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This Article Full Text (PDF) All Versions of this Article: 16/13/1805 01-0938fjev1    most recent Alert me when this article is cited Alert me if a correction is posted 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 Citing Articles Citing Articles via Google Scholar Google Scholar Articles by FAEHLING, M. Articles by WALTENBERGER, J. Search for Related Content PubMed PubMed Citation Articles by FAEHLING, M. Articles by WALTENBERGER, J.

Essential role of calcium in vascular endothelial growth factor A-induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole ¹

Department of Internal Medicine II and
^* Department of General Physiology, University of Ulm, 89081 Ulm, Germany

²Correspondence: Department of Internal Medicine II University of Ulm Robert-Koch-Str. 8, 89081 Ulm Germany. E-mail: johannes.waltenberger{at}medizin.uni-ulm.de

SPECIFIC AIM

Vascular endothelial growth factor A (VEGF-A) plays a major role in tumor angiogenesis and raises intracellular free calcium ([Ca²⁺]_i). The role of [Ca²⁺]_i in VEGF-A signal transduction is not fully clear. Carboxyamidotriazole (CAI), known as an inhibitor of calcium influx in vitro and of angiogenesis in vivo, is under clinical investigation as a tumoristatic agent. The present study was designed to elucidate the role of calcium in VEGF-A induced proliferation, especially the effects of CAI on VEGF-A-induced signaling were studied.

PRINCIPAL FINDINGS

1. VEGF-A-induces a biphasic increase of [Ca²⁺]_i
Stimulation of HUVEC with VEGF-A induces a biphasic calcium signal in single endothelial cells with an initial transient peak dependent on release from intracellular stores. This is followed by a sustained plateau phase of elevated [Ca²⁺]_i that is dependent on extracellular calcium and lasts for at least 1 h as long as VEGF-A is present. Thus, VEGF-A elicits a prolonged [Ca²⁺]_i signal that may affect both early and late events of VEGF-A-induced signaling, including proliferation (Fig. 1 A, B).

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of CAI on the calcium signal induced by VEGF-A (10 ng/mL). [Ca2+]i was measured in single HUVEC using Fura-2. The experiments shown are representative of 5–11 experiments (each studying 8–10 single cells) in each group. A) Incubation with CAI (3 µM) for 30 min suppressed the calcium signal induced by VEGF-A (bottom trace). For comparison, a normal response to VEGF-A added in the absence of CAI is shown (top trace). CAI (3 µM) added during the plateau phase was without effect (top trace). B) Prolonged presence of CAI (3 µM, up to 20 min) did not change the calcium plateau induced by VEGF-A. The plateau was abolished on addition of calcium-free buffer (1 mM EGTA). C) Effect of various concentrations of CAI on [Ca2+]i. No VEGF-A was added. D) Summary of VEGF-A effect after 30 min exposure to CAI (1 or 3 µM, respectively) or control (calcium solution). VEGF-A was added at time 0 and was present throughout the period shown. Data of single cells were analyzed after 0, 3, 5, 10, 15, 20 min. Peak [Ca2+]i concentration and the time-to-peak (from addition of VEGF-A to the maximum [Ca2+]i) were determined from each experiment; the corresponding peak calcium concentration is represented in the graph. [Ca2+]i with CAI is significantly different from [Ca2+]i with VEGF-A alone at all data points shown, including t = 0 (t-test). There is no significant difference between 1 µM CAI and 3 µM CAI.

2. VEGF-A stimulates generation of inositol-trisphosphate (IP₃)
VEGF-A increased the intracellular level of IP₃ in three independent experiments (radioimmunoassay). This is associated with the release of calcium from IP₃-sensitive intracellular stores accounting for the initial transient [Ca²⁺]_i peak. Store-operated calcium influx secondary to depletion of IP₃-sensitive intracellular calcium stores accounts for the VEGF-A-induced calcium plateau.

3. CAI inhibits VEGF-A-induced IP₃ formation and calcium signaling
CAI (10 µM) inhibits the VEGF-A-induced increase of IP₃ and both phases of the calcium response (Fig. 1) . This indicates that CAI, generally regarded as a "calcium influx inhibitor," not only inhibits calcium influx but also the release of calcium from intracellular stores (Fig. 1A, D ). Therefore, CAI does not act as a classical membrane channel blocker since CAI is without effect on the calcium signal when added during the influx-dependent plateau phase (Fig. 1A, B ). Almost complete inhibition of the calcium signal by CAI is reached at the lower micromolar level (Fig. 1D ). Inhibition of the calcium signal can be explained by inhibition of IP₃ formation.

