3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension.

BACKGROUND: Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure. METHODS: Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function. RESULTS: Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments. CONCLUSIONS: 3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

Prevalence of Urogenital Mycoplasma genitalium Infection at 2 US Army Medical Facilities.

BACKGROUND: Sexually transmitted infections (STIs) have a high incidence in the US Armed Forces and can adversely impact service members' ability to perform their duties. Better knowledge of Mycoplasma genitalium (MG) epidemiology in the military is needed to understand the potential impact of this emerging pathogen on force readiness. METHODS: We conducted cross-sectional analyses of data from US Army service members and other Military Health System beneficiaries participating in a trial of an STI/HIV behavioral intervention at Fort Liberty, NC, and Joint Base Lewis-McChord, WA. At enrollment, participants completed questionnaires and provided biological specimens for nucleic acid amplification testing for MG, Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (NG). We used principal component analysis and robust Poisson regression to examine associations between participant characteristics and prevalent urogenital MG. RESULTS: Among 432 participants enrolled between November 2020 and February 2023, 43 had MG (prevalence, 10.0%), of whom 13 had coinfection with another bacterial STI (all 13 were positive for CT, with 1 also positive for NG). The prevalence of MG was significantly higher among female (13.5%) versus male (7.6%; P = 0.048) participants and non-Hispanic Black (14.9%) versus non-Hispanic White participants (6.6%; P = 0.045). Single relationship status and increased number of recent sexual partners were correlated, and their component was associated with higher MG prevalence (adjusted prevalence ratio, 2.11; 95% confidence interval, 1.29-3.48). CONCLUSIONS: The high prevalence of urogenital MG among Military Health System beneficiaries highlights the importance of understanding the potential clinical sequelae of MG and conducting additional epidemiologic research in military settings.

Mortality and morbidity after combined heart and liver transplantation in the failing Fontan: An updated dual center retrospective study.

INTRODUCTION: As the adult Fontan population with Fontan associated liver disease continues to increase, more patients are being referred for transplantation, including combined heart and liver transplantation. METHODS: We report updated mortality and morbidity outcomes after combined heart and liver transplant in a retrospective cohort series of 40 patients (age 14 to 49 years) with Fontan circulation across two centers from 2006-2022. RESULTS: The 30-day, 1-year, 5-year and 10-year survival rate was 90%, 80%, 73% and 73% respectively. Sixty percent of patients met a composite comorbidity of needing either post-transplant mechanical circulatory support, renal replacement therapy or tracheostomy. Cardiopulmonary bypass time > 283 min (4.7 h) and meeting the composite comorbidity were associated with mortality by Kaplan Meier analysis. CONCLUSION: Further study to mitigate early mortality and the above comorbidities as well as the high risk of bleeding and vasoplegia in this patient population is warranted.

Post-Transplant Extracorporeal Membrane Oxygenation for Severe Primary Graft Dysfunction to Support the Use of Marginal Donor Hearts.

Severe primary graft dysfunction (PGD) is the leading cause of early postoperative mortality following orthotopic heart transplantation (OHT). Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) has been used as salvage therapy. This study aimed to evaluate the outcomes in adult OHT recipients who underwent VA-ECMO for severe PGD. We retrospectively reviewed 899 adult (≥18 years) patients who underwent primary OHT at our institution between 1997 and 2017. Recipients treated with VA-ECMO (19, 2.1%) exhibited a higher incidence of previous cardiac surgery (p = .0220), chronic obstructive pulmonary disease (p = .0352), and treatment with a calcium channel blocker (p = .0018) and amiodarone (p = .0148). Cardiopulmonary bypass (p = .0410) and aortic cross-clamp times (p = .0477) were longer in the VA-ECMO cohort and they were more likely to have received postoperative transfusion (p = .0013); intra-aortic balloon pump (IABP, p < .0001), and reoperation for bleeding or tamponade (p < .0001). The 30-day, 1-year, and overall survival after transplantation of non-ECMO patients were 95.9, 88.8, and 67.4%, respectively, compared to 73.7, 57.9, and 47.4%, respectively in the ECMO cohort. Fourteen (73.7%) of the ECMO patients were weaned after a median of 7 days following OHT (range: 1-12 days). Following OHT, VA-ECMO may be a useful salvage therapy for severe PGD and can potentially support the usage of marginal donor hearts.

