This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ziche, M. Articles by Presta, M. Search for Related Content PubMed PubMed Citation Articles by Ziche, M. Articles by Presta, M.

(Circulation Research. 1997;80:845-852.)
© 1997 American Heart Association, Inc.

Articles

Nitric Oxide Promotes Proliferation and Plasminogen Activator Production by Coronary Venular Endothelium Through Endogenous bFGF

Marina Ziche, Astrid Parenti, Fabrizio Ledda, Patrizia Dell'Era, Harris J. Granger, Carlo A. Maggi, , Marco Presta

From the Department of Pharmacology (M.Z., A.P., F.L.), University of Florence (Italy); the Department of Biomedical Sciences and Biotechnologies (P.D., M.P.), University of Brescia (Italy); the Microcirculation Research Institute and Department of Medical Physiology (H.J.G.), Texas A&M University, College Station; and the Pharmacology Department (C.A.M.), A. Menarini Pharmaceuticals, Florence, Italy.

Correspondence to Marina Ziche, MD, Department of Pharmacology, University of Florence, Viale Morgagni 65, 50134 Florence, Italy. E-mail ziche{at}stat.ds.unifi.it

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase–polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

Key Words: microvascular endothelium • plasminogen activator • autocrine proliferation • sodium nitroprusside • substance P

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Coronary angiogenesis is an important homeostatic response to inadequate nutrient delivery in myocardium under physiological stress and in pathophysiological disorders. Myocardial vascularity increases in response to chronic alveolar hypoxia (eg, high-altitude hypoxia) or cardiac hypermetabolism (eg, hyperthyroidism, augmented preload or afterload, increased inotropy) and after coronary ischemia.¹ A common denominator in all these chronic conditions is the sustained elevation of coronary blood flow in the regions that will later exhibit neovascularization. A number of molecules (eg, adenosine and vascular endothelial growth factor) released from hypoxic tissue have received attention as potential linkages between angiogenesis and myocardial function.^{2 3} However, the rate of coronary blood flow itself may be an important physical determinant of coronary angiogenesis.⁴

Continuous administration of coronary vasodilators leads to augmented myocardial vascularity, and a role for the endothelium-derived relaxing factor, NO, has been postulated.¹ Several vasoactive agents have been described to induce angiogenesis in vivo and endothelial cell proliferation and mobilization in vitro.^{5 6 7 8} Recently, we have reported that NO is responsible for the angiogenesis induced by SP, a neuropeptide whose effects at the microvascular level are endothelium dependent.^{9 10} The mechanism by which NO controls angiogenesis, however, has not been elucidated. A possible explanation is that NO could modulate the activity and/or the production of angiogenic factors as indicated for other peptides.¹¹

The steps required for new vessel growth are biologically complex and probably require coordinate regulation of contributing components, including proliferation and migration of endothelial cells and matrix degradation.¹² It is unclear whether a single agent that will stimulate all necessary components exists, although bFGF appears to be the most likely. bFGF is a potent angiogenic factor, and in vitro studies have demonstrated that bFGF can initiate the cellular responses associated with angiogenesis, including uPA production and cell division.^{13 14 15} However, this cytokine is not secreted in the classical sense and probably does not diffuse, so its role may be limited to stimulation of endothelial cell outgrowth in an autocrine fashion^{16 17 18} or as a result of endothelial disruption during wounding.¹⁹ bFGF has been detected in cardiac myocytes and cells of the coronary vasculature.^{20 21} These observations suggest that bFGF might induce coronary angiogenesis by an autocrine/paracrine mechanism. Indeed, cultured coronary endothelium exhibits the FGF receptor on its surface and expresses bFGF mRNA.²¹

In order to elucidate the mechanism of action of NO in angiogenesis, we investigated the interaction of NO and bFGF in signaling proliferation and uPA production in cultured coronary endothelium. The vasodilator NaNp, which provides an exogenous source of NO,²² and the neuropeptide SP, which induces NO production in endothelial cells,²³ were used to increase intracellular NO. In addition, insight into the basic question was provided by the use of blockers of NO synthase. The contribution of bFGF was determined from measurements of the levels of bFGF peptide and mRNA and the use of neutralizing antibodies.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Line and Culture Conditions
CVECs were obtained by a bead-perfusion technique through the coronary sinus, as previously reported.²⁴ These cells are endothelial in nature, as evidenced by labeling with antibody to factor VIII–related antigen and uptake of acetylated low-density lipoproteins. At confluence, they form a typical contact-inhibited monolayer with the usual cobblestone morphology. Cells were maintained in culture in DMEM supplemented with 2 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), and 10% FCS on gelatin-coated dishes. Cells were cloned, and each clone was subcultured up to a maximum of 25 passages. Passages between 15 and 20 were used in these experiments.

Proliferation Studies
CVECs were seeded onto 48-multiwell plates (4x10³ cells per well) in DMEM supplemented with 5% FCS and were allowed to adhere overnight. After 48 hours in serum-free medium (0.1% FCS), medium was removed, and cells were incubated with increasing concentrations of the agents for 2 to 3 days. In experiments in which L-NMMA, L-NNA, and L-NAME were used to inhibit NO synthase,²⁵ the drug used was added 1 hour before the agonist. In the experiments in which neutralizing antibodies were used, the cells were incubated simultaneously with the test substances and with the antibody. Irrelevant IgGs were used as a control. At the end of incubation time, the supernatants were removed from the multiwell plates, and cells were fixed by adding 1 mL of ice-cold methanol and kept at 4°C overnight. Cells were then stained with Diff-Quik (Mertz-Dade AG). Cell numbers were obtained by counting by microscopic examination at x100 magnification with the aid of an ocular grid (21 mm²). Each well was divided into 10 fields, and cells were counted by double-blind procedure in seven randomly selected fields. Data are expressed as total cell number per well and as percent increase over basal proliferation.

