Self-initiated lifestyle interventions lead to potential insight into an effective, alternative, non-surgical therapy for mitochondrial disease associated multiple symmetric lipomatosis.

BACKGROUND: A 56-year-old female, diagnosed as a carrier of the mitochondrial DNA mutation (MTTK c.8344A > G) associated with the MERRF (myoclonic epilepsy with ragged red fibers) syndrome, presented with a relatively uncommon but well-known phenotypic manifestation: severe multiple symmetric lipomatosis (MSL). After surgical resection of three kilograms of upper mid-back lipomatous tissue, the patient experienced a significant decline in her functional capacity and quality of life, which ultimately resulted in her placement on long-term disability. METHODS: Dissatisfied with the available treatment options centered on additional resection surgeries, given the high probability of lipoma regrowth, the patient independently researched and applied alternative therapies that centred on a carbohydrate-restricted diet and a supervised exercise program. RESULTS: The cumulative effect of her lifestyle interventions resulted in the reversal of her MSL and her previously low quality of life. She met all her personal goals by the one-year mark, including reduced size of the residual post-surgical lipomas, markedly enhanced exercise tolerance, and return to work. She continues to maintain her interventions and to experience positive outcomes at the two-year mark. INTERPRETATION: This case report documents the timing and nature of lifestyle interventions in relation to the reversal in growth pattern of her previously expanding and debilitating lipomas. The profound nature of the apparent benefit on lipoma growth demonstrates the intervention's potential as a new feasible non-surgical therapy for mitochondrial-disease-associated MSL, and justifies its systematic study. We also describe how this case has inspired the care team to re-examine its approach to involved patients.

Plasma Neutrophil Gelatinase-Associated Lipocalin Is Associated With Acute Kidney Injury and Clinical Outcomes in Neonates Undergoing Cardiopulmonary Bypass.

OBJECTIVES: Acute kidney injury is a frequent complication following neonatal cardiac surgery and is associated with significant morbidity and mortality. The objectives of this study were to determine if plasma neutrophil gelatinase-associated lipocalin levels were associated with acute kidney injury and clinical outcomes in neonates with congenital heart disease undergoing cardiopulmonary bypass. DESIGN: Retrospective single-center observational study. SETTING: A pediatric cardiac ICU within a tertiary-care academic hospital. PATIENTS: Patients age less than 30 days undergoing cardiac surgery requiring cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma neutrophil gelatinase-associated lipocalin peaked at 12 hours postcardiopulmonary bypass and more than doubled compared with preoperative levels. Higher preoperative and 24-hour postoperative neutrophil gelatinase-associated lipocalin levels were associated with acute kidney injury (r = 0.30, r = 0.49), longer duration of mechanical ventilation (r = 0.40, r = 0.51), ICU (r = 0.32, r = 0.33) and hospital lengths of stay (r = 0.28, r = 0.32), and total hospital charges (r = 0.35, r = 0.30; all p values < 0.05). CONCLUSIONS: Both preoperative and 24-hour postoperative plasma neutrophil gelatinase-associated lipocalin levels are associated with acute kidney injury and worse clinical outcomes in neonates undergoing cardiac surgery. Plasma neutrophil gelatinase-associated lipocalin levels may have a role in risk stratification for predicting postoperative renal dysfunction as well as providing a potential clinical trajectory in the postoperative period.

Elevated Wall Tension Leads to Reduced miR-133a in the Thoracic Aorta by Exosome Release.

Background Reduced miR-133a was previously found to be associated with thoracic aortic ( TA ) dilation, as seen in aneurysm disease. Because wall tension increases with vessel diameter (Law of Laplace), this study tested the hypothesis that elevated tension led to the reduction of miR-133a in the TA . Methods and Results Elevated tension (1.5 g; 150 mm Hg) applied to murine TA ex vivo reduced miR-133a tissue abundance compared with TA held at normotension (0.7 g; 70 mm Hg). Cellular miR-133a levels were reduced with biaxial stretch of isolated murine TA fibroblasts, whereas smooth muscle cells were not affected. Mechanisms contributing to the loss of miR-133a abundance were further investigated in TA fibroblasts. Biaxial stretch did not reduce primary miR-133a transcription and had no effect on the expression/abundance of 3 micro RNA -specific exoribonucleases. Remarkably, biaxial stretch increased exosome secretion, and exosomes isolated from TA fibroblasts contained more miR-133a. Inhibition of exosome secretion prevented the biaxial stretch-induced reduction of miR-133a. Subsequently, 2 in vivo models of hypertension were used to determine the effect of elevated wall tension on miR-133a abundance in the TA : wild-type mice with osmotic pump-mediated angiotensin II infusion and angiotensin II -independent spontaneously hypertensive mice. Interestingly, the abundance of miR-133a was decreased in TA tissue and increased in the plasma in both models of hypertension compared with a normotensive control group. Furthermore, miR-133a was elevated in the plasma of hypertensive human subjects, compared with normotensive patients. Conclusions Taken together, these results identified exosome secretion as a tension-sensitive mechanism by which miR-133a abundance was reduced in TA fibroblasts.

