ABSTRACT
Right heart failure is the leading cause of death in pulmonary hypertension (PH), and echocardiography is a commonly used tool for evaluating the risk hierarchy of PH. However, few studies have explored the dynamic changes in the structural and functional changes of the right heart during the process of PH. Previous studies have found that pulmonary circulation coupling right ventricular adaptation depends on the degree of pressure overload and other factors. In this study, we performed a time-dependent evaluation of right heart functional changes using transthoracic echocardiography in a SU5416 plus hypoxia (SuHx)-induced PH rat model. Rats were examined in 1-, 2-, 4-, and 6-week using right-heart catheterization, cardiac echocardiography, and harvested heart tissue. Our study found that echocardiographic measures of the right ventricle (RV) gradually worsened with the increase of right ventricular systolic pressure, and right heart hypofunction occurred at an earlier stage than pulmonary artery thickening during the development of PH. Furthermore, sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2), a marker of myocardial damage, was highly expressed in week 2 of SuHx-induced PH and had higher levels of expression of γ-H2AX at all timepoints, as well as higher levels of DDR-related proteins p-ATM and p53/p-p53 and p21 in week 4 and week 6. Our study demonstrates that the structure and function of the RV begin to deteriorate with DNA damage and cellular senescence during the early stages of PH development.
Subject(s)
Heart Failure , Hypertension, Pulmonary , Animals , Rats , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/diagnostic imaging , Tumor Suppressor Protein p53 , Heart Failure/chemically induced , Heart Failure/diagnostic imaging , Echocardiography , DNA Damage , Hypoxia/complicationsABSTRACT
Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.
Subject(s)
Endothelial Cells , Ion Channels , Mechanoreceptors , TRPC6 Cation Channel , Calcium/metabolism , Cations/metabolism , Diglycerides/metabolism , Diglycerides/pharmacology , Endothelial Cells/metabolism , Humans , Hypotonic Solutions/metabolism , Hypotonic Solutions/pharmacology , Ion Channels/genetics , Ion Channels/metabolism , Mechanoreceptors/metabolism , Mechanotransduction, Cellular/genetics , Mechanotransduction, Cellular/physiology , Pulmonary Artery/cytology , Pulmonary Artery/metabolism , TRPC6 Cation Channel/genetics , TRPC6 Cation Channel/metabolismABSTRACT
Concentric pulmonary vascular wall thickening due partially to increased pulmonary artery (PA) smooth muscle cell (PASMC) proliferation contributes to elevating pulmonary vascular resistance (PVR) in patients with pulmonary hypertension (PH). Although pulmonary vasoconstriction may be an early contributor to increasing PVR, the transition of contractile PASMCs to proliferative PASMCs may play an important role in the development and progression of pulmonary vascular remodeling in PH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) is a trigger for PASMC contraction and proliferation. Here, we report that upregulation of Piezo1, a mechanosensitive cation channel, is involved in the contractile-to-proliferative phenotypic transition of PASMCs and potential development of pulmonary vascular remodeling. By comparing freshly isolated PA (contractile PASMCs) and primary cultured PASMCs (from the same rat) in a growth medium (proliferative PASMCs), we found that Piezo1, Notch2/3, and CaSR protein levels were significantly higher in proliferative PASMCs than in contractile PASMCs. Upregulated Piezo1 was associated with an increase in expression of PCNA, a marker for cell proliferation, whereas downregulation (with siRNA) or inhibition (with GsMTx4) of Piezo1 attenuated PASMC proliferation. Furthermore, Piezo1 in the remodeled PA from rats with experimental PH was upregulated compared with PA from control rats. These data indicate that PASMC contractile-to-proliferative phenotypic transition is associated with the transition or adaptation of membrane channels and receptors. Upregulated Piezo1 may play a critical role in PASMC phenotypic transition and PASMC proliferation. Upregulation of Piezo1 in proliferative PASMCs may likely be required to provide sufficient Ca2+ to assure nuclear/cell division and PASMC proliferation, contributing to the development and progression of pulmonary vascular remodeling in PH.
