KLF2 and KLF4 control endothelial identity and vascular integrity Panjamaporn Sangwung 1, 2,#, Guangjin Zhou1,#, Lalitha Nayak1,3, E. Ricky Chan4, Sandeep Kumar5, Dong-Won Kang5, Rongli Zhang1, Xudong Liao1, Yuan Lu1, Keiki Sugi1, Hisashi Fujioka7, Hong Shi1, Stephanie D Lapping1, Chandra C. Ghosh8, Sarah J. Higgins8, Samir M. Parikh8, Hanjoon Jo5,6, and Mukesh K Jain1,2,9 Supplemental Figures and Tables 1 Supplemental Figure 1 A Klf2 Klf4 Nos3 Thbd 3 ** 3 ** 3 ** 3 ** NS ** * NS ** NS * NS Day 6 2 2 2 2 post-tamoxifen 1 1 1 1 expression Relative mRNA Relative mRNA 0 0 0 0 CRE -KO -KO CRE -KO -KO CRE -KO -KO CRE -KO -KO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO EC- EC- EC- EC- EC- EC- EC- EC- B Klf2 Klf4 Nos3 Thbd 3 ** 3 ** 3 ** 3 ** NS Day 5 ** ** ** ** NS NS * post-tamoxifen 2 2 2 2 1 1 1 1 expression Relative mRNA Relative mRNA 0 0 0 0 CRE -KO -KO CRE -KO -KO CRE -KO -KO CRE -KO -KO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO EC- EC- EC- EC- EC- EC- EC- EC- C Klf2 Klf4 Nos3 Thbd 3 ** 3 ** 3 ** 3 ** NS ** NS NS Day 4 ** NS NS NS 2 2 2 2 post-tamoxifen 1 1 1 1 expression Relative mRNA Relative mRNA 0 0 0 0 CRE -KO -KO CRE -KO -KO CRE -KO -KO CRE -KO -KO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO EC- EC- EC- EC- EC- EC- EC- EC- D CRE EC-DKO KLF4 TM GAPDH eNOS GAPDH Supplemental Figure 1. Expression of Klf2, Klf4, and their targets in primary cardiac microvascular EC. Relative mRNA expression levels of Klf2 (Kruppel-like family of transcription factor 2), Klf4, Nos3 (endothelial nitric oxide synthase), and Thbd (thrombomodulin) in primary cardiac microvascular EC isolated from mice at day 6 (A), day 5 (B), and day 4 (C) following tamoxifen (n=3-5 per genotype, each sample was pooled from 2 mice). (D) Representative western blot of primary cardiac microvascular EC using antibodies against KLF4, eNOS (endothelial nitric oxide synthase), TM (thrombomodulin), and GAPDH (endogenous glyceraldehyde 3-phosphate dehydrogenase). CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout, EC-Klf2-KO: EC-specific Klf2 knockout, EC-Klf4-KO: EC-specific Klf4 knockout. Sample in each group was pooled from 7-8 mice. *: P < 0.05, * *: P < 0.01, NS: Not significant. One-way ANOVA with Bonferroni's post hoc test. 2 Supplemental Figure 2 1.2 Baseline Day 6 post-tamoxifen 0.8 CRE 0.4 EKG (mV) 0.0 -0.4 1.2 0.8 EC-DKO 0.4 ST EKG (mV) 0.0 -0.4 -0.8 1.2 0.8 0.4 EC-Klf2-KO EKG (mV) 0.0 -0.4 -0.8 1.2 0.8 EC-Klf4-KO 0.4 EKG (mV) 0.0 -0.4 100 ms -0.8 Supplemental Figure 2. Electrocardiogram (EKG) recording. Representative EKG of CRE, EC-DKO, EC-Klf2-KO, and EC-Klf4-KO mice at baseline without tamoxifen (left) and day 6 post-tamoxifen injection (right). n=4-5 per genotype. CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout, EC-Klf2-KO: EC-specific Klf2 knockout, EC-Klf4-KO: EC-specific Klf4 knockout. 