Blood flow velocity is reduced in a tumor micro-dissemination in the visceral pleura in anesthetized open-chest rat lung.

BACKGROUND: Recently we developed a method to observe pulmonary micrometastasis by labeling cancer cells with green fluorescent protein (GFP). We applied the method for observation of micro-dissemination on the visceral pleura. MATERIALS AND METHODS: RCN9 rat colon cancer cells labeled with GFP were injected into the pleural cavity of Fischer F344 rats. Six weeks after injection, the chest wall was resected under general anesthesia and the lung surface was observed by real-time confocal laser-scanning microscopy. Blood flow was visualized by intravenous injection of fluorescein isothiocyanate-labeled red blood cells, by which blood flow velocity was measured. RESULTS: Dissemination was created in 4 out of 5 rats. Fifteen sites of micro-dissemination were observed (mean diameter, 35.8+/-13.3 microm). Blood flow velocity was 114.1+/-26.1 microm/s in the tumor tissue and 183.4+/-35.0 microm/s out of the tumor tissue. CONCLUSION: We were able to observe pleural micro-dissemination. Blood flow velocity was significantly lower in the tumor tissue.

In vivo observation of pulmonary micrometastasis of colon cancer in normal rats.

The initial kinetics of cancer cell metastasis to organs requires investigation to establish an effective strategy against malignant disease. In vivo observation of pulmonary micrometastasis at an extremely early stage is of particular importance, and it is desirable from a clinical perspective to use an animal model with a normal immune system. RCN-9 cells labeled with green fluorescent protein were injected into the liver parenchyma of Fischer F344 male rats and the lungs were observed using real-time confocal laser scanning microscopy from 3 to 10 weeks after injection. Metastasis at the single cell level was observed throughout this period, but the number of pulmonary micrometastases did not increase significantly with time. The largest metastasis was 300 mum in diameter, and the mean size of the metastases did not increase with time. There were two types of micrometastases in terms of shape: round and linear metastases, with the latter resembling the pulmonary microvasculature. The precise location of each pulmonary micrometastasis was revealed by acridine orange infusion. We could observe a single cancer cell and a small cancer mass in endothelial and interstitial locations in vivo, and we found proliferating cancer cells both inside and outside of microvessels. Most of the pulmonary micrometastases stayed dormant as a single cell or a cancer mass of less than 100 microm in diameter until 10 weeks after cancer-cell injection into the liver.

Effects of oncostatin M on secretion of vascular endothelial growth factor and reconstruction of liver-like structure by fetal liver cells in monolayer and three-dimensional cultures.

Vascular endothelial growth factor (VEGF) is crucial for the development and regeneration of the liver. However, there have been no reports about VEGF secretion by cultured fetal liver cells (FLCs). In the present study, the effects of oncostatin M (OSM), which strongly stimulates the growth and albumin secretion of FLCs, on VEGF secretion and morphological changes of long-term cultured FLCs were investigated under three-dimensional (3-D) and monolayer conditions. The cultured FLCs proliferated well and showed stable secretion of VEGF for up to 1 month under both monolayer and 3-D culture conditions. The addition of OSM to cultured cells strongly enhanced VEGF secretion. Compared with 3-D cultures, VEGF secretion per cell was higher in monolayer cultures. After 1 month in culture, the FLCs in 3-D cultures formed large aggregates like liver tissue, and FLCs also formed colonies and duct-like structures after several months of culture even under monolayer conditions. In conclusion, OSM stimulated the secretion of VEGF by cultured FLCs, which seemed to contribute to the development of a liver-like structure.

Evaluation of polydimethylsiloxane scaffolds with physiologically-relevant elastic moduli: interplay of substrate mechanics and surface chemistry effects on vascular smooth muscle cell response.

Polydimethylsiloxane (PDMS) is used extensively to study cell-substrate interactions because its mechanical properties are easily tuned in physiologically relevant ranges. However, changes in mechanical properties also modulate surface chemistry and cell response. Here, we correlate the mechanical and surface properties of PDMS to vascular smooth muscle cell (VSMC) behavior. We find that a 5-fold increase in base:crosslinker ratio leads to approximately 40-fold decrease in elastic modulus but no significant differences in surface wettability. However, when polyelectrolyte multilayers are adsorbed to promote cell adhesion, wettability varies inversely with substrate stiffness. Despite these differences in hydrophobicity, the amount of adsorbed protein remains the same. In the absence of serum, there is a 39% decrease in cell attachment and a 42% decrease in spreading as the elastic modulus decreases from 1.79 to 0.05 MPa. In the presence of serum or adsorbed fibronectin, the differences in attachment and spreading are diminished. This is not the case for the rate of serum-stimulated cell proliferation, which remains inversely dependent on crosslinker concentration. We conclude that for the range of crosslinker concentrations investigated, the surface properties dominate the initial cell attachment and spreading, whereas the mechanical properties influence the long-term cell growth.

Endovascular stent configuration affects intraluminal flow dynamics and in vitro endothelialization.

Neointimal hyperplasia influenced by intravascular hemodynamics is considered partly responsible for restenosis after endovascular stenting. To evaluate the effect of stent configuration on fluid flow behavior, we visualized flow near stents, and measured the proliferation of cultured endothelial cells (ECs). A single-coil stent (coil pitch; CP = 2.5, 5, or 10 mm) was inserted into a glass tube and perfused at 30-90 ml/min, while the flow pattern was determined by particle imaging velocimetry. The reduction of the flow velocity near the wall was correlated with the decrease in the coil interval of the stent. In perfusion cultures with stents, the proliferation of ECs was influenced by the local flow velocity distribution. When a stent with a CP value of 10 mm was used, the doubling time of ECs was 30.7 h, while the doubling time was 38.5 h when the CP was 5 mm. The doubling time of ECs was shorter at sites upstream of the stent wire where the velocity was higher than downstream of the wire. In conclusion, a single-coil stent can be used to modify hemodynamic factors, suggesting that improved stent design may facilitate rapid endothelialization after stent implantation.

Especially polymorphonuclear leukocytes, but also monomorphonuclear leukocytes, roll spontaneously in venules of intact rat skin: involvement of E-selectin.

White blood cells roll spontaneously in venules of intact, noninflamed rat skin. We investigated noninvasively in two experimental series which leukocyte subtypes participate in this phenomenon and the possible involvement of E-selectin. Male Lewis rats were anesthetized with sodium pentobarbital, and intravital video microscopy was performed on postcapillary venules in the nail-fold of a hind leg. In series 1 acridine yellow was infused for 15 min (50 mg per kg intravenously) to stain the leukocyte nuclei in situ. With the use of fluorescence microscopy rolling leukocytes could be classified unequivocally as polymorphonuclear (granulocytes) or monomorphonuclear (lymphocytes/monocytes) by the shape of their nucleus. Irrespective of vessel depth beneath the skin surface (25-45 microm), most identified rolling leukocytes were classified as granulocytes (72%-100%; median 89%). This percentage was independent of total rolling leukocyte flux, systemic leukocyte count, or their in vitro differentiation pattern. In series 2, rats were treated with either a synthetic, highly selective E-selectin blocking peptide or a control peptide (intravenously, 12 mg peptide per kg bolus, followed by 50 mg per kg per h). E-selectin blockade significantly reduced the leukocyte rolling level to about 50% of baseline (p <0.01), whereas the rolling velocity increased (p <0.01); the control peptide had no effect. In summary, most of the leukocytes rolling spontaneously in postcapillary venules of intact rat skin are granulocytes, despite the absence of an acute inflammatory reaction. One of the adhesion molecules involved in this phenomenon is E-selectin.