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Am J Physiol Heart Circ Physiol 279: H817-H824, 2000;
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Vol. 279, Issue 2, H817-H824, August 2000

Lack of hypoxic stimulation of VEGF secretion from neutrophils and platelets

Petra Koehne1, Carsten Willam2, Evelyn Strauss1, Ralf Schindler2, Kai-Uwe Eckardt2, and Christoph Bührer1

Departments of 1 Neonatology and 2 Nephrology and Medical Intensive Care, Charité, Campus Virchow-Klinikum, Humboldt University, D-13353 Berlin, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Low oxygen (O2) is the key stimulus for expression of vascular endothelial growth factor (VEGF) in several adherent cells. Whether hypoxia also directs the release of VEGF protein from neutrophils (polymorphonuclear neutrophils; PMN) and platelets has not been investigated. We therefore compared VEGF release of platelets, PMN, and human vascular smooth muscle cells (HSMC) in response to hypoxia with that to activators of cellular degranulation. In contrast to HSMC, VEGF release from PMN and platelets or VEGF mRNA expression in PMN was not stimulated under hypoxic conditions (1% O2). Hypo- or hyperthermia and acidosis, other conditions potentially associated with ischemic and inflammatory tissue injury, also did not stimulate VEGF secretion from PMN. However, stimulation of platelets with thrombin and of PMN with phorbol 12-myristate 13-acetate induced a time-dependent release of VEGF, peaking after 30 and 60 min, respectively. This was blocked by the degranulation inhibitor pentoxifylline but not by the protein-synthesis inhibitor cycloheximide. We conclude that rapid release of VEGF from platelets and PMN may occur independently of oxygenation during inflammation and hemostasis.

vascular endothelial growth factor; hypoxia; angiogenesis; intracellular vascular endothelial growth factor pool


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), also known as vascular permeability factor (VPF), is a potent endothelial cell mitogen (10) that promotes angiogenesis (31). In addition, VEGF acts as a chemoattractant for monocytes (4) and enhances vascular permeability (13). Of note, VEGF receptors are localized almost exclusively on endothelial cells (23). Thus VEGF is thought to play a major role in the normal host repair of damaged blood vessels and in neoangiogenesis; indeed, beneficial effects by use of VEGF as a therapeutic angiogenic agent were reported (30). Although encoded by a single gene, VEGF has several isoforms generated by alternative exon splicing. Of these, the 121-amino acid isoform, VEGF121, is a soluble glycoprotein that binds poorly to heparin and is freely secreted by the cell. VEGF165 (165-amino acid isoform), the most abundant form in virtually all human tissues, is a glycoprotein with some heparin-binding capacity, although ~70% is secreted by the cell. The larger two splice variants, VEGF189 and VEGF206 (189- and 206-amino acid isoforms, respectively), are highly basic polypeptides that bind heparin avidly and, after production, are held at the cell surface, bound to heparan sulfate proteoglycans rather than being secreted (14).

VEGF expression and secretion by a variety of anchorage-dependent cell types, including human vascular smooth muscle cells (HSMC), are well known (1, 2, 18, 32). More recently, release of VEGF from circulating blood cells such as T lymphocytes (8), mononuclear cells (15), polymorphonuclear neutrophils (PMN) (9), and platelets (22) has been described. Platelets release VEGF during platelet aggregation together with the release of beta -thromboglobulin, suggesting that VEGF resides in the alpha -granules of platelets (12). Aside from thrombin, other mediators of platelet activation, such as collagen, epinephrine, and ADP, are capable of inducing VEGF release in vitro (20). A granule-specific distribution of the intracellular pool of VEGF has also been observed in resting human PMN. VEGF release from human PMN was reported after phorbol 12-myristate 13-acetate (PMA)-, N-formylmethionyl-leucyl-phenylalanine, and tumor necrosis factor-alpha -induced cellular degranulation (9, 26).

