Supplementary Data. Plasmid -2486CAT containing the 2.5 kb fragment of VE-cadherin promoter region (-2486

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1 Supplementary Data Materials and Methods Generation and Genotyping of Transgenic Mice Plasmid -2486CAT containing the 2.5 kb fragment of VE-cadherin promoter region (-2486 to +24) was a gift from P. Huber. 1 The VE-cadherin promoter DNA followed by Cre recombinase cdna, and polyadenylation signal from SV40 were inserted into pbluescriptskii (Stratagene) and a resultant plasmid was named as pbs-ve-cad-cre. The 4.2 kb VE-cad-Cre transgene (Figure 1A) excised from pbs-ve-cad-cre was microinjected into fertilized C57BL/6J eggs. VE-cad-Cre transgenic founders were confirmed by both PCR and Southern analyses using tail DNA. We obtained three lines of VE-cad-Cre mice and crossed with either EGFP reporter mice or LacZ reporter mice. Since β galactosidase is fused with nuclear localization sequence, LacZ expression was found in the nucleus when examined by histochemical analysis. There was no obvious difference for reporter expression among the offspring of three lines. Therefore, line1 containing an estimated two copies of VE-cad-Cre gene (Supplementary Figure 1C) was used for all the following experiments. Tie2/EG mice were obtained by crossing Tie2-Cre mice (a gift from M. Yanagisawa, Texas Southwestern University) with CAG-CAT-EGFP. 2 In Vivo GFP imaging The GFP fluorescence from VE/EG mice were observed using SZX12 stereo-fluorescent microscope (Olympus, Japan). EGFP images were obtained through an XF2043 dichroic 1

2 filter (Omega Optical) and a set of an S484/15 excitation filter and an S515/30 emission filter (Chroma Technology) with a 75-W Xenon arc lamp. Histochemistry, Immunostaining, Immunofluorescence, and in Situ Hybridization X-gal staining of embryo of VE/Z was performed as described previously. 3 Wild type mouse embryos (E9.5) and heart tissue sections obtained from 5-week -old mice were immunostained with anti-ve-cadherin (BD Biosciences). Immunoreaction was visualized with horseradish peroxidase-conjugated secondary antibody. The frozen sections obtained from VE/Z mice were immunostained with anti-cd31 (BD Bioscience) and anti-α-smooth muscle actin (SMA) (Sigma). Those from VE/EG mice were immunostained with anti-cd31, anti-α-sma, and anti-gfp developed in our laboratory, followed by incubation with fluorescence-conjugated secondary antibodies. Frozen sections obtained from wild type sham-operated and infarcted were immunostained with anti-mouse VE-cadherin (A kind gift from Dr. Vestweber, University of Munster, Germany). Immunoreaction was detected with avidin-biotin-peroxidase comlex method followed by visualization with diaminobenzidin. X-gal stained embryo tissue sections were counter-stained with hematoxylin. Tissue sections were imaged on an Axiovert 25 inverted universal microscope equipped with an Axiocam HR color CCD camera (Zeiss) after immunostaining or histochemistry. Confocal imges were obtained by a LSM 510 META microscope (Carl Zeiss). Preparation of embryos or frozen sections and in situ hybridization with digoxigenin-labeled RNA probes were performed following manufacturer s instruction 2

3 (Roche Diagnostics). The probe was derived from the 1.7 kb amino-terminal fragment of mouse VE-cadherin cdna amplified by PCR using E13.5 mouse cdna library as a template. Sections were imaged as described above. Genomic PCR Analysis and Southern Analysis VE-cad-Cre transgenic founders were confirmed by both PCR and Southern analyses using tail DNA. The 5 and 3 primers, Cre-f, 5 -gttcgcaagaacctgatggaca-3 and Cre-r, 5 -ctagagcctgttttgcaggttc-3, respectively were chosen within the Cre cdna (Supplementary Figure 1A). For Southern analyses, genomic DNA digested with BglII were electrophoresed and transferred to the Hybond-N+ membrane (Amersham Biosciences). The membrane was hybridized with a radio-labeled probe produced by 0.6 kb EcoRV/BglII fragment of Cre transgene using random prime methods (Roche Diagnostics). Neovascularization Models (Corneal Assay and Myocardial Infarction Model) A pellet containing recombinant mouse VEGF165 (R & D Systems) was implanted into a corneal micro pocket. Neovascularization marked by EGFP in living mice was observed with a fluorescent microscope (SZX12, Olympus). 8-week-old VE/EG or VE/Z mice were anesthetized and ventilated by cannulating the trachea. The chest was opened by a left thoracotomy, and the left anterior descending coronary artery was permanently ligated. Infarcted mice were sacrificed 3 days after ligation and their organs were subjected to histological examination. The number of the capillary vessels that were positive for CD31 were counted in the at least 10 undamaged area of 5 sections of sham-operated or infarcted 3

