Endothelial PHD2 deficiency induces apoptosis resistance and inflammation via AKT activation and AIP1 loss independent of HIF2α.

In hypoxic and pseudohypoxic rodent models of pulmonary hypertension (PH), hypoxia-inducible factor (HIF) inhibition attenuates disease initiation. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PH in humans, indicating the involvement of other mechanisms. Given the association between endothelial cell dysfunction and PH, the effects of pseudohypoxia and its underlying pathways were investigated in primary human lung endothelial cells. PHD2 silencing or inhibition, while activating HIF2α, induced apoptosis-resistance and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activated AKT and ERK, inhibited JNK, and reduced AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affected these same kinase pathways and produced a similar dysfunctional endothelial cell phenotype, which was partially reversed by AKT inhibition. Consistent with these in vitro findings, AIP1 protein levels in lung endothelial cells were decreased in Tie2-Cre/Phd2 knockout mice compared with wild-type controls. Lung vascular endothelial cells from patients with pulmonary arterial hypertension (PAH) showed IFN/STAT activation. Lung tissue from both SU5416/hypoxia PAH rats and patients with PAH all showed AKT activation and dysregulated AIP1 expression. In conclusion, PHD2 deficiency in lung vascular endothelial cells drives an apoptosis-resistant and inflammatory phenotype, mediated by AKT activation and AIP1 loss independent of HIF signaling. Targeting these pathways, including PHD2, AKT, and AIP1, holds the potential for developing new treatments for endothelial dysfunction in PH.NEW & NOTEWORTHY HIF activation alone does not conclusively lead to human PH, suggesting that HIF-independent signaling may also contribute to hypoxia-induced PH. This study demonstrated that PHD2 silencing-induced pseudohypoxia in human lung endothelial cells suppresses apoptosis and activates STAT, effects that persist despite HIF2α inhibition or knockdown and are attributed to AKT and ERK activation, JNK inhibition, and AIP1 loss. These findings align with observations in lung endothelial cells and tissues from PAH rodent models and patients.

The Aconitate Decarboxylase 1/Itaconate Pathway Modulates Immune Dysregulation and Associates with Cardiovascular Disease Markers and Disease Activity in Systemic Lupus Erythematosus.

The Krebs cycle enzyme aconitate decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Previously, we reported that administration of 4-octyl itaconate to lupus-prone mice abrogated immune dysregulation and clinical features. In this study, we explore the role of the endogenous ACOD1/itaconate pathway in the development of TLR7-induced lupus (imiquimod [IMQ] model). We found that, in vitro, ACOD1 was induced in mouse bone marrow-derived macrophages and human monocyte-derived macrophages following TLR7 stimulation. This induction was partially dependent on type I IFN receptor signaling and on specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum levels of anti-dsDNA and proinflammatory cytokines, and enhanced kidney immune complex deposition and proteinuria, when compared with the IMQ-treated wild-type mice. Consistent with these results, Acod1-/- bone marrow-derived macrophages treated in vitro with IMQ showed higher proinflammatory features. Furthermore, itaconate serum levels in systemic lupus erythematosus patients were decreased compared with healthy individuals, in association with disease activity and specific perturbed cardiometabolic parameters. These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in systemic lupus erythematosus, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

Long-term engraftment and maturation of autologous iPSC-derived cardiomyocytes in two rhesus macaques.

Cellular therapies with cardiomyocytes produced from induced pluripotent stem cells (iPSC-CMs) offer a potential route to cardiac regeneration as a treatment for chronic ischemic heart disease. Here, we report successful long-term engraftment and in vivo maturation of autologous iPSC-CMs in two rhesus macaques with small, subclinical chronic myocardial infarctions, all without immunosuppression. Longitudinal positron emission tomography imaging using the sodium/iodide symporter (NIS) reporter gene revealed stable grafts for over 6 and 12 months, with no teratoma formation. Histological analyses suggested capability of the transplanted iPSC-CMs to mature and integrate with endogenous myocardium, with no sign of immune cell infiltration or rejection. By contrast, allogeneic iPSC-CMs were rejected within 8 weeks of transplantation. This study provides the longest-term safety and maturation data to date in any large animal model, addresses concerns regarding neoantigen immunoreactivity of autologous iPSC therapies, and suggests that autologous iPSC-CMs would similarly engraft and mature in human hearts.

BMPR2 Loss Activates AKT by Disrupting DLL4/NOTCH1 and PPARγ Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Asthmatic patients with high serum amyloid A have proinflammatory HDL: Implications for augmented systemic and airway inflammation.

