Stuart J. Hirst

Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis.

Protein kinase C (PKC) is a multifunctional, cyclic nucleotide-independent protein kinase that phosphorylates serine and threonine residues in many target proteins. This enzyme was identified in bovine cerebellum by Nishizuka and co-workers (Takai et al., 1977; Inoue et al., 1977) as a protein kinase that phosphorylated histone and protamine. Since its discovery, much interest has been shown in PKC and its role in signal transduction. Development (Otte et al., 1991), memory (Alkon, 1989), differentiation (Cutler et al., 1993), proliferation (Murray et al., 1993) and carcinogenesis (Ashendel, 1985) all are processes for which PKC has been implicated. Once thought to be a single protein, PKC is now known to comprise a large family of enzymes that differ in structure, cofactor requirements and function. Indeed, the PKC family is the largest serine/threonine-specific kinase family known (Parker, 1992) to which many cellular responses have been credited (Nishizuka, 1995). This enzyme multiplicity, together with variation in cellular and tissue distribution, and abundance might explain why so many signal transduction functions have been attributed to this kinase. Here we briefly describe the organization and regulation of PKC and review the current understanding of their role in the regulation of airways smooth muscle (ASM) tone and mitogenesis, which have been investigated in some detail.

Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca2+ in the cytosol ([Ca2+]i). A rise in [Ca2+]i is normalized by rapid reuptake of Ca2+ into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca2+ (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca2+]i, cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca2+]i following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca2+ re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca]i after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.

β2-Adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells

Airway smooth muscle (ASM) is a potential source of multiple pro‐inflammatory cytokines during airway inflammation. β‐Adrenoceptor agonist hyporesponsiveness is a characteristic feature of asthma, and interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α are implicated in its cause. Here, the capacity of β‐adrenoceptor agonists to prevent release of GM‐CSF, RANTES, eotaxin and IL‐8, elicited by IL‐1β or TNFα, was examined in human ASM cells. Isoprenaline (∼EC50 150 nM), a non‐selective β‐adrenoceptor agonist, and salbutamol (∼EC50 25 nM), a selective β2‐adrenoceptor agonist, attenuated release of GM‐CSF, RANTES and eotaxin, but not IL‐8 (EC50 >1 μM). The maximum extent of attenuation was RANTES eotaxin > GM‐CSF >> IL‐8, and was prevented by either propranolol (1 μM), a non‐selective β‐adrenoceptor antagonist, or ICI 118511 (IC50 15 nM), a selective β2‐adrenoceptor antagonist. The cyclic AMP‐elevating agents, dibutyryl cyclic AMP (∼EC50 135 μM), forskolin (∼EC50 530 nM) and cholera toxin (∼EC50 575 pg ml−1) abolished IL‐1β‐induced release of GM‐CSF, RANTES and eotaxin, but not IL‐8. IL‐1β (1 ng ml−1) attenuated early increases (up to 1 h) in cyclic AMP formation induced by salbutamol (1 μM), but not by forskolin (10 μM). The cyclo‐oxygenase inhibitor, indomethacin (1 μM) prevented later increases (3 – 12 h) in IL‐1β‐stimulated cyclic AMP content, but did not prevent the attenuation by salbutamol of IL‐1β‐induced cytokine release. We conclude in human ASM cells that activation of β2‐adrenoceptors and generation of cyclic AMP is negatively‐linked to the release, elicited by IL‐1β or TNFα, of eosinophil‐activating cytokines such as GM‐CSF, RANTES and eotaxin, but not IL‐8.

Multiple β1 Integrins Mediate Enhancement of Human Airway Smooth Muscle Cytokine Secretion by Fibronectin and Type I Collagen

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with interstitial collagen and fibronectin are major pathological features of airway remodeling in asthma. We have previously shown that these ECM components confer enhanced ASM proliferation in vitro, but their action on its newly characterized secretory function is unknown. Here, we examined the effects of fibronectin and collagen types I, III, and V on IL-1β-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1β-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that α2β1, α5β1, αvβ1, and αvβ3 integrins were required for up-regulation of IL-1β-dependent ASM secretory responses by fibronectin, while α2β1 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1β-dependent ASM secretory responses through a β1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma.

