Supplementary Data
András Iring et al., “Shear stress-induced endothelial adrenomedullin signaling regulates vascular tone and blood pressure”
Supplementary Tables
Supplementary Table 1. Target sequences of siRNAs used in the siRNA screen (Fig. 4A).
Gene Target sequence (5´-3´) ADORA2A CGGAACAGCTCCCAGGTCT ADORA2A GCTGTTAGATCCTCCATGT ADORA2A GGCTTTCCACGGGTTCAGA ADORA2B GAGACTTCCGCTACACTTT ADORA2B GAGCTCATGGATCACTCAA ADORA2B GATGCAGCCACGAACGTGA ADRB1 CAGATCTGGTCATGGGTCT ADRB1 GTGTCATCGCCCTGGACCA ADRB1 CCATCTCGGCGCTGGTGTC ADRB2 CAAGTTCTACTTGAAGGAA ADRB2 CAACTTCTGGTGTGAGTTT ADRB2 GTCATCACAGCCATTGCCA CALCRL GATCAGTTCTGATACGCAA CALCRL GATACTCTCCGTAGTGCAT CALCRL GATTTATGATTTACCTATA CCRL1 GTATGAAGTGATCTGTATA CCRL1 GCTACTTCATCACGGCAAA CCRL1 GCATCAAACATCTGCATTT CCRL2 GACCCTACAATATTGTACT CCRL2 GCTTCTTTACCGGACTTCA CCRL2 CCTGTTGCTCTACTCCATA CELSR1 CTATGAGGAGAATCGAGTA CELSR1 GACTGAAGGTCCAGACGCA CELSR1 CCAACATCGCCACGCTGAA CELSR3 CCTTTGTAACCAGAGAGAT CELSR3 CAGCTTATGATCCAGATGT CELSR3 GCAATACCGGGAGACGCTT CXCR7 GCATGAGTGTGGATCGCTA CXCR7 GCTACGACACGCACTGCTA CXCR7 CTTTGGAGCAGAATGCCAA 2
ELTD1 CTCTTCTAATTCAACTCTT ELTD1 CAAGTTTATTACTAATGAT ELTD1 GTACCATACAGCTATAGTA FZD1 GTAACCAATGCCAAACTTT FZD1 GATTAGCCACCGAAATAAA FZD1 CAGTGTTCCGCCGAGCTCA FZD2 CGCTTTGCGCGCCTCTGGA FZD2 GACATGCAGCGCTTCCGCT FZD2 CGCACTACACGCCGCGCAT FZD4 GTATGTGCTATAATATTTA FZD4 CCATTGTCATCTTGATTAT FZD4 CCAACATGGCAGTGGAAAT FZD5 GGATTTAAGGCCCAGTTTA FZD5 GACCATAACACACTTGCTT FZD5 CAAGTGATCCTGGGAAAGA FZD6 GCATTGTATCTCTTATGTA FZD6 GTGCTTACTGAGTGTCCAA FZD6 CCAATTACTGTTCCCAGAT FZD8 CCATCTGCCTAGAGGACTA FZD8 GGACTAGGCTATTGGCAGA FZD8 CAAAGATATACAGTCTGTA FZD9 CTGTCAAGGTCAGGCAAGT FZD9 CAGAGAAGTTCCAGTACGT FZD9 CGCTCTACGTGATCCAGGA GNAS CTGATTGACTGCGCCCAGT GPR107 CTGCTATTAACTTAGCGAA GPR107 CCTTGATATCGAGATCACA GPR107 GAAGTACCGTGGATTCAAA GPR116 CATTTATTCTTCCCAAGCT GPR116 GTGATATATTGCACTGAAA GPR116 GAATTGGAGTCAAATTGAA GPR124 CTGCACTACTCTTCACTGT GPR124 CACTGGTGATGGAGTCTGA GPR124 CCGTATTTGCGGGAGGCAT GPR125 CATATTTATTCAGTTGAAA GPR125 GCCATATACTCTATTCGGA GPR125 GTGTTTATCCCAAGTCTCT GPR126 CTACCTTACATCCAAGTCT GPR126 GAGGTTACATCTAGATGAA GPR126 CATTTAATGACTTCGACAT GPR137 GTACCACCCTGTTCTCTTT GPR137 CAAAGCGTCGGCCAGAGAT GPR137 CTGACTGCGGCCTACACCA GPR143 GGATCAAGACCCGATTCTT GPR143 CGAGTTTCGTGGACGACAT
