FAM222A, Part of the BET-Regulated Basal Endothelial Transcriptome, Is a Novel Determinant of Endothelial Biology and Angiogenesis-Brief Report.

BACKGROUND: BETs (bromodomain and extraterminal domain-containing epigenetic reader proteins), including BRD4 (bromodomain-containing protein 4), orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced proinflammatory, proatherosclerotic transcriptional responses; BET inhibitors, like JQ1, repress these effects and decrease atherosclerosis. While BET effects in pathogenic conditions have prompted therapeutic BET inhibitor development, BET action under basal conditions, including ECs, has remained understudied. To understand BET action in basal endothelial transcriptional programs, we first analyzed EC RNA-Seq data in the absence versus presence of JQ1 before using BET regulation to identify novel determinants of EC biology and function. METHODS: RNA-Seq datasets of human umbilical vein ECs without and with JQ1 treatment were analyzed. After identifying C12orf34, also known as FAM222A (family with sequence similarity 222 member A), as a previously unreported, basally expressed, potently JQ1-induced EC gene, FAM222A was studied in endothelial and angiogenic responses in vitro using small-interference RNA silencing and lentiviral overexpression, in vitro, ex vivo and in vivo, including aortic sprouting, matrigel plug assays, and murine neonatal oxygen-induced retinopathy. RESULTS: Resting EC RNA-Seq data indicate BETs direct transcriptional programs underlying core endothelial properties including migration, proliferation, and angiogenesis. BET inhibition in resting ECs also significantly induced a subset of mRNAs, including FAM222A-a unique BRD4-regulated gene with no reported EC role. Silencing endothelial FAM222A significantly decreased cellular proliferation, migration, network formation, aorta sprouting, and Matrigel plug vascularization through coordinated modulation of VEGF (vascular endothelial growth factor) and NOTCH mediator expression in vitro, ex vivo, in vivo; lentiviral FAM222A overexpression had opposite effects. In vivo, siFAM222A significantly repressed retinal revascularization in neonatal murine oxygen-induced retinopathy through similar angiogenic signaling modulation. CONCLUSIONS: BET control over the basal endothelial transcriptome includes FAM222A, a novel, BRD4-regulated, key determinant of endothelial biology and angiogenesis.

Novel Approaches to Imaging the Pulmonary Vasculature and Right Heart.

There is an increased appreciation for the importance of the right heart and pulmonary circulation in several disease states across the spectrum of pulmonary hypertension and left heart failure. However, assessment of the structure and function of the right heart and pulmonary circulation can be challenging, due to the complex geometry of the right ventricle, comorbid pulmonary airways and parenchymal disease, and the overlap of hemodynamic abnormalities with left heart failure. Several new and evolving imaging modalities interrogate the right heart and pulmonary circulation with greater diagnostic precision. Echocardiographic approaches such as speckle-tracking and 3-dimensional imaging provide detailed assessments of regional systolic and diastolic function and volumetric assessments. Magnetic resonance approaches can provide high-resolution views of cardiac structure/function, tissue characterization, and perfusion through the pulmonary vasculature. Molecular imaging with positron emission tomography allows an assessment of specific pathobiologically relevant targets in the right heart and pulmonary circulation. Machine learning analysis of high-resolution computed tomographic lung scans permits quantitative morphometry of the lung circulation without intravenous contrast. Inhaled magnetic resonance imaging probes, such as hyperpolarized 129Xe magnetic resonance imaging, report on pulmonary gas exchange and pulmonary capillary hemodynamics. These approaches provide important information on right ventricular structure and function along with perfusion through the pulmonary circulation. At this time, the majority of these developing technologies have yet to be clinically validated, with few studies demonstrating the utility of these imaging biomarkers for diagnosis or monitoring disease. These technologies hold promise for earlier diagnosis and noninvasive monitoring of right heart failure and pulmonary hypertension that will aid in preclinical studies, enhance patient selection and provide surrogate end points in clinical trials, and ultimately improve bedside care.

Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis.

AIMS: Blood eosinophil count and eosinophil cationic protein (ECP) concentration are risk factors of cardiovascular diseases. This study tested whether and how eosinophils and ECP contribute to vascular calcification and atherogenesis. METHODS AND RESULTS: Immunostaining revealed eosinophil accumulation in human and mouse atherosclerotic lesions. Eosinophil deficiency in ΔdblGATA mice slowed atherogenesis with increased lesion smooth muscle cell (SMC) content and reduced calcification. This protection in ΔdblGATA mice was muted when mice received donor eosinophils from wild-type (WT), Il4-/-, and Il13-/- mice or mouse eosinophil-associated-ribonuclease-1 (mEar1), a murine homologue of ECP. Eosinophils or mEar1 but not interleukin (IL) 4 or IL13 increased the calcification of SMC from WT mice but not those from Runt-related transcription factor-2 (Runx2) knockout mice. Immunoblot analyses showed that eosinophils and mEar1 activated Smad-1/5/8 but did not affect Smad-2/3 activation or expression of bone morphogenetic protein receptors (BMPR-1A/1B/2) or transforming growth factor (TGF)-ß receptors (TGFBR1/2) in SMC from WT and Runx2 knockout mice. Immunoprecipitation showed that mEar1 formed immune complexes with BMPR-1A/1B but not TGFBR1/2. Immunofluorescence double-staining, ligand binding, and Scatchard plot analysis demonstrated that mEar1 bound to BMPR-1A and BMPR-1B with similar affinity. Likewise, human ECP and eosinophil-derived neurotoxin (EDN) also bound to BMPR-1A/1B on human vascular SMC and promoted SMC osteogenic differentiation. In a cohort of 5864 men from the Danish Cardiovascular Screening trial and its subpopulation of 394 participants, blood eosinophil counts and ECP levels correlated with the calcification scores of different arterial segments from coronary arteries to iliac arteries. CONCLUSION: Eosinophils release cationic proteins that can promote SMC calcification and atherogenesis using the BMPR-1A/1B-Smad-1/5/8-Runx2 signalling pathway.

Intrapulmonary arterial contraction assay reveals region-specific deregulation of vasoreactivity to lung injuries.

Intrapulmonary arteries located in the proximal lung differ from those in the distal lung in size, cellular composition, and the surrounding microenvironment. However, whether these structural variations lead to region-specific regulation of vasoreactivity in homeostasis and following injury is unknown. Herein, we employ a two-step method of precision-cut lung slice (PCLS) preparation, which maintains almost intact intrapulmonary arteries, to assess contractile and relaxation responses of proximal preacinar arteries (PaAs) and distal intraacinar arteries (IaAs) in mice. We found that PaAs exhibited robust vasoconstriction in response to contractile agonists and significant nitric oxide (NO)-induced vasodilation. In comparison, IaAs were less contractile and displayed a greater relaxation response to NO. Furthermore, in a mouse model of pulmonary arterial hypertension (PAH) induced by chronic exposure to ovalbumin (OVA) allergen and hypoxia (OVA-HX), IaAs demonstrated a reduced vasocontraction despite vascular wall thickening with the emergence of new αSMA+ cells coexpressing markers of pericytes. In contrast, PaAs became hypercontractile and less responsive to NO. The reduction in relaxation of PaAs was associated with decreased expression of protein kinase G, a key component of the NO pathway, following chronic OVA-HX exposure. Taken together, the PCLS prepared using the modified preparation method enables functional evaluation of pulmonary arteries in different anatomical locations and reveals region-specific mechanisms underlying the pathophysiology of PAH in a mouse model.NEW & NOTEWORTHY Utilizing mouse precision-cut lung slices with preserved intrapulmonary vessels, we demonstrated a location-dependent structural and contractile regulation of pulmonary arteries in health and on noxious stimulations. For instance, chronic ovalbumin and hypoxic exposure increased pulmonary arterial pressure (PAH) by remodeling intraacinar arterioles to reduce vascular wall compliance while enhancing vasoconstriction in proximal preacinar arteries. These findings suggest region-specific mechanisms and therapeutic targets for pulmonary vascular diseases such as PAH.

