Pulmonary arterial hypertension (PAH) is a condition of unknown etiology whose pathological features include increased vascular resistance, perivascular inflammatory cell infiltration and pulmonary arteriolar remodeling. Although risk factors for PAH are poorly defined, recent studies indicate that obesity may be an important risk factor for this condition. The mechanisms leading to this association are largely unknown, but bioactive mediators secreted from adipose tissue have been implicated in this process. One of the most important mediators released from adipose tissue is the adipokine adiponectin. Adiponectin is highly abundant in the circulation of lean healthy individuals, and possesses well-described metabolic and antiinflammatory actions. Levels of adiponectin decrease with increasing body mass, and low levels are directly linked to the development of PAH in mice. Moreover, overexpression of adiponectin has been shown to protect mice from developing PAH in response to inflammation and hypoxia. Based on the findings from these studies, it is suggested that the effects of adiponectin are mediated, in part, through its antiinflammatory and antiproliferative properties. In this review, we discuss the emerging evidence demonstrating a role for adiponectin in lung vascular homeostasis and discuss how deficiency in this adipocyte-derived hormone might explain the recent association between obesity and PAH.

The past decade has seen increased application of 18-flurodeoxyglucose positron emission tomography ( ¹⁸ FDG-PET) imaging to help diagnose and monitor disease, particularly in oncology, vasculitis and atherosclerosis. Disordered glycolytic metabolism and infiltration of plexiform lesions by inflammatory cells has been described in idiopathic pulmonary arterial hypertension (IPAH). We hypothesized that increased ¹⁸ FDG uptake may be present in the lungs, large pulmonary arteries and right ventricle of patients with pulmonary hypertension, and that this uptake would be related to markers of immune activation. We imaged the thorax of 14 patients with pulmonary hypertension (idiopathic and chronic thromboembolic) and six controls by ¹⁸ FDG-PET/computed tomography (CT) and measured uptake in the lung parenchyma, large pulmonary arteries and right ventricle. ¹⁸ FDG uptake in the lungs and pulmonary arteries was normalized for venous blood activity to give a target-to-background ratio (TBR). Blood was contemporaneously drawn for high-sensitivity CRP - C-reactive protein (CRP) (hsCRP), N-Terminal Probrain natriuteric peptide (NT-ProBNP) and other inflammatory cytokines. IPAH patients had significantly higher lung parenchymal TBR (P=0.034) and right ventricle FDG uptake (P=0.007) than controls. Uptake in the main pulmonary arteries was similar in chronic thromboembolic pulmonary hypertension, IPAH and controls. There were no correlations between ¹⁸ FDG uptake and hsCRP or inflammatory cytokine levels. NT-ProBNP correlated with RV uptake in those with pulmonary hypertension (r=0.55, P=0.04). In this pilot study, we found increased ¹⁸ FDG uptake in the lung parenchyma and right ventricle of subjects with IPAH. The lung uptake might be useful as a surrogate marker of increased cellular metabolism and immune activation as underlying mechanisms in this disease. Further evaluation of the impact of targeted therapies in treatment-naïve patients and the significance of right ventricular uptake is suggested.

Schistosomiasis-associated pulmonary arterial hypertension (PAH) is one of the most common causes of pulmonary hypertension worldwide. A potential contributing mechanism to the pathogenesis of this disease is a localized immune reaction to retained and persistent parasite-derived antigens. We sought to identify Schistosoma-derived egg antigens present in the lungs of individuals who died of the disease. We obtained 18 lung samples collected at autopsy from individuals who died of schistosomiasis-associated PAH in Brazil. A rabbit polyclonal antibody was created to known Schistosoma mansoni-soluble egg antigen (SEA). Histologic assessment and immunostaining of the human tissue was performed, along with immunostaining and immunoblotting of lung tissue from mice experimentally infected with S. mansoni. All 18 lung samples had evidence of pulmonary vascular remodeling with plexiform lesions and arterial medial thickening, but no visible eggs were seen. The anti-SEA antibody detected S. mansoni egg antigens in visible eggs in mouse lung and human intestine specimens, but did not identify a significant amount of egg antigen in the human lung specimens. In mouse granulomas containing degraded eggs, we observed colocalization of egg antigens and macrophage lysosomes. In conclusion, there is unlikely to be a significant amount of persistent parasite-derived antigens within the lungs of individuals who die of schistosomiasis-associated PAH. This suggests that retained and persistent parasite proteins are not contributing to a localized immune response in the pathogenesis of this disease.