3
GPR143 CATCCTATTTCGAAAGACA GPR146 CCTACATGTCCAGCGTCTA GPR146 GCTTCTGTACCGCCACATC GPR146 GCATCCAGGCGACGATGCT GPR153 CCACCATCGTGGTCATCTA GPR153 CGCTTCATCGTGGCTGAGA GPR153 CCCTCATGGCCAACGATGA GPR157 CTCTATGAGACACTTAACA GPR157 CAAAGAATGTTACAGCACA GPR157 GAGATTGCTGATTTATTCA GPR160 CAATGTTTCTATTATATCT GPR160 CTCTTATACCGTGAACTCT GPR160 CTGAATTTCTCTAAAGCGA GPR161 CAGATGTCATGCTACTTGA GPR161 CAACTTCCTGCTGTCTGTA GPR161 GCTATACTGCATTCTGGCA GPR173 GTATCTTTGAGCATCGCTA GPR173 CAGATGGTGCCAGCCATCA GPR173 CCTTTATGGCTGTGCTCTT GPR176 CCATTGCCTTGGACAGGTA GPR176 GACATTTCCAGACAAGTAT GPR176 GTGTTCAAATCTGTCACCA GPR30 CTCTCTTCCTGCAGGTCAA GPR30 CCGAGAACGTCTTCATCAG GPR30 CTCACTCATCGAGGTGTTC GPR4 GCTCTTCCGCGACCGTTAT GPR4 CTTGCAAGCTCTTCGGGTT GPR4 CCTACCACGTGCTCCTGCT GPRC5A GGACCAACGTCAACATCTT GPRC5A CTTTCTGTCAGCCTCAGTT GPRC5A CCTTCATCATCACACTCGA GPRC5B CAACTACTTCGACACGTCA GPRC5B CCTCATCTACGACATGGTA GPRC5B GTCTCTTTGGGCTGACCTT HCRTR1 GCCATCTGCCTGCCGGCTA HCRTR1 GCTGGTATGCCATCTGCCA HCRTR1 CTGCTACCTGCCCATCAGT HRH2 GAAGCTACCAGGAAGCTTA HRH2 GTTTCTATGTTATGGCCCA HRH2 CTACTTCACCGTGTTCGTT LGR4 CTTACTGTTTGGTTCATTT LGR4 CAAGCAATGTGTAGTCTAT LGR4 GCATAAAGCTGAGGCCTGT LPAR6 GAAACAACAACATACATGA LPAR6 CTCTCAAGGATTGTAATTT
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LPAR6 CAATCTGAGTATCAAAGGA LPHN2 GTTACTGGGTCAGATGTAT LPHN2 CATACACTCTTCGATTTGA LPHN2 GACTATAAGAGCTATGGAA PROKR1 CAGCCTACTTCACCACTGA PROKR1 GTGGTATCGGCAACTTCAT PROKR1 CAGTAGACCAGCAGATCTA PTGER4 GTAAGAATCATCTTCTAAA PTGER4 GCACTATTAAAGTAATAAA PTGER4 GTTAATACGAGGTATAACA PTH2R CTCCTTATATTTACGACTT PTH2R CTCTATGTCATGTACACTA PTH2R GCTACAAACTACTATTGGA TPRA1 CTGCTGTGCGCAGACATCA TPRA1 CTCGGATTCTGATCCACCA TPRA1 CTCGTCAGCTCCTGTTTCT
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Supplementary Figures and Figure Legends
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Supplementary Figure 1. Validation of Gαs knock-down or knock out and effect of various knock-downs and knock-outs on flow- and ligand-induced signaling processes .
(A, B) BAECs were transfected with scrambled (control) siRNA or siRNA directed against G αs (encoded by GNAS) as indicated. (A) Quantitative RT-PCR analysis was performed to validate the efficiency of siRNA-mediated knock-down of GNAS.
