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.

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 (P¼0·0007), high portal plasma LPS concentrations (P¼0·005) and an elevated liver weight:body weight ratio (P¼ 0·01) in the N group compared with the parameters in the control group. TRP supplementation led to an increase in occludin concentrations (P¼ 0·0009) and consecutively reduced liver weight:body weight ratio (P¼ 0·009) as well as overall hepatic fat accumulation in the N group (P¼ 0·05). In addition, the N group exhibited reduced SERT protein expression (P¼ 0·002), which was normalised by TRP supplementation (P¼0·02). 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.

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 NA-FLD 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, serotoninreuptake transporter (SERT), occludin, and claudin-1. Tropisetron treatment significantly reduced liver fat content (Ϫ29%), liver inflammation (Ϫ56%), and liver cell necrosis (Ϫ59%) 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-␣ 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.

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.

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.