Profoundly lower muscle mass and rate of contractile protein synthesis in boys with Duchenne muscular dystrophy.

Boys with Duchenne muscular dystrophy (DMD) experience a progressive loss of functional muscle mass, with fibrosis and lipid accumulation. Accurate evaluation of whole-body functional muscle mass (MM) in DMD patients has not previously been possible and the rate of synthesis of muscle proteins remains unexplored. We used non-invasive, stable isotope-based methods from plasma and urine to measure the fractional rate of muscle protein synthesis (FSR) functional muscle mass (MM), and fat free mass (FFM) in 10 DMD (6-17 years) and 9 age-matched healthy subjects. An oral dose of D3 creatine in 70% 2 H2 O was administered to determine MM and FFM followed by daily 70% 2 H2 O to measure protein FSR. Functional MM was profoundly reduced in DMD subjects compared to controls (17% vs. 41% of body weight, P < 0.0001), particularly in older, non-ambulant patients in whom functional MM was extraordinarily low (<13% body weight). We explored the urine proteome to measure FSR of skeletal muscle-derived proteins. Titin, myosin light chain and gelsolin FSRs were substantially lower in DMD subjects compared to controls (27%, 11% and 40% of control, respectively, P < 0.0001) and were strongly correlated. There were no differences in muscle-derived sarcoplasmic proteins FSRs (creatine kinase M-type and carbonic anhydrase-3) measured in plasma. These data demonstrate that both functional MM, body composition and muscle protein synthesis rates can be quantified non-invasively and are markedly different between DMD and control subjects and suggest that the rate of contractile but not sarcoplasmic protein synthesis is affected by a lack of dystrophin. KEY POINTS: Duchenne muscular dystrophy (DMD) results in a progressive loss of functional skeletal muscle but total body functional muscle mass or rates of muscle protein synthesis have not previously been assessed in these patients. D3 -creatine dilution was used to measure total functional muscle mass and oral 2 H2 O was used to examine the rates of muscle protein synthesis non-invasively in boys with DMD and healthy controls using urine samples. Muscle mass was profoundly lower in DMD compared to control subjects, particularly in older, non-ambulant patients. The rates of contractile protein synthesis but not sarcoplasmic proteins were substantially lower in DMD. These results may provide non-invasive biomarkers for disease progression and therapeutic efficacy in DMD and other neuromuscular diseases.

Loss of microfibril-associated protein 5 (MFAP5) expression in colon cancer stroma.

MFAP5, a 25-kD microfibril-associated glycoprotein that is involved in elastic microfibril assembly, has been demonstrated to be significantly downregulated in tumor stroma by previous gene expression study. The aim of this study was to confirm the reduced expression of MFAP5 in colonic tumor stroma using immunohistochemistry and to explore the utility of MFAP5 as a marker to facilitate diagnosing an invasive component versus pseudoinvasion in colon polyps. In all 19 colon cancer resection cases evaluated, while there was intact MFAP5 immunoreactivity in the uninvolved normal connective tissue, there was marked reduction of MFAP5 immunoreactivity in the desmoplastic stroma surrounding the invasive component. The difference in MFAP5 expression levels was most pronounced within the tumor, while a more heterogeneous expression pattern was observed at the tumor invasive front. Reduction of MFAP5 staining was also observed in the stroma around mucin pools in 6 out of 9 sections from mucinous adenocarcinomas and in areas with high-grade dysplasia. For the polypectomy cases, intact expression of MFAP5 was seen in the stroma surrounding the displaced adenomatous glands in 9 out of 12 polyps with pseudoinvasion. Loss of expression of MFAP5 was observed in the stroma surrounding small foci of invasive adenocarcinoma in 8 of 10 malignant polyps. MFAP5 is a useful marker that may help distinguish normal connective tissue from stroma within invasive colonic adenocarcinoma. MFAP5 may facilitate the distinction between pseudoinvasion and true invasive cancer in colonic adenomatous polyps with a sensitivity of 80% (confidence interval 44-96%) and a specificity of 75% (confidence interval 43-93%) in this small cohort.

Carbonic Anhydrase Inhibition Ameliorates Inflammation and Experimental Pulmonary Hypertension.