Measurement of cGMP Levels
cGMP levels were measured on cell extracts from confluent cell monolayers by radioimmunoassay using iodinated tracer.²⁶ Cell monolayers were treated with 1 mmol/L 3-isobutyl-5-methylxanthine for 15 minutes before stimulation. After stimulation, cells were rinsed with PBS and removed by scraping in ice-cold 10% trichloroacetic acid. After centrifugation, cGMP levels were assayed in the supernatant; proteins were measured in the pellet by Bradford's procedure. Data are expressed as fmol/mg protein and represent mean±SEM of four determinations.

uPA Assay
The assay was performed on subconfluent cultured cells as described previously.²⁷ Briefly, CVECs were plated in 24-well dishes at 5x10⁴ cells/cm². After 24 hours, cells were washed twice with serum-free medium and incubated in fresh medium containing 0.4% FCS and different concentrations of bFGF or NaNp. Twenty-four hours later, cell-associated uPA activity was measured. To this purpose, cells were washed twice with PBS and were extracted with 100 µL of 0.05% Triton X-100 in 60 mmol/L Tris-HCl, pH 8.5 (T/T buffer). Aliquots of the cell extracts corresponding to 1 µg of protein were incubated in a microtiter plate with 4.2 ng of purified Glu-plasminogen and 42 nmol of plasmin chromogenic substrate H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide-acetate in 150 µL of T/T buffer. After incubation at 37°C, the plate was read at 405 nm with an automatic microplate reader. Human urokinase was used as a standard.

Fibrin Zymography
For determination of the molecular weight of cell-associated PA activity, CVEC extracts (15 µg of total protein) were run on SDS–10% polyacrylamide gel under nonreducing conditions. Then, proteins were electrophoretically transferred from the gel to a nitrocellulose membrane for 2 hours at 400 mA in 40 mmol/L sodium phosphate buffer, pH 6.5. Zymography of the proteins transferred to the membrane was carried out on a casein-agarose gel as described previously.²⁸ No lytic bands were observed in control gels made in the absence of plasminogen to identify possible plasminogen-independent caseinolytic activities.

Western Blot Analysis
CVECs were seeded in 10-cm-diameter Petri dishes in DMEM plus 10% FCS and allowed to grow to 90% confluence. Cells were starved from serum for 48 hours. The stimulation with NaNp, SP, or Sar9-SP was carried out in medium containing 0.1% FCS for 6 and 15 hours in the absence or in the presence of 200 µmol/L L-NMMA. After two washes with PBS, cells were lysed with 20 mmol/L Tris-HCl (pH 7.2), 150 mmol/L NaCl, 0.25% (vol/vol) Nonidet P-40, and 1 mmol/L phenyl-methylsulfonyl fluoride for 30 minutes. The lysate was then transferred into an Eppendorf tube and centrifuged at 5000 rpm for 1 minute. The supernatant was then kept at -20°C until use. The supernatants of the cells were precipitated with trichloroacetic acid, washed with ethanol, and suspended in reducing Laemmli buffer. All samples (700 µg of protein each) were boiled and run on 12% SDS-PAGE, blotted onto nitrocellulose filter, and immunostained with mouse monoclonal anti-bFGF antibodies (1:100). The antigen-antibody complexes were visualized using appropriate secondary antibodies and the ECL detection system, as recommended by the manufacturer (Amersham Corp). The cell extracts of parental and bFGF-transfected mouse aortic endothelial cells (MAE and MAE-pZIP-bFGF3F2 clone, respectively) were used as negative or positive control for the expression of all bFGF isoforms.²⁹

Total RNA Extraction and Reverse Transcription
After overnight starvation, CVECs (3.5x10⁶ per 10-cm plate) were incubated for 6 hours with test substances in the presence of 0.1% FCS. At the end of incubation, total RNA was isolated by the standard guanidine thiocyanate–phenol–chloroform extraction.³⁰ Total RNA from MAE and MAE-pZIP-bFGF3F2 clone was used as negative or positive control for the expression of bFGF mRNA.²⁹

cDNA was synthesized in 20 µL of reaction volumes containing 1 µg of total RNA, 2.5 µmol/L oligo dT₁₆, 0.5 mmol/L dNTP, 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl₂, and 200 U of M-MLV reverse transcriptase. After 60 minutes of incubation at 38°C, samples were heated at 95°C for 5 minutes and then rapidly chilled on ice.

Differential RT-PCR Analysis
Differential RT-PCR^{31 32} for bFGF was carried out by using 5 µL of cDNA and specific primers for bFGF with sequences as follows: sense (OBN 308), 5'-GGAGTGTGTGCTAACCGTTACCTGGCTATG-3'; antisense (OBN 309), 5'-TCAGCTCTTAGCAGACATTGGAAGAAAAAG-3'.³³ Calibration was performed by coamplification of the same cDNA samples with primers for GAPDH as internal standard, with sequences as follows: sense (GAPDH-L), 5'-CCATGGAGAAGGCTGGGG-3'; antisense (GAPDH-R), 5'-CAAAGTTGTCATGGATGACC-3'.³⁴ For PCR amplification, a Perkin-Elmer GeneAmp PCR System 2400 was used. The reaction mixture contained 10 nmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl₂, 50 mmol/L KCl, 0.25 mmol/L of each dNTP, 1 µmol/L of each primer, and 1.25 U Amplitaq DNA polymerase (Perkin-Elmer) in an 80-µL final volume. The PCR cycles were as follows: 30 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. After 30 cycles of amplification, aliquots of each sample product (20 µL) were electrophoresed on a 3% agarose gel and stained with ethidium bromide. The sizes of the amplification products were 194 and 242 bp for GAPDH and bFGF, respectively.

The intensities of the two bands corresponding to bFGF and GAPDH amplification products were measured with a charge-coupled device video camera (C3077/-01, Hamamatsu Photonics) connected with a video frame grabber (M4476, Hamamatsu). The video frame grabber is a plug-in board used in a Macintosh IIsi computer (Apple). For the acquisition of the image, we used Imagequest IQBase software (Hamamatsu). Image processing and analysis were performed with the free software IMAGE, version 1.28. The results are reported as the ratio between the bFGF and GAPDH amplification analysis.

Statistical Analysis
The data are reported as mean±SEM of at least three experiments. Statistical analysis was performed by using one-way ANOVA followed by Scheffé's test or by Student's t test for paired and/or unpaired data. A value of P<.05 was taken as significant.

