Premature atherosclerosis and thrombotic complications are major causes of morbidity and mortality in patients with systemic lupus erythematosus (SLE). However, the high incidence of these complications cannot be explained by traditional risk factors alone, suggesting direct effects of an activated immune system on hemostasis. The unexpected nucleotide sequence homology between SLE patient-derived autoantibodies against complement C1q (Fab anti-C1q) and von Willebrand factor (VWF) led us to investigate a potential interaction between the complement and hemostatic systems on the level of initiating molecules. VWF was found to bind to surface-bound C1q under static conditions. The binding could specifically be inhibited by Fab anti-C1q and C1q-derived peptides. Under shear stress the C1q-VWF interaction was enhanced, resembling the binding of VWF to collagen I. Additionally, we could show that C1q-VWF complexes induced platelet rolling and firm adhesion. Furthermore, we observed VWF binding to C1q-positive apoptotic microparticles and cholesterol crystals, as well as increased VWF deposition in C1q-positive glomeruli of SLE patients compared with control nephropathy. We show, to our knowledge for the first time, binding of VWF to C1q and thus a direct interaction between starter molecules of hemostasis and the classical pathway of complement. This direct interaction might contribute to the pathogenic mechanisms in complement-mediated, inflammatory diseases.

Background/ObjectivesInflammatory changes on the endothelium are responsible for leukocyte recruitment to plaques in atherosclerosis. Noninvasive assessment of treatment-effects on endothelial inflammation may be of use for managing medical therapy and developing novel therapies. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) with contrast enhanced ultrasound (CEU) could assess treatment effects on endothelial phenotype in early atherosclerosis.MethodsMice with atherosclerosis produced by gene deletion of the LDL-receptor and Apobec-1-editing protein were studied. At 12 weeks of age, mice received 8 weeks of regular chow or atorvastatin-enriched chow (10 mg/kg/day). At 20 weeks, CEU molecular imaging for aortic endothelial VCAM-1 expression was performed with VCAM-1-targeted (MBVCAM) and control microbubbles (MBCtr). Aortic wall thickness was assessed with high frequency ultrasound. Histology, immunohistology and Western blot were used to assess plaque burden and VCAM-1 expression.ResultsPlaque burden was reduced on histology, and VCAM-1 was reduced on Western blot by atorvastatin, which corresponded to less endothelial expression of VCAM-1 on immunohistology. High frequency ultrasound did not detect differences in aortic wall thickness between groups. In contrast, CEU molecular imaging demonstrated selective signal enhancement for MBVCAM in non-treated animals (MBVCAM 2±0.3 vs MBCtr 0.7±0.2, p<0.01), but not in statin-treated animals (MBVCAM 0.8±0.2 vs MBCtr 1.0±0.2, p = ns; p<0.01 for the effect of statin on MBVCAM signal).ConclusionsNon-invasive CEU molecular imaging detects the effects of anti-inflammatory treatment on endothelial inflammation in early atherosclerosis. This easily accessible, low-cost technique may be useful in assessing treatment effects in preclinical research and in patients.

OBJECTIVE Anti-oxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an NADPH-oxidase-inhibitor with anti-inflammatory properties. We used contrast enhanced ultrasound (CEU) molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation APPROACH AND RESULTS Genetically-modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4mg/kg/d I.P.) or saline. CEU molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MBV), to platelet GPIbα (MBPl), and control microbubbles (MBCtr). Aortic VCAM-1 was measured using Western Blot. Aortic ROS generation was measured using a lucigenin assay. Hydroethidine (HE) oxidation was used to assess aortic superoxide generation. Baseline signal for MBV (1.3±0.3 A.U.) and MBPl (1.5±0.5 A.U.) was higher than for MBCtr (0.5±0.2 A.U., p<0.01). In saline-treated animals, signal did not significantly change for any microbubble agent whereas short-term apocynin significantly (p<0.05) reduced VCAM-1 and platelet signal (MBV: 0.3±0.1, MBPl: 0.4±0.1 MBCtr: 0.3±0.2 A.U., p=0.6 between agents). Apocynin reduced aortic VCAM-1 expression by 50% (p<0.05). However, apocynin therapy did not reduce either ROS content, superoxide generation, or macrophage content. CONCLUSIONS Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content, and is not associated with a detectable change in oxidative burden.

Background-Cardiac tests for diagnosing myocarditis lack sensitivity or specificity. We hypothesized that contrastenhanced ultrasound molecular imaging could detect myocardial inflammation and the recruitment of specific cellular subsets of the inflammatory response in murine myocarditis. Methods and Results-Microbubbles (MB) bearing antibodies targeting lymphocyte CD4 (MB CD4 ), endothelial P-selectin (MB PSel ), or isotype control antibody (MB Iso ) and MB with a negative electric charge for targeting of leukocytes (MB Lc ) were prepared. Attachment of MB CD4 was validated in vitro using murine spleen CD4+ T cells. Twenty-eight mice were studied after the induction of autoimmune myocarditis by immunization with α-myosin-peptide; 20 mice served as controls. Contrast-enhanced ultrasound molecular imaging of the heart was performed. Left ventricular function was assessed by conventional and deformation echocardiography, and myocarditis severity graded on histology. Animals were grouped into no myocarditis, moderate myocarditis, and severe myocarditis. In vitro, attachment of MB CD4 to CD4+ T cells was significantly greater than of MB Iso . Of the left ventricular ejection fraction or strain and strain rate readouts, only longitudinal strain was significantly different from control animals in severe myocarditis. In contrast, contrast-enhanced ultrasound molecular imaging showed increased signals for all targeted MB versus MB Iso both in moderate and severe myocarditis, and MB CD4 signal correlated with CD4+ T-lymphocyte infiltration in the myocardium. Conclusions-Contrast-enhanced ultrasound molecular imaging can detect endothelial inflammation and leukocyte infiltration in myocarditis in the absence of a detectable decline in left ventricular performance by functional imaging.In particular, imaging of CD4+ T cells involved in autoimmune responses could be helpful in diagnosing myocarditis.(Circ Cardiovasc Imaging. 2016;9:e004720.