Clinical relevance of intracellular vascular endothelial growth factor levels in B-cell chronic lymphocytic leukemia

Strong evidence exists for an association between high vascular endothelial growth factor (VEGF) levels and poor prognoses in patients with solid tumors and acute leukemia. Using Western blot analysis and solid-phase radioimmunoassay, we measured cellular VEGF levels in B-cell chronic lymphocytic leukemia (CLL) samples from 225 patients and correlated these levels with disease characteristics and prognoses. The median VEGF level in CLL samples was 7.26 times the median level detected in normal peripheral blood mononuclear cells. Patients with lower levels of VEGF protein showed a trend toward shorter survival (P =.07). However, in a subgroup of CLL patients with good prognoses or early-stage disease (Rai stages 0-II, Binet stages A,B; beta2-M 相似文献   

In vitro and in vivo angiogenesis in PC12 pheochromocytoma cells is mediated by vascular endothelial growth factor.

Angiogenesis, the formation of new blood vessels from existing vascular endothelium, is essential for tumor growth. Vascular endothelial growth factor (VEGF) is an endotheliumspecific mitogen and regulator of angiogenesis. Angiogenesis has been associated to the malignant phenotype of pheochromocytomas and is readily observed in experimental pheochromocytomas. Although VEGF gene expression has already been demonstrated in the rat PC12 cell line, the detailed mechanisms of action are not known. We have, therefore, studied angiogenesis in the rat PC12 pheochromocytoma cell line in vitro and in vivo. VEGF gene expression and accumulation of VEGF protein in cytoplasm and conditioned medium of PC12 cells was found. Conditioned medium from PC12 cells significantly increased proliferation of VEGF-dependent endothelial cells from human umbilical veins, and this effect reversed upon addition of a neutralizing anti-VEGF antibody. Dexamethasone and nerve growth factor (NGF) increased VEGF mRNA expression and accumulation of VEGF protein of PC12 subclones with established metastatic activity in vivo. PC12 cells xenotransplanted to nude mice had marked VEGF expression and induced host angiogenesis, confirmed by the presence of CD34-positive endothelial cells in the experimental PC12 tumors. When NGF-primed PC12 cells were immobilized in Matrigel supplemented with rising concentrations of the growth factor and xenotransplanted, increasing NGF resulted in tumors with smaller areas of necrosis and increased vital tumor volume. These results suggest that VEGF is a mediator of angiogenesis in the PC12 pheochromocytoma cell line, and that dexamethasone and NGF affect VEGF expression. Our data further suggest that NGF may contribute to angiogenesis in experimental pheochromocytoma.     

In vitro production of anti-RBC antibodies and cytokines in chronic lymphocytic leukemia

B-chronic lymphocytic leukemia (B-CLL) patients have a high prevalence of autoimmune phenomena, mainly autoimmune hemolytic anemia (AIHA). Immunoregulatory cytokines play a role in the regulation of both autoimmunity and leukemic B-cell growth. Mitogen-stimulated direct antiglobulin test (MS-DAT) is a recently described test able to disclose latent anti-RBC autoimmunity in AIHA. We investigated the prevalence of anti-RBC autoimmunity by MS-DAT and the pattern of cytokine production by PHA-stimulated whole blood cultures from 69 B-CLL patients and 53 controls. Results showed that anti-RBC IgG values in unstimulated, PHA-, PMA-, and PWM-stimulated cultures were significantly higher in B-CLL patients compared with controls. In B-CLL, the prevalence of anti-RBC autoimmunity was 28.9% by MS-DAT, compared with 4.3% by the standard DAT. Production of IFN-gamma, IL-2, IL-13, TNF-alpha, sCD23, and sCD30 was significantly increased in all B-CLL patients compared with controls, whereas there was no difference in IL-4, IL-6, IL-10, and TGF-beta production. Multivariate analysis showed that IL-4 was significantly increased in MS-DAT-positive compared with -negative patients. Patients with autoantibody positivity displayed greater IFN-gamma production than negative patients. These data are in line with the hypothesis that autoimmune phenomena in B-CLL are associated with an imbalance towards a Th-2-like profile. The elevated prevalence of anti-RBC autoimmunity found by MS-DAT suggests that an underestimated latent autoimmunity exists in B-CLL.     

Transforming growth factor beta induces vascular endothelial growth factor elaboration from pleural mesothelial cells in vivo and in vitro.

