沙利度胺对人胰腺癌细胞细胞动力学和VEGF表达的影响

背景：研究显示沙利度胺及其类似物具有免疫调节、抗血管生成、抗炎等多方面的作用,其对多发性骨髓瘤和一些实体瘤的抗肿瘤作用与其免疫调节活性以及抗血管生成、抗增殖和促凋亡特性有关。目的：观察沙利度胺对人胰腺癌细胞细胞动力学和血管内皮生长因子（VEGF）表达的影响,探讨其用于胰腺癌临床辅助治疗的可能性。方法：以沙利度胺干预人胰腺癌细胞株Patu-898848h,MTT实验检测细胞增殖,流式细胞术检测细胞周期和细胞凋亡,RT—PCR检测VEGFmRNA表达。结果：经不同浓度沙利度胺干预的Patu-8988细胞,生长抑制率、G0／G1期细胞比例和细胞凋亡率均显著高于溶剂对照组（P〈0．05）,VEGF异构体VEGF121、VEGF165mRNA表达显著低于溶剂对照组（P〈0．05）。结论：沙利度胺能从多方面对胰腺癌生长产生抑制作用,包括直接抑制癌细胞增殖、诱导细胞周期G0／G1期阻滞和早期凋亡,以及通过抑制VEGF转录而抑制肿瘤血管发生。     

Modulation of endothelial cell network formation in vitro by molecular signaling of head and neck squamous cell carcinoma (HNSCC) exposed to cetuximab

Overexpression of EGFR plays a key-role in head and neck squamous cell carcinoma (HNSCC) and justifies the extensive use of cetuximab, a monoclonal anti-EGFR antibody, as well as EGFR-tyrosine kinase inhibitors (EGFR-TKI), which have been reported to inhibit tumor cell growth and the secretion of pro-angiogenic factors by tumor cells, such as VEGF and IL-8. Moreover, vessel normalization in tumors, suggesting a more complex mediation of endothelial cell growth control has also been observed in vivo. The present study was designed to investigate the angiogenic consequences of exposure of HNSCC tumor cell lines to cetuximab and intercellular signaling between tumor and endothelial cells by secretion of pro- and anti-angiogenic mediators in the conditioned media (CM). The results achieved showed that cetuximab decreased the secretion of VEGF by HNSCC cells and that exposure of human umbilical vein endothelial cells (HUVEC) to CM from HNSCC cells exposed to cetuximab induced an increase in endothelial cell network formation. Angiogenesis proteome profiling showed that cetuximab induced a complex alteration of the secretion of pro- and anti-angiogenic factors by HNSCC cells without enabling to identify a unique molecular marker. Expression of endothelial membrane receptors (VEGFR-2, EGFR, PECAM-1 and Notch-4) was investigated and only EGFR expression was found influenced when HUVEC were exposed to CM from cetuximab-exposed HNSCC cells. These results showed that the decrease in the secretion of pro-angiogenic agents like VEGF by HNSCC cells exposed to cetuximab could not be sufficient to justify its anti-angiogenic activity in vitro.     

TRAIL negatively regulates VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions

Solid tumors supply oxygen and nutrients required for angiogenesis by producing vascular endothelial growth factor (VEGF). Thus, inhibitors of VEGF signaling abrogate tumor angiogenesis, resulting in the suppression of tumor growth and metastasis. We here investigated the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on VEGF-induced angiogenesis. TRAIL inhibited VEGF-induced in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs) and in vivo neovascularization in chicken embryos and mice. TRAIL blocked VEGF-induced angiogenic signaling by inhibiting ERK, Src, FAK, paxillin, Akt, and eNOS. Further, TRAIL blocked intracellular Ca²⁺ elevation and actin reorganization in HUVECs stimulated with VEGF, without inhibiting VEGF receptor-2 tyrosine phosphorylation. TRAIL increased caspase-8 activity, without inducing caspase-9/-3 activation and apoptosis. Moreover, TRAIL resulted in cleavage of FAK into FAK-related non-kinase-like fragments in VEGF-stimulated HUVECs, which was blocked by a caspase-8 inhibitor and cellular caspase-8-like inhibitory protein. Biochemical and pharmacological inhibition of caspase-8 and FAK blocked the inhibitory effects of TRAIL on VEGF-stimulated anti-angiogenic signaling and events. In addition, caspase-8 knockdown also suppressed VEGF-mediated signaling and angiogenesis, suggesting that procaspase-8 plays a role of a non-apoptotic modulator in VEGF-induced angiogenic signaling. These results suggest that TRAIL inhibits VEGF-induced angiogenesis by increasing caspase-8 activity and subsequently decreasing non-apoptotic signaling functions of procaspase-8, without inducing caspase-3 activation and endothelial cell cytotoxicity. These data indicate that caspase-8 may be used as an anti-angiogenic drug for solid tumors resistant to TRAIL and anti-tumor drugs.     