Subject(s)
Hypertension, Pulmonary , Membrane Proteins/metabolism , Pulmonary Artery , Animals , Calcium Signaling/physiology , Cell Proliferation , Cells, Cultured , Humans , Hypertension, Pulmonary/metabolism , Ion Channels/genetics , Ion Channels/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/metabolism , Rats , Vascular RemodelingABSTRACT
Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.
Subject(s)
Endothelial Cells/metabolism , Hypertension, Pulmonary/metabolism , Ion Channels/biosynthesis , Mechanotransduction, Cellular/physiology , Pulmonary Artery/metabolism , Up-Regulation/physiology , Adult , Aged , Animals , Cells, Cultured , Endothelial Cells/drug effects , Female , Humans , Hypertension, Pulmonary/pathology , Indoles/pharmacology , Male , Mechanotransduction, Cellular/drug effects , Mice , Mice, Inbred C57BL , Middle Aged , Pulmonary Artery/drug effects , Pulmonary Artery/pathology , Pyrroles/pharmacology , Rats , Rats, Sprague-Dawley , Up-Regulation/drug effectsABSTRACT
Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Sustained vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and concentric arterial remodeling due partially to PASMC proliferation are the major causes for increased pulmonary vascular resistance and increased pulmonary arterial pressure in patients with precapillary pulmonary hypertension (PH) including PAH and PH due to respiratory diseases or hypoxemia. We and others observed upregulation of TRPC6 channels in PASMCs from patients with PAH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is a pivotal signaling cascade for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 antagonist has yet been developed and tested for treatment of PAH or PH. In this study, we sought to investigate whether block of receptor-operated Ca2+ channels using a nonselective blocker of cation channels, 2-aminoethyl diphenylborinate (2-APB, administered intraperitoneally) and a selective blocker of TRPC6, BI-749327 (administered orally) can reverse established PH in mice. The results from the study show that intrapulmonary application of 2-APB (40 µM) or BI-749327 (3-10 µM) significantly and reversibly inhibited acute alveolar hypoxia-induced pulmonary vasoconstriction. Intraperitoneal injection of 2-APB (1 mg/kg per day) significantly attenuated the development of PH and partially reversed established PH in mice. Oral gavage of BI-749327 (30 mg/kg, every day, for 2 wk) reversed established PH by â¼50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749327 both significantly inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. In summary, the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH/PH. BI-749327, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH and PH due to respiratory diseases or hypoxemia.
Subject(s)
Gene Expression Regulation/drug effects , Hypertension, Pulmonary/pathology , Muscle, Smooth, Vascular/pathology , Pulmonary Artery/pathology , TRPC6 Cation Channel/metabolism , Vasoconstriction , Animals , Boron Compounds/pharmacology , Calcium Signaling , Cells, Cultured , Humans , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/metabolism , Mice , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , TRPC6 Cation Channel/antagonists & inhibitors , TRPC6 Cation Channel/geneticsABSTRACT
The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.
Subject(s)
Kv1.2 Potassium Channel/genetics , Kv1.5 Potassium Channel/genetics , Pulmonary Arterial Hypertension/genetics , Receptors, Calcium-Sensing/genetics , Stromal Interaction Molecule 2/genetics , Calcium Channels/drug effects , Calcium Channels/genetics , Calcium Signaling/genetics , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Estrenes/pharmacology , HEK293 Cells , Humans , Indoles/pharmacology , Jagged-1 Protein/genetics , Membrane Potentials/drug effects , Myocytes, Smooth Muscle/metabolism , Pulmonary Arterial Hypertension/metabolism , Pulmonary Arterial Hypertension/pathology , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Pyrrolidinones/pharmacology , Receptors, Calcium-Sensing/drug effects , Receptors, Notch/genetics , Single-Cell AnalysisABSTRACT
Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel ß1 subunit (BKCaß1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaß1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.
