Organ site-dependent expression of basic fibroblast growth factor in human renal cell carcinoma cells.

We investigated the influence of organ microenvironment on the angiogenic phenotype in human renal cell carcinoma (HRCC) cells. HRCC line SN12C was established in vitro from a surgical specimen, and metastatic line SN12PM6 was isolated from a lung metastasis produced by parental cells implanted into the kidney of nude mice. SN12C (low metastasis) and SN12PM6 (high metastasis) cells were injected into the kidney or subcutis of nude mice. The kidney tumors were highly vascularized (as revealed by immunohistochemistry using antibodies against factor VIII), and metastatic, whereas the subcutaneous tumors were not. The expression of mRNA for basic fibroblast growth factor (bFGF) in kidney tumors was 10 to 20 times that found in subcutaneous tumors. Similar data were obtained at the protein level by using fluorescence activated cell sorting, immunohistochemistry, and enzyme-linked immunosorbent assay. bFGF was detected in the urine of mice with tumors in the kidney but not subcutaneous tumors. The level of bFGF in the serum of mice with kidney tumors was two to three times that in mice with subcutaneous tumors. The changes in bFGF expression in the tumors was transient. Collectively, these data indicate that the organ microenvironment can influence the expression level of bFGF in HRCC.     

Production of experimental malignant pleural effusions is dependent on invasion of the pleura and expression of vascular endothelial growth factor/vascular permeability factor by human lung cancer cells

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.     

Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the angiogenesis associated with the growth of many human and animal tumors. VPF/VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with two endothelial cell tyrosine kinase receptors, KDR and flt-1. We studied 16 cases of AIDS-associated Kaposi's sarcoma (KS), 2 cases of cutaneous angiosarcoma, and 6 cases of capillary hemangioma by in situ hybridization for expression of VPF/VEGF, KDR, and flt-1 mRNAs. We also performed immunohistochemical staining for VPF/VEGF protein in 15 cases. Tumor cells in KS and angiosarcoma strongly expressed KDR but not flt-1 mRNA. Endothelial cells in small stromal vessels in and around these tumors strongly expressed both KDR and flt-1 mRNAs. Tumor cells expressed VPF/VEGF mRNA strongly in only one case of KS, adjacent to an area of necrosis. This was also the only case in which the tumor cells stained substantially for VPF/VEGF protein. VPF/VEGF mRNA and protein were, however, strongly expressed by squamous epithelium in areas of hyperplasia and near areas of ulceration overlying tumors. VPF/VEGF mRNA was also expressed focally at lower levels by infiltrating inflammatory cells, probably macrophages. The strong expression of both KDR and flt-1 in small stromal vessels in and around tumors suggests that VPF/VEGF may be an important regulator of the edema and angiogenesis seen in these tumors. The strong expression of KDR by tumor cells in KS and angiosarcoma implies that VPF/VEGF may also have a direct effect on tumor cells. Tumor cells in four of six capillary hemangiomas strongly expressed both KDR and flt-1 mRNAs in contrast to the high level expression of only KDR observed in the malignant vascular tumors studied. Neither VPF/VEGF mRNA or protein were strongly expressed in capillary hemangiomas. VPF/VEGF and its receptors may play an important but as yet incompletely understood role in the pathogenesis of both benign and malignant vascular tumors.     

Analysis of the tumor vasculature and metastatic behavior of xenografts of human melanoma cell lines transfected with vascular permeability factor.

