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Akt-Dependent Glycolysis-Driven Lipogenesis Supports Proliferation and Survival of Human Pulmonary Arterial Smooth Muscle Cells in Pulmonary Hypertension.

ABSTRACT: Hyper-proliferation of pulmonary arterial vascular smooth muscle cells (PAVSMC) is an important pathological component of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Lipogenesis is linked to numerous proliferative diseases, but its role in PAVSMC proliferation in PAH remains to be elucidated. We found that early-passage human PAH PAVSMC had significant up-regulation of key fatty acids synthesis enzymes ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN), and increased unstimulated proliferation compared to control human PAVSMC. Treatment with an allosteric ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) significantly decreased proliferation and induced apoptosis of human PAH PAVSMC. Intracellular lipid content and proliferation of PAH PAVSMC were not reduced by incubation in lipid-depleted media but suppressed by a non-metabolizable analog of glucose 2-Deoxy-D-glucose (2-DG) and partially restored by addition of pyruvate. Protein kinase Akt was upregulated in human PAH PAVSMC in a sirtuin 7 (SIRT7)- and c-Jun N-terminal kinase (JNK)-dependent manner. Pharmacological inhibition of Akt down-regulated ACLY and ACC, significantly reduced intracellular lipid content, inhibited proliferation and induced apoptosis of human PAH PAVSMC. Taken together, these data demonstrate that human PAH PAVSMC have up-regulated lipogenesis, which is supported in an Akt- and glycolysis-dependent manner and is required for increased proliferation and survival. Our data suggest that there is a mechanistic link between glycolysis, lipogenesis, and the proliferation of human PAH PAVSMC and call for further studies to determine the potential attractiveness of a SIRT7/JNK-Akt-lipogenesis axis as a target pathway to inhibit PAVSMC hyper-proliferation in PAH.

SUBMITTER: Jiang L

PROVIDER: S-EPMC9274086 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RationalePulmonary arterial hypertension (PAH) is a rare progressive pulmonary vascular disorder associated with vascular remodeling and right heart failure. Vascular remodeling involves numerous signaling cascades governing pulmonary arterial smooth muscle cell (PASMC) proliferation, migration and differentiation. Glycogen synthase kinase 3beta (GSK3ß) is a serine/threonine kinase and can act as a downstream regulatory switch for numerous signaling pathways. Hence, we hypothesized that GSK3ß plays a crucial role in pulmonary vascular remodeling.MethodsAll experiments were done with lung tissue or isolated PASMCs in a well-established monocrotaline (MCT)-induced PAH rat model. The mRNA expression of Wnt ligands (Wnt1, Wnt3a, Wnt5a), upstream Wnt signaling regulator genes (Frizzled Receptors 1, 2 and secreted Frizzled related protein sFRP-1) and canonical Wnt intracellular effectors (GSK3ß, Axin1) were assessed by real-time polymerase chain reaction and protein levels of GSK3ß, phospho-GSK3ß (ser 9) by western blotting and localization by immunohistochemistry. The role of GSK3ß in PASMCs proliferation was assessed by overexpression of wild-type GSK3ß (WT) and constitutively active GSK3ß S9A by [(3)H]-thymidine incorporation assay.ResultsIncreased levels of total and phosphorylated GSK3ß (inhibitory phosphorylation) were observed in lungs and PASMCs isolated from MCT-induced PAH rats compared to controls. Further, stimulation of MCT-PASMCs with growth factors induced GSK3ß inactivation. Most importantly, treatment with the PDGFR inhibitor, Imatinib, attenuated PDGF-BB and FCS induced GSK3ß phosphorylation. Increased expression of GSK3ß observed in lungs and PASMC isolated from MCT-induced PAH rats was confirmed to be clinically relevant as the same observation was identified in human iPAH lung explants. Overexpression of GSK3ß significantly increased MCT-PASMCs proliferation by regulating ERK phosphorylation. Constitutive activation of GSK3ß (GSK3ß S9A, 9th serine replaced to alanine) inhibited MCT-PASMCs proliferation by decreasing ERK phosphorylation.ConclusionThis study supports a central role for GSK3ß in vascular remodeling processes and suggests a novel therapeutic opportunity for the treatment of PAH.

Project description:Background: We aimed to evaluate the expression of cysteine rich 61 (Cyr61) in patients with pulmonary arterial hypertension (PAH) as well as monocrotaline (MCT) induced PAH rat, and further investigate the effects and potential mechanisms of Cyr61 on the proliferation of pulmonary arterial smooth muscle cells (PASMCs). Methods and Results: Plasma samples were collected from 20 patients with idiopathic PAH, 20 connective tissue disease (CTD) associated PAH, 29 age-, gender- and disease matched CTD without PAH patients, and 28 healthy controls. ELISA was used to detect the level of Cyr61 in plasma. MCT-induced PAH (MCT-PAH) rat model was established by a single subcutaneous injection of MCT (60mg·kg-1). Lung tissues and pulmonary arteries of rats were collected, while the PASMCs were dissected and cultivated for in vitro experiments. Expression of Cyr61 in the lung tissues, pulmonary arteries and PASMCs were tested by immunohistochemical staining, western blot and quantitative real-time polymerase chain reaction. PASMCs from PAH rats were stimulated by exogenous recombinant Cyr61 protein and knocked down by small interfering RNA. Cell Counting Kit-8 assay was used to identify cell proliferation and the expression of p-AKT and AKT were analysed by western blot. The results showed plasma level of Cyr61 in PAH patients, especially CTD-PAH patients, were significant higher than that of CTD without PAH patients and healthy controls. Compared with wild rats, Cyr61 was overexpressed in the lung tissue, pulmonary arterial and PASMCs in PAH rats. Exogenous recombinant Cyr61 protein promoted the proliferation of PASMCs in a dose-dependent manner. While the expression of Cyr61 in PASMCs was inhibited by specific siRNA, cell proliferation was restrained and the expression of p-AKT declined. Conclusion: Plasma Cyr61 concentration in PAH patients was highly increased. Cyr61 could promote PASMCs proliferation via AKT pathway, indicating that Cyr61 may play a role in the pathogenesis of PAH.

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Akt-Dependent Glycolysis-Driven Lipogenesis Supports Proliferation and Survival of Human Pulmonary Arterial Smooth Muscle Cells in Pulmonary Hypertension.

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