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Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31 + /CD45 - /BrdU + ). This was accompanied by a reduction of total leukocyte count (CD45 + ), neutrophils (MPO + ), neutrophil extracellular traps (NETs) (MPO + CitH3 + ), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68 + /MRC1 - ) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68 + /MRC1 + ) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.

Objective: Arteriogenesis, describing the process of collateral artery growth, is activated by fluid shear stress (FSS). Since this vascular mechanotransduction may involve microRNAs (miRNAs), we investigated the FSS-induced expression of vascular cell miRNAs and their functional impact on collateral artery growth during arteriogenesis. Approach and Results: To this end, rats underwent femoral artery ligation and arteriovenous anastomosis to increase collateral blood flow to maximize FSS and trigger collateral vessel remodeling. Five days after surgery, a miRNA expression profile was obtained from collateral tissue, and upregulation of 4 miRNAs (miR-24-3p, miR-143-3p, miR-146a-5p, and miR-195-5p) was verified by quantitative polymerase chain reaction. Knockdown of miRNAs at the same time of the surgery in an in vivo mouse ligation and recovery model demonstrated that inhibition of miR-143-3p only severely impaired blood flow recovery due to decreased arteriogenesis. In situ hybridization revealed distinct localization of miR-143-3p in the vessel wall of growing collateral arteries predominantly in smooth muscle cells. To investigate the mechanotransduction of FSS leading to the increased miR-143-3p expression, cultured endothelial cells were exposed to FSS. This provoked the expression and release of TGF-β (transforming growth factor-β), which increased the expression of miR-143-3p in smooth muscle cells in the presence of SRF (serum response factor) and myocardin. COL5A2 (collagen type V-α2)-a target gene of miR-143-3p predicted by in silico analysis-was found to be downregulated in growing collaterals., Conclusions: These results indicate that the increased miR-143-3p expression in response to FSS might contribute to the reorganization of the extracellular matrix, which is important for vascular remodeling processes, by inhibiting collagen V-α2 biosynthesis.

Arteriogenesis is an intricate process in which increased shear stress in pre-existing arteriolar collaterals induces blood vessel expansion, mediated via endothelial cell activation, leukocyte recruitment and subsequent endothelial and smooth muscle cell proliferation. Extracellular RNA (eRNA), released from stressed cells or damaged tissue under pathological conditions, has recently been discovered to be liberated from endothelial cells in response to increased shear stress and to promote collateral growth. Until now, eRNA has been shown to enhance coagulation and inflammation by inducing cytokine release, leukocyte recruitment, and endothelial permeability, the latter being mediated by vascular endothelial growth factor (VEGF) signaling. In the context of arteriogenesis, however, eRNA has emerged as a transmitter of shear stress into endothelial activation, mediating the sterile inflammatory process essential for collateral remodeling, whereby the stimulatory effects of eRNA on the VEGF signaling axis seem to be pivotal. In addition, eRNA might influence subsequent steps of the arteriogenesis cascade as well. This article provides a comprehensive overview of the beneficial effects of eRNA during arteriogenesis, laying the foundation for further exploration of the connection between the damaging and non-damaging effects of eRNA in the context of cardiovascular occlusive diseases and of sterile inflammation.

Fluid shear stress in the vasculature is the driving force for natural bypass growth, a fundamental endogenous mechanism to counteract the detrimental consequences of vascular occlusive disease, such as stroke or myocardial infarction. This process, referred to as "arteriogenesis," relies on local recruitment of leukocytes, which supply growth factors to preexisting collateral arterioles enabling them to grow. Although several mechanosensing proteins have been identified, the series of mechanotransduction events resulting in local leukocyte recruitment is not understood. In a mouse model of arteriogenesis (femoral artery ligation), we found that endothelial cells release RNA in response to increased fluid shear stress and that administration of RNase inhibitor blocking plasma RNases improved perfusion recovery. In contrast, treatment with bovine pancreatic RNase A or human recombinant RNase1 interfered with leukocyte recruitment and collateral artery growth. Our results indicated that extracellular RNA (eRNA) regulated leukocyte recruitment by engaging vascular endothelial growth factor receptor 2 (VEGFR2), which was confirmed by intravital microscopic studies in a murine cremaster model of inflammation. Moreover, we found that release of von Willebrand factor (VWF) as a result of shear stress is dependent on VEGFR2. Blocking VEGFR2, RNase application, or VWF deficiency interfered with platelet-neutrophil aggregate formation, which is essential for initiating the inflammatory process in arteriogenesis. Taken together, the results show that eRNA is released from endothelial cells in response to shear stress. We demonstrate this extracellular nucleic acid as a critical mediator of mechanotransduction by inducing the liberation of VWF, thereby initiating the multistep inflammatory process responsible for arteriogenesis., (© 2019 by The American Society of Hematology.)

The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit(+)/CXCR-4(+) cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Cell injury releases nucleic acids supporting inflammation and stem cell activation. Here, the impact of extracellular ribonucleic acid, especially transfer RNA (ex-tRNA), on vasculogenesis and leukopoiesis of mouse embryonic stem (ES) cells was investigated., Approach and Results: ex-tRNA, whole cell RNA and ribosomal RNA (ex-rRNA) but not DNA increased CD31-positive vascular structures in embryoid bodies. Ex-tRNA and ex-rRNA increased numbers of VEGFR2(+), CD31(+) and VE-cadherin(+) vascular cells as well as CD18(+), CD45(+) and CD68(+) cells, indicating leukocyte/macrophage differentiation. This was paralleled by mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor-165 (VEGF165) and neuropilin 1 (NRP1), phosphorylation of phosphatidyl inositol 3-kinase (PI3K) and VEGF receptor 2 (VEGFR2) as well as mRNA expression of α-smooth muscle actin (α-SMA). ex-tRNA was taken up by endosomes, increased expression of the pro-angiogenic semaphorin B4 receptor plexin B1 as well as the ephrin-type B receptor 4 (EphB4) and ephrinB2 ligand and enhanced cell migration, which was inhibited by the VEGFR2 antagonist SU5614 and the PI3K inhibitor LY294002. This likewise abolished the effects of ex-tRNA on vasculogenesis and leukopoiesis of ES cells. Ex-tRNA increased NOX1, NOX2, NOX4 and DUOX2 mRNA and boosted the generation of superoxide and hydrogen peroxide which was inhibited by radical scavengers, the NADPH oxidase inhibitors apocynin, VAS2870, ML171, and plumbagin as well as shRNA silencing of NOX1 and NOX4., Conclusions: Our findings indicate that ex-tRNA treatment induces vasculogenesis and leukopoiesis of ES cells via superoxide/hydrogen peroxide generated by NADPH oxidase and activation of VEGFR2 and PI3K., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Angiogenesis and vascular development are essential for fetal development and growth, whereby early pregnancy loss and other pregnancy-related pathologies have been linked to aberrant vascular development. As Wnt signalling has been suggested to play a role in the vascularization of chorionic villi, we investigated the expression of Wnt family members in trophoblasts and stromal cells isolated from chorionic villi of early placenta and the influence of Wnt signalling on CD133(+)CD34(+)-hematopoietic progenitor (CD133(+)CD34(+)) cells. Wnt-5a was expressed in human placental stromal cells and to a lesser extent in human trophoblast cells. rWnt-5a impeded migration and induced adhesion of CD133(+)CD34(+) cells, in accordance with the expression of adhesion proteins, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). rWnt-5a-related regulation of the ICAM-1 and VCAM-1 expression were dependent on the release of Ca2+ and the activation of transcription factor - nuclear factor of activated T-cells (NF-AT). We propose that Wnt-5a is required during early placenta development to mediate adhesion and homing of CD133(+)CD34(+) cells., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Objective: Restoration of patency is a natural target of vascular remodeling after venous thrombosis that involves vascular endothelial cells and smooth muscle cells, as well as leukocytes. Acute pulmonary emboli usually resolve <6 months. However, in some instances, thrombi transform into fibrous vascular obstructions, resulting in occlusion of the deep veins, or in chronic thromboembolic pulmonary hypertension (CTEPH). We proposed that dysregulated thrombus angiogenesis may contribute to thrombus persistence., Approach and Results: Mice with an endothelial cell-specific conditional deletion of vascular endothelial growth factor receptor 2/kinase insert domain protein receptor were used in a model of stagnant flow venous thrombosis closely resembling human deep vein thrombosis. Biochemical and functional analyses were performed on pulmonary endarterectomy specimens from patients with CTEPH, a human model of nonresolving venous thromboembolism. Endothelial cell-specific deletion of kinase insert domain protein receptor and subsequent ablation of thrombus vascularization delayed thrombus resolution. In accordance with these findings, organized human CTEPH thrombi were largely devoid of vascular structures. Several vessel-specific genes, such as kinase insert domain protein receptor, vascular endothelial cadherin, and podoplanin, were expressed at lower levels in white CTEPH thrombi than in organizing deep vein thrombi and organizing thrombi from aortic aneurysms. In addition, red CTEPH thrombi attenuated the angiogenic response induced by vascular endothelial growth factor., Conclusions: In the present work, we propose a mechanism of thrombus nonresolution demonstrating that endothelial cell-specific deletion of kinase insert domain protein receptor abates thrombus vessel formation, misguiding thrombus resolution. Medical conditions associated with the development of CTEPH may be compromising early thrombus angiogenesis.

