Naomasa Kawaguchi

Myocardial Regeneration Therapy for Heart Failure Hepatocyte Growth Factor Enhances the Effect of Cellular Cardiomyoplasty

Background—We hypothesized that transfection of the gene for human hepatocyte growth factor (hHGF) combined with cellular cardiomyoplasty might regenerate the impaired myocardium. Methods and Results—We used a ligation model of proximal left anterior descending coronary artery (LAD) of Lewis rats. Two weeks after LAD ligation, 3 different treatments were conducted: (1) neonatal rat cardiomyocytes group (106 cells, T group, n=11), (2) HVJ-liposomes bearing the hHGF gene group (H group, n=10), and (3) combined (T-H group, n=10). The injection site was the scar area of myocardial infarction. For control, culture medium was injected (C group, n=13). Echocardiography demonstrated that cardiac performance was significantly ameliorated in the T-H group 4 and 8 weeks after injection. Contrast echocardiography also showed a marked increase in myocardial perfusion in the T-H group but not in the other groups. In the T-H group, neovascularization and a marked reduction of fibrosis were observed histologically. In an immunohistochemical study, strong staining for &bgr;1-integrin, &agr;-, and &bgr;-dystroglycan were found principally in the basement membrane of myocytes in the T-H group 8 weeks after transplantation, although there was weak immunoreactivity in the T group. Conclusions—hHGF gene transfection enhanced the cellular cardiomyoplasty possibly by stimulating angiogenesis, restoring the impaired ECM, and promoting the integration of the dissociated grafted myocytes. The combined effects might have lead to the improved cardiac performance. Thus, combined therapy may be a promising strategy for the treatment of heart failure caused by myocardial infarction.

Impaired myocardium regeneration with skeletal cell sheets--a preclinical trial for tissue-engineered regeneration therapy.

Background. We hypothesized that autologous skeletal cell (SC) sheets regenerate the infract myocardium in porcine heart as a preclinical trial. Methods and Results. The impaired heart was created by implantation of ameroid constrictor on left anterior descending for 4 weeks. SCs isolated from leg muscle were cultured and detached from the temperature-responsive domain-coated dishes as single monolayer cell sheet at 20°C. The following therapies were conducted: SC sheets (SC group, n=5); sham (C group n=5). Echocardiography demonstrated that cardiac performance was significantly improved in the SC group 3 and 6 months after operation (fractional area shortening, 3 months; SC vs. C=49.5±2.8 vs. 24.6±2.0%, P<0.05) and left ventricle dilatation was well attenuated in the SC group. Color kinesis index showed that distressed regional diastolic and systolic function in infarcted anterior wall was significantly recovered (SC vs. C=57.4±8.6 vs. 30.2±4.7%, P<0.05, diastolic: 58.5±4.5 vs. 35.4±6.6%, P<0.05, systolic). Factor VIII immunostains demonstrated that vascular density was significantly higher in the SC group than the C group. And % fibrosis and cell diameter were significantly lower in the SC group. And hematoxylin-eosin staining depicted that skeletal origin cells and well-developed-layered smooth muscle cells were detected in the implanted area. Positron emission tomography showed better myocardial perfusion and more viable myocardial tissue in the distressed myocardium receiving SC sheets compared with the myocardium receiving no sheets. Conclusions. SC sheet implantation improved cardiac function by attenuating the cardiac remodeling in the porcine ischemic myocardium, suggesting a promising strategy for myocardial regeneration therapy in the impaired myocardium.

In situ tissue regeneration using a novel tissue-engineered, small-caliber vascular graft without cell seeding

