Roberto Machado

Pulmonary hypertension and NO in sickle cell.

To the editor:nnWe are honored that our work merits the attention of Dr Bunn and colleagues[1][1] but disagree with some of their conclusions. Traditional risk factors for vaso-occlusive pain crisis, such as leukocytosis and high hemoglobin levels, incompletely predict vasculopathic events and

Hematologic, biochemical, and cardiopulmonary effects of l-arginine supplementation or phosphodiesterase 5 inhibition in patients with sickle cell disease who are on hydroxyurea therapy

Objectives:u2002 Fetal hemoglobin (HbF) induction involves NO‐cGMP signaling pathways. l‐arginine, an NO precursor, and the phosphodiesterase (PDE) 5 inhibitor sildenafil, which potentiates cGMP, were studied in adults with sickle cell disease (SCD) who were stably on HU.

Functional promoter variants in sphingosine 1-phosphate receptor 3 associate with susceptibility to sepsis-associated acute respiratory distress syndrome

The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor S1PR3 are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African- and ED samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Because of limited sample size, conclusive findings could not be drawn in African-descent ARDS subjects; however, significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two ED samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter; reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge; and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.

Hipertensão arterial pulmonar associada à anemia falciforme

Pulmonary hypertension is a common complication of sickle cell anemia. Despite the fact that the elevations in pulmonary artery pressures are slight, morbidity and mortality are high. In adult sickle cell anemia patients, pulmonary hypertension is emerging as a major risk factor for death. The pathogenesis of sickle cell anemia-related pulmonary hypertension is multifactorial, including hemolysis, impaired nitric oxide bioavailability, chronic hypoxemia, thromboembolism, chronic liver disease and asplenia. In the majority of patients, pulmonary arterial hypertension is the main cause of elevated pulmonary artery pressures. However, pulmonary venous hypertension also plays a role in a subgroup of patients. Specific data on the effects of treatment modalities for pulmonary hypertension in patients with sickle cell anemia are scarce. It is likely that all patients would benefit from maximization of sickle cell anemia therapy, and that patients with the severe form of the disease would benefit from treatment with selective pulmonary vasodilators and antiproliferative agents. Large trials evaluating the effects of treatment for pulmonary hypertension in the sickle cell anemia population are underway.

Conflicting Physiological and Genomic Cardiopulmonary Effects of Recruitment Maneuvers in Murine Acute Lung Injury

Low tidal volume ventilation, although promoting atelectasis, is a protective strategy against ventilator-induced lung injury. Deep inflation (DI) recruitment maneuvers restore lung volumes, but potentially compromise lung parenchymal and vascular function via repetitive overdistention. Our objective was to examine cardiopulmonary physiological and transcriptional consequences of recruitment maneuvers. C57/BL6 mice challenged with either PBS or LPS via aspiration were placed on mechanical ventilation (5 h) using low tidal volume inflation (TI; 8 μl/g) alone or in combination with intermittent DIs (0.75 ml twice/min). Lung mechanics during TI ventilation significantly deteriorated, as assessed by forced oscillation technique and pressure-volume curves. DI mitigated the TI-induced alterations in lung mechanics, but induced a significant rise in right ventricle systolic pressures and pulmonary vascular resistances, especially in LPS-challenged animals. In addition, DI exacerbated the LPS-induced genome-wide lung inflammatory transcriptome, with prominent dysregulation of a gene cluster involving vascular processes, as well as increases in cytokine concentrations in bronchoalveolar lavage fluid and plasma. Gene ontology analyses of right ventricular tissue expression profiles also identified inflammatory signatures, as well as apoptosis and membrane organization ontologies, as potential elements in the response to acute pressure overload. Our results, although confirming the improvement in lung mechanics offered by DI, highlight a detrimental impact in sustaining inflammatory response and exacerbating lung vascular dysfunction, events contributing to increases in right ventricle afterload. These novel insights should be integrated into the clinical assessment of the risk/benefit of recruitment maneuver strategies.

A novel p38 mitogen-activated protein kinase/Elk-1 transcription factor-dependent molecular mechanism underlying abnormal endothelial cell proliferation in plexogenic pulmonary arterial hypertension.

