Biomedical subjects
V M Miller
Publications and source records attributed to V M Miller.
Evaluation of a xenogeneic acellular collagen matrix as a small-diameter vascular graft in dogs--preliminary observations.
Autogenous veins are the materials of choice for arterial reconstruction. In the absence of autogenous material, prosthetic materials are used. However, vascular prostheses of less than 0.4 cm in diameter have low long-term patency. This study was designed to determine if cells would infiltrate an engineered xenogeneic biomaterial used as a small diameter arterial graft in dogs and, if so, to determine the phenotype of the infiltrating cells. Nine acellular xenogeneic grafts (0.4 cm in diameter, 5 cm long), composed of porcine collagen derived from the submucosa of the small intestine and type I bovine collagen, were implanted as end to-end interposition grafts in femoral arteries of five male mongrel dogs (total of nine grafts). All dogs received daily aspirin (325 mg). Patency of implanted grafts was monitored weekly by Duplex ultrasonography. After 9 weeks, or earlier in case of blood flow reduction by at least 75%, grafts were explanted and prepared for light or electron microscopy to evaluate cellularization. Eight of nine grafts remained patent up to 9 weeks. At explant, diameters were 0.31 +/- 0.02 cm at the midgraft, and 0.14 +/- 0.01 and 0.19 +/- 0.01 cm at the proximal and distal anastomoses. At explant, cells of mesenchymal origin (endothelial cells, smooth muscle cells, myofibroblasts) were embedded in the extracellular matrix of the graft scaffold. Minimal evidence of cellular inflammatory reaction and no aneurysmal dilatation or thrombus formation was detected. Variable degrees of hyperplasia were present at proximal and distal anastomoses. This preliminary study demonstrates that a collagen-based xenogeneic biomaterial provides a scaffold for cellularization when used for arterial reconstruction in dogs.
Early remodeling of saphenous vein grafts: proliferation, migration and apoptosis of adventitial and medial cells occur simultaneously with changes in graft diameter and blood flow.
BACKGROUND: This study was designed to determine how migration and proliferation of adventitial and medial cells correlate temporally with hemodynamic characteristics including changes in diameter and blood flow in vein grafts. METHODS: Male mongrel dogs underwent end-to-side reversed saphenous vein bypass grafting across a ligated femoral artery. Hemodynamic parameters were assessed by duplex ultrasound. Proliferating cells were labeled 24-48 h after grafting with 5-bromo-2'-deoxyuridine (BrdU). Grafts were removed on postoperative days 2, 5, 7 and 14. Immunohistochemistry was performed on graft sections with antibodies to nuclear antigen Ki-67 (MIB-1), BrdU and smooth muscle cell markers. Apoptosis was identified by modified TUNEL staining. Proliferating/apoptotic cells were quantified digitally. RESULTS: Mean luminal cross-sectional area, blood flow and velocity increased from day 2 to 7. Cell proliferation was evident in adventitia and media on day 2, maximum on day 5 and decreased significantly by day 14. Apoptosis was maximum on day 5. Early proliferating cells (BrdU labeled) localized in the adventitia and media on day 2 were more prevalent in the neointima on days 5-14 suggesting inward migration. On day 2, proliferating cells stained positive only for vimentin. By days 5-14, proliferating cells stained for alpha-smooth-muscle actin, a phenotype characteristic of myofibroblasts. CONCLUSION: These results indicate that cell proliferation and apoptosis occur simultaneously within the adventitia and media of the vein during the first week following grafting, when changes in diameter and blood flow are greatest. In addition, proliferating adventitial cells subsequently migrate inwards to contribute to the formation of neointima.
Cardioplegia alters porcine coronary endothelial cell growth and responses to aggregating platelets.
Experiments were designed to determine the viability of endothelial cells and their responses to products released by aggregating platelets following single flush perfusion of the coronary arteries with cardioplegic solutions used for cardiac transplantation. Porcine coronary arteries were perfused with crystalloid (Plegisol) or blood cardioplegic solutions; nonperfused hearts placed in 0.9% saline were used as controls. Immediately following perfusion and after 5-hour storage of the hearts in the same cardioplegic solution, rings were cut from the right coronary arteries and suspended in organ chambers for the measurement of isometric force. In some rings the endothelium was removed deliberately. The left circumflex coronary arteries were flushed with collagenase and the harvested endothelial cells were plated for cell culture. Left anterior descending coronary arteries were perfusion fixed with glutaraldehyde for scanning electron microscopy. In the organ chamber experiments, aggregating platelets and adenosine diphosphate caused relaxations in rings with endothelium. These relaxations were reduced immediately following crystalloid cardioplegia and were restored following 5-hour storage. Serotonin caused contractions in all rings. Rings without endothelium were more sensitive than rings with endothelium to the amine; this difference was augmented following 5-hour storage of the heart. Significantly fewer foci of endothelial cells grew in culture following 5-hour storage of the hearts in crystalloid cardioplegic solution compared to control (p < 0.05). There were no anatomical differences identified among groups by scanning electron microscopy. These results suggest that crystalloid cardioplegia alters the responses of coronary arteries to substances released by aggregating platelets and reduces the ability of endothelial cells to replicate. Such changes may contribute to altered vascular resistance following reperfusion of transplanted hearts and potentially to later structural changes in the coronary arteries.
Kininogenase activity of prostate-derived human glandular kallikrein (hK2) purified from seminal fluid.
