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Purpose: Blood vessel reconstruction is an increasing need of patients suffering from cardiovascular diseases. For the development of microvascular prostheses, efficient endothelialization is mandatory to prevent graft occlusion. Here, we assessed the impact of amnion-derived mesenchymal stem/stromal cells (hAMSC), known for their important angiogenic potential, on the integrity and stability of endothelial cells exposed to shear stress in vascular grafts.Methods: Human placental endothelial cells (hPEC) were cultured at the inner surface of an expanded polytetrafluoroethylene (ePTFE) graft positioned within a bioreactor and exposed to a minimal shear stress of 0.015 dyne/cm2or a physiological shear stress of 0.92 dyne/cm2. hAMSC attached to the outer graft surface were able to interact with human placental endothelial cells by paracrine factors.Results: Microscopical analysis and evaluation of glucose/lactate metabolism evidenced successful cell seeding of the graft: hPEC formed a stable monolayer, hAMSC showed a continuous growth during 72 h incubation. hAMSC improved the viability of hPEC exposed to 0.015 dyne/cm2as shown by a decreased lactate dehydrogenase release of 13% after 72 h compared to hPEC single culture. The viability-enhancing effect of hAMSC on hPEC was further improved by 13% under physiological shear stress. Angiogenesis array analysis revealed that hPEC exposed to physiological shear stress and hAMSC co-culture reduced the secretion of angiogenin, GRO, MCP-1, and TIMP-2.Conclusion: hAMSC exerted best survival-enhancing effects on hPEC under exposure to physiological shear stress and modulated endothelial function by paracrine factors. Our data support further studies on the development of grafts functionalized with hAMSC–derived secretomes to enable fast clinical application.

ABSTRACTMaternal gestational diabetes (GDM) is associated with hyperglycaemia and hyperinsulinemia in the fetal circulation which consequently may induce endothelial dysfunction in the feto-placental vasculature. In fact, feto-placental vasculature reveals various morphological changes in response to GDM. The cell adhesion molecules (CAMs) ICAM-1, VCAM-1 and E-selectin promote attachment and trans-endothelial migration of leukocytes, and are up regulated in inflammation and endothelial dysfunction. Thus, we hypothesized that the GDM environment upregulates ICAM-1, VCAM-1 and E-selectin in the feto-placental endothelium. We isolated primary feto-placental endothelial cells (fpEC) after normal (n=18) and GDM pregnancy (n=11) and analyzed mRNA (RT-qPCR) and protein expression (Immunoblot) of ICAM-1, VCAM-1 and E-selectin. While other CAMs were unchanged on mRNA and protein levels, ICAM-1 protein was decreased by GDM. Further analysis revealed also a decrease in the release of soluble ICAM-1 (sICAM-1), whose levels correlated negatively with maternal BMI. We conclude that this reduction of ICAM-1 protein species is the result of post-translational regulation, since ICAM-1 mRNA expression was unchanged. In fact, miRNAs targeting ICAM-1 were upregulated in GDM fpEC. Immunohistochemistry showed weaker ICAM-1 staining in the placental endothelium after GDM pregnancies, and demonstrated ICAM-1 binding partners CD11a and CD18 expressed on leukocytes in fetal circulation and on placental tissue macrophages. This study identified reduction of ICAM-1 protein in fpEC in GDM pregnancy, which was regulated post-transcriptionally. Low ICAM-1 protein production may represent a protective, placenta-specific mechanism to avoid leukocyte transmigration into the placenta in response to GDM.

Inflammation is a complex defense mechanism characterized by leukocyte migration from the vasculature into damaged tissues and subsequent deposition of extracellular matrix resulting in tissue repair. The inflammatory process is generally categorized into an acute, rapid response, and a persistent but slowly evolving chronic condition, which may progress into inflammatory diseases. An excessive deposition of extracellular matrix leads to overgrowth, hardening, and/or scarring of tissues, defined as fibrosis. The amnion has been used as biomaterial in medicine for over 100 years and has been proven valuable for the treatment of different pathological conditions including wound healing. In light of recent reports, this article will review the effects of the amnion and its cellular components within the inflammatory-fibrotic scenario and the factors described so far that could be involved in these immunomodulatory actions. As proof of principles, we will also discuss pre-clinical and clinical applications of the amnion where advantage has been taken of its anti-inflammatory and anti-fibrotic properties. It is conceivable that the local host environment in which the amnion is placed may have a profound role in influencing the production and function of soluble factors and the shift towards different steps in triggering healing. The healing effect depends on time, dosage, and location of cytokine/growth factor production by the amnion, together with the influence of the host microenvironment. Indeed, determining the specific cascade of events that may define the role of the amnion in a given clinical situation remains a challenge.

