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, 110 (2), 188-99

Carfilzomib Reverses Pulmonary Arterial Hypertension

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Carfilzomib Reverses Pulmonary Arterial Hypertension

Xinhong Wang et al. Cardiovasc Res.

Abstract

Aims: Pulmonary arterial hypertension (PAH) remains a lethal disease with pronounced narrowing of pulmonary vessels due to abnormal cell growth. Agents that can reduce the pulmonary vascular thickness thus have therapeutic potential. The present study investigated the efficacy of carfilzomib (CFZ), a proteasome inhibitor and a cancer chemotherapeutic drug, on reversing PAH.

Methods and results: In two rat models of PAH, SU5416/hypoxia and SU5416/ovalbumin, CFZ effectively reversed pulmonary vascular remodelling with the promotion of apoptosis and autophagy. In human pulmonary artery smooth muscle cells, knocking down mediators of autophagy attenuated CFZ-induced cell death. The cell death role of autophagy was promoted by the participation of tumour protein p53-inducible nuclear protein 1. CFZ increased the protein ubiquitination, and siRNA knockdown of ubiquitin inhibited cell death, suggesting that CFZ-induced cell death is ubiquitin-dependent. Mass spectrometry demonstrated the ubiquitination of major vault protein and heat shock protein 90 in response to CFZ. The siRNA knockdown of these proteins enhanced CFZ-induced cell death, revealing that they are cell survival factors. CFZ reduced right-ventricular pressure and enhanced the efficacy of a vasodilator, sodium nitroprusside. While no indications of CFZ toxicity were observed in the right ventricle of PAH rats, apoptosis was promoted in the left ventricle. Apoptosis was prevented by dexrazoxane or by pifithrin-α without interfering with the efficacy of CFZ to reverse pulmonary vascular remodelling.

Conclusion: The addition of anti-tumour agents such as CFZ along with cardioprotectants to currently available vasodilators may be a promising way to improve PAH therapy.

Keywords: Apoptosis; Carfilzomib; Proteasome inhibitor; Pulmonary heart disease; Pulmonary hypertension.

