Ethnic minorities with critical limb ischemia derive equal amputation risk reduction from autologous cell therapy compared with whites.

OBJECTIVE: Ethnic minorities (nonwhites) with critical limb ischemia (CLI) have historically performed worse compared with whites with regard to major amputation risk reduction and amputation-free survival (AFS) after peripheral vascular intervention. This post hoc analysis was completed to determine whether this precedent also extended to treatment of CLI without a suitable revascularization option with intramuscular injections of concentrated bone marrow aspirate (cBMA). METHODS: The treatment arm of the randomized, double-blind, multicenter MarrowStim PAD Kit for the Treatment of Critical Limb Ischemia in Subjects with Severe Peripheral Arterial Disease (MOBILE) trial was stratified by ethnicity and evaluated for demographics, comorbidities, and outcomes. The primary and therapeutic end point was 1-year AFS and major amputation, respectively. Noninferiority analysis was performed with the margin set at historically reported hazard ratios. RESULTS: Thirty-seven minority (African American, Hispanic, other) CLI patients (9 placebo, 28 cBMA) with no suitable revascularization option were randomized to cBMA or placebo at a 3:1 ratio during the MOBILE trial. At 1-year follow-up for the treatment group, overall AFS was 80%. Of the 28 minority patients randomized to cBMA intervention, an 89% AFS rate was observed compared with 77% in whites. Specifically, 22 of 24 (92%) African Americans survived amputation free at 1-year follow-up. Noninferiority testing confirmed no difference between whites and the ethnic minority treated with cBMA with respect to major amputation reduction; however, noninferiority could not be confirmed with regard to AFS. No significant differences favoring whites treated with cBMA were noted in the secondary end points of vascular quality of life, limb pain, ankle-brachial index, toe-brachial index, transcutaneous oximetry, and 6-minute walk testing. CONCLUSIONS: This post hoc analysis of the MOBILE trial demonstrates noninferiority of cBMA intervention in minorities with no-option CLI for the therapeutic end point of major amputation prevention. cBMA represents a novel treatment paradigm and should be explored for minorities with poor revascularization options who face impending amputation secondary to progressive CLI.

Osteopontin may be a driver of abdominal aortic aneurysm formation.

OBJECTIVE: Previous in vitro and animal studies have suggested that osteopontin (OPN), an inflammatory extracellular matrix protein, is involved in the formation and growth of abdominal aortic aneurysms (AAAs). However, the mechanism by which this occurs continues to be nebulous. The relationship between OPN and inflammation-suppressing lymphocytes present in the human AAA condition was investigated and presented herein. METHODS: Serum OPN concentrations were measured in healthy, risk factor-matched non-AAA and AAA patients by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to determine the source of OPN secretion using aortic tissue collected from multiorgan donors and AAA patients undergoing open surgical repair. Vascular smooth muscle cells (VSMCs) were exposed to various inflammatory mediators, and OPN expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction and ELISA. The inflammatory nature of OPN and the aortic wall was determined using a TR1 suppressor cell induction assay as a surrogate and characterized by ELISA and fluorescence-activated cell sorting. RESULTS: OPN was found to be elevated in both the plasma and aortic homogenate of AAA patients compared with controls. On immunohistochemistry, OPN localized to the tunica media of the diseased aorta but was minimally expressed in healthy aorta. In vitro, cigarette smoke extract was the most potent stimulator of OPN secretion by VSMCs and increased both messenger RNA and supernatant concentrations. OPN demonstrated an ability to inhibit the induction of interleukin 10-secreting TR1 lymphocytes, a depleted population in the AAA patient, from naive precursors. Last, neutralizing receptor targets of OPN in the setting of AAA homogenate coincubation abrogated the inhibition of TR1 induction. CONCLUSIONS: OPN, secreted by the VSMCs of the tunica media, is elevated in the circulating plasma and aortic wall of patients with AAA. It can inhibit the induction of the TR1 suppressor cell, leading to an overall proinflammatory state contributing to progressive aortic wall breakdown and dilation.

Rationale and Design of the ARREST Trial Investigating Mesenchymal Stem Cells in the Treatment of Small Abdominal Aortic Aneurysm.

BACKGROUND: Abdominal aortic aneurysms (AAAs) are a major source of morbidity and mortality despite continuing advances in surgical technique and care. Although the inciting factors for AAA development continue to be elusive, accumulating evidence suggests a significant periaortic inflammatory response leading to degradation and dilation of the aortic wall. Previous human trials have demonstrated safety and efficacy of mesenchymal stem cells (MSCs) in the treatment of inflammation-related pathologies such as rheumatoid arthritis, graft versus host disease, and transplant rejection. Therefore, herein, we describe the Aortic Aneurysm Repression with Mesenchymal Stem Cells (ARREST) trial, a phase I investigation into the safety of MSC infusion for patients with small AAA and the cells' effects on modulation of AAA-related inflammation. METHODS: ARREST is a phase I, single-center, double-blind, randomized controlled trial (RCT) investigating infusion both dilute and concentrated MSCs compared to placebo in 36 small AAA (35-45 mm) patients. Subjects will be followed by study personnel for 12 months to ascertain incidence of adverse events, immune cell phenotype expression, peripheral cytokine profile, and periaortic inflammation. Maximum transverse aortic diameter will be assessed regularly for 5 years by a combination of computed tomography and duplex sonography. RESULTS: Four patients have thus far been enrolled, randomized, and treated per protocol. We anticipate the conclusion of the treatment phase within the next 24 months with ongoing long-term follow-up. CONCLUSIONS: ARREST will be pivotal in assessing the safety of MSC infusion and provide preliminary data on the ability of MSCs to favorably modulate the pathogenic AAA host immune response. The data gleaned from this phase I trial will provide the groundwork for a larger, phase III RCT which may provide the first pharmaceutical intervention for AAA.