Wnt7a deficit is associated with dysfunctional angiogenesis in pulmonary arterial hypertension.

INTRODUCTION: Pulmonary arterial hypertension (PAH) is characterised by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. We hypothesised that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH. METHODS: Lung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a -/- mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx). RESULTS: Healthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with the formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced vascular endothelial growth factor (VEGF)-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signalling by facilitating Y1175 tyrosine phosphorylation in vascular endothelial growth factor receptor 2 (VEGFR2) through receptor tyrosine kinase-like orphan receptor 2 (ROR2), a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a -/- mice under either chronic hypoxia or SuHx, global Wnt7a +/- mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodelling. Similar to PAH, Wnt7a +/- PMVECs exhibited an insufficient angiogenic response to VEGF-A that improved with Wnt7a. CONCLUSIONS: Wnt7a promotes VEGF signalling in lung PMVECs and its loss is associated with an insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH.

High thermo-optic tunability in PECVD silicon-rich amorphous silicon carbide.

In this work, the thermo-optic coefficient (TOC) of the silicon-rich amorphous silicon carbide (a-SiC) thin film deposited by plasma-enhanced chemical vapor deposition (PECVD) was characterized. We found that the TOC of the film increases as its silicon content increases. A more than threefold improvement in the TOC was measured, reaching a TOC as high as 1.88×10-4 ∘C-1, which is comparable to that of crystalline silicon. An efficient thermo-optic phase shifter has also been demonstrated by integrating the silicon-rich a-SiC micro-ring structure with a NiCr heater. Tunability of 0.117 nm/mW was demonstrated, and a corresponding tuning efficiency P π as low as 4.2 mW has been measured at an optical wavelength of 1550 nm. These findings make silicon-rich a-SiC a good candidate material for thermo-optic applications in photonic integrated circuits.

Realization of a wide steering end-fire facet optical phased array using silicon rich silicon nitride.

The design, fabrication, and characterization of a 16-element optical phased array (OPA) using a high index (n = 3.1) silicon-rich silicon nitride (SRN) is demonstrated. We present one-dimensional beam steering with end-fire facet antennas over a wide steering range of >115° at a fixed wavelength of 1525 nm. A beam width of 6.3° has been measured at boresight, consistent with theory. We demonstrate SRN as a viable material choice for chip-scale OPA applications due to its high thermo-optic coefficient, high optical power handling capacity [negligible two-photon absorption (TPA)], wide transparency window, and CMOS compatibility.

Metabolomic and transcriptomic signatures of chemogenetic heart failure.

The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H2O2) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H2O2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart.NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H2O2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.

Silicon-organic hybrid thermo-optic switch based on a slot waveguide directional coupler.

We propose and demonstrate a passively biased 2 × 2 thermo-optic switch with high power efficiency and fast response time. The device benefits from the highly concentrated optical field of a slot waveguide mode and the strong thermo-optic effect of a nematic liquid crystal (NLC) cladding. The NLC fills the nano-slot region and is aligned by the subwavelength grating inside. The measured power consumption and thermal time constant are 0.58 mW and 11.8 µs, respectively, corresponding to a figure-of-merit of 6.8 mW µs. The proposed silicon-organic hybrid device provides a new solution to design thermo-optic actuators having low power consumption and fast operation speed.

Circulating BMP9 Protects the Pulmonary Endothelium during Inflammation-induced Lung Injury in Mice.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

Protocol paper: a multi-center, double-blinded, randomized, 6-month, placebo-controlled study followed by 12-month open label extension to evaluate the safety and efficacy of Saracatinib in Fibrodysplasia Ossificans Progressiva (STOPFOP).

