Pharmacology and Emerging Therapies for Group 3 Pulmonary Hypertension Due to Chronic Lung Disease.

Pulmonary hypertension (PH) frequently complicates chronic lung disease and is associated with high morbidity and poor outcomes. Individuals with interstitial lung disease and chronic obstructive pulmonary disease develop PH due to structural changes associated with the destruction of lung parenchyma and vasculature with concurrent vasoconstriction and pulmonary vascular remodeling similar to what is observed in idiopathic pulmonary arterial hypertension (PAH). Treatment for PH due to chronic lung disease is largely supportive and therapies specific to PAH have had minimal success in this population with exception of the recently FDA-approved inhaled prostacyclin analogue treprostinil. Given the significant disease burden of PH due to chronic lung diseases and its associated mortality, a great need exists for improved understanding of molecular mechanisms leading to vascular remodeling in this population. This review will discuss the current understanding of pathophysiology and emerging therapeutic targets and potential pharmaceuticals.

Non-Muscle MLCK Contributes to Endothelial Cell Hyper-Proliferation through the ERK Pathway as a Mechanism for Vascular Remodeling in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction, uncontrolled proliferation and migration of pulmonary arterial endothelial cells leading to increased pulmonary vascular resistance resulting in great morbidity and poor survival. Bone morphogenetic protein receptor II (BMPR2) plays an important role in the pathogenesis of PAH as the most common genetic mutation. Non-muscle myosin light chain kinase (nmMLCK) is an essential component of the cellular cytoskeleton and recent studies have shown that increased nmMLCK activity regulates biological processes in various pulmonary diseases such as asthma and acute lung injury. In this study, we aimed to discover the role of nmMLCK in the proliferation and migration of pulmonary arterial endothelial cells (HPAECs) in the pathogenesis of PAH. We used two cellular models relevant to the pathobiology of PAH including BMPR2 silenced and vascular endothelial growth factor (VEGF) stimulated HPAECs. Both models demonstrated an increase in nmMLCK activity along with a robust increase in cellular proliferation, inflammation, and cellular migration. The upregulated nmMLCK activity was also associated with increased ERK expression pointing towards a potential integral cytoplasmic interaction. Mechanistically, we confirmed that when nmMLCK is inhibited by MLCK selective inhibitor (ML-7), proliferation and migration are attenuated. In conclusion, our results demonstrate that nmMLCK upregulation in association with increased ERK expression may contribute to the pathogenesis of PAHby stimulating cellular proliferation and migration.

MicroRNA-212-5p, an anti-proliferative miRNA, attenuates hypoxia and sugen/hypoxia-induced pulmonary hypertension in rodents.

MicroRNAs (miRNA, miR-) play important roles in disease development. In this study, we identified an anti-proliferative miRNA, miR-212-5p, that is induced in pulmonary artery smooth muscle cells (PASMCs) and lungs of pulmonary hypertension (PH) patients and rodents with experimental PH. We found that smooth muscle cell (SMC)-specific knockout of miR-212-5p exacerbated hypoxia-induced pulmonary vascular remodeling and PH in mice, suggesting that miR-212-5p may be upregulated in PASMCs to act as an endogenous inhibitor of PH, possibly by suppressing PASMC proliferation. Extracellular vesicles (EVs) have been shown recently to be promising drug delivery tools for disease treatment. We generated endothelium-derived EVs with an enriched miR-212-5p load, 212-eEVs, and found that they significantly attenuated hypoxia-induced PH in mice and Sugen/hypoxia-induced severe PH in rats, providing proof of concept that engineered endothelium-derived EVs can be used to deliver miRNA into lungs for treatment of severe PH.

Hemin-Induced Endothelial Dysfunction and Endothelial to Mesenchymal Transition in the Pathogenesis of Pulmonary Hypertension Due to Chronic Hemolysis.

Pulmonary hypertension in sickle cell disease is an independent predictor of mortality, yet the pathogenesis of pulmonary vascular disease in chronic hemolytic disorders remains incompletely understood and treatment options are limited primarily to supportive care. The release of extracellular hemoglobin has been implicated in the development of pulmonary hypertension, and in this study we explored the direct effects of hemin, the oxidized moiety of heme, on the pulmonary artery endothelium. We found that low dose hemin exposure leads to significantly increased endothelial cell proliferation, migration, and cytokine release as markers of endothelial dysfunction. Protein expression changes in our pulmonary artery endothelial cells showed upregulation of mesenchymal markers after hemin treatment in conjunction with a decrease in endothelial markers. Endothelial to mesenchymal transition (EndoMT) resulting from hemin exposure was further confirmed by showing upregulation of the transcription factors SNAI1 and SLUG, known to regulate EndoMT. Lastly, given the endothelial dysfunction and phenotypic transition observed, the endothelial cytoskeleton was considered a potential novel target. Inhibiting myosin light chain kinase, to prevent phosphorylation of myosin light chain and cytoskeletal contraction, attenuated hemin-induced endothelial hyper-proliferation, migration, and cytokine release. The findings in this study implicate hemin as a key inducer of endothelial dysfunction through EndoMT, which may play an important role in pulmonary vascular remodeling during the development of pulmonary hypertension in chronic hemolytic states.

