Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA-expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung-conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-ß1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.

Netrin-1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin-Induced Pulmonary Fibrosis.

OBJECTIVE: Fibrocytes are collagen-producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)-related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells. METHODS: We performed biomechanical evaluation of decellularized scaffolds prepared from lung explants from healthy control subjects and patients with scleroderma, using tensile testing and biochemical and proteomic analysis. Cells obtained from healthy controls and patients with SSc-related ILD were cultured on these scaffolds, and CD45+pro-ColIα1+ cells meeting the criteria for fibrocytes were quantified. The contribution of the neuromolecule netrin-1 to fibrosis was assessed using neutralizing antibodies in this system and by administering bleomycin via inhalation to netrin-1(+/-) mice. RESULTS: Compared with control lung scaffolds, lung scaffolds from patients with SSc-related ILD showed aberrant anatomy, enhanced stiffness, and abnormal ECM composition. Culture of control cells in lung scaffolds from patients with SSc-related ILD increased production of pro-ColIα1+ cells, which was stimulated by enhanced stiffness and abnormal ECM composition. Cells from patients with SSc-related ILD demonstrated increased pro-ColIα1 responsiveness to lung scaffolds from scleroderma patients but not enhanced stiffness. Enhanced detection of netrin-1-expressing CD14(low) cells in patients with SSc-related ILD was observed, and antibody-mediated netrin-1 neutralization attenuated detection of CD45+pro-ColIα1+ cells in all settings. Netrin-1(+/-) mice were protected against bleomycin-induced lung fibrosis and fibrocyte accumulation. CONCLUSION: Factors present in the lung matrices of patients with scleroderma regulate fibrocyte accumulation via a netrin-1-dependent pathway. Netrin-1 regulates bleomycin-induced pulmonary fibrosis in mice. Netrin-1 might be a novel therapeutic target in SSc-related ILD.

Human microvascular pericyte basement membrane remodeling regulates neutrophil recruitment.

OBJECTIVE: Neutrophil extravasation at post-capillary venules, consisting of EC, PC, and the shared ECM, increases following fibrotic remodeling in the lung, liver, and skin. The role of fibrotic pericyte-derived ECM in regulating EC activation and neutrophil recruitment remains unexplored. METHODS: To elucidate the role of human pericyte-derived ECM in EC activation, we characterized PC-derived ECM following transforming growth factor-ß1, IL-1ß, CCL2, or bleomycin activation, and examined surface adhesion molecule expression and neutrophil recruitment by EC cultured on PC-ECM. RESULTS: Pro-inflammatory activation of PC-induced deposition of compositionally distinct ECM compared with non-activated control. Bleomycin activation induced fibronectin-rich and collagen-poor ECM remodeling by PC, facilitating increased neutrophil transendothelial migration when compared with non-activated pericyte ECM (49.9 ± 3.4% versus 29.7 ± 1.4%). Increases in fibronectin compared to collagen I, are largely responsible for ECM-regulated neutrophil recruitment, as EC cultured on fibronectin supported increased neutrophil transmigration compared to collagen I (51.6 ± 6.2% versus 28.0 ± 4.8%). We attribute this difference to increased expression of ICAM-1 and its redistribution to EC borders. CONCLUSIONS: This is the first demonstration of human pericyte sensitivity to inflammatory stimuli, inducing fibrotic matrix deposition that regulates EC adhesion molecule expression and neutrophil recruitment.

Microvascular targets for anti-fibrotic therapeutics.

Fibrosis is characterized by excessive extracellular matrix deposition and is the pathological outcome of repetitive tissue injury in many disorders. The accumulation of matrix disrupts the structure and function of the native tissue and can affect multiple organs including the lungs, heart, liver, and skin. Unfortunately, current therapies against the deadliest and most common fibrosis are ineffective. The pathogenesis of fibrosis is the result of aberrant wound healing, therefore, the microvasculature plays an important role, contributing through regulation of leukocyte recruitment, inflammation, and angiogenesis. Further exacerbating the condition, microvascular endothelial cells and pericytes can transdifferentiate into matrix depositing myofibroblasts. The contribution of the microvasculature to fibrotic progression makes its cellular components and acellular products attractive therapeutic targets. In this review, we examine many of the cytokine, matrix, and cellular microvascular components involved in fibrosis and discuss their potential as targets for fibrotic therapies with a particular focus on developing nanotechnologies.

Transendothelial migration enables subsequent transmigration of neutrophils through underlying pericytes.

During acute inflammation, neutrophil recruitment into extravascular tissue requires neutrophil tethering and rolling on cytokine-activated endothelial cells (ECs), tight adhesion, crawling towards EC junctions and transendothelial migration (TEM). Following TEM, neutrophils must still traverse the subendothelial basement membrane and network of pericytes (PCs). Until recently, the contribution of the PC layer to neutrophil recruitment was largely ignored. Here we analyze human neutrophil interactions with interleukin (IL)-1ß-activated human EC monolayers, PC monolayers and EC/PC bilayers in vitro. Compared to EC, PC support much lower levels of neutrophil binding (54.6% vs. 7.1%, respectively) and transmigration (63.7 vs. 8.8%, respectively) despite comparable levels of IL-8 (CXCL8) synthesis and display. Remarkably, EC/PC bilayers support intermediate levels of transmigration (37.7%). Neutrophil adhesion to both cell types is Mac-1-dependent and while ICAM-1 transduction of PCs increases neutrophil adhesion to (41.4%), it does not increase transmigration through PC monolayers. TEM, which increases neutrophil Mac-1 surface expression, concomitantly increases the ability of neutrophils to traverse PCs (19.2%). These data indicate that contributions from both PCs and ECs must be considered in evaluation of microvasculature function in acute inflammation.

Proteomic identification and functional validation of activins and bone morphogenetic protein 11 as candidate novel muscle mass regulators.

Myostatin is a secreted TGF-beta family member that controls skeletal muscle growth. Humans, cattle, and dogs carrying natural loss-of-function mutations in the myostatin gene and myostatin knockout mice exhibit significant increases in skeletal muscle mass. Treatment of adult mice with antimyostatin antibodies also resulted in significant muscle mass increases. However, myostatin-knockout mice that were treated with a soluble form of the activin type II receptor (ActRII) B increased their muscle mass by an additional 15-25%, indicating that there is at least one additional ligand, in addition to myostatin, that functions to limit muscle growth. Here, both soluble ActRII and -IIB fragment-crystallizable proteins were used to affinity purify their native ligands from human and mouse sera. Using mass spectrometry-based proteomics and in vitro binding assays we have identified and confirmed that a number of TGF-beta family members, including myostatin, activins-A, -B, and -AB, bone morphogenetic proteins (BMPs) -9, -10, and -11, bind to both ActRIIs. Many of these factors, such as BMPs-11, -9, and -10 were discovered in systemic circulation for the first time, indicating that these ligands may also act in an endocrine fashion. Using a promoter-specific gene reporter assay, we demonstrated that soluble ActRIIB fragment-crystallizable proteins can inhibit the canonical signaling induced by these ligands. In addition, like myostatin, these factors were able to block the differentiation of myoblast cells into myotubes. However, in addition to myostatin, only BMP-11, and activins-A, -B, and -AB could be blocked from inhibiting the myoblast-to-myotube differentiation with both soluble ActRIIs, thus implicating them as potential novel regulators of muscle growth.