RATIONALE: Serum amyloid A (SAA) is bound to high-density lipoproteins (HDL) in blood. Although SAA is increased in the blood of patients with asthma, it is not known whether this modifies asthma severity. OBJECTIVE: We sought to define the clinical characteristics of patients with asthma who have high SAA levels and assess whether HDL from SAA-high patients with asthma is proinflammatory. METHODS: SAA levels in serum from subjects with and without asthma were quantified by ELISA. HDLs isolated from subjects with asthma and high SAA levels were used to stimulate human monocytes and were intravenously administered to BALB/c mice. RESULTS: An SAA level greater than or equal to 108.8 µg/mL was defined as the threshold to identify 11% of an asthmatic cohort (n = 146) as being SAA-high. SAA-high patients with asthma were characterized by increased serum C-reactive protein, IL-6, and TNF-α; older age; and an increased prevalence of obesity and severe asthma. HDL isolated from SAA-high patients with asthma (SAA-high HDL) had an increased content of SAA as compared with HDL from SAA-low patients with asthma and induced the secretion of IL-6, IL-1ß, and TNF-α from human monocytes via a formyl peptide receptor 2/ATP/P2X purinoceptor 7 axis. Intravenous administration to mice of SAA-high HDL, but not normal HDL, induced systemic inflammation and amplified allergen-induced neutrophilic airway inflammation and goblet cell metaplasia. CONCLUSIONS: SAA-high patients with asthma are characterized by systemic inflammation, older age, and an increased prevalence of obesity and severe asthma. HDL from SAA-high patients with asthma is proinflammatory and, when intravenously administered to mice, induces systemic inflammation, and amplifies allergen-induced neutrophilic airway inflammation. This suggests that systemic inflammation induced by SAA-high HDL may augment disease severity in asthma.

Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

Unexpected sirolimus-stimulated airway hyperreactivity in lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a multisystem disease affecting primarily women, characterised in the lung by proliferation of LAM cells, abnormal smooth muscle-like cells with dysfunctional tuberous sclerosis complex genes. This dysfunction results in activation of mechanistic target of rapamycin (mTOR), leading to LAM cell proliferation. Sirolimus (rapamycin) is the only United States Food and Drug Administration-approved treatment for pulmonary LAM, resulting in decreased LAM cell growth/size and stabilised lung function [1].

Mono-ADP-ribosyltransferase 1 ( Artc1 )-deficiency decreases tumorigenesis, increases inflammation, decreases cardiac contractility, and reduces survival.

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD + to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1 -KO and Artc1/Arh1 -double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF- α . In a xenograft model using tumorigenic Arh1 -KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1 -KO and heterozygous recipient mice, with tumor infiltration by CD8 + T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1 -double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1 -KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1 -KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1 -KO was less than their Artc1 -WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility.

ADP-ribose-acceptor hydrolase 2 ( Arh2 ) deficiency results in cardiac dysfunction, tumorigenesis, inflammation, and decreased survival.

ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2 -knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2 -KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2 -Het mice showed results similar to those seen in Arh2 -KO mice except for cardiomegaly. Arh2 -KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2 -Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2 -KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.

Differential Effect of Dietary Supplementation with a Soybean Oil Enriched in Oleic Acid versus Linoleic Acid on Plasma Lipids and Atherosclerosis in LDLR-Deficient Mice.

Both monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) play important roles in lipid metabolism, and diets enriched with either of these two fatty acids are associated with decreased cardiovascular risk. Conventional soybean oil (CSO), a common food ingredient, predominantly contains linoleic acid (LA; C18:2), a n-6 PUFA. Recently, a modified soybean oil (MSO) enriched in oleic acid (C18:1), a n-9 MUFA, has been developed, because of its improved chemical stability to oxidation. However, the effect of the different dietary soybean oils on cardiovascular disease remains unknown. To test whether diets rich in CSO versus MSO would attenuate atherosclerosis development, LDL receptor knock-out (LDLR-KO) mice were fed a Western diet enriched in saturated fatty acids (control), or a Western diet supplemented with 5% (w/w) LA-rich CSO or high-oleic MSO for 12 weeks. Both soybean oils contained a similar amount of linolenic acid (C18:3 n-3). The CSO diet decreased plasma lipid levels and the cholesterol content of VLDL and LDL by approximately 18% (p < 0.05), likely from increased hepatic levels of PUFA, which favorably regulated genes involved in cholesterol metabolism. The MSO diet, but not the CSO diet, suppressed atherosclerotic plaque size compared to the Western control diet (Control Western diet: 6.5 ± 0.9%; CSO diet: 6.4 ± 0.7%; MSO diet: 4.0 ± 0.5%) (p < 0.05), independent of plasma lipid level changes. The MSO diet also decreased the ratio of n-6/n-3 PUFA in the liver (Control Western diet: 4.5 ± 0.2; CSO diet: 6.1 ± 0.2; MSO diet: 2.9 ± 0.2) (p < 0.05), which correlated with favorable hepatic gene expression changes in lipid metabolism and markers of systemic inflammation. In conclusion, supplementation of the Western diet with MSO, but not CSO, reduced atherosclerosis development in LDLR-KO mice independent of changes in plasma lipids.

