Naringin activates semaphorin 3A to ameliorate TGF-ß-induced endothelial-to-mesenchymal transition related to atrial fibrillation.

The loss of semaphorin 3A (Sema3A), which is related to endothelial-to-mesenchymal transition (EndMT) in atrial fibrosis, is implicated in the pathogenesis of atrial fibrillation (AF). To explore the mechanisms by which EndMT affects atrial fibrosis and assess the potential of a Sema3A activator (naringin) to prevent atrial fibrosis by targeting transforming growth factor-beta (TGF-ß)-induced EndMT, we used human atria, isolated human atrial endocardial endothelial cells (AEECs), and used transgenic mice expressing TGF-ß specifically in cardiac tissues (TGF-ß transgenic mice). We evaluated an EndMT marker (Twist), a proliferation marker (proliferating cell nuclear antigen; PCNA), and an endothelial cell (EC) marker (CD31) through triple immunohistochemistry and confirmed that both EndMT and EC proliferation contribute to atrial endocardial fibrosis during AF in TGF-ß transgenic mice and AF patient tissue sections. Additionally, we investigated the impact of naringin on EndMT and EC proliferation in AEECs and atrial fibroblasts. Naringin exhibited an antiproliferative effect, to which AEECs were more responsive. Subsequently, we downregulated Sema3A in AEECs using small interfering RNA to clarify a correlation between the reduction in Sema3A and the elevation of EndMT markers. Naringin treatment induced the expression of Sema3A and a concurrent decrease in EndMT markers. Furthermore, naringin administration ameliorated AF and endocardial fibrosis in TGF-ß transgenic mice by stimulating Sema3A expression, inhibiting EndMT markers, reducing atrial fibrosis, and lowering AF vulnerability. This suggests therapeutic potential for naringin in AF treatment.

Genetics in neovascular age-related macular degeneration susceptibility and treatment response to anti-VEGF intravitreal injection: A case series study.

BACKGROUND: To identify genotypes associated with neovascular age-related macular degeneration (nAMD) and investigate the associations between genotype variations and anti-vascular endothelial growth factor (VEGF) treatment response. METHODS: This observational, retrospective, case series study enrolled patients diagnosed with nAMD who received anti-VEGF treatment in National Taiwan University Hospital with at least one-year follow-up between 2012 and 2020. A genome-wide association study (GWAS) was conducted on enrolled patients and controls. Correlations between the genotypes identified from GWAS and the treatment response of functional/anatomical biomarkers, including visual acuity (VA), presence of intraretinal or subretinal fluid (SRF), serous or fibrovascular pigmented epithelium detachment (PED), and disruption of the ellipsoid zone (EZ), were analysed. RESULTS: In total, 182 patients with nAMD and 1748 controls were enrolled. GWAS revealed 16 single nucleotide polymorphisms (SNPs) as risk loci for nAMD, including seven loci in CFH and ARMS2/HTRA1 and nine novel loci, including rs117517872 and rs79835234(COPB2-DT), rs7525578(RAP1A), rs2123738(LOC105376755), rs1374879(CNTN3), rs3812692(SAR1A), rs117501587(PRKCA), rs9965945(CNDP1), and rs189769231(MATK). Our study revealed rs800292(CFH), rs11200638(HTRA1), and rs2123738(LOC105376755) correlated with poor treatment response in VA (P = 0.005), SRF (P = 0.044), and fibrovascular PED (P = 0.007), respectively. Rs9965945(CNDP1) was correlated with poor response in disruption of EZ (P = 0.046) and serous PED (P = 0.049). CONCLUSIONS: Among the 16 SNPs found in the GWAS, four loci-CFH, ARMS2/HTRA1, and two novel loci-were correlated with the susceptibility of nAMD and anatomical/functional responses after anti-VEGF treatment.

Osteopontin mediation of disturbed flow-induced endothelial mesenchymal transition through CD44 is a novel mechanism of neointimal hyperplasia in arteriovenous fistulae for hemodialysis access.