BACKGROUND: Fibrodysplasia Ossificans Progressiva (FOP) is a genetic, progressive and devastating disease characterized by severe heterotopic ossification (HO), loss of mobility and early death. There are no FDA approved medications. The STOPFOP team identified AZD0530 (saracatinib) as a potent inhibitor of the ALK2/ACVR1-kinase which is the causative gene for this rare bone disease. AZD0530 was proven to prevent HO formation in FOP mouse models. The STOPFOP trial investigates the repositioning of AZD0530, originally developed for ovarian cancer treatment, to treat patients with FOP. METHODS: The STOPFOP trial is a phase 2a study. It is designed as a European, multicentre, 6-month double blind randomized controlled trial of AZD0530 versus placebo, followed by a 12-month trial comparing open-label extended AZD0530 treatment with natural history data as a control. Enrollment will include 20 FOP patients, aged 18-65 years, with the classic FOP mutation (ALK2 R206H). The primary endpoint is objective change in heterotopic bone volume measured by low-dose whole-body computer tomography (CT) in the RCT phase. Secondary endpoints include 18F NaF PET activity and patient reported outcome measures. DISCUSSION: Clinical trials in rare diseases with limited study populations pose unique challenges. An ideal solution for limiting risks in early clinical studies is drug repositioning - using existing clinical molecules for new disease indications. Using existing assets may also allow a more fluid transition into clinical practice. With positive study outcome, AZD0530 may provide a therapy for FOP that can be rapidly progressed due to the availability of existing safety data from 28 registered clinical trials with AZD0530 involving over 600 patients. TRIAL REGISTRATION: EudraCT, 2019-003324-20. Registered 16 October 2019, https://www.clinicaltrialsregister.eu/ctr-search/trial/2019-003324-20/NL . CLINICALTRIALS: gov , NCT04307953 . Registered 13 March 2020.

Efficient and compact thermo-optic phase shifter in silicon-rich silicon nitride.

The design, fabrication, and characterization of low-loss ultra-compact bends in high-index (n=3.1 at λ=1550nm) plasma-enhanced chemical vapor deposition silicon-rich silicon nitride (SRN) were demonstrated and utilized to realize efficient, small footprint thermo-optic phase shifter. Compact bends were structured into a folded waveguide geometry to form a rectangular spiral within an area of 65×65µm2, having a total active waveguide length of 1.2 mm. The device featured a phase-shifting efficiency of 8mW/π and a 3 dB switching bandwidth of 15 KHz. We propose SRN as a promising thermo-optic platform that combines the properties of silicon and stoichiometric silicon nitride.

Exacerbated inflammatory signaling underlies aberrant response to BMP9 in pulmonary arterial hypertension lung endothelial cells.

Imbalanced transforming growth factor beta (TGFß) and bone morphogenetic protein (BMP) signaling are postulated to favor a pathological pulmonary endothelial cell (EC) phenotype in pulmonary arterial hypertension (PAH). BMP9 is shown to reinstate BMP receptor type-II (BMPR2) levels and thereby mitigate hemodynamic and vascular abnormalities in several animal models of pulmonary hypertension (PH). Yet, responses of the pulmonary endothelium of PAH patients to BMP9 are unknown. Therefore, we treated primary PAH patient-derived and healthy pulmonary ECs with BMP9 and observed that stimulation induces transient transcriptional signaling associated with the process of endothelial-to-mesenchymal transition (EndMT). However, solely PAH pulmonary ECs showed signs of a mesenchymal trans-differentiation characterized by a loss of VE-cadherin, induction of transgelin (SM22α), and reorganization of the cytoskeleton. In the PAH cells, a prolonged EndMT signaling was found accompanied by sustained elevation of pro-inflammatory, pro-hypoxic, and pro-apoptotic signaling. Herein we identified interleukin-6 (IL6)-dependent signaling to be the central mediator required for the BMP9-induced phenotypic change in PAH pulmonary ECs. Furthermore, we were able to target the BMP9-induced EndMT process by an IL6 capturing antibody that normalized autocrine IL6 levels, prevented mesenchymal transformation, and maintained a functional EC phenotype in PAH pulmonary ECs. In conclusion, our results show that the BMP9-induced aberrant EndMT in PAH pulmonary ECs is dependent on exacerbated pro-inflammatory signaling mediated through IL6.

