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1.
Circ Heart Fail ; 6(5): 953-63, 2013 Sep 01.
Article in English | MEDLINE | ID: mdl-23797369

ABSTRACT

BACKGROUND: Systemic sclerosis­associated pulmonary artery hypertension (SScPAH) has a worse prognosis compared with idiopathic pulmonary arterial hypertension (IPAH), with a median survival of 3 years after diagnosis often caused by right ventricular (RV) failure. We tested whether SScPAH or systemic sclerosis­related pulmonary hypertension with interstitial lung disease imposes a greater pulmonary vascular load than IPAH and leads to worse RV contractile function. METHODS AND RESULTS: We analyzed pulmonary artery pressures and mean flow in 282 patients with pulmonary hypertension (166 SScPAH, 49 systemic sclerosis­related pulmonary hypertension with interstitial lung disease, and 67 IPAH). An inverse relation between pulmonary resistance and compliance was similar for all 3 groups, with a near constant resistance×compliance product. RV pressure­volume loops were measured in a subset, IPAH (n=5) and SScPAH (n=7), as well as SSc without PH (n=7) to derive contractile indexes (end-systolic elastance [Ees] and preload recruitable stroke work [Msw]), measures of RV load (arterial elastance [Ea]), and RV pulmonary artery coupling (Ees/Ea). RV afterload was similar in SScPAH and IPAH (pulmonary vascular resistance=7.0±4.5 versus 7.9±4.3 Wood units; Ea=0.9±0.4 versus 1.2±0.5 mm Hg/mL; pulmonary arterial compliance=2.4±1.5 versus 1.7±1.1 mL/mm Hg; P>0.3 for each). Although SScPAH did not have greater vascular stiffening compared with IPAH, RV contractility was more depressed (Ees=0.8±0.3 versus 2.3±1.1, P<0.01; Msw=21±11 versus 45±16, P=0.01), with differential RV-PA uncoupling (Ees/Ea=1.0±0.5 versus 2.1±1.0; P=0.03). This ratio was higher in SSc without PH (Ees/Ea=2.3±1.2; P=0.02 versus SScPAH). CONCLUSIONS: RV dysfunction is worse in SScPAH compared with IPAH at similar afterload, and may be because of intrinsic systolic function rather than enhanced pulmonary vascular resistive and pulsatile loading.


Subject(s)
Arterial Pressure , Hypertension, Pulmonary/etiology , Myocardial Contraction , Pulmonary Artery/physiopathology , Scleroderma, Systemic/complications , Ventricular Dysfunction, Right/etiology , Ventricular Function, Right , Adult , Aged , Analysis of Variance , Cardiac Catheterization , Chi-Square Distribution , Compliance , Familial Primary Pulmonary Hypertension , Female , Humans , Hypertension, Pulmonary/diagnosis , Hypertension, Pulmonary/physiopathology , Linear Models , Lung Diseases, Interstitial/etiology , Lung Diseases, Interstitial/physiopathology , Male , Middle Aged , Nonlinear Dynamics , Predictive Value of Tests , Prognosis , Pulmonary Circulation , Risk Factors , United States , Vascular Resistance , Ventricular Dysfunction, Right/diagnosis , Ventricular Dysfunction, Right/physiopathology , Ventricular Pressure
2.
PLoS One ; 7(4): e34951, 2012.
Article in English | MEDLINE | ID: mdl-22545094

ABSTRACT

BACKGROUND: Gene expression profiling of peripheral blood mononuclear cells (PBMCs) is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells. METHODOLOGY/PRINCIPAL FINDINGS: The transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH), 19 patients with systemic sclerosis without pulmonary hypertension (SSc), 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH), and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD) were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease. CONCLUSIONS/SIGNIFICANCE: In PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression.


Subject(s)
Erythroid Cells/metabolism , Hypertension, Pulmonary/genetics , Leukocytes, Mononuclear/metabolism , Transcriptome , Adult , Aged , Female , Gene Expression Regulation , Hemodynamics , Humans , Hypertension, Pulmonary/physiopathology , Leukocytes, Mononuclear/pathology , Lung/metabolism , Lung/physiopathology , Male , Middle Aged
3.
Proc Natl Acad Sci U S A ; 109(4): 1239-44, 2012 Jan 24.
Article in English | MEDLINE | ID: mdl-22232678

ABSTRACT

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca(2+)](i)) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca(2+)](i), pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca(2+)](i). These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.