Pathogenic Mechanisms of Pulmonary Arterial Hypertension: Homeostasis Imbalance of Endothelium-Derived Relaxing and Contracting Factors.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease. Sustained pulmonary vasoconstriction and concentric pulmonary vascular remodeling contribute to the elevated pulmonary vascular resistance and pulmonary artery pressure in PAH. Endothelial cells regulate vascular tension by producing endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs). Homeostasis of EDRF and EDCF production has been identified as a marker of the endothelium integrity. Impaired synthesis or release of EDRFs induces persistent vascular contraction and pulmonary artery remodeling, which subsequently leads to the development and progression of PAH. In this review, the authors summarize how EDRFs and EDCFs affect pulmonary vascular homeostasis, with special attention to the recently published novel mechanisms related to endothelial dysfunction in PAH and drugs associated with EDRFs and EDCFs.

Unsupervised Clustering Reveals Sarcoidosis Phenotypes Marked by a Reduction in Lymphocytes Relate to Increased Inflammatory Activity on 18FDG-PET/CT.

Introduction: Sarcoidosis is a T-helper cell mediated disease characterized by granulomatous inflammation. We posited that unsupervised clustering of various features in sarcoidosis would establish phenotypes associated with inflammatory activity measured by 18FDG-PET/CT. Our goal was to identify unique features capable of distinguishing clusters and subsequently examine the relationship with FDG avidity to substantiate their potential use as markers for sarcoidosis inflammation. Methods: We performed a retrospective study of a diverse, but primarily African American, cohort of 58 subjects with biopsy proven sarcoidosis followed at the University of Illinois Bernie Mac Sarcoidosis Center and Center for Lung Health who underwent 18FDG-PET/CT scan. Demographic, therapeutic, radiographic, and laboratory data were utilized in unsupervised cluster analysis to identify sarcoidosis phenotypes. The association between clusters, their defining features, and quantitative measurements on 18FDG-PET/CT was determined. The relevance of these features as markers of 18FDG-PET/CT inflammatory activity was also investigated. Results: Clustering determined three distinct phenotypes: (1) a predominantly African American cluster with chronic, quiescent disease, (2) a predominantly African American cluster with elevated conventional inflammatory markers, advanced pulmonary disease and extrathoracic involvement, and (3) a predominantly Caucasian cluster characterized by reduced lymphocyte counts and acute disease. In contrast to the chronic quiescent cluster, Clusters 2 and 3 were defined by significantly greater FDG avidity on 18FDG-PET/CT. Despite similarly increased inflammatory activity on 18FDG-PET/CT, Clusters 2, and 3 differed with regards to extrathoracic FDG avidity and circulating lymphocyte profiles, specifically CD4+ T-cells. Notably, absolute lymphocyte counts and CD4+ T-cell counts were found to predict 18FDG-PET/CT inflammatory activity by receiver operating curve analysis with a 69.2 and 73.42% area under the curve, respectively. Conclusions: Utilizing cluster analysis, three distinct phenotypes of sarcoidosis were identified with significant variation in race, disease chronicity, and serologic markers of inflammation. These phenotypes displayed varying levels of circulating inflammatory cells. Additionally, reduction in lymphocytes, specifically CD4+ T-cells, was significantly related to activity on 18FDG-PET/CT. Though future studies are warranted, these findings suggest that peripheral lymphocyte counts may be considered a determinant of sarcoidosis phenotypes and an indicator of active inflammation on 18FDG-PET/CT.

A Review of Transcriptome Analysis in Pulmonary Vascular Diseases.

Transcriptome analysis is a powerful tool in the study of pulmonary vascular disease and pulmonary hypertension. Pulmonary hypertension is a disease process that consists of several unique pathologies sharing a common clinical definition, that of elevated pressure within the pulmonary circulation. As such, it has become increasingly important to identify both similarities and differences among the different classes of pulmonary hypertension. Transcriptome analysis has been an invaluable tool both in the basic science research on animal models as well as clinical research among the various different groups of pulmonary hypertension. This work has identified new potential candidate genes, implicated numerous biochemical and molecular pathways in diseased onset and progression, developed gene signatures to appropriately classify types of pulmonary hypertension and severity of illness, and identified novel gene mutations leading to hereditary forms of the disease.

Angiotensin-(1-7) in Paraventricular Nucleus Contributes to the Enhanced Cardiac Sympathetic Afferent Reflex and Sympathetic Activity in Chronic Heart Failure Rats.