The placental endothelium is unique among the entire human vasculature. The blood enriched in oxygen and nutrients is transported in the veins, whereas the arteries contain deoxygenated blood coming from the fetus. The placental vasculature has to develop rapidly to ensure adequate supply of the fetus. Therefore, factors present in the fetal circulation will stimulate placental angiogenesis. In the third trimester of pregnancy the placental endothelium is richly endowed with insulin receptors. In a pregnancy complicated by maternal diabetes, fetal hyperinsulinemia resulting from maternal and, hence, fetal hyperglycaemia induces changes in the placental vasculature such as increased growth and angiogenesis. This review will discuss general effects of insulin on endothelial cells and further focus on insulin effects on the placental endothelium. Isolation and culture of placental endothelial cells has allowed the identification of insulin effects in vitro. These include metabolic effects of insulin i.e. stimulation of glycogen synthesis, and modulation of angiogenesis on the placental arterial endothelium i.e. regulation of ephrin-B2 expression, an arterial specific signalling molecule implicated in sprouting. The effect of insulin on ephrin-B2 in placental arterial endothelial cells as well as their particularly high expression levels of insulin receptors and receptors for vascular endothelial growth factors indicate that placental angiogenesis is likely to emanate from the arterial compartment and is stimulated by insulin.

The location and physiological functions of acetylcholinesterase and butyrylcholinesterase in the placenta are still debated. In the present study the activities of both enzymes were studied histochemically in the rat and human placenta, using an optimized Karnovsky/ Roots method. Additionally, they were measured biochemically. Acetylcholinesterase was active in the syncytiotrophoblast, cytotrophoplast cells and the visceral and parietal yolk sac epithelial cells of the rat (n = 10) and in the syncytiotrophoblast, cytotrophoblast cells, endothelial cells and the media of fetal blood vessels of the human placenta (n = 9). Butyrylcholinesterase could not be detected histochemically. Biochemically measured levels at certain developmental stages of the placenta revealed maximum acetylcholinesterase activity in the 8th week p.m. human placentae (102.9 nmol·min-1 per mg protein), 35% lower activity in the 12th week p.m., and minimum (44.1 nmol-min-1 per mg protein) in term placentae. In contrast, maximum butyrylcholinesterase activity was measured in week 12 p.m. (106.9 nmol·min-1 per mg protein). In rat placentae, butyrylcholinesterase activity on gestational day 21 reached 150% of the level on gestational day 16. Acetylcholinesterase activity remained constant. In placentae of pre-eclamptic patients, acetylcholinesterase and butyrylcholinesterase activities were found to be increased by 16% and 45%, respectively. The results suggest that placental acetylcholinesterase can no longer be considered as derived from maternal blood, but is primarily located within rat and human placental tissue.

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM 15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (Fc?RII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.

Glucose is taken up by tumor cells via sodium-independent facilitated diffusion along a concentration gradient. To examine the regulation of this process by substrate concentration, we investigated the effect of hyperglycemia on the glucose-transport system of choriocarcinoma-derived JAR and JEG-3 cells by culturing them for 24, 48 and 96 hr in medium containing either 5.5 (normoglycemia) or 25 (hyperglycemia) mM d-glucose, respectively. Immunocytochemically, choriocarcinoma cells expressed the high-affinity glucose transporter isoforms GLUT1 and GLUT3. Based on initial uptake measurements using 3-O-[¹⁴C]methyl-d-glucose, kinetic parameters were calculated as Km= 15 mM and Vmax= 95 fmol/sec per cell for JAR and Km= 9 mM and Vmax= 64 fmol/sec per cell for JEG-3 cells. In JAR cells cultured under hyperglycemic conditions, uptake rates were significantly increased at 15, 20 and 25 mM exogenous d-glucose concentrations as compared with normoglycemic conditions. This effect was due to an increase in Vmax, whereas Km remained unchanged. Using Northern blotting, GLUT1 mRNA levels were higher but GLUT3 transcripts were reduced upon hyperglycemia. Western blotting revealed elevated GLUT1 and GLUT3 expression under hyperglycemic conditions. Hyperglycemia did not significantly influence the glucose-transport system of JEG-3 cells. We conclude that sustained hyperglycemia stimulates the glucose-transport system of JAR, but not of JEG-3, choriocarcinoma cells in vitro due to changes in GLUT1 and GLUT3 expression levels. We speculate that this mechanism may contribute to the beneficial effects of induced hyperglycemia as an adjuvant in tumor therapy. Int. J. Cancer 78:353–360, 1998.© 1998 Wiley-Liss, Inc.