High resolution imaging of collagen organisation and synthesis using a versatile collagen specific probe

Collagen is the protein primarily responsible for the load-bearing properties of tissues and collagen architecture is one of the main determinants of the mechanical properties of tissues. Visualisation of changes in collagen three-dimensional structure is essential in order to improve our understanding of collagen fibril formation and remodelling, e.g. in tissue engineering experiments. A recently developed collagen probe, based on a natural collagen binding protein (CNA35) conjugated to a fluorescent dye, showed to be much more specific to collagen than existing fluorescent techniques currently used for collagen visualisation in live tissues. In this paper, imaging with this fluorescent CNA35 probe was compared to imaging with second harmonic generation (SHG) and the imaging of two- and three-dimensional collagen organisation was further developed. A range of samples (cell culture, blood vessels and engineered tissues) was imaged to illustrate the potential of this collagen probe. This images of collagen organisation showed improved detail compared to images generated with SHG, which is currently the most effective method for viewing three-dimensional collagen organisation in tissues. In conclusion, the fluorescent CNA35 probe allows easy access to high resolution imaging of collagen, ranging from very young fibrils to more mature collagen fibres. Furthermore, this probe enabled real-time visualisation of collagen synthesis in cell culture, which provides new opportunities to study collagen synthesis and remodelling

High resolution imaging of collagen organisation and synthesis using a versatile collagen specific probe

Collagen is the protein primarily responsible for the load-bearing properties of tissues and collagen architecture is one of the main determinants of the mechanical properties of tissues. Visualisation of changes in collagen three-dimensional structure is essential in order to improve our understanding of collagen fibril formation and remodelling, e.g. in tissue engineering experiments. A recently developed collagen probe, based on a natural collagen binding protein (CNA35) conjugated to a fluorescent dye, showed to be much more specific to collagen than existing fluorescent techniques currently used for collagen visualisation in live tissues. In this paper, imaging with this fluorescent CNA35 probe was compared to imaging with second harmonic generation (SHG) and the imaging of two- and three-dimensional collagen organisation was further developed. A range of samples (cell culture, blood vessels and engineered tissues) was imaged to illustrate the potential of this collagen probe. This images of collagen organisation showed improved detail compared to images generated with SHG, which is currently the most effective method for viewing three-dimensional collagen organisation in tissues. In conclusion, the fluorescent CNA35 probe allows easy access to high resolution imaging of collagen, ranging from very young fibrils to more mature collagen fibres. Furthermore, this probe enabled real-time visualisation of collagen synthesis in cell culture, which provides new opportunities to study collagen synthesis and remodelling

Two-photon microscopy and its applications in (micro) vascular research

Currently many thermoregulatory models incorporate cutaneous perfusion, as it plays a major role in heat transport. The magnitude of perfusion is generally described as a function of temperature. Some physiological models, such as formulated by Stolwijk, Tanabe and Fiala, incorporate central tonus and local sensitivity explicitly. However, used perfusion control parameters have been estimated and are not based on physiological measurements. Therefore, their applicability might not be justified. In this research a method is proposed to measure central sympathetic vasoconstrictor tone and local cutaneous vasoconstrictor sensitivity. Together, the protocol was performed on males and females, young adults (18-28yr) and elderly (68-78yr) and on both glabrous and non-glabrous skin. Local cutaneous perfusion is measured by laser-Doppler flowmetry. Through iontophoresis of Bretylium tosylate (10mM) sympathetic nerve terminals are blocked, such that local endogenous release of sympathetic neurotransmitters is abolished. Afterwards a superphysiological dose noradrenaline (2.5mM) is administered through iontophoresis. The reduction in perfusion is used as a measure for vasoconstrictor sensitivity at maximal vasoconstrictor tone. This is repeated over a range of local temperatures. Then the whole body is cooled and the decrease in perfusion at a non-bretylium site is measured. Combining perfusion and local sensitivity, vasoconstrictor tone is described as a function of temperature

Arterial radius estimation from microscopic data using a new algorithm for circle parameter estimation

