Biomedical subjects

Kevin H Mayo

Publications and source records attributed to Kevin H Mayo.

At least 19 recordsLinked to original sources

PubMed2007

Early in vivo assessment of angiostatic therapy efficacy by molecular MRI.

Noninvasive diagnostic imaging methods to establish the efficacy of angiostatic therapies are becoming increasingly important with the first Food and Drug Administration approvals of such agents. Magnetic resonance molecular imaging is an imaging technique that allows the visualization of pathological processes in vivo with a better spatial resolution as compared with nuclear methods, such as photon emission tomography and single photon emission computed tomography. In this study, we used alpha(v)beta3 targeted bimodal liposomes to quantitate angiogenesis in a tumor mouse model with magnetic resonance imaging (MRI) and to evaluate the therapeutic efficacy of the angiogenesis inhibitors anginex and endostatin. The MRI findings were validated with fluorescence microscopy and showed a very good correlation with the microvessel density. In conclusion, this study provides evidence that molecular MRI can be used to noninvasively measure the efficacy of angiogenesis inhibitors during the course of therapy.

Angiogenesis Inhibitors↗

PubMed2006

Topomimetics of amphipathic beta-sheet and helix-forming bactericidal peptides neutralize lipopolysaccharide endotoxins.

Release of lipopolysaccharide (LPS) endotoxin from Gram negative bacterial membranes triggers macrophages to produce large quantities of cytokines that can lead to septic shock and eventual death. Agents that bind to and neutralize LPS may provide a means to clinically prevent septic shock upon bacterial infection. Previously, we reported the design of antibacterial helix peptide SC4 and beta-sheet-forming betapep peptides that neutralize LPS in vitro. We hypothesized that the ability of these and other such peptides to neutralize LPS rested in the common denominator of positively charged amphipathic structure. Here, we describe the design and synthesis of nonpeptide, calixarene-based helix/sheet topomimetics that mimic the folded conformations of these peptides in their molecular dimensions, amphipathic surface topology, and compositional properties. From a small library of topomimetics, we identified several compounds that neutralize LPS in the 10-8 M range, making them as effective as bactericidal/permeability increasing protein and polymyxin B. In an endotoxemia mouse model, three of the most in vitro effective topomimetics are shown to be at least partially protective against challenges of LPS from different bacterial species. NMR studies provide mechanistic insight by suggesting the site of molecular interaction between topomimetics and the lipid A component of LPS, with binding being mediated by electrostatic and hydrophobic interactions. This research contributes to the development of pharmaceutical agents against endotoxemia and septic shock.

Animals↗

PubMed2006

Epigenetic regulation of tumor endothelial cell anergy: silencing of intercellular adhesion molecule-1 by histone modifications.

Tumors can escape from immunity by repressing leukocyte adhesion molecule expression on tumor endothelial cells and by rendering endothelial cells unresponsive to inflammatory activation. This endothelial cell anergy is induced by angiogenic growth factors and results in reduced leukocyte-vessel wall interactions, thereby attenuating infiltration of leukocytes into the tumor. This report describes a novel mechanism of endothelial cell anergy regulation. We recently reported that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have angiostatic activity. Here, we studied whether epigenetic mechanisms regulate this angiogenesis-mediated escape from immunity. We found that DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine, as well as HDAC inhibitor trichostatin A, reexpressed intercellular adhesion molecule-1 (ICAM-1) on tumor-conditioned endothelial cells in vitro, resulting in restored leukocyte-endothelial cell adhesion. In addition, treatment with DNMT or HDAC inhibitors in vivo also restored ICAM-1 expression on tumor endothelial cells from two different mouse tumor models. Furthermore, leukocyte-vessel wall interactions in mouse tumors were increased by these compounds, as measured by intravital microscopy, resulting in enhanced leukocyte infiltration. We show that ICAM-1 down-regulation in tumor endothelial cells is associated with ICAM-1 promoter histone H3 deacetylation and loss of histone H3 Lys(4) methylation but not with DNA hypermethylation. In conclusion, our data show that ICAM-1 is epigenetically silenced in tumor endothelial cells by promoter histone modifications, which can be overcome by DNMT and HDAC inhibitors, suggesting a new molecular mechanism based on which novel therapeutic approaches for cancer can be pursued.