Figures

Figure 1
Figure 1
CFZ reduces the wall thickness of remodelled pulmonary arteries (PAs) and promotes apoptosis without affecting normal PAs in SU5416/hypoxia model of PAH. (A) Schematics of CFZ treatment in a rat model of PAH induced by SU5416 and hypoxia. Rats were injected with SU5416 and exposed to hypoxia for 3 weeks, and then placed in normoxia for 5 weeks. Rats were then injected with 6 mg/kg body weight CFZ, twice per week for 2 weeks. The rats were sacrificed after 3 days of the last injection. (B) Representative H&E staining of small PAs (diameters ranging from 50 to 100 μm). Scale bars, 50 μm. Bar graphs represent means ± SEM of percent wall thickness. (C) Representative TUNEL assay results. Scale bars, 50 μm. The bar graph represents means ± SEM of percent TUNEL positive cells in the small PAs after subtracting the background staining that were found in control lungs. (D and E) PAs were surgically isolated from rats, homogenized, and subjected to western blotting for cleaved caspase-3 and Bcl-xL. Bar graphs represent means± SEM. *Values significantly different from each other at P< 0.05. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6).
Figure 2
Figure 2
CFZ induces autophagic cell death. (A and B) Pulmonary arteries (PAs) were surgically isolated from rats subjected to SU5416/hypoxia treatment with remodelled PA and from normal rats, homogenized, and subjected to western blotting analysis for LC3B-II and p62 expression. Bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6). (C–E) HPASMCs were transfected with control siRNA and LC3B, AMPK or TP53INP1 siRNA. After 48 h, transfection medium were changed to starvation medium for 4 h, and then HPASMCs were treated with CFZ (0.3 μM) for 20 h. Bar graphs show the cell number determined by counting on a haemocytometer (LC3B siRNA, n = 6; AMPK siRNA, n = 4; TP53INP1, n = 4). *Values significantly different from each other at P< 0.05. (F) HPASMCs were treated with CFZ (0.3 μM) for 20 h, and cell lysates were immunoprecipitated (IP) with TP53INP1 antibody, followed by immunoblotting with the LC3B antibody. The bar graph represents means ± SEM. *Values significantly different from each other at P< 0.05(n = 3).
Figure 3
Figure 3
CFZ induces protein ubiquitination. (A) Isolated pulmonary arteries from the SU5416/hypoxia model of PAH were homogenized, and subjected to western blotting to monitor protein ubiquitination using a ubiquitin (Ub) antibody. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6). (B) Protein ubiquitination was monitored in isolated pulmonary arteries from the SU5416/ovalbumin model of PAH. (PAH, n = 6; PAH + CFZ, n = 6). Bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05. (C) HPASMCs were treated with various doses of CFZ for 20 h. Cell lysates were analysed for protein ubiquitination by western blotting. (D) Cells were treated with CFZ (0.3 μM) for various durations. Bar graphs represent means ± SEM. *Values significantly different from untreated control at P< 0.05 (n = 6).
Figure 4
Figure 4
CFZ-induced cell killing is ubiquitin-dependent. HPASMCs were transfected with control siRNA and Ub siRNA. (A) Knocking down efficiency was monitored by western blotting. (B) After transfection for 48 h, the transfection medium was changed to the starvation medium. Four hours later, cells were treated with CFZ (0.3 μM) for 20 h. The cell number was determined by counting on a haemocytometer. (C and D) Cell lysates were subjected to immunoblotting to analyse the expression of p62 and Bcl-xL. (E) Cell lysates were immunoprecipitated (IP) with TP53INP1 antibody, followed by immunoblotting with the LC3B antibody. The bar graphs represent means ± SEM. *Values significantly different from each other at P< 0.05 (n = 4).
Figure 5
Figure 5
Identifications of proteins that are ubiquitinated by CFZ. (A) HPASMCs were treated with CFZ (0.3 μM) for 20 h, and cell lysates were immunoprecipitated (IP) with the Ub antibody. Bands indicated by arrows in Coomassie Blue-stained gels were consistently increased by CFZ treatment. The bar graph represents means ± SEM (n = 3). Mass spectrometry identified that these bands contain MVP and HSP90. Ubiquitination of MVP was confirmed in (B) CFZ-treated HPASMCs (n = 6), (C) isolated pulmonary arteries from SU5416/ovalbumin model (n = 4), and (D) isolated pulmonary arteries from SU5416/hypoxia model (n = 4) by immunoprecipitation with Ub antibody followed by immunoblotting with MVP. The ubiquitination of HSP90 were confirmed in (E) CFZ-treated HPASMCs (n = 6), (F) isolated pulmonary arteries from SU5416/ovalbumin model (n = 6), and (G) isolated pulmonary arteries from SU5416/hypoxia model (n = 4) by immunoprecipitation with Ub antibody followed by immunoblotting with HSP90 antibody. (H and I) HPASMCs were transfected with siRNA to knockdown MVP or HSP90. Efficiency of siRNA knockdown is shown in the western blotting data. Cells were treated with CFZ (0.3 μM) for 20 h, and the cell number was determined by counting on a haemocytometer. (MVP siRNA, n = 5; HSP90 siRNA, n = 6). *Values significantly different from each other at P< 0.05.
Figure 6
Figure 6
CFZ decreases RV pressure in PAH rats and increases the susceptibility of vasodilators to reduce RV pressure. PAH rats (SU5416/ovalbumin) were injected with CFZ (6 mg/kg body weight), twice a week. Three days after the last injection, rats were anaesthetized and mechanically ventilated. A Millar catheter was inserted into the RV apex to record RV pressure. After stabilization of haemodynamic recording for 10 min, sodium nitroprusside (SNP; 10, 20, or 50 μg/kg body weight) was slowly administered through the jugular vein in a 100 μL injection volume. (A) Representative homodynamic recording. (B) The bar graph represents means ± SEM of RV systolic pressure (DMSO, n = 6; CFZ, n = 5). (C) The bar graph represents means ± SEM of the heart rate. (D) The bar graph represents means ± SEM of Fulton index. *P< 0.05.
Figure 7
Figure 7
Dexrazoxane (DEX) and pifithrin-α (PFT-α) protects the left ventricle (LV) from CFZ-induced apoptosis without affecting the efficacy of CFZ in reversing PA remodelling. (A) In SU5416/hypoxia model of PAH, cleaved caspase-3 formation in the LV was analysed by western blotting. *Values significantly different from each other at P< 0.05. (Control, n = 6; Control + CFZ, n = 6; PAH, n = 5; PAH + CFZ, n = 6). (B) PAH rats (SU5416/hypoxia) were divided into four groups to determine the protective effects of DEX or PFTα. DEX (50 mg/kg) or PFT-α (2.2 mg/kg) was injected intraperitoneally along with 6 mg/kg body weight CFZ, twice a week for 2 weeks. Rats were then sacrificed 3 days after the last injection. LV tissues were homogenized, and subjected to western blotting for cleaved capase-3 formation. (C) Pulmonary arteries (PA) from these rats were surgically isolated, homogenized, and subjected to western blotting analysis for cleaved caspase-3. (D) Apoptosis in remodelled small PA induced by CFZ was not affected by DEX or PFT-α as shown in TUNEL staining. (E) The reduction of remodelled PA thickness induced by CFZ was not affected by DEX or PFT-α by analysing H&E staining. *Values in bar graphs denote significantly different from values from untreated PAH rats at P< 0.05. (PAH, n = 5; PAH + CFZ, n = 6; PAH + CFZ + DEX, n = 5; PAH + CFZ + PFT-α, n = 6).

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