SUPPLEMENTAL MATERIAL       Mechanism of the susceptibility of remodeled pulmonary vessels to drug-induced cell killing  

   

Yasmine F. Ibrahim, M.D.,§ Chi-Ming Wong, Ph.D.,§ Ludmila Pavlickova, M.D., Lingling Liu, M.D., Lobsang Trasar, M.D., Geetanjali Bansal, Ph.D.

and Yuichiro J. Suzuki, Ph.D.*  

Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, DC 20057 USA

  §These authors contributed equally to this project.   *To whom correspondence should be addressed:

Prof. Yuichiro J. Suzuki Department of Pharmacology and Physiology Georgetown University Medical Center 3900 Reservoir Road NW Washington, DC 20057 USA

  TEL: (202) 687-8090 FAX: (202) 687-8825 e-mail: ys82@georgetown.edu

 

hypoxia+saline

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Supplemental Fig. S1: DNR induces apoptosis in remodeled pulmonary arteries. Rats were exposed to hypoxia for two weeks, and then injected with DNR. Rats were then placed back in hypoxia for three days. (A) The expression levels of cleaved caspase-3 were monitored by IHC in the fixed lung sections. Brown stains in IHC indicate apoptotic cells in pulmonary arterial walls. (B) Western blotting of cleaved caspase-3 and β-actin (as a control) in isolated pulmonary arteries. The bar graph represents means ± SEM (n = 3). * denotes the value significantly different from -DNR control at P<0.05.

A -DNR +DNR

lumen lumen

Supplemental Fig. S1

B

-DNR +DNR

Supplemental Fig. S2: DNR downregulates Bcl-xL and Bcl-2 expression in remodeled pulmonary arteries. Rats were exposed to hypoxia for two weeks, and then injected with DNR. Rats were then placed back in hypoxia. Three days after the injection, lungs were harvested. Tissues were immersed in buffered 10% paraformaldehyde and embedded in paraffin for IHC analysis of (A) Bcl-xL and (B) Bcl-2.

Supplemental Fig. S2

control hypoxic PH

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Supplemental Fig. S3: MG-132 upregulates annexin A1. PASMCs were treated with MG-132 (10 µM) for 1 h.  Cell lysates were prepared, and annexin A1 protein levels were monitored by western blotting. The bar graph represents means ± SEM of % control of the ratio of annexin A1 to β-actin protein expression. The symbol (*) denotes the value significantly different from the untreated control value at P<0.05 (n = 10).

Supplemental Fig. S3

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Supplemental Fig. S4: Mediators of pulmonary hypertension downregulate annexin A1. (A) PASMCs were pre-treated with deferoxamine (DFO) and then treated with endothein-1 (ET-1) for 30 min. Cell lysates were prepared and annexin A1 protein levels were monitored by western blotting. The bar graph represents means ± SEM of the ratio of annexin A1 to β-actin protein expression. The symbol (*) denotes the value significantly different from the untreated control value and the symbol (a) denotes the value significantly different from the ET-1 value at P < 0.05 (n = 7). (B) Rats were injected with DFO and subjected to chronic hypoxia at 10% O2 to promote pulmonary hypertension. Pulmonary arteries were isolated and homogenized. Annexin A1 protein levels were monitored by western blotting. The bar graph represents means ± SEM. The symbol (*) denotes the value significantly different from the normoxia control value and the symbol (a) denotes values significantly different from the hypoxia value at P < 0.05.

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Supplemental Fig. S4

Supplemental Fig. S5: DNR downregulates p62 expression in remodeled pulmonary arteries. Rats were exposed to hypoxia for two weeks, and then injected with DNR. Rats were then placed back in hypoxia. Three days after the injection, lungs were harvested. Tissues were immersed in buffered 10% paraformaldehyde and embedded in paraffin for IHC analysis of p62 in pulmonary arteries/arterioles (30 - 60 µm diameter).

Supplemental Fig. S5

control hypoxic PH D

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Supplemental Fig. S6

Differentiated HPASMCs

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Proliferating HPASMCs

-DNR

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Supplemental Fig. S6: Effects of DNR on differentiated and proliferating human PASMCs. [A] Differentiated and [B] proliferating human PASMCs were treated with DNR for 22 h and observed under a microscope. Representative images are shown.

 

Supplemental Fig. S7

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Supplemental Fig. S7: siRNA knockdown of LC3B. Cultured human pulmonary artery SMCs were treated with LC3B siRNA or control scrambled siRNA for 2 days. Cell lysates were prepared and LC3B protein levels were monitored by western blotting. G3PDH levels were also monitored as control. The bar graph represents means ± SEM of the ratio of LC3B to G3PDH expressed in arbitrary unit (a.u.). The symbol * denotes that values are significantly different from control siRNA values at P < 0.05 (n = 3).

*  

Supplemental Fig. S8: DNR upregulates parkin expression in normal pulmonary arteries. Rats were exposed to hypoxia for two weeks, and then injected with DNR. Rats were then placed back in hypoxia. Three days after the injection, lungs were harvested. Tissues were immersed in buffered 10% paraformaldehyde and embedded in paraffin for IHC analysis of parkin in pulmonary arteries/arterioles (30 - 60 µm diameter).

Supplemental Fig. S8

control hypoxic PH D

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Supplemental Fig. S9

control siRNA

parkin siRNA

β-actin

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control siRNA

parkin siRNA

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Supplemental Fig. S9: siRNA knockdown of parkin. Cultured human pulmonary artery SMCs were treated with parkin siRNA or control scrambled siRNA for 2 days. Cell lysates were prepared and parkin protein levels were monitored by western blotting. Levels of β-actin expression were also monitored as control. The bar graph represents means ± SEM of the ratio of parkin to β-actin expressed in arbitrary unit (a.u.). The symbol * denotes that values are significantly different from control siRNA values at P < 0.05 (n = 3).

Gata4 �

inactive

active

CBF/ NF-Y �

Bcl-2/Bcl-xL �

Antitumor drugs

CCAAT Box�

Annexin A1

Supplemental Fig. S10: Proposed mechanism for the drug-induced apoptosis of PASMCs via the regulation of GATA4 gene transcription. In remodeled pulmonary vessels with proliferating PASMCs, antitumor drugs upregulate annexin A1 expression. Annexin A1 binds to CBF/NF-Y and inhibits this transcription factor to activate GATA4 which serves as a cell survival factor via anti-apoptotic Bcl-xL and Bcl-2.

CBF/ NF-Y �

Annexin A1

Supplemental Fig. S10