BACKGROUND/AIMS: Cardiac sympathetic afferent reflex (CSAR) enhancement contributes to exaggerated sympathetic activation in chronic heart failure (CHF). The current study aimed to investigate the roles of angiotensin (Ang)-(1-7) in CSAR modulation and sympathetic activation and Ang-(1-7) signaling pathway in paraventricular nucleus of CHF rats. METHODS: CHF was induced by coronary artery ligation. Responses of renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) to epicardial application of capsaicin were used to evaluate CSAR in rats with anesthesia. RESULTS: Ang-(1-7) increased RSNA, MAP, CSAR activity, cAMP level, NAD(P)H oxidase activity and superoxide anion level more significantly in CHF than in sham-operated rats, while Mas receptor antagonist A-779 had the opposite effects. Moreover, Ang-(1-7) augmented effects of Ang II in CHF rats. The effects of Ang-(1-7) were blocked by A-779, adenylyl cyclase inhibitor SQ22536, protein kinase A inhibitor Rp-cAMP, superoxide anion scavenger tempol and NAD(P)H oxidase inhibitor apocynin. Mas and AT1 receptor protein expressions, Ang-(1-7) and Ang II levels in CHF increased. CONCLUSIONS: These results indicate that Ang-(1-7) in paraventricular nucleus enhances CSAR and sympathetic output not only by exerting its own effects but also by augmenting the effects of Ang II through Mas receptor in CHF. Endogenous Ang-(1-7)/Mas receptor activity contributes to CSAR enhancement and sympathetic activation in CHF, and NAD(P)H oxidase-derived superoxide anions and the cAMP-PKA signaling pathway are involved in mediating the effects of Ang-(1-7) in CHF.

Inhaled Antibiotics for Gram-Negative Respiratory Infections.

Gram-negative organisms comprise a large portion of the pathogens responsible for lower respiratory tract infections, especially those that are nosocomially acquired, and the rate of antibiotic resistance among these organisms continues to rise. Systemically administered antibiotics used to treat these infections often have poor penetration into the lung parenchyma and narrow therapeutic windows between efficacy and toxicity. The use of inhaled antibiotics allows for maximization of target site concentrations and optimization of pharmacokinetic/pharmacodynamic indices while minimizing systemic exposure and toxicity. This review is a comprehensive discussion of formulation and drug delivery aspects, in vitro and microbiological considerations, pharmacokinetics, and clinical outcomes with inhaled antibiotics as they apply to disease states other than cystic fibrosis. In reviewing the literature surrounding the use of inhaled antibiotics, we also highlight the complexities related to this route of administration and the shortcomings in the available evidence. The lack of novel anti-Gram-negative antibiotics in the developmental pipeline will encourage the innovative use of our existing agents, and the inhaled route is one that deserves to be further studied and adopted in the clinical arena.

Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension.

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca(2+)]cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced [Ca(2+)]cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced [Ca(2+)]cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.

Pulmonary hypertension associated with thalassemia syndromes.

Chronic hemolytic anemia has increasingly been identified as an important risk factor for the development of pulmonary hypertension (PH). Within the thalassemia syndromes, there are multiple mechanisms, both distinct and overlapping, by which PH develops and that differ among ß-thalassemia major or intermedia patients. PH in ß-thalassemia major correlates with the severity of hemolysis, yet in patients whose disease is well treated with chronic transfusion therapy, the development of PH can be related to cardiac dysfunction and the subsequent toxic effects of iron overload rather than hemolysis. ß-Thalassemia intermedia, on the other hand, has a higher incidence of PH owing to the low level of hemolysis that exists over years without the requirement for frequent transfusions, while splenectomy is shown to play an important role in both types. Standard therapies such as chronic transfusion have been shown to mitigate PH, and appropriate chelation therapy can avoid the toxic effects of iron overload, yet is not indicated in many patients. Limited evidence exists for the use of pulmonary vasodilators or other therapies, such as l-carnitine, to treat PH associated with thalassemia. Here, we review the most recent findings regarding the pathogenic mechanisms, epidemiology, presentation, diagnosis, and treatment of PH in thalassemia syndromes.

Notch Activation of Ca(2+) Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.

Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension.

Pulmonary vascular remodeling, mainly attributable to enhanced pulmonary arterial smooth muscle cell proliferation and migration, is a major cause for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary hypertension. The signaling cascade through Akt, comprised of three isoforms (Akt1-3) with distinct but overlapping functions, is involved in regulating cell proliferation and migration. This study aims to investigate whether the Akt/mammalian target of rapamycin (mTOR) pathway, and particularly which Akt isoform, contributes to the development and progression of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Compared with the wild-type littermates, Akt1(-/-) mice were protected against the development and progression of chronic HPH, whereas Akt2(-/-) mice did not demonstrate any significant protection against the development of HPH. Furthermore, pulmonary vascular remodeling was significantly attenuated in the Akt1(-/-) mice, with no significant effect noted in the Akt2(-/-) mice after chronic exposure to normobaric hypoxia (10% O2). Overexpression of the upstream repressor of Akt signaling, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and conditional and inducible knockout of mTOR in smooth muscle cells were also shown to attenuate the rise in right ventricular systolic pressure and the development of right ventricular hypertrophy. In conclusion, Akt isoforms appear to have a unique function within the pulmonary vasculature, with the Akt1 isoform having a dominant role in pulmonary vascular remodeling associated with HPH. The PTEN/Akt1/mTOR signaling pathway will continue to be a critical area of study in the pathogenesis of pulmonary hypertension, and specific Akt isoforms may help specify therapeutic targets for the treatment of pulmonary hypertension.

Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu) plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX), a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2) also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC). In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α) with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.