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At least 685 records · Page 38Linked to original sources
PubMed2005

Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity.

Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins alphavbeta3, alphavbeta5, and/or alpha5beta1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.

Angiogenesis Inhibitors↗
PubMed2004

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Cells, Cultured↗
PubMed2005

A novel homozygous mutation (1619delC) in GPIIb gene associated with Glanzmann thrombasthenia, the decay of GPIIb-mRNA and the synthesis of a truncated GPIIb unable to form complex with GPIIIa.

The absence of agonist-induced platelet aggregation and the lack of fibrinogen receptor (GPIIb/IIIa) on the platelet surface demonstrated that the severe hemorrhagic complications of a child of Romany descent were caused by Glanzmann thrombasthenia. DNA sequencing revealed a novel homozygous deletion of a cytosine (1619delC) in the GPIIb gene causing a frameshift and predicting a novel stop codon at position 533 following 24 altered amino acids. Both parents possessed the same deletion in heterozygous form. The amount of GPIIb mRNA in the patient's platelets was 0.06% of the amount measured in control platelets. Neither GPIIb nor its truncated form could be detected in the platelets of the patient by Western blotting, while a small amount of GPIIIa was demonstrated. Quantitative flowcytometric analysis showed an elevated number of vitronectin receptors, a component of which is GPIIIa, on the patient's platelets. The surface expression of vitronectin receptor on thrombasthenic, but not on normal platelets was further increased by activation with thrombin receptor agonist peptide. BHK cells transfected with wild type GPIIIa andmutated GPIIb failed to express any mature GPIIb or pro-GPIIb. Immunoprecipitation with a polyclonal antibody recognizing both GPIIb and GPIIIa recovered a 60 kDa truncated form of GPIIb. This band was absent when immunoprecipitation was carried out with an antibody recognizing GPIIIa, suggesting that the truncated protein, lacking calf-1, calf-2 domains and major part of the thigh domain, is unable to form complex with GPIIIa.

Animals↗
PubMed2006

Pericellular proteases in angiogenesis and vasculogenesis.

Pericellular proteases play an important role in angiogenesis and vasculogenesis. They comprise (membrane-type) matrix metalloproteinases [(MT-)MMPs], serine proteases, cysteine cathepsins, and membrane-bound aminopeptidases. Specific inhibitors regulate them. Major roles in initiating angiogenesis have been attributed to MT1-matrix metalloproteinase (MMP), MMP-2, and MMP-9. Whereas MT-MMPs are membrane-bound by nature, MMP-2 and MMP-9 can localize to the membrane by binding to alphavbeta3-integrin and CD44, respectively. Proteases switch on neovascularization by activation, liberation, and modification of angiogenic growth factors and degradation of the endothelial and interstitial matrix. They also modify the properties of angiogenic growth factors and cytokines. Neovascularization requires cell migration, which depends on the assembly of protease-protein complexes at the migrating cell front. MT1-MMP and urokinase (u-PA) form multiprotein complexes in the lamellipodia and focal adhesions of migrating cells, facilitating proteolysis and sufficient support for endothelial cell migration and survival. Excessive proteolysis causes loss of endothelial cell-matrix interaction and impairs angiogenesis. MMP-9 and cathepsin L stimulate the recruitment and action of blood- or bone-marrow-derived accessory cells that enhance angiogenesis. Proteases also generate fragments of extracellular matrix and hemostasis factors that have anti-angiogenic properties. Understanding the complexity of protease activities in angiogenesis contributes to recognizing new targets for stimulation or inhibition of neovascularization in disease.

ADAM Proteins↗
PubMed2004

Beta3-integrin mediates smooth muscle cell accumulation in neointima after carotid ligation in mice.

BACKGROUND: Pharmacological blockade of beta3-integrins inhibits neointimal lesion formation in nonmouse animal models of arterial injury. In contrast, beta3-integrin-deficient (beta3-/-) mice are not protected from neointimal lesion formation after arterial injury. We investigated this discrepancy in beta3-/- and wild-type (beta3+/+) mice using different models of injury. METHODS AND RESULTS: After disruption of the carotid with a transluminal probe, there was no significant difference in neointimal thickening between beta3-/- and beta3+/+ mice. However, after ligation of the carotid without medial disruption, there was reduced neointimal thickening in beta3-/- mice compared with beta3+/+ mice at intervals up to 3 months. Lesion reduction in beta3-/- mice was associated with fewer intimal smooth muscle cells (SMCs) without a difference in SMC apoptosis or proliferation rate compared with beta3+/+ mice, consistent with reduced SMC migration from the media into the intima of beta3-/- mice. Moreover, combined eccentric medial disruption and ligation of the carotid in beta3-/- mice resulted in neointimal lesion formation only at the site of medial disruption. Transplantation of bone marrow cells harvested from beta3+/+ mice into irradiated beta3-/- mice resulted in reduced neointimal lesion formation after carotid ligation injury, confirming the importance of alpha(v)beta3 and not alpha(IIb)beta3 in the attenuated response. CONCLUSIONS: The alpha(v)beta3-integrin mediates intimal SMC accumulation that contributes to neointimal thickening in the setting of arterial ligation.

