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Vasculogenic mimicry (VM) is a non-classical mechanism recently described in many tumors, whereby cancer cells, rather than endothelial cells, form blood vessels. Transgelin is an actin-binding protein that has been implicated in multiple stages of cancer development. In this study, we investigated the role of transgelin in VM and assessed its effect on the expression of endothelial and angiogenesis-related genes during VM in MDA-MB-231 breast cancer cells. We confirmed the ability of MDA-MB-231 cells to undergo VM through a tube formation assay. Flow cytometry analysis revealed an increase in the expression of the endothelial-related markers VE-cadherin and CD34 in cells that underwent VM, compared with those growing in a monolayer, which was confirmed by immunocytochemistry. We employed siRNA to silence transgelin, and knockdown efficiency was determined by western blot analyses. Downregulation of transgelin suppressed cell proliferation and tube formation, but increased IL-8 levels in Matrigel cultures. RT-PCR analyses revealed that the expression of IL-8, VE-cadherin, and CD34 was unaffected by transgelin knockdown, indicating that increased IL-8 expression was not due to enhanced transcriptional activity. More importantly, the inhibition of IL-8/CXCR2 signaling also resulted in suppression of VM with increased IL-8 levels, confirming that increased IL-8 levels after transgelin knockdown was due to inhibition of IL-8 uptake. Our findings indicate that transgelin regulates VM by enhancing IL uptake. These observations are relevant to the future development of efficient antivascular agents. Impact statement Vasculogenic mimicry (VM) is an angiogenic-independent mechanism of blood vessel formation whereby aggressive tumor cells undergo formation of capillary-like structures. Thus, interventions aimed at angiogenesis might not target the entire tumor vasculature. A more holistic approach is therefore needed in the development of improved antivascular agents. Transgelin, an actin-binding protein, has been associated with multiple stages of cancer development such as proliferation, migration and invasion, but little is known about its role in vasculogenic mimicry. We present here, an additional mechanism by which transgelin promotes malignancy by way of its association with the occurrence of VM. Although transgelin knockdown did not affect the transcript levels of most of the angiogenesis-related genes in this study, it was associated with the inhibition of the uptake of IL-8, accompanied by suppressed VM, indicating that transgelin is required for VM. These observations are relevant to the future development of efficient antivascular agents.

Background: Chemokines have been recognized as important modulators of angiogenesis, and they play critical roles in the development and metastasis of hepatocellular carcinoma (HCC), although their origins and latent molecular mechanisms remain elusive. The aim of this study was to investigate how activated hepatic stellate cells (a-HSCs) promote angiogenesis in HCC., Methods: A total of 22 HCC patients were enrolled randomly. We used immunohistochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA) to analyse the production of interleukin-8 (IL-8) in a-HSCs derived from HCC tissues. The angiogenic effects of IL-8 in vitro and in vivo were assessed by ELISA, real-time quantitative polymerase chain reaction, capillary tube formation assay, and chick embryo chorioallantoic membrane assay., Results: The present study showed that IL-8 was enriched predominantly in the tumour stroma of HCC tissues and was mainly derived from a-HSCs, rather than from hepatoma cells, in vivo and in vitro. Angiogenesis was most active at the invading edge, which was close to the a-HSCs. The angiogenic effect was dramatically attenuated by an IL-8 neutralizing antibody both in vitro and in vivo. Moreover, the IL-8 neutralizing antibody down-regulated Ser727-phosphorylated STAT3 levels in hepatoma cells treated with a-HSCs conditioned medium., Conclusions: These findings reveal that a-HSCs within the stroma of HCC contribute to tumour angiogenesis via IL-8.