Expression of GNAS in control siRNA treated cells was set as 100 % (n=4). (B) Cells were incubated with isoproterenol (10 µM, 10 min) or forskolin (10 µM, 10 min) and the intracellular cAMP levels were determined (n=6). (C-F) Human aorta endothelial cells (HAECs) (C and F) or BAECs (D and E) were transfected with scrambled
(control) siRNA or siRNA directed against G αs (C-F) or against CALCRL and adrenomedullin (ADM) (E) as indicated and were exposed to flow for 30 min or for the indicated time periods (15 dynes/cm 2 in C, E and F) or were incubated with VEGF
(50 ng/ml, 10 min) (D). Total and phosphorylated eNOS (C) and AKT (D) were analysed by immunoblotting as indicated. In E, nitrite concentration in the cell culture medium was determined by a chemiluminescent reaction using a Sievers 280i nitric oxide analyzer (n=4), and in F, nitrate and nitrite concentration in the cell culture medium was determined using a fluorimetric reaction (n=3). Graphs and bar diagrams show the densitometric evaluation (n=2 (C), n=3 (D)). (G) HAECs were transfected with scrambled siRNA (control) or siRNA directed against eNOS as indicated, and eNOS wild-type or the eNOS phospho-site mutant S1177A and S633A were expressed by lentiviral transduction. Shown is an immunoblot of cell lysate using antibodies against eNOS or β-tubulin. (H) Lung endothelial cells (Lung EC) were isolated by FACS from indicated mouse lines, and G αs and tubulin expression was analyzed by immunoblotting. Total lung lysates served as controls. (I) Plasma nitrite levels in wild-type (n=6) and EC-Calcrl-KO animals (n=4), EC-Adm-KO and
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EC-Gαs-KO mice (n=4) measured 10 days after induction using a Sievers 280i nitric oxide analyzer. Data presented are the mean ± SEM; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤
0.001; n.s., not significant (two-way ANOVA with Bonferroni’s post-hoc test (B and
E); one-way ANOVA with Tukey’s post-hoc test (C,D,F,I)).
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Supplementary Figure 2. Expression of GPCRs in BAECs and HUVECs.
RNA-sequencing was performed to determine gene expression of GPCRs in BAECs and HUVECs. Shown are the mean log 2 values of expression of GPCRs after normalization based on TPM. Values have been sorted based on the expression in
BAECs (n=4).
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Supplementary Figure 3. Validation of CALCRL, GPR146 and ADM knock-down in
BAECs, comparison of flow- and ADM-induced eNOS phosphorylation and role of
CALCRL and ADM in human endothelial cells.
(A and C) BAECs were transfected with scrambled (control) siRNA or siRNA directed against the indicated gene. Quantitative RT-PCR analysis was performed to validate the efficiency of siRNA-mediated knock-down of CALCRL (A, left panel), GPR146 (A, right panel), adrenomedullin (ADM) (C). Expression of the indicated gene in control siRNA treated cells was set as 100 % (n=3). (B) Comparison of flow-, ADM- and
ADM2-induced eNOS phosphorylation at serine 635. The graph shows the
11 densitometric evaluation (n=3). ( D and E) HAECs were transfected with scrambled
(control) siRNA or siRNA directed against CALCRL or ADM as indicated and were exposed to flow for 30 min or the indicated time periods (15 dynes/cm 2) and total and phosphorylated eNOS (D) as well as nitrate and nitrite concentration in the cell culture medium (E) were determined. Graphs and bar diagrams show the densitometric evaluation (n=3). Data represent the mean ± SEM; *, p ≤ 0.05 (one- way ANOVA with Tukey’s post-hoc test (B); two-way ANOVA with Bonferroni’s post- hoc test (D and E).
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Supplementary Figure 4. Validation of PIEZO1 knock-down in BAECs, function of
CALCRL and ADM in Yoda1-induced eNOS activation and role of Gαq/G α11 in flow- induced adrenomedullin release.
