Antiangiogenesis is produced by nontoxic doses of vinblastine

The effects of vinblastine (VBL) on endothelial cell functions involved in angiogenesis, namely proliferation, chemotaxis, spreading on fibronectin (FN), secretion of matrix-metalloproteinase-2 (MMP-2) and MMP-9, and morphogenesis on Matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo by using the chick embryo chorioallantoic membrane (CAM) model. In vitro, at noncytotoxic doses (0.1, 0.25, 0. 5, 0.75, and 1 pmol/L), VBL impacted all these functions, except secretion of MMPs, in a dose-dependent fashion. By contrast, proliferation of other primary cells such as fibroblasts and lymphoid tumor cells was not impacted. In vivo, VBL at 0.5, 0.75, and 1 pmol/L again displayed a dose-dependent antiangiogenic activity. Lack of cytotoxicity in vitro and in vivo was shown both morphologically, and also because the antiangiogenic effects were rapidly abolished when VBL was removed. Apoptosis was not induced. At the ultrastructural level, impairment of cell functions in vitro was associated with thin disturbance of the cytoskeleton, in the form of slight depolymerization and accumulation of microfilaments, which was equally reversible. Results suggest that VBL has an antiangiogenic component at very low, noncytotoxic doses, and that antiangiogenesis by VBL could be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases, Kaposi's sarcoma, and cancer.     

Human lymphoblastoid cells produce extracellular matrix-degrading enzymes and induce endothelial cell proliferation, migration, morphogenesis, and angiogenesis

Human lymphoproliferative diseases can be hypothesized to invade locally and to metastatize via mechanisms similar to those developed by a variety of solid tumors, i.e., the secretion of extracellular matrix-degrading enzymes and stimulation of angiogenesis. To assess this hypothesis, Namalwa, Raji, and Daudi cell lines (Burkitt’s lymphoma), LIK and SB cell lines (B-cell lymphoblastic leukemia), CEM and Jurkat cell lines (T-cell lymphoblastic leukemia), and U266 cell line (multiple myeloma) were evaluated for their capacity to produce matrix metalloproteinase-2 and -9, and urokinase-type plasminogen activator. These cell lines were also assessed for their ability: (1) to produce the angiogenic basic fibroblast growth factor and vascular endothelial growth factor; (2) to induce an angiogenic phenotype in cultured endothelial cells, represented by cell proliferation, chemotaxis, and morphogensis; (3) to stimulate angiogenesis in different in vivo experimental models. All cell lines expressed the mRNA for one or both metalloproteinases. Namalwa, Raji, LIK, SB, and U266 cells secreted the active form of both metalloproteinases, while Daudi, CEM, and Jurkat cells produced metalloproteinase-2 but not -9. In contrast, urokinase-type plasminogen activator was secreted only by SB cells. While Raji, LIK, SB, CEM, and Jurkat cells secreted both basic fibroblast growth factor and vascular endothelial growth factor, Daudi and U266 cells produced only the former, and Namalwa cells only the latter. Accordingly, the conditioned medium of all cell lines stimulated cell proliferation and/or chemotaxis in cultured endothelial cells, with the exception of that of Namalwa cells which was ineffective. The conditioned medium of CEM and Jurkat cells induced morphogenesis in cultured endothelial cells grown on a reconstituted basement membrane (Matrigel). Lastly, Namalwa, Raji, LIK, SB, U266, CEM, and Jurkat cells induced angiogenesis and mononuclear cell recruitment in the murine Matrigel sponge model and in a chick embryo chorioallantoic membrane assay. The extent of angiogenesis in both models was strictly correlated with the density of the mononuclear cell infiltrate. The results indicate that human lymphoproliferative disease cells possess both local and remote invasive ability via the secretion of matrix-degrading enzymes and the induction of angiogenesis which is fostered by host inflammatory cells and by an intervening ensemble of angiogenic factors.     

Determination of vascularization of B-cell non-Hodgkin's lymphomas with four markers

We evaluated the microvessel area (an index of angiogenesis) by using the factor VIII-related antigen (factor VIII-RA), and the expression of three components of the subendothelial basement membrane, namely: tenascin, laminin and type IV collagen. The four markers were assessed by immunohistochemical methods in 57 B-cell non-Hodgkin's lymphomas (B-NHL) and 28 benign lymphadenopathies. We found that microvessel area and the basement membrane markers had a different distribution pattern in relation to the histological type and degree of malignancy. In reactive and atypical lymphoid hyperplasias and in follicular low- and intermediate-grade B-NHL, microvessels were distributed within the interfollicular zones. By contrast, diffuse intermediate-grade and high-grade B-NHL were highly vascularized and microvessels were closely related to the neoplastic cells. Microvessel area was significantly associated with tenascin expression in all histological grades. Conversely, it was not correlated to laminin and type IV collagen expression, especially in diffuse intermediate-grade and high-grade B-NHL, where the expression of these markers was poor and fragmented. Our study suggests that angiogenesis and tenascin expression are associated phenomena in B-NHL, and that both increase with the malignancy grade. The prognostic value of angiogenesis and tenascin in B-NHL warrants further assessment in follow-up studies.     

Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma

To assess whether the progression of plasma cell tumors is accompanied by angiogenesis and secretion of matrix-degrading enzymes, bone marrow biopsy specimens from 20 patients with monoclonal gammopathy of undetermined significance (MGUS), 18 patients with nonactive multiple myeloma (MM), and 26 patients with active MM were evaluated for their angiogenic potential and matrix-metalloproteinase (MMP) production. A fivefold increase of the factor VIII+ microvessel area was measured by a planimetric method of point counting in the bone marrow of patients with active MM as compared with nonactive MM and MGUS patients (P <.01). When serum-free conditioned media (CM) of plasma cells isolated from the bone marrow of each patient were tested in vivo for their angiogenic activity in the chick embryo chorioallantoic membrane (CAM) assay, the incidence of angiogenic samples was significantly higher (P <. 01) in the active MM group (76%) compared with nonactive MM (33%) and MGUS (20%) groups. Moreover, a linear correlation (P <.01) was found between the extent of vascularization of the bone marrow of a given patient and the angiogenic activity exerted in the CAM assay by the plasma cells isolated from the same bone marrow. In vitro, a significantly higher fraction of the plasma cell CM samples from the active MM group stimulated human umbilical vein endothelial cell (HUVEC) proliferation (53%, P <.01), migration (42%, P <.05), and/or monocyte chemotaxis (38%, P <.05) when compared with nonactive MM and MGUS groups (ranging between 5% and 15% of the samples). Also, immunoassay of plasma cell extracts showed significantly higher (P <. 01) levels of the angiogenic basic fibroblast growth factor (FGF)-2 in the active MM patients than in nonactive MM and MGUS patients (153 +/- 59, 23 +/- 17, and 31 +/- 18 pg FGF-2/100 micrograms of protein, respectively). Accordingly, neutralizing anti-FGF-2 antibody caused a significant inhibition (ranging from 54% to 68%) of the biological activity exerted on cultured endothelial cells and in the CAM assay by plasma cell CM samples from active MM patients. Finally, in situ hybridization of bone marrow plasma cells and gelatin-zymography of their CM showed that active MM patients express significantly higher (P <.01) levels of MMP-2 mRNA and protein when compared with nonactive MM and MGUS patients, whereas MMP-9 expression was similar in all groups. Taken together, these findings indicate that the progression of plasma cell tumors is accompanied by an increase of bone marrow neovascularization. This is paralleled by an increased angiogenic and invasive potential of bone marrow plasma cells, which is dependent, at least in part, by FGF-2 and MMP-2 production. Induction of angiogenesis and secretion of MMPs by plasma cells in active disease may play a role in their medullary and extramedullary dissemination, raising the hypothesis that angiostatic/anti-MMP agents may be used for therapy of MM.     

TNP-470 and recombinant human interferon-alpha2a inhibit angiogenesis synergistically

The hypothesis that the combination of two known antiangiogenic agents TNP-470 and interferon (IFN)-alpha exerts synergistic effects has been investigated in vitro and in vivo. In vitro, TNP-470 and recombinant human IFN-alpha2a (rhIFN-alpha2a) resulted in a dose-dependent inhibition of proliferation of human umbilical vein endothelial cells (HUVECs) and EA.hy926 endothelial cells. Compared with the two agents used singly at their lowest or ineffective doses, combined treatment with the same doses inhibited more intensely in the absence of cytotoxicity and displayed similar behaviour on cell chemotaxis and capillary morphogenesis on Matrigel. However, the secretion of matrix metalloproteinase 2 (MMP-2) and MMP-9 was not influenced by the two agents, either alone or in combination, even when they were applied at their lowest efficacious doses or at higher cytotoxic doses. Experiments in vivo with the chick embryo chorioallantoic membrane (CAM)-sponge assay revealed the same dose-dependent inhibition and synergy. As the basic fibroblast growth factor (bFGF)-induced angiogenesis in the CAM-sponge model was strongly inhibited by the combined treatment, TNP-470 and rhIFN-alpha2a would appear to exert antiangiogenesis synergistically, perhaps by interfering with the bFGF-mediated pathway.     

Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with upgrading and myometrial invasion in endometrial carcinoma

BACKGROUND: Changes in angiogenesis and expression of extracellular matrix-degrading enzymes have been substantiated during tumour changeover and progression. METHODS: Tissues from 64 biopsies of endometrial carcinoma (EC) and 15 biopsies of normal (control) endometrium were investigated immunohistochemically to determine their microvessel number, and by in situ hybridisation to determine the expression of mRNA of the matrix metalloproteinase-2 (MMP-2, gelatinase A) and metalloproteinase-9 (MMP-9 gelatinase B). EC were grouped according to both histological grade (G) G1 to G3 and depth of myometrial (M) invasion M1 to M3. RESULTS: EC as a whole gave significantly higher counts over control endometria. Counts of the G1 group overlapped those of the control group, increased significantly in the G2 and even more in the G3 group. G3 biopsies in particular also displayed most microvessels widely scattered in the tumour tissue, in close association with tumour cells, and as winding and arborized tubes, often dilated in microaneurysmatic segments. Counts also increased in M2 and M3. Expression of the MMP-2 and MMP-9 mRNA, evaluated as percentages of positive biopsies and intensity of expression, were upregulated with the transition from control and G1 groups to G2 and G3, and in relation to advancing depth of invasion. In EC, MMP-2 and MMP-9 mRNA were also expressed by host stromal cells, including microvascular endothelial cells, fibroblasts and macrophages. In the control biopsies, poor expression of MMP-2 mRNA in few endothelial cells and no expression of MMP-9 mRNA were detected. CONCLUSION: These in situ data suggest that angiogenesis and degradation of extracellular matrix occur simultaneously with EC upgrading and advancing depth of invasion, and that EC cells and some host stromal cell populations cooperate in tumour progression.     

Bone marrow angiogenesis and mast cell density increase simultaneously with progression of human multiple myeloma

Immunohistochemical, cytochemical and ultrastructural data showing vivid angiogenesis and numerous mast cells (MCs) in the bone marrow of 24 patients with active multiple myeloma (MM) compared with 34 patients with non-active MM and 22 patients with monoclonal gammopathy of undetermined significance (MGUS) led us to hypothesize that angiogenesis parallels progression of MM, and that MCs participate in its induction via angiogenic factors in their secretory granules.     

A phase II study of three-weekly docetaxel and weekly trastuzumab in HER2-overexpressing advanced breast cancer

BACKGROUND: To test safety and activity of 3-weekly doses of docetaxel and a weekly dose of trastuzumab in women with HER2-overexpressing advanced breast cancer. PATIENTS AND METHODS: Forty-two women, median age 53 years (range 36-73 years), with HER2-overexpressing advanced breast cancer were enrolled in a study of docetaxel, 75 mg/m(2) q3w for 6 cycles, and trastuzumab, 4 mg/kg loading dose, 2 mg/kg weekly thereafter. Thirty-four patients (81%) had visceral metastatic involvement. Thirty-five patients had received prior chemotherapy as part of their treatment: adjuvant/neoadjuvant (26), metastatic (2) and both (7). Thirty-one patients had been previously exposed to an anthracycline and 11 to paclitaxel. Four patients had previously received high-dose chemotherapy followed by autologous stem cell transplant. RESULTS: 226 cycles (median 6, range 1-6) were administered. The median delivered dose intensity for docetaxel was 24 mg/m(2)/week (range 16-25 mg/m(2)/week). The intent to treat overall response rate was 67% (95% confidence interval, 52-79%). Median progression-free survival, time to treatment failure, and duration of response were 9, 8 and 12 months, respectively. Symptomatic cardiotoxicity (grade 3) occurred in 1 patient. The most common grade 3/4 toxicity was neutropenia (76% of the patients), although febrile neutropenia did not occur. CONCLUSIONS: Three-weekly doses of docetaxel and a weekly dose of trastuzumab is an active and safe combination in patients with HER2-overexpressing advanced breast cancer.     

Safety and activity of docetaxel and trastuzumab in HER2 overexpressing metastatic breast cancer: a pilot phase II study

We conducted a pilot phase II trial of trastuzumab administered concurrently with docetaxel in women with HER2-overexpressing advanced breast cancer. Twenty-five women with HER2-positive (3+ by immunohistochemistry = 16, 2+ = 9) metastatic breast cancer received docetaxel (75 mg/m every 3 weeks for 6 cycles) and trastuzumab (4 mg/kg loading dose, 2 mg/kg weekly thereafter). Twenty-three patients (92%) had visceral metastatic involvement. Twenty-three patients had received prior chemotherapy as part of adjuvant (18), metastatic (2), and both (3) treatment. The number of cycles administered was 121 (median 6, range 1-6). Symptomatic cardiotoxicity (GIII) occurred in one patient. The most common grade GIII/IV toxicity was neutropenia (80% of the cycles), although febrile neutropenia did not occur. No other GIII/IV toxicities were observed. Response rate was 70% (1 complete response and 15 partial responses) in 23 evaluable patients. The combination of docetaxel and trastuzumab is well tolerated and has clinically meaningful antitumor activity.