Tryptase Stimulation Research Articles

Tryptase activates enteric glial cells followed by affecting neuronal properties possibly via the stimuli-associated mediators

ObjectivesMast cell-derived tryptase causes neuronal elongation/sensitization leading to visceral hypersensitivity. However, effects of tryptase on enteric glial cells (EGCs) and subsequent interaction between EGCs and neurons remain unknown. MethodsWe evaluated proteins and mRNA expressions in EGC (CRL-2690, ATCC) after tryptase stimulation: nerve growth factor (NGF), netrin-1, and glial cell-derived neurotrophic factor (GDNF). We examined morphological changes in neurons (PC12 cells, CRL-1721.1) by co-incubation with the conditioned medium of EGCs after tryptase stimulation. ResultsEGC was activated by tryptase, and proliferated (by 1.8-fold) with cytoplasmic expansion and process elongation. Intercellular connections of EGC were more complexed. Tryptase induced mRNA expression (2.5-fold) and protein expression of NGF. Netrin-1 (3-fold) and GDNF (3-fold) mRNA expressions were increased at 30 min. Increase in netrin-1 continued until 6 h, whereas the latter decreased by 3 h. The conditioned medium of EGC after tryptase stimulation expanded neuronal cytoplasm (round or ramified shapes) and neurite outgrowth with elongation of cytoskeletal filaments in time-dependent and dose-dependent manners. These changes were similar to those after NGF stimulation. Growth cone proteins of neurons were also increased by the conditioned medium. ConclusionEGC activated by tryptase changes neuronal morphology (process elongation and cytoplasm expansion) possibly via the stimuli-associated mediators.

Intestinal mucosal injury induced by tryptase-activated protease-activated receptor 2 requires β-arrestin-2 in vitro.

Tryptase exacerbates intestinal ischemia-reperfusion injury, however, the direct role of tryptase in intestinal mucosal injury and the underlying mechanism remains largely unknown. Protease-activated receptor 2 (PAR‑2), commonly activated by tryptase, interacts with various adaptor proteins, including β‑arrestin‑2. The present study aimed to determine whether tryptase is capable of inducing intestinal mucosal cell injury via PAR‑2 activation and to define the role of β‑arrestin‑2 in the process of injury. The IEC‑6 rat intestinal epithelial cell line was challenged by tryptase stimulation. Cell viability, lactate dehydrogenase (LDH) activity and apoptosis were analyzed to determine the severity of cell injury. Injury was also evaluated following treatments with specific PAR‑2 and extracellular signal‑related kinases (ERK) inhibitors, and knockdown of β‑arrestin‑2. PAR‑2, ERK and β‑arrestin‑2 protein expression levels were evaluated. Tryptase treatment (100 and 1,000 ng/ml) resulted in IEC‑6 cell injury, as demonstrated by significant reductions in cell viability, accompanied by concomitant increases in LDH activity and levels of cleaved caspase‑3 protein expression. Furthermore, tryptase treatment led to a marked increase in PAR‑2 and phosphorylated‑ERK expression, and exposure to specific PAR‑2 and ERK inhibitors eliminated the changes induced by tryptase. Knockdown of β‑arrestin‑2 blocked tryptase‑mediated cell injury, whereas tryptase exerted no influence on β‑arrestin‑2 expression in IEC‑6 cells. These data indicate that tryptase may directly damage IEC‑6 cells via PAR-2 and the downstream activation of ERK, and demonstrate that the signaling pathway requires β-arrestin-2.

Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA2 activation. Incubation with the membrane phospholipid-derived PLA2 hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA2 activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS.

PGE2 Release from Tryptase‐Stimulated Rabbit Ventricular Myocytes is Mediated by Calcium‐Independent Phospholipase A2γ