N-acetylneuraminic acid (Neu5Ac) represents the most common terminal carbohydrate residue in many mammalian glycoconjugates and is directly involved in a number of different physiological as well as pathological cellular processes. Endogenous sialic acids derive from the biosynthetic precursor molecule N-acetyl-D-mannosamine (ManNAc). Interestingly, N-acyl-analogues of D-mannosamine (ManN) can also be incorporated and converted into corresponding artificial sialic acids by eukaryotic cells. Within this study, we optimized a protocol for the chemical synthesis of various peracetylated ManN derivatives resulting in yields of approximately 100%. Correct molecular structures of the obtained products ManNAc, N-propanoyl-ManN (ManNProp) and N-butyl-ManN (ManNBut) were verified by GC-, ESI-MS- and NMR-analyses. By applying these substances to human umbilical vein endothelial cells (HUVECs), we could show that each derivative was metabolized to the corresponding N-acylneuraminic acid variant and subsequently incorporated into nascent glycoproteins. To investigate whether natural and/or artificial sialic acid precursors are able to modulate the angiogenic capacity of HUVECs, a spheroid assay was performed. By this means, an increase in total capillary length has been observed when cells incorporated N-butylneuraminic acid (Neu5But) into their glycoconjugates. In contrast, the natural precursor ManNAc inhibited the growth of capillaries. Thus, sialic acid precursors may represent useful agents to modulate blood vessel formation.