Vascular endothelial growth factor (VEGF) increases vascular permeability and is important in pleural effusion formation. In studies using transforming growth factor beta (TGF-beta) to produce pleurodesis, we observed that although TGF-beta was more effective than talc or doxycycline, it induced transient production of large pleural effusions. We hypothesized that TGF-beta stimulates VEGF production in pleural tissues in vivo, and by mesothelial cells in vitro. New Zealand White rabbits (n = 33) were given TGF-beta(2) (1.7 or 5.0 microg), talc (400 mg/kg), doxycycline (10 mg/kg), or buffer intrapleurally. Pleural fluid was collected at 24 h. Intrapleural injection of TGF-beta(2) induced a dose-dependent increase in VEGF production. The pleural fluid VEGF level was 2-fold higher in rabbits given 5.0 microg of TGF-beta(2) than in those given 1.7 microg of TGF-beta(2) and 3-fold higher than in those given buffer. VEGF levels were higher after the injection of TGF-beta(2) than after administration of talc or doxycycline. The pleural fluid VEGF correlated significantly with the volume of pleural effusions (r = 0.79, p < 0.00001). In vitro, TGF-beta(2) stimulated a dose-dependent increase in VEGF production from murine pleural mesothelial cells. At 4 and 24 h, TGF-beta(2), but not talc or doxycycline, induced a significant increase in VEGF, when compared with controls. The mesothelial cell VEGF production was significantly reduced by anti-TGF-beta antibody in the TGF-beta(2), talc, and control (buffer and medium) groups. In conclusion, mesothelial cells are an important source of VEGF. TGF-beta increases the VEGF production by mesothelial cells in vivo and in vitro.     

Transforming growth factor-beta stimulates vascular endothelial growth factor production by folliculostellate pituitary cells

TGF-beta isoforms are expressed in the anterior pituitary and modulate the growth and function of endocrine pituitary cells. Recently, TGF-beta has been shown to stimulate growth and basic fibroblast growth factor secretion in nonendocrine folliculostellate (FS) pituitary cells. We therefore studied whether the production of FS cell-derived vascular endothelial growth factor (VEGF), the most important regulator of vascular permeability and angiogenesis, is affected by TGF-beta. We observed by RT-PCR that TtT/GF cells, which are FS mouse pituitary tumor cells, synthesize TGF-beta1, -beta2, and -beta3. They also express TGF-beta receptors types 1 and 2, as well as Smad2, Smad3, and Smad4 proteins, which are essential for TGF-betabinding and signaling. Stimulation of TtT/GF cells with either TGF-beta1 or TGF-beta3 induced a rapid translocation of Smad2 into the cell nuclei. Both TGF-beta isoforms dose dependently stimulated VEGF production in TtT/GF cells, but not in lactosomatotroph GH3 cells. Time-course studies and suppression of TGF-beta-induced VEGF production by cycloheximide suggest that TGF-beta induces de novo synthesis of VEGF in folliculostellate cells, which is completely blocked by dexamethasone. In primary rat pituitary cell cultures, TGF-beta1 and -beta3 stimulated VEGF production. TGF-beta stimulation of VEGF production by folliculostellate cells could modulate intrapituitary vascular permeability and integrity as well as angiogenesis in an auto-/paracrine manner.     

Circulating endothelial cells in patients with chronic lymphocytic leukemia

Angiogenesis is increased in B-cell chronic lymphocytic leukemia (B-CLL). We wanted to quantify and characterize the circulating endothelial cells (CECs) in patients with B-CLL and correlate with plasma angiogenesis-related factors. Using a four-color flow cytometry, we prospectively analyzed the CEC in the whole blood of 20 healthy controls and 20 patients with B-CLL. We quantified (CD45−/CD31+/CD146+) and characterized the CECs according to whether they were apoptotic (annexin stain) or activated (CD106+). We also measured plasma levels of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and thrombospondin-1 (TSP-1). Most patients (90%) had Rai stages 0–2 at the time of diagnosis. As a group, B-CLL patients had higher number of CECs (median of 26.5 cells/ml) compared (P = 0.04) to healthy controls (18.5 cells/ml). However, only four (20%) patients had elevated CEC counts, defined as ≥2 SD of the control mean (≥53 cells/ml). The proportions of apoptotic (P = 0.83) and activated (P = 0.12) CECs were similar in both groups. B-CLL patients had higher FGF-2 (P < 0.001), lower TSP-1 (P = 0.004), and similar VEGF (P = 0.27) plasma levels. The number of CECs was not associated with Rai stage, absolute lymphocyte count, or levels of angiogenesis-related factors. CECs are increased in only a small fraction of B-CLL patients in our cohort with low rates of apoptosis and activation. While no correlation was found between CECs and clinical features, more studies in a larger patient sample size and advanced disease are necessary. Supported by the Gundersen Lutheran Center for Cancer and Blood Disorders and the Gundersen Lutheran Medical Foundation. The Greater Richland Area Cancer Elimination (GRACE), Inc. and the Gundersen Lutheran Medical Foundation funded this study.     

