Mutagenesis on a complex mouse genetic background by site-specific nucleases.

Mouse models with complex genetic backgrounds are increasingly used in preclinical research to accurately model human disease and to enable temporal and cell-specific evaluation of genetic manipulations. Backcrossing mice onto these complex genetic backgrounds takes time and leads to significant wastage of animals. In this study, we aimed to evaluate whether site-specific nucleases could be used to generate additional genetic mutations in a complex genetic background, using the REVERSA mouse model of atherosclerosis, a model harbouring four genetically altered alleles. The model is comprised of a functional null mutation in the Ldlr gene in combination with a ApoB100 allele, which, after high-fat diet, leads to the rapid development of atherosclerosis. The regression of the pathology is achieved by inducible knock-out of the Mttp gene. Here we report an investigation to establish if microinjection of site-specific nucleases directly into zygotes prepared from the REVERSA could be used to investigate the role of the ATP binding cassette transporter G1 (ABCG1) in atherosclerosis regression. We show that using this approach we could successfully generate two independent knockout lines on the REVERSA background, both of which exhibited the expected phenotype of a significant reduction in cholesterol efflux to HDL in bone marrow-derived macrophages. However, loss of Abcg1 did not impact atherosclerosis regression in either the aortic root or in aortic arch, demonstrating no important role for this transporter subtype. We have demonstrated that site-specific nucleases can be used to create genetic modifications directly onto complex disease backgrounds and can be used to explore gene function without the need for laborious backcrossing of independent strains, conveying a significant 3Rs advantage.

Hif-2α programmes oxygen chemosensitivity in chromaffin cells.

The study of transcription factors that determine specialised neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electro-physiologically excitable cells that link the oxygen content of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by the Type I cells of the carotid body and recent work has revealed one isoform of the transcription factor HIF, HIF-2α, to have a non-redundant role in the development and function of that organ. Here we show that the activation of HIF-2α, including isolated overexpression alone, is sufficient to induce oxygen chemosensitivity in the otherwise unresponsive adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues that resemble the foetal organ of Zuckerkandl and also manifest oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of classes of genes that are ordinarily characteristic of the carotid body, including G-protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking two major oxygen sensing systems.

Novel Role of 5-Methyl-(6S)-Tetrahydrofolate in Mediating Endothelial Cell Tetrahydrobiopterin in Pregnancy and Implications for Gestational Hypertension.

BACKGROUND: Folate intake during pregnancy is essential for fetal development and maternal health. However, the specific effects of folic acid (FA) and 5-methyl-(6S)-tetrahydrofolate (5-MTHF) on the prevention and treatment of hypertensive disorders of pregnancy remain unclear. We investigated whether FA and 5-MTHF have different effects on endothelial cell tetrahydrobiopterin (BH4) metabolism in pregnancy and the possible consequences for endothelial NO generation, maternal blood pressure, and fetal growth. METHODS: We analyzed the maternal blood pressure in pregnant wild-type (Gch1fl/fl) and Gch1fl/fl Tie2cre mice treated with either FA or 5-MTHF starting before pregnancy, mid-pregnancy or late pregnancy. BH4, superoxide, and NO bioavailability were determined in mouse and human models of endothelial cell BH4 deficiency by high-performance liquid chromatography. RESULTS: In vitro studies in mouse and human endothelial cells showed that treatment with 5-MTHF, but not FA, elevated BH4 levels, reduced superoxide production, and increased NO synthase activity. In primary endothelial cells isolated from women with hypertensive pregnancies, exposure to 5-MTHF, but not FA, restored the reduction in BH4 levels and NO synthase activity. In vivo studies in mice revealed that oral treatment with 5-MTHF, but not FA, prevented and treated hypertension in pregnancy when administered either before or during pregnancy, respectively, and normalized placental and fetal growth restriction if administered from mid-gestation onward. CONCLUSIONS: Collectively, these studies identify a critical role for 5-MTHF in endothelial cell function in pregnancy, related to endothelial cell BH4 availability and NO synthase activity. Thus, 5-MTHF represents a novel therapeutic agent that may potentially improve endothelial function in hypertensive disorders of pregnancy by targeting endothelial cell BH4.

Role of Human Epicardial Adipose Tissue-Derived miR-92a-3p in Myocardial Redox State.

BACKGROUND: Visceral obesity is directly linked to increased cardiovascular risk, including heart failure. OBJECTIVES: This study explored the ability of human epicardial adipose tissue (EAT)-derived microRNAs (miRNAs) to regulate the myocardial redox state and clinical outcomes. METHODS: This study screened for miRNAs expressed and released from human EAT and tested for correlations with the redox state in the adjacent myocardium in paired EAT/atrial biopsy specimens from patients undergoing cardiac surgery. Three miRNAs were then tested for causality in an in vitro model of cardiomyocytes. At a clinical level, causality/directionality were tested using genome-wide association screening, and the underlying mechanisms were explored using human biopsy specimens, as well as overexpression of the candidate miRNAs and their targets in vitro and in vivo using a transgenic mouse model. The final prognostic value of the discovered targets was tested in patients undergoing cardiac surgery, followed up for a median of 8 years. RESULTS: EAT miR-92a-3p was related to lower oxidative stress in human myocardium, a finding confirmed by using genetic regulators of miR-92a-3p in the human heart and EAT. miR-92a-3p reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase-derived superoxide (O2.-) by targeting myocardial expression of WNT5A, which regulated Rac1-dependent activation of NADPH oxidases. Finally, high miR-92a-3p levels in EAT were independently related with lower risk of adverse cardiovascular events. CONCLUSIONS: EAT-derived miRNAs exert paracrine effects on the human heart. Indeed miR-92a-3p suppresses the wingless-type MMTV integration site family, member 5a/Rac1/NADPH oxidase axis and improves the myocardial redox state. EAT-derived miR-92a-3p is related to improved clinical outcomes and is a rational therapeutic target for the prevention and treatment of obesity-related heart disease.

Crucial neuroprotective roles of the metabolite BH4 in dopaminergic neurons.

Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.

Cardiomyocyte tetrahydrobiopterin synthesis regulates fatty acid metabolism and susceptibility to ischaemia-reperfusion injury.

NEW FINDINGS: What is the central question of this study? What are the physiological roles of cardiomyocyte-derived tetrahydrobiopterin (BH4) in cardiac metabolism and stress response? What is the main finding and its importance? Cardiomyocyte BH4 has a physiological role in cardiac metabolism. There was a shift of substrate preference from fatty acid to glucose in hearts with targeted deletion of BH4 synthesis. The changes in fatty-acid metabolic profile were associated with a protective effect in response to ischaemia-reperfusion (IR) injury, and reduced infarct size. Manipulating fatty acid metabolism via BH4 availability could play a therapeutic role in limiting IR injury. ABSTRACT: Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) synthases in which its production of NO is crucial for cardiac function. However, non-canonical roles of BH4 have been discovered recently and the cell-specific role of cardiomyocyte BH4 in cardiac function and metabolism remains to be elucidated. Therefore, we developed a novel mouse model of cardiomyocyte BH4 deficiency, by cardiomyocyte-specific deletion of Gch1, which encodes guanosine triphosphate cyclohydrolase I, a required enzyme for de novo BH4 synthesis. Cardiomyocyte (cm)Gch1 mRNA expression and BH4 levels from cmGch1 KO mice were significantly reduced compared to Gch1flox/flox (WT) littermates. Transcriptomic analyses and protein assays revealed downregulation of genes involved in fatty acid oxidation in cmGch1 KO hearts compared with WT, accompanied by increased triacylglycerol concentration within the myocardium. Deletion of cardiomyocyte BH4 did not alter basal cardiac function. However, the recovery of left ventricle function was improved in cmGch1 KO hearts when subjected to ex vivo ischaemia-reperfusion (IR) injury, with reduced infarct size compared to WT hearts. Metabolomic analyses of cardiac tissue after IR revealed that long-chain fatty acids were increased in cmGch1 KO hearts compared to WT, whereas at 5 min reperfusion (post-35 min ischaemia) fatty acid metabolite levels were higher in WT compared to cmGch1 KO hearts. These results indicate a new role for BH4 in cardiomyocyte fatty acid metabolism, such that reduction of cardiomyocyte BH4 confers a protective effect in response to cardiac IR injury. Manipulating cardiac metabolism via BH4 could play a therapeutic role in limiting IR injury.

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2.

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hypertrophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocardiography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Resolution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell-cycle defects and mitochondrial dysfunction. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteasomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteasomal system is activated; a mechanism that has been implicated in cardiomyopathies previously. In parallel, a lack of functional alpha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell-cycle defects, the likely cause of the death of the embryos. The defects also have wide-ranging morphological consequences.

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.

Mood symptoms in cervical dystonia: Relationship with motor symptoms and quality of life.

Background: Cervical dystonia (CD) has a high prevalence of anxiety and depression. The relationship between motor severity, mood symptoms and QoL is unclear and how to adequately assess these is also unknown. Instruments like the BAI, BDI and HADS are often used but items within these relating to somatic symptoms might influence the results. Methods: Patients with idiopathic cervical dystonia (CD) were included. The BAI, BDI, HADS, CIDP58 and TWSTRS2- severity score were used for assessment of motor, mood and QoL symptoms. Pearson's correlations between motor and non-motor symptom scores were assessed. The psychometric properties of the psychiatric tools were measured and principal component analysis performed after identifying items that could correspond to somatic symptoms. Results: 201 participants were included. 42% of participants had either significant depression or anxiety symptoms or both when measured by BAI and BDI and 51% of patients met criteria on HADS. HADS-A and HADS-D, BAI and BDI were poorly correlated with TWSTRS2-S. The HADS-A and HADS-D both showed strong correlation with the sleep subdomain of CDIP58. Psychometric and principal component analysis on 149/201 participants did not reveal factor loadings consistent with the a priori somatic groupings. However mean scores were higher for somatic items. Conclusion: A good score on the CDIP58, a commonly used tool, does not indicate mild disease severity or minimal mood symptoms. Minimal motor symptoms, similarly, also does not imply a positive QoL. Clinicians should be mindful on ideal methods for performing a holistic assessment of CD patients. This likely warrants a combination of motor, QoL and mood assessment tools.

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury.

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1fl/fl with Tie2cre mice, generating Gch1fl/flTie2cre mice and littermate controls. GTP cyclohydrolase protein and BH4 levels were reduced in heart tissues from Gch1fl/flTie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH4. Deficiency in coronary endothelial cell BH4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1fl/flTie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH4 led to mild cardiac hypertrophy in Gch1fl/flTie2cre hearts. Endothelial cell BH4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1/BH4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury.NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

PHACTR1 modulates vascular compliance but not endothelial function: a translational study.

AIMS: The non-coding locus at 6p24 located in Intron 3 of PHACTR1 has consistently been implicated as a risk allele in myocardial infarction and multiple other vascular diseases. Recent murine studies have identified a role for Phactr1 in the development of atherosclerosis. However, the role of PHACTR1 in vascular tone and in vivo vascular remodelling has yet to be established. The aim of this study was to investigate the role of PHACTR1 in vascular function. METHODS AND RESULTS: Prospectively recruited coronary artery disease (CAD) patients undergoing bypass surgery and retrospectively recruited spontaneous coronary artery dissection (SCAD) patients and matched healthy volunteers were genotyped at the PHACTR1 rs9349379 locus. We observed a significant association between the PHACTR1 loci and changes in distensibility in both the ascending aorta (AA = 0.0053 ± 0.0004, AG = 0.0041 ± 0.003, GG = 0.0034 ± 0.0009, P < 0.05, n = 58, 54, and 7, respectively) and carotid artery (AA = 12.83 ± 0.51, AG = 11.14 ± 0.38, GG = 11.69 ± 0.66, P < 0.05, n = 70, 65, and 18, respectively). This association was not observed in the descending aorta or in SCAD patients. In contrast, the PHACTR1 locus was not associated with changes in endothelial cell function with no association between the rs9349379 locus and in vivo or ex vivo vascular function observed in CAD patients. This finding was confirmed in our murine model where the loss of Phactr1 on the pro-atherosclerosis ApoE-/- background did not alter ex vivo vascular function. CONCLUSION: In conclusion, we have shown a role for PHACTR1 in arterial compliance across multiple vascular beds. Our study suggests that PHACTR1 has a key structural role within the vasculature.

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

[Figure: see text].

Developmental role of PHD2 in the pathogenesis of pseudohypoxic pheochromocytoma.

Despite a general role for the HIF hydroxylase system in cellular oxygen sensing and tumour hypoxia, cancer-associated mutations of genes in this pathway, including PHD2, PHD1, EPAS1 (encoding HIF-2α) are highly tissue-restricted, being observed in pseudohypoxic pheochromocytoma and paraganglioma (PPGL) but rarely, if ever, in other tumours. In an effort to understand that paradox and gain insights into the pathogenesis of pseudohypoxic PPGL, we constructed mice in which the principal HIF prolyl hydroxylase, Phd2, is inactivated in the adrenal medulla using TH-restricted Cre recombinase. Investigation of these animals revealed a gene expression pattern closely mimicking that of pseudohypoxic PPGL. Spatially resolved analyses demonstrated a binary distribution of two contrasting patterns of gene expression among adrenal medullary cells. Phd2 inactivation resulted in a marked shift in this distribution towards a Pnmt-/Hif-2α+/Rgs5+ population. This was associated with morphological abnormalities of adrenal development, including ectopic TH+ cells within the adrenal cortex and external to the adrenal gland. These changes were ablated by combined inactivation of Phd2 with Hif-2α, but not Hif-1α. However, they could not be reproduced by inactivation of Phd2 in adult life, suggesting that they arise from dysregulation of this pathway during adrenal development. Together with the clinical observation that pseudohypoxic PPGL manifests remarkably high heritability, our findings suggest that this type of tumour likely arises from dysregulation of a tissue-restricted action of the PHD2/HIF-2α pathway affecting adrenal development in early life and provides a model for the study of the relevant processes.

