Human Muscle Cultures Research Articles

Distinct human skeletal muscle-derived CD90 progenitor subsets for myo-fibro-adipogenic disease modeling and treatment in multiplexed conditions.

Chronic muscle injuries, such as massive rotator cuff tears, are associated with progressive muscle wasting, fibrotic scarring, and intramuscular fat accumulation. While progenitor cell subsets are usually studied in culture conditions that drive either myogenic, fibrogenic, or adipogenic differentiation, it is still unknown how combined myo-fibro-adipogenic signals, which are expected to occur in vivo, modulate progenitor differentiation. We therefore evaluated the differentiation potential of retrospectively generated subsets of primary human muscle mesenchymal progenitors in multiplexed conditions in the presence or absence of 423F drug, a modulator of gp130 signaling. We identified a novel CD90+CD56- non-adipogenic progenitor subset that maintained a lack of adipogenic potential in single and multiplexed myo-fibro-adipogenic culture conditions. CD90-CD56- demarcated fibro-adipogenic progenitors (FAP) and CD56+CD90+ progenitors were typified as myogenic. These human muscle subsets exhibited varying degrees of intrinsically regulated differentiation in single and mixed induction cultures. Modulation of gp130 signaling via 423F drug mediated muscle progenitor differentiation in a dose-, induction-, and cell subset-dependent manner and markedly decreased fibro-adipogenesis of CD90-CD56- FAP. Conversely, 423F promoted myogenesis of CD56+CD90+ myogenic subset, indicated by increased myotube diameter and number of nuclei per myotube. 423F treatment eliminated FAP-derived mature adipocytes from mixed adipocytes-FAP cultures but did not modify the growth of non-differentiated FAP in these cultures. Collectively, these data demonstrate that capability of myogenic, fibrogenic, or adipogenic differentiation is largely dependent on the intrinsic features of cultured subsets, and that the degree of lineage differentiation varies when signals are multiplexed. Moreover, our tests performed in primary human muscle cultures reveal and confirm the potential triple-therapeutic effects of 423F drug which simultaneously attenuates degenerative fibrosis, fat accumulation and promotes myo-regeneration.

Analysis of human satellite cell dynamics on cultured adult skeletal muscle myofibers

BackgroundMaintaining stem cells in physiologically relevant states is necessary to understand cell and context-specific signalling paradigms and to understand complex interfaces between cells in situ. Understanding human stem cell function is largely based on tissue biopsies, cell culture, and transplantation into model organisms.MethodsHere, we describe a method to isolate post-mortem intact human muscle myofibers and culture muscle stem cells within the niche microenvironment to assay cellular dynamics, stem cell identity, stem cell hierarchy, and differentiation potential.ResultsWe show human myofiber culture maintains complex cell-cell contacts and extracellular niche composition during culture. Human satellite cells can be cultured at least 8 days, which represents a timepoint of activation, differentiation, and de novo human myofiber formation. We demonstrate that adult human muscle stem cells undergo apicobasal and planar cell divisions and express polarized dystrophin and EGFR. Furthermore, we validate that stimulation of the EGFR pathway stimulates the generation of myogenic progenitors and myogenic differentiation.ConclusionsThis method provides proof of principle evidence for the use of human muscle to evaluate satellite cell dynamics and has applications in pre-clinical evaluation of therapeutics targeting muscle repair.

Persistent upregulation of the β-tubulin tubb6, linked to muscle regeneration, is a source of microtubule disorganization in dystrophic muscle.

