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Objects: To investigate the value of CT-based 3D templating software for pre-operative planning in patients with acetabular dysplasia undergoing total hip arthroplasty (THA) with a minimum ...

Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism. Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1β (IL-1β)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3'-untranslated region (3'-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured. Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3'-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1β-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects. Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.

Depressed calcium handling by the sarcoplasmic reticulum (SR) Ca-ATPase and its regulator phospholamban (PLN) is a key characteristic of human and experimental heart failure. Accumulating evidence indicates that increases in the relative levels of PLN to Ca-ATPase in failing hearts and resulting inhibition of Ca sequestration during diastole, impairs contractility. Here, we identified a genetic variant in the PLN promoter region, which increases its expression and may serve as a genetic modifier in dilated cardiomyopathy (DCM). The variant AF177763.1:g.203A>C (at position -36 bp relative to the PLN transcriptional start site) was found only in the heterozygous form in 1 out of 296 normal subjects and in 22 out of 381 cardiomyopathy patients (heart failure at age of 18-44 years, ejection fraction=22+/-9%). In vitro analysis, using luciferase as a reporter gene in rat neonatal cardiomyocytes, indicated that the PLN-variant increased activity by 24% compared to the wild type. Furthermore, the g.203A>C substitution altered the specific sequence of the steroid receptor for the glucocorticoid nuclear receptor (GR)/transcription factor in the PLN promoter, resulting in enhanced binding to the mutated DNA site. These findings suggest that the g.203A>C genetic variant in the human PLN promoter may contribute to depressed contractility and accelerate functional deterioration in heart failure.

The transcription factor NF-kappaB regulates a wide variety of biological effects in diverse cell types and organs, particularly stress and adaptive responses. Recently, it has become recognized that NF-kappaB and its upstream regulator tumor necrosis factor (TNF)-alpha regulate specific antithetical effects. For instance, in the heart, NF-kappaB has been found to be required for development of late preconditioning against myocardial infarction and yet is critically involved in mediating cell death after ischemia/reperfusion injury. There remains a bias that NF-kappaB is a "general" transcription factor that is activated by a plethora of stimuli, including neurohormonal, pathophysiological, and stress stimuli, and affects regulation of numerous downstream genes. The question has become, how can such a "general" transcription factor be critically involved in mediating specific effects? An emerging hypothesis is that NF-kappaB is part of a complicated signaling network or web, and that different combinatorial interactions between various activated signaling pathway components produce specific outcomes. This idea is supported by the large number of interactions discovered in the past 14 years between NF-kappaB and other signaling pathways at multiple levels. Notwithstanding the complexities of signal-induced activation of NF-kappaB, since it is a transcription factor, specific effects of NF-kappaB activation must be underlain by the activation and/or suppression of distinct subsets of NF-kappaB-dependent genes. At this level, selectivity is conferred by the expression of specific NF-kappaB subunits, their post translational modifications, and by combinatorial interactions between NF-kappaB and other transcription factors and coactivators that form specific enhanceosome complexes in association with particular promoters. These enhanceosome complexes represent another level of signaling integration whereby the activities of multiple upstream pathways converge to impress a distinct pattern of gene expression upon the NF-kappaB-dependent transcriptional network. Understanding how the overall cellular signaling network translates NF-kappaB activation into the regulation of specific subsets of NF-kappaB-dependent genes will lead to a mechanistic understanding of how NF-kappaB mediates diverse and paradoxical biological effects.

