ABSTRACT
Asthma is an obstructive respiratory disease characterised by chronic inflammation with airway hyperresponsiveness. In asthmatic airways, there is an increase in airway smooth muscle (ASM) cell bulk, which differs from non-asthmatic ASM in characteristics. This study aimed to assess the usefulness of hTERT immortalisation of human ASM cells as a research tool. Specifically we compared proliferative capacity, inflammatory mediator release and extracellular matrix (ECM) production in hTERT immortalised and parent primary ASM cells from asthmatic and non-asthmatic donors. Our studies revealed no significant differences in proliferation, IL-6 and eotaxin-1 production, or CTGF synthesis between donor-matched parent and hTERT immortalised ASM cell lines. However, deposition of ECM proteins fibronectin and fibulin-1 was significantly lower in immortalised ASM cells compared to corresponding primary cells. Notably, previously reported differences in proliferation and inflammatory mediator release between asthmatic and non-asthmatic ASM cells were retained, but excessive ECM protein deposition in asthmatic ASM cells was lost in hTERT ASM cells. This study shows that hTERT immortalised ASM cells mirror primary ASM cells in proliferation and inflammatory profile characteristics. Moreover, we demonstrate both strengths and weaknesses of this immortalised cell model as a representation of primary ASM cells for future asthma pathophysiological research.
Subject(s)
Myocytes, Smooth Muscle/physiology , Phenotype , Respiratory System/cytology , Telomerase/metabolism , Asthma/pathology , Cell Proliferation , Cells, Cultured , Extracellular Matrix/metabolism , Humans , Immunologic Factors/metabolism , Telomerase/geneticsABSTRACT
BACKGROUND: Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. OBJECTIVES: To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. METHODS: Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. RESULTS: We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. CONCLUSIONS: Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.
Subject(s)
Asthma/genetics , Asthma/metabolism , Myocytes, Smooth Muscle/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, Peptide/genetics , Acetylcholine/pharmacology , Animals , Case-Control Studies , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Humans , Male , Membrane Glycoproteins , Membrane Proteins/pharmacology , Mice , Mice, Inbred BALB C , Muscle Contraction/drug effects , Myocytes, Smooth Muscle/physiology , Oligonucleotide Array Sequence Analysis , Polymorphism, Single Nucleotide , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/metabolism , Respiratory System/cytology , Spider Venoms/pharmacology , Transcription, GeneticABSTRACT
BACKGROUND: Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. OBJECTIVE: To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. METHODS: Airway smooth muscle cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and Western blotting. Receptor signalling and function were determined by mRNA knockdown and intracellular calcium mobilization experiments. RESULTS: S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilization and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. CONCLUSION: S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma.
Subject(s)
Airway Remodeling/drug effects , Lysophospholipids/pharmacology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Sphingosine/analogs & derivatives , Adrenal Cortex Hormones/pharmacology , Airway Remodeling/genetics , Asthma/genetics , Asthma/metabolism , Asthma/pathology , Bronchi/drug effects , Bronchi/metabolism , Bronchi/pathology , Calcium/metabolism , Case-Control Studies , Cells, Cultured , Cluster Analysis , Drug Resistance , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/metabolism , Signal Transduction , Sphingosine/pharmacology , Sphingosine-1-Phosphate Receptors , rho-Associated Kinases/metabolismABSTRACT
Free calcium ions within the cytosol serve as a key secondary messenger system for a diverse range of cellular processes. Dysregulation of cytosolic Ca(2+) handling in airway smooth muscle (ASM) has been implicated in asthma, and it has been hypothesised that this leads, at least in part, to associated changes in both the architecture and function of the lung. Significant research is therefore directed towards furthering our understanding of the mechanisms which control ASM cytosolic calcium, in addition to those regulating the sensitivity of its downstream effector targets to calcium. Key aspects of the recent developments in this field were discussed at the 8th Young Investigators' Symposium on Smooth Muscle (2013, Groningen, The Netherlands), and are outlined in this review.
Subject(s)
Calcium/physiology , Muscle, Smooth/physiology , Respiratory Physiological Phenomena , Animals , Asthma/physiopathology , Humans , Muscle Contraction/physiology , Muscle, Smooth/drug effects , Muscle, Smooth/physiopathology , Respiratory Physiological Phenomena/drug effects , Second Messenger Systems/physiologyABSTRACT
Contractile G-protein-coupled receptors (GPCRs) have emerged as key regulators of smooth muscle contraction, both under healthy and diseased conditions. This brief review will discuss some key topics and novel insights regarding GPCR-mediated airway and vascular smooth muscle contraction as discussed at the 7th International Young Investigators' Symposium on Smooth Muscle (2011, Winnipeg, Manitoba, Canada) and will in particular focus on processes driving Ca(2+)-mobilization and -sensitization.