Inflammation and vascular smooth muscle cell (VSMC) phenotypic switching are causally linked to pulmonary arterial hypertension (PAH) pathogenesis. Carbonic anhydrase inhibition induces mild metabolic acidosis and exerts protective effects in hypoxic pulmonary hypertension. Carbonic anhydrases and metabolic acidosis are further known to modulate immune cell activation. To evaluate if carbonic anhydrase inhibition modulates macrophage activation, inflammation, and VSMC phenotypic switching in severe experimental pulmonary hypertension, pulmonary hypertension was assessed in Sugen 5416/hypoxia (SU/Hx) rats after treatment with acetazolamide or ammonium chloride (NH4Cl). We evaluated pulmonary and systemic inflammation and characterized the effect of carbonic anhydrase inhibition and metabolic acidosis in alveolar macrophages and bone marrow-derived macrophages (BMDMs). We further evaluated the treatment effects on VSMC phenotypic switching in pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs) and corroborated some of our findings in lungs and pulmonary arteries of patients with PAH. Both patients with idiopathic PAH and SU/Hx rats had increased expression of lung inflammatory markers and signs of PASMC dedifferentiation in pulmonary arteries. Acetazolamide and NH4Cl ameliorated SU/Hx-induced pulmonary hypertension and blunted pulmonary and systemic inflammation. Expression of carbonic anhydrase isoform 2 was increased in alveolar macrophages from SU/Hx animals, classically (M1) and alternatively (M2) activated BMDMs, and lungs of patients with PAH. Carbonic anhydrase inhibition and acidosis had distinct effects on M1 and M2 markers in BMDMs. Inflammatory cytokines drove PASMC dedifferentiation, and this was inhibited by acetazolamide and acidosis. The protective antiinflammatory effect of acetazolamide in pulmonary hypertension is mediated by a dual mechanism of macrophage carbonic anhydrase inhibition and systemic metabolic acidosis.

FGF receptors control alveolar elastogenesis.

Alveologenesis, the final step of lung development, is characterized by the formation of millions of alveolar septa that constitute the vast gas-exchange surface area. The genetic network driving alveologenesis is poorly understood compared with earlier steps in lung development. FGF signaling through receptors Fgfr3 and Fgfr4 is crucial for alveologenesis, but the mechanisms through which they mediate this process remain unclear. Here we show that in Fgfr3;Fgfr4 (Fgfr3;4) global mutant mice, alveolar simplification is first observed at the onset of alveologenesis at postnatal day 3. This is preceded by disorganization of elastin, indicating defects in the extracellular matrix (ECM). Although Fgfr3 and Fgfr4 are expressed in the mesenchyme and epithelium, inactivation in the mesenchyme, but not the epithelium, recapitulated the defects. Expression analysis of components of the elastogenesis machinery revealed that Mfap5 (also known as Magp2), which encodes an elastin-microfibril bridging factor, is upregulated in Fgfr3;4 mutants. Mfap5 mutation in the Fgfr3;4 mutant background partially attenuated the alveologenesis defects. These data demonstrate that, during normal lung maturation, FGF signaling restricts expression of the elastogenic machinery in the lung mesenchyme to control orderly formation of the elastin ECM, thereby driving alveolar septa formation to increase the gas-exchange surface.

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice.

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria.

Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility.

Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity.

Effects of miRNA-145 on airway smooth muscle cells function.

The pathological changes of airway smooth muscle (ASM) contribute to airway remodeling during asthma. Here, we investigated the effect of miR-145 on ASM function. We found that miR-145 was aberrantly more highly expressed in ASM cells exposed to cytokine stimulation that mimic the airway conditions of patients with asthma. Repression of miR-145 resulted in decreased ASM cell proliferation and migration in a dose-dependent manner and down-regulation of type I collagen and contractile protein MHC in ASM cells. qRT-PCR and Western blot analysis demonstrated that miR-145 negatively regulated the expression of downstream target Krüppel-like factor 4 (KLF4) protein, and overexpression of KLF4 attenuated the effects of miR-145 on ASM cells. Further studies showed that KLF4 significantly up-regulated the expression of p21 and down-regulated matrix metalloproteinase (MMP-2 and MMP-9). In conclusion, miR-145 overexpression in ASM cells significantly inhibited KLF4, and subsequently affected downstream p21, MMP-2, and MMP-9 expressions, eventually leading to enhanced proliferation and migration of ASM cells in vitro.

