Nobuya Zempo

Neovascularization induced by autologous bone marrow cell implantation in peripheral arterial disease.

Neovascularization has recently been used as a new treatment for severe ischemic disease. We tried to induce therapeutic neovascularization by autologous bone marrow cell implantation (BMCI) in eight selected patients with chronic peripheral arterial disease (PAD), in whom traditional treatments had failed. Improvement of subjective symptoms was seen in seven patients after treatment. Of three limbs with toe or finger ulceration, complete healing was achieved in two, while the other one became less severe after treatment. No relative toxicity was observed in any of the patients. BMCI might be a feasible treatment for selected patients with chronic PAD.

Enhanced Expression of Matrix Metalloproteinase-9 in Abdominal Aortic Aneurysms

Abstract. Abdominal aortic aneurysms (AAAs) are characterized by structural alterations of the aortic wall resulting from degradation of collagen and elastin. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, show strong elastinolytic activity. We examined the levels of mRNA for MMP-2, MMP-9, membrane type (MT)-MMP-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 in AAAs (n= 8), atherosclerotic occlusive diseases (AOD) (n= 8), and normal subjects (n= 8) using the reverse transcription-polymerase chain reaction (RT-PCR). We also analyzed the gelatinolytic activity of these metalloproteinases using gelatin zymography. The levels of MMP-2 and MMP-9 mRNA were increased in the AAA group compared with those in the AOD group and normal subjects. The levels for TIMP-1 and TIMP-2 mRNA in the AAA group were also higher than those in the AOD and normal groups. Only in the case of MT-MMP-1 was the difference between AAA and AOD not statistically significant. By gelatin zymography with the same samples used for RT-PCR, gelatinolytic activity of MMP-9 was elevated in all AAA tissues. The 62-kDa form of MMP-2 was elevated in both the AAA and AOD groups and did not differ significantly between them. Linear regression analysis demonstrated a significant positive correlation between mRNA levels of MMPs and those of TIMPs. These observations suggest that aneurysm formation in patients with atherosclerosis is related to the degree of MMP-9 expression.

The influence of tumour resection on angiostatin levels and tumour growth--an experimental study in tumour-bearing mice.

The phenomenon of primary neoplasms inhibiting the growth of their metastatic lesions is thought to be related to endogenous angiogenesis inhibitors. The aim of this experiment was to investigate the influence of tumour resection on angiostatin levels and tumour growth using a tumour-bearing mouse model. A primary Lewis lung cancer tumour model was established in C57BL/6 mice and these mice were divided into two groups 10 days after the tumour cells were inoculated. In the surgical resection group (S group) the tumour was resected, but in the control group (C group) a sham operation was performed. The level of angiostatin in the sera was analysed 5 days after the operation by western blotting. To observe tumour growth, four Lewis lung cancer models were established in these mice from both the S and C groups. An immunohistochemical analysis of the tumour tissues was conducted to estimate the proliferation and apoptotic rates of the tumour cells, as well as the amount of neoangiogenesis in the tumours. Angiostatin was observed in the tumour-bearing mice, but disappeared within 5 days after the tumour had been resected. Increased tumour growth was observed in all of the tumour models in the S group compared with the C group and the differences were significant. A significantly higher intratumour vessel density and proliferation cell index, but a significantly lower apoptotic index were also found in the S group compared with the C group. These findings demonstrated that angiostatin was generated directly from the tumour tissue. Furthermore, tumour resection accelerates the growth of other tumours and this is probably related to multiple factors including increased neoangiogenesis, increased tumour cell proliferation, and decreased apoptosis.

Enhanced Tumor Necrosis Factor-α Expression in Small Sized Abdominal Aortic Aneurysms

Circulating levels of tumor necrosis factor α (TNF-α) are elevated in the patients with abdominal aortic aneurysm (AAA). We investigated TNF-α expression and cellular infiltration in the walls of AAAs of different sizes. Twenty-seven surgical specimens of AAAs were categorized according to the maximum aneurysm diameter into a small size group (less than 50 mm in diameter, n = 8; S group), a medium-sized group (50 to 59 mm in diameter, n = 11; M group), and a large size group (larger than 59 mm in diameter, n = 8; L group). The level of TNF-α and interleukin-1β(IL-1β) in the aneurysm wall was measured by ELISA. Immunohistochemical staining was performed to observe the TNF-α expression and the infiltration of macrophages and lymphocytes in aneurysm walls. Enzyme-linked immunosorbent assay showed that the level of TNF-α in the S group (5.47 ± 3.48 pg/mg protein) was significantly higher (p < 0.05) than that in the M group (2.70 ± 1.33 pg/mg protein) or the L group (1.82 ± 1.21 pg/mg protein). No significant difference in IL-1β was observed between the S, M, and L groups. Immunohistochemical analysis also showed that TNF-α was expressed strongly in the S group but was negative or weakly positive in the M and L groups. Furthermore, the expression of TNF-α was seen mainly where the aneurysm wall showed atheromatous change and macrophage infiltration. These results indicated that the expression of TNF-α in the aneurysm wall was enhanced in small AAAs, and this enhancement might be related to the infiltration of macrophages.

