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Am J Physiol Heart Circ Physiol 280: H885-H891, 2001;
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Vol. 280, Issue 2, H885-H891, February 2001

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor

Outi Saijonmaa1,2, Tuulikki Nyman1, Riikka Kosonen1, and Frej Fyhrquist1,2

1 Minerva Institute for Medical Research and 2 Department of Internal Medicine, Helsinki University Central Hospital, SF-00250 Helsinki, Finland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 × 10-6 mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 × 10-5 mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor Nomega -nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 µM) and the selective COX-2 inhibitor NS-398 (5 µM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 µM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 × 10-3 mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.

human umbilical vein endothelial cells; regulation; signal transduction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ANGIOTENSIN-CONVERTING ENZYME (ACE) is a dipeptidyl carboxypeptidase widely distributed on the luminal surface of the vascular endothelium. ACE catalyzes the proteolytic cleavage of angiotensin I to angiotensin II (ANG II) and has bradykinin degrading activity (24). Thus ACE participates in the control of vascular resistance by generating ANG II and degrading bradykinin. ANG II also acts as a vascular growth factor participating in angiogenesis, vascular remodeling, and response to vascular wall injury (8). Increased ACE accumulation in atherosclerotic blood vessel walls has been reported (7, 18). Furthermore, ACE inhibitors are effective in both reducing experimental atherogenesis (2) and the reduction of left ventricular hypertrophy of hypertensive patients (5).

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells (10). It is involved in several endothelium-specific functions, such as migration, proliferation, and angiogenesis. VEGF is postulated to be the major growth factor responsible for hypoxia-stimulated angiogenesis (10). Angiogenesis is needed for physiological functions such as embryonic development and female reproductive functions but also occurs in pathological conditions such as atherosclerosis, diabetic retinopathy, and tumor growth (10). VEGF also has additional biological activities including increased vascular permeability and vasodilation (10). Recent reports suggest an interaction between VEGF and the renin-angiotensin system. ANG II was shown to potentiate VEGF-induced angiogenic activity in bovine retinal microcapillary endothelial cells (19). Furthermore, an ACE inhibitor, captopril, suppressed neovascularization and growth of experimental tumors in rats (29). According to a recent report (17), long-term use of ACE inhibitors may protect against cancer.

In the present study, we report that VEGF induces ACE expression and enzyme amount in cultured human endothelial cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial Cell Culture

Endothelial cells were prepared from human umbilical cord veins according to Jaffe et al. (16). Veins were cannulated, washed with phosphate-buffered saline (PBS), and treated with 0.5% collagenase (Sigma, St. Louis, MO) in PBS for 15 min at room temperature, and cells were then collected by centrifugation. Cells were grown to confluence in 0.2% gelatin (Sigma)-coated cell culture flasks (Costar, Cambridge, MA) in medium-199 (GIBCO-BRL, Belmont, CA) supplemented with 20% fetal calf serum (GIBCO-BRL), 20 µg/ml endothelial cell growth supplement (Sigma), 12 U/ml heparin (Sigma), 100 U/ml penicillin G, 100 µg/ml streptomycin (GIBCO-BRL), and 2 mM L-glutamine (GIBCO-BRL) at 37°C in humidified 5% CO2 in air. The cells were detached with 0.125% trypsin-0.02% Na2EDTA solution (GIBCO-BRL) and subcultured on 48-well cell culture plates (Costar) coated with 0.2% gelatin solution. The cells were identified as endothelial cells by their typical cobblestone appearance and the presence of von Willebrand factor, demonstrated by an immunofluorescence method using rabbit immunoglobulin to human von Willebrand factor (Dakopatts, Glostrup, Denmark). More than 90% of the cells were stained positively.

Experimental Design

Confluent subcultures at passages 1-2 were incubated for 4-72 h in medium-199 supplemented with 5% fetal calf serum with VEGF165 (0.6 × 10-6 mmol/l). The incubation time of 24 h was chosen for the following studies. Cell cultures were incubated with or without the following substances: VEGF165 (0.07-1.2 × 10-6 mmol/l), placenta growth factor (PIGF; 0.69-2.75 × 10-6 mmol/l), herbimycin (4.35 × 10-5 mmol/l), zaprinast (0.1 mmol/l), Nomega -nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l), GF109203X {GFX; 2- [1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)- maleimide, 2.5 × 10-3 mmol/l}, indomethacin (5 µM), NS-398 [N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; 5 µM], or resveratrol (5 µM). Cells were preincubated with herbimycin, zaprinast, L-NAME, indomethacin, NS-398, resveratrol, or GFX for 30 min before VEGF was added. To downregulate protein kinase C (PKC), cells were preincubated with phorbol 12-myristate 13-acetate (PMA; 10-3 mmol/l) for 24 h before the experiment. Downregulation of PKC was confirmed by using PMA (10-3 mmol/l) as a negative control during the experiment. GFX was from Calbiochem (San Diego, CA); other substances were from Sigma. After the incubation time, the ACE assay was performed as described in ACE Inhibitor Binding Assay. The effect of test substances on cellular viability and growth were tested by a CellTiter 96 cell proliferation/cytotoxicity assay kit (Promega, Madison, WI).

