Effects of a soft robotic exosuit on the quality and speed of overground walking depends on walking ability after stroke.

BACKGROUND: Soft robotic exosuits can provide partial dorsiflexor and plantarflexor support in parallel with paretic muscles to improve poststroke walking capacity. Previous results indicate that baseline walking ability may impact a user's ability to leverage the exosuit assistance, while the effects on continuous walking, walking stability, and muscle slacking have not been evaluated. Here we evaluated the effects of a portable ankle exosuit during continuous comfortable overground walking in 19 individuals with chronic hemiparesis. We also compared two speed-based subgroups (threshold: 0.93 m/s) to address poststroke heterogeneity. METHODS: We refined a previously developed portable lightweight soft exosuit to support continuous overground walking. We compared five minutes of continuous walking in a laboratory with the exosuit to walking without the exosuit in terms of ground clearance, foot landing and propulsion, as well as the energy cost of transport, walking stability and plantarflexor muscle slacking. RESULTS: Exosuit assistance was associated with improvements in the targeted gait impairments: 22% increase in ground clearance during swing, 5° increase in foot-to-floor angle at initial contact, and 22% increase in the center-of-mass propulsion during push-off. The improvements in propulsion and foot landing contributed to a 6.7% (0.04 m/s) increase in walking speed (R2 = 0.82). This enhancement in gait function was achieved without deterioration in muscle effort, stability or cost of transport. Subgroup analyses revealed that all individuals profited from ground clearance support, but slower individuals leveraged plantarflexor assistance to improve propulsion by 35% to walk 13% faster, while faster individuals did not change either. CONCLUSIONS: The immediate restorative benefits of the exosuit presented here underline its promise for rehabilitative gait training in poststroke individuals.

Multifocal pupillographic objective perimetry for assessment of early diabetic retinopathy and generalised diabetes-related tissue injury in persons with type 1 diabetes.

BACKGROUND: To examine the potential utility of five multifocal pupillographic objective perimetry (mfPOP) protocols, in the assessment of early diabetic retinopathy (DR) and generalised diabetes-related tissue injury in subjects with type 1 diabetes (T1D). METHODS: Twenty-five T1D subjects (age 41.8 ± 12.1 (SD) years, 13 male) with either no DR (n = 13) or non-proliferative DR (n = 12), and 23 age and gender-matched control subjects (age 39.7 ± 12.9 years, 9 male) were examined by mfPOP using five different stimulus methods differing in visual field eccentricity (central 30° and 60°), and colour (blue, yellow or green test-stimuli presented on, respectively, a blue, yellow or red background), each assessing 44 test-locations per eye. In the T1D subjects, we assessed 16 metabolic status and diabetes complications variables. These were summarised as three principal component analysis (PCA) factors. DR severity was assessed using Early Treatment of Diabetic Retinopathy Study (ETDRS) scores. Area under the curve (AUC) from receiver operator characteristic analyses quantified the diagnostic power of mfPOP response sensitivity and delay deviations for differentiating: (i) T1D subjects from control subjects, (ii) T1D subjects according to three levels of the identified PCA-factors from control subjects, and (iii) TID subjects with from those without non-proliferative DR. RESULTS: The two largest PCA-factors describing the T1D subjects were associated with metabolic variables (e.g. body mass index, HbA1c), and tissue-injury variables (e.g. serum creatinine, vibration perception). Linear models showed that mfPOP per-region response delays were more strongly associated than sensitivities with the metabolic PCA-factor and ETDRS scores. Combined mfPOP amplitude and delay measures produced AUCs of 90.4 ± 8.9% (mean ± SE) for discriminating T1D subjects with DR from control subjects, and T1D subjects with DR from those without of 85.9 ± 8.8%. The yellow and green stimuli performed better than blue on most measures. CONCLUSIONS/INTERPRETATION: In T1D subjects, mfPOP testing was able to identify localised visual field functional abnormalities (retinal/neural reflex) in the absence or presence of mild DR. mfPOP responses were also associated with T1D metabolic status, but less so with early stages of non-ophthalmic diabetes complications.

Canine ACL rupture: a spontaneous large animal model of human ACL rupture.

