Kouichi Morioka

Transmyocardial laser revascularization induced angiogenesis correlated with the expression of matrix metalloproteinases and platelet-derived endothelial cell growth factor

OBJECTIVE Transmyocardial laser revascularization (TMLR) has been widely evaluated as a treatment for ischemic myocardium. However, its mechanism remains unclear. One mechanism is angiogenesis. This study examines the relationship between TMLR and angiogenesis from the viewpoint of matrix metalloproteinases and platelet-derived endothelial cell growth factor. METHODS The left anterior descending coronary artery (LAD) was ligated permanently in 12 beagle dogs. TMLR was accomplished in six of the 12 dogs using a carbon dioxide laser. No laser treatment was done in the six control dogs. Two weeks after the initial operation, dogs were euthanized and transmural samples (each of approximately 0.5 g) were cut from the center of the infarcted LAD territory, right ventricular wall, left circumflex artery perfuse area and interventricular septum except the LAD perfuse area. They were snap-frozen in liquid nitrogen for matrix metalloproteinases and platelet-derived endothelial cell growth factor activity analysis. Hematoxylin and eosin staining, double immunohistologic staining with anti-proliferating cell nuclear antigen and von Willebrand factor antibody, and immunohistologic staining with antibody against platelet-derived endothelial cell growth factor were performed for histologic studies. The activities of matrix metalloproteinases were examined by gelatin zymography. The activity of platelet-derived endothelial cell growth factor was examined by a spectrophotometric method. RESULTS The channels were found to be infiltrated with granulation tissue and fibrosis. In the laser group, the active matrix metalloproteinase-2 and platelet-derived endothelial cell growth factor activity in the area of the left anterior descending coronary artery was significantly higher than in the control group (P<0.0001 and P=0.037, respectively). Within the channel remnants or close to these areas, the number of von Willebrand factor positive microvessels and proliferating cell nuclear antigen with correlating von Willebrand factor positive microvessels were significantly higher than in the control group (P=0.001 and P=0.0006, respectively). These increases in microvessels significantly correlated with the expression of matrix metalloproteinases and platelet-derived endothelial cell growth factor. CONCLUSION Based on these findings it was concluded that transmyocardial laser revascularization induced angiogenesis correlated with the expression of active matrix metalloproteinases-2 and platelet-derived endothelial cell growth factor.

Effects of Depletion of Leukocytes and Platelets on Cardiac Dysfunction After Cardiopulmonary Bypass

BACKGROUND This study examined the effects of the depletion of leukocytes and platelets from circulated blood on cardiac function after cardiopulmonary bypass in 37 patients who underwent coronary artery bypass grafting or aortic valve replacement. METHODS Leukocytes and platelets were removed continuously using a blood cell separator, beginning immediately after the start of the operation and ending 1 hour after the release of the aortic cross-clamp in 19 patients (LPD group), but not in the remaining 18 patients (control group). Blood cell counts and levels of thromboxane B2, 6-keto-prostaglandin F1alpha, leukocyte elastase, complements C3a and C4a, thrombin-antithrombin III complex, and D-dimer were determined periodically during and after the operation. The cardiac index, the difference between the central and peripheral core temperatures, and the doses of catecholamines and vasodilators required to support the circulation in the early postoperative period also were assessed. RESULTS Leukocyte and platelet counts and levels of leukocyte elastase, thromboxane B2, thromboxane2/6-ketoprostaglandin F1alpha, thrombin-antithrombin III complex, and D-dimer were significantly lower in the LPD group than in the control group before and after the release of the aortic cross-clamp and during the perioperative period. There were no significant differences in the levels of 6-keto-prostaglandin F1alpha or complements C3a and C4a between the two groups. The catecholamine dose was significantly lower in the LPD group than in the control group (1.1 +/- 2.5 versus 5.0 +/- 5.2 mg/kg, respectively). Fewer patients required the use of nitroprusside as a vasodilator in the LPD group than in the control group (1/19 versus 12/18, respectively). CONCLUSIONS The depletion of leukocytes and platelets using a blood cell separator prevents the deterioration of cardiac function after cardiac operations using cardiopulmonary bypass.

