Miriam Stölting

Endothelin-1 (ET-1) levels are altered in atherosclerosis, while the roles of the endothelin receptors ET_AR and ET_BR during the pathogenesis of atherosclerosis remain unclear. Therefore, the focus of this study was to clarify how endothelin receptors are expressed in advanced human atherosclerotic plaques and how this is related to atherosclerotic risk factors. Ex vivo expression analysis was performed by quantitative real-time PCR (qRT-PCR) of 98 atherosclerotic plaques and controls that were obtained from adult patients undergoing vascular surgery. Correlation analyses of atherosclerosis-promoting factors were accomplished using a linear regression model. We found an overall reduced expression of ET receptors and smooth muscle actin (SMA), a marker of healthy vascular smooth muscle cells, in atherosclerotic plaques, whereas the levels of ET-1 and matrix metalloproteinase 2 (MMP-2), a marker of atherosclerosis progression, remained unchanged. Reduced expression was predominantly correlated with hypertension, which affects both receptors as well as SMA. Age, body mass index (BMI) and gender also correlated with either ET_AR, ET_BR or SMA expression in advanced plaques. In contrast, no effect of diabetes mellitus or smoking was found, indicating an ancillary effect of those risk factors. The results of our study indicate that endothelin receptor expression during the pathogenesis of atherosclerosis is predominantly correlated with hypertension.

Variations in vascular wall shear stress are often presumed to result in the formation of atherosclerotic lesions at specific arterial regions, where continuous laminar flow is disturbed. The influences of altered blood flow dynamics and oscillations on the integrity of endothelial cells and the endothelial layer have been extensively studied in vitro and in vivo. Under pathological conditions, the Arg-Gly-Asp (RGD) motif binding integrin α_vβ₃ has been identified as a relevant target, as it induces endothelial cell activation. Animal models for in vivo imaging of endothelial dysfunction (ED) mainly rely on genetically modified knockout models that develop endothelial damage and atherosclerotic plaques upon hypercholesterolemia (ApoE^−/− and LDLR^−/−), thereby depicting late-stage pathophysiology. The visualization of early ED, however, remains a challenge. Therefore, a carotid artery cuff model of low and oscillating shear stress was applied in CD-1 wild-type mice, which should be able to show the effects of altered shear stress on a healthy endothelium, thus revealing alterations in early ED. Multispectral optoacoustic tomography (MSOT) was assessed as a non-invasive and highly sensitive imaging technique for the detection of an intravenously injected RGD-mimetic fluorescent probe in a longitudinal (2–12 weeks) study after surgical cuff intervention of the right common carotid artery (RCCA). Images were analyzed concerning the signal distribution upstream and downstream of the implanted cuff, as well as on the contralateral side as a control. Subsequent histological analysis was applied to delineate the distribution of relevant factors within the carotid vessel walls. Analysis revealed a significantly enhanced fluorescent signal intensity in the RCCA upstream of the cuff compared to the contralateral healthy side and the downstream region at all time points post-surgery. The most obvious differences were recorded at 6 and 8 weeks after implantation. Immunohistochemistry revealed a high degree of α_v-positivity in this region of the RCCA, but not in the left common carotid artery (LCCA) or downstream of the cuff. In addition, macrophages could be detected by CD68 immunohistochemistry in the RCCA, showing ongoing inflammatory processes. In conclusion, MSOT is capable of delineating alterations in endothelial cell integrity in vivo in the applied model of early ED, where an elevated expression of integrin α_vβ₃ was detected within vascular structures.

(1) Background: The prognosis of cancer is dependent on immune cells in the tumor microenvironment (TME). The protein S100A9 is an essential regulator of the TME, associated with poor prognosis. In this study, we evaluated early therapy effects on the TME in syngeneic murine breast cancer via S100A9-specific in vivo imaging. (2) Methods: Murine 4T1 cells were implanted orthotopically in female BALB/c mice (n = 59). Tumor size-adapted fluorescence imaging was performed before and 5 days after chemo- (Doxorubicin, n = 20), anti-angiogenic therapy (Bevacizumab, n = 20), or placebo (NaCl, n = 19). Imaging results were validated ex vivo (immunohistochemistry, flow cytometry). (3) Results: While tumor growth revealed no differences (p = 0.48), fluorescence intensities (FI) for S100A9 in Bevacizumab-treated tumors were significantly lower as compared to Doxorubicin (2.60 vs. 15.65 AU, p < 0.0001). FI for Doxorubicin were significantly higher compared to placebo (8.95 AU, p = 0.01). Flow cytometry revealed shifts in monocytic and T-cell cell infiltrates under therapy, correlating with imaging. (4) Conclusions: S100A9-specific imaging enables early detection of therapy effects visualizing immune cell activity in the TME, even before clinically detectable changes in tumor size. Therefore, it may serve as a non-invasive imaging biomarker for early therapy effects.

Cardiovascular disease remains the most frequent cause of death worldwide. Atherosclerosis, an underlying cause of cardiovascular disease, is an inflammatory disorder associated with endothelial dysfunction. The endothelin system plays a crucial role in the pathogenesis of endothelial dysfunction and is involved in the development of atherosclerosis. We aimed to reveal the expression levels of the endothelin-A receptor (ET_AR) in the course of atherogenesis to reveal possible time frames for targeted imaging and interventions. We used the ApoE^−/− mice model and human specimens and evaluated ET_AR expression by quantitative rtPCR (qPCR), histology and fluorescence molecular imaging. We found a significant upregulation of ET_AR after 22 weeks of high-fat diet in the aortae of ApoE^−/− mice. With regard to translation to human disease, we applied the fluorescent probe to fresh explants of human carotid and femoral artery specimens. The findings were correlated with qPCR and histology. While ET_AR is upregulated during the progression of early atherosclerosis in the ApoE^−/− mouse model, we found that ET_AR expression is substantially reduced in advanced human atherosclerotic plaques. Moreover, those expression changes were clearly depicted by fluorescence imaging using our in-house designed ET_AR-Cy 5.5 probe confirming its specificity and potential use in future studies.