Long-term exposure to muscarinic agonists decreases expression of contractile proteins and responsiveness of rabbit tracheal smooth muscle cells.

BACKGROUND: Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs). METHODS: ASMCs were serum starved before treatment with muscarinic agonists. Cell phenotype was studied by optical microscopy and indirect immunofluorescence, using smooth muscle α-actin, desmin and SM-Myosin Heavy Chain (SM-MHC) antibodies. [N-methyl-3H]scopolamine binding studies were performed in order to assess M3 muscarinic receptor expression on isolated cell membranes. Contractility studies were performed on isolated ASMCs treated with muscarinic agonists. Proliferation was estimated using methyl-[3H]thymidine incorporation, MTT or cell counting methods. Involvement of PI3K and MAPK signalling pathways was studied by cell incubation with the pathway inhibitors LY294002 and PD98059 respectively. RESULTS: Prolonged culture of ASMCs with acetylcholine, carbachol or FBS, reduced the expression of α-actin, desmin and SM-MHC compared to cells cultured in serum free medium. Treatment of ASMCs with muscarinic agonists for 3-15 days decreased muscarinic receptor expression and their responsiveness to muscarinic stimulation. Acetylcholine and carbachol induced DNA synthesis and increased cell number, of ASMCs that had acquired a contractile phenotype by 7 day serum starvation. This effect was mediated via a PI3K and MAPK dependent mechanism. CONCLUSIONS: Prolonged exposure of rabbit ASMCs to muscarinic agonists decreases the expression of smooth muscle specific marker proteins, down-regulates muscarinic receptors and decreases ASMC contractile responsiveness. Muscarinic agonists are mitogenic, via the PI3K and MAPK signalling pathways.

Mammalian target of rapamycin (mTOR) inhibition with rapamycin improves cardiac function in type 2 diabetic mice: potential role of attenuated oxidative stress and altered contractile protein expression.

Elevated mammalian target of rapamycin (mTOR) signaling contributes to the pathogenesis of diabetes, with increased morbidity and mortality, mainly because of cardiovascular complications. Because mTOR inhibition with rapamycin protects against ischemia/reperfusion injury, we hypothesized that rapamycin would prevent cardiac dysfunction associated with type 2 diabetes (T2D). We also investigated the possible mechanisms and novel protein targets involved in rapamycin-induced preservation of cardiac function in T2D mice. Adult male leptin receptor null, homozygous db/db, or wild type mice were treated daily for 28 days with vehicle (5% DMSO) or rapamycin (0.25 mg/kg, intraperitoneally). Cardiac function was monitored by echocardiography, and protein targets were identified by proteomics analysis. Rapamycin treatment significantly reduced body weight, heart weight, plasma glucose, triglyceride, and insulin levels in db/db mice. Fractional shortening was improved by rapamycin treatment in db/db mice. Oxidative stress as measured by glutathione levels and lipid peroxidation was significantly reduced in rapamycin-treated db/db hearts. Rapamycin blocked the enhanced phosphorylation of mTOR and S6, but not AKT in db/db hearts. Proteomic (by two-dimensional gel and mass spectrometry) and Western blot analyses identified significant changes in several cytoskeletal/contractile proteins (myosin light chain MLY2, myosin heavy chain 6, myosin-binding protein C), glucose metabolism proteins (pyruvate dehydrogenase E1, PYGB, Pgm2), and antioxidant proteins (peroxiredoxin 5, ferritin heavy chain 1) following rapamycin treatment in db/db heart. These results show that chronic rapamycin treatment prevents cardiac dysfunction in T2D mice, possibly through attenuation of oxidative stress and alteration of antioxidants and contractile as well as glucose metabolic protein expression.

Changes in the expression of smooth muscle contractile proteins in TNBS- and DSS-induced colitis in mice.

