Corosolic acid attenuates platelet-derived growth factor signaling in macrophages and smooth muscle cells of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease that is characterized by vascular remodeling of the pulmonary artery. Pulmonary vascular remodeling is primarily caused by the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are facilitated by perivascular inflammatory cells including macrophages. Corosolic acid (CRA) is a natural pentacyclic triterpenoid that exerts anti-inflammatory effects. In the present study, the effects of CRA on the viability of macrophages were examined using monocrotaline (MCT)-induced PAH rats and human monocyte-derived macrophages. Although we previously reported that CRA inhibited signal transducer and activator of transcription 3 (STAT3) signaling and ameliorated pulmonary vascular remodeling in PAH, the inhibitory mechanism remains unclear. Therefore, the underlying mechanisms were investigated using PASMCs from idiopathic PAH (IPAH) patients. In MCT-PAH rats, CRA inhibited the accumulation of macrophages around remodeled pulmonary arteries. CRA reduced the viability of human monocyte-derived macrophages. In IPAH-PASMCs, CRA attenuated cell proliferation and migration facilitated by platelet-derived growth factor (PDGF)-BB released from macrophages and PASMCs. CRA also downregulated the expression of PDGF receptor ß and its signaling pathways, STAT3 and nuclear factor-κB (NF-κB). In addition, CRA attenuated the phosphorylation of PDGF receptor ß and STAT3 following the PDGF-BB simulation. The expression and phosphorylation levels of PDGF receptor ß after the PDGF-BB stimulation were reduced by the small interfering RNA knockdown of NF-κB, but not STAT3, in IPAH-PASMCs. In conclusion, CRA attenuated the PDGF-PDGF receptor ß-STAT3 and PDGF-PDGF receptor ß-NF-κB signaling axis in macrophages and PASMCs, and thus, ameliorated pulmonary vascular remodeling in PAH.

Up-regulated expression of two-pore domain K+ channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension.

Background: Pulmonary arterial hypertension (PAH) is a severe and rare disease in the cardiopulmonary system. Its pathogenesis involves vascular remodeling of the pulmonary artery, which results in progressive increases in pulmonary arterial pressure. Chronically increased pulmonary arterial pressure causes right ventricular hypertrophy and subsequent right heart failure. Pulmonary vascular remodeling is attributed to the excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), which are induced by enhanced Ca2+ signaling following the up-/down-regulation of ion channel expression. Objectives: In the present study, the functional expression of two-pore domain potassium KCNK channels was investigated in PASMCs from idiopathic PAH (IPAH) patients and experimental pulmonary hypertensive (PH) animals. Results: In IPAH-PASMCs, the expression of KCNK1/TWIK1 and KCNK2/TREK1 channels was up-regulated, whereas that of KCNK3/TASK1 and KCNK6/TWIK2 channels was down-regulated. The similar up-regulated expression of KCNK1 and KCNK2 channels was observed in the pulmonary arterial smooth muscles of monocrotaline-induced PH rats, Sugen 5416/hypoxia-induced PH rats, and hypoxia-induced PH mice. The facilitated proliferation of IPAH-PASMCs was suppressed by the KCNK channel blockers, quinine and tetrapentylammonium. The migration of IPAH-PASMCs was also suppressed by these channel blockers. Furthermore, increases in the proliferation and migration were inhibited by the siRNA knockdown of KCNK1 or KCNK2 channels. The siRNA knockdown also caused membrane depolarization and subsequent decrease in cytosolic [Ca2+]. The phosphorylated level of c-Jun N-terminal kinase (JNK) was elevated in IPAH-PASMCs compared to normal-PASMCs. The increased phosphorylation was significantly reduced by the siRNA knockdown of KCNK1 or KCNK2 channels. Conclusion: Collectively, these findings indicate that the up-regulated expression of KCNK1 and KCNK2 channels facilitates the proliferation and migration of PASMCs via enhanced Ca2+ signaling and JNK signaling pathway, which is associated with vascular remodeling in PAH.