Background: Plexiform lesions comprising proliferative endothelial cells are hallmarks of pulmonary arterial hypertension. Results: Granzyme B cleaves intersectin-1s and generates a fragment with endothelial cell proliferative potential via phosphorylation of p38MAPK and Elk-1 transcription factor. Conclusion: Granzyme B cleavage of intersectin-1s and subsequent p38MAPK/Elk-1 activation are critical for endothelial cell proliferation. Significance: The novel pathogenic p38MAPK/Elk-1 signaling may explain the formation of plexiform lesions. Plexiform lesions (PLs), the hallmark of plexogenic pulmonary arterial hypertension (PAH), contain phenotypically altered, proliferative endothelial cells (ECs). The molecular mechanism that contributes to EC proliferation and formation of PLs is poorly understood. We now show that a decrease in intersectin-1s (ITSN-1s) expression due to granzyme B (GrB) cleavage during inflammation associated with PAH and the high p38/Erk1/2MAPK activity ratio caused by the GrB/ITSN cleavage products lead to EC proliferation and selection of a proliferative/plexiform EC phenotype. We used human pulmonary artery ECs of PAH subjects (ECPAH), paraffin-embedded and frozen human lung tissue, and animal models of PAH in conjunction with microscopy imaging, biochemical, and molecular biology approaches to demonstrate that GrB cleaves ITSN-1s, a prosurvival protein of lung ECs, and generates two biologically active fragments, an N-terminal fragment (GrB-EHITSN) with EC proliferative potential and a C-terminal product with dominant negative effects on Ras/Erk1/2. The proliferative potential of GrB-EHITSN is mediated via sustained phosphorylation of p38MAPK and Elk-1 transcription factor and abolished by chemical inhibition of p38MAPK. Moreover, lung tissue of PAH animal models and human specimens and ECPAH express lower levels of ITSN-1s compared with controls and the GrB-EHITSN cleavage product. Moreover, GrB immunoreactivity is associated with PLs in PAH lungs. The concurrent expression of the two cleavage products results in a high p38/Erk1/2MAPK activity ratio, which is critical for EC proliferation. Our findings identify a novel GrB-EHITSN-dependent pathogenic p38MAPK/Elk-1 signaling pathway involved in the poorly understood process of PL formation in severe PAH.

Health Disparities in Patients with Pulmonary Arterial Hypertension: A Blueprint for Action. An Official American Thoracic Society Statement

Background: Health disparities have a major impact in the quality of life and clinical care received by minorities in the United States. Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disorder that affects children and adults and that, if untreated, results in premature death. The impact of health disparities in the diagnosis, treatment, and clinical outcome of patients with PAH has not been systematically investigated. Objectives: The specific goals of this research statement were to conduct a critical review of the literature concerning health disparities in PAH, identify major research gaps and prioritize direction for future research. Methods: Literature searches from multiple reference databases were performed using medical subject headings and text words for pulmonary hypertension and health disparities. Members of the committee discussed the evidence and provided recommendations for future research. Results: Few studies were found discussing the impact of health disparities in PAH. Using recent research statements focused on health disparities, the group identified six major study topics that would help address the contribution of health disparities to PAH. Representative studies in each topic were discussed and specific recommendations were made by the group concerning the most urgent questions to address in future research studies. Conclusions: At present, there are few studies that address health disparities in PAH. Given the potential adverse impact of health disparities, we recommend that research efforts be undertaken to address the topics discussed in the document. Awareness of health disparities will likely improve advocacy efforts, public health policy and the quality of care of vulnerable populations with PAH.

Modulation of Intersectin-1s Lung Expression Induces Obliterative Remodeling and Severe Plexiform Arteriopathy in the Murine Pulmonary Vascular Bed

Murine models of pulmonary arterial hypertension (PAH) that recapitulate the plexiform and obliterative arteriopathy seen in PAH patients and help in defining the molecular mechanisms involved are missing. Herein, we investigated whether intersectin-1s (ITSN) deficiency and prolonged lung expression of an ITSN fragment with endothelial cell (EC) proliferative potential (EHITSN), present in the lungs of PAH animal models and human patients, induce formation of plexiform/obliterative lesions and defined the molecular mechanisms involved. ITSN-deficient mice (knockout/heterozygous and knockdown) were subjected to targeted lung delivery of EHITSN via liposomes for 20 days. Immunohistochemistry and histological and morphometric analyses revealed a twofold increase in proliferative ECs and a 1.35-fold increase in proliferative α-smooth muscle actin-positive cells in the lungs of ITSN-deficient mice, transduced with the EHITSN relative to wild-type littermates. Treated mice developed severe medial wall hypertrophy, intima proliferation, and various forms of obliterative and plexiform-like lesions in pulmonary arteries, similar to PAH patients. Hemodynamic measurements indicated modest increases in the right ventricular systolic pressure and right ventricle hypertrophy. Transcriptional and protein assays of lung tissue indicated p38MAPK-dependent activation of Elk-1 transcription factor and increased expression of c-Fos gene. This unique murine model of PAH-like plexiform/obliterative arteriopathy induced via a two-hit pathophysiological mechanism without hypoxia provides novel druggable targets to ameliorate and, perhaps, reverse the EC plexiform phenotype in severe human PAH.