Subject(s)
Digoxin/pharmacology , Hypertension, Pulmonary/prevention & control , Hypoxia-Inducible Factor 1/metabolism , Hypoxia/complications , Transcriptional Activation/physiology , Analysis of Variance , Animals , Blood Pressure/drug effects , Calcium/metabolism , Digoxin/blood , Hypertension, Pulmonary/etiology , Hypertrophy, Right Ventricular/prevention & control , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Mice , Microscopy, Confocal , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/cytology , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transcriptional Activation/drug effects
4.
BMC Med Genomics ; 4: 78, 2011 Nov 02.
Article in English | MEDLINE | ID: mdl-22047531

ABSTRACT

BACKGROUND: MicroRNAs are ~22-nt long regulatory RNAs that serve as critical modulators of post-transcriptional gene regulation. The diversity of miRNAs in endothelial cells (ECs) and the relationship of this diversity to epithelial and hematologic cells is unknown. We investigated the baseline miRNA signature of human ECs cultured from the aorta (HAEC), coronary artery (HCEC), umbilical vein (HUVEC), pulmonary artery (HPAEC), pulmonary microvasculature (HPMVEC), dermal microvasculature (HDMVEC), and brain microvasculature (HBMVEC) to understand the diversity of miRNA expression in ECs. RESULTS: We identified 166 expressed miRNAs, of which 3 miRNAs (miR-99b, miR-20b and let-7b) differed significantly between EC types and predicted EC clustering. We confirmed the significance of these miRNAs by RT-PCR analysis and in a second data set by Sylamer analysis. We found wide diversity of miRNAs between endothelial, epithelial and hematologic cells with 99 miRNAs shared across cell types and 31 miRNAs unique to ECs. We show polycistronic miRNA chromosomal clusters have common expression levels within a given cell type. CONCLUSIONS: EC miRNA expression levels are generally consistent across EC types. Three microRNAs were variable within the dataset indicating potential regulatory changes that could impact on EC phenotypic differences. MiRNA expression in endothelial, epithelial and hematologic cells differentiate these cell types. This data establishes a valuable resource characterizing the diverse miRNA signature of ECs.


Subject(s)
Endothelial Cells/metabolism , Gene Expression Profiling/methods , MicroRNAs/genetics , Blood Cells/cytology , Blood Cells/metabolism , Cell Differentiation , Child , Chromosomes, Human/genetics , Cluster Analysis , Computational Biology , Endothelial Cells/cytology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Female , Genetic Variation , Humans , Organ Specificity , Reproducibility of Results
5.
Circulation ; 124(23): 2533-42, 2011 Dec 06.
Article in English | MEDLINE | ID: mdl-22082675

ABSTRACT

BACKGROUND: Pulmonary hypertension (PH) is a lethal syndrome associated with the pathogenic remodeling of the pulmonary vasculature and the emergence of apoptosis-resistant cells. Apoptosis repressor with caspase recruitment domain (ARC) is an inhibitor of multiple forms of cell death known to be abundantly expressed in striated muscle. We show for the first time that ARC is expressed in arterial smooth muscle cells of the pulmonary vasculature and is markedly upregulated in several experimental models of PH. In this study, we test the hypothesis that ARC expression is essential for the development of chronic hypoxia-induced PH. METHODS AND RESULTS: Experiments in which cells or mice were rendered ARC-deficient revealed that ARC not only protected pulmonary arterial smooth muscle cells from hypoxia-induced death, but also facilitated growth factor-induced proliferation and hypertrophy and hypoxia-induced downregulation of selective voltage-gated potassium channels, the latter a hallmark of the syndrome in humans. Moreover, ARC-deficient mice exhibited diminished vascular remodeling, increased apoptosis, and decreased proliferation in response to chronic hypoxia, resulting in marked protection from PH in vivo. Patients with PH have significantly increased ARC expression not only in remodeled vessels but also in the lumen-occluding lesions associated with severe disease. CONCLUSIONS: These data show that ARC, previously unlinked to pulmonary hypertension, is a critical determinant of vascular remodeling in this syndrome.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/physiopathology , Hypoxia/metabolism , Hypoxia/physiopathology , Muscle Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Cell Death/physiology , Cell Division/physiology , Cells, Cultured , Chronic Disease , Disease Models, Animal , Humans , Hypertension, Pulmonary/pathology , Hypoxia/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/genetics , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/physiology , Potassium Channels, Voltage-Gated/genetics , Potassium Channels, Voltage-Gated/physiology , Pulmonary Circulation/physiology , Rats , Up-Regulation/physiology , Vasoconstriction/physiology
6.
J Rheumatol ; 38(11): 2410-8, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21965638

