Androgen deprivation therapy duration is significantly associated with Testosterone recovery in Japanese patients with prostate cancer.

OBJECTIVE: Due to the fear generated by COVID-19 in Spring 2020, many patients postponed their scheduled outpatient visits. To differentiate those patients with prostate cancer (PCa) whose androgen deprivation therapy (ADT) injection treatment can be postponed, we investigated the characteristics of testosterone (T) recovery in Japanese patients after they received combined ADT and radiation therapy (RT). METHODS: We included 81 patients with PCa treated with ADT and RT at Keio University Hospital from January 2013 to December 2018. T-recovery was defined as the time interval between the last ADT injection and 3-6 months after T-normalization. The Kaplan-Meier method was used to estimate time to T-recovery. Cox proportional hazards models identified T-recovery predictors. RESULTS: The 50% cumulative incidence of T-recovery was 7.0 months for the 6-short-term group (defined as patients having ≤6 months of ADT therapy) versus 13.0 months for the 6-long-term group (>6 months of therapy) (p < 0.001). The incidence was 7.0 months for the 12 short-term-ADT (ST) group versus 18.0 months for the 12 long-term-ADT (LT) group (p < 0.001). Multivariate analysis revealed that a shorter duration of ADT was associated with a shorter time to T-recovery (hazard ratio, 0.253; 95% CI, 0.138-0.465; p < 0.001). No other factors were significant predictors of T-recovery. CONCLUSION: Androgen deprivation therapy duration is significantly associated with T-recovery in Japanese patients with PCa. If a patient undergoes ADT for more than 6 or 12 months, it is possible to postpone their outpatient visits for 13 and 18 months, respectively.

Urodynamics findings pre- and post-untethering surgery in children with filum lipoma: A single-institution experience.

OBJECTIVES: We aimed to investigate the changes in urodynamics and voiding cystourethrogram parameters on pre- and post-untethering surgery in patients aged under 2 years with filum lipoma. METHODS: Sixty-two patients were enrolled in this study. The changes in urodynamics and voiding cystourethrogram parameters were compared before untethering surgery and 6 months after untethering surgery. These parameters were bladder volume, bladder deformity, vesicoureteral reflux during voiding cystourethrogram, detrusor overactivity, bladder compliance, and post-void residual volume in urodynamics. RESULTS: Bladder volume during voiding cystourethrogram and bladder compliance increased significantly from 89.8 ± 49.5 mL to 114.5 ± 50.5 mL (P = 0.0069) and 10.2 ± 6.2 mL/mmH2 O to 17.0 ± 13.3 mL/mmH2 O (P = 0.0008), respectively, at 6-month follow-up. Six patients required combination management with clean intermittent catheterization at 25.1 ± 8.2 months (14.3 ± 6.5-months follow-up) because of elevated post-void residual volumes. CONCLUSIONS: According to voiding cystourethrogram results, bladder function and urodynamics in patients with filum lipoma significantly improved after untethering surgery. Non-invasive assessment based on measurements of post-void residual should be considered as a postoperative follow-up method.

COVID-19 mRNA Vaccine-induced Pneumonitis.

A 65-year-old man experienced cough and shortness of breath 3 days after receiving the first dose of the Pfizer-BioNTech coronavirus disease 2019 (COVID-19) vaccine. Chest X-ray revealed bilateral infiltrates, and the desaturation deteriorated rapidly. The symptoms and radiographic abnormalities rapidly improved after the initiation of corticosteroid therapy. Intradermal testing of the Pfizer-BioNTech COVID-19 vaccine showed a delayed positive reaction. Based on these findings, the patient was diagnosed with COVID-19 vaccine-induced pneumonitis. The timing of the onset of pneumonitis after vaccination and the results of intradermal testing suggest that Type IV hypersensitivity against COVID-19 vaccine may have been responsible for this clinical condition.

Quantitative Analysis of Iodine Image of Dual-energy Computed Tomography at Rest: Comparison With 99mTc-Tetrofosmin Stress-rest Single-photon Emission Computed Tomography Myocardial Perfusion Imaging as the Reference Standard.