Vascular permeability factor (VPF) is an important mediator of vascular development in tumors. Some human melanoma cell lines have a low VPF expression level in culture, but this level is upregulated when growing as a tumor in nude mice. Other melanoma lines have a constitutively high VPF expression. To compare the biological behavior of tumors with these two expression patterns, a human melanoma cell line with an inducible VPF expression was transfected with VPF expression constructs. In this way, several lines were obtained that constitutively produce either the soluble VPF121 or the matrix-associated VPF189 variant at levels of 4 to 30 times the VPF level in mature tumors derived from the parental line. The recombinant VPF RNA, which lacks most of the 5' noncoding sequences present in the endogenous VPF mRNA, was much more efficiently translated than the endogenous messenger. Upon injection in nude mice, all VPF-transfected lines developed tumors with aberrations in vascularization and in distribution of matrix components. In these tumors the blood vessels were hyperpermeable for an i.v. injected protein tracer. Transfection did not influence the in vitro growth rate of the cell lines, but the tumors from the VPF-transfected lines had higher growth rates in vivo than tumors from the parental line or the vector-transfected line. Although the incidence of lung metastasis was similar in all lines, the number of metastases per affected lung was significantly increased in mice carrying VPF-transfectant tumors. We conclude that the pattern and the level of VPF expression in a tumor are important determinants of the architecture and functionality of the vascular bed, but that overexpression of VPF does not necessarily lead to an increase of microvascular density or metastatic spread. The role of VPF in melanoma progression is obviously complex and may be difficult to derive in its generality from a single experimental model.     

Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is an angiogenic cytokine with potential for the treatment of tissue ischemia. To investigate the properties of the new blood vessels induced by VPF/VEGF, we injected an adenoviral vector engineered to express murine VPF/VEGF164 into several normal tissues of adult nude mice or rats. A dose-dependent angiogenic response was induced in all tissues studied but was more intense and persisted longer (months) in skin and fat than in heart or skeletal muscle (< or =3 weeks). The initial response (within 18 hours) was identical in all tissues studied and was characterized by microvascular hyperpermeability, edema, deposition of an extravascular fibrin gel, and the formation of enlarged, thin-walled pericyte-poor vessels ("mother" vessels). Mother vessels developed from preexisting microvessels after pericyte detachment and basement membrane degradation. Mother vessels were transient structures that evolved variably in different tissues into smaller daughter vessels, disorganized vessel tangles (glomeruloid bodies), and medium-sized muscular arteries and veins. Vascular structures closely resembling mother vessels and each mother vessel derivative have been observed in benign and malignant tumors, in other examples of pathological and physiological angiogenesis, and in vascular malformations. Together these data suggest that VPF/VEGF has a role in the pathogenesis of these entities. They also indicate that the angiogenic response induced by VPF/VEGF is heterogeneous and tissue specific. Finally, the muscular vessels that developed from mother vessels in skin and perimuscle fat have the structure of collaterals and could be useful clinically in the relief of tissue ischemia.     

Protective effect of vascular endothelial growth factor/vascular permeability factor 165 and 121 on glomerular endothelial cell injury in the rat

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) promotes the repair of injured vessels by stimulating angiogenesis. VEGF/VPF reportedly has cytoprotective activity but no study has shown the protective effect of VEGF/VPF on glomerular endothelial cells. We examined whether recombinant VEGF/VPF121 and VEGF/VPF165 isoforms could prevent injury of glomerular endothelial cells. Mild glomerular injury was induced in rats by an intravenous-injection of a limited dose of anti-Thy-1.1 antibody to obtain lesions similar to those found in the human disease. Recombinant VEGF/VPF165, VEGF/VPF121 or BSA was administered 4 h before the injection of the antibody, and once daily for 3 days. In the BSA-injected rats, mesangial cell lysis and endothelial cell injury in dilated capillary tufts were evident without endothelial cell apoptosis on days 1-4. Thereafter, cell proliferation and repair began and remodeling of the glomeruli was completed by day 28. Macrophages but not polymorphonuclear leukocytes accumulated significantly in the glomeruli on days 1-4. Treatment with VEGF/VPF isoform protected endothelial cells but not mesangial cells from destruction on day 1, and accelerated the repair of both types of cells, which was completed by day 18, 10 days earlier than that of the control animals. The results indicate that VEGF/VPF121 or VEGF/VPF165 can protect glomerular endothelial cells against injury, independent of apoptosis-inhibition activity, thereby promoting reconstruction of glomeruli. The protective effect of VEGF/VPF on endothelial cells suggests that it could provide therapeutic benefit for certain kidney diseases.     

