Loss of developmentally derived Irf8+ macrophages promotes hyperinnervation and arrhythmia in the adult zebrafish heart.

Recent developments in cardiac macrophage biology have broadened our understanding of the critical functions of macrophages in the heart. As a result, there is further interest in understanding the independent contributions of distinct subsets of macrophage to cardiac development and function. Here, we demonstrate that genetic loss of interferon regulatory factor 8 (Irf8)-positive embryonic-derived macrophages significantly disrupts cardiac conduction, chamber function, and innervation in adult zebrafish. At 4 months post-fertilization (mpf), homozygous irf8st96/st96 mutants have significantly shortened atrial action potential duration and significant differential expression of genes involved in cardiac contraction. Functional in vivo assessments via electro- and echocardiograms at 12 mpf reveal that irf8 mutants are arrhythmogenic and exhibit diastolic dysfunction and ventricular stiffening. To identify the molecular drivers of the functional disturbances in irf8 null zebrafish, we perform single cell RNA sequencing and immunohistochemistry, which reveal increased leukocyte infiltration, epicardial activation, mesenchymal gene expression, and fibrosis. Irf8 null hearts are also hyperinnervated and have aberrant axonal patterning, a phenotype not previously assessed in the context of cardiac macrophage loss. Gene ontology analysis supports a novel role for activated epicardial-derived cells (EPDCs) in promoting neurogenesis and neuronal remodeling in vivo. Together, these data uncover significant cardiac abnormalities following embryonic macrophage loss and expand our knowledge of critical macrophage functions in heart physiology and governing homeostatic heart health.

Deep learning automation of radiographic patterns for hallux valgus diagnosis.

Artificial intelligence (AI) and deep learning are becoming increasingly powerful tools in diagnostic and radiographic medicine. Deep learning has already been utilized for automated detection of pneumonia from chest radiographs, diabetic retinopathy, breast cancer, skin carcinoma classification, and metastatic lymphadenopathy detection, with diagnostic reliability akin to medical experts. In the World Journal of Orthopedics article, the authors apply an automated and AI-assisted technique to determine the hallux valgus angle (HVA) for assessing HV foot deformity. With the U-net neural network, the authors constructed an algorithm for pattern recognition of HV foot deformity from anteroposterior high-resolution radiographs. The performance of the deep learning algorithm was compared to expert clinician manual performance and assessed alongside clinician-clinician variability. The authors found that the AI tool was sufficient in assessing HVA and proposed the system as an instrument to augment clinical efficiency. Though further sophistication is needed to establish automated algorithms for more complicated foot pathologies, this work adds to the growing evidence supporting AI as a powerful diagnostic tool.

Experimentally Validated Finite Element Analysis of Thoracic Spine Compression Fractures in a Porcine Model.

Vertebral compression fractures (VCFs) occur in 1 to 1.5 million patients in the US each year and are associated with pain, disability, altered pulmonary function, secondary vertebral fracture, and increased mortality risk. A better understanding of VCFs and their management requires preclinical models that are both biomechanically analogous and accessible. We conducted a study using twelve spinal vertebrae (T12-T14) from porcine specimens. We created mathematical simulations of vertebral compression fractures (VCFs) using CT scans for reconstructing native anatomy and validated the results by conducting physical axial compression experiments. The simulations accurately predicted the behavior of the physical compressions. The coefficient of determination for stiffness was 0.71, the strength correlation was 0.88, and the failure of the vertebral bodies included vertical splitting on the lateral sides or horizontal separation in the anterior wall. This finite element method has important implications for the preventative, prognostic, and therapeutic management of VCFs. This study also supports the use of porcine specimens in orthopedic biomechanical research.

Long-term statin therapy is associated with severe coronary artery calcification.

BACKGROUND: Atherosclerosis and consequent risk of cardiovascular events or mortality can be accurately assessed by quantifying coronary artery calcium score (CACS) derived from computed tomography. HMG-CoA-reductase inhibitors (statins) are the primary pharmacotherapy used to reduce cardiovascular events, yet there is growing data that support statin use may increase coronary calcification. We set out to determine the likelihood of severe CACS in the context of chronic statin therapy. METHODS: We established a retrospective, case-control study of 1,181 U.S. veterans without coronary artery disease (CAD) from a single site, the Providence VA Medical Center. Duration of statin therapy for primary prevention was divided into 5-year categorical increments. The primary outcome was CACS derived from low-dose lung cancer screening computed tomography (LCSCT), stratified by CACs severity (none = 0; mild = 1-99; moderate = 100-399; and severe ≥400 AU). Statin duration of zero served as the referent control. Ordinal logistic regression analysis determined the association between duration of statin use and CACS categories. Proportional odds assumption was tested using likelihood ratio test. Atherosclerotic cardiovascular disease (ASCVD) risk score, body mass index, and CKD (glomerular filtration rate of <60 ml/min/1.73 m2) were included in the adjustment models. RESULTS: The mean age of the study population was 64.7±7.2 years, and 706 (60%) patients were prescribed a statin at baseline. Duration of statin therapy was associated with greater odds of having increased CACS (>0-5 years, OR: 1.71 [CI: 1.34-2.18], p<0.001; >5-10 years, OR: 2.80 [CI: 2.01-3.90], p<0.001; >10 years, OR: 5.30 [CI: 3.23-8.70], p<0.001), and the relationship between statin duration and CACS remained significant after multivariate adjustment (>0-5 years, OR: 1.49 [CI: 1.16-1.92], p = 0.002; >5-10 years, OR: 2.38 [CI: 1.7-3.35], p<0.001; >10 years, OR: 4.48 [CI: 2.7-7.43], p<0.001). CONCLUSIONS: Long-term use of statins is associated with increased likelihood of severe CACS in patients with significant smoking history. The use of CACS to interpret cardiovascular event risk may require adjustment in the context of chronic statin therapy.

