Ma, et al. Supplemental Data

Transcription

1 Ma, et al Supplemental Data Title: Calpain mediates pulmonary vascular remodeling in rodent models of pulmonary hypertension and its inhibition attenuates pathologic features of disease Authors: Wanli Ma, Weihong Han, Peter A. Greer, Rubin M. Tuder, Haroldo A. Toque, Kevin K. W. Wang, R. William Caldwell, and Yunchao Su Corresponding author: Yunchao Su, MD, Ph.D., Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Health Sciences University, th Street, Augusta, GA Tel: (76) Fax: (76)

2 mice KO mice normoxia hypoxia normoxia hypoxia α-actin Calpain-4 DAPI Overlay Supplemental Figure 1. Induction of calpain-4 knockout in the smooth muscle of pulmonary arterioles of mice with hypoxic pulmonary hypertension. 5 days after regimen of tamoxifen administration, mice and ER-Cre +/- Capn4 flox/flox mutant mice were exposed to room air (normoxia) or 1% oxygen (hypoxia) for 3 weeks. (A): Lung slides from ER-Cre +/- Capn4 flox/flox mutant and mice exposed to normoxia or hypoxia were double-stained for α-actin (red) and calpain-4 (green) and then counter stained with DAPI. Images are representative of 6 independent experiments. Magnification x4.

3 floxed CAPN4PZ 4.3 kb 3.2 kb mice Knockout mice B.8 Relative density of mice Knockout mice Supplemental Figure 2. Southern blot analysis of lung tissues after tamoxifen induction. To assess the efficiency of Cre-mediated deletion of the loxp-flanked Calpain-4 gene segment in ER-Cre +/- Capn4 flox/+ mice, Southern blot analysis was done with genomic DNA from lung tissues of and knockout mice as described previously by us (Genesis. 26;44(6):297-33). After PstI digestion, fragments corresponding to floxed CAPN4 PZ allele, and excised CAPN4 P genes were predicted at sizes of 3.2, and 4.3 kb, respectively. (A): Tamoxifen treatment caused the appearance of a 4.3-kb band and the weakening of the 3.2-kb band in the lungs of ER-Cre +/- Capn4 flox/+ mice. (B) is a bar graph depicting the changes in floxed CAPN4 PZ allele. Results are expressed as mean ± SE; n=6. P <.5 vs. mice.

4 C Active TGFβ1 content (ng/mg protein) E EGF content (ng/mg protein) Total TGFβ1 content (ng/mg protein) EGF active TGF β1 Total TGF β1 normoxia hypoxia KO KO+hypoxia normoxia hypoxia KO KO+hypoxia normoxia hypoxia KO KO+hypoxia B PDGF-BB content (ng/mg protein) D Active TGFβ1 content (pg/ml) PDGF-BB normoxia hypoxia KO KO+hypoxia active TGF β1 before normalized to total protein normoxia hypoxia KO KO+hypoxia Supplemental Figure 3. Alterations in the contents of EGF, PDGF-BB, active TGFβ1 and total TGFβ1 in the lungs of hypoxia-induced pulmonary hypertension. The contents of EGF (A), PDGF (B), active TGFβ1 (C and D), and total TGFβ1 (E) in the homogenates of lungs of ER-Cre+/- Capn4flox/flox mutant and mice exposed to normoxia or hypoxia for 3 weeks were assayed using ELISA. Results are expressed as mean ± SE; n=8 experiments. P <.5 vs. ; P<.5 vs. KO (knock-out mice) only.

5 α-actin Collagen-I overlay B α-actin SBDP overlay MCT 2 weeks MCT 2 weeks MCT 3 weeks MCT 3 weeks MCT 3 weeks + MCT 3 weeks + Supplemental Figure 4. Effects of calpain inhibitor on SBDP and collagen-1 in the smooth muscle of pulmonary arterioles of rats with MCT-induced pulmonary hypertension. Male Sprague-Dawley rats with age of 8 weeks were injected subcutaneously without or with MCT (6 mg/kg). After 2 weeks, rats and MCT-injected rats (MCT 2 weeks) were subjected to determination of pulmonary hypertension and pulmonary vascular remodeling. At same time (the beginning of third week), another groups of rats (MDL 2817) and MCT-injected rats (MCT 3 weeks+) started to receive calpain inhibitor (2 mg/kg, i.p.) once daily. A second group of MCT-injected rats received same volume of vehicle (MCT 3 weeks). Pulmonary hypertension and pulmonary vascular remodeling were accessed one week later (3 weeks after MCT injection). (A): Lung slides were double-stained for α-actin (red) and collagen-i (green). (B): Lung slides were double-stained for α-actin (red) and SBDP (green). Images are representative of 6 independent experiments. Magnification x4.

6 MDL +MDL small unit (calpain-4) calpain-1 calpain-2 calpastatin p-smad2/3 B Relative density (ratio to GAPDH) MDL +MDL total Smad2/3 collagen-i. small unit calpain-1 calpain-2 calpastatin C Relative density (Ratio to GAPDH) collagen-i MDL MDL GAPDH D Relative density (ratio to total Smad2/3) p-smad2/3 MDL +MDL Supplemental Figure 5. Protein contents of calpain-1, calpain-2, calpain-4, calpastatin, p-smad2/3, total Smad2/3 and collagen-i in the lungs of rats with MCT-induced pulmonary hypertension. Rats were treated as described in Online Figure 2. Then protein contents of calpain-1, calpain-2, calpain-4, calpastatin, p-smad2/3, total Smad2/3 and collagen-i in lung homogenates were analyzed using Western blot analysis. (A) is representative immuno-blot from 6 experiments. (B), (C) and (D) are bar graphs depicting the changes in calpain-1, calpain-2, calpain-4, calpastatin, p-smad2/3, total Smad2/3 and collagen-i quantified by scanning densitometry. Results are expressed as mean ± SE; n=6 experiments. P <.5 vs. ; P<.5 vs. group; P <.5 vs. MCT 2 weeks; P<.5 vs. MCT 3 weeks. MDL=MDL2617.

