Title: Smooth muscle cell-specific Tgfbr1 deficiency promotes aortic aneurysm formation by stimulating multiple signaling events Pu Yang 1, 3, radley M. Schmit 1, Chunhua Fu 1, Kenneth DeSart 1, S. Paul Oh 2, Scott. erceli 1, 4, Zhihua Jiang 1 Supplementary Data Tgfbr1 f/f ;R26R + ;Myh11 -CreER + T_d5 α β γ Ex3 β + γ 3 1 iko 2 f/f WT 192 320 Tgfbr2 f/f ;R26R + ;Myh11 -CreER + Tgfbr1 f/f.tgfbr2 f/f ;R26R + ; Myh11-CreER + α γ x y z Ex2 x z α + γ x + y x + z iko f/f WT 310 550 434 600 Supplementary Figure 1. The Myh11-driven Cre-loxP system facilitates efficient deletion of Tgfbr1 and Tgfbr2 in aortic SMCs. () X-gal staining. Note the robust recombination-events in the medial layer of the Ts. Scale bars: 100 μm. () Genotyping results obtained with genomic DN extracted from explanted SMCs and primers illustrated in the cartoon. Upper row, Tgfbr1 deletion; Lower row, Tgfbr2 deletion. 1
SMCs 10% FS TGFβ1 psmd2 SMD2 β-actin psmd3 SMD3 β-actin WT Tgfbr1 iko Tgfbr2 iko + + + + + + + + + mrn Expression (Relative to untreated WT SMCs) CTGF 6 5 3 2 0 TGFβ1 + + SMCs WT Tgfbr1 iko 2.0 1.0 0.5 TGFβ1 + + SMCs WT Tgfbr2 iko 2.4 1.8 1.2 0.6 4.0 3.0 2.0 1.0 Col1a2 + + WT Tgfbr1 iko + + WT Tgbfr2 iko 20 15 10 5 0 6.0 4.5 3.0 Elastin + + WT Tgfbr1 iko + + WT Tgfbr2 iko C SMCs perk WT Tgfbr1 iko Tgfbr2 iko TGFβ1 + + + 44 42 2.4 1.8 1.2 0.6 P=29 ERK 44 42 TGFβ1 SMCs + + + WT Tgfbr1 iko Tgfbr2 iko Supplementary Figure 2. Tgfbr1 iko or Tgfbr2 iko abrogates TGF-β-stimulated SMD and ERK signaling in SMCs. () Western blotting assays for SMD2 and SMD3 phosphorylation. The addition of serum to TGF-β1 treatment was intended to create a more physiological context for TGF-β stimulation. () Expression of TGF-β responsive genes in SMCs with the indicated genotype. Levels of mrn were determined with qrt-pcr. (C) ctivation of ERK pathway in SMCs following TGF-β1 stimulation. ssays were performed in triplicates. SMCs were stimulated with TGF-β1 (1.0ng/ml) for one and twenty-four hours for protein and mrn assays, respectively. 2
a a b b c c 1 2 3 4 5 6 a b b d d 1 3 Supplementary Figure 3. ortic pathologies detected on gross examination in Tgfbr1 iko aortas at d28. Evans blue (5% in saline) was injected through tail vein to mice 30 minutes before tissue collection. () Thoracic aortas. rrows a, b, and c indicate the pathologies of aneurysmal dilation, intramural hematoma, and contained rupture, respectively. () Magnified view of the aorta #1 and #3. rrow d points to areas with Evans blue extravasation through intimal/medial tears. 3
C Movat Masson SEM En face Tgfbr1 iko, d10 Tgfbr1 f/f, d10 Tgfbr1 iko, d13 Tgfbr1 f/f, d13 rch rch Root d10 Movat Masson Medial Thickness (mm) 8 p=ns 6 4 2 0 Tgfbr1f/f d2 d5 d10 Tgfbr1 iko, d13 Tgfbr1 f/f, d13 Supplementary Figure 4. Early pathology of Tgfbr1 iko aortas features isolated intimal/medial tears and intramural hematoma. () Histology of Ts with indicated genotype on d10. rrows point to elastic fiber breaks. () Medial thickness measured for Tgfbr1 f/f (d10, n=7) and Tgfbr1 iko (n=6, 7, and 12 at d2, d5, and d10, respectively) Ts. Data were analyzed using one-way NOV. (C) Histology of Ts with indicated genotype on d13. En face indicates luminal en face microscopy. rrows point to areas with Evans blue extravasation. SEM denotes scanning electron microscopy. Masson s and Movat s staining images show areas of Evans blue extravasation correlates to intimal/medial tears (arrows). Scale bars: 100 μm. 4
C D E @ # Supplementary Figure 5. ortic aneurysms located in the SR region displays similar pathological features as those located in the T region. Images represent Masson s trichrome staining of cross sections of the SR specimens collected from different animals on d28. Panel represents normal histology of Tgfbr1 f/f SR segments while panels -E show medial depletion (, arrows), intramural hematoma (C, arrows), rupture (D), and aortic dissection (E, #: true lumen; @: false lumen) detected in Tgfbr1 iko SR segments, respectively. Scale bars: 100 μm. 5