Inflammation is associated with cardiovascular disease, including myocardial infarction, atherosclerosis, myocarditis and congestive heart failure. Mast cells have been implicated in inflammation, but their precise role in cardiac inflammation remains unclear. Mast cells contain a variety of pre-formed granule-associated mediators, including tryptase. We have previously demonstrated that the majority of the phospholipase A(2) (PLA(2)) activity in isolated rabbit ventricular myocytes is membrane-associated, calcium-independent and selective for plasmalogen phospholipids. We hypothesized that tryptase stimulation of rabbit ventricular myocytes would increase iPLA(2) activity, leading to increased arachidonic acid and prostaglandin E(2) (PGE(2)) release. Isolated rabbit ventricular myocytes were stimulated with tryptase and iPLA(2) activity, arachidonic acid and PGE(2) release were measured. Tryptase stimulation increased iPLA(2) activity after 5 min. Activation of iPLA(2) was accompanied by increased arachidonic acid and PGE(2) release in tryptase-stimulated myocytes. However no increase in platelet activating factor was observed with tryptase stimulation. To distinguish between different iPLA(2) isoforms in the myocardium, we pretreated ventricular myocytes with the (R)- and (S)-enantiomers of bromoenol lactone (BEL) to selectively inhibit iPLA(2)γ and β respectively. Pretreatment with (R)-BEL resulted in complete inhibition of tryptase-stimulated iPLA(2) activity, arachidonic acid and PGE(2) release, suggesting the iPLA(2)γ is the predominant myocardial isoform activated by tryptase. These studies demonstrate that PGE(2) release from tryptase stimulated rabbit ventricular myocytes is mediated primarily by iPLA(2)γ.

Activation of calcium‐independent phospholipase A 2 following protease‐activated receptor cleavage in mouse cardiomyocytes

Serine proteases such as thrombin and tryptase can regulate target cells by activating G-protein coupled protease-activated receptors (PAR). The role for PAR activation is not well defined and could be pro- or anti-inflammatory. We determined whether activation of calcium-independent phospholipase A2 (iPLA2) following PAR cleavage in cardiac myocytes contributes to PAF production and PGE2 release. We stimulated HL-1 mouse cardiomyocytes with thrombin or tryptase and measured arachidonic acid and prostaglandin E2 (PGE2) release, plus platelet-activating factor (PAF) production. Thrombin (0,1 IU/ml) or tryptase (20 ng/ml) stimulation resulted in a significant increase in arachidonic acid and PGE2 release that was maximal at 5 mins. Pretreatment of HL-1 cells with (R)-bromoenol lactone ((R)-BEL), a selective iPLA2γ inhibitor, completely blocked thrombin- and tryptase-stimulated arachidonic acid and PGE2 release. Similarly, increases in PAF production observed in HL-1 cells stimulated with thrombin (1 IU/ml, 10 mins) or tryptase (20 ng/ml, 10 mins) were inhibited by BEL pretreatment. These data suggest that there is an increase in iPLA2γ activity in mouse cardiac myocytes following PAR activation, that results in increased arachidonic acid and PGE2 release, and increased PAF production, mediators that may play a role in cardiac inflammation.

Activation of group VI phospholipase A2 isoforms in cardiac endothelial cells

The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A(2) (iPLA(2)β) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA(2)β-knockout (iPLA(2)β-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I(2) (PGI(2)), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI(2) release and increased PAF production. Selective inhibition of iPLA(2)β with (S)-bromoenol lactone (5 μM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA(2)β-KO endothelial cells did not result in significant PAF production and inhibited AA and PGI(2) release. Stimulation of cardiac endothelial cells from group VIB (iPLA(2)γ)-KO mice increased PAF production to levels similar to those of WT cells but significantly attenuated PGI(2) release. These results indicate that cardiac endothelial cell PAF production is dependent on iPLA(2)β activation and that both iPLA(2)β and iPLA(2)γ may be involved in PGI(2) release.

Urothelial Cell Platelet-activating Factor Production Mediated by Calcium-independent Phospholipase A 2γ

