Ryohei Yozu

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca2+ oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2′-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis

The avascularity of cardiac valves is abrogated in several valvular heart diseases (VHDs). This study investigated the molecular mechanisms underlying valvular avascularity and its correlation with VHD. Chondromodulin-I, an antiangiogenic factor isolated from cartilage, is abundantly expressed in cardiac valves. Gene targeting of chondromodulin-I resulted in enhanced Vegf-A expression, angiogenesis, lipid deposition and calcification in the cardiac valves of aged mice. Echocardiography showed aortic valve thickening, calcification and turbulent flow, indicative of early changes in aortic stenosis. Conditioned medium obtained from cultured valvular interstitial cells strongly inhibited tube formation and mobilization of endothelial cells and induced their apoptosis; these effects were partially inhibited by chondromodulin-I small interfering RNA. In human VHD, including cases associated with infective endocarditis, rheumatic heart disease and atherosclerosis, VEGF-A expression, neovascularization and calcification were observed in areas of chondromodulin-I downregulation. These findings provide evidence that chondromodulin-I has a pivotal role in maintaining valvular normal function by preventing angiogenesis that may lead to VHD.

Periostin advances atherosclerotic and rheumatic cardiac valve degeneration by inducing angiogenesis and MMP production in humans and rodents

Valvular heart disease (VHD) is the term given to any disease process involving one or more of the heart valves. The condition can be congenital or acquired, for example as a result of atherosclerosis or rheumatic fever. Despite its clinical importance, the molecular mechanisms underlying VHD remain unknown. We investigated the pathophysiologic role and molecular mechanism of periostin, a protein that plays critical roles in cardiac valve development, in degenerative VHD. Unexpectedly, we found that periostin levels were drastically increased in infiltrated inflammatory cells and myofibroblasts in areas of angiogenesis in human atherosclerotic and rheumatic VHD, whereas periostin was localized to the subendothelial layer in normal valves. The expression patterns of periostin and chondromodulin I, an angioinhibitory factor that maintains cardiac valvular function, were mutually exclusive. In WT mice, a high-fat diet markedly increased aortic valve thickening, annular fibrosis, and MMP-2 and MMP-13 expression levels, concomitant with increased periostin expression; these changes were attenuated in periostin-knockout mice. In vitro and ex vivo studies revealed that periostin promoted tube formation and mobilization of ECs. Furthermore, periostin prominently increased MMP secretion from cultured valvular interstitial cells, ECs, and macrophages in a cell type-specific manner. These findings indicate that, in contrast to chondromodulin I, periostin plays an essential role in the progression of cardiac valve complex degeneration by inducing angiogenesis and MMP production.

Administration of granulocyte colony-stimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair.

The administration of granulocyte colony‐stimulating factor (G‐CSF) after myocardial infarction (MI) improves cardiac function and survival rates in mice. It was also reported recently that bone marrow (BM)‐derived c‐kit+ cells or macrophages in the infarcted heart are associated with improvement of cardiac remodeling and function. These observations prompted us to examine whether BM‐derived hematopoietic cells mobilized by G‐CSF administration after MI play a beneficial role in the infarct region. A single hematopoietic stem cell from green fluorescent protein (GFP)‐transgenic mice was used to reconstitute hematopoiesis in each experimental mouse. MI was then induced, and the mice received G‐CSF for 10 days. In the acute phase, a number of GFP+ cells showing the elongated morphology were found in the infarcted area. Most of these cells were positive for vimentin and α‐smooth muscle actin but negative for CD45, indicating that they were myofibroblasts. The number of these cells was markedly enhanced by G‐CSF administration, and the enhanced myofibroblast‐rich repair was considered to lead to improvements of cardiac remodeling, function, and survival rate. Next, G‐CSF‐mobilized monocytes were harvested from the peripheral blood of GFP‐transgenic mice and injected intravenously into the infarcted mice. Following this procedure, GFP+ myofibroblasts were observed in the infarcted myocardium. These results indicate that cardiac myofibroblasts are hematopoietic in origin and could arise from monocytes/macrophages. MI leads to the recruitment of monocytes, which differentiate into myofibroblasts in the infarct region. Administration of G‐CSF promotes this recruitment and enhances cardiac protection. Disclosure of potential conflicts of interest is found at the end of this article.

