De novo hem- and lymphangiogenesis by endothelial progenitor and mesenchymal stem cells in immunocompetent mice.

Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350 pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.

VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.

VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41+ cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41+ cells in the BM and a significant increase in diploid CD41+ cells, whereas the number of polyploid CD41+ cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.

DNA methylation regulates expression of VEGF-R2 (KDR) and VEGF-R3 (FLT4).

BACKGROUND: Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGF-Rs) are important regulators for angiogenesis and lymphangiogenesis. VEGFs and VEGF-Rs are not only expressed on endothelial cells but also on various subtypes of solid tumors and leukemias contributing to the growth of the malignant cells. This study was performed to examine whether VEGF-R2 (KDR) and VEGF-R3 (FLT4) are regulated by DNA methylation. METHODS: Real-time (RT) PCR analysis was performed to quantify KDR and FLT4 expression in some ninety leukemia/lymphoma cell lines, human umbilical vein endothelial cells (HUVECs) and dermal microvascular endothelial cells (HDMECs). Western blot analyses and flow cytometric analyses confirmed results at the protein level. After bisulfite conversion of DNA we determined the methylation status of KDR and FLT4 by DNA sequencing and by methylation specific PCR (MSP). Western blot analyses were performed to examine the effect of VEGF-C on p42/44 MAPK activation. RESULTS: Expression of KDR and FLT4 was observed in cell lines from various leukemic entities, but not in lymphoma cell lines: 16% (10/62) of the leukemia cell lines expressed KDR, 42% (27/65) were FLT4 positive. None of thirty cell lines representing six lymphoma subtypes showed more than marginal expression of KDR or FLT4. Western blot analyses confirmed KDR and FLT4 protein expression in HDMECs, HUVECs and in cell lines with high VEGF-R mRNA levels. Mature VEGF-C induced p42/44 MAPK activation in the KDR- /FLT4(+) cell line OCI-AML1 verifying the model character of this cell line for VEGF-C signal transduction studies. Bisulfite sequencing and MSP revealed that GpG islands in the promoter regions of KDR and FLT4 were unmethylated in HUVECs, HDMECs and KDR(+) and FLT4(+) cell lines, whereas methylated cell lines did not express these genes. In hypermethylated cell lines, KDR and FLT4 were re-inducible by treatment with the DNA demethylating agent 5-Aza-2'deoxycytidine, confirming epigenetic regulation of both genes. CONCLUSIONS: Our data show that VEGF-Rs KDR and FLT4 are silenced by DNA methylation. However, if the promoters are unmethylated, other factors (e.g. transactivation factors) determine the extent of KDR and FLT4 expression.

Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo.

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of ß(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

Adipose tissue-derived soluble fms-like tyrosine kinase 1 is an obesity-relevant endogenous paracrine adipokine.

Adipose tissue growth depends on angiogenesis. We tested the hypothesis that adipose tissue produces factors relevant to angiogenesis. We obtained fat biopsies in 2 different patient cohorts, cultured adipose-derived stem cells and studied mature adipocytes. We performed microarray, RT-PCR, and Western blotting; studied a rat obesity/metabolic syndrome model; and conducted viral gene transfer experiments in leptin-deficient mice. The microarray identified the splice variant of the vascular endothelial growth factor receptor, the soluble fms-like tyrosine kinase 1 (sFlt-1), as an antiangiogenesis candidate. We verified the expression findings and found that sFlt-1 was secreted by isolated mature human adipocytes. Tumor necrosis factor-α decreased sFlt-1 expression in mature adipocytes, whereas hypoxia had no effect. Separating cells from adipose tissue showed that the highest sFlt-1 expression was present in adipose-tissue nonfat cells rather than in the adipocytes themselves. We also found that sFlt-1 expression and sFlt-1 release by adipose-tissue explants were inversely correlated with body mass index of the corresponding patients but was directly correlated with adiponectin expression. In the obesity/metabolic syndrome rat model, we observed that circulating sFlt-1 levels and sFlt-1 expression in adipose tissue were also inversely correlated with body weight. To model our putative antiangiogenic factor further, we next overexpressed sFlt-1 by viral transfer in a mouse genetic model of leptin deficiency and observed that the transfected mice gained less weight than controls. We suggest that sFlt-1 could act as a paracrine factor inhibiting adipose tissue growth. Local sFlt-1 may regulate angiogenic potential and thereby influence adipose tissue mass.