Acetylation↗

PubMed2006

Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy.

We describe that galectin-1 (gal-1) is a receptor for the angiogenesis inhibitor anginex, and that the protein is crucial for tumor angiogenesis. gal-1 is overexpressed in endothelial cells of different human tumors. Expression knockdown in cultured endothelial cells inhibits cell proliferation and migration. The importance of gal-1 in angiogenesis is illustrated in the zebrafish model, where expression knockdown results in impaired vascular guidance and growth of dysfunctional vessels. The role of gal-1 in tumor angiogenesis is demonstrated in gal-1-null mice, in which tumor growth is markedly impaired because of insufficient tumor angiogenesis. Furthermore, tumor growth in gal-1-null mice no longer responds to antiangiogenesis treatment by anginex. Thus, gal-1 regulates tumor angiogenesis and is a target for angiostatic cancer therapy.

Animals↗

PubMed2006

Anti-angiogenesis and anti-tumor activity of recombinant anginex.

Anginex, a synthetic 33-mer angiostatic peptide, specifically inhibits vascular endothelial cell proliferation and migration along with induction of apoptosis in endothelial cells. Here we report on the in vivo characterization of recombinant anginex and use of the artificial anginex gene for gene therapy approaches. Tumor growth of human MA148 ovarian carcinoma in athymic mice was inhibited by 80% when treated with recombinant anginex. Histological analysis of the tumors showed an approximate 2.5-fold reduction of microvessel density, suggesting that angiogenesis inhibition is the cause of the anti-tumor effect. Furthermore, there was a significant correlation between the gene expression patterns of 16 angiogenesis-related factors after treatment with both recombinant and synthetic anginex. To validate the applicability of the anginex gene for gene therapy, stable transfectants of murine B16F10 melanoma cells expressing recombinant anginex were made. Supernatants of these cells inhibited endothelial cell proliferation in vitro. Furthermore, after subcutaneous injection of these cells in C57BL/6 mice, an extensive delay in tumor growth was observed. These data show that the artificial anginex gene can be used to produce a recombinant protein with similar activity as its synthetic counterpart and that the gene can be applied in gene therapy approaches for cancer treatment.

Angiogenesis Inhibitors↗

PubMed2006

Design of nonpeptidic topomimetics of antiangiogenic proteins with antitumor activities.

The inhibition of angiogenesis is a promising avenue for cancer treatment. Although some angiostatic compounds are in the process of development and testing, these often prove ineffective in vivo or have unwanted side effects. We have designed, synthesized, and evaluated a small library of nonpeptidic, calixarene-based protein surface topomimetics that display chemical substituents to approximate the molecular dimensions and amphipathic features (hydrophobic and positively charged residues) of the antiangiogenic peptide anginex, which, like many antiangiogenic proteins, consists primarily of an antiparallel beta-sheet structure as the functional unit. Two of the topomimetics (0118 and 1097) were potent angiogenesis inhibitors in vitro, as determined by endothelial cell proliferation, migration, and chick embryo chorioallantoic membrane assays. Moreover, both compounds were highly effective at inhibiting tumor angiogenesis and growth in two mouse models (MA148 human ovarian carcinoma and B16 murine melanoma). Our results demonstrate the feasibility of designing nonpeptidic protein surface topomimetics as novel pharmaceutical agents for clinical intervention against cancer through angiostatic or other mechanisms.

Angiogenesis Inhibitors↗

PubMed2006

Gene expression of tumor angiogenesis dissected: specific targeting of colon cancer angiogenic vasculature.

