naphthoquinones and Hypertension--Pulmonary

An emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments.. Pulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry.. We detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results.. In PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

Topics: Alternative Splicing; Animals; Antagomirs; Cattle; Cell Proliferation; Endothelium, Vascular; Fibroblasts; Glycolysis; Heterogeneous-Nuclear Ribonucleoproteins; Histone Deacetylase Inhibitors; Humans; Hypertension, Pulmonary; Interleukin-1beta; Macrophages; Mice; Mice, Inbred C57BL; MicroRNAs; Naphthoquinones; Polypyrimidine Tract-Binding Protein; Protein Isoforms; Pyruvate Kinase; RNA Interference

To test the hypothesis that chronic hypoxic pulmonary hypertension (CH-PH) is associated with increased survivin and decreased voltage-gated potassium (KV) channels expression in pulmonary arteries, rats were randomized as: normoxia (N); normoxia + YM155, survivin suppressor (NY); hypoxia (H); hypoxia + YM155 (HY). HY group had significantly reduced pulmonary arterial pressure, right ventricular weight and right ventricular hypertrophy compared with H group. Survivin mRNA and protein were detected in pulmonary arteries of rats with CH-PH, but not rats without CH-PH. YM155 downregulated survivin protein and mRNA. KV channel expression and activity were upregulated after YM155 treatment. Survivin may play a role in the pathogenesis of CH-PH.

Topics: Animals; Chronic Disease; Disease Models, Animal; Gene Expression Regulation; Hypertension, Pulmonary; Hypoxia; Imidazoles; Male; Microtubule-Associated Proteins; Muscle, Smooth, Vascular; Naphthoquinones; Patch-Clamp Techniques; Potassium Channels, Voltage-Gated; Pulmonary Wedge Pressure; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; RNA; Survivin

Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein Receptors, Type II; Calcium; Cardiotonic Agents; Cell Proliferation; Cells, Cultured; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; In Situ Nick-End Labeling; Interleukin-6; Male; Muscle, Smooth, Vascular; Naphthoquinones; NFATC Transcription Factors; Potassium Channels, Voltage-Gated; Proliferating Cell Nuclear Antigen; Rats; rho-Associated Kinases

We hypothesized that 5-lipoxygenase and cyclooxygenase products might be mediators of cardiopulmonary and systemic vascular effects induced by a 4 h continuous infusion of platelet-activating factor (PAF, 10 ng/kg/min) in anesthetized pigs. Indomethacin (cyclooxygenase inhibitor) potentiated and CGS 8515 (5-lipoxygenase inhibitor) attenuated PAF-induced increases in total peripheral resistance (TPR) from 2.5 to 4 h. However, the 5-lipoxygenase inhibitor failed to modify pulmonary vasoconstriction and hypertension caused by PAF. Except for a delay in onset (approximately 44 s) and rate of development of pulmonary hypertension during the initial 10 min of PAF infusion, the pulmonary hemodynamic changes were also not attenuated by indomethacin. On the other hand, at 4 h, the PAF-induced pulmonary hypertension and systemic vasoconstriction were completely or partially reversed, respectively, by WEB 2086 (PAF receptor antagonist). The PAF-induced increases in plasma thromboxane B2 (TXB2) were blocked by indomethacin but not by CGS 8515, and at 4 h the 5-lipoxygenase inhibitor potentiated the levels of TXB2 in pigs treated with PAF. The plasma concentrations of 6-keto-PGF1 alpha and leukotriene B4 (LTB4) were not modified by PAF or CGS 8515 + PAF. We conclude that PAF-induced increases in TPR (2.5-4 h) are potentiated by indomethacin and are dependent on 5-lipoxygenase products other than LTB4. Although the early pulmonary vascular response (< 10 min) to PAF is dependent on cyclooxygenase products, the sustained response (after 10 min) cannot be explained by either 5-lipoxygenase or cyclooxygenase products but may be mediated directly by PAF receptors.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Azepines; Blood; Calcimycin; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Hemodynamics; Hypertension, Pulmonary; In Vitro Techniques; Indomethacin; Infusions, Intra-Arterial; Leukotriene B4; Lipoxygenase Inhibitors; Naphthoquinones; ortho-Aminobenzoates; Platelet Activating Factor; Swine; Thromboxane B2; Triazoles