Metabolic Impact of Rapamycin (Sirolimus) and B-Estradiol Using Mouse Embryonic Fibroblasts as a Model for Lymphangioleiomyomatosis.

INTRODUCTION: Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease that predominantly affects women of childbearing age. Exogenous rapamycin (sirolimus) has been shown to improve clinical outcomes and was recently approved to treat LAM, whereas estrogen (E2) is implicated in disease progression. No consistent metabolic model currently exists for LAM, therefore wild-type mouse embryonic fibroblasts (MEF +/+) and TSC2 knockout cells (MEF -/-) were used in this study as a model for LAM. METHODS: Oxygen consumption rates (OCR) and redox potential were measured to determine metabolic state across control cells, MEF +/+ and -/- cells treated with rapamycin (Rapa), and MEF +/+ and -/- cells treated with E2. An XF96 extracellular flux analyzer from Seahorse Bioscience® was used to measure OCR, and a RedoxSYS™ ORP was used to measure redox potential. RESULTS: OCR of MEF -/- cells treated with rapamycin (MEF -/- Rapa) versus MEF -/- control were significantly lower across all conditions. The static oxidation reduction potential of the MEF -/- Rapa group was also lower, approaching significance. The coupling efficiency and ratio of ATP-linked respiration to maximum respiration were statistically lower in MEF -/- Rapa compared to MEF +/+ Rapa. There were no significant metabolic findings across any of the MEF cells treated with E2. MEF -/- control cells versus MEF +/+ control cells were not found to significantly differ. CONCLUSION: MEF cells are thought to be a feasible metabolic model for LAM, which has implications for future pharmacologic and biologic testing.

Cardiac Regeneration in the Human Left Ventricle After CorMatrix Implantation.

CorMatrix is an organic extracellular matrix (ECM) derived from porcine small intestine submucosa and is used for pericardial closure and cardiac tissue repair. During explantation of a HeartMate II (Thoratec Corp, Pleasanton, CA) left ventricular assist device (LVAD) because of infection, CorMatrix was used to repair the left ventricular apex and aorta. Three months later, a HeartWare HVAD (HeartWare International, Inc, Framingham, MA) was implanted for recurrent heart failure. Excised apical CorMatrix samples showed cardiac tissue remodeling with viable cardiomyoblasts similar to native myocardium. Excised CorMatrix from the aorta showed organization of collagen and elastin similar to native aortic tissue.

Chronic Myocardial Ischemia Leads to Loss of Maximal Oxygen Consumption and Complex I Dysfunction.

BACKGROUND: Cardiomyocytes rely heavily on mitochondrial energy production through oxidative phosphorylation. Chronic myocardial ischemia may cause mitochondrial dysfunction and affect ATP formation. Metabolic changes due to ischemia alters cardiac bioenergetics and hence myocardial function and overall bioenergetic state. Here, we evaluate differences in functional status of respiratory complexes in mitochondrial isolates extracted from left atrial appendage tissue (LAA) from patients undergoing cardiac surgery, with and without chronic ischemia. METHODS: Mitochondrial isolates were extracted from LAA in ischemic coronary artery bypass grafting patients (n = 8) and non-ischemic control patients (n = 6) undergoing other cardiac surgery (valve repair/replacement). Coupling and electron transport chain assays were performed using Seahorse XFe 96 (Agilent Technologies, Santa Clara, CA) analyzer. Oxygen consumption rates were measured to calculate respiration states. RESULTS: Respiratory control rate (RCR) in ischemic patients was significantly lower than control patients (6.17 ± 0.27 vs 7.11 ± 0.31, respectively; p < 0.05). This is the result of minimal, non-significant state 3ADP and state 4O changes in chronic ischemia. Complex I respiration is diminished in ischemic tissue (99.1 ± 14.9 vs 257.8 ± 65.2 in control; p < 0.01). Maximal complex I/II respiration ratio was significantly lower in ischemic patients (58.9% ± 5.5% vs 90.9% ± 8.8%; p < 0.05), a difference that was also seen in complex I/IV ratios (p < 0.05). There was no significant difference in complex II/IV ratios between groups. CONCLUSIONS: Ischemic patients have aberrant mitochondrial function, highlighted by a lowered RCR. All ratios involving complex I were affected, suggesting that the insufficient ATP formation is predominantly due to complex I dysfunction. Complex II and IV respiration may be impaired as well, but to a lesser extent.

Improved metabolism and redox state with a novel preservation solution: implications for donor lungs after cardiac death (DCD).