Crucial to designing angiostatic and vascular targeting agents is the identification of target molecules. Because angiogenesis is not limited to pathologic conditions, careful evaluation of putative therapeutic targets is warranted to prevent adverse effects associated with impaired physiologic angiogenesis. To identify tumor-specific angiogenesis markers, we compared transcriptional profiles of angiogenic endothelial cells isolated from malignant and nonmalignant tissues with those of resting endothelial cells. We identified 17 genes that showed specific overexpression in tumor endothelium but not in angiogenic endothelium of normal tissues, creating a therapeutic window for tumor vasculature-specific targeting. Antibody targeting of 4 cell-surface-expressed or secreted products (vimentin, CD59, HMGB1, IGFBP7) inhibited angiogenesis in vitro and in vivo. Finally, targeting endothelial vimentin in a mouse tumor model significantly inhibited tumor growth and reduced microvessel density. Our results demonstrate the usefulness of the identification and subsequent targeting of specific tumor endothelial markers for anticancer therapy.

Animals↗

PubMed2006

Anti-angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte-endothelium interactions and infiltration in tumors.

Tumor escape from immunity, as well as the failure of several anti-cancer vaccination and cellular immunotherapy approaches, is suggested to be due to the angiogenesis-mediated suppression of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel wall interactions. We hypothesized that inhibition of angiogenesis would overcome this escape from immunity. We investigated this in vivo by means of intravital microscopy and ex vivo by immunohistochemistry in two mouse tumor models. Angiogenesis inhibitors anginex, endostatin, and angiostatin, and the chemotherapeutic agent paclitaxel were found to significantly stimulate leukocyte-vessel wall interactions by circumvention of EC anergy in vivo, i.e., by the up-regulation of endothelial adhesion molecules in tumor vessels. This was confirmed by in vitro studies of cultured EC at the protein and mRNA levels. The new angiostatic designer peptide anginex was most potent at overcoming EC anergy; the enhanced leukocyte-vessel interactions led to an increase in the numbers of tumor infiltrating leukocytes. While anginex inhibited tumor growth and microvessel density significantly, the amount of infiltrated leukocytes (CD45), as well as the number of CD8+ cytotoxic T lymphocytes, was enhanced markedly. The current results suggest that immunotherapy strategies can be improved by combination with anti-angiogenesis.

Angiostatins↗

PubMed2005

Cloning an artificial gene encoding angiostatic anginex: From designed peptide to functional recombinant protein.

Anginex, a designed peptide 33-mer, is a potent angiogenesis inhibitor and anti-tumor agent in vivo. Anginex functions by inhibiting endothelial cell (EC) proliferation and migration leading to detachment and apoptosis of activated EC's. To better understand tumor endothelium targeting properties of anginex and enable its use in gene therapy, we constructed an artificial gene encoding the biologically exogenous peptide and produced the protein recombinantly in Pichia pastoris. Mass spectrometry shows recombinant anginex to be a dimer and circular dichroism shows the recombinant protein folds with beta-strand structure like the synthetic peptide. Moreover, like parent anginex, the recombinant protein is active at inhibiting EC growth and migration, as well as inhibiting angiogenesis in vivo in the chorioallantoic membrane of the chick embryo. This study demonstrated that it is possible to produce a functionally active protein version of a rationally designed peptide, using an artificial gene and the recombinant protein approach.

Angiogenesis Inhibitors↗

PubMed2005

Anginex synergizes with radiation therapy to inhibit tumor growth by radiosensitizing endothelial cells.

We have demonstrated that the designed peptide anginex displays potent antiangiogenic activity. The aim of our study was to investigate the effect of anginex on established tumor vasculature as an adjuvant to radiation therapy of solid tumors. In the MA148 human ovarian carcinoma athymic mouse model, anginex (10 mg/kg) in combination with a suboptimal dose of radiation (5 Gy once weekly for 4 weeks) caused tumors to regress to an impalpable state. In the more aggressive SCK murine mammary carcinoma model, combination of anginex and a single radiation dose of 25 Gy synergistically increased the delay in tumor growth compared to the tumor growth delay caused by either treatment alone. Immunohistochemical analysis also demonstrated significantly enhanced effects of combined treatment on tumor microvessel density and tumor or endothelial cell proliferation and viability. In assessing physiologic effects of anginex, we observed a reduction in tumor perfusion and tumor oxygenation in SCK tumors after 5-7 daily treatments with anginex with no reduction in blood pressure. To test anginex as a radiosensitizer, additional studies using SCK tumors were performed. Three daily i.p. injections of anginex were able to enhance the effect of 2 radiation doses of 10 Gy, resulting in 50% complete responses, whereas the known antiangiogenic agent angiostatin did not enhance the radiation response of SCK tumors. Mechanistically, it appears that anginex functions as an endothelial cell-specific radiosensitizer because anginex showed no effect on in vitro radiosensitivity of SCK or MA148 tumor cells, whereas anginex significantly enhanced the in vitro radiosensitivity of 2 endothelial cell types. This work supports the idea that the combination of the antiangiogenic agent anginex and radiation may lead to improved clinical outcome in treating cancer patients.

