R. de Crom

Angiogenic Murine Endothelial Progenitor Cells Are Derived From a Myeloid Bone Marrow Fraction and Can Be Identified by Endothelial NO Synthase Expression

Objective—Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization and are therefore of great interest for autologous cell therapies to treat ischemic vascular disease. However, the origin and functional properties of these EPCs are still in debate. Methods and Results—Here, ex vivo expanded murine EPCs were characterized in terms of phenotype, lineage potential, differentiation from bone marrow (BM) precursors, and their functional properties using endothelial NO synthase (eNOS)–green fluorescent protein transgenic mice. Despite high phenotypic overlap with macrophages and dendritic cells, EPCs displayed unique eNOS expression, endothelial lineage potential in colony assays, and angiogenic characteristics, but also immunologic properties such as interleukin-12p70 production and low levels of T-cell stimulation. The majority of EPCs developed from an immature, CD31+Ly6C+ myeloid progenitor fraction in the BM. Addition of myeloid growth factors such as macrophage–colony-stimulating factor (M-CSF) and granulocyte/macrophage (GM)-CSF stimulated the expansion of spleen-derived EPCs but not BM-derived EPCs. Conclusion—The close relationship between EPCs and other myeloid lineages may add to the complexity of using them in cell therapy. Our mouse model could be a highly useful tool to characterize EPCs functionally and phenotypically, to explore the origin and optimize the isolation of EPC fractions for therapeutic neovascularization.

Appraisal of hepatic lipase and lipoprotein lipase activities in mice.

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.

High density lipoprotein-binding proteins in porcine liver. Isolation and histological localization.

The antiatherogenic properties of high density lipoproteins (HDLs) are thought to reside in their involvement in the reverse cholesterol transport pathway. Specific HDL-binding proteins could play a key role in this process. Two HDL-binding proteins of approximately 90 and 180 kd were identified in porcine liver by ligand blotting and were purified to apparent homogeneity by a combination of protein extraction, DEAE-cellulose chromatography, Con A-Sepharose chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding of 125I-HDL by these proteins could be actively competed for by unlabeled HDL but not by low density lipoprotein. Polyclonal antisera have been raised against these two proteins. Each antiserum recognized only one of the HDL-binding proteins, indicating that they are not immunologically related. Moreover, striking differences in localization were observed in immunohistochemical studies. The 90-kd protein is located within the hepatocellular plates, while the 180-kd protein is present along the lining of the sinusoids. These results suggest functional differences between the two HDL-binding proteins described.

Structural relation between HDL-binding proteins in porcine liver

We have found strong evidence for a relation between three high-density lipoprotein (HDL)-binding proteins of 90, 110, and 180 kDa in porcine liver that were detected by ligand blotting. Because HDL-binding proteins with identical molecular masses were detected in human liver, all subsequent experiments were performed with porcine liver proteins. An antiserum raised against a highly purified preparation of the 90-kDa HDL-binding protein, designated 90-PC, showed cross-immunoreactivity with the 110- and 180-kDa HDL-binding proteins. Purified protein preparations of the 90-, 110-, and 180-kDa HDL-binding proteins were obtained and analyzed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. Under nonreducing conditions these preparations showed protein bands with the expected molecular masses. Reduction of these preparations resulted in protein bands of 90 kDa. Ligand blotting experiments with 125I-HDL showed protein bands of 90, 110, and 180 kDa under nonreducing conditions and a 90-kDa protein band in all three preparations under reducing conditions. Immunoblotting experiments with 90-PC antiserum resulted in a similar pattern. The three protein preparations were then subjected to cyanogen bromide cleavage and the resulting peptides separated on gel. Immunoblotting with the 90-PC antibody revealed a pattern of protein bands that was remarkably similar in all three protein preparations. Immunohistochemical localization studies with the 90-PC antibody showed that the HDL-binding proteins were present both at the borders of the sinusoids as well as within the hepatocellular plates.(ABSTRACT TRUNCATED AT 250 WORDS)

Ex Vivo and In Vivo Regulation of Arginase in Response to Wall Shear Stress

Background: Alterations of wall shear stress can predispose the endothelium to the development of atherosclerotic plaques. We evaluated the modulation of arginase by wall shear stress. Material and methods: We perfused isolated carotid arterial segments to either unidirectional high mean shear stress (HSS) or low mean and oscillating shear stress (OSS) for 3 days. Vascular function was analyzed by diameter measurement, arginase expression and localization by western blot and immunohistochemistry, respectively. These effects were also evaluated in right carotid artery of apolipoprotein E (apoE-/-) deficient mice, fed with high cholesterol diet, which was exposed to HSS, LSS and OSS flow conditions by the placement of a shear stress modifier for 9 weeks. ApoE-/- mice received either the arginase inhibitor nor-Noha (20mg/kg, 5 days/week) or placebo for 9 weeks. Plaque size and I/M ratio were determined by histology. Results: Our data from ex vivo perfusion showed that exposure of carotid segments to both low and oscillatory flow conditions significantly increase arginase II protein expression and activity as compared to high shear stress athero-protective flow condition. Long-term treatment with nor-Noha effectively decreased arginase activity at LSS and OSS regions, which in turn was accompanied by a decreased I/M ratio and the size of atherosclerotic lesion. In the lesion, inhibition of arginase decreased the number of CD68 positive cells at LSS and OSS zones. Exposure of carotid artery to OSS induced a more pronounced activation of arginase as compared to HSS. Conclusions: Arginase is modulated by patterns of wall shear stress. Long-term treatment of apoE- /- mice with arginase inhibitor decreased carotid I/M ratio and atherosclerotic lesion at LSS and OSS regions. Therefore, inhibition of arginase by nor-Noha may emerge as a distinct way to target atherosclerosis disease.

Shear stress and the IVUS derived vessel wall thickness

The role of shear stress in atherosclerosis has been well documented in vitro. In this chapter a novel technique and several applications will be described. The technique consists of a combination of 3D imaging of blood vessels and Computational Fluid Dynamics. The 3D imaging techniques consist either of 3D-IVUS or a combination of 3D IVUS and Angiography (‘ANGUS’). In the applications of these techniques it will be demonstrated that shear stress pays a role in the prediction of plaque location in in-stent restenosis, in vascular remodeling after balloon angioplasty, and in plaque location in primary atherosclerosis. Attempts to locally increase shear stress by a newly developed flow divider indicate that shear stress accounts for 50% of restenosis.