Friedreich's ataxia reveals a mechanism for coordinate regulation of oxidative metabolism via feedback inhibition of the SIRT3 deacetylase.

Friedreich's ataxia (FRDA) is the most common inherited human ataxia and is caused by a deficiency in the mitochondrial protein frataxin. Clinically, patients suffer from progressive spinocerebellar degeneration, diabetes and a fatal cardiomyopathy, associated with mitochondrial respiratory chain defects. Recent findings have shown that lysine acetylation regulates mitochondrial function and intermediary metabolism. However, little is known about lysine acetylation in the setting of pathologic energy stress and mitochondrial dysfunction. We tested the hypothesis that the respiratory chain defects in frataxin deficiency alter mitochondrial protein acetylation. Using two conditional mouse models of FRDA, we demonstrate marked hyperacetylation of numerous cardiac mitochondrial proteins. Importantly, this biochemical phenotype develops concurrently with cardiac hypertrophy and is caused by inhibition of the NAD(+)-dependent SIRT3 deacetylase. This inhibition is caused by an 85-fold decrease in mitochondrial NAD(+)/NADH and direct carbonyl group modification of SIRT3, and is reversed with excess SIRT3 and NAD(+) in vitro. We further demonstrate that protein hyperacetylation may be a common feature of mitochondrial disorders caused by respiratory chain defects, notably, cytochrome oxidase I (COI) deficiency. These findings suggest that SIRT3 inhibition and consequent protein hyperacetylation represents a negative feedback mechanism limiting mitochondrial oxidative pathways when respiratory metabolism is compromised, and thus, may contribute to the lethal cardiomyopathy in FRDA.

A TAT-frataxin fusion protein increases lifespan and cardiac function in a conditional Friedreich's ataxia mouse model.

Friedreich's ataxia (FRDA) is the most common inherited human ataxia and results from a deficiency of the mitochondrial protein, frataxin (FXN), which is encoded in the nucleus. This deficiency is associated with an iron-sulfur (Fe-S) cluster enzyme deficit leading to progressive ataxia and a frequently fatal cardiomyopathy. There is no cure. To determine whether exogenous replacement of the missing FXN protein in mitochondria would repair the defect, we used the transactivator of transcription (TAT) protein transduction domain to deliver human FXN protein to mitochondria in both cultured patient cells and a severe mouse model of FRDA. A TAT-FXN fusion protein bound iron in vitro, transduced into mitochondria of FRDA deficient fibroblasts and reduced caspase-3 activation in response to an exogenous iron-oxidant stress. Injection of TAT-FXN protein into mice with a conditional loss of FXN increased their growth velocity and mean lifespan by 53% increased their mean heart rate and cardiac output, increased activity of aconitase and reversed abnormal mitochondrial proliferation and ultrastructure in heart. These results show that a cell-penetrant peptide is capable of delivering a functional mitochondrial protein in vivo to rescue a very severe disease phenotype, and present the possibility of TAT-FXN as a protein replacement therapy.

Cardiomyopathy of Friedreich's ataxia: use of mouse models to understand human disease and guide therapeutic development.

Friedreich's ataxia is a multisystem disorder of mitochondrial function affecting primarily the heart and brain. Patients experience a severe cardiomyopathy that can progress to heart failure and death. Although the gene defect is known, the precise function of the deficient mitochondrial protein, frataxin, is not known and limits therapeutic development. Animal models have been valuable for understanding the basic events of this disease. A significant need exists to focus greater attention on the heart disease in Friedreich's ataxia, to understand its long-term outcome, and to develop new therapeutic strategies using existing medications and approaches. This review discusses some key features of the cardiomyopathy in Friedreich's ataxia and potential therapeutic developments.

Rab10 associates with primary cilia and the exocyst complex in renal epithelial cells.

Rab10, a mammalian homolog of the yeast Sec4p protein, has previously been associated with endocytic recycling and biosynthetic membrane transport in cultured epithelia and with Glut4 translocation in adipocytes. Here, we report that Rab10 associates with primary cilia in renal epithelia in culture and in vivo. In addition, we find that Rab10 also colocalizes with exocyst proteins at the base of nascent cilia, and physically interacts with the exocyst complex, as detected with anti-Sec8 antibodies. These data suggest that membrane transport to the primary cilum may be mediated by interactions between Rab10 and an exocyst complex located at the cilium base.

Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells.

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.

Rab10 regulates membrane transport through early endosomes of polarized Madin-Darby canine kidney cells.

Rab10, a protein originally isolated from Madin-Darby Canine Kidney (MDCK) epithelial cells, belongs to a family of Rab proteins that includes Rab8 and Rab13. Although both Rab8 and Rab13 have been found to mediate polarized membrane transport, the function of Rab10 in mammalian cells has not yet been established. We have used quantitative confocal microscopy of polarized MDCK cells expressing GFP chimeras of wild-type and mutant forms of Rab10 to analyze the function of Rab10 in polarized cells. These studies demonstrate that Rab10 is specifically associated with the common endosomes of MDCK cells, accessible to endocytic probes internalized from either the apical or basolateral plasma membrane domains. Expression of mutant Rab10 defective for either GTP hydrolysis or GTP binding increased recycling from early compartments on the basolateral endocytic pathway without affecting recycling from later compartments or the apical recycling pathway. These results suggest that Rab10 mediates transport from basolateral sorting endosomes to common endosomes.