We developed and tested a new method for automatic determination of vessel wall diameters from image stacks obtained using Two-photon Laser Scanning Microscopy (TPLSM) on viable arteries in perfusion flow chambers. To this extent, a new method has been proposed for estimating the parameters of a circle describing the inner diameter of the blood vessels. The new method is based on the Hough Transform and the observation that three points that are not co-linear uniquely define a circle. By only storing the estimated center location, the computational and memory costs of the Hough Transform can be greatly reduced. We tested the algorithm on twenty images and compared the result with a ground-truth established by human volunteers, and a standard Least Squares technique. With errors of 3-5%, the algorithm allows accurate estimation of the vessel diameters from image stacks containing only small parts of the vessel cross section. Combined with TPLSM imaging of anatomical vessel wall properties, potentially, the algorithm enables the correlation of structural and functional properties of large intact arteries simultaneously, without requirements for additional experiments

Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy

We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E-/- mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E-/- mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis. © 2007 BC Decker Inc

Improved Quantification of Cell Density in the Arterial Wall-A Novel Nucleus Splitting Approach Applied to 3D Two-Photon Laser-Scanning Microscopy

Accurate information on vascular smooth muscle cell (VSMC) content, orientation, and distribution in blood vessels is indispensable to increase understanding of arterial remodeling and to improve modeling of vascular biomechanics. We have previously proposed an analysis method to automatically characterize VSMC orientation and transmural distribution in murine carotid arteries under well-controlled biomechanical conditions. However, coincident nuclei, erroneously detected as one large nucleus, were excluded from the analysis, hampering accurate VSMC content characterization and distorting transmural distributions. In the present study, therefore, we aim to (1) improve the previous method by adding a "nucleus splitting" procedure to split coinciding nuclei, (2) evaluate the accuracy of this novel method, and (3) test this method in a mouse model of VSMC apoptosis. After euthanasia, carotid arteries from SM22 alpha-hDTR Apoe(-/-) and control Apoe(-/-) mice were bluntly dissected, excised, mounted in a biaxial biomechanical tester and brought to in vivo axial stretch and a pressure of 100 mmHg. Nuclei and elastin fibers were then stained using Syto-41 and Eosin-Y, respectively, and imaged using 3D two-photon laser scanning microscopy. Nuclei were segmented from images and coincident nuclei were split. The nucleus splitting procedure determines the likelihood that voxel pairs within coincident nuclei belong to the same nucleus and utilizes these likelihoods to identify individual nuclei using spectral clustering. Manual nucleus counts were used as a reference to assess the performance of our splitting procedure. Before and after splitting, automatic nucleus counts differed -26.6 +/- 9.90% (p < 0.001) and -1.44 +/- 7.05% (p = 0.467) from the manual reference, respectively. Whereas the slope of the relative difference between the manual and automated counts as a function of the manual count was significantly negative before splitting (p = 0.008), this slope became insignificant after splitting (p = 0.653). Smooth muscle apoptosis led to a 33.7% decrease in VSMC density (p = 0.008). Nucleus splitting improves the accuracy of automated cell content quantification in murine carotid arteries and overcomes the progressively worsening problem of coincident nuclei with increasing cell content in vessels. The presented image analysis framework provides a robust tool to quantify cell content, orientation, shape, and distribution in vessels to inform experimental and advanced computational studies on vascular structure and function

Properties and fate of human mesenchymal stem cells upon miRNA let-7f-promoted recruitment to atherosclerotic plaques.