Thin filament-associated proteins such as calponin, caldesmon, tropomyosin, and smoothelin are thought to regulate acto-myosin interaction and thus, muscle contraction. However, the effect of inflammation on the expression of thin filament-associated proteins is not known. The aim of the present study is to determine the changes in the expression of calponin, caldesmon, tropomyosin, and smoothelin in colonic smooth muscle from trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulphate (DSS)-induced colitis in mice. Expression of h-caldesmon, h2-calponin, α-tropomyosin, and smoothelin-A was measured by qRT-PCR and Western blot. Contraction in response to acetylcholine in dispersed muscle cells was measured by scanning micrometry. mRNA and protein expression of α-actin, h2-calponin, h-caldesmon, smoothelin, and α-tropomyosin in colonic muscle strips from mice with TNBS- or DSS-induced colitis was significantly increased compared to control animals. Contraction in response to acetylcholine was significantly decreased in muscle cells isolated from inflamed regions of TNBS- or DSS-treated mice compared to control mice. Our results show that increase in the expression of thin filament-associated contractile proteins, which inhibit acto-myosin interaction, could contribute to decrease in smooth muscle contraction in inflammation.

Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer.

BACKGROUND: Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer. METHODS: We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature. RESULTS: Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p = 0.082). CONCLUSIONS: We have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.

Skeletal muscle adaptation in response to mechanical stress in p130cas-/- mice.

Mammalian skeletal muscles undergo adaptation in response to changes in the functional demands upon them, involving mechanical-stress-induced cellular signaling called "mechanotransduction." We hypothesized that p130Cas, which is reported to act as a mechanosensor that transduces mechanical extension into cellular signaling, plays an important role in maintaining and promoting skeletal muscle adaptation in response to mechanical stress via the p38 MAPK signaling pathway. We demonstrate that muscle-specific p130Cas-/- mice express the contractile proteins normally in skeletal muscle. Furthermore, muscle-specific p130Cas-/- mice show normal mechanical-stress-induced muscle adaptation, including exercise-induced IIb-to-IIa muscle fiber type transformation and hypertrophy. Finally, we provide evidence that exercise-induced p38 MAPK signaling is not impaired by the muscle-specific deletion of p130Cas. We conclude that p130Cas plays a limited role in mechanical-stress-induced skeletal muscle adaptation.

TIPE2 deficiency accelerates neointima formation by downregulating smooth muscle cell differentiation.

Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a key role in the development of atherosclerosis. However, the mechanisms that mediate VSMC phenotypic switching are unclear. We report here that TIPE2, the tumor necrosis factor (TNF) α-induced protein 8-like 2 (TNFAIP8L2), plays an atheroprotective role by regulating phenotypic switching of VSMCs in response to oxidized low-density lipoprotein (ox-LDL) stimuli. TIPE2-deficient VSMCs treated with ox-LDL expressed lower levels of contractile proteins such as SMαA, SM-MHC and calponin, whereas the proliferation, migration and the synthetic capacity for growth factors and cytokines were increased remarkably. Furthermore, TIPE2 inhibited VSMCs proliferation by preventing G 1/S phase transition. Interestingly, these effects of TIPE2 on VSMCs were dependent on P38 and ERK1/2 kinase signals. As a result, neointima formation was accelerated in the carotid arteries of TIPE2-deficient mice. These results indicate that TIPE2 is a potential inhibitor of atherosclerosis.

Muscarinic receptor stimulation augments TGF-ß1-induced contractile protein expression by airway smooth muscle cells.

Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-ß(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M(2) and M(3) receptors were stimulated with methacholine (MCh), TGF-ß(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-α-actin and calponin by TGF-ß(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-α-actin stress fibers by TGF-ß(1). MCh did not augment TGF-ß(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-ß(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3ß and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-ß(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD.

Distinct regional and subcellular localization of the actin-binding protein filamin A in the mature rat brain.

Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain. Here we characterize in detail the neuroanatomical distribution and subcellular localization of FLNa in the mature rat brain, by using two antisera directed against epitopes at either the N' or the C' terminus of the protein, further validated by mRNA expression. FLNa was widely and selectively expressed throughout the brain, and the intensity of immunoreactivity was region dependent. The most intensely FLNa-labeled neurons were found in discrete neuronal systems, including basal forebrain structures, anterior nuclear group of thalamus, and hypothalamic parvocellular neurons. Pyramidal neurons in neocortex and hippocampus and magnocellular cells in basolateral amygdaloid nucleus were also intensely FLNa immunoreactive, and strong FLNa labeling was evident in the pontine and medullary raphe nuclei and in sensory and spinal trigeminal nuclei. The subcellular localization of FLNa was evaluated in situ as well as in primary hippocampal neurons. Punctate expression was found in somata and along the dendritic shaft, but FLNa was not detected in dendritic spines. These subcellular distribution patterns were recapitulated in hippocampal and neocortical pyramidal neurons in vivo. The characterization of the expression and subcellular localization of FLNa may provide new clues to the functional roles of this cytoskeletal protein in the adult brain.

Cilostazol promotes vascular smooth muscles cell differentiation through the cAMP response element-binding protein-dependent pathway.

OBJECTIVE: Cilostazol, a potent type 3 phosphodiesterase inhibitor, has recently been found to reduce neointimal formation by inhibiting vascular smooth muscle cell (VSMC) proliferation. The aim of this study is to investigate whether cilostazol exerts an action on phenotypic modulation of VSMCs, another important process in the pathogenesis of neointimal formation. METHODS AND RESULTS: Cilostazol may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of smooth muscle cell differentiation marker contractile proteins. The upregulation of contractile proteins by cilostazol involves the cAMP/protein kinase A (PKA) signaling pathway, because the cAMP analog mimicked and specific cAMP/PKA inhibitors opposed the effect of cilostazol. Furthermore, cilostazol-activated cAMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Deletion and mutational analysis of the contractile protein promoters along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE is essential for cilostazol-induced contractile protein expression. Transfection of dominant-negative CREB (mutated Ser133) plasmid in VSMCs blocked cilostazol-stimulated contractile protein expression. In vivo, cilostazol upregulated contractile proteins and induced the activation of CREB in the neointima of balloon-injured arteries. CONCLUSIONS: Cilostazol promotes VSMC differentiation through the cAMP/PKA/CREB signaling cascade.

Anillin expression is a marker of favourable prognosis in patients with renal cell carcinoma.

The clinical course of renal cell carcinoma (RCC) can be difficult to predict. In this study we evaluated the prognostic value of anillin and Ki-67 in predicting survival in RCC. Immunohistochemical analysis using anillin and Ki-67 antibodies was performed on tissue microarrays constructed from paraffin-embedded specimens from 152 patients with primary RCC. The mean follow-up time was 90 months. Levels of anillin and Ki-67 staining were correlated with clinical factors, pathological features and survival. Anillin expression in cytoplasmic and nuclear fractions of the RCC cell line 786-O was examined using Western blot analysis. Cytoplasmic anillin immunopositivity was detected in 121 (83%) tumours. Nuclear anillin expression was present in 40 (27%) tumours and increased Ki-67 activity in 98 (66%) tumours. A positive association was found between nuclear anillin and Ki-67 proliferation activity (p=0.005). The mean RCC-specific survival times for anillin immunopositive and immunonegative tumours were 158 (95% CI 143-173) and 109 (78-141) months, respectively, with p=0.03. Increased Ki-67 activity showed a tendency towards a poorer prognosis, although this was not statistically significant. In the Cox regression analysis for cytoplasmic anillin, nuclear anillin or Ki-67 rate, and age, gender, stage and nuclear grade, the only significant factor in RCC-specific survival was stage (p<0.001). Western blot analysis showed anillin expression in both nuclear and cytosolic fractions of the RCC cell line. To conclude, anillin expression can be observed both in the cytoplasm and nuclei in patients with RCC. Cytoplasmic anillin expression is a marker of favourable prognosis in RCC patients.

A positive relationship between filamin and VEGF in patients with lung cancer.