Subject(s)
Down-Regulation/genetics , Familial Primary Pulmonary Hypertension/genetics , MicroRNAs/genetics , Potassium Channels, Voltage-Gated/genetics , Adolescent , Adult , Cells, Cultured , Familial Primary Pulmonary Hypertension/metabolism , Female , Humans , Male , Membrane Potentials/genetics , Middle Aged , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/metabolism , RNA, Messenger/genetics , Up-Regulation/genetics , Vasoconstriction/genetics , Young AdultABSTRACT
OBJECTIVE: Regular exercise is an effective nonpharmacological means of preventing and controlling hypertension. However, the molecular mechanisms underlying its effects remain undetermined. The hypothesis that hypertension increases the functional coupling of large-conductance Ca(2+)-activated K(+) (BKCa) channels with ryanodine receptors in spontaneously hypertensive rats (SHR) as a compensatory response to an increase in intracellular Ca(2+) concentration in cerebral artery smooth muscle cells was assessed here. It was further hypothesized that exercise training would prevent this increase in functional coupling. APPROACH AND RESULTS: SHR and Wistar-Kyoto (WKY) rats were randomly assigned to sedentary groups (SHR-SED and WKY-SED) and exercise training groups (SHR-EX and WKY-EX). Cerebral artery smooth muscle cells displayed spontaneous transient outward currents at membrane potentials more positive than -40 mV. The amplitude of spontaneous transient outward currents together with the spontaneous Ca(2+) sparks in isolated cerebral artery smooth muscle cells was significantly higher in SHR-SED than in WKY-SED. Moreover, hypertension displayed increased whole-cell BKCa, voltage-gated Ca(2+) channel, but decreased KV currents in cerebral artery smooth muscle cells. In SHRs, the activity of the single BKCa channel increased markedly, and the protein expression of BKCa (ß1, but not α-subunit) also increased, but KV1.2 decreased significantly. Exercise training ameliorated all of these functional and molecular alterations in hypertensive rats. CONCLUSIONS: These data indicate that hypertension leads to enhanced functional coupling of ryanodine receptors-BKCa to buffer pressure-induced constriction of cerebral arteries, which attributes not only to an upregulation of BKCa ß1-subunit function but also to an increase of Ca(2+) release from ryanodine receptors. However, regular aerobic exercise efficiently prevents augmented coupling and so alleviates the pathological compensation and restores cerebral arterial function.
Subject(s)
Blood Pressure , Cerebral Arteries/metabolism , Exercise Therapy , Hypertension/therapy , Large-Conductance Calcium-Activated Potassium Channel beta Subunits/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Receptor Cross-Talk , Ryanodine Receptor Calcium Release Channel/metabolism , Animals , Calcium Signaling , Cerebral Arteries/physiopathology , Disease Models, Animal , Hypertension/metabolism , Hypertension/physiopathology , Kv1.2 Potassium Channel/metabolism , Membrane Potentials , Muscle, Smooth, Vascular/physiopathology , Rats, Inbred SHR , Rats, Inbred WKY , Signal Transduction , Up-Regulation , VasoconstrictionABSTRACT
Acute myeloid leukemia (AML) is a prevalent hematological tumor associated with a high morbidity and mortality rate. CDK9, functioning as a pivotal transcriptional regulator, facilitates transcriptional elongation through phosphorylation of RNA polymerase II, which further governs the protein levels of Mcl-1 and c-Myc. Therefore, CDK9 has been considered as a promising therapeutic target for AML treatment. Here, we present the design, synthesis, and evaluation of CDK9 inhibitors bearing a flavonoid scaffold. Among them, compound 21a emerged as a highly selective CDK9 inhibitor (IC50 = 6.7 nM), exhibiting over 80-fold selectivity towards most other CDK family members and high kinase selectivity. In Mv4-11 cells, 21a effectively hindered cell proliferation (IC50 = 60 nM) and induced apoptosis by down-regulating Mcl-1 and c-Myc. Notably, 21a demonstrated significant inhibition of tumor growth in the Mv4-11 xenograft tumor model. These findings indicate that compound 21a holds promise as a potential candidate for treating AML.
Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Flavonoids/pharmacology , Flavonoids/therapeutic use , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Leukemia, Myeloid, Acute/pathology , Apoptosis , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Cell Line, Tumor , Cyclin-Dependent Kinase 9/metabolismABSTRACT
Objectives: Depression is the leading cause of ill health and disability worldwide, and has become one of the key issues in the public health sector. Social participation is one of the most important measures to support the rapidly aging population and can reduce the risk of depression in the elderly. This study aims to explore the causal relationship between different types of social participation and depression in the elderly, and reduce the level of depression in the elderly by promoting social participation. Methods: In order to accurately evaluate the causal relationship between participation in different types of social activities and depression in the elderly, this paper uses propensity score matching (PSM) for analysis based on counterfactual framework. The specific matching methods used are: k-nearest neighbor matching method, kernel matching method and radius matching method. Results: In this study, 31.4% of the elderly have depression, and the proportion of female elderly is significantly higher. Participation in different social activities has different effects on depression in the elderly. Taking into account sample selection bias, participation in friend-making, exercise, and recreational activities can effectively reduce the risk of depression in the elderly. Compared with other social activities, participation in exercise and recreational activities are more helpful to reduce the risk of depression in the elderly. Participating in exercise activities only reduces the risk of depression in 60-69-year-olds, while participating in economic activities increases the risk of depression in the elderly aged 70 and over. Compared with the male elderly, participation in friend-making, exercise, and recreational activities results in the female elderly having stronger resistance to the risk of depression. Conclusions: Depression was prevalent among the elderly. Through PSM analysis, different types of social participation have different effects on depression in the elderly. In order to maximize the positive effects of different types of social participation on depression in the elderly, it is necessary to provide differentiated social support environment for the elderly. Expanding the research on the relationship between social participation and depression of the elderly will help to formulate more reasonable public health policies and improve the mental health of the elderly.
Subject(s)
Disabled Persons , Social Participation , Aged , Aged, 80 and over , Aging , China/epidemiology , Exercise , Female , Humans , MaleABSTRACT
Pulmonary arterial hypertension (PAH) is a severe disease characterized by sustained vasoconstriction, concentric wall thickening and vascular remodeling leading to increased pulmonary vascular resistance, causing right heart failure and death. Acute alveolar hypoxia causes pulmonary vasoconstriction, while sustained hypoxia causes pulmonary hypertension (PH). Activation of Notch signaling is implicated in the development of PAH and chronic hypoxia induced PH via partially its enhancing effect on Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs). Pharmacological experiments and genetic approach using animal models of experimental PH (e.g., chronic hypoxia-induced PH) have been routinely utilized to study pathogenic mechanisms of PAH/PH and identify novel therapeutic targets. In this chapter, we describe protocols to investigate the role of Notch by measuring pulmonary hemodynamics in vivo and pulmonary arterial pressure ex vivo in mouse models of experimental PH. Using these experimental protocols, one can study the role of Notch or Notch signaling pathway in the pathogenic mechanisms of pulmonary vascular disease and develop novel therapies by targeting Notch ligands and receptors.
Subject(s)
Hypertension, Pulmonary , Muscle, Smooth, Vascular , Animals , Cell Proliferation , Cells, Cultured , Hypoxia/metabolism , Mice , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery , Vascular RemodelingABSTRACT
A series of novel biphenyl-based scaffold derivatives were identified as selective histone deacetylase 6 (HDAC6) inhibitors through an in-house compound library screening approach. The biological evaluation indicated that most of target compounds exhibited moderate to good inhibitory activity and selectivity against HDAC6. Especially, compound C10 was identified as a potent and highly selective HDACs inhibitor, with HDAC1 IC50 value of 3600 nM, HDAC6 IC50 value of 23 nM, and the HDAC1/6 selectivity index of 157. Moreover, C10 displayed robust anti-proliferative activity, induced cancer cells apoptosis, increased the level of acetylated α-tubulin and inhibited cancer cells migration in vitro. C10 showed significant antitumor efficacy (TGI: 75%) in CT26 colon carcinoma xenograft model in mice with no considerable toxicity in vivo. More importantly, C10 could also activate antitumor immunity so as to synergistically exert antitumor effects in vivo. Overall, our findings have provided a new avenue for design, development and investigation into the mechanism underlying the antitumor efficacy of selective HDAC6 inhibitors.