Increased Serum Interleukin-8: Correlation with Poor Prognosis in Patients with Postoperative Multiple Organ Failure

Abstract. This study investigated whether cytokines and colony-stimulating factors can predict prognosis in patients with postoperative multiple organ failure (MOF). We evaluated 14 patients with postoperative MOF who underwent operation for cardiovascular disease. Seven patients recovered from MOF (survivors) and seven did not recover and died (nonsurvivors). The white blood cell (WBC) count, granulocyte colony-stimulating factor, monocytic colony-stimulating factor, interleukin-6 (IL-6), and IL-8 were measured on the day the patients were judged to be in MOF and each week thereafter until the patients recovered or died. Survivors and nonsurvivors were equivalent in terms of age, gender, proportion of use of extracorporeal circulation, operation time, volume of blood transfusion, time from operation to the onset of MOF, the MOF score, proportion of bacteremia, duration of MOF, and number of failed organs. The mean duration of MOF was less than 2 weeks in both groups; therefore the measurements were compared on the first day of MOF and 1 week later. No significant differences between the two groups in terms of WBC counts, colony-stimulating factors, and IL-6 levels were noted. However, the serum level of IL-8 was significantly higher in nonsurvivors than in survivors. Patients with a high serum levels of IL-8 at the time of MOF had a poor prognosis.

The preoperative administration of lentinan ameliorated the impairment of natural killer activity after cardiopulmonary bypass

The aim of this study was to determine whether the preoperative administration of lentinan, which is used clinically to activate T cell function in cancer patients, prevents the impairment of lymphocyte function after cardiopulmonary bypass (CPB). A total of 25 adults undergoing coronary artery bypass grafting were enrolled in this study. Lentinan (2 mg) was given to 10 randomly selected patients 7 d before surgery, while the other 15 patients were considered as a control. The white blood cell count, percentage of lymphocytes, subsets of lymphocytes, and natural killer cell activity were measured preoperatively, immediately after CPB and 1, 3, and 6 d after surgery. The white blood cell counts and the percentage of lymphocytes were not significantly different between the two groups; however, the percentage of CD4-positive cells in the lentinan group recovered to normal more rapidly than in the control group. Although natural killer cell activity was impaired in the control group after CPB, it maintained a nearly normal level in the lentinan group. The preoperative administration of lentinan for patients undergoing CPB ameliorated the impairment of natural killer activity and promoted the rapid recovery of CD4-positive cells.

Matrix Metalloproteinase Expressions in Arteriosclerotic Aneurysmal Disease

Medial degeneration of extracellular matrix (ECM) proteins in the wall of abdominal aortas results in smooth muscle cell destruction, a loss of architectural integrity, and abdominal aortic aneurysm (AAA) formation. It has been theorized that an imbalance between proteinases and their naturally occurring inhibitors is the cause of these observed histologic abnormalities. Therefore, the purpose of this investigation was to determine if differences in the matrix metalloproteinase (MMP) -2 and -9, tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA) protein and activity levels existed between infrarenal AAA and normal abdominal aortic tissue specimens. Between November 1995 and January 1997, 10 patients undergoing elective infrarenal AAA repair had a portion of their aneurysm walls snap frozen in liquid nitrogen and processed for subsequent western blot or zymographic analysis. Tissue specimens from 6 normal abdominal aortas obtained from fresh cadaver specimens were similarly processed and served as controls. Protein levels for MMP-2, MMP-9, TIMP-1, uPA, and tPA were analyzed by western blotting. The degree of MMP-2 and MMP-9 gelatinolytic activity was analyzed by zymography. Detection and immunolocalization for MMP-2, MMP-9 and CD68 was performed on tissue sections of AAA and normal infrarenal abdominal aortas fixed in 10% formalin. MMP-9 and tPA protein levels were increased in AAAs compared to controls by western blotting. However, uPA levels were slightly increased in controls. No differences in TIMP-1 protein levels were identified. Similarly, zymography demonstrated increased MMP-2 and MMP-9 gelatinolytic activity in AAAs compared to controls (p<0.05). CD68-positive cells (macrophages) in the adventitia and media demonstrated immunoreactivity to MMP-9. This investigation demonstrated increased MMP-9 proteinase activity and tPA protein levels in the walls of AAAs, as well as inflammatory leukocyte invasion of the adventitia and media compared to controls. These data suggest that leukocyte-derived MMP-9 is associated with aortic wall degeneration and aneurysm formation. Furthermore, activation of MMP-9 may be caused by increased tPA levels in the walls of AAAs.