转染VEGF165基因抑制兔动脉损伤后内膜增生

目的：观察局部转染血管内皮生长因子165（VEGF165）基因对损伤动脉内膜增生的影响并探讨可能机制。 方法： 采用显微外科手术方法，建立兔右髂外动脉损伤模型。将105只新西兰大白兔随机分为3组，每组35只。A组为生理盐水对照组，B组为脂质体介导的pBudCE4.1转染组，C组为脂质体介导的pBudCE4.1/VEGF165转染组。用微注射器将各种转染液注入损伤的血管壁，每组按实验终点（术后2、3、7、14和28 d）分为5个亚组，每个亚组7只兔。于术后各实验终点，取损伤段的血管用于病理组织学检查、电镜观察、RT-PCR和免疫组化染色检查。 结果： 术后各时点C组血管内膜厚度和内膜面积均显著小于A组和B组（P<0.01），术后28 d时C组管腔狭窄率平均比A组和B组低51.6%和49.8％。术后各时点C组血管壁的VEGF165基因的mRNA表达量和血管壁VEGF165阳性细胞率明显高于A组和B组（P<0.01）。C组血管壁血管内皮细胞（VEC）修复和生长增殖速度明显快于A组和B组。A组和B组间无明显差异。 结论： 局部转染VEGF165基因可抑制血管新生内膜增生及血管再狭窄，为将来血管内膜增生的基因治疗奠定基础。     

Tumor Growth of FGF or VEGF Transfected MCF-7 Breast Carcinoma Cells Correlates with Density of Specific Microvessels Independent of the Transfected Angiogenic Factor

We have previously shown that fibroblast growth factor (FGF)-1-, FGF-4-, or vascular endothelial growth factor (VEGF/VPF)-transfected MCF-7 breast carcinoma cells growing as tumors in nude mice are tamoxifen resistant and/or estrogen independent. These transfectants provide opportunity for study of in situ tumor-induced angiogenesis promoted by the individual angiogenic factors under growth-promoting versus growth-inhibiting hormonal conditions. In the present study, vessels in tumors harvested at varying times after tumor cell injection were immunohistochemically highlighted and vessel morphology and topography were scored on a scale of 0 to 4 by blinded observers. In tumors produced by all cell lines under all growth-promoting hormonal conditions, there was significantly increased abundance (P < 0.05) of edge-associated and intratumor microvessels, but not of stromally located microvessels, when compared with tumor nodules harvested under growth-inhibiting conditions, regardless of the identity of the angiogenic factor or the hormonal treatment. Image analysis of bromodeoxyuridine (BrdU)-labeled nuclei of tumors produced by all cell lines under all hormonal conditions harvested at early time points showed that mean labeling indices were highest for hormonal conditions that produced the most robust growth in that particular cell line, implying that a high BrdU labeling index is a predictor of future tumor growth in individual tumors. These results confirm previous studies that established the importance of neovascularization for tumor growth and provide validation for use of these cell lines to study the process of angiogenesis in vivo. Study of gene expression in endothelial cells in edge-associated or intratumor vessels using this model might reveal mechanisms important in tumor-induced angiogenesis in human breast cancer.     

Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis.

