Lisa Corbett

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)

Vascular Endothelial Growth Factor Induction by Prostaglandin E2 in Human Airway Smooth Muscle Cells Is Mediated by E Prostanoid EP2/EP4 Receptors and SP-1 Transcription Factor Binding Sites

Prostaglandin E2 (PGE2) can increase thelial vascular endogrowth factor A (VEGF-A) production but the mechanisms involved are unclear. Here we characterized the transcriptional mechanisms involved in human airway smooth muscle cells (HASMC). PGE2 increased VEGF-A mRNA and protein but not mRNA stability. PGE2 stimulated the activity of a transiently transfected 2068-bp (–2018 to +50) VEGF-A promoter-driven luciferase construct. Functional 5′ deletional analysis mapped the PGE2 response element to the 135-bp sequence (–85/ +50) of the human VEGF-A promoter. PGE2-induced luciferase activity was reduced in cells transfected with a 135-bp VEGF promoter fragment containing mutated Sp-1 binding sites but not in cells transfected with a construct containing mutated EGR-1 binding sites. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed binding of Sp-1 to the VEGF promoter. PGE2 increased phosphorylation of Sp-1 and luciferase activity of a transfected Sp-1 reporter construct. PGE receptor agonists EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) mimicked the effect of PGE2, and reverse transcription-PCR, Western blotting, and flow cytometry confirmed the presence of EP2 and EP4 receptors. VEGF protein release and Sp-1 reporter activity were increased by forskolin and isoproterenol, which increase cytosolic cAMP, and the cAMP analogue, 8-bromoadenosine-3′,5′-cyclophosphoric acid. These studies suggest that PGE2 increases VEGF transcriptionally and involves the Sp-1 binding site via a cAMP-dependent mechanism involving EP2 and EP4 receptors.

Cyclooxygenase-2 Expression by Nonsteroidal Anti-inflammatory Drugs in Human Airway Smooth Muscle Cells: Role of Peroxisome Proliferator-Activated Receptors

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to modulate cyclooxygenase (COX)-2 expression, but the mechanisms involved are controversial and may be cell specific. We show in this study that indomethacin (Indo), flurbiprofen (Flur), and the selective COX-2 inhibitor NS-398 induced COX-2 expression and markedly enhanced IL-1β-induced COX-2 expression in human airway smooth muscle (HASM) cells. These effects were not reversed by exogenous PGE2, suggesting that they are prostanoid-independent. Indeed, PGE2 also induced and enhanced IL-1β-induced COX-2 expression. Peroxisome proliferator-activated receptor (PPAR) α and PPARγ (not PPARβ) were expressed in HASM cells. PPARγ activators ciglitizone (Cig) and 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), but not the PPARα activator WY-14643, mimicked the effect of NSAIDs on COX-2 expression. Treatment with Flur, NS-398, Cig, and 15d-PGJ2 alone, but not Indo and WY-14643, elevated COX activity; however, neither enhanced IL-1β-induced COX activity. Pretreatment with dexamethasone suppressed COX-2 expression, PGE2 release, and COX activity induced by NS-398, Cig, IL-1β, alone or in combination. Unlike IL-1β, NS-398 and Cig did not cause NF-κB (p65) nuclear translocation, nor did they further enhance IL-1β-induced NF-κB translocation, but they stimulated PPARγ translocation. Indo, NS-398, Flur, and 15d-PGJ2, but not WY-14643, induced transcriptional activity of a COX-2 reporter construct containing the peroxisome proliferator response element (PPRE) on their own and enhanced the effect of IL-1β, but had no effect on a COX-2 reporter construct lacking the PPRE. The results suggest that COX-2 expression by NSAIDs is biologically functional, prostanoid-independent, and involves PPARγ activation, and provide the first direct evidence that the PPRE in the promoter is required for NSAID-induced COX-2 expression.

Mast Cell β-Tryptase Selectively Cleaves Eotaxin and RANTES and Abrogates Their Eosinophil Chemotactic Activities

Recent studies have shown that a lack of eosinophils in asthmatic airway smooth muscle (ASM) bundles in contrast to the large number of mast cells is a key feature of asthma. We hypothesized that this is caused by β-tryptase, the predominant mast cell-specific protease, abrogating the eosinophil chemotactic activities of ASM cell-derived eosinophil chemoattractants such as eotaxin and RANTES. We studied the effect of β-tryptase on the immunoreactivities of human ASM cell-derived and recombinant eotaxin and other recombinant chemokines that are known to be produced by human ASM cells. We report in this study that purified β-tryptase markedly reduced the immunoreactivity of human ASM cell-derived and recombinant eotaxin, but had no effect on eotaxin mRNA expression. The effect was mimicked by recombinant human β-tryptase in the presence of heparin and was reversed by heat inactivation and the protease inhibitor leupeptin, suggesting that the proteolytic activity of tryptase is required. β-Tryptase also exerted similar effects on recombinant RANTES, but not on the other chemokines and cytokines that were screened. Furthermore, a chemotaxis assay revealed that recombinant eotaxin and RANTES induced eosinophil migration concentration-dependently, which was abrogated by pretreatment of these chemokines with β-tryptase. Another mast cell protease chymase also markedly reduced the immunoreactivity of eotaxin, but had no effect on RANTES and other chemokines and did not affect the influence of β-tryptase on RANTES. These findings suggest that mast cell β-tryptase selectively cleaves ASM-derived eotaxin and RANTES and abrogates their chemotactic activities, thus providing an explanation for the eosinophil paucity in asthmatic ASM bundles.

