Endothelial cell vasodilator dysfunction mediates progressive pregnancy-induced hypertension in endothelial cell tetrahydrobiopterin deficient mice.

BACKGROUND AND PURPOSE: Pregnancy-associated vascular remodelling is essential for both maternal and fetal health. We have previously shown that maternal endothelial cell tetrahydrobiopterin (BH4) deficiency causes poor pregnancy outcomes. Here, we investigated the role and mechanisms of endothelial cell-mediated vasorelaxation function in these outcomes. EXPERIMENTAL APPROACH: The vascular reactivity of mouse aortas and uterine arteries from non-pregnant and pregnant endothelial cell-specific BH4 deficient mice (Gch1fl/flTie2cre mice) was assessed by wire myography. Systolic blood pressure was assessed by tail cuff plethysmography. KEY RESULTS: In late pregnancy, systolic blood pressure was significantly higher (∼24 mmHg) in Gch1fl/flTie2cre mice compared with wild-type littermates. This was accompanied by enhanced vasoconstriction and reduced endothelial-dependent vasodilation in both aorta and uterine arteries from pregnant Gch1fl/flTie2cre mice. In uterine arteries loss of eNOS-derived vasodilators was partially compensated by upregulation of intermediate and large-conductance Ca2+-activated K+ channels. In rescue experiments, oral BH4 supplementation alone did not rescue vascular dysfunction and pregnancy-induced hypertension in Gch1fl/flTie2cre mice. However, combination with the fully reduced folate, 5-methyltetrahydrofolate (5-MTHF), restored endothelial cell vasodilator function and blood pressure. CONCLUSIONS AND IMPLICATIONS: We identify a critical requirement for maternal endothelial cell Gch1/BH4 biosynthesis in endothelial cell vasodilator function in pregnancy. Targeting vascular Gch1 and BH4 biosynthesis with reduced folates may provide a novel therapeutic target for the prevention and treatment of pregnancy-related hypertension.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology.

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Endothelial GTPCH (GTP Cyclohydrolase 1) and Tetrahydrobiopterin Regulate Gestational Blood Pressure, Uteroplacental Remodeling, and Fetal Growth.

[Figure: see text].

When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping.

BACKGROUND: Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model. RESULTS: We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1-3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role. CONCLUSIONS: A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.

Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology.

Macrophages are mononuclear phagocytes derived from haematopoietic progenitors that are widely distributed throughout the body. These cells participate in both innate and adaptive immune responses and lie central to the processes of inflammation, development, and homeostasis. Macrophage physiology varies depending on the environment in which they reside and they exhibit rapid functional adaption in response to external stimuli. To study macrophages in vitro, cells are typically cultured ex vivo from the peritoneum or alveoli, or differentiated from myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). BMDMs represent an efficient and cost-effective means of studying macrophage biology. However, the inherent sensitivity of macrophages to biochemical stimuli (such as cytokines, metabolic intermediates, and RNS/ROS) makes it imperative to control experimental conditions rigorously. Therefore, the aim of this study was to establish an optimised and standardised method for the isolation and culture of BMDMs. We used classically activated macrophages isolated from WT and nitric oxide (NO)-deficient mice to develop a standardised culture method, whereby the constituents of the culture media are defined. We then methodically compared our standardised protocol to the most commonly used method of BMDM culture to establish an optimal protocol for the study of nitric oxide (NO)-redox biology and immunometabolism in vitro.

Correction: Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

[This corrects the article DOI: 10.1371/journal.pone.0190558.].

Adipose tissue-derived WNT5A regulates vascular redox signaling in obesity via USP17/RAC1-mediated activation of NADPH oxidases.

Obesity is associated with changes in the secretome of adipose tissue (AT), which affects the vasculature through endocrine and paracrine mechanisms. Wingless-related integration site 5A (WNT5A) and secreted frizzled-related protein 5 (SFRP5), adipokines that regulate noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized that WNT5A released from AT exerts endocrine and paracrine effects on the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a cohort of 1004 humans with atherosclerosis, obesity was associated with increased WNT5A bioavailability in the circulation and the AT, higher expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human arterial wall, and increased vascular oxidative stress due to activation of NADPH oxidases. Plasma concentration of WNT5A was elevated in patients with coronary artery disease compared to matched controls and was independently associated with calcified coronary plaque progression. We further demonstrated that WNT5A induces arterial oxidative stress and redox-sensitive migration of vascular smooth muscle cells via Frizzled 2-mediated activation of a previously uncharacterized pathway involving the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and the GTPase RAC1. Our study identifies WNT5A and its downstream vascular signaling as a link between obesity and vascular disease pathogenesis, with translational implications in humans.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation.

Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH4) modulates IL-1ß production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage.

Phospholipid membranes drive abdominal aortic aneurysm development through stimulating coagulation factor activity.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, ∼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.

Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac 19F MRS/MRI and temporal tracking of progenitor cells.

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and 19F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENEHD-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine 19F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold 19F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac 19F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with 19F MRI.

Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Endothelial Cell Tetrahydrobiopterin Modulates Sensitivity to Ang (Angiotensin) II-Induced Vascular Remodeling, Blood Pressure, and Abdominal Aortic Aneurysm.

