Combinatorial selection of a single stranded DNA thioaptamer targeting TGF-beta1 protein

A phosphorothioate single-stranded DNA aptamer (thioaptamer) targeting transforming growth factor-beta1 (TGF-beta1) was isolated by in-vitro combinatorial selection. The aptamer selection procedure was designed to modify the backbone of single-stranded DNA aptamers, where 5' of both A and C are phosphorothioates, since this provides enhanced nuclease resistance as well as higher affinity than that of a phosphate counterpart. The thioaptamer selected from a combinatorial library (5x10(14) sequences) binds to TGF-beta1 protein with an affinity of 90 nM. In this report, sequence, predicted secondary structure, and binding affinity of the selected thioaptamer (T18_1_3) are presented.     

Combinatorial selection of a RNA thioaptamer that binds to Venezuelan equine encephalitis virus capsid protein

A phosphorothioate RNA aptamer (thioaptamer) targeting the capsid protein of Venezuelan equine encephalitis virus (VEEV) was isolated by in vitro combinatorial selection. The selected thioaptamer had a strong binding affinity (approximately 7nM) and high specificity for the target protein. For the binding to the protein, the overall tertiary structure of the thioaptamer is required. We introduce two theoretical methods to examine the effect of phosphorothioate modification on the enhancement of binding affinity and one experimental method to examine the nature of the multiple bands of thioaptamer in a native gel.     

Delivery of the p38 MAPkinase inhibitor SB202190 to angiogenic endothelial cells: development of novel RGD-equipped and PEGylated drug-albumin conjugates using platinum(II)-based drug linker technology

Endothelial cells play an important role in inflammatory disorders, as they control the recruitment of leukocytes into inflamed tissue and the formation of new blood vessels. Activation of p38MAP kinase results in the production of proinflammatory cytokines and the expression of adhesion molecules. P38MAP kinase inhibitors are therefore considered important candidates for the treatment of inflammatory disorders. In the present study, we propose a novel strategy to counteract these processes by delivery of the p38MAP kinase inhibitor SB202190 into angiogenic endothelial cells. A drug-targeting conjugate was developed by conjugation of SB202190 to human serum albumin (HSA) using a novel platinum-based linker. Specificity for angiogenic endothelial cells was introduced by conjugation of cyclic RGD-peptides via bifunctional polyethylene glycol linkers. The final products contained an average of nine SB202190 and six RGDPEG groups per albumin. The platinum-based linker displayed high stability in buffers and culture medium, but released SB202190 slowly upon competition with sulfur-containing ligands like glutathione. RGDPEG-SB-HSA bound to alpha(v3)-integrin expressing endothelial cells (human umbilical cord vein endothelial cells) with low nanomolar affinity and was subsequently internalized. When HUVEC were treated with TNF to induce inflammatory events, pretreatment with RGDPEG-SB-HSA partially inhibited proinflammatory gene expression (IL-8, E-selectin; 30% inhibition) and secretion of cytokines (IL-8, 34% inhibition). We conclude that the developed RGDPEG-SB-HSA conjugates provide a novel means to counteract inflammation disorders such as rheumatoid arthritis.     

E-selectin, thymus- and activation-regulated chemokine/CCL17, and intercellular adhesion molecule-1 are constitutively coexpressed in dermal microvessels: a foundation for a cutaneous immunosurveillance system

The success of the cutaneous immune system reflects its ability to rapidly and efficiently recruit leukocytes to areas of trauma and infection. Skin-homing memory T cells expressing cutaneous lymphocyte-associated Ag tether on the walls of postcapillary venules in inflamed skin via interaction with endothelial E-selectin and roll in response to the shear stress imparted by flowing blood. Rolling cells sample the vascular surface for chemoattractant compounds (e.g., thymus- and activation-regulated chemokine/CCL17 interacting with CCR4 on the leukocyte surface) and, if successfully stimulated, progress to firm arrest and transmigration mediated by LFA-1 and vascular ICAM-1. Although it is established that this sequence of events draws T cells into inflamed skin, the mechanisms directing trafficking of T cells to noninflamed skin are less well characterized. We hypothesized that basal expression and colocalization of E-selectin, chemokine (e.g., CCL17), and ICAM-1 in dermal vessels could serve to recruit T cells to noninflamed human skin. Immunohistochemical staining for E-selectin and CD31 demonstrated E-selectin expression in a restricted subset of dermal vessels in noninflamed human skin from three different sites. Confocal multicolor immunofluorescence imaging revealed a nonuniform distribution of E-selectin in dermal vessels as well as colocalization of E-selectin with CCL17 and ICAM-1. Coexpression of these molecules on blood vessels in noninflamed skin provides the basis for a model of cutaneous immunosurveillance system active in the absence of pathologic inflammation.     