ABSTRACT

OBJECTIVE: The tricuspid annular plane systolic excursion (TAPSE) strongly reflects right ventricular (RV) function and predicts survival in idiopathic pulmonary arterial hypertension (PAH). But its role in systemic sclerosis (SSc)-associated PAH has not been established. Our objective was to validate the TAPSE in the assessment of RV function and prediction of survival in SSc-PAH. METHODS: Fifty consecutive patients with SSc-PAH who underwent echocardiography with TAPSE measurement within 1 h of clinically indicated right heart catheterization were followed prospectively. The relationship between TAPSE and measures of RV function and measures of survival was assessed. RESULTS: The majority of the cohort were women in New York Heart Association class III/IV with severe PAH (mean cardiac index 2.4 ± 0.8 l/min/m(2)). RV function was significantly impaired (mean cardiac index 2.1 ± 0.7 vs 2.9 ± 0.8 l/min/m(2); p < 0.01) and RV afterload was significantly greater (mean pulmonary vascular resistance 11.1 ± 5.1 vs 5.8 ± 2.5 Wood units; p < 0.01) in subjects with a TAPSE ≤ 1.7 cm. The proportion surviving in the low TAPSE group was significantly lower [0.56 (95% CI 0.37-0.71) and 0.46 (95% CI 0.28-0.62) vs 0.87 (95% CI 0.55-0.96) and 0.79 (95% CI 0.49-0.93), 1- and 2-year survival, respectively]. TAPSE ≤ 1.7 cm conferred a nearly 4-fold increased risk of death (HR 3.81, 95% CI 1.31-11.1, p < 0.01). CONCLUSION: TAPSE is a robust measure of RV function and strongly predicts survival in patients with PAH-SSc. Future studies are needed to identify the responsiveness of TAPSE to PAH-specific therapy and to assess its diagnostic utility in PAH-SSc.


Subject(s)
Heart Ventricles/diagnostic imaging , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/physiopathology , Outcome Assessment, Health Care , Scleroderma, Systemic/complications , Scleroderma, Systemic/physiopathology , Tricuspid Valve/diagnostic imaging , Ventricular Function, Right/physiology , Aged , Blood Pressure/physiology , Cohort Studies , Female , Follow-Up Studies , Humans , Hypertension, Pulmonary/mortality , Kaplan-Meier Estimate , Male , Middle Aged , Predictive Value of Tests , Prospective Studies , Regional Blood Flow/physiology , Reproducibility of Results , Scleroderma, Systemic/mortality , Sensitivity and Specificity , Survival Rate , Systole/physiology , Tricuspid Valve/physiology , Ultrasonography , Vascular Resistance/physiology
7.
J Clin Invest ; 121(6): 2301-12, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21537080

ABSTRACT

The cardiac pathological response to sustained pressure overload involves myocyte hypertrophy and dysfunction along with interstitial changes such as fibrosis and reduced capillary density. These changes are orchestrated by mechanical forces and factors secreted between cells. One such secreted factor is TGF-ß, which is generated by and interacts with multiple cell types. Here we have shown that TGF-ß suppression in cardiomyocytes was required to protect against maladaptive remodeling and involved noncanonical (non-Smad-related) signaling. Mouse hearts subjected to pressure overload and treated with a TGF-ß-neutralizing Ab had suppressed Smad activation in the interstitium but not in myocytes, and noncanonical (TGF-ß-activated kinase 1 [TAK1]) activation remained. Although fibrosis was greatly reduced, chamber dysfunction and dilation persisted. Induced myocyte knockdown of TGF-ß type 2 receptor (TßR2) blocked all maladaptive responses, inhibiting myocyte and interstitial Smad and TAK1. Myocyte knockdown of TßR1 suppressed myocyte but not interstitial Smad, nor TAK1, modestly reducing fibrosis without improving chamber function or hypertrophy. Only TßR2 knockdown preserved capillary density after pressure overload, enhancing BMP7, a regulator of the endothelial-mesenchymal transition. BMP7 enhancement also was coupled to TAK1 suppression. Thus, myocyte targeting is required to modulate TGF-ß in hearts subjected to pressure overload, with noncanonical pathways predominantly affecting the maladaptive hypertrophy/dysfunction.