In dysfunctional arteriovenous fistulae (AVF) for hemodialysis access, neointimal hyperplasia (NH) is prone to occur in the region exposed to disturbed flow. We hypothesized that disturbed flow contributes to NH in AVF by inducing endothelial mesenchymal transition (EndMT) through activation of the osteopontin/CD44 axis. In rats with aortocaval fistula, a rodent model of AVF, we demonstrated development of EndMT and expression of osteopontin and CD44 specifically in the vicinity of the arteriovenous junction using immunostaining. Duplex scan confirmed this region was exposed to a disturbed flow. A mixed ultrastructural phenotype of endothelium and smooth muscle cells was found in luminal endothelial cells of the arteriovenous junction by electron microscopy ascertaining the presence of EndMT. Endothelial lineage tracing using Cdh5-Cre/ERT2;ROSA26-tdTomato transgenic mice showed that EndMT was involved in NH of AVF since the early stage and that the endothelial-derived cells contributed to 24% of neointimal cells. In human umbilical vein endothelial cells (HUVECs) in culture, osteopontin treatment induced EndMT, which was suppressed by CD44 knockdown. Exposure to low oscillatory wall shear stress using a parallel-plate system induced EndMT in HUVECs, also suppressed by osteopontin or CD44 knockdown. In AVF of CD44 knockout mice, EndMT was mitigated and NH decreased by 35% compared to that in wild-type mice. In dysfunctional AVF of patients with uremia, expressions of osteopontin, CD44, and mesenchymal markers in endothelial cells overlying the neointima was also found by immunostaining. Thus, the osteopontin/CD44 axis regulates disturbed flow-induced EndMT, plays an important role in neointimal hyperplasia of AVF, and may act as a potential therapeutic target to prevent AVF dysfunction.

CD44 regulates Epac1-mediated ß-adrenergic-receptor-induced Ca2+-handling abnormalities: implication in cardiac arrhythmias.

BACKGROUND: Sustained, chronic activation of ß-adrenergic receptor (ß-AR) signaling leads to cardiac arrhythmias, with exchange proteins directly activated by cAMP (Epac1 and Epac2) as key mediators. This study aimed to evaluate whether CD44, a transmembrane receptor mediating various cellular responses, participates in Epac-dependent arrhythmias. METHODS: The heart tissue from CD44 knockout (CD44-/-) mice, cultured HL-1 myocytes and the tissue of human ventricle were used for western blot, co-immunoprecipitaiton and confocal studies. Line-scanning confocal imaging was used for the study of cellular Ca2+ sparks on myocytes. Optical mapping and intra-cardiac pacing were applied for arrhythmia studies on mice's hearts. RESULTS: In mice, isoproterenol, a ß-AR agonist, upregulated CD44 and Epac1 and increased the association between CD44 and Epac1. Isoproterenol upregulated the expression of phospho-CaMKII (p-CaMKII), phospho-ryanodine receptor (p-RyR), and phospho-phospholamban (p-PLN) in mice and cultured myocytes; these effects were attenuated in CD44-/- mice compared with wild-type controls. In vitro, isoproterenol, 8-CPT-cAMP (an Epac agonist), and osteopontin (a ligand of CD44) significantly upregulated the expression of p-CaMKII, p-RyR, and p-PLN; this effect was attenuated by CD44 small interfering RNA (siRNA). In myocytes, resting Ca2+ sparks were induced by isoproterenol and overexpressed CD44, which were prevented by inhibiting CD44. Ex vivo optical mapping and in vivo intra-cardiac pacing studies showed isoproterenol-induced triggered events and arrhythmias in ventricles were prevented in CD44-/- mice. The inducibility of ventricular arrhythmias (VAs) was attenuated in CD44-/- HF mice compared with wild-type HF controls. In patients, CD44 were upregulated, and the association between CD44 and Epac1 were increased in ventricles with reduced contractility. CONCLUSION: CD44 regulates ß-AR- and Epac1-mediated Ca2+-handling abnormalities and VAs. Inhibition of CD44 is effective in reducing VAs in HF, which is potentially a novel therapeutic target for preventing the arrhythmias and sudden cardiac death in patients with diseased hearts.

HYPERREFLECTIVE FOCI AS IMPORTANT PROGNOSTIC INDICATORS OF PROGRESSION OF RETINITIS PIGMENTOSA.

