Functional and molecular determinants of right ventricular response to severe pulmonary hypertension in a large animal model.

Right ventricular (RV) failure is the major determinant of outcome in pulmonary hypertension (PH). Calves exposed to 2-wk hypoxia develop severe PH and unlike rodents, hypoxia-induced PH in this species can lead to right heart failure. We, therefore, sought to examine the molecular and structural changes in the RV in calves with hypoxia-induced PH, hypothesizing that we could identify mechanisms underlying compensated physiological function in the face of developing severe PH. Calves were exposed to 14 days of environmental hypoxia (equivalent to 4,570 m/15,000 ft elevation, n = 29) or ambient normoxia (1,525 m/5,000 ft, n = 25). Cardiopulmonary function was evaluated by right heart catheterization and pressure volume loops. Molecular and cellular determinants of RV remodeling were analyzed by cDNA microarrays, RealTime PCR, proteomics, and immunochemistry. Hypoxic exposure induced robust PH, with increased RV contractile performance and preserved cardiac output, yet evidence of dysregulated RV-pulmonary artery mechanical coupling as seen in advanced disease. Analysis of gene expression revealed cellular processes associated with structural remodeling, cell signaling, and survival. We further identified specific clusters of gene expression associated with 1) hypertrophic gene expression and prosurvival mechanotransduction through YAP-TAZ signaling, 2) extracellular matrix (ECM) remodeling, 3) inflammatory cell activation, and 4) angiogenesis. A potential transcriptomic signature of cardiac fibroblasts in RV remodeling was detected, enriched in functions related to cell movement, tissue differentiation, and angiogenesis. Proteomic and immunohistochemical analysis confirmed RV myocyte hypertrophy, together with localization of ECM remodeling, inflammatory cell activation, and endothelial cell proliferation within the RV interstitium. In conclusion, hypoxia and hemodynamic load initiate coordinated processes of protective and compensatory RV remodeling to withstand the progression of PH.NEW & NOTEWORTHY Using a large animal model and employing a comprehensive approach integrating hemodynamic, transcriptomic, proteomic, and immunohistochemical analyses, we examined the early (2 wk) effects of severe PH on the RV. We observed that RV remodeling during PH progression represents a continuum of transcriptionally driven processes whereby cardiac myocytes, fibroblasts, endothelial cells, and proremodeling macrophages act to coordinately maintain physiological homeostasis and protect myocyte survival during chronic, severe, and progressive pressure overload.

A Time- and Compartment-Specific Activation of Lung Macrophages in Hypoxic Pulmonary Hypertension.

Studies in various animal models suggest an important role for pulmonary macrophages in the pathogenesis of pulmonary hypertension (PH). Yet, the molecular mechanisms characterizing the functional macrophage phenotype relative to time and pulmonary localization and compartmentalization remain largely unknown. In this study, we used a hypoxic murine model of PH in combination with FACS to quantify and isolate lung macrophages from two compartments over time and characterize their programing via RNA sequencing approaches. In response to hypoxia, we found an early increase in macrophage number that was restricted to the interstitial/perivascular compartment, without recruitment of macrophages to the alveolar compartment or changes in the number of resident alveolar macrophages. Principal component analysis demonstrated significant differences in overall gene expression between alveolar and interstitial macrophages (IMs) at baseline and after 4 and 14 d hypoxic exposure. Alveolar macrophages at both day 4 and 14 and IMs at day 4 shared a conserved hypoxia program characterized by mitochondrial dysfunction, proinflammatory gene activation, and mTORC1 signaling, whereas IMs at day 14 demonstrated a unique anti-inflammatory/proreparative programming state. We conclude that the pathogenesis of vascular remodeling in hypoxic PH involves an early compartment-independent activation of lung macrophages toward a conserved hypoxia program, with the development of compartment-specific programs later in the course of the disease. Thus, harnessing time- and compartment-specific differences in lung macrophage polarization needs to be considered in the therapeutic targeting of macrophages in hypoxic PH and potentially other inflammatory lung diseases.

Increased prevalence of EPAS1 variant in cattle with high-altitude pulmonary hypertension.

High-altitude pulmonary hypertension (HAPH) has heritable features and is a major cause of death in cattle in the Rocky Mountains, USA. Although multiple genes are likely involved in the genesis of HAPH, to date no major gene variant has been identified. Using whole-exome sequencing, we report the high association of an EPAS1 (HIF2α) double variant in the oxygen degradation domain of EPAS1 in Angus cattle with HAPH, mean pulmonary artery pressure >50 mm Hg in two independent herds. Expression analysis shows upregulation of 26 of 27 HIF2α target genes in EPAS1 carriers with HAPH. Of interest, this variant appears to be prevalent in lowland cattle, in which 41% of a herd of 32 are carriers, but the variant may only have a phenotype when the animal is hypoxemic at altitude. The EPAS1 variant will be a tool to determine the cells and signalling pathways leading to HAPH.

Adventitial fibroblasts induce a distinct proinflammatory/profibrotic macrophage phenotype in pulmonary hypertension.

Macrophage accumulation is not only a characteristic hallmark but is also a critical component of pulmonary artery remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Using multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, and primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive pulmonary arteries (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL-6 and STAT3, HIF1, and C/EBPß signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL-4/IL-13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation, complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, and deficiency in C/EBPß or HIF1 attenuated fibroblast-driven macrophage activation. These findings challenge the current paradigm of IL-4/IL-13-STAT6-mediated alternative macrophage activation as the sole driver of vascular remodeling in PH, and uncover a cross-talk between adventitial fibroblasts and macrophages in which paracrine IL-6-activated STAT3, HIF1α, and C/EBPß signaling are critical for macrophage activation and polarization. Thus, targeting IL-6 signaling in macrophages by completely inhibiting C/EBPß or HIF1α or by partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL-6 and absent IL-4/IL-13 signaling.

