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Angiogenesis in gestational vascular complications
Thrombosis Research 127 Suppl. 3 (2011) S64–S66
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Angiogenesis in gestational vascular complications Simcha Yagel* Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Centers, Mt. Scopus, Jerusalem, Israel
article info
abstract
Keywords: sFlt1-14 VEGF Heparanase Preeclampsia
Vascular endothelial growth factor (VEGF) is a key player in vasculogenesis and angiogenesis in the embryo, and essential in neovascularization in adults. Natural VEGF inhibitors such as soluble VEGF receptors, among them the soluble VEGF-trapping receptor Flt1 (sFlt1), participate in VEGF regulation. Decreased levels of VEGF and increased levels of sFlt1 have been implicated in the pathophysiology of preeclampsia. We discovered a soluble receptor, sFlt1-14, qualitatively different from sFlt1 and a potent VEGF inhibitor. It is generated in a cell type specific fashion, primarily in nonendothelial cells, most notably in vascular smooth muscle cells. We showed that increased production of soluble VEGF receptors in pregnancy is owing to expression of sFlt1-14, from the end of the first trimester to term. This expression is markedly elevated in preeclampsia, and is expressed chiefly by syncitial knots. In subsequent studies we found that sFlt1 is a strong heparin binder: this capability enables it to stay attached to blood vessels and to the placenta. Ex vivo, sFlt1 can be heparin displaced to medium from aortic segments and placental villi. In vivo, pregnant women treated with the low molecular weight heparin (LMWH) have elevated sFlt1 levels in their circulations. Interestingly, LMWH raised VEGF levels over and above the increase in sFlt1 levels in these patients. Heparanaseoverexpressing non-pregnant as well as pregnant transgenic mice present elevated levels of sFlt1 in their circulations. Ex vivo prevention of heparanase maturation through cathepsin L inhibition, or targeting heparanase directly with a neutralizing antibody, both resulted in a marked reduction in sFlt1 secretion to medium of normal and preeclamptic placental explants. These findings uncover a new level of regulation that controls sFlt1 bio-distribution, and directs it to function in the vicinity of its producing cell. Heparanase or LMWH has the ability to liberate sFlt1 from its retention, so this process may be a potential target for preeclampsia treatment. © 2011 Elsevier Ltd. All rights reserved.
Vascular endothelial growth factor (VEGF) is a key player in vasculogenesis and angiogenesis in the embryo, and essential in neovascularization in adults. It plays additional roles in nonvascular development and in homeostasis. Owing to its potent effect on blood vessels VEGF is normally kept in precise balance in the body. Natural VEGF inhibitors such as soluble VEGF receptors, among them the soluble VEGF-trapping receptor Flt1 (sFlt1), participate in VEGF regulation. Decreased levels of VEGF and increased levels of sFlt1 have been implicated in the pathophysiology of preeclampsia. However, there is still controversy surrounding the roles of sFlt1 and VEGF in the development of preeclampsia [1–7]. We began our investigations into the possible connection of sFlt1 to preeclampsia from two cases of dichorionic biamniotic twins managed in our department, which served to test the concept of sFlt1 involvement in PE. The first case was a G1P0, carrying one healthy fetus and one with severe intra-uterine growth restriction (IUGR) with absence * Correspondence: Prof. Simcha Yagel. Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Centers, Jerusalem, Israel. Tel.: +972 2 5844111; fax: +972 2 5815370. E-mail address: [email protected] (S. Yagel). 0049-3848 /$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
of diastolic flow in the umbilical artery. The mother developed severe preeclampsia, and in light of her worsening clinical state was delivered by emergency cesarean. In the maternal serum in the course of her pregnancy we discovered high levels of sFlt1 and low levels of VEGF for gestational age. Post-partum analysis of the placentas revealed multiple syncitial knots in the IUGR placenta, and high levels of sFlt1 in his umbilical cord blood, and relatively low levels of VEGF, while these indices were normal in the cotwin. The IUGR placenta revealed high levels of sFlt1 by in situ hybridization. The second case was a woman in her first pregnancy, with one healthy fetus and one with severe IUGR and absent diastolic flow in the umbilical artery. She developed severe preeclampsia at 29 weeks gestation. We found excessive levels of sFlt1 in the maternal serum. The IUGR fetus was also diagnosed with gross abnormality of the cerebellum. In light of these findings selective feticide was performed on the IUGR twin. Symptoms of preeclampsia subsequently disappeared, including maternal hypertension and proteinuria, and a dramatic decrease in serum sFlt1 levels.