Sutureless anastomosis of blood vessels using cyanoacrylate adhesives.

On the assumption that the remaining suture threads of the anastomotic line play an important role in the progression of anastomotic neointimal hyperplasia, we performed an experimental study on the sutureless anastomosis of blood vessels. An expanded polytetrafluoroethylene graft, 5 mm in diameter and 2 cm in length, was implanted on the abdominal aorta of mongrel adult using one of three methods of anastomosis, namely; a continuous suture, a stay suture, or sutureless anastomosis. Overall patency rates were 83.3 per cent, 91.7 per cent and 75.0 per cent respectively. The thickness of the pannus in the distal anastomotic line after 12 months was 107 μm in one graft in the continuous suture group, 106 μm and 222 μm in 2 grafts each in the stay suture group, and 41 μm and 117 μm in 2 grafts each in the sutureless group. Because there were cases of patency even after 12 months with a very small pannus thickness, sutureless anastomosis is considered to be a useful method of preventing anastomotic neointimal hyperplasia.

Digitonin enhances the antitumor effect of cisplatin during isolated lung perfusion.

BACKGROUND The antitumor effect of isolated lung perfusion with cisplatin was limited because the intracellular platinum concentration did not increase sufficiently. To solve this problem, digitonin, a detergent, was chosen to increase cell permeability and enhance intracellular uptake and antitumor effect. This study was designed to investigate toxicity, pharmacokinetics, and efficacy of isolated lung perfusion with the combined use of digitonin and cisplatin in Fischer 344 rats. METHODS Systemic and local toxicities of isolated lung perfusion treatment were evaluated on the basis of body weight change, survival rate, and histologic findings. The maximal tolerated dose of digitonin was determined by assessing survival on day 21 after contralateral pneumonectomy, body weight change, and histologic findings. Pharmacokinetics were observed in a solitary lung tumor nodule model by measuring platinum concentration in tumor and normal lung tissue. The antitumor effect was evaluated by the number of tumor nodules in the left lung 21 days after isolated lung perfusion. Isolated lung perfusion was performed 7 days after 1.0 x 10(6) methylcholanthrene sarcoma cells were injected into the external jugular vein. RESULTS The maximal tolerated dose of digitonin was 20 micromol/L. Platinum concentration of tumor nodules in the digitonin-cisplatin-treated rats was 20% higher than in the cisplatin-only group (5.48 +/- 0.64 microg/g tissue versus 4.50 +/- 1.09 microg/g tissue; p = 0.067). The number of pulmonary nodules decreased significantly by digitonin use (1.3 +/- 1.5 versus 9.7 +/- 2; p < 0.0001). CONCLUSIONS Isolated lung perfusion with digitonin and cisplatin in combination was performed safely and enhanced the antitumor effect. These drugs in combination show promise for enhancing the effect of clinical isolated lung perfusion.

Evaluation of brain oxygenation during selective cerebral perfusion by near-infrared Spectroscopy

BACKGROUND Although selective cerebral perfusion (SCP) has been used for cerebral protection in aortic arch operations, the appropriate perfusion conditions of SCP are unclear. METHODS We used near-infrared spectroscopy, which evaluates brain ischemia noninvasively and continuously, to determine whether perfusion with SCP (core temperature, 20 degrees C; flow rate, 10 mL.kg-1.min-1) was acceptable in terms of oxyhemoglobin and deoxyhemoglobin in patients having SCP for aortic arch operations (SCP group, n = 6) versus patients having cardiopulmonary bypass (CPB) for coronary artery bypass grafting (CPB group, n = 6). RESULTS There were no significant differences in age (65 +/- 10 versus 63 +/- 12 years), CPB time (199 +/- 67 versus 199 +/- 52 minutes), changes in hematocrit (-12.9% +/- 3.7% versus -12.5% +/- 6.0%), lowest blood pressure (43 +/- 7 versus 45 +/- 10 mm Hg), or highest central venous pressure (8 +/- 2 versus 9 +/- 4 mm Hg) between the SCP and CPB groups. In the SCP group, the maximum decrease in oxyhemoglobin level and the maximum increase in deoxyhemoglobin level were -5.0 to -11.4 mumol/L and -0.1 to 3.9 mumol/L, respectively; in the CPB group, the respective changes were -3.2 to -14.2 mumol/L and -0.4 to 3.6 mumol/L. Changes of oxyhemoglobin and deoxyhemoglobin levels in the SCP group were almost within the range of those in the CPB group. There were no brain complications in either group. CONCLUSIONS As described here, SCP is acceptable and safe for brain protection in aortic arch procedures.