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The zoonotic pathogen Bartonella henselae ( Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature. [ABSTRACT FROM AUTHOR]

Unlabelled: Essentials Mechanism of thrombin-induced inflammation is not fully understood. Thrombin induced monocyte adhesion and barrier loss require Angiopoietin-2 (Ang-2). Ang-2 mediates vessel leakage and monocyte adhesion through SHP-2/p38MAPK pathway. Calcium dependent SHP2/p38MAPK activation regulates Ang-2 expression through a feedback loop., Summary: Background Thrombin imparts an inflammatory phenotype to the endothelium by promoting increased monocyte adhesion and vascular permeability. However, the molecular players that govern these events are incompletely understood. Objective The aim of this study was to determine whether Angiopoietin-2 (Ang-2) has a role, if any, in regulating inflammatory signals initiated by thrombin. Methods Assessment of vascular leakage by Miles assay was performed by intra-dermal injection on the foot paw. Surface levels of intercellular adhesion molecule-1 (ICAM-1) were determined by flow cytometry. Overexpression, knockdown and phosphorylation of proteins were determined by Western blotting. Results In time-course experiments, thrombin-stimulated Ang-2 up-regulation, peaked prior to the expression of adhesion molecule ICAM-1 in human umbilical vein-derived endothelial cells (HUVECs). Knockdown of Ang-2 blocked both thrombin-induced monocyte adhesion and ICAM-1 expression. In addition, Ang-2(-/-) mice displayed defective vascular leakage when treated with thrombin. Introducing Ang-2 protein in Ang-2(-/-) mice failed to recover a wild-type phenotype. Mechanistically, Ang-2 appears to regulate the thrombin-activated calcium spike that is required for tyrosine phosphatase SHP2 and p38 MAPK activation. Further, down-regulation of SHP2 attenuated both thrombin-induced Ang-2 expression and monocyte adhesion. Down-regulation of the adaptor protein Gab1, a co-activator of SHP2, as well as overexpression of the Gab1 mutant incapable of interacting with SHP2 (YFGab1), inhibited thrombin-mediated effects, including downstream activation of p38 MAPK, which in turn was required for Ang-2 expression. Conclusions The data establish an essential role of the Gab1/SHP2/p38MAPK signaling pathway and Ang-2 in regulating thrombin-induced monocyte adhesion and vascular leakage., (© 2016 International Society on Thrombosis and Haemostasis.)

One of the major challenges in tissue engineering of bone substitutes remains vascularization of the transplant. We have developed a three-dimensional collagen-based coculture system to assess interactions between human endothelial cells (hECs) and human osteoblasts (hOBs) in vitro. Human umbilical vein endothelial cells (HUVECs) were grown as three-dimensional multicellular spheroids and seeded in a collagen matrix to assess sprouting of the spheroids, that is, formation of tubelike structures resembling early capillaries. Direct cell contact between hOBs and HUVECs was established by incorporating hOBs into the EC spheroids, thus forming heterogeneous cospheroids. Spatial organization of cospheroids and sprout configuration were assessed by immunohistochemical wholemount staining techniques and confocal laser microscopy. Cumulative sprout length of spheroids was quantitatively analyzed by digital imaging planimetry. In this model HUVECs and hOBs formed heterogeneous cospheroids with distinct spatial organization. The ability of HUVEC spheroids to form tubelike structures on angiogenic stimulation with vascular endothelial growth factor and basic fibroblast growth factor was suppressed in heterogeneous HUVEC/hOB cospheroids. The model system introduced in this study may be useful to assess the mechanisms involved in regulating angiogenesis during bone formation and to further investigate the mechanisms by which heterotypic cell-cell interactions inhibit endothelial tube formation for applications in bone tissue engineering.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.

