Intercellular adhesion molecule-1 in extravasation of normal mononuclear and leukaemia cells.

Interaction of intercellular adhesion molecules (ICAMs) with their receptors has a key role in normal leucocyte adhesion and migration, whereas in leukaemia this has not been well established. In this study, we have evaluated the roles of different adhesion molecules in normal and leukaemia cell extravasation in a novel organotypic model for vessel wall and measured plasma ICAM-1 and -2 levels in acute leukaemia patients at diagnosis and during chemotherapy. We found that both normal mononuclear cells and blast cells from acute leukaemia patients, as well as retinoic acid-treated promyelocytic leukaemia cells, rapidly extravasated through endothelial cell layers into the underlying collagen matrix. ICAM-1 antibody prevented the extravasation, while antibodies to other adhesion molecules showed little (CD18, ICAM-2) or no inhibition (CD11a and ICAM-3). Soluble ICAM-1 (sICAM-1) protein had no effect. We also observed increased plasma sICAM-1 and -2 levels in leukaemia patients and found that they correlated only weakly with the white blood cell count. No correlation was found between sICAM-1 or -2 levels and the response to therapy. Although elevated sICAM-2 levels decreased rapidly during chemotherapy, sICAM-1 levels did not. Because sICAM-1 protein had no effect on leukaemia cell extravasation in vitro, it is probable that the increased plasma sICAM-1 levels in leukaemia patients may not play a role in leukaemia cell infiltration. However, as we showed that ICAM-1 mediated leukaemia cell extravasation on the cell surface, it is possible that cellular ICAM-1 has an important role in disease progression.

Soluble urokinase receptor levels correlate with number of circulating tumor cells in acute myeloid leukemia and decrease rapidly during chemotherapy.

The importance of plasminogen activation, mediated by urokinase (uPA) and its receptor (uPAR), is well established in many physiologica and pathological processes, such as in cell migration and tumor-cell invasion. Recently, additional functions have been described for uPA and uPAR, particularly in cell adhesion and chemotaxis. The amounts of uPA and uPAR in various tumor types and in the plasma/serum samples of cancer patients have been shown to correlate with survival prognosis, indicating the relevance of these molecules in malignancy. We previously showed that in acute myeloid leukemia, a high level of plasma soluble uPAR (suPAR) at diagnosis correlates with poor response to chemotherapy. However, in this case, as in other cancers, the origin of suPAR is unknown. Therefore, we have now analyzed uPAR in cells, plasma, and urine of patients with acute leukemia (n = 35) at 0, 5, 14, 28, and 56 days after start of chemotherapy. In response to cytotoxic treatment, suPAR levels decreased rapidly, and the decreasing plasma suPAR (p-suPAR levels correlated highly with decreasing numbers of circulating tumor cells, suggesting that the elevated p-suPAR was produced by circulating tumor cells. Moreover, the p-suPAR level appeared to correlate with the amount of uPAR in tumor cell lysates at diagnosis. Our results also show for the first time that in lysates of circulating tumor cells, studied by immunoprecipitation and immunoblotting, uPAR was partly in fragmented form, whereas only full-length uPAR was found in normal leukocytes. We also detected fragmented suPAR in peripheral blood plasma, in urine, and especially in the plasma compartment of bone-marrow aspirates of acute myeloid leukemia patients, in a pattern differing considerably from that found in healthy individuals. Because proteolytic cleavage of uPAR induces a potent chemotactic response in vitro, it is possible that these fragments may play a role in the pathophysiology of acute leukemia.

Blast cell-surface and plasma soluble urokinase receptor in acute leukemia patients: relationship to classification and response to therapy.

Plasminogen activation in leukemia has been less well characterized than in other malignancies. However, the increased tendency to bleeding and tissue infiltration by leukemic cells are processes in which plasminogen activation may be involved. We have examined plasma and the peripheral blood mononuclear cell fraction from 80 patients including 53 patients with newly diagnosed acute leukemia and 27 patients with other hematological disorders as well as 21 healthy controls. In 28 of 29 examined patients with acute myeloid leukemia (AML) and in two of three patients with hybrid leukemia we found urokinase receptor (uPAR) on the cell surface, while most (7/9) samples from patients with acute lymphoblastic leukemia (ALL) were negative for uPAR. The plasma mean value for soluble uPAR (suPAR) was significantly elevated in patients with AML and ALL. In AML the highest values were found in patients who had residual disease after several cycles of chemotherapy. Compared to controls the uPA antigen levels in patient plasmas were elevated and decreased along with uPAR during treatment. Our results suggest that cell surface uPAR may be a useful marker for leukemia classification and in our material a high level of plasma suPAR correlated with resistance to chemotherapy in AML.