S. Yagel / Thrombosis Research 127 (2011) S64–S66
Fig. 1. sFlt1-14 is a splicing variant of VEGF receptor 1 (Flt-1).
These two cases demonstrate the importance of sFlt1 in the pathophysiology of preeclampsia, as has also been noted by others. We aimed to characterize sFlt1 on the molecular level, and to determine its source. Previously the source of sFlt1 in maternal serum was undetermined, whether it was secreted by the placenta, or whether as part of the pathology of preeclampsia it was produced by other maternal cells. We demonstrated the existence of a splicing variant of VEGF receptor 1 (Flt-1): sFlt1-14 (Fig. 1). We showed that this variant of a natural VEGF inhibitor, possibly protecting non-endothelial cells from the effects of VEGF, is implicated in the development of preeclampsia. sFlt1-14 strongly inhibits VEGF signaling. We showed that recombinant sFlt1 and sFlt1-14 proteins were produced in HeLa cells transfected with the respective expression vector. Western blotting detected soluble Flt1 receptors in cells or released into medium using antibody directed against an extracellular epitope. We exposed porcine aortic ECs that were engineered to stably express Flk1, to VEGF. Flk1 phosphorylation served as a measure of the extent of VEGF signaling. We observed an inhibitory effect of preincubation of VEGF with sFlt1 or sFlt1-14. SFlt1-14 differs from sFlt1 in the source cells that produce it, and it functions as a potent VEGF inhibitor. It is generated primarily in non-endothelial cells. In vascular smooth muscle cells (VSMC) Flt1 mRNA is converted to sFlt1-14, while endothelial cells of the same vessels express sFlt1. We showed that sFlt1-14 expression by VSMC’s is upregulated on coculture with endothelial cells or by direct exposure to VEGF. The increase in soluble VEGF receptors in pregnancy stems from induced expression of placental sFlt1-14, from the end of the first trimester to term. In women with preeclampsia sFlt1-14 expression is dramatically increased. The source of this excess seems to be the syncitial knots. These knots are clusters of degenerative syncitial trophoblast in the placenta. sFlt1-14 was identified by in situ hybridization in syncitial knots (Fig. 2). Expression of sFlt1 was lower in healthy syncytiotrophoblast in comparison to knots from PE placentas [8]. No sFlt1-14 was detected in placental ECs [9,10].