PURPOSE: Dual-energy computed tomography (DECT) can be used for visual determination of iodine distribution in the myocardium (iodine image); however, the accuracy and reproducibility of the process remains debatable. Because of the low contrast-to-noise ratio of CT, we hypothesized that quantitative measurement may be more accurate for detecting myocardial ischemia. In this study, we evaluated our quantitative method by comparing it with visual analysis using Tc-tetrofosmin (TF) stress-rest single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) as the reference standard. MATERIALS AND METHODS: Forty-three patients who had a significant stenosis on cardiac rest DECT and had received Tc-TF stress-rest SPECT MPI within 1 month were retrospectively analyzed. The regions of interest were set on iodine images in accordance with the American Heart Association (AHA) 17-segment model (a total of 731 segments). The regions of interest values were divided by the amount of iodine (mg) per unit weight (kg) and defined as perfusion value (perfusion value analysis). All segments were also visually analyzed and receiver operating characteristic curve analysis performed to identify the superior analysis. RESULTS: The receiver operating characteristic curve analysis showed that perfusion value analysis is significantly superior to visual analysis [the area under the curve: 0.921 (95% confidence interval, 0.860-0.981) versus 0.685 (95% confidence interval, 0.580-0.791), respectively, P<0.05], with 93.8% sensitivity, 99.1% specificity, 98.9% accuracy, 83.3% positive predictive value, and 99.7% negative predictive value (P<0.01). CONCLUSIONS: Quantitative analysis of the iodine image of rest DECT, called perfusion value analysis, is more accurate than visual analysis when compared with Tc-TF SPECT MPI as the reference standard.

Fibroblast growth factor 23 inhibits osteoblastic gene expression and induces osteoprotegerin in vascular smooth muscle cells.

BACKGROUND AND AIMS: Elevated fibroblast growth factor 23 (FGF23) levels are associated with cardiovascular mortality in patients with chronic kidney disease. However, both clinical and basic research have demonstrated conflicting evidence regarding the pathophysiological role of FGF23 in vascular calcification. The aim of this study was to determine the role of FGF23 in the osteoblastic gene expression in vascular smooth muscle cells (SMCs). METHODS AND RESULTS: We transduce human aortic SMCs (HASMCs) expressing klotho and FGF receptors with the adenovirus expressing human FGF23 (Ad-FGF23). We observed significant decreases in the expression of osteoblast-marker genes including BMP2, BMP4, MSX2, RUNX2 and ALP, as well as reduced calcification. Notably, Ad-FGF23 increased mRNA and protein levels of osteoprotegerin (OPG), and human OPG promoter was activated by FGF23. Moreover, in HASMCs overexpressing klotho, FGF23 upregulated OPG expression, whereas depletion of klotho by siRNA attenuated FGF23-induced OPG expression. Furthermore, in 73 consecutive patients with type 2 diabetes mellitus undergoing cardiac computed tomography to determine coronary calcium scores (CCSs), serum FGF23 levels were positively correlated with OPG independent of phosphate and estimated glomerular filtration rate (eGFR, r = 0.65, p < 0.01). Serum FGF23 levels were significantly elevated in patients with high CCSs (≧100) compared to those with low CCSs (<100). CONCLUSIONS: Our in vitro results indicate that FGF23 suppresses osteoblastic gene expression and induces OPG expression in HASMCs. Together with our cross-sectional clinical assessment, the present study lends support to our hypothesis that FGF23 counteracts osteogenic conversion of vascular SMCs as a part of a compensatory mechanism to mitigate vascular calcification.

Reversible Cardiomyopathy After Epirubicin Administration.

Anthracycline-containing chemotherapy can cause irreversible and progressive left ventricular dysfunction. Epirubicin, which is widely used for breast cancer chemotherapy, is an anthracycline that has less cardiac toxicity than doxorubicin. The present report describes the case of a 70-year-old woman with breast cancer who developed severe congestive heart failure and severe cardiac dysfunction at 6 weeks from epirubicin final administration. Left ventricular function gradually improved after intensive treatment for heart failure and recovered completely within 2 months. To the best of our knowledge, this is the first report to describe epirubicin-induced subacute reversible cardiotoxicity.