Vascular morphogenesis through mechanisms of vasculogenesis, angiogenesis and intussusception is associated primarily with embryonic and fetal development and is down-regulated in the healthy adult. Physiological angiogenesis in the adult is restricted to the female reproductive system where it occurs cyclically in the ovary and the uterus as well as pregnancy-associated in the placenta and in the mammary gland. Of all the different organs, the cyclic corpus luteum of the ovary is the organ site with the strongest physiological angiogenesis. The hormonally regulated cyclic processes in the corpus luteum are characterized by discrete phases of blood vessel growth, vessel maturation and vessel regression. This chapter discusses the morphological changes of the vasculature in the cyclic corpus luteum in relation to the regulating molecular mechanisms. These data establish the dynamic processes in the ovarian corpus luteum as a unique system for studying all steps of the angiogenic cascade, including vessel maturation and vessel regression. Inhibition of angiogenesis impairs the normal ovarian cycle, reflecting that angiogenesis is rate-limiting for ovulation and growth of the corpus luteum and may, thus, be a potential target for therapeutic intervention in the reproductive function.

Microvessel density (MVD) counting techniques have been widely used to assess the vasculature in tumors. MVD counts assess the presence of blood vessels but do not give an indication of the degree of angiogenesis and the functional status of the tumor neovasculature. To analyze angiogenesis and the functional status of the tumor vascular bed, we have quantitated endothelial cell proliferation and the recruitment of pericytes in human tumors [glioblastomas (n = 30), renal cell carcinomas (n = 22), colon carcinomas (n = 18), mammary carcinomas (n = 24), lung carcinomas (n = 15), and prostate carcinomas (n = 19)]. These findings were compared to the physiological angiogenesis in the cyclic bovine ovarian corpus luteum. Tissue sections were examined applying double-labeling immunohistochemical techniques to detect proliferating endothelial cells and to colocalize endothelial cells and pericytes. The following parameters were quantitated: (a) MVD count; (b) proliferating capillary index (PCI); (c) proliferating tumor versus endothelial cell index; and (d) microvessel pericyte coverage index (MPI). Based on endothelial cell proliferation, angiogenesis was found to be present in all tumors with characteristic and significant differences between the tumor types (glioblastomas, PCI = 9.6 +/- 6.1%; renal cell carcinomas, PCI = 9.4 +/- 5.2%; colon carcinomas, PCI = 7.8 +/- 5.2%; mammary carcinomas, PCI = 5.0 +/- 4.8%; lung carcinomas, PCI = 2.6 +/- 2.5%; prostate carcinomas, PCI = 2.0 +/- 1.4%). There was a considerable degree of heterogeneity in the intensity of angiogenesis within each tumor group, as indicated by large standard deviations. Even in the most angiogenic tumors, angiogenesis was found to be 4 to 20 times less intense as compared with the physiological angiogenesis in the growing ovarian corpus rubrum (PCI = 40.6 +/- 6.2%). Varying degrees of pericyte recruitment to the tumor microvasculature were determined in the different tumor types (glioblastomas, MPI = 12.7 +/- 7.9%; renal cell carcinomas, MPI = 17.9 +/- 7.8%; colon carcinomas, MPI = 65.4 +/- 10.5%; mammary carcinomas, MPI = 67.3 +/- 14.2%; lung carcinomas, MPI = 40.8 +/- 14.5%; prostate carcinomas, MPI = 29.6 +/- 9.5%). The data demonstrate distinct quantitative variations in the intensity of angiogenesis in malignant human tumors. Furthermore, the varying degrees of pericyte recruitment indicate differences in the functional status of the tumor vasculature in different tumors that may reflect varying degrees of maturation of the tumor vascular bed.

