Inhibition of Hsp110-STAT3 interaction in endothelial cells alleviates vascular remodeling in hypoxic pulmonary arterial Hypertension model

Abstract

Background: Pulmonary arterial hypertension (PAH) is a progressive and devastating disease characterized by pulmonary vascular remodeling which is associated with the malignant phenotypes of pulmonary vascular cells. Recently, the effects of heat shock protein 110 (Hsp110) in human arterial smooth muscle cells were reported. However, the underlying roles and mechanisms of Hsp110 in human pulmonary arterial endothelial cells (HPAECs) that was disordered firstly at the early stage of PAH remain unknown.

Methods: In this research, the expression of Hsp110 in PAH human patients and rat models was investigated, and the Hsp110 localization was determined both in vivo and in vitro. The roles and mechanism of elevated Hsp110 in excessive cell proliferation and migration of HPAECs were assessed respectively exposed to hypoxia. Small molecule inhibitors targeting Hsp110-STAT3 interaction were screened via fluorescence polarization, anti-aggregation and western blot assays. Moreover, the effects of compound 6 on HPAECs abnormal phenotypes in vitro and pulmonary vascular remodeling of hypoxia-indued PAH rats in vivo by interrupting Hsp110-STAT3 interaction were evaluated.

Results: Our studies demonstrated that Hsp110 expression was increased in the serum of patients with PAH, as well as in the lungs and pulmonary arteries of PAH rats, when compared to their respective healthy subjects. Moreover, Hsp110 levels were significantly elevated in HPAECs under hypoxia and mediated its aberrant phenotypes. Furthermore, boosted Hsp110-STAT3 interaction resulted in abnormal proliferation and migration via elevating p-STAT3 and c-Myc in HPAECs. Notably, we successfully identified compound 6 as potent Hsp110-STAT3 interaction inhibitor, which effectively inhibited HPAECs proliferation and migration, and significantly ameliorated right heart hypertrophy and vascular remodeling of rats with PAH.

Conclusions: Our studies suggest that elevated Hsp110 plays a vital role in HPAECs and inhibition of the Hsp110-STAT3 interaction is a novel strategy for improving vascular remodeling. In addition, compound 6 could serve as a promising lead compound for developing first-in-class drugs against PAH.

Keywords: Heat shock protein 110; Protein-protein interaction; Pulmonary arterial Hypertension; Pulmonary arterial endothelial cell; Vascular remodeling.

Fig. 1

High expression and localization of Hsp110 in PAH. (A) Serum levels of Hsp110 in healthy subjects (n = 10), and PAH patients (n = 10). (B) Western blots of HSPs including Hsp110, Hsp90 and Hsp70 in lung tissues from PAH rat models (n = 6 each). (C) Statistical analysis of HSPs levels in lung tissues. (D) Western blots of HSPs in pulmonary arteries from hypoxia-induced PAH rat model (n = 6 each). (E) Statistical analysis of HSPs levels in pulmonary arteries. (F) Representative immunostaining micrographs of lung sections in hypoxia-induced PAH rats. Staining was performed for Hsp110. Scale bar: 50 μm (left), Scale bar: 20 μm (right). (G) Representative immunofluorescence staining of lung tissues for Hsp110 (pink), α-SMA (α-smooth muscle actin; green) and CD31 (red) from normoxia or hypoxia treated rats. Nuclei were counterstained with DAPI (blue). Scale bars: 25 μm. Results are expressed as the mean ± standard error; *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group

Fig. 2

Expression of Hsp110 in HPAECs under hypoxia and its effect on hypoxia-induced proliferation and migration. (A) Western blots of Hsp110 in pulmonary artery cell lines under hypoxia, including HPAECs, HLF-1 and HPASMCs. Con: control, Hyp: Hypoxia (n = 3 each). (B) Statistical analysis of Hsp110 expression in pulmonary artery cell lines. (C) The knockdown efficiency of siRNA against Hsp110 in PAECs. (D) Images of proliferating nuclei labeled with 5-ethynyl-2’-deoxyuridine (EdU) in HPAECs under hypoxia with knockdown of Hsp110. Scale bars: 200 μm. (E) Statistical analysis of EdU assay. (F) Representative migration images from the trans-well assay in HPAECs under hypoxia with knockdown of Hsp110. Scale bars: 1000 μm. (G) Statistical analysis of trans-well assay. Results are expressed as the mean ± standard error, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group, ^###P < 0.001 versus the hypoxia group