Reagents
FCS was from Hyclone. Diff-Quik was from Mertz-Dade AG. All reagents for cell culture, NaNp, L-NMMA, and L-NNA were purchased from Sigma Chemical Co. Human urokinase and L-NAME were from Calbiochem. SP and Sar9-SP were from Peninsula Laboratories. Glu-plasminogen was from Kabi AB. Plasmin chromogenic substrate H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide-acetate was purchased from American Diagnostics. Recombinant bFGF and suramin were kindly provided by Dr N. Mongelli, Farmitalia-Pharmacia, Milan, Italy. Rabbit polyclonal anti-bFGF antibody was a generous gift of Dr D.B. Rifkin (New York University Medical Center, New York City) and was affinity-purified by bFGF–Affi-Gel affinity chromatography. The unbound IgG from the bFGF affinity column is referred to as irrelevant IgG and used as a control. Affinity-purified antibody, but not irrelevant IgG, recognizes recombinant bFGF in immunoblot and dot blot analysis and quenches the activity of the growth factor in mitogenic assays on cultured endothelial cells (data not shown). The mouse monoclonal anti-bFGF antibody (Upstate Biotechnology, Inc) was a kind gift of Dr F. Cozzolino, National Research Council (CNR), Rome, Italy. The radioimmunoassay kit for cGMP measurement was from Amersham. Mononucleotides dATP, dCTP, dGTP, dTTP, and the DNA marker (XIV) were from Boehringer-Mannheim. RT was from GIBCO BRL, Life Technologies Italia Srl. Oligo dT₁₆ and AmpliTaq DNA polymerase were from Perkin-Elmer. Primers for GAPDH were kindly provided by Dr A. Peri, University of Florence (Italy).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
bFGF Promotes Cell Growth and uPA Activity in CVECs
bFGF has been shown to induce cell proliferation and uPA production in endothelial cells of different origin and to stimulate angiogenesis in the myocardium. On this basis, the ability of bFGF to stimulate also the growth of cultured coronary endothelial cells while increasing their invasive capacity, as expressed by the production of the proteolytic enzyme uPA, was assessed in parallel experiments. bFGF induced a dose-dependent proliferation of sparse CVECs with maximal activity at 10 ng/mL. After a 24-hour exposure of subconfluent monolayers to bFGF, a dose-dependent increase in cell-associated uPA activity was also observed (Fig 1a). The maximal effect was obtained at 30 ng/mL bFGF. The ED₅₀s for the induction of both cell proliferation and uPA production were 3 ng/mL. The role of endogenous NO in mediating bFGF-induced growth and uPA production was investigated by preincubation of CVECs with three different NO synthase inhibitors (L-NAME, L-NMMA, and L-NNA). The effectiveness of NO synthase inhibition in this cell type was confirmed by the block of SP-induced cGMP elevation (Table 1). As shown in Table 2, the inhibitors did not affect cell proliferation and uPA upregulation induced by the maximal effective concentration of bFGF. Consistent with these observations, bFGF did not affect cGMP levels, ruling out the involvement of NO generation in mediating the biological activity of the growth factor.

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of bFGF and NaNp on uPA activity and proliferation in CVECs. uPA production () and cell proliferation () were assessed in parallel experiments. Cell-associated PA activity was measured in subconfluent CVECs after 24 hours of exposure to increasing concentrations of bFGF (a) and NaNp (b) by a spectrophotometric assay using a plasmin chromogenic substrate (see "Materials and Methods" for details). Proliferation was measured after 48 hours of exposure to the growth factor (a) and to NaNp (b) by the number of total cells counted at the end of incubation. Data are expressed as percent increase over basal response and represent the results of at least three experiments run in duplicate. The first significant value is reported. **P<.01 vs basal. Panel c shows fibrin zymography of CVEC extracts. CVEC cells were treated for 24 hours with 30 ng/mL bFGF or 100 µmol/L NaNp or were left untreated (control [C]). Then, cell extracts (15 µg of protein) were analyzed by SDS-PAGE, followed by zymography on a casein-agarose gel. The lytic zone corresponding to bovine uPA is marked by the arrow. Molecular weights are in thousands.

View this table: [in this window] [in a new window]   Table 1. Effect of NO Synthase Inhibitors on cGMP Levels in CVECs

View this table: [in this window] [in a new window]   Table 2. Effect of NO Synthase Inhibitors on Proliferation and uPA Activity of CVECs Exposed to bFGF

NaNp Promotes uPA Upregulation in CVECs
We then investigated whether NO by itself could affect the growth and invasive capacity of cultured coronary endothelial cells. The exogenous administration of the NO donor NaNp produced a dose-dependent increase of uPA activity, as demonstrated both by the enzymatic chromogenic assay (Fig 1b) and by fibrin zymography (Fig 1c). The maximal effect for both uPA upregulation and cell proliferation was produced by 10 µmol/L NaNp. At this concentration, NaNp increased uPA levels by 3-fold compared with levels in unstimulated control cells; cell number increased by 45.6% over basal (8x10³ cells per well [basal control] versus 12x10³ cells per well [in the presence of NaNp]). The effect of NaNp on CVECs was compared with that produced by bFGF. The extent of the maximally effective dose of NaNp in triggering uPA activity was comparable to that observed in CVECs treated with 1 to 3 ng/mL of bFGF. At higher concentrations (100 µmol/L and 1 mmol/L), the NO donor drug produced a plateau phase of the dose-response curve. Fibrin zymography of the extracts of CVECs exposed for 24 hours to 100 µmol/L NaNp revealed one major band with an apparent M_r of 49 000, corresponding to the expected M_r of bovine uPA. This band, more intense in cells treated with bFGF 30 ng/mL, was barely detectable in the cell extract of control untreated CVECs (Fig 1c)