Caveolin-1 forms a complex with P2X7 receptor and tunes P2X7-mediated ATP signaling in mouse bone marrow-derived macrophages.

The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1ß secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1ß secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1ß secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1ß secretion and cell death in macrophages.

Pimaric acid reduces vasoconstriction via BKCa channel activation and VDCC inhibition in rat pulmonary arterial smooth muscles.

Pulmonary vessels play a pivotal role in oxygen circulation. We previously demonstrated that pimaric acid (PiMA) activated large-conductance Ca2+-activated K+ (BKCa) channels and inhibited voltage-dependent Ca2+ channels (VDCCs). In the present study, PiMA attenuated vasoconstriction induced by high K+ or endothelin-1 in rat pulmonary arterial smooth muscles (PASMs). PiMA also reduced high K+-induced cytosolic [Ca2+] increase in PASM cells. PiMA increased BKCa currents and decreased VDCC currents. BKCa channels and VDCCs were formed by the α/ß1 and α1C/α1D/ß2/ß3 subunits, respectively. These results indicate that PiMA induces vasorelaxation through the dual effects of BKCa channel activation and VDCC inhibition in PASMs.

Mitofusin 1 and 2 differentially regulate mitochondrial function underlying Ca2+ signaling and proliferation in rat aortic smooth muscle cells.

Mitochondria play a substantial role in cytosolic Ca2+ buffering and energy metabolism. We recently demonstrated that mitofusin 2 (Mfn2) regulated Ca2+ signaling by tethering mitochondria and sarcoplasmic reticulum (SR), and thus, facilitated mitochondrial function and the proliferation of vascular smooth muscle cells (VSMCs). However, the physiological role of mitofusin 1 (Mfn1) on Ca2+ signaling and mitochondrial function remains unclear. Herein, the roles of Mfn1 and Mfn2 in mitochondrial function underlying Ca2+ signaling, ATP production, and cell proliferation were examined in rat aortic smooth muscle A10 cells. Following an arginine vasopressin-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt), Mfn2 siRNA (siMfn2) reduced cytosolic Ca2+ removal and mitochondrial Ca2+ uptake. However, Mfn1 siRNA (siMfn1) attenuated mitochondrial Ca2+ uptake, facilitated Ca2+ removal from mitochondria, and resulted in increased [Ca2+]cyt, which was mediated by the downregulation of mitochondrial Ca2+ uniporter (MCU) expression and the upregulation of mitochondrial Na+/Ca2+ exchanger (NCLX) expression. Furthermore, siMfn1 increased the mitochondrial membrane potential, ATP production by adenine nucleotide translocase (ANT), and cell proliferation, whereas siMfn2 exhibited the opposite responses. In conclusion, Mfn1 modulates the expressions of MCU, NCLX, and ANT, and Mfn2 tethers mitochondria to SR, which demonstrates their different mitochondrial functions for Ca2+ signaling, ATP production, and the proliferation of VSMCs.

Corosolic acid ameliorates vascular remodeling in pulmonary arterial hypertension via the downregulation of STAT3 signaling.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that is characterized by vascular remodeling of the pulmonary artery. PAH remodeling is primarily caused by the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Therefore, an inhibitory mechanism is expected as a target for the treatment of PAH. Corosolic acid (CRA) is a pentacyclic triterpenoid extracted from the leaves of Banaba (Lagerstroemia speciosa) that exerts anti-diabetic, anti-inflammatory, and anti-tumor effects. In the present study, the effects of CRA on PAH remodeling were examined using PASMCs from idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats. CRA inhibited the excessive proliferation of IPAH-PASMCs in a concentration-dependent manner (IC50 = 14.1 µM). It also reduced the migration of IPAH-PASMCs. The CRA treatment downregulated the expression of signal transducer and activator of transcription 3 (STAT3) in IPAH-PASMCs. In MCT-PH rats, the administration of CRA (1 mg/kg/day) attenuated increases in right ventricular systolic pressure, pulmonary vascular remodeling, and right ventricular hypertrophy. CRA also decreased the expression of STAT3 in pulmonary arterial smooth muscles from MCT-PH rats. In conclusion, the anti-proliferative and anti-migratory effects of CRA in PASMCs ameliorated PAH remodeling by downregulating STAT3 signaling pathways.