Tetramerization of STAT5 promotes autoimmune-mediated neuroinflammation.

Signal tranducer and activator of transcription 5 (STAT5) plays a critical role in mediating cellular responses following cytokine stimulation. STAT proteins critically signal via the formation of dimers, but additionally, STAT tetramers serve key biological roles, and we previously reported their importance in T and natural killer (NK) cell biology. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knockin (DKI) mouse strain, we report here that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The mild EAE phenotype observed in DKI mice correlates with the impaired extravasation of pathogenic T-helper 17 (Th17) cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated STAT5 tetramerization regulates the production of CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune neuroinflammation.

Clearance of pegylated interferon by Kupffer cells limits NK cell activation and therapy response of patients with HBV infection.

Pegylated interferon-α (PEG-IFN-α), where IFN-α is attached to polyethylene glycol (PEG), is an approved treatment for chronic hepatitis B virus (HBV) infection, a disease that causes liver-related morbidity and mortality in 257 million people worldwide. It is unknown why only a minority of patients respond to PEG-IFN-α. Using sequential blood samples and liver biopsies of patients with chronic HBV infection before, during, and after PEG-IFN-α treatment, we find that patients with early natural killer (NK) cell activation after PEG-IFN-α injection experienced greater liver inflammation, lysis of HBV-infected hepatocytes, and hepatitis B surface antigen (HBsAg) decline than those without. NK cell activation was associated with induction of interferon-stimulated genes and determined by PEG-IFN-α pharmacokinetics. Patients with delayed increases in PEG-IFN-α concentrations had greater amounts of PEG-specific immunoglobulin M (IgM) immune complexes in the blood and more PEG and IgM detected in the liver than patients with rapid increase in PEG-IFN-α concentration. This was associated with reduced NK cell activation. These results indicate that the immunomodulatory functions of PEG-IFN-α, particularly activation of NK cells, play a pivotal role in the response to treatment and further demonstrate that these functions are affected by PEG-IFN-α pharmacokinetics. Accelerated clearance of antibody-complexed pegylated drugs by Kupffer cells may be important beyond the field of HBV therapeutics. Thus, these findings may contribute to improving the efficacy of pegylated drugs that are now being developed for other chronic diseases and cancer.

Type I interferon activation and endothelial dysfunction in caveolin-1 insufficiency-associated pulmonary arterial hypertension.

Interferonopathies, interferon (IFN)-α/ß therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1-/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH.

X-ray microtomosynthesis of unstained pathology tissue samples.

In pathology protocols, a tissue block, such as one containing a mouse brain or a biopsy sample from a patient, can produce several hundred thin sections. Substantial time may be required to analyse all sections. In cases of uncertainty regarding which sections to focus on, noninvasive scout imaging of intact blocks can help in guiding the pathology procedure. The scouting step is ideally done in a time window of minutes without special sample preparation that may interfere with the pathology procedures. The challenge is to obtain some visibility of unstained tissue structures at sub-10 µm resolution. We explored a novel x-ray tomosynthesis method as a way to maximise contrast-to-noise ratio, a determinant of tissue visibility. It provided a z-stack of thousands of images at 7.3 µm resolution (10% contrast, half-period of 68.5 line pairs/mm), in scans of 5-15 minutes. When compared with micro-CT scans, the straight-line tomosynthesis scan did not need to rotate the sample, which allowed flat samples, such as paraffin blocks, to be kept as close as possible to the x-ray source. Thus, given the same hardware, scan time and resolution, this mode maximised the photon flux density through the sample, which helped in maximising the contrast-to-noise ratio. The tradeoff of tomosynthesis is incomplete 3D information. The microtomosynthesis scanner has scanned 110 unstained human and animal tissue samples as part of their respective pathology protocols. In all cases, the z-stack of images showed tissue structures that guided sectioning or provided correlative structural information. We describe six examples that presented different levels of visibility of soft tissue structures. Additionally, in a set of coronary artery samples from an HIV patient donor, microtomosynthesis made a new discovery of isolated focal calcification in the internal elastic lamina of coronary wall, which was the onset of medial calcific sclerosis in the arteries.

