Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases

The success of solid tumor growth and metastasis is dependent upon angiogenesis. Neovascularization within the tumor is regulated, in part, by a dual and opposing system of angiogenic and angiostatic factors. We now report that IP-10, a recently described angiostatic factor, as a potent angiostatic factor that regulates non-small cell lung cancer (NSCLC)-derived angiogenesis, tumor growth, and spontaneous metastasis. We initially found significantly elevated levels of IP-10 in freshly isolated human NSCLC samples of squamous cell carcinoma (SCCA). In contrast, levels of IP-10 were equivalent in either normal lung tissue or adenocarcinoma specimens. The neoplastic cells in specimens of SCCA were the predominant cells that appeared to express IP-10 by immunolocalization. Neutralization of IP-10 in SCCA tumor specimens resulted in enhanced tumor-derived angiogenic activity. Using a model of human NSCLC tumorigenesis in SCID mice, we found that NSCLC tumor growth was inversely correlated with levels of plasma or tumor- associated IP-10. IP-10 in vitro functioned as neither an autocrine growth factor nor as an inhibitor of proliferation of the NSCLC cell lines. Reconstitution of intratumor IP-10 for a period of 8 wk resulted in a significant inhibition of tumor growth, tumor-associated angiogenic activity and neovascularization, and spontaneous lung metastases, whereas, neutralization of IP-10 for 10 wk augmented tumor growth. These findings support the notion that tumor-derived IP-10 is an important endogenous angiostatic factor in NSCLC.     

Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung cancer.

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.     

ELR+ CXC chemokines and their receptors (CXC chemokine receptor 1 and CXC chemokine receptor 2) as new therapeutic targets

ELR+ CXC chemokines, by direct interaction with their cell surface receptors CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2), are believed to be crucially involved in the direct migration and activation of leukocytes. ELR+ CXC chemokines are supposed to play a key role in several inflammatory diseases and this makes ELR+ CXC chemokines and their receptors attractive therapeutic targets. The first aim of this review is to discuss the potential pathological role of ELR+ CXC chemokines in different pathologies, including ulcerative colitis (UC), ischaemia/reperfusion injury (RI), bronchiolitis obliterans syndrome (BOS) and tumor progression. Moreover, the most recently described inhibitors of ELR+ CXC chemokines and their therapeutic indications will be reviewed. Finally, the mode of action and the potential therapeutical use of reparixin, a new potent and selective inhibitor of CXCR1/2 activity, and its chemical derivatives are also discussed.     

Role of CXCR2/CXCR2 ligands in vascular remodeling during bronchiolitis obliterans syndrome

Angiogenesis and vascular remodeling support fibroproliferative processes; however, no study has addressed the importance of angiogenesis during fibro-obliteration of the allograft airway during bronchiolitis obliterans syndrome (BOS) that occurs after lung transplantation. The ELR(+) CXC chemokines both mediate neutrophil recruitment and promote angiogenesis. Their shared endothelial cell receptor is the G-coupled protein receptor CXC chemokine receptor 2 (CXCR2). We found that elevated levels of multiple ELR(+) CXC chemokines correlated with the presence of BOS. Proof-of-concept studies using a murine model of BOS not only demonstrated an early neutrophil infiltration but also marked vascular remodeling in the tracheal allografts. In addition, tracheal allograft ELR(+) CXC chemokines were persistently expressed even in the absence of significant neutrophil infiltration and were temporally associated with vascular remodeling during fibro-obliteration of the tracheal allograft. Furthermore, in neutralizing studies, treatment with anti-CXCR2 Abs inhibited early neutrophil infiltration and later vascular remodeling, which resulted in the attenuation of murine BOS. A more profound attenuation of fibro-obliteration was seen when CXCR2(-/-) mice received cyclosporin A. This supports the notion that the CXCR2/CXCR2 ligand biological axis has a bimodal function during the course of BOS: early, it is important for neutrophil recruitment and later, during fibro-obliteration, it is important for vascular remodeling independent of neutrophil recruitment.     

The angiostatic activity of interferon-inducible protein-10/CXCL10 in human melanoma depends on binding to CXCR3 but not to glycosaminoglycan.