Almost any growth of tumors is to some extent associated with an inflammatory reaction which may be anti-tumorigenic by acting directly on tumor cells or protumorigenic cells presumably by inducing tumor-associated angiogenesis. In this study, we have analyzed the angiogenesis-inducing capacity of monocyte chemoattractant protein-1 (MCP-1), a key regulatory molecule of monocyte trafficking to sites of inflammation. MCP-1 was found to be potently angiogenic when implanted into rabbit cornea, exerting potency similar to the specific angiogenic vascular endothelial growth factor (VEGF)-A(121). MCP-1-induced angiogenesis in the cornea is associated with prominent recruitment of macrophages, whereas VEGF-A(121)-induced corneal angiogenesis is devoid of inflammatory cell recruitment. Based on these findings, we studied MCP-1 expression and macrophage recruitment in human invasive ductal mammary carcinomas in comparison with the physiological angiogenic processes in bovine ovarian corpus luteum. Macrophage recruitment was always associated with MCP-1 expression. High macrophage counts in mammary tumors corresponded with poor prognosis. In contrast, physiological ovarian angiogenesis was associated with only minimal inflammatory recruitment of macrophages. Our data show that MCP-1 is an indirect inflammation-associated inducer of angiogenesis and demonstrate distinct qualitative differences between tumor angiogenesis in human mammary tumors and physiological angiogenesis in the ovary., (Copyright 1999 Wiley-Liss, Inc.)

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.

Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when cells are allowed to establish cell-cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell-cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.

Cyclic angiogenic processes in the ovarian corpus luteum (CL) of monovulatory species are characterized by distinct phases of blood vessel growth, vessel maturation, and vessel regression. To characterize molecular and cellular systems that may play a role in regulating blood vessel maturation, we have (a) analyzed the spatiotemporal expression of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1 (Flt-1) and VEGF-R2 (Flk-1) throughout the ovarian cycle, (b) examined the recruitment of pericytes during vessel maturation, and (c) quantitatively measured the ratio of angiopoietin-2 (Ang-2) to angiopoietin-1 (Ang-1) throughout the ovarian cycle. The data indicate that the VEGF/VEGF-receptor system is expressed not only during ovarian angiogenesis, but also with similar intensity in the nonangiogenic midstage CL. In fact, VEGF is expressed through most of the ovarian cycle, only being down-regulated during luteolysis, which leads to regression of the CL neovasculature. Pericytes are recruited soon after the induction of CL angiogenesis following the front of invading endothelial cells. Based on a double-staining immunohistochemistry technique, we developed a microvessel maturation index (MMI) that reflects the percentage of the capillary neovasculature that is associated with pericytes. The MMI in the angiogenic corpus rubrum is approximately 0.60. This value is not significantly higher in the nonangiogenic midstage CL but increases to close to 0.90 during CL regression. Lastly, an RT-PCR analysis of Ang-1 and Ang-2 expression revealed that both molecules are expressed throughout the ovarian cycle. The quantitative Ang-2/Ang-1 ratio does, however, change from 1.34 in the angiogenic CL and 1.07 in the midstage CL to 7.59 during CL regression, reflecting the strong overexpression of Ang-2 over Ang-1 during blood vessel regression. Taken together, the data support a model of a transiently maturated vasculature in the midstage CL, which is characterized by VEGF and pericyte contact-mediated endothelial cell survival and an induction of blood vessel regression during luteolysis that is characterized by the down-regulation of VEGF and the up-regulation of Ang-2.

Analysis of endothelial cell (EC) chemokine receptor expression by RT-PCR revealed that EC essentially do not express CC-chemokine receptors whereas they express all known CXC-chemokine receptors. Endotheliotropic functions of ligands for CXCR-1, CXCR-2, and CXCR-3 have previously been described. We have consequently performed a detailed analysis of endothelial CXCR-4 expression. CXCR-4 is constitutively expressed by quiescent, resting EC. Cytokine stimulation revealed that bFGF upregulates endothelial CXCR-4 expression, whereas TNF alpha downregulates endothelial CXCR-4 expression. Expression of CXCR-4 mRNA as well as protein is also upregulated in autocrine activated, migrating bovine aortic endothelial cells (BAEC). Furthermore, migrating BAEC preferentially present CXCR-4 on the cell surface as evidenced by cytochemistry and FACS analysis. Lastly, the monospecific CXCR-4 ligand SDF-1 was found to act as a potent inducer of EC chemotaxis. In summary, the data indicate that the CXCR-4/SDF-1 receptor ligand interaction may be an important regulator of activated endothelial cell functions as they occur during vascular remodeling and angiogenesis.