High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.

Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels.

BACKGROUND: Postnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. RESULTS: In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels.Mouse lung microvascular endothelial cells (MLMVECs) were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony). These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs) in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. CONCLUSION: The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

Neuroblastoma progression correlates with downregulation of the lymphangiogenesis inhibitor sVEGFR-2.

PURPOSE: Tumor progression correlates with the induction of a dense supply of blood vessels and the formation of peritumoral lymphatics. Hemangiogenesis and lymphangiogenesis are potently regulated by members of the vascular endothelial growth factor (VEGF) family. Previous studies have indicated the upregulation of VEGF-A and -C in progressed neuroblastoma, however, quantification was performed using semiquantitative methods, or patients who had received radiotherapy or chemotherapy were studied. EXPERIMENTAL DESIGN: We have analyzed primary neuroblastoma from 49 patients using real-time reverse transcription-PCR and quantified VEGF-A, -C, and -D and VEGF receptors (VEGFR)-1, 2, 3, as well as the soluble form of VEGFR2 (sVEGFR-2), which has recently been characterized as an endogenous inhibitor of lymphangiogenesis. None of the patients had received radiotherapy or chemotherapy before tumor resection. RESULTS: We did not observe upregulation of VEGF-A, -C, and -D in metastatic neuroblastoma, but found significant downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic stages III, IV, and IVs. In stage IV neuroblastoma, there were tendencies for the upregulation of VEGF-A and -D and the downregulation of the hemangiogenesis/lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2 in MYCN-amplified tumors. Similarly, MYCN transfection of the neuroblastoma cell line SH-EP induced the upregulation of VEGF-A and -D and the switching-off of sVEGFR-2. CONCLUSION: We provide evidence for the downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic neuroblastoma stages, which may promote lymphogenic metastases. Downregulation of hemangiogenesis and lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2, and upregulation of angiogenic activators VEGF-A and VEGF-D in MYCN-amplified stage IV neuroblastoma supports the crucial effect of this oncogene on neuroblastoma progression.

Reduction of circulating soluble Flt-1 alleviates preeclampsia-like symptoms in a mouse model.

Preeclampsia (PE) is characterized by widespread endothelial damage with hypertension, proteinuria, glomeruloendotheliosis and elevated soluble Flt-1 (sFlt-1), a natural occurring antagonist of vascular endothelial growth factor (VEGF). Cancer patients receiving anti-VEGF therapy exhibit similar symptoms. We suggested that a decrease in circulating sFlt-1 would alleviate the symptoms associated with PE. Adenoviral (Adv) overexpression of sFlt-1 induced proteinuria, caused glomerular damage and increase in blood pressure in female Balb/c mice. Circulating level of sFlt-1 above 50 ng/ml plasma induced severe vascular damage and glomerular endotheliosis. Albumin concentration in urine was elevated up to 30-fold, compared to control AdvGFP-treated animals. The threshold of kidney damage was in the range of 20-30 ng/ml sFlt-1 in plasma (8-15 ng/ml in urine). Co-administration of AdvsFlt-1 with AdvVEGF to neutralize circulating sFlt-1 resulted in more than a 70% reduction in free sFlt-1 in plasma, more than 80% reduction in urine and rescued the damaging effect of sFlt-1 on the kidneys. This demonstrates that below a critical threshold sFlt-1 fails to elicit damage to the fenestrated endothelium and that co-expression of VEGF is able to rescue effects mediated by sFlt-1 overexpression.

Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth.

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).

Mandibular bone repair by implantation of rhBMP-2 in a slow release carrier of polylactic acid--an experimental study in rats.

