor Is More Important Than Basic Fibroblastic Growth Factor During Ischemic Wound Healing Arch Surg, February 1, 1999; 134(2): 200 - 205. [Abstract] [Full Text] [PDF]
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A. Rivard, M. Silver, D. Chen, M. Kearney, M. Magner, B. Annex, K. Peters, and J. M. Isner Rescue of Diabetes-Related Impairment of Angiogenesis by Intramuscular Gene Therapy with Adeno-VEGF Am. J. Pathol., February 1, 1999; 154(2): 355 - 363. [Abstract] [Full Text] [PDF]
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A. Rivard, J.-E. Fabre, M. Silver, D. Chen, T. Murohara, M. Kearney, M. Magner, T. Asahara, and J. M. Isner Age-Dependent Impairment of Angiogenesis Circulation, January 12, 1999; 99(1): 111 - 120. [Abstract] [Full Text] [PDF]
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 S. Noda and H. B. Barner Arterial conduits Ann. Thorac. Surg., January 1, 1999; 67(1): 285 - 286. [Full Text] [PDF]
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Y.-X. Chen, Y. Nakashima, K. Tanaka, S. Shiraishi, K. Nakagawa, and K. Sueishi Immunohistochemical Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor in Atherosclerotic Intimas of Human Coronary Arteries Arterioscler Thromb Vasc Biol, January 1, 1999; 19(1): 131 - 139. [Abstract] [Full Text] [PDF]
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 T. Korff and H. G. Augustin Integration of Endothelial Cells in Multicellular Spheroids Prevents Apoptosis and Induces Differentiation J. Cell Biol., November 30, 1998; 143(5): 1341 - 1352. [Abstract] [Full Text] [PDF]
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M. A. Ramos, M. Kuzuya, T. Esaki, S. Miura, S. Satake, T. Asai, S. Kanda, T. Hayashi, and A. Iguchi Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions Arterioscler Thromb Vasc Biol, July 1, 1998; 18(7): 1188 - 1196. [Abstract] [Full Text] [PDF]
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 L. P. Reynolds, J. D. Kirsch, K. C. Kraft, and D. A. Redmer Time-Course of the Uterine Response to Estradiol-17ß in Ovariectomized Ewes: Expression of Angiogenic Factors Biol Reprod, July 1, 1998; 59(3): 613 - 620. [Abstract] [Full Text]
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G. Seghezzi, S. Patel, C. J. Ren, A. Gualandris, G. Pintucci, E. S. Robbins, R. L. Shapiro, A. C. Galloway, D. B. Rifkin, and P. Mignatti Fibroblast Growth Factor-2 (FGF-2) Induces Vascular Endothelial Growth Factor (VEGF) Expression in the Endothelial Cells of Forming Capillaries: An Autocrine Mechanism Contributing to Angiogenesis J. Cell Biol., June 29, 1998; 141(7): 1659 - 1673. [Abstract] [Full Text] [PDF]
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M. Oberhoff, S. Novak, C. Herdeg, A. Baumbach, A. Kranzhofer, A. Bohnet, B. Horch, H. Hanke, K. K. Haase, and K. R. Karsch Local and systemic delivery of low molecular weight heparin stimulates the reendothelialization after balloon angioplasty Cardiovasc Res, June 1, 1998; 38(3): 751 - 762. [Abstract] [Full Text] [PDF]
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T. Murohara, J. R. Horowitz, M. Silver, Y. Tsurumi, D. Chen, A. Sullivan, and J. M. Isner Vascular Endothelial Growth Factor/Vascular Permeability Factor Enhances Vascular Permeability Via Nitric Oxide and Prostacyclin Circulation, January 13, 1998; 97(1): 99 - 107. [Abstract] [Full Text] [PDF]
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J. Waltenberger Modulation of Growth Factor Action : Implications for the Treatment of Cardiovascular Diseases Circulation, December 2, 1997; 96(11): 4083 - 4094. [Abstract] [Full Text]
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T. Bombardini, E. Picano, and T. Bombardini The Coronary Angiogenetic Effect of Heparin: Experimental Basis and Clinical Evidence Angiology, November 1, 1997; 48(11): 969 - 976. [Abstract] [PDF]
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R. Natarajan, W. Bai, L. Lanting, N. Gonzales, and J. Nadler Effects of high glucose on vascular endothelial growth factor expression in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, November 1, 1997; 273(5): H2224 - H2231. [Abstract] [Full Text] [PDF]
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D. S. Winlaw Angiogenesis in the Pathobiology and Treatment of Vascular and Malignant Diseases Ann. Thorac. Surg., October 1, 1997; 64(4): 1204 - 1211. [Abstract] [Full Text]
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 A. Pedram, M. Razandi, R.-M. Hu, and E. R. Levin Vasoactive Peptides Modulate Vascular Endothelial Cell Growth Factor Production and Endothelial Cell Proliferation and Invasion J. Biol. Chem., July 4, 1997; 272(27): 17097 - 17103. [Abstract] [Full Text] [PDF]
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 R. O SCHLINGEMANN and V. W M VAN HINSBERGH Role of vascular permeability factor/vascular endothelial growth factor in eye disease Br J Ophthalmol, June 1, 1997; 81(6): 501 - 512. [Full Text] [PDF]
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J. Waltenberger, U. Mayr, S. Pentz, and V. Hombach Functional Upregulation of the Vascular Endothelial Growth Factor Receptor KDR by Hypoxia Circulation, October 1, 1996; 94(7): 1647 - 1654. [Abstract] [Full Text]
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M. Fujita, M. Ikemoto, M. Kishishita, H. Otani, R. Nohara, T. Tanaka, S.-i. Tamaki, A. Yamazato, and S. Sasayama Elevated Basic Fibroblast Growth Factor in Pericardial Fluid of Patients With Unstable Angina Circulation, August 15, 1996; 94(4): 610 - 613. [Abstract] [Full Text]
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 M. Papetti and I. M. Herman Mechanisms of normal and tumor-derived angiogenesis Am J Physiol Cell Physiol, May 1, 2002; 282(5): C947 - C970. [Abstract] [Full Text] [PDF]
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