Induction of Angiogenesis by Airway Smooth Muscle From Patients with Asthma

RATIONALE Airway remodeling in asthma involves accumulation of airway smooth muscle (ASM) and increased vascularity due to angiogenesis. Bronchial blood vessels and ASM are found in close proximity, and ASM releases multiple proinflammatory mediators, including vascular endothelial growth factor (VEGF). OBJECTIVES We examined whether release of proangiogenic mediators is increased in ASM from subjects with asthma and whether this is translated to induction of angiogenesis. METHODS Biopsy-derived ASM cells were cultured from 12 subjects with mild asthma, 8 with moderate asthma, and 9 healthy control subjects. Angiogenesis induced by cell-conditioned medium (CM) from ASM was evaluated in a tubule formation assay. Anti-CD31-labeled tubules were quantified by image analysis. Angiogenic factors in CM were quantified by antibody arrays and by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS Induction of angiogenesis by CM from unstimulated ASM was increased in subjects with mild asthma (twofold) and moderate asthma (threefold), compared with healthy CM (P < 0.001). Levels of angiogenic factors (VEGF, angiopoietin [Ang]-1, angiogenin) were similarly elevated in CM from subjects with asthma compared with that from healthy subjects (P < 0.05), whereas antiangiogenic factors (endostatin, Ang-2) were unchanged. VEGF, Ang-1, and angiogenin in combination increased vascularity (twofold, P < 0.01) in cultured intact biopsies. Selective VEGF immunodepletion abolished enhanced tubule formation by CM from asthmatic ASM (P < 0.01), but CM depletion of Ang-1 or angiogenin had no effect. CONCLUSIONS ASM cultured from subjects with mild or moderate asthma, but not from healthy control subjects, promotes angiogenesis in vitro. This proangiogenic capacity resides in elevated VEGF release and suggests that ASM regulates airway neovascularization in asthma.

Some structural determinants of the antiproliferative effect of heparin-like molecules on human airway smooth muscle

Accumulation of airway smooth muscle (ASM) and its infiltration by mast cells are key pathological features of airway remodelling in asthma. Heparin, a major component of mast cell granules, inhibits ASM proliferation by an unknown mechanism. Here, unfractionated heparins and related glycosaminoglycans having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule that were required for its antiproliferative activity in cultured human ASM cells. Proliferation induced by 10% fetal bovine serum (FBS) was abrogated by two unfractionated commercial heparin preparations (Sigma and Multiparin) and this effect was reproduced with each of three low‐molecular weight heparin preparations (3, 5 and 6 kDa, respectively), demonstrating that antiproliferative activity resided in at least a 3 kDa heparin fraction. N‐desulphated 20% re‐acetylated (N‐de) heparin (anticoagulant) and O‐desulphated heparin (O‐de) (non‐anticoagulant) fractions also inhibited FBS‐dependent proliferation (rank potency: Sigma heparin>O‐de>N‐de) suggesting that the antiproliferative action of heparin involved N‐sulphation but was independent of its anticoagulant activity. Other sulphated molecules with variable anionic charge (dextran sulphate, fucoidan, chondroitin sulphates A or B, heparan sulphate) inhibited proliferation to varying degrees, as did the non‐sulphated molecules hyaluronic acid and poly‐L‐glutamic acid. However, nonsulphated dextran had no effect. In summary, attenuation of FBS‐dependent proliferation of human ASM by heparin involves but does not depend upon sulphation, although loss of N‐sulphation reduces antiproliferative activity. This antiproliferative effect is independent of anionic charge and the anticoagulant actions of heparin.

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)-1β, and tumor necrosis factor (TNF)-α 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-1β (0.5 ng/mL) and TNF-α (10ng/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 8h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1μM dexamethasone abolished enhanced release of VEGF by TNF-α. 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.