To determine the effect of phospholipase A(2) (PLA(2)) inhibitors on urothelial cell platelet-activating factor (PAF) production in response to tryptase stimulation. Urothelial cells isolated from normal human ureters were immortalized with the human papillomavirus type 16E6E7 cell line (TEU-2 cells). PLA(2) activity in TEU-2 cells was measured using (16:0, [(3)H]18:1) plasmenylcholine and phosphatidylcholine substrates in the presence and absence of calcium. [(3)H]PAF production was measured in TEU-2 cells prelabeled with [(3)H] acetic acid. PAF-acetylhydrolase activity was measured by determining the amount of [(3)H] acetate hydrolyzed from [(3)H]PAF incubated with TEU-2 cellular protein. Adherence of human polymorphonuclear leukocyte (PMN) to TEU-2 cells was assessed by measuring myeloperoxidase activity in adherent PMNs after incubation with TEU-2 cells. Most PLA(2) activity measured in TEU-2 cells was determined to be membrane-associated, calcium-independent PLA(2) and selective for plasmenylcholine substrate. Stimulation of TEU-2 cells with tryptase results in increased production of PAF and increased PMN adherence that were inhibited completely by pretreatment with the membrane-associated, calcium-independent PLA(2)γ-selective inhibitor (R)-bromoenol lactone. Pretreatment with the cytosolic PLA(2) inhibitor methyl arachidonyl fluorophosphonate resulted in potentiation of tryptase-stimulated PAF production and PMN adherence to TEU-2 cells that is a result of PAF-acetylhydrolase inhibition. Tryptase stimulation of TEU-2 cells results in activation of membrane-associated, calcium-independent PLA(2)γ, leading to an increase in PAF production and increased PMN adherence. Inhibition of TEU-2 cell PAF-acetylhydrolase activity with methyl arachidonyl fluorophosphonate potentiated tryptase-stimulated PAF production and PMN adherence.

Endothelial Cell Prostaglandin I2 and Platelet-Activating Factor Production Are Markedly Attenuated in the Calcium-Independent Phospholipase A2β Knockout Mouse

Damage and activation of lung endothelium can lead to interstitial edema, infiltration of inflammatory cells into the interstitium and airways, and production of inflammatory metabolites, all of which propagate airway inflammation in a variety of diseases. We have previously determined that stimulation of human microvascular endothelial cells from lung (HMVEC-L) results in activation of a calcium-independent phospholipase A(2) (iPLA(2)), and this leads to arachidonic acid release and production of prostaglandin I(2) (PGI(2)) and platelet-activating factor (PAF). We stimulated lung endothelial cells isolated from iPLA(2)beta-knockout (KO) and wild type (WT) mice with thrombin and tryptase to determine the role of iPLA(2)beta in endothelial cell membrane phospholipid hydrolysis. Thrombin or tryptase stimulation of WT lung endothelial cells resulted in increased arachidonic acid release and production of PGI(2) and PAF. Arachidonic acid release and PGI(2) production by stimulated iPLA(2)beta-KO endothelial cells were significantly reduced compared to WT. Measured PLA(2) activity and PGI(2) production by iPLA(2)beta-KO cells were suppressed by pretreatment with (R)-bromoenol lactone (R-BEL), which is a selective inhibitor of iPLA2gamma. In contrast to the increase in PAF production induced by stimulation of WT endothelial cells, none was observed for KO cells, and this suggests that endothelial PAF production is entirely dependent on iPLA(2)beta activity. Because inflammatory cell recruitment involves the interaction of endothelial cell PAF with PAF receptors on circulating cells, these data suggest that iPLA(2)beta may be a suitable therapeutic target for the treatment of inflammatory lung diseases.

PGE2 release from tryptase‐stimulated rabbit ventricular myocytes is mediated by calcium‐independent phospholipase A2γ

Inflammation is associated with several cardiovascular diseases and mast cells have been implicated in many of these, but their precise role remains unclear. We hypothesize that tryptase, a pre formed mast cell mediator, stimulates rabbit ventricular myocyte calcium independent phospholipase A2 (iPLA2) activity, resulting in increased arachidonic acid and prostaglandin E2 (PGE2) release. Tryptase stimulation (20 ng/ml) increased iPLA2 activity after 5 mins (3.56 ± 0.12 to 5.87 ± 0.31 nmol/mg protein/min, n=6, p<0.01). This was accompanied by increased arachidonic acid (1.02 ± 0.12 to 5.02 ± 0.21%, n=6, p<0.01) and PGE2 release (4.50 ± 0.30 to 16.70 ± 0.59 ng/mg protein, n=8, p<0.01) in stimulated myocytes. To distinguish between different myocardial iPLA2 isoforms, we pretreated ventricular myocytes with the R‐ and S‐ enantiomers of bromoenol lactone (BEL, 5 μM, 10 mins) to selectively inhibit iPLA2γ and β respectively. (R)‐BEL pretreatment resulted in complete inhibition of tryptase‐stimulated iPLA2 activity, arachidonic acid and PGE2 release, suggesting the iPLA2γ is activated in response to tryptase. Pretreatment with (S)‐BEL had little effect on tryptase‐stimulated responses. Previous studies suggest that PGE2 may be cardioprotective. Thus tryptase release from activated mast cells may play a protective role in cardiac inflammation via iPLA2γ activation and increased PGE2 release from myocytes.