Subject(s)
Hypertrophy, Left Ventricular/physiopathology , Myocytes, Cardiac/physiology , Transforming Growth Factor beta1/physiology , Ventricular Remodeling/physiology , Animals , Aortic Diseases/complications , Aortic Diseases/physiopathology , Bone Morphogenetic Protein 7/physiology , Connective Tissue Growth Factor/physiology , Constriction, Pathologic/complications , Constriction, Pathologic/physiopathology , Coronary Circulation , Gene Knockdown Techniques , Hypertrophy, Left Ventricular/diagnostic imaging , Hypertrophy, Left Ventricular/etiology , Hypertrophy, Left Ventricular/prevention & control , Ligation , MAP Kinase Kinase Kinases/physiology , Male , Mice , Mice, Inbred C57BL , Myocardium/pathology , Myocytes, Cardiac/pathology , Pressure , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Receptor, Transforming Growth Factor-beta Type I , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/physiology , Signal Transduction , Smad Proteins/physiology , Transforming Growth Factor beta1/antagonists & inhibitors , Ultrasonography
8.
Cardiovasc Res ; 90(2): 353-63, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21421555

ABSTRACT

AIMS: It has been well demonstrated that phosphodiesterase-5A (PDE5A) is expressed in smooth muscle cells and plays an important role in regulation of vascular tone. The role of endothelial PDE5A, however, has not been yet characterized. The present study was undertaken to determine the presence, localization, and potential physiologic significance of PDE5A within vascular endothelial cells. METHODS AND RESULTS: We demonstrate primary location of human, mouse, and bovine endothelial PDE5A at or near caveolae. We found that the spatial localization of PDE5A at the level of caveolin-rich lipid rafts allows for a feedback loop between endothelial PDE5A and nitric oxide synthase (NOS3). Treatment of human endothelium with PDE5A inhibitors resulted in a significant increase in NOS3 activity, whereas overexpression of PDE5A using an adenoviral vector, both in vivo and in cell culture, resulted in decreased NOS3 activity and endothelium-dependent vasodilation. The molecular mechanism responsible for these interactions is primarily regulated by cGMP-dependent second messenger. PDE5A overexpression also resulted in a significant decrease in protein kinase 1 (PKG1) activity. Overexpression of PKG1 rapidly activated NOS3, whereas silencing of the PKG1 gene with siRNA inhibited both NOS3 phosphorylation (S1179) and activity, indicating a novel role for PKG1 in direct regulation of NOS3. CONCLUSION: Our data collectively suggest another target for PDE5A inhibition in endothelial dysfunction and provide another physiologic significance for PDE5A in the modulation of endothelial-dependent flow-mediated vasodilation. Using both in vitro and in vivo models, as well as human data, we show that inhibition of endothelial PDE5A improves endothelial function.


Subject(s)
Caveolae/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Endothelial Cells/enzymology , Nitric Oxide Synthase Type III/metabolism , Vasodilation/physiology , Animals , Aorta/cytology , Aorta/enzymology , Cattle , Cells, Cultured , Coronary Vessels/cytology , Coronary Vessels/enzymology , Cyclic GMP-Dependent Protein Kinase Type I , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/genetics , Endothelial Cells/cytology , Humans , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Membrane Microdomains/metabolism , Mice , Pulmonary Artery/cytology , Pulmonary Artery/enzymology , Signal Transduction/physiology , Umbilical Veins/cytology , Umbilical Veins/enzymology
9.
Am J Respir Crit Care Med ; 182(2): 252-60, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20339143

