Changes in Pulmonary Arterial Wall Mechanical Properties and Lumenal Architecture with Induced Vascular Remodeling

To explore and quantify pulmonary arterial remodeling we used various methods including micro-CT, high-resolution 3-dimensional x-ray imaging, to examine the structure and function of intact pulmonary vessels in isolated rat lungs. The rat is commonly used as an animal model for studies of pulmonary hypertension (PH) and the accompanying vascular remodeling, where vascular remodeling has been defined primarily by changes in the vessel wall composition in response to hypertension inducing stimuli such as chronic hypoxic exposure (CHE) or monocrotaline (MCT) injection. Little information has been provided as to how such changes affect the vessel wall mechanical properties or the lumenal architecture of the pulmonary arterial system that actually account for the hemodynamic consequences of the remodeling. In addition, although the link between primary forms of pulmonary hypertension and inherited genetics is well established, the role that genetic coding plays in hemodynamics and vascular remodeling is not. Therefore, we are utilizing Fawn-Hooded (FH), Sprague-Dawley (SD) and Brown Norway (BN)rat strains along with unique imaging methods to parameterize both vessel distensibility and lumenal morphometry using a principal pulmonary arterial pathway analysis based on self-consistency. We have found for the hypoxia model, in addition to decreased body weight, increased hematocrit, increased right ventricular hypertrophy, the distensibility of the pulmonary arteries is shown to decrease significantly in the presence of remodeling

Emerging role of angiogenesis in adaptive and maladaptive right ventricular remodeling in pulmonary hypertension

Right ventricular (RV) function is the primary prognostic factor for both morbidity and mortality in pulmonary hypertension (PH). RV hypertrophy is initially an adaptive physiological response to increased overload; however, with persistent and/or progressive afterload increase, this response frequently transitions to more pathological maladaptive remodeling. The mechanisms and disease processes underlying this transition are mostly unknown. Angiogenesis has recently emerged as a major modifier of RV adaptation in the setting of pressure overload. A novel paradigm has emerged that suggests that angiogenesis and angiogenic signaling are required for RV adaptation to afterload increases and that impaired and/or insufficient angiogenesis is a major driver of RV decompensation. Here, we summarize our current understanding of the concepts of maladaptive and adaptive RV remodeling, discuss the current literature on angiogenesis in the adapted and failing RV, and identify potential therapeutic approaches targeting angiogenesis in RV failure

The serotonin transporter promotes a pathological estrogen metabolic pathway in pulmonary hypertension via cytochrome P450 1B1 pulmonary circulation

Pulmonary arterial hypertension (PAH) is a devastating vasculopathy that predominates in women and has been associated with dysregulated estrogen and serotonin signaling. Overexpression of the serotonin transporter (SERT+) in mice results in an estrogen-dependent development of pulmonary hypertension (PH). Estrogen metabolism by cytochrome P450 1B1 (CYP1B1) contributes to the pathogenesis of PAH, and serotonin can increase CYP1B1 expression in human pulmonary arterial smooth muscle cells (hPASMCs). We hypothesized that an increase in intracellular serotonin via increased SERT expression may dysregulate estrogen metabolism via CYP1B1 to facilitate PAH. Consistent with this hypothesis, we found elevated lung CYP1B1 protein expression in female SERT+ mice accompanied by PH, which was attenuated by the CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS). Lungs from female SERT+ mice demonstrated an increase in oxidative stress that was marked by the expression of 8-hydroxyguanosine; however, this was unaffected by CYP1B1 inhibition. SERT expression was increased in monocrotaline-induced PH in female rats; however, TMS did not reverse PH in monocrotaline-treated rats but prolonged survival. Stimulation of hPASMCs with the CYP1B1 metabolite 16α-hydroxyestrone increased cellular proliferation, which was attenuated by an inhibitor (MPP) of estrogen receptor alpha (ERα) and a specific ERα antibody. Thus, increased intracellular serotonin caused by increased SERT expression may contribute to PAH pathobiology by dysregulation of estrogen metabolic pathways via increased CYP1B1 activity. This promotes PASMC proliferation by the formation of pathogenic metabolites of estrogen that mediate their effects via ERα. Our studies indicate that targeting this pathway in PAH may provide a promising antiproliferative therapeutic strategy