Based on previous findings that early growth response 1 (Egr-1) participates in leukocyte recruitment and cell proliferation in vitro, this study was designed to investigate its mode of action during arteriogenesis in vivo. In a model of peripheral arteriogenesis, Egr-1 was significantly upregulated in growing collaterals of wild-type (WT) mice, both on mRNA and protein level. Egr-1(-/-) mice demonstrated delayed arteriogenesis after femoral artery ligation. They further showed increased levels of monocytes and granulocytes in the circulation, but reduced levels in adductor muscles under baseline conditions. After femoral artery ligation, elevated numbers of macrophages were detected in the perivascular zone of collaterals in Egr-1(-/-) mice and mRNA of leukocyte recruitment mediators was upregulated. Other Egr family members (Egr-2 to -4) were significantly upregulated only in Egr-1(-/-) mice, suggesting a mechanism of counterbalancing Egr-1 deficiency. Moreover, splicing factor-1, downregulated in WT mice after femoral artery ligation in the process of increased vascular cell proliferation, was upregulated in Egr-1(-/-) mice. αSM-actin on the other hand, significantly downregulated in WT mice, showed no differential expression in Egr-1(-/-) mice. While cell cycle regulator cyclin E and cdc20 were upregulated in Egr-1(-/-) mice, cyclin D1 expression decreased below the detection limit in collaterals, and the proliferation marker ki67 was not differentially expressed. In conclusion, compensation for deficiency in Egr-1 function in leukocyte recruitment can presumably be mediated by other transcription factors; however, Egr-1 is indispensable for effective vascular cell cycle progression in arteriogenesis.

3-Deazaadenosine (c3Ado) is a potent inhibitor of S-adenosylhomocysteine hydrolase, which regulates cellular methyltransferase activity. In the present study, we sought to determine the effect of c3Ado on vascular smooth muscle cell (VSMC) function and neointima formation in vivo. c3Ado dose-dependently prevented the proliferation and migration of human coronary VSMCs in vitro. This was accompanied by an increased expression of the cyclin-dependent kinase inhibitors p21(WAF1/Cip1), p27(Kip1), a decreased expression of G(1)/S phase cyclins, and a lack of retinoblastoma protein hyperphosphorylation. In accordance with these findings, fluorescence-activated cell-sorting analysis of propidium iodide-stained cells indicated a cell cycle arrest in the G(0)/G(1) phase. Importantly, c3Ado did not affect the number of viable (trypan blue exclusion) or apoptotic cells (TUNEL). Mechanistically, c3Ado prevented FCS-induced Ras carboxyl methylation and membrane translocation and activity by inhibiting isoprenylcysteine carboxyl methyltransferase and reduced FCS-induced extracellular signal-regulated kinase (ERK)1/2 and Akt phosphorylation in a dose-dependent manner. Conversely, rescuing signal transduction by overexpression of a constitutive active Ras mutant abrogated c3Ado's effect on proliferation. For in vivo studies, the femoral artery of C57BL/6 mice was dilated and mice were fed a diet containing 150 microg of c3Ado per day. c3Ado prevented dilation-induced Ras activation, as well as ERK1/2 and Akt phosphorylation in vivo. At day 21, VSMC proliferation (proliferating-cell nuclear antigen [PCNA]-positive cells), as well as the neointima/media ratio (0.7+/-0.2 versus 1.6+/-0.4; P<0.05) were significantly reduced, without any changes in the number of apoptotic cells. Our data indicate that c3Ado interferes with Ras methylation and function and thereby with mitogenic activation of ERK1/2 and Akt, preventing VSMC cell cycle entry and proliferation and neointima formation in vivo. Thus, therapeutic inhibition of S-adenosylhomocysteine hydrolase by c3Ado may represent a save and effective novel approach to prevent vascular proliferative disease.

Placental vascular development begins very early in pregnancy and is characterized by construction of a primitive vascular network in a low-oxygen environment. In vitro three-component assays of this process are scarce. In this study, a complex three-dimensional spheroid model for in vitro studies of placental vasculogenesis with regard to cell-cell interactions between cytotrophoblasts (CTs), villous stromal cells and endothelial precursor cells was established. Microscopic and immunohistochemical analyses of the spheroids showed structural and differentiation patterns resembling the structure and differentiation of early placental chorionic villous tissue (in regard to the expression of multiple markers cytokeratin-7, vimentin, CD34, CD31). The authenticity of this model to in vivo events allowed investigation of placental vascular development and trophoblast invasion under physiological and pathological conditions. Particularly enhanced spheroidal expression of SDF-1alpha and its receptor CXCR4, the major chemokine system in embryonic vasculogenesis, in a low-oxygen environment was detected. In addition, our model confirmed previously described invasive phenotype of trophoblasts through collagen under low- (physiologic), but not high- (pathologic) oxygen concentrations. Therefore, the three-dimensional spheroid model consisting of major placental cell types proved to be an appropriate system to investigate early placental vessel development under both physiological and pathological conditions.

