Georgiana Cheng

Clinical and Echocardiographic Characteristics of Papillary Fibroelastomas A Retrospective and Prospective Study in 162 Patients

Background—Cardiac papillary fibroelastoma (CPF) is a primary cardiac neoplasm that is increasingly detected by echocardiography. The clinical manifestations of this entity are not well described. Methods and Results—In a 16-year period, we identified patients with CPF from our pathology and echocardiography databases. A total of 162 patients had pathologically confirmed CPF. Echocardiography was performed in 141 patients with 158 CPFs, and 48 patients had CPFs that were not visible by echocardiography (<0.2 cm), leaving an echocardiographic subgroup of 93 patients with 110 CPFs. An additional 45 patients with a presumed diagnosis of CPF were identified. The mean age of the patients was 60±16 years of age, and 46.1% were male. Echocardiographically, the mean size of the CPFs was 9±4.6 mm; 82.7% occurred on valves (aortic more than mitral), 43.6% were mobile, and 91.4% were single. During a follow-up period of 11±22 months, 23 of 26 patients with a prospective diagnosis of CPF that was confirmed by pathological examination had symptoms that could be attributable to embolization. In the group of 45 patients with a presumed diagnosis of CPF, 3 patients had symptoms that were likely due to embolization (incidence, 6.6%) during a follow-up period of 552±706 days. Conclusions—CPFs are generally small and single, occur most often on valvular surfaces, and may be mobile, resulting in embolization. Because of the potential for embolic events, symptomatic patients, patients undergoing cardiac surgery for other lesions, and those with highly mobile and large CPFs should be considered for surgical excision.

Persistent over-expression of specific CC class chemokines correlates with macrophage and T-cell recruitment in mdx skeletal muscle

Prior studies and the efficacy of immunotherapies provide evidence that inflammation is mechanistic in pathogenesis of Duchenne muscular dystrophy. To identify putative pro-inflammatory mechanisms, we evaluated chemokine gene/protein expression patterns in skeletal muscle of mdx mice. By DNA microarray, reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, and immunoblotting, convergent evidence established the induction of six distinct CC class chemokine ligands in adult MDX: CCL2/MCP-1, CCL5/RANTES, CCL6/mu C10, CCL7/MCP-3, CCL8/MCP-2, and CCL9/MIP-1gamma. CCL receptors, CCR2, CCR1, and CCR5, also showed increased expression in mdx muscle. CCL2 and CCL6 were localized to both monocular cells and muscle fibers, suggesting that dystrophic muscle may contribute toward chemotaxis. Temporal patterns of CCL2 and CCL6 showed early induction and maintained expression in mdx limb muscle. These data raise the possibility that chemokine signaling pathways coordinate a spatially and temporally discrete immune response that may contribute toward muscular dystrophy. The chemokine pro-inflammatory pathways described here in mdx may represent new targets for treatment of Duchenne muscular dystrophy.

Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice

Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin‐glycoprotein complex (DGC)‐competent and DGC‐deficient myofibers distributed in a fiber type‐specific pattern. Up‐regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type‐specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and α‐syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, α7β1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Temporal and spatial mRNA expression patterns of TGF-β1, 2, 3 and TβRI, II, III in skeletal muscles of mdx mice

Abstract To address potential regulatory roles of TGF-β1 in muscle inflammation and fibrosis associated with dystrophin deficiency, we performed quantitative RT-PCR and in situ hybridization to characterize the temporal and spatial mRNA expression patterns of TGF-β1 and other TGF-β subfamily members, TGF-β2 and TGF-β3, as well as their receptors, in quadriceps and diaphragm muscles of mdx mice. TGF-β1 mRNA was markedly upregulated in the endomysial inflammatory cells and regenerating fibers of mdx quadriceps and diaphragm, with the mRNA levels correlated with the degree of endomysial inflammation. Upregulation of TGF-β2, β3, and their receptors was also appreciated but to a much lesser degree. While high levels of TGF-β1 mRNA remained in the aging mdx quadriceps but not the diaphragm, progressive fibrosis only occurred in the diaphragm. Our data support a regulatory role for TGF-β1 in muscle inflammation in mdx mice. It also suggests different susceptibility of quadriceps and diaphragm muscles to fibrosis induced by TGF-β1 signaling pathway.

Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice

To address whether mdx mice with haploinsufficiency of utrophin (mdx/utrn+/-) develop more severe skeletal muscle inflammation and fibrosis than mdx mice, to represent a better model for Duchenne muscular dystrophy (DMD), we performed qualitative and quantitative analysis of skeletal muscle inflammation and fibrosis in mdx and mdx/utrn+/- littermates. Inflammation was significantly worse in mdx/utrn+/- quadriceps at age 3 and 6 months and in mdx/utrn+/- diaphragm at age 3 but not 6 months. Fibrosis was more severe in mdx/utrn+/- diaphragm at 6 months, and at this age, mild fibrosis was noted in quadriceps of mdx/utrn+/- but not mdx mice. The findings indicate that utrophin compensates, although insufficiently, for the effects of dystrophin loss with regard to inflammation and fibrosis of both quadriceps and diaphragm muscles in mdx mice. With more severe muscle dystrophy than mdx mice and a longer life span than utrophin-dystrophin-deficient (dko) mice, mdx/utrn+/- mice provide a better mouse model for testing potential therapies for muscle inflammation and fibrosis associated with DMD.