Left atrial strain provides incremental value for embolism risk stratification over CHA2DS2-VASc score and indicates prognostic impact in patients with atrial fibrillation.

BACKGROUND: The aim of this study was to investigate whether left atrial (LA) strain has incremental value over the CHA2DS2-VASc score for stratifying the risk for embolism in patients with atrial fibrillation (AF) and whether LA strain predicts poststroke mortality. METHODS: Consecutive patients with paroxysmal or persistent AF with acute embolism (82 patients) or without (204 controls) were prospectively enrolled. Global peak LA longitudinal strain during ventricular systole (LAS) was assessed during AF rhythm. Global LAS was compared between the groups in the first cross-sectional study. Then, the 82 patients with acute embolism were prospectively followed during the second prospective cohort study. RESULTS: Global LAS was lower in patients with acute embolism than in controls (P < .001). Global LAS < 15.4% differentiated patients with acute embolism from controls, with an area under the curve of 0.83 (P < .0001). In multivariate analysis, global LAS was independently associated with acute embolism (odds ratio, 0.74; 95% confidence interval, 0.67-0.82; P < .001) and had an incremental value over the CHA2DS2-VASc score (P < .0001). Furthermore, 26 patients with acute embolisms died during a median follow-up period of 425 days. Global LAS independently predicted mortality after embolism. CONCLUSIONS: In this observational study, LA strain provided incremental diagnostic information over that provided by the CHA2DS2-VASc score, suggesting that LA strain analysis could improve the current risk stratification of embolism in patients with AF. LA strain can also predict poststroke mortality.

The time-adjusted gradual replacement injection method enables better visualization of the right heart.

BACKGROUND: Despite the improvement of cardiac CT, right heart visualization remains challenging. OBJECTIVE: We herein describe a new method, called the time-adjusted gradual replacement injection protocol. The aim of this study was to compare this protocol with the split-bolus injection protocol. METHODS: Fifty-two patients who had undergone dual-source cardiac CT were retrospectively recruited. Twenty-six patients were injected by using the split-bolus injection protocol, and 26 patients were injected by using the time-adjusted gradual replacement injection protocol. For this method, we injected contrast medium for 10 seconds at a flow rate of 0.07 × body weight mL/s, then gradually replaced the contrast material with saline until 2 seconds before finishing the scans. The CT attenuation was measured in 4 chambers, the aorta, and the coronary arteries. The visualization of the anatomic structures and the occurrence and severity of streak artifacts were scored for the cardiac structures in the heart. For the analyses, either Welch t-test or Student t-test was performed. RESULTS: In the right heart, the CT values and visualization scores were significantly higher in the time-adjusted replacement injection group than in the split-bolus injection group, whereas the artifact scores were comparable between the 2 groups. The CT values, visualization scores, and artifact scores of the left heart were not significantly different between the 2 groups. CONCLUSIONS: In this study, the time-adjusted gradual replacement injection protocol provided excellent attenuation for visualization of the right heart. This method may help to accurately evaluate the right cardiac anatomy and thereby identify any potential diseases.

Capillary endothelial fatty acid binding proteins 4 and 5 play a critical role in fatty acid uptake in heart and skeletal muscle.

