Yasuhiro Shudo

Sustained Release of Engineered Stromal Cell–Derived Factor 1-α From Injectable Hydrogels Effectively Recruits Endothelial Progenitor Cells and Preserves Ventricular Function After Myocardial Infarction

Background— Exogenously delivered chemokines have enabled neovasculogenic myocardial repair in models of ischemic cardiomyopathy; however, these molecules have short half-lives in vivo. In this study, we hypothesized that the sustained delivery of a synthetic analog of stromal cell–derived factor 1-&agr; (engineered stromal cell–derived factor analog [ESA]) induces continuous homing of endothelial progenitor cells and improves left ventricular function in a rat model of myocardial infarction. Methods and Results— Our previously designed ESA peptide was synthesized by the addition of a fluorophore tag for tracking. Hyaluronic acid was chemically modified with hydroxyethyl methacrylate to form hydrolytically degradable hydrogels through free-radical–initiated crosslinking. ESA was encapsulated in hyaluronic acid hydrogels during gel formation, and then ESA release, along with gel degradation, was monitored for more than 4 weeks in vitro. Chemotactic properties of the eluted ESA were assessed at multiple time points using rat endothelial progenitor cells in a transwell migration assay. Finally, adult male Wistar rats (n=33) underwent permanent ligation of the left anterior descending (LAD) coronary artery, and 100 µL of saline, hydrogel alone, or hydrogel+25 µg ESA was injected into the borderzone. ESA fluorescence was monitored in animals for more than 4 weeks, after which vasculogenic, geometric, and functional parameters were assessed to determine the therapeutic benefit of each treatment group. ESA release was sustained for 4 weeks in vitro, remained active, and enhanced endothelial progenitor cell chemotaxis. In addition, ESA was detected in the rat heart >3 weeks when delivered within the hydrogels and significantly improved vascularity, ventricular geometry, ejection fraction, cardiac output, and contractility compared with controls. Conclusions— We have developed a hydrogel delivery system that sustains the release of a bioactive endothelial progenitor cell chemokine during a 4-week period that preserves ventricular function in a rat model of myocardial infarction.

Novel regenerative therapy using cell-sheet covered with omentum flap delivers a huge number of cells in a porcine myocardial infarction model

OBJECTIVE A key challenge to applying cell transplantation to treat severely damaged myocardium is in delivering large numbers of cells with minimum cell loss. We developed a new implantation method using skeletal myoblast (SMB) sheets, wrapped with an omentum flap as a blood supply to deliver huge numbers of SMBs to the damaged heart. We examined whether this method could be used to deliver a large amount of cells to deteriorated porcine myocardium. METHODS Cell sheets were obtained by culturing mini-pig autologous SMB cells on temperature-responsive culture dishes. Myocardial infarction was induced by placing an ameroid constrictor around the left anterior descending artery. The mini-pigs were divided into 4 treatment groups (n = 6 in each): cell sheets with omentum, cell sheets only, omentum only, and sham operation. Each animal implant consisted of 30 cell sheets (1.5 × 10(7) cells per sheet). Six 5-layer constructs were each placed on a different area, immediately adjacent to but not overlapping one another, to cover the infarct and border regions. RESULTS The new regenerative cell delivery system using SMB sheets covered and wrapped with omentum resulted in (1) a significantly reduced infarct size causing, at least in part, a thin scar with thick well-vascularized cardiac tissue; (2) increased angiogenesis, as determined by a significantly higher vascular density; and (3) improved cardiac function, as determined by echocardiography, compared with the conventional method (SMB sheet implantation). CONCLUSIONS This cell delivery system shows potential for repairing the severely failed heart.

Continuous Flow Left Ventricular Assist Device Implant Significantly Improves Pulmonary Hypertension, Right Ventricular Contractility, and Tricuspid Valve Competence

Continuous flow left ventricular assist devices (CF LVAD) are being implanted with increasing frequency for end‐stage heart failure. At the time of LVAD implant, a large proportion of patients have pulmonary hypertension, right ventricular (RV) dysfunction, and tricuspid regurgitation (TR). RV dysfunction and TR can exacerbate renal dysfunction, hepatic dysfunction, coagulopathy, edema, and even prohibit isolated LVAD implant. Repairing TR mandates increased cardiopulmonary bypass time and bicaval cannulation, which should be reserved for the time of orthotopic heart transplantation. We hypothesized that CF LVAD implant would improve pulmonary artery pressures, enhance RV function, and minimize TR, obviating need for surgical tricuspid repair.