Adventitial delivery of platelet-derived endothelial cell growth factor gene prevented intimal hyperplasia of vein graft

BACKGROUND Platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP) reportedly inhibits vascular smooth muscle cells (VSMCs) migration and proliferation. We hypothesized that adventitial administration of the PD-ECGF/TP gene will suppress intimal hyperplasia and prevent vein graft failure. METHODS The study used 68 female rabbits. Rabbit jugular vein was autogenously transplanted into carotid artery with a cuff anastomotic technique. To define vascular wall gene transfer efficiency, poloxamer hydrogel (20%) containing plasmid vector encoding the LacZ gene and different concentrations of trypsin (0%, 0.1%, 0.25%, and 0.5%, n = 5 for each group) was applied to the adventitia of the vein graft. Gene transfer efficiency was evaluated 7 days later by X-gal staining. An additional 48 rabbits received poloxamer hydrogel (20%) containing 0.25% trypsin and the human PD-ECGF/TP gene, LacZ gene, or saline. Intima thickness was evaluated at 2 and 8 weeks after grafting (n = 8 for each group at each time point). Transgene expression was examined by reverse transcriptase-polymerase chain reaction, immunoblotting assay, and immunohistochemical staining. Immunohistochemical staining was also used to determine VSMC proliferation, heme oxygenase-1 expression, and macrophage infiltration. RESULTS Incorporation of trypsin into the poloxamer hydrogel significantly increased vessel wall gene transfer. Trypsin at 0.25% and 0.5% resulted in higher gene transfer at the same level without effecting intimal hyperplasia and inflammation; thus, trypsin at 0.25% concentration was used for subsequent experiments. Compared with the LacZ and saline groups, grafts receiving the PD-ECGF/TP gene significantly reduced intimal thickness at 2 and 8 weeks after treatment. The ratio of proliferative VSMC was lower in PD-ECGF/TP treated grafts. Histologic examination of the PD-ECGF/TP transgene grafts demonstrated high expression of heme oxygenase-1, which has been reported to inhibit VSMC proliferation, suggesting that heme oxygenase-1 may be important in the inhibition effect of PD-ECGF/TP on VSMC. No neoplastic or morphologic changes were found in the remote organs. CONCLUSIONS A safe and highly efficient gene transfer method was developed by using poloxamer hydrogel and a low concentration of trypsin. Neointimal hyperplasia was significantly reduced by adventitial application of the PD-ECGF/TP gene to the vein graft. Our data suggest that adventitial delivery of the PD-ECGF/TP gene after grafting may be promising method for preventing vein graft failure.

Postoperative inflammatory reactions of impregnated Dacron grafts.

The clinical efficacy of Dacron grafts that are impregnated with collagen or gelatin has been well demonstrated, but inflammatory reactions during the first few postoperative weeks have been reported. We experienced two patients, an 87-year-old man with a reconstruction of an abdominal aorta and a 7-year-old boy with a reconstruction of aortic arch with a collagen-impregnated graft (Hemashield), who continued to demonstrate a high fever with a high serum level of C-reactive protein (CRP) and immunogloblin for more than 5 months. The body temperature, the white blood cell (WBC) counts, and the serum level of CRP were compared on the seventh and 14th postoperative day among the 37 patients who underwent a reconstruction of either a thoracic or abdominal aorta using the Hemashield graft (Hemashield group) and a nonimpregnated graft (control group) in our hospital. An elevation of body temperature above 38°C was seen 29% of the patients in the Hemashield group and 0% in the control group. No significant differences were seen in the WBC counts, but the serum level of CRP was significantly higher on the 14th postoperative day in the Hemashield group. We should therefore pay careful attention to inflammatory reactions after the implantation of the impregnated grafts.