A Phase II Clinical Trial of Low-Dose Inhaled Carbon Monoxide in Idiopathic Pulmonary Fibrosis

Background Preclinical studies have demonstrated that low‐dose carbon monoxide (CO) can abrogate experimental lung fibrosis. To test the therapeutic role of inhaled CO, we designed a clinical study in patients with idiopathic pulmonary fibrosis (IPF). Methods We conducted a multicenter, phase IIa, double‐blinded, sham‐controlled, clinical trial. Patients with IPF were randomized to treatment with inhaled CO at 100 to 200 parts per million or to inhaled 21% oxygen for 2 h daily, twice weekly, for 12 weeks. The primary study end point was the difference in change in matrix metalloproteinase‐7 (MMP7) serum concentration after 12 weeks of treatment. Secondary end points included pulmonary function test measures, 6‐min walk distance, rates of adverse events, acute exacerbation, hospitalization and death, and quality of life measures. Results Fifty‐eight subjects were randomized to treatment with inhaled CO (n = 29) or placebo (n = 29). Despite modest increases in CO blood levels, the change in MMP7 concentrations after 12 weeks of treatment did not significantly differ between the study arms (MMP7 difference at week 12, −0.90 ng/mL; 95% CI, −4.18 to 2.38 ng/mL). No differences were observed in physiologic measures, incidence of acute exacerbations, hospitalization, death, or patient‐reported outcomes. Importantly, no differences in distribution of adverse events were noted between the treatment arms. Conclusions Inhaled CO is well tolerated and can be safely administered to patients with IPF in the ambulatory setting; however, inhaled CO did not result in significant changes in study end points. Our findings support testing the efficacy of inhaled therapies in future IPF clinical trials. Trial Registry ClinicalTrials.gov; No.: NCT01214187; URL: www.clinicaltrials.gov.

Mechanistic insights and characterization of cardiomyopathy due to Sickle Cell Disease

Summary We sought to characterize the features of sickle cell cardiomyopathy and to identify causative mechanisms using comprehensive cardiac magnetic resonance, echocardiography, and arterial tonometry. We found that sickle cell cardiomyopathy is characterized by 4-chamber dilation, myocardial fibrosis, abnormal myocardial perfusion reserve, diastolic dysfunction, and only rarely myocardial iron overload. Left ventricular dilation and myocardial fibrosis are associated with increased blood transfusion requirements; where as, diastolic dysfunction is due to increased aortic stiffness. Background Cardiovascular disease is an important cause of morbidity and mortality in adults with sickle cell disease (SCD). We sought to characterize the features of SCD cardiomyopathy and to identify causative mechanisms. Methods Stable adults with SCD (n=38) and matched controls (n=13) were prospectively recruited to undergo 1) multiparametric cardiovascular magnetic resonance (CMR) (i. e. steady state free precession cine, regadenoson first pass myocardial perfusion, phase sensitive inversion recovery late gadolinium enhancement (LGE), and myocardial and hepatic multi-echo time (TE) single breathhold T2* imaging), 2) echocardiography (TTE), and 3) applanation tonometry (pulse wave analysis). Chamber size and function were measured from CMR using method of disks. Myocardial perfusion reserve index (MPRi) was calculated from time intensity curves generated from first pass perfusion images as the stress to rest ratio of mid-ventricular myocardium upslope (normalized by the left ventricular cavity upslope). LGE was considered present if the signal intensity (SI) was >5 standard deviations above normal remote myocardium and if seen in 2 consecutive slices or 2 imaging planes. Myocardial and hepatic T2* times were calculated using the formula ΔTE/ ln(SI TE2/SI TE1). Presence of diastolic dysfunction (DD) was determined using American Society of Echocardiography criteria. Aortic augmentation index was determined using standard tonometry methods.