Combination of Dichloroacetate and Atorvastatin Regulates Excessive Proliferation and Oxidative Stress in Pulmonary Arterial Hypertension Development via p38 Signaling.

Pulmonary arterial hypertension (PAH) is a lethal disease generally characterized by pulmonary artery remodeling. Mitochondrial metabolic disorders have been implicated as a critical regulator of excessively proliferative- and apoptosis-resistant phenotypes in pulmonary artery smooth muscle cells (PASMCs). Dichloroacetate (DCA) is an emerging drug that targets aerobic glycolysis in tumor cells. Atorvastatin (ATO) is widely used for hyperlipemia in various cardiovascular diseases. Considering that DCA and ATO regulate glucose and lipid metabolism, respectively, we hypothesized that the combination of DCA and ATO could be a potential treatment for PAH. A notable decrease in the right ventricular systolic pressure accompanied by reduced right heart hypertrophy was observed in the DCA/ATO combination treatment group compared with the monocrotaline treatment group. The DCA/ATO combination treatment alleviated vascular remodeling, thereby suppressing excessive PASMC proliferation and macrophage infiltration. In vitro, both DCA and ATO alone reduced PASMC viability by upregulating oxidative stress and lowering mitochondrial membrane potential. Surprisingly, when combined, DCA/ATO was able to decrease the levels of reactive oxygen species and cell apoptosis without compromising PASMC proliferation. Furthermore, suppression of the p38 pathway through the specific inhibitor SB203580 attenuated cell death and oxidative stress at a level consistent with that of DCA/ATO combination treatment. These observations suggested a complementary effect of DCA and ATO on rescuing PASMCs from a PAH phenotype through p38 activation via the regulation of mitochondrial-related cell death and oxidative stress. DCA in combination with ATO may represent a novel therapeutic strategy for PAH treatment.

Galectin-3 Mediates Endothelial-to-Mesenchymal Transition in Pulmonary Arterial Hypertension.

Galectin-3 (Gal-3) is highly expressed in fibrotic tissue related to diverse etiologies. endothelial-to-mesenchymal transition (EndoMT), A less well studied phenomenon serves as a critical process in pulmonary vascular remodeling associated with the development of pulmonary arterial hypertension (PAH). EndoMT is hypothesized to contribute to the over-proliferation of αSMA positive cells. We aim to investigate the potential role of Gal-3 in regulating EndoMT in PAH. We observed an upregulation in both Gal-3 and αSMA expression in the monocrotaline (MCT) and Hypoxia PAH model, accompanied with intimal thickening. For more profound vascular remodeling and endothelial layer lesion in former model, we employed Gal-3 knockdown and overexpression lentivirus methodology to the MCT rats to determine the mechanisms underlying abnormal endothelial cell transition in PAH. PAH was evaluated according to right ventricular systolic pressure, right heart hypertrophy and pulmonary artery remodeling. A reduction in Gal-3 was protective against the development of PAH, while Gal-3 upregulation aggravated pulmonary vascular occlusion. In addition, Gal-3 deficiency suppressed pulmonary vascular cell proliferation and macrophage infiltration. Finally, we revealed that in endothelial cells treated with tumor necrosis factor α and hypoxia (representing an in vitro model of PAH), inhibition of Gal-3 by siRNA was able to abolish the associated upregulation of αSMA. These observations suggesting Gal-3 serves as a critical mediator in PAH by regulating EndoMT. Inhibition of Gal-3 may represent a novel therapeutic target for PAH treatment.

microRNA-423-3p exosomes derived from cardiac fibroblasts mediates the cardioprotective effects of ischaemic post-conditioning.

AIMS: A recent study reported the cardioprotective effects mediated by cardiac fibroblasts (CFs) during acute phase of ischaemia-reperfusion injury (IRI). Little is known about whether exosomes/microvesicles mediate this beneficial effect and whether ischaemia post-conditioning (Postcon) can regulate this process. Here, we aimed to investigate the cardioprotective effect of CFs-exosomes/microvesicles and whether Postcon can regulate this effect. METHODS AND RESULTS: By using transwells co-culture system, we found that hypoxia-reoxygenation (H/R) significantly increased the exosomes/microvesicles secretion of CFs and CFs protected H9C2 cells against H/R injury and Postcon could amplify these effects. Inhibition of CFs exosomes/microvesicles secretion led to a significant abrogation on the amplified protective effect of H/R-Postcon. We further demonstrated that Postcon enhanced the cardioprotective effect of CFs-exosomes/microvesicles both in vitro and in vivo. To detect the underlying mechanism, exosomes/microvesicles microRNAs were analysed by RNA sequencing and quantitative polymerase chain reaction, our results revealed that miR-423-3p expression was selectively enhanced by Postcon in CFs exosomes/microvesicles. By co-culture H9C2 cells with CFs-exosomes/microvesicles enriching with miR-423-3p, we demonstrated that H/R-Postcon exerted cardioprotective effects by upregulation of miR-423-3p in CFs-exosomes/microvesicles. RNA-fluorescence in situ hybridization and qPCR demonstrated that the decreasing of miR-423-3p is closely related to IRI, by inhibited miR-423-3p expression with its antagomir in vivo, we demonstrated that miR-423-3p plays an essential mediate role in I/R-Postcon-induced cardioprotection against I/R in vivo, Postcon may exert cardioprotective effect by upregulation of miR-423-3p in CFs exosomes/microvesicles. Gain- and loss-of-function approaches suggested that rescuing the down-regulated miR-423-3p might be a potential strategy to protect the cardiomyocytes against H/R. Using computational predictions tools and luciferase reporter assay, we demonstrated that miR-423-3p regulates the expression of Ras-related protein Rap-2c (RAP2C) in H9C2 cells, and knockdown of RAP2C by siRNA obviously increased cell viability and reduced apoptosis in H9C2 cells under H/R. CONCLUSIONS: In conclusion, we demonstrated, for the first time, that CFs participate in cardioprotective effects via an exosomes/microvesicles pathway during the acute phase of IRI and Postcon can enhance this effect by upregulating the expression of CFs exosomes/microvesicles miR-423-3p, which targets the downstream effector RAP2C.