Upregulated ClC3 Channels/Transporters Elicit Swelling-Activated Cl- Currents and Induce Excessive Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling of the pulmonary artery, which is mainly attributed to the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) comprising the medial layer of pulmonary arteries. The activity of ion channels associated with cytosolic Ca2+ signaling regulates the pathogenesis of PAH. Limited information is currently available on the role of Cl- channels in PASMCs. Therefore, the functional expression of ClC3 channels/transporters was herein investigated in the PASMCs of normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). Expression analyses revealed the upregulated expression of ClC3 channels/transporters at the mRNA and protein levels in IPAH-PASMCs. Hypoosmotic perfusion (230 mOsm) evoked swelling-activated Cl- currents (ICl-swell) in normal-PASMCs, whereas 100 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) exerted the opposite effects. The small interfering RNA (siRNA) knockdown of ClC3 did not affect ICl-swell. On the other hand, ICl-swell was larger in IPAH-PASMCs and inhibited by DIDS and the siRNA knockdown of ClC3. IPAH-PASMCs grew more than normal-PASMCs. The growth of IPAH-PASMCs was suppressed by niflumic acid and DIDS, but not by 9-anthracenecarboxylic acid or T16Ainh-A01. The siRNA knockdown of ClC3 also inhibited the proliferation of IPAH-PASMCs. Collectively, the present results indicate that upregulated ClC3 channels/transporters are involved in ICl-swell and the excessive proliferation of IPAH-PASMCs, thereby contributing to the pathogenesis of PAH. Therefore, ClC3 channels/transporters have potential as a target of therapeutic drugs for the treatment of PAH.

Increased TMEM16A-Mediated Ca2+-Activated Cl- Currents in Portal Vein Smooth Muscle Cells of Caveolin 1-Deficient Mice.

Ca2+-activated Cl- (ClCa) channels regulate membrane excitability and myogenic tone in vascular smooth muscles. TMEM16A-coding proteins are mainly responsible for functional ClCa channels in vascular smooth muscles, including portal vein smooth muscles (PVSMs). Caveolae are cholesterol-rich and Ω-shaped invaginations on the plasma membrane that structurally contributes to effective signal transduction. Caveolin 1 (Cav1) accumulates in caveolae to form functional complexes among receptors, ion channels, and kinases. The present study examined the functional roles of Cav1 in the expression and activity of ClCa channels in the portal vein smooth muscle cells (PVSMCs) of wild-type (WT) and Cav1-knockout (KO) mice. Contractile experiments revealed that the amplitude of spontaneous PVSM contractions was larger in Cav1-KO mice than WT mice. Under whole-cell patch-clamp configurations, ClCa currents were markedly inhibited by 1 µM Ani9 (a selective TMEM16A ClCa channel blocker) in WT and Cav1-KO PVSMCs. However, Ani9-sensitive ClCa currents were significantly larger in Cav1-KO PVSMCs than in WT PVSMCs. Expression analyses showed that TMEM16A expression levels were higher in Cav1-KO PVSMs than in WT PVSMs. Therefore, the caveolar structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.

Mitofusin 2 positively regulates Ca2+ signaling by tethering the sarcoplasmic reticulum and mitochondria in rat aortic smooth muscle cells.