Human interferon-inducible protein 10 (IP-10; HGMW-approved gene symbol CXCL10) is an ELR(-) CXC chemokine that contains binding domains for both the chemokine receptor CXCR3 and glycosaminoglycans. IP-10 has been recently demonstrated to be a potent angiostatic protein in vivo. Whether IP-10 exerts its angiostatic function through binding to CXCR3, glycosaminoglycans, or both, is not clear. To clarify this issue, we created expression constructs for mutants of IP-10 that exhibit partial (IP-10C) or total (IP-10C22) loss of binding to CXCR3 or loss of binding to glycosaminoglycans (IP-10H and IP-10C22H). The A375 human melanoma cell line was transfected with these expression vectors, and stable clones were selected and inoculated subcutaneously into nude mice. As expected, tumor cells secreting wild-type IP-10 showed remarkable reduction in tumor growth compared to control vector-transfected tumor cells. Surprisingly, mutation of IP-10 resulting in partial loss of receptor binding (IP-10C), or loss of GAG binding (IP-10H), did not significantly alter the ability to inhibit tumor growth. This tumor growth inhibition was associated with a reduction in microvessel density, leading to the observed increase in both tumor cell apoptosis and necrosis. In contrast, expression of the IP-10C22 mutant failed to inhibit melanoma tumor growth. These data suggest that CXCR3 receptor binding, but not glycosaminoglycan binding, is essential for the tumor angiostatic activity of IP-10. We conclude that the arginine 22 amino acid residue of IP-10 is essential for both CXCR3 binding and angiostasis.     

Cytokine- and chemokine-based gene therapy for cancer

Cytokines are protein/glycoprotein messengers of the immune system and have distinct autocrine and paracrine functions to modulate immunity. Recombinant cytokine proteins have been employed as biological drugs for cancer, viral and autoimmune targets. Unfortunately, systemic delivery of pharmacological doses of proteins often results in severe side effects and toxicities. As these therapeutic proteins tend to have very short half-lives and are complex to manufacture and deliver, many investigators are evaluating the genetic delivery of cytokine genes. Here, some of the promising cytokines currently under investigation for cancer therapies are examined, including interleukin (IL)-2, IL-4, IL-12, IL-24, interferon (IFN)-alpha, IFN beta, IFN gamma, granulocyte-monocyte colony-stimulating factor and tumor necrosis factor (TNF)-alpha. Chemokines are smaller chemotactic cytokines which induce migration of leukocytes, activate inflammatory responses, and are implicated in the regulation of tumor development and growth. Chemokines can modulate tumor growth via regulation of tumor-associated angiogenesis, by activation of host immunological responses or by direct inhibition of tumor cell proliferation. In this review, chemokines that have been proposed as antitumor drugs will be discussed, including Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG and SDF-1 alpha from the human CXC and C-C chemokine families.     

Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice.

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.     

CXCR4和VEGF-C在非小细胞肺癌中的表达及与淋巴结转移之间的关系

目的：探讨趋化因子受体4（CXCR 4）和血管内皮生长因子C（VEGF-C）在人非小细胞肺癌（N SCLC）组织中的表达及其与淋巴结转移的关系。方法：应用免疫组化SP法检测103例N SCLC和20例癌旁正常肺组织中CXCR 4和VEGF-C的表达。结果：CXCR 4和VEGF-C的表达与N SCLC患者的临床分期及有无淋巴结转移密切相关（P〈0.05）,而与患者的性别、年龄、肿瘤大小、肿瘤分化程度和组织学类型无关（P〉0.05）。CXCR 4阳性表达与VEGF-C阳性表达呈显著正相关（P〈0.05）。结论：CXCR 4和VEGF-C的表达可作为评估N SCLC转移与复发的一个观测指标。     

Combined CXC chemokine and interleukin-12 gene transfer enhances antitumor immunity

It has been shown that intratumor administration of an adenovirus vector expressing IL-12 produces a potent T cell-mediated response that leads to significant tumor regression in a murine breast cancer model. IP-10 and MIG are CXC chemokines that recruit mononuclear cells in vivo. In addition to their chemotactic roles, IP-10 and MIG inhibit angiogenesis. We tested whether the addition of IP-10 or MIG may both enhance the antitumor immune response of IL-12 through T cell recruitment and inhibit tumor growth through angiostasis. Adenovirus vectors expressing IP-10 or MIG and/or IL-12 were administered intratumorally in a murine model of mammary adenocarcinoma and fibrosarcoma. Administration of IP-10 or MIG in combination with IL-12 resulted in considerable tumor regression and increased survival time of tumor-bearing animals as compared with IP-10, MIG, IL-12 alone or control-treated animals, with the IP-10 IL-12 combination being most effective. These results suggest augmenting the antitumor immune response and inhibiting tumor angiogenesis with adenoviral vectors expressing IP-10 in combination with IL-12 is a novel way to enhance tumor regression.     

Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity

Endothelial cell receptors for the angiostatic chemokines IFN-gamma-inducible protein of 10 kDa (IP-10) and monokine induced by IFN-gamma (Mig) have not yet been identified, and the mechanisms responsible for the effects of these chemokines on angiogenesis are still unclear. IP-10 and Mig share a common functional receptor on activated T lymphocytes, named CXC chemokine receptor 3 (CXCR3). Using in situ hybridization and immunohistochemistry, we show that CXCR3 is expressed by a small percentage of microvascular endothelial cells in several human normal and pathological tissues. Primary cultures of human microvascular endothelial cells (HMVECs) likewise express CXCR3, although this expression is limited to the S/G2-M phase of their cell cycle. Both IP-10 and Mig, as well as the IFN-gamma-inducible T-cell alpha chemoattractant (I-TAC), which all share high-affinity binding for CXCR3, block HMVEC proliferation in vitro, an effect that can be inhibited by an anti-CXCR3 antibody. These data provide definitive evidence of CXCR3 expression by HMVEC and open new avenues for therapeutic interventions in all conditions in which an angiostatic effect may be beneficial.     

Expression of CXCR3 on mononuclear cells and CXCR3 ligands in patients with metastatic renal cell carcinoma in response to systemic IL-2 therapy

Chemokines play an important role in regulating tumor-mediated immunity, angiogenesis, and tumor cell metastasis. The chemokine receptor, CXCR3, is expressed in various human tumors, including renal cell carcinoma (RCC). CXCR3 is also associated with antiangiogenic effects in multiple tumors, and we hypothesized that interleukin-2 (IL-2) treatment of patients with metastatic clear cell RCC could augment CXCR3 levels on circulating mononuclear cells and correlate to outcome. The kinetics of CXCR3 expression on circulating mononuclear cells and its ligands (CXCL9, CXCL10, and CXCL11) in plasma were evaluated in 20 patients with metastatic clear cell RCC during cycles 1 and 2 of high dose IL-2 therapy. Subpopulations of peripheral blood mononuclear cells (PBMCs) were studied by dual color flow cytometry. Angiogenic ligands were measured and an "angiogenic ratio" was calculated prehigh and posthigh dose IL-2. CXCR3 expression on PBMC at baseline was similar in patients with metastatic RCC and normal controls. PBMC CXCR3 expression increased during treatment, and peaked during cycle 2. Plasma from RCC patients displayed similar baseline levels of CXCR3 ligands to normal controls. However, the angiogenic ratio was significantly increased in patients with metastatic RCC at baseline. Plasma levels of CXCR3 ligands increased during treatment, resulting in a reversal in the angiogenic ratio to favor angiostatic chemokines. The CXCR3/CXCR3 ligand biologic axis and angiogenic ratio may be important biomarkers in clear cell RCC patients who are undergoing high dose IL-2 therapy.     

An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig,and I-TAC,and acts as functional receptor for platelet factor 4

The chemokines CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC regulate lymphocyte chemotaxis, mediate vascular pericyte proliferation, and act as angiostatic agents, thus inhibiting tumor growth. These multiple activities are apparently mediated by a unique G protein-coupled receptor, termed CXCR3. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, including a powerful angiostatic effect, but its specific receptor is still unknown. Here, we describe a distinct, previously unrecognized receptor named CXCR3-B, derived from an alternative splicing of the CXCR3 gene that mediates the angiostatic activity of CXCR3 ligands and also acts as functional receptor for CXCL4. Human microvascular endothelial cell line-1 (HMEC-1), transfected with either the known CXCR3 (renamed CXCR3-A) or CXCR3-B, bound CXCL9, CXCL10, and CXCL11, whereas CXCL4 showed high affinity only for CXCR3-B. Overexpression of CXCR3-A induced an increase of survival, whereas overexpression of CXCR3-B dramatically reduced DNA synthesis and up-regulated apoptotic HMEC-1 death through activation of distinct signal transduction pathways. Remarkably, primary cultures of human microvascular endothelial cells, whose growth is inhibited by CXCL9, CXCL10, CXCL11, and CXCL4, expressed CXCR3-B, but not CXCR3-A. Finally, monoclonal antibodies raised to selectively recognize CXCR3-B reacted with endothelial cells from neoplastic tissues, providing evidence that CXCR3-B is also expressed in vivo and may account for the angiostatic effects of CXC chemokines.     