Plasma levels of basic fibroblast growth factor and vascular endothelial growth factor and their association with IgVH mutation status in patients with B-cell chronic lymphocytic leukemia

The mutation status of genes encoding the variable region of immunoglobulin heavy chains (IgV(H)) is a strong predictor of disease progression and survival in B-cell chronic lymphocytic leukemia (B-CLL). We investigated whether there is an association between the concentration of both vascular endothelial growth factor and basic fibroblast growth factor and IgV(H) mutation status in 49 untreated B-CLL patients.     

Prognostic significance of cellular vascular endothelial growth factor expression in chronic phase chronic myeloid leukemia

The impact of elevated vascular endothelial growth factor (VEGF) expression on the course of chronic myeloid leukemia (CML) is unknown. By radioimmunoassay, we measured pretreatment cellular VEGF protein in bone marrow samples from 184 (148 chronic and 36 accelerated/blastic phases) CML patients and found the levels to be 1.6-fold higher than in 31 normal control bone marrow samples (P =.000 01). No significant differences were found in VEGF levels by different phases of CML (P =.1). VEGF levels correlated with older age (P =.01) and higher platelet count (P =.0003), but also with smaller spleen size (P =.004), lower white blood cell count (P =.0006), and lower percentage of peripheral blasts (P =.04). With the use of Cox proportional hazard model and VEGF levels as a continuous variable, high VEGF levels correlated with shorter survival of patients in chronic CML (P =.008). Multivariate analysis showed that VEGF was not independent of the synthesis stage (P =.09). These data suggest that VEGF plays a role in the biology of CML and that VEGF inhibitors should be investigated in CML.     

Transgenic overexpression of vascular endothelial growth factor-B isoforms by endothelial cells potentiates postnatal vessel growth in vivo and in vitro

Vascular endothelial growth factors (VEGFs) play significant roles in endothelial growth, survival, and function, and their potential use as therapeutic agents to promote the revascularization of ischemic tissues in being avidly explored. VEGF-A has received most attention, as it is a potent stimulator of vascular growth. Results in clinical trials of VEGF-A as a therapeutic agent have fallen short of high expectations because of serious edematous side effects caused by its activity in promoting vascular permeability. VEGF-B, a related factor, binds some of the VEGF-A receptors but not to VEGF receptor 2, which is implicated in the vascular permeability promoting activity of VEGF-A. Despite little in vitro evidence to date for the ability of Vegf-B to directly promote angiogenesis, recent data indicate that it may promote postnatal vascular growth in mice, suggesting that it may have potential therapeutic application. We have specifically studied the effects of VEGF-B on vascular growth in vivo and on angiogenesis in vitro by analyzing transgenic mice in which individual isoforms (VEGFB167Tg and VEGFB186Tg) of VEGF-B are overexpressed in endothelial cells. VEGFB167Tg and VEGFB186Tg mice displayed enhanced vascular growth in the Matrigel assay in vivo and during cutaneous wound healing. In the aortic explant assay, explants from VEGFB167Tg and VEGFB186Tg mice displayed elevated vascular growth, suggesting a direct effect of VEGF-B isoforms in potentiating angiogenesis. These data support the use of VEGF-B as a therapeutic agent to promote vascular growth, in part, by potentiating angiogenesis. Furthermore, the lack of vascular permeability activity associated with either transgenic overexpression of the VEGF-B gene in endothelial cells or application of VEGF-B protein to the skin of mice in the Miles assay indicates that use of VEGF-B as a therapy should not be associated with edematous side effects.     