Because conditions leading to neoangiogenesis such as tissue hypoxia or tumor growth are frequently associated with accumulation and activation of white blood cells and platelets, VEGF release from these cells may play an important role. However, the mechanisms of VEGF secretion are not well characterized for circulating blood cells. In anchorage-dependent cells, hypoxia is a potent stimulus for VEGF release (24). PMN have also been found to be able to respond to hypoxia by increasing the release of certain proteins, including interleukin-8 and interleukin-1beta (6). Whether they produce VEGF in response to hypoxia and whether the mechanisms of VEGF release from platelets and PMN are sensitive to changes in oxygenation have not been investigated. Moreover, the time course and magnitude of VEGF release from platelets and PMN have not been directly related to those of resident cells.

We have therefore studied VEGF release in parallel in vascular HSMC, PMN, and platelets to compare the release in response to hypoxia with that to activators of cellular degranulation and with preformed stores of VEGF. In addition, we tested whether hypoxia induces VEGF mRNA expression in PMN and whether VEGF release from PMN depends on protein synthesis. Furthermore, as PMN fighting infection may encounter an acidic environment and hyperthermia in addition to hypoxia, we also investigated VEGF release by PMN under these conditions.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

DMSO, Triton X-100, HEPES, PMA, pentoxifylline [PTX; 3,7-dimethyl-1-(5-oxohexyl)-xanthine], cycloheximide {3-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide}, thrombin, agarose, and the reagents for lactate dehydrogenase (LDH) quantification were purchased from Sigma Chemical (Deisenhofen, Germany). The human VEGF immunoassay for the quantitative determination of human VEGF concentrations and the human VEGF-specific PCR primer pair were obtained from R&D Systems (Wiesbaden-Nordenstadt, Germany). Tissue culture plastic ware was obtained from Becton-Dickinson (Heidelberg, Germany). DMEM, RPMI 1640, PBS, FCS, trypsin-EDTA solution, guanidine isothiocyanate, and antibiotics were from GIBCO Life Technologies (Berlin, Germany). Collagenase was purchased from Roth (Karlsruhe, Germany). L-Glutamine was acquired from Biochrom (Berlin, Germany). The monoclonal antibody against anti-smooth muscle alpha -actin was obtained from Progen Biotechnik (Bad Nauheim, Germany). Ficoll-Paque was purchased from Pharmacia Biotech (Freiburg, Germany). Cesium chloride was obtained from Paesel (Frankfurt, Germany). Avian myeloblastosis virus (AMV) reverse transcriptase, dATP, dCTP, dGTP, and dTTP were purchased from Promega (Serva, Heidelberg, Germany). Ampli-Taq DNA polymerase was from Perkin Elmer-Cetus (Emeryville, CA). Digoxigenin-11-dUTP and DIG Luminescent Detection Kit for Nucleic Acids were obtained from Boehringer-Mannheim (Mannheim, Germany). X-ray film was purchased from Kodak (Stuttgart, Germany). The nylon membrane was obtained from Amersham (Braunschweig, Germany).

Methods

Neutrophil preparation. PMN were obtained from healthy adult volunteers after informed consent was given, as described previously (28). Briefly, clotting was prevented by sodium citrate, and PMN were purified under sterile conditions by centrifugation on Ficoll-Paque cushions. Contaminating erythrocytes were removed by hypotonic lysis. Cells were resuspended in RPMI 1640, supplemented with 0.5% (20 mM) HEPES, at a final concentration of 1 × 107 cells/ml. After stimulation with PMA (50 ng/ml), treatment with PTX (500 µmol/l) and cycloheximide (10 µg/ml), or incubation under hypoxic conditions for various periods of time, the culture supernatants were removed, centrifuged at 2,100 rpm for 10 min at 4°C, and stored at -20°C for further VEGF and LDH quantification.

Isolation of human platelets. Platelets were isolated from buffy coats of healthy blood donors (after informed consent) by centrifugation for 10 min at 1,100 rpm. Cells were resuspended in fresh serum-free RPMI 1640 medium, supplemented with 0.5% (20 mM) HEPES, at a final concentration of 5 × 108 cells/ml for the experiments. After incubation for various time intervals up to 24 h under different conditions, the culture supernatants were removed, centrifuged at 2,100 rpm for 10 min at 4°C, and stored at -20°C until further VEGF and LDH quantification.