Animals↗
PubMed2006

Model of vasculogenesis from embryonic stem cells for vascular research and regenerative medicine.

Embryonic stem (ES) cells are highlighted as promising cell sources for regenerative medicine. Here, we focused on providing the platform that forced ES cells to reproduce the vascular organization process, leading to efficiency and safety evaluation as preclinical testing of biological agents. Murine ES cell-derived embryoid bodies on matrigel, but not collagen or gelatin, could be differentiated into sprouting blood vessels without the addition of growth factors. The expression of endothelial cell marker CD31 and smooth muscle marker alpha-smooth muscle actin was partially colocalized and started to increase 7 days after culture on matrigel, accompanied by the induction of a number of growth factors, such as vascular endothelial growth factor, fibroblast growth factor-2, hepatocyte growth factor, transforming growth factor-beta, and angiopoietin-1. Moreover, notch-related genes, such as Del1 or Del4 (delta-like 1/4) and hey1 or hey2 (hairy/enhancer of split related TRPW motif 1/2), were upregulated in a similar time course. The treatment of neutralizing antibodies against these growth factors failed to inhibit the differentiation into the sprouting blood vessels, whereas arginine-glycine-aspartic peptide, a selective inhibitor for the alphavbeta3-integrins, did inhibit differentiation. An anticancer drug to inhibit angiogenesis, TNP-470, also blocked the vascular formation in this model. ES cells could reproduce the vascular organization process on the biosynthetic scaffolds, such as matrigel, without the addition of growth factors. In the future, a human ES-based tissue model would be an optional tool for the screening of pharmaceutical drugs for vascular disease.

Animals↗
PubMed2006

SB-267268, a nonpeptidic antagonist of alpha(v)beta3 and alpha(v)beta5 integrins, reduces angiogenesis and VEGF expression in a mouse model of retinopathy of prematurity.

PURPOSE: To determine whether SB-267268, a nonpeptidic antagonist of the alpha(v)beta3 and alpha(v)beta5 integrins, attenuates angiogenesis in a murine model of retinopathy of prematurity (ROP) and alters the expression of vascular endothelial growth factor (VEGF) and its second receptor (VEGF-R2). METHODS: In receptor binding, SB-267268 exhibited nanomolar potency for human, monkey, and murine alpha(v)beta3 and alpha(v)beta5. SB-267268 inhibited the attachment of alpha(v)beta3-transfected HEK293 cells to microtiter plate wells precoated with RGD-containing matrix proteins, and vitronectin-mediated human and rat aortic smooth-muscle-cell migration. At postnatal day (P)12, C57BL/6 mice were exposed to 80% oxygen for 7 days followed by 7 days in room air (angiogenic period). Between P12 and P17, ROP mice were administered sterile saline (vehicle intraperitoneal [i.p.]) or SB-267268 (60 mg/kg bi-daily, i.p.). Shams were exposed to room air from P0 and administered either vehicle or SB-267268 during P12 to 17. In at least 3 randomly chosen paraffin sections from each eye, the number of blood vessel profiles in the inner retina were counted. In situ hybridization for VEGF and VEGFR-2 was performed on at least 8 randomly chosen paraffin sections from each eye. RESULTS: SB-267268 reduced pathologic angiogenesis in ROP mice by approximately 50% and had no effect on developmental retinal angiogenesis in shams. Both VEGF and VEGFR-2 mRNA were upregulated in the inner retina of ROP mice and reduced with SB-267268. CONCLUSIONS: Nonpeptidic inhibition of alpha(v)beta3 and alpha(v)beta5 integrins is effective in ROP and may be a suitable anti-angiogenic therapy for other ischemic retinal pathologies.

Angiogenesis Inhibitors↗
PubMed1997

Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis.