Angiogenesis Inhibitors↗

PubMed2005

Angiostatic agents prevent the development of endometriosis-like lesions in the chicken chorioallantoic membrane.

A prospective study was performed to determine the effects of the angiostatic compounds anti-hVEGF antibody, TNP-470, endostatin, and anginex on the vascularization and on endometriosis-like lesion formation in the chicken chorioallantoic membrane model. Endometriosis-like lesion formation was significantly impaired after treatment with angiostatic agents, which was associated with decreased vessel densities in the surrounding chorioallantoic membrane and more necrosis in the endometriosis-like lesions.

Angiogenesis Inhibitors↗

PubMed2004

The nociceptin pharmacophore site for opioid receptor binding derived from the NMR structure and bioactivity relationships.

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is also implicated in the perception of pain and visual, auditory, and olfactory functions. In this study, we investigated the NMR solution structure of nociceptin in membrane-like environments (trifluoroethanol and SDS micelles) and found it to have a relatively stable helix conformation from residues 4-17 with functionally important N-terminal residues being folded aperidoically on top of the helix. In functional assays for receptor binding and calcium flux, alanine-scanning variants of nociceptin indicated that functionally important residues generally followed helix periodicity, consistent with the NMR structural model. Structure-activity relationships allowed identification of pharmacophore sites that were used in small molecule data base searches, affording hits with demonstrated nociceptin receptor binding affinities.

Alanine↗

PubMed2004

Platelet factor 4 and interleukin-8 CXC chemokine heterodimer formation modulates function at the quaternary structural level.

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.

Animals↗

PubMed2004

Acylation of SC4 dodecapeptide increases bactericidal potency against Gram-positive bacteria, including drug-resistant strains.

We have conjugated dodecyl and octadecyl fatty acids to the N-terminus of SC4, a potently bactericidal, helix-forming peptide 12-mer (KLFKRHLKWKII), and examined the bactericidal activities of the resultant SC4 'peptide-amphiphile' molecules. SC4 peptide-amphiphiles showed up to a 30-fold increase in bactericidal activity against Gram-positive strains (Staphylococcus aureus, Streptococcus pyogenes and Bacillus anthracis), including S. aureus strains resistant to conventional antibiotics, but little or no increase in bactericidal activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fatty acid conjugation improved endotoxin (lipopolysaccharide) neutralization by 3- to 6-fold. Although acylation somewhat increased lysis of human erythrocytes, it did not increase lysis of endothelial cells, and the haemolytic effects occurred at concentrations 10- to 100-fold higher than those required for bacterial cell lysis. For insight into the mechanism of action of SC4 peptide-amphiphiles, CD, NMR and fluorescence spectroscopy studies were performed in micelle and liposome models of eukaryotic and bacterial cell membranes. CD indicated that SC4 peptide-amphiphiles had the strongest helical tendencies in liposomes mimicking bacterial membranes, and strong membrane integration of the SC4 peptide-amphiphiles was observed using tryptophan fluorescence spectroscopy under these conditions; results that correlated with the increased bactericidal activities of SC4 peptide-amphiphiles. NMR structural analysis in micelles demonstrated that the two-thirds of the peptide closest to the fatty acid tail exhibited a helical conformation, with the positively-charged side of the amphipathic helix interacting more with the model membrane surface. These results indicate that conjugation of a fatty acid chain to the SC4 peptide enhances membrane interactions, stabilizes helical structure in the membrane-bound state and increases bactericidal potency.