OBJECTIVE Various types of natural and synthetic scaffolds with arterial tissue cells or differentiated stem cells have recently attracted interest as potential small-caliber vascular grafts. It was thought that the synthetic graft with the potential to promote autologous tissue regeneration without any seeding would be more practical than a seeded graft. In this study, we investigated in situ tissue regeneration in small-diameter arteries using a novel tissue-engineered biodegradable vascular graft that did not require ex vivo cell seeding. METHODS Small-caliber vascular grafts (4 mm in diameter) were fabricated by compounding a collagen microsponge with a biodegradable woven polymer tube that was constructed in a plain weave pattern with a double layer of polyglycolic acid (core) and poly-L-lactic acid (sheath) fibers. We implanted these tissue-engineered vascular grafts bilaterally into the carotid arteries of mongrel dogs (body weight, 20-25 kg). No anticoagulation regimen was used after implantation. We sacrificed the dogs 2, 4, 6, and 12 months (n = 4 in each group) after implantation and evaluated the explants histologically and biochemically. RESULTS All of the tissue-engineered vascular grafts were patent with no signs of thrombosis or aneurysm at any time. Histologic and biochemical examinations showed excellent in situ tissue regeneration with an endothelial cell monolayer, smooth muscle cells, and a reconstructed vessel wall with elastin and collagen fibers. CONCLUSION Our study indicated that this novel tissue-engineered vascular graft promoted in situ tissue regeneration and did not require ex vivo cell seeding, thereby conferring better patency on small-caliber vascular prostheses.

Survivin Determines Cardiac Function by Controlling Total Cardiomyocyte Number

Background— Survivin inhibits apoptosis and regulates cell division in many organs, but its function in the heart is unknown. Methods and Results— We show that cardiac-specific deletion of survivin resulted in premature cardiac death. The underlying cause was a dramatic reduction in total cardiomyocyte numbers as determined by a stereological method for quantification of cells per organ. The resulting increased hemodynamic load per cell led to progressive heart failure as assessed by echocardiography, magnetic resonance imaging, positron emission tomography, and invasive catheterization. The reduction in total cardiomyocyte number in α-myosin heavy chain (MHC)–survivin−/− mice was due to an ≈50% lower mitotic rate without increased apoptosis. This occurred at the expense of DNA accumulation because survivin-deficient cardiomyocytes displayed marked DNA polyploidy indicative of consecutive rounds of DNA replication without cell division. Survivin small interfering RNA knockdown in neonatal rat cardiomyocytes also led to polyploidization and cell cycle arrest without apoptosis. Adenoviral overexpression of survivin in cardiomyocytes inhibited doxorubicin-induced apoptosis, induced DNA synthesis, and promoted cell cycle progression. The phenotype of the αMHC-survivin−/− mice also allowed us to determine the minimum cardiomyocyte number sufficient for normal cardiac function. In human cardiomyopathy, survivin was potently induced in the failing heart and downregulated again after hemodynamic support by a left ventricular assist device. Its expression positively correlated with the mean cardiomyocyte DNA content. Conclusions— We suggest that the ontogenetically determined cardiomyocyte number may be an independent factor in the susceptibility to cardiac diseases. Through its profound impact on both cardiomyocyte replication and apoptosis, survivin may emerge as a promising new target for myocardial regeneration.

Role of angiotensin II in high fructose-induced left ventricular hypertrophy in rats.

Recent studies suggest the linkage of hypertension and insulin resistance. High fructose diet is known to induce hyperinsulinemia and hypertension in rats. In a previous study, however, high fructose (66%) diet failed to elevate blood pressure but increased left ventricular weight in Sprague-Dawley rats. In the present study, we investigated the precise mechanism of high fructose diet-induced changes in the cardiovascular system in rats. Intake of fructose-enriched diet for 2 weeks increased serum insulin and plasma angiotensin II levels. Urinary excretion of sodium and norepinephrine was not changed. Blood pressure measured directly through an indwelling catheter was not increased, but left ventricular weight and protein content were increased by high fructose diet. To further elucidate the role of the renin-angiotensin system, an angiotensin II type 1 receptor antagonist, TCV-116, was given orally at 1 mg/kg per day with either normal or high fructose diet. Concomitant administration of TCV-116 did not affect plasma glucose or serum insulin levels. Plasma angiotensin II was increased, but neither urinary sodium nor norepinephrine was changed by TCV-116. TCV-116 similarly decreased blood pressure in rats on normal and high fructose diets. Increase in left ventricular weight induced by high fructose diet was prevented by the concomitant administration of TCV-116. On the other hand, left ventricular weight in control rats was not changed by TCV-116. In conclusion, increased plasma angiotensin II may account for the left ventricular hypertrophy induced by high fructose diet, whereas hemodynamic change, sodium retention, and the sympathetic nervous system do not play an important role.