Plasminogen activation in human leukemia and in normal hematopoietic cells.

The active process of pericellular proteolysis is central in tumor invasion, and in particular the essential role of the urokinase-type plasminogen activator (uPA) is well established. uPA-mediated plasminogen activation facilitates cell migration and invasion through extracellular matrices by dissolving connective tissue components. uPA, its receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) are enriched in several types of tumors. The importance of proteolysis and especially plasminogen activation is less clear in hematopoietic malignancies than in solid tumors. However, patients with leukemia have an increased tendency to bleeding, not always attributable to thrombocytopenia, and tissue infiltration by leukemic cells, processes in which plasminogen activation may be involved. Several studies have indicated that plasminogen activators (PAs) are highly expressed by cultured leukemia cells. Furthermore, differing from adherent tumor cells, leukemic cells have an enhanced capacity to activate pro-uPA and mainly the active form of uPA is released to culture medium. Ex vivo studies have shown that uPAR, uPA and its inhibitors can be found on the surface of normal blood cells and on the blast cell surfaces from patients with acute leukemia as well as from plasma samples. Elevated levels of PAs and their inhibitors have been detected in leukemic cell lysates. Few studies have tried to demonstrate a correlation between prognosis of leukemia and levels of plasminogen activators. More in vivo studies are needed to show, if any of the factors of the plasminogen activation process can be used as tools in subclassification or as markers for prognosis in leukemia. This review article will focus on the in vivo studies of plasminogen activation in leukemia and will present several in vitro findings on PAs in normal leukocytes and leukemic cell lines.

Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3.

Lymphatic vessels have been difficult to study in detail in normal and tumor tissues because of the lack of molecular markers. Here, monoclonal antibodies against the extracellular domain of the vascular endothelial growth factor-C receptor that we have named VEGFR-3 were found to specifically stain endothelial cells of lymphatic vessels and vessels around tumors such as lymphoma and in situ breast carcinoma. Interestingly, the spindle cells of several cutaneous nodular AIDS-associated Kaposi's sarcomas and the endothelium around the nodules were also VEGFR-3 positive. The first specific molecular marker for the lymphatic endothelium should provide a useful tool for the analysis of lymphatic vessels in malignant tumors and their metastases and the cellular origin and differentiation of Kaposi's sarcomas.

Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells.

We generated a panel of monoclonal antibodies against the extracellular domain of the Tie receptor tyrosine kinase and studied its expression in human haemopoietic and tumour cell lines and in samples from leukaemia patients. Most of the erythroblastic/megakaryoblastic (6/8), 2/7 myeloid and 3/6 B-lymphoblastic leukaemia cell lines were Tie-positive. The erythroblastic/megakaryoblastic leukaemia cell lines also expressed the related Tie-2/Tek gene and, surprisingly, its recently cloned ligand gene angiopoietin-1, which was located in chromosome 8q23.1. In addition, 16% of freshly isolated leukaemia samples were Tie positive. Peripheral blood mononuclear cells were Tie negative, but a few Tie positive cells were found in immunoperoxidase staining of mobilized peripheral blood stem cells. Long-term culture of isolated umbilical cord blood CD34+ Tie+ and CD34+ Tie- cells indicated that the Tie+ fraction contained a slightly higher frequency of cobblestone area forming cells (CAFC). Thus, Tie is expressed on haemopoietic progenitor cells and some leukaemic blasts. The coexpression of Tie-2 and angiopoietin-1 in megakaryoblastic leukaemia cell lines suggests the existence of an autocrine ligand/receptor signalling loop in these cells.

Functional and phenotypic characterization of cord blood and bone marrow subsets expressing FLT3 (CD135) receptor tyrosine kinase.