Fibrin fragment E stimulates the proliferation,migration and differentiation of human microvascular endothelial cells in vitro

Various factors involved in haemostasis also regulate the development of new blood vessels by a process called angiogenesis. Enzymatic cleavage of fibrin yields a variety of fibrin degradation products, particularly in areas of intense angiogenesis such as in healing wounds and active atherosclerotic plaques. One of these, fibrin fragment E (FnE), is a potent angiogenic factor in the chick chorioallantoic membrane assay of angiogenesis. Here, we extend these studies to show that FnE stimulates the proliferation, migration and differentiation of human dermal microvascular endothelial cells (HuDMECs) in vitro, both in the absence and presence of such additional endothelial growth factors as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We also show that these stimulatory effects occur at concentrations of the protein known to be present in angiogenic tissues in vivo. FnE enhanced the angiogenic effects of VEGF or bFGF, indicating a possible synergy between the signalling pathways used by these three angiogenic factors. This revised version was published online in June 2006 with corrections to the Cover Date.     

The sponge/Matrigel angiogenesis assay

It has become increasingly clear that definitive tests for angiogenesis require in vivo assays. Recently, the Matrigel plug assay has become the method of choice for many studies involving in vivo testing for angiogenesis. In this assay, test angiogenesis-inducing compounds such as bFGF or tumor cells are introduced into cold liquid Matrigel which, after subcutaneous injection, solidifies and permits penetration by host cells and the formation of new blood vessels. Assessment of angiogenesis in the Matrigel plug is achieved either by measuring hemoglobin or by scoring selected regions of histological sections for vascular density. We now describe a modification of the Matrigel plug assay which permits a more precise visualization of the angiogenic reaction, provides directional information, requires no histological analysis, and lends itself to photographic documentation and image analysis protocols. We illustrate the assay by describing dose- and time-dependent responses to tumors of murine and human origin, to angiogenesis-inducing factors such as bFGF (FGF-2) and VEGF and to anti-angiogenic agents such as endostatin. The method has been used as well to demonstrate blood vessel recruitment by embryonic chick aortic arch rudiments. Additionally it has been able to detect strain-dependent differences in susceptibility to angiogenic stimulation.     

Mechanism and its regulation of tumor－induced angiogenesis

Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into cancerous growths, supplying nutrients and oxygen and removing waste products. The process of angiogenesis plays an important role in many physiological and pathological conditions. Solid tumors depend on angiogenesis for growth and metastasis in a hostile environment. In the prevascular phase, the tumor is rarely larger than 2 to 3 mm^3 and may contain a million or more cells. Up to this size, tumor cells can obtain the necessary oxygen and nutrient supplies required for growth and survival by simple passive diffusion. The properties of tumors to release and induce several angiogenic and antiangiogenic factors which play crucial roles in regulating endothelial cell (EC) proliferation, migration, apoptosis or survival, cell-cell and cell-matrix adhesion through different intracellular signalinq are thought to be the essential mechanisms during tumor-induced angiogenesis. Tumor angiogenesis actually starts with tumor cells releasing molecules that send signals to surrounding normal host tissue. This signaling activates certain genes in the host tissue that, in turn, make proteins to encourage growth of new blood vessels. In this review, we focus the mechanisms of tumor-induced angiogenesis, with an emphasis on the regulatory role of several angiogenic and anti-angiogenic agents during the angiogenic process in tumors. Advances in understanding the mechanisms of tumor angiogenesis have led to the development of several most effective anti-angiogenic and anti-metastatic therapeutic agents and also have provided several techniques for the regulation of cancer′s angiogenic switch. The suggestion is made that standard cytotoxic chemotherapy and angiogenesis inhibitors used in combination may produce complementary therapeutic benefits in the treatment of cancer.     

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.     