Combination of MIG (CXCL9) chemokine gene therapy with low-dose cisplatin improves therapeutic efficacy against murine carcinoma

MIG (monokine induced by interferon-gamma) is a CXC chemokine ligand (CXCL9) that can potently inhibit angiogenesis, and displays thymus-dependent antitumor effects. The effectiveness of a treatment combining gene therapy with plasmid-borne MIG (pORF-MIG) and low-dose cisplatin chemotherapy was determined using colon carcinoma (CT26) and Lewis lung carcinoma (LL/2c) murine models. The program was carried out via intramuscular delivery of pORF-MIG at 100 mug/mouse twice a week for 4 weeks, and/or intraperitoneal delivery of cisplatin at 0.6 mg/kg/mouse every 3 days for 48 days. Tumor volume and survival time were evaluated after treatment. CD31 immunohistochemical staining in tumor tissues and alginate capsule models in vivo was used to evaluate angiogenesis. Induction of apoptosis and cytotoxic T-lymphocyte (CTL) activity were also assessed. The combination of pORF-MIG and low-dose cisplatin produced significant antitumor activity, with complete tumor regression in 4/10 of CT26 colon carcinomas and 3/10 of LL/2c lung carcinomas, low vascularity, in alginate capsules, apparently degraded tumor microvessel density, and increased induction of apoptotic and CTL activities compared with either treatment alone. This study suggests that the combination of pORF-MIG plus cisplatin augments the inhibition of angiogenesis and the induction of apoptosis or CTL activity, all of which enhance antitumor activity. These findings may prove useful in further explorations of the application of combinatorial approaches to the treatment of solid tumors.     

Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration, Metastasis and Neovascularization

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.     

瘦素在人非小细胞肺癌中的表达及其与肿瘤血管生成的关系

目的：研究瘦素在正常肺组织及非小细胞肺癌（NSCLC）组织中的表达,并探讨其与肿瘤血管生成、转移、分期、分型的关系。方法：用免疫组织化学SP法检测60例NSCLC组织和10例癌旁正常肺组织中瘦素与血管内皮生长因子（VEGF）的表达情况。结果：瘦素在NSCLC组织及正常肺组织中均有表达,且在NSCLC组织中异质性高表达（P=0.049）。NSCLC组织中瘦素的表达与肿瘤的分化程度（P=0.024）、有无淋巴结转移（P=0.010）、临床TNM分期（P=0.024）均存在相关性。瘦素和VEGF在NSCLC组织中的表达与肿瘤淋巴结转移及肿瘤分化程度都存在相关性;在NSCLC组织中,瘦素呈表达强阳性者,其VEGF的表达率阳性者也明显增加,两者表达呈正相关（r=0.970,P〈0.01）。结论：瘦素在肺癌组织中呈异质性高表达,且与NSCLC的临床病理特征密切相关;瘦素有可能协同VEGF或通过上调VEGF的表达而促进NSCLC中肿瘤血管的生成,在NSCLC的浸润和转移过程中起作用。     

环氧合酶-2对非小细胞肺癌血管生成及预后的影响

目的研究环氧合酶-2（COX-2）在非小细胞肺癌（NSCLC）中的表达及其对血管生成和预后的影响。方法随访手术切除且临床资料完整的48例非小细胞肺癌患者,采用免疫组织化学技术检测活检标本中COX-2、血管内皮生长因子（VEGF）表达和微血管密度（MVD）。通过生存曲线法比较阳性组和阴性组患者的预后情况。结果COX-2在非小细胞肺癌组织中阳性表达率为66.7%（32/48）,与VEGF表达68.8%（33/48）呈显著正相关（r=0.687,P〈0.01）,COX-2与淋巴结转移、组织分型、肺癌分期及较短生存期具有相关性。MVD随COX-2、VEGF表达增强而增高。结论COX-2的高表达可使VEGF表达上调,促进肿瘤血管形成,增加微血管密度。COX-2在非小细胞肺癌组织中高表达与肿瘤血管生成密切相关,可作为预测非小细胞肺癌预后的一种有用指标。     

Prevention of angiogenesis by naked DNA IL-12 gene transfer: angioprevention by immunogene therapy