4 mice. Ten undamaged area in which capillaries were observed as rings were used for the analysis. Mononuclear Cell Preparation Mononuclear cells in bone marrow cells and peripheral blood cells were prepared by density separation in Ficoll-Paque PLUS (Amersham Biosciences) and hemolysed with ammonium chloride buffer. Mononuclear cells were filtered through a 35 μm cell strainer. Real-Time PCR for quantitative analysis of VE-cadherin mrna CD45-posive bone marrow cells from wild type infarcted B57/BL6J or sham-operated mice were obtained by cell sorting (FACS Aria, BD Biosciences). One μg of total RNA prepared using Trizol was subejectred to reverse-transcription PCR (Invitrogen). VE-cadherin mrna was quantitated by real-time PCR (SYBR Green,Qiagen) using forward primer 5'- TCAACGCATCTGTGCCAGAGAT-3', reverse primer 5'-CACGATTTGGTACAAGACAGTG-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mrna was also quantitated simultaneously. Supplementary Figure 1. Genotyping of VE-cad-Cre mouse. A, Schematic illustration of the transgene of VE-cadherin promoter followed by Cre recombinase cdna (VE-cad-Cre) and a primer set for detection of Cre recombinase gene (Cre-f and Cre-r). B, VE-cad-Cre transgene was examined by PCR using tail DNA of transgenic mice. The expected size for the partial DNA derived from the transgene is 350 bp. p, positive control 4

5 (PCR using pbs-ve-cad-cre DNA as a template). n, negative control (PCR using pbluescript as a template. C, Southern blot analysis of VE-cad-Cre transgene. Tail DNA digested with BglII was electrophoresed along with pbs-ve-cad-cre DNA equivalent to a single copy (1c) and 10 copies (10c). VE-cad-Cre transgene was detected at 2.4 kb. Cre-, the mouse without VE-cad-Cre transgene; Cre+, the mouse with VE-cad-Cre transgene. Supplementary Figure 2. Sustained EGFP expression after birth in Tie2/EG mice. Similar to Figure 2, EGFP expression in the heart and lung of Tie2/EG were examined under a stereo-fluorescent microscope (A, C, E, and G). Tissue sections obtained from Tie2/EG mice were immunostained with anti-gfp (green) and anti-cd31 (red) (B, D, F, and H). Macroscopically EGFP expression in the heart and lung was observed both at neonatal stage (3 d) and 5 weeks (5 w) after birth. EGFP-positive cells are also CD31-positve in both heart and lung (arrows). I, LacZ-expression driven VE-cadherin promoter was examined in the heart of a littermate lacking Cre gene (Cre-), neonate VE/Z mouse heart (3 d), and 5-week-old VE/Z mouse heart (5 w). The arrow and the arrowheads indicate LacZ-stained endothelial cells of a coronary artery and capillary, respectively. Bar = 50 μm. Supplementary Figure 3. Cardiac vessel-specific activation of VE-cadherin promoter by circulating factors. Para biotic pairs of a wild type mouse (gray) and a VE/Z mouse (blue) was created as indicated. Tissues were examined for LacZ expression from the mouse indicated by the broken line box. A, Paradises itself does not activate VE-cadherin 5

6 promoter of heart vessels in the VE/Z mouse. B, C, and D, The wild type mouse of a Para biotic pair was coronary-legated (cross). Sections were stained with X-gal. B, Note that LacZ-positive cells are observed exclusively in the coronary arteries. C and D, No LacZ stained cell in the vasculature of other organs. Bars (in A and B) =20 μm; Bars ( in C and D) = 50 μm. Cardiac ischemia may produces heart vessel-specific factors that activate VE-cadherin promoter. This notion is supported by the data that in the mouse without infarction connected to the mice with infarction, VE-cadherin promoter is only activated in the heart vessels of the mice without infarction and is not activated in other organs. Thus, cardiac ischemia is crucial for inducing VE-cadherin promoter in both pre-existing cells and mobilized cells to the ischemic heart. Supplementary Figure 4. The capillary vessels are increased in the infarcted mice. Capillary vessels detected by CD31-positive ring were counted as described in Materials and Methods of Supplementary data. Supplementary Figure 5. VE-cadherin mrna was increased two times greater in CD45-positive bone marrow cells upon cardiac ischemia than in CD45-positive bome marrow cells in sham-operated mice. VE-cadherin mrna is analyzed by real-time PCR by comparing the amount of VE-cadherin mrna products with that of GAPDH mrna products. Reference 6

7 1. Gory S, Vernet M, Laurent M, Dejana E, Dalmon J, Huber P. The vascular endothelial-cadherin promoter directs endothelial-specific expression in transgenic mice. Blood. 1999;93: Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol. 2001;230: Schlaeger TM, Qin Y, Fujiwara Y, Magram J, Sato TN. Vascular endothelial cell lineage-specific promoter in transgenic mice. Development. 1995;121:

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12 VE-cadherin/GAPDH CD45-Sham CD45-AMI1 Supplementary Figure 5 12