PURPOSE: To investigate the presence and clinical relevance of hyperreflective foci (HRFs) in retinitis pigmentosa. METHODS: Seventy seven retinitis pigmentosa cases were retrospectively reviewed. The 10-mm wide cross-line macular scans in optical coherence tomography were acquired. Hyperreflective foci were classified according to the location in optical coherence tomography: outer layers within the macula (HRF-outer-central), macular border beyond the central 3 mm (HRF-outer-perifoveal), and choroid (HRF-choroidal). The visual acuity at baseline, at 12 months, and other fundus characteristics were collected. RESULTS: The mean logMAR best-corrected visual acuity decreased from 0.59 ± 0.66 (20/78 in Snellen) to 0.74 ± 0.81 (20/106 in Snellen) in 1 year. Sixty-six (42.9%), 105 (68.2%), and 98 (63.6%) eyes were classified to HRF-outer-central, HRF-outer-perifoveal, and HRF-choroidal group, respectively. Hyperreflective foci were positively correlated with poorer vision, central macular thinning, and ellipsoid zone disruption (all P < 0.001). Worse vision was associated with older age, macular involvement, and the coexistence of two or three HRF groups (P = 0.014, 0.047, 0.019, <0.001, respectively). Hyperreflective foci developed more frequently in patients with thick choroid than in those with thin choroid. The coexistence of three HRF groups was correlated with quicker visual deterioration (P = 0.034). CONCLUSION: Hyperreflective foci are common in retinitis pigmentosa and can be a negative prognostic indicator of macular thickness and visual preservation. Thick choroid was associated with all groups of HRFs, especially HRF-choroidal.

Cathepsin S Inhibition Suppresses Experimental Systemic Lupus Erythematosus-Associated Pulmonary Arterial Remodeling.

Patients with systemic lupus erythematosus (SLE) associated with pulmonary arterial hypnertension (PAH) receive targeted therapy for PAH to decrease pulmonary arterial systolic pressure and significantly prolong their survival. Cysteine cathepsin proteases play critical roles in the progression of cardiovascular disease. Inhibition of cathepsin S (Cat S) has been shown to improve SLE and lupus nephritis. However, the effect of Cat S inhibitors on SLE-associated PAH (SLE-PAH) remains unclear, and there is no animal model for translational research on SLE-PAH. We hypothesized that the inhibition of Cat S may affect PAH development and arterial remodeling associated with SLE. A female animal model of SLE-PAH, female MRL/lpr (Lupus), was used to evaluate the role of pulmonary arterial remodeling in SLE. The key finding of the research work is the establishment of an animal model of SLE associated with PAH in female MRL/lpr mice that is able to evaluate pulmonary arterial remodeling starting from the age of 11 weeks to 15 weeks. Cat S protein level was identified as a marker of experimental SLE. Pulmonary hypertension in female MRL/lpr (Lupus) mice was treated by administering the selective Cat S inhibitor Millipore-219393, which stimulated peroxisome proliferator-activated receptor-gamma (PPARγ) in the lungs to inhibit Cat S expression and pulmonary arterial remodeling. Studies provide an animal model of female MRL/lpr (Lupus) associated with PAH and a deeper understanding of the pathogenesis of SLE-PAH. The results may define the role of cathepsin S in preventing progressive and fatal SLE-PAH and provide approaches for therapeutic interventions in SLE-PAH.

Sirolimus Suppresses Phosphorylation of Cofilin and Reduces Interstitial Septal Thickness in Sporadic Lymphangioleiomyomatosis.

Sporadic lymphangioleiomyomatosis (S-LAM) is a rare lung disease characterized by the proliferation of smooth muscle-like LAM cells and progressive cystic destruction. Sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, has a proven efficacy in patients with LAM. However, the therapeutic mechanisms of sirolimus in LAM remain unclear. We aimed to evaluate sirolimus-related lung parenchymal changes and the potential effect in LAM cells and modulating pathological cystic destruction. Lung specimens were examined for histopathological changes by HMB45 staining and compared the LAM patients treated with and without sirolimus. We detected the overexpression of mTOR, HMB45, and phosphorylation of cofilin (p-cofilin) in LAM patients. Sirolimus showed efficacy in patients with LAM, who exhibited a reduced expression of mTOR and p-cofilin as well as reduced interstitial septal thickness. In addition, sirolimus suppresses mTOR and p-cofilin, thus suppressing the migration and proliferation of LAM cells isolated from the patient's lung tissue. This study demonstrates that interstitial septal thickness, as determined by histological structural analysis. Sirolimus effectively reduced the expression of p-cofilin and interstitial septal thickness, which may be a novel mechanism by sirolimus. Moreover, we develop a new method to isolate and culture the LAM cell, which can test the possibility of medication in vitro and impact this current study has on the LAM field. The development of approaches to interfere with mTOR-cofilin1-actin signaling may result in an option for S-LAM therapy.