Mitochondria buffer cytosolic Ca2+ increases following Ca2+ influx from extracellular spaces, and Ca2+ release from intracellular Ca2+ store sites under physiological circumstances. Therefore, close contact of mitochondria with the sarcoplasmic reticulum (SR) is required for maintaining Ca2+ homeostasis. Mitofusin 2 (Mfn2) localizes in both mitochondrial and SR membranes and is hypothesized to optimize the distance and Ca2+ transfer between these organelles. However, the physiological significance of Mfn2 in vascular smooth muscle cells (VSMCs) is poorly understood. In the present study, the role of Mfn2 in the physical and functional couplings between SR and mitochondria was examined in rat aortic smooth muscle cells (rASMCs) by confocal and electron microscope imaging. When Mfn2 was knocked down using siRNA in rASMCs, the mean distance between these organelles was extended from 16.2 to 21.6 nm. The increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) induced by 100 nM arginine vasopressin (AVP) was not affected by Mfn2 siRNA knockdown, whereas cytosolic Ca2+ removal was slower after Mfn2 knockdown. Following the AVP-induced [Ca2+]cyt increase, mitochondrial Ca2+ uptake and Ca2+ refill into the SR were attenuated by Mfn2 knockdown. In addition, Mfn2-knockdown cells exhibited a loss of mitochondrial membrane potential (ΔΨmito) and lower ATP levels in mitochondria. Moreover, Mfn2 knockdown inhibited cell proliferation. In contrast, Mfn2 overexpression increased ΔΨmito and cell growth. This study strongly suggests that Mfn2 is responsible for SR-mitochondria Ca2+ signaling by tethering mitochondria to SR, thereby regulating ATP production and proliferation of VSMCs.

Involvement of small-conductance Ca2+-activated K+ (SKCa2) channels in spontaneous Ca2+ oscillations in rat pinealocytes.

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 µM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 µM DCEBIO), but not by an IKCa channel opener (1 µM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 µM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.

Ca2+ Signaling and Proliferation via Ca2+-Sensing Receptors in Human Hepatic Stellate LX-2 Cells.

Hepatic stellate cells (HSCs) play a significant role in the development of chronic liver diseases. Hepatic damage activates HSCs and results in hepatic fibrosis. The functions of activated HSCs require an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt). However, the regulatory mechanisms underlying Ca2+ signaling in activated HSCs remain largely unknown. In the present study, functional analyses of Ca2+-sensing receptors (CaSRs) were performed using activated human HSCs, LX-2. Expression analyses revealed that CaSR proteins were expressed in α-smooth muscle actin-positive LX-2 cells. Extracellular Ca2+ restoration (from 0 to 2.2 mM) increased [Ca2+]cyt in these cells. The extracellular Ca2+-induced increase in [Ca2+]cyt was reduced by the CaSR antagonists, NPS2143 and Calhex 231. Furthermore, the growth of LX-2 cells was blocked by NPS2143 and Calhex 231 in concentration-dependent manners (IC50 = 6.0 and 9.5 µM, respectively). LX-2 cell proliferation was also attenuated by NPS2143 and Calhex 231. In conclusion, CaSRs are functionally expressed in activated HSCs and regulate Ca2+ signaling and cell proliferation. The present results provide insights into the molecular mechanisms underlying hepatic fibrosis and will contribute to the development of potential therapeutic targets.

SKF96365 activates calcium-sensing receptors in pulmonary arterial smooth muscle cells.

In pulmonary arterial smooth muscle cells (PASMCs), an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) is involved in many physiological processes such as cell contraction and proliferation. However, chronic [Ca2+]cyt increases cause pulmonary vasoconstriction and vascular remodeling, resulting in pulmonary arterial hypertension (PAH). Therefore, [Ca2+]cyt signaling plays a substantial role in the regulation of physiological and pathological functions in PASMCs. In the present study, the effects of SKF96365 on [Ca2+]cyt were examined in PASMCs from normal subjects and idiopathic pulmonary arterial hypertension (IPAH) patients. SKF96365 is widely used as a blocker of non-selective cation channels. SKF96365 did not affect the resting [Ca2+]cyt in normal-PASMCs. However, SKF96365 increased [Ca2+]cyt in IPAH-PASMCs in a concentration-dependent manner (EC50 = 18 µM). The expression of Ca2+-sensing receptors (CaSRs) was higher in IPAH-PASMCs than in normal-PASMCs. The SKF96365-induced [Ca2+]cyt increase was inhibited by CaSR antagonists, NPS2143 and Calhex 231. The CaSR-mediated [Ca2+]cyt increase was facilitated by SKF96365 and the activation was blocked by NPS2143 or Calhex 231. In addition, the SKF96365-induced [Ca2+]cyt increase was reduced by siRNA knockdown of CaSRs. Taken together, SKF96365 activates CaSRs in IPAH-PASMCs and promotes [Ca2+]cyt signaling.