Dynamic changes in lung microRNA profiles during the development of pulmonary hypertension due to chronic hypoxia and monocrotaline

<b>Objective</b>: MicroRNAs (miRNAs) are small noncoding RNAs that have the capacity to control protein production through binding "seed" sequences within a target mRNA. Each miRNA is capable of potentially controlling hundreds of genes. The regulation of miRNAs in the lung during the development of pulmonary arterial hypertension (PAH) is unknown.<p></p> <b>Methods and Results</b>: We screened lung miRNA profiles in a longitudinal and crossover design during the development of PAH caused by chronic hypoxia or monocrotaline in rats. We identified reduced expression of Dicer, involved in miRNA processing, during the onset of PAH after hypoxia. MiR-22, miR-30, and let-7f were downregulated, whereas miR-322 and miR-451 were upregulated significantly during the development of PAH in both models. Differences were observed between monocrotaline and chronic hypoxia. For example, miR-21 and let-7a were significantly reduced only in monocrotaline-treated rats. MiRNAs that were significantly regulated were validated by quantitative polymerase chain reaction. By using in vitro studies, we demonstrated that hypoxia and growth factors implicated in PAH induced similar changes in miRNA expression. Furthermore, we confirmed miR-21 downregulation in human lung tissue and serum from patients with idiopathic PAH.<p></p> <b>Conclusion</b>: Defined miRNAs are regulated during the development of PAH in rats. Therefore, miRNAs may contribute to the pathogenesis of PAH and represent a novel opportunity for therapeutic intervention.<p></p&gt

Transient but not genetic loss of miR-451 attenuates the development of pulmonary arterial hypertension

<b>Rationale:</b> MicroRNAs are small non-coding RNAs involved in the regulation of gene expression and have recently been implicated in the development of pulmonary arterial hypertension (PAH). Previous work established that miR-451 is up-regulated in rodent models of PAH.<p></p> <b>Objectives:</b> The role of miR-451 in the pulmonary circulation is unknown. We therefore sought to assess the involvement of miR-451 in the development of pulmonary arterial hypertension.<p></p> <b>Methods:</b> Silencing of miR-451 was performed in vivo using miR-451 knockout mice and an antimiR targeting mature miR-451 in rats. Coupled with exposure to hypoxia, indices of pulmonary arterial hypertension were assessed. The effect of modulating miR-451 on human pulmonary artery smooth muscle cell proliferation and migration was analysed.<p></p> <b>Measurements and Main Results:</b> We observed a reduction in systolic right ventricular pressure in hypoxic rats pre-treated with antimiR-451 compared to hypoxia alone (47.7 ± 1.36mmHg and 56.0 ± 2.03mmHg respectively, p<0.01). In miR-451 knockout mice following exposure to chronic hypoxia, no significant differences were observed compared to wild type hypoxic mice. In vitro analysis demonstrated that over-expression of miR-451 in human pulmonary artery smooth muscle cells promoted migration under serum-free conditions. No effect on cellular proliferation was observed.<p></p> <b>Conclusions:</b> Transient inhibition of miR-451 attenuated the development of pulmonary arterial hypertension in hypoxia exposed rats. Genetic deletion of miR-451 had no beneficial effect on indices of pulmonary arterial hypertension, potentially due to pathway redundancy compensating for the loss of miR-451.<p></p&gt

Quantification of Pulmonary Arterial Wall Distensibility Using Parameters Extracted from Volumetric Micro-CT Images

