Inflamm. Res. (2015) 64:497–500 DOI 10.1007/s00011-015-0830-0

Inflammation Research

ORIGINAL RESEARCH PAPER

Diverse activity of human secretory phospholipases A2 on the migration of human vascular smooth muscle cells W. Pruzanski1,3 • J. Kopilov1 • A. Kuksis2

Received: 22 April 2015 / Accepted: 29 April 2015 / Published online: 22 May 2015  Springer Basel 2015

Abstract Objective Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC). Material We investigated the impact of sPLA2 IIA, V, and X and of oleic acid, linoleic acid and lysophosphatidylcholine on the migration of human VSMC. Methods Recombinant human sPLA2’s and Boyden’s chamber method were applied. Results sPLA2, IIA but not V or X enhanced migration of VSMC in a dose/time dependent manner. Oleic and linoleic acids, and lysophosphatidylcholine markedly enhanced migration. Conclusions These results imply that sPLA2 IIA, which is known to be present in the arterial wall in the vicinity of VSMC, as well as products of lipid hydrolysis induced by sPLA2, enhance the migration of VSMC, and thus may contribute to atherogenic process. Keywords Phospholipase A2  Vascular smooth muscle cells  Migration

Introduction The role of vascular smooth muscle cells (VSMC) in atherogenesis has been extensively studied [1–6]. Proliferation of VSMC has been implicated in the atherogenetic process [2, 3]. We have shown that mitogenic activity of human VSMC is enhanced to various extents by a group of human secretory phospholipases A2, (sPLA2), IIA, V and X, [7–9]. sPLA2 has been shown to induce hydrolysis of human lipoproteins, releasing a variety of proinflammatory agents, which per se may play an important role in atherogenesis [7, 9, 10]. There is little information regarding the role of sPLA2 in migration of various cells. The first study showing that human pancreatic sPLA2 and synovial fluid sPLA2 induce migration of vascular epithelial cells was reported by Rizzo et al. [11]. Until now, there have been no comparative reports on the role of recombinant human groups IIA, V and X sPLA2 on migration of human VSMC. We report that sPLA2 IIA used as an attractant is inducing migration of VSMC, whereas sPLA2 V and X are inactive. It shows the diversity in the activity of various secretory sPLA2’s and the differences in the effect on the migration versus mitogenesis of the VSMC.

Responsible editor: John Di Battista. & W. Pruzanski [email protected] 1

Department of Medicine, University of Toronto, Toronto, Canada

2

Banting and Best Research Institute, University of Toronto, Toronto, Canada

3

Rosedale Medical Centre, 600 Sherbourne Street, Suite 602, Toronto, ON M4X 1W4, Canada

Materials and methods Human vascular smooth muscle cells (VSMC) of male and female origin were grown and rendered quiescent as described in detail [7]. Recombinant human group IIA sPLA2 (sPLA2 gr IIA) was prepared by Dr. J. Browning, Biogen, Cambridge, Massachusetts, USA. Recombinant human group V sPLA2’s (sPLA2 gr V) was prepared by Dr. Kwang Pyo Kim, University of Chicago, IL, USA.

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Recombinant human group X PLA2 (sPLA2 gr X) was prepared by Dr. G. Lambeau, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moleculaire et Cellulaire, Sophia Antipolis, France. Oleic and linoleic acids, and lysophosphatidylcholine were obtained from Sigma-Aldrich, Oakville, ON Canada. To check concentration of lysophosphatidylcholine in quiescent VSMC (4 9 105 cells/well), the cells were incubated with sPLA2’s (1 lg/ml) for 24 h, washed and sonicated. Lipids were extracted by Bligh–Dyer method and tested for PtdCho and lysoPtdCho. Two experiments in triplicate gave identical results. Control VSMC and the cells exposed to sPLA2’s IIA and X contained PtdCho between 84.0 and 88.2 mol %, and lysoPtdCho between 5.7 and 8.2 mol %. Cells exposed to sPLA2’s gr V showed reduction in PtdCho to 66.0 mol % and increase in lysoPtdCho to 15.7 mol %. Supernatants did not contain lysoPtdCho or PtdCho. Migration of VSMC was assayed by the modified Boyden chamber method [12] using FALCON 24-well Insert System with 8-lm polyethylene terephthalate (PET) transparent membranes. Sub-confluent quiescent VSMC were trypsinized and suspended in 231 Medium with Vitamin C, ITS and PSA. 8 9 104 cells in 150 ll were placed in the upper compartment of the chamber and 750 ll of the same medium with or without various agents was placed in the lower compartment. The chambers were incubated for various periods of time at 37 C and 5 % CO2 to allow cell migration. After the incubation, the non-migrated cells were removed from the upper side of the filter with a cotton applicator. The filters were fixed with 70 % ethanol and stained with hematoxilin (Harris Hematoxilin Solution Modified, Sigma Diagnostic, St. Louis, MO, USA). Migration was quantified by counting cells that migrated to the lower surface of the filter. Four or more random fields in each filter were examined. Migration was expressed as the mean ± SD (SEM) of cells. Three experiments were done with each sPLA2 and with each concentration. Each experiment was done in duplicate and often triplicate. Migration of VSMC through the membranes induced by platelet-derived growth factor homodimer bb (PDGF-bb) 10 ng/ml (Sigma-Aldrich, Canada) was used as a positive control attractant.