BACKGROUND: Anterior cruciate ligament (ACL) rupture in humans is a common condition associated with knee pain, joint instability, and secondary osteoarthritis (OA). Surgical treatment with an intraarticular graft provides reasonable outcomes at mid and long-term follow-up. Non-modifiable and modifiable factors influence risk of ACL rupture. The etiology, mechanobiology, causal biomechanics, and causal molecular pathways are not fully understood. The dog model has shared features of ACL rupture that make it a valuable spontaneous preclinical animal model. In this article, we review shared and contrasting features of ACL rupture in the two species and present information supporting spontaneous canine ACL rupture as a potentially useful preclinical model of human ACL rupture with a very large subject population. RESULTS: ACL rupture is more common in dogs than in humans and is diagnosed and treated using similar approaches to that of human patients. Development of OA occurs in both species, but progression is more rapid in the dog, and is often present at diagnosis. Use of client-owned dogs for ACL research could reveal impactful molecular pathways, underlying causal genetic variants, biomechanical effects of specific treatments, and opportunities to discover new treatment and prevention targets. Knowledge of the genetic contribution to ACL rupture is more advanced in dogs than in humans. In dogs, ACL rupture has a polygenetic architecture with moderate heritability. Heritability of human ACL rupture has not been estimated. CONCLUSION: This article highlights areas of One Health research that are particularly relevant to future studies using the spontaneous canine ACL rupture model that could fill gaps in current knowledge.

Risk factors for nonunion following tibiotalocalcaneal arthrodesis: A systematic review and meta-analysis.

BACKGROUND: The goal of this study is to review the literature to identify risk factors for nonunion after tibiotalocalcaneal arthrodesis (TTCA) and stratify them based on strength of evidence. METHODS: Five databases were searched from inception to May 17th, 2020. Abstracts and full-text articles were screened for those that included risk factors predictive of nonunion following TTCA. RESULTS: Eight studies involving 624 patients were included and 33 potential risk factors for nonunion were identified. Strong evidence supported prior peripheral neuropathic conditions as risk factors for nonunion following surgery (OR: 2.86, 95% CI: 1.56-5.23). CONCLUSION: TTCA is an effective salvage procedure but is associated with high nonunion rates. The results of our meta-analysis suggest that prior peripheral neuropathic conditions have strong evidence for failure to achieve union. Surgeons should be cognizant of these risks when performing TTCA and carefully monitor patients with the aforementioned comorbidity to achieve successful results.

Recapitulating the Binding Affinity of Nrf2 for KEAP1 in a Cyclic Heptapeptide, Guided by NMR, X-ray Crystallography, and Machine Learning.

Macrocycles, including macrocyclic peptides, have shown promise for targeting challenging protein-protein interactions (PPIs). One PPI of high interest is between Kelch-like ECH-Associated Protein-1 (KEAP1) and Nuclear Factor (Erythroid-derived 2)-like 2 (Nrf2). Guided by X-ray crystallography, NMR, modeling, and machine learning, we show that the full 20 nM binding affinity of Nrf2 for KEAP1 can be recapitulated in a cyclic 7-mer peptide, c[(D)-ß-homoAla-DPETGE]. This compound was identified from the Nrf2-derived linear peptide GDEETGE (KD = 4.3 µM) solely by optimizing the conformation of the cyclic compound, without changing any KEAP1 interacting residue. X-ray crystal structures were determined for each linear and cyclic peptide variant bound to KEAP1. Despite large variations in affinity, no obvious differences in the conformation of the peptide binding residues or in the interactions they made with KEAP1 were observed. However, analysis of the X-ray structures by machine learning showed that locations of strain in the bound ligand could be identified through patterns of subangstrom distortions from the geometry observed for unstrained linear peptides. We show that optimizing the cyclic peptide affinity was driven partly through conformational preorganization associated with a proline substitution at position 78 and with the geometry of the noninteracting residue Asp77 and partly by decreasing strain in the ETGE motif itself. This approach may have utility in dissecting the trade-off between conformational preorganization and strain in other ligand-receptor systems. We also identify a pair of conserved hydrophobic residues flanking the core DxETGE motif which play a conformational role in facilitating the high-affinity binding of Nrf2 to KEAP1.

Sequential iodine-mediated diallylsilane rearrangement/asymmetric dihydroxylation: Synthesis and reactions of enantioenriched oxasilacycles.

Products from an iodine-mediated diallylsilane rearrangement were taken into an asymmetric dihydroxylation (AD) reaction resulting in the formation of diastereomeric 6-membered oxasilacycles. Removal of the epimeric stereocenter among this mixture of diastereomers by elimination of iodine produced a single enantioenriched cyclic allyl silyl ether. The resulting allyl silane was then successfully engaged in several further transformations, providing an alternative means to prepare useful intermediates for enantioselective synthesis.