Cell culture. Vascular HSMC were prepared as described previously (3). HSMC were obtained from umbilical veins, deendothelialized with collagenase. Small tissue cubes of 2-4 mm were cut from the deendothelialized inner layer and placed intimal surface down into the dish. The cell culture medium contained DMEM, 15% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 200 mM/l L-glutamine. Dishes were placed in a humidified incubator (5% CO2-95% air) at 37°C. After 5-8 days of incubation, HSMC started to proliferate. The tissue pieces were removed when surrounding space was covered by cells. At confluence, cells were detached using 0.25% trypsin in 0.02% EDTA and split at a 1:3 ratio. Media were changed twice weekly. The purity and identity of the HSMC cultures were verified by use of monoclonal antibody against anti-smooth muscle alpha -actin. For all experiments, confluent 35-mm dishes of third-passage HSMC were used (2 × 105 cells). The cells were washed with PBS before the experiments. During the experiments, the content of FCS was reduced to 1%, and the used HSMC culture medium was supplemented with 0.5% (20 mM) HEPES. The culture supernatants were removed after the incubation period and stored at -20°C until VEGF and LDH quantification.

Hypoxia, hypothermia, hyperthermia, and acidosis. VEGF release by HSMC, human PMN, and platelets was studied under normoxic or hypoxic conditions at a temperature of 37°C. Additionally, the effects of hypothermia, hyperthermia, and acidic pH on PMN release of VEGF were evaluated under normoxic conditions, comparing only the 30- and 120-min time points. Normoxia was defined as 95% air-5% CO2. Hypoxia was characterized as 1% O2-5% CO2-94% N2. A temperature of 34°C was used for hypothermia and 40°C for hyperthermia. Different thermal conditions and O2 tensions were achieved with a humidified single-chamber incubator (model no. 3165; Forma Scientific, Labotect, Göttingen, Germany). Variations in pH were accomplished by addition of 70 µl of different HEPES buffer solutions per milliliter of RPMI 1640 medium before PMN incubation and were reaffirmed by pH analysis of culture medium at the end of the incubation period (model ABL 505; Radiometer, Willich, Germany). HEPES buffer solutions (1 M HEPES) of pH 5.3, 6.8, and 7.4 were needed to keep medium pH at 6.8, 7.1, and 7.4, respectively.

Stimulation experiments with thrombin or PMA. Thrombin was solubilized in distilled sterile water (stock concentration 1 U/µl), and PMA was solubilized in DMSO (stock concentration 1 mg/5 ml). Both drugs were further diluted in cell culture medium. Platelets were stimulated with thrombin at a final concentration of 1 U/ml. PMN were incubated with PMA at a concentration of 50 ng/ml.

Cycloheximide and PTX treatment. Cycloheximide was solubilized in DMSO (stock concentration 1 µg/µl), whereas PTX was solubilized in distilled sterile water (stock concentration 50 mmol/l). Both drugs were further diluted in cell culture medium. Cycloheximide was used at a concentration of 10 µg/ml in the experiments, and PTX was used at a final concentration of 500 µmol/l. PMN and platelets were preincubated with PTX for 30 min at 37°C and then further incubated with PMA or thrombin for time periods between 0.5 and 24 h. Only PMN were preincubated with cycloheximide for 30 min at 37°C before 50 ng/ml PMA were added for the indicated times. In HSMC, PTX or cycloheximide was added directly before hypoxia started.

Detergent lysis. Solubilization of integral membrane proteins was achieved by the addition of the nonionic detergent Triton X-100. For the treatment of the three different cell types, Triton X-100 was diluted in cell culture medium and used at a final concentration of 1%.