Galectin-3 mediates pulmonary vascular remodeling in hypoxia-induced pulmonary arterial hypertension.

Pulmonary vascular adventitia serves as a key regulator of pulmonary vascular remodeling in the pathogenesis of pulmonary arterial hypertension (PAH). Excessive proliferation and differentiation of pulmonary adventitial fibroblasts (PAFs) are proven to be crucial in the pathogenesis of PAH. Galectin-3 (Gal-3) is known as a key fibroblasts activating factor which is involved in the fibrogenesis of several diseases, such as pulmonary fibrosis, vascular fibrosis, and heart failure. Therefore, we seek to investigate the potential role of Gal-3 in regulating PAF cells in the pathogenesis of PAH. Gal-3 plasma concentration was significantly higher in PAH patients. Gal-3 was upregulated in pulmonary artery adventitia of hypoxia-induced PAH rats. Inhibition of Gal-3 with N-Acetyl-D-lactosamine (N-Lac) ameliorated PAH and pulmonary vascular remodeling. Gal-3 can stimulate the proliferation, differentiation, and collagen synthesis of PAFs, which was reversed by N-Lac. Transforming growth factor ß1 increased Gal-3 expression in PAFs, whereas N-Lac significantly suppressed transforming growth factor ß1-induced proliferation, differentiation, and collagen synthesis of PAFs. Gal-3 serves as a critical regulator in the pathogenesis of PAH by regulating the proliferation, differentiation, and extracellular matrix deposition synthesis of PAFs. Inhibition of Gal-3 may represent a novel therapeutic target for PAH treatment.

Galectin-3 mediates the pulmonary arterial hypertension-induced right ventricular remodeling through interacting with NADPH oxidase 4.

Pulmonary arterial hypertension (PAH) is a progressive disorder that affects both pulmonary vasculature and the heart. The response of the right ventricle (RV) to the increased afterload is an important determinant of the PAH final outcome. Galectin-3 (Gal-3), a novel biomarker in left cardiac remodeling, takes part in multiple pathophysiological processes including the inflammation, fibrosis, immunity, and oxidative stress. The levels of Gal-3 are elevated in PAH patients, although the exact mechanisms underlie the PAH-induced right ventricular structural changes remain unclear. Our results showed that the serum Gal-3 and NADPH oxidase 4 (Nox4) levels were significantly elevated and correlated in 26 human PAH patients when compared with 14 age- and sex-matched healthy controls. In the monocrotaline-induced PAH rat models of right ventricular hypertrophy and fibrosis, the Gal-3 and Nox4 expressions were both significantly upregulated compared with the controls. Moreover, the Gal-3 positive areas were co-localized with the collagen III-specific staining and the Gal-3 and Nox4 were partly co-localized in the intercellular area. The exogenous Gal-3 recombinant protein stimulated the proliferation, differentiation, collagen deposition, and Nox4 expression of cardiac fibroblasts. These simulations were blocked by the Gal-3 knockdown. The profibrotic effects of transforming growth factor-ß1 (TGF-ß1) on the cardiac fibroblasts were partially mediated by the Gal-3. Subsequently, our results showed that Gal-3 mediated the TGF-ß1-induced cardiac fibrotic process through interacting with the Nox4 and Nox4-derived oxidative stress. Therefore, Gal-3 plays an important role in the PAH-induced right ventricular remodeling through interacting with the Nox4 and Nox4-derived oxidative stress. Gal-3 may become a RV-specific diagnostic and therapeutic target for clinics.

Exosomes Derived from Human Pulmonary Artery Endothelial Cells Shift the Balance between Proliferation and Apoptosis of Smooth Muscle Cells.

BACKGROUND: The overproliferation of pulmonary vascular cells is noted in pulmonary hypertension. The role of exosomes from pulmonary artery endothelial cells (PAEC) in the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMC) remains unclear. METHODS: Exosomes were isolated and purified from the culture medium of PAEC using a commercial kit. Lipopolysaccharide (LPS), hypoxia, and hydrogen peroxide were utilized to induce PAEC injury. Coculture of PAEC and PASMC was conducted using Transwell plates, and GW4869 was applied to inhibit exosome release. The proliferation and apoptosis level of PASMC was assayed by MTT assay, apoptosis staining, and cleaved caspase-3 immunoblotting. Plasma exosomes were isolated by differential ultracentrifugation. RESULTS: LPS or hypoxia enhance exosome release from PAEC. Release of PAEC-derived exosomes positively correlates with LPS concentration. The coculture of LPS-disposed PAEC with PASMC leads to overproliferation and apoptosis resistance in PASMC, and the exosome inhibitor GW4869 can partly cancel out this effect. Exosomes derived from PAEC could be internalized into PASMC, and thus promote proliferation and induce apoptosis resistance in PASMC. Idiopathic pulmonary arterial hypertension patients exhibit a higher circulation level of endothelium-derived exosomes. CONCLUSIONS: Inflammation and hypoxia could induce PAEC to release exosomes. PAEC- derived exosomes are involved in overproliferation and apoptosis resistance in PASMC, by which they may contribute to the pathogenesis of pulmonary hypertension.