The Clinical Impact of Mean Vessel Size and Solidity in Breast Carcinoma Patients

Angiogenesis quantification, through vessel counting or area estimation in the most vascular part of the tumour, has been found to be of prognostic value across a range of carcinomas, breast cancer included. We have applied computer image analysis to quantify vascular properties pertaining to size, shape and spatial distributions in photographed fields of CD34 stained sections. Aided by a pilot (98 cases), seven parameters were selected and validated on a separate set from 293 breast cancer patients.Two new prognostic markers were identified through continuous Cox regression with endpoints Breast Cancer Specific Survival and Distant Disease Free Survival: The average size of the vessels as measured by their perimeter (p = 0.003 and 0.004, respectively), and the average complexity of the vessel shapes measured by their solidity (p = 0.004 and 0.004). The Hazard ratios for the corresponding median-dichotomized markers were 2.28 (p = 0.005) and 1.89 (p = 0.016) for the mean perimeter and 1.80 (p = 0.041) and 1.55 (p = 0.095) for the shape complexity. The markers were associated with poor histologic type, high grade, necrosis, HR negativity, inflammation, and p53 expression (vessel size only).Both markers were found to strongly influence the prognostic properties of vascular invasion (VI) and disseminated tumour cells in the bone marrow. The latter being prognostic only in cases with large vessels (p = 0.004 and 0.043) or low complexity (p = 0.018 and 0.024), but not in the small or complex vessel groups (p>0.47). VI was significant in all groups, but showed greater hazard ratios for small and low complexity vessels (6.54–11.2) versus large and high complexity vessels (2.64–3.06).We find that not only the overall amount of produced vasculature in angiogenic hot-spots is of prognostic significance, but also the morphological appearance of the generated vessels, i.e. the size and shape of vessels in the studied hot spots.     

Thioaptamer interactions with prion proteins: sequence-specific and non-specific binding sites

Binding of nucleic acids to the prion protein (PrP) created a conundrum that required distinguishing between non-specific interactions and biologically important polynucleotides. In the process of developing selective ligands for PrP, we found using a single-stranded DNA thioaptamer library that the binding of thioaptamers to PrP occurs on at least two different sites on the protein. Selection against recombinant (rec) PrP of Syrian hamster (SHa) sequence 90-231 folded into an alpha-helical-rich conformation identified a 12-base consensus sequence within a series of 20 thioaptamers, all of which consist of 40 bases. Each thioaptamer was comprised of both normal and thio-dA modified bases. One thioaptamer designated 97 bound to recSHaPrP with affinity of 0.58(+/-0.1) nM; lower affinities for bovine (Bo), and human (Hu) were found, establishing that binding is dependent on the primary structure of PrP. High affinity binding of thioaptamer 97 to PrP was found to be mediated through the dodecyl sequence GACACAAGCCGA within the consensus region with five critical backbone modifications 5' to each dA residue. A control oligonucleotide with an equivalent number of phosphorothioates to thioaptamer 97 and a scrambled consensus sequence could not distinguish among the three PrP sequences. Control oligonucleotides bearing non-selected sequences bound to PrP at a sequence-independent DNA-binding site. In contrast, the high-affinity binding of thioaptamer 97 to PrP depends on (1) backbone modifications, (2) oligonucleotide sequence, and (3) PrP sequence.     

