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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00642

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A functional overlap of plasminogen and MMPs regulates vascularization during placental development

Helene Solberg1, Julie Rinkenberger2, Keld Danø1, Zena Werb2 and Leif R. Lund1,*

1 Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, 2100 Copenhagen, Denmark
2 Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA



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  		Fig. 1. Expression of MT1-MMP and uPAR during mouse embryo implantation. Implantation sites (7.5 dpc and 8.5 dpc) and 12.5 dpc placentas from wild-type mice were analyzed by in situ hybridization for MT1-MMP mRNA (A,E,I,M) and immunohistochemistry for uPAR (C,G,K,O). As controls, a sense probe for MT1-MMP (B,F,J,N) and preimmune rabbit IgG (D,H,L,P), were included. At 7.5 (A) and 8.5 (E) dpc MT1-MMP signal was seen in cells bordering the remnants of the uterus lumen (white arrows), as well as in the undifferentiated decidua cells and the maternal vessels (thin red arrows). In addition, at 8.5 dpc a few cells present at the mesometrial side close to the embryo contained mRNA for MT1-MMP (curved red arrows). At later stages of placental development (12.5 dpc), MT1-MMP was still expressed by the undifferentiated decidual cells, as well as in the maternal vessels (thin red arrows). In addition, MT1-MMP mRNA was detected in cells lying in close proximity to spongiotrophoblast cells (bold red arrow) (I). At 7.5 dpc (C) and 8.5 dpc (G) uPAR immunoreactivity was detected in the decidual cells, as well as in the maternal vessels. Weak staining could also be seen in the trophoblast cells. At 12.5 dpc, placentas the remaining mesometrial decidua cells, as well as the giant trophoblast cells, were positive for uPAR immunoreactivity. uPAR staining was also seen in the maternal and embryonic vessels as well as in the spongiotrophoblast cells (K). The areas boxed in E-H are shown at higher magnification in MP, respectively. am, antimesometrial; d, decidua; e, embryo; ec, ectoplacental cone; l, labyrinth layer; m, mesometrial; s, spongiotrophoblast layer. Scale bars: 500 µm in A-L; 100 µm in M-P.

 


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  		Fig. 2. Effect of galardin treatment on 7.5 and 8.5 dpc implantation sites and 12.5 dpc placenta from wild-type and Plg-deficient mice. (A) Hematoxylin and Eosin stained sections of 7.5 and 8.5 dpc implantation sites and 12.5 dpc placentas from mock-treated wild-type mice (parts a,e,i), galardin-treated wild-type mice (parts b,f,j), mock-treated Plg-deficient mice (parts c,g,k) and galardin-treated Plg-deficient mice (parts d,h,l). Typical implantation sites from mock-treated wild-type mice and most of the galardin-treated wild-type and Plg-deficient mice had an elongated egg shape (parts a-c), whereas 60% of the implantation sites from the galardin-treated Plg-deficient mice were round (part d) (as they were in 33% of the mock-treated Plg-deficient mice and the galardin-treated wild-type mice). At 8.5 dpc, 50% of the embryos from galardin-treated Plg-deficient mice were runted and at a developmental stage resembling 7.5 dpc embryo from mock-treated wild-type mice (part h). Sections of 12.5 dpc placentas from Plg-deficient and wild-type mice treated with galardin revealed that the tissue was loosely associated and the decidua and spongiotrophoblast layers separated upon collection (arrows in parts j,l). Placentas from galardin-treated Plg-deficient mice had a less developed and thinner labyrinth layer than in mock- and galardin-treated wild-type and in Plg-deficient mice. Length of bars (in parts il) indicates the size of the labyrinth layer. (B) Histograms of the decidua length at 7.5 dpc±s.d. for each genotype. (C). Histograms of the labyrinth width at 12.5 dpc± s.d. for each genotype. am, antimesometrial; d, decidua; e, embryo; ec, ectoplacental cone; l, labyrinth layer; m, mesometrial; s, spongiotrophoblast layer. Scale bar: 500 µm.