The class III receptor tyrosine kinase FLT3/FLK2 (FLT3; CD135) represents an important molecule involved in early steps of hematopoiesis. Here we compare cell-surface expression of FLT3 on bone marrow (BM) and cord blood (CB) cells using monoclonal antibodies (MoAbs) specific for the extracellular domain of human FLT3. Flow cytometric analysis of MACS-purified BM and CB cells showed that 63% to 82% of BM CD34+ and 88% to 95% of the CB CD34+ cells coexpress FLT3. Clonogenic assays and morphological characterization of FACS-sorted BM CD34+ cells demonstrate that colony-forming unit-granulocyte-macrophage (CFU-GM) and immature myelo-monocytic precursor cells are enriched in the subpopulation staining most brightly with the FLT3 MoAb whereas the majority of the burst-forming units-erythroid (BTU-E) and small cells with lymphoid morphology are found in the FLT3- population. In contrast, statistically indistinguishable proportions of CFU-granulocyte-erythrocyte-megakaryocyte-macrophage (CFU-GEMM) and more primitive cobblestone area forming cells (CAFC) were detected in both fractions, albeit the FLT3+ fraction consistently showed more CAFC activity than the FLT3- fraction. Although in both, BM and CB the majority of CD34+CD117+ (KIT+), CD34+CD90+ (Thy-1+), and CD34+CD109+ cells coexpress FLT3, three-color phenotypic analyses are consistent with the functional findings and suggest that the most primitive cells defined as CD34+CD38-, CD34+CD71low, CD34+HLA-DR-, CD34+CD117low, CD34+CD90+, and CD34+CD109+ express low levels of cell-surface FLT3 and were therefore not enriched to a statistically significant extent with the bright versus negative sorting scheme. Thus, clear segregation of the most primitive progenitors from BM CD34+ cells was confounded by low apparent levels of FLT3 cell-surface expression on these cells, whereas myeloid progenitors unambiguously segregated with the FLT3 brightest cells and erythroid progenitors with the FLT3 dimmest. Additional phenotypic analyses using MoAbs against progenitor/stem cell markers including the mucinlike molecule MGC-24v (CD164), the receptor tyrosine kinases TIE, FMS (CD115), and KIT (CD117) further illustrate the differences in surface antigen expression profiles of BM and CB CD34+ cells. Notably, CD115 is rarely detected on CB CD34+ cells, whereas 20% to 25% of the BM CD34+FLT3+ cells are CD115+. Furthermore, 80% to 95% of the CB CD34+CD117+ but only 60% to 75% of the BM CD34+CD117+ cells coexpress FLT3. Only a negligible amount of CD34+CD19+ are detected in CB, while in BM 20% to 30% of CD34+CD19+ presumed pro/pre-B cells coexpress FLT3. In contrast, the majority of the CD34+CD164+ and CD34+TIE+ subsets in both CB and BM coexpress FLT3. Analysis of unseparated cells showed that FLT3 expression is not restricted to CD34+ subsets. About 65% to 70% of lymphocyte-gated BM CD34-FLT3+ cells are positive for the monocytic marker CD115 whereas 25% to 30% of these cells consist of CD10 expressing B-cell precursors. Finally, CD34- monocytes in BM, CB, and PB express FLT3 whereas granulocytes are FLT3-. Our data show that detectable FLT3 appears first at low levels on the surface of primitive multilineage progenitor cells and disappears during defined stages of B-cell development, but is upregulated and maintained during monocytic maturation.

Expression of Mad, an antagonist of Myc oncoprotein function, in differentiating keratinocytes during tumorigenesis of the skin.

The Myc oncoprotein is associated with cell proliferation and is often down-regulated during cell differentiation. The related Mad transcription factor, which antagonises Myc activity, is highly expressed in epidermal keratinocytes. Mad also inhibits cell proliferation in vitro. To study Mad expression in keratinocyte proliferation and differentiation, we have analysed Mad RNA expression in regenerating and hyperproliferative epidermal lesions and epidermal tumours of varying degrees of differentiation using the RNA in situ hybridisation and RNAase protection techniques. Mad was strongly expressed in differentiating suprabasal keratinocytes in healing dermal wounds and in benign hyperproliferative conditions, but also in squamous cell carcinomas, in which the keratinocytes retain their differentiation potential. However, Mad expression was lost in palisading basal carcinoma cells and poorly differentiated squamous cell carcinomas, which lacked the epithelial differentiation marker syndecan-1. We therefore suggest that Mad expression is closely associated with epithelial cell differentiation, and that this association is retained in epithelial tumours of the skin.

The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells.

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting-gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.

Myeloid lineage involvement in acute lymphoblastic leukemia: a morphology antibody chromosomes (MAC) study.