The aim of the present study was to test the hypothesis that human recombinant bone morphogenic protein 2 (rhBMP-2) implanted in a slow release carrier of polylactic acid (PLA) can repair a non-healing defect in the rat mandible and maintain the thickness of an augmented volume. p-DL-lactic acid discs were produced and loaded with 48 and 96 microg rhBMP-2 and inserted into non-healing defects of the mandible of 45 Wistar rats. Fifteen rats received implants with 96 microg rhBMP-2 (Group 2), 48 microg rhBMP-2 (Group 1) and blank implants without BMP (Group 0) each on one side of the mandible. Unfilled defects of the same size on the contralateral sides of the mandibles served as empty controls. After 6, 13 and 26 weeks, implants of each group were retrieved from five animals each and submitted to flat panel detector computed tomography. Bone formation and thickness of augmentation was assessed by computer-assisted histomorphometry. In Group 2 significantly more bone was produced than in Group 1. Implants of Group 1 induced significantly more bone than the blank controls only after 6 weeks, whereas the difference was not significant after 13 and 26 weeks. Differences between Group 2 and Group 1 were clearly significant after 26 weeks. The thickness of bone tissue was maintained in Group 2 whereas it decreased in Group 1 and was negligible in Group 0. It is concluded that the PLA implants with 96 microg rhBMP-2 were able to bridge a non-healing defect in the rat mandible and maintained the thickness of an augmented volume. However, continuous supply of osteogenic signals appears to be required to compensate for adverse effects during polymer degradation.

Similarities and differences of human and experimental mouse lymphangiomas.

Lymphangioma is a disfiguring malformation of early childhood. A mouse lymphangioma model has been established by injecting Freund's incomplete adjuvant (FIA) intraperitoneally, but has not been compared with the human disease. We show that, in accordance with studies from the 1960s, the mouse model represents an oil-granuloma, made up of CD45-positive leukocytes and invaded by blood and lymph vessels. Several markers of lymphatic endothelial cells are expressed in both mouse and human, like CD31, Prox1, podoplanin, and Lyve-1. However, the human disease affects all parts of the lymphovascular tree. We observed convolutes of lymphatic capillaries, irregularly formed collectors with signs of disintegration, and large lymph cysts. We observed VEGFR-2 and -3 expression in both blood vessels and lymphatics of the patients, whereas in mouse VEGFR-2 was confined to activated blood vessels. The experimental mouse FIA model represents a vascularized oil-granuloma rather than a lymphangioma and reflects the complexity of human lymphangioma only partially.

Elevated expression of VEGFR-3 in lymphatic endothelial cells from lymphangiomas.

BACKGROUND: Lymphangiomas are neoplasias of childhood. Their etiology is unknown and a causal therapy does not exist. The recent discovery of highly specific markers for lymphatic endothelial cells (LECs) has permitted their isolation and characterization, but expression levels and stability of molecular markers on LECs from healthy and lymphangioma tissues have not been studied yet. We addressed this problem by profiling LECs from normal dermis and two children suffering from lymphangioma, and also compared them with blood endothelial cells (BECs) from umbilical vein, aorta and myometrial microvessels. METHODS: Lymphangioma tissue samples were obtained from two young patients suffering from lymphangioma in the axillary and upper arm region. Initially isolated with anti-CD31 (PECAM-1) antibodies, the cells were separated by FACS sorting and magnetic beads using anti-podoplanin and/or LYVE-1 antibodies. Characterization was performed by FACS analysis, immunofluorescence staining, ELISA and micro-array gene analysis. RESULTS: LECs from foreskin and lymphangioma had an almost identical pattern of lymphendothelial markers such as podoplanin, Prox1, reelin, cMaf and integrin-alpha1 and -alpha9. However, LYVE-1 was down-regulated and VEGFR-2 and R-3 were up-regulated in lymphangiomas. Prox1 was constantly expressed in LECs but not in any of the BECs. CONCLUSION: LECs from different sources express slightly variable molecular markers, but can always be distinguished from BECs by their Prox1 expression. High levels of VEGFR-3 and -2 seem to contribute to the etiology of lymphangiomas.