Deep tear (4) T_1 Medial thinning (3) T_2 Medial thinning (3) Deep tear (5) Intramural hematoma (5) Sample Intimal/ medial tears Intramural hematoma Medial thinning/ depletion Total score T_1 4 0 3 7 T_2 5 5 3 13 C Supplementary Figure 6. Examples of using the proposed scoring system to quantify aortic wall degeneration. Masson s staining images () and () show T specimens collected from two individual Tgfbr1 iko animals. Pathologies present each specimen are specified in the text-boxes with a score for that pathology given in the parenthesis. Scores assigned to each specimen are summarized in the table and the total score is utilized to estimate the severity of aneurysmal degeneration. (C) Magnified view of the boxed area in, showing presence of blood cells in space between elastic laminae. 6
1.6 psmd2_d5 1.6 psmd2_d13 (Relative to Tgfbr1f/f) 1.2 0.8 0.4 P<01 P=19 (Relative to Tgfbr1f/f) 1.2 0.8 0.4 P<01 P=49 Tgfbr1f/f Tgfbr1iko Tgfbr2iko Tgfbr1iko.Tgfbr2iko Tgfbr1f/f Tgfbr1iko Tgfbr2iko Tgfbr1iko.Tgfbr2iko Tgfbr1 f/f Tgfbr1 iko _d5 Tgfbr1 iko _ d13 Negative control L Supplementary Figure 7. Loss of SMC specific Tgfbr1, Tgfbr2, or both disrupts the SMD2-mediated TGF-β signaling. () Quantification of the psmd2 immunoblots for Tgfbr1 f/f, Tgfbr1 iko, Tgfbr2 iko, and Tgfbr1 iko.tgfbr2 iko Ts (n=5 in each group) at the indicated time points. Statistics were obtained with one-way NOV. () Fluorescent IHC assays for psmd2 in Ts. Red, psmd2; Green, auto-fluorescence of the elastic laminae. White dash lines indicate the medial-adventitial boarder. Specimens stained with isotype-matched IgGs served as a negative control. Note the reduced intensity of psmd2 staining in Tgfbr1 iko Ts compared to the Tgfbr1 f/f Ts. 7
6.0 psmd1/5/8_d5 6.0 psmd1/5/8_d13 (Relative to Tgfbr1f/f) 4.5 3.0 P=NS (Relative to Tgfbr1f/f) 4.5 3.0 * *P<01 Tgfbr1f/f Tgfbr1iko Tgfbr2iko Tgfbr1iko.Tgfbr2iko Tgfbr1f/f Tgfbr1iko Tgfbr2iko Tgfbr1iko.Tgfbr2iko Supplementary Figure 8. Loss of SMC specific Tgfbr1, Tgfbr2, or both Tgfbr1and Tgfbr2 increases the level of psmd1/5/8 in the aortic wall. ar graphs show quantification of the psmd1/5/8 immunoblots produced by Tgfbr1 f/f, Tgfbr1 iko, Tgfbr2 iko, and Tgfbr1 iko.tgfbr2 iko Ts (n=5 in each group) at the indicated time points. Statistics were obtained with one-way NOV. perk ERK Veh RDE kda 44 42 44 42 perk/erk +/- SEM 1.2 0.9 0.6 0.3 P=07 β-actin 42 Veh RDE Supplementary Figure 9. Treatment with RDE-119 inhibits ERK phosphorylation in Tgfbr1 iko Ts. Levels of perk were determined with western blotting assays using total protein extracted from Ts of Tgfbr1 iko animals treated with RDE-119 (RDE) or vehicle dissolvent (Veh) for two weeks(n=6 per group). Density of each perk immunoblot was quantified and normalized to the total ERK. Data were analyzed using unpaired t-test. 8
Los Hyd Pro SP (mmhg) +/- SEM 120 100 80 60 40 20 0 d0 d14 d28 SP Drop (mmhg) +/- SEM 40 30 20 10 0 P=0.17 Los Hyd Pro Supplementary Figure 10. Mice receiving different treatments show similar changes in the systolic blood pressure (SP). Tgfbr1 iko mice were treated with Losartan (Los, n=10), Hydralazine (Hyd, n=12), or Propranolol (Pro, n=10). P=0.17, one-way NOV. Tgfbr1 iko -Pla. Tgfbr1 iko -Los 2.5 perk_d5 2.0 perk_d13 d5 perk4 2/44 β-actin (Relative to Pla) 2.0 1.0 P=NS 1.0 P=NS perk4 2/44 0.5 0.5 d13 β-actin Pla Los Pla Los Supplementary Figure 11. The abundance of perk in Tgfbr1 iko aortas is not impacted by treatment with losartan at the early stage. () Western blots of perk for Ts that were treated with placebo (Pla) or losartan (Los) at the indicated time points. () Quantification of the blots shown in panel. Data were expressed as fold-change relative to placebo-treated controls and analyzed using the unpaired t-test. 9
Tgfbr1 iko T1R ERK Tgfbr2 SMD Wall Degeneration Wall Homeostasis T Supplementary Figure 12. hypothetical model for the Tgfbr1 iko -driven aortic aneurysm development. Lines connect molecular and cellular events promoted by baseline TGFR1 signaling (shown in gray) or activated as a result of Tgfbr1 iko (shown in black). Terminal arrows indicate activation or enhancement while terminal straight lines denote inhibition. Solid lines indicate cell-autonomous regulation. Dash lines indicate pathways with details to be defined. 10