Inhibition of calcium-independent phospholipase A2 prevents inflammatory mediator production in pulmonary microvascular endothelium.

Inhalation of allergens can result in mast cell degranulation and release of granule contents, including tryptase, in the lung. Injury to human pulmonary microvascular endothelial cells (HMVEC-L) can also result in activation of the coagulation cascade and thrombin generation. We hypothesize that these proteases activate calcium-independent phospholipase A2 (iPLA2), in HMVEC-L, leading to the production of membrane phospholipids-derived inflammatory mediators. Both thrombin and tryptase stimulation of HMVEC-L increased iPLA2 activity that was inhibited by pretreatment with the iPLA2 selective inhibitor bromoenol lactone (BEL). Arachidonic acid and prostaglandin I2 (PGI2) release were also increased in tryptase and thrombin stimulated cells and inhibited by BEL pretreatment. Pretreating the endothelial cells with AACOCF3 a cytosolic PLA2 inhibitor did not inhibit tryptase or thrombin induced arachidonic acid and PGI2 release. In addition thrombin and tryptase also increased HMVEC-L platelet activating factor (PAF) production that significantly contributes to the recruitment and initial adherence of polymorphonuclear neutrophils (PMN) to the endothelium. Tryptase or thrombin stimulated increase in PMN adherence to the endothelium was inhibited by pretreatment of HMVEC-L with BEL or pretreatment of PMN with CV3988, a PAF receptor specific antagonist. Collectively, these data support our hypothesis that iPLA2 activity is responsible for membrane phospholipid hydrolysis in response to tryptase or thrombin stimulation in HMVEC-L. Therefore selective inhibition of iPLA2 may be a pharmacological target to inhibit the early inflammation in pulmonary vasculature that occurs as a consequence of mast cell degranulation or acute lung injury.

Tryptase activates calcium-independent phospholipase A2and releases PGE2in airway epithelial cells

Human small airway epithelial cells (HSAEC) form the boundary between the external environmental allergens and the internal lung milieu. Mast cells are present in human lung tissue interspersed within the pulmonary epithelium and can secrete a host of pre- and newly formed mediators from their granules, which may propagate small airway inflammation. In this study, tryptase stimulation of HSAEC increased membrane-associated, calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) activity, resulting in increased arachidonic acid and PGE(2) release. These responses were inhibited by pretreating HSAEC with the iPLA(2)-selective inhibitor bromoenol lactone. The tryptase-stimulated PGE(2) production was inhibited by treating HSAEC with the cyclooxygenase (COX)-1-selective inhibitor SC-560 and the nonselective COX inhibitor aspirin but not by the COX-2-selective inhibitor CAY10404, indicating that the early release of arachidonic acid is metabolized by constitutive COX-1 to form PGE(2) in tryptase-stimulated HSAEC. Additionally, platelet-activating factor production and neutrophil adherence to tryptase-stimulated HSAEC was also increased. This complex response can set up a cascade of inflammatory mediator production in small airways. We speculate that selective inhibition of iPLA(2)gamma-mediated phospholipid hydrolysis may prove beneficial in inflammatory airway diseases.

Potential mechanism for recruitment and migration of CD133 positive cells to areas of vascular inflammation

Objective Mast cells are found in large numbers in atherosclerotic plaques. The present study was conducted to determine whether tryptase stimulation of human coronary artery endothelial cells (HCAEC) would lead to an increase in transmigration of CD133 positive cells (CD133+). In vitro these cells can differentiate into mast cells under the influence of specific cytokines and growth factors. Methods and Results CD133+ cells were isolated from umbilical cord blood. They express mRNA for several adhesion molecules that are also utilized in neutrophil migration and can migrate across an HCAEC monolayer. Migration increased significantly when HCAEC were stimulated with tryptase and decreased when CD133+ cells were pretreated with CV3988, a platelet activating factor receptor (PTAFR) antagonist. Following long-term cell culture, these cells stained positively for the presence of tryptase, a mast cell enzyme. Conclusion CD133+ cells can be utilized as a mast cell precursor population. The transendothelial migration is facilitated by the presence of tryptase and may utilize the PAF/PTAFR interaction in a manner similar to that involved in neutrophil transmigration. Following transmigration, a subset of these progenitor cells may mature into mast cells in the subendothelial space and play a role in propagation of the inflammatory process in atherosclerosis.