Systemic administration of the nitric oxide (NO) synthase inhibitor Nomega-nitro--arginine methyl ester (L-NAME) to rabbits bearing a corneal implant blocked vascular endothelial growth factor (VEGF), but not basic fibroblast growth factor (bFGF)-induced angiogenesis. L-NAME completely blocked angiogenesis induced by VEGF-transfected MCF-7 breast carcinoma cells and the cells remained dormant in the cornea. Postcapillary endothelial cell migration and growth induced by VEGF were blocked by both the NO synthase inhibitor Nomega-mono-methyl--arginine and by the guanylate cyclase inhibitor LY 83583. We conclude that NO is a downstream imperative of VEGF-, but not bFGF-induced angiogenesis, and propose that the NO synthase/guanylate cyclase pathway is a potential target for controlling tumor angiogenesis in response to VEGF. Our studies support recent evidence that VEGF and bFGF induce angiogenesis by different mechanistic pathways using the alphavbeta5 and alphavbeta3 integrins, respectively.

Animals↗
PubMed1997

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

Cells, Cultured↗
PubMed2004

Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin.

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.

Adolescent↗
PubMed2006

Blockade of alpha v beta3 and alpha v beta5 integrins by RGD mimetics induces anoikis and not integrin-mediated death in human endothelial cells.

Alpha v integrins are thought to play an important role in tumor angiogenesis. However, discrepancies between findings with Arg-Gly-Asp (RGD) mimetics, which block angiogenesis in animal models, and knockout mice, in which loss of some alpha v integrins enhances tumor angiogenesis, raise questions concerning the function of these integrins and the precise role of alpha v substrate mimetics in antiangiogenic therapies. We have examined the effects of a novel non-peptide RGD mimetic, S 36578-2, on human endothelial cells to elucidate its antagonist activity and to identify possible agonist functions. S 36578-2 is highly selective for alpha v beta3 and alpha v beta5 integrins and induces detachment, caspase-8 activation, and apoptosis in human umbilical endothelial cells (HUVECs) plated on vitronectin. Importantly, the compound has no effect on the morphology or survival of cells plated on interstitial matrix components such as fibronectin, and it does not potentiate the apoptotic process in suspended cells. Identical results were obtained with a cyclic RGD peptide with similar target specificity. In microvascular endothelial cells, S 36578-2-induced death was also linked to its antiadhesive effect, with established lines markedly more resistant than primary cultures to the antiadhesive and proapoptotic effects. Altogether, these findings have important implications for the development of this class of antiangiogenics.

Anoikis↗
PubMed2006

Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages.

Chronic obstructive pulmonary disease (COPD) is associated with increased apoptosis and defective phagocytosis in the airway. As uncleared cells can undergo secondary necrosis and perpetuate inflammation, strategies to improve clearance would have therapeutic significance. There is evidence that the 15-member macrolide antibiotic azithromycin has anti-inflammatory properties. Its effects may be increased in the lung due to its ability to reach high concentrations in alveolar macrophages (AMs). The present study investigated the effects of low-dose (500 ng x mL(-1)) azithromycin on the phagocytosis of apoptotic bronchial epithelial cells and neutrophils by AMs. Flow cytometry was applied to measure phagocytosis and receptors involved in AM recognition of apoptotic cells. Cytokines were investigated using cytometric bead array. Baseline phagocytosis was reduced in COPD subjects compared with controls. Azithromycin significantly improved the phagocytosis of epithelial cells or neutrophils by AMs from COPD subjects by 68 and 38%, respectively, often up to levels comparable with controls. The increase in phagocytosis was partially inhibited by phosphatidylserine, implicating the phosphatidylserine pathway in the pro-phagocytic effects of azithromycin. Azithromycin had no effect on other recognition molecules (granulocyte-macrophage colony-stimulating factor, CD44, CD31, CD36, CD91, alphavbeta3 integrin). At higher doses, azithromycin decreased levels of pro-inflammatory cytokines. Thus, low-dose azithromycin therapy could provide an adjunct therapeutic option in chronic obstructive pulmonary disease.

Anti-Bacterial Agents↗
PubMed2004

Polyphosphoinositides-dependent regulation of the osteoclast actin cytoskeleton and bone resorption.