γ-Hydroxybutyric acid and 5-fluorouracil, metabolites of UFT, inhibit the angiogenesis induced by vascular endothelial growth factor

UFT, a drug composed of uracil and tegafur at the molar ratio of 4:1, is an orally active agent for the treatment of a wide variety of malignant tumours. Using a murine dorsal air sac (DAS) assay, we have previously shown that UFT and its metabolites, γ-hydroxybutyric acid (GHB) and 5-fluorouracil (5-FU), inhibited the angiogenesis induced by murine renal cell carcinoma. Here we report that UFT was more effective than other fluorinated pyrimidines such as 5-FU and doxifluridine (5^′-DFUR) in blocking the angiogenic responses elicited by five human cancer cell lines which produced high levels of vascular endothelial growth factor (VEGF), but no detectable fibroblast growth factor-2 (FGF-2) in vitro. In contrast, UFT was unable to block the angiogenic response to one human gastric cancer cell line which produced both VEGF and FGF-2 in vitro. However, the production or secretion of VEGF by these cells was unaffected by GHB and 5-FU treatment. Interestingly, GHB suppressed the chemotactic migration and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by VEGF, without inhibiting their DNA synthesis. Since GHB did not affect the FGF-2-driven activities in HUVECs, its action appears to be VEGF-selective. On the other hand, 5-FU inhibited DNA synthesis and migration of HUVECs stimulated by both VEGF and FGF-2, and tube formation driven by VEGF, suggesting that 5-FU is cytotoxic to endothelial cells. The inhibitory effects of 5-FU, and especially those GHB, were reproduced under in vivo condition using the DAS assay. The VEGF-mediated angiogenesis was significantly inhibited by UFT, 5-FU, and especially by GHB. We propose that the selective inhibitory effects of GHB on VEGF-mediated responses of endothelial cells are involved in the anti-angiogenic activity of UFT. This revised version was published online in June 2006 with corrections to the Cover Date.     

Suppression of growth of pancreatic cancer cell and expression of vascular endothelial growth factor by gene silencing with RNA interference

OBJECTIVE: To explore the anti‐angiogenesis and tumor cell growth suppressive effects resulted from gene silencing by RNAi in BxPC‐3 human pancreatic cancer cells. METHODS: The designation and transfection of vascular endothelial growth factor (VEGF)‐siRNA lentivirus was carried out in vitro. Real‐time PCR and western blot were conducted to measure the expression levels of VEGF mRNA and protein. Flow cytometry was employed to evaluate cell apoptosis and cell death. A lactate dehydrogenase (LDH) assay was used to assess the cytotoxicity of VEGF‐siRNA. A 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide (MTT) assay was used to picture the cellular growth. For the in vivo study, BxPC‐3 cells were injected subcutaneously into nude mice to form xenografts. The mice were divided into three groups according to the intervention used. The control group, the negative control group and the knockdown group of mice were injected with saline, an empty lentivirus vehicle and lentivirus carrying VEGF‐siRNA, respectively. None of the mice died during the study. When these mice were killed, the xenografts were collected and the tumor sizes of the different groups were compared. Finally, immunohistochemistry was used to assess the VEGF expression level and microvascular density. RESULTS: After the transfection of VEGF‐siRNA lentivirus, the cellular expression of VEGF mRNA decreased to 50% of the control and the VEGF protein in the BxPC‐3 cells decreased to 30% of the control. Apoptosis and cell death increased after transfection of the VEGF‐siRNA lentivirus. The LDH assay showed high cytotoxicity induced by VEGF‐siRNA lentivirus transfection. The MTT assay showed slower cellular growth in the knockdown cells. Tumor growth suppression was observed in nude mice that had received the VEGF‐siRNA lentivirus transfection, and the tumor sizes of the xenografts in this group were clearly smaller than those in other two groups. VEGF expression and microvascular density were significantly decreased. CONCLUSION: Vascular endothelial growth factor gene silencing via VEGF‐siRNA can effectively inhibit the production of VEGF and exert an anti‐angiogenesis and tumor cell growth suppressive effect both in vitro and in vivo.     

Inhibition of both paracrine and autocrine VEGF/ VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias

Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.     