In healthy adult skeletal muscle fibers microtubules form a three-dimensional grid-like network. In the mdx mouse, a model of Duchenne muscular dystrophy (DMD), microtubules are mostly disordered, without periodicity. These microtubule defects have been linked to the mdx mouse pathology. We now report that increased expression of the beta 6 class V β-tubulin (tubb6) contributes to the microtubule changes of mdx muscles. Wild-type muscle fibers overexpressing green fluorescent protein (GFP)-tubb6 (but not GFP-tubb5) have disorganized microtubules whereas mdx muscle fibers depleted of tubb6 (but not of tubb5) normalize their microtubules, suggesting that increasing tubb6 is toxic. However, tubb6 increases spontaneously during differentiation of mouse and human muscle cultures. Furthermore, endogenous tubb6 is not uniformly expressed in mdx muscles but is selectively increased in fiber clusters, which we identify as regenerating. Similarly, mdx-based rescued transgenic mice that retain a higher than expected tubb6 level show focal expression of tubb6 in subsets of fibers. Tubb6 is also upregulated in cardiotoxin-induced mouse muscle regeneration, in human myositis and DMD biopsies, and the tubb6 level correlates with that of embryonic myosin heavy chain, a regeneration marker. In conclusion, modulation of a β-tubulin isotype plays a role in muscle differentiation and regeneration. Increased tubb6 expression and microtubule reorganization are not pathological per se but reflect a return to an earlier developmental stage. However, chronic elevation of tubb6, as occurs in the mdx mouse, may contribute to the repeated cycles of regeneration and to the pathology of the disease.

LBOS 02-04 BLOOD PRESSURE-ASSOCIATED POLYMORPHISMS IN SLC4A7 (SODIUM/BICARBONATE CO-TRANSPORTER NBCn1) ARE LINKED TO GENE EXPRESSION AND INTRACELLULAR pH REGULATION

Objective: Chromosome 3p24.1 containing the SLC4A7 gene has been identified by genome-wide association studies as one of the genomic loci that influence blood pressure. SLC4A7 encodes electroneutral Na+/HCO3– co-transporter (NBCn1) which regulates intracellular pH (pHi) in various tissues including vascular smooth muscle and endothelium, with knockout models demonstrating an altered blood pressure phenotype. We conducted a functional study of blood pressure-associated single nucleotide polymorphisms at the SLC4A7 locus in primary cultures of human vascular smooth muscle and endothelial cells. Design and method: Primary cultures of human umbilical vein endothelial cells and artery smooth muscle cells of different SLC4A7 genotypes were used. DNA-protein interactions were identified by formaldehyde-associated isolation of regulatory elements (FAIRE) and electrophoretic mobility shift assays (EMSA). Gene expression was quantified by allelic imbalance analyses and protein immunoblots. NBCn1 protein function was evaluated using the baseline pHi and rate of dimethyl-amiloride-insensitive, Na+/HCO3–-dependent pHi recovery following ammonium-prepulse-induced intracellular acidification. A10 cells overexpressing 326E or 326K SLC4A7 variants were used to verify primary culture results. Results: FAIRE identified the BP risk allele as associated with preferentially open chromatin, and EMSA identified allele-dependent DNA-protein interactions. Consistent with this, the risk allele was associated with increased NBCn1 mRNA, protein expression, availability at the cell membrane and subsequently higher Na+/HCO3–-dependent, dimethlyamiloride-insensitive net base flux and steady-state pHi compared to protective allele homozygotes. However, in the presence of Na+/H+ exchange activity, the genotypic effect of NBCn1 on net acid extrusion and steady-state pHi was detected only in vascular smooth muscle cells but not endothelial cells. There was no discernable effect of the missense polymorphism resulting in the amino acid substitution E326K. Conclusions: These findings provide an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus and blood pressure, revealing an important genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells.