The relative expression of α1- and α2-Na+/K+-ATPase isoforms found in vascular smooth muscle is developmentally regulated and under hormonal and neurogenic control. The physiological roles of these isoforms in vascular function are not known. It has been postulated that the α1-isoform serves a “housekeeping” role, whereas the α2-isoform localizes to a subsarcolemmal compartment and modulates contractility. To test this hypothesis, isoform-specific gene-targeted mice in which the mRNA for either the α1- or the α2-Na+/K+-ATPase isoform was ablated were utilized. Both of these knockouts, [Formula: see text] and [Formula: see text], are lethal; the latter dies at birth, which allows this neonatal aorta to be studied. Isometric force in [Formula: see text]-aorta was more sensitive to contractile agonists and less sensitive to the vasodilators forskolin and sodium nitroprusside (SNP) than wild-type (WT) aorta; [Formula: see text]-aortas had intermediate values. In contrast, neonatal [Formula: see text]-aorta was similar to WT. Western blot analysis indicated a population of 70% α1- and 30% α2-isoforms in the WT. Thus in terms of the total Na+/K+-ATPase protein, the [Formula: see text]-aorta (at 70%) would be similar to the [Formula: see text]-aorta (at 65%) but with a dramatically different phenotype. These data suggest that individual α-isoforms of the Na+/K+-ATPase differ functionally and that the α2-isoform couples more strongly to activation-relaxation pathways. Three-dimensional image-acquisition and deconvolution analyses suggest that the α2-isoform is distributed differently than the α1-isoform. Importantly, these isoforms do not localize to the same regions.

The Na,K-ATPase is composed of two subunits, alpha and beta, and each subunit consists of multiple isoforms. In the case of alpha, four isoforms, alpha1, alpha2, alpha3, and alpha4 are present in mammalian cells. The distribution of these isoforms is tissue- and developmental-specific, suggesting that they may play specific roles, either during development or coupled to specific physiological processes. In order to understand the functional properties of each of these isoforms, we are using gene targeting, where animals are produced lacking either one copy or both copies of the corresponding gene or have a modified gene. To date, we have produced animals lacking the alpha1 and alpha2 isoform genes. Animals lacking both copies of the alpha1 isoform gene are not viable, while animals lacking both copies of the alpha2 isoform gene make it to birth, but are either born dead or die very soon after. In the case of animals lacking one copy of the alpha1 or alpha2 isoform gene, the animals survive and appear healthy. Heart and EDL muscle from animals lacking one copy of the alpha2 isoform exhibit an increase in force of contraction, while there is reduced force of contraction in both muscles from animals lacking one copy of the alpha1 isoform gene. These studies indicate that the alpha1 and alpha2 isoforms carry out different physiological roles. The alpha2 isoform appears to be involved in regulating Ca(2+) transients involved in muscle contraction, while the alpha1 isoform probably plays a more generalized role. While we have not yet knocked out the alpha3 or alpha4 isoform genes, studies to date indicate that the alpha4 isoform is necessary to maintain sperm motility. It is thus possible that the alpha2, alpha3, and alpha4 isoforms are involved in specialized functions of various tissues, helping to explain their tissue- and developmental-specific regulation.

1The transcript of the Na+-Ca2+ exchanger gene NCX1 undergoes alternative splicing to produce tissue-specific isoforms. The cloned NCX1 isoforms were expressed in Xenopus oocytes and studied using a two-electrode voltage clamp method to measure Na+-Ca2+ exchanger activity. 2The cardiac isoform (NCX1.1) expressed in oocytes was less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform (NCX1.3). 3The cardiac isoform of NCX1 is more sensitive to activation by protein kinase A (PKA) than the renal isoform which may be explained by preferential phosphorylation. The cardiac isoform of NCX1 is phosphorylated to a greater extent than the renal isoform. 4The action of PKA phosphorylation which increases the activity of the cardiac isoform of the Na+-Ca2+ exchanger in oocytes was confirmed in adult rat ventricular cardiomyocytes by measuring Na+-dependent Ca2+ flux. 5We conclude that alternative splicing of NCX1 confers distinct functional characteristics to tissue-specific isoforms of the Na+-Ca2+ exchanger.

