VE-statin, an endothelial repressor of smooth muscle cell migration

Abstract

The recruitment and proliferation of smooth muscle cells and pericytes are two key events for the stabilization of newly formed capillaries during angiogenesis and, when out of control in the adult, are the main causes of arteriosclerosis. We have identified a novel gene, named VE-statin for vascular endothelial-statin, which is expressed specifically by endothelial cells of the developing mouse embryo and in the adult, and in early endothelial progenitors. The mouse and human VE-statin genes have been located on chromosome 2 and 9, respectively, they span >10 kbp and are transcribed in two major variants arising from independent initiation sites. The VE-statin transcripts code for a unique protein of 30 kDa that contains a signal peptide and two epidermal growth factor (EGF)-like modules. VE-statin is found in the cellular endoplasmic reticulum and secreted in the cell supernatant. Secreted VE-statin inhibits platelet-derived growth factor (PDGF)-BB-induced smooth muscle cell migration, but has no effects on endothelial cell migration. VE-statin is the first identified inhibitor of mural cell migration specifically produced by endothelial cells.

Fig. 1. Sequence of the mouse VE-statin cDNA and protein. The complete 1371 bp sequence of VE-statin-a. VE-statin-b differs from VE-statin-a in the first 169 bp (italics). The coding sequence is shown in upper case, the minimal Kozak sequence is boxed, the arrowhead indicates the signal peptide potential cleavage site, and EGF-like modules are underlined.

Fig. 2. Structure of the mouse and human VE-statin genes. (A) Schematic representation of the human and mouse VE-statin genes. Numbers and horizontal lines indicate the exons. Exons 1a and 1b code for VE-statin-a and VE-statin-b, respectively. The ATG start codon is located in exon 3 and the stop codon in exon 10. Polyadenylation signals were found in the mouse (ATAATAAAGC) and human (ACAATAAAAA) genomic sequences. SINE = short interspersed nuclear elements. (B) RNase protection assay of VE-statin-a and VE-statin-b transcripts was performed on tRNA (Ctrl) or on mouse embryo E10.5 and H5V endothelial cell RNA. VE-statin-a has several (at least six) start sites which span ∼60 bp (arrowheads), whereas VE-statin-b has a unique start site. Numbers on the left correspond to size markers. (C) FISH analysis of the mouse and human chromosomes using VE-statin-specific fluorescent probes. In both species, a unique signal was found on each corresponding pair of chromosomes (arrowheads).

Fig. 3. Analysis of VE-statin transcript expression. (A) RT–PCR analysis of expression of the transcripts of VE-statin (total), VE-statin-a, VE-statin-b, db1 and gapdh in total RNA of E10.5 mouse embryo and heart, and in various cell lines. (B) Northern blot analysis of expression of VE-statin-a (top) and VE-statin-b (bottom) in various mouse tissues (arrowheads). Numbers indicate the size of markers (kb).