The inhibition of angiogenesis is considered to be one of the most promising strategies that might lead to the development of novel antineoplastic therapies. This concept is supported by the dramatic results of gene inactivation experiments in mice that have identified several vascular endothelium related molecules as rate limiting for embryonic angiogenesis. Likewise, a number of recent animal studies have shown that angiogenesis inhibitors can prevent metastasis and shrink established experimental tumours to small dormant microtumours. In this review, Hellmut Augustin illustrates differences between developmental angiogenesis, physiological angiogenesis in the adult, and pathological angiogenesis in experimental animal tumours and natural human tumours. He then summarizes the experimental approaches to antiangiogenic therapies and finally discusses critical issues that need to be considered in translating these novel therapeutic strategies into clinical practice.

Vessel density counting has been performed in a variety of tumors and used as predictive parameter for the tumor's malignant behavior (metastasis, five year survival). A number of studies have reported conflicting results on the predictive value of vessel density counts. We have quantitated the number of microvessels in routine pathology specimens of paraffin embedded mammary tumors and related these findings to the histopathological diagnosis. Average vessel density counts of vascular hot spots of malignant and benign mammary tumors were similar (34 +/- 15 vs. 31 +/- 10), though significantly higher as in the adjacent normal mammary tissue (12 +/- 5). Analysis of individual tumors, however, showed that significantly more malignant than benign tumors had vessel density counts beyond a defined cut-off value (50 microvessels/HPF). The results suggest that high counts may indeed serve as an independent prognostic parameter. In contrast, low counts may also be observed in malignant tumors and may, thus, not be used as negative prognostic factor.

Objective: Circulating levels of endothelial cell adhesion molecules are elevated in women with preeclampsia. The aim of the present study was to determine levels of these molecules in the fetal circulation of normal pregnancies and pregnancies complicated by preeclampsia., Study Design: Fetal plasma samples from the umbilical vein and peripheral maternal plasma and serum sample were collected at delivery from women with preeclampsia and women with normal pregnancy. Women with non-proteinuric pregnancy-induced hypertension (PIH) were excluded from the study. A sandwich ELISA technique was employed to quantitate concentrations of soluble ICAM-1 (CD54), VCAM-1 (CD106), and E-selectin (CD62E)., Results: The normal values of soluble endothelial cell adhesion molecules in the fetal circulation were determined as 162+/-45 ng/ml for ICAM-1, 1612+/-582 ng/ml for VCAM-1, and 154+/-58 ng/ml for E-selectin. They were found to markedly differ from the corresponding normal values in the maternal circulation (sICAM-1: 247+/-65 ng/ml; sVCAM-1: 715+/-170 ng/ml; sE-selectin: 34+/-14 ng/ml). The concentrations of sICAM-1, sVCAM-1, and sE-selectin were significantly elevated in women with preeclampsia compared to healthy control pregnant women. In contrast, there was no difference in the circulating fetal concentrations of these molecules between normal pregnancies and pregnancies complicated by preeclampsia., Conclusions: Normal values of sICAM-1, sVCAM-1, and sE-selectin in fetal circulation are markedly different from the values obtained for healthy adults. Plasma concentrations of these molecules are elevated in women with preeclampsia but not in the fetal circulation of preeclamptic pregnancies suggesting that based on the analysis of soluble adhesion molecules the fetal circulation may not be affected by the factor(s) that lead to disturbed endothelial cell function in women with preeclampsia.