Using a new class of nontoxic and degradable glycopolymer-based vehicles termed poly(glycoamidoamine)s, we demonstrate virus-like delivery efficacy of oligodeoxynucleotide (ODN) decoys to cardiomyoblasts (H9c2), primary cardiomyocytes, and the mouse heart. These glycopolymers bind and compact ODN decoys into nanoparticle complexes that are internalized by the cell membrane and mediate nuclear uptake of DNA in 90+% of cultured primary cardiomyocytes and 87% of the mouse myocardium. Experimental results reveal that decoys delivered via these glycopolymers block the activation of the transcription factor NF-κB, a major contributor to ischemia/reperfusion injury. Decoy complexes formed with glycopolymer T4 significantly blocked downstream gene expression of Cox-2 and limited myocardial infarction in vivo, phenocopying a transgenic mouse model. These promising delivery vehicles may facilitate high-throughput genetic approaches in animal models. Additionally, the low toxicity, biodegradation, and outstanding delivery efficacy suggest that these nanomedicines may be clinically applicable for gene regulatory therapy.

NF-kappaB is a pleiotropic transcription factor implicated in the regulation of diverse biological phenomena, including apoptosis, cell survival, cell growth, cell division, innate immunity, cellular differentiation, and the cellular responses to stress, hypoxia, stretch and ischemia. In the heart, NF-kappaB has been shown to be activated in atherosclerosis, myocarditis, in association with angina, during transplant rejection, after ischemia/reperfusion, in congestive heart failure, dilated cardiomyopathy, after ischemic and pharmacological preconditioning, heat shock, burn trauma, and in hypertrophy of isolated cardiomyocytes. Regulation of NF-kappaB is complicated; in addition to being activated by canonical cytokine-mediated pathways, NF-kappaB is activated by many of the signal transduction cascades associated with the development of cardiac hypertrophy and response to oxidative stress. Many of these signaling cascades activate NF-kappaB by activating the IkappaB kinase (IKK) complex a major component of the canonical pathway. These signaling interactions occur largely via signaling crosstalk involving the mitogen-activated protein kinase/extracellular signalregulated kinase kinases (MEKKs) that are components of mitogen activated protein kinase (MAPK) signaling pathways. Additionally, there are other signaling factors that act more directly to activate NF-kappaB via IkappaB or by direct phosphorylation of NF-kappaB subunits. Finally, there are combinatorial interactions at the level of the promoter between NF-kappaB, its coactivators, and other transcription factors, several of which are activated by MAPK and cytokine signaling pathways. Thus, in addition to being a major mediator of cytokine effects in the heart, NF-kappaB is positioned as a signaling integrator. As such, NF-kappaB functions as a key regulator of cardiac gene expression programs downstream of multiple signal transduction cascades in a variety of physiological and pathophysiological states. We show that genetic blockade of NF-kappaB reduces infarct size in the murine heart after ischemia/reperfusion (I/R), implicating NF-kappaB as a major determinant of cell death after I/R. These results support the concept that NF-kappaB may be an important therapeutic target for specific cardiovascular diseases.

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the alpha2 isoform is widely expressed in neurons, unlike in the adult brain, in which alpha2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of alpha2 isoform expression in neurons is interesting in light of our examination of mice lacking the alpha2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-Bötzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase alpha2 isoform could be important in the modulation of neuronal activity in the neonate.

Cardiovascular disease represents a promising clinical target for novel gene therapy and oligonucleotide treatments because of the lack of effective pharmacological agents that specifically block myocardial injury and infarction post ischemia and reperfusion (I/R). It has been shown that activation of a transcription factor, NF-kappaB following I/R, may mediate myocardial cell death.1 Previous studies2 indicate that the delivery of a naked double stranded oligonucleotide sequence (termed a DNA decoy) containing the NF-kappaB DNA binding sequence can effectively block activation of NF-kappaB in the heart in vivo. That study revealed that a reduction in NF-kappaB signaling leads to a decrease in myocardial necrosis. Thus, a significant reduction in the area of myocardial infarction was observed.2 However, one of the greatest challenges with DNA delivery has been developing a suitable delivery system that can effectively increase the DNA decoy efficacy and the area of delivery in the myocardium.