Muscle Cell Proliferation During Muscle Healing Can Be Stimulated By Mast Cell

Mature skeletal muscle can regenerate because myoblasts persist in adult muscle in the form of satellite and stem cells. In injured muscles, resolution of inflammation leads to the secretion of mitogenic and growth factors activating these cells which proliferate to repair muscle. Mast cells were shown to contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase, a protease secreted by activated mast cells. Tryptase binds to a protease-activated receptor (PAR-2), which induces the expression of cyclo-oxygenase-2 (COX-2) and the production of cyclopentenone prostaglandins, which interact with nuclear receptors and activate cell proliferation. PURPOSE: To evaluate if mast cell tryptase can stimulate skeletal muscle cell proliferation and determine if this effect is dependent on PAR-2 receptors. METHODS: L6 muscle cells were first cultured in medium containing 10% serum and then submitted to proliferation assays with various tryptase concentrations in 1% serum. Control group received medium only. Cell number was determined with CellTiter assay 48h post-seeding. To show that the effect of tryptase on proliferation was related to PAR-2 activation, L6 cells were incubated with the synthetic PAR-2 agonist SLIGRL and control wells with the reverse sequence peptide. Western blot analyses were used to study the impact of tryptase stimulation and cell detachment procedure on COX-2 expression in L6 cells. RESULTS: Cell proliferation was significantly increased by tryptase when compared to control (43.6 ± 2.8%, p<0.05) and in a dose-dependent manner. The synthetic agonist SLIGRL was less potent but also stimulated proliferation (13.3 ± 2.7%), confirming the implication of PAR-2 in this signaling cascade. Our results showed that COX-2 expression was increased several folds in these experiments but this effect was likely related to the cell detachment procedure per se rather than being induced by tryptase; no correlation was found with the increase in proliferation observed. CONCLUSION: Tryptase can accelerate proliferation of L6 muscle cells in vitro and this effect is dependent on the activation of PAR-2. The implication of COX-2 in this mechanism has yet to be confirmed and is presently under investigation. Supported by grants from NSERC and CIHR.

Loss of prostaglandin E2 release from immortalized urothelial cells obtained from interstitial cystitis patient bladders

Interstitial cystitis (IC) is associated with increased activated mast cell numbers in the bladder and impairment of the barrier function of the urothelium. We stimulated immortalized urothelial cells derived from the inflamed region of IC bladders (SR22A or SM28 abn) or from healthy bladders (PD07i or PD08i) with tryptase and measured phospholipase A(2) (PLA(2)) activity and the resultant release of arachidonic acid and prostaglandin E(2) (PGE(2)). Tryptase stimulation of either PD07i or SR22A resulted in similar increases in PLA(2) activity and arachidonic acid release. However, tryptase stimulation of SR22A and SM28 abn did not result in a significant increase in PGE(2) release compared with the increase in PGE(2) release from tryptase-stimulated PD07i and PD08i cells. Expression of mRNA for cyclooxygenase-2 and PGE synthase was lower and mRNA for 15-hydroxyprostaglandin dehydrogenase was higher in SR22A compared with PD07i, suggesting that both decreased synthesis and increased metabolism are responsible for the lack of a PGE(2) response in tryptase-stimulated SR22A cells. Since PGE(2) is a cytoprotective eicosanoid, the failure to produce this metabolite in cells isolated from the IC bladder may represent an increased susceptibility to damage by proinfammatory stimuli.