High-altitude pulmonary hypertension (HAPH) is a consequence of chronic alveolar hypoxia, leading to hypoxic vasoconstriction and remodeling of the pulmonary circulation. Brisket disease in cattle is a naturally occurring animal model of hypoxic pulmonary hypertension. Genetically susceptible cattle develop severe pulmonary hypertension and right heart failure at altitudes >7,000 ft. No information currently exists regarding the identity of the pathways and gene(s) responsible for HAPH or influencing severity. We hypothesized that initial insights into the pathogenesis of the disease could be discovered by a strategy of (1) sequencing of functional candidates revealed by single nucleotide polymorphism (SNP) analysis and (2) gene expression profiling of affected cattle compared with altitude-matched normal controls, with gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA). We isolated blood from a single herd of Black Angus cattle of both genders, aged 12-18 months, by jugular vein puncture. Mean pulmonary arterial pressures were 85.6±13 mmHg STD in the 10 affected and 35.3±1.2 mmHg STD in the 10 resistant cattle, P<0.001. From peripheral blood mononuclear cells, DNA was hybridized to an Affymetrix 10K Gene Chip SNP, and RNA was used to probe an Affymetrix Bovine genome array. SNP loci were remapped using the Btau 4.0 bovine genome assembly. mRNA data was analyzed by the Partek software package to identify sets of genes with an expression that was statistically different between the two groups. GSEA and IPA were conducted on the refined expression data to identify key cellular pathways and to generate networks and conduct functional analyses of the pathways and networks. Ten SNPs were identified by allelelic association and four candidate genes were sequenced in the cohort. Neither endothelial nitric oxide synthetase, NADH dehydrogenase, TG-interacting factor-2 nor BMPR2 were different among affected and resistant cattle. A 60-gene mRNA signature was identified that differentiated affected from unaffected cattle. Forty-six genes were overexpressed in the affected and 14 genes were downregulated in the affected cattle by at least 20%. GSEA and Ingenuity analysis identified respiratory diseases, inflammatory diseases and pathways as the top diseases and disorders (P<5.14×10 ^-14 ), cell development and cell signaling as the top cellular functions (P<1.20×10 ^-08 ), and IL6, TREM, PPAR, NFkB cell signaling (P<8.69×10 ^-09 ) as the top canonical pathways associated with this gene signature. This study provides insights into differences in RNA expression in HAPH at a molecular level, and eliminates four functional gene candidates. Further studies are needed to validate and refine these preliminary findings and to determine the role of transcribed genes in the development of HAPH.

In patients with pulmonary arterial hypertension (PAH), right ventricular mass (RVM) correlates linearly with pulmonary artery pressure, and decreases with successful treatment. Accurate measurement of RVM currently requires cardiovascular magnetic resonance (CMR) imaging. We therefore tested the relationship between RVM and a simple, 12 lead ECG-derived value, the Butler-Leggett (BL) score. This has previously been validated in patients with RV hypertrophy (RVH) due to mitral stenosis. We also tested the diagnostic accuracy of the BL score in detecting RVH. The Scottish Pulmonary Vascular Unit database was reviewed retrospectively. Twenty-eight patients with PAH were identified, in whom CMR and ECG data had been recorded no more than 28 days apart. All had completed a comprehensive clinical assessment, including right heart catheterization. CMR-derived absolute RVM and RV mass index (RVMI=RV mass/LV mass) were correlated against BL score. The ability of this score to detect RVH was tested using 2 x 2 contingency tables. RVM and RVMI correlated with BL score (r=0.77, P<0.001 and r=0.78, P<0.001, respectively). A BL score >0.7 mV proved a highly specific but insensitive indicator of RVH, based on either absolute RVM (sensitivity 74%, specificity 100%) or a high RVMI (sensitivity 61%, specificity 100%). The BL score, which can be defined using a standard 12-lead ECG, correlates with RVM and RVMI in patients with PAH. A score >0.7 mV was a highly specific but insensitive indicator of RVH in these patients.