NaNp Effects on CVECs Are Mediated by Endogenous bFGF
Endogenous bFGF has been reported to regulate DNA synthesis and uPA activity in endothelial cells through an autocrine mechanism of action. We therefore investigated whether endogenous bFGF could account for the observed prolonged responses of CVECs exposed to the NO donor drug NaNp. As shown in Fig 2a, Western blot analysis of the CVEC extracts after a 6-hour exposure to NaNp revealed an increase in the expression of three immunoreactive bands with an apparent molecular mass of 18, 22, and 24 kD, respectively. These immunoreactive bands were identified as the typical bFGF isoforms,³⁵ since they comigrated with bFGF isoforms expressed by endothelial cells transfected with bFGF cDNA (Fig 2a), were recognized by both monoclonal and polyclonal anti-bFGF antibodies, and did not cross-react with irrelevant IgG (data not shown). On this basis, we then investigated whether endogenous bFGF could account for the increase in cell proliferation and uPA activity produced by NaNp. To this purpose, CVECs were incubated with NaNp in the presence or in the absence of neutralizing anti-bFGF antibodies. These antibodies neutralized the mitogenic and uPA-inducing activity exerted by recombinant bFGF. Under the same experimental conditions, anti-bFGF antibodies significantly inhibited cell proliferation and uPA upregulation induced by NaNp, whereas irrelevant antibodies were totally ineffective (Fig 2b and 2c). uPA activity was reduced by 50±2% and cell proliferation was reduced by 80±7% in NaNp-treated cultures incubated with anti-bFGF antibodies. No effect was exerted by these antibodies on cell proliferation and uPA activity in control cultures. In agreement with these observations, uPA upregulation elicited by 100 µmol/L NaNp was inhibited by 88% when assayed in the presence of 500 µg/mL suramin, a molecule that blocks bFGF interaction with its receptor.²¹ Taken together, these observations implicate endogenous bFGF as the mediator of the increase in cell proliferation and uPA production exhibited by CVECs in response to exogenous NO.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of NaNp on endogenous bFGF production, uPA activity, and proliferation in CVECs. a, Western blot analysis of cell lysates (700 µg per lane) from CVECs treated with 100 µmol/L of NaNp. bFGF production was assessed after 6 hours of exposure of the cell monolayers to the drug. C indicates control; ZIP, 700 µg of bFGF-transfected MAE-ZIP-bFGF3F2 cell extract. bFGF isoforms are indicated by arrowheads. Molecular weights are in thousands. b, PA activity was assessed as described in Fig 1. Neutralizing anti-bFGF antibodies (1:50) or nonimmune IgGs (NI) were added 10 minutes before the addition of bFGF (3 ng/mL) or NaNp (100 µmol/L). Data are expressed as percentage of maximal response obtained with bFGF or NaNp alone. **P<.01 and ***P<.001 vs controls (n=3). c, Proliferation was assessed after 3 days of exposure to the stimuli. The neutralizing anti-bFGF MAb or NI were added at a dose of 5 µg/mL 10 minutes before the addition of test substances (10 ng/mL bFGF or 10 µmol/L NaNp). Data are expressed as percent increase over basal proliferation (8x103 cells per well). ***P<.001 vs controls (n=4).

NO-Dependent Modulation of Endogenous bFGF Accounts for the Proliferative Effect of SP
The role of endogenous bFGF as a mediator of NO effect was then investigated on peptides promoting endothelial cell growth by endogenous NO, like the neuropeptide SP. CVECs were exposed to SP or to Sar9-SP, the selective agonist for the NK-1 receptor, which accounts for the biological activity of SP on vascular endothelium. Both molecules promoted microvascular proliferation with a similar potency (Fig 3a). As for NaNp-induced proliferation, the growth of CVECs in response to SP was prevented by neutralizing monoclonal anti-bFGF antibodies (Fig 3b). Accordingly, the exposure of CVECs to the maximal proliferative concentration (10 nmol/L) of SP or of Sar9-SP induced a significant increase in the expression of all bFGF isoforms (Fig 3c). The increase was detectable after 6 hours of exposure (data not shown) and maximal after 15 hours of treatment.

View larger version (16K): [in this window] [in a new window]   Figure 3. SP and Sar9-SP induce proliferation and bFGF production in CVECs. a, Proliferation of CVECs in response to different concentrations of SP () or of the selective NK-1 receptor agonist Sar9-SP (). Proliferation was assessed as described in Fig 1. Data are expressed as percent increase over basal response. The first significant value is reported. **P<.01 vs basal (n=3). b, CVEC proliferation in response to 1 and 10 nmol/L SP (open bars) was challenged with anti-bFGF MAb (5 µg/mL) (hatched bars). Growth was evaluated as described in Fig 2c. Data are expressed as percent increase over basal proliferation (8x103 cells per well) and represent the results of three experiments run in duplicate. ***P<.001 vs controls. c, Western blot analysis of cell lysates (700 µg per lane) obtained from CVECs treated with 10 nmol/L of SP or Sar9-SP. bFGF production was measured after 15 hours of exposure of the cell monolayers to the agents. C indicates control. Molecular weights are in thousands.

To assess whether bFGF upregulation by SP reflected an increase of the steady state levels of bFGF mRNA, semiquantitative differential RT-PCR analysis of CVEC total RNA was performed. As shown in Fig 4a, 6 hours of exposure to SP doubled bFGF mRNA induction compared with the control GAPDH transcript. The treatment of the cells with the NO synthase inhibitor L-NMMA fully prevented bFGF upregulation by SP at mRNA and protein levels (Fig 4a and 4b), leading to the conclusion that bFGF expression was linked to NO production. In both RT-PCR and Western blot assay, parental MAE and MAE-pZIP-bFGF3F2 clone were used as negative and positive controls, respectively, for bFGF expression.²⁹

View larger version (50K): [in this window] [in a new window]   Figure 4. Modulation of SP-induced bFGF expression by NO synthase inhibitor in CVECs. a, Differential RT-PCR analysis of total RNA from CVECs treated for 6 hours with 10 nmol/L SP in the absence and in the presence of 200 µmol/L L-NMMA. bFGF and GAPDH coamplification products were electrophoresed on a 3% agarose gel and stained with ethidium bromide, and the intensity of the bands was analyzed by image analysis. Lanes are as follows: 1, no treatment (basal); 2, L-NMMA–treated cells (+L-NMMA); 3, SP-treated cells (SP); 4, SP plus L-NMMA–treated cells (+L-NMMA); 5, negative control MAE; 6, positive control bFGF-transfected MAE-pZIP-bFGF3F2 cells (ZIP); and 7, molecular weight standards (M). Numbers are the ratio between optical density values obtained for 194-bp bFGF and 242-bp GAPDH amplification products. Results are representative of three independent experiments. b, Western blot analysis of CVEC lysates. Cells were treated for 15 hours as described in panel a, and extracts (700 µg per lane) were probed with anti-bFGF antibody. bFGF isoforms are indicated by arrowheads. Molecular weights are in thousands.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present report, three novel observations are documented. First, it is shown that uPA activity is increased in parallel with DNA synthesis in coronary postcapillary venular endothelium exposed to the NO donor drug NaNp. Second, it is demonstrated that the expression of the angiogenic factor bFGF is increased in endothelial cells treated with NaNp and with the NO-dependent vasodilating peptide SP and modulates the degradative and proliferative effect of both vasodilators. Third, it is shown that the expression of the growth factor is under the control of the NO pathway via an autocrine/paracrine loop.