Recombinant thrombomodulin inhibits thrombin-induced vascular endothelial growth factor production in neuronal cells

Thrombin is a serine protease which is generated from its precursor prothrombin by the activation of the blood coagulation cascade. Thrombin converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a central role in thrombosis and hemostasis by regulating platelet aggregation and blood coagulation. Here, we show that thrombn enhanced vascular endothelial growth factor (VEGF) production in a dose- and time-dependent manner in the supernatant of cultured PC-12 cells, as determined by enzyme-linked immunosorbent assay (ELISA). Thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) exerted an effect similar to thrombin on VEGF production. Thrombin-induced VEGF production was significantly attenuated by recombinant human thrombomodulin (rTM) and its minimal functional domain E456. Furthermore, the antioxidant N-acetyl-L-cysteine (NAC) markedly inhibited thrombin-induced VEGF production. Thus, rTM and NAC apparently inhibited the effect of thrombin on VEGF production in neuronal cells.     

Intracellular tumor necrosis factor production by T- and B-cells in B-cell chronic lymphocytic leukemia

BACKGROUND AND OBJECTIVES: The pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) has been linked with the production and activity of certain growth factors. Tumor necrosis factor (TNF-alpha) is important for the growth and survival of B-CLL cells. TNF-alpha promotes the proliferation of the malignant cell clone and is believed to play a role in the progression of B-CLL. The aim of our study was to examine the level of production and intracellular expression of TNF-alpha by T- and B-cells in B-CLL in correlation with stage of disease and clinical parameters. DESIGN AND METHODS: Using a three-color flow cytometry technique we analyzed intracellular TNF-alpha expression by B-cells (CD19+) and by T-cell subsets (CD3+/CD4+ and CD3+/CD8+) in peripheral blood (PB) and bone marrow (BM) from 40 patients with B-CLL and 24 healthy controls. RESULTS: A higher number of TNF-a-positive B-cells were found in PB and BM in B-CLL patients than in normal controls. In BM this difference was statistically significant (p<0.007). Likewise, in PB and BM the percentage of T-cells expressing TNF-alpha was significantly higher in B-CLL patients than in normal controls (PB: p<0.00001; BM: p<0.007). CD3+/CD4+ cells from patients displayed a lower level of intracellular TNF-alpha expression than did CD3+/CD8+ cells (PB: p<0.0001; BM: p<0.04). The number of T-cells expressing TNF-alpha in B-CLL patients was higher in those with stages III-IV than in patients with early stage disease (PB: p<0.007; BM: p<0.01). Additionally, PB and BM T-cell subsets from patients in stages III-IV had a statistically significant higher level of cytoplasmic TNF-a expression than the corresponding cells from healthy controls (PB: p<0.02; BM: p<0.05). In PB the percentage of CD4+ and CD8+ T-cells expressing cytoplasmic TNF-alpha positively correlated with the stage of disease, total tumor mass (TTM) score and lymphocytosis. In BM only the percentage of CD8 T-cells positively correlated with TTM score and lymphocytosis. The expression of TNF-alpha in leukemic B-cells did not correlate with any progression parameters of disease. INTERPRETATION AND CONCLUSIONS: The results obtained suggest that malignant B-cells are exposed to large numbers of T-cells able to synthesize and secrete TNF-alpha. Moreover, T-cells even though fewer than B-cells may be an important source of TNF-a in advanced stages of disease. This indicates that the TNF-alpha can be associated with progression of B-CLL and may be implicated in some side-effects associated with this disease.     

Proliferation of B cells from chronic lymphocytic leukemia is selectively promoted by B cell growth factor

B cell activation was studied in B cells from B cell chronic lymphocytic leukemia (B-CLL) patients. After in vitro stimulation, these B cells showed extensive proliferation in the presence of high-molecular-weight B cell growth factor (BCGF). In contrast, this effect was not observed upon addition of recombinant interleukin-2 (IL-2). In agreement, upon stimulation, B cells expressed Bac-1 antigen but failed to acquire the IL-2 receptor. These results demonstrate that the utilization of the BCGF pathway can be segregated from that of IL-2 in B cells from B-CLL patients.     