Proliferative pulmonary vascular remodeling is the pathologic hallmark of pulmonary arterial hypertension (PAH) that ultimately leads to right heart failure and death. Highly proliferative endothelial cells known as endothelial colony-forming cells (ECFC) participate in vascular homeostasis in health as well as in pathological angiogenic remodeling in disease. ECFC are distinguished by the capacity to clonally proliferate from a single cell. The presence of ECFC in the human pulmonary arteries and their role in PAH pathogenesis is largely unknown. In this study, we established a simple technique for isolating and growing ECFC from cultured pulmonary artery endothelial cells (PAEC) to test the hypothesis that ECFC reside in human pulmonary arteries and that the proliferative vasculopathy of PAH is related to greater numbers and/or more proliferative ECFC in the pulmonary vascular wall. Flow cytometric forward and side scatter properties and aggregate correction were utilized to sort unmanipulated, single PAEC to enumerate ECFC in primary PAEC cultures derived from PAH and healthy lungs. After 2 weeks, wells were assessed for ECFC formation. ECFC derived from PAH PAEC were more proliferative than control. A greater proportion of PAH ECFC formed colonies following subculturing, demonstrating the presence of more ECFC with high proliferative potential among PAH PAEC. Human androgen receptor assay showed clonality of progeny, confirming that proliferative colonies were single cell-derived. ECFC expressed CD31, von Willebrand factor, endothelial nitric oxide synthase, caveolin-1 and CD34, consistent with an endothelial cell phenotype. We established a simple flow cytometry method that allows ECFC quantification using unmanipulated cells. We conclude that ECFC reside among PAEC and that PAH PAEC contain ECFC that are more proliferative than ECFC in control cultures, which likely contributes to the proliferative angiopathic process in PAH.

We have shown previously that acute hypoxia downregulates protein kinase G (PKG) expression and activity in ovine fetal pulmonary vessels and pulmonary arterial smooth muscle cells (SMC). Here, we report that acute hypoxia also reduces the expression of leucinezipper-positive MYPT1 (LZ ⁺ MYPT1), a subunit of myosin light chain (MLC) phosphatase, in ovine fetal pulmonary arterial SMC. We found that in hypoxia, there is greater interaction between LZ ⁺ MYPT1 and RhoA and Rho kinase 1 (ROCK1)/Rho kinase 2 (ROCK2) and decreased interaction between LZ ⁺ MYPT1 and PKG, resulting in increased MLC ₂₀ phosphorylation, a higher pMLC ₂₀ /MLC ₂₀ ratio and SMC contraction. In normoxic SMC PKG overexpression,LZ ⁺ MYPT1 expression is upregulated while PKG knockdown had an opposite effect. LZ ⁺ MYPT1 overexpression enhanced the interaction between PKG and LZ ⁺ MYPT1. Overexpression of a mutant LZ ^- MYPT1 isoform in SMC mimicked the effects of acute hypoxia and decreased pMLC ₂₀ /MLC ₂₀ ratio. Collectively, our data suggest that hypoxia downregulates LZ ⁺ MYPT1 expression by suppressing PKG levels, reduces the interaction of LZ ⁺ MYPT1 with PKG and promotes LZ ⁺ MYPT1 interaction with RhoA or ROCK1/ROCK2, thereby promoting pulmonary arterial SMC contraction.

Pulmonary vein stenosis (PVS) post radiofrequency ablation for chronic atrial fibrillation poses a diagnostic challenge for the clinician. PVS presents with nonspecific symptoms, signs and radiographic features, and may be associated with significant pulmonary vascular involvement. Interestingly, others have described variation of the pulmonary artery wedge pressure between sites of the lung as a clue to pulmonary veno-occlusive disorders. We report, to the best of our knowledge, the first case that describes the regional loss of V waves while recording the mean pulmonary artery wedge pressure (mPawp) as well as the difference in pulmonary artery wedge pressure gradients as the main diagnostic clues for PVS.