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.

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.

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.

Serotonin receptor type 3 antagonists improve obesity-associated fatty liver disease in mice.

Obesity is a major cause for nonalcoholic fatty liver disease (NAFLD). Previous studies suggested that alterations in intestinal motility and permeability contribute to the development of NAFLD. Serotonin and serotonin receptor type 3 (5-HT(3)R) are key factors in the regulation of intestinal motility and permeability. Therefore, we studied the effect of the 5-HT(3)R antagonists tropisetron and palonosetron on the development of NAFLD in leptin-deficient obese mice. Four-week-old ob/ob mice and lean controls were treated for 6 weeks orally with tropisetron or palonosetron at 0.2 mg/kg per day. We determined markers of liver damage and inflammation, portal endotoxin levels, and duodenal concentrations of serotonin, serotonin-reuptake transporter (SERT), occludin, and claudin-1. Tropisetron treatment significantly reduced liver fat content (-29%), liver inflammation (-56%), and liver cell necrosis (-59%) in ob/ob mice. The beneficial effects of tropisetron were accompanied by a decrease in plasma alanine aminotransferase and portal vein plasma endotoxin levels, an attenuation of enhanced MyD88 and tumor necrosis factor-α mRNA expression in the liver, and an increase of tight junction proteins in the duodenum. Tropisetron treatment also caused a reduction of elevated serotonin levels and an increase of SERT in the duodenum of ob/ob mice. Palonosetron had similar effects as tropisetron with regard to the reduction of liver fat and other parameters. Tropisetron and palonosetron are effective in attenuating NAFLD in a genetic mouse model of obesity. The effect involves the intestinal nervous system, resulting in a reduction of endotoxin influx into the liver and subsequently of liver inflammation and fat accumulation.