Study Question: How does hypoxia-mediated down-regulation of dual specificity phosphatase-2 (DUSP2) promote endometriotic lesion development?, Summary Answer: Inhibition of DUSP2 by hypoxia enhances endometriotic lesion growth via promoting interleukin-8 (IL-8)-dependent angiogenesis., What Is Known Already: Angiogenesis is a prerequisite for the development of endometriosis. DUSP2 is down-regulated in endometriotic stromal cells in a hypoxia inducible factor-1α-dependent manner. Down-regulation of DUSP2 contributes to the pathological process of endometriosis., Study Design, Size, Duration: A laboratory study recruiting 20 patients of reproductive age with endometriosis and normal menstrual cycles, and an autoimplant-induced mouse model of endometriosis using 13 mice in a 28-day treatment., Participants/materials, Setting, Methods: IL-8 mRNA levels were assayed in endometrial stromal cells maintained in normoxic or hypoxic (1% O2) conditions, with or without DUSP2 knockdown. Promoter activity and chromatin immunoprecipitation (ChIP) assays were conducted to characterize the regulation of IL-8 by DUSP2. Conditioned media from cells maintained in normoxic or hypoxic conditions, and cells with/without DUSP2 knockdown were collected to investigate the angiogenic capacity using an in vitro tube formation assay. Reparixin, an IL-8 receptor blocker, was administered to investigate the role of IL-8 in hypoxia-mediated angiogenesis and the development of endometriotic-like lesions in an autotransplanted mouse model., Main Results and the Role of Chance: IL-8 mRNA was increased by both hypoxia and DUSP2 knockdown in endometrial stromal cells in an extracellular signal-regulated protein kinase-dependent manner (P < 0.05 versus control). Promoter activity and ChIP assays demonstrated that expression of IL-8 was regulated by CCAAT/enhancer binding protein α (P < 0.05 versus control). Furthermore, conditioned media collected from hypoxia-exposed or DUSP2 knockdown endometrial stromal cells promoted tube formation, which was abolished by co-treatment with reparixin (P < 0.05 versus control). Results from the autotransplanted mouse model demonstrated that number of blood vessels and size of endometriotic-like lesions were markedly reduced in recipient mice treated with reparixin (P < 0.05 versus control)., Limitations, Reasons for Caution: This study was conducted in primary human cell cultures and a mouse model, therefore may not fully reflect the situation in vivo., Wider Implications of the Findings: This is the first study to highlight the potential application of an IL-8 receptor blocker as a therapeutic target to treat endometriosis. This study demonstrates IL-8 as a key angiogenic factor regulated by hypoxia/DUSP2, which suggests an alternative mechanism through which hypoxia may promote angiogenesis., Study Funding/competing Interests: This study was funded by the National Science Council of Taiwan (NSC101-2314-B-006-043-MY2). The author declares that there is no conflict of interest., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Notch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether Notch signalling mediates vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2)-induced vascular function., Methods: Notch-1 intracellular domain (Notch-1 IC), Notch-4 IC, Delta-like-ligand 4, Hes-related transcriptional repressors-1 and 2 (Hrt-1, Hrt-2) mRNA and/or protein expression was measured by Real-time PCR and/or western blot. VEGF/Ang2 induced EC function was assessed using transwell invasion chambers, matrigel tube formation assays and wound repair scratch assays±Notch-1 siRNA or an γ-secretase inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanly))-S-phenylglycine-t-Butyl Ester (DAPT) in RA synovial explants or human microvascular EC. Interleukin (IL)-6 and IL-8 were measured by ELISA and MMP2 and 9 by gelatine zymography., Results: Notch-1 IC and Notch-4 IC protein expressions were demonstrated in RA and psoriatic arthritis synovial biopsies, with minimal expression observed in Osteoarthritis (OA). VEGF and Ang2 induced Notch-1 IC/ Notch-4 IC protein expression in synovial explant cultures and human microvascular EC levels were further potentiated by VEGF/Ang2 stimulation in combination. Notch-1, Delta-like-ligand 4, and Hrt-2 mRNA expression were significantly induced by VEGF and Ang2 alone and in combination. Furthermore VEGF/Ang2-induced EC invasion, angiogenesis and migration were inhibited by Notch-1 siRNA or DAPT. Conditioned media from VEGF/Ang2 stimulated RA synovial explants induced EC tube formation, an effect that was inhibited by DAPT. Finally, DAPT significantly decreased VEGF/Ang2 induced IL-6, IL-8, MMP2 and 9 expressions in RA synovial explants., Conclusions: Notch-1 mediates VEGF/Ang2-induced angiogenesis and EC invasion in inflammatory arthritis.

The expression of lysophosphatidic acid (LPA)-specific receptors in cervical cancer has not been clearly defined. In this study, we identified LPA1, LPA2 and LPA3 receptors' mRNA in SiHa, HeLa and CaSki cell lines by RT-PCR. These receptors were not associated with tumor cell proliferation in vitro. We then used a xenograph animal model to evaluate the effects of these receptors on in vivo cervical cancer tumorigenicity. When SiHa cells with different receptor expression patterns were seeded on the backs of SCID mice, the resulting knockout of both LPA2 and LPA3 significantly attenuated tumor growth; this decrease in tumor growth was found to be linked with decreased angiogenesis (microvessel density), suggesting that LPA2 and LPA3 are crucial for in vivo tumor growth through an angiogenic mechanism. We further investigated this mechanism of LPA receptor 2/3-mediated angiogenic capability by analyzing angiogenic factors in protein lysates from receptor knockout tumors, by detecting interleukin (IL-8) mRNA expression after treating with siRNA, by evaluating the biological role of LPA-enhanced IL-8 via endothelial cell tube formation, monolayer permeability, migration and cell growth assays, and by IL-8 knockout xenograft mice modeling. We found that the angiogenesis is mediated through IL-8. Finally, we evaluated the regulation pathways involved in LPA-induced IL-8 expression. We found that LPA receptor 2/3-mediated IL-8 expression occurs through Gi/PI3K/AKT, Gi/PKC and IκB/NF-κB signaling. In conclusion, we propose that LPA2 and LPA3 might play an important role in cervical cancer tumor growth through IL-8-dependent angiogenesis., (Copyright © 2011 UICC.)