Subject(s)
Antineoplastic Agents , Animals , Antineoplastic Agents/pharmacology , Biphenyl Compounds , Cell Proliferation , Dose-Response Relationship, Drug , Histone Deacetylase 1/metabolism , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/pharmacology , Humans , Mice , Molecular Structure , Structure-Activity RelationshipABSTRACT
Pulmonary arterial hypertension is a progressive and fatal disease and rodents with experimental pulmonary hypertension (PH) are often used to study pathogenic mechanisms, identify therapeutic targets, and develop novel drugs for treatment. Here we describe a hands-on set of experimental approaches including ex vivo lung angiography and histology and in vivo right heart catheterization (RHC) to phenotypically characterize pulmonary hemodynamics and lung vascular structure in normal mice and mice with experimental PH. We utilized Microfil polymer as contrast in our ex vivo lung angiogram to quantitatively examine pulmonary vascular remodeling in mice with experimental PH, and lung histology to estimate pulmonary artery wall thickness. The peripheral lung vascular images were selected to determine the total length of lung vascular branches, the number of branches and the number of junctions in a given area (mm-2). We found that the three parameters determined by angiogram were not significantly different among the apical, middle, and basal regions of the mouse lung from normal mice, and were not influenced by gender (no significant difference between female and male mice). We conducted RHC in mice to measure right ventricular systolic pressure, a surrogate measure for pulmonary artery systolic pressure and right ventricle (RV) contractility (RV ± dP/dtmax) to estimate RV function. RHC, a short time (4-6 min) procedure, did not alter the lung angiography measurements. In summary, utilizing ex vivo angiogram to determine peripheral vascular structure and density in the mouse lung and utilizing in vivo RHC to measure pulmonary hemodynamics are reliable readouts to phenotype normal mice and mice with experimental PH. Lung angiogram and RHC are also reliable approaches to examine pharmacological effects of new drugs on pulmonary vascular remodeling and hemodynamics.
ABSTRACT
Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+) cyt ] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.
ABSTRACT
BACKGROUND AND PURPOSE: Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH). EXPERIMENTAL APPROACH: Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2 ) for 4 weeks were used as model of HPH for in vivo experiments. KEY RESULTS: Halofuginone increased voltage-gated K+ (Kv ) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 µM) inhibited receptor-operated Ca2+ entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca2+ entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 µM). Intraperitoneal administration of halofuginone (0.3 mg·kg-1 , for 2 weeks) partly reversed established PH in mice. CONCLUSION AND IMPLICATIONS: Halofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor-operated and store-operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.
Subject(s)
Hypertension, Pulmonary , Pharmaceutical Preparations , Animals , Calcium , Hypertension, Pulmonary/drug therapy , Hypoxia/drug therapy , Mice , Myocytes, Smooth Muscle , Phosphatidylinositol 3-Kinases , Piperidines , Pulmonary Artery , QuinazolinonesABSTRACT
Hypoxic Pulmonary Vasoconstriction (HPV) is an important physiological mechanism of the lungs that matches perfusion to ventilation thus maximizing O2 saturation of the venous blood within the lungs. This study emphasizes on principal pathways in the initiation and modulation of hypoxic pulmonary vasoconstriction with a primary focus on the role of Ca2+ signaling and Ca2+ influx pathways in hypoxic pulmonary vasoconstriction. We used an ex vivo model, isolated perfused/ventilated mouse lung to evaluate hypoxic pulmonary vasoconstriction. Alveolar hypoxia (utilizing a mini ventilator) rapidly and reversibly increased pulmonary arterial pressure due to hypoxic pulmonary vasoconstriction in the isolated perfused/ventilated lung. By applying specific inhibitors for different membrane receptors and ion channels through intrapulmonary perfusion solution in isolated lung, we were able to define the targeted receptors and channels that regulate hypoxic pulmonary vasoconstriction. We show that extracellular Ca2+ or Ca2+ influx through various Ca2+-permeable channels in the plasma membrane is required for hypoxic pulmonary vasoconstriction. Removal of extracellular Ca2+ abolished hypoxic pulmonary vasoconstriction, while blockade of L-type voltage-dependent Ca2+ channels (with nifedipine), non-selective cation channels (with 30 µM SKF-96365), and TRPC6/TRPV1 channels (with 1 µM SAR-7334 and 30 µM capsazepine, respectively) significantly and reversibly inhibited hypoxic pulmonary vasoconstriction. Furthermore, blockers of Ca2+-sensing receptors (by 30 µM NPS2143, an allosteric Ca2+-sensing receptors inhibitor) and Notch (by 30 µM DAPT, a γ-secretase inhibitor) also attenuated hypoxic pulmonary vasoconstriction. These data indicate that Ca2+ influx in pulmonary arterial smooth muscle cells through voltage-dependent, receptor-operated, and store-operated Ca2+ entry pathways all contribute to initiation of hypoxic pulmonary vasoconstriction. The extracellular Ca2+-mediated activation of Ca2+-sensing receptors and the cell-cell interaction via Notch ligands and receptors contribute to the regulation of hypoxic pulmonary vasoconstriction.