GTPCH (GTP cyclohydrolase 1, encoded by Gch1) is required for the synthesis of tetrahydrobiopterin; a critical regulator of endothelial NO synthase function. We have previously shown that mice with selective loss of Gch1 in endothelial cells have mild vascular dysfunction, but the consequences of endothelial cell tetrahydrobiopterin deficiency in vascular disease pathogenesis are unknown. We investigated the pathological consequence of Ang (angiotensin) II infusion in endothelial cell Gch1 deficient (Gch1fl/fl Tie2cre) mice. Ang II (0.4 mg/kg per day, delivered by osmotic minipump) caused a significant decrease in circulating tetrahydrobiopterin levels in Gch1fl/fl Tie2cre mice and a significant increase in the Nω-nitro-L-arginine methyl ester inhabitable production of H2O2 in the aorta. Chronic treatment with this subpressor dose of Ang II resulted in a significant increase in blood pressure only in Gch1fl/fl Tie2cre mice. This finding was mirrored with acute administration of Ang II, where increased sensitivity to Ang II was observed at both pressor and subpressor doses. Chronic Ang II infusion in Gch1fl/fl Tie2ce mice resulted in vascular dysfunction in resistance mesenteric arteries with an enhanced constrictor and decreased dilator response and medial hypertrophy. Altered vascular remodeling was also observed in the aorta with an increase in the incidence of abdominal aortic aneurysm formation in Gch1fl/fl Tie2ce mice. These findings indicate a specific requirement for endothelial cell tetrahydrobiopterin in modulating the hemodynamic and structural changes induced by Ang II, through modulation of blood pressure, structural changes in resistance vessels, and aneurysm formation in the aorta.

Roles for endothelial cell and macrophage Gch1 and tetrahydrobiopterin in atherosclerosis progression.

Aims: GTP cyclohydrolase I catalyses the first and rate-limiting reaction in the synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS). Both eNOS and iNOS have been implicated in the progression of atherosclerosis, with opposing effects in eNOS and iNOS knockout mice. However, the pathophysiologic requirement for BH4 in regulating both eNOS and iNOS function, and the effects of loss of BH4 on the progression of atherosclerosis remains unknown. Methods and results: Hyperlipidemic mice deficient in Gch1 in endothelial cells and leucocytes were generated by crossing Gch1fl/flTie2cre mice with ApoE-/- mice. Deficiency of Gch1 and BH4 in endothelial cells and myeloid cells was associated with mildly increased blood pressure. High fat feeding for 6 weeks in Gch1fl/flTie2CreApoE-/- mice resulted in significantly decreased circulating BH4 levels, increased atherosclerosis burden and increased plaque macrophage content. Gch1fl/flTie2CreApoE-/- mice showed hallmarks of endothelial cell dysfunction, with increased aortic VCAM-1 expression and decreased endothelial cell dependent vasodilation. Furthermore, loss of BH4 from pro-inflammatory macrophages resulted in increased foam cell formation and altered cellular redox signalling, with decreased expression of antioxidant genes and increased reactive oxygen species. Bone marrow chimeras revealed that loss of Gch1 in both endothelial cells and leucocytes is required to accelerate atherosclerosis. Conclusion: Both endothelial cell and macrophage BH4 play important roles in the regulation of NOS function and cellular redox signalling in atherosclerosis.

Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

PURPOSE: To a) achieve cardiac 19F-Magnetic Resonance Imaging (MRI) of perfluoro-crown-ether (PFCE) labeled cardiac progenitor stem cells (CPCs) and bone-derived bone marrow macrophages, b) determine label concentration and cellular load limits, and c) achieve spectroscopic and image-based quantification. METHODS: Theoretical simulations and experimental comparisons of spoiled-gradient echo (SPGR), rapid acquisition with relaxation enhancement (RARE), and steady state at free precession (SSFP) pulse sequences, and phantom validations, were conducted using 19F MRI/Magnetic Resonance Spectroscopy (MRS) at 9.4 T. Successful cell labeling was confirmed using flow cytometry and confocal microscopy. For CPC and macrophage concentration quantification, in vitro and post-mortem cardiac validations were pursued with the use of the transfection agent FuGENE. Feasibility of fast imaging is demonstrated in murine cardiac acquisitions in vivo, and in post-mortem murine skeletal and cardiac applications. RESULTS: SPGR/SSFP proved favorable imaging sequences yielding good signal-to-noise ratio values. Confocal microscopy confirmed heterogeneity of cellular label uptake in CPCs. 19F MRI indicated lack of additional benefits upon label concentrations above 7.5-10 mg/ml/million cells. The minimum detectable CPC load was ~500k (~10k/voxel) in two-dimensional (2D) acquisitions (3-5 min) using the butterfly coil. Additionally, absolute 19F based concentration and intensity estimates (trifluoroacetic-acid solutions, macrophages, and labeled CPCs in vitro and post-CPC injections in the post-mortem state) scaled linearly with fluorine concentrations. Fast, quantitative cardiac 19F-MRI was demonstrated with SPGR/SSFP and MRS acquisitions spanning 3-5 min, using a butterfly coil. CONCLUSION: The developed methodologies achieved in vivo cardiac 19F of exogenously injected labeled CPCs for the first time, accelerating imaging to a total acquisition of a few minutes, providing evidence for their potential for possible translational work.