OBJECTIVE: Fatty acids (FAs) are the major substrate for energy production in the heart. Here, we hypothesize that capillary endothelial fatty acid binding protein 4 (FABP4) and FABP5 play an important role in providing sufficient FAs to the myocardium. APPROACH AND RESULTS: Both FABP4/5 were abundantly expressed in capillary endothelium in the heart and skeletal muscle. The uptake of a FA analogue, 125I-15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid, was significantly reduced in these tissues in double-knockout (DKO) mice for FABP4/5 compared with wild-type mice. In contrast, the uptake of a glucose analogue, 18F-fluorodeoxyglucose, was remarkably increased in DKO mice. The expression of transcripts for the oxidative catabolism of FAs was reduced during fasting, whereas transcripts for the glycolytic pathway were not altered in DKO hearts. Notably, metabolome analysis revealed that phosphocreatine and ADP levels were significantly lower in DKO hearts, whereas ATP content was kept at a normal level. The protein expression levels of the glucose transporter Glut4 and the phosphorylated form of phosphofructokinase-2 were increased in DKO hearts, whereas the phosphorylation of insulin receptor-ß and Akt was comparable between wild-type and DKO hearts during fasting, suggesting that a dramatic increase in glucose usage during fasting is insulin independent and is at least partly attributed to the post-transcriptional and allosteric regulation of key proteins that regulate glucose uptake and glycolysis. CONCLUSIONS: Capillary endothelial FABP4/5 are required for FA transport into FA-consuming tissues that include the heart. These findings identify FABP4/5 as promising targets for controlling the metabolism of energy substrates in FA-consuming organs that have muscle-type continuous capillary.

Incremental diagnostic value of la strain with leg lifts in heart failure with preserved ejection fraction.

OBJECTIVES: The purposes of this study were to examine left atrial (LA) functional reserve in patients with heart failure (HF) with preserved ejection fraction (HFpEF) and to determine whether LA strain has an incremental diagnostic value over clinical and conventional echocardiographic parameters. BACKGROUND: Patients with HFpEF have multiple cardiovascular reserve abnormalities. Although the LA is dysfunctional in HFpEF, the diagnostic value of LA strain remains unknown. METHODS: The LA at rest and during passive leg lifts was echocardiographically assessed in 40 patients with HFpEF and in 46 patients with hypertension without HF (HT controls). Global peak atrial longitudinal strain during ventricular systole (global LAS) and booster strain during atrial contraction (global LAB) were assessed using speckle tracking. RESULTS: Patients with HFpEF had an enlarged LA and reduced LA emptying fraction compared with HT controls at rest, while LA stroke volume (SV) was similar between the groups. During leg lifts, increases in LA reservoir and contractile function (i.e., global LAS and LAB) were blunted in HFpEF patients compared with HT controls, resulting in impaired LASV responses. Global LAS and LAB during leg lifts accurately differentiated HFpEF from HT controls (areas under the curve: 0.95 and 0.92, respectively). Resting global LAS had a significant incremental diagnostic value over clinical (age and sex) and conventional echocardiographic parameters (E/E' ratio, left ventricular mass index, and maximum LA volume index) (global chi-square: 49.6 vs. 30.8; p < 0.0001). The diagnostic value was further improved by adding global LAS during leg lifts (global chi-square: 72.2 vs. 49.6; p < 0.0001). CONCLUSIONS: An enlarged LA compensates for LA dysfunction and maintains LASV at rest in patients with HFpEF. However, depressed LA reserve affects LA performance during leg lifts. Evaluation of LA function, including LA strain using leg lifts, might provide incremental diagnostic value for HFpEF.

Peroxisome proliferator-activated receptor-γ in capillary endothelia promotes fatty acid uptake by heart during long-term fasting.

BACKGROUND: Endothelium is a crucial blood-tissue interface controlling energy supply according to organ needs. We investigated whether peroxisome proliferator-activated receptor-γ (PPARγ) induces expression of fatty acid-binding protein 4 (FABP4) and fatty acid translocase (FAT)/CD36 in capillary endothelial cells (ECs) to promote FA transport into the heart. METHODS AND RESULTS: Expression of FABP4 and CD36 was induced by the PPARγ agonist pioglitazone in human cardiac microvessel ECs (HCMECs), but not in human umbilical vein ECs. Real-time PCR and immunohistochemistry of the heart tissue of control (Pparg(fl/null)) mice showed an increase in expression of FABP4 and CD36 in capillary ECs by either pioglitazone treatment or 48 hours of fasting, and these effects were not found in mice deficient in endothelial PPARγ (Pparg(▵)(EC)(/null)). Luciferase reporter constructs of the Fabp4 and CD36 promoters were markedly activated by pioglitazone in HCMECs through canonical PPAR-responsive elements. Activation of PPARγ facilitated FA uptake by HCMECs, which was partially inhibited by knockdown of either FABP4 or CD36. Uptake of an FA analogue, (125)I-BMIPP, was significantly reduced in heart, red skeletal muscle, and adipose tissue in Pparg(▵)(EC)(/null) mice as compared with Pparg(fl/null) mice after olive oil loading, whereas those values were comparable between Pparg(fl/null) and Pparg(▵)(EC)(/null) null mice on standard chow and a high-fat diet. Furthermore, Pparg(▵)(EC)(/null) mice displayed slower triglyceride clearance after olive oil loading. CONCLUSIONS: These findings identified a novel role for capillary endothelial PPARγ as a regulator of FA handing in FA-metabolizing organs including the heart in the postprandial state after long-term fasting.

