Elaine Holland

Human airway smooth muscle cells secrete vascular endothelial growth factor: up-regulation by bradykinin via a protein kinase C and prostanoid-dependent mechanism

Bronchial vascular remodeling is an important feature of the pathology of chronic asthma, but the responsible mechanisms and main sources of an‐giogenic factors are unclear. Here we report that human airway smooth muscle cells express vascular endo‐thelial growth factor (VEGF)121,165,189,206 splice variants and secrete VEGF protein constitutively. VEGF protein secretion was increased by the proinflammatory asthma mediator bradykinin through post‐transcrip‐tional mechanisms. Bradykinin‐induced VEGF secretion was dependent on the B2 bradykinin receptor activation of protein kinase C and generation of endogenous prostanoids. This is the first report that bradykinin can increase VEGF secretion in any biological system and the first to show that airway smooth muscle cells produce VEGF. Our results suggest a novel role for human airway smooth muscle in contributing to bronchial mucosal angiogenesis in chronic asthma by secretion of VEGF and suggest a wider role for mesen‐chymal cell products in mediating angiogenesis in inflammatory and allergic diseases.—Knox, A. J., Corbett, L., Stocks, J., Holland, E., Zhu, Y. M., Pang, L. Human airway smooth muscle cells secrete vascular endothelial growth factor: up‐regulation by bradykinin via a protein kinase C and prostanoid‐dependent mechanism. FASEB J. 15, 2480–2488 (2001)

Garlic as an inhibitor of Pseudomonas aeruginosa quorum sensing in cystic fibrosis--a pilot randomized controlled trial.

Pseudomonas aeruginosa forms biofilms in the cystic fibrosis lung. Quorum sensing (QS) controls biofilm maturation, immune evasion, antibiotic tolerance and virulence factor production. Garlic shows QS inhibitory activity in vitro and in animal models. We report the first clinical trial in man of a QS inhibitor.

Role of cyclo‐oxygenase‐2 induction in interleukin‐1β induced attenuation of cultured human airway smooth muscle cell cyclic AMP generation in response to isoprenaline

1 Airway smooth muscle (ASM) in human asthma shows reduced relaxation and cyclic AMP generation in response to β‐adrenoceptor agonists. IL‐β attenuates cyclic AMP generation but the underlying mechanism is unclear. We have reported that IL‐1β induces cyclo‐oxygenase‐2 (COX‐2) in human ASM cells and results in a marked increase in prostanoid generation with PGE2 and PGI2 as the major products. 2 We investigated the role of COX‐2 induction and prostanoid release (measured as PGE2) in IL‐1β induced attenuation of cyclic AMP generation in response to the β‐adrenoceptor agonist isoprenaline (ISO). 3 Pre‐treatment of human ASM cells with IL‐1β significantly attenuated cyclic AMP generation in response to high concentrations of ISO (1.0–10.0 μm) in a time‐ and concentration‐dependent manner. The effect was accompanied by a high concentration of PGE2 release. The non‐selective COX inhibitor indomethacin (Ind), the selective COX‐2 inhibitor NS‐398, the protein synthesis inhibitors cycloheximide (CHX) and actinomycin D and the steroid dexamethasone (Dex) all abolished the PGE2 release and prevented the attenuated cyclic AMP generation. 4 COX substrate arachidonic acid time‐ and concentration‐dependently mimicked IL‐1β induced attenuation and the effect was prevented by the non‐selective COX inhibitors Ind and flurbiprofen, but not by NS‐398, CHX and Dex. 5 In contrast to IL‐1β, TNFα and IFNγ, which are ineffective in inducing COX‐2 and releasing PGE2 from human ASM cells, did not affect the cyclic AMP formation. 6 Our study demonstrates that COX‐2 induction and the consequent release of prostanoids plays a crucial role in IL‐1β induced attenuation of human ASM cell cyclic AMP response to ISO.

Production of PGE2 by bovine cultured airway smooth muscle cells and its inhibition by cyclo-oxygenase inhibitors.

1 Prostaglandin E2 (PGE2) is thought to be an important inhibitory modulator of inflammatory processes in the airway. It inhibits inflammatory cell function and cholinergic neurotransmission in vitro and roles have been postulated in vivo in refractoriness and in the mechanism of action of the diuretic agent, frusemide. 2 The production of PGE2 by bovine cultured airway smooth muscle cells has been studied under a range of conditions. The effects of cyclo‐oxygenase inhibitors (flurbiprofen, indomethacin, acetyl salicylic acid) on serum‐induced production of PGE2 were assessed over a range of concentrations (10−7–10−4 m). 3 Serum‐stimulated production of PGE2 in control wells ranged from 350 to 800 ng PGE2 ml−1 in cells from different animals. All three cyclo‐oxygenase inhibitors inhibited PGE2 production with an order of potency, flurbiprofen > indomethacin > acetyl salicylic acid. Log IC50 values were − 6.24 for flurbiprofen, − 5.23 for indomethacin and − 3.50 for acetyl salicylic acid. 4 PGE2 production was stimulated by arachidonic acid (10−5 m) or addition of the proinflammatory mediator, bradykinin (10−8–10−5 m). 5 Incubation of cells for 24 h with 5 bromo deoxyuridine (BRDU) (10−4 m) to prevent DNA synthesis did not alter PGE2 production in response to serum, suggesting that it was not a function of proliferation per se. 6 Our study suggests that airway smooth muscle may be an important source of PGE2. Production of PGE2 may be a novel feedback mechanism whereby airway smooth muscle cells can negatively modulate airways inflammation. The differing potencies of the cyclo‐oxygenase inhibitors may explain the contrasting effect of these drugs in recent studies in asthma.

