Highlights d Artery endothelial cells (ECs) of neonatal hearts have a unique response to injury d Injury stimulates artery cell migration and reassembly into collateral arteries d CXCL12-CXCR4 signaling guides artery reassembly, facilitating heart regeneration d Adult artery ECs can be induced to undergo artery reassembly with exogenous CXCL12

Background: Pulmonary arterial hypertension (PAH) is a life-threatening disorder of the pulmonary circulation associated with loss and impaired regeneration of microvessels. Reduced pericyte coverage of pulmonary microvessels is a pathological feature of PAH and is caused partly by the inability of pericytes to respond to signaling cues from neighboring pulmonary microvascular endothelial cells (PMVECs). We have shown that activation of the Wnt/planar cell polarity pathway is required for pericyte recruitment, but whether production and release of specific Wnt ligands by PMVECs are responsible for Wnt/planar cell polarity activation in pericytes is unknown. Methods: Isolation of pericytes and PMVECs from healthy donor and PAH lungs was carried out with 3G5 or CD31 antibody–conjugated magnetic beads. Wnt expression profile of PMVECs was documented via quantitative polymerase chain reaction with a Wnt primer library. Exosome purification from PMVEC media was carried out with the ExoTIC device. Hemodynamic profile, right ventricular function, and pulmonary vascular morphometry were obtained in a conditional endothelium-specific Wnt5a knockout ( Wnt5a ECKO ) mouse model under normoxia, chronic hypoxia, and hypoxia recovery. Results: Quantification of Wnt ligand expression in healthy PMVECs cocultured with pericytes demonstrated a 35-fold increase in Wnt5a, a known Wnt/planar cell polarity ligand. This Wnt5a spike was not seen in PAH PMVECs, which correlated with an inability to recruit pericytes in Matrigel coculture assays. Exosomes purified from media demonstrated an increase in Wnt5a content when healthy PMVECs were cocultured with pericytes, a finding that was not observed in exosomes of PAH PMVECs. Furthermore, the addition of either recombinant Wnt5a or purified healthy PMVEC exosomes increased pericyte recruitment to PAH PMVECs in coculture studies. Although no differences were noted in normoxia and chronic hypoxia, Wnt5a ECKO mice demonstrated persistent pulmonary hypertension and right ventricular failure 4 weeks after recovery from chronic hypoxia, which correlated with significant reduction, muscularization, and decreased pericyte coverage of microvessels. Conclusions: We identify Wnt5a as a key mediator for the establishment of pulmonary endothelium-pericyte interactions, and its loss could contribute to PAH by reducing the viability of newly formed vessels. We speculate that therapies that mimic or restore Wnt5a production could help prevent loss of small vessels in PAH.

Idiopathic pulmonary fibrosis (IPF) is a life-threatening progressive lung disorder with limited therapeutic options. While interleukin-10 (IL-10) is a potent anti-inflammatory and anti-fibrotic cytokine, its utility in treating lung fibrosis has been limited by its short half-life. We describe an innovative hydrogel-based approach to deliver recombinant IL-10 to the lung for the prevention and reversal of pulmonary fibrosis in a mouse model of bleomycin-induced lung injury. Our studies show that a hyaluronan and heparin-based hydrogel system locally delivers IL-10 by capitalizing on the ability of heparin to reversibly bind IL-10 without bleeding or other complications. This formulation is significantly more effective than soluble IL-10 for both preventing and reducing collagen deposition in the lung parenchyma after 7 days of intratracheal administration. The antifibrotic effect of IL-10 in this system is dependent on suppression of TGFβ driven collagen production by lung fibroblasts and myofibroblasts. We conclude that hydrogelbased delivery of IL-10 to the lung is a promising therapy for fibrotic lung disorders.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a user-friendly system that can address imaging applications with varied requirements in terms of resolution (mesoscopic to sub-micrometer), sample geometry (size, shape, and number), and compatibility with tissue-clearing protocols and sample holders of various refractive indices. We present a 'hybrid' system that combines a novel non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet architecture for versatile multi-scale volumetric imaging. Main TextRecent advances in tissue-clearing protocols greatly reduce optical scattering, aberrations, and background uorescence, enabling deep-tissue imaging with high resolution and contrast. These approaches have yielded new insights in many elds, including neuroscience, developmental biology, and anatomic pathology [1][2][3][4][5][6][7][8][9][10][11]. Light-sheet microscopy has emerged as a preferred means for highresolution volumetric imaging of cleared tissues due to its unrivaled speed and low photobleaching [12,13]. Many variants of light-sheet microscopes have been developed in recent years by academic researchers and commercial entities to tackle a diverse range of imaging applications (Error! Reference source not found. and Error! Reference source not found.) [14][15][16][17][18]. Whereas individual light-sheet systems are well-suited for a subset of cleared-tissue applications, trade-offs are inevitable. In particular, no current light-sheet microscope can satisfy all of the following requirements: (1) user-friendly mounting

Reduced endothelial-pericyte interactions are linked to progressive small vessel loss in pulmonary arterial hypertension (PAH), but the molecular mechanisms underlying this disease remain poorly understood. To identify relevant gene candidates associated with aberrant pericyte behavior, we performed a transcriptome analysis of patient-derived donor control and PAH lung pericytes followed by functional genomics analysis. Compared with donor control cells, PAH pericytes had significant enrichment of genes involved in various metabolic processes, the top hit being PDK4, a gene coding for an enzyme that suppresses mitochondrial activity in favor of glycolysis. Given reports that link reduced mitochondrial activity with increased PAH cell proliferation, we hypothesized that increased PDK4 is associated with PAH pericyte hyperproliferation and reduced endothelial-pericyte interactions. We found that PDK4 gene and protein expression was significantly elevated in PAH pericytes and correlated with reduced mitochondrial metabolism, higher rates of glycolysis, and hyperproliferation. Importantly, reducing PDK4 levels restored mitochondrial metabolism, reduced cell proliferation, and improved endothelial-pericyte interactions. To our knowledge, this is the first study that documents significant differences in gene expression between human donor control and PAH lung pericytes and the link between mitochondrial dysfunction and aberrant endothelial-pericyte interactions in PAH. Comprehensive characterization of these candidate genes could provide novel therapeutic targets to improve endothelial-pericyte interactions and prevent small vessel loss in PAH.

Drug-induced pulmonary arterial hypertension (D-PAH) is a form of World Health Organization Group 1 pulmonary hypertension (PH) defined by severe small vessel loss and obstructive vasculopathy, which leads to progressive right heart failure and death. To date, 16 different compounds have been associated with D-PAH, including anorexigens, recreational stimulants, and more recently, several Food and Drug Administration-approved medications. Although the clinical manifestation, pathology, and hemodynamic profile of D-PAH are indistinguishable from other forms of pulmonary arterial hypertension, its clinical course can be unpredictable and to some degree dependent on removal of the offending agent. Because only a subset of individuals develop D-PAH, it is probable that genetic susceptibilities play a role in the pathogenesis, but the characterization of the genetic factors responsible for these susceptibilities remains rudimentary. Besides aggressive treatment with PH-specific therapies, the major challenge in the management of D-PAH remains the early identification of compounds capable of injuring the pulmonary circulation in susceptible individuals. The implementation of pharmacovigilance, precision medicine strategies, and global warning systems will help facilitate the identification of high-risk drugs and incentivize regulatory strategies to prevent further outbreaks of D-PAH. The goal for this review is to inform clinicians and scientists of the prevalence of D-PAH and to highlight the growing number of common drugs that have been associated with the disease.

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Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH), but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. Although no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV; rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared with healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.