Multiple papillary fibroelastomas of the aortic valve detected by real time three-dimensional transesophageal echocardiographic images.

Papillary fibroelastoma (PFE) is a well-known primary cardiac tumor, but multiple PFEs are rare. We report an interesting case with multiple PFEs that were clearly demonstrated and evaluated with real time three-dimensional (RT3D) transesophageal echocardiography (TEE). A 77-year-old woman was referred to our institution with a diagnosis of osteoarthritis of the hip. Transthoracic echocardiography showed an abnormal structure on the aortic valve. Although two-dimensional TEE revealed typical characteristics of multiple PFE, RT3D TEE clearly demonstrated their number and location on the right and non-coronary cusp of the aortic valve. These results were subsequently confirmed by surgery and pathological findings. RT3D TEE is an exceptionally useful tool for pre-surgical evaluation of PFE. .

Pulmonary hypertension with extensive calcification in small pulmonary vessels and alveolar capillary wall in a chronic hemodialysis patient.

We report a case of a 41-year-old male with end-stage renal disease receiving chronic hemodialysis who was referred to this hospital because of dyspnea. He had been on a regular dialysis for 20 years due to chronic glomerulonephritis. His transthoracic echocardiography revealed severe pulmonary hypertension (PH), and cardiac catheterization confirmed this diagnosis. From clinical examination and review of the chest X-ray and computed tomography images, we thought PH was due to multifactorial mechanisms typical of hemodialysis patients. However, microscopic examination of lung tissue from autopsy specimen revealed extensive calcium deposits not only in alveolar septal wall but also in alveolar capillaries and small vessels, which had diffuse intimal thickening causing the narrowing of the lumens. These pathological findings suggest that pulmonary vascular calcification contributed to the PH in this patient. .

Pressure mediated hypertrophy and mechanical stretch up-regulate expression of the long form of leptin receptor (ob-Rb) in rat cardiac myocytes.

BACKGROUND: Hyperleptinemia is known to participate in cardiac hypertrophy and hypertension, but the relationship between pressure overload and leptin is poorly understood. We therefore examined the expression of leptin (ob) and the leptin receptor (ob-R) in the pressure-overloaded rat heart. We also examined gene expressions in culture cardiac myocytes to clarify which hypertension-related stimulus induces these genes. RESULTS: Pressure overload was produced by ligation of the rat abdominal aorta, and ob and ob-R isoform mRNAs were measured using a real-time polymerase chain reaction (PCR). We also measured these gene expressions in neonatal rat cardiac myocytes treated with angiotensin II (ANGII), endothelin-1 (ET-1), or cyclic mechanical stretch. Leptin and the long form of the leptin receptor (ob-Rb) gene were significantly increased 4 weeks after banding, but expression of the short form of the leptin receptor (ob-Ra) was unchanged. ob-Rb protein expression was also detected by immunohistochemistry in hypertrophied cardiac myocytes after banding. Meanwhile, plasma leptin concentrations were not different between the control and banding groups. In cultured myocytes, ANGII and ET-1 increased only ob mRNA expression. However, mechanical stretch activated both ob and ob-Rb mRNA expression in a time-dependent manner, but ob-Ra mRNA was unchanged by any stress. CONCLUSIONS: We first demonstrated that both pressure mediated hypertrophy and mechanical stretch up-regulate ob-Rb gene expression in heart and cardiac myocytes, which are thought to be important for leptin action in cardiac myocytes. These results suggest a new local mechanism by which leptin affects cardiac remodeling in pressure-overloaded hearts.