Placental vascular development is essential for fetal growth and development. Inadequate placental vascular development is associated with early pregnancy losses and other pregnancy related pathologies. In addition to the ubiquitous, well-characterized angiogenic factors like vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), some pregnancy-specific factors (e.g. human chorionic gonadotropin (hCG), insulin-like growth factor-II (IGF-II) or alpha fetoprotein (AFP) were recently described to play a possible regulatory role in this process. In the present study we described an improved separation method for human placental microvascular endothelial cells (HPMVEC) and their functional characterization. Using the combination of enzymatic digestion and multistep immunomagnetic sorting with CD31 antibodies a model for villous vascularization was established. Isolated cells took up ac-dil-LDL, spontaneously formed capillary-like structures, and expressed common endothelial markers such as vascular endothelial growth factor receptor-2 (VEGFR-2), angiopoetin-1 and -2, Tie-2, CD144, thrombomodulin, and von Willebrand factor (vWF) as shown by RT-PCR, flow cytometry and Western blot analysis. The expression of the hCG/LH receptor in the placental vascular tree was verified both in vitro and in vivo. hCG stimulated proliferation of HPMVEC in a dose specific manner. While hCG alone had no significant effect on endothelial cell apoptosis, the combination of VEGF-A and hCG protected HPMVEC from staurosporine-induced apoptosis. hCG significantly stimulated sprout formation when compared to controls in a spheroid angiogenesis assay. Our results demonstrate a modified and reproducible method allowing studies of placental vascular development and provide new insights into the possible role of trophoblastic factors in this process.

The extracellular adherence protein (Eap), a broad-spectrum adhesin secreted by Staphylococcus aureus, was previously shown to curb acute inflammatory responses, presumably through its binding to endothelial cell (EC) ICAM-1. Examining the effect of Eap on endothelial function in more detail, we here show that, in addition, Eap functions as a potent angiostatic agent. Concomitant treatment of EC with purified Eap resulted in the complete blockage of the mitogenic and sprouting responses elicited by vascular endothelial growth factor (VEGF)165 or basic fibroblast growth factor (bFGF). Moreover, the induction of tissue factor and decay-accelerating factor were repressed by Eap, as determined by qRT-polymerase chain reaction (qRT-PCR), with a corresponding reduction in Egr-1 protein up-regulation seen. This angiostatic activity was accompanied by a corresponding inhibition in ERK1/2 phosphorylation, while activation of p38 was not affected. Inhibition occurred downstream of tyrosine kinase receptor activation, as comparable effects were seen on TPA-induced ERK1/2 phosphorylation. Similar to previously described angiostatic agents like angiopoietin-1 or the 16-kDa prolactin fragment, Eap blockage of the Ras/Raf/MEK/ERK cascade was localized by pull-down assay at the level of Ras activation. Eap's combined anti-inflammatory and antiangiogenic properties render this bacterial protein not only an important virulence factor during S. aureus infection but open new perspectives for therapeutic applications in pathological neovascularization.