Unlabelled: A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem-like cells, called tumor-initiating cells (TICs). We previously identified a TIC population derived from hepatocellular carcinoma (HCC) that is characterized by membrane expression of CD133. Here, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome-wide microarray analysis. A significantly dysregulated interleukin-8 (IL-8) signaling network was identified in CD133(+) liver TICs obtained from HCC clinical samples and cell lines. IL-8 was found to be overexpressed at both the genomic and proteomic levels in CD133(+) cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL-8 secretion in CD133(+) liver TICs to exhibit a greater ability to self-renew, induce tumor angiogenesis, and initiate tumors. In further support of these observations, IL-8 repression in CD133(+) liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL-8 functional network identified neurotensin (NTS) and CXCL1 to be preferentially expressed in CD133(+) liver TICs. Addition of exogenous NTS resulted in concomitant up-regulation of IL-8 and CXCL1 with simultaneous activation of p-ERK1/2 and RAF-1, both key components of the mitogen-activated protein kinase (MAPK) pathway. Enhanced IL-8 secretion by CD133(+) liver TICs can in turn activate an IL-8-dependent feedback loop that signals through the MAPK pathway. Further, in its role as a liver TIC marker CD133 also plays a functional part in regulating tumorigenesis of liver TICs by way of regulating NTS, IL-8, CXCL1, and MAPK signaling., Conclusion: CD133(+) liver TICs promote angiogenesis, tumorigenesis, and self-renewal through NTS-induced activation of the IL-8 signaling cascade., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Angiogenesis is implicated in many pathological conditions, including cancer progression. Novel approaches have enabled an understanding of how endothelial cellular processes are regulated in vivo during angiogenesis. Key players in angiogenesis are vascular endothelial growth factors (VEGFs) and Notch ligands. However, mechanisms of angiogenic responses by other proangiogenic factors in vivo are largely unknown. Research using cultured endothelial cells has shown that c-Fes is involved in the activation of phosphoinositide 3-kinase (PI3-kinase) downstream of a variety of cytokine receptors. The PI3-kinase/c-Akt pathway regulates cell survival, migration, and morphological differentiation of endothelial cells during angiogenesis, and c-Fes thus may be a potential target of anti-cancer therapy, especially for patients with anti-VEGF refractory cancer. In addition, a number of experiments have shown that a bone marrow-derived monocytic lineage regulates angiogenesis, and c-Fes is also expressed in these cells. Roles for c-Fes during angiogenesis will be the focus of extensive research in the future.

Three-dimensional culture alters cancer cell signaling; however, the underlying mechanisms and importance of these changes on tumor vascularization remain unclear. A hydrogel system was used to examine the role of the transition from 2D to 3D culture, with and without integrin engagement, on cancer cell angiogenic capability. Three-dimensional culture recreated tumor microenvironmental cues and led to enhanced interleukin 8 (IL-8) secretion that depended on integrin engagement with adhesion peptides coupled to the polymer. In contrast, vascular endothelial growth factor (VEGF) secretion was unaffected by 3D culture with or without substrate adhesion. IL-8 diffused greater distances and was present in higher concentrations in the systemic circulation, relative to VEGF. Implantation of a polymeric IL-8 delivery system into GFP bone marrow-transplanted mice revealed that localized IL-8 up-regulation was critical to both the local and systemic control of tumor vascularization in vivo. In summary, 3D integrin engagement within tumor microenvironments regulates cancer cell angiogenic signaling, and controlled local and systemic blockade of both IL-8 and VEGF signaling may improve antiangiogenic therapies.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.

Tumor-derived growth factors and cytokines stimulate neoangiogenesis from surrounding capillaries to support tumor growth. Recent studies have revealed that macrophage migration inhibitory factor (MIF) expression is increased in lung cancer, particularly non-small cell lung carcinomas (NSCLC). Because MIF has important autocrine effects on normal and transformed cells, we investigated whether autocrine MIF and its only known family member, D-dopachrome tautomerase (D-DT), promote the expression of proangiogenic factors CXCL8 and vascular endothelial growth factor in NSCLC cells. Our results demonstrate that the expression of CXCL8 and vascular endothelial growth factor are strongly reliant upon both the individual and cooperative activities of the two family members. CXCL8 transcriptional regulation by MIF and D-DT appears to involve a signaling pathway that includes the activation of JNK, c-jun phosphorylation, and subsequent AP-1 transcription factor activity. Importantly, HUVEC migration and tube formation induced by supernatants from lung adenocarcinoma cells lacking either or both MIF and D-DT are substantially reduced when compared with normal supernatants. Finally, we demonstrate that the cognate MIF receptor, CD74, is necessary for both MIF- and D-DT-induced JNK activation and CXCL8 expression, suggesting its potential involvement in angiogenic growth factor expression. This is the first demonstration of a biological role for D-DT, and its synergism with MIF suggests that the combined therapeutic targeting of both family members may enhance current anti-MIF-based therapies.

