Endothelial lined skeletal muscle ventricles: open and percutaneous seeding techniques.

Twelve bilateral skeletal muscle ventricles (SMVs) were constructed in six dogs by wrapping each latissimus dorsi muscle around a cylindrical, plastic mandrel (volume 30 cc). After 6 to 10 weeks, five dogs had one of their SMVs seeded with allogeneic cultured canine endothelial cells (8 x 10(6) cells/pouch) via an open technique, whil the contralateral SMV was seeded by percutaneous injection of cells into the space around the mandrel. After 1 week, the SMVs were excised. Viable, adherent endothelial cells were present in all seeded pouches; this was confirmed via fluorescent microscopy with several endothelial cell markers; KLH-2, dilacetylated low-density lipoprotein and antibodies to von Willebrand factor. The inner lining of the SMVs were also examined with scanning and transmission electron microscopy; the highest concentration of cells were seen at the apex where a continuous endothelial monolayer was observed. No significant difference in the distribution or the morphology of the endothelial lining was noted between the open and percutaneous seeding techniques. These data show that SMVs can be seeded with an endothelial monolayer using both open and percutaneous techniques.

Skeletal muscle ventricles seeded with autogenous endothelium.

Skeletal muscle ventricles (SMVs) are muscular pumping chambers constructed from skeletal muscle. Previously, SMVs were connected to the systemic circulation with vascular conduits and used to assist the heart. In this study, SMVs were constructed from the latissimus dorsi muscle in eight dogs. The SMVs were seeded with autologous endothelial cells, but not connected to the circulation. Endothelial cells were harvested enzymatically from autogenous external jugular vein and grown in tissue culture. After 9 weeks, 6 electrically conditioned SMVs were seeded with endothelial cells by injecting 4-5 ml of culture medium containing 5-8 x 10(6) autogenous endothelial cells into each SMV lumen adjacent to the mandrel. Conditioning was stopped at the time of endothelial seeding. One week after seeding, electrical conditioning was resumed. Two weeks after seeding, the animals were killed and the SMVs excised. Histologic examination confirmed the presence of a confluent monolayer of cells covering 80-100% of the luminal surface in each seeded SMV. The endothelial nature of the cells lining the SMV lumen was established by fluorescent microscopy. Endothelial cells were pre labeled with the cellular marker PKH before seeding; the SMVs were also incubated with the endothelial marker dil-acetylated LDL. Endothelial cells also were identified by staining with fluorescently labeled antibodies to von Willebrand factor. Based upon these data, electrically conditioned SMVs can be seeded successfully with a near-complete, autologous endothelial monolayer. Additionally, this endothelial monolayer can be maintained on the luminal surface of a contracting SMV. In-circulation studies will determine whether endothelial cell seeding of SMVs can decrease or eliminate the incidence of thromboembolism.

Microgravity decreases tyrosine hydroxylase expression in rat adrenals.

During spaceflight, alterations in blood and urinary catecholamine (CA) levels have been observed, yet the cellular/molecular mechanisms leading to these changes are not known. We used molecular, immunological, and biochemical approaches to analyze in situ the expression of catecholamine enzymes in adrenal medullary chromaffin cells of rats flown for 6 days on board Space Shuttle mission STS-54. Exposure to microgravity (10(-6) g) resulted in a 35% inhibition of both the expression and the specific activity of tyrosine hydroxylase (TH), the rate-limiting step in the cascade of CA synthesis. By contrast, the expression, specific activity, and immunoreactivity of other catecholamine-synthesizing enzymes, e.g., phenylethanolamine-N-methyl-transferase (PNMT), were not altered. The total tissue CA contents were reduced, concomitant with a decrease in the epinephrine:norepinephrine ratio. These results are in line with reports of other gravity-sensitive cellular effects and suggest that the inhibition of TH expression might be due to a direct effect of microgravity on PKC-dependent signal transduction pathways in chromaffin cells.

Morphology and integrity of endothelial cell monolayers inside a ventricle shaped perfusion chamber.

The long-term maintenance of patients with failing hearts on cardiac prostheses requires prevention of device related thromboembolic events. This challenge is being addressed by endothelialization of the blood sacs. However, the practice of establishing and maintaining a durable endothelial cell monolayer inside a beating prosthesis has not been fully realized. Thus, before exposing endothelial cell monolayers to the hemodynamics inside an artificial heart, the authors studied the effect of various flow patterns in a ventricle shaped chamber on the integrity and morphology of the endothelium. After 20 hours of superfusion by pulsatile flow, there were no denudation signs in the jet, where shear stress was 1.5 dynes/cm2. However, there was measurable damage to the monolayer close to the periphery of the eddies (turbulent flow) at 0.15 dynes/cm2. In either case, there were no signs of cell alignment with the flow, but there were changes in cell morphology compared with that of static control. These findings suggest that adjustment of endothelial cells in response to frictional forces occurs even at low shear stresses and that random velocity fluctuations might jeopardize the integrity of endothelial cell monolayers.

Experimental studies of pulsatile flow and endothelial cell adaptation in ventricle shaped cell culture chambers.

The authors' long-term research goal is to minimize the risk of thromboembolic complications in cardiac prostheses by lining blood contacting surfaces with a functional monolayer of autologous endothelial cells. These cells recognize changes in hemodynamics and can adapt effectively to experimentally manipulated flow conditions. By implication, the morphology of endothelial cells, in conjunction with their function, might serve as an indicator of the flow patterns in a particular location. It was hypothesized that, by understanding flow patterns at a given site, the local morphology and function of the endothelial cells in such a region could be predicted. To test this hypothesis, a series of ventricle shaped flow chambers were designed and perfused with pulsatile flow. The flow field in the chambers was studied by computer aided dye visualization and nuclear scintigraphy. The results showed that the large scale motion of the fluid in the cavity was highly coherent and consisted of distinct flow patterns. The temporal and spatial characteristics of the flow patterns, and their implications with respect to endothelial cell endurance in this in vitro environment, were examined in detail.