ACE Inhibitor Binding Assay

The ACE amount in intact endothelial cells was measured by an inhibitor binding assay developed and characterized in our laboratory (23). Briefly, a lisinopril analog, 351A [p-hydroxybenzamidine derivative of N-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline; Merck Sharp & Dohme, Rahway, NJ], was labeled with 125I (IMS 30, Amersham, Buckinghamshire, UK) using the chloramine T method, as described elsewhere (11). 125I-labeled 351A is specifically bound to ACE (11, 15). After the incubation of human umbilical vein endothelial cells (HUVECs) with test substances, cell monolayers were washed with PBS. Typically, 20,000 counts per minute (cpm) of label per well in the culture medium was added to HUVECs cultured on 48-well plates. After incubation at 37°C for 2 h, cells were washed twice with PBS, detached with 0.1 M NaOH, and then counted in a gamma-counter. The amount of ACE is given as the inhibitor (125I-labeled 351A) bound in counts per minute per 105 cells, which is proportional to the amount of ACE on the cell membrane (23). The method was previously shown to correspond to the enzyme activity method by Watanabe et al. (11, 30).

ACE mRNA Measurement

Endothelial cells grown on gelatin-coated cell culture flasks were incubated with VEGF (0.3 × 10 -6 mmol/l). Total RNA from endothelial cells was isolated using the RNA STAT-60 RNA isolation reagent (Tel-Test, Friendswood, TX).

Preparation of antisense 32P-labeled riboprobes. ACE and beta -actin probes were generated by RT-PCR using human endothelial cell RNA. The T7 promoter sequence was appended to the antisense PCR primers and incorporated into the PCR product. The primer sites for beta -actin were located at nucleotides 158-176 and 314-331 (21) and for ACE at nucleotides 492-512 and 746-764 (26). 32P-labeled riboprobes were transcribed with T7 RNA polymerase using a Maxiscript in vitro transcription kit (Ambion, Austin, TX). The transcription with T7 polymerase generated a 273-bp riboprobe for ACE (protected length 267 bp) and a 179-bp riboprobe for beta -actin (protected length 173 bp). The transcription reaction was incubated for 60 min at 37°C, and the DNA was digested with DNase for 15 min at 37°C. The full length 32P-labeled riboprobes were separated by electrophoresis on a 8 M urea-5% polyacrylamide gel, excised from the gel, and eluted into 300 µl of buffer [RNase Protection Assay (RPA) II kit, Ambion] overnight. The specific activities of the probes were 5.3 × 108 cpm/µg and 0.4 × 108 cpm/µg for ACE and beta -actin, respectively.

RNase protection assay. Solution hybridization RPA was carried out using a RPA II Kit (Ambion) following the manufacturer's instructions. In brief, the sample RNA (10 µg) with 1 × 105 cpm of gel-purified high-specific activity ACE riboprobe and 4 × 104 cpm of beta -actin riboprobe were coprecipitated for 30 min at -20°C. The resulting pellet was resuspended in 20 µl of hybridization buffer, denatured, and incubated overnight at 42°C. After hybridization, the samples were digested with a 1:100 dilution of RNase (combination of RNase A and RNase T1 in Ambion digestion buffer) for 30 min at 37°C. The protected RNA was precipitated and resuspended in 8 µl of gel loading buffer. The samples were denatured and resolved by electrophoresis on 5% polyacrylamide-8 M urea gels. Quantification of the radioactive bands was performed by densitometric analysis using Advanced Image Data Analyzer (AIDA 2.0) software after scanning of a phosphoimager screen (Bio-Imaging Analyzer BAS-5000, Fuji, Tokyo, Japan). The results for ACE mRNA were normalized for the amount of beta -actin mRNA measured simultaneously in each sample.

Statistical Evaluation

Results are expressed as means ± SE of eight replicate determinations from three to four separate experiments. Analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test was applied.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACE Protein and mRNA

The membrane-bound ACE amount was measured in intact endothelial cell cultures by an inhibitor binding assay previously developed and characterized in our laboratory (23). Time course experiments showed the time dependency of the stimulation of ACE, with maximum stimulation measured after 24-h incubation (Fig. 1A). Incubation of endothelial cells with VEGF (0.07-1.2 × 10-6 mmol/l) for 24 h dose dependently increased the ACE amount (Fig. 1B).