Unique wreath-like smooth muscle proliferation of the pulmonary vasculature in pulmonary veno-occlusive disease versus pulmonary arterial hypertension.

BACKGROUND/PURPOSE: Pulmonary veno-occlusive disease (PVOD) is a rare but fatal cause of pulmonary hypertension reported to be linked to mutations of eukaryotic initiation factor 2 alpha kinase 4 (EIF2AK4), also known as general control nonderepressible 2 kinase (GCN2). PVOD is difficult to diagnose and often initially misdiagnosed as other types of idiopathic pulmonary arterial hypertension (IPAH). To rapidly and correctly identify PVOD patients and explore the possible pathogenesis, we thoroughly investigated histopathological features and GCN2 protein levels in non-PAH, PVOD and PAH patients. METHODS: Lung specimens were examined for histopathological changes, including those of pulmonary arteries and veins, by Masson's trichrome, modified Verhoeff's and α-SMA staining in the PVOD, IPAH, and non-PAH groups. GCN2 and α-SMA expression in lung tissue was examined by immunohistochemistry and western blotting. RESULTS: PVOD and IPAH patients showed significant intimal and medial thickening of muscular pulmonary arteries compared with non-PAH patients. PVOD patients had more prominent intimal and medial thickening of muscular pulmonary veins than the other two groups. Interestingly, specialized muscle bundles surrounding the tunica adventitia of the pulmonary artery and vein were observed in PVOD patients. A significant decrease in GCN2 expression in the PVOD group was confirmed by immunohistochemistry and western blotting. CONCLUSION: Our study is the first to show remarkable histological structures, including the wreath-like arrangement of a hyperplastic muscle bundle in the adventitia of pulmonary arteries, in PVOD patients as a diagnostic clue and to disclose the biological difference between PAH and PVOD in a Taiwanese population.

Remodeling Matrix Synthesis in a Rat Model of Aortocaval Fistula and the Cyclic Stretch: Impaction in Pulmonary Arterial Hypertension-Congenital Heart Disease.

Pulmonary arterial hypertension-congenital heart disease (PAH-CHD) is characterized by systemic to pulmonary arterial shunts and sensitively responds to volume overload and stretch of the vascular wall leading to pulmonary vascular remodeling. We hypothesized that the responses of pulmonary artery smooth muscle cells (PASMCs) to mechanical stress-associated volume overload may promote vascular remodeling in PAH-CHD. Here, we show that significantly increased collagen was in the PA adventitial layer by trichrome staining in PAH-CHD patients and an aortocaval fistula (ACF) rat model in which chronic vascular volume overload induced-PAH. We assessed the gene expression profiles of SMC markers, extracellular matrix, and collagen in isolated SMCs from pulmonary and thoracic vessels with cyclic stretch-triggered responses by real-time PCR analysis. The data corresponded to collagen deposition, which modulated pulmonary vascular remodeling in clinical and experimental PAH-ACF cases as well as in cyclic stretch-triggered SMCs in an in vitro model. We observe that collagen I A2 (COLIA2) is expressed in the control rat, but collagen I A1 (COLIA1) and Notchs remarkably increase in the lungs of ACF rats. Interestingly, closing the left-to-right shunt that leads to a reduced blood volume in the PA system of ACF rats (ACFRs) decreased the expression of COLIA1 and increased that of collagen I A2(COLIA2). This study contributes to the stretch-induced responses of SMCs and provides important future directions for therapies aimed at preventing abnormal matrix protein synthesis in volume overload-induced pulmonary hypertension (PH).

Ubiquitin Pathway Is Associated with Worsening Left Ventricle Function after Mitral Valve Repair: A Global Gene Expression Study.