Enzyme immunoassay for VEGF. Quantitative VEGF measurements in samples from conditioned medium were performed with the use of a commercially available ELISA kit, according to the recommendations of the manufacturer. The human VEGF immunoassay recognized the soluble isoforms VEGF121 and VEGF165 in supernatants of PMN, platelets, and HSMC. The assay had a minimum detectable concentration of 5 pg/ml VEGF (for cell culture supernatants) and did not cross-react with other known cytokines, in particular, platelet-derived growth factor (PDGF). Optical density at 450 nm was measured on a Titertek Multiscan MC plate reader (Flow Laboratories, Helsinki, Finland), and VEGF concentration was determined by linear regression from a standard curve, using the kit VEGF165 as standard and GraphPad software (San Diego, CA) for analysis.

LDH determination. LDH activity in samples was determined with a kinetic enzymatic assay at 340 nm. The detection limit for LDH was 4.7 U/l.

Measurement of medium osmolality. Osmolality was measured in RPMI 1640 medium after addition of different HEPES buffer solutions by use of an osmometer (model no. 030; Osmomat, Gonotec, Berlin, Germany), according to the recommendations of the manufacturer.

PCR. Semiquantitative PCR was used to assess VEGF mRNA expression under different conditions in PMN and HSMC. Immediately after the incubation period, culture supernatants were removed and PMN and HSMC were lysed in 4 M guanidine isothiocyanate. Total RNA was extracted by ultracentrifugation (Sorvall Ultra Pro 80 ultraspeed centrifuge; Sorvall Instruments, DuPont, Newtown, CT) through 5.7 M cesium chloride, quantitated by determination of optical density at 260 and 280 nm (Ultraspec 2000 spectrophotometer, Pharmacia Biotech), precipitated in ethanol, and adjusted to 100 pg/ml. Reverse transcription of 1 µg RNA into cDNA was performed by use of random hexamers and AMV reverse transcriptase and 1× PCR buffer (20 mM Tris · HCl, pH 8.3, 50 mM KCl, 2 mM MgCl, and 100 µg/ml bovine serum albumin). The cDNA was amplified by PCR in a total volume of 50 µl with the use of 2.5 U Ampli-Taq DNA polymerase; 100 µM dATP, dCTP, and dGTP; 50 µM dTTP; and 0.5 µM of each primer in 1× PCR buffer. Incorporation of digoxigenin was performed by addition of 10 µM digoxigenin-11-dUTP to the PCR reaction mixture. Cycles included 1 min each at 95°C, 55°C, and 72°C in a microprocessor-driven thermal cycler (Landgraf, Hannover, Germany).

Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows (product size 486 bp): 5'-CCA TGG AGA AGG CTG GG and CAA AGT TGT CAT GGA TGA CC-3'. Primers for human VEGF were as follows (GenBank X62568; product size 441 bp, amplifying VEGF isoform 165): 5'-AGG GCA GAA TCA TCA CGA AG and CGC TCC GTC GAA CTC AAT TT-3'.

Aliquots were taken after different numbers of cycles (usually after 24, 28, and 33 cycles) and analyzed on 1.5% agarose gels, and the PCR products were stained with ethidium bromide. The PCR products were transferred to nylon membranes by capillary blotting with the use of 20× standard sodium citrate as blotting buffer. The nylon membranes were fixed by ultraviolet light, and the digoxigenin-UTP that had been incorporated into the PCR products was visualized by staining with anti-digoxigenin antibody conjugated to alkaline phosphatase (27). Luminescence of the substrate (Lumigen PPD) was documented by short exposure to X-ray film.

Calculations and statistics. Results were expressed as means ± SE (n = no. of experiments). Individual groups were compared by Student's t-test. For comparison of more than two groups, significant differences revealed by one-way ANOVA (Kruskal Wallis) were further analyzed by post hoc Student's t-test (SPSS 9.0 for Windows 95; SPSS, Chicago, IL). Significance was considered attained when P < 0.05. Densitometric calculations of film images were performed with the analysis program Scion Image (Scion, Frederick, MD).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Hypoxia on the VEGF Release in Smooth Muscle Cells, Neutrophils, and Platelets

As shown in Fig. 1, incubation of HSMC under hypoxic conditions induced a significant increase of VEGF concentration in the culture supernatants. After 8 h, a 47.5-fold increase was evident; this continued to rise 1.32-fold after 12 h and was only slightly further increased by 1.09-fold during the remainder of the 24 h of the experiment. In the control experiment, VEGF levels secreted by HSMC under normoxic conditions did not exceed 23.5 ± 7.2 pg/ml during the whole time course. Detergent lysis with Triton X-100 had no effect on VEGF release in resting HSMC. The incubation of human PMN and platelets under hypoxic conditions for time periods between 0.5 and 24 h did not result in a significant increase of soluble VEGF in the supernatants compared with the normoxic controls (Figs. 2 and 3).