ABSTRACT

RATIONALE: Pulmonary arterial hypertension (PAH) related to systemic sclerosis (SSc) has a poorer prognosis compared with other forms of PAH for reasons that remain unexplained. OBJECTIVES: To identify risk factors of mortality in a well-characterized cohort of patients with PAH related to systemic sclerosis (SSc-PAH). METHODS: Seventy-six consecutive patients with SSc (64 women and 12 men; mean age 61 +/- 11 yr) were diagnosed with PAH by heart catheterization in a single center, starting in January 2000, and followed over time. Kaplan-Meier estimates were calculated and mortality risk factors were analyzed. MEASUREMENTS AND MAIN RESULTS: Forty (53%) patients were in World Health Organization functional class III or IV. Mean pulmonary artery pressure was 41 +/- 11 mm Hg, pulmonary vascular resistance (PVR) was 8.6 +/- 5.6 Wood units, and cardiac index was 2.4 +/- 0.7 L/min/m(2). Median follow-up time was 36 months, with 42 deaths observed. Survival estimates were 85%, 72%, 67%, 50%, and 36% at 1, 2, 3, 4, and 5 years, respectively. Multivariate analysis identified PVR (hazard ratio [HR], 1.10; 95% confidence interval [CI], 1.03-1.18; P < 0.01), stroke volume index (HR, 0.94; 95% CI, 0.89-0.99; P = 0.02), and pulmonary arterial capacitance (HR, 0.43; 95% CI, 0.20-0.91; P = 0.03) as strong predictors of survival. An estimated glomerular filtration rate less than 60 ml/min/1.73 m(2) portended a threefold risk of mortality. CONCLUSIONS: Our results suggest that specific components of right ventricular dysfunction and renal impairment contribute to increased mortality in SSc-PAH. Understanding the mechanisms of right ventricular dysfunction in response to increased afterload should lead to improved targeted therapy in these patients.


Subject(s)
Hypertension, Pulmonary/mortality , Scleroderma, Systemic/mortality , Cohort Studies , Female , Follow-Up Studies , Glomerular Filtration Rate/physiology , Humans , Hypertension, Pulmonary/physiopathology , Kaplan-Meier Estimate , Male , Middle Aged , Multivariate Analysis , Pericardial Effusion/epidemiology , Pulmonary Circulation/physiology , Risk Factors , Scleroderma, Systemic/physiopathology , Severity of Illness Index , Stroke Volume/physiology , Vascular Resistance/physiology , Ventricular Dysfunction/epidemiology
10.
Circ Res ; 105(1): 12-5, 2009 Jul 02.
Article in English | MEDLINE | ID: mdl-19520971

ABSTRACT

Cardiac myocyte targeted MerCreMer transgenic mice expressing tamoxifen-inducible Cre driven by the alpha-myosin heavy chain promoter are increasingly used to control gene expression in the adult heart. Here, we show tamoxifen-mediated MerCreMer (MCM) nuclear translocation can induce severe transient dilated cardiomyopathy in mice with or without loxP transgenes. The cardiomyopathy is accompanied by marked reduction of energy/metabolism and calcium-handling gene expression (eg, PGC1-alpha, peroxisome proliferator-activated alpha, SERCA2A), all fully normalized with recovery. MCM-negative/flox-positive controls display no dysfunction with tamoxifen. Nuclear Cre translocation and equally effective gene knockdown without cardiomyopathy is achievable with raloxifene, suggesting toxicity is not simply from Cre. Careful attention to controls, reduced tamoxifen dosing and/or use of raloxifene is advised with this model.


Subject(s)
Cardiomyopathy, Dilated/etiology , Integrases/genetics , Myocytes, Cardiac , Tamoxifen/pharmacology , Active Transport, Cell Nucleus , Animals , Energy Metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Integrases/drug effects , Integrases/metabolism , Mice , Mice, Transgenic , Tamoxifen/administration & dosage
11.
Chest ; 135(6): 1455-1461, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19118270

ABSTRACT

BACKGROUND: Pulmonary hypertension (PH) is a common complication of sarcoidosis that is associated with increased mortality. The pathogenesis of PH in sarcoidosis is uncertain, and the role of pulmonary arterial hypertension (PAH)-specific therapies remains to be determined. METHODS: We conducted a retrospective study of patients with sarcoidosis and PH at two referral centers. New York Heart Association (NYHA) functional class, exercise capacity, hemodynamic data, pulmonary function tests, and survival were collected and analyzed. RESULTS: Twenty-two sarcoidosis patients treated with PAH-specific therapies were identified. After a median of 11 months of follow-up, NYHA class was improved in nine subjects. Mean 6-min walk distance (n = 18) increased by 59 m (p = 0.032). Patients with a higher FVC experienced a greater increment in exercise capacity. Among 12 patients with follow-up hemodynamic data, mean pulmonary artery pressure was reduced from 48.5 +/- 4.3 to 39.4 +/- 2.8 mm Hg (p = 0.008). The 1- and 3-year transplant-free survival rates were 90% and 74%, respectively. CONCLUSIONS: PAH-specific therapy may improve functional class, exercise capacity, and hemodynamics in PH associated with sarcoidosis. Prospective, controlled trials of PAH therapies for sarcoidosis are warranted to verify this apparent benefit. Mortality among the study population was high, highlighting the need for urgent evaluation at a lung transplant center.