The CC chemokine monocyte chemoattractant protein (MCP)-1 is induced by inflammatory cytokines and acts as a potent regulator of monocyte trafficking. Monocytes adhere preferentially to migrating endothelial cells in vitro and to endothelial cells at the migration front in vivo after aortic balloon denudation injury. Based on these findings, we analyzed MCP-1 expression in migrating and resting bovine aortic endothelial (BAE) cells and identified prominently upregulated levels of MCP-1 expression in migrating BAE cells. Stimulation of resting BAE cells with 5 ng/mL bFGF resulted in a fourfold induction of MCP-1 mRNA expression. The time course of bFGF-induced MCP-1 mRNA expression indicated a rapid and direct stimulation of MCP-1 expression that was detectable 30 minutes after stimulation. Levels of basal MCP-1 expression, as well as upregulated levels of MCP-1 in migrating BAE cells, were downregulated by addition of a neutralizing anti-bFGF monoclonal antibody (1.0 microgram/mL). Digestion of conditioned media of resting BAE cells with collagenase led to a dose-dependent induction of MCP-1 expression in resting BAE cells, which was inhibited > 50% by addition of neutralizing anti-bFGF antibody. Confirmation of the Northern blot experiments by ELISA-based quantitation of MCP-1 protein levels identified threefold to sixfold higher levels of MCP-1 in the supernatants of bFGF-stimulated BAE cells than in unstimulated resting BAE cells. Finally, analysis of MCP-1 expression by in situ hybridization carried out on en face preparations of aortas demonstrated that MCP-1 expression is dramatically upregulated in regenerating endothelial cells in vivo after balloon denudation. Though not establishing a direct causal relation between the preferential adhesion of monocytes to migrating endothelial cells, these findings strongly suggest that autocrine-activated endothelial cell-derived MCP-1 may play a critical role in recruiting monocytes. They furthermore support the concept that bFGF acts as an autocrine regulator of endothelial cell activity and may imply an involvement of bFGF as a mediator of inflammatory cell trafficking.

Applying an RNA display strategy to identify genes of autocrine activated endothelial cells, we identified among others the Ras-related protein TC21/R-Ras2 as a differentially expressed gene of bovine aortic endothelial cells (BAEC). Migrating BAEC express prominently upregulated steady state levels of TC21/R-Ras2 mRNA (Northern blot, in situ hybridization) and protein (Western blot). Growth factor stimulation identified TC21/R-Ras2 as aFGF, bFGF, and EGF inducible molecule of BAEC. Exposure to actinomycin D revealed a half life time of TC21/R-Ras2 mRNA of > 2 h. These results strongly suggest that transcriptional regulation of Ras molecules contributes to their signal transduction capacity and a possible role of TC21/R-Ras2 in the signal transduction of autocrine activated endothelial cells.

Objective: The aim of the current study was to determine levels of circulating endothelial cell adhesion molecules during preeclampsia and to assess their predictive value as diagnostic markers for the early identification of pregnant women at risk of developing preeclampsia., Study Design: Plasma samples were obtained from women with preeclampsia; the syndrome of hemolysis, elevated liver enzymes, and low platelets; uncomplicated pregnancy-induced hypertension; and women with normal pregnancy. In addition, longitudinal plasma profiles of pregnant women were randomly collected to determine individual profiles of circulating endothelial cell adhesion molecules. A sandwich enzyme-linked immunosorbent assay technique was used to quantitate concentrations of soluble intercellular adhesion molecule-1 (CD54), vascular cell adhesion molecule-1 (CD106), E-selectin (CD62E), platelet endothelial cell adhesion molecule (CD31), and P-selectin (CD62P)., Results: Plasma levels of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and platelet endothelial cell adhesion molecule-1 were significantly elevated in women with preeclampsia compared with healthy control pregnant women. Longitudinal analysis of soluble plasma intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels during pregnancy revealed that these molecules (1) show little variation in healthy pregnant women, (2) do not vary during normal pregnancy, and (3) are significantly elevated in women with preeclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelets compared with control pregnant women and those with uncomplicated pregnancy-induced hypertension. Analysis of soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels in longitudinal profiles of pregnant women identified significantly elevated levels of these molecules in the plasma of preeclampsia-prone women 3 to 15 weeks before the onset of clinical symptoms., Conclusion: Elevated soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 measurements during pregnancy can be considered as major risk factors. Elevated levels of these substances in the plasma of pregnant women with preeclampsia support the concept of a primary endothelial cell involvement in the pathogenesis of preeclampsia. Although currently based on a limited database, significantly elevated levels of soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the plasma of otherwise healthy pregnant women suggest a very high predictive value of these molecules for the earliest identification of women at risk of developing preeclampsia.