Prostaglandin E2 induces expression of MAPK phosphatase 1 (MKP-1) in airway smooth muscle cells

Prostaglandin E2 (PGE2) is a prostanoid with diverse actions in health and disease. In chronic respiratory diseases driven by inflammation, PGE2 has both positive and negative effects. An enhanced understanding of the receptor-mediated cellular signalling pathways induced by PGE2 may help us separate the beneficial properties from unwanted actions of this important prostaglandin. PGE2 is known to exert anti-inflammatory and bronchoprotective actions in human airways. To date however, whether PGE2 increases production of the anti-inflammatory protein MAPK phosphatase 1 (MKP-1) was unknown. We address this herein and use primary cultures of human airway smooth muscle (ASM) cells to show that PGE2 increases MKP-1 mRNA and protein upregulation in a concentration-dependent manner. We explore the signalling pathways responsible and show that PGE2-induces CREB phosphorylation, not p38 MAPK activation, in ASM cells. Moreover, we utilize selective antagonists of EP2 (PF-04418948) and EP4 receptors (GW 627368X) to begin to identify EP-mediated functional outcomes in ASM cells in vitro. Taken together with earlier studies, our data suggest that PGE2 increases production of the anti-inflammatory protein MKP-1 via cAMP/CREB-mediated cellular signalling in ASM cells and demonstrates that EP2 may, in part, be involved.

Abstract 210: Biosynthesis of D-series Resolvin by Isolated Vascular Cells and Tissues

Introduction: Resolvin-D1 (RvD1) and other specialized pro-resolving lipid mediators (SPM) are synthesized in-vivo from docosahexaenoic acid (DHA) through transcellular pathways involving leukocytes. We investigated if vascular tissues, in the absence of inflammatory cells, can contribute to the local production of SPM. Methods: Primary cultures of human saphenous vein endothelial (EC) and smooth muscle (SMC) cells were supplemented with DHA in cell culture media (10% serum) for 4h-24h. Freshly harvested rabbit aorta was incubated intact or following gentle EC denudation in medium with or without DHA for 48h. RvD1 levels were quantified by ELISA, and lipoxygenase (LO) expression by western blotting. Results: In the absence of DHA supplementation, EC and SMC produced undetectable levels of RvD1. DHA treatment produced a dose and time-dependent increase in RvD1 production by EC and SMC (10.1 ±1.0 pg, 7.4 ±0.2 pg respectively; 1000nM DHA; 24h; Fig A, B). 5-LO expression was demonstrated in both cell types, however DHA induced increased 5-LO expression in EC (Fig C) but not in SMC. DHA-treated intact rabbit aorta segments produced 0.24±0.05 pg RvD1/mg tissue versus 0.13±0.01 pg RvD1/mg tissue in media alone. Moreover, EC-denuded aortas produced significantly less RvD1 (Fig D). Conclusions: Human vascular cells and rabbit vascular tissue can biosynthesize RvD1 de novo from its precursor DHA, signifying a potentially important local source of SPM in the vasculature.

SERCA1 protein expression in muscle of patients with Brody disease and Brody syndrome and in cultured human muscle fibers

Brody disease is an inherited myopathy associated with a defective function of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) protein. Mutations in the ATP2A1 gene have been reported only in some patients. Therefore it has been proposed to distinguish patients with ATP2A1 mutations, Brody disease (BD), from patients without mutations, Brody syndrome (BS). We performed a detailed study of SERCA1 protein expression in muscle of patients with BD and BS, and evaluated the alternative splicing of SERCA1 in primary cultures of normal human muscle and in infant muscle. SERCA1 reactivity was observed in type 2 muscle fibers of patients with and without ATP2A1 mutations and staining intensity was similar in patients and controls. Immunoblot analysis showed a significant reduction of SERCA1 band in muscle of BD patients. In addition we demonstrated that the wild type and mutated protein exhibits similar solubility properties and that RIPA buffer improves the recovery of the wild type and mutated SERCA1 protein. We found that SERCA1b, the SERCA1 neonatal form, is the main protein isoform expressed in cultured human muscle fibers and infant muscle. Finally, we identified two novel heterozygous mutations within exon 3 of the ATP2A1 gene from a previously described patient with BD.

