Identification of VEGF-independent cytokines in proliferative diabetic retinopathy vitreous.

PURPOSE: To identify inflammatory cytokines significantly elevated and independent of VEGF levels in the vitreous of proliferative diabetic retinopathy (PDR) patients that may serve as novel diagnostic factors or therapeutic targets. METHODS: Thirty-nine cytokines and chemokines were measured from the vitreous of 72 patients undergoing vitrectomy (29 controls and 43 PDR) via a magnetic bead-based immunoassay. Patient information, including sex, age, history of smoking, cancer diagnosis and treatment, and presence of diabetes and hypertension were also collected. Univariate and multivariate logistic regression analyses were performed to assess the association of cytokine concentrations and patient demographics with disease. RESULTS: Nineteen cytokines were significantly elevated in the vitreous of PDR patients compared with controls, including five novel cytokines that have not previously been associated with PDR: sCD40L, GM-CSF, IFNα2, IL-12p40, and MCP-3. Sixteen cytokines were found to be statistically independent of VEGF. Of these, 14 show a statistically significant interaction with VEGF, while two do not. With regards to patient demographics, age and hypertension were statistically significant risk factors with the odds of disease decreasing with increasing age and increasing 3-fold for hypertensive patients. CONCLUSIONS: This is the first report of a comprehensive multiplex analysis to identify novel VEGF-independent cytokines associated with PDR. Of the 39 inflammatory cytokines tested, 16 are predictive of disease risk, independent of VEGF levels. These PDR-associated cytokines represent potential targets in the treatment of PDR, both in conjunction with anti-VEGF therapy, as well as for patients that are nonresponders to such therapy.

MEK genomics in development and disease.

The mitogen-activated protein kinase kinases (the MAPK/ERK kinases; MKKs or MEKs) and their downstream substrates, the extracellular-regulated kinases have been intensively studied for their roles in development and disease. Until recently, it had been assumed any mutation affecting their function would have lethal consequences. However, the identification of MEK1 and MEK2 mutations in developmental syndromes as well as chemotherapy-resistant tumors, and the discovery of genomic variants in MEK1 and MEK2 have led to the realization the extent of genomic variation associated with MEKs is much greater than had been appreciated. In this review, we will discuss these recent advances, relating them to what is currently understood about the structure and function of MEKs, and describe how they change our understanding of the role of MEKs in development and disease.

Opportunities and challenges in tumor angiogenesis research: back and forth between bench and bed.

Angiogenesis is essential for tumor growth and metastasis. Many signaling pathways are involved in regulating tumor angiogenesis, with the vascular endothelial growth factor pathway being of particular interest. The recognition of the heterogeneity in tumor vasculature has led to better predictions of prognosis through differential analyses of the vasculature. However, the clinical benefits from antiangiogenic therapy are limited, because many antiangiogenic agents cannot provide long-term survival benefits, suggesting the development of drug resistance. Activation of the hypoxia and c-Met pathways, as well as other proangiogenic factors, has been shown to be responsible for such resistance. Vessel co-option could be another important mechanism. For future development, research to improve the efficacy of antiangiogenic therapy includes (a) using tumor-derived endothelial cells for drug screening; (b) developing the drugs focusing on specific tumor types; (c) developing a better preclinical model for drug study; (d) developing more accurate biomarkers for patient selection; (e) targeting the c-Met pathway or other pathways; and (f) optimizing the dose and schedule of antiangiogenic therapy. In summary, the future of antiangiogenic therapy for cancer patients depends on our efforts to develop the right drugs, select the right patients, and optimize the treatment conditions.

Persistent inhibition of oxygen-induced retinal neovascularization by anthrax lethal toxin.

PURPOSE: To evaluate the role of mitogen-activated protein kinase kinase (MKK) signaling in a mouse model of oxygen-induced retinopathy (OIR) that mimics retinopathy of prematurity (ROP). METHODS: Postnatal day 7 mice were exposed to elevated oxygen for 5 days to induce retinopathy. Anthrax lethal toxin (LeTx), an MKK inhibitor, was injected into the vitreous after restoration to normoxia, and its effects on vascular growth were analyzed by whole mount immunofluorescence and confocal microscopy. Pericyte coverage was determined by PDGFR-ß and α-SMA staining. Macrophage presence was determined by F4/80 staining. Vitreal cytokine secretion was measured by ELISA and multianalyte profiling. RESULTS: Intravitreal injection of LeTx over a restricted time interval after return to normoxic conditions blocked the progression of OIR. This block was independent of vascular endothelial growth factor (VEGF) release and did not alter the release of cytokines and growth factors associated with OIR. VEGFR2 expression and activation were similarly unaffected. LeTx had no statistically significant effect on macrophage recruitment. LeTx sensitivity correlated with vessel maturity, extent of hypoxia, and growth of the deep vascular plexus network. CONCLUSIONS: Correlation among pericyte coverage, deep vascular plexus growth, and hypoxia after LeTx treatment indicate immature vessels in a hypoxic environment are preferentially sensitive to LeTx-mediated MKK inhibition. The persistence of VEGF without concomitant induction of neovascular growth or revascularization of vaso-obliterated zones suggests MKK inhibition causes an inability of the cells that are present, or a failure to recruit cells able, to respond to proangiogenic stimuli. These results indicate the inhibition of MKK signaling presents a novel strategy for the inhibition of vascular retinopathies such as OIR and ROP.

Perturbation of mouse retinal vascular morphogenesis by anthrax lethal toxin.

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.

Cytotoxicity of the matrix metalloproteinase-activated anthrax lethal toxin is dependent on gelatinase expression and B-RAF status in human melanoma cells.

Anthrax lethal toxin (LeTx) shows potent mitogen-activated protein kinase pathway inhibition and apoptosis in melanoma cells that harbor the activating V600E B-RAF mutation. LeTx is composed of two proteins, protective antigen and lethal factor. Uptake of the toxin into cells is dependent on proteolytic activation of protective antigen by the ubiquitously expressed furin or furin-like proteases. To circumvent nonspecific LeTx activation, a substrate preferably cleaved by gelatinases was substituted for the furin LeTx activation site. Here, we have shown that the toxicity of this matrix metalloproteinase (MMP)-activated LeTx is dependent on host cell surface MMP-2 and MMP-9 activity as well as the presence of the activating V600E B-RAF mutation, making this toxin dual specific. This additional layer of tumor cell specificity would potentially decrease systemic toxicity from the reduction of nonspecific toxin activation while retaining antitumor efficacy in patients with V600E B-RAF melanomas. Moreover, our results indicate that cell surface-associated gelatinase expression can be used to predict sensitivity among V600E B-RAF melanomas. This finding will aid in the better selection of patients that will potentially respond to MMP-activated LeTx therapy.

Biological and biochemical characterization of anthrax lethal factor, a proteolytic inhibitor of MEK signaling pathways.

The secretion of factors that block critical intracellular signaling pathways is a common strategy used by pathogenic bacteria for disabling host defenses and causing disease. Anthrax lethal toxin (LeTx) has been shown to cleave and inactivate mitogen-activated protein kinase (MAPK) kinases (MKKs or MEKs) and to inhibit MKK signaling. Cleavage of MKKs by LeTx prevents activation of their downstream substrates, the MAPKs. Because MAPK pathways regulate a variety of crucial cellular functions including proliferation, survival, differentiation, adhesion, and motility, LeTx has become a focus of study as an investigative tool as well as for the treatment and prevention of diseases due to malfunctions in MAPK signaling. This chapter describes methods for expressing and purifying the components of LeTx and focuses on techniques available for assessing its activity.

LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin.

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)-deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.

The role of the human papillomavirus type 18 E7 oncoprotein during the complete viral life cycle.

The role of the human papillomavirus oncoprotein E7 in carcinogenesis has been extensively studied. While the role of HPV E7 in the viral life cycle has also been studied, certain disparities exist, indicating that genotype differences may influence the role that E7 plays in the viral life cycle. In this study, we investigated the role of HPV18 E7 in the viral life cycle in order to gain a further understanding of this issue. To determine the role that HPV18 E7 plays in the viral life cycle, a translation termination substitution mutant of E7 in the context of the full HPV18 genome was created. We introduced linearized HPV18 E7-deficient genomic DNA into primary keratinocytes, where it recircularized and was maintained episomally at a range of five to several hundred copies of HPV genomic DNA. The mutant genomes failed to amplify following epithelial stratification and differentiation in organotypic culture. Moreover, virion morphogenesis did not occur. We found that the expression of HPV16 or HPV18 E7 in trans was able to rescue the amplification defect but not the defect in virion morphogenesis. These studies indicate that HPV18 E7 plays a critical role in the productive stage of the viral life cycle. In addition, these studies add further proof to the hypothesis that genotype differences exist for the role of E7 during the viral life cycle.

Analysis of tumor-host interactions by gene expression profiling of lung adenocarcinoma xenografts identifies genes involved in tumor formation.

Tumor cell lines are relied on extensively for cancer investigations, yet cultured cells in an in vitro environment differ considerably in behavior compared with those of the same cancer cells that proliferate and form tumors in vivo. To uncover gene expression changes related to tumor formation, gene expression profiles of human lung adenocarcinoma (A549) cells grown as lung tumors in immune-compromised mice were compared with profiles of the same cells grown in vitro. Additionally, profiles of uninvolved adjacent mouse tissue were determined. A profound interplay between cancer cells and the host was shown that affected a complex protein interaction network involving processes of extracellular interaction, growth factor signaling, hemostasis, immune response, and transcriptional regulation. Growth in vivo of A549 cells, which carry an activating k-ras mutation, induced changes in gene expression that corresponded highly to a pattern characteristic of human lung tumors with k-ras mutation. Cytokines interleukin-4, interleukin-6, and IFN-gamma each induced distinct in vitro genomic responses in cancer cells that emulated many of the changes in gene expression observed in vivo. Genes that were both selectively induced in vivo and overexpressed in human lung adenocarcinoma tumors included CSPG2, which has not been associated previously with tumor formation. Knockdown in A549 of CSPG2 by RNA interference significantly inhibited tumor growth in vivo but not in vitro. Thus, analysis of tumor xenografts by gene expression profiling has the potential for identifying genes involved in tumor development that may not be expressed in cancer cells grown in vitro.

Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector.

While recent studies have demonstrated that retroviral vectors can be used to stably express short hairpin RNA (shRNA) to inhibit gene expression, these studies have utilized replication-defective retroviruses. We describe the creation of a replication-competent, Gateway-compatible retroviral vector capable of expressing shRNA that inhibits the expression of specific genes.

Induction of the upstream regulatory region of human papillomavirus type 31 by dexamethasone is differentiation dependent.

Glucocorticoids have been shown to play a role in the transforming abilities of human papillomaviruses (HPVs), and glucocorticoid response elements (GREs) have been identified in the upstream regulatory regions (URRs) of various HPV types. These findings have made glucocorticoids potential therapeutic targets for HPV infection. We have previously shown that the URR of HPV type 31 (HPV31) is insensitive to induction by the synthetic glucocorticoid dexamethasone (dex) in monolayer culture, despite the identification of three potential GREs in the 5' region of the URR. Due to the fact that the HPV life cycle is intimately linked to the differentiation of the host tissue, we chose to determine whether the URR of HPV31 was inducible by dex under differentiating conditions. Upon suspension of cells in a semisolid medium of methylcellulose, we found that the URR of HPV31 was inducible by dex. The three GREs appear to play roles as independent repressors of this inducibility. By 5' deletion analysis, the element(s) responsible for this induction was localized to nucleotides (nt) 7238 to 7557. Furthermore, we found that the region between nt 7883 and 7900 appears to act as a repressor of dex inducibility. These findings indicate that epithelial differentiation has a profound effect on the action of dex on the URR of HPV31, suggesting that glucocorticoids play an important role in the differentiation-dependent life cycle of HPV.

Comparison of the basal and glucocorticoid-inducible activities of the upstream regulatory regions of HPV18 and HPV31 in multiple epithelial cell lines.

Steroid hormone receptors have been shown to bind to response elements in the upstream regulatory region (URR) of human papillomavirus (HPV) in a ligand-dependent manner to affect viral promoter activity. To better understand how the enhancer activity of the URR differs between high risk HPV types, we chose to compare the basal and glucocorticoid-dependent activities of the URRs of HPV18 and HPV31. We found that the URR of HPV18 is a stronger enhancer than the URR of HPV31 in six different cell lines of epithelial origin. Furthermore, the activity of the URR of HPV31 was not inducible by the synthetic glucocorticoid dexamethasone (dex) in any cell line tested, while the URR of HPV18 was dex-inducible in the majority of these lines. These studies indicate significant differences between the URRs of high risk HPV types.

The upstream regulatory region of human papillomavirus type 31 is insensitive to glucocorticoid induction.

The upstream regulatory region (URR) of various types of human papillomaviruses (HPVs) has been shown to contain functional glucocorticoid response elements (GREs), including HPV type 11 (HPV11), HPV16, and HPV18. Glucocorticoids have been demonstrated to induce the transcriptional activity of the early promoters of these HPV types. Although it has been assumed that the URR of HPV31 contains at least one GRE, no functionality has been demonstrated. We attempt to show here inducibility of the URR of HPV31 by the synthetic glucocorticoid dexamethasone (dex). By sequence analysis we identified three potential GREs in the URR of HPV31. Gel shift analysis indicated that each of these three sites has the potential to be a functional GRE. However, constructs containing the full-length URR, 5' deletions of the URR, and an internal fragment of the URR containing all three putative GREs were only weakly inducible by dex. Linker scanning mutants, whereby each potential GRE was replaced individually, in double combination, or in triple combination by a unique polylinker, had no effect on dex inducibility. Replacement of each of the three HPV31 GREs with the GRE of HPV18 failed to induce a response to dex. Placement of the HPV18 GRE into the URR of HPV31 in a region similar to its location in the HPV18 URR was also unable to result in a strong dex induction of the HPV31 URR. These data suggest that the lack of dex inducibility is due to the overall context of the HPV31 URR and may be dependent on the requirements of the major early promoter for transcriptional activation. Finally, replacement of the HPV18 GRE with each of the HPV31 GREs in HPV18 only showed weak inducibility, indicating that the three GREs of HPV31 are in fact only weak inducers of dex. Overall, these data suggest that dex responsiveness, along with oncogenic potential, may provide a possible explanation for the classification of HPV31 as an intermediate-risk virus and demonstrate the complexity of transcriptional regulation of the URR of HPV.

Infectious virions produced from a human papillomavirus type 18/16 genomic DNA chimera.

The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.

Genetic analysis of cis regulatory elements within the 5' region of the human papillomavirus type 31 upstream regulatory region during different stages of the viral life cycle.

The function of the 5' region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5' region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the viral life cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the viral life cycle. This study identified (i) viral cis elements that regulate transcription in the presence and absence of any viral gene products or viral DNA replication, (ii) the role of host tissue differentiation in viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5' region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the viral life cycle.