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  		Fig. 1.   Time course of vascular endothelial growth factor (VEGF) release in human vascular smooth muscle cells (HSMC) exposed to hypoxia. HSMC (2 × 105 cells) in culture were either incubated with conditioned media in 1% O2-5% CO2-94% N2 for 2, 4, 8, 12, or 24 h or maintained in 95% air. A maximum VEGF increase in HSMC supernatants of 68-fold resulted after 24-h incubation under hypoxic conditions. Line graph also demonstrates effect of Triton X-100 (Triton X) on VEGF secretion by these cells. Values are means ± SE; no. of experiments (n) = 7-13 at each time point. ** P < 0.001 vs. normoxic control.



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  		Fig. 2.   Kinetics of phorbol 12-myristate 13-acetate (PMA)-induced VEGF release in human polymorphonuclear neutrophils (PMN). PMN (107/ml) were incubated with 50 ng/ml of PMA for indicated periods of time at normoxic conditions in RPMI 1640 medium. Effect of detergent lysis with 1% Triton X-100 on VEGF release of PMN is indicated. VEGF values are compared with effect of hypoxia and normoxic controls at same time points. Values are means ± SE; n = 5-15 at each time point. * P < 0.05 vs. normoxic control.



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  		Fig. 3.   Kinetics of thrombin- and Triton X-100-induced VEGF release in human platelets. Platelets (5 × 108/ml) were incubated in serum-free RPMI 1640 medium with 1 U/ml of thrombin or lysed with 1% Triton X-100 for indicated periods of time at normoxic conditions. VEGF values are compared with effect of hypoxia and normoxic controls at same time points. Values are means ± SE; n = 5-12 at each time point. * P < 0.05 vs. normoxic control.

Effect of Hypoxia on the VEGF mRNA Expression in Smooth Muscle Cells and Neutrophils

In semiquantitative PCR, mRNA expression for VEGF in HSMC roughly doubled after 8 h and tripled after 12 or 24 h of hypoxia compared with respective normoxic controls (Fig. 4). The ratio between VEGF mRNA signal and GAPDH signal of the same exposure time and condition was calculated after densitometric analysis and served as an indicator for VEGF mRNA increase. In HSMC, the ratios were 1.0, 2.21, 1.16, 3.14, 1.11, and 2.52 after 8-h normoxia, 8-h hypoxia, 12-h normoxia, 12-h hypoxia, 24-h normoxia, and 24-h hypoxia, respectively. In HSMC (but not in PMN), a faint second amplification product ~50- to 100-bp shorter than the main VEGF message amplification product (VEGF165) and probably representing a splice variant was present and increased in parallel with the main VEGF message in response to hypoxia. In PMN, no significant differences of normoxic and hypoxic mRNA expression were seen after 8, 12, or 24 h of exposure. Consequently, the ratio of VEGF mRNA and GAPDH signal was in a range between 0.88 and 1.0. The density of the shown GAPDH bands was homogenous at all time intervals for both cell types. GAPDH bands of HSMC did show a 1.0-, 0.95-, 0.92-, 1.02-, 1.04-, and 1.11-fold expression compared with the first band. GAPDH of PMN showed a 1.0-, 1.0-, 1.06-, 1.05-, 1.0-, and 1.0-fold expression compared with the first band.


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  		Fig. 4.   Detection of VEGF mRNA expression (top lanes) in HSMC and PMN after 8, 12, and 24 h of incubation under normoxic (N) or hypoxic (H) conditions by semiquantitative PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; bottom lanes) served as internal control. After 8 h of hypoxia, VEGF mRNA expression in HSMC had approximately doubled and after 12 and 24 h had tripled. In contrast to HSMC, amount of VEGF mRNA remained constant in PMN after 8, 12, and 24 h of incubation under normoxic or hypoxic conditions.