Subject(s)
Antihypertensive Agents/administration & dosage , Hemodynamics/drug effects , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/etiology , Sarcoidosis/complications , Administration, Inhalation , Administration, Oral , Aged , Bosentan , Cohort Studies , Drug Therapy, Combination , Epoprostenol/administration & dosage , Female , Follow-Up Studies , Hemodynamics/physiology , Humans , Hypertension, Pulmonary/mortality , Iloprost/administration & dosage , Kaplan-Meier Estimate , Linear Models , Male , Middle Aged , Piperazines/administration & dosage , Probability , Purines/administration & dosage , Respiratory Function Tests , Retrospective Studies , Risk Assessment , Sarcoidosis/diagnosis , Sarcoidosis/mortality , Severity of Illness Index , Sildenafil Citrate , Statistics, Nonparametric , Sulfonamides/administration & dosage , Sulfones/administration & dosage , Survival Rate , Treatment Outcome
12.
Transl Res ; 151(4): 197-207, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18355767

ABSTRACT

We hypothesize that pulmonary arterial hypertension (PAH)-associated genes identified by expression profiling of peripheral blood mononuclear cells (PBMCs) from patients with idiopathic pulmonary arterial hypertension (IPAH) can also be identified in PBMCs from scleroderma patients with PAH (PAH-SSc). Gene expression profiles of PBMCs collected from IPAH (n = 9), PAH-SSc (n = 10) patients, and healthy controls (n = 5) were generated using HG_U133A_2.0 GeneChips and were processed by the RMA/GCOS_1.4/SAM_1.21 data analysis pipeline. Disease severity in consecutive patients was assessed by functional status and hemodynamic measurements. The expression profiles were analyzed using PAH severity-stratification, and identified candidate genes were validated with real-time polymerase chain reaction (PCR). Transcriptomics of PBMCs from IPAH patients was highly comparable with that of PMBCs from PAH-SSc patients. The PBMC gene expression patterns significantly correlate with right atrium pressure (RA) and cardiac index (CI), which are known predictors of survival in PAH. Array stratification by RA and CI identified 364 PAH-associated candidate genes. Gene ontology (GO) analysis revealed significant (Z(score) > 1.96) alterations in angiogenesis genes according to PAH severity: matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF) were significantly upregulated in mild as compared with severe PAH and healthy controls, as confirmed by real-time PCR. These data demonstrate that PBMCs from patients with PAH-SSc carry distinct transcriptional expression. Furthermore, our findings suggest an association between angiogenesis-related gene expression and severity of PAH in PAH-SSc patients. Deciphering the role of genes involved in vascular remodeling and PAH development may reveal new treatment targets for this devastating disorder.


Subject(s)
Genetic Predisposition to Disease , Hypertension, Pulmonary/genetics , Matrix Metalloproteinase 9/genetics , Pulmonary Artery , Scleroderma, Systemic/genetics , Vascular Endothelial Growth Factor A/genetics , Aged , Female , Gene Expression Profiling , Humans , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Pilot Projects , Pulmonary Artery/physiopathology , Scleroderma, Systemic/physiopathology , Severity of Illness Index , Up-Regulation/genetics
13.
Am J Respir Crit Care Med ; 177(8): 896-905, 2008 Apr 15.
Article in English | MEDLINE | ID: mdl-18202349

ABSTRACT

RATIONALE: Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-beta receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-beta family signaling in PH and pulmonary vascular remodeling remains unclear. OBJECTIVES: To determine whether inhibition of TGF-beta signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH). METHODS: We have used an orally active small-molecule TGF-beta receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH. MEASUREMENTS AND MAIN RESULTS: The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-beta signaling as documented by psmad2 expression. Inhibition of TGF-beta signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-beta signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling. CONCLUSIONS: These findings provide evidence that increased TGF-beta signaling participates in the pathogenesis of experimental severe PH.