(A) BAECs were transfected with scrambled (control) siRNA or siRNA directed against the indicated gene. Quantitative RT-PCR analysis was performed to validate
13 the efficiency of siRNA-mediated knock-down of PIEZO1. Expression of the indicated gene in control siRNA treated cells was set as 100 % (n=3). (B and C) BAECs were transfected with control siRNA, siRNA directed against CALCRL and ADM or were pretreated with the adrenomedullin receptor antagonist AM(22-52) and were exposed to Yoda1 (1 µM) for 30 min. Total and phosphorylated eNOS was determined by immunoblotting. Graphs show the densitometric evaluation of blots (n=2). (D) BAECs were incubated in the absence or presence of the Ca 2+ ionophore A23187 (10 µM, 1 min). Thereafter the adrenomedullin concentration in the cell culture medium was determined (n=5). (E) BAECs were transfected with scrambled (control) siRNA or siRNA directed against G αq/G α11 as indicated. Cells were then exposed to flow (15 dynes/cm 2) for the indicated time periods or to Yoda1 (1 µM) and the adrenomedullin concentration in the cell culture medium was determined (n=3). The lower panel shown G αq/G α11 knock-down efficiency. Data represent the mean ± SEM; *, p ≤ 0.05;
**, p ≤ 0.01; ***, p ≤ 0.001 (one-way ANOVA with Tukey’s post-hoc test (B,D,E); two- way ANOVA with Bonferroni’s post-hoc test (C)).
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Supplementary Figure 5. Generation of mice carrying a floxed allele of the gene
Calcrl.
(A) Mouse embryonic stem (ES) cells generated from the strain C57BL/6N carrying the shown targeted allele of the gene encoding CALCRL were obtained from KOMP repository ( Calcrl tm1a(KOMP)Wtsi ). ES cells were used to generate chimeric mice, and germ-line transmission was verified after crossing chimeras with a Flp deleter mouse line. Mice carrying the indicated floxed allele were then used to generate inducible endothelium-specific CALCRL-deficient mice ( Tek-CreER T2 ;Calcrl fl/fl , referred to as
EC-Calcrl-KO mice). Shown is the targeted Calcrl allele before and after Flp- and
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Cre-mediated recombination resulting in the floxed and null allele, respectively. (B)
Lung endothelial cells (left panel) and aortic vascular smooth muscle cells (right panel) were isolated from wild-type and EC-Calcrl-KO mice 10 days after induction with tamoxifen. Quantitative RT-PCR analysis was performed against exon 7 to validate the loss of CALCRL. The qPCR primer sequence used against murine Calcrl exon 7 was as follows: 5´-TAGGACCTGGGACGGATG-3´ (forward primer), 5´-
TCCTGAAAATAGTCAGGGCAGT-3´ (reverse primer). Expression of CALCRL in wild-type mice was set as 100 % (n=4 (endothelial cells); n=2 (smooth muscle cells)).
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Supplementary Figure 6. Basic characterization of aortic segments from induced
EC-Calcrl-KO and EC-Adm-KO mice.
(A,B) Effect of increasing concentration of phenylephrine and acetylcholine on the vascular tension of aortic segments from wild-type, EC-Calcrl-KO animals (n=8, WT; n=10, EC-Calcrl-KO) and EC-Adm-KO mice (n=13, WT; n=11, EC-Adm-KO). In experiments testing the effect of acetylcholine, vessels were pre-contracted with
10 µM phenylephrine.
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Supplementary Figure 7A
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Supplementary Figure 7B
Supplementary Figure 7. Representative flow cytometric plots of Tie2-expressing bone marrow- (7A) or spleen-derived cells (7B) from Tie2CreER T2+ ;Rosa26 flox-mTRed-
Stop-flox-mGFP animals.
All mice were treated with 1 mg/ml tamoxifen per day for 5 consecutive days to induce expression of GFP under the Tie2 promoter. Two weeks after the last
19 injection, animals were sacrificed and bone marrow cells were obtained from tibia and femur, and spleens were isolated and mechanically dissociated into a single-cell suspension (n=4 mice per genotype). Cell suspension was filtered through a 70-µm nylon filter and washed. The spleen cell suspension was subjected to an additional step of erythrocyte lysis. Cells were then washed, centrifuged and stained for flow cytometry. Cells were acquired on a FACS Canto II flow cytometer (BD) and analysis was performed using Diva software. Hematopoietic lineages analyzed represent T cells (CD4 and CD8), B cells (CD45 and B220) and myeloid cells (CD11b, Ly6C,
Ly6G and F4/80). Cre-negative littermates served as controls.