The molecular mechanism for worsening left ventricular (LV) function after mitral valve (MV) repair for chronic mitral regurgitation remains unknown. We wished to assess the LV transcriptome and identify determinants associated with worsening LV function post-MV repair. A total of 13 patients who underwent MV repair for chronic primary mitral regurgitation were divided into two groups, preserved LV function (N = 8) and worsening LV function (N = 5), for the study. Specimens of LV from the patients taken during surgery were used for the gene microarray study. Cardiomyocyte cell line HL-1 cells were transfected with gene-containing plasmids and further evaluated for mRNA and protein expression, apoptosis, and contractile protein degradation. Of 67,258 expressed sequence tags, microarrays identified 718 genes to be differentially expressed between preserved-LVF and worsening-LVF, including genes related to the protein ubiquitination pathway, bone morphogenetic protein (BMP) receptors, and regulation of eIF4 and p70S6K signaling. In addition, worsening-LVF was associated with altered expressions of genes pathologically relevant to heart failure, such asdownregulated apelin receptors and upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). HL-1 cardiomyocyte cells transfected with ubiquitination-related genes demonstrated activation of the protein ubiquitination pathwaywith an increase in the ubiquitin activating enzyme E1 (UAE-E1). It also led to increased apoptosis, downregulated and ubiquitinated X-linked inhibitor of apoptosis protein (XIAP), and reduced cell viability. Overexpression of ubiquitination-related genes also resulted in degradation and increased ubiquitination of α-smooth muscle actin (SMA). In conclusion, worsening-LVF presented differential gene expression profiles from preserved-LVF after MV repair. Upregulation of protein ubiquitination-related genes associated with worsening-LVF after MV repair may exert adverse effects on LV through increased apoptosis and contractile protein degradation.

Tachycardia-induced CD44/NOX4 signaling is involved in the development of atrial remodeling.

Atrial fibrillation (AF) is associated with oxidative stress and Ca2+-handling abnormalities in atrial myocytes. Our prior study has demonstrated the involvement of CD44, a membrane receptor for hyaluronan (HA), in the pathogenesis of AF. This study further evaluated whether CD44 and its related signaling mediate atrial tachycardia-induced oxidative stress and Ca2+-handling abnormalities. Tachypacing in atrium-derived myocytes (HL-1 cell line) induced the activation of CD44-related signaling, including HA and HA synthase (HAS) expression. Blocking HAS/HA/CD44 signaling attenuated tachypacing-induced oxidative stress (NADPH oxidase [NOX] 2/4 expression) and Ca2+-handling abnormalities (oxidized Ca2+/calmodulin-dependent protein kinase II [ox-CaMKII] and phospho-ryanodine receptor type 2 [p-RyR2] expression) in HL-1 myocytes. Furthermore, a direct association between CD44 and NOX4 was documented in tachy-paced HL-1 myocytes and atrial tissues from AF patients. In vitro, Ca2+ spark frequencies in atrial myocytes isolated from CD44-/- mice were lower than those from wild-type mice. Furthermore, administration of an anti-CD44 blocking antibody in atrial myocytes isolated from wild-type mice diminished the frequency of Ca2+ spark. Ex vivo tachypacing models of CD44-/- mice exhibited a lower degree of oxidative stress and expression of ox-CaMKII/p-RyR2 in their atria than those of wild-type mice. In vivo, burst atrial pacing stimulated a less inducibility of AF in CD44-/-mice than in wild-type mice. In conclusion, atrial tachypacing-induced Ca2+-handling abnormalities are mediated via CD44/NOX4 signaling, which provides a possible explanation for the development of AF.