7 C Active TGFβ1 content (ng/mg protein) E EGF content (ng/mg protein) Total TGFβ1 content (ng/mg protein) EGF + active TGF β1 + Total TGF β1 B PDGF-BB content (ng/mg protein) D Active TGFβ1 content (pg/ml) PDGF-BB + Supplemental Figure 6. Alterations in the contents of EGF, PDGF, active TGFβ1 and total TGFβ1 in the lungs of rats with MCT-induced pulmonary hypertension. Rats were treated as described in Online Figure 2. The contents of EGF (A), PDGF-BB (B), active TGFβ1 (C and D) and total TGFβ1 (E) in the homogenates of lungs were assayed using ELISA. Results are expressed as mean ± SE; n=6 experiments. P <.5 vs. ; P<.5 vs. ; P <.5 vs. MCT 2 weeks; P<.5 vs. MCT 3 weeks. active TGF β1 before normalized to total protein

8 1 5 anti-tgfβ antibody (μg/ml) TGFβ1 (ng/ml) Collagen-I GAPDH Supplemental Figure 7. Neutralizing effect of anti-tgfβ antibody on collagen synthesis in PASMC. PASMC were incubated without or with active TGFβ1 (1. ng/ml) in the absence or presence of anti-tgfβ neutralizing antibody (1. μg/ml) for 24 h after which collagen content was measured using Western blot analysis. The images are representative immuno-blots from 3 experiments.

9 Positive, H 2 O 2 3 mm Relative cell number 95.8% M1 4.2% M2 Relative cell number 68.4% M1 31.6% M FITC-active caspase 3 FITC-active caspase 3, 2 μm PDGF-BB, 1 ng/ml Relative cell number 96.5% M1 3.5% M2 Relative cell number 95.1% M1 4.9% M FITC-active caspase 3 FITC-active caspase 3 Supplemental Figure 8. PDGF and does not cause apoptosis in PASMC. PASMC were incubated with PDGF-BB (1 ng/ml, 24 h), (2 μm, 24 h), or H 2 O 2 (3. mm, 24 h, positive ) after which active caspase 3 was assayed using a FITC-active caspase 3 apoptosis kit from BD Biosciences. Data are representative of 3 independent experiments. M1 = mean fluorescence intensity of normal cells. M2 = mean fluorescence intensity of apoptotic cells.

10 EGF + EGF PTEN PP2A-B56- α PP2A-B56- β Collagen-I GAPDH Supplemental Figure 9. EGF does not affect the protein contents of PTEN and PP2A subunits B56-α and B56-β in PASMC. PASMC were incubated with EGF (1 ng/ml) in the presence and absence of (2 μm) for 24 h after which the protein contents of PTEN, PP2A subunits B56-α and B56-β, and intracellular collagen-i were measured using Western blot analysis. The images are representative immuno-blots from 3 experiments.

11 Relaxation (%) Knockout C % of KCl-induced contraction Knockout E Relaxation (%) Knockout Log [Acetylcholine]: M Log [Phenylephrine]: M Log [SNP]: M B mice Knockout mice D mice Knockout mice Supplemental Figure 1. (A) and (B): knockout of calpain-4 decreases endothelium-dependent relaxation. (C) and (D): knockout of calpain-4 decreases smooth muscle contraction induced by phenylephrine. (E): knockout of calpain- 4 does not affect NO donor SNP-induced relaxation. Results are expressed as mean ± SE; n=4-5 experiments. P <.5 vs. mice.

12 Cells passed membrance (cells/mm 2 ) Vehicle PDGF-BB B PDGF-BB + PDGF-BB CTGF Alk5 GAPDH Supplemental Figure 11. Effects of PDGF-BB and on cell migration and protein contents of CTGF and Alk5 in PASMC. (A) Migration of PASMC was measured using Boyden chamber in the presence or absence of PDGF-BB (1 ng/ml) or (2 μm). Results are expressed as mean ± SE; n=3 experiments. P<.5 vs. ; P<.5 vs. in Vehicle group. (B) PASMC were incubated with PDGF-BB (1 ng/ml) in the presence or absence of (2 μm) for 24 h after which the protein contents of CTGF and Alk5 were measured using Western blot analysis. The images are representative immuno-blots from 3 experiments.

13 Hypoxia and MCT EGF/PDGF EGF/PDGF receptor Cell plasma membrane SIS3 ( ) SB ( ) Calpain activation Smad4 Smad2/3 TGFβ1 TGFβ1 Nucleus Calpain ( ) sirna Knockout Transcription Collagen and Proliferation latent TGFβ1 Pulmonary vascular remodeling Supplemental Figure 12. A schematic pathway illustrating the role of calpain in pulmonary vascular remodeling of pulmonary hypertension. PDGF and EGF released in hypoxia- or MCT-induced pulmonary hypertension cause activation of calpain which translocates to Golgi and induces activation of TGFβ. TGFβ/p-Smad signaling leads to collagen synthesis and smooth muscle proliferation which contributes to pulmonary vascular remodeling and pulmonary hypertension.