VPF/VEGF is a multifunctional cytokine that contributes to angiogenesis by both direct and indirect mechanisms. On the one hand, VPF/VEGF stimulates the ECs lining nearby microvessels to proliferate, to migrate, and to alter their pattern of gene expression. On the other hand, VPF/VEGF renders these same microvascular ECs hyperpermeable so that they spill plasma proteins into the extravascular space, leading to the clotting of extravasated fibrinogen with deposition of a fibrin gel. Extravascular fibrin serves as a provisional matrix that favors and supports the ingrowth of new blood vessels and other mesenchymal cells that generate mature, vascularized stroma. These same principles apply in tumors, in several examples of non-neoplastic pathology, and in physiological processes that involve angiogenesis and new stroma generation. In all of these examples, microvascular hyperpermeability and the introduction of a provisional, plasma-derived matrix precede and accompany the onset of EC division and new blood vessel formation. It would seem, therefore, that tumors have "borrowed" fundamental mechanisms that developed in multicellular organisms for purposes of tissue defense, renewal, and repair. VPF/VEGF, therefore has taught us something new about angiogenesis; namely, that vascular hyperpermeability and consequent plasma protein extravasation are important, perhaps essential, elements in its generation. However, this finding raises a paradox. While VPF/VEGF induces vascular hyperpermeability, other potent angiogenic factors apparently do not, at least in subtoxic concentrations that are more than sufficient to induce angiogenesis. Nonetheless, wherever angiogenesis has been studied, the newly generated vessels have been found to be hyperpermeable. How, therefore, do angiogenic factors other than VPF/VEGF lead to the formation of new and leaky blood vessels? We do not as yet have a complete answer to this question. One possibility is that at least some angiogenic factors mediate their effect by inducing or stimulating the expression of VPF/VEGF. In fact, there is already one clear example of this. TGF-alpha is a potent angiogenic factor but does not itself increase microvascular permeability. However, TGF-alpha strikingly upregulates VPF/VEGF expression in cultured keratinocytes and is thought to be responsible, at least in part, for the overexpression of VPF/VEGF in psoriasis. Moreover, overexpression of TGF-alpha, along with that of the EGF receptor with which it interacts, is characteristic of many malignant tumors, raising the possibility that TGF-alpha acts to stimulate VPF/VEGF expression in other types of epithelial cells and in this manner induces angiogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

VEGF165反义RNA对人食管鳞癌细胞影响的实验研究

目的 :观察血管内皮生长因子16 5 (VEGF16 5 )反义RNA对人食管鳞癌细胞EC10 9的影响 ,探讨其治疗食管癌的可行性。方法 :采用亚克隆技术 ,构建并鉴定VEGF16 5 反义RNA的真核表达载体。以重组质粒转染人食管鳞癌细胞EC10 9后 ,将其接种于裸鼠皮下 ,分别利用原位杂交、激光共聚焦、图象分析及微血管计数等方法 ,观察转染前后EC10 9细胞的生物学性状和致瘤性。结果 :成功地构建了VEGF16 5 反义RNA的真核表达载体 ,并在EC10 9细胞中获得表达。转染细胞中VEGF16 5 的表达下降 75% ,其生物学性状不受外源基因表达的影响 ,但其在裸鼠皮下的致瘤性和和瘤组织中血管的生成明显下降。VEGF16 5 反义RNA转染组、空载体转染组和对照组中肿瘤的体积 ,分别为 (82 0± 112 .5)mm3 、(793 0± 10 3 5)mm3 和 (7850± 950 )mm3(P <0 .0 1) ;微血管的密度分别为 (8.5± 1.2 ) /mm2 、(44.3± 9.4) /mm2 和 (46.4± 12 .6) /mm2 (P <0 .0 1)。结论 :VEGF16 5反义RNA能够明显减少食管鳞癌细胞内VEGF16 5 的表达 ,具有抑制肿瘤生长和血管生成的作用 ,可望用于实体肿瘤的辅助治疗。     

Host Microvasculature Influence on Tumor Vascular Morphology and Endothelial Gene Expression

