Angiotensin-converting enzyme activity by canine pulmonary microvascular and central pulmonary artery endothelial cells exposed to hypoxia.

To compare the amount of angiotensin-converting enzyme (ACE) activity in pulmonary artery endothelial cells from different sites and to examine the effect of severe hypoxia (less than 1% of O(2) in 5% CO(2) and 95% N(2)) on the ACE activity expressed by these cells, endothelial cells were harvested and cultured from canine main pulmonary artery by scraping the luminal surface of the artery and from canine pulmonary artery microvessels by infusing chilled buffer with microcarrier beads and 0.02% ethylenediamine tetraacetic acid (EDTA). ACE activity in cell lysates and culture medium was evaluated by fluorometric assay with hippuryl-L-histidyl-L-leucine as a substrate. ACE activity in cell lysates and postculture medium of pulmonary microvascular endothelial cells (PMVEC) was higher than in cell lysates and culture medium of central pulmonary artery endothelial cells (PAEC). However, hypoxia suppressed cellular ACE activity in both PAEC and PMVEC. The degree of suppression of ACE activity by hypoxia, which was determined as (ACE activity in normoxia - ACE activity in hypoxia)/ACE activity in normoxia x 100(%), was larger in PMVEC than in PAEC. The pulmonary microvasculature may be a greater source of ACE than central pulmonary artery, and the ACE activity of pulmonary microvascular endothelial cells seem to be sensitive to hypoxia, although the small diameter of the vessels improves conditions for interaction of blood-borne substance with endothelial enzymes.

Atrial myocytes cultured from explanted human adult myocardium demonstrate DNA replication.

The current theory of myocardial development holds that after a limited number of divisions, the myocardiocytes of the developing heart are irreversibly withdrawn from the generation cycle. It is, therefore, considered impossible to grow adult human myocardiocytes in culture, making it necessary for studies of cardiac muscle in culture to be carried out using animal or fetal human models. Recently, we developed a method for isolating, culturing, and characterizing myocardiocytes derived from explanted adult human atrial myocardium. A highly pure fraction (93%) of one of four morphologically discrete cell populations was separated using selective attachment techniques. These cells possessed features consistent with those seen in animal and fetal myocardiocytes. Using immunoperoxidase stains, these cells stained positive for actin, myoglobin, and atrial natriuretic peptide, proving the cells are myocardial muscle cells. Electron microscopy showed numerous bundles of myofibrils with interspersed dense Z-bodies and pleomorphic mitochondria. Bromo-deoxyuridine incorporation confirmed that the cells were replicating their DNA. Thus, cell morphology, immunoperoxidase stains, electron microscopy, and cell proliferation testing showed these cells to be myocardiocytes undergoing DNA replication and mitosis. We must now reconsider our current thinking about myocardial development and investigate what factors contribute to the inhibition of myocardial cell proliferation after injury in vivo.

Effect of papaverine on endothelial cell harvest from canine external jugular veins.

Endothelial cell seeding procedures have been developed to line prosthetic bypass grafts used in peripheral vascular disease; however, because of current inefficient cell harvest techniques a high ratio of vein-to-graft area is necessary. This study was done to determine if the use of papaverine, a smooth muscle cell relaxant, would affect the number or viability of endothelial cells harvested from canine external jugular veins. Using a 0.12 mg/ml solution of papaverine in tissue culture medium to bathe the veins during dissection and excision, the viable cell yield was 2.20 +/- 1.16 (cells x 10(4)/cm2). A control group of veins using standard dissection technique gave a yield of 0.97 +/- 0.40 (p = 0.025). A second group of veins dissected while bathed in tissue culture medium alone gave a yield of 1.82 +/- 0.75, compared to a yield of 2.73 +/- 0.45 for papaverine harvested veins (p = 0.009). Percent cell viability was not significantly different for any of the groups: 73, 70, and 76% for papaverine, control and media only veins, respectively. The papaverine-harvested cells and those harvested with medium alone grew to 95% confluence in tissue culture in 9.8 +/- 1.1 and 9.9 +/- 0.9 days, respectively. Compared to conventional surgical techniques, use of papaverine more than doubled the endothelial cell yield from excised vein segments without adversely affecting viability or rate of growth in cell culture.