Tank-Binding-Kinase 1 (TBK-1) has been proposed as a putative mediator in tumor angiogenesis. It was the aim of our study to gain insight into TBK-1s role in tumor angiogenesis and tumor-associated microvascular inflammation. TBK-1 overexpressing KB 3-1 cells were generated and their growth characteristics were analyzed. Expression of TBK-1, VEGF, RANTES and Il-8 were quantified using qPCR and western blot analysis. Intravital microscopy using the dorsal skinfold chamber model in nude mice addressed total (TIVD) and functional intratumoral vascular density (FIVD), perfusion index, vessel diameter and leukocyte sticking. Transfection of KB-3 cells resulted in significantly increased TBK-1, RANTES and IL-8 expression without affecting cellular growth. Supernatants from TBK-1 overexpressing clones induced HUVEC proliferation. Intravital microscopy identified an increase in leukocyte sticking paralleled by significantly increased TIVD and FIVD as a result of increased VEGF expression. Therefore, TBK-1 represents a novel mediator of tumor angiogenesis and exerts proinflammatory effects via upregulation of inflammatory cytokines. The TBK-1 pathway might be an important cross-link between angiogenesis and inflammation representing a possible target for anti-tumor therapy.

In this study, we investigated the anti-angiogenic potential of nitric oxide (NO) donors and anti-integrin compounds against neutrophil-activating protein-2 (NAP-2), interleukin-8 (IL-8), and basic fibroblast growth factor (FGF-2)-induced angiogenesis. In vivo, recombinant human NAP-2 and FGF-2 induce a potent and comparable angiogenic response in the chick embryo chorioallantoic membrane (CAM). We demonstrate that NO donors and anti-integrin agents are capable of abrogating either NAP-2- or FGF-2-induced angiogenesis in the CAM model. The NO donor, S-nitroso N-acetyl penicillamine (SNAP), blocked either NAP-2- or FGF-2-mediated angiogenesis in the CAM. Similarly, angiogenesis stimulated with NAP-2 or FGF-2 was blocked by antagonist of the alphavbeta3 integrin in the CAM model. However, the inhibition of NAP-2 and IL-8 by the anti-integrin compound is significantly less than the inhibition observed with FGF-2 as the angiogenic stimulus. Similarly, the ability of these mechanisms to also inhibit endothelial cell differentiation was demonstrated. Taken together, these data illustrate the involvement of multiple pathways in the mechanisms of action for the alpha-chemokine- and cytokine-mediated angiogenesis. These approaches may be a useful tool for the inhibition of angiogenesis associated with human tumor growth or with neovascular, ocular, and inflammatory diseases where chemokines and cytokines are involved.

Angiogenesis is an essential step in initial tumor development and metastasis. Consequently, compounds that inhibit angiogenesis would be useful in treating cancer. A variety of antitumor effects mediated by 1alpha, 25-dihydroxyvitamin D3 (1,25-VD) have been reported, one of which is anti-angiogenesis; however, detailed mechanisms remain unclear. We have demonstrated that 1,25-VD inhibits prostate cancer (PCa) cell-induced human umbilical vein endothelial cell migration and tube formation, two critical steps involved in the angiogenesis. An angiogenesis factor, interleukin-8 (IL-8), secreted from PCa cell was suppressed by 1,25-VD at both mRNA and protein levels. Mechanistic dissection found that 1,25-VD inhibits NF-kappaB signal, one of the most important IL-8 upstream regulators. The 1,25-VD-mediated NF-kappaB signal reduction was shown to result from the blocking of nuclear translocation of p65, a subunit of the NF-kappaB complex, and was followed by attenuation of the NF-kappaB complex binding to DNA. The role of IL-8 in PCa progression was further examined by PCa tissue microarray analyses. We found that IL-8 expression was elevated during PCa progression, which suggests that IL-8 may play a role in tumor progression mediated through its stimulation on angiogenesis. These findings indicate that 1,25-VD could prevent PCa progression by interrupting IL-8 signaling, which is required in tumor angiogenesis, and thus applying vitamin D in PCa treatment may be beneficial for controlling disease progression.

The gram-negative bacterium Bartonella henselae is capable of causing angiogenic lesions as a result of infection. Previously, it has been shown that B. henselae infection can result in production of the chemokine interleukin-8 (IL-8). In this study, we demonstrated that monocytes, endothelial cells, and hepatocytes produce IL-8 in response to B. henselae infection. We also investigated the role of IL-8 in B. henselae-induced endothelial cell proliferation and capillary tube formation. Both in vitro angiogenesis assays were IL-8 dependent. B. henselae-mediated inhibition of apoptosis, as indicated by gene expression of Bax and Bcl-2, was also shown to be IL-8 dependent in endothelial cells. Furthermore, infection of endothelial cells with B. henselae stimulated upregulation of the IL-8 chemokine receptor CXCR2. Infection of human endothelial cells by B. henselae resulting in IL-8 production likely plays a central role in the ability of this organism to cause angiogenesis during infection.