Comprehensive Ex Vivo Comparison of 5 Clinically Used Conduit Configurations for Valve-Sparing Aortic Root Replacement Using a 3-Dimensional-Printed Heart Simulator.

BACKGROUND: Many graft configurations are clinically used for valve-sparing aortic root replacement, some specifically focused on recapitulating neosinus geometry. However, the specific impact of such neosinuses on valvular and root biomechanics and the potential influence on long-term durability are unknown. METHODS: Using a custom 3-dimenstional-printed heart simulator with porcine aortic roots (n=5), the anticommissural plication, Stanford modification, straight graft (SG), Uni-Graft, and Valsalva graft configurations were tested in series using an incomplete counterbalanced measures design, with the native root as a control, to mitigate ordering effects. Hemodynamic and videometric data were analyzed using linear models with conduit as the fixed effect of interest and valve as a fixed nuisance effect with post hoc pairwise testing using Tukey's correction. RESULTS: Hemodynamics were clinically similar between grafts and control aortic roots. Regurgitant fraction varied between grafts, with SG and Uni-Graft groups having the lowest regurgitant fractions and anticommissural plication having the highest. Root distensibility was significantly lower in SG versus both control roots and all other grafts aside from the Stanford modification (P≤0.01 for each). All grafts except SG had significantly higher cusp opening velocities versus native roots (P<0.01 for each). Relative cusp opening forces were similar between SG, Uni-Graft, and control groups, whereas anticommissural plication, Stanford modification, and Valsalva grafts had significantly higher opening forces versus controls (P<0.01). Cusp closing velocities were similar between native roots and the SG group, and were significantly lower than observed in the other conduits (P≤0.01 for each). Only SG and Uni-Graft groups experienced relative cusp closing forces approaching that of the native root, whereas relative forces were >5-fold higher in the anticommissural plication, Stanford modification, and Valsalva graft groups. CONCLUSIONS: In this ex vivo modeling system, clinically used valve-sparing aortic root replacement conduit configurations have comparable hemodynamics but differ in biomechanical performance, with the straight graft most closely recapitulating native aortic root biomechanics.

Signalosome-Regulated Serum Response Factor Phosphorylation Determining Myocyte Growth in Width Versus Length as a Therapeutic Target for Heart Failure.

BACKGROUND: Concentric and eccentric cardiac hypertrophy are associated with pressure and volume overload, respectively, in cardiovascular disease both conferring an increased risk of heart failure. These contrasting forms of hypertrophy are characterized by asymmetrical growth of the cardiac myocyte in mainly width or length, respectively. The molecular mechanisms determining myocyte preferential growth in width versus length remain poorly understood. Identification of the mechanisms governing asymmetrical myocyte growth could provide new therapeutic targets for the prevention or treatment of heart failure. METHODS: Primary adult rat ventricular myocytes, adeno-associated virus (AAV)-mediated gene delivery in mice, and human tissue samples were used to define a regulatory pathway controlling pathological myocyte hypertrophy. Chromatin immunoprecipitation assays with sequencing and precision nuclear run-on sequencing were used to define a transcriptional mechanism. RESULTS: We report that asymmetrical cardiac myocyte hypertrophy is modulated by SRF (serum response factor) phosphorylation, constituting an epigenomic switch balancing the growth in width versus length of adult ventricular myocytes in vitro and in vivo. SRF Ser103 phosphorylation is bidirectionally regulated by RSK3 (p90 ribosomal S6 kinase type 3) and PP2A (protein phosphatase 2A) at signalosomes organized by the scaffold protein mAKAPß (muscle A-kinase anchoring protein ß), such that increased SRF phosphorylation activates AP-1 (activator protein-1)-dependent enhancers that direct myocyte growth in width. AAV are used to express in vivo mAKAPß-derived RSK3 and PP2A anchoring disruptor peptides that block the association of the enzymes with the mAKAPß scaffold. Inhibition of RSK3 signaling prevents concentric cardiac remodeling induced by pressure overload, while inhibition of PP2A signaling prevents eccentric cardiac remodeling induced by myocardial infarction, in each case improving cardiac function. SRF Ser103 phosphorylation is significantly decreased in dilated human hearts, supporting the notion that modulation of the mAKAPß-SRF signalosome could be a new therapeutic approach for human heart failure. CONCLUSIONS: We have identified a new molecular switch, namely mAKAPß signalosome-regulated SRF phosphorylation, that controls a transcriptional program responsible for modulating changes in cardiac myocyte morphology that occur secondary to pathological stressors. Complementary AAV-based gene therapies constitute rationally-designed strategies for a new translational modality for heart failure.