We have previously demonstrated that vascular endothelial growth factor-165 (VEGF), a tumor-secreted angiogenic factor, can acutely and chronically induce fenestrations in microvascular endothelium (Cancer Res 1997, 57:765–772). Because the morphology and function of microvascular endothelium differs from tissue to tissue, we undertook studies to examine whether the neovasculature in tumors also differed depending upon tumor location. Four tumor types implanted in the brain or subcutis in nude mice were studied: a murine rhabdomyosarcoma (M1S), a murine mammary carcinoma (EMT), and two human glioblastomas (U87 and U251). In addition, we studied Chinese hamster ovary cells stably transfected with human VEGF₁₆₅. As previously reported, tumors grown in the subcutaneous space had a microvasculature that was fenestrated and had open endothelial gaps. The identical tumors when grown in the brain also had fenestrated endothelium and vessels with open endothelial gaps, but they were drastically reduced in occurrence. Open endothelial gaps were not seen in all tumors implanted in the brain (EMT and M1S), although fenestrated endothelium was always seen. VEGF and VEGF receptors were measured in tumors from both locations by immunoblotting and competitive polymerase chain reaction, respectively. VEGF amount was not significantly different between the tumor locations. Interestingly, total tumor vascular mRNA expression of both Flk-1 and Flt-1 was greater in tumor vessels derived from the brain compared with tumor vessels derived from subcutaneous tissues. These results demonstrate that the host microvascular environment determines the morphology and function of the tumor vasculature and that endothelia from different tissues vary in their ability to express the VEGF receptors given identical stimuli.     

Modification of the primary tumor microenvironment by transforming growth factor alpha-epidermal growth factor receptor signaling promotes metastasis in an orthotopic colon cancer model

The transforming growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) signaling pathway appears to play a critical role in colon cancer progression, but the cellular and molecular mechanisms that contribute to metastasis remain unknown. KM12C colon cancer cell clones expressing high (C9) or negligible (C10) levels of TGFalpha were implanted into the cecal walls of nude mice. C9 tumors formed autocrine and paracrine EGFR networks, whereas C10 tumors were unable to signal through EGFR. The tumor microenvironment of C9, but not C10, contained cells enriched in vascular endothelial growth factor (VEGF) A, interleukin-8, and matrix metalloproteinases-2 and -9 and had a high vascular surface area. C9 tumors recruited a macrophage population that co-expressed F4/80 and lymphatic vessel endothelial hyaluronic acid receptor and produced VEGFC. The mean lymphatic density of C9 tumors was threefold higher than that of C10 tumors. C9, but not C10, tumor cells metastasized to regional lymph nodes in all mice and to the liver in 5 of 10 mice. Forced expression of TGFalpha in C10 tumor cells led to the generation of autocrine and paracrine EGFR signaling, macrophage recruitment, enhanced blood and lymphatic vascular surface areas, and increased lymphatic metastasis. Collectively, these data show that activation of TGFalpha-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFalpha-positive colon cancers.     

Blockade of epidermal growth factor receptor signaling on tumor cells and tumor-associated endothelial cells for therapy of human carcinomas

The purpose of this study was to determine whether the expression of epidermal growth factor receptor (EGF-R) and activated EGF-R by tumor-associated endothelial cells is influenced by interaction with specific growth factors in the microenvironment. Different human carcinoma cell lines expressing EGF-R with low or high levels of EGF/transforming growth factor (TGF)-alpha were implanted into orthotopic organs of nude mice. In the EGF/TGF-alpha-positive bladder cancer (253J-BV), pancreatic cancer (L3.6pl), and renal cancer (RBM1-IT) but not in the EGF/TGF-alpha-negative renal cancer SN12-PM6, tumor-associated endothelial cells expressed EGF-R and activated EGF-R. Mice were implanted with human 253J-BV bladder tumors (EGF+) or human SN12-PM6 renal tumors (EGF-). Treatment with oral PKI 166 (a specific inhibitor of EGF-R phosphorylation) alone, intraperitoneal paclitaxel alone (253J-BV), gemcitabine alone (SN12-PM6), or combination of PKI 166 and chemotherapy produced a 60%, 32%, or 81% reduction in the volume of 253J-BV bladder tumors, respectively, and 26%, 23%, or 51% reduction in the volume of SN12-PM6 kidney tumors, respectively. Immunohistochemical analyses demonstrated down-regulation of activated EGF-R in EGF/TGF-alpha-positive and EGF/TGF-alpha-negative lesions from mice treated with PKI 166, although apoptosis of tumor-associated endothelial cells was found only in EGF/TGF-alpha-positive tumors. Collectively, these data suggest that expression of activated EGF-R by tumor-associated endothelial cells provides an important target for therapy.     

Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine that is overexpressed in many tumors, in healing wounds, and in rheumatoid arthritis. VPF/VEGF is thought to induce angiogenesis and accompanying connective tissue stroma in two ways: 1), by increasing microvascular permeability, thereby modifying the extracellular matrix and 2), as an endothelial cell mitogen. VPF/VEGF has been reported in animal corpora lutea and we investigated the possibility that it might be present in human ovaries and have a role in corpus luteum formation. We here report that VPF/VEGF mRNA and protein are expressed by human ovarian granulosa and theca cells late in follicle development and, subsequent to ovulation, by granulosa and theca lutein cells. Therefore, VPF/VEGF is ideally positioned to provoke the increased permeability of thecal blood vessels that occurs shortly before ovulation. VPF/VEGF likely also contributes to the angiogenesis and connective tissue stroma generation that accompany corpus luteum/corpus albicans formation. Finally, VPF/VEGF was overexpressed in the hyperthecotic ovarian stroma of Stein-Leventhal syndrome in which it may also have a pathophysiological role.     

Glomeruloid microvascular proliferation follows adenoviral vascular permeability factor/vascular endothelial growth factor-164 gene delivery

Glomeruloid bodies are a defining histological feature of glioblastoma multiforme and some other tumors and vascular malformations. Little is known about their pathogenesis. We injected a nonreplicating adenoviral vector engineered to express vascular permeability factor/vascular endothelial growth factor-164 (VPF/VEGF(164)) into the ears of athymic mice. This vector infected local cells that strongly expressed VPF/VEGF(164) mRNA for 10 to 14 days, after which expression gradually declined. Locally expressed VPF/VEGF(164) induced an early increase in microvascular permeability, leading within 24 hours to edema and deposition of extravascular fibrin; in addition, many pre-existing microvessels enlarged to form thin-walled, pericyte-poor, "mother" vessels. Glomeruloid body precursors were first detected at 3 days as focal accumulations of rapidly proliferating cells in the endothelial lining of mother vessels, immediately adjacent to cells expressing VPF/VEGF(164). Initially, glomeruloid bodies were comprised of endothelial cells but subsequently pericytes and macrophages also participated. As they enlarged by endothelial cell and pericyte proliferation, glomeruloid bodies severely compromised mother vessel lumens and blood flow. Subsequently, as VPF/VEGF(164) expression declined, glomeruloid bodies devolved throughout a period of weeks by apoptosis and reorganization into normal-appearing microvessels. These results provide the first animal model for inducing glomeruloid bodies and indicate that VPF/VEGF(164) is sufficient for their induction and necessary for their maintenance.     

Regulation of tumor growth and metastasis by interleukin-10: the melanoma experience.