Interleukin-1β Differentially Regulates β2 Adrenoreceptor and Prostaglandin E2-mediated cAMP Accumulation and Chloride Efflux from Calu-3 Bronchial Epithelial Cells ROLE OF RECEPTOR CHANGES, ADENYLYL CYCLASE, CYCLO-OXYGENASE 2, AND PROTEIN KINASE A

Here we tested the effect of interleukin-1β, a pro-inflammatory cytokine, on cAMP accumulation and chloride efflux in Calu-3 airway epithelial cells in response to ligands binding to adenylyl cyclase-coupled receptors such as the β2 adrenoreceptor and EP prostanoid receptors. Interleukin-1β significantly increased isoprenaline-induced cAMP accumulation by increasing β2 adrenoreceptor numbers via a protein kinase A-dependent mechanism. In contrast, interleukin-1β significantly impaired prostaglandin E2-induced cAMP accumulation by induction of cyclo-oxygenase-2, prostaglandin E2 production, and a resulting down-regulation of adenylyl cyclase. The cAMP changes were all mirrored by alterations in chloride efflux assessed using the fluorescent chloride probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide with interleukin-1β increasing chloride efflux in response to isoprenaline and reducing the response to prostaglandin E2. Studies with glibenclamide confirmed that chloride efflux was via the cystic fibrosis transmembrane conductance regulator. Calu-3 expresses EP4 receptors, but not EP2, and receptor expression is reduced by interleukin-1β. Collectively, these results provide mechanistic insight into how interleukin-1β can differentially regulate cAMP generation and chloride efflux in response to different adenylyl cyclase-coupled ligands in the same cell. These findings have important implications for diseases involving inflammation and abnormal ion flux such as cystic fibrosis.

Impaired cAMP production in human airway smooth muscle cells by bradykinin: role of cyclooxygenase products

Interleukin (IL)-1beta impairs human airway smooth muscle (ASM) cell cAMP responses to isoproterenol (Iso). We investigated if bradykinin (BK) could cause a similar effect and the role of cyclooxygenase (COX) products in this event, since we have recently reported that BK, like IL-1beta, also causes COX-2 induction and prostanoid release in human ASM cells. BK pretreatment significantly attenuated Iso-induced cAMP generation in a time- and concentration-dependent manner. cAMP generation by prostaglandin (PG) E2 but not by forskolin was also impaired. The COX inhibitor indomethacin completely prevented the impairment, whereas the selective COX-2 inhibitors NS-398 and nimesulide, protein synthesis inhibitors cycloheximide and actinomycin D, and steroid dexamethasone were all partially effective. The impairment was mimicked by the B2 agonist [Tyr(Me)8]BK, the Ca2+ ionophore A-23187, and PGE2 and prevented by the B2 antagonist HOE-140, but anti-IL-1beta serum was ineffective. The results indicate that BK impairs human ASM cell responses to Iso, and the effect is largely mediated by B2 receptor-related COX product release via both COX isoforms and is independent of IL-1beta.

Regulation of guanosine 3′:5′-cyclic monophosphate in ovine tracheal epithelial cells

Guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C‐type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)). Both 10−710−3 m and 10−710−3 m SNAP generated a concentration‐dependent marked elevation in cyclic GMP production when incubated with 10−3 m 3‐isobutyl‐1‐methylxanthine (IBMX) (both greater than 25×baseline values with highest drug concentration). The increase in production of cyclic GMP in response to 10−6 m SNP and 10−5 m SNAP was markedly inhibited by both 5×10−5 m haemoglobin (102% and 92% inhibition) and 5×10−5 m methylene blue (82% and 84% inhibition). The increase in cyclic GMP in response to 10−3 m SNP was measured following co‐incubation with the phosphodiesterase inhibitors 10−710−3 m IBMX, 10−710−4 m milrinone and 10−710−4 m SKF 96231. Only 10−410−3 m IBMX significantly increased cyclic GMP levels. Cyclic GMP production was also significantly elevated from baseline by 10−5 m ANP, 10−5 m BNP, 10−5 m CNP and 200 iu ml−1 of E. coli STa toxin in the presence of 10−3 m IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (24 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.