Long-term cardiovascular inflammation and fibrosis in a murine model of vasculitis induced by Lactobacillus casei cell wall extract.

Background: Kawasaki disease (KD), an acute febrile illness and systemic vasculitis, is the leading cause of acquired heart disease in children in industrialized countries. KD leads to the development of coronary artery aneurysms (CAA) in affected children, which may persist for months and even years after the acute phase of the disease. There is an unmet need to characterize the immune and pathological mechanisms of the long-term complications of KD. Methods: We examined cardiovascular complications in the Lactobacillus casei cell wall extract (LCWE) mouse model of KD-like vasculitis over 4 months. The long-term immune, pathological, and functional changes occurring in cardiovascular lesions were characterized by histological examination, flow cytometric analysis, immunofluorescent staining of cardiovascular tissues, and transthoracic echocardiogram. Results: CAA and abdominal aorta dilations were detected up to 16 weeks following LCWE injection and initiation of acute vasculitis. We observed alterations in the composition of circulating immune cell profiles, such as increased monocyte frequencies in the acute phase of the disease and higher counts of neutrophils. We determined a positive correlation between circulating neutrophil and inflammatory monocyte counts and the severity of cardiovascular lesions early after LCWE injection. LCWE-induced KD-like vasculitis was associated with myocarditis and myocardial dysfunction, characterized by diminished ejection fraction and left ventricular remodeling, which worsened over time. We observed extensive fibrosis within the inflamed cardiac tissue early in the disease and myocardial fibrosis in later stages. Conclusion: Our findings indicate that increased circulating neutrophil counts in the acute phase are a reliable predictor of cardiovascular inflammation severity in LCWE-injected mice. Furthermore, long-term cardiac complications stemming from inflammatory cell infiltrations in the aortic root and coronary arteries, myocardial dysfunction, and myocardial fibrosis persist over long periods and are still detected up to 16 weeks after LCWE injection.

Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase.

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R-/-), B2- (B2R-/-) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R-/-). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R-/- presented similar level of vascular dysfunction as found in B1R-/- or B2R-/- mice. In accordance, aortic rings from B1R-/- or B2R-/- mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R-/- and B2R-/- mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R-/- or B2R-/- mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.

Cardioprotective Action of Ginkgo biloba Extract against Sustained ß-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway.

Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba-mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic ß-adrenergic receptor stimulation (ß-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M2) and downregulation of ß1-AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo, we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M2/NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba.

Increased Nitric Oxide Bioavailability and Decreased Sympathetic Modulation Are Involved in Vascular Adjustments Induced by Low-Intensity Resistance Training.

Resistance training is one of the most common kind of exercise used nowadays. Long-term high-intensity resistance training are associated with deleterious effects on vascular adjustments. On the other hand, is unclear whether low-intensity resistance training (LI-RT) is able to induce systemic changes in vascular tone. Thus, we aimed to evaluate the effects of chronic LI-RT on endothelial nitric oxide (NO) bioavailability of mesenteric artery and cardiovascular autonomic modulation in healthy rats. Wistar animals were divided into two groups: exercised (Ex) and sedentary (SED) rats submitted to the resistance (40% of 1RM) or fictitious training for 8 weeks, respectively. After LI-RT, hemodynamic measurements and cardiovascular autonomic modulation by spectral analysis were evaluated. Vascular reactivity, NO production and protein expression of endothelial and neuronal nitric oxide synthase isoforms (eNOS and nNOS, respectively) were evaluated in mesenteric artery. In addition, cardiac superoxide anion production and ventricle morphological changes were also assessed. In vivo measurements revealed a reduction in mean arterial pressure and heart rate after 8 weeks of LI-RT. In vitro studies showed an increased acetylcholine (ACh)-induced vasorelaxation and greater NOS dependence in Ex than SED rats. Hence, decreased phenylephrine-induced vasoconstriction was found in Ex rats. Accordingly, LI-RT increased the NO bioavailability under basal and ACh stimulation conditions, associated with upregulation of eNOS and nNOS protein expression in mesenteric artery. Regarding autonomic control, LI-RT increased spontaneous baroreflex sensitivity, which was associated to reduction in both, cardiac and vascular sympathetic modulation. No changes in cardiac superoxide anion or left ventricle morphometric parameters after LI-RT were observed. In summary, these results suggest that RT promotes beneficial vascular adjustments favoring augmented endothelial NO bioavailability and reduction of sympathetic vascular modulation, without evidence of cardiac overload.