Because interleukin-10 (IL-10) has potent immunosuppressive and anti-inflammatory properties and is produced by some cancers, including melanoma, we hypothesized that its production by tumor cells may contribute to the escape from immune surveillance. To test this hypothesis, we transfected human A375P melanoma cells that do not express IL-10 with the murine IL-10 gene and subsequently analyzed for changes in tumor growth and metastasis in nude mice. Surprisingly, IL-10 gene transfer resulted in a loss of metastasis and significant inhibition of tumor growth. In addition, the growth of other murine or human melanoma cells was also inhibited when they were admixed with IL-10-transfected cells before injection into nude mice. We provide evidence that IL-10 exerts its antitumor and antimetastatic activity by inhibiting angiogenesis in vivo. The in vivo decrease in neovascularization found in IL-10-secreting tumors is most likely due to the ability of IL-10 to downregulate the synthesis of vascular endothelial growth factor (VEGF), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and matrix metalloproteinase-9 (MMP-9) in tumor-associated macrophages. Other studies have shown that IL-10 inhibits tumor metastasis through a natural killer (NK) cell-dependent mechanism. The inhibitory effects of IL-10 on tumor growth and metastasis were also demonstrated in other tumor models, including breast cancers. Furthermore, administration of rIL-10 into mice resulted in inhibition of tumor metastasis. Because IL-10 has little toxicity when given systemically to human volunteers, its efficacy as an antimetastatic agent should be further explored, both as an independent and in combination with other inhibitors of neovascularization.     

Respiratory syncytial virus stimulation of vascular endothelial cell growth Factor/Vascular permeability factor

We hypothesized that respiratory syncytial virus (RSV)-induced pathologies could be mediated, in part, by vascular active cytokines elaborated during virus infection. To address this hypothesis, we determined whether RSV stimulated vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF) elaboration in vitro. Supernatants from unstimulated A549 cells and normal human bronchial epithelial cells contained modest levels of VEGF. In contrast, supernatants from RSV-infected cells contained elevated levels of VEGF/VPF. This stimulation was seen after as little as 2 h, was still prominent after 48 h, and, by immunoblot, was specific for the 165- and 121-amino acid isoforms of VEGF/VPF. It was not associated with significant cell cytotoxicity or alterations in VEGF messenger RNA. It did, however, require new protein biosynthesis. In accordance with these findings, the 165- and 121-amino acid isoforms of VEGF/VPF were also found in the nasal washings from patients with RSV infections. These studies demonstrate that RSV is a potent stimulator of VEGF/VPF elaboration and that, in vitro, this stimulation is mediated via a noncytotoxic translational and/or post-translational biosynthetic mechanism. VEGF/VPF may play an important role in the pathogenesis of RSV-induced disorders.     

血管内皮生长因子C及其受体3在非小细胞肺癌组织中的表达及意义

目的　探讨血管内皮生长因子C(VEGF C)和受体 (VEGFR) 3在人非小细胞肺癌(NSCLC)组织中的表达及其与微血管、微淋巴管形成、淋巴转移、预后之间的关系。方法　对 76例人NSCLC及相应癌旁组织行VEGF C、VEGFR 3及CD34免疫组织化学染色链霉素抗生物素蛋白 过氧化物酶 (SP)法检测 ,进行淋巴管密度计数、微血管密度 (MVD)计数 ,并结合临床和病理资料进行分析。结果　NSCLC中 ,VEGF C的表达与肺癌分化程度负相关 (P =0 0 0 9)。VEGF C和VEGFR 3的表达水平与淋巴结转移呈正相关 (分别P =0 0 0 8,P =0 0 13) ,与淋巴浸润呈正相关 (分别P =0 0 2 7,P =0 0 2 0 )。VEGF C的表达与VEGFR 3在肺癌细胞中的表达呈正相关 (P =0 0 0 9)。VEGF C与淋巴管密度 (P =0 0 0 6 )、MVD(P =0 0 4 6 )呈正相关。淋巴管密度与淋巴结转移 (P =0 0 10 )、淋巴浸润 (P =0 0 19)、TNM分期 (P =0 0 15 )呈正相关 ,MVD与血行转移 (P <0 0 0 1)、TNM分期 (P <0 0 0 1)呈正相关。VEGF C阳性表达与生存时间、5年生存率呈负相关 (P <0 0 0 1)。结论　NSCLC中 ,VEGF C通过自分泌方式作用于受体VEGFR 3,促进肺癌组织生长 ,抑制分化。VEGF C促使肺癌内淋巴管形成 ,促进肺癌淋巴结转移。VEGF C和VEGFR 3表达增高、淋巴管密度增加     