Acute Exercise and Ca2+ Stimulation Regulate Enzymes Involved in DNA Methylation in Human Skeletal Muscle

Adaptations to exercise occur at the organ, cellular, protein, and molecular levels. At the molecular level, epigenetics are defined as non-sequence changes to the DNA that regulate gene expression. Two types of epigenetics modifications are chromatin remodeling and DNA methylation and while recent studies clearly demonstrate that exercise results in covalent modifications of histone in the chromatin, less is known about whether exercise changes DNA methylation at specific promoters. Changes in DNA methylation occur through DNA methyltransferase enzymes and putative demethylase enzymes. PURPOSE: To determine whether exercise is capable of altering mRNA levels of enzymes that modify DNA methylation levels. METHODS: Healthy young men (n=8) performed 3 hours of cycle ergometer exercise at 60% of VO2max and muscle biopsies were taken at 0 (pre), 3 (immediately post), 4.5, 6, 12, and 24 hours. Primary muscle cultures were established in vitro from healthy young males, subjected to a variety of stimuli and inhibitors. Both studies utilized SYBR green qPCR to examine the mRNA levels of Dnmt3a, Dnmt3b, GADD45b, and GADD45a. RESULTS: In vivo the mRNA level of Dnmt3b was decreased by 50% (p<0.05) immediately following exercise, while GADD45b (17 fold) and GADD45a (6 fold) both peaked at 6 hrs (p<0.05). In culture, stimulation of proliferating myoblasts with either AICAR, epinephrine, of ionomycin (a Ca2+ ionophore) failed to alter mRNA levels of the Dnmt3a/b, while ionomycin increased GADD45b (∼4-5 fold) and GADD45a (∼7-9 fold). Similarly, in differentiated myotubes GADD45b and GADD45a were upregulated (3 and 2 fold respectively, p<0.05)) by Ca ionomycin, however Dnmt3a and Dnmt3b were both downregulated by about 40% following stimulation by ionomycin. Stimulation by ionomycin while inhibiting p38/MAPK during further increased GADD45b and increased Dnmt3a/b to control levels. CONCLUSIONS: In vivo exercise and in vitro stimulation of primary human muscle cultures can increase the mRNA of GADD45b/a, a putative DNA demethylase, while simultaneously decreasing Dnmt3a/b, two de novo DNA methyltransferases. Thus, future experiments are addressing whether simultaneous changes in DNA methylation of promoters of genes regulated during acute exercise also occurs.

In AβPP‐overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AβPP751 and GSK3β activation: effects mitigated by lithium and apparently relevant to sporadic inclusion‐body myositis

Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-beta (Abeta)-precursor protein 751 (AbetaPP751), Abeta, phosphorylated tau, and other 'Alzheimer-characteristic' proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer's disease (AD) patients and AD transgenic-mouse models, phosphorylation of neuronal AbetaPP695 (p-AbetaPP) on Thr668 (equivalent to T724 of AbetaPP751) is considered detrimental because it increases generation of cytotoxic Abeta and induces tau phosphorylation. Activated glycogen synthase kinase3beta (GSK3beta) is involved in phosphorylation of both AbetaPP and tau. Lithium, an inhibitor of GSK3beta, was reported to reduce levels of both the total AbetaPP and p-AbetaPP in AD animal models. In relation to s-IBM, we now show for the first time that (1) In AbetaPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3beta activity and AbetaPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated-AbetaPP, (ii) total amount of AbetaPP, (iii) Abeta oligomers, and (iv) GSK3beta activity; and (c) lithium improves proteasome function. (2) In biopsied s-IBM muscle fibers, GSK3beta is significantly activated and AbetaPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3beta inhibitors, might benefit s-IBM patients.

