Loss of TGFß-Mediated Repression of Angiopoietin-2 in Pericytes Underlies Germinal Matrix Hemorrhage Pathogenesis.

BACKGROUND: TGF (transforming growth factor)-ß pathway is central to blood-brain barrier development as it regulates cross talk between pericytes and endothelial cells. Murine embryos lacking TGFß receptor Alk5 (activin receptor-like kinase 5) in brain pericytes (mutants) display endothelial cell hyperproliferation, abnormal vessel morphology, and gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH), leading to perinatal lethality. Mechanisms underlying how ALK5 signaling in pericytes noncell autonomously regulates endothelial cell behavior remain elusive. METHODS: Transcriptomic analysis of human brain pericytes with ALK5 silencing identified differential gene expression. Brain vascular cells isolated from mutant embryonic mice with GMH-IVH and preterm human IVH brain samples were utilized for target validation. Finally, pharmacological and genetic inhibition was used to study the therapeutic effects on GMH-IVH pathology. RESULTS: Herein, we establish that the TGFß/ALK5 pathway robustly represses ANGPT2 (angiopoietin-2) in pericytes via epigenetic remodeling. TGFß-driven SMAD (suppressor of mothers against decapentaplegic) 3/4 associates with TGIF1 (TGFß-induced factor homeobox 1) and HDAC (histone deacetylase) 5 to form a corepressor complex at the Angpt2 promoter, resulting in promoter deacetylation and gene repression. Moreover, murine and human germinal matrix vessels display increased ANGPT2 expression during GMH-IVH. Isolation of vascular cells from murine germinal matrix identifies pericytes as a cellular source of excessive ANGPT2. In addition, mutant endothelial cells exhibit higher phosphorylated TIE2 (tyrosine protein kinase receptor). Pharmacological or genetic inhibition of ANGPT2 in mutants improves germinal matrix vessel morphology and attenuates GMH pathogenesis. Importantly, genetic ablation of Angpt2 in mutant pericytes prevents perinatal lethality, prolonging survival. CONCLUSIONS: This study demonstrates that TGFß-mediated ANGPT2 repression in pericytes is critical for maintaining blood-brain barrier integrity and identifies pericyte-derived ANGPT2 as an important pathological target for GMH-IVH.

The age of bone marrow dictates the clonality of smooth muscle-derived cells in atherosclerotic plaques.

Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to ~70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin ß3 resulting in increased tumor necrosis factor [TNF]-α signaling. TNFα signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin ß3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies.

Vascular pathobiology of pulmonary hypertension.

Pulmonary hypertension (PH), increased blood pressure in the pulmonary arteries, is a morbid and lethal disease. PH is classified into several groups based on etiology, but pathological remodeling of the pulmonary vasculature is a common feature. Endothelial cell dysfunction and excess smooth muscle cell proliferation and migration are central to the vascular pathogenesis. In addition, other cell types, including fibroblasts, pericytes, inflammatory cells and platelets contribute as well. Herein, we briefly note most of the main cell types active in PH and for each cell type, highlight select signaling pathway(s) highly implicated in that cell type in this disease. Among others, the role of hypoxia-inducible factors, growth factors (e.g., vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor-ß and bone morphogenetic protein), vasoactive molecules, NOTCH3, Kruppel-like factor 4 and forkhead box proteins are discussed. Additionally, deregulated processes of endothelial-to-mesenchymal transition, extracellular matrix remodeling and intercellular crosstalk are noted. This brief review touches upon select critical facets of PH pathobiology and aims to incite further investigation that will result in discoveries with much-needed clinical impact for this devastating disease.

MEKK3-TGFß crosstalk regulates inward arterial remodeling.

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFß-signaling both in vitro and in vivo. Endothelial-specific TGFßR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFßR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

Macrophage-derived PDGF-B induces muscularization in murine and human pulmonary hypertension.