Embryonic development and malformation of lymphatic vessels.

In the human, malformations of lymphatic vessels can be observed as lymphangiectasia, lymphangioma and lymphangiomatosis, with a prevalence of 1.2-2.8 per thousand. Their aetiology is unknown and a causal therapy does not exist. We investigated the origin of lymphatic endothelial cells (LECs) in avian and murine embryos, and compared the molecular profile of LECs from normal and malformed lymphatics of children. In avian embryos, Prox1+ lymphangioblasts are located in the confluence of the cranial and caudal cardinal veins, where the jugular lymph sac (JLS) forms. Cell lineage studies show that the JLS is of venous origin. In contrast, the lymphatics of the dermis are derived from mesenchymal lymphangioblasts located in the dermatomes, suggesting a dual origin of LECs in avian embryos. The same may hold true for murine embryos, where Lyve1+ LEC precursors are found in the cardinal veins, and in the mesenchyme. The mesenchymal cells express the pan-leukocyte marker CD45, indicating a cell type with lymphendothelial and leukocyte characteristics. In the human, such cells might give rise to Kaposi's sarcoma. Microarray analyses of LECs from lymphangiomas of children show a large number of regulated genes, such as VEGFR3. Our studies show that lymphvasculogenesis and lymphangiogenesis occur simultaneously in the embryo, and suggest a function for VEGFR3 in lymphangiomas.

The association between vascular endothelial growth factor-C, its corresponding receptor, VEGFR-3, and prognosis in primary breast cancer: a study with 193 cases.

Lymphangiogenesis plays an important role in several normal and pathological conditions, such as wound healing, pathogen infection, inflammation or the metastasis formation of endothelial malignancies. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are important and specific regulatory factors for lymphatic endothelial proliferation and lymphangiogenesis. Both growth factors mediate their biological activity mainly by VEGF receptor-3 (VEGFR-3, Flt-4). In this study, we measured intratumoral levels of VEGF-C and VEGFR-3 through enzyme-linked immunosorbent assay (ELISA) in 193 primary breast cancer tissues and examined their prognostic values. A significant correlation was found between the VEGF-C and VEGFR-3 protein levels. High VEGF-C levels were associated with low-grade tumors and a smaller size. Univariate analysis showed that high VEGF-C was significantly associated with a favourable prognosis for disease-free survival (DFS) and overall survival (OS). No significant prognostic value of VEGFR-3 was detected. Multivariate analysis confirmed the independent prognostic value of VEGF-C. The intratumoral VEGF-C level is a significant prognostic indicator of primary breast cancer. An investigation of the mechanisms of VEGF-C protein processing in human cancer tissue should be carried out in the future.

Regulation of soluble vascular endothelial growth factor receptor (sFlt-1/sVEGFR-1) expression and release in endothelial cells by human follicular fluid and granulosa cells.

BACKGROUND: During the female reproductive cycle, follicular development and corpus luteum formation crucially depend on the fast generation of new blood vessels. The importance of granulosa cells and follicular fluid in controlling this angiogenesis is still not completely understood. Vascular endothelial growth factor (VEGF) produced by granulosa cells and secreted into the follicular fluid plays an essential role in this process. On the other hand, soluble VEGF receptor-1 (sFlt-1) produced by endothelial cells acts as a negative modulator for the bioavailability of VEGF. However, the regulation of sFlt-1 production remains to be determined. METHODS: We analyzed the influence of human follicular fluid obtained from FSH-stimulated women as well as of human granulosa cell conditioned medium on sFlt-1 production in and release from human umbilical vein endothelial cells (HUVEC) in vitro. Soluble Flt-1 gene expression was determined by RT-PCR analysis, amount of sFlt-1-protein was quantified by Sandwich-ELISA. RESULTS: Human follicular fluid as well as granulosa cell-conditioned medium significantly inhibit the production of sFlt-1 by endothelial cells on a posttranscriptional level. Treatment of cultured granulosa cells with either hCG or FSH had not impact on the production of sFlt-1 inhibiting factors. We further present data suggesting that this as yet unknown sFlt-1 regulating factor secreted by granulosa cells is not heat-sensitive, not steroidal, and it is of low molecular mass (< 1000 Da). CONCLUSION: We provide strong support that follicular fluid and granulosa cells control VEGF availability by down regulation of the soluble antagonist sFlt-1 leading to an increase of free, bioactive VEGF for maximal induction of vessel growth in the ovary.