CSC-3436 inhibits TWIST-induced epithelial-mesenchymal transition via the suppression of Twist/Bmi1/Akt pathway in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer deaths worldwide, especially in male. With poor prognosis, significant portions of patients with HNSCC die due to cancer recurrence and tumor metastasis after chemotherapy and targeted therapies. The HNSCC FaDu cell ectopic expression of Twist, a key transcriptional factor of epithelial-mesenchymal transition (EMT), which triggers EMT and results in the acquisition of a mesenchymal phenotype, was used as the cell model. Our results demonstrated that treatment with newly synthesized 2-(3-hydroxyphenyl)-5-methylnaphthyridin-4-one (CSC-3436), a flavonoid derivative, elicited changes in its cell morphology, upregulated E-cadherin messenger RNA and protein expression, downregulated N-cadherin, vimentin, and CD133 (a marker associated with tumor-initiating cells) in FaDu-pCDH-Twist cells. Moreover, CSC-3436 exposure reduced B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) expression regulated by Twist and further suppressed the direct co-regulation of E-cadherin by Twist and Bmi1. Interestingly, CSC-3436 reduced EMT, cancer stemness, and migration/invasion abilities through the inhibition of the Twist/Bmi1-Akt/ß-catenin pathway. Most importantly, our findings provided new evidence that CSC-3436 played a crucial role in therapeutic targeting to Bmi1 and its molecular pathway in HNSCC, and it will be valuable in prognostic prediction and treatment.

Cathepsin S promotes the development of pulmonary arterial hypertension.

Cysteine cathepsin proteases play critical roles in cardiovascular disease progression and are implicated in extracellular matrix (ECM) degradation. Patients with pulmonary arterial hypertension (PAH) exhibit increased elastase production by pulmonary arterial smooth muscle cells (PASMCs), which is related to the degradation of elastic fibers and pulmonary vascular remodeling. However, the mechanism by which cathepsins regulate the ECM and PASMC proliferation in PAH remains unclear. We hypothesized that cathepsin proteases in PASMCs promote the development of PAH. Here, we show overexpression of cathepsin S (Cat S) and degradation of elastic laminae in the lungs of patients with idiopathic PAH and in the PASMCs of monocrotaline-induced PAH model (MCT-PAH) rats. In addition, pulmonary hypertension can be treated in MCT-PAH rats by administering a selective Cat S inhibitor, Millipore-219393, which stimulates peroxisome proliferator-activated receptor-γ (PPARγ) to inhibit the expression of Cat S, thus suppressing the proliferation and migration of MCT-PAH PASMCs. We then reduced Cat S or PPARγ expression by using small interfering RNA in human PASMCs to demonstrate a mechanistic link between Cat S signaling and PPARγ protein, and the results suggest that PPARγ is upstream of Cat S signaling. In conclusion, the activity of Cat S in pulmonary vascular remodeling and degradation of elastin fibers through the disruption of PPARγ is pathophysiologically significant in PAH.

Atrial fibrillation and its arrhythmogenesis associated with insulin resistance.

BACKGROUND: Insulin resistance (IR) is considered as a risk factor for atrial fibrillation (AF) even before diabetes develops. The pathophysiology and underlying mechanism are largely unclear. METHODS: We investigated the corresponding mechanism in two IR models of rats fed 15-week high-fat (HFa) and high-fructose/cholesterol (HFr) diets. AF was evaluated and induced by burst atrial pacing. Isolated atrial myocytes were used for whole-cell patch clamp and calcium assessment. Ex vivo whole heart was used for optical mapping. Western blot and immunofluorescence were used for quantitative protein evaluation. RESULTS: Both HFa and HFr rat atria were vulnerable to AF evaluated by burst atrial pacing. Isolated atrial myocytes from HFa and HFr rats revealed significantly increased sarcoplasmic reticulum calcium content and diastolic calcium sparks. Whole-heart mapping showed prolonged calcium transient duration, conduction velocity reduction, and repetitive ectopic focal discharge in HFa and HFr atria. Protein analysis revealed increased TGF-ß1 and collagen expression; increased superoxide production; abnormal upregulation of calcium-homeostasis-related proteins, including oxidized CaMKIIδ, phosphorylated-phospholamban, phosphorylated-RyR-2, and sodium-calcium exchanger; and increased Rac1 activity in both HFa and HFr atria. We observed that inhibition of CaMKII suppressed AF in both HF and HFr diet-fed rats. In vitro palmitate-induced IR neonatal cardiomyocytes and atrial fibroblasts expressed significantly more TGF-ß1 than did controls, suggesting paracrine and autocrine effects on both myocytes and fibroblasts. CONCLUSIONS: IR engenders both atrial structural remodeling and abnormal intracellular calcium homeostasis, contributing to increased AF susceptibility. The inhibition of CaMKII may be a potential therapeutic target for AF in insulin resistance.

