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ABSTRACT Two-photon microscopy is an innovative technology that has high potential to combine the examination of soft and hard tissues in vitro and in vivo. Calcium phosphates are widely used substitutes for bone tissue engineering, since they are degradable and consequently replaced by newly formed tissue. It is well known that osteoclasts are responsible for the resorption processes during bone remodelling. We hypothesize that also macrophages are actively involved in the resorption process of calcium phosphate scaffolds and addressed this question in in vitro culture systems by two-photon laser scanning microscopy. Beta-tricalcium phosphate specimens were incubated with (1) macrophages, (2) interleukin-4 activated macrophages, and (3) osteoclasts for up to 21 days. Interestingly, macrophages degraded beta-tricalcium phosphate specimens in an equivalent fashion compared to osteoclasts and significantly more than IL-4 activated macrophages. An average of ∼32% of the macrophages was partially filled with ceramic material while this was 18% for osteoclasts and 9% for IL-4 activated macrophages. For the first time by applying two-photon microscopy, our studies show the previously unrecognized potential of macrophages to phagocytose ceramic material, which is expected to have implication on osteoconductive scaffold design. Microsc. Res. Tech. 77:143-152, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The article discusses a research study which evaluated the role of lysophosphatidic acid (LPA) receptors in promoting chemokine CXCL12-dependent vascular repair by smooth muscle progenitor cells (SPC). A review of the related literature on LPA and CXCL12 expression is presented. It describes the methods employed for creating mouse models of neointima formation, for bone marrow transplantation and for blood chemistry. Presented in details are the research findings.

At the moment, many researchers are using in vitro techniques to investigate chemokine-driven leukocyte adhesion/recruitment, for example, by using a transwell or flow chamber system. Here we describe a more physiologically relevant, sophisticated, and highly flexible method to study leukocyte adhesion ex vivo in fresh murine carotid arteries under arterial flow conditions. This model mimics an in vivo situation and allows the combination of leukocytes and arteries isolated from different donors in one experiment, generating information on both vascular and leukocyte adhesive properties of both donors. This method provides a versatile, highly physiologically relevant model to investigate leukocyte adhesion., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recruitment of leukocytes into arteries is a hallmark event throughout all stages of atherosclerosis and hence stands out as a primary therapeutic target. To understand the molecular mechanisms of arterial leukocyte subset infiltration, real-time visualization of recruitment processes of leukocyte subsets at high resolution is a prerequisite. In this review we provide a balanced overview of optical imaging modalities in the more commonly used experimental models for atherosclerosis (e.g., mouse models) allowing for in vivo display of recruitment processes in large arteries and further detail strategies to overcome hurdles inherent to arterial imaging. We further provide a synopsis of techniques allowing for non-toxic, photostable labeling of target structures. Finally, we deliver a short summary of ongoing developments including the emergence of novel labeling approaches, the use of superresolution microscopy, and the potentials of opto-acoustic microscopy and intravascular 2-dimensional near-infrared fluorescence microscopy.

Smooth muscle cells (SMCs) play a pivotal role in regulating vascular tone in arteries, and are therefore an essential part of constitutive models of the artery wall. In the present study, we developed a method to quantify in 3D the orientation of SMCs in the intact artery wall. We stained cell nuclei in excised mouse carotid arteries mounted between micropipettes and imaged these in 3D using two-photon laser scanning microscopy. A clustering method was used to identify individual nuclei. Orientations of these nuclei (as a representative of the SMC orientations) were found by calculating the inertia matrix eigenvectors. Subsequently, SMC locations and orientations were converted to cylindrical and spherical coordinate systems, respectively. We found SMCs to be arranged in two distinct layers. For each of these layers, SMC orientations were described by a Bingham distribution. Distributions showed a statistically significant helical and transversal angular component in both inner and outer layers. In conclusion, this study demonstrates that SMC orientation can be quantified in 3D, and shows a distinct helical as well as transversal orientation. The acquired distribution data are essential to improve current constitutive models of the artery wall, by describing physiological SMC orientation and dispersion.

The application of optical nanoparticles in cardiovascular research is increasing because of the high spatiotemporal resolution and high sensitivity of optical techniques as compared with other imaging platforms. The major cause of cardiovascular events is atherosclerosis, which is a chronic inflammation of the arterial wall. Interestingly, the composition rather than the size of nonstenotic atherosclerotic plaques and severe plaques with >90% stenosis are indicators for high-risk vulnerability to rupture and acute cardiovascular events. Optical techniques may be highly suitable for discriminating, at subcellular resolution, the different stages of plaque progression by targeting bright and nontoxic optical nanoparticles toward distinct molecular epitopes in order to distinguish vulnerable from stable atherosclerotic plaques. Several optical techniques including two-photon laser scanning microscopy (TPLSM), optical coherence tomography (OCT), and photoacoustic imaging (PAI) have been applied for (in vivo) characterization of atherosclerotic plaques, in addition to investigate their feasibility in the clinical setting. Optical nanoparticles, however, have predominantly been used in optical molecular imaging of tumors, but their application in cardiovascular research is increasing. In this review, we first describe shortly the basics of the mentioned optical techniques. Then, we detail on the most-extensively studied optical nanoparticles and relatively new optical nanoparticles that hold promise for in vivo applications in atherosclerosis research., (Copyright © 2011 John Wiley & Sons, Inc.)

Rationale: The chemokine CXCL12 (CXC motif ligand 12) and its receptor CXCR 4 (CXC motif receptor 4) direct the recruitment of smooth muscle progenitor cells (SPCs) in neointima formation after vascular injury. Lysophosphatidic acid (LPA) induces CXCL12 and neointimal accumulation of smooth muscle cells (SMCs) in uninjured arteries. Thus, we hypothesize that LPA may regulate CXCL12-mediated vascular remodelling., Objectives: We evaluated the role of LPA receptors in initiating CXCL12-dependent vascular repair by SPCs., Methods and Results: Wire-induced carotid injury was performed in apolipoprotein E(-/-) mice on western-type diet. LPA receptor expression was studied by immunostaining and quantitative RT-PCR. LPA receptors LPA(1) and LPA(3) were detected in the media of uninjured arteries and in the injury-induced neointima. LPA(3) mRNA was upregulated and LPA(1) mRNA downregulated at one week after injury. The LPA(1/3) antagonist Ki16425 inhibited neointima formation by 71% and reduced both relative neointimal SMCs and the macrophage content. Additionally, neointimal hypoxia-inducible factor-1alpha and CXCL12 expression, the injury-induced peripheral stem cell antigen-1 (Sca-1)(+)/Lin(-) SPC mobilization, and the neointimal recruitment of Sca-1(+)SMCs were inhibited by Ki16425. In wild type mice, LPA20:4 increased CXCL12 and hypoxia-inducible factor-1alpha expression in carotid arteries as early as 1 day following short-term endoluminal incubation. LPA20:4-induced SPC mobilization and neointima formation were blocked by Ki16425, LPA(1)- and LPA(3)-specific small interfering (si)RNA, and the CXCR4 antagonist POL5551. Ki16425 reduced LPA20:4-mediated neointimal recruitment of SPC as demonstrated by 2-photon microscopy in bone marrow chimeric mice after repopulation with SM22-LacZ transgenic, hematopoietic cells. Moreover, POL5551 decreased the neointimal accumulation of CXCR4(+) SMCs., Conclusions: LPA(1) and LPA(3) promote neointima formation through activation of CXCL12-mediated mobilization and recruitment of SPCs.

A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.