Stimulation of PAR-1 or PAR-4 promotes similar pattern of VEGF and endostatin release and pro-angiogenic responses mediated by human platelets

Background: Platelets mediate angiogenesis through the secretion of several factors, including the pro-angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic endostatin. Although previous findings indicated that these molecules are packed into different alpha-granules and selectively released by specific stimulation of protease-activated receptor (PAR)-1 or PAR-4, recent evidences are against this hypothesis. Objectives: To elucidate the controversies about the VEGF and endostatin release and the overall angiogenic effect of PARs-stimulated platelets. Methods: VEGF and endostatin were quantified by enzyme linked-immunosorbent assay (ELISA). Endothelial proliferation (pNPP assay), wound healing (scratch assay) and tubule formation (matrigel) of human microvascular endothelial cells (HMEC-1) and endothelial progenitor cells (EPC) were determined using supernatants from PAR-1- or PAR-4-stimulated platelets. Results: Activation of washed platelets (WPs) by PAR-1- or PAR-4-activating peptide (AP) promoted the VEGF and endostatin secretion in a concentration-dependent manner, being PAR-1-AP more potent than PAR-4-AP. The release of both molecules was abrogated by pre-incubation of platelets with PAR antagonists. Activation of platelet-rich plasma (PRP) with either PAR-1-AP or PAR-4-AP induced a significant VEGF secretion. Quantification of platelet-endostatin secretion was not possible in PRP due to the high levels of plasmatic endostatin vs. platelet content. Releasates from PAR-1- or PAR-4-activated WPs promoted similar pattern of angiogenic responses of HMEC-1 or EPC. Moreover, proliferation of HMEC-1 mediated by PAR-stimulated PRP releasates was delayed and significantly lower compared with that induced by PAR-stimulated WPs. Conclusions: Our results are in contrast with the previously described differential release of VEGF and endostatin induced by the selective PAR-1 or PAR-4 stimulation, and support the notion that while circulating endostatin accounts for the maintenance of a systemic anti-angiogenic state, locally, the release of platelet alpha-granule content promotes angiogenesis.     

Neutrophil granulocyte derived MMP-9 is a VEGF independent functional component of the angiogenic switch in pancreatic ductal adenocarcinoma

Background  

Vascular endothelial growth factor (VEGF) that is secreted by tumor cells plays a key role in angiogenesis. Matrix metalloproteinase 9 (MMP-9) is produced by inflammatory cells, such as stromal granulocytes (PMN), remodels the extracellular matrix and is known to promote angiogenesis indirectly by interacting with VEGF. The aim of this study was to determine the role of PMN-derived MMP-9, its interaction with VEGF, and the efficacy of anti-angiogenic therapy targeting MMP-9 with oral Doxycycline and VEGF with Bevacizumab in pancreatic cancer (PDAC).     

Angiotensin‐I converting enzyme inhibitors suppress angiogenesis and growth of esophageal carcinoma xenografts

It has recently been suggested that angiotensin‐I converting enzyme (ACE) inhibitors decrease the risk of cancer. However, studies to date have not investigated esophageal carcinoma. Therefore, we investigated the inhibitory effect of ACE inhibitors on growth of esophageal carcinoma xenografts. We used the EC9706 cell line, which expresses the highest vascular endothelial growth factor (VEGF) mRNA level, to establish xenografts in 21 BALB/c nude mice. The mice were then randomly allocated to receive normal saline, perindopril (4 mg/kg), or benazepril (6 mg/kg). Five weeks later, the nude mice were sacrificed and all tumors were dissected and weighed. The number of microvessels was counted by immunostaining endothelial cells for CD31 and the microvessel density was assessed. The EC9706 cell line showed the highest expression of VEGF mRNA of four esophageal squamous cell carcinoma lines. After treatment, the average tumor inhibitory rate in the benazepril group was 45.4%, which was significantly higher than that of the control group (P < 0.05). Similar findings were observed when we used tumor weight as an index for tumor growth inhibition (P < 0.05). By contrast, there was no significant difference between the perindopril group and the control group (P > 0.05). The benazepril group appeared to show less vascularization than the control group (P < 0.05), but we did not find a significant difference between the perindopril group and the control group (P > 0.05). The EC9706 cell line showed the highest expression of VEGF mRNA level of the four esophageal squamous cell carcinoma cell lines examined. Benazepril inhibited the growth of esophageal carcinoma in vivo. The potential mechanism of benazepril seems to involve suppression of new vessel formation. Therefore, benazepril could be used as an effective agent for the treatment of esophageal carcinoma.     