PMA-Induced Release of VEGF by Neutrophils

Stimulation of human PMN with 50 ng/ml PMA resulted in a rapid release of VEGF (Fig. 2), with VEGF concentrations peaking 60 min after addition of PMA. Maximum VEGF concentrations after PMA were equivalent to ~69% of VEGF concentrations in supernatants of PMN lysed by the detergent, Triton X-100. Apparent VEGF concentrations in supernatants of detergent-lysed PMN but not of PMA-stimulated PMN decreased over time and were similar to controls after 24 h. PMN exhibited a baseline VEGF release also in the absence of specific stimulation or detergent lysis.

Thrombin-Induced Release of VEGF by Platelets

Stimulation of human platelets with thrombin at a concentration of 1 U/ml resulted in a rapid release of VEGF, reaching its maximum of 607.6 ± 106.4 pg/ml within 30 min (Fig. 3). The amount of VEGF released in response to 30 min of incubation with thrombin corresponded to 80% of VEGF released by detergent lysis. VEGF concentrations decreased slightly in supernatants of thrombin-stimulated or detergent-lysed platelets during the 24-h experimental time course but remained significantly higher than in controls. Similar to PMN, a spontaneous VEGF release was also observed in unstimulated platelet controls.

Effect of PTX and Cycloheximide on the PMA-Induced VEGF Release in Neutrophils

Treatment of PMN with 500 µmol/l PTX, an inhibitor of cellular degranulation, 30 min before the addition of PMA attenuated the effect of PMA. A significant reduction of PMA-induced VEGF release (by 53%) resulted after 1 h (Fig. 5, top) and 2 h of coincubation with PMN and PTX. In contrast, preincubation of PMN with cycloheximide did not affect the PMA-induced VEGF release (data not shown).


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  		Fig. 5.   Inhibitory effect of pentoxifylline (PTX; 500 µmol/l) on PMA-induced VEGF release in PMN (top) and on thrombin-induced VEGF release in human platelets (bottom) after 1 h of simultaneous incubation at 37°C under normoxia. Values are means ± SE; n = 5-8 at each time point. * P < 0.05 vs. normoxic control. # P < 0.05 vs. PMA or thrombin alone.

Effect of PTX on the Thrombin-Induced VEGF Release in Platelets

Pretreatment of platelets with 500 µmol/l PTX blocked the release of VEGF into the medium throughout the whole time course. After 30 min, 1 h (Fig. 5, bottom), and 2 h of coincubation with thrombin and PTX, VEGF concentrations in the supernatants were significantly reduced to 34, 27, and 25% of thrombin-treated samples, respectively.

Effect of PTX and Cycloheximide on the Hypoxia-Induced VEGF Release in Smooth Muscle Cells

The increased expression of VEGF observed in HSMC under hypoxic conditions was completely blocked in the presence of the protein synthesis inhibitor cycloheximide at the 12-h (Fig. 6) and 24-h time points. Under normoxic conditions, cycloheximide did not significantly reduce the VEGF content in the supernatants of HSMC compared with controls (data not shown). In contrast, addition of PTX for the incubation period had no influence on the hypoxia-mediated VEGF increase.


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  		Fig. 6.   VEGF concentration in supernatants of HSMC after 12 h of incubation. Influence of PTX (500 µmol/l) and cycloheximide (10 µg/ml) on VEGF induction under hypoxic conditions is compared. Values are means ± SE; n = 5-13 at each time point. ** P < 0.001 vs. normoxic control. ## P < 0.001 vs. hypoxia.