We looked for clonal chromosomal abnormalities in myeloid cell lineages in the bone marrow aspirates from six children with acute lymphoblastic leukemia (ALL). The study was carried out using a combination of MAC (morphology, antibody, chromosomes) and in situ hybridization procedures. In patients whose leukemic cells expressed only lymphoid antigens, we found chromosomal aberrations in CD10- and CD20/22-positive lymphoid cells. Mature CD22+ and CD3+ lymphocytes did not have the chromosomal aberrations. In one patient whose leukemic cells also expressed myeloid-associated antigens, the clonal chromosome aberrations were seen not only in the CD10+ and CD19+ blasts, but also in glycophorin A-positive morphologically nonleukemic erythroblasts.

Expression of the mad gene during cell differentiation in vivo and its inhibition of cell growth in vitro.

Mad is a basic region helix-loop-helix leucine zipper transcription factor which can dimerize with the Max protein and antagonize transcriptional activation by the Myc-Max transcription factor heterodimer. While the expression of Myc is necessary for cell proliferation, the expression of Mad is induced upon differentiation of at least some leukemia cell lines. Here, the expression of the mad gene has been explored in developing mouse tissues. During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain. At later stages mad expression was detected in neuroretina, epidermis, and whisker follicles, and in adult mice mad was expressed at variable levels in most organs analyzed. Interestingly, in the skin mad was highly expressed in the differentiating epidermal keratinocytes, but not in the underlying proliferating basal keratinocyte layer. Also, in the gut mad mRNA was abundant in the intestinal villi, where cells cease proliferation and differentiate, but not in the crypts, where the intestinal epithelial cells proliferate. In the testis, mad expression was associated with the completion of meiosis and early development of haploid cells. In cell culture, Mad inhibited colony formation of a mouse keratinocyte cell line and rat embryo fibroblast transformation by Myc and Ras. The pattern of mad expression in tissues and its ability to inhibit cell growth in vitro suggests that Mad can cause the cessation of cell proliferation associated with cell differentiation in vivo.

BMX, a novel nonreceptor tyrosine kinase gene of the BTK/ITK/TEC/TXK family located in chromosome Xp22.2.

The Bmx sequence was identified and cloned during our search for novel tyrosine kinase genes expressed in human bone marrow cells. Bmx cDNA comprises a long open reading frame of 675 amino acids, containing one SH3, one SH2 and one tyrosine kinase domain, which are about 70% identical with Btk, Itk and Tec and somewhat less with Txk tyrosine kinase sequences. The amino terminal sequences of these four tyrosine kinases are about 40% identical and each contains a so-called pleckstrin homology domain. The 2.7 kb Bmx mRNA was expressed in endothelial cells and several human tissues by Northern blotting and an 80 kD Bmx polypeptide was detected in human endothelial cells. Immunoprecipitates of COS cells transfected with a Bmx expression vector and NIH3T3 cells expressing a Bmx retrovirus contained a tyrosyl phosphorylated Bmx polypeptide of similar molecular weight. The BMX gene was located in chromosomal band Xp22.2 between the DXS197 and DXS207 loci. Interestingly, chromosome X also contains the closest relative of BMX, the BTK gene, implicated in X-linked agammaglobulinemia. The BMX gene thus encodes a novel nonreceptor tyrosine kinase, which may play a role in the growth and differentiation of hematopoietic cells.

Power of the MAC (morphology-antibody-chromosomes) method in distinguishing reactive and clonal cells: report of a patient with acute lymphatic leukemia, eosinophilia, and t(5;14).

We present a patient with acute lymphatic leukemia, eosinophilia, and a 5;14-translocation, a rare but well-documented condition. In order to clarify whether granulocytes were involved in the disease, we applied the MAC (Morphology-Antibody-Chromosomes) technique to samples of the bone marrow and, during a central nervous system relapse, to those of the cerebrospinal fluid. The karyotype of the blast cells was 47,XY, + X,t(5;14)(q31;q32),i(7)(q10). Interphase cytogenetic study by in situ hybridization with an X-specific alphoid probe revealed the abnormality in CD10, CD19, and TdT (terminal deoxynucleotidyl transferase) positive lymphoid cells, whereas CD13 positive, Sudan black B positive, eosinophilic, and basophilic granulocytes as well as monocytes and small lymphocytes did not have the abnormality. Our results show that the eosinophilic and basophilic granulocytes in this subtype of acute leukemia do not belong to the malignant clone but are reactive. This study also confirmed the usefulness of the MAC technique in distinguishing neoplastic and reactive cells in malignancy.

Expression of tie receptor tyrosine kinase in leukemia cell lines.