S65
We have preliminary data showing that sFlt1 is secreted to the maternal circulation inside microparticles produced by syncitiotrophoblasts. Another question arose, that while sFlt1 needs to be retained by VSMCs to grant them VEGF protection, it can be found far from its placental producing cells. What enables sFlt1 to be retained by or released from its producing cell? VEGF R2 is capable of heparan binding, which modulates its signaling. Flt1 lg-like domain 4 is capable of heparan binding, but this capability does not modulate its signaling. Working from the hypothesis that this heparan binding capability operates to enable sFlt1 local retention, we aimed to show this in a physiological context. Heparan sulfate (HS) proteoglycan binds to sFlt1 producing cells. Both sFlt1 isoforms are excellent heparan binders, and sFlt1’s ability to strongly bind heparan might affect its bioavailability. Heparin can compete with HS for sFlt1 binding. To demonstrate this, we utilized the displaceable sFlt1 pool in the murine aortic arch. We showed that heparin displaced sFlt1 from aorta explants to medium. Another natural arena for sFlt1-HS retention is the placenta. We showed that heparin displaced sFlt1 from human term placental villi. Normally, this large fraction of sFlt1 is not freely diffused to the maternal circulation, rather it is contained by the placenta. When LMWH is administered for coagulation prophylaxis to gravidae at risk of coagulation disorders, serum sFlt1 levels were found to be increased as compared to normal untreated pregnant controls. Interestingly, VEGF levels also increased in these patients, as much as two to four times more than sFlt1, ensuring a positive VEGF/sFlt1 ratio. To investigate whether there is a physiological mechanism that controls sFlt1 retention or release, we examined heparanase. Heparanase is an endoglucosidase that can be found in the placenta, that cleaves HS chains and releases bound proteins. It is a potentially active mechanism for the release of placental sFlt1 (Fig. 3). We compared the levels of sFlt1 in the serum of nonpregnant wild-type mice to those of heparanase-overexpressing transgenic (HTG) mice. We found that sFlt1 levels in the serum of HTG mice was nearly double that of wild-type mice. In pregnant wild-type and HTG mice the difference was more much marked and increased exponentially as pregnancy advanced.
Fig. 3. Heparanase cleaves HS chains and releases bound placental sFlt1.
Fig. 2. sFlt1-14 was identified by in situ hybridization in syncitial knots.
In short, heparanase can release sFlt1 from the placental reservoir. We next investigated whether targeting heparanase can reduce sFlt1 secretion in placenta explants. When heparanase was neutralized in placental villi explants, we observed a 35% reduction in sFlt1 secretion to medium as compared to controls, in placentas from normal and preeclamptic pregnancies. We also observed the effect of cathepsin L inhibition on sFlt1 secretion to placental villi explants in medium, and observed a similar effect, both in normal and PE placentas [11,12]. These results need to be verified in larger samples. In summary, sFlt1 is a strong heparin binder. A large reservoir of sFlt1 is contained in the placenta. Heparanse is involved in the release of sFlt1 by the placenta, and could potentially be targeted to reduce sFlt1 secretion in PE.
S66
S. Yagel / Thrombosis Research 127 (2011) S64–S66
Conflict of Interest Statement The author declares no conflict of interest. References [1] Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature 2006;443:993–7. [2] Cao L, Jiao X, Zuzga DS, Liu Y, Fong DM, Young D, et al. VEGF links hippocampal activity with neurogenesis, learning and memory. Nat Genet 2004;36:827–35. [3] Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–42. [4] Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672–83. [5] Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649–58.
[6] Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling – in control of vascular function. Nat Rev Mol Cell Biol 2006;7:359–71. [7] Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell 2007;130:691–703. [8] Sela S, Itin A, Natanson-Yaron S, Greenfield C, Goldman-Wohl D, Yagel S, et al. A novel human-specific soluble vascular endothelial growth factor receptor 1: cell-type-specific splicing and implications to vascular endothelial growth factor homeostasis and preeclampsia. Circ Res 2008;102:1566–74. [9] Tenney B, Parker, F. The placenta in toxemia of pregnancy. Am J Obstet Gynecol 1940;39:1000–5. [10] Heazell AE, Moll SJ, Jones CJ, Baker PN, Crocker IP. Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species. Placenta 2007; 28(Suppl A):S33–40. [11] Haimov-Kochman R, Friedmann Y, Prus D, Goldman-Wohl DS, Greenfield C, Anteby EY, et al. Localization of heparanase in normal and pathological human placenta. Mol Hum Reprod 2002;8:566–73. [12] Abboud-Jarrous G, Atzmon R, Peretz T, Palermo C, Gadea BB, Joyce JA, et al. Cathepsin L is responsible for processing and activation of proheparanase through multiple cleavages of a linker segment. J Biol Chem 2008;283: 18167–76.