Hyaluronan deposition and correlation with inflammation in a murine ovalbumin model of asthma.

Asthma is a chronic inflammatory disease of the airways characterized by airway remodeling, which includes changes in the extracellular matrix (ECM). However the role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in asthma as well as in many other biological processes. Our study investigates the processes involved in HA synthesis, deposition, localization and degradation during an acute and chronic murine model of ovalbumin (OVA)-induced allergic pulmonary inflammation. Mice were sensitized, challenged to OVA and sacrificed at various time points during an 8-week challenge protocol. Bronchoalveolar lavage (BAL) fluids, blood, and lung tissue were collected for study. RNA, HA, protein and histopathology were analyzed. Analyses of lung sections and BAL fluids revealed an early deposition and an increase in HA levels within 24 h of antigen exposure. HA levels peaked at day 8 in BAL, while inflammatory cell recovery peaked at day 6. Hyaluronan synthase (HAS)1 and HAS2 on RNA levels peaked within 2 h of antigen exposure, while hyaluronidase (HYAL)1 and HYAL2 on RNA levels decreased. Both inflammatory cell infiltrates and collagen deposition co-localized with HA deposition within the lungs. These data support a role for HA in the pathogenesis of inflammation and airway remodeling in a murine model of asthma. HA deposition appears largely due to up regulation of HAS1 and HAS2. In addition, HA appears to provide the scaffolding for inflammatory cell accumulation as well as for new collagen synthesis and deposition.

Impaired respiratory function in mdx and mdx/utrn+/− mice

Muscle fibrosis is a prominent pathological feature that directly causes muscle dysfunction in Duchenne muscular dystrophy (DMD). The DMD mouse models, mdx mice and mdx mice with haploinsufficiency of the utrophin gene (mdx/utrn+/−), display progressive diaphragm fibrosis. We performed unrestrained whole‐body plethysmography (WBP) in mdx and mdx/utrn+/− mice, and compared them with wild‐type controls. Respiratory function gauged by respiratory frequency, tidal volume, minute volume, peak inspiratory flow, and peak expiratory flow was significantly impaired in the mdx mice. Consistent with more severe diaphragm fibrosis in the mdx/utrn+/− mice, respiratory impairment was worse than in mdx mice at 6 months. WBP is useful for monitoring in vivo respiratory function of mdx and mdx/utrn+/− mice, and it may serve as an outcome measurement for therapies that target diaphragm fibrosis. The mdx/utrn+/− mouse model may be better than the mdx model for testing antifibrotic therapies, especially at the severe stage. Muscle Nerve 43: 263–267, 2011

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

Background: TSG-6 transfers heavy chains (HCs) from the inter-α-inhibitor to hyaluronan (HA), increasing its avidity for leukocytes. Results: Recombinant TSG-6 increased leukocyte adhesion to HA and its accumulation in airway cells. Conclusion: In addition to its ability to transfer HCs to HA, TSG-6 amplifies HA synthesis. Significance: TSG-6 is a potent regulator of HA synthesis and is likely to be involved in a variety of inflammatory diseases. We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6−/−, HAS1/3−/−, and CD44−/− mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

TSG-6 protein is crucial for the development of pulmonary hyaluronan deposition, eosinophilia, and airway hyperresponsiveness in a murine model of asthma.

Background: TSG-6 is important in the organization of hyaluronan (HA). Results: Lack of TSG-6 results in diminished HA accumulation, inflammation, and airway hyperresponsiveness. Conclusion: TSG-6 is essential for the pathological manifestations in a murine model of asthma. Significance: TSG-6 is likely to contribute to the pathogenesis of asthma. Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6−/− mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6−/− mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6−/− mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.

An Altered Phenotype in a Conditional Knockout of Pitx2 in Extraocular Muscle

PURPOSE To determine the temporal and spatial expression of Pitx2, a bicoid-like homeobox transcription factor, during postnatal development of mouse extraocular muscle and to evaluate its role in the growth and phenotypic maintenance of postnatal extraocular muscle. METHODS Mouse extraocular muscles of different ages were examined for the expression of Pitx2 by RT-PCR, q-PCR, and immunostaining. A conditional mutant mouse strain, in which Pitx2 function is inactivated at postnatal day (P)0, was generated with a Cre-loxP strategy. Histology, immunostaining, real-time PCR, in vitro muscle contractility, and in vivo ocular motility were used to study the effect of Pitx2 depletion on extraocular muscle. RESULTS All three Pitx2 isoforms were expressed by extraocular muscle and at higher levels than in other striated muscles. Immunostaining demonstrated the presence of Pitx2 mainly in extraocular muscle myonuclei. However, no obvious expression patterns were observed in terms of anatomic region (orbital versus global layer), innervation zone, or muscle fiber types. The mutant extraocular muscle had no obvious pathology but had altered muscle fiber sizes. Expression levels of myosin isoforms Myh1, Myh6, Myh7, and Myh13 were reduced, whereas Myh2, Myh3, Myh4, and Myh8 were not affected by postnatal loss of Pitx2. In vitro, Pitx2 loss made the extraocular muscles stronger, faster, and more fatigable. Eye movement recordings found saccades to have a lower peak velocity. CONCLUSIONS Pitx2 is important in maintaining the mature extraocular muscle phenotype and regulating the expression of critical contractile proteins. Modulation of Pitx2 expression can influence extraocular muscle function with long-term therapeutic implications.