3 Supplemental Figure 3 A Cardiac function ** ** p=0.06484 NS NS NS NS NS NS 60 80 ** 40 ** 40 60 30 40 FS (%) EF (%) 20 20 CO (ml/min) 20 10 0 0 0 CRE -KO -KO CRE -KO -KO CRE -KO -KO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC-DKO EC- EC- EC- EC- EC- EC- B C Chamber size ** NS * ** NS NS NS 5 5 NS 5 NS 4 4 4 3 3 3 2 2 2 LVIDs (mm) LVIDs LVIDd (mm) LVIDd LV/BW (mg/g) LV/BW 1 1 1 0 0 0 -KO -KO CRE CRE -KO -KO -KO -KO Klf2 Klf4 CRE EC-DKO Klf2 Klf4 EC-DKO Klf2 Klf4 EC- EC- EC-DKO EC- EC- EC- EC- Supplemental Figure 3. Echocardiographic analysis at day 6 after tamoxifen administration. (A) Fractional shortening (FS), ejection fraction (EF), and cardiac output (CO) in CRE (n=3), EC-Klf2-KO (4), EC-Klf4-KO (n=4), and EC-DKO mice (n=5). (B) Left ventricle (LV)-to-body weight (BW) ratio. (C) LV internal dimension at end diastole (LVIDd), and at end systole (LVIDs). CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout, EC-Klf2-KO: EC-specific Klf2 knockout, EC-Klf4-KO: EC-specific Klf4 knockout. Data are presented as mean ± SEM. *: P < 0.05, * *: P < 0.01. NS: Not significant. One-way ANOVA with Bonferroni's post hoc test. 4 Supplemental Figure 4 EC-Klf2-KO EC-Klf4-KO Brain Lung Heart Skin Supplemental Figure 4. Gross post-mortem examination in EC-Klf2-KO and EC-Klf4-KO mice. Representative gross anatomy pictures of brain, lungs, heart, and subcutaneous tissues from EC-Klf2-KO (EC-specific Klf2 knockout) and EC-Klf4-KO (EC-specific Klf4 knockout) mice at day 6 post-tamoxifen (n=3-4 per genotype). 5 Supplemental Figure 5 A CRE EC-DKO B Day 4 post-tamoxifen Lung Kidney Heart 0.5 **0.5 0.5 NS Eosin 0.4 0.4 0.4 (Lung) 0.3 0.3 0.3 0.2 0.2 0.2 Permeability 0.1 0.1 0.1 (corrected E620) 0.0 0.0 0.0 CRE EC-DKO CRE EC-DKO CRE EC-DKO EM C Day 5 post-tamoxifen (Lung) Lung Kidney Heart 0.5 0.5 0.5 0.4 **0.4 * 0.4 NS 0.3 0.3 0.3 0.2 0.2 0.2 0.1 0.1 0.1 Permeability Evans (corrected E620) 0.0 0.0 0.0 blue CRE EC-DKO CRE EC-DKO CRE EC-DKO (Lung) D Day 6 post-tamoxifen Lung Kidney Heart 0.5 0.5 0.5 * * * 0.4 0.4 0.4 0.3 0.3 0.3 Evans 0.2 0.2 0.2 blue (Kidney) 0.1 0.1 0.1 Permeability 0.0 0.0 0.0 (corrected E620) CRE EC-DKO CRE EC-DKO CRE EC-DKO Supplemental Figure 5. Endothelial-specific deletion of Klf2 and Klf4 results in vascular leak. (A, top) Representative pictures of lung Eosin staining show erythrocyte leakage (white arrow) in extravascular tissues at day 6 post-tamoxifen. (n=3-4 per genotype). Scale bar is 200 μm. (A, middle) Representative electron microscopic (EM) images of lung indicate discontinuity of endothelial monolayer at day 6 post-tamoxifen (n=3-4 per genotype). Scale bar is 2 μm. Endothelial cells (EC): red arrowhead, extravascular erythrocytes: white arrow, leucocytes: blue arrow. (A, bottom) Representative pictures of Evans blue vascular permeability assay for the lungs and kidneys at day 6 post-tamoxifen (n=7-8 per genotype). (B-D) Quantification of extravasated Evans Blue dye in the lungs, kidneys, and heart at day 4 (B, n=6-8 per genotype), day 5 (C, n=6-9 per genotype), and day 6 (D, n=7-8 per genotype) post-tamoxifen. CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout. Data are presented as mean ± SEM. *: P < 0.05, * *: P < 0.01, NS: Not significant. 