In previous studies, we demonstrated a role for NO in angiogenesis.⁹ The present investigation was undertaken to determine by which mechanism NO induces angiogenesis. The results presented here document the fact that in microvascular endothelium the elevation of intracellular NO induces the endogenous production of an angiogenic factor that makes the capillary endothelium acquire the feature of an "angiogenic endothelium" (as for increased proliferative and degradative capacity).

Proteinase production and growth of endothelium are necessary steps for the promotion of angiogenesis, and among the proteinases involved in angiogenesis, uPA plays a central role.¹³ Data in the literature have indicated a direct role for NO in endogenous fibrinolysis. Indeed, NO inhibits plasminogen activator inhibitor production by platelets and endothelial cells,³⁶ and an increase in NO and uPA production by cultured endothelium has been described during elevated shear stress.^{37 38} In coronary postcapillary venular endothelium, we found increased uPA activity and DNA synthesis in response to bFGF and to the NO donor drug NaNp. Western blot analysis revealed that NaNp treatment induced the endothelial cells to produce endogenous bFGF. Neutralizing anti-bFGF antibodies blocked uPA production and DNA synthesis after nipride treatment. Thus, NaNp induced the endothelial cells to produce bFGF and to respond to the endogenous growth factor. At the coronary level, the beneficial effect of nitrovasodilators has mainly been viewed as linked to the hemodynamic changes (improved vasodilatation and increased blood flow of the microcirculation). The data reported in the present study show that in response to nipride, coronary endothelium also undergoes modifications of the proteolytic balance and of angiogenic factor expression. Thus, the nitrovasodilator induces vasodilation as an early response and can favor neovascular growth as a late event.

Coronary angiogenesis occurring in the border zone of the ischemic myocardium contributes to limit the damage to the myocardium and the infarct size.³⁹ To this aim, intracoronary bFGF is proposed to enhance angiogenesis in this pathological condition.⁴⁰ During inflammation and injury, primary sensory neurons modulate vasodilation and exert trophic functions by releasing neuropeptides such as SP.⁴¹ In previous studies, we have shown that migration and proliferation of endothelial cells by tachykinins involve activation of the NK-1 receptor, NO production, and increase in cGMP levels.^{7 8 23} The present study documents that either SP or the selective NK-1 receptor agonist Sar9-SP likewise stimulates production of endogenous bFGF, ultimately leading to proliferation of coronary microvascular endothelial cells. Our findings demonstrate that NO synthase inhibitors prevent both cell proliferation⁹ and bFGF upregulation (present study) induced by SP. Thus, bFGF, with the intermediate step of NO production/release, is the final mediator of the angiogenic proliferative response to SP in coronary microvasculature.

Our results indicate that the effect of exogenous bFGF on CVEC proliferation and uPA activity is independent of NO synthase activation, in keeping with the observation that exogenous bFGF can elicit angiogenesis in vivo despite the block of NO production by capillary endothelium.⁹ On the other hand, our data demonstrate that elevation of NO levels in coronary endothelium increases endogenous bFGF production and that NO synthase inhibitors block bFGF upregulation. A direct correlation has been reported on bFGF levels in endothelial cells and their invasive behavior with angiogenesis in vivo.¹⁸ Thus, the present study expands previous observations on the autocrine role played by endogenous bFGF in regulating the proliferative behavior and protease production of vascular endothelium^{17 18 42 43} and discloses the existence of an autocrine control exerted by NO.

Angiogenesis involves the proliferation of endothelial cells under the control of local peptide growth factors and physical forces. Vasodilation is a component of angiogenesis, leading to the possibility that elevated shear rate may play a role as a stimulus for neovascularization.⁴⁴ The NO synthase/guanylate cyclase is an ideal signaling mechanism for integrating both chemical and physical influences and is involved in angiogenesis.⁹ Our data show that NO contributes to the angiogenic process by inducing endogenous bFGF in capillary endothelium. Thus, the experimental evidence reported in the present study provides insight into the mechanisms through which vasodilation is coupled to angiogenesis, revealing a feedback loop controlled by the endothelium via NO and bFGF.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor CVEC = coronary venular endothelial cell MAb = monoclonal antibody L-NAME = N-nitro-L-arginine methyl ester L-NMMA = N-monomethyl-L-arginine L-NNA = N-nitro-L-arginine MAE = mouse aortic endothelial cells NaNp = sodium nitroprusside PCR = polymerase chain reaction RT = reverse transcriptase Sar9-SP = [Sar9]-SP sulfone SP = substance P uPA = urokinase-type plasminogen activator ZIP = mouse aortic endothelial cells expressing bFGF

	Acknowledgments

 
This study was supported by funds from the Italian Ministry for the University and for Scientific and Technological Research, the National Research Council of Italy (grant No. 95.02983.CT14), AIRC (Special Project "Angiogenesis"), and European Communities (Biomed 2 Programme "Angiogenesis and Cancer," PL 950669) to Dr Ziche; by MERIT Award HL-21498 from the National Heart, Lung, and Blood Institute and the Texas Advanced Technology and Research Program to Dr Granger; and by the National Research Council of Italy (grant No. 95.02925.CT14), AIRC (Special Project "Angiogenesis"), and European Communities (Human Capital Mobility Project "Mechanisms for the Regulation of Angiogenesis") to Dr Presta. We wish to thank Dr L. Morbidelli for technical assistance.