The Relationship Between Maladaptive Personality and Social Role Impairment in Depressed Older Adults in Primary Care.

Objective: Personality pathology is associated with impaired social functioning in adults, though further evidence is needed to examine the individual contributions of personality traits and processes to social functioning in depressed older adults. This study is a secondary analysis examining the relationship between maladaptive personality traits and processes and social role impairment in depressed older adults in primary care. Methods: Participants (N = 56) were 77% female and ranged in age between 55-89 (M = 66.82, SD = 8.75). Personality pathology was measured by maladaptive traits (NEO-FFI) and processes (Inventory of Interpersonal Problems; IIP-PD-15). Individual variable as well as combined predictive models of social role impairment were examined. Results: Higher neuroticism (ß = 0.30, p < .05), lower agreeableness (ß = -0.35 p < .001) and higher IIP-PD-15 (ß = 0.28, p < .01) scores predicted greater impairment in social role functioning. A combined predictive model of neuroticism and IIP-PD-15 scores predicted unique variance in social role impairment (R2 = .71). Conclusion: These results link select personality traits and interpersonal processes to social role impairment, suggesting that these are indicators of personality pathology in older adults. Clinical Implications: These findings lend preliminary support for clinical screening of personality pathology in depressed older adults utilizing both personality trait and process measures.

Rapid identification of homozygosity and site of wild relative introgressions in wheat through chromosome-specific KASP genotyping assays.

For future food security, it is important that wheat, one of the most widely consumed crops in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is currently being introduced into wheat through introgressions from its wild relatives. For trait discovery, it is necessary that each introgression is homozygous and hence stable. Breeding programmes rely on efficient genotyping platforms for marker-assisted selection (MAS). Recently, single nucleotide polymorphism (SNP)-based markers have been made available on high-throughput Axiom® SNP genotyping arrays. However, these arrays are inflexible in their design and sample numbers, making their use unsuitable for long-term MAS. SNPs can potentially be converted into Kompetitive allele-specific PCR (KASP™) assays that are comparatively cost-effective and efficient for low-density genotyping of introgression lines. However, due to the polyploid nature of wheat, KASP assays for homoeologous SNPs can have difficulty in distinguishing between heterozygous and homozygous hybrid lines in a backcross population. To identify co-dominant SNPs, that can differentiate between heterozygotes and homozygotes, we PCR-amplified and sequenced genomic DNA from potential single-copy regions of the wheat genome and compared them to orthologous copies from different wild relatives. A panel of 620 chromosome-specific KASP assays have been developed that allow rapid detection of wild relative segments and provide information on their homozygosity and site of introgression in the wheat genome. A set of 90 chromosome-nonspecific assays was also produced that can be used for genotyping introgression lines. These multipurpose KASP assays represent a powerful tool for wheat breeders worldwide.

Exploiting the genome of Thinopyrum elongatum to expand the gene pool of hexaploid wheat.

KEY MESSAGE: One hundred and thirty four introgressions from Thinopyrum elongatum have been transferred into a wheat background and were characterised using 263 SNP markers. Species within the genus Thinopyrum have been shown to carry genetic variation for a very wide range of traits including biotic and abiotic stresses and quality. Research has shown that one of the species within this genus, Th. elongatum, has a close relationship with the genomes of wheat making it a highly suitable candidate to expand the gene pool of wheat. Homoeologous recombination, in the absence of the Ph1 gene, has been exploited to transfer an estimated 134 introgressions from Th. elongatum into a hexaploid wheat background. The introgressions were detected and characterised using 263 single nucleotide polymorphism markers from a 35 K Axiom® Wheat-Relative Genotyping Array, spread across seven linkage groups and validated using genomic in situ hybridisation. The genetic map had a total length of 187.8 cM and the average chromosome length was 26.8 cM. Comparative analyses of the genetic map of Th. elongatum and the physical map of hexaploid wheat confirmed previous work that indicated good synteny at the macro-level, although Th. elongatum does not contain the 4A/5A/7B translocation found in wheat.

Multivariate genome-wide association analysis identifies novel and relevant variants associated with anterior cruciate ligament rupture risk in the dog model.