Acylation↗

PubMed2004

Measuring protein self-diffusion in protein-protein mixtures using a pulsed gradient spin-echo technique with WATERGATE and isotope filtering.

Here we report a modified pulsed gradient spin-echo (PGSTE) pulse sequence to measure diffusion coefficients. This approach incorporates WATERGATE combined with isotopic filtering into a standard PGSTE experiment. Doing this eliminates much of the disadvantages from the combination of diffusion encoding and heteronuclear selection intervals and allows for facile modification of the diffusion pulse sequence with flexibility of the time period between RF pulses. The new diffusion pulse sequence is demonstrated using an 15N-labeled peptide and an 15N-labeled protein in a mixture with a protein of similar size.

Algorithms↗

PubMed2004

A simple method to measure 13CH2 heteronuclear dipolar cross-correlation spectral densities.

Here, we report a method to simultaneously determine CH2 cross-correlation spectral densities and T1 relaxation times in the laboratory and rotating frames. To accomplish this, we have employed an indirect approach that is based on measurement of differences in relaxation rates acquired with and without cross-correlation terms. The new method, which can be employed using multidimensional NMR and standard relaxation pulse sequences, is validated experimentally by investigation of a selectively 13C-enriched hexadecapeptide and the uniformly 13C-enriched immunoglobulin-binding domain of streptococcal protein G (GB1). Use of this approach makes determination of CH2 cross-correlation spectral densities in uniformly 13C-enriched proteins now routine and provides novel information concerning their internal motions.

Bacterial Proteins↗

PubMed2004

Structure/function studies of an endotoxin-neutralizing peptide derived from bactericidal/permeability-increasing protein.

BACKGROUND: Bactericidal/permeability-increasing protein, BPI, has a beta-turn with alternating cationic and hydrophobic residues in its lipopolysaccharide (endotoxin, LPS)-binding domain. A peptide, betapep25, was designed with 9 residues of the LPS-binding domain of BPI flanked by beta-turn-inducing elements. Thereafter, we sought to use single amino acid substitutions to identify residues that are important for the biological activities of betapep25. METHODS: Single alanine or norleucine replacement "walkthrough" peptides based on betapep25 were generated and tested for their ability to kill P aeruginosa and to neutralize endotoxin. RESULTS: Substitution of all lysines inhibited bactericidal activity. Inhibition of LPS-neutralizing activity was seen in 9 peptides in which an alanine or norleucine was substituted for each of 4 of the basic residues and 1 hydrophobic residue from the LPS-binding region of BPI and 4 hydrophobic residues from the beta-turn-inducing regions flanking the LPS-binding region on the carboxy-terminal side. Intriguingly, these last 4 substitutions resulted in peptides that exhibited increased bactericidal activity compared to betapep25. CONCLUSIONS: These results demonstrate the importance of both cationic and hydrophobic amino acid residues to bactericidal and endotoxin-neutralizing activities. These perturbations of biological activity should be considered in the design of synthetic peptide endotoxin antagonists.

Amino Acid Sequence↗

PubMed2003

Design of a partial peptide mimetic of anginex with antiangiogenic and anticancer activity.

Based on structure-activity relationships of the angiostatic beta-sheet-forming peptide anginex, we have designed a mimetic, 6DBF7, which inhibits angiogenesis and tumor growth in mice. 6DBF7 is composed of a beta-sheet-inducing dibenzofuran (DBF)-turn mimetic and two short key amino acid sequences from anginex. This novel antiangiogenic molecule is more effective in vivo than parent anginex. In a mouse xenograft model for ovarian carcinoma, 6DBF7 is observed to reduce tumor growth by up to 80%. It is suggested that the activity is based on antiangiogenesis, because in vitro tube formation is inhibited, and because treatment of tumor-bearing mice led to a significant reduction in microvessel density within the tumor. This partial peptide mimetic is the first endothelial cell-specific molecule designed as a substitute for an angiostatic inhibitory peptide.

Alanine↗