Contents lists available at ScienceDirect
Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t h r o m r e s
Angiogenesis in gestational vascular complications Simcha Yagel* Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Centers, Mt. Scopus, Jerusalem, Israel
article info
abstract
Keywords: sFlt1-14 VEGF Heparanase Preeclampsia
Vascular endothelial growth factor (VEGF) is a key player in vasculogenesis and angiogenesis in the embryo, and essential in neovascularization in adults. Natural VEGF inhibitors such as soluble VEGF receptors, among them the soluble VEGF-trapping receptor Flt1 (sFlt1), participate in VEGF regulation. Decreased levels of VEGF and increased levels of sFlt1 have been implicated in the pathophysiology of preeclampsia. We discovered a soluble receptor, sFlt1-14, qualitatively different from sFlt1 and a potent VEGF inhibitor. It is generated in a cell type specific fashion, primarily in nonendothelial cells, most notably in vascular smooth muscle cells. We showed that increased production of soluble VEGF receptors in pregnancy is owing to expression of sFlt1-14, from the end of the first trimester to term. This expression is markedly elevated in preeclampsia, and is expressed chiefly by syncitial knots. In subsequent studies we found that sFlt1 is a strong heparin binder: this capability enables it to stay attached to blood vessels and to the placenta. Ex vivo, sFlt1 can be heparin displaced to medium from aortic segments and placental villi. In vivo, pregnant women treated with the low molecular weight heparin (LMWH) have elevated sFlt1 levels in their circulations. Interestingly, LMWH raised VEGF levels over and above the increase in sFlt1 levels in these patients. Heparanaseoverexpressing non-pregnant as well as pregnant transgenic mice present elevated levels of sFlt1 in their circulations. Ex vivo prevention of heparanase maturation through cathepsin L inhibition, or targeting heparanase directly with a neutralizing antibody, both resulted in a marked reduction in sFlt1 secretion to medium of normal and preeclamptic placental explants. These findings uncover a new level of regulation that controls sFlt1 bio-distribution, and directs it to function in the vicinity of its producing cell. Heparanase or LMWH has the ability to liberate sFlt1 from its retention, so this process may be a potential target for preeclampsia treatment. © 2011 Elsevier Ltd. All rights reserved.
Vascular endothelial growth factor (VEGF) is a key player in vasculogenesis and angiogenesis in the embryo, and essential in neovascularization in adults. It plays additional roles in nonvascular development and in homeostasis. Owing to its potent effect on blood vessels VEGF is normally kept in precise balance in the body. Natural VEGF inhibitors such as soluble VEGF receptors, among them the soluble VEGF-trapping receptor Flt1 (sFlt1), participate in VEGF regulation. Decreased levels of VEGF and increased levels of sFlt1 have been implicated in the pathophysiology of preeclampsia. However, there is still controversy surrounding the roles of sFlt1 and VEGF in the development of preeclampsia [1–7]. We began our investigations into the possible connection of sFlt1 to preeclampsia from two cases of dichorionic biamniotic twins managed in our department, which served to test the concept of sFlt1 involvement in PE. The first case was a G1P0, carrying one healthy fetus and one with severe intra-uterine growth restriction (IUGR) with absence * Correspondence: Prof. Simcha Yagel. Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Centers, Jerusalem, Israel. Tel.: +972 2 5844111; fax: +972 2 5815370. E-mail address: [email protected] (S. Yagel). 0049-3848 /$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
of diastolic flow in the umbilical artery. The mother developed severe preeclampsia, and in light of her worsening clinical state was delivered by emergency cesarean. In the maternal serum in the course of her pregnancy we discovered high levels of sFlt1 and low levels of VEGF for gestational age. Post-partum analysis of the placentas revealed multiple syncitial knots in the IUGR placenta, and high levels of sFlt1 in his umbilical cord blood, and relatively low levels of VEGF, while these indices were normal in the cotwin. The IUGR placenta revealed high levels of sFlt1 by in situ hybridization. The second case was a woman in her first pregnancy, with one healthy fetus and one with severe IUGR and absent diastolic flow in the umbilical artery. She developed severe preeclampsia at 29 weeks gestation. We found excessive levels of sFlt1 in the maternal serum. The IUGR fetus was also diagnosed with gross abnormality of the cerebellum. In light of these findings selective feticide was performed on the IUGR twin. Symptoms of preeclampsia subsequently disappeared, including maternal hypertension and proteinuria, and a dramatic decrease in serum sFlt1 levels.
