Vascular Calcifying Progenitor Cells Possess Bidirectional Differentiation Potentials

Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1⁺ (Sca-1⁺/PDGFRα⁺ and Sca-1⁺/PDGFRα⁻) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1⁻ (Sca-1⁻/PDGFRα⁺ and Sca-1⁻/PDGFRα⁻) progenitor cells. Among Sca-1⁺ progenitor populations, Sca-1⁺/PDGFRα⁻ cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1⁺/PDGFRα⁺ cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1⁺/PDGFRα⁻ cells preferentially differentiated into osteoclast-like cells. Sca-1⁺ progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1⁺ calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1⁺ progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1⁺/PDGFRα⁻ cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1⁺/PDGFRα⁻ cells into Apoe^−/− mice increased the severity of calcified atherosclerotic plaques. However, Sca-1⁺/PDGFRα⁻ cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1⁺/PDGFRα⁻ cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.     

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated m.14484T>C (MT-ND6) mutation in Chinese families

Mitochondrial m.14484T>C (MT-ND6) mutation has been associated with Leber's hereditary optic neuropathy. Previous investigations revealed that the m.14484T>C mutation is a primary factor underlying the development of optic neuropathy but is not sufficient to produce a clinical phenotype. However, mitochondrial haplogroups have been proposed to modulate the phenotypic manifestation of the m.14484T>C mutation. Here, we performed the clinical, genetic evaluation and complete mitochondrial genome sequence analysis of 41 Han Chinese pedigrees carrying the m.14484T>C mutation. These families exhibited a wide range of penetrances and expressivities of optic neuropathy. The average ratio between affected male/female matrilineal relatives from 41 families was 2:1. The penetrance of optic neuropathy in these Chinese pedigrees ranged from 5.6% to 100%, with the average of 23.8%. Furthermore, the age-of-onset for optic neuropathy varied from 4 to 44 years, with the average of 19.3 years. Sequence analysis of their mitochondrial genomes identified distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups, indicating that the m.14484T>C mutation occurred through recurrent origins and founder events. We showed that mitochondrial haplogroups M9, M10 and N9 increased the penetrance of optic neuropathy in these Chinese families. In particular, these mitochondrial haplogroup specific variants: m.3394T>C (MT-ND1), m.14502T>C (MT-ND4) and m.14693A>G (MT-TE) enhanced the penetrance of visual loss in these Chinese families. These data provided the direct evidence that mitochondrial modifiers modulate the variable penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the m.14484T>C mutation.     

Formation of the death domain complex between FADD and RIP1 proteins in vitro

Fas-associated death domain (FADD) protein is an adapter molecule that bridges the interactions between membrane death receptors and initiator caspases. The death receptors contain an intracellular death domain (DD) which is essential to the transduction of the apoptotic signal. The kinase receptor-interacting protein 1 (RIP1) is crucial to programmed necrosis. The cell type interplay between FADD and RIP1, which mediates both necrosis and NF-κB activation, has been evaluated in other studies, but the mechanism of the interaction of the FADD and RIP1 proteins remain poorly understood. Here, we provided evidence indicating that the DD of human FADD binds to the DD of RIP1 in vitro. We developed a molecular docking model using homology modeling based on the structures of FADD and RIP1. In addition, we found that two structure-based mutants (G109A and R114A) of the FADD DD were able to bind to the RIP1 DD, and two mutations (Q169A and N171A) of FADD DD and four mutations (G595, K596, E620, and D622) of RIP1 DD disrupted the FADD–RIP1 interaction. Six mutations (Q169A, N171A, G595, K596, E620, and D622) lowered the stability of the FADD–RIP1 complex and induced aggregation that structurally destabilized the complex, thus disrupting the interaction.     

LORETA Current Source Density for Duration Mismatch Negativity and Neuropsychological Assessment in Early Schizophrenia

Introduction

Patients with schizophrenia elicit cognitive decline from the early phase of the illness. Mismatch negativity (MMN) has been shown to be associated with cognitive function. We investigated the current source density of duration mismatch negativity (dMMN), by using low-resolution brain electromagnetic tomography (LORETA), and neuropsychological performance in subjects with early schizophrenia.

Methods

Data were obtained from 20 patients meeting DSM-IV criteria for schizophrenia or schizophreniform disorder, and 20 healthy control (HC) subjects. An auditory odd-ball paradigm was used to measure dMMN. Neuropsychological performance was evaluated by the brief assessment of cognition in schizophrenia Japanese version (BACS-J).

Results

Patients showed smaller dMMN amplitudes than those in the HC subjects. LORETA current density for dMMN was significantly lower in patients compared to HC subjects, especially in the temporal lobes. dMMN current density in the frontal lobe was positively correlated with working memory performance in patients.

Conclusions

This is the first study to identify brain regions showing smaller dMMN current density in early schizophrenia. Further, poor working memory was associated with decreased dMMN current density in patients. These results are likely to help understand the neural basis for cognitive impairment of schizophrenia.     

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)₃ or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.