LDH Concentration in Supernatants of Neutrophils, Platelets, and Smooth Muscle Cells

LDH concentrations in the samples were determined as an indicator for cellular damage. In the supernatants of HSMC, LDH levels remained unchanged under normoxic as well as under hypoxic conditions throughout the 24-h experiments and did not exceed the basal LDH content of the 2-h control (32.7 ± 10.3 U/ml). Incubation with PTX or cycloheximide produced no increase in LDH release. Triton X-100 was the only drug that induced a significant LDH increase after a 12-h (68.0 ± 7.2 U/ml) and 24-h (59.0 ± 3.1 U/ml) incubation period. Compared with HSMC, basal LDH concentrations were markedly higher in the supernatants of platelets (175.9 ± 36.4 U/ml after 2 h). Hypoxia and PTX did not alter the LDH amount in the medium during the 24-h period. A significant reduction of LDH content occurred in the medium after incubation of platelets with Triton X-100 at all time intervals (25.1 ± 10.9 U/ml after 2 h). Thrombin did not significantly elevate the LDH level until after an incubation period of 24 h (352.9 ± 22.0 U/ml). In PMN, LDH release did not significantly rise above the 30-min control level (105.6 ± 21.5 U/ml) during further incubation under normoxic or hypoxic conditions. In contrast to platelets and HSMC, LDH levels remained fairly constant under incubation with Triton X-100. Stimulation of PMN with PMA lead to a significant rise of LDH content in the medium after 12 h (226.5 ± 24.4 U/ml) and 24 h (279.1 ± 38.3 U/ml). Similar effects were seen under coincubation with PMA and cycloheximide or PMA and PTX, whereas incubation with cycloheximide or PTX alone induced no further LDH increase above control levels in the supernatants of PMN.

Effect of Temperature and pH on VEGF Release of Neutrophils

Table 1 presents the VEGF release characteristics of PMN in response to relevant acidic pH in combination with hypo- and hyperthermia at two different time points. Cells incubated at 37°C and pH 7.4 served as controls. Higher concentrations of HEPES buffer itself had no effect on the VEGF release from PMN controls after 30 and 120 min, as comparison with VEGF concentrations in supernatants of controls with 0.5% (20 mM) HEPES buffer from our time-course experiments revealed (189.5 ± 23.7 pg/ml after 30 min and 204.4 ± 19.8 pg/ml after 120 min). Neither hypo- nor hyperthermia influenced VEGF secretion of PMN at the two time points. In contrast, acidosis of increasing severity led to a significant reduction of VEGF secretion, independent of temperature, at the 30-min time interval. Interestingly, specific stimulation of PMN with PMA resulted in a two- to fourfold VEGF increase under all conditions (data not shown). Evaluation of LDH concentrations in the supernatants showed increasing LDH levels in parallel with growing acidosis.

                              
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  		Table 1.   VEGF and LDH concentrations in medium samples of neutrophils in response to temperature and pH changes


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VEGF is an endothelial cell-specific mitogen and has been shown to be a potent angiogenic factor mainly through its paracrine action, and several nonendothelial cell types have been found to release VEGF. The main finding of the present study is that the characteristics of VEGF secretion from two different types of circulating blood cells, PMN and platelets, are grossly different from those of vascular smooth muscle cells.

Most importantly, hypoxia, which is a potent stimulus of VEGF secretion from smooth muscle cells (24) (Fig. 1) and epithelial cells (19, 25) and is believed to be a key element in the guidance of new vessel formation, had no effect on VEGF secretion from PMN and platelets (Figs. 2 and 3). In adherent cells, a reduction in O2 availability stimulates VEGF through both transcriptional activation via increased expression of hypoxia-inducible transcription factors (HIF) (11) and a stabilization of VEGF mRNA (16). This was confirmed in our experiments, showing that increased accumulation of VEGF in the supernatant of HSMC is paralleled by an increase in steady-state mRNA levels. The increase in VEGF secretion, however, was more marked than the rise in mRNA and did not persist at the same rate despite continuously elevated mRNA levels (Figs. 1 and 4), suggesting that time-dependent alterations also occur in protein translation and/or secretion. The initial O2-sensing mechanism responsible for the hypoxic induction of VEGF has not been identified unequivocally, and it is not known whether it only affects gene expression or can also induce other O2-dependent cellular responses. Obviously, mechanisms dependent on changes in gene expression cannot operate in anuclear platelets. But in other cell types, it has been found that mechanisms do exist by which hypoxia can increase the release of preformed proteins from secretory granules, e.g., catecholamines in chromaffin cells of the adrenal medulla (21). PMN, in contrast to platelets, are capable of regulated gene expression, but during hypoxia not only the secretion of VEGF but also the levels of VEGF mRNA were unchanged (Figs. 2 and 4). This is particularly noteworthy, because the expression of other genes in PMN, including interleukin-1beta and interleukin-8, has been found to be induced by hypoxia in vitro (6), indicating the presence and functional potential of O2-dependent gene expression in neutrophils. The mechanisms of O2-dependent regulation of these cytokines may be different, however, from the HIF-dependent regulation of VEGF in other cell types, and whether HIF is activated in hypoxic PMN has not yet been determined.