Fig. 4. In situ expression pattern of VE-statin, flk-1 and db1. (Top panel) In situ hybridization analysis of expression of VE-statin (A, D and G), flk-1 (B, E and H) and db1 (C, F and I) in E7.5 (A–C) and E10.5 (D–I) mouse embryos. At E7.5, VE-statin (A) and flk-1 (B) are expressed in the primitive blood islands of the yolk sac (arrows). flk-1 is also expressed in the intra-embryonic mesoderm (B, asterisk). db1 expression is not detected at this stage (C). At E10.5, VE-statin (D) and vegfr-2 (E) are similarly expressed in the forming blood vessels. A higher magnification of the caudal part of the embryo shows the parallel expression patterns of VE-statin (G) and flk-1 (H) in the endothelium (dorsal aorta, arrow; hind limb vessels, arrowhead). At this stage, db1 is ubiquitously expressed in the embryo, with a higher expression in the neuroepithelium (F, arrow). db1 expression is never detected in the endothelium (I). Bars represent 100 µm in (A–C) and (G–I), and 1 mm in (D–F). (Bottom panel) VE-statin expression analysis in E10.5 (J–L) and E13.5 (M–P) embryos, in 3-day-old pups (Q) and in adult tissues (R). At E10.5, VE-statin expression is detected in the third and fourth branchial arch arteries (J, arrow and arrowhead, respectively), in the umbilical vein and artery (K) and in the endothelial cell precursors of the cephalic mesenchyme (L). At E13.5, VE-statin is expressed in endothelial cells of the endocardium (M), in the primitive pulmonary vascular network (N), in the blood vessels surrounding the ribs (O) and in the vascular network associated with the pigmented layer of the retina (P). After birth, VE-statin expression is detected in the renal artery (Q, arrow) and vein (arrowhead), in the glomerular capillary network (Q, asterisk) and in the peritubular capillaries (Q, open arrow). In the pregnant female, VE-statin is strongly expressed in the endothelial cells of the blood vessels of the mesometrial deciduum (R, arrow). a = atrium; cm = cephalic mesenchyme; gl = glomerulus; Li = liver; Lu = lung; md = mesometrial deciduum; n = neuroepithelium; Ra = renal artery; rb = rib; Rv = renal vein; tu = renal tubules; ua = umbilical artery; uv = umbilical vein; v = ventricule. Bars represent 100 µm.

Fig. 4. In situ expression pattern of VE-statin, flk-1 and db1. (Top panel) In situ hybridization analysis of expression of VE-statin (A, D and G), flk-1 (B, E and H) and db1 (C, F and I) in E7.5 (A–C) and E10.5 (D–I) mouse embryos. At E7.5, VE-statin (A) and flk-1 (B) are expressed in the primitive blood islands of the yolk sac (arrows). flk-1 is also expressed in the intra-embryonic mesoderm (B, asterisk). db1 expression is not detected at this stage (C). At E10.5, VE-statin (D) and vegfr-2 (E) are similarly expressed in the forming blood vessels. A higher magnification of the caudal part of the embryo shows the parallel expression patterns of VE-statin (G) and flk-1 (H) in the endothelium (dorsal aorta, arrow; hind limb vessels, arrowhead). At this stage, db1 is ubiquitously expressed in the embryo, with a higher expression in the neuroepithelium (F, arrow). db1 expression is never detected in the endothelium (I). Bars represent 100 µm in (A–C) and (G–I), and 1 mm in (D–F). (Bottom panel) VE-statin expression analysis in E10.5 (J–L) and E13.5 (M–P) embryos, in 3-day-old pups (Q) and in adult tissues (R). At E10.5, VE-statin expression is detected in the third and fourth branchial arch arteries (J, arrow and arrowhead, respectively), in the umbilical vein and artery (K) and in the endothelial cell precursors of the cephalic mesenchyme (L). At E13.5, VE-statin is expressed in endothelial cells of the endocardium (M), in the primitive pulmonary vascular network (N), in the blood vessels surrounding the ribs (O) and in the vascular network associated with the pigmented layer of the retina (P). After birth, VE-statin expression is detected in the renal artery (Q, arrow) and vein (arrowhead), in the glomerular capillary network (Q, asterisk) and in the peritubular capillaries (Q, open arrow). In the pregnant female, VE-statin is strongly expressed in the endothelial cells of the blood vessels of the mesometrial deciduum (R, arrow). a = atrium; cm = cephalic mesenchyme; gl = glomerulus; Li = liver; Lu = lung; md = mesometrial deciduum; n = neuroepithelium; Ra = renal artery; rb = rib; Rv = renal vein; tu = renal tubules; ua = umbilical artery; uv = umbilical vein; v = ventricule. Bars represent 100 µm.