Proliferation of endothelial and tumor epithelial cells by progestin-induced vascular endothelial growth factor from human breast cancer cells: paracrine and autocrine effects

Angiogenesis, the formation of new blood vessels, is essential for tumor expansion, and vascular endothelial growth factor (VEGF) is one of the most potent angiogenic growth factors known. We have previously shown that natural and synthetic progestins, including those used in hormone replacement therapy and oral contraception, induce the synthesis and secretion of VEGF in a subset of human breast cancer cells in a progesterone receptor-dependent manner. We now report that conditioned medium from progestin-treated breast tumor cells can induce the proliferation of endothelial cells in a paracrine manner and induce the proliferation of tumor epithelial cells in a paracrine and an autocrine manner. The use of an anti-VEGF antibody and SU-1498, an inhibitor of VEGF receptor-2 (VEGFR-2 or flk/kdr) tyrosine kinase activity, demonstrated that these effects involve interactions between VEGF and VEGFR-2. Also, blockage of progestin-induced VEGF by the antiprogestin RU-486 (mifepristone) eliminated VEGF-induced proliferative effects. The ability of VEGF to increase the proliferation of endothelial cells and tumor cells, including those that do not release VEGF in response to progestins, suggests that these effects are mediated by amplification of the progestin signal, which culminates in angiogenesis and tumor growth. These novel findings suggest that targeting the release of VEGF from tumor epithelial cells as well as blocking interactions between VEGF and VEGFR-2 on both endothelial and tumor epithelial cells may facilitate the development of new antiangiogenic therapies for progestin-dependent breast tumors. Furthermore, these data indicate that it would be useful to develop selective progesterone receptor modulators that prevent the release of angiogenic growth factors from breast cancer cells.     

Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma

Neuroblastoma is the most common pediatric abdominal tumor and principally a p53 wild-type, highly vascular, aggressive tumor, with limited response to anti-VEGF therapies alone. MDM2 is a key inhibitor of p53 and a positive activator of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) activity with an important role in neuroblastoma pathogenesis. We hypothesized that concurrent inhibition of both MDM2 and VEGF signaling would have cooperative anti-tumor effects, potentiating anti-angiogenic strategies for neuroblastoma and other p53 wild-type tumors. We orthotopically implanted SH-SY5Y neuroblastoma cells into nude mice (n = 40) and treated as follows: control, bevacizumab, Nutlin-3a, combination of bevacizumab plus Nutlin-3a. Expression of HIF-1α and VEGF were measured by qPCR, Western blot, and ELISA. Tumor apoptosis was measured by immunohistochemistry and caspase assay. Angiogenesis was evaluated by immunohistochemistry for vascular markers (CD-31, type-IV collagen, αSMA). Both angiogenesis and metastatic burden were digitally quantified. In vitro, Nutlin-3a suppresses HIF-1α expression with subsequent downregulation of VEGF. Bevacizumab plus Nutlin-3a leads to significant suppression of tumor growth compared to control (P < 0.01) or either agent alone. Combination treated xenograft tumors display a marked decrease in endothelial cells (P < 0.0001), perivascular basement membrane (P < 0.04), and vascular mural cells (P < 0.004). Nutlin-3a alone and in combination with bevacizumab leads to significant tumor apoptosis (P < 0.0001 for both) and significant decrease in incidence of metastasis (P < 0.05) and metastatic burden (P < 0.03). Bevacizumab plus Nutlin-3a cooperatively inhibits tumor growth and angiogenesis in neuroblastoma in vivo with dramatic effects on tumor vascularity. Concomitantly targeting VEGF and p53 pathways potently suppresses tumor growth, and these results support further clinical development of this approach.     

Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: Role of vascular endothelial growth factor

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone–ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone–ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.     

Improved quantification of angiogenesis in the rat aortic ring assay

In vitro angiogenesis assays are essential for the identification of potential angiogenic agents and screening for pharmacological inhibitors. Among these assays, the rat aortic ring model developed by Nicosia bridges the gap between in vivo and in vitro models. The quantification of angiogenesis on this system must be applicable to characterise vascular networks of various states of complexity. We present here an improved computer-assisted image analysis which allows: (1) the determination of the aortic ring area and its factor shape; (2) the number of microvessels, the total number of branchings, the maximal microvessel length and the microvessel distribution; (3) the total number of isolated fibroblast-like cells and their distribution. We show that this method is suitable to quantify spontaneous angiogenesis as well as to analyse a complex microvascular network induced by various concentrations of vascular endothelial growth factor (VEGF). In addition, by evaluating a new parameter, the fibroblast-like cell distribution, our results show that: (1) during spontaneous angiogenic response, maximal fibroblast-like cell migration delimits microvascular outgrowth; and (2) the known angiogenic inhibitor Batimastat prevents endothelial cell sprouting without completely blocking fibroblast-like cell migration. Finally, this new method of quantification is of great interest to better understand angiogenesis and to test pro- or anti-angiogenic agents. This revised version was published online in June 2006 with corrections to the Cover Date.