Masao Yoshitatsu

A surface-bound form of human C1 esterase inhibitor improves xenograft rejection.

BACKGROUND The purpose of the present study was to investigate the effect of the C1 esterase inhibitor (C1-INH) molecule against human complement attack on a swine endothelial cell (SEC) membrane. Human C1-INH functions as an inhibitor for complement reaction in the first step of the classical pathway in the fluid phase. METHODS A surface-bound form of human C1-INH (C1-INH-PI) consisting of a full-length coding sequence of C1-INH and a glycosylphosphatidylinositol (GPI) anchor of the decay-accelerating factor (CD55) was constructed, and stable Chinese hamster ovarian tumor (CHO) cell lines and SEC lines expressing C1-INH-PI were then prepared by transfection of the constructed cDNA. The basic function of the transfected molecules on the xenosurface was investigated using CHO transfectants for the sake of convenience. The efficacy of C1-INH-mediated protection of SEC from human complement was then assessed as an in vitro hyperacute rejection model of a swine-to-human discordant xenograft. RESULTS Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-PI showed a medium level of expression of these molecules. The C1-INH levels were significantly reduced as a result of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, suggesting that the molecules were present as the PI-anchor form. Approximately 51.3 x 10(4) and 13.3 x 10(4) molecules of C1-INH-PI blocked human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively. In addition, the complement-inhibiting activity of human C1-INH molecules is not homologously restricted. CONCLUSIONS The results suggest that the surface-bound form of C1-INH represents a good candidate as a safeguard against hyperacute rejection of xenografts.

Combined autologous cellular cardiomyoplasty using skeletal myoblasts and bone marrow cells for human ischemic cardiomyopathy with left ventricular assist system implantation: Report of a case

Myocardial regeneration therapy shows great promise as a treatment for heart failure. We recently introduced combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells as a treatment for human ischemic cardiomyopathy. We report the results of our first clinical trial of this technique, used to treat a patient with severe heart failure caused by ischemic cardiomyopathy who was being managed with a left ventricular assist system (LVAS). After combined cell transplantation, the patient showed signs of improved cardiac performance and angiogenesis, and reduced fibrosis.

Advanced left-atrial fibrosis is associated with unsuccessful maze operation for valvular atrial fibrillation

OBJECTIVE Atrial dilatation and fibrosis are considered to be important factors in the occurrence and maintenance of atrial fibrillation (AF). However, the relationship between those structural remodeling and postoperative sinus conversions after a maze operation has been rarely studied. The purpose of this study was to determine whether pathological evaluation of atrial tissues was useful for predicting an unsuccessful maze operation in patients with valvular AF. METHODS Between March 2006 and June 2007, left-atrial tissues in the posterior wall and right-atrial appendage were obtained from 47 consecutive patients (24 patients with chronic AF, and 23 with sinus rhythm) undergoing mitral valve surgery (MVS). A concomitant maze operation was performed for all patients with chronic AF. Atrial cell diameters were measured using hematoxylin and eosin staining, and quantitative assessment of atrial fibrosis was performed with Masson trichrome staining using an image analyzer (Image Processor for Analytical Pathology, Sumika Technoservice Co., Hyogo, Japan). RESULTS Successful MVS was performed for all patients and there were no complications associated with tissue sampling. Patients with chronic AF had more advanced histological features in both atria as compared with those with sinus rhythm. Sixteen of 24 patients, who underwent a maze operation, had successfully restored sinus rhythm (successful maze group), while that in the remaining eight was not restored (unsuccessful maze group). Patients in the unsuccessful maze group had a larger left-atrial dimension and cardiothoracic ratio as compared with those in the successful group, whereas the duration of AF was not significantly different. Patients in the unsuccessful maze group also had greater hypertrophy of cardiomyocytes and more extensive intercellular fibrosis in the left atrium, while there were no differences for right-atrial pathological features between the groups. Multivariate logistic analysis confirmed that a larger amount of left-atrial fibrosis (>15%) was significantly associated with an unsuccessful maze operation. CONCLUSIONS The present results suggested that advanced fibrosis in the left atrium, but not in the right atrium, might be significantly associated with an unsuccessful maze operation in patients with valvular AF.

Prevention of hyperacute rejection by phosphatidylinositol-anchored mini-complement receptor type 1.

Complement receptor type 1 (CR1, CD35) contains both factor I cofactor activity and convertase decay accelerating activity, but is, in general, thought to be an extrinsic regulator of complement activation. In this study, we prepared a phosphatidylinositol (PI)-anchored mini-CR1, which is composed of the short consensus repeat (SCR) 8-11 of CR1 and the PI anchor of DAF, and expressed it stably on a swine endothelial cell (SEC) line. We then examined the intrinsic regulatory activity of the mini-CR1, with respect to complement-mediated cell lysis as an in vitro hyperacute rejection model of a swine to human discordant xenograft. Flowcytometric profiles of the stable SEC lines with mini-CR1 showed a moderate level of expression for this molecule. Mini-CR1 blocked human complement-mediated cell lysis by approximately 50-70% on SEC. From the data of this study and our previous studies, mini-CR1 was more effective than membrane cofactor protein (MCP, CD46), and as effective as decay accelerating factor (DAF, CD55) in this system. The results suggest that PI-anchored mini-CR1 represents a useful molecule for clinical xenotransplantation.

