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.Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.

Podosomes are highly dynamic structures that are involved in cell adhesion and extracellular matrix remodeling. They present as intracellular columns composed of an actin-rich core region and a surrounding ring-like structure containing focal adhesion proteins, actin binders as well as cell signaling molecules. A key player in podosome biogenesis is the scaffolding protein cortactin, which is thought to control actin assembly at the core region. We show that the zona occludens protein 1 (ZO-1), a pivotal tight junction protein and known binding partner of cortactin, is a component of podosomes. In the smooth muscle cell line A7r5, phorbol ester treatment induced a rapid relocation of ZO-1 from the cell cortex and cytosolic pools toward newly formed podosomes. Podosomal localization was also observed for the known ZO-1-binding proteins l-afadin, α-catenin, and phospho-connexin 43. Truncation studies revealed that the actin-binding domain but not the association with cortactin is necessary for ZO-1 recruitment to podosomes. Moreover, impaired ZO-1 expression leads to significantly reduced podosome formation and concomitant decreased matrix degradation at podosomes. Our findings demonstrate that besides their known function in tight junction assembly and intercellular communication, zona occludens proteins and their binding partners may play a novel role in podosome formation and associated function, thus regulating cell adhesion and matrix remodeling.

Purpose: Extracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis. Procedures: Extracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell-derived versus extracellular vesicles from healthy serum.

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 (7 T) 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. Visualization of atherosclerosis using MR imaging is an emerging tool to gain deeper and more dynamic insights into biological processes of atherosclerosis with a high spatial and temporal resolution as well as high sensitivity. An advantage of MR imaging is the ability to acquire anatomical, functional and biological information simultaneously. Noninvasive characterization and assessment of plaque burden, characterization of plaque features (molecular and anatomical) and monitoring of plaque progression are crucial to predict plaque rupture, which causes myocardial infarction or stroke. Various molecular MRI approaches have tried to characterize specific aspects of atherosclerosis. Endothelial permeability, one of the key features of early vascular dysfunction, can be assessed by using albumin-affine gadolinium (Gd) chelates such as Gadofosveset trisodium 1,2. Influx of inflammatory cells such as proinflammatory macrophages can be traced by using iron-oxide nanoparticles, which has been similarly successful both in mice and humans. Additionally, the complex process of vascular remodeling has been successfully investigated by targeting tropoelastin, elastin and other extracellular proteins by more or less specific gadolinium agents in mice and rabbits 3-5. All approaches rely on alteration of the MR signal induced either by shortening T1 relaxation (in case of gadolinium) or shortening T2* (in case of iron-oxide nanoparticles), generated by accumulation of a

The CXC chemokine receptor 3 (CXCR3) has been linked to autoimmune and inflammatory disease, allograft rejection, and ischemic nephropathy. CXCR3 is expressed on endothelial and smooth muscle cells. Although a recent study posited that antagonizing of CXCR3 function may reduce atherosclerosis, the role of CXCR3 in controlling physiological vascular functions remains unclear. This study demonstrates that disruption of CXCR3 leads to elevated mean arterial pressures in anesthetized and conscious mice, respectively. Stimulation of isolated resistance vessels with various vasoconstrictors showed increased contractibility in CXCR3-/- mice in response to angiotensin II (ANG II) and a decreased vasodilatation in response to acetylcholine (ACh). The increased contractibility was related to higher ANG II type 1 receptor (AT1R) expression, whereas the decreased vasodilatation was related to lower M3-ACh receptor expression in the mesenteric arteries of CXCR3-/- mice compared with wild-type mice. The vasodilatatory response to ACh could be antagonized by the nonselective ACh receptor antagonist atropine and the selective M3 receptor antagonist 4-DAMP, but not by M1, M2, and M4 receptor antagonists. Additionally, EMSA studies revealed that transcription factors SP-1 and EGR-1 interact as a complex with the murine AT1R promoter region. Furthermore, we could show increased expression of SP-1 in CXCR3-/- mice indicating an imbalanced SP-1 and EGR-1 complex formation which causes increased AT1R expression and hypertension. The data indicate that CXCR3 receptor is important in vascular contractility and hypertension, possibly through upregulated AT1R expression.