Transendothelial migration of CD133+ hematopoietic progenitor cells isolated from human umbilical cord blood

Transmigration of hematopoietic progenitor cells across the vascular endothelium and subsequent maturation in the tissue helps to maintain the pool of resident mast cells. We determined the potential of CD133+ cells isolated from umbilical cord blood to migrate across an endothelial cell monolayer. CD133+ cells possess mRNA for several adhesion molecules that are critical in the process of neutrophil transmigration. Stem cell factor &amp; its receptor CD117 are required for homing &amp; differentiation of mast cells in the tissue. We detected the presence of CD34, CD117, CD29, &amp; CD18 on the surface of CD133+ cells using flow cytometry. Tryptase stimulation leads to increased endothelial cell platelet activating factor (PAF) production that may facilitate the interaction between the endothelium &amp; the PAF receptor (PTAFR) on circulating CD133+ cells. Treatment with the PTAFR antagonist CV3988 significantly reduced the percentage of transmigrating CD133+ cells across a tryptase stimulated endothelial monolayer. This suggests that CD133+ cell migration depends on the PAF/PTAFR interaction in a manner similar to that involved in neutrophil transmigration. Following transmigration there is the potential for a subset of these cells, in the presence of SCF &amp; IL‐6, to mature into tissue mast cells.This work was supported by AHA Heartland Affiliate Grant # 0610118Z.

Impaired prostaglandin E2 (PGE2) production in urothelial cells from an interstitial cystitis patient

SR22 cells are from the inflamed area of the bladder of a patient with interstitial cystitis (IC), a disease characterized by increased activated mast cells in the bladder wall and compromised urothelial function. Since the urinary content of tryptase is increased in IC, this study determined whether there was impairment of prostaglandin E2 (PGE2) release from SR22 cells in response to tryptase. Responses in SR22 cells were compared to responses observed in PD07i cells derived from a healthy bladder. Interestingly, although PAR‐2 density was significantly lower in SR22 cells, tryptase stimulation of either SR22 or PD07i resulted in a similar increase in phospholipase A2 activity and arachidonic acid release. However, SR22 cells did not show any increase in PGE2 release in response to tryptase, whereas there was a significant increase in PGE2 release from tryptase‐stimulated PD07i. The levels of COX‐1 and COX‐2 mRNA expression were equivalent, but mRNA expression of PGE synthase was considerably lower and of hydroxyprostaglandin dehydrogenase was considerably higher in SR22 compared to PD07i, suggesting that both decreased production and increased metabolism could responsible for the lack of PGE2 response. Since PGE2 is cytoprotective, these data may explain why IC is associated with impaired ability to resist cell damage and warrants further investigation.Supported by DK66119 (JM)

Prostaglandin production in human coronary artery endothelial cells is modulated differentially by selective phospholipase A 2 inhibitors

Atherosclerotic plaque formation is a dynamic process involving repeated injury and inflammation of the endothelium. We have demonstrated previously that thrombin and tryptase stimulation of human coronary artery endothelial cells (HCAEC) leads to increased phospholipase A 2 (PLA 2) activity and generation of membrane phospholipid derived inflammatory metabolites, including eicosanoids and platelet activating factor. Thus, our hypothesis is that selective PLA 2 inhibitors have therapeutic potential as anti-inflammatory agents. Stimulation of confluent HCAEC monolayers with thrombin or tryptase resulted in a concentration and time-dependent increase in both prostaglandin E 2 (PGE 2) and prostacyclin (PGI 2) production. Pretreatment with PX-18 to inhibit secretory PLA 2 or BEL to inhibit calcium-independent PLA 2 prior to thrombin or tryptase stimulation resulted in a significant inhibition of both PGI 2 and PGE 2 release. However, pretreatment with methyl arachidonyl fluorophosphonate (MAFP), a widely used inhibitor of cytosolic PLA 2 isoforms, resulted in a significant potentiation of both thrombin and tryptase stimulated PGI 2 and PGE 2 release as a consequence of increased free arachidonic acid production. We conclude that the use of selective PLA 2 inhibitors may be of therapeutic benefit in the development and progression of atherosclerosis, however, the development of such an agent requires rigorous screening.