Received October 7, 1996; accepted March 10, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Hudlicka O, Brown M, Egginton S. Angiogenesis in skeletal and cardiac muscle. Physiol Rev. 1992;72:369-417.[Free Full Text]

2. Dusseau JW, Hutchins PM, Malbasa DS. Stimulation of angiogenesis by adenosine on chick chorioallantoic membrane. Circ Res. 1986;59:163-170.[Abstract/Free Full Text]

3. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843-845.[Medline] [Order article via Infotrieve]

4. Ziada AMAR, Hudlicka O, Tyler KR, Wright AJA. The effect of long-term vasodilatation on capillary growth and performance in rabbit heart and skeletal muscle. Cardiovasc Res. 1984;18:724-732.[Medline] [Order article via Infotrieve]

5. Fernandez LA, Twickler J, Mead A. Neovascularization produced by angiotensin II. J Lab Clin Med. 1985;105:141-145.[Medline] [Order article via Infotrieve]

6. Meininger CJ, Shelling ME, Granger HJ. Adenosine and hypoxia stimulate proliferation and migration of endothelial cells. Am J Physiol. 1988;255:H554-H562.[Abstract/Free Full Text]

7. Ziche M, Morbidelli L, Pacini M, Geppetti P, Alessandri G, Maggi CA. Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells. Microvasc Res. 1990;40:264-278.[Medline] [Order article via Infotrieve]

8. Ziche M, Morbidelli L, Geppetti P, Maggi CA, Dolara P. Substance P induces migration of capillary endothelial cells: a novel NK-1 selective receptor mediated activity. Life Sci. 1991;48:PL7-PL11.[Medline] [Order article via Infotrieve]

9. Ziche M, Morbidelli L, Masini E, Amerini S, Granger HJ, Maggi CA, Geppetti P, Ledda F. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J Clin Invest. 1994;94:2036-2044.

10. D'Orleans-Juste P, Dion S, Mizrahi J, Regoli D. Effects of peptides and nonpeptides on isolated arterial smooth muscles: role of endothelium. Eur J Pharmacol. 1985;114:9-21.[Medline] [Order article via Infotrieve]

11. Kourembanas S, McQuillan LP, Leung GK, Faller DV. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J Clin Invest. 1993;92:99-104.

12. Folkman J. Angiogenesis: initiation and control. Ann N Y Acad Sci. 1982;401:212-227.[Medline] [Order article via Infotrieve]

13. Mignatti P, Mazzieri R, Rifkin DB. Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor. J Cell Biol. 1991;113:1193-1201.[Abstract/Free Full Text]

14. Presta M, Moscatelli D, Joseph-Silverstein J, Rifkin DB. Purification from a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis and migration. Mol Cell Biol. 1986;6:4060-4066.[Abstract/Free Full Text]

15. Mignatti P, Tsuboi R, Robbins E, Rifkin DB. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. J Cell Biol. 1989;108:671-682.[Abstract/Free Full Text]

16. Schweigerer L, Neufeld G, Freidman J, Abraham JA, Fiddes JC, Gospodarowicz D. Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature. 1987;325:257-259.[Medline] [Order article via Infotrieve]

17. Sato Y, Rifkin DB. Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis and DNA synthesis. J Cell Biol. 1988;107:1199-1205.[Abstract/Free Full Text]

18. Tsuboi R, Sato Y, Rifkin DB. Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells. J Cell Biol. 1990;110:511-517.[Abstract/Free Full Text]

19. McNeil PL, Muthukrishnan L, Warder E, D'Amore PA. Growth factors are released by mechanically wounded cells. J Cell Biol. 1989;109:811-822.[Abstract/Free Full Text]

20. Speir E, Tanner V, Gonzales AM, Farris J, Baird A, Casscells W. Acid and basic fibroblast growth factors in adult rat heart myocytes: localization, regulation in culture, and effects on DNA synthesis. Circ Res. 1992;71:251-259.[Abstract/Free Full Text]

21. Hawker JRH, Granger J. Tyrosine kinase inhibitors impair fibroblast growth factor signaling in coronary endothelial cells. Am J Physiol. 1993;266:H107-H120.

22. Feelish M, Noack E. Nitric oxide (NO) formation from nitrovasodilators occurs independently of hemoglobin or non-heme iron. Eur J Pharmacol. 1987;142:465-469.[Medline] [Order article via Infotrieve]

23. Ziche M, Morbidelli L, Parenti A, Amerini S, Granger HJ, Maggi CA. Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial cells. Life Sci. 1993;53:1105-1112.[Medline] [Order article via Infotrieve]

24. Schelling ME, Meininger CJ, Hawker JR, Granger HJ. Venular endothelial cells from bovine heart. Am J Physiol. 1988;254:H1211-H1217.[Abstract/Free Full Text]

25. Moncada S, Palmer RMJ, Higgs A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991;43:109-142.[Medline] [Order article via Infotrieve]

26. Morbidelli L, Chang C-H, Douglas JG, Granger HJ, Ledda F, Ziche M. Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. Am J Physiol. 1996;270:H411-H415.[Abstract/Free Full Text]

27. Presta M, Maier JAM, Ragnotti G. The mitogenic signaling pathway but not the plasminogen activator-inducing pathway of basic fibroblast growth factor is mediated through protein kinase C in fetal bovine aortic endothelial cells. J Cell Biol. 1989;109:1877-1884.[Abstract/Free Full Text]

28. Colombi M, Barlati S, Magdelenat H, Fiszer-Szagarz B. Relationship between multiple forms of plasminogen activator in human breast tumors and plasma and presence of metastasis in lymph nodes. Cancer Res. 1986;44:2971-2975.[Abstract/Free Full Text]

29. Gualandris A, Rusnati M, Belleri M, Nelli EM, Bastaki M, Molinari-Tosatti MP, Bonardi F, Parolini S, Albini A, Morbidelli L, Ziche M, Corallini A, Possati L, Vacca A, Ribatti D, Presta M. Basic fibroblast growth factor overexpression in mouse endothelial cells: an autocrine model of angiogenesis and angioproliferative diseases. Cell Growth Differ. 1996;7:147-160.[Abstract]

30. Chomczynsky P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]

31. Norman JG, Fink GW, Sexton C, Carter G. Transgenic animals demonstrate a role for the IL-1 receptor in regulating IL-1ß gene expression at steady-state and during the systemic stress induced by acute pancreatitis. J Surg Res. 1996;63:231-236.[Medline] [Order article via Infotrieve]

32. Fink GW, Norman JG. Intrapancreatic interleukin-1ß gene expression by specific leukocyte populations during acute pancreatitis. J Surg Res. 1996;63:369-373.[Medline] [Order article via Infotrieve]

33. Abraham JA, Mergia A, Whang JL, Tumolo A, Friedman J, Hjerrild KA, Gospodarovicz D, Fiddes JC. Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science. 1986;233:545-548.[Abstract/Free Full Text]

34. Peri A, Cordella-Miele E, Miele L, Mukherjee AB. Tissue-specific expression of the gene coding for human Clara cells 10-kD protein, a phospholipase A2-inhibitory protein. J Clin Invest. 1993;92:2099-2109.