A microscopy version of the imaging method for 3D luggage screening has been adapted to image unstained pathology samples. Pathology tests of tissue samples are used for clinical diagnosis and for biomedical research. The tissue samples are often embedded in paraffin blocks and sectioned into many thin slices, which are then stained with the appropriate agents for light microscopy. Since each tissue block can produce several hundred thin sections, much time and labour is required to analyse all sections. Noninvasive scout imaging of intact blocks can help in guiding the pathology procedure. The scouting step is ideally done in a time window of minutes without special sample preparation that may interfere with the pathology procedures. The challenge is to obtain some visibility of unstained tissue structures at sufficient resolution. X-ray imaging is a promising tool to meet the challenge since x-rays can penetrate thick samples that are opaque to visible light. With x-ray imaging, a determinant of tissue visibility is the flux density of photons that illuminate the sample. We explored a novel x-ray tomosynthesis method as a way to maximise this factor. It provided a stack of thousands of cross-sectional images at 7.3 µm resolution (half-period of 68.5 line pairs/mm) in scans of 5-15 minutes. When compared with micro-CT scans (a widely used laboratory technology), this method did not need to rotate the sample, which allowed flat samples such as paraffin blocks to be kept as close as possible to the x-ray source. Thus, given the same hardware, scan time and resolution, this method maximised the photon flux density through the sample, which helped in improving the visibility of unstained tissue under x-ray. The tradeoff of the method is incomplete 3D information. Over 100 unstained human and animal tissue samples have been scanned with this method as part of their respective pathology protocols. In all cases, the stack of cross-sectional images showed tissue structures that guided pathology analysis or provided correlative structural information. We describe six examples that presented different levels of tissue visibility. Additionally, in a set of coronary artery samples from an HIV patient donor, microtomosynthesis made a new discovery of isolated focal calcification in the internal elastic lamina of coronary wall, which was the onset of medial calcific sclerosis in the arteries.

Bacillus anthracis edema toxin inhibits hypoxic pulmonary vasoconstriction via edema factor and cAMP-mediated mechanisms in isolated perfused rat lungs.

Bacillus anthracis edema toxin (ET) inhibited lethal toxin-stimulated pulmonary artery pressure (Ppa) and increased lung cAMP levels in our previous study. We therefore examined whether ET inhibits hypoxic pulmonary vasoconstriction (HPV). Following baseline hypoxic measures in isolated perfused lungs from healthy rats, compared with diluent, ET perfusion reduced maximal Ppa increases (mean ± SE percentage of maximal Ppa increase with baseline hypoxia) during 6-min hypoxic periods (FIO2 = 0%) at 120 min (16 ± 6% vs. 51 ± 6%, P = 0.004) and 180 min (11.4% vs. 55 ± 6%, P = 0.01). Protective antigen-mAb (PA-mAb) and adefovir inhibit host cell edema factor uptake and cAMP production, respectively. In lungs perfused with ET following baseline measures, compared with placebo, PA-mAb treatment increased Ppa during hypoxia at 120 and 180 min (56 ± 6% vs. 10 ± 4% and 72 ± 12% vs. 12 ± 3%, respectively, P ≤ 0.01) as did adefovir (84 ± 10% vs. 16.8% and 123 ± 21% vs. 26 ± 11%, respectively, P ≤ 0.01). Compared with diluent, lung perfusion with ET for 180 min reduced the slope of the relationships between Ppa and increasing concentrations of endothelin-1 (ET-1) (21.12 ± 2.96 vs. 3.00 ± 0.76 × 108 cmH2O/M, P < 0.0001) and U46619, a thromboxane A2 analogue (7.15 ± 1.01 vs. 3.74 ± 0.31 × 107 cmH2O/M, P = 0.05) added to perfusate. In lungs isolated from rats after 15 h of in vivo infusions with either diluent, ET alone, or ET with PA-mAb, compared with diluent, the maximal Ppa during hypoxia and the slope of the relationship between change in Ppa and ET-1 concentration added to the perfusate were reduced in lungs from animals challenged with ET alone (P ≤ 0.004) but not with ET and PA-mAb together (P ≥ 0.73). Inhibition of HPV by ET could aggravate hypoxia during anthrax pulmonary infection.NEW & NOTEWORTHY The most important findings here are edema toxin's potent adenyl cyclase activity can interfere with hypoxic pulmonary vasoconstriction, an action that could worsen hypoxemia during invasive anthrax infection with lung involvement. These findings, coupled with other studies showing that lethal toxin can disrupt pulmonary vascular integrity, indicate that both toxins can contribute to pulmonary pathophysiology during infection. In combination, these investigations provide a further basis for the use of antitoxin therapies in patients with worsening invasive anthrax disease.