The tie receptor tyrosine kinase mRNA was originally identified as an amplified product in reverse transcription-polymerase chain reaction analysis of human K562 leukemia cell RNA. In situ hybridization analysis revealed that the corresponding mouse gene is expressed predominantly in endothelial cells. We have explored tie mRNA and protein expression in tumor cell lines. The 4.4 kb tie mRNA was expressed at high levels in five of five human megakaryoblastic leukemia cell lines studied and in two IL-3-dependent mouse myeloid leukemia cell lines, but not in 42 other leukemia cell lines representing various hematopoietic lineages. Increased expression of tie mRNA and protein was observed upon treatment of the megakaryoblastic leukemia cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA), known to enhance megakaryoblastic markers. Among several cell lines from solid tumors, two fibrosarcomas, one rhabdomyosarcoma and one melanoma cell line were positive for tie mRNA. These results suggest that among hematopoietic lineages tie is predominantly expressed in cells with megakaryoblastic properties and that the tie tyrosine kinase is a receptor for a regulatory factor specific for megakaryoblasts, endothelial cells, and occasional tumor cell lines derived from mesenchymal tissues.

L-myc and N-myc in hematopoietic malignancies.

The myc proto-oncogenes encode nuclear DNA-binding phosphoproteins which regulate cell proliferation and differentiation. The c-myc gene is implicated in hematopoietic malignancies on the basis of its frequent deregulation in naturally occurring leukemias and lymphomas. Recent evidence suggests that also the N-myc and L-myc genes may have a role in normal and malignant hematopoiesis. N-myc and to a certain degree L-myc can substitute for c-myc in transformation assays in vitro, and their overexpression can block the differentiation of leukemia cell lines. Immunoglobulin heavy chain enhancer (IgH) -driven overexpression of N-myc or L-myc genes cause lymphatic and myeloid tumors, respectively, in transgenic mice. Furthermore, the L-myc and N-myc genes are expressed in several human leukemias and leukemia cell lines, L-myc predominantly in myeloid and N-myc both in myeloid and in some lymphoid leukemias. All N/L-myc positive leukemias and leukemia cell lines coexpress the c-myc gene, thus exemplifying a lack of negative cross-regulation between the different myc genes in leukemia cells. Taken together, these data suggest that L-myc and N-myc may participate in the growth regulation of hematopoietic cells.

Abundant urokinase activity on the surface of mononuclear cells from blood and bone marrow of acute leukemia patients.

We have examined the mononuclear cell fraction from 35 individuals, 18 with hematologic malignancies and 17 healthy controls for the presence of cell surface-associated plasminogen activator (PA) activity. PA activity was found on the cell surface of 10 out of 12 samples from patients with acute leukemia. In addition to active urokinase (uPA) found on the cell surface in four out of five acute myeloid leukemia patients, tissue-type PA activity was detected in the same samples (3 of 5). Two out of four samples from acute lymphoid leukemia displayed only uPA activity and three out of three samples from biphenotypic leukemia were also clearly uPA-positive. Plasmin activity was not detected in any of the samples. PA activity was not found on the surface of mononuclear cells from either patients with chronic lymphoid leukemia or healthy controls and, in this respect, the cell surface-bound uPA activity behaved as a marker for acute leukemia. The finding of PA activity on the cell surface in acute leukemia suggests that there may be continuous generation of plasmin with consequent consumption of plasma plasmin inhibitors.

Alternative mRNA forms and open reading frames of the max gene.

The max gene encodes a heterodimeric partner of Myc. We have recently identified an alternative max mRNA (delta max) that contains an additional internal exon introducing an in-frame translational termination. Here we have studied the expression of human max mRNAs by Northern blotting analysis. In addition to the major 2.3-kb mRNA form, four bands were identified. Our results indicate that these bands represent differentially spliced mRNA forms, which contain altogether three open reading frames. In addition to the previously identified Max and delta Max proteins, sequence analysis of a 3.5-kb mRNA form predicted a protein that resembles delta Max in structure. Like delta Max, this protein enhanced the number of transformed foci in the ras-myc co-transformation assay. Although the 3.5-kb mRNA represents a minor form in actively proliferating cells, a shift from the major 2.3-kb mRNA to the 3.5-kb form was observed in response to high cell density or acidification of the growth medium. Our results indicate the presence of several differentially spliced mRNA forms of the max gene, and suggest a possible mechanism for the production of functionally distinct Max proteins.

FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines.

The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.