Clinical Characteristics of Patients Hospitalized with Coronavirus Disease, Thailand.

Among 11 patients in Thailand infected with severe acute respiratory syndrome coronavirus 2, we detected viral RNA in upper respiratory specimens a median of 14 days after illness onset and 9 days after fever resolution. We identified viral co-infections and an asymptomatic person with detectable virus RNA in serial tests. We describe implications for surveillance.

Surgical excision of a free floating ascending aortic thrombus.

BACKGROUND: A 73-year-old female presented with leg claudication and chest pain. A mobile mass in the ascending aorta was found. RESULTS: The mass was removed through a transverse aortotomy on circulatory arrest via sternotomy. CONCLUSION: Free-floating ascending aortic thrombus is a rare source of peripheral embolization. We advocate for emergent surgical resection to prevent further embolization and stroke.

A modified implantation technique for temporary right ventricular assist device: Enabling ambulation and less invasive decannulation.

This report describes our unique temporary right ventricular assist device (RVAD) implantation technique, which enables early mobilization even during biventricular support and subsequent less invasive RVAD removal without needing resternotomy upon recovery.

Emergence and Spread of kelch13 Mutations Associated with Artemisinin Resistance in Plasmodium falciparum Parasites in 12 Thai Provinces from 2007 to 2016.

Artemisinin-based combination therapy (ACT) is the most effective and widely used treatment for uncomplicated Plasmodium falciparum malaria and is a cornerstone for malaria control and prevention globally. Resistance to artemisinin derivatives has been confirmed in the Greater Mekong Subregion (GMS) and manifests as slow parasite clearance in patients and reduced ring stage susceptibility to artemisinins in survival assays. The P. falciparumkelch13 gene mutations associated with artemisinin-resistant parasites are now widespread in the GMS. We genotyped 277 samples collected during an observational study from 2012 to 2016 from eight provinces in Thailand to identify P. falciparum kelch13 mutations. The results were combined with previously reported genotyping results from Thailand to construct a map illustrating the evolution of P. falciparum kelch13 mutations from 2007 to 2016 in that country. Different mutant alleles were found in strains with different geographical origins. The artemisinin resistance-conferring Y493H and R539T mutations were detected mainly in eastern Thailand (bordering Cambodia), while P574L was found only in western Thailand and R561H only in northwestern Thailand. The C580Y mutation was found across the entire country and was nearing fixation along the Thai-Cambodia border. Overall, the prevalence of artemisinin resistance mutations increased over the last 10 years across Thailand, especially along the Thai-Cambodia border. Molecular surveillance and therapeutic efficacy monitoring should be intensified in the region to further assess the extent and spread of artemisinin resistance.

Developmental Defects Mediated by the P1/HC-Pro Potyviral Silencing Suppressor Are Not Due to Misregulation of AUXIN RESPONSE FACTOR 8.