β‐Arrestins specifically constrain β2‐adrenergic receptor signaling and function in airway smooth muscle

Chronic use of inhaled beta-agonists by asthmatics is associated with a loss of bronchoprotective effect and deterioration of asthma control. Beta-agonist-promoted desensitization of airway smooth muscle beta-2-adrenergic receptors, mediated by G protein-coupled receptor kinases and arrestins, is presumed to underlie these effects, but such a mechanism has never been demonstrated. Using in vitro, ex vivo, and in vivo murine models, we demonstrate that beta-arrestin-2 gene ablation augments beta-agonist-mediated airway smooth muscle relaxation, while augmenting beta-agonist-stimulated cyclic adenosine monophosphate production. In cultures of human airway smooth muscle, small interfering RNA-mediated knockdown of arrestins also augments beta-agonist-stimulated cyclic adenosine monophosphate production. Interestingly, signaling and function mediated by m2/m3 muscarinic acetylcholine receptors or prostaglandin E(2) receptors were not affected by either beta-arrestin-2 knockout or arrestin knockdown. Thus, arrestins are selective regulators of beta-2-adrenergic receptor signaling and function in airway smooth muscle. These results and our previous findings, which demonstrate a role for arrestins in the development of allergic inflammation in the lung, identify arrestins as potentially important therapeutic targets for obstructive airway diseases.

Glucocorticoid Modulation of Insulin Signaling in Human Subcutaneous Adipose Tissue

Glucocorticoid (GC) excess is characterized by central obesity, insulin resistance, and in some cases, type 2 diabetes. However, the impact of GC upon insulin signaling in human adipose tissue has not been fully explored. We have examined the effect of GC upon insulin signaling in both human sc primary preadipocyte cultures and a novel human immortalized sc adipocyte cell line (Chub-S7) and contrasted this with observations in primary cultures of human skeletal muscle. This is an in vitro study characterizing the impact of GC upon insulin signaling in human tissues. Biopsy specimens were from healthy volunteers who gave their full and informed written consent. Combinations of treatments, including GC, RU38486, and wortmannin, were used. Insulin signaling cascade gene and protein expression and insulin-stimulated glucose uptake were determined. In human adipocytes, pretreatment with GC induced a dose-dependent [1.0 (control); 1.2 +/- 0.1 (50 nm); 2.2 +/- 0.2 (250 nm), P < 0.01 vs. control; 3.4 +/- 0.2 (1000 nm), P < 0.001 vs. control] and time-dependent [1.0 (1 h); 3.2 +/- 2.0 (6 h); 9.1 +/- 5.9 (24 h), P < 0.05 vs. 1 h; 4.5 +/- 2.2 (48 h)] increase in insulin-stimulated protein kinase B/akt phosphorylation. In addition, whereas insulin receptor substrate (IRS)-1 protein expression did not change, IRS-1 tyrosine phosphorylation increased. Furthermore, GC induced IRS-2 mRNA expression (2.8-fold; P < 0.05) and increased insulin-stimulated glucose uptake [1.0 (control) 1.8 +/- 0.1 (insulin) vs. 2.8 +/- 0.2 (insulin + GC); P < 0.05]. In contrast, in primary cultures of human muscle, GC decreased insulin-stimulated glucose uptake [1.0 (control) 1.9 +/- 0.2 (insulin) vs. GC 1.3 +/- 0.1 (insulin + GC); P < 0.05]. We have demonstrated tissue-specific regulation of insulin signaling by GC. Within sc adipose tissue, GCs augment insulin signaling, yet in muscle GCs cause insulin resistance. We propose that enhanced insulin action in adipose tissue increases adipocyte differentiation, thereby contributing to GC-induced obesity.