Prostanoid EP4 agonist L-902,688 activates PPARγ and attenuates pulmonary arterial hypertension.

Prostacyclin agonists that bind the prostacyclin receptor (IP) to stimulate cAMP synthesis are effective vasodilators for the treatment of idiopathic pulmonary arterial hypertension (IPAH), but this signaling may occur through nuclear peroxisome proliferator-activated receptor-γ (PPARγ). There is evidence of scant IP and PPARγ expression but stable prostanoid EP4 receptor (EP4) expression in IPAH patients. Both IP and EP4 functionally couple with stimulatory G protein (Gs), which activates signal transduction. We investigated the effect of an EP4-specific agonist on pulmonary arterial remodeling and its regulatory mechanisms in pulmonary arterial smooth muscle cells (PASMCs). Immunoblotting evealed IP, EP4, and PPARγ expression in human pulmonary arterial hypertension (PAH) and monocrotaline (MCT)-induced PAH rat lung tissue. Isolated PASMCs from MCT-induced PAH rats (MCT-PASMCs) were treated with L-902,688, a selective EP4 agonist, to investigate the anti-vascular remodeling effect. Scant expression of IP and PPARγ but stable expression of EP4 was observed in IPAH patient lung tissues and MCT-PASMCs. L-902,688 inhibited IP-insufficient MCT-PASMC proliferation and migration by activating PPARγ in a time- and dose-dependent manner, but these effects were reversed by AH-23848 (an EP4 antagonist) and H-89 [a protein kinase A (PKA) inhibitor], highlighting the crucial role of PPARγ in the activity of this EP4 agonist. L-902,688 attenuated pulmonary arterial remodeling in hypoxic PAH mice and MCT-induced PAH rats; therefore, we conclude that the selective EP4 agonist L-902,688 reverses vascular remodeling by activating PPARγ. This study identified a novel EP4-PKA-PPARγ pathway, and we propose EP4 as a potential therapeutic target for PAH.

Photocytotoxic Copper(II) Complexes with Schiff-Base Scaffolds for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising and minimally invasive method for the treatment of superficial diseases, and photosensitizers with high phototoxicity indices (defined as (IC50dark )/(IC50irradiation )) are essential for the development of ideal photosensitizing properties for this technology. Herein, we report a series of photocytotoxic copper(II) complexes [Cu(R QYMP)(dppn)] (R QYMP=N,N,O-tridentate Schiff-base derivatives, dppn=benzo[i]dipyrido[3,2-a;2',3'-c]phenazine), the structures of which have been confirmed by mass spectrometry and FTIR spectroscopy. X-ray crystallography revealed that the CuN4 O core of the [Cu(cumyl QYMP)(dppn)](ClO4 ) complex (3) has a distorted square-pyramidal geometry. Phototoxicity indices of 329 against human squamous cell carcinoma (SCC15) and 296 against basal cell carcinoma (BCC) cell lines have been determined with [Cu(3-OMe QYMP)(dppn)](ClO4 ) (4). This can be attributed to the formation of reactive oxygen species, cell apoptosis, and caspase-3 activation, indicating high potential of complex 4 as a photosensitizer candidate in PDT. Thus, copper complexes bearing suitable Schiff-base ligands with a dppn co-ligand may be considered for the design of efficient metal-based anticancer agents for PDT.

Codependence of Bone Morphogenetic Protein Receptor 2 and Transforming Growth Factor-ß in Elastic Fiber Assembly and Its Perturbation in Pulmonary Arterial Hypertension.

OBJECTIVE: We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells previously reported, PA elastic fibers are susceptible to degradation because of their abnormal assembly. APPROACH AND RESULTS: Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-ß1 (TGFß1). Thus, we considered whether BMPs like TGFß1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA smooth muscle cells and adventitial fibroblasts, TGFß1 increased elastin mRNA, but the elevation in elastin protein was dependent on BMPR2; TGFß1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFß1-stimulated elastic fiber assembly was impaired in idiopathic (I) PAH-PA adventitial fibroblast versus control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PA adventitial fibroblast by TGFß1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PA adventitial fibroblasts stimulated with BMP4 or TGFß1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension. CONCLUSIONS: Disrupting BMPR2 impairs TGFß1- and BMP4-mediated elastic fiber assembly and is of pathophysiologic significance in PAH.

