Nobuyuki Hirose

The local injection of peritoneal macrophages induces neovascularization in rat ischemic hind limb muscles.

Macrophages play a pivotal role in the development of newly formed vascular networks, in addition to their normal immunological functions. This research focuses on peritoneal macrophages as a novel source in cell implantation therapy for ischemic diseases. In this study, production of angiogenic growth factors by peritoneal macrophages and its in vivo effect of neovascularization were evaluated. Mononuclear cells from the peritoneal cavity (P-MNCs) enriched with macrophages were isolated and stimulated with hypoxia and interleukin-1β (IL-1β) to mimic an ischemic tissue environment in vitro. Expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) of mRNA in P-MNCs was apparently enhanced by hypoxic stimulation, and the production of VEGF protein was also augmented by hypoxia and IL-1β. A rat ischemic hind limb model was created and P-MNCs (8 × 106/limb) were injected into the ischemic muscles. The blood flow, which was assessed using the colored microsphere method, showed that the percentage blood flow was significantly increased by P-MNCs injection 4 weeks after surgery (48.3 ± 16.8% in noninjected ischemic limb vs. 84.3 ± 13.0% in the P-MNCs-injected limb). A histological analysis revealed that the number of capillaries detected by alkaline phosphatase staining was increased in the P-MNCs group 4 weeks after injection. Furthermore, the number of α-smooth muscle actin-positive vessels also showed a significant increase following P-MNC injection. The injected P-MNCs labeled with fluorescence were detected in the interstitial space of ischemic muscles, and VEGF protein expression of the implanted cells was confirmed by immunohistochemistry. These results indicate that peritoneal macrophages stimulate capillary formation and arteriogenesis in the ischemic limbs, possibly through the production of angiogenic growth factors. These findings suggest that the physiological angiogenic property of peritoneal macrophages could therefore be utilized for neovascularization in cell implantation therapy.

Bilirubin Oxidation Provoked by Nitric Oxide Radicals Predicts the Progression of Acute Cardiac Allograft Rejection

Bilirubin, a strong intrinsic antioxidant, quenches free radicals produced under inflammatory conditions. The oxidized bilirubin metabolites, i.e. biopyrrins, are immediately excreted into urine and can indicate the intensity of oxidation in vivo. Our preliminary studies suggested the involvement of reactive nitrogen species (RNS) in generation of biopyrrins. However, little is known about biological significance of bilirubin oxidation by RNS. Here, we analyzed the correlation between bilirubin oxidation and nitric oxide (NO) radicals during rat acute cardiac allograft rejection. In allograft recipients, urinary biopyrrins steeply increased on day 3 prior to the increase in myocardial tissue damage marker, serum troponin‐T. In contrast, no significant changes in urinary biopyrrins were evident in recipients of isografts or cyclosporine‐A treated allografts. Urinary nitrotyrosine, a marker of oxidation by NO radicals also increased on day 3, while administration of a NO synthase inhibitor, NG‐monomethyl‐L‐arginine apparently diminished the elevation of urinary biopyrrins as well as nitrotyrosine. Immunohistochemistry revealed enhanced local expression of heme oxygenase‐1, biopyrrins and nitrotyrosine in allografts in accordance with the cellular infiltrates, suggesting that changes in urinary biopyrrins reflect the bilirubin oxidation in grafts undergoing rejection. These results indicate that locally evoked bilirubin oxidation by NO radicals can predict the progression of rejection.

Avoidance of Coronary Sinus Injury During Retrograde Cardioplegia.

Coronary sinus injury is a rare but critical complication of retrograde cardioplegia. Even after detection, successful repair with a pericardial patch is not always attained. Malalignment of the infusion cannula relative to the coronary sinus is likely to be partially responsible for the injury. Monitoring the location of the cannula tip can help prevent injury. Transesophageal echocardiography can indicate the vascular structure within which the cannula is located. This finding may be useful in preventing injury to the coronary sinus or middle cardiac vein.