Background-Atherosclerotic plaque destabilization triggers clinical cardiovascular disease and thus represents an attractive therapeutic target. Weakening of tissue through the action of matrix-degrading enzymes, called matrix metalloproteinases (MMPs), released by resident macrophages was previously implicated in unstable vascular syndromes. Methods and Results-We used a hypercholesterolemic rabbit model of atherosclerosis to investigate the gelatinolytic activity associated with macrophage-derived foam cells (FCs). Gelatinolytic activity and expression of MMP-9 but not of MMP-2 cosegregated with macrophage FCs in aortic lesions. Macrophage-derived gelatinases were further investigated in vitro. MMP-9 was identified as the main macrophage-derived gelatinase in cells isolated from aortic lesions and from granuloma induced in the same rabbits to increase cell yield. Importantly, detection of activated MMP-9 in the FC culture medium supports the notion that these cells can independently initiate processing of secreted MMP zymogens to active enzymes. We further examined whether FC gelatinolytic activity is dependent on the presence of reactive oxygen species (ROS). We found that treatment (1 to 5 days) with 1 to 10 mmol/L N-acetyl-L-cysteine (NAC), an ROS scavenger, decreased not only gelatinolytic activity but also gelatinase expression by FCs. Similarly, NAC treatment of explanted lesions abolished in situ gelatinolytic activity and MMP-9 expression. Conclusions-Macrophage FCs are an abundant source of gelatinolytic activity that can be inhibited in vitro and in situ by NAC. This newly described action of antioxidant therapy might prove useful to inhibit matrix degradation and to improve vascular stability. (Circulation. 1998;97:2445-2453.)

Arteries remodel in response to environmental changes. We investigated whether mechanical strain modulates production of matrix metalloproteinase (MMP)-2 and -9 by cultured vascular smooth muscle cells (SMC). MMP-2 and MMP-9 expression were tested using human saphenous vein SMC cultured on silicone membranes at rest or subjected to physiological levels (5%) of stationary or cyclical (1 Hz) uniaxial strain. Compared with control, stationary strain significantly increased MMP-2 mRNA levels at all time points, whereas cyclic strain decreased it after 48 h. Both secreted and cell-associated pro-MMP-2 levels were increased by stationary strain at all times (P < 0.01), whereas cyclic strain decreased secreted levels after 48 h (P < 0.02). MMP-9 mRNA levels and pro-MMP-9 protein were increased after 48 h of stationary stretch (P < 0.01) compared with both no strain and cyclic strain. Our study indicates that vascular SMC show a selective response to different types of strain. We suggest that local increases in stationary mechanical strain resulting from stenting, hypertension, or atherosclerosis may lead to enhanced matrix degradation by SMC.

S U M M A R YWe examined the distribution of cell adhesion-related molecules (CAMs) among mouse embryonic stem (ES) cells and the spatial distribution on cell surfaces before and during differentiation. The cell-cell heterogeneity of SSEA-1, PECAM-1, and ICAM-1 among the undifferentiated cells in the ES cell colonies was evident by immunohistochemistry and immuno-SEM, supporting the flow cytometry findings. In contrast, most undifferentiated ES cells strongly expressed CD9. SSEA-1 was located preferentially on the edge of low protuberances and microvilli and formed clusters or linear arrays of 3-20 particles. PECAM-1 and ICAM-1 were randomly localized on the free cell surfaces, whereas CD9 was preferentially localized on the microvilli or protuberances, especially in the cell periphery. Both the SSEA-1 ϩ fraction and the SSEA-1 Ϫ fraction of magnetic cell sorting (MACS) formed undifferentiated colonies after plating. Flow cytometry showed that these populations reverted separately again to a culture with a mixed phenotype. Differentiation induced by retinoic acid downregulated the expression of all CAMs. Immuno-SEM showed decreases of SSEA-1 in the differentiated ES cells, although some clustering still remained. Our findings help to elucidate the significance of these molecules in ES cell maintenance and differentiation and suggest that cell surface antigens may be useful for defining the phenotype of undifferentiated and differentiated ES cells.

Pluripotent embryonic stem (ES) cells can be a source of hepatocytes for bioartificial livers or transplantation. In this study, embryoid bodies (EBs) were formed from ES cells cultured in polypropylene conical tubes. The EBs were then inserted into a collagen scaffold three-dimensional culture system and stimulated with exogenous growth factors and hormones to induce hepatic histogenesis. The EB-derived cells expressed liver-specific genes, and albumin-positive cells formed cord-like structures that were not present in two-dimensional monolayer culture systems. However, these albumin- positive cells were not cytokeratin 18 positive. Electron microscopy showed immature hepatocyte- like cells having tight junctions, rough endoplasmic reticulum, and intercellular canaliculi. The scaffold including EB-derived hepatocyte-like cells was transplanted into the median lobes of partially hepatectomized nude mice. After 7 and 14 days, cells positive for both albumin and cytokeratin 18 appeared in the transplant and formed clustered aggregates. Thus the collagen scaffold three-dimensional culture system and the liver regeneration environment induced hepatocyte-like cells and hepatic lobule-like aggregates from EBs. Therefore, differentiating EBs in the scaffold culture system may be useful in developing bioartificial livers, secondary livers, and as pharmaceutical models.

