Faculty Bibliography

Major depressive disorder is a common psychiatric disorder that is thought to be triggered by both genetic and environmental factors. Depressive symptoms are an important public health problem and contribute to vulnerability to major depression. Although a substantial number of genetic and epigenetic studies have been performed to date, the detailed etiology of depression remains unclear and there are no validated biomarkers. DNA methylation is one of the major epigenetic modifications that play diverse roles in the etiology of complex diseases. In this study, we performed an epigenome-wide association study (EWAS) of DNA methylation on subjects with (N = 20) or without (N = 27) depressive symptoms in order to examine whether different levels of DNA methylation were associated with depressive tendencies. Employing methylation-array technology, a total of 363,887 methylation sites across the genomes were investigated and several candidate CpG sites associated with depressive symptoms were identified, especially annotated to genes linked to a G-protein coupled receptor protein signaling pathway. These data provide a strong impetus for validation studies using a larger cohort and support the possibility that G-protein coupled receptor protein signaling pathways are involved in the pathogenesis of depression.

The purpose of this study was to examine the influence of topical vancomycin on cell migration and survival of tissue healing cells. Human osteoblasts, myoblasts and fibroblasts were exposed to vancomycin at concentrations of 1, 3, 6, or 12 mg/cm² for either a 1-h or 48-h (continuous) duration. Continuous exposure to all vancomycin concentrations significantly reduced cell survival (<22% cells survived) and migration in osteoblasts and myoblasts (P < 0.001). 1-h vancomycin exposure reduced osteoblast and myoblast survival and migration only at 12 mg/cm² (P < 0.001). Further in vivo studies are warranted to optimize the dosage of intrawound vancomycin.

ATP-sensitive K+ (KATP) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of KATP channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of KATP channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate KATP channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of KATP channel trafficking to increase surface KATP channel density. EHD2 had no effect on cardiac Na+ channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on KATP channel surface expression. EHD2 did not directly affect KATP channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing KATP channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated KATP channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with KATP channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal KATP channel surface expression: role in cardioprotection.

While therapeutic modulation of miRNAs provides a promising approach for numerous diseases, the promiscuous nature of miRNAs raises concern over detrimental off-target effects. miR-33 has emerged as a likely target for treatment of cardiovascular diseases. However, the deleterious effects of long-term anti-miR-33 therapies and predisposition of miR-33^-/-mice to obesity and metabolic dysfunction exemplify the possible pitfalls of miRNA-based therapies. Our work provides an in-depth characterization of miR-33^-/-mice and explores the mechanisms by which loss of miR-33 promotes insulin resistance in key metabolic tissues. Contrary to previous reports, our data do not support a direct role for SREBP-1-mediated lipid synthesis in promoting these effects. Alternatively, in adipose tissue of miR-33^-/-mice, we observe increased pre-adipocyte proliferation, enhanced lipid uptake, and impaired lipolysis. Moreover, we demonstrate that the driving force behind these abnormalities is increased food intake, which can be prevented by pair feeding with wild-type animals.

In amyotrophic lateral sclerosis (ALS) and animal models of ALS, includingSOD1-G93Amice, disassembly of the neuromuscular synapse precedes motor neuron loss and is sufficient to cause a decline in motor function that culminates in lethal respiratory paralysis. We treatedSOD1-G93Amice with an agonist antibody to MuSK, a receptor tyrosine kinase essential for maintaining neuromuscular synapses, to determine whether increasing muscle retrograde signaling would slow nerve terminal detachment from muscle. The agonist antibody, delivered after disease onset, slowed muscle denervation, promoting motor neuron survival, improving motor system output, and extending the lifespan ofSOD1-G93Amice. These findings suggest a novel therapeutic strategy for ALS, using an antibody format with clinical precedence, which targets a pathway essential for maintaining attachment of nerve terminals to muscle.

Rationale: Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and/or methods that block liver lipoprotein secretion. Objective: We sought to develop a more direct and time-effective method to create and then reverse hypercholesterolemia as well as atherosclerosis via transient knockdown of the hepatic LDL receptor (LDLR) followed by its rapid restoration. Methods and Results: We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels due to recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, i.e. regression of inflammatory cell content, after a period of 2-3 weeks. Conclusions: We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. While cholesterol reduction decreased early en-face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.

Rationale: Genome-Wide Association Studies identified single nucleotide polymorphisms (SNPs) near the SORT1 locus strongly associated with decreased plasma low-density lipoprotein cholesterol (LDL-C) levels and protection from atherosclerotic cardiovascular disease and myocardial infarction. The minor allele of the causal SORT1 SNP locus creates a putative C/EBPα binding site in the SORT1 promoter, thereby increasing sortilin expression by 12-fold in liver, which is rich in this transcription factor. Our previous studies in mice have showed reductions in plasma LDL-C and its principal protein component, apolipoprotein B (apoB) with increased SORT1 expression, and in vitro studies suggested that sortilin promoted the presecretory lysosomal degradation of apoB associated with the LDL precursor, very-low density lipoprotein (VLDL). Objective: To determine directly that SORT1 overexpression results in apoB degradation and to identify the mechanisms by which this reduces apoB and VLDL secretion by the liver, thereby contributing to understanding the clinical phenotype of lower LDL-C levels. Methods and Results: Pulse-chase studies directly established that SORT1 overexpression results in apoB degradation. As noted above, previous work implicated a role for lysosomes in this degradation. Through in vitro and in vivo studies, we now demonstrate that the sortilin-mediated route of apoB to lysosomes is unconventional and intersects with autophagy. Increased expression of sortilin diverts more apoB away from secretion, with both proteins trafficking to the endosomal compartment in vesicles that fuse with autophagosomes to form amphisomes. The amphisomes then merge with lysosomes. Furthermore, we show that sortilin itself is a regulator of autophagy and that its activity is scaled to the level of apoB synthesis. Conclusions: These results strongly suggest that an unconventional lysosomal targeting process dependent on autophagy degrades apoB that was diverted from the secretory pathway by sortilin, and provide a mechanism contributing to the reduced LDL-C found in individuals with SORT1 overexpression.

The key stages of fracture healing are proliferation, differentiation, and remodeling. Each stage of fracture healing is regulated by specific growth factors. Orthopaedic surgeons should understand the basic biologic principles of fracture repair and the therapeutic targets that can augment the natural regenerative capacity of the human body. In addition, orthopaedic surgeons should be aware of the key regulators in fracture healing and their potential uses in the field of orthopaedics.