Subject(s)
Hypertension, Pulmonary/physiopathology , Protein Serine-Threonine Kinases/physiology , Receptors, Transforming Growth Factor beta/physiology , Transforming Growth Factor beta/physiology , Animals , Disease Models, Animal , Endothelial Cells , Hepatocytes , Hypertension, Pulmonary/chemically induced , Male , Monocrotaline/administration & dosage , Monocrotaline/toxicity , Plant Extracts/administration & dosage , Plant Extracts/toxicity , Rats , Rats, Sprague-Dawley , Receptor, Transforming Growth Factor-beta Type I , Signal Transduction , Tumor Cells, Cultured
14.
Circ Heart Fail ; 1(4): 213-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19808294

ABSTRACT

BACKGROUND: Pulmonary hypertension (PH) secondary to left heart failure portends a poor prognosis and is a relative contraindication to heart transplantation at many centers. We tested the hypothesis that when PH persists after adequate left ventricle unloading via recent left ventricular assist device (LVAD) therapy, phosphodiesterase type 5A inhibition would decrease PH in this population. METHODS AND RESULTS: We performed an open-label clinical trial using control patients not receiving therapy. Between 1999 and 2007, 138 consecutive patients undergoing cardiac transplantation evaluation with advanced left ventricular dysfunction, an elevated pulmonary capillary wedge pressure, and PH (defined by a pulmonary vascular resistance (PVR) >3 Woods Units), were treated with LVAD therapy. Fifty-eight of these patients reduced their pulmonary capillary wedge pressure to a value <15 mm Hg (11.8+/-2.0 mm Hg from baseline 23.2+/-6.2 mm Hg) 1 to 2 weeks after LVAD implantation, but despite this improvement, the PVR of these patients was only minimally affected (5.65+/-3.00 to 5.39+/-1.78 Wood Units). Twenty-six consecutive patients from this group with persistently elevated PVR were started on oral phosphodiesterase type 5A inhibition with sildenafil and titrated to an average of dose of 51.9 mg by mouth 3 times per day. The average PVR in the sildenafil-treated group fell from 5.87+/-1.93 to 2.96+/-0.92 Wood Units (P<0.001) and the mean pulmonary artery pressure fell from 36.5+/-8.6 to 24.3+/-3.6 mm Hg (P<0.0001) and was significantly lower when compared with the 32 LVAD recipients not receiving sildenafil at weeks 12 to 15 after the initial post-LVAD hemodynamic measurements (13 to 17 weeks post-LVAD implantation). In addition, hemodynamic measurements of right ventricular function in sildenafil-treated patients was also improved compared with patients not receiving sildenafil. CONCLUSIONS: In patients with persistent PH after recent LVAD placement, phosphodiesterase type 5A inhibition in this open-label trial resulted in a significant decrease in PVR when compared with control patients.


Subject(s)
Heart-Assist Devices , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/etiology , Phosphodiesterase 5 Inhibitors , Phosphodiesterase Inhibitors/therapeutic use , Ventricular Dysfunction, Left/complications , Ventricular Dysfunction, Left/therapy , Adult , Female , Hemodynamics/drug effects , Humans , Hypertension, Pulmonary/physiopathology , Male , Middle Aged , Treatment Outcome , Vascular Resistance/drug effects , Ventricular Dysfunction, Left/physiopathology
15.
J Cell Biochem ; 95(4): 849-58, 2005 Jul 01.
Article in English | MEDLINE | ID: mdl-15838879

ABSTRACT

The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pull down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFalpha- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFalpha- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.


Subject(s)
Endothelium/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Myosin-Light-Chain Kinase/metabolism , Actins/metabolism , Amino Acid Sequence , Binding Sites , Cells, Cultured , Humans , Macrophage Migration-Inhibitory Factors/chemistry , Macrophage Migration-Inhibitory Factors/genetics , Molecular Sequence Data , Myosin-Light-Chain Kinase/chemistry , Myosin-Light-Chain Kinase/genetics , Protein Binding , Protein Structure, Tertiary , Saccharomyces cerevisiae , Two-Hybrid System Techniques , Umbilical Cord/metabolism
16.
J Cell Biochem ; 94(2): 351-64, 2005 Feb 01.
Article in English | MEDLINE | ID: mdl-15526279

