How Different Albumin-Binders Drive Probe Distribution of Fluorescent RGD Mimetics

The biodistribution of medical imaging probes depends on the chemical nature of the probe and the preferred metabolization and excretion routes. Especially targeted probes, which have to reach a certain (sub)cellular destination, have to be guided to the tissue of interest. Therefore, small molecular probes need to exhibit a well-balanced polarity and lipophilicity to maintain an advantageous bioavailability. Labelled antibodies circulate for several days due to their size. To alter the biodistribution behavior of probes, different strategies have been pursued, including utilizing serum albumin as an inherent transport mechanism for small molecules. We describe here the modification of an existing fluorescent RGD mimetic probe targeted to integrin alpha(v)beta(3) with three different albumin binding moieties (ABMs): a diphenylcyclohexyl (DPCH) group, a p-iodophenyl butyric acid (IPBA) and a fatty acid (FA) group with the purpose to identify an optimal ABM for molecular imaging applications. All three modifications result in transient albumin binding and a preservation of the target binding capability. Spectrophotometric measurements applying variable amounts of bovine serum albumin (BSA) reveal considerable differences between the compounds concerning their absorption and emission characteristics and hence their BSA binding mode. In vivo the modified probes were investigated in a murine U87MG glioblastoma xenograft model over the course of 1 wk by fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT). While the unmodified probe was excreted rapidly, the albumin-binding probes were accumulating in tumor tissue for at least 5 days. Considerable differences between the three probes in biodistribution and excretion characteristics were proved, with the DPCH-modified probe showing the highest overall signal intensities, while the FA-modified probe exhibits a low but more specific fluorescent signal. In conclusion, the modification of small molecular RGD mimetics with ABMs can precisely fine-tune probe distribution and offers potential for future clinical applications

Lasp-1 Regulates Podosome Function

Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins

Development, synthesis and evaluation of novel fluorescent Endothelin-B receptor probes

Delahaye J, Stölting M, Geyer C, et al. Development, synthesis and evaluation of novel fluorescent Endothelin-B receptor probes. European Journal of Medicinal Chemistry. 2023;258: 115568.The endothelin (ET) signaling system is comprised of three endothelin peptides (ET-1, -2 and -3) and two corresponding endothelin-A and -B receptors (ETAR and ETBR), which belong to the G-protein coupled receptor (GPCR) superfamily. The endothelin axis, as this system is also referred to, contributes to the maintenance of vascular tone, functions as regulator of inflammation and proliferation and helps in balancing water homeostasis. In pathological settings, the ET axis is known to contribute to endothelial activation in cardiovascular diseases, to cell proliferation, chemoresistance and metastasis in cancer and to inflammation and fibrosis in renal disease. Antagonists of ETAR and ETBR, either subtype-specific compounds or substances with high affinity to both receptors, have been developed for more than 30 years. In the preclinical context, in vivo imaging of endothelin receptor expression has been utilized to assess ET-axis contribution to e.g. cancer or myocardial infarction. In this work, we present the development and synthesis of two novel ETBR-specific fluorescent probes, based on the available antagonists BQ788 and IRL2500 and their preliminary evaluation in a breast cancer context. Copyright © 2023 Elsevier Masson SAS. All rights reserved

Podosome lifetime is altered in Lasp-1 knockdown macrophages.

<p>Primary human macrophages were treated with Lasp-1-specific siRNA (Oligo A or C) or control siRNA. (<b>A</b>) Examples for podosome dynamics included in the analysis. Shown are single podosomes of cells co-transfected with siRNA and Lifeact-TagGFP2. Podosome lifetime is defined as 1) appearance to dissolution, 2) appearance to fission, or 3) fusion to dissolution. (<b>B–D</b>) Measurement of podosome lifetime from cells treated with Lasp-1-specific siRNA, compared to controls. Double asterisks indicate values highly significant different from controls with <i>P</i><0.004 (means+SD, n = 3×4) (B). (C, D) Graph showing detailed podosome lifetime analysis, data from (B), of cells transfected with Lasp-1-specific siRNA (Oligo A (C; purple); Oligo C (D; brown), compared to controls (C, D; grey; (means+SE; n = 3×4;)).</p

Mass Spectrometry Imaging of atherosclerosis-affine Gadofluorine following Magnetic Resonance Imaging

Molecular imaging of atherosclerosis by Magnetic Resonance Imaging (MRI) has been impaired by a lack of validation of the specific substrate responsible for the molecular imaging signal. We therefore aimed to investigate the additive value of mass spectrometry imaging (MSI) of atherosclerosis-affine Gadofluorine P for molecular MRI of atherosclerotic plaques. Atherosclerotic Ldlr(-/-) mice were investigated by high-field MRI (7T) at different time points following injection of atherosclerosis-affine Gadofluorine P as well as at different stages of atherosclerosis formation (4, 8, 16 and 20 weeks of HFD). At each imaging time point mice were immediately sacrificed after imaging and aortas were excised for mass spectrometry imaging: Matrix Assisted Laser Desorption Ionization (MALDI) Imaging and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. Mass spectrometry imaging allowed to visualize the localization and measure the concentration of the MR imaging probe Gadofluorine P in plaque tissue ex vivo with high spatial resolution and thus adds novel and more target specific information to molecular MR imaging of atherosclerosis

Lasp-1 is a component of the podosome ring structure.