BACKGROUND: Anterior cruciate ligament rupture (ACLR) is a debilitating and potentially life-changing condition in humans, as there is a high prevalence of early-onset osteoarthritis after injury. Identification of high-risk individuals before they become patients is important, as post-treatment lifetime burden of ACLR in the USA ranges from $7.6 to $17.7 billion annually. ACLR is a complex disease with multiple risk factors including genetic predisposition. Naturally occurring ACLR in the dog is an excellent model for human ACLR, as risk factors and disease characteristics in humans and dogs are similar. In a univariate genome-wide association study (GWAS) of 237 Labrador Retrievers, we identified 99 ACLR candidate loci. It is likely that additional variants remain to be identified. Joint analysis of multiple correlated phenotypes is an underutilized technique that increases statistical power, even when only one phenotype is associated with the trait. Proximal tibial morphology has been shown to affect ACLR risk in both humans and dogs. In the present study, tibial plateau angle (TPA) and relative tibial tuberosity width (rTTW) were measured on bilateral radiographs from purebred Labrador Retrievers that were recruited to our initial GWAS. We performed a multivariate genome wide association analysis of ACLR status, TPA, and rTTW. RESULTS: Our analysis identified 3 loci with moderate evidence of association that were not previously associated with ACLR. A locus on Chr1 associated with both ACLR and rTTW is located within ROR2, a gene important for cartilage and bone development. A locus on Chr4 associated with both ACLR and TPA resides within DOCK2, a gene that has been shown to promote immune cell migration and invasion in synovitis, an important predictor of ACLR. A third locus on Chr23 associated with only ACLR is located near a long non-coding RNA (lncRNA). LncRNA's are important for regulation of gene transcription and translation. CONCLUSIONS: These results did not overlap with our previous GWAS, which is reflective of the different methods used, and supports the need for further work. The results of the present study are highly relevant to ACLR pathogenesis, and identify potential drug targets for medical treatment.

Autonomous multi-joint soft exosuit with augmentation-power-based control parameter tuning reduces energy cost of loaded walking.

BACKGROUND: Soft exosuits are a recent approach for assisting human locomotion, which apply assistive torques to the wearer through functional apparel. Over the past few years, there has been growing recognition of the importance of control individualization for such gait assistive devices to maximize benefit to the wearer. In this paper, we present an updated version of autonomous multi-joint soft exosuit, including an online parameter tuning method that customizes control parameters for each individual based on positive ankle augmentation power. METHODS: The soft exosuit is designed to assist with plantarflexion, hip flexion, and hip extension while walking. A mobile actuation system is mounted on a military rucksack, and forces generated by the actuation system are transmitted via Bowden cables to the exosuit. The controller performs an iterative force-based position control of the Bowden cables on a step-by-step basis, delivering multi-articular (plantarflexion and hip flexion) assistance during push-off and hip extension assistance in early stance. To individualize the multi-articular assistance, an online parameter tuning method was developed that customizes two control parameters to maximize the positive augmentation power delivered to the ankle. To investigate the metabolic efficacy of the exosuit with wearer-specific parameters, human subject testing was conducted involving walking on a treadmill at 1.50 m s- 1 carrying a 6.8-kg loaded rucksack. Seven participants underwent the tuning process, and the metabolic cost of loaded walking was measured with and without wearing the exosuit using the individualized control parameters. RESULTS: The online parameter tuning method was capable of customizing the control parameters, creating a positive ankle augmentation power map for each individual. The subject-specific control parameters and resultant assistance profile shapes varied across the study participants. The exosuit with the wearer-specific parameters significantly reduced the metabolic cost of load carriage by 14.88 ± 1.09% (P = 5 × 10- 5) compared to walking without wearing the device and by 22.03 ± 2.23% (P = 2 × 10- 5) compared to walking with the device unpowered. CONCLUSION: The autonomous multi-joint soft exosuit with subject-specific control parameters tuned based on positive ankle augmentation power demonstrated the ability to improve human walking economy. Future studies will further investigate the effect of the augmentation-power-based control parameter tuning on wearer biomechanics and energetics.

The Effects of Walking Workstations on Biomechanical Performance.