ABSTRACT
Wogonin, a natural product isolated from the plant Scutellaria baicalensis, has been shown to be a potent and selective inhibitor of CDK9. With the purpose of investigating the activity and selectivity of this chemical scaffold, several series of wogonin derivatives were prepared and screened for CDK9 inhibition and cellular antiproliferative activity. Among these compounds, the drug-like compound 51 showed potent activity against CDK9 (IC50â¯=â¯19.9â¯nM) and MV4-11â¯cell growth (IC50â¯=â¯20â¯nM). In addition, compound 51 showed much improved physicochemical properties, such as water solubility, compared with the parent compound wogonin. The follow-up studies showed that the compound 51 is selective toward CDK9-overexpressing cancer cells over normal cells. Preliminary mechanism studies on the anticancer effect indicated that 51 inhibited the proliferation of MV4-11â¯cells via caspase-dependent apoptosis. In addition, highlighted compound 51 showed significant antitumor activity in mouse acute myeloid leukemia (AML) models without producing apparent toxic effects in vivo, which gave us a new tool for further investigation of CDK9-targeted inhibitor as a potential antitumor drug especially for AML.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Design , Flavanones/pharmacology , Protein Kinase Inhibitors/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 9 , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Flavanones/chemical synthesis , Flavanones/chemistry , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
BACKGROUND AND PURPOSE: Regular physical activity is an effective non-pharmacological therapy for prevention and control of hypertension. However, the underlying mechanisms are not fully understood. Accumulating evidence shows that the elevated vascular tone in hypertension is a consequence of the 'ion channel remodelling' that occurs during sustained high BP. The present study investigated the effects of aerobic exercise on the electrical remodelling of L-type Ca(2+) (Cav 1.2) and large-conductance Ca(2+) -activated K(+) (KCa 1.1) channels in mesenteric arteries (MAs) from spontaneously hypertensive rats (SHRs). EXPERIMENTAL APPROACH: SHRs and normotensive (Wistar-Kyoto) rats were subjected to aerobic training or kept sedentary, and vascular mechanical and functional properties were evaluated. KEY RESULTS: Exercise did not affect the heart weight, but reduced the heart rate and body weight in SHR. In mesenteric arterial myocytes, exercise normalized the increased Cav 1.2 and KCa 1.1 current density in SHRs. Exercise also ameliorated the increased open probability and mean open time of the single KCa 1.1 channel in hypertension. The isometric contraction study revealed that both nifedipine (Cav 1.2 channel blocker) and NS11021 (KCa 1.1 channel activator) induced concentration-dependent vasorelaxation in MAs precontracted with noradrenaline. Exercise normalized the increased sensitivity of tissues to nifedipine and NS11021 in SHR. Furthermore, protein expression of the Cav 1.2 α1C -subunit together with the KCa 1.1 α- and ß1-subunit was significantly increased in SHRs; and exercise ameliorated these molecular alterations in hypertension. CONCLUSIONS AND IMPLICATIONS: Chronic exercise reduces BP and restores vascular function in MAs from SHR, which might be related to the correction of the Cav 1.2 and KCa 1.1 channel remodelling during hypertension.