IL-12 is thought to induce a cytokine cascade with antiangiogenic effects mediated by IFN-gamma and angiostatic CXCR3 chemokine ligands. Naked DNA intramuscular injection of an expression vector plasmid producing IL-12 resulted in significant, well-tolerated elevation of serum IL-12 levels. Injection of the IL-12 plasmid at least 2 days, and up to 20 days, before subcutaneous injection of matrigel with angiogenic factors resulted in strong prevention of angiogenesis in both C57/bl and nude mice. A single injection of the IL-12 plasmid contemporarily with the matrigel or 2 days after resulted in partial, statistically not significant, inhibition. Control plasmid injection did not affect either angiogenesis or angiogenesis inhibition by IL-12 protein in vivo. Angiogenesis inhibition was observed in NK cell-depleted C57/bl and nude mice as well as in IFN-gamma(-/-) and CXCR3(-/-) knockout mice, indicating that NK- and/or T-cell-initiated IFN-gamma-chemokine cascades were not involved in the angiogenesis inhibition observed in vivo. Finally, IL-12 plasmid DNA gene transfer significantly prevented the growth and vascularization of highly angiogenic KS-Imm Kaposi's sarcoma and TS/A murine mammary carcinoma tumors in nude and/or syngeneic mice. These data suggest that a preventive gene therapy approach using antiangiogenic cytokines can effectively inhibit tumor angiogenesis and KS, representing an example of angioimmunoprevention.     

CXC chemokine expression after stimulation with interferon-gamma in primary rat hepatocytes in culture

Monokine-induced by gamma interferon (MIG) and gamma-interferon-inducible protein (IP-10) are members of the CXC chemokine family that have been shown to be induced by interferon-gamma (IFNy) in some cell types. The purpose of this investigation was to determine whether IFNgamma influences CXC chemokine production, particularly MIG and IP-10, in primary rat hepatocytes in culture. Previous experiments in our laboratory have demonstrated that pharmacologic doses of IFNgamma in an in vivo model of hepatic ischemia/reperfusion-induced liver injury resulted in increased hepatic levels of IP-10 and MIG and decreased hepatic levels of macrophage inflammatory protein-2, Kupffer cells, and epithelial neutrophil-activating protein, with a concomitant decrease in neutrophil-mediated hepatic injury. In the current investigation, MIG and IP-10 mRNA and protein were up-regulated in primary rat hepatocytes in vitro in response to IFNgamma. Although MIG and IP-10 mRNA were both somewhat increased at early time points, larger increases in these chemokines were seen at later time points, specifically at 24, 48, and 72 h of incubation as compared to controls. Levels of Kupffer cells and macrophage inflammatory protein-2 mRNA after IFNgamma were negligible and similar to those seen in controls. These findings were confirmed by enzyme-linked immunosorbent assay analysis. These studies demonstrate that IFNgamma in vitro up-regulates the production of MIG and IP-10, at both the mRNA and protein levels.     

Rho-kinase signaling regulates pulmonary infiltration of neutrophils in abdominal sepsis via attenuation of CXC chemokine formation and Mac-1 expression on neutrophils

Excessive neutrophil infiltration is a major component in septic lung injury, although the signaling mechanisms behind pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. Herein, we hypothesized that Rho-kinase activity may play a significant role in pulmonary neutrophil recruitment and tissue damage in abdominal sepsis. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 (0.5 or 5 mg/kg) before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid and lung tissue were harvested for analysis of neutrophil infiltration, as well as edema and CXC chemokine formation. Blood was collected for analysis of Mac-1 on neutrophils and CD40L on platelets as well as soluble CD40L and matrix metalloproteinase 9 (MMP-9) in plasma. Cecal ligation and puncture triggered significant pulmonary damage characterized by neutrophil infiltration, increased levels of CXC chemokines, and edema formation in the lung. Furthermore, CLP upregulated Mac-1 expression on neutrophils, decreased CD40L on platelets, and increased soluble CD40L and MMP-9 in the circulation. Interestingly, inhibition of Rho-kinase dose-dependently decreased CLP-induced neutrophil expression of Mac-1, formation of CXC chemokines and edema, as well as neutrophil infiltration and tissue damage in the lung. Moreover, Rho-kinase inhibition significantly reduced sepsis-provoked gene expression of CXC chemokines in alveolar macrophages. In contrast, Rho-kinase inhibition had no effect on platelet shedding of CD40L or plasma levels of MMP-9 in septic mice. In conclusion, these data demonstrate that the Rho-kinase signaling pathway plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of CXC chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis.     

Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer

The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.