 


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  		Fig. 3A. MMP9 expression, fibrin accumulation, and vascularization in implantation sites from galardin-treated wild-type and Plg-deficient mice. (A) At 7.5 dpc in situ hybridization for MMP9 mRNA showed expression in the giant trophoblast cells (black arrows). Few giant trophoblasts were observed in the ectoplacental cone in 7.5 dpc implantation sites from galardin-treated Plg-deficient mice (part d) compared with implantation sites from mock- and galardin-treated wild-type mice and from mock-treated Plg-deficient mice (parts a-c). Fibrin(ogen) was detected by immunohistochemical staining using rabbit anti-fibrin(ogen) (parts e-h). No difference in the fibrin(ogen) staining pattern was observed among implantation sites from the four groups of mice. Endothelial cells were detected by in situ hybridization for PECAM mRNA (parts i-l). A decrease in the vascularization was seen in implantation sites from some galardin-treated Plg-deficient mice (part l), when compared with implantation sites from mock- and galardin-treated wild-type mice and from mock-treated Plg-deficient mice (parts i-k) (see Table 2). The areas boxed in parts i and l are shown at higher magnification in parts m and n, respectively. am, antimesometrial; d, decidua; e, embryo; m, mesometrial. Scale bar: 500 µm in parts a-l; 50 µm in parts m,n.

 


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  		Fig. 3B. MMP9 expression, fibrin accumulation, and vascularization in implantation sites from galardin-treated wild-type and Plg-deficient mice. (B) Expression of MMP-9 at 8.5 dpc was detected by in situ hybridization (parts a-d) and was present in the giant trophoblast cells (black arrows) that are identified by their expression of PL-1 mRNA (red arrows) (parts e-h). The spatial distribution of the giant trophoblast cells in 8.5 dpc implantation sites from galardin-treated Plg-deficient mice (part d), was similar to that of mock-treated wild-type mice 7.5 dpc implantation sites (data not shown). Endothelial cells were detected by in situ hybridization for PECAM mRNA (parts i-l). The vascularization of the decidua in 8.5 dpc galardin-treated Plg-deficient mice (parts l,n) was like that found at 7.5 dpc mock-treated wild-type mice (Fig. 3A, parts i,m). The areas boxed in parts i and l are shown at higher magnification in parts m and n, respectively. am, antimesometrial; d, decidua; e, embryo; m, mesometrial. Scale bar: 500 µm in parts a-l; 100 µm in parts m,n.

 


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  		Fig. 4. Effect of galardin treatment on 12.5 dpc placentas from wild-type and galardin-treated Plg-deficient mice. No difference in the distribution of the spongiotrophoblast cells identified by 4311 expression is observed between placentas from galardin-treated Plg-deficient mice (D) and placentas from mock- or galardin-treated wild-type mice or mock-treated Plg-deficient mice (A-C). Trophoblast giant cells were identified by in situ hybridization for PL-1 mRNA (brightfield, E-H,M,P; darkfield, I-L). The spatial distributions of PL1-positive cells in the central part of the placenta are shown in higher magnification of the boxed area of placentas from galardin-treated Plg-deficient mice (H,P), or from mock-treated wild-type mice (E,M). In mock-treated wild-type mice (E,I,M), galardin-treated wild-type mice (F,J) and mock-treated Plg-deficient mice (G,K) PL-1 positive trophoblast giant cells are primarily found towards the periphery of the placenta, whereas in galardin-treated Plg-deficient mice (H,L,Q) PL1-positive trophoblast giant cells are also found in the central part of the placenta (black and blue arrows). In I-L, white arrows point towards the central part of the placenta. Immunohistochemical staining for the endothelial cell marker CD34 (black arrows) (N,Q) revealed that the labyrinth layer in 33% of the placentas from galardin-treated Plg-deficient mice was non-vascularized (Q). Immunohistochemical staining for fibrin(ogen) in placentas from mock-treated wild-type mice (O) or from galardin-treated Plg-deficient mice (R) showed large amounts of fibrin deposition surrounding the central arteries in the middle of the placentas (black arrows). No other differences in the amount of fibrin deposition were observed in placentas from galardin-treated Plg-deficient mice, when compared with placentas from the other three groups of mice (O; data not shown). The areas boxed in E and H are shown at higher magnification in M-O and P-R respectively to demonstrate the distribution of the three different markers. D, decidua; L, labyrinth layer; S, spongiotrophoblast layer. Scale bar: 500 µm in A-H; 100 µm in I-N.

 

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© The Company of Biologists Ltd 2003