35. Florkiewicz RZ, Sommer A. Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons. Proc Natl Acad Sci U S A. 1989;86:3978-3981.[Abstract/Free Full Text]

36. Drummer S, Ludke S, Spannagl M, Schramm W, Gerzer R. The nitric oxide donor SIN-1 is a potent inhibitor of plasminogen activator inhibitor release from stimulated platelets. Thromb Res. 1991;63:553-556.[Medline] [Order article via Infotrieve]

37. Ohno M, Gibbons GH, Dzau VJ, Cooke JP. Shear stress elevates endothelial cGMP: role of a potassium channel and G protein coupling. Circulation. 1993;88:193-197.[Abstract/Free Full Text]

38. Diamond SL, Eskin SG, McIntire LV. Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science. 1989;243:1483-1485.[Abstract/Free Full Text]

39. Granger HJ, Ziche M, Howker JR Jr, Meininger CJ, Czisny LE, Zaweja DC. Molecular and cellular basis of myocardial angiogenesis. Cell Mol Biol Res. 1994;40:81-85.[Medline] [Order article via Infotrieve]

40. Scharper W, Ito WD. Molecular mechanisms in coronary collateral vessel growth. Circ Res. 1996;79:911-919.[Free Full Text]

41. Maggi CA, Borsini F, Santicioli P, Geppetti P, Abelli L, Evangelista S, Manzini S, Theodorsson Norheim E, Somma V, Amenta F, Bacciarelli C, Meli A. Cutaneous lesions in capsaicin pretreated rats. Naunyn Schmiedebergs Arch Pharmacol. 1987;336:538-545.[Medline] [Order article via Infotrieve]

42. Itoh H, Mukoyama M, Pratt RE, Dzau VJ. Specific blockade of basic fibroblast growth factor gene expression in endothelial cells by antisense oligonucleotide. Biochem Biophys Res Commun. 1992;188:1205-1213.[Medline] [Order article via Infotrieve]

43. Pepper MS, Sappino A-P, Stocklin R, Montesano R, Orci L, Vassalli J-D. Upregulation of urokinase receptor expression on migrating endothelial cells. J Cell Biol. 1993;122:673-684.[Abstract/Free Full Text]

44. Clark ER, Clark EL. Microscopic observations on the growth of blood capillaries in the living mammal. Am J Anat. 1939;64:251-299.

This article has been cited by other articles:

				S. R. Mallery, J. C. Zwick, P. Pei, M. Tong, P. E. Larsen, B. S. Shumway, B. Lu, H. W. Fields, R. J. Mumper, and G. D. Stoner Topical Application of a Bioadhesive Black Raspberry Gel Modulates Gene Expression and Reduces Cyclooxygenase 2 Protein in Human Premalignant Oral Lesions Cancer Res., June 15, 2008; 68(12): 4945 - 4957. [Abstract] [Full Text] [PDF]

				Y. Matsumoto, S. Ueda, S.-i. Yamagishi, K. Matsuguma, R. Shibata, K. Fukami, H. Matsuoka, T. Imaizumi, and S. Okuda Dimethylarginine Dimethylaminohydrolase Prevents Progression of Renal Dysfunction by Inhibiting Loss of Peritubular Capillaries and Tubulointerstitial Fibrosis in a Rat Model of Chronic Kidney Disease J. Am. Soc. Nephrol., May 1, 2007; 18(5): 1525 - 1533. [Abstract] [Full Text] [PDF]

				H. Konishi, K. Sydow, and J. P. Cooke Dimethylarginine Dimethylaminohydrolase Promotes Endothelial Repair After Vascular Injury J. Am. Coll. Cardiol., March 13, 2007; 49(10): 1099 - 1105. [Abstract] [Full Text] [PDF]

				S. Donnini, R. Solito, A. Giachetti, H. J. Granger, M. Ziche, and L. Morbidelli Fibroblast Growth Factor-2 Mediates Angiotensin-Converting Enzyme Inhibitor-Induced Angiogenesis in Coronary Endothelium J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 515 - 522. [Abstract] [Full Text] [PDF]

				R. P. Singh, G. Deep, M. Chittezhath, M. Kaur, L. D. Dwyer-Nield, A. M. Malkinson, and R. Agarwal Effect of silibinin on the growth and progression of primary lung tumors in mice. J Natl Cancer Inst, June 21, 2006; 98(12): 846 - 855. [Abstract] [Full Text] [PDF]

				J. Zheng, Y. Wen, J. L. Austin, and D.-b. Chen Exogenous Nitric Oxide Stimulates Cell Proliferation via Activation of a Mitogen-Activated Protein Kinase Pathway in Ovine Fetoplacental Artery Endothelial Cells Biol Reprod, February 1, 2006; 74(2): 375 - 382. [Abstract] [Full Text] [PDF]

				S.-h. Lee, M. Nishino, T. Mazumdar, G. E. Garcia, M. Galfione, F. L. Lee, C. L. Lee, A. Liang, J. Kim, L. Feng, et al. 16-kDa Prolactin Down-Regulates Inducible Nitric Oxide Synthase Expression through Inhibition of the Signal Transducer and Activator of Transcription 1/IFN Regulatory Factor-1 Pathway Cancer Res., September 1, 2005; 65(17): 7984 - 7992. [Abstract] [Full Text] [PDF]

				M. Camozzi, S. Zacchigna, M. Rusnati, D. Coltrini, G. Ramirez-Correa, B. Bottazzi, A. Mantovani, M. Giacca, and M. Presta Pentraxin 3 Inhibits Fibroblast Growth Factor 2-Dependent Activation of Smooth Muscle Cells In Vitro and Neointima Formation In Vivo Arterioscler Thromb Vasc Biol, September 1, 2005; 25(9): 1837 - 1842. [Abstract] [Full Text] [PDF]

				J. Jacobi, K. Sydow, G. von Degenfeld, Y. Zhang, H. Dayoub, B. Wang, A. J. Patterson, M. Kimoto, H. M. Blau, and J. P. Cooke Overexpression of Dimethylarginine Dimethylaminohydrolase Reduces Tissue Asymmetric Dimethylarginine Levels and Enhances Angiogenesis Circulation, March 22, 2005; 111(11): 1431 - 1438. [Abstract] [Full Text] [PDF]

				V Achan, H. Ho, C Heeschen, M Stuehlinger, J. Jang, M Kimoto, P Vallance, and J. Cooke ADMA regulates angiogenesis: genetic and metabolic evidence Vascular Medicine, February 1, 2005; 10(1): 7 - 14. [Abstract] [PDF]