Prolonged sitting has been associated with negative health effects. Walking workstations have become increasingly popular in the workplace. There is a lack of research on the biomechanical effect of walking workstations. This study analyzed whether walking while working alters normal gait patterns. A total of 9 participants completed 4 walking trials at 2.4 and 4.0 km·h-1: baseline walking condition, walking while performing a math task, a reading task, and a typing task. Biomechanical data were collected using standard motion capture procedures. The first maximum vertical ground reaction force, stride width, stride length, minimum toe clearance, peak swing hip abduction and flexion angles, peak swing and stance ankle dorsiflexion, and knee flexion angles were analyzed. Differences between conditions were evaluated using analysis of variance tests with Bonferroni correction (P ≤ .05). Stride width decreased during the reading task at both speeds. Although other parameters exhibited significant differences when multitasking, these changes were within the normal range of gait variability. It appears that for short periods, walking workstations do not negatively impact gait in healthy young adults.

Necrotic Cell Sensor Clec4e Promotes a Proatherogenic Macrophage Phenotype Through Activation of the Unfolded Protein Response.

BACKGROUND: Atherosclerotic lesion expansion is characterized by the development of a lipid-rich necrotic core known to be associated with the occurrence of complications. Abnormal lipid handling, inflammation, and alteration of cell survival or proliferation contribute to necrotic core formation, but the molecular mechanisms involved in this process are not properly understood. C-type lectin receptor 4e (Clec4e) recognizes the cord factor of Mycobacterium tuberculosis but also senses molecular patterns released by necrotic cells and drives inflammation. METHODS: We hypothesized that activation of Clec4e signaling by necrosis is causally involved in atherogenesis. We addressed the impact of Clec4e activation on macrophage functions in vitro and on the development of atherosclerosis using low-density lipoprotein receptor-deficient (Ldlr-/-) mice in vivo. RESULTS: We show that Clec4e is expressed within human and mouse atherosclerotic lesions and is activated by necrotic lesion extracts. Clec4e signaling in macrophages inhibits cholesterol efflux and induces a Syk-mediated endoplasmic reticulum stress response, leading to the induction of proinflammatory mediators and growth factors. Chop and Ire1a deficiencies significantly limit Clec4e-dependent effects, whereas Atf3 deficiency aggravates Clec4e-mediated inflammation and alteration of cholesterol efflux. Repopulation of Ldlr-/- mice with Clec4e-/- bone marrow reduces lipid accumulation, endoplasmic reticulum stress, and macrophage inflammation and proliferation within the developing arterial lesions and significantly limits atherosclerosis. CONCLUSIONS: Our results identify a nonredundant role for Clec4e in coordinating major biological pathways involved in atherosclerosis and suggest that it may play similar roles in other chronic inflammatory diseases.

Effects of DNA damage in smooth muscle cells in atherosclerosis.

RATIONALE: DNA damage and the DNA damage response have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double-stranded breaks (DSBs) are hypothesized to promote plaque progression and instability, in part, by promoting cell senescence, apoptosis, and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. OBJECTIVE: To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. METHODS AND RESULTS: Human atherosclerotic plaque VSMCs showed increased expression of multiple DNA damage response proteins in vitro and in vivo, particularly the MRE11/RAD50/NBS1 complex that senses DSB repair. Oxidative stress-induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated 2 novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with apolipoprotein E(-/-) mice. SM22α-NBS1/apolipoprotein E(-/-) VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/apolipoprotein E(-/-) VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. CONCLUSIONS: Human atherosclerotic plaque VSMCs show increased DNA damage, including DSBs and DNA damage response activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability.

Vascular Smooth Muscle Cell Senescence Promotes Atherosclerosis and Features of Plaque Vulnerability.

BACKGROUND: Although vascular smooth muscle cell (VSMC) proliferation is implicated in atherogenesis, VSMCs in advanced plaques and cultured from plaques show evidence of VSMC senescence and DNA damage. In particular, plaque VSMCs show shortening of telomeres, which can directly induce senescence. Senescence can have multiple effects on plaque development and morphology; however, the consequences of VSMC senescence or the mechanisms underlying VSMC senescence in atherosclerosis are mostly unknown. METHODS AND RESULTS: We examined the expression of proteins that protect telomeres in VSMCs derived from human plaques and normal vessels. Plaque VSMCs showed reduced expression and telomere binding of telomeric repeat-binding factor-2 (TRF2), associated with increased DNA damage. TRF2 expression was regulated by p53-dependent degradation of the TRF2 protein. To examine the functional consequences of loss of TRF2, we expressed TRF2 or a TRF2 functional mutant (T188A) as either gain- or loss-of-function studies in vitro and in apolipoprotein E(-/-) mice. TRF2 overexpression bypassed senescence, reduced DNA damage, and accelerated DNA repair, whereas TRF2(188A) showed opposite effects. Transgenic mice expressing VSMC-specific TRF2(T188A) showed increased atherosclerosis and necrotic core formation in vivo, whereas VSMC-specific TRF2 increased the relative fibrous cap and decreased necrotic core areas. TRF2 protected against atherosclerosis independent of secretion of senescence-associated cytokines. CONCLUSIONS: We conclude that plaque VSMC senescence in atherosclerosis is associated with loss of TRF2. VSMC senes cence promotes both atherosclerosis and features of plaque vulnerability, identifying prevention of senescence as a potential target for intervention.

