Targeted delivery of protein arginine deiminase-4 inhibitors to limit arterial intimal NETosis and preserve endothelial integrity.

AIMS: Recent evidence suggests that 'vulnerable plaques', which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage. METHODS AND RESULTS: We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity. CONCLUSIONS: NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.

Association of Somatic GNAQ Mutation With Capillary Malformations in a Case of Choroidal Hemangioma.

Importance: Choroidal hemangiomas are defined by a thickened choroid owing to vessel overgrowth, which may increase the intraocular pressure and lead to glaucoma. Choroidal hemangioma and glaucoma often co-occur in patients with Sturge-Weber syndrome, a rare neurocutaneous disorder characterized by capillary malformations. Objective: To determine whether the mutation found in most capillary malformations, GNAQ R183Q (c.548G>A), was present in the choroidal hemangioma of a patient with Sturge-Weber syndrome. Design, Setting, and Participant: Using laser-capture microdissection, choroidal blood vessels were isolated from paraffin-embedded tissue sections, and genomic DNA was extracted for mutational analysis. Choroidal sections were analyzed in parallel. A patient with choroidal hemangioma and Sturge-Weber syndrome who had undergone enucleation was analyzed in this study at Boston Children's Hospital. Negative controls were choroidal tissue from an eye with retinoblastoma and unaffected lung tissue; brain tissue from a different patient with Sturge-Weber syndrome served as a positive control. Infantile hemangioma was analyzed as well. Data were analyzed in 2018. Main Outcomes and Measures: The mutant allelic frequency of GNAQ R183 and GNAQ Q209L/H/P was determined by droplet digital polymerase chain reaction on isolated genomic DNA. The infantile hemangioma marker glucose transporter-1 was visualized by immunofluorescent staining of tissue sections. Results: The GNAQ R183Q mutation was present in the patient's choroidal vessels (21.1%) at a frequency similar to that found in brain tissue from a different patient with Sturge-Weber syndrome (25.1%). In contrast, choroidal vessels from a case of retinoblastoma were negative for the mutation (0.5%), as was lung tissue (0.2%). The patient's choroidal tissue was negative for the 3 GNAQ mutations associated with congenital hemangioma and for the infantile hemangioma marker glucose transporter-1. Conclusions and Relevance: The results suggest that a more accurate description for choroidal hemangioma in patients with Sturge-Weber syndrome is choroidal capillary malformation. This finding may explain why propranolol, used to treat infantile hemangiomas, has been largely ineffective in patients with choroidal hemangioma. Further studies are needed to corroborate this finding.

Human microvasculature-on-a chip: anti-neovasculogenic effect of nintedanib in vitro.

Idiopathic pulmonary fibrosis is characterized by a progressive scarring and stiffening of the peripheral lung tissue that decreases lung function. Over the course of the disease, the lung microvasculature undergoes extensive remodeling. There is increased angiogenesis around fibrotic foci and an absence of microvessels within the foci. To elucidate how the anti-fibrotic drug nintedanib acts on vascular remodeling, we used an in vitro model of perfusable microvessels made with primary endothelial cells and primary lung fibroblasts in a microfluidic chip. The microvasculature model allowed us to study the impact of nintedanib on permeability, vascularized area, and cell-cell interactions. The anti-vasculogenic impact of nintedanib was visible at the minimal concentrations of 10 nM, showing a significant increase in vessel permeability. Furthermore, nintedanib decreased microvessel density, diameter, and influenced fibroblast organization around endothelial microvessels. These results show that nintedanib acts on the endothelial network formation and endothelial-perivascular interactions. Advanced in vitro microvasculature models may thus serve to pinpoint the mechanistic effect of anti-fibrotic drugs on the microvascular remodeling in 3D and refine findings from animal studies.

Increased PD-L1 expression and IL-6 secretion characterize human lung tumor-derived perivascular-like cells that promote vascular leakage in a perfusable microvasculature model.

Pericytes represent important support cells surrounding microvessels found in solid organs. Emerging evidence points to their involvement in tumor progression and metastasis. Although reported to be present in the human lung, their specific presence and functional orientation within the tumor microenvironment in non-small cell lung cancer (NSCLC) has not yet been adequately studied. Using a multiparameter approach, we prospectively identified, sorted and expanded mesenchymal cells from human primary NSCLC samples based on co-expression of CD73 and CD90 while lacking hematopoietic and endothelial lineage markers (CD45, CD31, CD14 and Gly-A) and the epithelial marker EpCAM. Compared to their normal counterpart, tumor-derived Lineage-EpCAM-CD73+CD90+ cells showed enhanced expression of the immunosuppressive ligand PD-L1, a higher constitutive secretion of IL-6 and increased basal αSMA levels. In an in vitro model of 3D microvessels, both tumor-derived and matched normal Lineage-EpCAM-CD73+CD90+ cells supported the assembly of perfusable vessels. However, tumor-derived Lineage-EpCAM-CD73+CD90+ cells led to the formation of vessels with significantly increased permeability. Together, our data show that perivascular-like cells present in NSCLC retain functional abnormalities in vitro. Perivascular-like cells as an eventual target in NSCLC warrants further investigation.

Digital Quantification of Proteins and mRNA in Single Mammalian Cells.

Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells.

Primary Human Lung Pericytes Support and Stabilize In Vitro Perfusable Microvessels.

The formation of blood vessels is a complex tissue-specific process that plays a pivotal role during developmental processes, in wound healing, cancer progression, fibrosis, and other pathologies. To study vasculogenesis and vascular remodeling in the context of the lung, we developed an in vitro microvascular model that closely mimics the human lung microvasculature in terms of three-dimensional architecture, accessibility, functionality, and cell types. Human pericytes from the distal airway were isolated and characterized using flow cytometry. To assess their role in the generation of normal microvessels, lung pericytes were mixed in fibrin gel and seeded into well-defined microcompartments together with primary endothelial cells (human umbilical cord vein endothelial cells). Patent microvessels covering an area of 3.1 mm(2) formed within 3-5 days and were stable for up to 14 days. Soluble signals from the lung pericytes were necessary to establish perfusability, and pericytes migrated toward endothelial microvessels. Cell-cell communication in the form of adherens and tight junctions, as well as secretion of basement membrane were confirmed using transmission electron microscopy and immunocytochemistry on chip. Direct coculture of pericytes with endothelial cells decreased the microvascular permeability by one order of magnitude from 17.8×10(-6) to 2.0×10(-6) cm/s and led to vessels with significantly smaller and less variable diameter. Upon phenylephrine administration, vasoconstriction was observed in microvessels lined with pericytes, but not in endothelial microvessels only. Perfusable microvessels were also generated with human lung microvascular endothelial cells and lung pericytes. Human lung pericytes were thus shown to have a prominent influence on microvascular morphology, permeability, vasoconstriction, and long-term stability in an in vitro microvascular system. This biomimetic platform opens new possibilities to test functions and interactions of patient-derived cells in a physiologically relevant microvascular setting.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells.

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.