Vascular adhesion protein 1 mediates binding of immunotherapeutic effector cells to tumor endothelium

Tumor-infiltrating lymphocytes (TIL) can be used as an immunotherapeutic tool to treat cancer. Success of this therapy depends on the homing and killing capacity of in vitro-activated and -expanded TIL. Vascular adhesion protein 1 (VAP-1) is an endothelial molecule that mediates binding of lymphocytes to vessels of inflamed tissue. Here, we studied whether VAP-1 is involved in binding of TIL, lymphokine-activated killer (LAK) cells, and NK cells to vasculature of the cancer tissue. We demonstrated that VAP-1 is expressed on the endothelium of cancer vasculature. The intensity and number of positive vessels varied greatly between the individual specimens, but it did not correlate with the histological grade of the cancer. Using an in vitro adhesion assay we showed that VAP-1 mediates adhesion of TIL, LAK, and NK cells to cancer vasculature. Treatment of the tumor sections with anti-VAP-1 Abs diminished the number of adhesive cells by 60%. When binding of different effector cell types was compared, it was evident that different cancer tissues supported the adhesion of TIL to a variable extent and LAK cells were more adhesive than TIL and NK cells to tumor vasculature. These data suggest that VAP-1 is an important interplayer in the antitumor response. Thus, by up-regulating the expression of VAP-1 in tumor vasculature, it can be possible to improve the effectiveness of TIL therapy.     

Gangliosides expressed on breast cancer cells are E-selectin ligands

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe^x) and sialyl Lewis A (sLe^a) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin–Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin–Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.     

L-Selectin from Human, but Not from Mouse Neutrophils Binds Directly to E-Selectin

L-Selectin on neutrophils as well as inducible E- and P-selectin on endothelium are involved in the recruitment of neutrophils into inflamed tissue. Based on cell attachment assays, L-selectin was suggested to function as a carbohydrate presenting ligand for E- and P-selectin. However, previous affinity isolation experiments with an E-selectin–Ig fusion protein had failed to detect L-selectin among the isolated E-selectin ligands from mouse neutrophils. We show here that L-selectin from human neutrophils, in contrast to mouse neutrophils, can be affinity-isolated as a major ligand from total cell extracts using E-selectin–Ig as affinity probe. Binding of human L-selectin to E-selectin was direct, since purified L-selectin could be reprecipitated with E-selectin–Ig. Recognition of L-selectin was abolished by sialidase-treatment, required Ca²⁺, and was resistant to treatment with endoglycosidase F. Binding of L-selectin to a P-selectin–Ig fusion protein was not observed. In agreement with the biochemical data, the anti–Lselectin mAb DREG56 inhibited rolling of human neutrophils on immobilized E-selectin–Ig but not on P-selectin–Ig. No such inhibitory effect was seen with the anti–mouse L-selectin mAb MEL14 on mouse neutrophils. Rolling of E-selectin transfectants on purified and immobilized human L-selectin was inhibited by mAb DREG56. We conclude that L-selectin on human neutrophils is a major glycoprotein ligand among very few glycoproteins that can be isolated by an E-selectin affinity matrix. The clear difference between human and mouse L-selectin suggests that E-selectin–binding carbohydrate moieties are attached to different protein scaffolds in different species.     

A novel <Emphasis Type="Italic">Flk1</Emphasis>-TVA transgenic mouse model for gene delivery to angiogenic vasculature

The genes that regulate the formation of blood vessels in adult tissues represent promising therapeutic targets because angiogenesis plays a role in many diseases, including cancer. We wished to develop a mouse model allowing characterization of gene function in adult angiogenic vasculature while minimizing effects on embryonic vasculature or adult quiescent vasculature. Here we describe a transgenic mouse model that allows expression of proteins in the endothelial cells of newly forming blood vessels in the adult using a selective retroviral gene delivery system. We generated transgenic mouse lines that express the TVA receptor for the RCAS avian-specific retrovirus from Flk1 gene regulatory elements that drive expression in proliferating endothelial cells. Several of these Flk1-TVA lines expressed TVA mRNA in the embryonic vasculature and TVA protein in new blood vessels growing into subcutaneous extracellular matrix implants in adult mice. In a Flk1-TVA line that was crossed with the MMTV-PyMT transgenic mammary tumor model, tumor endothelial cells also expressed the TVA protein. Furthermore, endothelial cells in extracellular matrix implants and the tumors of Flk1-TVA mice were susceptible to RCAS infection, as determined by expression of green fluorescent protein encoded by the virus. The Flk1-TVA mouse model in conjunction with the RCAS gene delivery system will be useful to study molecular mechanisms underlying adult forms of angiogenesis.     

Ultrasound Molecular Imaging of Secreted Frizzled Related Protein-2 Expression in Murine Angiosarcoma

Angiosarcoma is a biologically aggressive vascular malignancy with a high metastatic potential. In the era of targeted medicine, knowledge of specific molecular tumor characteristics has become more important. Molecular imaging using targeted ultrasound contrast agents can monitor tumor progression non-invasively. Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. We hypothesize that SFRP2-directed imaging could be a novel approach to imaging the tumor vasculature. To develop an SFRP2 contrast agent, SFRP2 polyclonal antibody was biotinylated and incubated with streptavidin-coated microbubbles. SVR angiosarcoma cells were injected into nude mice, and when tumors were established the mice were injected intravenously with the SFRP2 -targeted contrast agent, or a control streptavidin-coated contrast agent. SFRP2 -targeted contrast agent detected tumor vasculature with significantly more signal intensity than control contrast agent: the normalized fold-change was 1.6±0.27 (n = 13, p = 0.0032). The kidney was largely devoid of echogenicity with no significant difference between the control contrast agent and the SFRP2-targeted contrast agent demonstrating that the SFRP2-targeted contrast agent was specific to tumor vessels. Plotting average pixel intensity obtained from SFRP2-targeted contrast agent against tumor volume showed that the average pixel intensity increased as tumor volume increased. In conclusion, molecularly-targeted imaging of SFRP2 visualizes angiosarcoma vessels, but not normal vessels, and intensity increases with tumor size. Molecular imaging of SFRP2 expression may provide a rapid, non-invasive method to monitor tumor regression during therapy for angiosarcoma and other SFRP2 expressing cancers, and contribute to our understanding of the biology of SFRP2 during tumor development and progression.     

Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels

A tumor-homing peptide, F3, selectively binds to endothelial cells in tumor blood vessels and to tumor cells. Here, we show that the cell surface molecule recognized by F3 is nucleolin. Nucleolin specifically bound to an F3 peptide affinity matrix from extracts of cultured breast carcinoma cells. Antibodies and cell surface biotin labeling revealed nucleolin at the surface of actively growing cells, and these cells bound and internalized fluorescein-conjugated F3 peptide, transporting it into the nucleus. In contrast, nucleolin was exclusively nuclear in serum-starved cells, and F3 did not bind to these cells. The binding and subsequent internalization of F3 were blocked by an antinucleolin antibody. Like the F3 peptide, intravenously injected antinucleolin antibodies selectively accumulated in tumor vessels and in angiogenic vessels of implanted "matrigel" plugs. These results show that cell surface nucleolin is a specific marker of angiogenic endothelial cells within the vasculature. It may be a useful target molecule for diagnostic tests and drug delivery applications.     

Confocal Laser Endomicroscopy for the Morphometric Evaluation of Microvessels in Human Colorectal Cancer Using Targeted Anti-CD31 Antibodies

Introduction

Numerous anti-angiogenic agents are currently developed to limit tumor growth and metastasis. While these drugs offer hope for cancer patients, their transient effect on tumor vasculature is difficult to assess in clinical settings. Confocal laser endomicroscopy (CLE) is a novel endoscopic imaging technology that enables histological examination of the gastrointestinal mucosa. The aim of the present study was to evaluate the feasibility of using CLE to image the vascular network in fresh biopsies of human colorectal tissue. For this purpose we have imaged normal and malignant biopsy tissue samples and compared the vascular network parameters obtained with CLE with established histopathology techniques.

Materials and Methods

Fresh non-fixed biopsy samples of both normal and malignant colorectal mucosa were stained with fluorescently labeled anti-CD31 antibodies and imaged by CLE using a dedicated endomicroscopy system. Corresponding biopsy samples underwent immunohistochemical staining for CD31, assessing the microvessel density (MVD) and vascular areas for comparison with CLE data, which were measured offline using specific software.

Results

The vessels were imaged by CLE in both normal and tumor samples. The average diameter of normal vessels was 8.5±0.9 µm whereas in tumor samples it was 13.5±0.7 µm (p = 0.0049). Vascular density was 188.7±24.9 vessels/mm² in the normal tissue vs. 242.4±16.1 vessels/mm² in the colorectal cancer samples (p = 0.1201). In the immunohistochemistry samples, the MVD was 211.2±42.9/mm² and 351.3±39.6/mm² for normal and malignant mucosa, respectively. The vascular area was 2.9±0.5% of total tissue area for the normal mucosa and 8.5±2.1% for primary colorectal cancer tissue.

Conclusion

Selective imaging of blood vessels with CLE is feasible in normal and tumor colorectal tissue by using fluorescently labeled antibodies targeted against an endothelial marker. The method could be translated into the clinical setting for monitoring of anti-angiogenic therapy.     

Selection of cell specific peptides in a rat carotid injury model using a random peptide-presenting bacterial library

Cell specific peptides are possible candidates to enable targeted delivery of drugs and therapeutic genes in vivo. This study explores the utility of using a peptide-presenting bacterial library (pFliTrx) for the selection of new cell specific peptides, which bind to vascular cells of perfused tissues or organs. The balloon-injured rat carotid artery served as a model. Following perfusion of injured vascular segments with pFliTrx, 36 single clones could be identified. In radioligand binding studies, one of them, peptide P36, binds predominantly to perfused injured versus control vessel segments. It was additionally found that P36 binds with a 700-fold higher affinity in vitro to endothelial cells stimulated by treatment with LPS and TNF-alpha compared with unstimulated endothelial cells. The amino acid sequence of P36 reveals high homology to alpha(4)beta(1)-integrin, which mediates leukocyte migration from the vasculature at sites of inflammation via binding to cellular adhesion molecules, such as VCAM. In summary, this study demonstrates, that high specific peptides directed against injured vascular cells can be selected using a random peptide-presenting bacterial library.     

Parasite Biomass-Related Inflammation,Endothelial Activation,Microvascular Dysfunction and Disease Severity in Vivax Malaria

Plasmodium vivax can cause severe malaria, however its pathogenesis is poorly understood. In contrast to P. falciparum, circulating vivax parasitemia is low, with minimal apparent sequestration in endothelium-lined microvasculature, and pathogenesis thought unrelated to parasite biomass. However, the relationships between vivax disease-severity and total parasite biomass, endothelial autocrine activation and microvascular dysfunction are unknown. We measured circulating parasitemia and markers of total parasite biomass (plasma parasite lactate dehydrogenase [pLDH] and PvLDH) in adults with severe (n = 9) and non-severe (n = 53) vivax malaria, and examined relationships with disease-severity, endothelial activation, and microvascular function. Healthy controls and adults with non-severe and severe falciparum malaria were enrolled for comparison. Median peripheral parasitemia, PvLDH and pLDH were 2.4-fold, 3.7-fold and 6.9-fold higher in severe compared to non-severe vivax malaria (p = 0.02, p = 0.02 and p = 0.015, respectively), suggesting that, as in falciparum malaria, peripheral P. vivax parasitemia underestimates total parasite biomass, particularly in severe disease. P. vivax schizonts were under-represented in peripheral blood. Severe vivax malaria was associated with increased angiopoietin-2 and impaired microvascular reactivity. Peripheral vivax parasitemia correlated with endothelial activation (angiopoietin-2, von-Willebrand-Factor [VWF], E-selectin), whereas markers of total vivax biomass correlated only with systemic inflammation (IL-6, IL-10). Activity of the VWF-cleaving-protease, ADAMTS13, was deficient in proportion to endothelial activation, IL-6, thrombocytopenia and vivax disease-severity, and associated with impaired microvascular reactivity in severe disease. Impaired microvascular reactivity correlated with lactate in severe vivax malaria. Findings suggest that tissue accumulation of P. vivax may occur, with the hidden biomass greatest in severe disease and capable of mediating systemic inflammatory pathology. The lack of association between total parasite biomass and endothelial activation is consistent with accumulation in parts of the circulation devoid of endothelium. Endothelial activation, associated with circulating parasites, and systemic inflammation may contribute to pathology in vivax malaria, with microvascular dysfunction likely contributing to impaired tissue perfusion.