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

The formation of a new microvasculature is essential for the long-term survival and function of the islet graft. In this study we examined endothelium of isolated pancreatic islets by stimulation with growth factors, different culture conditions, and genetic modification. We also inspected the effect of immunosuppressives used in human transplantation on angiogenesis. Isolated islets were embedded in a three-dimensional fibrin or Matrigel matrix. The effect of hyperglycemia, hypoxia, and the addition of VEGF and bFGF was investigated. We exposed islets from transgenic mice expressing the VEGF gene (RIP1VEGF-A) to high glucose (16.7 mmol/L) medium and tested the immunosuppressive agents rapamycin (100 ng/ml) and FK506 (100 ng/ml). To quantify angiogenesis the percentage of sprouting islets was determined. New endothelial capillary-like structures protruded from isolated pancreatic islets. Addition of VEGF to the islets and transgenic RIP-VEGF islets showed a two- to threefold increase of sprouting islets compared to control. Hypoxic culture conditions stimulated angiogenesis, resulting in a twofold increase of capillary sprouting. Rapamycin and FK506 proved to be potent inhibitors of angiogenesis in this system, because a decrease of sprouting islets of more than 20% by both agents was observed. Isolated pancreatic islets are capable of forming new capillary structures and are susceptible to pro- and antiangiogenic stimuli.

Angiogenesis, the growth of new blood vessels, is a complex biological process that is orchestrated by several growth factors and components of the extracellular matrix, including fibronectin (FN) and its receptor the integrin alpha5beta1. Angiogenesis is a critical part of inflammation and wound repair, but the mechanism by which vascular proliferation and migration is regulated by inflammatory cells is not completely understood. We have previously shown that human neutrophil peptides (HNPs), also known as alpha-defensins, which are secreted in high concentrations when neutrophils are activated, bind specifically to FN in the extracellular matrix and inhibit plasminogen activation. Therefore, we asked whether HNPs act as a link between inflammation and angiogenesis. Alpha5beta1-mediated endothelial cell adhesion and migration to FN, both under control conditions and under stimulation by vascular endothelial growth factor (VEGF), were inhibited specifically and in a dose-dependent manner by HNPs, whereas endothelial cell adhesion and migration to other components of the extracellular matrix, such as vitronectin, collagen, or fibrinogen/fibrin were not. Consistent with this finding, HNPs bound to and promoted the binding of fibronectin to alpha5beta1 integrin in arginine-glycine-aspartic acid (RGD)-independent manner. HNPs also completely inhibited VEGF-induced proliferation and induced apoptosis of endothelial cells in a dose-dependent manner. Moreover, HNPs inhibited capillary tube formation in three-dimensional fibrin-matrices as well as neovascularization in vivo in the chicken chorioallantoic membrane assay. Taken together, these data indicate that HNPs can regulate angiogenesis by affecting endothelial cell adhesion and migration in an FN-dependent manner as well as endothelial cell proliferation. These findings provide new insight into the role of inflammatory cells in angiogenesis and might provide a platform for developing a novel class of anti-angiogenesis drugs.

The molecular coordination between angiogenesis and vascular remodeling is a critical step for the development of a functional vasculature in the placenta and the uterus during pregnancy. The oncodevelopmental albumin homolog alpha-fetoprotein (AFP) is mainly synthesized in the developing fetus, and its expression has been found to be associated with highly vascularized tumors in the adult. In this study, we investigated the angiogenic activity of AFP and its possible role in the fetomaternal unit. Immunohistochemical studies revealed that the AFP-binding protein(s) is expressed in blood vessels of chorionic villi from placentae of the second and the third but not of the first trimester during pregnancy. At low concentrations, AFP directly stimulates or enhances, respectively, vascular endothelial growth factor-induced proliferation and sprout formation of endothelial cells isolated from the placenta and the uterus possibly by a MAPK-dependent pathway. Furthermore, AFP enhances blood vessel formation in a chick chorioallantoic membrane assay in vivo. Interestingly, AFP has no proliferative or migratory effects on endothelial cells isolated from the umbilical vein in the absence of vascular endothelial growth factor. These data indicate that AFP may act as a specific proangiogenic factor of endothelial cells within the fetomaternal unit during advanced stages in pregnancy.