Relation between connective tissue growth factor and cardiac sympathetic nerve activity in heart failure in DCM patients.

Cardiac fibrosis is an important process of myocardial remodeling. Connective tissue growth factor (CTGF) is a cytokine that plays a key role in the occurrence of progressive fibrosis and excessive scarring. CTGF levels are increased in the failing heart. In addition, sympathetic nerve activity is enhanced in the failing heart, and exacerbates heart failure. To clarify the relation between cardiac sympathetic nerve activity and CTGF, we studied 35 (M/F = 28/7 patients) aged 57 ± 15 years with dilated cardiomyopathy (DCM). Cardiac sympathetic nerve activity was estimated from the total defect score (TDS) and from the H/M ratio and washout rate (WR) on I-123-MIBG imaging. Cardiac symptoms (NYHA class), exercise capacity (specific activity scale: SAS), brain natriuretic peptide (BNP), hemodynamics, and CTGF were assessed. There was a significant correlation between the CTGF and WR on I-123-MIBG (r = 0.45, P = 0.008). Also, a higher plasma CTGF level was associated with a lower SAS score (r = 0.51, P = 0.002), but not the TDS, H/M ratio, or BNP concentration. Moreover, a higher NYHA class and pulmonary artery wedge pressure were associated with a higher plasma CTGF level. The plasma CTGF level can be strongly related with cardiac sympathetic nerve activity in heart failure in DCM patients.

Sequestering aromatic molecules with a spin-crossover Fe(II) microporous coordination polymer.

All in a spin: A series of three-dimensional porous coordination polymer {Fe(dpe)[Pt(CN)(4)]}⋅G (dpe = 1,2-di(4-pyridyl)ethylene; G = phenazine, anthracene, or naphthalene) exhibiting spin crossover and host-guest functions is reported. The magnetic properties of the framework are very sensitive to the chemical nature (aromatic or hydroxilic solvents) and the size of the included guest molecules.

Stearoyl-CoA desaturase-1 (SCD1) augments saturated fatty acid-induced lipid accumulation and inhibits apoptosis in cardiac myocytes.

Mismatch between the uptake and utilization of long-chain fatty acids in the myocardium leads to abnormally high intracellular fatty acid concentration, which ultimately induces myocardial dysfunction. Stearoyl-Coenzyme A desaturase-1 (SCD1) is a rate-limiting enzyme that converts saturated fatty acids (SFAs) to monounsaturated fatty acids. Previous studies have shown that SCD1-deficinent mice are protected from insulin resistance and diet-induced obesity; however, the role of SCD1 in the heart remains to be determined. We examined the expression of SCD1 in obese rat hearts induced by a sucrose-rich diet for 3 months. We also examined the effect of SCD1 on myocardial energy metabolism and apoptotic cell death in neonatal rat cardiac myocytes in the presence of SFAs. Here we showed that the expression of SCD1 increases 3.6-fold without measurable change in the expression of lipogenic genes in the heart of rats fed a high-sucrose diet. Forced SCD1 expression augmented palmitic acid-induced lipid accumulation, but attenuated excess fatty acid oxidation and restored reduced glucose oxidation. Of importance, SCD1 substantially inhibited SFA-induced caspase 3 activation, ceramide synthesis, diacylglycerol synthesis, apoptotic cell death, and mitochondrial reactive oxygen species (ROS) generation. Experiments using SCD1 siRNA confirmed these observations. Furthermore, we showed that exposure of cardiac myocytes to glucose and insulin induced SCD1 expression. Our results indicate that SCD1 is highly regulated by a metabolic syndrome component in the heart, and such induction of SCD1 serves to alleviate SFA-induced adverse fatty acid catabolism, and eventually to prevent SFAs-induced apoptosis.