Prostate-specific human glandular kallikrein (hK2) is an active enzyme in human seminal fluid. It is one of three serine proteases in the human kallikrein gene family, which includes hK1 (tissue kallikrein) and hK3 (prostate-specific antigen [PSA]). In order to examine kininogenase activity (i.e., production of kinin by these enzymes), we tested for bradykinin and/or Lys-bradykinin release upon incubation of hK2 and for other kallikreins with high-molecular weight kininogen (HMWK), which contains the nonapeptide bradykinin. Kinins are important regulatory peptides (especially for vascular permeability), and they may have a role in enhancing sperm motility. High-molecular weight kininogen is the substrate for plasma kallikrein (PKa potent kinin-generating enzyme circulating in blood, not of the same gene family) and for hK1. Glandular kallikrein and protein-C inhibitor (PCI)-hK2 complex, a serpin protease inhibitor that binds hK2, were purified to homogeneity by affinity and size-exclusion chromatography. About one-half of the hK2 is found in complex with PCI. The kallikrein enzymes were incubated with HMWK, and the resulting cleavage products were analyzed for kinin activity using enzyme immunoassay, high-performance liquid chromatography and mass spectrometry, and in vitro bioassay. Our results show that hK2 cleaves HMWK to produce bradykinin, not Lys-bradykinin (like hK1), and the resultant heavy (56-kDa) and light (42-kDa) chains of HMWK show similar electrophoretic mobility to those cleaved by PK. Prostate-specific antigen (hK3) had no kinin-generating activity. We also identified three other internal cleavage sites for hK2 in HMWK (Arg427, Arg437, and Arg457) that yielded two peptides, one of which is identical to a PK-cleaved peptide. Glandular kallikrein is about 500-fold less active than is PK or tissue kallikrein, but it may play a physiologically important role in bradykinin release in seminal fluid.
Functional changes in canine saphenous veins after cryopreservation.
This study was designed to determine the functional characteristics of the endothelium, smooth muscle and nerve terminals of cryopreserved veins. Freshly harvested and cryopreserved canine saphenous veins were cut into rings. In some rings, the endothelium was removed. Cryopreserved veins were stored at -196 degrees C for at least 3 weeks prior to use. All rings contracted in a concentration-dependent manner to depolarization with KCl and to alpha-adrenergic stimulation; the maximal tensions were significantly less in cryopreserved than in freshly harvested veins. Calcium ionophore A23187 caused greater relaxations in rings with than without endothelium in freshly harvested and cryopreserved veins. These relaxations were reduced significantly by methylene blue and NG-monomethyl-L-arginine (L-NMMA) only in fresh veins. Cocaine-sensitive uptake of H3-norepinephrine was reduced following cryopreservation. Immediately after cryopreservation, the production of prostacyclin was elevated. The calcium ionophore A23187 stimulated production of prostacyclin only in freshly harvested veins. Tissue content of endothelin did not change following cryopreservation. These results suggest that cryopreservation of canine saphenous veins alters nerve terminals and decreases the ability of the smooth muscle to contract. The endothelium releases an endothelium-derived relaxing factor and prostanoids following cryopreservation but the ability to synthesize nitric oxide is probably reduced. These changes following cryopreservation may affect patency of the veins when used as arterial grafts.
Modified sapheno-arterial anastomosis.
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Activation of polymorphonuclear leukocyte oxygen radical production during acute lung rejection in dogs: inhibition by an antiadhesion molecule monoclonal antibody.
Monoclonal antibodies against leukocyte adhesion molecules reduce the severity of reperfusion injury and, in some cases, prevent acute allograft rejection. These effects are assumed to be due to prevention of leukocyte-endothelial adhesion. Experiments were designed to study activation of polymorphonuclear leukocytes during acute rejection of canine single lung allografts and to test the ability of an anti-beta 2-integrin monoclonal antibody (R15.7/H4) to regulate leukocyte metabolism independent of leukocyte-endothelial adhesion. Peripheral blood polymorphonuclear leukocytes were obtained by Ficoll-Hypaque centrifugation from unoperated control dogs (n = 10), from dogs with a single lung autotransplant (n = 4), and from dogs after single lung allotransplantation during unmodified rejection (n = 12). Oxygen radicals were measured with luminal-enhanced chemiluminescence of unstimulated polymorphonuclear leukocytes, polymorphonuclear leukocytes stimulated by opsonized-zymosan, and polymorphonuclear leukocytes incubated with anti-beta 2-integrin beta-chain (CD18) monoclonal antibodies before exposure to opsonized zymosan. Polymorphonuclear leukocyte viability was unaffected by the monoclonal antibodies. Polymorphonuclear leukocytes from unoperated and autotransplant dogs did not produce oxygen radicals unless stimulated by opsonized zymosan. Polymorphonuclear leukocytes from rejecting animals produced oxygen radicals in the absence of zymosan, and when stimulated, production of oxygen radicals was significantly greater than that of polymorphonuclear leukocytes from unoperated and autotransplant dogs. Production of oxygen radicals was inhibited significantly by the monoclonal antibodies in polymorphonuclear leukocytes from all three groups. This study shows that rejection activates polymorphonuclear leukocytes and increases the potential to produce activated oxygen species. In addition to inhibiting leukocyte adhesion to endothelial cells, anti-beta 2-integrin monoclonal antibodies appears to suppress production of oxygen radicals by polymorphonuclear leukocytes.