Stiffening, or loss of distensibility, of arterial vessel walls is among the manifestations of a number of vascular diseases including pulmonary arterial hypertension. We are attempting to quantify the mechanical properties of vessel walls of the pulmonary arterial tree using parameters derived from high-resolution volumetric x-ray CT images of rat lungs. The pulmonary arterial trees of the excised lungs are filled with a contrast agent. The lungs are imaged with arterial pressures spanning the physiological range. Vessel segment diameters are measured from the inlet to the periphery, and distensibilities calculated from diameters as a function of pressure. The method shows promise as an adjunct to other morphometric techniques such as histology and corrosion casting. It possesses the advantages of being nondestructive, characterizing the vascular structures while the lungs are imaged rapidly and in a near-physiological state, and providing the ability to associate mechanical properties with vessel location in the intact tree hierarchy

Novel signaling pathways in pulmonary arterial hypertension (2015 Grover Conference Series)

The proliferative endothelial and smooth muscle cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Mutations in bone morphogenetic protein type 2 receptor (BMPR2) have been identified as the most common genetic cause of PAH and females with BMPR2 mutations are 2.5 times as likely to develop heritable forms of PAH than males. Higher levels of estrogen have also been observed in males with PAH, implicating sex hormones in PAH pathogenesis. Recently, the estrogen metabolite 16α-OHE1 (hydroxyestrone) was implicated in the regulation of miR29, a microRNA involved in modulating energy metabolism. In females, decreased miR96 enhances serotonin’s effect by upregulating the 5-hydroxytryptamine 1B (5HT1B) receptor. Because PAH is characterized as a quasi-malignant disease, likely due to BMPR2 loss of function, altered signaling pathways that sustain this cancer-like phenotype are being explored. Extracellular signal–regulated kinases 1 and 2 and p38 mitogen-activated protein kinases (MAPKs) play a critical role in proliferation and cell motility, and dysregulated MAPK signaling is observed in various experimental models of PAH. Wnt signaling pathways preserve pulmonary vascular homeostasis, and dysregulation of this pathway could contribute to limited vascular regeneration in response to injury. In this review, we take a closer look at sex, sex hormones, and the interplay between sex hormones and microRNA regulation. We also focus on MAPK and Wnt signaling pathways in the emergence of a proproliferative, antiapoptotic endothelial phenotype, which then orchestrates an angioproliferative process of vascular remodeling, with the hope of developing novel therapies that could reverse the phenotype

Dexamethasone induces apoptosis in pulmonary arterial smooth muscle cells

BACKGROUND: Dexamethasone suppressed inflammation and haemodynamic changes in an animal model of pulmonary arterial hypertension (PAH). A major target for dexamethasone actions is NF-κB, which is activated in pulmonary vascular cells and perivascular inflammatory cells in PAH. Reverse remodelling is an important concept in PAH disease therapy, and further to its anti-proliferative effects, we sought to explore whether dexamethasone augments pulmonary arterial smooth muscle cell (PASMC) apoptosis. METHODS: Analysis of apoptosis markers (caspase 3, in-situ DNA fragmentation) and NF-κB (p65 and phospho-IKK-α/β) activation was performed on lung tissue from rats with monocrotaline (MCT)-induced pulmonary hypertension (PH), before and after day 14–28 treatment with dexamethasone (5 mg/kg/day). PASMC were cultured from this rat PH model and from normal human lung following lung cancer surgery. Following stimulation with TNF-α (10 ng/ml), the effects of dexamethasone (10(−8)–10(−6) M) and IKK2 (NF-κB) inhibition (AS602868, 0–3 μM (0-3×10(−6) M) on IL-6 and CXCL8 release and apoptosis was determined by ELISA and by Hoechst staining. NF-κB activation was measured by TransAm assay. RESULTS: Dexamethasone treatment of rats with MCT-induced PH in vivo led to PASMC apoptosis as displayed by increased caspase 3 expression and DNA fragmentation. A similar effect was seen in vitro using TNF-α-simulated human and rat PASMC following both dexamethasone and IKK2 inhibition. Increased apoptosis was associated with a reduction in NF-κB activation and in IL-6 and CXCL8 release from PASMC. CONCLUSIONS: Dexamethasone exerted reverse-remodelling effects by augmenting apoptosis and reversing inflammation in PASMC possibly via inhibition of NF-κB. Future PAH therapies may involve targeting these important inflammatory pathways

Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung.

There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH