Synchronous down-modulation of miR-17 family members is an early causative event in the retinal angiogenic switch.

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3' UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response.

Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer.

Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

Cooperative effects of aminopeptidase N (CD13) expressed by nonmalignant and cancer cells within the tumor microenvironment.

Processes that promote cancer progression such as angiogenesis require a functional interplay between malignant and nonmalignant cells in the tumor microenvironment. The metalloprotease aminopeptidase N (APN; CD13) is often overexpressed in tumor cells and has been implicated in angiogenesis and cancer progression. Our previous studies of APN-null mice revealed impaired neoangiogenesis in model systems without cancer cells and suggested the hypothesis that APN expressed by nonmalignant cells might promote tumor growth. We tested this hypothesis by comparing the effects of APN deficiency in allografted malignant (tumor) and nonmalignant (host) cells on tumor growth and metastasis in APN-null mice. In two independent tumor graft models, APN activity in both the tumors and the host cells cooperate to promote tumor vascularization and growth. Loss of APN expression by the host and/or the malignant cells also impaired lung metastasis in experimental mouse models. Thus, cooperation in APN expression by both cancer cells and nonmalignant stromal cells within the tumor microenvironment promotes angiogenesis, tumor growth, and metastasis.

Vascular ligand-receptor mapping by direct combinatorial selection in cancer patients.

Molecules differentially expressed in blood vessels among organs or between damaged and normal tissues, are attractive therapy targets; however, their identification within the human vasculature is challenging. Here we screened a peptide library in cancer patients to uncover ligand-receptors common or specific to certain vascular beds. Surveying ~2.35 x 10(6) motifs recovered from biopsies yielded a nonrandom distribution, indicating that systemic tissue targeting is feasible. High-throughput analysis by similarity search, protein arrays, and affinity chromatography revealed four native ligand-receptors, three of which were previously unrecognized. Two are shared among multiple tissues (integrin α4/annexin A4 and cathepsin B/apolipoprotein E3) and the other two have a restricted and specific distribution in normal tissue (prohibitin/annexin A2 in white adipose tissue) or cancer (RAGE/leukocyte proteinase-3 in bone metastases). These findings provide vascular molecular markers for biotechnology and medical applications.

On the synergistic effects of ligand-mediated and phage-intrinsic properties during in vivo selection.

Phage display has been used as a powerful tool in the discovery and characterization of ligand-receptor complexes that can be utilized for therapeutic applications as well as to elucidate disease mechanisms. While the basic properties of phage itself have been well described, the behavior of phage in an in vivo setting is not as well understood due to the complexity of the system. Here, we take a dual approach in describing the biophysical mechanisms and properties that contribute to the efficacy of in vivo phage targeting. We begin by considering the interaction between phage and target by applying a kinetic model of ligand-receptor complexation and internalization. The multivalent display of peptides on the pIII capsid of phage is also discussed as an augmenting factor in the binding affinity of phage-displayed peptides to cellular targets accessible in a microenvironment of interest. Lastly, we examine the physical properties of the total phage particle that facilitate improved delivery and targeting in vivo compared to free peptides.

MicroRNAs and ultraconserved genes as diagnostic markers and therapeutic targets in cancer and cardiovascular diseases.

MicroRNAs (miRNAs), approximately 19-25 nucleotides in length, are posttranscriptional regulators of protein expression that target and inhibit translation of messenger (m) RNAs. Recent research on miRNAs has produced a plethora of new material on the role of miRNAs in disease. Deregulation or ablation of miRNA expression has led to major pathologies including heart disease and cancer. Signatures of differential miRNA expression have been uncovered for nearly every disease. Recent research has focused on exploitation of the selectivity of these signatures as markers of disease and for therapeutic applications. The significance of additional mechanisms of abnormal posttranscriptional regulation, such as ultraconserved genes (UCGs), has recently been recognized. This review focuses on the identification of aberrant posttranscriptional regulators (miRNAs and UCGs) in cancer and cardiovascular disease and addresses the applications of this work towards diagnosis and therapy.

Combinatorial ligand-directed lung targeting.

Phage display of random peptide libraries is a powerful, unbiased method frequently used to discover ligands for virtually any protein of interest and to reveal functional protein-protein interaction partners. Moreover, in vivo phage display permits selection of peptides that bind specifically to different vascular beds without any previous knowledge pertaining to the nature of their corresponding receptors. Vascular targeting exploits molecular differences inherent in blood vessels within given organs and tissues, as well as diversity between normal and angiogenic blood vessels. Over the years, our group has identified phage capable of homing to lung blood vessels based on screenings using immortalized lung endothelial cells combined with in vivo selections after intravenous administration of combinatorial libraries. Peptides targeting lung vasculature have been extensively characterized and a lead homing peptide has shown interesting biological properties, bringing novel insights as to the implications of lung endothelial cell apoptosis in the pathogenesis of emphysema. We have also designed and developed targeted nanoparticles with imaging capabilities and/or drug delivery functions by combining phage display technology and elemental gold (Au) nanoparticles, constituting a promising platform for the development of therapeutic agents for imaging and treatment of lung disorders. Given the important role of the endothelium in the pathogenesis and progression of several diseases associated with the airways, ligand-directed discovery of lung vascular markers is an important milestone toward the development of future targeted therapies.