Background: Angiogenic factor seems necessary for the development of hepatocellular carcinoma (HCC), which is a hypervascular malignancy. This study examined the expression of interleukin (IL)-8, a potent angiogenic factor, in HCC samples., Methods: We measured IL-8 expression by using reverse transcriptase-polymerase chain reaction in clinical HCC tissues from 45 patients who underwent surgical resection. We then assessed correlations between IL-8 expression and microvessel growth or clinicopathologic factors. We also elucidated the in vitro effect of IL-8 on HepG2 development by using fluorometric assays of proliferation, chemotaxis, and invasion., Results: The expression of IL-8 did not significantly correlate with the microvessel count in HCC tissues, but the incidence of microscopic vessel invasion was significantly higher in IL-8-positive than in IL-8-negative tissues. Thus, more IL-8 was expressed in HCCs at pathologic stage III/IV than in those at stage I/II. Assays in vitro showed that IL-8 stimulates HepG2 chemotactic and invasive activities rather than cell proliferation., Conclusions: The expression of IL-8 in human HCC has more relevance to metastatic potential, such as vessel invasion, than to angiogenesis or cell proliferation.

Hypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis. It represents an attractive therapeutic target in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis. In HIF-1alpha knockdown DLD-1 colon cancer cells (DLD-1(HIF-kd)), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1alpha (DLD-1(HIF-wt)). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1(HIF-kd) but not DLD-1(HIF-wt) cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-kappaB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1(HIF-kd) but not DLD-1(HIF-wt) xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1alpha may be most effective when IL-8 is simultaneously targeted.

Abstract Interleukin (IL)-8, a cytokine of the CXC chemokine family that was originally classified as a neutrophil chemoattractant, is now reported to play an important role in tumor progression and metastasis in a variety of human cancers, including lung cancers. IL-8 biologic activity in tumors and the tumor microenvironment may contribute to tumor progression through its potential function in the regulation of angiogenesis, cancer cell growth and survival, tumor cell motion, leukocyte infiltration and modification of immune responses. Recently, infiltrating macrophages in tumor stroma have been considered to be able to stimulate cancer growth, enhance angiogenesis and promote metastasis, and has prognostic significance in several human cancers. Accumulating evidence also shows that cancer cells and stromal cell interaction can stimulate cancer cells, as well as stromal cells in the expression of IL-8 and other growth factors. Here, we summarize current information about IL-8 biology in human lung cancers and focus on its effect on tumor angiogenesis, regulation of IL-8 expression in tumors, its prognostic significances, the role of tumor infiltrating macrophages in the production of IL-8 in cancer cells and the tumor microenvironment, gene expression profiles after cancer cell-stromal cell interaction, and the effect of a variety anti- inflammatory drugs on the modification of IL-8 and other gene expressions in cancer cells and the tumor microenvironment in lung cancers.

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., K(d), approximately 2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal disorder. Fibroplasia and deposition of extracellular matrix are dependent, in part, on angiogenesis and vascular remodeling. We obtained open lung biopsies from patients undergoing thoracic surgery for reasons other than interstitial lung disease (control) (n = 78) and from patients with IPF (n = 91). We found that levels of epithelial neutrophil-activating peptide 78 (ENA-78) were greater from tissue specimens of IPF patients, as compared with control subjects. When ENA-78 was depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced. Immunolocalization of ENA-78 demonstrated that hyperplastic Type II pneumocytes and macrophages were the predominant cellular sources of ENA-78. These findings support the notion that ENA-78 may be an important additional factor that regulates angiogenic activity in IPF.

Anti-angiogenesis is likely to develop into a novel therapeutic approach for patients with solid malignancies. Most current clinical trials evaluate anti-angiogenic drugs aimed primarily against single angiogenesis stimulators. Here, we show that a single solid malignancy, i.e., a human embryonal rhabdomyosarcoma, produces in vivo at least three biologically active angiogenesis stimulators (vascular endothelial growth factor, basic fibroblast growth factor and interleukin-8). This suggests that tumour angiogenesis results from the activity of multiple, rather than a single angiogenesis stimulator(s). We, furthermore, show that a combination of anti-angiogenic drugs is more effective in inhibiting tumour-induced endothelial cell growth than a single agent. Our results imply that clinical anti-angiogenic strategies for the treatment of solid malignancies may be most effective when multiple rather than single antiangiogenic drugs are used.