Comparison of early postoperative quality of life in minimally invasive versus conventional valve surgery

AbstractPurpose. Minimally invasive cardiac surgery (MICS), an approach in which full sternotomy is avoided and the surgical incision is minimal, has been shown to produce less postoperative discomfort and to enable earlier mobilization and discharge than conventional cardiac surgery (CCS). This study was performed to retrospectively evaluate quality of life following MICS in comparison with CCS valve surgery. Methods. Sixty-six patients scheduled for MICS and 50 patients scheduled for CCS for isolated aortic or mitral valve surgery from January 1999 to June 2001 were enrolled in the study. The clinical records for the two groups were compared across intraoperative parameters and those associated with postoperative quality of life. Results. The aortic clamp and cardiopulmonary bypass times in the MICS group were longer than those in the CCS group (144 ± 42 and 224 ± 58 min vs 112 ± 21 and 179 ± 27 min, P ≪ 0.001). Postoperative pain medication (rectal buprenorphine and intramuscular pethidine) was administered to 18 of the 66 MICS patients (27%), as compared with 26 of the 50 CCS patients (52%, P = 0.007). Postoperative delirium was less frequent in the MICS group than the CCS group (26% vs 44%, P = 0.039). Initial postoperative food intake and urine catheter removal were possible earlier in the MICS than in the CCS group. MICS patients had shorter stays in the intensive care unit than CCS patients (37.4 ± 7.3 vs 45.9 ± 8.7 h, P ≪ 0.001). Conclusion. Although longer aortic clamp and cardiopulmonary bypass times remain a problem in MICS procedures, our results suggest that MICS, as compared with CCS, facilitates earlier recovery of daily activities and provides improved quality of life in the early postoperative period.

Role of vascular endothelial growth factor-A in development of abdominal aortic aneurysm

AIMS Increased angiogenesis, chronic inflammation, and extracellular matrix degradation are the major pathological features of abdominal aortic aneurysm (AAA). We sought to elucidate the role of vascular endothelial growth factor (VEGF)-A, a potent angiogenic and proinflammatory factor, in the development of AAA. METHODS AND RESULTS Human AAA samples showed increased VEGF-A expression, neovascularization, and macrophage infiltration compared with normal aortic walls. AAA was induced in mice by periaortic application of CaCl(2). AAA mice were treated with soluble VEGF-A receptor (sFlt)-1 or phosphate-buffered saline and sacrificed 6 weeks after the operation. Treatment with sFlt-1 resulted in reduced aneurysm size, restored wavy structure of the elastic lamellae, reduced Mac-2(+) monocytes/macrophages, CD3(+) T-lymphocytes, and CD31(+) vessels, and attenuated matrix metalloproteinase (MMP)-2 and 9 activity in periaortic tissue of AAA. Increased aortic mRNA expression of monocyte chemotactic protein-1, tumour necrosis factor-α, and intercellular adhesion molecule-1 in AAA was attenuated by sFlt-1 treatment. CONCLUSION VEGF-A was overexpressed in the aortic wall of human and experimental AAA. Treatment with sFlt-1 inhibited AAA development in mice, in association with reduced neoangiogenesis, infiltration of inflammatory cells, MMP activity, and extracellular matrix degradation. These findings suggest a crucial role of VEGF-A in the development of AAA.

Omentopexy enhances graft function in myocardial cell sheet transplantation

Myocardial cell sheets (MCS) are a potentially valuable tool for tissue engineering aimed at heart regeneration. Several methods have recently been established for the fabrication of MCS. However, the lack of a sufficient blood supply has inhibited functional recovery of the MCS. To address this challenge, we combined MCS transplantation with omentopexy (OP), which utilizes omental tissue as a surgical flap. Rats were divided into five groups: sham, myocardial infarction (MI), MCS transplantation, OP, and MCS+OP. Histologic analysis revealed that MCS+OP drastically reversed MI-induced cardiac remodeling. Echocardiography revealed that MCS increased cardiac function, while OP had a synergistic beneficial effect with MCS transplantation. Immunofluorescence imaging showed that OP increased the survival of transplanted cardiomyocytes, and increased the blood supply through enhancement of angiogenesis and migration of small arteries into the MCS. Taken together, we concluded that OP is a promising strategy for the enhancement of graft function in MCS transplantation.

Tumor necrosis factor-α converting enzyme is a key mediator of abdominal aortic aneurysm development