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  		Fig. 1.   A: time course effect of vascular endothelial growth factor (VEGF; 0.6 × 10-6 mmol/l) on the angiotensin-converting enzyme (ACE) amount. B: dose-dependent increase of the ACE amount in endothelial cells treated with VEGF for 24 h. Bars indicate means ± SE. Significant differences: *P < 0.05 and ***P < 0.001 vs. control.

To study whether an increased ACE protein amount in VEGF-treated cells correlated with increased ACE mRNA, a RPA was used to measure mRNA levels. Accordingly, increased ACE mRNA levels were induced by VEGF (0.3 × 10-6 mmol/l) treatment for 24 h (Fig. 2).


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  		Fig. 2.   Effect of VEGF (0.3 × 10-6 mmol/l) on ACE mRNA expression after 24 h incubation detected by a RNase protection assay. Gel bands were visualized by scanning with a phosphoimager screen and quantitated by densinometry. A: bars are relative ACE mRNA levels normalized to beta -actin. B: ACE mRNA gel bands from a typical experiment.

The other member of the VEGF family of growth factors, PIGF (0.69-2.75 × 10-6 mmol/l), did not modify the ACE amount after 4-h or 24-h incubation (data not shown).

Tyrosine Kinase Inhibition

VEGF receptors reportedly possess intrinsic tyrosine kinase activity (6, 27). To investigate whether the stimulatory effect of VEGF was mediated via the activation of tyrosine kinases, we used the tyrosine kinase inhibitor herbimycin. Pretreatment of endothelial cells with herbimycin (4.35 × 10-5 mmol/l) inhibited the stimulatory effect of VEGF (Fig. 3).


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  		Fig. 3.   Effect of herbimycin (4.35 × 10-5 mmol/l) treatment on the VEGF (0.15 × 10-6 mmol/l)-induced ACE amount in endothelial cells after 24-h incubation. Herbimycin was added 30 min before VEGF. Bars indicate means ± SE. Significant differences: *P < 0.05 and ***P < 0.001 vs. control; dagger dagger dagger P < 0.001 vs. VEGF.

Role of cGMP

We (23) have previously shown that cGMP is an intracellular mediator regulating ACE. To study whether cGMP is related to the VEGF stimulatory effect on ACE, endothelial cell cultures were coincubated for 24 h with VEGF and the selective cGMP phosphodiesterase type V inhibitor zaprinast (0.1 mmol/l), which inhibits breakdown of intracellular cGMP. As shown in Fig. 4, ACE stimulation was potentiated by coincubation of the cells with VEGF and zaprinast.


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  		Fig. 4.   Potentiation of the VEGF (0.15 × 10-6 mmol/l)-stimulated ACE amount by coincubation of cell cultures with VEGF (0.15 × 10-6 mmol/l) and the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l) for 24 h. Bars indicate means ± SE. Significant differences: *P < 0.05 and ***P < 0.001 vs. control; dagger dagger dagger P < 0.001 vs. VEGF.

Role of Nitric Oxide

Nitric oxide (NO) production leads to increased intracellular cGMP levels. To study whether the stimulatory effect of VEGF on ACE was mediated by NO, the NO synthase inhibitor L-NAME was used. Treatment of HUVECs with L-NAME (5.4 mmol/l) for 24 h slightly decreased the basal ACE amount. Preincubation of endothelial cells for 30 min with L-NAME (5.4 mmol/l) and then coincubation with VEGF for further 24 h partly inhibited the stimulatory effect of VEGF (Fig. 5).


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  		Fig. 5.   Effect of Nomega -nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) on the VEGF (0.15 × 10-6 mmol/l)-stimulated ACE amount after 24-h incubation. L-NAME was added 30 min before VEGF. Bars indicate means ± SE. Significant differences: ***P < 0.001 vs. control; dagger dagger dagger P < 0.001 vs. VEGF.

Role of Prostacyclin

To study the role of PGI2 in VEGF-mediated ACE induction, endothelial cells were pretreated with cyclooxygenase (COX) inhibitors. The nonselective COX inhibitor indomethacin (5 µM) and the selective COX-2 inhibitor NS-398 (5 µM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 µM) was without effect (Fig. 6).


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  		Fig. 6.   Effect of the cyclooxygenase inhibitors indomethacin (5 µM), NS-398 (5 µM), and resveratrol (5 µM) on the VEGF (0.3 × 10-6 mmol/l)-stimulated ACE amount after 24-h incubation. The inhibitors were added 30 min before VEGF. Bars indicate means ± SE. Significant differences: **P < 0.01 vs. control.