S. Yagel / Thrombosis Research 127 (2011) S64–S66
Fig. 1. sFlt1-14 is a splicing variant of VEGF receptor 1 (Flt-1).
These two cases demonstrate the importance of sFlt1 in the pathophysiology of preeclampsia, as has also been noted by others. We aimed to characterize sFlt1 on the molecular level, and to determine its source. Previously the source of sFlt1 in maternal serum was undetermined, whether it was secreted by the placenta, or whether as part of the pathology of preeclampsia it was produced by other maternal cells. We demonstrated the existence of a splicing variant of VEGF receptor 1 (Flt-1): sFlt1-14 (Fig. 1). We showed that this variant of a natural VEGF inhibitor, possibly protecting non-endothelial cells from the effects of VEGF, is implicated in the development of preeclampsia. sFlt1-14 strongly inhibits VEGF signaling. We showed that recombinant sFlt1 and sFlt1-14 proteins were produced in HeLa cells transfected with the respective expression vector. Western blotting detected soluble Flt1 receptors in cells or released into medium using antibody directed against an extracellular epitope. We exposed porcine aortic ECs that were engineered to stably express Flk1, to VEGF. Flk1 phosphorylation served as a measure of the extent of VEGF signaling. We observed an inhibitory effect of preincubation of VEGF with sFlt1 or sFlt1-14. SFlt1-14 differs from sFlt1 in the source cells that produce it, and it functions as a potent VEGF inhibitor. It is generated primarily in non-endothelial cells. In vascular smooth muscle cells (VSMC) Flt1 mRNA is converted to sFlt1-14, while endothelial cells of the same vessels express sFlt1. We showed that sFlt1-14 expression by VSMC’s is upregulated on coculture with endothelial cells or by direct exposure to VEGF. The increase in soluble VEGF receptors in pregnancy stems from induced expression of placental sFlt1-14, from the end of the first trimester to term. In women with preeclampsia sFlt1-14 expression is dramatically increased. The source of this excess seems to be the syncitial knots. These knots are clusters of degenerative syncitial trophoblast in the placenta. sFlt1-14 was identified by in situ hybridization in syncitial knots (Fig. 2). Expression of sFlt1 was lower in healthy syncytiotrophoblast in comparison to knots from PE placentas [8]. No sFlt1-14 was detected in placental ECs [9,10].
