Novel cell-free strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation

BACKGROUND: Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. METHODOLOGY/PRINCIPAL FINDINGS: EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. CONCLUSIONS/SIGNIFICANCE: Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.

Cell-based therapy facilitates venous thrombus resolution

Endothelial progenitor cells (EPC) are involved in many healing processes in cardiovascular diseases and can be found in spontaneously resolving venous thrombi. The purpose of the present study was to investigate whether the therapeutic administration of EPC might enhance the resolution of venous thrombi. For this purpose, venous thrombosis was induced in the infrarenal inferior vena cava (IVC) in 28 athymic nude rats. Culture expanded EPC derived from human peripheral blood mononuclear cells were injected intravenously two and four days after thrombus induction. Recanalisation of the IVC and thrombus organisation were assessed by laser Doppler measurements of the blood flow and immunohistochemical detection of endothelialised luminal structures in the thrombus. EPC transplantation resulted in significantly enhanced thrombus neovascularisation (capillary density: 186.6 +/- 26.7/HPF vs. 78 +/- 12.3/HPF, p<0.01; area covered by capillaries: 8.9 +/- 1.7 microm(2) vs. 2.5 +/- 1.3 microm(2), p<0.01) and was accompanied by a substantial increase in intra-thrombus blood flow (perfusion ratio: 0.7 +/- 0.07 vs. 0.3 +/- 0.08, p<0.02). These results were paralleled by augmented macrophage recruitment into resolving thrombi in the animals treated with EPC (39.4 +/- 4.7/HPF vs. 11.6 +/- 1.9/HPF, p<0.01). Our data suggest that EPC transplantation might be of clinical value to facilitate venous thrombus resolution in cases where current therapeutic options have limited success.

Mitochondrial Outer Membrane Proteome of Trypanosoma brucei Reveals Novel Factors Required to Maintain Mitochondrial Morphology

Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

Isotopes Trace Biogeochemistry and Sources of Cu and Zn in an intertidal soil

River floodplain soils are sinks and potential sources for toxic trace metals like Cu and Zn. We hypothesize that stable Cu and Zn isotope ratios reflect both the mobilization and the sources of metals. We determined the soil properties, the concentrations and partitioning of Cu and Zn, and variations in δ65Cu and δ66Zn values in a core obtained from an Aquic Udifluvent developed on a freshwater intertidal mudflat of the River Elbe, Germany. The core was sampled at 2 cm intervals to a depth of 34 cm, which corresponds to approximately 9 yr of sedimentation. Elevated concentrations of Cu (up to 320 μg g−1) and Zn (up to 2080 μg g−1) indicated anthropogenic pollution. At the time of sampling the redox conditions changed from oxic (Eh 200 to 400 mV, above 22 cm deep) to strongly anoxic conditions (-100 to -200 mV, below 22 cm deep). The δ65Cu values varied systematically with depth (from -0.02 to 0.16‰) and were correlated with the Fe, C, and N concentrations. Although pre-depositional variations cannot be ruled out, the systematic variation with depth suggests post-sedimentation fractionation of δ65Cu in response to seasonally variable organic matter deposition and redox conditions. In contrast, the δ66ZnIRMM values were uniform (from -0.07 to 0.01‰) throughout the core, indicating that the Zn isotopes did not significantly fractionate after deposition and that the Zn sources were homogeneous throughout the sedimentation.

Valgus Hip With High Antetorsion Causes Pain Through Posterior Extraarticular FAI

BACKGROUND: Valgus hips with increased antetorsion present with lack of external rotation and posterior hip pain that is aggravated with hip extension and external rotation. This may be the result of posterior femoroacetabular impingement (FAI). QUESTIONS/PURPOSES: We asked whether (1) the range of motion (ROM); (2) the location of anterior and posterior bony collision zones; and (3) the prevalence of extraarticular impingement differ between valgus hips with increased antetorsion compared with normal hips and hips with idiopathic FAI. METHODS: Surface models based on CT scan reconstructions of 13 valgus hips with increased antetorsion, 22 hips with FAI, and 27 normal hips were included. Validated three-dimensional collision detection software was used to quantify the simulated hip ROM and the location of impingement on the acetabular and the femoral sides. RESULTS: Hips with coxa valga and antetorsion showed decreased extension, external rotation, and adduction, whereas internal rotation in 90° of flexion was increased. Impingement zones were more anteroinferior on the femur and posteroinferior on the acetabular (pelvic) side; and the zones were more frequently extraarticular, posterior, or to a lesser degree anterior against the inferior iliac spine. We found a higher prevalence of extraarticular impingement for valgus hips with increased antetorsion. CONCLUSIONS: Valgus hips with increased antetorsion predispose to posterior extraarticular FAI and to a lesser degree anteroinferior spine impingement. LEVEL OF EVIDENCE: Level II, prognostic study. See Guidelines for Authors for a complete description of levels of evidence.