Role of PKC

The involvement of PKC in ACE regulation was studied. Activation of PKC with the phorbol ester PMA (1 µM) increased ACE by 160% from the control level (Fig. 7), indicating that PKC activation was involved in ACE induction.


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  		Fig. 7.   Effect of protein kinase C (PKC) downregulation on VEGF-induced ACE production. In control experiments, cell cultures were treated with VEGF (0.15 × 10-6mmol/l) or phorbol 12-myristate 13-acetate (PMA; 10-3 mmol/l) for 24 h. To downregulate PKC, cell cultures were preincubated with PMA (10-3 mmol/l) for 24h and then incubated further with VEGF (0.15 × 10-6 mmol/l) for 24 h. Downregulation of PKC was confirmed by using PMA as a negative control during the experiment. Bars indicate means ± SE. Significant differences: ***P < 0.001 vs. control.

To study the role of PKC in VEGF signaling, activation of PKC was inhibited by downregulation with PMA pretreatment or by inhibiting PKC with the selective inhibitor GFX. Downregulating of PKC by pretreatment of endothelial cells for 24 h with PMA (1 µM) abolished the VEGF-induced ACE increase (Fig. 7). Furthermore, pretreatment of endothelial cell cultures with GFX (2.5 × 10-3 mmol/l) for 30 min and then coincubation with VEGF (0.3 × 10-6 mmol/l) for further 24 h inhibited the ACE increase (Fig. 8), indicating that PKC was involved in VEGF-induced ACE production.


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  		Fig. 8.   Effect of the selective PKC inhibitor GF109203X (GFX; 2.5 × 10-3 mmol/l) on the VEGF (0.15 × 10-6 mmol/l)-stimulated ACE amount after 24-h incubation. GFX was added 30 min before VEGF. Bars indicate means ± SE. Significant differences: *P < 0.05 and ***P < 0.001 vs. control; dagger dagger dagger P < 0.001 vs. VEGF.

Growth or Toxicity Effects

None of the test substances incubated with confluent endothelial cell cultures had toxic or growth effects, as tested by a CellTiter 96 cell proliferation/cytotoxicity assay kit (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Here, we show that VEGF, a regulator of vasculogenesis and angiogenesis, increases ACE at both mRNA and protein levels in cultured human endothelial cells. The maximum increase of ACE was reached after a 24-h incubation time. The concentrations of VEGF that stimulated ACE were in the range reported to induce mitogenesis, chemotaxis, and other signaling events in HUVECs (1). The induction of ACE by VEGF is a new observation, which suggests an interaction between VEGF and the renin-angiotensin system at a level not demonstrated earlier. Interaction of ANG II and VEGF has been suggested. A recent report (19) showed that ANG II potentiates VEGF-dependent cell growth and tube formation of retinal microvascular endothelial cells through induction of the VEGF receptor KDR/Flk-1. Furthermore, ANG II induced VEGF mRNA expression in rat heart endothelial cells (3). The finding that VEGF induces ACE production, which probably lead to increased ANG II production, suggests potentiating interaction of these systems.

VEGF mediates its effects through endothelial cell-specific receptors. Two of these receptors have been identified: namely, the phosphotyrosine kinase receptors Flt-1 and KDR/Flk-1. Both of these receptors are expressed in HUVECs (13). KDR/Flk-1 receptors are involved primarily in mitogenesis, whereas Flt-1 receptors are required for endothelial cell morphogenesis (10). Here, we show that induction of ACE by VEGF was inhibited by pretreatment of HUVECs with herbimycin, a potent protein tyrosine kinase inhibitor. This suggests that activation of tyrosine kinases is involved in ACE induction. Because HUVECs express both KDR/Flk-1 and Flt-1 receptors, it is at present unclear which of these receptors is responsible for mediating ACE induction. The finding that PIGF, a member of VEGF family growth factors which mediates its effects only through Flt-1 receptors (10), did not induce ACE suggests that ACE induction by VEGF was mediated by KDR/Flk-1 receptors.

VEGF mediates its biological effects in endothelial cells via various intracellular signaling pathways. However, these pathways are still unclear. We focused on VEGF signaling pathways involved in ACE induction.

NO production and subsequent intracellular cGMP elevation contribute to the angiogenic effect of VEGF in HUVECs (14, 20). We (23) previously reported that cGMP is involved in ACE induction in HUVECs. In the present study, we show that treatment ofHUVECs with the selective cGMP phosphodiesterase inhibitor zaprinast, which inhibits breakdown of cGMP, potentiated the stimulatory effect of VEGF. These data suggest that intracellular cGMP was increased by VEGF and involved in VEGF-induced ACE stimulation.