Downregulation of Ca2+-Activated Cl- Channel TMEM16A Mediated by Angiotensin II in Cirrhotic Portal Hypertensive Mice.

Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca2+-activated Cl- (ClCa) channels. TMEM16A is mainly responsible for ClCa channel conductance in vascular smooth muscle cells, including portal vein smooth muscle cells (PVSMCs). In the present study, the functional roles of TMEM16A channels were examined using two experimental portal hypertensive models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with non-cirrhotic portal hypertension. Expression analyses revealed that the expression of TMEM16A was downregulated in BDL-PVSMs, but not in PPVL-PVSMs. Whole-cell ClCa currents were smaller in BDL-PVSMCs than in sham- and PPVL-PVSMCs. The amplitude of spontaneous contractions was smaller and the frequency was higher in BDL-PVSMs than in sham- and PPVL-PVSMs. Spontaneous contractions sensitive to a specific inhibitor of TMEM16A channels, T16Ainh-A01, were reduced in BDL-PVSMs. Furthermore, in normal PVSMs, the downregulation of TMEM16A expression was mimicked by the exposure to angiotensin II, but not to bilirubin. This study suggests that the activity of ClCa channels is attenuated by the downregulation of TMEM16A expression in PVSMCs associated with cirrhotic portal hypertension, which is partly mediated by increased angiotensin II in cirrhosis.

Involvement of TREK1 channels in the proliferation of human hepatic stellate LX-2 cells.

Activation of hepatic stellate cells (HSCs) causes hepatic fibrosis and results in chronic liver diseases. Although activated HSC functions are facilitated by an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), the pathophysiological roles of ion channels are largely unknown. In the present study, functional analyses of the two-pore domain K+ (K2P) channels, which regulate the resting membrane potential and [Ca2+]cyt, were performed using the human HSC line, LX-2. Expression analyses revealed that TREK1 (also known as KCNK2 and K2P2.1) channels are expressed in LX-2 cells. Whole-cell K+ currents were activated by 10 µM arachidonic acid and the activation was abolished by 100 µM tetrapentylammonium, which are pharmacological characteristics of TREK1 channels. The siRNA knockdown of TREK1 channels caused membrane depolarization and reduced [Ca2+]cyt. In addition, TREK1 knockdown downregulated the gene expression of collage type I and platelet-derived growth factor. Furthermore, TREK1 knockdown inhibited the proliferation of LX-2 cells. In conclusion, the activity of TREK1 channels determines the resting membrane potential and [Ca2+]cyt, which play a role in extracellular matrix production and cell proliferation in HSCs. This study may help elucidate the molecular mechanism underlying hepatic fibrosis in HSCs and provide a potential therapeutic target for hepatic fibrosis.

Comparative analysis of age in monocrotaline-induced pulmonary hypertensive rats.

Pulmonary arterial hypertension (PAH) is a rare, progressive, and fatal cardiovascular/lung disease. The incidence rate is affected by age. Monocrotaline (MCT, 60 mg/kg)-treated rats are widely used as an experimental PAH model. Here, we found that young rats died at a mean of 23.4 days after MCT injection, whereas adult rats survived for over 42 days. However, young (7-week-old) and adult (20-week-old) MCT-treated rats developed PAH, and had upregulated Ca2+-sensing receptor and transient receptor potential canonical subfamily 6 channel expression in pulmonary arteries. The present study provides novel information for elucidating the mechanism underlying the age difference in PAH patients.