Impaired angiogenesis and extracellular matrix metabolism in autosomal-dominant hyper-IgE syndrome.

There are more than 7000 described rare diseases, most lacking specific treatment. Autosomal-dominant hyper-IgE syndrome (AD-HIES, also known as Job's syndrome) is caused by mutations in STAT3. These patients present with immunodeficiency accompanied by severe nonimmunological features, including skeletal, connective tissue, and vascular abnormalities, poor postinfection lung healing, and subsequent pulmonary failure. No specific therapies are available for these abnormalities. Here, we investigated underlying mechanisms in order to identify therapeutic targets. Histological analysis of skin wounds demonstrated delayed granulation tissue formation and vascularization during skin-wound healing in AD-HIES patients. Global gene expression analysis in AD-HIES patient skin fibroblasts identified deficiencies in a STAT3-controlled transcriptional network regulating extracellular matrix (ECM) remodeling and angiogenesis, with hypoxia-inducible factor 1α (HIF-1α) being a major contributor. Consistent with this, histological analysis of skin wounds and coronary arteries from AD-HIES patients showed decreased HIF-1α expression and revealed abnormal organization of the ECM and altered formation of the coronary vasa vasorum. Disease modeling using cell culture and mouse models of angiogenesis and wound healing confirmed these predicted deficiencies and demonstrated therapeutic benefit of HIF-1α-stabilizing drugs. The study provides mechanistic insights into AD-HIES pathophysiology and suggests potential treatment options for this rare disease.

Macrophage fatty acid oxidation inhibits atherosclerosis progression.

Atherosclerosis is a chronic disorder of the vessel wall. One key regulator of disease progression is lipid handling in macrophages. However, the role of macrophage mitochondrial-dependent fatty acid ß-oxidation (FAO) in atherosclerosis is not well defined. To address this, we focused on carnitine palmitoyltransferase (CPT) 1 and 2, which play an essential role in the transport of long chain fatty acids (FAs) into the mitochondria. Using conditional alleles of these mitochondrial enzymes, we have generated myeloid-specific Cpt1a and Cpt2 knockout mutants (CPT1a M-KO and CPT2 M-KO). In culture, macrophages derived from CPT1a and CPT2 M-KO mice have impaired FAO, enhanced expression of the CD36 scavenger receptor, increased uptake of oxidized low-density lipoprotein (oxLDL), and augmented transformation into cholesterol-rich foam cells. In line with these in vitro observations, in the atherosclerosis-susceptible apolipoprotein E (ApoE) KO background, CPT2 M-KO mice demonstrated augmented atherosclerosis, accompanied by increased accumulation of aortic macrophages with elevated CD36 expression. These data suggest that macrophage FAO is athero-protective and that augmenting FAO may potentially slow atherosclerotic progression.

Efficient differentiation of cardiomyocytes and generation of calcium-sensor reporter lines from nonhuman primate iPSCs.

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy, but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with >85% purity in 10 days, using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes, we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies, including in vivo tissue regeneration models and drug screening.

Chronic skin inflammation accelerates macrophage cholesterol crystal formation and atherosclerosis.

Inflammation is critical to atherogenesis. Psoriasis is a chronic inflammatory skin disease that accelerates atherosclerosis in humans and provides a compelling model to understand potential pathways linking these diseases. A murine model capturing the vascular and metabolic diseases in psoriasis would accelerate our understanding and provide a platform to test emerging therapies. We aimed to characterize a new murine model of skin inflammation (Rac1V12) from a cardiovascular standpoint to identify novel atherosclerotic signaling pathways modulated in chronic skin inflammation. The RacV12 psoriasis mouse resembled the human disease state, including presence of systemic inflammation, dyslipidemia, and cardiometabolic dysfunction. Psoriasis macrophages had a proatherosclerotic phenotype with increased lipid uptake and foam cell formation, and also showed a 6-fold increase in cholesterol crystal formation. We generated a triple-genetic K14-RacV12-/+/Srb1-/-/ApoER61H/H mouse and confirmed psoriasis accelerates atherogenesis (~7-fold increase). Finally, we noted a 60% reduction in superoxide dismutase 2 (SOD2) expression in human psoriasis macrophages. When SOD2 activity was restored in macrophages, their proatherogenic phenotype reversed. We demonstrate that the K14-RacV12 murine model captures the cardiometabolic dysfunction and accelerates vascular disease observed in chronic inflammation and that skin inflammation induces a proatherosclerotic macrophage phenotype with impaired SOD2 function, which associated with accelerated atherogenesis.