Titanium oxide nanotubes with Ca ions on their surfaces were prepared as 2 mm cylindrical inserts and placed into surgically created bone defects in the femurs of Wistar rats. On day 3, fibroblast-like cells were present on the surface of the nanotube inserts and fibers were observed by scanning electron microscopy (SEM). On day 7, cells with alkaline phosphatase activity were present and identified as osteoblasts by SEM and transmission electron microscopy. New bone matrices were observed in and around the porous nanotube inserts by light microscopy. Compared with clinically used hydroxyapatite and tricalcium phosphate, beta-titanium oxide nanotubes promote faster acquisition and development of osteoblasts and bone tissues and have better bone regenerating ability after one week.

To evaluate the immunological functions of the greater omentum in the peritoneal cavity, the localization of cell adhesion molecules (CAMs) on mesothelial cells and leukocytes in the omental milky spots were studied in normal and lipopolysaccharide (LPS)-stimulated mice by means of immunoelectron microscopy. The milky spots featured numerous leukocytes among the dome-shaped mesothelial cells, even in the normal stable state. Leukocyte integrins LFA-1, Mac-1, and VLA-4 were preferentially localized to microvilli and ruffles of macrophages and lymphocytes. The mesothelial cells of the milky spots showed higher ICAM-1 levels than did those of other omental regions, and fibronectin was detected in the stomata. The number of leukocytes markedly increased following an increase in proliferating cell nuclear antigen (PCNA)-positive cells in the milky spots after LPS stimulation. The mesothelial cells contained VCAM-1 newly restricted to the microvilli and increasing amounts of ICAM-1. These results show that the omental milky spots are active sites for leukocyte migration and peritoneal leukocyte supply because of the presence of adhesion molecules and active cell proliferation. Proliferative active leukocytes and those that have migrated from vessels pass through the stomata via an interaction of VLA-4 and fibronectin, adhere to the microvilli of the activated mesothelial cell surface as the result of an interaction between ICAM-1/VCAM-1 and integrins, and exude into the peritoneal cavity. Much of the exudation and adhesion of leukocytes seen in the milky spots of LPS-stimulated mice may be attributable to an increase in cell proliferation and in the amounts of ICAM-1 and VCAM-1.

Ureteric bud epithelial cells and metanephric mesenchymal cells that comprise the metanephric kidney primordium are capable of producing nephrons and collecting ducts through reciprocal inductive interaction. Once these cells are induced from pluripotent embryonic stem (ES) cells, they have the potential to become powerful tools in the regeneration of kidney tissues. In this study, we investigated these renal primordial cells and structures in mouse ES cell outgrowths and their transplants. Gene expression essential for early kidney development was examined by RT-PCR in embryoid body (EB) outgrowths and their transplants in adult mice. Histochemical detection of kidney primordial structures and gene expression analysis coupled with laser microdissection were performed in transplant tissues. RT-PCR analysis detected gene expression of Pax-2, Lim-1, c-Ret, Emx2, Sall1, WT-1, Eya-1, GDNF, and Wnt-4 in the EB outgrowths from days 6-9 of expansion onward, and also in the teratoma tissues 14 and 28 days after transplantation. Histochemical analysis 14 days after transplantation showed that some ducts were positive for Pax-2, endo A cytokeratin, kidney-specific cadherin, and Dolichos biflorus agglutinin and that dichotomous branching of these ducts had occurred. These staining patterns and morphological features are intrinsic for mesonephric ducts and ureteric buds. In long-term survival of 28 days, Pax-2-immunoreactivity disappeared in some renal primordia-like structures, indicating their differentiation. Some ducts were accompanied by mesonephric nephron-like convoluted tubules. RT-PCR analysis of those structures collected by microdissection confirmed that they expressed kidney development-related genes. In conclusion, these data suggest the potential of ES cells to produce renal primordial duct structures and provides an insight into the regeneration of kidney tissues.

The morphology of human embryonic stem (ES) cells changes with their colonial growth. For a better understanding of the growth of ES cell colonies in culture, we determined their cytochemical and ultrastructural characteristics focusing on images of living cells under a phase contrast microscope. During the initial growth stages, the colonies exhibited a mosaic appearance with discernible cell-cell borders. PAS staining coupled with amylase digestion demonstrated that the bright granules and dark deposits in the cytoplasm contained glycogen. Ultrastructurally they were glycogen accumulations, and clustered open spaces associated with various amounts of glycogen. Although intercellularly heterogeneous, these structures were detectable throughout colony growth. As the colonies grew, compaction towards the centre emerged and increased, accompanied by heterogeneous increases in coarse particles with or without a halo. TUNEL showed these particles to consist at least in part of apoptotic cells/bodies. Transmission electron microscopy indicated that most apoptotic cells had been phagocytosed by intact ES cells.Spontaneous differentiation was detected occasionally in the periphery of the colonies. The presence of PASpositive fibrous structures not susceptible to amylase digestion and laminin-immunoreactivity indicated the accumulation of extracellular matrix in the peripheral differentiated areas. These findings made it possible to determine the growth stage of human ES cell colonies.