AIMS: Atherosclerosis is a chronic inflammatory disease of the arteries leading to the formation of atheromatous plaques. Human mesenchymal stem cells (hMSCs) are recruited from the circulation into plaques where in response to their environment they adopt a phenotype with immunomodulatory properties. However, the mechanisms underlying hMSC function in these processes are unclear. Recently, we described that miRNA let-7f controls hMSC invasion guided by inflammatory cytokines and chemokines. Here, we investigated the role of let-7f in hMSC tropism to human atheromas and the effects of the plaque microenvironment on cell fate and release of soluble factors. METHODS AND RESULTS: Incubation of hMSCs with LL-37, an antimicrobial peptide abundantly found in plaques, increased biosynthesis of let-7f and N-formyl peptide receptor 2 (FPR2), enabling chemotactic invasion of the cells towards LL-37, as determined by qRT-PCR, flow cytometry, and cell invasion assay analysis. In an Apoe &nbsp;-/- mouse model of atherosclerosis, circulating hMSCs preferentially adhered to athero-prone endothelium. This property was facilitated by elevated levels of let-7f in the hMSCs, as assayed by ex vivo artery perfusion and 2-photon laser scanning microscopy. Exposure of hMSCs to homogenized human atheromatous plaque material considerably induced the production of various cytokines, chemokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases, as studied by PCR array and Western blot analysis. Moreover, exposure to human plaque extracts elicited differentiation of hMSCs into cells of the myogenic lineage, suggesting a potentially plaque-stabilizing effect. CONCLUSIONS: Our findings indicate that let-7f promotes hMSC tropism toward atheromas through the LL-37/FPR2 axis and demonstrate that hMSCs upon contact with human plaque environment develop a potentially athero-protective signature impacting the pathophysiology of atherosclerosis. TRANSLATIONAL PERSPECTIVE: Human mesenchymal stem cells (hMSCs) represent a promising therapeutic approach in various pathophysiological processes associated with inflammation including atherosclerosis. The current knowledge about the mechanisms of hMSC tropism towards human atherosclerotic plaques and their beneficial effects at the site is poor. Bridging this gap is essential for clinical application of hMSCs. Our work provides insight into the contribution of microRNA let-7f in hMSC recruitment to atheroprone areas, where hMSCs display athero-protective potential by releasing immunomodulatory factors and differentiating towards plaque-stabilizing cells. Our findings highlight circulating hMSCs as a possible therapeutic strategy for the stabilization of atherosclerotic plaques

Endothelial ENPP2 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 2) Increases Atherosclerosis in Female and Male Mice

Background: Maladapted endothelial cells (ECs) secrete ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin)-a lysophospholipase D that generates lysophosphatidic acids (LPAs). ENPP2 derived from the arterial wall promotes atherogenic monocyte adhesion induced by generating LPAs, such as arachidonoyl-LPA (LPA20:4), from oxidized lipoproteins. Here, we aimed to determine the role of endothelial ENPP2 in the production of LPAs and atherosclerosis. Methods: We quantified atherosclerosis in mice harboring loxP-flanked Enpp2 alleles crossed with Apoe(-/-) mice expressing tamoxifen-inducible Cre recombinase under the control of the EC-specific bone marrow X kinase promoter after 12 weeks of high-fat diet feeding. Results: A tamoxifen-induced EC-specific Enpp2 knockout decreased atherosclerosis, accumulation of lesional macrophages, monocyte adhesion, and expression of endothelial CXCL (C-X-C motif chemokine ligand) 1 in male and female Apoe(-/-) mice. In vitro, ENPP2 mediated the mildly oxidized LDL (low-density lipoprotein)-induced expression of CXCL1 in aortic ECs by generating LPA20:4, palmitoyl-LPA (LPA16:0), and oleoyl-LPA (LPA18:1). ENPP2 and its activity were detected on the endothelial surface by confocal imaging. The expression of endothelial Enpp2 established a strong correlation between the plasma levels of LPA16:0, stearoyl-LPA (LPA18:0), and LPA18:1 and plaque size and a strong negative correlation between the LPA levels and ENPP2 activity in the plasma. Moreover, endothelial Enpp2 knockout increased the weight of high-fat diet-fed male Apoe(-/-) mice. Conclusions: We demonstrated that the expression of ENPP2 in ECs promotes atherosclerosis and endothelial inflammation in a sex-independent manner. This might be due to the generation of LPA20:4, LPA16:0, and LPA18:1 from mildly oxidized lipoproteins on the endothelial surface