Angiogenesis, the formation of new vessels from pre-existing vasculature, is critical to ascites development and metastasis in ovarian cancer. Many growth factors important to ovarian cancer invasion are also prominent in its associated angiogenesis. Deregulation of normal angiogenic processes occurs with the cancer's acquisition of the ability to secrete pro-angiogenic factors. The local imbalance of endogenous angio-stimulators and angio-inhibitors promotes vascularization and vascular leak. Assessment of these pro-angiogenic growth factors and enumeration of tumour-associated microvessels have been shown to be prognosticators of ovarian cancer outcome, and may also be surrogates of ovarian cancer tumour burden and/or ascites formation. The process of angiogenesis has been targeted for therapeutics development. Ovarian cancer is a primary cancer against which these new agents are being tested. Thus, further understanding of the molecular and cell biology of angiogenesis in the context of ovarian cancer offers important directions for estimation of patient outcome and for patient treatment.

Angiogenesis is a significant prognostic factor in melanoma, but the angiogenic factors controlling the neovascularization are not well defined. The purpose of this study was to investigate whether the angiogenesis and metastasis of melanoma are promoted by vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), platelet-derived endothelial cell growth factor (PD-ECGF), and/or basic fibroblast growth factor (bFGF). Cells from human melanoma lines (A-07, D-12, R-18, and U-25) transplanted to BALB/c nu/nu mice were used as tumor models. Expression of angiogenic factors was studied by ELISA, Western blotting, and immunohistochemistry. Angiogenesis was assessed by using an intradermal angiogenesis assay. Lung colonization and spontaneous lung metastasis were determined after i.v. and intradermal inoculation of tumor cells, respectively. The specific roles of VEGF, IL-8, PD-ECGF, and bFGF in tumor angiogenesis, lung colonization, and spontaneous metastasis were assessed in mice treated with neutralizing antibody. The melanoma lines expressed multiple angiogenic factors, and each line showed a unique expression pattern. Multiple angiogenic factors promoted angiogenesis in the most angiogenic melanoma lines, whereas angiogenesis in the least angiogenic melanoma lines was possibly promoted solely by VEGF. Tumor growth, lung colonization, and spontaneous metastasis were controlled by the rate of angiogenesis and hence by the angiogenic factors promoting the angiogenesis. Lung colonization and spontaneous metastasis in A-07 were inhibited by treatment with neutralizing antibody against VEGF, IL-8, PD-ECGF, or bFGF. Each of these angiogenic factors may promote metastasis in melanoma, because inhibition of one of them could not be compensated for by the others. Our observations suggest that efficient antiangiogenic treatment of melanoma may require identification and blocking of common functional features of several angiogenic factors.

The growth and spread of tumour cells depends on adequate vasculature. We have previously reported that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. To provide evidence for a causal role of IL-8 in angiogenesis and tumorigenicity of human gastric cancer, we used the lipofectin method to stably transfect the human TMK-1 gastric carcinoma cells (low endogenous IL-8) with an IL-8 expression vector or control vector. Transfection with IL-8 did not affect the proliferation of cultured cells, yet the culture supernatants of the transfected (but not control) cells stimulated proliferation of human umbilical vein endothelial cells. The IL-8-transfected and control cells were injected into the gastric wall of nude mice. IL-8-transfected cells produced rapidly growing, highly vascular neoplasms as compared to control cells. These results provide direct evidence for the role of IL-8 in the angiogenesis and tumorigenicity of human gastric carcinomas.

Expression of interleukin-8 (IL-8) by human melanoma cells correlates with their metastatic potential in vivo. Moreover, UVB irradiation of primary cutaneous melanoma induces IL-8 mRNA and protein production and increases both tumor growth and metastasis in nude mice. Although IL-8 has been shown to be an angiogenic factor, the biological consequences of increased IL-8 production by melanoma cells and the role of IL-8 in the metastatic process remains unclear. The purpose of this review is to determine the role of IL-8 in tumor growth and metastasis of human melanoma. Transfection of nonmetastatic and IL-8-negative melanoma cells with the IL-8 gene rendered them highly tumorigenic and increased their metastatic potential in nude mice. The IL-8-transfected cells displayed upregulation of MMP-2 expression and activity and increased invasiveness through Matrigel-coated filters. Activation of MMP-2 by IL-8 can enhance the invasion of host stroma by the tumor cells and increase angiogenesis and, hence, metastasis. In addition to UVB, IL-8 can also be upregulated by hypoxia conditions, suggesting that the environment plays a major role in regulating IL-8 expression and metastasis. The studies summarized in this review suggest that in melanoma, IL-8 may serve as the angiogenic factor distinguishing benign from malignant cells.

Among angiogenic factors, VEGF secreted from activated macrophages under the influence of ovarian steroids, IL-8 expressed in endometrial stromal cells, and basic FGF expressed in endometriotic tissue and PD-ECGF expressed in lining epithelial cells independently of the sex steroidal milieu might contribute to the characteristic advancement of angiogenic lesions in endometriosis in individual manners. Copyrightz1999S.KargerAG,Basel

We report here the role of the CXC chemokine, epithelial neutrophil activating peptide (ENA-78), as an angiogenic factor in human non-small cell lung cancer (NSCLC). In freshly isolated human specimens of NSCLC, elevated levels of ENA-78 were found that strongly correlated with the vascularity of the tumors. In a SCID mouse model of human NSCLC tumorigenesis, expression of ENA-78 in developing tumors correlated with tumor growth in two different NSCLC cell lines. Furthermore, passive immunization of NSCLC tumor-bearing mice with neutralizing anti-ENA-78 antibodies reduced tumor growth, tumor vascularity, and spontaneous metastases, while having no effect on the proliferation of NSCLC cells either in vitro or in vivo. These findings suggest that ENA-78 is an important angiogenic factor in human NSCLC.