Development of an enzyme-linked immunoreceptor assay (ELIRA) for quantification of the biological activity of recombinant human bone morphogenetic protein-2.

Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.

Vascular endothelial growth factor and its soluble receptor, Flt-1, are not correlated to erythropoietin in diabetics with normal or reduced renal function.

BACKGROUND AND AIMS: Recombinant erythropoietin upregulates the expression of the vascular endothelial growth factor (VEGF) receptors, Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), in endothelial cells. The integrity of the VEGF system seems to be crucial for the regulation of endothelial permeability and thus for the avoidance of renal protein leakage. As albuminuria/proteinuria is a hallmark of diabetic nephropathy, we examined cross-sectionally in 35 type 1 and 37 type 2 diabetic patients with various degrees of renal dysfunction and albuminuria whether there was an interrelationship between intrinsic erythropoietin (EPO) and VEGF/Flt-1. METHODS AND RESULTS: In patients with plasma creatinine values < or =1.5 (n = 53) or >1.5 mg/dL (n = 19), the mean serum EPO was 5.6 +/- 4.4 and 10.2 +/- 7.0 mU/mL (P = 0.02), respectively. In the two groups, urinary and serum VEGF(165) concentrations were similarly distributed (mean 94.3 +/- 91.8 vs 108 +/- 72.2 ng/L and 91.7 +/- 76.8 vs 91.9 +/- 74.9 ng/L, respectively; both P = NS). The mean urinary Flt-1 for the two groups amounted to 0.14 +/- 0.35 and 0.51 +/- 0.93 ng/mL (P = 0.045), respectively. No correlation between VEGF or Flt-1 and EPO was apparent. CONCLUSION: Our data suggest that in vivo EPO does not affect the functionality and/or production of components of the VEGF/Flt-1 system in diabetics with normal or reduced renal function.

Immunodetection and quantification of vascular endothelial growth factor receptor-3 in human malignant tumor tissues.

Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D), are the major molecules involved in developmental and pathological lymphangiogenesis. Here we describe for the first time the development of a specific indirect enzyme-linked immunosorbent assay (ELISA) for the quantification of VEGFR-3 in different human cell and tissue lysates. A combination of the goat polyclonal anti-VEGFR-3 antibody and the mouse monoclonal anti-human VEGFR-3 antibody was used. The assay was highly sensitive and reproducible with a detection range of 0.2-25 ng/ml. The assay was specific for VEGFR-3, with no cross-reactivity to VEGFR-1 or VEGFR-2. Complex formation with VEGF-C and VEGF-D had no effect on the sensitivity of the assay. The VEGFR-3 concentration in the lysates of cultured human dermal microvascular endothelial cells was 14-fold higher than in the lysates from human umbilical vein endothelial cells. In human kidney, breast, colon, gastric and lung cancer tissues the protein levels of VEGFR-3 were in the range of 0.6-16.7 ng/mg protein. Importantly, the level of VEGFR-3 protein detected in the ELISA correlated significantly with the number of VEGFR-3 positive vessels observed in histochemical sections, suggesting that the ELISA assay may be a reliable surrogate of measuring VEGFR-3-positive vessel density. The protein levels of VEGFR-3 in 27 renal cell carcinoma samples had a significant correlation with the levels of VEGF-C (p<0.001), or biological active, free VEGF-A (p<0.0001), but not with VEGFR-1 or total VEGF-A. This assay provides a useful tool for the investigations of the expression levels of VEGFR-3 in physiological and pathological processes, particular in cancer and in lymphangiogenesis-related disease.