Excess macrophages and smooth muscle cells (SMCs) characterize many cardiovascular diseases, but crosstalk between these cell types is poorly defined. Pulmonary hypertension (PH) is a lethal disease in which lung arteriole SMCs proliferate and migrate, coating the normally unmuscularized distal arteriole. We hypothesized that increased macrophage platelet-derived growth factor-B (PDGF-B) induces pathological SMC burden in PH. Our results indicate that clodronate attenuates hypoxia-induced macrophage accumulation, distal muscularization, PH, and right ventricle hypertrophy (RVH). With hypoxia exposure, macrophage Pdgfb mRNA was upregulated in mice, and LysM­Cre mice carrying floxed alleles for hypoxia-inducible factor 1a, hypoxia-inducible factor 2a, or Pdgfb had reduced macrophage Pdgfb and were protected against distal muscularization and PH. Conversely, LysM­Cre von-Hippel Lindaufl/fl mice had increased macrophage Hifa and Pdgfb and developed distal muscularization, PH, and RVH in normoxia. Similarly, Pdgfb was upregulated in macrophages from human idiopathic or systemic sclerosis-induced pulmonary arterial hypertension patients, and macrophage-conditioned medium from these patients increased SMC proliferation and migration via PDGF-B. Finally, in mice, orotracheal administration of nanoparticles loaded with Pdgfb siRNA specifically reduced lung macrophage Pdgfb and prevented hypoxia-induced distal muscularization, PH, and RVH. Thus, macrophage-derived PDGF-B is critical for pathological SMC expansion in PH, and nanoparticle-mediated inhibition of lung macrophage PDGF-B has profound implications as an interventional strategy for PH.

Macrophage-derived netrin-1 drives adrenergic nerve-associated lung fibrosis.

Fibrosis is a macrophage-driven process of uncontrolled extracellular matrix accumulation. Neuronal guidance proteins such as netrin-1 promote inflammatory scarring. We found that macrophage-derived netrin-1 stimulates fibrosis through its neuronal guidance functions. In mice, fibrosis due to inhaled bleomycin engendered netrin-1-expressing macrophages and fibroblasts, remodeled adrenergic nerves, and augmented noradrenaline. Cell-specific knockout mice showed that collagen accumulation, fibrotic histology, and nerve-associated endpoints required netrin-1 of macrophage but not fibroblast origin. Adrenergic denervation; haploinsufficiency of netrin-1's receptor, deleted in colorectal carcinoma; and therapeutic α1 adrenoreceptor antagonism improved collagen content and histology. An idiopathic pulmonary fibrosis (IPF) lung microarray data set showed increased netrin-1 expression. IPF lung tissues were enriched for netrin-1+ macrophages and noradrenaline. A longitudinal IPF cohort showed improved survival in patients prescribed α1 adrenoreceptor blockade. This work showed that macrophages stimulate lung fibrosis via netrin-1-driven adrenergic processes and introduced α1 blockers as a potentially new fibrotic therapy.

Understanding alveolarization to induce lung regeneration.

BACKGROUND: Gas exchange represents the key physiological function of the lung, and is dependent upon proper formation of the delicate alveolar structure. Malformation or destruction of the alveolar gas-exchange regions are key histopathological hallmarks of diseases such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis; all of which are characterized by perturbations to the alveolo-capillary barrier structure. Impaired gas-exchange is the primary initial consequence of these perturbations, resulting in severe clinical symptoms, reduced quality of life, and death. The pronounced morbidity and mortality associated with malformation or destruction of alveoli underscores a pressing need for new therapeutic concepts. The re-induction of alveolarization in diseased lungs is a new and exciting concept in a regenerative medicine approach to manage pulmonary diseases that are characterized by an absence of alveoli. MAIN TEXT: Mechanisms of alveolarization first need to be understood, to identify pathways and mediators that may be exploited to drive the induction of alveolarization in the diseased lung. With this in mind, a variety of candidate cell-types, pathways, and molecular mediators have recently been identified. Using lineage tracing approaches and lung injury models, new progenitor cells for epithelial and mesenchymal cell types - as well as cell lineages which are able to acquire stem cell properties - have been discovered. However, the underlying mechanisms that orchestrate the complex process of lung alveolar septation remain largely unknown. CONCLUSION: While important progress has been made, further characterization of the contributing cell-types, the cell type-specific molecular signatures, and the time-dependent chemical and mechanical processes in the developing, adult and diseased lung is needed in order to implement a regenerative therapeutic approach for pulmonary diseases.