Adaptation of the maternal uterine vasculature is essential for normal fetal and placental development in which angiogenesis is considered one of the most critical adaptive changes during pregnancy. Highly expressed in cytotrophoblasts and maternal endothelial cells during pregnancy, IGF-II promotes cell migration and regulates fetal and placental growth. We hypothesized that IGF-II regulates uterine angiogenesis during pregnancy. Both uterine vasculature and isolated uterine microvascular endothelial cells expressed high levels of IGF-II and IGF-II/mannose-6 phosphate receptor mRNA as shown by in situ hybridization. Physiological concentrations of IGF-II significantly increased vessel formation, as shown by a three-dimensional angiogenesis assay in vitro or a chicken chorionallantoic membrane assay in vivo. The angiogenic response of IGF-II could be reversed by the addition of beta-galactosidase or rabbit-antihuman IGF-II/M6P receptor antiserum, whereas blocking antibodies against IGF-I receptor or insulin receptor influenced IGF-II-induced sprout formation. IGF-II promoted migration of endothelial cells (10-250 ng/ml) tested in a modified Boyden chamber, but no stimulating effect on proliferation was observed. The application of several intracellular signal transduction molecules and their inhibitors indicated that protein kinase C and G(i) protein might play a role in the IGF-II-induced angiogenesis. Our results suggest an important angiogenic role of IGF-II in the vascular adaptation to pregnancy.

Transplantation of pancreatic islets reconstitutes glucose homeostasis in diabetes mellitus. Before transplantation, islets are disrupted from the surrounding blood vessels by the isolation procedure, with the grafted tissue being subject to ischemic damage. The survival of transplanted islets is assumed to depend on effective revascularization. Perfusion studies suggest that newly formed microvessels supplying the graft with nutrients are exclusively rebuilt by the host. It is generally not known whether isolated islets contain endothelial cells (EC), which potentially participate in the revascularization process. Therefore, we tried to detect immature EC in isolated islets by transformation with polyoma middle T antigen. Endothelioma cells were generated, implicating the presence of de-differentiated EC within isolated islets. When embedded in a fibrin gel, the islets developed cellular cords consisting of EC, whereas FGF-2 and VEGF stimulated the formation of cord-like structures. Furthermore, we studied the presence of donor EC in islet grafts by using transgenic mice with an EC lineage-specific promotor-LacZ reporter construct (Tie-2LacZ). Following islet transplantation, Tie-2LacZ-positive EC of both donor and recipient were identified in the vicinity of or within the graft up to 3 wk after transplantation. In conclusion, EC and/or their progenitors with angiogenic capacity reside within isolated islets of different species, and their proliferative potential can be stimulated by various inducers. These graft-related endothelia persist after islet transplantation and are integrated within newly formed microvessels.

Angiogenesis and vascular remodeling are crucial processes in tumor invasion and metastasis as well as in embryo implantation and normal development of the placenta. We have previously shown that hCG expressed in trophoblast and various malignant tumors promotes cellular motility and that uterine endothelium expresses hCG/LH receptor in vivo. In this study hCG was proposed to promote angiogenesis. A three-dimensional in vitro angiogenesis system consisting of uterine microvascular endothelial cells seeded on microcarriers and entrapped in a fibrin matrix was used to study the influence of hCG on neovascularization. Physiological concentrations of hCG (5-50,000 mU/ml) significantly increased in vitro capillary formation (up to 2.5-fold) and migration of endothelial cells in a Boyden chamber assay (up to 3.6-fold) in a dose-dependent manner, whereas hCG had no effect on cell proliferation. In vivo, hCG induced neovascularization in the chicken chorioallantoic membrane assay comparable to the activity of vascular endothelial growth factor. hCG-secreting tumors (choriocarcinoma, endometrium, and ovarian carcinoma) promoted in vitro neovascularization (up to 3-fold), whereas hCG-neutralizing antibody, pertussis toxin (G protein inhibitor), or GRGDTP peptide (integrin antagonist), respectively, abolished both tumor- and hCG-induced capillary sprout formation. Our data indicate a novel function for hCG in uterine adaptation to early pregnancy as well as in tumor development and underline the importance of hCG as an as yet unrecognized angiogenic factor.