				J. Sun and J. K. Liao Induction of Angiogenesis by Heat Shock Protein 90 Mediated by Protein Kinase Akt and Endothelial Nitric Oxide Synthase Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2238 - 2244. [Abstract] [Full Text] [PDF]

				J. L. Unthank, K. M. Sheridan, and M. C. Dalsing Collateral Growth in the Peripheral Circulation: A Review Vascular and Endovascular Surgery, July 1, 2004; 38(4): 291 - 313. [Abstract] [PDF]

				M. Rusnati, M. Camozzi, E. Moroni, B. Bottazzi, G. Peri, S. Indraccolo, A. Amadori, A. Mantovani, and M. Presta Selective recognition of fibroblast growth factor-2 by the long pentraxin PTX3 inhibits angiogenesis Blood, July 1, 2004; 104(1): 92 - 99. [Abstract] [Full Text] [PDF]

				J. W. Crott, S.-W. Choi, J. M. Ordovas, J. S. Ditelberg, and J. B. Mason Effects of dietary folate and aging on gene expression in the colonic mucosa of rats: implications for carcinogenesis Carcinogenesis, January 1, 2004; 25(1): 69 - 76. [Abstract] [Full Text] [PDF]

				K. Kawasaki, R. S. Smith Jr., C.-M. Hsieh, J. Sun, J. Chao, and J. K. Liao Activation of the Phosphatidylinositol 3-Kinase/Protein Kinase Akt Pathway Mediates Nitric Oxide-Induced Endothelial Cell Migration and Angiogenesis Mol. Cell. Biol., August 15, 2003; 23(16): 5726 - 5737. [Abstract] [Full Text] [PDF]

				D. L. Brutsaert Cardiac Endothelial-Myocardial Signaling: Its Role in Cardiac Growth, Contractile Performance, and Rhythmicity Physiol Rev, January 1, 2003; 83(1): 59 - 115. [Abstract] [Full Text] [PDF]

				C. J. Sullivan and J. B. Hoying Flow-Dependent Remodeling in the Carotid Artery of Fibroblast Growth Factor-2 Knockout Mice Arterioscler Thromb Vasc Biol, July 1, 2002; 22(7): 1100 - 1105. [Abstract] [Full Text] [PDF]

				J. P. Cooke and D. W. Losordo Nitric Oxide and Angiogenesis Circulation, May 7, 2002; 105(18): 2133 - 2135. [Full Text] [PDF]

				T. Padro, R. M. Mesters, B. Dankbar, H. Hintelmann, R. Bieker, M. Kiehl, W. E. Berdel, and J. Kienast The catalytic domain of endogenous urokinase-type plasminogen activator is required for the mitogenic activity of platelet-derived and basic fibroblast growth factors in human vascular smooth muscle cells J. Cell Sci., January 5, 2002; 115(9): 1961 - 1971. [Abstract] [Full Text] [PDF]

				D. Wothe, A. Hohimer, M. Morton, K. Thornburg, G. Giraud, and L. Davis Increased coronary blood flow signals growth of coronary resistance vessels in near-term ovine fetuses Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R295 - R302. [Abstract] [Full Text] [PDF]

				P. G. Lloyd, H. T. Yang, and R. L. Terjung Arteriogenesis and angiogenesis in rat ischemic hindlimb: role of nitric oxide Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2528 - H2538. [Abstract] [Full Text] [PDF]

				A. Parenti, L. Brogelli, S. Donnini, M. Ziche, and F. Ledda ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2 Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H99 - H107. [Abstract] [Full Text] [PDF]

				T. R. Quinlan, D. Li, V. E. Laubach, E. G. Shesely, N. Zhou, and R. A. Johns eNOS-deficient mice show reduced pulmonary vascular proliferation and remodeling to chronic hypoxia Am J Physiol Lung Cell Mol Physiol, October 1, 2000; 279(4): L641 - L650. [Abstract] [Full Text] [PDF]

				J. J. Jang, H.-K. V. Ho, H. H. Kwan, L. F. Fajardo, and J. P. Cooke Angiogenesis Is Impaired by Hypercholesterolemia : Role of Asymmetric Dimethylarginine Circulation, September 19, 2000; 102(12): 1414 - 1419. [Abstract] [Full Text] [PDF]

				J. A. Whitney, Z. German, T. S. Sherman, I. S. Yuhanna, and P. W. Shaul Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells Am J Physiol Lung Cell Mol Physiol, January 1, 2000; 278(1): L131 - L138. [Abstract] [Full Text] [PDF]

				L. E. Davis, A. R. Hohimer, and M. J. Morton Myocardial blood flow and coronary reserve in chronically anemic fetal lambs Am J Physiol Regulatory Integrative Comp Physiol, July 1, 1999; 277(1): R306 - R313. [Abstract] [Full Text] [PDF]

				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]

				J.-E. Fabre, A. Rivard, M. Magner, M. Silver, and J. M. Isner Tissue Inhibition of Angiotensin-Converting Enzyme Activity Stimulates Angiogenesis In Vivo Circulation, June 15, 1999; 99(23): 3043 - 3049. [Abstract] [Full Text] [PDF]

				S. Babaei, K. Teichert-Kuliszewska, J.-C. Monge, F. Mohamed, M. P. Bendeck, and D. J. Stewart Role of Nitric Oxide in the Angiogenic Response In Vitro to Basic Fibroblast Growth Factor Circ. Res., May 19, 1998; 82(9): 1007 - 1015. [Abstract] [Full Text] [PDF]

				J. D. Hood, C. J. Meininger, M. Ziche, and H. J. Granger VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells Am J Physiol Heart Circ Physiol, March 1, 1998; 274(3): H1054 - H1058. [Abstract] [Full Text] [PDF]

				A. Parenti, L. Morbidelli, X.-L. Cui, J. G. Douglas, J. D. Hood, H. J. Granger, F. Ledda, and M. Ziche Nitric Oxide Is an Upstream Signal of Vascular Endothelial Growth Factor-induced Extracellular Signal-regulated Kinase1/2 Activation in Postcapillary Endothelium J. Biol. Chem., February 13, 1998; 273(7): 4220 - 4226. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ziche, M. Articles by Presta, M. Search for Related Content PubMed PubMed Citation Articles by Ziche, M. Articles by Presta, M.