2-tailed Student’s t test. 6 Supplemental Figure 6 NS ***CRE EC-DKO 5 4 3 2 Relative mRNA expression Relative mRNA 1 0 F3 Serpine1 F2rl3 Supplemental Figure 6. Expression of F3, Serpine1, and F2rl3 mRNA in primary cardiac microvascular EC at day 6 post-tamoxifen. n=3-4 per genotype, each sample was pooled from 2 mice. F3: tissue factor, Serpine1: serine (or cysteine) peptidase inhibitor, clade E, member 1, F2rl3: coagulation factor II receptor-like 3, CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout. *: P < 0.05, * *: P < 0.01, NS: Not significant. 2-tailed Student’s t test. 7 Supplemental Figure 7 EC-DKO EC-DKO EC-DKO EM (Brain) Supplemental Figure 7. Representative electron microscopic (EM) images of brain indicate degeneration of EC and extravascular erythrocytes in the EC-DKO mice at day 6 post-tamoxifen. Scale bar is 2 μm. Endothelial cells (EC): red arrowhead, extravascular erythrocytes: white arrow, leucocytes: blue arrow, vessel lumen: black arrowhead (n=3-4 per genotype). EC-DKO: EC-specific Klf2 and Klf4 double knockout. 8 Supplemental Table 1. Differentially expressed genes (q<0.05) in cardiac microvascular EC obtained from EC-DKO in comparison to CRE mice at day 6 post-tamoxifen. Ensembl_ID Gene Description EC-DKOvsCRE.log2FC EC-DKOvsCRE.RawP EC-DKOvsCRE.FDR ENSMUSG00000099397 Gm7809 predicted gene 7809 6.520828668 2.75E‐181 7.75E-177 ENSMUSG00000051855 Mest mesoderm specific transcript 3.334038311 2.80E‐113 3.94E-109 ENSMUSG00000031886 Ces2e carboxylesterase 2E -3.950756039 2.80E‐91 2.62E-87 ENSMUSG00000024743 Syt7 synaptotagmin VII -3.222926334 1.53E‐87 1.08E-83 ENSMUSG00000028766 Alpl alkaline phosphatase, liver/bone/kidney -3.05284643 1.51E‐80 8.51E-77 ENSMUSG00000037010 Apln apelin 2.944609555 2.96E‐74 1.39E-70 ENSMUSG00000031628 Casp3 caspase 3 2.776995883 1.45E‐68 5.82E-65 ENSMUSG00000028364 Tnc tenascin C 5.209422965 6.36E‐63 2.24E-59 ENSMUSG00000024501 Dpysl3 dihydropyrimidinase-like 3 2.381613989 7.96E‐62 2.49E-58 ENSMUSG00000020151 Ptprr protein tyrosine phosphatase, receptor type, R -3.666146488 7.70E‐61 2.17E-57 ENSMUSG00000053835 H2-T24 histocompatibility 2, T region locus 24 -2.626013971 1.35E‐60 3.45E-57 ENSMUSG00000046186 Cd109 CD109 antigen 2.489258617 2.24E‐60 5.24E-57 ENSMUSG00000094655 Gm25360 predicted gene, 25360 2.418946823 5.78E‐55 1.25E-51 ENSMUSG00000074272 Ceacam1 carcinoembryonic antigen-related cell adhesion molecule 1 -2.384575452 2.51E‐54 5.04E-51 ENSMUSG00000006014 Prg4 proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein) -3.007843345 3.61E‐53 6.77E-50 ENSMUSG00000026582 Sele selectin, endothelial cell 2.217786815 3.05E‐52 5.36E-49 ENSMUSG00000021822 Plau plasminogen activator, urokinase 2.243213549 5.79E‐49 9.58E-46 ENSMUSG00000001473 Tubb6 tubulin, beta 6 class V 3.218962042 1.16E‐48 1.81E-45 ENSMUSG00000041552 Ptchd1 patched domain containing 1 4.126628073 7.48E‐48 1.05E-44 ENSMUSG00000005124 Wisp1 WNT1 inducible signaling pathway protein 1 3.563401963 7.25E‐48 1.05E-44 ENSMUSG00000031465 Angpt2 angiopoietin 2 2.149078948 7.67E‐46 1.03E-42 ENSMUSG00000053062 Jam2 junction adhesion molecule 2 -2.085030593 1.05E‐45 1.35E-42 ENSMUSG00000020053 Igf1 insulin-like growth factor 1 2.760058282 3.09E‐43 3.64E-40 ENSMUSG00000024713 Pcsk5 proprotein convertase subtilisin/kexin type 5 -2.189606108 3.11E‐43 3.64E-40 ENSMUSG00000018593 Sparc secreted acidic