Vascular endothelial growth factor-165 overexpression stimulates angiogenesis and induces cyst formation and macrophage infiltration in human ovarian cancer xenografts

Vascular endothelial growth factor (VEGF) is suggested to be an important regulator of angiogenesis in ovarian cancer. We have evaluated the effects of VEGF overexpression on the histology and growth rate of human ovarian cancer xenografts. OVCAR-3 human ovarian cancer cells were stably transfected with an expression vector encoding the 165-amino acid isoform of VEGF. As subcutaneous xenografts, moderately and highly VEGF(165)-overexpressing OVCAR-3 cells formed tumors with large cysts. Immunohistochemistry demonstrated an increase in the number of CD31-positive microvessels, some of which were larger in diameter than those in the parental tumors, as well as extensive vascular rimming around the cysts. Weakly VEGF(165)-overexpressing tumors also contained an increased number of CD31-positive microvessels and occasional vascular rimming, but cysts were not present. Immunohistochemistry further revealed the presence of monocytes and macrophages in both parental and VEGF(165)-overexpressing xenografts. Interestingly, the number of monocytes/macrophages was greatly increased in moderately and highly VEGF(165)-overexpressing xenografts and large areas populated with monocytes/macrophages were detected within the tumor stroma. Although the higher number of CD31-positive cells would suggest a better vascularization pattern in VEGF(165)-overexpressing xenografts, tumor growth rates were not increased when compared with that of parental xenografts. These data provide functional evidence for a role of VEGF(165) in cyst formation and monocyte/macrophage infiltration.     

Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors.

The overexpression in tumor cells of (proto)-oncogenic receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or ErbB2/neu (also known as HER-2) is generally thought to contribute to the development of solid tumors primarily through their effects on promoting uncontrolled cell proliferation. However, agents that antagonize the function of the protein products encoded by these (proto)-oncogenes are known to behave in vivo in a cytotoxic-like manner. This implies that such oncogenes may regulate critical cell survival functions, including angiogenesis. The latter could occur as a consequence of regulation of relevant growth factors by such oncogenes. We therefore sought to determine whether EGFR or ErbB2/neu may contribute to tumor angiogenesis by examining their effects on the expression of vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF), one of the most important of all known inducers of tumor angiogenesis. We found that in vitro treatment of EGFR-positive A431 human epidermoid carcinoma cells, which are known to be heavily dependent on VEGF/VPF in vivo as an angiogenesis growth factor, with the C225 anti-EGFR neutralizing antibody caused a dose-dependent inhibition of VEGF protein expression. Prominent suppression of VEGF/VPF expression in vivo, as well as a significant reduction in tumor blood vessel counts, were also observed in established A431 tumors shortly after injection of the antibody as few as four times into nude mice. Transformation of NIH 3T3 fibroblasts with mutant ErbB2/neu, another EGFR-like oncogenic tyrosine kinase, resulted in a significant induction of VEGF/VPF, and the magnitude of this effect was further elevated by hypoxia. Moreover, treatment of ErbB2/neu-positive SKBR-3 human breast cancer cells in vitro with a specific neutralizing anti-ErbB2/neu monoclonal antibody (4D5) resulted in a dose-dependent reduction of VEGF/VPF protein expression. Taken together, the results suggest that oncogenic properties of EGFR and ErbB2/neu may, at least in part, be mediated by stimulation of tumor angiogenesis by up-regulating potent angiogenesis growth factors such as VEGF/VPF. These genetic changes may cooperate with epigenetic/environmental effects such as hypoxia to maximally stimulate VEGF/VPF expression. Therapeutic disruption of EGFR or ErbB2/neu protein function in vivo may therefore result in partial suppression of angiogenesis, a feature that could enhance the therapeutic index of such agents in vivo and endow them with anti-tumor effects, the magnitude of which may be out of proportion with their observed cytostatic effects in monolayer tissue culture.