NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells - PubMed (original) (raw)

NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells

Ehrin N Johnson et al. J Biol Chem. 2003.

Abstract

Mice deficient for the transcription factor NFATc1 fail to form pulmonary and aortic valves, a defect reminiscent of some types of congenital human heart disease. We examined the mechanisms by which NFATc1 is activated and translocated to the nucleus in human pulmonary valve endothelial cells to gain a better understanding of its potential role(s) in post-natal valvular repair as well as valve development. Herein we demonstrate that activation of NFATc1 in human pulmonary valve endothelial cells is specific to vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2. VEGF-induced NFATc1 nuclear translocation was inhibited by either cyclosporin A or a calcineurin-specific peptide inhibitor; these findings suggest that VEGF stimulates NFATc1 nuclear import in human pulmonary valve endothelial cells by a calcineurin-dependent mechanism. Importantly, both cyclosporin A and the calcineurin-specific peptide inhibitor reduced VEGF-induced human pulmonary valve endothelial cell proliferation, indicating a functional role for NFATc1 in endothelial growth. In contrast, VEGF-induced proliferation of human dermal microvascular and human umbilical vein endothelial cells was not sensitive to cyclosporin A. Finally, NFATc1 was detected in the endothelium of human pulmonary valve leaflets by immunohistochemistry. These results suggest VEGF-induced NFATc1 activation may be an important mechanism in cardiac valve maintenance and function by enhancing endothelial proliferation.

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Figures

FIG. 1. HPVECs express endothelial-specific markers

HPVEC were stained by indirect immunofluorescence using rabbit control IgG (A), rabbit anti-human von Willebrand Factor (vWF) (B), goat anti-human CD31/PECAM-1 (C), or mouse anti-human E-selectin (D). In panel D, HPVEC were stimulated with 1 µg/ml LPS for 3 h to induce E-selectin expression. Photographs were taken at 630× magnification.

FIG. 2. VEGF stimulates NFATc1 nuclear translocation by a calcineurin-dependent mechanism

A, HPVEC were stimulated for 30 min with no growth factor (a and b), 50 ng/ml VEGF (c and d), 50 ng/ml bFGF (e), or 50 ng/ml Flt-1sel (f) or 50 ng/ml KDR-sel-2 (g and h). HPVECs were incubated with a mouse isotyped-matched control IgG1 (a) or with mouse anti-human NFATc1 (b–h), followed by anti-mouse-conjugated FITC. In panels d and h, HPVEC were preincubated with 5 µ

CsA for 2 h prior to addition of VEGF and KDR-sel-2, respectively. Photographs were taken at 630× magnification. In B, cell lysates from HPVEC in EBM-2 with growth factors (lane 1), EBM-2 without growth factors for 24 h (lanes 2–6) were analyzed for expression of NFATc1 by Western blot. Cells were stimulated with VEGF for 15 min (lanes 3 and 4) or 24 h (lanes 5 and 6) in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of 5 µm CsA. α-Tubulin levels in each cell lysate are shown as a control. C, expression of NFATc1, NFATc2, NFATc3, and NFATc4 in HPVECs treated without (panels a–d) or with (panels d–h) VEGF was analyzed as described in A using family member-specific mAbs. D, cell lysates from human lymphoma cell lines (lanes 1 and 2), HPVEC (lane 3), HUVEC (lane 4), and HDMEC (lane 5) were analyzed by Western blot using family member-specific mAbs against NFATc1, NFATc2, and NFATc3. α-Tubulin levels in the cell lysates are shown as a control.

FIG. 3. VEGF induces proliferation by a cyclosporin-sensitive mechanism in HPVEC but not in HDMEC or HUVEC

A, quiescent HPVEC were stimulated with 10 ng/ml VEGF or with 10 ng/ml of the VEGF receptor-selective variants, VEGFwt, KDR-sel-2, or Flt-sel, in the absence (open bars) or presence (black bars) of 5 µ

CsA. Cell proliferation was assayed by [3H]thymidine incorporation. Data represent mean ± S.D. of a representative experiment (n = 3), each performed in triplicate. Asterisks denote a statistically significant (p < 0.05) difference. B, HDMEC and HUVEC were stimulated with 10 ng/ml VEGF in the absence (open bars) or presence (black bars) of 5 µ

CsA. Data are plotted as -fold induction. The change in VEGF-mediated proliferation of these cell types after addition of CsA was not statistically significant.

FIG. 4. VIVIT inhibits VEGF-induced proliferation of HPVEC

HPVEC were retrovirally transduced with GFP or GFP-VIVIT. The brightest GFP-positive cells (fluorescence signal > 102) were sorted for analysis of NFATc1 localization in GFP-positive (A and B) and GFP-VIVIT-positive (C and D) cells and for proliferation assays (E). GFP-positive and GFP-VIVIT-positive sorted cells are shown in A and C, respectively. For indirect immunofluorescence, the same cells were stimulated with 10 ng/ml VEGF for 30 min followed by staining with anti-human NFATc1 mAb, followed by a Texas Red-conjugated anti-mouse IgG (B and D). Photographs were taken at 630× magnification. For proliferation assays (E), quiescent HPVEC were stimulated with 10 ng/ml VEGF or KDR-sel-2 for 24 h and assayed for [3H]thymidine incorporation. Data represent mean ± S.D. of a representative experiment (n = 3), each performed in triplicate. Asterisks denote a statistically significant (p < 0.05) difference.

FIG. 5. Expression and localization of NFATc1 in human pulmonary valve leaflets

Serial sections of formalin-fixed paraffin-embedded sections human pulmonic valve leaflets were immunostained with anti-human NFATc1 mAb (A) or with anti-human CD31 mAb (B), and endothelial marker. The inset in A shows a section stained with an isotype-matched control mouse IgG1.

FIG. 6. Model for VEGF-induced proliferation in cardiac valve endothelium

These results and previous studies (8) demonstrate that two pathways present during valvulogenesis are also active in post-natal heart valves. In this study, we show that a VEGF-mediated pathway, signaling through KDR/VEGF-R2, results in intranuclear NFATc1 and valvular endothelial cell proliferation. A TGF-β-mediated pathway induces differentiation to a mesenchymal phenotype (8). Whether or not cross-talk between these two pathways, as has been shown in the mouse (27), occurs in post-natal valves and affects post-natal valve function or repair warrants investigation.

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NFATc1 mediates vascular endothelial growth factor-induced proliferation of human pulmonary valve endothelial cells - PubMed (original) (raw)