Does Stringent Restrictive Annuloplasty for Functional Mitral Regurgitation Cause Functional Mitral Stenosis and Pulmonary Hypertension

Background— It remains controversial whether restrictive mitral annuloplasty (RMA) for functional mitral regurgitation (MR) can induce functional mitral stenosis (MS) that may cause postoperative residual pulmonary hypertension (PH). Methods and Results— One hundred eight patients with left ventricular (LV) dysfunction and severe MR underwent RMA with stringent downsizing of the mitral annulus. Systolic pulmonary artery pressure (PAP) and mitral valve performance variables were determined by Doppler echocardiography prospectively and 1 month after RMA. Fifty-eight patients underwent postoperative hemodynamic measurements. Postoperative echocardiography showed a mean pressure half-time of 92±14 ms, a transmitral mean gradient of 2.9±1.1 mm Hg, and a mitral valve effective orifice area of 2.4±0.4 cm2, consistent with functional MS. Doppler-derived systolic PAP was 32±8 mm Hg, which correlated weakly with the transmitral mean gradient (&rgr;=0.23, P=0.02). Postoperative cardiac catheterization also showed significant improvements in LV volume and systolic function, pulmonary capillary wedge pressure, cardiac index, and systolic PAP; the latter was associated with LV end-diastolic pressure [standardized partial regression coefficient (SPRC)=0.51], pulmonary vascular resistance (SPRC=0.47), cardiac index (SPRC=0.37), and transmitral pressure gradient (SPRC=0.20). In a multivariate Cox proportional hazard model, postoperative PH (systolic PAP >40 mm Hg), but not mitral valve performance variables, was strongly associated with adverse cardiac events. Conclusions— RMA for functional MR resulted in varying degrees of functional MS. However, our data were more consistent with the residual PH being caused by LV dysfunction and pulmonary vascular disease than by the functional MS. The residual PH, not functional MS, was the major predictor of post-RMA adverse cardiac events.

Spatially Oriented, Temporally Sequential Smooth Muscle Cell-Endothelial Progenitor Cell Bi-Level Cell Sheet Neovascularizes Ischemic Myocardium

Background— Endothelial progenitor cells (EPCs) possess robust therapeutic angiogenic potential, yet may be limited in the capacity to develop into fully mature vasculature. This problem might be exacerbated by the absence of a neovascular foundation, namely pericytes, with simple EPC injection. We hypothesized that coculturing EPCs with smooth muscle cells (SMCs), components of the surrounding vascular wall, in a cell sheet will mimic the native spatial orientation and interaction between EPCs and SMCs to create a supratherapeutic angiogenic construct in a model of ischemic cardiomyopathy. Methods and Results— Primary EPCs and SMCs were isolated from Wistar rats. Confluent SMCs topped with confluent EPCs were spontaneously detached from the Upcell dish to create an SMC-EPC bi-level cell sheet. A rodent ischemic cardiomyopathy model was created by ligating the left anterior descending coronary artery. Rats were then immediately divided into 3 groups: cell-sheet transplantation (n=14), cell injection (n=12), and no treatment (n=13). Cocultured EPCs and SMCs stimulated an abundant release of multiple cytokines in vitro. Increased capillary density and improved blood perfusion in the borderzone elucidated the significant in vivo angiogenic potential of this technology. Most interestingly, however, cell fate–tracking experiments demonstrated that the cell-sheet EPCs and SMCs directly migrated into the myocardium and differentiated into elements of newly formed functional vasculature. The robust angiogenic effect of this cell sheet translated to enhanced ventricular function as demonstrated by echocardiography. Conclusions— Spatially arranged EPC-SMC bi-level cell-sheet technology facilitated the natural interaction between EPCs and SMCs, thereby creating structurally mature, functional microvasculature in a rodent ischemic cardiomyopathy model, leading to improved myocardial function.

Impact of untreated mild-to-moderate mitral regurgitation at the time of isolated aortic valve replacement on late adverse outcomes.