Second, and similar to the effect of hypoxia on VEGF secretion, hyperthermia, hypothermia, and acidosis, conditions frequently encountered during inflammation, also did not induce VEGF release from PMN. Rather, low pH actually delayed and inhibited VEGF secretion from PMN (Table 1).

Finally, the different mechanisms of VEGF release do also have considerable impact on the kinetics of VEGF secretion. After stimulation in a cell type-specific fashion, platelets and PMN are capable of releasing significant amounts of VEGF, which others have shown to be stored in secretory granules (9, 20, 22, 29, 33, 34). In PMN, rapid VEGF release amounting to ~65-70% of total VEGF content within 60 min (Fig. 2), as determined by detergent lysis, was triggered by direct activation of protein kinase C, a key enzyme of leukocytes involved in inflammatory activation. Likewise, thrombin, a specific activator of platelets, induced a rapid release of ~75% of total VEGF within 30 min (Fig. 3). That the release occurs from preformed intracellular stores is supported by the observation that VEGF secretion was markedly inhibited by pentoxyfilline, an inhibitor of cellular degranulation (Fig. 5) (5). In addition, in PMN we were unable to detect any subsequent de novo synthesis. Both PMN and platelets showed an early spontaneous VEGF release, most likely reflecting inadvertent degranulation by the experimental procedures themselves. The continuous decrease of VEGF levels in supernatants of detergent-lysed PMN but not of PMA-stimulated cells during time-course experiments was probably due to the release of larger quantities of VEGF-degrading proteases from PMN into the culture medium under Triton X-100-induced lysis. Similarly, the significant decrease of LDH concentration seen after 24 h of PMN detergent lysis with Triton X-100 is attributable to the same proteases. The lesser degree of VEGF degradation in the supernatants of platelets during our time-course experiments is probably due to a lack of proteases released by these cells. Nevertheless, it also appears possible that a slight decrease of VEGF secretion by platelets in response to the specific stimulator thrombin at 12-24 h may have been related to a loss of platelet viability, because the experimental setup did not allow for constant agitation of platelets. The significant reduction of LDH content in the medium after detergent lysis of platelets at all time intervals was probably due to the release of an LDH-degrading factor from the platelets. In contrast to PMN and platelets, HSMC contained no stores of VEGF, resulting in a markedly different time pattern of VEGF release by these cells. Instead, the release was dependent on de novo protein synthesis, and an increase occurred only after a lag period of several hours after the onset of hypoxia (Fig. 1).

It is tempting to speculate that these entirely different patterns of VEGF secretion reflect different and possibly complementary roles of resident cells and circulating blood cells in the initiation of the effects that are triggered by VEGF in tissue ischemia and inflammation (7, 17). The rapid release after leukocyte activation or platelet aggregation might be responsible for the early onset of these effects, which could then be maintained by the secretion of newly formed VEGF from vascular smooth muscle cells. Clearly, the relevance of platelet- and PMN-derived VEGF is dependent on its local release and the overall balance with other angiogenic or anti-angiogenic factors.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Koehne, Dept. of Neonatology, Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany (E-mail: petra.koehne{at}charite.de).

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. §1734 solely to indicate this fact.

Received 18 May 1999; accepted in final form 8 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(2):H817-H824
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