Increasing evidence suggests that autocrine endothelial cell activity contributes significantly to the angiogenic cascade once the endothelial cells are initially activated by exogenous stimuli. We have employed the differential RNA-display technique to identify endothelial cell genes that are expressed under autocrine control as a result of the cells' release from growth arrest. Among the differentially expressed genes was the activin-binding and- neutralizing glycoprotein follistatin (FS), which was expressed by migrating endothelial cells and down-regulated once the cells had reached growth arrest. Cytokine exposure identified FS as a basic fibroblast growth factor (bFGF)-inducible gene. In contrast, activin-beta A, an inhibitor of endothelial cell proliferation, was constitutively expressed by migrating and resting endothelial cells. Exogenous recombinant FS induced proliferation of human umbilical vein endothelial cells and low bFGF-expressing bovine aortic endothelial cells. In vivo, FS was moderately angiogenic in the rabbit cornea. However, FS implantation in the cornea in combination with subcritical concentrations of bFGF induced a strong angiogenic response. The data demonstrate that FS by itself and particularly in synergy with bFGF induces angiogenesis. Furthermore, differential expression by endothelial cells suggests a critical role of the FS/activin-beta A system in regulating autocrine endothelial cell activity.

Angiogenesis occurs as a cyclically regulated process in the ovary and the uterus. After ovulation, there is massive sprouting of blood vessels in the growing corpus luteum (CL) during the first third of the ovarian cycle. During luteolysis and for several weeks thereafter, all newly formed vessels regress. Here we have systematically analyzed regression of blood vessels during luteolysis to identify mechanisms of blood vessel regression. Blood vessel counts are highest in the midcycle CL and drop rapidly after the onset of luteolysis. After a rapid phase of tissue dissociation, blood vessel regression proceeds slowly over several weeks in the residual CL. Endothelial cells in regressing vessels acquire a distinctly rounded and condensed phenotype. Ultrastructural analysis of blood vessel regression processes in the cyclic CL suggests two major mechanisms of blood vessel regression: a) detachment of rounded endothelial cells from their basement membrane, leaving areas devoid of covering endothelial cell monolayer, and b) contraction and occlusion of arterioles and small arteries with pronounced proliferation of smooth muscle cells. In situ detection of nucleosomal fragmentation products demonstrates numerous apoptotic luteal cells, but only a few apoptotic endothelial cells in the regressing CL. Induction of apoptosis in cultured endothelial cell monolayers by RGD peptides demonstrated that endothelial cells detach from their adhesive surface before fully becoming positive for nucleosomal fragmentation products. These data indicate that cyclic angiogenic processes in the ovary offer a suitable experimental system to analyze mechanisms of blood vessel growth and regression, and suggest that detachment of endothelial cells before apoptosis as well as contractive occlusion of blood vessels may be critical determinants of blood vessel regression.