EP4 Agonist L-902,688 Suppresses EndMT and Attenuates Right Ventricular Cardiac Fibrosis in Experimental Pulmonary Arterial Hypertension.

Right ventricular (RV) hypertrophy is characterized by cardiac fibrosis due to endothelial-mesenchymal transition (EndMT) and increased collagen production in pulmonary arterial hypertension (PAH) patients, but the mechanisms for restoring RV function are unclear. Prostanoid agonists are effective vasodilators for PAH treatment that bind selective prostanoid receptors to modulate vascular dilation. The importance of prostanoid signaling in the RV is not clear. We investigated the effects of the EP4-specific agonist L-902,688 on cardiac fibrosis and TGF-ß-induced EndMT. EP4-specific agonist treatment reduced right ventricle fibrosis in the monocrotaline (MCT)-induced PAH rat model. L-902,688 (1 µM) attenuated TGF-ß-induced Twist and α-smooth muscle actin (α-SMA) expression, but these effects were reversed by AH23848 (an EP4 antagonist), highlighting the crucial role of EP4 in suppressing TGF-ß-induced EndMT. These data indicate that the selective EP4 agonist L-902,688 attenuates RV fibrosis and suggest a potential approach to reducing RV fibrosis in patients with PAH.

Rosuvastatin suppresses atrial tachycardia-induced cellular remodeling via Akt/Nrf2/heme oxygenase-1 pathway.

Atrial fibrillation (AF) is associated with structural remodeling in atrial myocytes. Emerging evidence suggests that statin has a protective effect on AF through cholesterol-independent mechanisms. The aim of this study is to investigate whether heme oxygenase-1 (HO-1), a potent antioxidant system, mediates the suppressive effect of statin on atrial tachycardia-induced structural remodeling. Treatment of cultured atrium-derived myocytes (HL-1 cell line) with rosuvastatin enhanced HO-1 expression/activity and attenuated tachypacing-induced oxidative stress and myofibril degradation. Heme oxygenase-1 inhibitors and small-interfering RNA for HO-1 blocked the inhibitory effect of rosuvastatin on tachypacing-stimulated changes, suggesting the crucial role of HO-1 in mediating the effect of rosuvastatin. Time-dependent experiments and loss-of-function study demonstrated that Akt/Nrf2 pathways lay to the up-stream of HO-1 in this signaling cascade. Furthermore, the involvement of Akt/Nrf2/HO-1 pathway in the antioxidant effect of rosuvastatin was documented in an ex vivo tachypacing model. The suppressive effect of statin on atrial tachypacing-induced cellular remodeling is mediated via the activation of Akt/Nrf2/HO-1 signaling, which provides a possible explanation for the protective effect of statin on AF.

A Bayesian approach to differential edges with probabilistic interactions: applications in association and classification.

Motivation: Differential network (D-Net) analysis has attracted great attention in systems biology for its ability to identify genetic variations in response to different conditions. Current approaches either estimate the condition-specific networks separately followed by post-procedures to determine the differential edges or estimate the D-Net directly. Both types of analysis overlook the probabilistic inference and can only provide deterministic inference of the edges. Results: Here, we propose a Bayesian solution and translate the probabilistic estimation in the regression model to an inferential D-Net analysis for genetic association and classification studies. The proposed PRobabilistic Interaction for Differential Edges (PRIDE) focuses on inferring the D-Net with uncertainty so that the existence of the differential edges can be evaluated with probability and even prioritized if comparison among these edges is of interest. The performance of the proposed model is compared with state-of-the-art methods in simulations and is demonstrated in glioblastoma and breast cancer studies. The proposed PRIDE performs comparably to or outperforms most existing tools under deterministic evaluation criteria. Additionally, it offers the unique advantages, including prioritizing the differential edges with probabilities, highlighting the relative importance of hub nodes, and identifying potential sub-networks in a D-Net. Availability and implementation: All the data analyzed in this research can be downloaded at https://xenabrowser.net/datapages/. The R code for implementing PRIDE is available at https://github.com/YJGene0806/PRIDE_Code.