OBJECTIVE The impact of untreated mild-to-moderate mitral regurgitation (MR) on patients undergoing isolated aortic valve replacement (AVR) is uncertain. The aim of this study is to investigate its long-term effects on outcomes. METHOD We retrospectively reviewed 193 consecutive patients undergoing isolated AVR between 1993 and 2007. The mean age of the study group was 64+/-12 years, 59% were male and the mean preoperative ejection fraction was 59+/-12%. The pathologic aetiology and degree of MR was determined on preoperative echocardiogram. Patients were stratified into preoperative no/trivial MR (group I; n=134) versus mild-to-moderate MR (group II; n=59). The aetiology of MR in group II was either organic (n=35, 60%) or functional (n=24, 41%). Survival and functional outcome were compared between the two groups and analyses for predictors of adverse events were performed by the Cox proportional hazard model. RESULTS Operative mortality was 2.6% (n=5). In group II, mean degree of MR significantly decreased from 2.1+/-0.3 to 1.6+/-0.8 during the late period (p=0.003). The improvement in MR grade was more obvious in patients with functional aetiology. Although the actuarial survival was not significantly different between groups, freedom from re-admission for heart failure at 10 years was significantly lower in group II than in group I (23% vs 83%; p=0.002). Multivariate analysis demonstrated that independent predictors of heart failure were presence of mild-to-moderate MR (p=0.012, odds ratio (OR) 3.8) and left ventricular ejection fraction (p=0.004, OR 0.95). CONCLUSION Despite the significant reduction after isolated AVR, preoperative mild-to-moderate MR is an independent risk factor impacting long-term functional outcome. Our results suggested that the concomitant mitral valve surgery for mild-to-moderate MR is warranted, especially in patients with reduced left ventricular function.

Mechanism of Beneficial Effects of Restrictive Mitral Annuloplasty in Patients With Dilated Cardiomyopathy and Functional Mitral Regurgitation

Background— Restrictive mitral annuloplasty (RMA) often leads to reverse left ventricular (LV) remodeling in patients with advanced cardiomyopathy and functional mitral regurgitation. However, the mechanism responsible for its favorable effects on LV ejection performance has been poorly understood. We evaluated systolic wall stress using cineangiographic multidetector computed tomography (MDCT) and our developed software system to assess stress-shortening relations before and after RMA. Methods and Results— Twenty-four patients with dilated cardiomyopathy underwent 64-row MDCT before and 2 months after RMA. All patients underwent stringent downsizing annuloplasty with a semirigid complete ring. Reconstructed images were used to calculate LV end-diastolic index (EDVI) and end-systolic volume index (ESVI), LV ejection fraction, and regional and global end-systolic wall stress (ESS). After RMA, LVEDVI and LVESVI decreased from 151±52 to 131±53 mL/m2 (P=0.0001) and from 114±48 to 92±50 mL/m2 (P=0.0001), respectively. Global ESS decreased from 157±43 to 139±50 kdyne/cm2 (P=0.01), and LV ejection fraction improved from 27±8.0 to 33±13% (P=0.0007). There were significant correlations between change in LVEDVI and LVESVI (r=0.88, P<0.0001) and change in LVESVI and global ESS (r=0.68, P=0.0002). Moreover, the magnitude of increase in LV ejection fraction significantly correlated with the degree of reduction in global ESS (r=−0.61, P=0.002). Patients without significant reverse LV remodeling had significantly higher preoperative and postoperative global ESS than those with it. Conclusions— Our study suggests that decrease in afterload after reduction in volume overload was responsible for postoperative reverse LV remodeling process after RMA.

Assessment of Changes in Mitral Valve Configuration With Multidetector Computed Tomography: Impact of Papillary Muscle Imbrication and Ring Annuloplasty

Background— The optimal surgical procedures in functional mitral regurgitation remain controversial. We applied papillary muscle imbrication (PMI) combined with undersized mitral annuloplasty (UMAP). Multidetector computed tomography (MDCT) provides images of different phases of the cardiac cycle, allowing an assessment of the geometry. In the present study, we evaluated the mitral valve configuration and subvalvular apparatus before and after UMAP and/or PMI using MDCT imaging. Methods and Results— We studied 26 patients with functional mitral regurgitation (3+ to 4+) with an ejection fraction ≥35% who underwent diagnostic MDCT examinations before and early after the operation. Of these, 15 underwent UMAP and PMI (UMAP+PMI group) and 11 underwent UMAP (UMAP group). The annular anteroposterior diameter, tenting height, tenting area, and interpapillary muscle distance at end-systole were quantified. The annular anteroposterior diameter, tenting height, and tenting area were significantly decreased after the operation in both groups. Whereas the average change in annular anteroposterior diameter, tenting area, and interpapillary muscle distance did not differ between the 2 groups, the average change in tenting height was greater in the UMAP+PMI group than in the UMAP group (5.1±1.3 versus 3.8±2.3 mm, P=0.036). There was a significant correlation between the change in interpapillary muscle distance and the change in tenting height in the UMAP+PMI group (r=0.788, P=0.0005). Conclusions— Our results examined with MDCT indicated that UMAP combined with PMI improved leaflet tethering compared with UMAP, reflecting differences in the effects of the surgical procedures used, and suggested that concomitant PMI might be beneficial in some cases.