Relationship between hemostatic markers and platelet indices in patients with aortic aneurysm.

The purpose of this study was to investigate whether platelet indices [platelet count, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet distribution width (PDW)] could serve as diagnostic tools to evaluate the potential significance of platelet heterogeneity on thrombus formation in patients with aortic aneurysm (AA). Blood samples were obtained from 54 patients with AA (mean age 73 years; 40 males and 14 females), and from 120 age-matched controls (AC; mean age 74 years; 61 males and 59 females). Blood platelet indices were measured using an automated counter for all AC (n = 120) and AA (n = 54). Plasma thrombin-antithrombin III complex (TAT), α2-plasmin inhibitor-plasmin complex (PIC), D-dimer, von Willebrand factor antigen (vWF:Ag) and interleukin-6 (IL-6) were also measured in part of AC and AA. In AA patients, TAT, PIC, D-dimer, vWF:Ag and IL-6 levels were significantly (p ≤0.0005) higher than in AC. In the patients, TAT was significantly inversely correlated with platelet count (ρ = –0.302, p = 0.038, n = 48), and significantly positively correlated with MPV (ρ = 0.329, p = 0.0373, n = 48), P-LCR (ρ = 0.361, p = 0.0134, n = 48) and PDW (ρ = 0.315, p = 0.0466, n = 48). PIC was negatively correlated with platelet count and inversely correlated with MPV, P-LCR and PDW. vWF:Ag was not correlated with platelet count, and inversely correlated with MPV, P-LCR and PDW in the patients. IL-6 was positively correlated with platelet count, and significantly inversely correlated with MPV, P-LCR and PDW in the patients. In AC, vWF:Ag was inversely correlated with platelet count and significantly positively correlated with MPV, P-LCR and PDW. However, PIC, TAT and IL-6 were not correlated with platelet indices in AC. D-dimer was not at all correlated with platelet indices both in AA and AC. In conclusion, the correlation between platelet indices and plasma hemostatic factor levels, e.g. TAT, PIC, D-dimer, vWF:Ag and IL-6, will be important factors for the understanding of platelet heterogeneity in patients with AA.

Relationship between Hemostatic Markers and Circulating Biochemical Markers of Collagen Metabolism in Patients with Aortic Aneurysm

Our objective was to determine the relationship between plasma levels of hemostatic molecular markers – D-dimer and thrombin-antithrombin III complex (TAT) – and circulating biochemical markers of collagen metabolism – aminoterminal propeptide of type III procollagen (PIIIP) and carboxyterminal propeptide of type I procollagen (PICP) – in patients with aortic aneurysm. The subjects were 43 patients with aortic aneurysm (AA; mean age 71 years) and 26 age-matched controls (mean age 75 years). The mean D-dimer, TAT and PIIIP levels were higher in the patients than in the controls (p < 0.0001, 0.0001 and 0.012, respectively), while the mean PICP level was similar to that in the controls. Increased D-dimer had a significant correlation with PIIIP (r = 0.412, p = 0.006) and PICP (r = 0.342, p = 0.0246), while TAT correlated with PIIIP (r = 0.3194, p = 0.0374), but not with PICP. There was also a significant correlation (r = 0.306, p = 0.0463) between PIIIP and PICP. As shown by the significant positive correlations among D-dimer, TAT and PIIIP, accelerated fibrinolysis and thrombogenesis induce an increase of collagen degradation and procollagen synthesis in atherosclerotic lesions. These findings show that D-dimer and TAT, especially the former, may be useful markers to monitor the progression and predict the prognosis of AA.

Evaluation of Platelet Indexes in Patients with Aortic Aneurysm

The purpose of this study was to investigate whether platelet indexes [platelet count, mean platelet volume (MPV), platelet-large cell ratio (P-LCR), and platelet distribution width (PDW)] could serve as diagnostic tools to evaluate the potential significance of platelet heterogeneity on thrombus formation. Blood samples were obtained from 54 patients with aortic aneurysm (AA; mean age 73 years; 40 males, 14 females), from 120 age-matched controls (AC; mean age 74 years; 61 males, 59 females), and from 38 young controls (YC; mean age 30 years; 20 males, 18 females). We measured the blood platelet indexes using an automated counter, as well as plasma D-dimer and thrombin-antithrombin III complex (TAT) using ELISA. The AA patients were divided into two groups, group A (platelet count more than 150 × 109/l, n = 36) and group B (platelet count below 150 × 109/l, n = 18). There was no difference in the platelet count between AC and YC. However, P-LCR was significantly higher (p = 0.0113) in AC. MPV and PDW were also higher, but not significantly so. The platelet count was not different between group A and AC. MPV (p = 0.0024 and <0.0001, respectively), P-LCR (p < 0.0012 and <0.0001, respectively) and PDW (p = 0.0006 and 0.0005, respectively) were significantly lower in group A than in group B and AC. The platelet indexes were significantly lower in the 54 AA patients than in the AC. There were significant inverse relationships between the platelet count and other indexes in the AC and 54 AA patients; however, no relationships were observed in group A, B and YC. The D-dimer and TAT levels were significantly higher in group B than in groups A and YC. In conclusion, these findings suggest that large platelets decrease rather than increase in patients with AA.