Regulation of membrane type-1 matrix metalloproteinase activity and intracellular localization in clinical thoracic aortic aneurysms.

OBJECTIVE: Membrane type-1 matrix metalloproteinase (MT1-MMP) is elevated during thoracic aortic aneurysm (TAA) development in mouse models, and plays an important role in the activation of matrix metalloproteinase (MMP)-2 and the release of matrix- bound transforming growth factor-ß. In this study, we tested the hypothesis that MT1-MMP is subject to protein kinase C (PKC)-mediated regulation, which alters intracellular trafficking and activity with TAAs. METHODS: Levels of MMP-2, native and phosphorylated MT1-MMP, and PKC-δ were measured in aortic tissue from patients with small TAAs (<5 cm; n = 8) and large TAAs (>6.5 cm; n = 8), and compared with values measured in normal controls (n = 8). Cellular localization of green fluorescent protein (GFP)-tagged MT1-MMP was assessed in aortic fibroblasts isolated from control and 4-week TAA mice. The effects of PKC-mediated phosphorylation on MT1-MMP cellular localization and function (active MMP-2 vs phospo-Smad2 abundance) were assessed after treatment with a PKC activator (phorbol-12-myristate-13-acetate [PMA], 100 nM) with and without a PKC-δ-specific inhibitor (röttlerin, 3 µM). RESULTS: Compared with controls, MT1-MMP abundance was increased in aortas from both TAA groups. Active MMP-2 was increased only in the large TAA group. The abundances of phosphorylated MT1-MMP and activated PKC-δ were enhanced in the small TAA group compared with the large TAA group. MT1-MMP was localized on the plasma membrane in aortic fibroblasts from control mice and in endosomes from TAA mice. Treatment with PMA induced MT1-MMP-GFP internalization, enhanced phospho-Smad2, and reduced MMP-2 activation, whereas röttlerin pretreatment inhibited these effects. CONCLUSIONS: Phosphorylation of MT1-MMP mediates its activity through directing cellular localization, shifting its role from MMP-2 activation to intracellular signaling. Thus, targeted inhibition of MT1-MMP may have therapeutic relevance as an approach to attenuating TAA development.

Differential hypertensive protease expression in the thoracic versus abdominal aorta.

BACKGROUND: Hypertension (HTN), which is a major risk factor for cardiovascular morbidity and mortality, can drive pathologic remodeling of the macro- and microcirculation. Patterns of aortic pathology differ, however, suggesting regional heterogeneity of the pressure-sensitive protease systems triggering extracellular matrix remodeling in the thoracic (TA) and abdominal aortas (AA). This study tested the hypothesis that the expression of two major protease systems (matrix metalloproteinases [MMPs] and cathepsins) in the TA and AA would be differentially affected with HTN. METHODS: Normotensive (BPN3) mice at 14-16 weeks of age underwent implantation of osmotic infusion pumps for 28-day angiotensin II (AngII) delivery (1.46 mg/kg/day; BPN3+AngII; n = 8) to induce HTN. The TA and AA were harvested to determine levels of MMP-2, MMP-9, and membrane type 1-MMP, and cathepsins S, K, and L were evaluated in age-matched BPN3 (n = 8) control and BPH2 spontaneously hypertensive mice (non-AngII pathway; n = 7). Blood pressure was monitored via CODA tail cuff plethysmography (Kent Scientific Corporation, Torrington, Conn). Quantitative real-time polymerase chain reaction and immunoblotting/zymography were used to measure MMP and cathepsin messenger RNA expression and protein abundance, respectively. Target protease values were compared within each aortic region via analysis of variance. RESULTS: Following 28 days infusion, the BPN3+AngII mice had a 17% increase in systolic blood pressure, matching that of the BPH2 spontaneously hypertensive mice (both P < .05 vs BPN3). MMP-2 gene expression demonstrated an AngII-dependent increase in the TA (P < .05), but MMP-9 was not altered with HTN. Expression of tissue inhibitor of metalloproteinases-1 was markedly increased in TA of BPN3+AngII mice, but tissue inhibitor of metalloproteinases-2 demonstrated decreased expression in the AA of both hypertensive groups (P < .05). Only cathepsin K responded to AngII-induced HTN with significant elevation in the TA of those mice, but expression of cathepsin L and cystatin C was inhibited in AA of both hypertensive groups (P < .05). Apoptotic markers were not significantly elevated in any experimental group. CONCLUSIONS: By using two different models of HTN, this study has identified pressure-dependent as well as AngII-dependent regional alterations in aortic gene expression of MMPs and cathepsins that may lead to differential remodeling responses in each of the aortic regions. Further studies will delineate mechanisms and may provide targeted therapies to attenuate down-stream aortic pathology based on demonstrated regional heterogeneity.