Acute tumour response to a bispecific Ang-2-VEGF-A antibody: insights from multiparametric MRI and gene expression profiling.

BACKGROUND: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). METHODS: Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg(-1) dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 10(6) mm(2) s(-1)), vascular perfusion/permeability (K(trans), min(-1)) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed. RESULTS: Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour K(trans) (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis. CONCLUSIONS: Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.

Regulatory B cell-specific interleukin-10 is dispensable for atherosclerosis development in mice.

OBJECTIVE: To determine the role of regulatory B cell-derived interleukin (IL)-10 in atherosclerosis. APPROACH AND RESULTS: We created chimeric Ldlr(-/-) mice with a B cell-specific deficiency in IL-10, and confirmed that purified B cells stimulated with lipopolysaccharide failed to produce IL-10 compared with control Ldlr(-/-) chimeras. Mice lacking B-cell IL-10 demonstrated enhanced splenic B-cell numbers but no major differences in B-cell subsets, T cell or monocyte distribution, and unchanged body weights or serum cholesterol levels compared with control mice. After 8 weeks on high-fat diet, there were no differences in aortic root or aortic arch atherosclerosis. In addition to plaque size, plaque composition (macrophages, T cells, smooth muscle cells, and collagen) was similar between groups. CONCLUSIONS: In contrast to its prominent regulatory role in many immune-mediated diseases and its proposed modulatory role in atherosclerosis, B cell-derived IL-10 does not alter atherosclerosis in mice.

MHC Class II-restricted antigen presentation by plasmacytoid dendritic cells drives proatherogenic T cell immunity.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immune responses and are important regulators of immuno-inflammatory diseases. However, their role in atherosclerosis remains elusive. METHODS AND RESULTS: Here, we used genetic approaches to investigate the role of pDCs in atherosclerosis. Selective pDC deficiency in vivo was achieved using CD11c-Cre × Tcf4(-/flox) bone marrow transplanted into Ldlr(-/-) mice. Compared with control Ldlr(-/-) chimeric mice, CD11c-Cre × Tcf4(-/flox) mice had reduced atherosclerosis levels. To begin to understand the mechanisms by which pDCs regulate atherosclerosis, we studied chimeric Ldlr(-/-) mice with selective MHCII deficiency on pDCs. Significantly, these mice also developed reduced atherosclerosis compared with controls without reductions in pDC numbers or changes in conventional DCs. MHCII-deficient pDCs showed defective stimulation of apolipoprotein B100-specific CD4(+) T cells in response to native low-density lipoprotein, whereas production of interferon-α was not affected. Finally, the atheroprotective effect of selective MHCII deficiency in pDCs was associated with significant reductions of proatherogenic T cell-derived interferon-γ and lesional T cell infiltration, and was abrogated in CD4(+) T cell-depleted animals. CONCLUSIONS: This study supports a proatherogenic role for pDCs in murine atherosclerosis and identifies a critical role for MHCII-restricted antigen presentation by pDCs in driving proatherogenic T cell immunity.

Mitochondrial DNA damage can promote atherosclerosis independently of reactive oxygen species through effects on smooth muscle cells and monocytes and correlates with higher-risk plaques in humans.

BACKGROUND: Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. METHODS AND RESULTS: mtDNA damage occurred early in the vessel wall in apolipoprotein E-null (ApoE(-/-)) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE(-/-) mice deficient for mitochondrial polymerase-γ proofreading activity (polG(-/-)/ApoE(-/-)). polG(-/-)/ApoE(-/-) mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG(-/-)/ApoE(-/-) mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG(-/-)/ApoE(-/-) bone marrow increased the features of plaque vulnerability, and polG(-/-)/ApoE(-/-) monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. CONCLUSIONS: We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.