Preclinical Evaluation of the Engineered Stem Cell Chemokine Stromal Cell–Derived Factor 1α Analog in a Translational Ovine Myocardial Infarction ModelNovelty and Significance

Rationale: After myocardial infarction, there is an inadequate blood supply to the myocardium, and the surrounding borderzone becomes hypocontractile. Objective: To develop a clinically translatable therapy, we hypothesized that in a preclinical ovine model of myocardial infarction, the modified endothelial progenitor stem cell chemokine, engineered stromal cell–derived factor 1&agr; analog (ESA), would induce endothelial progenitor stem cell chemotaxis, limit adverse ventricular remodeling, and preserve borderzone contractility. Methods and Results: Thirty-six adult male Dorset sheep underwent permanent ligation of the left anterior descending coronary artery, inducing an anteroapical infarction, and were randomized to borderzone injection of saline (n=18) or ESA (n=18). Ventricular function, geometry, and regional strain were assessed using cardiac MRI and pressure–volume catheter transduction. Bone marrow was harvested for in vitro analysis, and myocardial biopsies were taken for mRNA, protein, and immunohistochemical analysis. ESA induced greater chemotaxis of endothelial progenitor stem cells compared with saline (P<0.01) and was equivalent to recombinant stromal cell–derived factor 1&agr; (P=0.27). Analysis of mRNA expression and protein levels in ESA-treated animals revealed reduced matrix metalloproteinase 2 in the borderzone (P<0.05), with elevated levels of tissue inhibitor of matrix metalloproteinase 1 and elastin in the infarct (P<0.05), whereas immunohistochemical analysis of borderzone myocardium showed increased capillary and arteriolar density in the ESA group (P<0.01). Animals in the ESA treatment group also had significant reductions in infarct size (P<0.01), increased maximal principle strain in the borderzone (P<0.01), and a steeper slope of the end-systolic pressure–volume relationship (P=0.01). Conclusions: The novel, biomolecularly designed peptide ESA induces chemotaxis of endothelial progenitor stem cells, stimulates neovasculogenesis, limits infarct expansion, and preserves contractility in an ovine model of myocardial infarction.

Normalization of Postinfarct Biomechanics Using a Novel Tissue-Engineered Angiogenic Construct

Background— Cell-mediated angiogenic therapy for ischemic heart disease has had disappointing results. The lack of clinical translatability may be secondary to cell death and systemic dispersion with cell injection. We propose a novel tissue-engineered therapy, whereby extracellular matrix scaffold seeded with endothelial progenitor cells (EPCs) can overcome these limitations using an environment in which the cells can thrive, enabling an insult-free myocardial cell delivery to normalize myocardial biomechanics. Methods and Results— EPCs were isolated from the long bones of Wistar rat bone marrow. The cells were cultured for 7 days in media or seeded at a density of 5×106 cells/cm2 on a collagen/vitronectin matrix. Seeded EPCs underwent ex vivo modification with stromal cell–derived factor-1&agr; (100 ng/mL) to potentiate angiogenic properties and enhance paracrine qualities before construct formation. Scanning electron microscopy and confocal imaging confirmed EPC–matrix adhesion. In vitro vasculogenic potential was assessed by quantifying EPC cell migration and vascular differentiation. There was a marked increase in vasculogenesis in vitro as measured by angiogenesis assay (8 versus 0 vessels/hpf; P=0.004). The construct was then implanted onto ischemic myocardium in a rat model of acute myocardial infarction. Confocal microscopy demonstrated a significant migration of EPCs from the construct to the myocardium, suggesting a direct angiogenic effect. Myocardial biomechanical properties were uniaxially quantified by elastic modulus at 5% to 20% strain. Myocardial elasticity normalized after implant of our tissue-engineered construct (239 kPa versus normal=193, P=0.1; versus infarct=304 kPa, P=0.01). Conclusions— We demonstrate restoration and normalization of post–myocardial infarction ventricular biomechanics after therapy with an angiogenic tissue-engineered EPC construct.