Pulmonary artery endothelial cell phenotypic alterations in a large animal model of pulmonary arteriovenous malformations after the Glenn shunt.

BACKGROUND: Longevity of the superior cavopulmonary connection (SCPC) is limited by the development of pulmonary arteriovenous malformations (PAVM). The goal of this study was to determine whether phenotypic changes in pulmonary artery endothelial cells (PAEC) that favor angiogenesis occur with PAVM formation. METHODS: A superior vena cava to right pulmonary artery connection was constructed in 5 pigs. Pulmonary arteries were harvested at 6 to 8 weeks after surgery to establish cultures of PAEC and smooth muscle cells, to determine cell proliferation, gene expression, and tubule formation. Abundance of proteins related to angiogenesis was measured in lung tissue. RESULTS: Contrast echocardiography revealed right-to-left shunting, consistent with PAVM formation. While the proliferation of smooth muscle cells from the right pulmonary artery (shunted side) and left pulmonary artery (nonshunted side) were similar, right PAEC proliferation was significantly higher. Expression profiles of genes encoding cellular signaling proteins were higher in PAECs from the right pulmonary artery versus left pulmonary artery. Protein abundance of angiopoietin-1, and Tie-2 (angiopoietin receptor) were increased in the right lung (both p < 0.05). Tubule formation was increased in endothelial cells from the right pulmonary artery compared with the left pulmonary artery (404 ± 16 versus 199 ± 71 tubules/mm(2), respectively; p < 0.05). CONCLUSIONS: These findings demonstrate that PAVMs developed in a clinically relevant animal model of SCPC concomitantly with differential changes in PAEC proliferative ability and phenotype. Moreover, there was a significant increase in the angiopoietin/Tie-2 complex in the right lung, which may provide novel therapeutic targets to attenuate PAVM formation after a SCPC.

Differential membrane type 1 matrix metalloproteinase substrate processing with ischemia-reperfusion: relationship to interstitial microRNA dynamics and myocardial function.

OBJECTIVES: Membrane type 1 matrix metalloproteinase (MT1-MMP) is critical to a number of proteolytic and profibrotic events. However, upstream regulation of MT1-MMP with myocardial ischemia-reperfusion remains poorly understood. MicroRNAs regulate post-transcriptional events, and in silico mapping has identified a conserved sequence in MT1-MMP for microRNA-133a. This study tested the hypothesis that changes in microRNA-133a regulation occur with myocardial ischemia-reperfusion, which contributes to time- and region-dependent changes in MT1-MMP activity and processing of MT1-MMP substrates. METHODS: Yorkshire pigs (n = 12) underwent ischemia-reperfusion (90 minutes ischemia and 120 minutes reperfusion), where regional preload recruitable stroke work (sonomicrometry), interstitial MT1-MMP activity (microdialysis), Smad2 abundance (immunoblotting), and interstitial microRNA-133a (polymerase chain reaction) were determined within the ischemia-reperfusion and remote regions. Human left ventricular fibroblasts were transduced with microRNA-133a and anti-microRNA-133a (lentivirus) to determine the effects on MT1-MMP protein abundance. RESULTS: With ischemia-reperfusion, regional preload recruitable stroke work decreased from steady state (139 ± 20 mm Hg to 44 ± 11 mm Hg, P < .05) within the ischemia-reperfusion region. MT1-MMP activity increased in both regions. Phosphorylated Smad2 increased within the ischemia-reperfusion region. Both in vitro and in vivo interstitial levels of microRNA-133a decreased with ischemia and returned to steady-state levels with reperfusion. In vitro transduction of microRNA-133a in left ventricular fibroblasts decreased MT1-MMP levels. CONCLUSIONS: Modulation of MT1-MMP activity and microRNA-133a exportation into the myocardial interstitium occurred in the setting of acute myocardial ischemia-reperfusion. In addition, changes in microRNA-133a expression in left ventricular fibroblasts resulted in an inverse modulation of MT1-MMP abundance. Therefore, targeting of microRNA-133a represents a potentially novel means for regulating the cascade of profibrotic events after ischemia-reperfusion.