Culture of Human Skeletal Muscle Myoblasts: Timing Appearance and Localization of Dystrophin-Glycoprotein Complex and Vinculin-Talin-Integrin Complex

The dystrophin-glycoprotein complex together with the vinculin-talin-integrin complex plays an important role in muscle function; in fact the mutations of their elements lead to diverse forms of muscular dystrophies. The relationship between the elements of dystrophin-glycoprotein complex and vinculin-talin-integrin and the time course of their formation are still not known in detail. In order to better understand this relationship we studied their expression during development in normal human skeletal muscle culture. Using a standardized muscle cell culture procedure, this study was performed to analyze the timing, appearance and the localization of some proteins of the dystrophin-glycoprotein complex and vinculin-talin-integrin complex during cellular proliferation (myoblast) and differentiation (4, 7, 15 and 21 days). The indirect immunofluorescence technique was used and cells were examined using a Meta Zeiss LSM510 confocal laser scanning inverted microscope. We examined the progressive appearance of the following proteins: α, β, γ, δ-sarcoglycans, β-dystroglycan, dystrophin, talin, vinculin and integrin isoform α₇/β₁. Immunofluorescence of these proteins, in satellite cells entering myogenic differentiation, revealed different patterns of localization depending on the time of culture. We showed that nondifferentiated cultures of human myoblasts expressed a perinuclear distribution of all proteins tested. During myoblast differentiation into myotubes (4 days) immunofluorescence gradually increased and was located in the whole cytoplasm. Subsequently, at day 7, a strong and homogeneous cytoplasmic labelling of all proteins was seen. At 15 days the distribution of the proteins was on the membrane. At this time some myotubes displayed a significant degree of precostameric banding pattern. As fusion proceeded at 21 days, the cytodistribution progressively changed and appeared along fibrillar longitudinal structures, and myotubes showed a clear periodic distribution (costameres). In conclusion, in normal human muscle cultures DGC and vinculin-talin-integrin proteins are first localized in the perinuclear region, then they diffuse in the cytoplasm and finally form at the plasma membrane into typical rib-like structures that are sarcolemma-associated.

Mitochondrial Signals for the Nucleus

What's a transcription factor of the basic helix-loop-helix leucine zipper family doing in the cytoplasm? Sans et al. explored why nearly all of the transcription factor MondoA and its binding partner protein called Mlx is found in the cytoplasm but the protein appears to function as a transcription factor in the nucleus. The authors found that in primary cultures of human muscle or erythroblast cells, MondoA and Mlx were associated with the mitochondria. Inhibition of nuclear shuttling of the proteins with an inhibitor of the CRM1 nuclear export factor caused MondoA (detected by confocal immunofluorescence microscopy) to accumulate in the nucleus. Thus, the authors suspected that MondoA could function to carry signals from the mitochondria to the nucleus. They obtained further circumstantial evidence for such a role from microarray experiments designed to detect genes transcribed in response to MondoA and Mlx. MondoA-Mlx regulated a broad range of targets, which included many genes associated with carbohydrate metabolism. Chromatin immunoprecipitation experiments showed that hexokinase II, 6-phospofructo-2-kinase/fructose-2,6-bisphosphatase 3, and lactate dehydrogenase A--three enzymes important for glycolysis--are direct targets for MondoA-Mlx. Indeed, depletion of MondoA by RNA interference caused a decrease in the rate of glycolysis (measured as the rate of conversion of 5-[ 3 H]glucose to 3 H 2 O). Thus, although other functions are not excluded, the authors propose that MondoA may function as an intracellular metabolic sensor. C. L. Sans, D. J. Satterwhite, C. A. Stoltzman, K. T. Breen, D. E. Ayer, MondoA-Mlx heterodimers are candidate sensors of cellular energy status: Mitochondrial localization and direct regulation of glycolysis. Mol. Cell. Biol. 26 , 4863-4871 (2006). [Abstract] [Full Text]

Sandford Lloyd Skinner (1933–2005)

