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

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.

Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2.

We investigated the hydrolysis of the minor glycerophospholipids of human HDL(3), total HDL and LDL using human group IIA, V and X secretory phospholipases A(2) (sPLA(2)s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA(2)s, and to a lesser extent by group IIA sPLA(2), the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA(2)s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA(2)s, although the group V sPLA(2) attacked it more readily than group X sPLA(2) (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA(2) hydrolysis remained minimal at 10-15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA(2) at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA(2)s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA(2) digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA(2)s. Since these sPLA(2)s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function.

Differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups IIA, V and X secretory phospholipases A2.

Human groups IIA, V and X secretory phospholipases A2 (sPLA2s) were incubated with human HDL3, total HDL and LDL over a range of enzyme and substrate concentrations and exposure times. The residual phosphatidylcholines (PtdChos) were assayed by high performance liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The enzymes varied markedly in their rates of hydrolysis of the different molecular species and in the production of lysoPtdCho. The sPLA2s were compared at a concentration of 1 microg/ml and an incubation time of 4 h, when all three enzymes showed significant activity. The groups V and X sPLA2 were up to 20 times more reactive than group IIA sPLA2. Group X sPLA2 hydrolyzed arachidonate and linoleate containing species preferentially, while group V hydrolyzed the linoleates in preference to polyunsaturates. In all instances, the arachidonoyl and linoleoyl palmitates were hydrolyzed in preference to the corresponding stearates by group X sPLA2. The group IIA enzyme appeared to hydrolyze randomly all diacyl molecular species. The minor alkylacyl and alkenylacyl glycerophosphocholines (GroPChos) were poor substrates for groups V and X sPLA2s and these phospholipids tended to accumulate. The present study demonstrates a preferential release of arachidonate from plasma lipoprotein PtdCho by group X sPLA2, as well as a relative resistance of polyunsaturated PtdChos to hydrolysis by group V enzyme, which had not been previously documented. The use of lipoprotein PtdCho as substrate with LC/ESI-MS identification of hydrolyzed molecular species eliminates much of the uncertainty about sPLA2 specificity arising from past analyses of fatty acid release from unknown or ill-defined sources.

Mitogenic effect of lipoproteins on human vascular smooth muscle cells: the impact of hydrolysis by gr II A phospholipase A(2).

Multifactorial interaction among lipoproteins, vascular wall cells, and inflammatory mediators has been recognized as the basis of atherogenesis. In the arterial wall high-density lipoprotein (HDL) and human secretory phospholipase A(2) (sPLA(2)) colocalize with vascular smooth muscle cells and concentrate in the atherosclerotic lesions. It has been shown that gr IIA sPLA(2) hydrolyzes lipoproteins, altering their structure and releasing active agents such as lyso-phosphatidylcholine (PtdCho) and free fatty acids. We investigated the impact of normal HDL(3) (NHDL(3)), acute phase HDL(3) (APHDL(3)), and low-density lipoprotein (LDL), both unhydrolyzed and sPLA(2)-hydrolyzed, and some products of hydrolysis, such as lyso-PtdCho, oleic and linoleic acid, on [(3)H] thymidine incorporation by DNA of cultured human vascular smooth muscle cells (VSMC). NHDL(3) markedly enhanced mitogenic activity of VSMC in a dose- and time-dependent manner. Doubling of thymidine incorporation was usually achieved by 40 microg/ml of NHDL(3) after 4 hours of incubation. APHDL(3) had invariably a stronger inducing effect on the mitogenic activity than NHDL(3); 40 microg/ml more than tripled [(3)H] thymidine incorporation after 4 hours of incubation. NHDL(3) preincubated with human apo serum amyloid A apolipoprotein-induced higher mitogenic activity in VSMC than NHDL(3) alone. Hydrolysis of NHDL(3), APHDL(3), or LDL by gr IIA sPLA(2) markedly enhanced mitogenic activity of VSMC as compared with unhydrolyzed lipoproteins. sPLA(2) concentrations that can be found in atherosclerotic vascular walls markedly enhanced lipoprotein-induced mitogenic activity of VSMC. sPLA(2) per se did not affect thymidine incorporation and VSMC did not release sPLA(2) into the medium. There was no evidence for hydrolysis of the wall of VSMC by gr IIA sPLA(2). The presence of the products of hydrolysis of lipoproteins such as oleic and linoleic acids and lyso-PtdCho or their combinations with NHDL(3) explains in part markedly enhanced mitogenic activity of VSMC. It is conceivable that sPLA(2,) which is known to colocalize with lipoproteins in the vascular wall in the domain of VSMC, is capable of induction of the mitogenic activity in these cells in vivo and should be considered as a proatherogenic enzyme.