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Letters to the Editor |
1 Istituto Istologia e Analisi Laboratorio2
Istituto Scienze Morfologiche3
Centro Citometria e Citomorfologia Università Studi "Carlo Bo", Urbino (PU), Italy
4 Istituti Ortopedici Rizzoli-Consiglio Nazionale delle Ricerche (ITOI-CNR), Bologna, Italy
aAddress correspondence to this author at: Istituto Istologia e Analisi Laboratorio, Via E. Zeppi, snc, Università Studi "Carlo Bo", 61029 Urbino (PU), Italy. Fax 39-0722-322370; e-mail f.mannello{at}uniurb.it.
To the Editor:
Matrix metalloproteinases (MMPs) are zinc/calcium-dependent effectors/mediators of physiologic and pathologic reproductive processes (1); MMPs may alter fetal homeostasis and be involved in pathologic syndromes of pregnancy (2). No information is available on MMPs circulating in healthy umbilical cord plasma (UCP). To characterize the effect(s) of umbilical cord blood (UCB) sampling and to identify possible cellular source(s) of MMP-2 (EC 3.4.24.24) and MMP-9 (EC 3.4.24.35) circulating in UCB, we investigated their concentrations, biochemical characteristics, and isoform distributions by immunoassay and gelatin zymography.
We collected UCB (n = 20) immediately after delivery from women with uncomplicated healthy pregnancies; mean (SD) gestation age at delivery was 38 (2) weeks. We used plastic tubes containing lithium heparin, dipotassium EDTA, or buffered sodium citrate (9NC); sera were obtained from clot-activator-gel tubes (SST; Becton Dickinson). After centrifugation at 1000g for 15 min, supernatants were collected. Leukocyte subpopulations were tested for their subsets and gelatinase content after sedimentation on Lympholyte® gradient (Cedarlane) (3).
Umbilical cord vein sections (n = 5) were excised, separated from the Wharton’s jelly, homogenized, and centrifuged at 8000g for 30 min; the supernatants were then analyzed (4).
Gelatinase calibrators were prepared from healthy capillary peripheral blood (3). MMP molecular isoforms were analyzed under nonreducing conditions on gelatin-copolymerized polyacrylamide gels (5). Western blotting was performed with anti-human MMP-2 and -9 monoclonal antibodies (clones 75-7F7 and GE-213, respectively; Calbiochem). We measured UCP gelatinases by BiotrakTM MMP-2 and MMP-9 assays (Amersham Pharmacia) (5). Mean (SE) values of three independent experiments performed in duplicate were calculated; statistical analysis was performed with the Student t-test and Mann–Whitney U-test. P values <0.05 were considered statistically significant.
Mean (SE) UCP gelatinase concentrations differed among the collection methods, with the highest concentrations in SST tubes [192 (18) and 46 (4) µg/L for MMP-2 and -9, respectively] and the lowest concentrations obtained from 9NC tubes [102 (8) and 5 (1) µg/L for MMP-2 and -9, respectively]. MMP-2 concentrations appeared unaffected by anticoagulants, and MMP-2 was constitutively present in all UCB samples at 124 (11) µg/L.
Gelatin zymography of whole UCB revealed a constant band at Mr 72 000 and additional isoforms at Mr 92 000, 130 000, and 225 000; these were biochemically recognized and immunologically identified as proMMP-2 and proMMP-9, respectively (Fig. 1A
). UCB gelatinases are MMPs circulating as latent proenzymes in zymogenic activatable isoforms.
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MMP patterns in UCP varied with different anticoagulants: samples collected in 9NC tubes showed mainly proMMP-2 at Mr 72 000 [102 (8) µg/L] and little MMP-9 [5 (1) µg/L], whereas UCP from samples collected in dipotassium EDTA and lithium heparin, as well as serum, had additional proMMP-9 isoforms of Mr 92 000, 130 000, and 225 000 [28 (3) µg/L; Fig. 1B
]. MMP-9 isoforms were significantly higher in serum than in plasma [46 (4) vs 14 (2) µg/L, respectively; P <0.01]; platelet aggregation during clotting may produce these differences (3). Anticoagulants added to the zymography buffer (concentrations equivalent to amounts in VacutainerTM Tubes) almost completely inhibited the UCP gelatinases only in samples collected in dipotassium EDTA [residual activity, 4 (1)%; P <0.01; Fig. 1A
, lane 3], whereas other anticoagulants did not affect MMP activity (measured activity, 97–104% of the expected).
Cytometric analyses of UCB buffy coats collected with different anticoagulants revealed no differences in leukocyte subset recovery. Leukocyte subpopulations zymographically produced only MMP-9 isoforms [55 (7) µg/L; Fig. 1B
, lanes 5 and 6]. UCB leukocytes thus apparently are the source of UCP MMP-9.
Umbilical cord vein extracts contained gelatinases mainly at Mr 72 000 [71 (5) µg/L] and negligible MMP-9 activity, unaltered by anticoagulants except for dipotassium EDTA [residual activity, 3 (0.5)%]. Western blotting and biochemical analyses identified the gelatinase as latent activatable proMMP-2 (Fig. 1C
).
We conclude that (a) anticoagulants alter UCB MMPs: unlike buffered sodium citrate, lithium heparin and dipotassium EDTA may induce the "release" of MMPs from UCB leukocytes, explaining the different UCP MMP profiles depending on the anticoagulant used (3); (b) umbilical cord leukocytes produce/secrete only MMP-9; (c) umbilical cord vein tissue is a source of the MMP-2 circulating in UCP; and (d) UCB MMPs may represent useful tools to identify pregnancy-associated syndromes (1)(2).
To avoid preanalytical misinterpretations and to optimize the diagnostic validity of UCB MMPs as biomarkers, we recommend the use of buffered sodium citrate, whereas use of serum or lithium-heparin and dipotassium-EDTA plasma, in which both MMP content and zymography are affected, should be avoided.
References
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