Sandford Lloyd (“Sandy”) Skinner, one of the pillars of the renin-angiotensin system, died on May 29, 2005, after a long illness. Sandy was a gentleman, scientist, teacher, and family man who touched the lives of the many who were fortunate to know him.⇓ Sandy was born in Clare, South Australia, on August 6, 1933. He graduated MBBS from The University of Adelaide in 1957. Following his medical studies, he researched the effects of vasoactive substances on the circulation and respiration of humans in the Department of Physiology, The University of Adelaide, for which he was awarded an MD degree in 1962. He also reported the artifactual elevation of plasma potassium levels during venepuncture.1 His first postdoctoral position during 1962–1963 was at the Cleveland Clinic working with Irvine Page and James McCubbin. This was the start of his life-long passion for the renin-angiotensin system. At the Cleveland Clinic he performed seminal studies demonstrating the role of the baroreceptor mechanism of control of renin release.2 Importantly, his studies demonstrated that renin release could be stimulated by reduction in renal perfusion pressure within the physiological range without significant change in renal blood flow, thus demonstrating that renin release was caused by a baroreceptor mechanism and not renal ischemia. He also showed that renal compression could stimulate renin release. These studies were important in showing that variation in renin release may have …

Proinflammatory Cytokines Upregulate mRNA Expression and Secretion of Vascular Endothelial Growth Factor in Cultured Human Airway Smooth Muscle Cells

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1beta (0.5 ng/mL) and TNF-alpha (10 ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8 h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1 microM dexamethasone abolished enhanced release of VEGF by TNF-alpha. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.

Expression profiling in spinal muscular atrophy reveals an RNA binding protein deficit

Spinal muscular atrophy is a common neuromuscular disorder caused by deletions or mutations within the survival motor neuron gene. The reason for specific motor neuron loss within the disease is still unclear. Expression profiling has been carried out in two models of spinal muscular atrophy; the heterozygote mouse model and human primary muscle cultures from a spinal muscular atrophy patient. A group of RNA binding proteins are up-regulated in spinal muscular atrophy motor neurons. One such protein, BRUNOL3, is highly expressed within spinal cord and muscle and also at the same developmental stage as survival motor neuron. The differential expression of Brunol3 has been confirmed with real-time RT-PCR in spinal cord and muscle of three different models of spinal muscular atrophy. BRUNOL3 has been shown to co-localise with survival motor neuron in the nuclei of neuronal cells and to co-immunoprecipitate with Smn in mouse brain. This is the first time that a link has been established between RNA binding proteins and survival motor neuron within motor neurons.

IgA sensitizes airway smooth muscle to histamine

Rationale IgA is an important defense against infectious pathogens, which are common triggers of asthma symptoms. We recently reported that primary cell cultures of human airway smooth muscle (ASM) cells express the IgA receptor (pIgR) that binds polymeric IgA (pIgA) but not monomeric IgA (mIgA), and that pIgA increases cytosolic calcium - a necessary prequel to smooth muscle contraction and therefore bronchoconstriction. In this study, we asked if pIgA sensitizes ASM to histamine-induced responses. Methods Cytosolic calcium was measured in Fura2-loaded human ASM cells stimulated first with histamine 10 −6M and then treated for 2h with buffer, pIgA or mIgA. Post-incubation histamine-induced cytosolic calcium concentrations were compared to the pre-incubation response. Tension was measured in isolated canine tracheal ASM strips first in response to histamine 10 −5M and then treated for 4h with buffer, pIgA or mIgA. Post-incubation histamine-induced tension was compared to the pre-incubation response. Results Incubation with pIgA (0.12 μg/ml) significantly augmented the histamine-induced increase in cytosolic calcium in human ASM cells compared to mIgA (12 μg/ml) or buffer (p<0.05). Similarly, incubation with pIgA (6 μg/ml) appreciably enhanced histamine-induced increases in tension (by 2g) in canine tracheal ASM strips compared to mIgA 3 μg/ml (0.36g) or buffer (0.35g; p<0.05). Conclusions Incubation with pIgA sensitizes human ASM cells and canine tracheal ASM strips to histamine. Because IgA is an important epithelial defense against viral and bacterial attachment, functional responses to IgA in ASM may account, at least in part, for the worsening of symptoms during upper respiratory tract infections in patients with allergic asthma.