Protein kinase C-ε mediates bradykinin-induced cyclooxygenase-2 expression in human airway smooth muscle cells1

We previously reported that proinflammatory mediator bradykinin (BK) induces cyclooxygenase (COX)‐2 expression in human airway smooth muscle (HASM), but the mechanism is unknown in any biological system. Here, we studied the role of specific protein kinase C (PKC) isozyme(s) in COX‐2 expression. Among the eight PKC isozymes present in HASM cells, the Ca2+‐independent PKC‐δ and ‐ε and the Ca2+‐dependent PKC‐α and ‐βI were translocated to the nucleus upon BK stimulation. BK‐induced COX‐2 expression and prostaglandin E2 (PGE2) accumulation were mimicked by the direct PKC activator phorbol 12‐myristate 13‐acetate (PMA) and inhibited by the broad spectrum PKC inhibitor bisindolylmaleimide I. However, the selective Ca2+‐dependent PKC isozyme inhibitor Go 6976 had no effect. Furthermore, the membrane‐permeable calcium chelator BAPTA‐AM had no effect on BK‐induced COX‐2 expression and COX activity despite its inhibition of PGE2 accumulation, suggesting the involvement of Ca2+‐independent PKC isozymes. Rottlerin, a PKC‐δ inhibitor, also had no effect, likely implicating PKC‐ε. BK‐stimulated transcriptional activation of a COX‐2 promoter reporter construct was enhanced by overexpression of wild‐type PKC‐ε and abolished by a dominant negative PKC‐ε, but it was not affected by wild‐type or dominant negative PKC‐α or ‐δ. Collectively, our results demonstrate that PKC‐ε mediates BK‐induced COX‐2 expression in HASM cells.

Cytokines upregulate vascular endothelial growth factor secretion by human airway smooth muscle cells: Role of endogenous prostanoids

Here, we report that vascular endothelial growth factor (VEGF)‐A secretion by human airway smooth muscle cells was increased by interleukin 1 beta (IL‐1β) and transforming growth factor beta (TGFβ). IL‐1β and TGFβ induced cyclo‐oxygenase (COX)‐2 protein and increased prostaglandin E2 (PGE2). Both IL‐1β and TGFβ increased VEGF‐A165 mRNA and VEGF promoter luciferase construct activity, in addition VEGF‐A protein was inhibited by actinomycin D suggesting transcriptional regulation. The COX inhibitors indomethacin and NS398 inhibited IL‐1β but not TGFβ mediated VEGF‐A production. Furthermore, the effect of the COX inhibitors was overcome by adding exogenous PGE2. In conclusion, IL‐1β increases VEGF‐A secretion by COX‐2 derived PGE2 production whereas TGFβ uses COX‐independent pathways.

Expression of Prostaglandin E Synthases in Periodontitis: Immunolocalization and Cellular Regulation

The inflammatory mediator prostaglandin E(2) (PGE(2)) is implicated in the pathogenesis of chronic inflammatory diseases including periodontitis; it is synthesized by cyclooxygenases (COX) and the prostaglandin E synthases mPGES-1, mPGES-2, and cPGES. The distribution of PGES in gingival tissue of patients with periodontitis and the contribution of these enzymes to inflammation-induced PGE(2) synthesis in different cell types was investigated. In gingival biopsies, positive staining for PGES was observed in fibroblasts and endothelial, smooth muscle, epithelial, and immune cells. To further explore the contribution of PGES to inflammation-induced PGE(2) production, in vitro cell culture experiments were performed using fibroblasts and endothelial, smooth muscle, and mast cells. All cell types expressed PGES and COX-2, resulting in basal levels of PGE(2) synthesis. In response to tumor necrosis factor (TNF-α), IL-1β, and cocultured lymphocytes, however, mPGES-1 and COX-2 protein expression increased in fibroblasts and smooth muscle cells, accompanied by increased PGE(2), whereas mPGES-2 and cPGES were unaffected. In endothelial cells, TNF-α increased PGE(2) production only via COX-2 expression, whereas in mast cells the cytokines did not affect PGE(2) enzyme expression or PGE(2) production. Furthermore, PGE(2) production was diminished in gingival fibroblasts derived from mPGES-1 knockout mice, compared with wild-type fibroblasts. These results suggest that fibroblasts and smooth muscle cells are important sources of mPGES-1, which may contribute to increased PGE(2) production in the inflammatory condition periodontitis.