Morphological analysis and preoperative simulation of a double-chambered right ventricle using 3-dimensional printing technology

We present a case of a double-chambered right ventricle in adulthood, in which we tried a detailed morphological assessment and preoperative simulation using 3-dimensional (3D) heart models for improved surgical planning. Polygonal object data for the heart were constructed from computed tomography images of this patient, and transferred to a desktop 3D printer to print out models in actual size. Medical staff completed all of the work processes. Because the 3D heart models were examined by hand, observed from various viewpoints and measured by callipers with ease, we were able to create an image of the complete form of the heart. The anatomical structure of an anomalous bundle was clearly observed, and surgical approaches to the lesion were simulated accurately. During surgery, we used an incision on the pulmonary infundibulum and resected three muscular components of the stenosis. The similarity between the models and the actual heart was excellent. As a result, the operation for this rare defect was performed safely and successfully. We concluded that the custom-made model was useful for morphological analysis and preoperative simulation.

Changes in left anterior descending coronary artery flow profiles after coronary artery bypass grafting examined by means of transthoracic Doppler echocardiography

OBJECTIVE We sought to investigate the changes of velocity profiles in the left anterior descending coronary artery after coronary artery bypass grafting using transthoracic Doppler echocardiography. METHODS Forty-five patients who received a bypass graft to the left anterior descending coronary artery were studied. Before coronary artery bypass grafting, Doppler velocity profiles of the distal left anterior descending coronary artery were recorded with transthoracic Doppler echocardiography. Peak systolic velocity, mean systolic velocity, peak diastolic velocity, mean diastolic velocity, total velocity time integral, systolic velocity time integral, and diastolic velocity time integral were measured. Three weeks after coronary artery bypass grafting, left anterior descending coronary artery antegrade flow in the distal portion of the anastomosis was obtained by using the same method. Coronary angiography was performed before and 3 weeks after coronary artery bypass grafting. RESULTS The overall success rate of measuring the left anterior descending coronary artery flow was 60.0% preoperatively and 80.0% postoperatively. In 25 patients, in whom all parameters were obtained both before and after coronary artery bypass grafting, the following increased significantly after coronary artery bypass grafting: peak systolic velocity (14.86 +/- 7.50 vs 25.07 +/- 17.02 cm/s, P =.0045), mean systolic velocity (9.86 +/- 5.42 vs 18.03 +/- 12.94 cm/s, P =.0026), peak diastolic velocity (24.26 +/- 12.54 vs 48.28 +/- 31.66 cm/s, P =.0021), mean diastolic velocity (14.94 +/- 6.65 vs 30.36 +/- 20.71 cm/s, P =.0022), diastolic velocity time integral (7.22 +/- 2.88 vs 15.55 +/- 10.39 cm, P =.0009), total velocity time integral (10.50 +/- 4.48 vs 19.27 +/- 12.63 cm, P =.0034), and diastolic-to-systolic velocity time integral ratio (3.09 +/- 1.53 vs 4.97 +/- 2.75, P =.0044). Angiography showed graft patency and no significant change in left anterior descending coronary artery stenosis in all patients. CONCLUSIONS Transthoracic Doppler echocardiography showed a significant increase in some parameters in left anterior descending coronary artery flow after coronary artery bypass grafting. Measurement of left anterior descending coronary artery flow by means of transthoracic Doppler echocardiography might be a noninvasive method to evaluate the effect of bypass grafting on the left anterior descending coronary artery.

Attempts to Prepare Suitable Complement Regulatory Molecules for Clinical Xenotransplantation

Transgenic pigs with human complement regulatory proteins (CRP) have been reported in several institutes. However, transgenic animals, the grafts from which achieve perfect complement inactivation, have not yet been developed. Half-finished complement regulation by expressed decay accelerating factor (DAF) CD55 or DAF + CD59, along with large doses of drugs, such as steroids, avoid hyperacute rejection but create new problems, such as acute vascular rejection. In addition, DAF was identified as the receptor for Echovirus and Coxsackievirus, while membrane cofactor protein (MCP) molecules are receptors for measles viruses and Streptococcus. As a result, we constructed several membrane-bound forms of serum CRPs, such as C4bp-PI, fH-PI, fl-PI, and C1 INH-PI, as well as mini-CRI, and assessed their functions in human complement regulation on the surfaces of xenomembranes. These CRPs are able to regulate complement activation in different ways and are all effective in down regulation. Therefore, it appears reasonable to employ not only physical