We examined interleukin-8 (IL-8) production in 17 lung cancer cell lines, IL-8 expression in tumor specimens and IL-8's contribution to tumor-induced angiogenesis in vivo. Eight of 13 non-small cell lung cancer cell lines constitutively produced high levels of IL-8. Four small cell lung cancer cell lines produced little or no IL-8. Immunohistochemical analysis of transbronchial biopsy specimens revealed IL-8 staining within adenocarcinomas (22/32), squamous cell carcinomas (12/21) and large cell carcinomas (2/3), but not within most small cell carcinomas (1/22). Anti-IL-8 antisera blocked tumor angiogenesis by two IL-8 producing cell lines in a mouse model.

Idiopathic pulmonary fibrosis (IPF) is a chronic and often fatal disorder. Fibroplasia and deposition of extracellular matrix are dependent, in part, on angiogenesis. We postulated that an imbalance exists in the expression of angiogenic (IL-8) vs angiostatic (IFN-gamma-inducible protein (IP-10)) CXC chemokines, which favors net angiogenesis in IPF. To test this hypothesis, we obtained open lung biopsies either from normal patients undergoing thoracic surgery for reasons other than interstitial lung disease (control) or from patients with IPF. We found that levels of IL-8 were greater from tissue specimens of IPF patients then from those of controls. In contrast, IP-10 levels were higher from tissue specimens obtained from control subjects than from those from IPF patients. When IL-8 or IP-10 was depleted from IPF tissue specimens, tissue-derived angiogenic activity was markedly reduced or enhanced, respectively. Immunolocalization of IL-8 demonstrated that the pulmonary fibroblast (PF) of IPF lung was the predominant cellular source of IL-8. Isolated PF from IPF patients constitutively produced more IL-8 and less IP-10 than control PF. Conditioned media from IPF-PFs demonstrated constitutive angiogenic activity that was attributable, in part, to IL-8. Depletion of IP-10 from IPF-PF CM resulted in an increase in corneal neovascularization. These findings support the notion that IL-8 and IP-10 are important factors that regulate angiogenic activity in IPF.

The salient feature of solid tumor growth is the strict dependence on local angiogenesis. We have previously demonstrated that IL-8 is an angiogenic factor present in freshly isolated specimens of human non-small cell lung cancer (NSCLC). Using a model of human NSCLC tumorigenesis in SCID mice, we now report that IL-8 acts as a promoter of human NSCLC tumor growth through its angiogenic properties. Passive immunization with neutralizing antibodies to IL-8 resulted in more than 40% reduction in tumor size and was associated with a decline in tumor-associated vascular density and angiogenic activity. IL-8 did not act as an autocrine growth factor for NSCLC proliferation. The reduction in primary tumor size in response to neutralizing antibodies to IL-8 was also accompanied by a trend toward a decrease in spontaneous metastasis to the lung. These data support the notion that IL-8 plays a significant role in mediating angiogenic activity during tumorigenesis of human NSCLC, thereby offering a potential target for immunotherapy against solid tumors.

In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma-dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fibroblast growth factor (bFGF) and 10 ng/ml of interleukin-8 (IL-8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co-cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor-beta mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor-alpha mRNA. By contrast, IN157 cells had much higher levels of IL-8 mRNA than IN301 cells. IN301-dependent tubular morphogenesis was inhibited by anti-VEGF or anti-bFGF antibody, and the inhibition was almost complete when anti-VEGF and anti-bFGF antibodies were present. On the other hand, IN157-dependent tubular morphogenesis was inhibited by anti-IL-8 antibody, but not by anti-VEGF or anti-bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL-8.