Thermo-optic properties of silicon-rich silicon nitride for on-chip applications.

We demonstrate the thermo-optic properties of silicon-rich silicon nitride (SRN) films deposited using plasma-enhanced chemical vapor deposition (PECVD). Shifts in the spectral response of Mach-Zehnder interferometers (MZIs) as a function of temperature were used to characterize the thermo-optic coefficients of silicon nitride films with varying silicon contents. A clear relation is demonstrated between the silicon content and the exhibited thermo-optic coefficient in silicon nitride films, with the highest achievable coefficient being as high as (1.65±0.08) ×10-4 K-1. Furthermore, we realize an SRN multi-mode interferometer (MMI) based thermo-optic switch with over 20 dB extinction ratio and total power consumption for two-port switching of 50 mW.

Highly sensitive silicon photonic temperature sensor based on liquid crystal filled slot waveguide directional coupler.

A highly sensitive silicon photonic temperature sensor based on silicon-on-insulator (SOI) platform has been proposed and demonstrated. A two-mode nano-slot waveguide device structure cladded with a nematic liquid crystal (LC), E7, was adopted to facilitate strong light-matter interaction and achieve high sensitivity. The fabricated sensor was characterized by measuring the optical transmission spectra at different ambient temperatures. The extracted temperature sensitivities of the E7-filled device are 0.810 nm/°C around room temperature and 1.619 nm/°C near 50°C, which match well with simulation results based on a theoretical analysis. The results obtained represent the highest experimentally demonstrated temperature sensitivity for a silicon-waveguide temperature sensor on SOI platform. The slot waveguide directional coupler device configuration provides submicron one-dimensional spatial resolution and flexible selection in LC materials for designing temperature sensitivity and operational temperature range required by specific applications.

Bone Morphogenetic Protein 9 Is a Mechanistic Biomarker of Portopulmonary Hypertension.

RATIONALE: BMP9 (bone morphogenetic protein 9) is a circulating endothelial quiescence factor with protective effects in pulmonary arterial hypertension (PAH). Loss-of-function mutations in BMP9, its receptors, and downstream effectors have been reported in heritable PAH. OBJECTIVES: To determine how an acquired deficiency of BMP9 signaling might contribute to PAH. METHODS: Plasma levels of BMP9 and antagonist soluble endoglin were measured in group 1 PAH, group 2 and 3 pulmonary hypertension (PH), and in patients with severe liver disease without PAH. MEASUREMENTS AND MAIN RESULTS: BMP9 levels were markedly lower in portopulmonary hypertension (PoPH) versus healthy control subjects, or other etiologies of PAH or PH; distinguished PoPH from patients with liver disease without PAH; and were an independent predictor of transplant-free survival. BMP9 levels were decreased in mice with PH associated with CCl4-induced portal hypertension and liver cirrhosis, but were normal in other rodent models of PH. Administration of ALK1-Fc, a BMP9 ligand trap consisting of the activin receptor-like kinase-1 extracellular domain, exacerbated PH and pulmonary vascular remodeling in mice treated with hypoxia versus hypoxia alone. CONCLUSIONS: BMP9 is a sensitive and specific biomarker of PoPH, predicting transplant-free survival and the presence of PAH in liver disease. In rodent models, acquired deficiency of BMP9 signaling can predispose to or exacerbate PH, providing a possible mechanistic link between PoPH and heritable PAH. These findings describe a novel experimental model of severe PH that provides insight into the synergy between pulmonary vascular injury and diminished BMP9 signaling in the pathogenesis of PAH.