Fig. 3

The regulatory mechanism of Hsp110 overexpression and knockdown on downstream STAT3 signaling pathway. (A) Western blots of Hsp110, p-STAT3, STAT3 and c-Myc in HPAECs after Hsp110 overexpression. (B) Western blots of Hsp110, p-STAT3, STAT3 and c-Myc in HPAECs under hypoxia with knockdown of Hsp110. (C) Statistical analysis of Hsp110 overexpression. (D) Statistical analysis of p-STAT3/STAT3 ratio after Hsp110 overexpression. (E) Statistical analysis of c-Myc levels after Hsp110 overexpression. (F) Statistical analysis of Hsp110 expression under hypoxia with knockdown of Hsp110. (G) Statistical analysis of p-STAT3/STAT3 ratio under hypoxia with knockdown of Hsp110. (H) Statistical analysis of c-Myc levels under hypoxia with knockdown of Hsp110. Results are expressed as the mean ± standard error, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 versus negative control (NC) or the control group, ^###P < 0.001 versus the hypoxia group

Fig. 4

Compound screening targeting Hsp110 molecular chaperone function and Hsp110-STAT3 PPI. (A) The chemical structures of pyrazolo[3,4-b] pyridine derivatives evaluated in this study. (B) Compound screening results targeting the ratio of p-STAT3/STAT3 at 10 µM. (C) Compound screening results targeting the expression of c-Myc at 10 µM. (D) Compound screening results for Hsp110 anti-aggregation activity at 100 µM. (E) The time dependent effect of compound 6 on Hsp110 anti-aggregation activity at 100 µM. (F) SPR assay demonstrating the stable fit of the interaction between compound 6 and Hsp110 (Kd = 4.04 µM). (G) Compound 6 treatment increases the protease susceptibility of the Hsp110 in cell lysates as determined by the DARTS assay. (H) Statistical analysis of Hsp110 levels in DARTS assay. (I-J) Co-IP results indicating compound 6 inhibits the Hsp110-STAT3 PPI both in HCT116 cells and HPAECs. Results are expressed as the mean ± standard error, n = 3; *P < 0.05, **P < 0.01 versus the compound 6 treatment group

Fig. 5

Compound 6 inhibits the abnormal proliferation and migration via disrupting the interaction between Hsp110 and STAT3 under hypoxia. (A) Cell viability was assessed by the CCK8 method after treatment with compound 6 or KNK437 under hypoxia for 48 hours. (B) The effect of different concentrations of compound 6 on the migration of HPAECs at different time points were examined by scratch assays. (C) Images of proliferating nuclei labeled with 5-ethynyl-2’-deoxyuridine (EdU) in HPAECs treatment with compound 6 under hypoxia. Scale bars: 200 μm. (D) Statistical analysis of EdU assay. (E) Representative migration images from the trans-well assay in HPAECs treated with compound 6 under hypoxia. Scale bars: 200 μm. (F) Statistical analysis of trans-well assay. (G) Western blots of Hsp110, Hsp90, Hsp70, p-STAT3, STAT3, c-Myc in HPAECs treatment with compound 6 or KNK437 under hypoxia. (H-L): Statistical analysis of western blotting results. Results are expressed as the mean ± standard error, n = 3; **P < 0.01, ***P < 0.001 versus the control group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the hypoxia group

Fig. 6

Compound 6 prevents the development of hypoxia-induced PAH. (A) mPAP waveform. (B) Statistical analysis of mPAP. (C) Statistical analysis of body weight (D) The representative photographs of hematoxylin and eosin (H&E) staining lung sections from each group. Scale bar: 100 μm. (E) RV/(LV + S) statistic. (F) RV/tibia length statistic. (G) Statistical analysis of PAMT%. Data are expressed as mean ± standard error, n = 6. ***P < 0.001 versus the control group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the hypoxia group. mPAP: mean pulmonary artery pressure; RV/(LV + S): right ventricle/(left ventricle + interventricular septum); PAMT: pulmonary artery medial thickness

Fig. 7

Compound 6 inhibited Hsp110-STAT3 PPI in hypoxia-induced PAH rats. (A): Western blots of Hsp110, p-STAT3, STAT3, c-Myc, PCNA in pulmonary arteries of PAH rats after compound 6 or KNK437 treatment. (B) Statistical analysis of Hsp110 expression in rat pulmonary arteries. (C) Statistical analysis of p-STAT3/STAT3 ratio in rat pulmonary arteries. (D) Statistical analysis of c-Myc levels in rat pulmonary arteries. (E) Statistical analysis of PCNA levels in rat pulmonary arteries. Data are expressed as mean ± standard error, n = 6. ***P < 0.001 versus the control group, ^##P < 0.01, ^###P < 0.001 versus the hypoxia group

Fig. 8

The molecular mechanism of Hsp110 in endothelial dysfunction and the mode of action of compound 6 in treating PAH. The abnormal up-regulation of Hsp110 induced by hypoxia plays an important role in the pathogenesis of pulmonary vascular remodeling and PAH development. In HPAECs, compound 6 interferes with the interaction between Hsp110 and STAT3 through direct binding to Hsp110, decreasing the protein levels of p-STAT3 and c-Myc, thereby inhibiting the abnormal proliferation and migration of endothelial cells and affecting endothelial function, ultimately improving pulmonary vascular remodeling