In vitro evaluation of vascular endothelial growth factor (VEGF)-eluting stents

BACKGROUND: Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen. It promotes re-endothelialisation of the damaged vessel surface seen after stenting. Stent thrombosis and in-stent restenosis are partly related to endothelial denudation caused by stent implantation. We propose using hydrocarbon polymer-coated stents immersed in VEGF to speed re-endothelialisation and reduce the risk of stent thrombosis and restenosis. METHODS: Stents (3 x 20 mm) were immersed in VEGF solutions and maximal VEGF absorption calculated. VEGF release from these stents was measured in a perfusion circuit. Delivery of VEGF to arterial wall was measured. Sterile VEGF-loaded stents were cultured with human umbilical vein endothelial cells. RESULTS: 18.5 +/- 4.1 microg VEGF was absorbed, 80% of which was released over 9 days; 11 +/- 6.8% of the initial VEGF loaded was delivered to the vessel wall. Cells exposed to VEGF-eluting stents showed an 11% increase in growth relative to controls. CONCLUSION: VEGF may be a candidate for stent-based delivery and may increase the rate of endothelialisation in vivo.     

In vitro leukocyte thymidine uptake and prognosis in chronic lymphocytic leukemia.

Among 60 patients with chronic lymphocytic leukemia, higher in vitro uptake of tritiated (3H) thymidine by leukocytes of a standard volume of peripheral blood was associated with a higher lymphocyte count, a more advanced stage, greater frequency of functional impairment and shorter survival. Appropriate analyses demonstrated that leukocyte thymidine uptake correlated with survival independently of these other disease features. Relative thymidine uptake (radioactivity per 10(3) lymphocytes) did not prove to be a useful prognostic parameter. Among 33 patients not receiving antileukemic therapy at the time of study, 15 of 17 (88 per cent) of those with higher thymidine uptake values, but only 3 of 16 (19 per cent) of those with lower values, were treated during a median follow-up period of four and a half years (p less than 0.001). Seven of the former group, but none of the latter group, died during the first three years of follow-up (p less than 0.01). We conclude that thymidine uptake by circulating leukocytes constitutes a relatively accurate index of the proliferating leukemic cell mass in this disease and provides useful prognostic information.     

Receptors for tumor necrosis factor on neoplastic B cells from chronic lymphocytic leukemia are expressed in vitro but not in vivo

Recombinant tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces proliferation of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL). To gain insight into the mechanisms involved in regulating TNF responsiveness, we have examined TNF receptor expression on neoplastic B-CLL cells. We have demonstrated that freshly isolated neoplastic B cells from patients with CLL did not express TNF receptors. After 1 day of incubation in culture medium, TNF receptors were detectable in the range of 540 to 1,500/cell. Kinetic experiments revealed that receptor expression was half-maximal after 3 hours of culturing and required de novo protein synthesis. The Scatchard plots of TNF-alpha binding indicated a single set of high-affinity TNF receptors with a dissociation constant of 70 pmol/L. TNF receptor expression in vitro was found in all examined cases. All cytokines tested, with the exception of IL-2, did not influence the expression of TNF receptors. The TNF receptor expression is enhanced in B-CLL cells cultured in the presence of interleukin-2 when compared with the receptor expression of cells cultured in medium alone. Our data suggest that neoplastic B-CLL cells in patients with stable disease do not express TNF receptors in vivo and that an unknown mechanism suppressing TNF receptor expression in vivo may play a role in growth regulation of neoplastic B cells.     

c-fos-induced growth factor/vascular endothelial growth factor D induces angiogenesis in vivo and in vitro

c-fos-induced growth factor/vascular endothelial growth factor D (Figf/Vegf-D) is a secreted factor of the VEGF family that binds to the vessel and lymphatic receptors VEGFR-2 and VEGFR-3. Here we report that Figf/Vegf-D is a potent angiogenic factor in rabbit cornea in vivo in a dose-dependent manner. In vitro Figf/Vegf-D induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in primary human umbilical cord vein endothelial cells (HUVECs) and in an immortal cell line derived from Kaposi's sarcoma lesion (KS-IMM). The treatment of HUVECs with Figf/Vegf-D induces dose-dependent cell growth. Figf/VEGF-D also induces HUVEC elongation and branching to form an extensive network of capillary-like cords in three-dimensional matrix. In KS-IMM cells Figf/Vegf-D treatment results in dose-dependent mitogenic and motogenic activities. Taken together with the previous observations that Figf/Vegf-D expression is under the control of the nuclear oncogene c-fos, our data uncover a link between a nuclear oncogene and angiogenesis, suggesting that Figf/Vegf-D may play a critical role in tumor cell growth and invasion.