Mast cell tryptase may play a protective role in early inflammation in human small airway epithelial cells

Small airway epithelial cells are the primary line of defense against inhaled environmental pathogens. Mast cell numbers are increased in asthmatic airways and release of mast cell mediators may regulate early inflammation. This study investigated the activation of calcium-independent phospholipase A2 (iPLA2) in response to tryptase stimulation of human small airways epithelial cells (HSAEC). Our results demonstrate that by 10 minutes tryptase stimulation lead to an increase in iPLA2 activity (2.5 ± 0.2 to 4.8 ± 0.18 nmol/mg protein/min, p<0.01, n=6)) and a subsequent increase in the release of arachidonic acid (0.6 ± 0.1 to 4.0 ± 0.1 %, p<0.01, n=6) and prostaglandin E2 (PGE2) (2107 ± 58 to 5344 ± 48 pg/mg protein, p<0.01, n=6). The increased iPLA2 activity was completely inhibited by pretreating the cells with bromoenol lactone (BEL, a selective iPLA2 inhibitor). BEL also inhibited the tryptase-induced arachidonic acid and PGE2 release from HSAEC. Previous studies have demonstrated that PGE2 can be beneficial in endothelial repair and thus our data suggest a protective action for mast cell degranulation. This work was supported by AHA Heartland Affiliate Grant# 0610118Z.

The Therapeutic Potential of PhospholipaseA2 Inhibitors in Cardiovascular Disease

Leukocyte recruitment and the expression of pro-inflammatory cytokines are prevalent characteristics of early atherogenesis. Recently, several inflammatory mediators have been linked to atheroma formation and inflammatory pathways have been shown to promote thrombosis. The discovery of mast cells, activated T lymphocytes and macrophages in atherosclerotic lesions, the detection of human leukocyte antigen class II expression, and the finding of local secretion of several cytokines all suggest the involvement of immune and inflammatory mechanisms in the pathogenesis of atherosclerosis. Recent research suggests activation of protease activated receptors (PAR) on the surface of endothelial cells may play a role in general mechanisms of inflammation. In previous studies, our laboratory has demonstrated that thrombin (which activates PAR-1) and tryptase (which activates PAR-2) stimulation of endothelial cells results in activation of calcium-independent phospholipase A(2) (iPLA(2)). iPLA(2) plays a critical role in the synthesis of membrane phospholipid-derived inflammatory mediators such as arachidonic acid, platelet activating factor (PAF), and prostaglandins, all demonstrated to be central in both the initiation and propagation of the inflammatory response. Activation of iPLA(2) results in release of choline lysophospholipids from endothelial cells, these metabolites may contribute to the initiation of ventricular arrhythmias following myocardial ischemia as a direct result of incorporation into the myocyte sarcolemma. This biochemical event represents a direct link between occlusion of a coronary vessel and the nearly immediate initiation of arrhythmogenesis often seen in myocardial ischemia.

Protease activation of calcium-independent phospholipase A 2 leads to neutrophil recruitment to coronary artery endothelial cells

Introduction Thrombin or tryptase cleavage of protease-activated receptors (PAR) on human coronary artery endothelial cells (HCAEC) results in activation of a membrane-associated, calcium-independent phospholipase A 2 (iPLA 2) that selectively hydrolyzes plasmalogen phospholipids. Atherosclerotic plaque rupture results in a coronary ischemic event in which HCAEC in the ischemic area would be exposed to increased thrombin concentrations in addition to tryptase released by activated mast cells present in the plaque. Materials and methods HCAEC were stimulated with thrombin or tryptase in the absence or presence of bromoenol lactone (BEL), a selective iPLA 2 inhibitor, and iPLA 2 activation, accumulation of biologically active membrane phospholipid-derived metabolites, upregulation of cell surface P-selectin expression and neutrophil adherence were measured. Results HCAEC exposed to thrombin or tryptase stimulation demonstrated an increase in iPLA 2 activity and arachidonic acid release. Additionally, stimulated HCAEC demonstrated increased platelet-activating factor (PAF) production and cell surface P-selectin expression, resulting in increased adhesion of neutrophils to HCAEC monolayers. Pretreatment with bromoenol lactone to inhibit iPLA 2, blocked membrane phospholipid-derived metabolite production, increased cell surface P-selectin expression and neutrophil adherence. Conclusions The similar biochemical and cellular responses in HCAEC exposed to thrombin or tryptase stimulation suggest that the cleavage of two separate PAR serve to extend the range of proteases to which the cells respond rather than resulting in separate intracellular events. This suggests that in conditions such as thrombosis and atherosclerosis that multiple mechanisms can activate the inflammatory response.