Lung cancer is the leading cause of malignancy-related mortality in the U.S. and is predicted to increase over the remainder of this decade. Despite attempts to advance early diagnosis and use combination therapies, the clinical response of this cancer yields an overall 5-year survival rate of less than 15%. Clearly, new strategies for therapy are indicated. Although carcinogenesis is complex, tumor growth beyond 1-2 mm3 is dependent on angiogenesis. One of the potential mechanisms that allows for tumorigenesis is dysregulation of the balance of angiogenic and angiostatic factors that favors net neovascularization within the primary tumor. Numerous studies have investigated the role of a variety of molecules in the regulation of angiogenesis. Recently, interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be an angiogenic factor. In contrast, platelet factor 4 (PF4), another C-X-C chemokine, has been shown to have angiostatic properties. It is interesting that the major structural difference between IL-8 and PF4 is the presence of the NH2-terminal ELR (Glu-Leu-Arg) motif that precedes the first cysteine amino acid residue of IL-8 and is important in ligand/receptor interactions. We hypothesize that angiogenesis associated with tumorigenesis is dependent on members of the C-X-C chemokine family acting as either angiogenic or angiostatic factors. This paradigm predicts that the biological balance in the expression of these C-X-C chemokines dictates whether the neoplasm grows and develops metastatic potential or regresses. In this review we discuss our recent laboratory findings that support this contention and suggest that further elucidation of the biology of C-X-C chemokines in the context of neovascularization of nonsmall cell lung cancer will permit novel targeted therapy aimed specifically at attenuating tumor growth and metastasis.

Background: We have previously described a method of quantitating angiogenesis by using a simple 133Xe clearance technique for repeated measurement of relative blood flow changes through s.c. sponge implants over a period of 14 days. The quantitative requirement of this bioassay is that the measurements of 6-minute 133Xe clearance should provide a fast and reliable means to detect relative blood flow changes in the neovasculature, so a more vigorous validation of the use of the 133Xe clearance technique as an indicator of angiogenesis is needed., Experimental Design: Four different techniques were used: (a) to measure absolute blood flow in the sponges using 113Sn microspheres; (b) to quantitate the levels of hemoglobin and total protein in the implants; (c) to determine the amount of neovasculature in the sponges by the carmine dye method; and (d) to carry out histologic and morphometric analysis of sponge implants. To confirm parallel changes in 133Xe clearance and in the other techniques, the effects of selected angiogenic promoters and inhibitors were also investigated., Results: There was a good correlation between 133Xe clearance from the sponges and absolute blood flow (r = 0.952, p < 0.01); the levels of hemoglobin (r = 0.982, p < 0.01) and total protein (r = 0.962, p < 0.01); the amount of carmine dye (r = 0.974, p < 0.01); the fibrovascular growth areas (r = 0.992, p < 0.01); and the vascular density (r = 0.997, p < 0.01) in the implants. Daily administration of 3 pmol of IL-1 alpha or IL-8 caused intense neovascularization. When given alone, lower doses of IL-1 alpha (0.3 pmol) or bradykinin (10 pmol) produced no apparent effect. However, co-administration of these doses to a single sponge together caused an increase in the rate of angiogenesis similar to that seen with a higher dose of IL-1 alpha (3 pmol) acting alone. In contrast, daily co-administration of a potent and selective protein kinase C inhibitor, calphostin C (4 micrograms), inhibited the neovascular response elicited by 3 pmol of IL-1 alpha. Furthermore, daily doses of 5 micrograms of dexamethasone for 14 days inhibited sponge-induced angiogenesis., Conclusions: The results clearly show that the 133Xe clearance technique not only gives an indication of the rate of perfusion of the sponges with blood but also gives a good estimate of its functional vascularity. Thus, the measurement of 133Xe clearance in the sponge implant provides a simple and objective method for routine studies of modifiers of angiogenesis.

We investigated the role of interleukin 8 (IL-8) in mediating angiogenesis in human bronchogenic carcinoma. Increased quantities of IL-8 were detected in tumor tissue as compared with normal lung tissue. Immunohistochemical staining of tumors revealed primary localization of IL-8 to individual tumor cells and demonstrated the capacity of tumor to elaborate IL-8. Functional studies that used tissue homogenates of tumors demonstrated the induction of both in vitro endothelial cell chemotaxis and in vivo corneal neovascularization. It is important to note that the addition of neutralizing antisera to IL-8 to these assays resulted in the marked and specific attenuation of these responses. Our observations definitively establish IL-8 as a primary mediator of angiogenesis in bronchogenic carcinoma and offer a potential target for immunotherapies against solid malignancies.

A rabbit corneal pocket model was used to demonstrate that physiologic concentrations of human recombinant (r) IL-8 may induce corneal neovascularization. Computer-assisted analysis of sequential fluorescein angiograms showed that rIL-8 doses ranging from 2 to 40 ng/cornea (P = 0.01), but not high dose rIL-8 (400 ng/cornea), results in neovascularization within 14 days. Repeat fluorescein angiograms 6 weeks after placing angiogenic doses of rIL-8 demonstrated significant regression (P = 0.01) of the vascularity present at 2 weeks, suggesting that IL-8 angiogenesis undergoes dynamic modulation similar to that normally seen in wound healing. To our knowledge, this is the first study showing an angiogenic role for IL-8, a finding that emphasizes the interplay between inflammation and wound healing. Our results imply that corneal-derived IL-8 may be important in corneal neovascularization, in particular, and that IL-8 may modulate wound healing in general. Finally, these results raise the possibility that corneal-derived cytokines, such as IL-8, may obfuscate the effects of agents tested in experimental corneal pocket models.