Macrophage IL-1ß promotes arteriogenesis by autocrine STAT3- and NF-κB-mediated transcription of pro-angiogenic VEGF-A.

Peripheral artery disease (PAD) leads to considerable morbidity, yet strategies for therapeutic angiogenesis fall short of being impactful. Inflammatory macrophage subsets play an important role in orchestrating post-developmental angiogenesis, but the underlying mechanisms are unclear. Here, we find that macrophage VEGF-A expression is dependent upon the potent inflammatory cytokine, IL-1ß. IL-1ß promotes pro-angiogenic VEGF-A165a isoform transcription via activation and promoter binding of STAT3 and NF-κB, as demonstrated by gene-deletion, gain-of-function, inhibition, and chromatin immunoprecipitation assays. Conversely, IL-1ß-deletion or inhibition of STAT3 or NF-κB increases anti-angiogenic VEGF-A165b isoform expression, indicating IL-1ß signaling may also direct splice variant selection. In an experimental PAD model of acute limb ischemia, macrophage IL-1ß expression is required for pro-angiogenic VEGF-A expression and for VEGF-A-induced blood flow recovery via angio- or arteriogenesis. Though further study is needed, macrophage IL-1ß-dependent transcription of VEGF-A via STAT3 and NF-κB may have potential to therapeutically promote angiogenesis in the setting of PAD.

Rac GTPase Signaling in Immune-Mediated Mechanisms of Atherosclerosis.

Coronary artery disease caused by atherosclerosis is a major cause of morbidity and mortality around the world. Data from preclinical and clinical studies support the belief that atherosclerosis is an inflammatory disease that is mediated by innate and adaptive immune signaling mechanisms. This review sought to highlight the role of Rac-mediated inflammatory signaling in the mechanisms driving atherosclerotic calcification. In addition, current clinical treatment strategies that are related to targeting hypercholesterolemia as a critical risk factor for atherosclerotic vascular disease are addressed in relation to the effects on Rac immune signaling and the implications for the future of targeting immune responses in the treatment of calcific atherosclerosis.

Statins Disrupt Macrophage Rac1 Regulation Leading to Increased Atherosclerotic Plaque Calcification.

OBJECTIVE: Calcification of atherosclerotic plaque is traditionally associated with increased cardiovascular event risk; however, recent studies have found increased calcium density to be associated with more stable disease. 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitors or statins reduce cardiovascular events. Invasive clinical studies have found that statins alter both the lipid and calcium composition of plaque but the molecular mechanisms of statin-mediated effects on plaque calcium composition remain unclear. We recently defined a macrophage Rac (Ras-related C3 botulinum toxin substrate)-IL-1ß (interleukin-1 beta) signaling axis to be a key mechanism in promoting atherosclerotic calcification and sought to define the impact of statin therapy on this pathway. Approach and Results: Here, we demonstrate that statin therapy is independently associated with elevated coronary calcification in a high-risk patient population and that statins disrupt the complex between Rac1 and its inhibitor RhoGDI (Rho GDP-dissociation inhibitor), leading to increased active (GTP bound) Rac1 in primary monocytes/macrophages. Rac1 activation is prevented by rescue with the isoprenyl precursor geranylgeranyl diphosphate. Statin-treated macrophages exhibit increased activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), increased IL-1ß mRNA, and increased Rac1-dependent IL-1ß protein secretion in response to inflammasome stimulation. Using an animal model of calcific atherosclerosis, inclusion of statin in the atherogenic diet led to a myeloid Rac1-dependent increase in atherosclerotic calcification, which was associated with increased serum IL-1ß expression, increased plaque Rac1 activation, and increased plaque expression of the osteogenic markers, alkaline phosphatase and RUNX2 (Runt-related transcription factor 2). CONCLUSIONS: Statins are capable of increasing atherosclerotic calcification through disinhibition of a macrophage Rac1-IL-1ß signaling axis.