Results Control 4 and 8 h migrations of human VSMC to the medium and migration towards the positive attractant PDGF-bb 10 ng/ml are shown in Tables 1 and 2. PDGF-bb attracted the cells proportionately to the cell number in the upper chamber, concentration of the factor in the lower

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chamber and the duration of migration (not shown). For example, 10 ng/ml of PDGF-bb attracted 141 ± 10.9 cells, whereas 20 ng/ml attracted 260 ± 12 cells (n = 2). To define the role of sPLA2’s as attractants in cell migration, various concentrations of the enzymes were placed in the lower chamber of the modified Boyden chambers. sPLA2’s IIA 50 ng/ml induced significantly higher migration than control (p \ 0.01) whereas, sPLA2’s V and X were inactive (Table 1). The induction of migration by sPLA2’s IIA was both concentration and the duration of migration dependent (Table 1). We also checked three products of HDL hydrolysis, namely lysophosphatidylcholine (LysoPC), oleic acid (OA) and linoleic acid (LA) for the impact on cell migration. It was found that OA and LA (10 micromol) enhanced migration as compared to the control (p \ 0.01) (Table 2). The activity of OA and LA was dose dependent up to the tested concentration of 50 micromol. LysoPC in concentrations from 10 to 25 micromol increased migration as well (Table 2). The concentration above 25 micromol was toxic to the cells.

Discussion Atherogenesis is a complex process involving a variety of enzymatic and immunological factors [1, 13–15]. Proliferation and migration of the vascular smooth muscle cells (VSMC) have been recognized as an important inducer of atherogenesis. We reported that human secretory phospholipases A2, groups IIA, V and X induce either directly and/or indirectly through the hydrolysis of lipids, mitogenesis of human VSMC [7–9]. There is still paucity of information about the factors inducing migration of VSMC. It was reported that vascular endothelial growth factor, (VEGF) which is overexpressed in the intima of atherosclerotic human coronary arteries activated migration, but not proliferation of VSMC [16]. Reactive oxygen species and NF-Jb were required for VEGF-mediated migration [17]. The same group also reported that TNF-a induces VSMC migration. Migration was accompanied by activation of several signaling pathways, including NF-Jb. Suppression of NF-Jb inhibited TNF-a mediated migration [18]. Several other factors such as apolipoprotein [19] or activated protein tyrosine kinase, [20] activated migration as well. There is almost no information about the impact of sPLA2’s on VSMC migration. Kanemasa et al. [21] reported in 1992 that pancreatic group I PLA2 activated migration of the rat embryonic aorta VSMC. Rizzo et al. reported in 2000 that human synovial fluid induced endothelial cells migration. Pretreatment of synovial fluid with pBPB, a known inhibitor of sPLA2, inhibited

Diverse activity of human secretory phospholipases A2…

499

Table 1 Migration of VSMC induced by sPLA2’s Duration of migration Agent

4h

p

b

8h

30.3 ± 4.5a

58.1 ± 6.4

159.0 ± 22.0

185.0 ± 29.0

\0.001

sPLA2 II A (25 ng/ml)

37.4 ± 5.0

72.0 ± 18.3

\0.05

sPLA2 II A (50 ng/ml)

62.1 ± 13.5

100.9 ± 9.8

sPLA2 V (50 ng/ml)

30.3 ± 4.9

65.8 ± 8.3

N/S

sPLA2 X (50 ng/ml)

32.9 ± 7.1

67.4 ± 7.5

N/S

Control PDGF (10 lg/ml)



\0.01

probable that interaction of sPLA2 with lipoproteins takes place in vivo and affects VSMC. Our study showed that recombinant sPLA2 gr IIA, but not V or X enhanced migration of VSMC in a dose-time related fashion. Oleic and linoleic acid, and lyso PC enhanced migration as well. These results show discordance in the effect of various secretory sPLA2’s on VSMC migration versus mitogenesis, and require further investigation of signaling mechanisms and inhibitory factors [29].

Mean ± SD of 3 experiments in duplicate a

Number of migrating cells

b

p compared to controls

References

Table 2 Migration of VSMC induced by hydrolysis products Agent

Number of migrating cells

pa

Control

33.5 ± 1.8

\0.001

PDGF-BB(10 ng/ll) Oleic acid

118.1 ± 6.2

10 lM

67.3 ± 3.4

\0.001

25 lM

105.9 ± 4.1

\0.001

50 lM

159.5 ± 6.0

\0.001

Linoleic acid 10 lM

93.2 ± 3.02

\0.001

25 lM

120.1 ± 6.8

\0.001

50 lM

167.5 ± 8.6

\0.001

Lysophosphatidylcholine 10 lM

53.7 ± 2.2

\0.001

25 lM

76.3 ± 2.2

\0.001

50 lM

Toxic

*Mean ± SD of 6 experiments in duplicate; Time of migration: 4 h a

p compared to control

migration. No pure sPLA2 was used in their experiments [11], however, we reported that synovial fluid contains sPLA2 group IIA [22]. Rizzo et al. [11] also reported that arachidonic acid and lysophosphatidylcholine, known hydrolysis products of lipids by sPLA2’s, stimulated migration. The significance of the above studies is related to the fact that secretory phospholipases A2 are located in the arterial wall, more so in the atherosclerotic areas and that these enzymes are located in the vicinity of VSMC [3, 23– 28]. We and others reported that human sPLA2’s hydrolyse human lipoproteins releasing arachidonic, oleic and linoleic acids and other proinflammatory agents, and converting Pc into lysoPC [8, 10]. Since lipoproteins are located in the vascular wall, especially in the atherosclerotic areas, in the vicinity of sPLA2 and VSMC, it is highly

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Diverse activity of human secretory phospholipases A2 on the migration of human vascular smooth muscle cells.

Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC)...
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