ABSTRACT

Tumor necrosis factor (TNFalpha-) generates both apoptotic and survival signals with endothelial cell (EC) survival dependent on nuclear factor kappa-B (NFkappaB) activation, a regulator of anti-apoptotic genes. We previously demonstrated that increased EC contractility, rearrangement of the actin cytoskeleton, and increased myosin light chain (MLC) phosphorylation occurs as a consequence of TNFalpha-induced activation of EC MLC kinase (EC MLCK) and is required for bovine lung EC apoptosis. As the association between MLCK and pro-survival signals such as NFkappaB activation is unknown, we studied the role of MLCK in the regulation of NFkappaB-dependent transactivation in bovine pulmonary artery EC. Both TNFalpha-induced increase in NFkappaB dependent transactivation measured by NFkappaB luciferase reporter assay (approximately fivefold) and nuclear translocation of NFkappaB were significantly inhibited by MLCK-selective inhibitors, KT5926 (60% inhibition of luciferase activity) and ML7 (50% decrease). Furthermore, our data revealed that inhibition of MLCK attenuated the TNFalpha-induced IkappaB phosphorylation, translocation of p65, NFkappaB-DNA binding, and NFkappaB transcriptional activity. Molecular approaches to either reduce EC MLCK expression (AdV EC MLCK antisense construct) or to reduce kinase activity (kinase-dead EC MLCK ATPdel mutant) produced similar attenuation of the TNFalpha-induced NFkappaB response. In contrast, a constitutively active MLCK mutant (EC MLCK1745) enhanced TNFalpha-induced luciferase activity. Together, these novel observations indicate that TNFalpha-induced cytoskeletal rearrangement driven by MLCK activity is necessary for TNFalpha-dependent NFkappaB activation and amplification of pro-survival signals.


Subject(s)
Apoptosis , Cytoskeleton/metabolism , Endothelial Cells/enzymology , Myosin-Light-Chain Kinase/physiology , NF-kappa B/metabolism , Pulmonary Artery/cytology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Azepines/pharmacology , Carbazoles/pharmacology , Cattle , Cell Nucleus/metabolism , Cells, Cultured , Electrophoretic Mobility Shift Assay , Endothelial Cells/cytology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation , I-kappa B Proteins/metabolism , Indoles/pharmacology , Luciferases/metabolism , Mutation , Myosin-Light-Chain Kinase/antagonists & inhibitors , NF-KappaB Inhibitor alpha , NF-kappa B/genetics , Naphthalenes/pharmacology , Phosphorylation , Protein Transport , Pulmonary Artery/enzymology , Sequence Deletion
17.
FASEB J ; 17(3): 407-16, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12631580

ABSTRACT

Cytoskeletal proteins are key participants in the cellular progression to apoptosis. Our previous work demonstrated the critical dependence of actomyosin rearrangement and MLC phosphorylation in TNF-alpha-induced endothelial cell apoptosis. As these events reflect the activation of the multifunctional endothelial cell (EC) MLCK isoform, we assessed the direct role of EC MLCK in the regulation of TNF-alpha-induced apoptosis. Bovine pulmonary artery endothelial cells expressing either an adenovirus encoding antisense MLCK cDNA (Ad.GFP-AS MLCK) or a dominant/negative EC MLCK construct (EC MLCK-ATPdel) resulted in marked reductions in MLCK activity and TNF-alpha-mediated apoptosis. In contrast, a constitutively active EC MLCK lacking the carboxyl-terminal autoinhibitory domains (EC MLCK-1745) markedly enhanced the apoptotic response to TNF-alpha. Immunostaining in GFP-EC MLCK-expressing cells revealed colocalization of caspase 8 and EC MLCK along actin stress fibers after TNF-alpha. TNF-alpha induced the caspase-dependent cleavage of EC MLCK-1745 in transfected endothelial cells, which was confirmed by mass spectroscopy with in vitro cleavage by caspase 3 at LKKD (D1703). The resulting MLCK fragments displayed significant calmodulin-independent kinase activity. These studies convincingly demonstrate that novel interactions between the apoptotic machinery and EC MLCK exist that regulate the endothelial contractile apparatus in TNF-alpha-induced apoptosis.


Subject(s)
Apoptosis , Caspases/metabolism , Endothelium, Vascular/enzymology , Myosin-Light-Chain Kinase/metabolism , Pulmonary Artery/cytology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/analysis , Cattle , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Myosin-Light-Chain Kinase/analysis , Myosin-Light-Chain Kinase/physiology , Stress Fibers/chemistry
18.
J Cell Biochem ; 88(3): 623-34, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12532337

ABSTRACT

Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(19) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated growth rate of mutant MLCK-transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility.


Subject(s)
Cell Division/physiology , Fibroblasts/metabolism , Muscle Contraction/physiology , Mutation , Myosin-Light-Chain Kinase/genetics , Myosin-Light-Chain Kinase/metabolism , Animals , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Cell Line , Fibroblasts/cytology , Humans , Phosphorylation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Stress Fibers/metabolism , Thrombin/metabolism
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