Runx2/Smad3 complex negatively regulates TGF-ß-induced connective tissue growth factor gene expression in vascular smooth muscle cells.

AIM: Connective tissue growth factor (CTGF), a direct target gene of transforming growth factor-ß (TGF-ß) signaling, plays an important role in the development of atherosclerosis. We previously showed that Runx2, a key transcription factor in osteoblast differentiation, regulates osteogenic conversion and dedifferentiation of vascular smooth muscle cells (VSMCs). In this study, we investigated the hypothesis that Runx2 modulates CTGF gene expression via the regulation of TGF-ß signaling. METHODS AND RESULTS: Expression of the Runx2 gene was decreased, and CTGF mRNA levels were reciprocally increased by TGF-ß in a time-dependent manner in cultured human aortic smooth muscle cells (HASMCs) and C3H10T1/2 cells. Forced expression of Runx2 decreased and the reduction of Runx2 expression by small interfering RNA enhanced both basal and TGF-ß-stimulated CTGF gene expression in HASMCs. Site-directed mutation analysis of the CTGF promoter indicated that transcriptional repression by Runx2 was mediated by the Smad-binding element (SBE) under basal and TGF-ß-stimulated conditions. Data obtained from immunoblots of Runx2-, Smad3- or Smad4-transfected cells and chromatin immunoprecipitation analysis indicated that Runx2 interacts with Smad3 at the SBE. Immunohistochemistry revealed that the expression of Runx2 and CTGF was distinct and almost mutually exclusive in human atherosclerotic plaque. CONCLUSIONS: These results for the first time demonstrate that Runx2/Smad3 complex negatively regulates endogenous and TGF-ß-induced CTGF gene expression in VSMCs. Thus, the induction of Runx2 expression contributes to the phenotypic modulation of VSMCs, in which the TGF-ß/Smad pathway plays a major role.

Notch induces myofibroblast differentiation of alveolar epithelial cells via transforming growth factor-{beta}-Smad3 pathway.

Notch is an ancient cell-signaling system that regulates the specification of cell fate. This study examined the role of Notch in the epithelial-mesenchymal transition (EMT) and myofibroblast differentiation of cultured RLE-6TN cells (i.e., rat alveolar epithelial cells). The activation of Notch, either by ectopic expression of the Notch intracellular domain or by the co-culture of RLE-6TN cells with L-Jagged1 cells, induces the expression of smooth muscle α-actin (SMA) and other mesenchymal marker genes (collagen I and vimentin), and reduces the expression of epithelial marker genes (E-cadherin, occludin, and zonula occludens-1). The pharmacologic inhibition of the endogenous Notch signal significantly inhibited the transforming growth factor-ß (TGF-ß)-induced expression of SMA. Cell migratory capacity was increased by Notch. Luciferase assays revealed that the CC(A/T)(6)GG (CArG) box and the TGF-ß control element (TCE) are required for Notch-induced SMA gene transcription. DNA microarray analysis revealed that members of the TGF-ß family as well as Jagged1 were induced in RLE-6TN cells by Notch. Western blot analysis showed that Notch induced the phosphorylation of Smad3, and the TGF-ß receptor type I/activin receptor-like kinase 5 (ALK5) kinase inhibitor SB431542 markedly reduced the Notch-induced expression of SMA. Enzyme-linked immunosorbent assays confirmed the production of TGF-ß1 from RLE-6TN cells by Notch. Immunohistochemistry of a bleomycin-induced model of pulmonary fibrosis and lung specimens from patients with idiopathic interstitial pneumonias showed that Notch was strongly expressed in myofibroblasts, identified as SMA-positive cells. These data indicate that Notch induces myofibroblast differentiation through a TGF-ß-Smad3 pathway that activates SMA gene transcription in a CArG-dependent and TCE-dependent manner in alveolar epithelial cells. Our data also imply that Notch induces the EMT phenotype, with increased migratory behavior in pulmonary fibrosis.