Fig. 5. Expression of the VE-statin protein. (A) Homology alignment of the human (top) and mouse (bottom) VE-statin protein sequences. Identical amino acids at matching positions are boxed in black; conservative substitutions are boxed in grey. Legends are as in Figure 1. (B) The complete VE-statin-a (a) and VE-statin-b (b) cDNA or control empty vector (Ctrl) were used in in vitro translation experiments. A single VE-statin protein migrating at the predicted size of 30 kDa is produced in both cases. (C) Top two lanes: RT–PCR analysis of expression of VE-statin 24 h after transfection of 3T3 cells with the pcDNA3 control (Ctrl) or pVE-statin-HA (VE-stat) vectors. Bottom lane: immunoblot analysis of expression of the VE-statin protein in extracts of pcDNA3- (Ctrl) and pVE-statin-HA- (VE-stat) transfected cells. (D) Cells were transfected with the pVE-statin–GFP (left) or pVE-statin-HA (right) expression vector, stained the next day with Hoechst reagent and visualized for direct fluorescence (GFP, left) or incubated with anti-HA- (green) and anti-protein disulfide isomerase- (red) specific antibodies. Incubation with secondary antibodies yielded no fluorescent signal (not shown).

Fig. 6. VE-statin is a secreted protein. (A) 3T3 fibroblasts were transfected or not (nt) with the pcDNA3 vector (–) or pVE-statin-HA expression vector (+). The next day, the culture medium was removed, fresh medium was added and the cells were cultured further for 6 h. Secreted VE-statin (arrowhead) was immunoprecipitated from the filtered medium and detected by western blotting. ns = non-specific signal. (B) Pulse–chase analysis of VE-statin secretion. 3T3 cells were transfected in conditions similar to those in (A). The following day, they were metabolically labelled for 1 h and chased for up to 8 h in the absence (medium) or presence of the secretion inhibitor brefeldin A (bref A). Supernatants and cell extracts (cells) were collected at the indicated times. VE-statin was immunoprecipitated, and analysed by SDS–PAGE and autoradiography. The results are representative of three independent experiments performed in similar conditions.

Fig. 7. VE-statin inhibits PDGF-induced AoSMC migration, not proliferation. (A) AoSMCs were plated at low density and cultured in control (filled circles) or VE-statin-containing (filled squares) medium, in the presence of 50 ng/ml PDGF-BB and in the absence (dotted line) or presence of 2% donor calf serum (solid line). Data are presented as mean ± 95% confidence intervals. (B) AoSMC were cultured until a confluent monolayer was formed. The monolayers were then wounded with a razor blade and the cells cultured in serum-free control (Ctrl) or VE-statin-containing medium in the absence (–, open bars) or presence of 80 ng/ml PDGF-BB (+, filled bars). The cells were cultured for 2 days and the monolayers observed by phase contrast microscopy. The bar indicates the location of the wound. (C) The migration rates in (B) were measured by counting the number of cells that had migrated beyond the wound mark after 48 h. A range of 3–7 identical fields per sample were recorded using a digital camera and analysed. The data are presented as mean ± 95% confidence intervals; they are representative of two experiments performed using two independent batches of conditioned medium. (D) AoSMCs (58 000 cells/cm²) were plated in the upper chamber of culture inserts in control (Ctrl) or VE-statin-containing, serum-free medium and in the absence (–, open bars) or presence of 80 ng/ml PDGF-BB (+, filled bars). Two days later, the cells that had crossed the porous membrane were counted. The data are presented as mean ± 95% confidence intervals; they are representative of three experiments performed using three independent batches of conditioned medium. (E) AoSMCs were plated in the upper chamber of culture inserts in conditions similar to those in (D), but incubated in VE-statin-depleted conditioned medium. The removal of VE-statin restores the stimulatory effects of PDGF on AoSMC migration. The data are presented as mean ± 95% confidence intervals; they are representative of two experiments performed using two independent batches of conditioned medium. (F) Primary HUVECs were assayed for migration using the Boyden chamber assay as above in control (Ctrl) or VE-statin-containing medium and in the absence (–, open bars) or presence of 10 ng/ml VEGF (+, filled bars) used as chemoattractant. The data are presented as mean ± 95% confidence intervals; they are representative of two independent experiments.