A novel mouse Cre-driver line targeting Perilipin 2-expressing cells in the neonatal lung.

Pulmonary diseases such as chronic obstructive pulmonary disease, lung fibrosis, and bronchopulmonary dysplasia are characterized by the destruction or malformation of the alveolar regions of the lung. The underlying pathomechanisms at play are an area of intense interest since these mechanisms may reveal pathways suitable for interventions to drive reparative processes. Lipid-laden fibroblasts (lipofibroblasts) express the Perilipin 2 (Plin2) gene-product, PLIN2, commonly called adipose-differentiation related protein (ADRP). These cells are also thought to play a role in alveolarization and repair after injury to the alveolus. Progress in defining the functional contribution of lipofibroblasts to alveolar generation and repair is hampered by a lack of in vivo tools. The present study reports the generation of an inducible mouse Cre-driver line to target cells of the ADRP lineage. Robust Cre-mediated recombination in this mouse line was detected in mesenchymal cells of the postnatal lung, and in additional organs including the heart, liver, and spleen. The generation and validation of this valuable new tool to genetically target, manipulate, and trace cells of the ADRP lineage is critical for assessing the functional contribution of lipofibroblasts to lung development and repair.

Stereological monitoring of mouse lung alveolarization from the early postnatal period to adulthood.

Postnatal lung maturation generates a large number of small alveoli, with concomitant thinning of alveolar septal walls, generating a large gas exchange surface area but minimizing the distance traversed by the gases. This demand for a large and thin gas exchange surface area is not met in disorders of lung development, such as bronchopulmonary dysplasia (BPD) histopathologically characterized by fewer, larger alveoli and thickened alveolar septal walls. Diseases such as BPD are often modeled in the laboratory mouse to better understand disease pathogenesis or to develop new interventional approaches. To date, there have been no stereology-based longitudinal studies on postnatal mouse lung development that report dynamic changes in alveoli number or alveolar septal wall thickness during lung maturation. To this end, changes in lung structure were quantified over the first 22 mo of postnatal life of C57BL/6J mice. Alveolar density peaked at postnatal day (P)39 and remained unchanged at 9 mo (P274) but was reduced by 22 mo (P669). Alveoli continued to be generated, initially at an accelerated rate between P5 and P14, and at a slower rate thereafter. Between P274 and P669, loss of alveoli was noted, without any reduction in lung volume. A progressive thinning of the alveolar septal wall was noted between P5 and P28. Pronounced sex differences were observed in alveoli number in adult (but not juvenile) mice, when comparing male and female mouse lungs. This sex difference was attributed exclusively to the larger volume of male mouse lungs.

Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial lung disease with unknown etiology. Due to a lack of effective treatment, IPF is associated with a high mortality rate. The hallmark feature of this disease is the accumulation of activated myofibroblasts that excessively deposit extracellular matrix proteins, thus compromising lung architecture and function and hindering gas exchange. Here we investigated the origin of activated myofibroblasts and the molecular mechanisms governing fibrosis formation and resolution. Genetic engineering in mice enables the time-controlled labeling and monitoring of lipogenic or myogenic populations of lung fibroblasts during fibrosis formation and resolution. Our data demonstrate a lipogenic-to-myogenic switch in fibroblastic phenotype during fibrosis formation. Conversely, we observed a myogenic-to-lipogenic switch during fibrosis resolution. Analysis of human lung tissues and primary human lung fibroblasts indicates that this fate switching is involved in IPF pathogenesis, opening potential therapeutic avenues to treat patients.

Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development.

A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cell populations during late lung development in mice. PDGFRα(+) cells (detected using a PDGFRα(GFP) reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα(+) cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα(+) cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα(+) cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRα(Cre) line, it was observed that PDGFRα(+) cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα(+) cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization.