Coronary Spasm: Ethnic and Sex Differences.

Coronary spasm (CS), which may occur at the epicardial (focal or diffuse spasm) and/or microvascular (microvascular spasm) level, is a well-established cause of myocardial ischaemia, in particular in patients with anginal chest pain despite unobstructed coronary arteries. The diagnosis of CS can be confirmed during coronary angiography by an additional provocation test with vasoactive substances such as acetylcholine. Due to partially inconsistent data from large clinical studies, especially between Asian and white CS patients, ethnic differences concerning the prevalence and angiographic patterns of CS seem to exist. Furthermore, several studies in patients with coronary vasomotor disorders pointed towards differences among male and female CS patients. This article gives an overview of ethnic- and sex-related differences in patients with CS.

Characterization and implications of intracoronary hemodynamic assessment during coronary spasm provocation testing.

BACKGROUND: Current diagnostic criteria for coronary spasm are based on patient's symptoms, ECG shifts and epicardial vasoconstriction during acetylcholine (ACh) spasm testing. AIMS: To assess the feasibility and diagnostic value of coronary blood flow (CBF) and resistance (CR) assessment as objective parameters during ACh testing. METHODS: Eighty-nine patients who underwent intracoronary reactivity testing including ACh testing with synchronous Doppler wire-based measurements of CBF and CR were included. Coronary microvascular and epicardial spasm, respectively, were diagnosed based on COVADIS criteria. RESULTS: Patients were 63 ± 13 years old, predominantly female (69%) and had preserved LV ejection fraction (64 ± 8%). Overall, assessment of CBF and CR during ACh testing revealed a decrease in CBF of 0.62 (0.17-1.53)-fold and an increase of CR of 1.45 [0.67-4.02]-fold in spasm patients compared to 2.08 (1.73-4.76) for CBF and 0.45 (0.44-0.63) for CR in patients without coronary spasm (both p < 0.01). Receiver operating characteristic revealed a high diagnostic ability of CBF and CR (AUC 0.86, p < 0.001, respectively) in identifying patients with coronary spasm. However, in 21% of patients with epicardial spasm and 42% of patients with microvascular spasm a paradoxical response was observed. CONCLUSIONS: This study demonstrates feasibility and potential diagnostic value of intracoronary physiology assessments during ACh testing. We observed opposite responses of CBF and CR to ACh in patients with positive vs. negative spasm test. While a decrease in CBF and an increase in CR during ACh seem pathognomonic for spasm, some patients with coronary spasm demonstrate paradoxical ACh response demanding further scientific investigations.

Deletion of endothelial leptin receptors in mice promotes diet-induced obesity.

Obesity promotes endothelial dysfunction. Endothelial cells not only respond, but possibly actively promote the development of obesity and metabolic dysfunction. Our aim was to characterize the role of endothelial leptin receptors (LepR) for endothelial and whole body metabolism and diet-induced obesity. Mice with tamoxifen-inducible, Tie2.Cre-ERT2-mediated deletion of LepR in endothelial cells (End.LepR knockout, KO) were fed high-fat diet (HFD) for 16 weeks. Body weight gain, serum leptin levels, visceral adiposity and adipose tissue inflammation were more pronounced in obese End.LepR-KO mice, whereas fasting serum glucose and insulin levels or the extent of hepatic steatosis did not differ. Reduced brain endothelial transcytosis of exogenous leptin, increased food intake and total energy balance were observed in End.LepR-KO mice and accompanied by brain perivascular macrophage accumulation, whereas physical activity, energy expenditure and respiratory exchange rates did not differ. Metabolic flux analysis revealed no changes in the bioenergetic profile of endothelial cells from brain or visceral adipose tissue, but higher glycolysis and mitochondrial respiration rates in those isolated from lungs. Our findings support a role for endothelial LepRs in the transport of leptin into the brain and neuronal control of food intake, and also suggest organ-specific changes in endothelial cell, but not whole-body metabolism.

Protein Tyrosine Phosphatase 1B Deficiency in Vascular Smooth Muscle Cells Promotes Perivascular Fibrosis following Arterial Injury.

BACKGROUND: Smooth muscle cell (SMC) phenotype switching plays a central role during vascular remodeling. Growth factor receptors are negatively regulated by protein tyrosine phosphatases (PTPs), including its prototype PTP1B. Here, we examine how reduction of PTP1B in SMCs affects the vascular remodeling response to injury. METHODS: Mice with inducible PTP1B deletion in SMCs (SMC.PTP1B-KO) were generated by crossing mice expressing Cre.ERT2 recombinase under the Myh11 promoter with PTP1Bflox/flox mice and subjected to FeCl3 carotid artery injury. RESULTS: Genetic deletion of PTP1B in SMCs resulted in adventitia enlargement, perivascular SMA+ and PDGFRß+ myofibroblast expansion, and collagen accumulation following vascular injury. Lineage tracing confirmed the appearance of Myh11-Cre reporter cells in the remodeling adventitia, and SCA1+ CD45- vascular progenitor cells increased. Elevated mRNA expression of transforming growth factor ß (TGFß) signaling components or enzymes involved in extracellular matrix remodeling and TGFß liberation was seen in injured SMC.PTP1B-KO mouse carotid arteries, and mRNA transcript levels of contractile SMC marker genes were reduced already at baseline. Mechanistically, Cre recombinase (mice) or siRNA (cells)-mediated downregulation of PTP1B or inhibition of ERK1/2 signaling in SMCs resulted in nuclear accumulation of KLF4, a central transcriptional repressor of SMC differentiation, whereas phosphorylation and nuclear translocation of SMAD2 and SMAD3 were reduced. SMAD2 siRNA transfection increased protein levels of PDGFRß and MYH10 while reducing ERK1/2 phosphorylation, thus phenocopying genetic PTP1B deletion. CONCLUSION: Chronic reduction of PTP1B in SMCs promotes dedifferentiation, perivascular fibrosis, and adverse remodeling following vascular injury by mechanisms involving an ERK1/2 phosphorylation-driven shift from SMAD2 to KLF4-regulated gene transcription.

Epicardial and microvascular coronary artery spasm in biopsy-proven viral myocarditis.

BACKGROUND: Coronary spasm has been suggested to be the underlying mechanism of chest pain in patients with myocarditis and unobstructed coronary arteries. Here we sought to investigate a potential association between virus type and coronary spasm endotype in patients with biopsy-proven viral myocarditis. METHODS: A total of 618 consecutive patients with unobstructed coronary arteries who underwent endomyocardial biopsy between 2008 and 2018 were screened. Viral myocarditis defined as (immuno-)histological evidence of myocardial inflammation and proof of viral genome by PCR was confirmed in 114 patients. Of these, 34 patients had undergone additional intracoronary acetylcholine (ACh) testing and served as the final study cohort. RESULTS: Patients in this study were 51 ± 27 years old, 41% were female and mean left ventricular ejection fraction was 58 ± 23%. Most frequently, virus DNA was detected by PCR from parvovirus B19 (PVB19, 59%) and human herpesvirus 6 (HHV6, 26%). ACh testing revealed epicardial spasm in 10 patients (29%) and microvascular spasm in 11 patients (32%). The rate of coronary spasm was higher in patients with PVB19-associated myocarditis compared to those with HHV6-associated myocarditis (80% vs. 33%, p = 0.031). In particular, there was a higher prevalence of microvascular spasm in patients with PVB19 compared to HHV6 infection (45% vs. 0%, p = 0.018). CONCLUSION: Coronary spasm is a frequent finding in patients with biopsy-proven viral myocarditis supporting the hypothesis that coronary spasm may contribute to chest pain in these patients. We observed a particular association of microvascular spasm with PVB19 infection.

Coronary artery spasm and impaired myocardial perfusion in patients with ANOCA: Predictors from a multimodality study using stress CMR and acetylcholine testing.

BACKGROUND: Functional coronary disorders such as coronary spasm and microvascular dysfunction (including microvascular spasm and impaired microvascular dilatation) are frequent findings among patients with angina and non-obstructed coronary arteries (ANOCA). In this study, we investigated a potential association of coronary spasm and myocardial perfusion abnormalities as well as predictors of such functional coronary disorders in ANOCA patients using a multimodality diagnostic strategy including adenosine stress CMR and intracoronary acetylcholine testing. METHODS: We enrolled 129 patients with ANOCA who underwent acetylcholine testing and adenosine stress perfusion CMR. Patients were allocated to 3 groups according to their spasm testing result with regard to standardized COVADIS criteria: 1) epicardial spasm, 2) microvascular spasm, and 3) no spasm. The myocardial perfusion reserve index (MPRI) was semiquantitatively determined from adenosine stress perfusion CMR. Multivariate regression analyses were performed to identify predictors of coronary functional disorders. RESULTS: Patients with epicardial spasm had lower MPRI than patients without, whereas MPRI was preserved in patients with microvascular spasm. Multivariate analyses revealed age, previous myocardial infarction, LVEF and epicardial spasm as independent predictors of diminished MPRI, whereas previous PCI was associated with epicardial spasm, and female sex was a strong predictor of microvascular spasm. CONCLUSIONS: Our results demonstrate coexistence of different functional coronary disorder endotypes involving the macro- and microvascular level of the coronary circulation in patients with ANOCA. We demonstrate that epicardial spasm is associated with diminished myocardial perfusion reserve and report further predictors of coronary functional disorders.

Invasive Diagnosis of Coronary Functional Disorders Causing Angina Pectoris.

Coronary vasomotion disorders represent a frequent cause of angina and/or dyspnoea in patients with non-obstructed coronary arteries. The highly sophisticated interplay of vasodilatation and vasoconstriction can be assessed in an interventional diagnostic procedure. Established parameters characterising adequate vasodilatation are coronary blood flow at rest, and, after drug-induced vasodilation, coronary flow reserve, and microvascular resistance (hyperaemic microvascular resistance, index of microcirculatory resistance). An increased vasoconstrictive potential is diagnosed by provocation testing with acetylcholine or ergonovine. This enables a diagnosis of coronary epicardial and/or microvascular spasm. Ischaemia associated with microvascular spasm can be confirmed by ischaemic ECG changes and the measurement of lactate concentrations in the coronary sinus. Although interventional diagnostic procedures are helpful for determining the mechanism of the angina, which may be the key to successful medical treatment, they are still neither widely accepted nor applied in many medical centres. This article summarises currently well-established invasive methods for the diagnosis of coronary functional disorders causing angina pectoris.

Repurposing Riociguat for Treatment of Refractory Angina Resulting From Coronary Spasm.

Coronary spasm is a frequent cause of angina despite unobstructed coronary arteries, and symptom control with recommended drugs is limited. We report the case of a 77-year-old woman who had refractory angina despite conventional antianginal treatment. Repurposing riociguat, a soluble guanylate cyclase stimulator, resulted in improvement of symptoms and prevention of spasm. (Level of Difficulty: Intermediate.).

Safety assessment and results of coronary spasm provocation testing in patients with myocardial infarction with unobstructed coronary arteries compared to patients with stable angina and unobstructed coronary arteries.

BACKGROUND: Coronary spasm is an established cause for myocardial infarction with unobstructed coronary arteries, and can be diagnosed using intracoronary acetylcholine testing. However, it has been questioned whether such testing is feasible and safe in the acute phase. The aim of this study was to assess the frequency of coronary spasm and the safety of the acetylcholine test in patients with myocardial infarction with unobstructed coronary arteries compared to patients with stable angina and unobstructed coronaries. METHODS: One hundred and eighty selected patients (52% women, mean age 62±13 years) with either myocardial infarction with unobstructed coronary arteries (n=80) or stable angina and unobstructed coronaries (n=100) were enrolled from 2007-2018. All patients underwent the acetylcholine test according to a standardised protocol immediately after diagnostic angiography. Apart from assessment of clinical, demographic and risk factor data, side effects and complications during the acetylcholine test were recorded. RESULTS: Overall, epicardial spasm was found in 26% with a higher prevalence among the myocardial infarction with unobstructed coronary arteries compared to the stable angina patients (35% vs 19%, p=0.017). Microvascular spasm was found in 42% with a higher prevalence among the stable patients compared to the myocardial infarction with unobstructed coronary arteries cohort (53% vs 29%, p=0.0014). There were no statistically significant differences in the rate of side effects (16% vs 14%, p=0.674) or complications (1% vs 2.5%, p=0.438) between the two groups. None of the patients experienced irreversible complications. CONCLUSION: Coronary spasm is a frequent cause for myocardial infarction with unobstructed coronary arteries. Spasm provocation testing using acetylcholine is feasible in such patients in the acute phase. The complication rate during acetylcholine testing in myocardial infarction with unobstructed coronary arteries patients is low and comparable to patients with stable angina.

Coronary Artery Spasm: The Interplay Between Endothelial Dysfunction and Vascular Smooth Muscle Cell Hyperreactivity.

Patients with angina pectoris, the cardinal symptom of myocardial ischaemia, yet without significant flow-limiting epicardial artery stenosis represent a diagnostic and therapeutic challenge. Coronary artery spasm (CAS) is an established cause for anginal chest pain in patients with angiographically unobstructed coronary arteries. CAS may occur at the epicardial level and/or in the microvasculature. Although the underlying pathophysiological mechanisms of CAS are still largely unclear, endothelial dysfunction and vascular smooth muscle cell (VSMC) hyperreactivity seem to be involved as major players, although their contribution to induce CAS is still seen as controversial. This article will look at the role and possible mechanistic interplay between an impaired endothelial and VSMC function in the pathogenesis of CAS.

Diagnosis of coronary microvascular dysfunction in the clinic.

The coronary microcirculation plays a pivotal role in the regulation of coronary blood flow and cardiac metabolism. It can adapt to acute and chronic pathologic conditions such as coronary thrombosis or long-standing hypertension. Due to the fact that the coronary microcirculation cannot be visualized in human beings in vivo, its assessment remains challenging. Thus, the clinical importance of the coronary microcirculation is still often underestimated or even neglected. Depending on the clinical condition of the respective patient, several non-invasive (e.g. transthoracic Doppler-echocardiography assessing coronary flow velocity reserve, cardiac magnetic resonance imaging, positron emission tomography) and invasive methods (e.g. assessment of coronary flow reserve (CFR) and microvascular resistance (MVR) using adenosine, microvascular coronary spasm with acetylcholine) have been established for the assessment of coronary microvascular function. Individual patient characteristics, but certainly also local availability, methodical expertise and costs will influence which methods are being used for the diagnostic work-up (non-invasive and/or invasive assessment) in a patient with recurrent symptoms and suspected coronary microvascular dysfunction. Recently, the combined invasive assessment of coronary vasoconstrictor as well as vasodilator abnormalities has been titled interventional diagnostic procedure (IDP). It involves intracoronary acetylcholine testing for the detection of coronary spasm as well as CFR and MVR assessment in response to adenosine using a dedicated wire. Currently, the IDP represents the most comprehensive coronary vasomotor assessment. Studies using the IDP to better characterize the endotypes observed will hopefully facilitate development of tailored and effective treatments.

Coronary Microvascular Spasm as the Underlying Cause of the Angiographic Slow Flow Phenomenon.

Slow coronary flow is frequently seen during angiography in patients with angina and unobstructed coronary arteries. However, the pathophysiology of this finding remains largely unclear. We report a case of a 52-year-old woman with slow coronary flow caused by acetylcholine-induced microvascular spasm, as confirmed by intracoronary flow measurements. (Level of Difficulty: Beginner.).

Safety assessment and results of coronary spasm provocation testing in patients with myocardial infarction with unobstructed coronary arteries compared to patients with stable angina and unobstructed coronary arteries.

BACKGROUND: Coronary spasm is an established cause for myocardial infarction with unobstructed coronary arteries, and can be diagnosed using intracoronary acetylcholine testing. However, it has been questioned whether such testing is feasible and safe in the acute phase. The aim of this study was to assess the frequency of coronary spasm and the safety of the acetylcholine test in patients with myocardial infarction with unobstructed coronary arteries compared to patients with stable angina and unobstructed coronaries. METHODS: One hundred and eighty selected patients (52% women, mean age 62±13 years) with either myocardial infarction with unobstructed coronary arteries (n=80) or stable angina and unobstructed coronaries (n=100) were enrolled from 2007-2018. All patients underwent the acetylcholine test according to a standardised protocol immediately after diagnostic angiography. Apart from assessment of clinical, demographic and risk factor data, side effects and complications during the acetylcholine test were recorded. RESULTS: Overall, epicardial spasm was found in 26% with a higher prevalence among the myocardial infarction with unobstructed coronary arteries compared to the stable angina patients (35% vs 19%, p=0.017). Microvascular spasm was found in 42% with a higher prevalence among the stable patients compared to the myocardial infarction with unobstructed coronary arteries cohort (53% vs 29%, p=0.0014). There were no statistically significant differences in the rate of side effects (16% vs 14%, p=0.674) or complications (1% vs 2.5%, p=0.438) between the two groups. None of the patients experienced irreversible complications. CONCLUSION: Coronary spasm is a frequent cause for myocardial infarction with unobstructed coronary arteries. Spasm provocation testing using acetylcholine is feasible in such patients in the acute phase. The complication rate during acetylcholine testing in myocardial infarction with unobstructed coronary arteries patients is low and comparable to patients with stable angina.

Lysed Erythrocyte Membranes Promote Vascular Calcification.

BACKGROUND: Intraplaque hemorrhage promotes atherosclerosis progression, and erythrocytes may contribute to this process. In this study we examined the effects of red blood cells on smooth muscle cell mineralization and vascular calcification and the possible mechanisms involved. METHODS: Erythrocytes were isolated from human and murine whole blood. Intact and lysed erythrocytes and their membrane fraction or specific erythrocyte components were examined in vitro using diverse calcification assays, ex vivo by using the murine aortic ring calcification model, and in vivo after murine erythrocyte membrane injection into neointimal lesions of hypercholesterolemic apolipoprotein E-deficient mice. Vascular tissues (aortic valves, atherosclerotic carotid artery specimens, abdominal aortic aneurysms) were obtained from patients undergoing surgery. RESULTS: The membrane fraction of lysed, but not intact human erythrocytes promoted mineralization of human arterial smooth muscle cells in culture, as shown by Alizarin red and van Kossa stain and increased alkaline phosphatase activity, and by increased expression of osteoblast-specific transcription factors (eg, runt-related transcription factor 2, osterix) and differentiation markers (eg, osteopontin, osteocalcin, and osterix). Erythrocyte membranes dose-dependently enhanced calcification in murine aortic rings, and extravasated CD235a-positive erythrocytes or Perl iron-positive signals colocalized with calcified areas or osteoblast-like cells in human vascular lesions. Mechanistically, the osteoinductive activity of lysed erythrocytes was localized to their membrane fraction, did not involve membrane lipids, heme, or iron, and was enhanced after removal of the nitric oxide (NO) scavenger hemoglobin. Lysed erythrocyte membranes enhanced calcification to a similar extent as the NO donor diethylenetriamine-NO, and their osteoinductive effects could be further augmented by arginase-1 inhibition (indirectly increasing NO bioavailability). However, the osteoinductive effects of erythrocyte membranes were reduced in human arterial smooth muscle cells treated with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide or following inhibition of NO synthase or the NO receptor soluble guanylate cyclase. Erythrocytes isolated from endothelial NO synthase-deficient mice exhibited a reduced potency to promote calcification in the aortic ring assay and after injection into murine vascular lesions. CONCLUSIONS: Our findings in cells, genetically modified mice, and human vascular specimens suggest that intraplaque hemorrhage with erythrocyte extravasation and lysis promotes osteoblastic differentiation of smooth muscle cells and vascular lesion calcification, and also support a role for erythrocyte-derived NO.

Endothelial Leptin Receptor Deletion Promotes Cardiac Autophagy and Angiogenesis Following Pressure Overload by Suppressing Akt/mTOR Signaling.

BACKGROUND: Cardiac remodeling is modulated by overnutrition or starvation. The adipokine leptin mediates energy balance between adipose tissue and brain. Leptin and its receptors are expressed in the heart. METHODS AND RESULTS: To examine the importance of endothelial leptin signaling in cardiac hypertrophy, transverse aortic constriction was used in mice with inducible endothelium-specific deletion of leptin receptors (End.LepR-KO) or littermate controls (End.LepR-WT). End.LepR-KO was associated with improved left ventricular function (fractional shortening, 28.4% versus 18.8%; P=0.0114), reduced left ventricular dilation (end-systolic inner left ventricular diameter, 3.59 versus 4.08 mm; P=0.0188) and lower heart weight (133 versus 173 mg; P<0.0001) 20 weeks after transverse aortic constriction. Histology and quantitative polymerase chain reaction analysis confirmed reduced cardiomyocyte hypertrophy. STAT3 (signal transducer and activator of transcription) activation was reduced, and Akt (protein kinase B) and mTOR (mammalian target of rapamycin) phosphorylation after transverse aortic constriction were blunted in End.LepR-KO hearts. Elevated LC3 (microtubule associated protein 1 light chain 3)-I/-II conversion ( P=0.0041) and increased (LC3II-positive) endothelial cells ( P=0.0042) in banded hearts of End.LepR-KO mice suggested improved cardiac angiogenesis because of activated autophagy. Microscopy confirmed autophagosome accumulation after genetic or small interfering RNA-mediated LepR downregulation. Enhanced sprouting angiogenesis was observed in endothelial cells ( P<0.0001) and aortic rings ( P=0.0060) from End.LepR-KO mice, and murine and human endothelial sprouting angiogenesis was reduced after mTOR inhibition using rapamycin or autophagy inhibition using 3-methyladenine. Banded End.LepR-KO mouse hearts exhibited less apoptosis ( P=0.0218), inflammation ( P=0.0251), and fibrosis ( P=0.0256). Reduced endothelial autophagy was also observed in myocardial biopsies of heart failure patients with cardiac fibrosis. CONCLUSIONS: Our findings suggest that endothelial leptin signaling contributes to cardiac fibrosis and functional deterioration by suppressing endothelial autophagy and promoting endothelial dysfunction in a chronic pressure overload model.

Inducible Knockdown of Endothelial Protein Tyrosine Phosphatase-1B Promotes Neointima Formation in Obese Mice by Enhancing Endothelial Senescence.

AIMS: Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of receptor tyrosine kinase signaling. In this study, we determined the importance of PTP1B expressed in endothelial cells for the vascular response to arterial injury in obesity. RESULTS: Morphometric analysis of vascular lesions generated by 10% ferric chloride (FeCl3) revealed that tamoxifen-inducible endothelial PTP1B deletion (Tie2.ERT2-Cre × PTP1Bfl/fl; End.PTP1B knockout, KO) significantly increased neointima formation, and reduced numbers of (endothelial lectin-positive) luminal cells in End.PTP1B-KO mice suggested impaired lesion re-endothelialization. Significantly higher numbers of proliferating cell nuclear antigen (PCNA)-positive proliferating cells as well as smooth muscle actin (SMA)-positive or vascular cell adhesion molecule-1 (VCAM1)-positive activated smooth muscle cells or vimentin-positive myofibroblasts were detected in neointimal lesions of End.PTP1B-KO mice, whereas F4/80-positive macrophage numbers did not differ. Activated receptor tyrosine kinase and transforming growth factor-beta (TGFß) signaling and oxidative stress markers were also significantly more abundant in End.PTP1B-KO mouse lesions. Genetic knockdown or pharmacological inhibition of PTP1B in endothelial cells resulted in increased expression of caveolin-1 and oxidative stress, and distinct morphological changes, elevated numbers of senescence-associated ß-galactosidase-positive cells, and increased expression of tumor suppressor protein 53 (p53) or the cell cycle inhibitor cyclin-dependent kinase inhibitor-2A (p16INK4A) suggested senescence, all of which could be attenuated by small interfering RNA (siRNA)-mediated downregulation of caveolin-1. In vitro, senescence could be prevented and impaired re-endothelialization restored by preincubation with the antioxidant Trolox. INNOVATION: Our results reveal a previously unknown role of PTP1B in endothelial cells and provide mechanistic insights how PTP1B deletion or inhibition may promote endothelial senescence. CONCLUSION: Absence of PTP1B in endothelial cells impairs re-endothelialization, and the failure to induce smooth muscle cell quiescence or to protect from circulating growth factors may result in neointimal hyperplasia.

Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

OBJECTIVE: Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. APPROACH AND RESULTS: Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. CONCLUSIONS: Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the cardiovascular risk factor obesity may have relevant therapeutic implications.

Endothelial deletion of protein tyrosine phosphatase-1B protects against pressure overload-induced heart failure in mice.

AIMS: Cardiac angiogenesis is an important determinant of heart failure. We examined the hypothesis that protein tyrosine phosphatase (PTP)-1B, a negative regulator of vascular endothelial growth factor (VEGF) receptor-2 activation, is causally involved in the cardiac microvasculature rarefaction during hypertrophy and that deletion of PTP1B in endothelial cells prevents the development of heart failure. METHODS AND RESULTS: Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in mice with endothelial-specific deletion of PTP1B (End.PTP1B-KO) and controls (End.PTP1B-WT). Survival up to 20 weeks after TAC was significantly improved in mice lacking endothelial PTP1B. Serial echocardiography revealed a better systolic pump function, less pronounced cardiac hypertrophy, and left ventricular dilation compared with End.PTP1B-WT controls. Histologically, banded hearts from End.PTP1B-KO mice exhibited increased numbers of PCNA-positive, proliferating endothelial cells resulting in preserved cardiac capillary density and improved perfusion as well as reduced hypoxia, apoptotic cell death, and fibrosis. Increased relative VEGFR2 and ERK1/2 phosphorylation and greater eNOS expression were present in the hearts of End.PTP1B-KO mice. The absence of PTP1B in endothelial cells also promoted neovascularization following peripheral ischaemia, and bone marrow transplantation excluded a major contribution of Tie2-positive haematopoietic cells to the improved angiogenesis in End.PTP1B-KO mice. Increased expression of caveolin-1 as well as reduced NADPH oxidase-4 expression, ROS generation and TGFß signalling were observed and may have mediated the cardioprotective effects of endothelial PTP1B deletion. CONCLUSIONS: Endothelial PTP1B deletion improves cardiac VEGF signalling and angiogenesis and protects against chronic afterload-induced heart failure. PTP1B may represent a useful target to preserve cardiac function during hypertrophy.

Effect of tryptophan supplementation on diet-induced non-alcoholic fatty liver disease in mice.

Intestinal serotonin (5-hydroxytrypamine, 5-HT) metabolism is thought to play a role in gut functions by regulating motility, permeability and other functions of the intestine. In the present study, we investigated the effect of tryptophan (TRP), the precursor of 5-HT, supplementation on intestinal barrier functions and non-alcoholic fatty liver disease (NAFLD). An established mouse model of NAFLD induced by feeding a fructose-rich diet (N group) was used in the present study. TRP was administered orally for 8 weeks to C57BL/6J control or NAFLD mice. NAFLD-related liver parameters (hepatic TAG and Oil Red O staining), intestinal barrier parameters (tight-junction protein occludin and portal plasma lipopolysaccharides (LPS)) and 5-HT-related parameters (5-HT, 5-HT transporter (SERT) and motility) were measured. We observed reduced duodenal occludin protein concentrations (P= 0·0007), high portal plasma LPS concentrations (P= 0·005) and an elevated liver weight:body weight ratio (P= 0·01) in the N group compared with the parameters in the control group. TRP supplementation led to an increase in occludin concentrations (P= 0·0009) and consecutively reduced liver weight:body weight ratio (P= 0·009) as well as overall hepatic fat accumulation in the N group (P= 0·05). In addition, the N group exhibited reduced SERT protein expression (P= 0·002), which was normalised by TRP supplementation (P= 0·02). For the first time, our data indicate that oral TRP supplementation attenuates experimental NAFLD in mice. The underlying mechanisms are not clear, but probably involve stabilisation of the intestinal barrier in the upper small intestine and amelioration of the dysregulated intestinal serotonergic system.