4. CAI inhibit VEGF-A-stimulated proliferation of endothelial cells
CAI concentration-dependently and reversibly inhibits VEGF-A-stimulated DNA synthesis ([H]thymidine incorporation and cell count) with half-maximal inhibition at the lower micromolar level (Fig. 2 ). Endothelial cell proliferation is inhibited by low concentrations of extracellular or intracellular free Ca²⁺, emphasizing the importance of Ca²⁺ signaling for VEGF-A action.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of CAI on VEGF-A-induced proliferation of HUVEC. A) CAI inhibits DNA synthesis ([H]thymidine incorporation) stimulated by VEGF-A (10 ng/mL, n=4). Control was proliferation of HUVEC in low-serum medium. *P < 0.05 compared with VEGF-A alone. B) CAI inhibits proliferation (cell count) stimulated by VEGF-A. HUVEC were exposed to CAI at the given concentration (µM) plus VEGF-A (10 ng/mL). After 24 h, cells were counted (n=3). Control was cell count in low-serum medium. *P < 0.05 compared with VEGF alone. C) Reversibility of CAI-induced inhibition of VEGF-induced proliferation. HUVEC were incubated with CAI at the concentration indicated in low-serum medium for 24 h. VEGF-A was added 30 min after washout of CAI with low-serum medium (n=6). D) Dependence of VEGF-A-induced DNA synthesis on extracellular free Ca2+. [ H]thymidine incorporation stimulated by VEGF-A (10 ng/mL) was inhibited by reduction of free Ca2+ (n=5). Control was [ H]thymidine incorporation in low-serum medium in the presence of 1.6 mM free Ca2+. The free Ca2+ concentration in the extracellular medium was adjusted by addition of the Ca2+ chelator EGTA (0–4 mM) to the culture medium. E) Inhibition of DNA synthesis stimulated by VEGF-A (10 ng/mL) by chelation of intracellular free Ca2+ using the membrane-permeant Ca2+ chelator BAPTA-AM. Cells were preincubated with BAPTA-AM for 30 min before addition of VEGF-A (10 ng/mL). Control was [ H]thymidine incorporation in low-serum medium in the absence of VEGF-A (n=3).

5. CAI inhibits VEGF-A-induced nitric oxide (NO) formation
To establish a link between inhibition of Ca²⁺ signaling by CAI and its cellular consequences, we functionally analyzed several components of the VEGF-A-activated signal transduction cascade. CAI inhibits the VEGF-A-induced increase of intracellular cGMP concentration in a concentration-dependent fashion as determined by radioreceptor assay. We used cGMP levels as a surrogate for NO formation since cGMP is a direct product of NO-induced activation of guanylate cyclase. The inhibition of VEGF-A-induced NO formation by CAI can explain why inhibition of calcium signaling can lead to inhibition of VEGF-A-induced endothelial cell proliferation.

6. CAI does not inhibit VEGF-A-induced tyrosine phosphorylation of VEGFR-2, activation of signaling molecules such as ERK1/2, PLC, and Akt, or translocation of NFAT from the cytosol to the nucleus
The lack of inhibition of ERK1/2 activation by CAI indicates that VEGF-A activates ERK1/2 in a calcium-independent fashion. Therefore, ERK1/2 activation, which under physiological conditions strongly correlates with endothelial-cell proliferation, is not sufficient for VEGF-A-induced endothelial proliferation in the absence of calcium signaling.

CONCLUSIONS AND SIGNIFICANCE

VEGF-A elicits a biphasic [Ca²⁺]_i signal closely related to elevation of intracellular IP₃. This suggests that IP₃ triggers the calcium signal by releasing calcium from IP₃-sensitive intracellular stores and activation of store-operated calcium influx secondary to depletion of IP₃-sensitive intracellular calcium stores (Fig. 3 ). Ca²⁺ signaling is essential for VEGF-A induced proliferation, since either CAI or low extracellular or intracellular free calcium inhibits VEGF-A-induced proliferation of HUVEC.

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic representation of the VEGF signaling pathways addressed in this paper.

Our findings suggest the following mechanism of action of CAI: CAI inhibits VEGF-A-induced IP₃ formation, which results in inhibition of release of calcium from intracellular stores and inhibition of calcium influx. Phosphorylation of PLC by VEGF-A is not inhibited by CAI. Therefore, the inhibition of IP₃ formation may be due to impaired availability of phospholipids for metabolic attack after CAI administration. Inhibition of the VEGF-A-induced calcium signal by CAI is associated with inhibition of NO formation and proliferation. The inhibitory action of CAI on VEGF-A-induced IP₃ formation, calcium metabolism, and NO release can account for the inhibition of endothelial proliferation and explain the antiangiogenic action of CAI. The functional relevance of these findings for the ongoing clinical trials using CAI is supported by the fact that the inhibiting concentrations used in our experiments are of the same level as the therapeutic plasma levels in vivo.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0938fje; to cite this article, use FASEB J. (September 19, 2002) 10.1096/fj.01-0938fje

This Article Full Text (PDF) All Versions of this Article: 16/13/1805 01-0938fjev1    most recent Alert me when this article is cited Alert me if a correction is posted 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 Citing Articles Citing Articles via Google Scholar Google Scholar Articles by FAEHLING, M. Articles by WALTENBERGER, J. Search for Related Content PubMed PubMed Citation Articles by FAEHLING, M. Articles by WALTENBERGER, J.