Induction of angiogenic responses by multiple factors, a crucial step in tumor growth and metastasis, is not completely understood. Recently, involvement of the cytoskeletal actin-binding proteins in angiogenesis has been suggested as a target for anti-neovascular cancer therapy in vitro. In this study, the expression of filamin A (FLNA) and vascular endothelial growth factor (VEGF) in paraffin-embedded tumor samples from patients with well-characterized lung tumors was immunohistochemically analyzed and compared with clinical variables and survival outcome. A positive expression of FLNA and VEGF was detected in the cytoplasm of tumor cells in 66 (48.2%) and 69 (50.4%) of the 137 patients with lung cancer, respectively (p<0.0001). A significant difference was observed between FLNA expression and VEGF expression. Although our findings do not suggest that the expression of FLNA alone plays an independent prognostic role, the angiogenesis pathway mediated by FLNA appears to be responsible for controlling the growth of lung tumors.

Alzheimer's disease-linked presenilin mutation (PS1M146L) induces filamin expression and γ-secretase independent redistribution.

Presenilin mutations are linked to the early onset familial Alzheimer's disease (FAD) and lead to a range of neuronal changes, indicating that presenilins interact with multiple cellular pathways to regulate neuronal functions. In this report, we demonstrate the effects of FAD-linked presenilin 1 mutation (PS1M146L) on the expression and distribution of filamin, an actin cross-linking protein that interacts with PS1 both physically and genetically. By using immunohistochemical methods, we evaluated hippocampal dentate gyrus for alterations of proteins involved in synaptic plasticity. Among many proteins expressed in the hippocampus, calretinin, glutamic acid decarboxylase (GAD67), parvalbumin, and filamin displayed distinct changes in their expression and/or distribution patterns. Striking anti-filamin immunoreactivity was associated with the polymorphic cells of hilar region only in transgenic mice expressing PS1M146L. In over 20% of the PS1M146L mice, the hippocampus of the left hemisphere displayed more pronounced upregulation of filamin than that of the right hemisphere. Anti-filamin labeled the hilar neurons only after the PS1M146L mice reached after four months of age. Double labeling immunohistochemical analyses showed that anti-filamin labeled neurons partially overlapped with cholecystokinin (CCK), somatostatin, GAD67, parvalbumin, and calretinin immunoreactive neurons. In cultured HEK293 cells, PS1 overexpression resulted in filamin redistribution from near cell peripheries to cytoplasm. Treatment of CHO cells stably expressing PS1 with WPE-III-31C or DAPT, selective γ-secretase inhibitors, did not suppress the effects of PS1 overexpression on filamin. These studies support a γ-secretase-independent role of PS1 in modulation of filamin-mediated actin cytoskeleton.

Caspase-3 knock-down reverses contractile dysfunction induced by sepsis in adult rat ventricular myocytes.

BACKGROUND AND PURPOSE: The present study tested the hypothesis that selective caspase-3 (C-3) knock-out would regulate the contractile actions of noradrenaline (NA) in the dysfunction of adult rat ventricular myocytes (ARVMs) induced by sepsis. Here, we have studied the contractile response of ARVMs, transfected with C-3 small interfering RNA (C-3 siRNA), to NA. EXPERIMENTAL APPROACH: Single ARVMs were isolated from the hearts of male Sprague-Dawley rats 3 days after induction of sepsis, and from sham-treated rats. The sham and septic ARVMs were treated with NA (10 microM) alone or after transfection with C-3 siRNA or non-silencing RNA (2 microM). Mechanical properties were measured digitally, and immunoblotting and immunocytochemical analyses were carried out. KEY RESULTS: The NA-induced increase in peak shortening (PS) was less in septic ARVMs and transfection with C-3 siRNA produced a significant increase in this PS. Immunocytochemical and immunoblot analyses revealed that NA exacerbated sepsis-induced up-regulation of C-3. Transfection of septic ARVMs with C-3 siRNA exhibited a decreased expression of C-3 fluorescence after NA. In septic ARVMs, we also observed a down-regulation of contractile proteins (alpha-actin, myosin light chain-1 and tropomyosin) along with DNA damage. Transfection of septic ARVMs with C-3 siRNA produced an increase in the expression of contractile proteins, and a decrease in DNA damage. CONCLUSIONS AND IMPLICATIONS: These data suggest that C-3 knock-down improved the loss of contractile response to NA in septic ARVMs, suggesting that C-3 regulated contractile dysfunction induced by sepsis in ARVMs.