A self-renewing, tissue-engineered vascular graft for arterial reconstruction

OBJECTIVE Various tissue-engineered vascular grafts have been studied to overcome the clinical disadvantages of conventional prostheses. Previous tissue-engineered vascular grafts have generally required preoperative cellular manipulation or use of bioreactors to improve performance, and their mechanical properties have been insufficient. We focused on the concept of in situ cellularization and developed a tissue-engineered vascular graft for arterial reconstruction that would facilitate renewal of autologous tissue without any pretreatment. METHODS The graft comprised an interior of knitted polyglycolic acid compounded with collagen to supply a scaffold for tissue growth and an exterior of woven poly-L-lactic acid for reinforcement. All components were biocompatible and biodegradable, with excellent cellular affinity. The grafts, measuring 10 mm in internal diameter and 30 mm in length, were implanted into porcine aortas, and their utility was evaluated to 12 months after grafting. RESULTS All explants were patent throughout the observation period, with no sign of thrombus formation or aneurysmal change. Presence in the neomedia of endothelialization with proper integrity and parallel accumulation of functioning smooth muscle cells, which responded to vasoreactive agents, was confirmed in an early phase after implantation. Sufficient collagen synthesis and lack of elastin were quantitatively demonstrated. Dynamic assessment and long-term results of the in vivo study indicated adequate durability of the implants. CONCLUSION The graft showed morphologic evidence of good in situ cellularization, satisfactory durability to withstand arterial pressure for 12 postoperative months, and the potential to acquire physiologic vasomotor responsiveness. These results suggest that our tissue-engineered vascular graft shows promise as an arterial conduit prosthesis.

Cardiac lesions and their reversibility after long term administration of methamphetamine.

In order to clarify the effect of methamphetamine (MA) on myocardium, histological, immunohistochemical and electron microscopic changes in the myocardium of rats were examined following daily intraperitoneal administration of MA at a dose of 1 mg per kg body weight for 4, 8, and 12 weeks before sacrifice. Normal saline (NS) was similarly injected for the same period before sacrifice to constitute a control group. Light microscopic changes found in the myocardium of the MA-treated group included atrophy, hypertrophy, patchy cellular infiltration, eosinophilic degeneration and disarray, edema myolysis, fibrosis, and the appearance of vacuoles. Ultrastructurally, nuclei and normal mitochondria had various shapes and there were dilated T tubules and sarcoplasmic reticulum, the accumulation of glycogen granules and fat droplets. Intra- and extra-cellular edema and intramyocytic vacuoles were often found. Withdrawal of MA at the twelfth week in another group of rats evidenced gradual recovery of the myocardial changes, commencing at 3 weeks after withdrawal. Optimism is therefore generated about the possibility of the affected hearts in MA-abuse patients returning towards the normal state if they give up the drug.

Aged Garlic Extract Inhibits Angiogenesis and Proliferation of Colorectal Carcinoma Cells

Because colorectal cancer is likely to develop in many people at some point during their lives, prevention has become a high priority. Diet and nutrition play an important role during the multistep colon carcinogenic process. Garlic has been traditionally used as a spice and is well known for its medicinal properties; several studies have indicated its pharmacologic functions, including its anticarcinogenic properties. However, the mechanisms by which garlic can prevent colorectal cancer remain to be elucidated. This study investigated the effect of aged garlic extract (AGE) on the growth of colorectal cancer cells and their angiogenesis, which are important microenvironmental factors in carcinogenesis. AGE suppressed the proliferation of 3 different colorectal cancer cell lines-HT29, SW480, and SW620-in the same way, but its effects on the invasive activities of these 3 cell lines were different. the invasive activities of SW480 and SW620 cells were inhibited by AGE, whereas AGE had no effect on the invasive activity of Ht29 cells. The action of AGE appears to be dependent on the type of cancer cell. On the other hand, AGE enhanced the adhesion of endothelial cells to collagen and fibronectin and suppressed cell motility and invasion. AGE also inhibited the proliferation and tube formation of endothelial cells potently. These results suggest that AGE could prevent tumor formation by inhibiting angiogenesis through the suppression of endothelial cell motility, proliferation, and tube formation. AGE would be a good chemopreventive agent for colorectal cancer because of its antiproliferative action on colorectal carcinoma cells and inhibitory activity on angiogenesis.