The adventitia: essential regulator of vascular wall structure and function.

The vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. It is the most complex compartment of the vessel wall and is composed of a variety of cells, including fibroblasts, immunomodulatory cells (dendritic cells and macrophages), progenitor cells, vasa vasorum endothelial cells and pericytes, and adrenergic nerves. In response to vascular stress or injury, resident adventitial cells are often the first to be activated and reprogrammed to influence the tone and structure of the vessel wall; to initiate and perpetuate chronic vascular inflammation; and to stimulate expansion of the vasa vasorum, which can act as a conduit for continued inflammatory and progenitor cell delivery to the vessel wall. This review presents the current evidence demonstrating that the adventitia acts as a key regulator of vascular wall function and structure from the outside in.

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension.

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a "constitutively activated" phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the "activated" highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, α(V)ß(3) and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH.

Emergence of fibroblasts with a proinflammatory epigenetically altered phenotype in severe hypoxic pulmonary hypertension.

Persistent accumulation of monocytes/macrophages in the pulmonary artery adventitial/perivascular areas of animals and humans with pulmonary hypertension has been documented. The cellular mechanisms contributing to chronic inflammatory responses remain unclear. We hypothesized that perivascular inflammation is perpetuated by activated adventitial fibroblasts, which, through sustained production of proinflammatory cytokines/chemokines and adhesion molecules, induce accumulation, retention, and activation of monocytes/macrophages. We further hypothesized that this proinflammatory phenotype is the result of the abnormal activity of histone-modifying enzymes, specifically, class I histone deacetylases (HDACs). Pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (termed PH-Fibs) expressed a constitutive and persistent proinflammatory phenotype defined by high expression of IL-1ß, IL-6, CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES), CCR7, CXCR4, GM-CSF, CD40, CD40L, and VCAM-1. The proinflammatory phenotype of PH-Fibs was associated with epigenetic alterations as demonstrated by increased activity of HDACs and the findings that class I HDAC inhibitors markedly decreased cytokine/chemokine mRNA expression levels in these cells. PH-Fibs induced increased adhesion of THP-1 monocytes and produced soluble factors that induced increased migration of THP-1 and murine bone marrow-derived macrophages as well as activated monocytes/macrophages to express proinflammatory cytokines and profibrogenic mediators (TIMP1 and type I collagen) at the transcriptional level. Class I HDAC inhibitors markedly reduced the ability of PH-Fibs to induce monocyte migration and proinflammatory activation. The emergence of a distinct adventitial fibroblast population with an epigenetically altered proinflammatory phenotype capable of recruiting, retaining, and activating monocytes/macrophages characterizes pulmonary hypertension-associated vascular remodeling and thus could contribute significantly to chronic inflammatory processes in the pulmonary artery wall.

The rat Ruby ( R) locus is Rab38: identical mutations in Fawn-hooded and Tester-Moriyama rats derived from an ancestral Long Evans rat sub-strain.

Hermansky-Pudlak syndrome (HPS) is a group of rare, recessive disorders in which oculocutaneous albinism, progressive pulmonary fibrosis, bleeding diathesis, and other abnormalities result from defective biogenesis of multiple cytoplasmic organelles. Seven different HPS genes are known in humans; in mouse, at least 16 loci are associated with HPS-like mutant phenotypes. In the rat, only two HPS models are known, Fawn-hooded (FH) and Tester Moriyama (TM), non-complementing strains in which HPS-like hypopigmentation and platelet storage pool deficiency result from a mutation of the Ruby (red eyed dilution; R) locus on Chromosome (Chr) 1. We have identified the R locus as the Rab38 gene, establishing that rat R is homologous to mouse chocolate ( cht). Further, we show that FH and TM rats have identical Rab38 Met1Ile mutations, occurring on an identical Chr 1 marker allele haplotype, indicating that these two strains derive from a common ancestor. This ancestor appears to have been a sub-strain of the outbred Long Evans (LE) strain, and several modern LE sub-strains carry the Rab38 Met1Ile R mutation on the same Chr 1 marker haplotype. These findings have significant implications for the many past and ongoing studies that involve the FH and LE-derivative rat strains. Hermansky-Pudlak syndrome (HPS; MIM 203300) is a group of autosomal recessive diseases in which oculocutaneous albinism (OCA), progressive and fatal pulmonary fibrosis, and bleeding diathesis due to platelet storage pool deficiency result from defects in the biogenesis of specific cytoplasmic organelles and granules: melanosomes, lysosomes, and platelet dense granules. In humans, seven different HPS genes are known. In the mouse, at least 16 loci associated with HPS-like mutant phenotypes are known, seven of which are homologous to the human HPS loci.

Hyperoxia-induced DNA damage causes decreased DNA methylation in human lung epithelial-like A549 cells.

The effect of hyperoxia on levels of DNA damage and global DNA methylation was examined in lung epithelial-like A549 cells. DNA damage was assessed by the single-cell gel electrophoresis (comet assay) and DNA methylation status by the cytosine extension assays. Cells exposed to ionizing radiation (0, 1, 2, 4, or 8 Gy) showed increasing rates of percentage of DNA in the tail and tail length with increasing radiation dose. When cells were exposed to room air (normoxia) for 1 day and 95% O2 (hyperoxia) for 1, 2, 3, 4, and 5 days, data indicated that hyperoxia caused time-dependent increases in levels of (a) single strand breaks, (b) double strand breaks, and (c) 8-oxoguanine. Decreased DNA methylation also was observed at day 5 of hyperoxic exposure, suggesting that hyperoxia-induced DNA damage can influence patterns of DNA methylation in a lung-derived cell line.