Angiogenesis occurs during embryogenesis and is a down-regulated process in the healthy adult that is almost exclusively linked to pathological conditions such as tumor growth, wound healing, and inflammation. Physiological angiogenic processes in the adult are restricted to the female reproductive system where they occur cyclically during the ovarian and uterine cycle as well as during pregnancy. By systematically analyzing the phenotypic changes of endothelial cells during bovine corpus luteum (CL) formation and regression, we have established a physiological model of blood vessel growth and regression. Quantitation of vessel density, percentage of vessels with lumen, and ratio of Bandeiraea simplicifolia-I to von Willebrand Factor-positive endothelial cells were established as parameters of angiogenesis. Sprouting endothelial cells invade the growing CL and continue to grow throughout the first third of the ovarian cycle. Thereafter the mature CL is characterized by a dense network of vessels with gradually decreasing vessel density. During luteolysis and for several weeks thereafter (regressing and residual CL) all newly formed vessels regress, which is accompanied by gradual foreshortening and rounding of endothelial cells and subsequent detachment. Based on histochemical detection of nucleosomal fragmentation products physiological blood vessel regression in the cyclic CL does not appear to involve endothelial cell apoptosis. Lectin histochemical analysis revealed a distinct alteration of endothelial cell glycoconjugate expression during ovarian angiogenesis comparable with the distinct pattern of hyperglycosylation of cultured migrating endothelial cells (up-regulation of binding sites for Lycopersicon esculentum lectin, wheat germ agglutinin, neuraminidase-treated peanut agglutinin, and Ricinus communis agglutinin-I on sprouting ECs). Northern blot analysis of glycosyltransferases during the different stages of angiogenesis revealed an up-regulation of beta-galactoside alpha 2,6-sialyltransferase and alpha 1,3-galactosyltransferase mRNA expression during the angiogenic stages of CL formation. These data establish the ovarian angiogenesis model as a suitable experimental system to study the functional and phenotypic properties of endothelial cells in sprouting and regressing blood vessels and provide additional evidence for the importance of endothelial cell surface glycoconjugates during angiogenesis.

Endothelial cells line the inside of all blood vessels forming a structurally and functionally highly heterogenous population of cells. Here we describe the application of the differential RNA display technique to the analysis of the heterogeneity of endothelial cells. Multiple fragment cDNAs from quiescent resting and from activated migrating endothelial cells were amplified by RT-PCR using random 10mer 5' primers and T11XY 3' primers. Labelled amplification products were displayed on a sequencing gel. Expression patterns of more than 5000 bands of the two cell populations were approximately 98% identical. Of the differentially expressed bands, 26 fragment cDNAs were reamplified, sequenced, and used as probes for Northern blots. Approximately 50% of the analyzed fragment cDNAs could be confirmed as being differentially expressed by Northern blot analysis. Among the differentially expressed cDNAs was follistatin, which was exclusively expressed by migrating and not by quiescent arrested endothelial cells. Stimulation by exogenous bFGF, however, induced follistatin expression in arrested endothelial cells. These experiments support the use of the differential RNA display technique as a rapid cloning strategy for the identification of differentially expressed genes and suggest a role of the follistatin/activin system in the autocrine control of endothelial cell growth and differentiation.

Endothelial cells line the inside of all blood vessels, forming a structurally and functionally heterogeneous population of cells. Their complexity and diversity has long been recognized, yet very little is known about the molecules and regulatory mechanisms that mediate the heterogeneity of different endothelial cell populations. The constitutive organ- and microenvironment-specific phenotype of endothelial cells controls internal body compartmentation, regulating the trafficking of circulating cells to distinct vascular beds. In contrast, surface molecules associated with the activated cytokine-inducible endothelial phenotype play a critical role in pathological conditions including inflammation, tumor angiogenesis, and wound healing. Differentiation of the endothelial cell phenotypes appears to follow similar mechanisms to the differentiation of hematopoietic cells, with the exception that endothelial cells maintain transdifferentiating competence. The present review offers a scheme of endothelial cell differentiation and discusses the possible applications of differentially expressed endothelial cell molecules as targets for directed therapeutic intervention.