S65
We have preliminary data showing that sFlt1 is secreted to the maternal circulation inside microparticles produced by syncitiotrophoblasts. Another question arose, that while sFlt1 needs to be retained by VSMCs to grant them VEGF protection, it can be found far from its placental producing cells. What enables sFlt1 to be retained by or released from its producing cell? VEGF R2 is capable of heparan binding, which modulates its signaling. Flt1 lg-like domain 4 is capable of heparan binding, but this capability does not modulate its signaling. Working from the hypothesis that this heparan binding capability operates to enable sFlt1 local retention, we aimed to show this in a physiological context. Heparan sulfate (HS) proteoglycan binds to sFlt1 producing cells. Both sFlt1 isoforms are excellent heparan binders, and sFlt1’s ability to strongly bind heparan might affect its bioavailability. Heparin can compete with HS for sFlt1 binding. To demonstrate this, we utilized the displaceable sFlt1 pool in the murine aortic arch. We showed that heparin displaced sFlt1 from aorta explants to medium. Another natural arena for sFlt1-HS retention is the placenta. We showed that heparin displaced sFlt1 from human term placental villi. Normally, this large fraction of sFlt1 is not freely diffused to the maternal circulation, rather it is contained by the placenta. When LMWH is administered for coagulation prophylaxis to gravidae at risk of coagulation disorders, serum sFlt1 levels were found to be increased as compared to normal untreated pregnant controls. Interestingly, VEGF levels also increased in these patients, as much as two to four times more than sFlt1, ensuring a positive VEGF/sFlt1 ratio. To investigate whether there is a physiological mechanism that controls sFlt1 retention or release, we examined heparanase. Heparanase is an endoglucosidase that can be found in the placenta, that cleaves HS chains and releases bound proteins. It is a potentially active mechanism for the release of placental sFlt1 (Fig. 3). We compared the levels of sFlt1 in the serum of nonpregnant wild-type mice to those of heparanase-overexpressing transgenic (HTG) mice. We found that sFlt1 levels in the serum of HTG mice was nearly double that of wild-type mice. In pregnant wild-type and HTG mice the difference was more much marked and increased exponentially as pregnancy advanced.
Fig. 3. Heparanase cleaves HS chains and releases bound placental sFlt1.
Fig. 2. sFlt1-14 was identified by in situ hybridization in syncitial knots.
In short, heparanase can release sFlt1 from the placental reservoir. We next investigated whether targeting heparanase can reduce sFlt1 secretion in placenta explants. When heparanase was neutralized in placental villi explants, we observed a 35% reduction in sFlt1 secretion to medium as compared to controls, in placentas from normal and preeclamptic pregnancies. We also observed the effect of cathepsin L inhibition on sFlt1 secretion to placental villi explants in medium, and observed a similar effect, both in normal and PE placentas [11,12]. These results need to be verified in larger samples. In summary, sFlt1 is a strong heparin binder. A large reservoir of sFlt1 is contained in the placenta. Heparanse is involved in the release of sFlt1 by the placenta, and could potentially be targeted to reduce sFlt1 secretion in PE.
S66
S. Yagel / Thrombosis Research 127 (2011) S64–S66
Conflict of Interest Statement The author declares no conflict of interest. References [1] Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature 2006;443:993–7. [2] Cao L, Jiao X, Zuzga DS, Liu Y, Fong DM, Young D, et al. VEGF links hippocampal activity with neurogenesis, learning and memory. Nat Genet 2004;36:827–35. [3] Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–42. [4] Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672–83. [5] Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111:649–58.
[6] Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling – in control of vascular function. Nat Rev Mol Cell Biol 2006;7:359–71. [7] Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, et al. Autocrine VEGF signaling is required for vascular homeostasis. Cell 2007;130:691–703. [8] Sela S, Itin A, Natanson-Yaron S, Greenfield C, Goldman-Wohl D, Yagel S, et al. A novel human-specific soluble vascular endothelial growth factor receptor 1: cell-type-specific splicing and implications to vascular endothelial growth factor homeostasis and preeclampsia. Circ Res 2008;102:1566–74. [9] Tenney B, Parker, F. The placenta in toxemia of pregnancy. Am J Obstet Gynecol 1940;39:1000–5. [10] Heazell AE, Moll SJ, Jones CJ, Baker PN, Crocker IP. Formation of syncytial knots is increased by hyperoxia, hypoxia and reactive oxygen species. Placenta 2007; 28(Suppl A):S33–40. [11] Haimov-Kochman R, Friedmann Y, Prus D, Goldman-Wohl DS, Greenfield C, Anteby EY, et al. Localization of heparanase in normal and pathological human placenta. Mol Hum Reprod 2002;8:566–73. [12] Abboud-Jarrous G, Atzmon R, Peretz T, Palermo C, Gadea BB, Joyce JA, et al. Cathepsin L is responsible for processing and activation of proheparanase through multiple cleavages of a linker segment. J Biol Chem 2008;283: 18167–76.