Mechanisms of cytokine effects on G protein-coupled receptor-mediated signaling in airway smooth muscle.

Numerous in vitro and in vivo studies have implicated the cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) as mediators of airway inflammation and therefore potentially important substances in the pathogenesis of asthma. In this study, we examined the mechanisms by which IL-1 beta and TNF-alpha affect inhibition of cell growth, G protein-coupled receptor (GPCR) desensitization, and the recently reported adenylyl cyclase sensitization in human airway smooth muscle (HASM) cultures. Our findings demonstrate that adenylyl cyclase sensitization is independent of cytokine-mediated cyclooxygenase type 2 (COX-2) and prostaglandin E(2) (PGE(2)) induction, whereas COX-2 induction appears to be required for both growth inhibition and GPCR desensitization. However, GPCR desensitization was highly dependent on the presence of EGF during chronic treatment with cytokines, which could be explained by a synergistic effect of EGF on cytokine-mediated COX-2 and PGE(2) induction. Interestingly, various agents (including inhibitors of p42/p44 and p38 mitogen-activated protein kinase signaling) were significantly more effective in inhibiting cytokine-mediated PGE(2) induction, GPCR desensitization, and cell growth inhibition than in inhibiting COX-2 induction. These data demonstrate disparity in the requirement and sufficiency of COX-2 induction in promoting different functional effects of IL-1 beta and TNF-alpha in HASM.

Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V.

Adenylyl cyclases are a nine-member family of differentially regulated enzymes responsible for the synthesis of cAMP. cAMP is an important second messenger that contributes to the regulation of airway smooth muscle tone. However, little is known regarding the expression and regulation of adenylyl cyclase isoforms in airway smooth muscle cells. Nondegenerate specific primers were designed for all nine known isoforms of human adenylyl cyclase. RT-PCR experiments were performed using total RNA extracted from whole human brain (positive control), whole rat brain (negative control), whole human trachea, human airway smooth muscle, and primary cultures of human airway smooth muscle cells. Seven of the nine known isoforms of adenylyl cyclase (isoforms I, III-VII, and IX) were expressed at the mRNA level in both human airway smooth muscle and primary cultures of human airway smooth muscle cells. Immunoblot and adenylyl cyclase functional assay indicated that isoform V is likely among the functionally predominant isoforms of adenylyl cyclase in human airway smooth muscle. These results suggest that multiple isoforms of adenylyl cyclase enzymes are coexpressed in human airway smooth muscle cells and that isoform V is among the functionally important isoforms.

Cathepsins are upregulated by IFN-gamma/STAT1 in human muscle culture: a possible active factor in dermatomyositis.

The aim of this work was to study which genes upregulated by the IFN-gamma/STAT1 system in human muscle might be involved in the process of muscle fiber atrophy in dermatomyositis (DM). These proteins included proteases (cathepsins B and L, calpain), proteins implicated in apoptosis and cell cycle (Bcl-x(l), Fas, p21), structural proteins (beta-actin, utrophin, desmin), and other proteins whose expression is known to be modified by IFN-gamma (neural cell adhesion molecule (N-CAM), major histocompatibility complex-I (MHC-I)). We performed immunocytochemistry, Western blot, and semiquantitative reverse transcriptase-polymerase chain reaction using human muscle cultures. We found upregulation of cathepsins B and L, bcl-x(l) and p21 while N-CAM, calpain, utrophin, desmin, beta-actin and Fas remained at basal levels. Immunohistochemistry on frozen sections from biopsies of patients with different muscle diseases showed upregulation of cathepsin L and calpain in perifascicular muscle fibers in DM. In view of these results, the increased expression of cathepsins L and B after IFN-gamma stimulation in muscle cultures and its inhibition using fludarabine, a STAT1 blocker, further support our previous studies and suggest that the increased expression of cathepsins detected in perifascicular muscle fibers in DM is mediated by IFN-gamma/STAT1 and contributes to their atrophy.