Lungs donated after cardiac death (DCD) are an underutilized resource for a dwindling donor lung transplant pool. Our study investigates the potential of a novel preservation solution, Somah, to better preserve statically stored DCD lungs, for an extended time period, when compared to low-potassium dextran solution (LPD). We hypothesize that Somah is a metabolically superior organ preservation solution for hypothermic statically stored porcine DCD lungs, possibly improving lung transplant outcomes. Porcine DCD lungs (n = 3 per group) were flushed with and submerged in cold preservation solution. The lungs were stored up to 12 h, and samples were taken from lung tissue and the preservation medium throughout. Metabolomic and redox potential were analyzed using high performance liquid chromatography, mass spectrometry, and RedoxSYS®, comparing substrate and pathway utilization in both preservation solutions. Glutathione reduction was seen in Somah but not in LPD during preservation. Carnitine, carnosine, and n-acetylcarnosine levels were elevated in the Somah medium compared with LPD throughout. Biopsies of Somah exposed lungs demonstrated similar trends after 2 h, up to 12 h. Adenosine gradually decreased in Somah medium over 12 h, but not in LPD. An inversely proportional increase in inosine was found in Somah. Higher oxidative stress levels were measured in LPD. Our study suggests suboptimal metabolic preservation in lungs stored in LPD. LPD had poor antioxidant potential, cytoprotection, and an insufficient redox potential. These findings may have immediate clinical implications for human organs; however, further investigation is needed to evaluate DCD lung preservation in Somah as a viable option for transplant.

Acellular porcine heart matrices: whole organ decellularization with 3D-bioscaffold & vascular preservation.

Regenerative medicine, particularly decellularization-recellularization methods via whole-organ tissue engineering, has been increasingly studied due to the growing donor organ shortage. Though numerous decellularization protocols exist, the ideal decellularization protocol for optimal recellularization is unclear. This study was performed to optimize existing heart decellularization protocols and compare current methods using the detergents SDS (sodium dodecyl sulfate), Triton X-100, OGP (octyl ß-D-glucopyranoside), and CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) through retrograde aortic perfusion via aortic cannulation of a whole porcine heart. The goal of decellularization is to preserve extracellular matrix integrity and architecture, which was analyzed in this study through histology, microscopy, DNA analysis, hydroxyproline content analysis, materials analysis and angiography. Effective decellularization was determined by analyzing the tissue organization, geometry, and biological properties of the resultant extracellular matrix scaffold. Using these parameters, optimal decellularization was achieved between 90 and 120 mmHg pressure with 3% SDS as a detergent. Relevance for patients: This study provides important information about whole heart decellularization, which will ultimately contribute to heart bioengineering.

Adipose-derived human stem/stromal cells: comparative organ specific mitochondrial bioenergy profiles.

BACKGROUND: Adipose-derived stem/stromal cells (ASCs) isolated from the stromal vascular fraction are a source of mesenchymal stem cells that have been shown to be beneficial in many regenerative medicine applications. ASCs are an attractive source of stem cells in particular, due to their lack of immunogenicity. This study examines differences between mitochondrial bioenergetic profiles of ASCs isolated from adipose tissue of five peri-organ regions: pericardial, thymic, knee, shoulder, and abdomen. RESULTS: Flow cytometry showed that the majority of each ASC population isolated from the adipose tissue of 12 donors, with an n = 3 for each tissue type, were positive for MSC markers CD90, CD73, and CD105, and negative for hematopoietic markers CD34, CD11B, CD19, and CD45. Bioenergetic profiles were obtained for ASCs with an n = 4 for each tissue type and graphed together for comparison. Mitochondrial stress tests provided the following measurements: basal respiration rate (measured as oxygen consumption rate [pmol O2/min], ATP production, proton leak, maximal respiration, respiratory control ratio, coupling efficiency, and non-mitochondrial respiration. Glycolytic stress tests provided the following measurements: basal glycolysis rate (measured as extracellular acidification rate [mpH/min]), glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. CONCLUSIONS: The main goal of this manuscript was to provide baseline reference values for future experiments and to compare bioenergetic potentials of ASCs isolated from adipose tissue harvested from different anatomical locations. Through an investigation of mitochondrial respiration and glycolysis, it was demonstrated that bioenergetic profiles do not significantly differ by region due to depot-dependent and donor-dependent variability. Thus, although the physiological function, microenvironment and anatomical harvest site may directly affect the characteristics of ASCs isolated from different organ regions, the ultimate utility of ASCs remains independent of the anatomical harvest site.

First in Man: Sternal Reconstruction with Autologous Stem Cells.

Sternal nonunion is associated with high morbidity and treated using rigid plate and screw fixation. This is the first reported example of successful sternal reconstruction using adipose-derived stromal vascular fraction (SVF) stem cells in addition to traditional techniques. Mesenchymal stem cells, one component of the SVF, play an important role in bone healing and were therefore used to promote remedial processes in a patient with sternal nonunion. A 3D printed model of the patient's sternum was used for preoperative planning of the plating. Intraoperatively, SVF was isolated using ultrasonic cavitation and previously planned sternal plating was completed. A total of 300 million cells were delivered via both local injection and intravenously before chest closure. The patient's pain dramatically decreased, commensurate with healed areas of nonunion by 3 months and maintained at 6 months postoperatively, supported by three-dimensional computed tomography imaging. Utilizing autologous stem cells from the SVF in conjunction with existing plating techniques may provide an optimal platform to stabilize the sternum and promote bone healing, although additional study is recommended.