Plant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA pathway. A recent report has attributed viral suppressor-mediated developmental defects to up-regulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence was that the developmental defects in transgenic Arabidopsis (Arabidopsis thaliana) expressing viral suppressors were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. We have reinvestigated the role of ARF8 in viral suppressor-mediated developmental defects, using two independent arf8 mutations and the P1/HC-Pro potyviral suppressor of silencing. Progeny of a cross between P1/HC-Pro transgenic Arabidopsis and the arf8-6 T-DNA insertion mutant showed little effect on the P1/HC-Pro phenotype in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the phenotype in the F2 generation. However, the loss of phenotype in the F2 generation was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA and the appearance of 35S promoter small interfering RNAs. Furthermore, an independent loss-of-function point mutation, Arf8-8, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together, these data argue against the previously reported role of increased ARF8 expression in developmental defects caused by P1/HC-Pro.

Biochemically engineered stromal cell-derived factor 1-alpha analog increases perfusion in the ischemic hind limb.

BACKGROUND: Despite promising therapeutic innovation over the last decade, peripheral arterial disease remains a prevalent morbidity, as many patients are still challenged with peripheral ischemia. We hypothesized that delivery of engineered stromal cell-derived factor 1-alpha (ESA) in an ischemic hind limb will yield significant improvement in perfusion. METHODS: Male rats underwent right femoral artery ligation, and animals were randomized to receive a 100 µL injection of saline (n = 9) or 6 µg/kg dosage of equal volume of ESA (n = 12) into the ipsilateral quadriceps muscle. Both groups of animals were also given an intraperitoneal injection of 40 µg/kg of granulocyte macrophage colony-stimulating factor (GMCSF). Perfusion was quantified using a laser Doppler imaging device preoperatively, and on postoperative days 0, 7, and 14. Immunohistochemistry was performed to quantify angiogenesis on day 14, and an mRNA profile was evaluated for angiogenic and inflammatory markers. RESULTS: Compared with the saline/GMCSF group at day 14, the ESA/GMCSF-injected animals had greater reperfusion ratios (Saline/GMCSF, 0.600 ± 0.140 vs ESA/GMCSF, 0.900 ± 0.181; group effect P = .006; time effect P < .0001; group×time effect P < .0001), elevated capillary density (10×; Saline/GMCSF, 6.40 ± 2.01 vs ESA/GMCSF, 18.55 ± 5.30; P < .01), and increased mRNA levels of vascular endothelial growth factor-A (Saline/GMCSF [n = 6], 0.298 ± 0.205 vs ESA/GMCSF [n = 8], 0.456 ± 0.139; P = .03). CONCLUSIONS: Delivery of ESA significantly improves perfusion in a rat model of peripheral arterial disease via improved neovasculogenesis, a finding which may prove beneficial in the treatment strategy for this debilitating disease.

Isolation and trans-differentiation of mesenchymal stromal cells into smooth muscle cells: Utility and applicability for cell-sheet engineering.

BACKGROUND: Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) have shown potential to differentiate into various cell types, including smooth muscle cells (SMCs). The extracellular matrix (ECM) represents an appealing and readily available source of SMCs for use in tissue engineering. In this study, we hypothesized that the ECM could be used to induce MSC differentiation to SMCs for engineered cell-sheet construction. METHODS: Primary MSCs were isolated from the BM of Wistar rats, transferred and cultured on dishes coated with 3 different types of ECM: collagen type IV (Col IV), fibronectin (FN), and laminin (LM). Primary MSCs were also included as a control. The proportions of SMC (a smooth muscle actin [aSMA] and SM22a) and MSC markers were examined with flow cytometry and Western blotting, and cell proliferation rates were also quantified. RESULTS: Both FN and LM groups were able to induce differentiation of MSCs toward smooth muscle-like cell types, as evidenced by an increase in the proportion of SMC markers (aSMA; Col IV 42.3 ± 6.9%, FN 65.1 ± 6.5%, LM 59.3 ± 7.0%, Control 39.9 ± 3.1%; P = 0.02, SM22; Col IV 56.0 ± 7.7%, FN 74.2 ± 6.7%, LM 60.4 ± 8.7%, Control 44.9 ± 3.6%) and a decrease in that of MSC markers (CD105: Col IV 64.0 ± 5.2%, FN 57.6 ± 4.0%, LM 60.3 ± 7.0%, Control 85.3 ± 4.2%; P = 0.03). The LM group showed a decrease in overall cell proliferation, whereas FN and Col IV groups remained similar to control MSCs (Col IV, 9.0 ± 2.3%; FN, 9.8 ± 2.5%; LM, 4.3 ± 1.3%; Control, 9.8 ± 2.8%). CONCLUSIONS: Our findings indicate that ECM selection can guide differentiation of MSCs into the SMC lineage. Fibronectin preserved cellular proliferative capacity while yielding the highest proportion of differentiated SMCs, suggesting that FN-coated materials may be facilitate smooth muscle tissue engineering.