Skeletal myoblast sheet transplantation improves the diastolic function of a pressure-overloaded right heart

OBJECTIVE The development of right ventricular dysfunction has become a common problem after surgical repair of complex congenital heart disease. A recent study reported that tissue-engineered skeletal myoblast sheet transplantation improves left ventricular function in patients with dilated and ischemic cardiomyopathy. Therefore myoblast sheet transplantation might also improve ventricular performance in a rat model of a pressure-overloaded right ventricle. METHODS Seven-week-old male Lewis rats underwent pulmonary artery banding. Four weeks after pulmonary artery banding, myoblast sheet transplantation to the right ventricle was performed in the myoblast sheet transplantation group (n = 20), whereas a sham operation was performed in the sham group (n = 20). RESULTS Four weeks after performing the procedure, a hemodynamic assessment with a pressure-volume loop showed a compensatory increase in systolic function in both groups. However, only the myoblast sheet transplantation group showed a significant improvement in the diastolic function: end-diastolic pressure (sham vs myoblast sheet transplantation, 10.3 +/- 3.1 vs 5.0 +/- 3.7 mm Hg; P < .001), time constant of isovolumic relaxation (11.1 +/- 2.5 vs 7.6 +/- 1.2 ms, P < .001), and end-diastolic pressure-volume relationship (16.1 +/- 4.5 vs 7.6 +/- 2.4/mL, P < .005). The right ventricular weight and cell size similarly increased in both groups. A histologic assessment demonstrated significantly suppressed ventricular fibrosis and increased capillary density in the myoblast sheet transplantation group in comparison with those in the sham group. Reverse transcription-polymerase chain reaction demonstrated an increased myocardial gene expression of hepatocyte growth factor and vascular endothelial growth factor in the myoblast sheet transplantation group but not in the sham group. CONCLUSIONS Skeletal myoblast sheet transplantation improved the diastolic dysfunction and suppressed ventricular fibrosis with increased capillary density in a rat model of a pressure-overloaded right ventricle. This method might become a novel strategy for the myocardial regeneration of right ventricular failure in patients with congenital heart disease.

FK506-induced kidney tubular cell injury

Some renal changes associated with cyclosporine, such as tubular vacuolization and glomerular thrombosis, have also been reported with FK506. Furthermore, FK506 therapy is associated with a decrease in glomerular filtration rate and renal plasma flow and an increase in renal vascular resistance. We studied the in vitro tubular cell sensitivity to FK506 in comparison with CsA, the ultrastructural changes induced by FK506 and CsA, and the effect of both drugs on tubular cell growth in vitro. We also investigated whether FK506 and CsA induced endothelin-1 (ET-1) secretion of cultured tubular cells and whether this stimulatory effect coincided with a change in the endothelin systemic synthesis. Exposure of tubular cells to high concentrations of FK506 or CsA (10, 50, 100 μM) induced a time- and dose-dependent cell injury in vitro. The damage induced by FK506 and CsA was characterized by a direct cytotoxic effect on tubular cells, as expressed by release of 3H thymidine from prelabeled cells, N-acetyl-β-D-glu-cosaminidase release, and cell detachment. Ultrastructural changes (vacuolizations, swelling, and mitochondrial enlargement) and inhibition of the growth of cultured tubular cells were also observed at high concentrations of FK506 and CsA. Low concentrations of FK506 and CsA (1, 0.1, 0.01, 0.001 %mUM) were not cytotoxic and induced only a minimal inhibitory effect on the growth of tubular cells in vitro. We demonstrated that FK506 (1, 0.1, 0.01 μM) time-dependently stimulated the secretion of endothelin by cultured tubular cells. CsA 10, 1, 0.1, 0.01 also exerted an enhancing effect on ET-1 secretion in cultured tubular cells. We observed that the concentration of CsA that induced the most important enhancing effect was 10 or 100 times higher than that required for FK506 to observe the same effect. The concentrations of FK506 or CsA that induced ET-1 secretion were not cytolytic for tubular cells in vitro. FK506− or CsA-treated rats showed an increase in serum level of ET-1 in comparison with the control. Through the stimulatory effect on endothelin secretion by tubular cells, FK506 and CsA may induce a perturbation of renal hemodynamics. Concentrations of FK506 and CsA, higher than established serum levels but close to those reached in tissues, are cytotoxic for tubular cells and induced ultrastructural changes and a significant delayed regeneration.