Potassium currents in human freshly isolated bronchial smooth muscle cells

1 K+ currents were studied in smooth muscle cells enzymatically dissociated from human bronchi, by use of the patch‐clamp technique. 2 In whole‐cell recordings a depolarization‐induced, 4‐aminopyridine (4‐AP)‐sensitive current was observed in only 26 of 155 cells, and in 20 of these 26 cells its amplitude at a test potential of 0 mV was less than 100 pA. 3 In the majority of cells depolarization to −40 mV or more positive potentials induced a noisy outward current which activated within milliseconds and showed almost no inactivation even during a 5 s depolarizing voltage step. This current was insensitive to 4‐AP (up to 5 mM) but was strongly inhibited in the presence of tetraethylammonium (TEA, 1 mM), charybdotoxin (ChTX, 100 nM) or iberiotoxin (IbTX, 50 nM) in the bath. The same current was also recorded by the nystatin‐perforated patch technique. 4 Single channels with a conductance of about 210 pS were recorded in cell‐attached patch, inside‐out patch, outside‐out patch and whole‐cell recording configurations. Channel open state probability in inside‐out patches was 0.5 at a membrane potential of 4±14 mV (mean±s.d., n= 13) mV even with a free Ca2+ concentration on the cytosolic side of the patch of less than 0.1 nM. Open state probability increased with depolarization and internal Ca2+ concentration. Single channels could be reversibly blocked by externally applied TEA, ChTX and IbTX. 5 In current‐clamp recordings with 100 nM free Ca2+ in the intracellular solution both TEA and ChTX caused substantial concentration‐dependent depolarization. 6 These results suggest that in human bronchial smooth muscle cells, in marked contrast to other species, the majority of the outward current induced by depolarization is not due to a delayed rectifier, but to the activity of a large conductance, ChTX‐sensitive K+ channel. The Ca2+– and voltage‐dependency of this channel may well allow a sufficiently high open state probability for it to play a part in the regulation of the resting membrane potential.

Matrix Metalloproteinase-19 Deficiency Promotes Tenascin-C Accumulation and Allergen-Induced Airway Inflammation

Matrix metalloproteinases (MMPs) recently appeared as key regulators of inflammation, allowing the recruitment and clearance of inflammatory cells and modifying the biological activity of many peptide mediators by cleavage. MMP-19 is newly described, and it preferentially cleaves matrix proteins such as collagens and tenascin-C. The role of MMP-19 in asthma has not been described to date. The present study sought to assess the expression of MMP-19 in a murine asthma model, and to address the biological effects of MMP-19 deficiency in mice. Allergen-exposed, wild-type mice displayed increased expression of MMP-19 mRNA and an increased number of MMP-19-positive cells in the lungs, as detected by immunohistochemistry. After an allergen challenge of MMP-19 knockout (MMP-19(-/-)) mice, exacerbated eosinophilic inflammation was detected in bronchoalveolar lavage fluid and bronchial tissue, along with increased airway responsiveness to methacholine. A shift toward increased T helper-2 lymphocyte (Th2)-driven inflammation in MMP-19(-/-) mice was demonstrated by (1) increased numbers of cells expressing the IL-33 receptor T(1)/ST(2) in lung parenchyma, (2) increased IgG(1) levels in serum, and (3) higher levels of IL-13 and eotaxin-1 in lung extracts. Tenascin-C was found to accumulate in peribronchial areas of MMP-19(-/-) after allergen challenges, as assessed by Western blot and immunohistochemistry analyses. We conclude that MMP-19 is a new mediator in asthma, preventing tenascin-C accumulation and directly or indirectly controlling Th2-driven airway eosinophilia and airway hyperreactivity. Our data suggest that MMP-19 may act on Th2 inflammation homeostasis by preventing the accumulation of tenascin protein.

Contractility and phenotype of human bronchiole smooth muscle after prolonged fetal bovine serum exposure.

Culture of dispersed airway smooth muscle (ASM) cells with fetal bovine serum (FBS) induces rapid growth and modulation from a contractile to a synthetic phenotype, but this may be an artificial situation due to loss of tissue architecture. In this study, structural, functional and biochemical changes of ASM were examined in human intact bronchiole ring segments (200 to 600 μ m internal diameter) after organ culture for up to 6 days in 10% FBS or in D-STIM, an FBS-free medium formulated to maintain a contractile phenotype. ASM content was unchanged after culture for 3 or 6 days with D-STIM or FBS compared with fresh tissues. However, by 6 days culture with FBS reduced the maximum developed contraction to several agonists (carbachol, histamine, and KCl). Smooth muscle (sm)- α -actin and sm-myosin heavy chain (MHC) isoform 1 expression was similar for all culture conditions, though FBS reduced calponin and metavinculin content. Nonmuscle (nm) proteins, including total vinculin, β -actin, and nm-MHC, were unchanged. Thus, ASM in human intact bronchioles maintains its functional, biochemical, and morphometric properties after culture in FBS-free (D-STIM) medium for at least 6 days, and for 3 days when cultured with FBS. These findings in organ culture may reflect more closely the in vivo situation in which tissue architecture is better preserved over cell culture, and may provide a basis for examining long-term effects of trophic or contractile stimuli on intact ASM in vitro that contribute to airway hyperresponsiveness and remodeling.