<p>(<b>A, B</b>) Confocal micrographs of primary human macrophages transfected with constructs encoding (A) EGFP-Lasp-1 and (B) EYFP-Vinculin (green), respectively, and stained with vinculin- or Lasp-1-specific antibodies (with Alexa488- or Alexa 568-conjugated secondary antibody) for endogenous vinculin or Lasp-1, respectively, and Cy5-conjugated phalloidin for F-actin (blue). White box indicates detail images below. Bars represent 10 µm. (<b>C, D</b>) 3D reconstruction of single podosomes of primary human macrophages. Left panels: xyz mode, right panels: xzy mode for the same podosome. (C) Cells were transfected with EGFP-Lasp-1 and stained with Alexa568-phalloidin for F-actin (podosome core) or vinculin-specific antibody (with Alexa568-labeled secondary antibody) for vinculin (podosome ring structure; red). (D) Both untransfected cells (stained with Alexa488-phalloidin for F-actin; green) and cells transfected with EYFP-vinculin (green) were stained with Lasp-1-specific antibody (with Alexa568-labeled secondary antibody) for endogenous Lasp-1 (red). (<b>E</b>) Confocal micrographs of a rat smooth muscle cell (A7r5), stained with Alexa-594 phalloidin for F-actin (red, upper panel) or zyxin-specific antibody (with Alexa594-labeled secondary antibody) for endogenous zyxin (red, lower panel) and co-stained with Lasp-1-specific antibody (with Alexa488-labeled secondary antibody) for endogenous Lasp-1 (green). Lasp-1 and its binding partner zyxin colocalize at F-actin-rich podosomes (merge) in PDBu-treated A7r5 cells. Bars represent 25 µm.</p

Lasp-1 mutants lacking either the LIM domain, the NEBU repeats and the Linker region, or the SH3 domain are still localized at podosomes.

<p>(<b>A</b>) Domain structure of Lasp-1 comprising an aminoterminal LIM domain, two nebulin (NEBU) repeats and an adjacent Linker region mediating actin binding, and a carboxyterminal SH3 (Src homology 3) domain that binds zyxin and palladin (N = aminoterminus, C = carboxyterminus). (<b>B</b>) Confocal micrographs of primary human macrophages transfected with EGFP-Lasp-1 deletion constructs (green) and stained with vinculin-specific antibody (with Alexa568-conjugated secondary antibody) for vinculin (red) and Cy5- phalloidin for F-actin. Constructs include EGFP-Lasp-1 wildtype (wt), EGFP-Lasp-1ΔLIM (lacking the LIM domain), EGFP-Lasp-1ΔSH3 (lacking the SH3 domain), and EGFP-Lasp-1ΔN-L (lacking the NEBU repeats and the Linker region). White boxes indicate detail images below. Bars represent 10 µm.</p

Lasp-1 recruitment is associated with early stages of podosome biogenesis.

<p>(<b>A</b>) Colocalization of recombinant EGFP-Lasp-1 and recombinant mRFP-actin at podosomes in PDBu-treated A7r5 cells. The white box marks the enlargement shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035340#pone-0035340-g003" target="_blank">Fig. 3B</a>. Bar represents 25 µm. (<b>B</b>) Appearance of EGFP-Lasp-1 during assembly and disassembly of podosomes (white boxes) in PDBu-treated cells over a period of 4 min. Inserts in the pictures taken at 0 s, 2 min. and 4 min. show the merged EGFP-Lasp-1 (green) and mRFP-actin (red) signals in two individual podosomes. Bar represents 2 µm. (<b>C</b>) Colocalization of EGFP-Lasp-1 (upper row) and DsRed-cortactin (middle row, merged pictures in the lower row) during initial stages of podosome assembly in PDBu-treated A7r5 cells over a period of 2 min. An individual podosome is marked by a red circle. Bars represent 25 µm (left panel) or 2 µm (right panels), respectively.</p

Monitoring Endothelin-A Receptor Expression during the Progression of Atherosclerosis

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 (ETAR) 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 ETAR expression by quantitative rtPCR (qPCR), histology and fluorescence molecular imaging. We found a significant upregulation of ETAR 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 ETAR is upregulated during the progression of early atherosclerosis in the ApoE−/− mouse model, we found that ETAR expression is substantially reduced in advanced human atherosclerotic plaques. Moreover, those expression changes were clearly depicted by fluorescence imaging using our in-house designed ETAR-Cy 5.5 probe confirming its specificity and potential use in future studies