Promoting safer sex in the context of heterosexual anal intercourse: A scoping review.

AIMS AND OBJECTIVES: To locate and summarise existing literature regarding safer sex practices specific to heterosexual anal intercourse and identify promising health promotion strategies. BACKGROUND: Much of the literature regarding anal intercourse and safer sex is related to men who have sex with men. However, some studies suggest there are more women than men engaging in unprotected receptive anal intercourse. The risks associated with this sexual practice have been well documented, although many healthcare providers fail to ask about anal intercourse while addressing safer sex in the heterosexual population. DESIGN: The study was based on Arksey and O'Malley's 2005 five-step methodology. METHODS: A search was conducted of MEDLINE; CINAHL; PsycInfo; Cochrane; and PubMed. Databases were searched from 1990-2020. The 72 studies selected were classified according to their main area of focus. A grey literature search was also included. This scientific submission has been assessed for accuracy and completeness using the PRISMA-ScR guideline criteria (File S1). RESULTS: The literature in this area is heterogeneous in terms of method and topic. Prevalence and incidence (n = 26) in addition to sexually transmitted infection risks (n = 26) related to heterosexual anal intercourse are well understood. However, there is limited information on condom use (n = 6), factors that influence heterosexual anal intercourse (n = 10) and health promotion strategies for this population and practice (n = 4). Two websites that mentioned heterosexual anal intercourse risk reduction activities were included. CONCLUSIONS: Although heterosexual anal intercourse appears to be an increasingly common sexual practice, very little is known about health promotion strategies nurses might use for encouraging safer sex in this population. RELEVANCE TO CLINICAL PRACTICE: Increased awareness of the prevalence and risks of heterosexual anal intercourse could enhance nurses' harm reduction strategies. Screening for sexually transmitted infections may be based on incorrect assumptions about sexual practices or due to stigma linked with anal intercourse. Reducing unprotected heterosexual anal intercourse will reduce sexually transmitted infections and their long-term sequelae.

Insulin-induced vascular redox dysregulation in human atherosclerosis is ameliorated by dipeptidyl peptidase 4 inhibition.

Recent clinical trials have revealed that aggressive insulin treatment has a neutral effect on cardiovascular risk in patients with diabetes despite improved glycemic control, which may suggest confounding direct effects of insulin on the human vasculature. We studied 580 patients with coronary atherosclerosis undergoing coronary artery bypass surgery (CABG), finding that high endogenous insulin was associated with reduced nitric oxide (NO) bioavailability ex vivo in vessels obtained during surgery. Ex vivo experiments with human internal mammary arteries and saphenous veins obtained from 94 patients undergoing CABG revealed that both long-acting insulin analogs and human insulin triggered abnormal responses of post-insulin receptor substrate 1 downstream signaling ex vivo, independently of systemic insulin resistance status. These abnormal responses led to reduced NO bioavailability, activation of NADPH oxidases, and uncoupling of endothelial NO synthase. Treatment with an oral dipeptidyl peptidase 4 inhibitor (DPP4i) in vivo or DPP4i administered to vessels ex vivo restored physiological insulin signaling, reversed vascular insulin responses, reduced vascular oxidative stress, and improved endothelial function in humans. The detrimental effects of insulin on vascular redox state and endothelial function as well as the insulin-sensitizing effect of DPP4i were also validated in high-fat diet-fed ApoE-/- mice treated with DPP4i. High plasma DPP4 activity and high insulin were additively related with higher cardiac mortality in patients with coronary atherosclerosis undergoing CABG. These findings may explain the inability of aggressive insulin treatment to improve cardiovascular outcomes, raising the question whether vascular insulin sensitization with DPP4i should precede initiation of insulin treatment and continue as part of a long-term combination therapy.

A key role for the novel coronary artery disease gene JCAD in atherosclerosis via shear stress mechanotransduction.

AIMS: Genome-wide association studies (GWAS) have consistently identified an association between coronary artery disease (CAD) and a locus on chromosome 10 containing a single gene, JCAD (formerly KIAA1462). However, little is known about the mechanism by which JCAD could influence the development of atherosclerosis. METHODS AND RESULTS: Vascular function was quantified in subjects with CAD by flow-mediated dilatation (FMD) and vasorelaxation responses in isolated blood vessel segments. The JCAD risk allele identified by GWAS was associated with reduced FMD and reduced endothelial-dependent relaxations. To study the impact of loss of Jcad on atherosclerosis, Jcad-/- mice were crossed to an ApoE-/- background and fed a high-fat diet from 6 to16 weeks of age. Loss of Jcad did not affect blood pressure or heart rate. However, Jcad-/-ApoE-/- mice developed significantly less atherosclerosis in the aortic root and the inner curvature of the aortic arch. En face analysis revealed a striking reduction in pro-inflammatory adhesion molecules at sites of disturbed flow on the endothelial cell layer of Jcad-/- mice. Loss of Jcad lead to a reduced recovery perfusion in response to hind limb ischaemia, a model of altered in vivo flow. Knock down of JCAD using siRNA in primary human aortic endothelial cells significantly reduced the response to acute onset of flow, as evidenced by reduced phosphorylation of NF-КB, eNOS, and Akt. CONCLUSION: The novel CAD gene JCAD promotes atherosclerotic plaque formation via a role in the endothelial cell shear stress mechanotransduction pathway.

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.

Nox2 contributes to age-related oxidative damage to neurons and the cerebral vasculature.

Oxidative stress plays an important role in aging-related neurodegeneration. This study used littermates of WT and Nox2-knockout (Nox2KO) mice plus endothelial cell-specific human Nox2 overexpression-transgenic (HuNox2Tg) mice to investigate Nox2-derived ROS in brain aging. Compared with young WT mice (3-4 months), aging WT mice (20-22 months) had obvious metabolic disorders and loss of locomotor activity. Aging WT brains had high levels of angiotensin II (Ang II) and ROS production; activation of ERK1/2, p53, and γH2AX; and losses of capillaries and neurons. However, these abnormalities were markedly reduced in aging Nox2KO brains. HuNox2Tg brains at middle age (11-12 months) already had high levels of ROS production and activation of stress signaling pathways similar to those found in aging WT brains. The mechanism of Ang II-induced endothelial Nox2 activation in capillary damage was examined using primary brain microvascular endothelial cells. The clinical significance of Nox2-derived ROS in aging-related loss of cerebral capillaries and neurons was investigated using postmortem midbrain tissues of young (25-38 years) and elderly (61-85 years) adults. In conclusion, Nox2 activation is an important mechanism in aging-related cerebral capillary rarefaction and reduced brain function, with the possibility of a key role for endothelial cells.

Effect of irradiation and bone marrow transplantation on angiotensin II-induced aortic inflammation in ApoE knockout mice.

BACKGROUND AND AIMS: Angiotensin II (Ang II) infusion promotes the development of aortic aneurysms and accelerates atherosclerosis in ApoE-/- mice. In order to elucidate the role of hematopoietic cells in these pathologies, irradiation and bone marrow transplantation (BMT) are commonly utilized. The aim of this study was to investigate the effects of irradiation and BMT on abdominal and thoracic aortic aneurysm formation and acute leukocyte recruitment in the aortic root and descending aorta, in an experimental mouse model of aortic aneurysm formation. METHODS: ApoE-/- mice were either lethally irradiated and reconstituted with ApoE-/- bone marrow or non-irradiated. Following engraftment, mice were treated with Ang II to induce aortic inflammation and accelerate atherosclerosis. RESULTS: Ang II infusion (0.8 mg/kg/day) in BMT mice resulted in reduced aortic aneurysms and atherosclerosis with decreased leukocyte infiltration in the aorta compared to non-BMT mice, when receiving the same dose of Ang II. Furthermore, the reduced aortic infiltration in BMT mice was accompanied by increased levels of monocytes in the spleen and bone marrow. A dose of 3 mg/kg/day Ang II was required to achieve a similar incidence of aneurysm formation as achieved with 0.8 mg/kg/day in non-BMT mice. CONCLUSIONS: This study provides evidence that BMT can alter inflammatory cell recruitment in experimental mouse models of aortic aneurysm formation and atherosclerosis and suggests that irradiation and BMT have a considerably more complex effect on vascular inflammation, which should be evaluated.

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.