Enthalpy of collagen interfibrillar bonds in fetal membranes.

During pregnancy, the fetal membrane (FM) is subjected to mechanical stretching that may result in preterm labor. The structural integrity of the FM is maintained by its collagenous layer. The disconnection and reconnection of molecular bonds between collagen fibrils are the fundamental processes that govern the irreversible mechanical and supermolecular changes in the FM. Here, we study the activation enthalpy of interfibrillar bonds in ex-vivo human FM. We analyze the strain-rate and temperature dependence of the irreversible deformations in FM subjected to inflation tests, which apply mechanical conditions similar to those experienced by the FM prior to and during the initiation of labor contractions. The obtained activation enthalpy of interfibrillar bonds matches the typical enthalpy values of polyvalent ionic bonds, implying on another important role that ions like Ca and Mg may play in the gestation and labor.

Collagen bundling and alignment in equibiaxially stretched human amnion.

We study irreversible collagen arrangement processes in ex-vivo human amnions subjected to inflation tests, which simulate the mechanical conditions prior to and during the initiation of labor uterine contractions. The investigation is focused on the center of the membrane where the stresses are maximal and equibiaxial. Second harmonic generation reveals an unexpected collagen rearrangement in the compact layer that is responsible for the structural integrity of the fetal membrane. The observed bundling and alignment of the collagen fibers indicate a deviation from the expected equibiaxial stress state. The statistical analysis of the fiber orientations provides information on two driving forces for collagen alignment: microscale flaws and macroscale deviation from the equibiaxial strain. As the pressure increases, the macroscale effect becomes dominant, and a high density of fibers that are aligned along a specific direction is observed. A model that explains these observations and relates them to the material properties is presented. The results of this study indicate that a temporal increase in intrauterine pressure or uterine cervix dilatation causes irreversible changes in collagen molecular connections that may lead to biological changes, such as the initiation of term and preterm labor.

Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165.

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.

Vascular endothelial growth factor (VEGF) and its receptors.

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells. Five VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene. These isoforms differ in their molecular mass and in biological properties such as their ability to bind to cell-surface heparan-sulfate proteoglycans. The expression of VEGF is potentiated in response to hypoxia, by activated oncogenes, and by a variety of cytokines. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. In vivo VEGF induces angiogenesis as well as permeabilization of blood vessels, and plays a central role in the regulation of vasculogenesis. Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors. The various VEGF forms bind to two tyrosine-kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1), which are expressed almost exclusively in endothelial cells. Endothelial cells express in addition the neuropilin-1 and neuropilin-2 coreceptors, which bind selectively to the 165 amino acid form of VEGF (VEGF165). This review focuses on recent developments that have widened considerably our understanding of the mechanisms that control VEGF production and VEGF signal transduction and on recent studies that have shed light on the mechanisms by which VEGF regulates angiogenesis.

Selective binding of VEGF121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells.

VEGF121 and VEGF165 are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF165 contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF165 a heparin binding capability which VEGF121 lacks. 125I-VEGF165 bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while 125I-VEGF121 bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of 125I-VEGF121 to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF165 and VEGF121 inhibited the binding of 125I-VEGF121 to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF165 by 2-3-fold. These results contrast with previous observations which have indicated that the binding of 125I-VEGF165 to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF165 is susceptible to oxidative damage caused by oxidants such as H2O2 or chloramine-T. VEGF121 was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF165 but not the receptor binding ability of damaged VEGF121. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF165 may enable the restoration of damaged VEGF165 function in processes such as inflammation or wound healing.

Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms.

The 121-amino acid form of vascular endothelial growth factor (VEGF121) and the 165-amino acid form (VEGF165) are mitogenic for vascular endothelial cells and induce angiogenesis in vivo. VEGF165 possesses a heparin binding ability and in the absence of heparin-like molecules does not bind efficiently to the VEGF receptors of vascular endothelial cells. The binding of 125I-VEGF165 to the VEGF receptors of endothelial cells, and the heparin-dependent binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1, were inhibited by the angiogenesis inhibitor platelet factor-4 (PF4). In contrast, PF4 was not able to inhibit the binding of VEGF121, a VEGF isoform which lacks a heparin binding capacity, to the VEGF receptors of the cells or to KDR/flk-1. These results indicate that PF4 may inhibit VEGF165 binding to VEGF receptors by disrupting the interaction of VEGF165 with cell surface heparan sulfates. Since PF4 mutants lacking a heparin binding ability retain their anti-angiogenic activity, alternative inhibitory mechanisms were also examined. 125I-PF4 bound with high affinity (Kd 5 x 10(-9) M) to VEGF165-coated wells. The binding of 125I-PF4 to the VEGF165-coated wells was inhibited by several types of heparin binding proteins, including unlabeled PF4 and unlabeled VEGF165. The binding was not inhibited by proteins which lack a heparin binding capacity, nor was it inhibited by VEGF121. Heparinase did not inhibit the binding of 125I-PF4 to VEGF165, indicating that heparin-like molecules are not required. These experiments suggest that PF4 can bind to heparin binding proteins such as VEGF165 leading to an inhibition of their receptor binding ability. In agreement with these results, we have observed that PF4 inhibits efficiently the VEGF165 induced proliferation of vascular endothelial cells. Unexpectedly, PF4 also inhibited efficiently the VEGF121-induced proliferation of the cells, indicating that PF4 can disrupt VEGF receptor mediated signal transduction using an unknown mechanism which does not interfere with VEGF121 binding.