Preclinical evaluation of the engineered stem cell chemokine stromal cell-derived factor 1α analog in a translational ovine myocardial infarction model.

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 1α 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 1α (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.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine for treatment of uncomplicated malaria in children in Zaire and Uíge Provinces, angola.

The development of resistance to antimalarials is a major challenge for global malaria control. Artemisinin-based combination therapies, the newest class of antimalarials, are used worldwide but there have been reports of artemisinin resistance in Southeast Asia. In February through May 2013, we conducted open-label, nonrandomized therapeutic efficacy studies of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) in Zaire and Uíge Provinces in northern Angola. The parasitological and clinical responses to treatment in children with uncomplicated Plasmodium falciparum monoinfection were measured over 28 days, and the main outcome was a PCR-corrected adequate clinical and parasitological response (ACPR) proportion on day 28. Parasites from treatment failures were analyzed for the presence of putative molecular markers of resistance to lumefantrine and artemisinins, including the recently identified mutations in the K13 propeller gene. In the 320 children finishing the study, 25 treatment failures were observed: 24 in the AL arms and 1 in the DP arm. The PCR-corrected ACPR proportions on day 28 for AL were 88% (95% confidence interval [CI], 78 to 95%) in Zaire and 97% (91 to 100%) in Uíge. For DP, the proportions were 100% (95 to 100%) in Zaire, and 100% (96 to 100%) in Uíge. None of the treatment failures had molecular evidence of artemisinin resistance. In contrast, 91% of AL late-treatment failures had markers associated with lumefantrine resistance on the day of failure. The absence of molecular markers for artemisinin resistance and the observed efficacies of both drug combinations suggest no evidence of artemisinin resistance in northern Angola. There is evidence of increased lumefantrine resistance in Zaire, which should continue to be monitored.

Shear-Thinning Supramolecular Hydrogels with Secondary Autonomous Covalent Crosslinking to Modulate Viscoelastic Properties In Vivo.

Clinical percutaneous delivery of synthetically engineered hydrogels remains limited due to challenges posed by crosslinking kinetics - too fast leads to delivery failure, too slow limits material retention. To overcome this challenge, we exploit supramolecular assembly to localize hydrogels at the injection site and introduce subsequent covalent crosslinking to control final material properties. Supramolecular gels were designed through the separate pendant modifications of hyaluronic acid (HA) by the guest-host pair cyclodextrin and adamantane, enabling shear-thinning injection and high target site retention (>98%). Secondary covalent crosslinking occurred via addition of thiols and Michael-acceptors (i.e., methacrylates, acrylates, vinyl sulfones) on HA and increased hydrogel moduli (E=25.0±4.5kPa) and stability (>3.5 fold in vivo at 28 days). Application of the dual-crosslinking hydrogel to a myocardial infarct model showed improved outcomes relative to untreated and supramolecular hydrogel alone controls, demonstrating its potential in a range of applications where the precise delivery of hydrogels with tunable properties is desired.

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 × 10(6) cells/cm(2) on a collagen/vitronectin matrix. Seeded EPCs underwent ex vivo modification with stromal cell-derived factor-1α (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.

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.