Metabolic Regulation of Adipose Tissue Macrophage Function in Obesity and Diabetes.

SIGNIFICANCE: Obesity and diabetes are associated with chronic activation of inflammatory pathways that are important mechanistic links between insulin resistance (IR), type 2 diabetes (T2D), and cardiovascular disease pathogenesis. The development of these metabolic diseases is associated with changes in both the number and phenotype of adipose tissue macrophages (ATMs). Emerging lines of evidence have shown that ATMs release proinflammatory cytokines similar to classically activated M1 macrophages, which directly contribute to IR or T2D. In contrast, adipose tissue (AT) from lean healthy individuals contains macrophages with a less inflammatory M2 phenotype. Recent Advances: Recent research has shown that macrophage phenotype is linked to profound changes in macrophage cellular metabolism. CRITICAL ISSUES: This review focuses on the role of macrophages in AT inflammation and obesity, and the metabolic changes in macrophage function that occur with activation that underpin their role in the pathogenesis of IR and T2D. We highlight current targets for altering macrophage metabolism from both within the field of metabolic disease and AT biology and more widely within inflammatory biology. FUTURE DIRECTIONS: As our knowledge of macrophage metabolic programming in AT builds, there will be increasing scope for targeting this aspect of macrophage biology as a therapeutic strategy in metabolic diseases. Antioxid. Redox Signal. 29, 297-312.

Endothelium-derived extracellular vesicles promote splenic monocyte mobilization in myocardial infarction.

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1-positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion- and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.

A key role for tetrahydrobiopterin-dependent endothelial NOS regulation in resistance arteries: studies in endothelial cell tetrahydrobiopterin-deficient mice.

BACKGROUND AND PURPOSE: The cofactor tetrahydrobiopterin (BH4) is a critical regulator of endothelial NOS (eNOS) function, eNOS-derived NO and ROS signalling in vascular physiology. To determine the physiological requirement for de novo endothelial cell BH4 synthesis for the vasomotor function of resistance arteries, we have generated a mouse model with endothelial cell-specific deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme for BH4 biosynthesis, and evaluated BH4-dependent eNOS regulation, eNOS-derived NO and ROS generation. EXPERIMENTAL APPROACH: The reactivity of mouse second-order mesenteric arteries was assessed by wire myography. High performance liquid chromatography was used to determine BH4, BH2 and biopterin. Western blotting was used for expression analysis. KEY RESULTS: Gch1fl/fl Tie2cre mice demonstrated reduced GTPCH protein and BH4 levels in mesenteric arteries. Deficiency in endothelial cell BH4 leads to eNOS uncoupling, increased ROS production and loss of NO generation in mesenteric arteries of Gch1fl/fl Tie2cre mice. Gch1fl/fl Tie2cre mesenteric arteries had enhanced vasoconstriction to U46619 and phenylephrine, which was abolished by L-NAME. Endothelium-dependent vasodilatations to ACh and SLIGRL were impaired in mesenteric arteries from Gch1fl/fl Tie2cre mice, compared with those from wild-type littermates. Loss of eNOS-derived NO-mediated vasodilatation was associated with increased eNOS-derived H2 O2 and cyclooxygenase-derived vasodilator in Gch1fl/fl Tie2cre mesenteric arteries. CONCLUSIONS AND IMPLICATIONS: Endothelial cell Gch1 and BH4-dependent eNOS regulation play pivotal roles in maintaining vascular homeostasis in resistance arteries. Therefore, targeting vascular Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of microvascular dysfunction in patients with cardiovascular disease.

Tracking Monocyte Recruitment and Macrophage Accumulation in Atherosclerotic Plaque Progression Using a Novel hCD68GFP/ApoE-/- Reporter Mouse-Brief Report.

OBJECTIVE: To create a model of atherosclerosis using green fluorescent protein (GFP)-targeted monocytes/macrophages, allowing analysis of both endogenous GFP+ and adoptively transferred GFP+ myeloid cells in arterial inflammation. APPROACH AND RESULTS: hCD68GFP reporter mice were crossed with ApoE-/- mice. Expression of GFP was localized to macrophages in atherosclerotic plaques and in angiotensin II-induced aortic aneurysms and correlated with galectin 3 and mCD68 expression. Flow cytometry confirmed GFP+ expression in CD11b+/CD64+, CD11c+/MHC-IIHI, and CD11b+/F4/80+ myeloid cells. Adoptive transfer of GFP+ monocytes demonstrated monocyte recruitment to both adventitia and atherosclerotic plaque, throughout the aortic root, within 72 hours. We demonstrated the biological utility of hCD68GFP monocytes by comparing the recruitment of wild-type and CCR2-/- monocytes to sites of inflammation. CONCLUSIONS: hCD68GFP/ApoE-/- mice provide a new approach to study macrophage accumulation in atherosclerotic plaque progression and to identify cells recruited from adoptively transferred monocytes.