OBJECTIVE Tumor necrosis factor (TNF)-α is known to be elevated in plasma and the aorta in abdominal aortic aneurysm (AAA) patients. We sought to clarify the role of TNF-α converting enzyme (Tace), which cleaves the transmembrane precursor of TNF-α, in AAA development. METHODS We obtained aortic sample of AAA during surgical operation to assess the histological features and protein expression of human AAA. AAA was induced in mice with temporal systemic deletion of Tace by the inducible Mx-1 Cre transgene (TaceMx1) and in wild-type littermates (CON) by periaortic application of CaCl(2) (AAA/TaceMx1, AAA/CON). RESULTS Tace expression was increased in human AAA samples as compared with normal aorta. Six weeks postoperatively, aortic diameter in AAA/TaceMx1 was decreased than in AAA/CON in association with attenuated TNF-α expression and extracellular matrix disruption. Increased activities of matrix metalloproteinase (MMP)-9 and MMP-2, numbers of Mac-2-positive macrophages, CD3-positive T lymphocytes and CD31-positive vessels in periaortic tissues, mRNA expression of CD68, monocyte chemotactic protein-1, TNF-α, vascular endothelial growth factor-A, p47 and glutathione peroxidases, and protein expression of phospho-c-Jun N-terminal kinase in AAA were all attenuated by Tace deletion. Protein expression of transforming growth factor (TGF)-β1 was upregulated by Tace deletion in sham-operated mice. TGF-β1 expression was further increased in AAA/TaceMx1. CONCLUSIONS Tace was overexpressed in the aortic wall in human and experimental AAA. Temporal systemic deletion of Tace prevented AAA development in association with attenuating inflammation, oxidative stress, neoangiogenesis and extracellular matrix disruption, suggesting a crucial role of Tace in AAA development.

Local Tenomodulin Absence, Angiogenesis, and Matrix Metalloproteinase Activation Are Associated With the Rupture of the Chordae Tendineae Cordis

Background— Rupture of the chordae tendineae cordis (CTC) is a well-known cause of mitral regurgitation. Despite its importance, the mechanisms by which the CTC is protected and the cause of its rupture remain unknown. CTC is an avascular tissue. We investigated the molecular mechanisms underlying the avascularity of CTC and the correlation between avascularity and CTC rupture. Methods and Results— We found that tenomodulin, which is a recently isolated antiangiogenic factor, was expressed abundantly in the elastin-rich subendothelial outer layer of normal rodent, porcine, canine, and human CTC. Conditioned medium from cultured CTC interstitial cells strongly inhibited tube formation and mobilization of endothelial cells; these effects were partially inhibited by small-interfering RNA against tenomodulin. The immunohistochemical analysis was performed on 12 normal and 16 ruptured CTC obtained from the autopsy or surgical specimen. Interestingly, tenomodulin was locally absent in the ruptured areas of CTC, where abnormal vessel formation, strong expression of vascular endothelial growth factor-A and matrix metalloproteinases, and infiltration of inflammatory cells were observed, but not in the normal or nonruptured area. In anesthetized open-chest dogs, the tenomodulin layer of tricuspid CTC was surgically filed, and immunohistological analysis was performed after several months. This intervention gradually caused angiogenesis and expression of vascular endothelial growth factor-A and matrix metalloproteinases in the core collagen layer in a time-dependent manner. Conclusions— These findings provide evidence that tenomodulin is expressed universally in normal CTC in a concentric pattern and that local absence of tenomodulin, angiogenesis, and matrix metalloproteinase activation are associated with CTC rupture.

Mortality and morbidity after total arch replacement using a branched arch graft with selective antegrade cerebral perfusion

BACKGROUND The early outcome after aortic arch surgery has improved. However, some operative survivors have died as a result of postoperative problems soon after discharge. This study determines the factors affecting mortality within 1 year of total arch replacement. METHODS Between July 1993 and November 2001, 103 patients (mean age 65 +/- 11 years, 26 women, 35 dissections) underwent total arch replacement through a median sternotomy using a branched arch graft with selective cerebral perfusion. Eighteen operations including 14 acute dissections were performed on an emergency basis. Concomitant procedures were root replacement in 5 patients, mitral valve replacement in 1, coronary artery bypass in 14, and open endovascular stent-graft in 9. The average time (minutes) for bypass, aortic cross-clamp, selective cerebral perfusion, and distal arrest were respectively 273 +/- 79, 163 +/- 54, 145 +/- 36, and 69 +/- 22. RESULTS Mechanical heart support was necessary in 3 patients. Stroke occurred in 9 patients, transient neurologic dysfunction in 7, and paraplegia/paraparesis in 4. The only independent determinant for postoperative stroke was a history of stroke (odds ratio 16.3, 95% confidence interval: 2.8 to 93.8). Thirty-one patients required ventilator support for more than 5 days. Hemodialysis was needed in 5 patients. Sternal infection or mediastinitis occurred in 6 patients. The in-hospital mortality was 12% (12 of 103). The actuarial survival rate at 1 year was 83%, and was 67% at 5 years. For the 1-year mortality independent determinants were emergency surgery (odds ratio 5.3, 95% confidence interval: 1.6 to 17.9) and age 75 years or older (odds ratio 4.0, 95% confidence interval: 1.1 to 13.9). CONCLUSIONS Total arch replacement using a branched arch graft with selective antegrade cerebral perfusion has a favorable 1-year mortality rate except for patients undergoing emergency surgery and for elderly patients.