To study the involvement of NO in ACE induction, endothelial cells were incubated with the NO synthase inhibitor L-NAME. L-NAME treatment did not modulate basal ACE production, which suggests that ACE is insensitive to modulation by low levels of NO spontaneously produced by endothelial cells. L-NAME treatment partially inhibited the stimulatory effect of VEGF, which suggests that NO is a mediator involved in ACE induction by VEGF but also that other intracellular mechanisms are involved.

It has been suggested that NO and PGI2 act in parallel in mediating the angiogenic, permeability, and vasodilation effects of VEGF (12). We therefore studied whether PGI2 was involved in VEGF-induced ACE production. PGI2 is formed by two types of COXs: a constitutive form, COX-1, and an inducible form, COX-2, which can be induced by growth factors and various inflammatory agents (25). When HUVECs were pretreated with the nonselective COX inhibitor indomethacin or the selective COX-2 inhibitor NS-398, the stimulatory effect of VEGF was potentiated, whereas pretreatment of cell cultures with the COX-1 selective inhibitor resveratrol did not modulate the stimulatory effect of VEGF. These results suggest that VEGF-induced PGI2 production, which has an inhibitory effect on ACE, counterbalances the stimulatory effect of VEGF mediated by other pathways. To our knowledge, the involvement of PGI2 on ACE regulation has not been reported before.

VEGF has been reported to activate PKC in endothelial cells, an effect that was abolished by the PKC inhibitors GFX and H-7 (31). On the other hand, the PKC activator PMA was shown to be a potent ACE stimulator (28). We show here that the VEGF-induced ACE increase was abolished by PKC downregulation or by pretreatment of HUVECs with GFX at a concentration reported to completely block PKC activation in HUVECs (1). These data suggest that PKC activation was an essential step in ACE induction by VEGF. The possible interaction of PKC, NO, and cGMP as intracellular mediators involved in ACE production remains to be clarified.

The interaction of ACE with VEGF is of special interest in view of the important role played by the renin-angiotensin system in many cardiovascular disorders in which angiogenesis is induced. This includes myointimal proliferation after vascular injury, atherosclerosis, and diabetic angiopathy. Association of enhanced vascular ACE expression with the development of coronary atherosclerosis in humans has been reported (7, 18). Furthermore, ACE inhibitors have vasculoprotective effects that may contribute to the prevention of coronary atherosclerosis in humans and animal models (2, 5). On the other hand, induction of VEGF in human atherosclerotic lesions and in animal models of arterial injury has been described (4, 22). Combined treatment with ACE inhibitors and inhibitors of VEGF, if made available, may be of value in the prevention of atherosclerosis.

ANG II has been reported to induce expression of VEGF and its receptors in endothelial cells. Conversely, the induction of ACE by VEGF shown here may lead to increased local production of ANG II. Thus synergy between VEGF and the renin-angiotensin system may be an important mechanism underlying angiogenesis in hypoxic or neoplastic tissues. A recent report (17) on a significantly decreased risk of incident or fatal cancer in patients treated for hypertension with an ACE inhibitor compared with those treated with antihypertensive drugs other than ACE inhibitors raises the interesting possibility that ACE may be involved in the development of cancer. Our observation that VEGF, thought to be a major inducer of angiogenesis and vasculogenesis in neoplastic tissue (10), is a powerful inducer of ACE in HUVECs points to the possible synergy between VEGF and the local renin-angiotensin system to promote vascularization of tumors. If true, such a synergy could be a future target of pharmacological intervention.

However, the role of ACE in angiogenesis is still unclear. Data causing difficulties of interpretation concerning ACE inhibition and angiogenesis have been published ranging from inhibition of angiogenesis to increased angiogenesis after treatment with ACE inhibitors (8, 9, 17, 29).

In summary, VEGF increased ACE at mRNA and protein levels in cultured human endothelial cells. Interaction of VEGF and ACE may play a role in the pathophysiological processes of the vascular wall.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Sigrid Jusélius Foundation, the Magnus Ehrnrooth Foundation, and the Liv och Hälsa Foundation and by Helsinki University Central Hospital Research Funds.


    FOOTNOTES

Address for reprint requests and other correspondence: O. Saijonmaa, Minerva Institute for Medical Research, Tukholmankatu 2, SF-00250 Helsinki, Finland (E-mail: Outi.Saijonmaa{at}helsinki.fi).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 November 1999; accepted in final form 15 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(2):H885-H891
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