Impingement adversely affects 10-year survivorship after periacetabular osteotomy for DDH

BACKGROUND Although periacetabular osteotomy (PAO) for developmental dysplasia of the hip (DDH) provides conceptual advantages compared with other osteotomies and reportedly is associated with joint survivorship of 60% at 20 years, the beneficial effect of proper acetabular reorientation with concomitant arthrotomy and creation of femoral head-neck offset on 10-year hip survivorship remains unclear. QUESTIONS/PURPOSES We asked the following questions: (1) Does the 10-year survivorship of the hip after PAO improve with proper acetabular reorientation and a spherical femoral head; (2) does the Merle d'Aubigné-Postel score improve; (3) can the progression of osteoarthritis (OA) be slowed; and (4) what factors predict conversion to THA, progression of OA, or a Merle d'Aubigné-Postel score less than 15 points? METHODS We retrospectively reviewed 147 patients who underwent 165 PAOs for DDH with two matched groups: Group I (proper reorientation and spherical femoral head) and Group II (improper reorientation and aspherical femoral head). We compared the Kaplan-Meier survivorship, Merle d'Aubigné-Postel scores, and progression of OA in both groups. A Cox regression analysis (end points: THA, OA progression, or Merle d'Aubigné-Postel score less than 15) was performed to detect factors predicting failure. The minimum followup was 10 years (median, 11 years; range, 10-14 years). RESULTS An increased survivorship was found in Group I. The Merle d'Aubigné-Postel score did not differ. Progression of OA in Group I was slower than in Group II. Factors predicting failure included greater age, lower preoperative Merle d'Aubigné-Postel score, and the presence of a Trendelenburg sign, aspherical head, OA, subluxation, postoperative acetabular retroversion, excessive acetabular anteversion, and undercoverage. CONCLUSIONS Proper acetabular reorientation and the creation of a spherical femoral head improve long-term survivorship and decelerate OA progression in patients with DDH.

Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization

In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration.

The Samoa Hurricane, 15-16 March 1889

Analysing historical weather extremes such as the tropical cyclone in Samoa in March 1889 could add to our understanding of extreme events. However, up to now the availability of suitable data was limiting the analysis of historical extremes, particularly in remote regions. The new “Twentieth Century Reanalysis” (20CR), which provides six-hourly, three-dimensional data for the entire globe back to 1871, might provide the means to study this and other early events. While its suitability for studying historical extremes has been analysed for events in the northern extratropics (see other papers in this volume), the representation of tropical cyclones, especially in early times, remains unknown. The aim of this paper is to study to the hurricane that struck Samoa on 15-16 March 1889. We analyse the event in 20CR as well as in contemporary observations. We find that the event is not reproduced in the ensemble mean of 20CR, nor is it within the ensemble spread. We argue that this is due to the paucity of data assimilated into 20CR. A preliminary compilation of historical observations from ships for that period, in contrast, provides a relatively consistent picture of the event. This shows that more observations would be available and implies that future versions of surface-based reanalyses might profit from digitizing further observations in the tropical region.

Usefulness of inducible ventricular tachycardia to predict long-term adverse outcomes in arrhythmogenic right ventricular cardiomyopathy

The role of the electrophysiologic (EP) study for risk stratification in patients with arrhythmogenic right ventricular cardiomyopathy is controversial. We investigated the role of inducible sustained monomorphic ventricular tachycardia (SMVT) for the prediction of an adverse outcome (AO), defined as the occurrence of cardiac death, heart transplantation, sudden cardiac death, ventricular fibrillation, ventricular tachycardia with hemodynamic compromise or syncope. Of 62 patients who fulfilled the 2010 Arrhythmogenic Right Ventricular Cardiomyopathy Task Force criteria and underwent an EP study, 30 (48%) experienced an adverse outcome during a median follow-up of 9.8 years. SMVT was inducible in 34 patients (55%), 22 (65%) of whom had an adverse outcome. In contrast, in 28 patients without inducible SMVT, 8 (29%) had an adverse outcome. Kaplan-Meier analysis showed an event-free survival benefit for patients without inducible SMVT (log-rank p = 0.008) with a cumulative survival free of an adverse outcome of 72% (95% confidence interval [CI] 56% to 92%) in the group without inducible SMVT compared to 26% (95% CI 14% to 50%) in the other group after 10 years. The inducibility of SMVT during the EP study (hazard ratio [HR] 2.99, 95% CI 1.23 to 7.27), nonadherence (HR 2.74, 95% CI 1.3 to 5.77), and heart failure New York Heart Association functional class II and III (HR 2.25, 95% CI 1.04 to 4.87) were associated with an adverse outcome on univariate Cox regression analysis. The inducibility of SMVT (HR 2.52, 95% CI 1.03 to 6.16, p = 0.043) and nonadherence (HR 2.34, 95% CI 1.1 to 4.99, p = 0.028) remained as significant predictors on multivariate analysis. This long-term observational data suggest that SMVT inducibility during EP study might predict an adverse outcome in patients with arrhythmogenic right ventricular cardiomyopathy, advocating a role for EP study in risk stratification.