BACKGROUND: Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin alphavbeta3 increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function. RESULTS: Transduction of TAT/full-length gelsolin and PBDs containing gelsolin peptides into osteoclasts demonstrated: 1) F-actin enriched patches; 2) disruption of actin ring; 3) an increase in the association polyphosphoinositides (PPIs) with the transduced peptides containing PBDs. The above-mentioned effects were more pronounced with gelsolin peptide containing 2 tandem repeats of PBDs (PBD (2)). Binding of PPIs to the transduced peptides has resulted in reduced levels of PPIs association with the endogenous gelsolin, and thereby disrupted the actin remodeling processes in terms of podosome organization in the clear zone area and actin ring formation. These peptides also exhibited a dominant negative effect in the formation of WASP-Arp2/3 complex indicating the role of phosphoinositides in WASP activation. The TAT-PBD gelsolin peptides transduced osteoclasts are functionally defective in terms of motility and bone resorption. CONCLUSIONS: Taken together, these data demonstrate that transduction of PBD gelsolin peptides into osteoclasts produced a dominant negative effect on actin assembly, motility, and bone resorption. These findings indicate that phosphoinositide-mediated signaling mechanisms regulate osteoclast cytoskeleton, podosome assembly/disassembly, actin ring formation and bone resorption activity of osteoclasts.

Actin Cytoskeleton↗
PubMed2005

Unsatisfactory gene transfer into bone-resorbing osteoclasts with liposomal transfection systems.

BACKGROUND: Bone-resorbing osteoclasts are multinucleated cells that are formed via fusion of their hematopoietic stem cells. Many of the details of osteoclast formation, activation and motility remain unsolved. Therefore, there is an interest among bone biologists to transfect the terminally differentiated osteoclasts and follow their responses to the transgenes in vitro. Severe difficulties in transfecting the large, adherent osteoclasts have been encountered, however, making the use of modern cell biology tools in osteoclast research challenging. Transfection of mature osteoclasts by non-viral gene transfer systems has not been reported. RESULTS: We have systematically screened the usefulness of several commercial DNA transfection systems in human osteoclasts and their mononuclear precursor cell cultures, and compared transfection efficacy to adenoviral DNA transfection. None of the liposome-based or endosome disruption-inducing systems could induce EGFP-actin expression in terminally differentiated osteoclasts. Instead, a massive cell death by apoptosis was found with all concentrations and liposome/DNA-ratios tested. Best transfection efficiencies were obtained by adenoviral gene delivery. Marginal DNA transfection was obtained by just adding the DNA to the cell culture medium. When bone marrow-derived CD34-positive precursor cells were transfected, some GFP-expression was found at the latest 24 h after transfection. Large numbers of apoptotic cells were found and those cells that remained alive, failed to form osteoclasts when cultured in the presence of RANKL and M-CSF, key regulators of osteoclast formation. In comparison, adenoviral gene delivery resulted in the transfection of CD34-positive cells that remained GFP-positive for up to 5 days and allowed osteoclast formation. CONCLUSION: Osteoclasts and their precursors are sensitive to liposomal transfection systems, which induce osteoclast apoptosis. Gene transfer to mononuclear osteoclast precursors or differentiated osteoclasts was not possible with any of the commercial transfection systems tested. Osteoclasts are non-dividing, adherent cells that are difficult to grow as confluent cultures, which may explain problems with transfection reagents. Large numbers of alphavbeta3 integrin on the osteoclast surface allows adenovirus endocytosis and infection proceeds in dividing and non-dividing cells efficiently. Viral gene delivery is therefore currently the method of choice for osteoclast transfection.

Antigens, CD34↗
PubMed2004

Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single-centre study.

Psoriasis and psoriatic arthritis are inflammatory diseases that respond well to anti-tumour necrosis factor-alpha therapy. To evaluate the effects of anti-tumour necrosis factor-alpha treatment on expression of adhesion molecules and angiogenesis in psoriatic lesional skin and synovial tissue, we performed a prospective single-centre study with infliximab therapy combined with stable methotrexate therapy. Eleven patients with both active psoriasis and psoriatic arthritis received infusions of infliximab (3 mg/kg) at baseline, and at weeks 2, 6, 14 and 22 in an open-label study. In addition, patients continued to receive stable methotrexate therapy in dosages ranging from 5 to 20 mg/week. Clinical assessments, including Psoriasis Area and Severity Index (PASI) and Disease Activity Score (DAS), were performed at baseline and every 2 weeks afterward. In addition, skin biopsies from a target psoriatic plaque and synovial tissue biopsies from a target joint were taken before treatment and at week 4. Immunohistochemical analysis was performed to detect the number of blood vessels, the expression of adhesion molecules and the presence of vascular growth factors. Stained sections were evaluated by digital image analysis. At week 16, the mean PASI was reduced from 12.3 +/- 2.4 at baseline to 1.8 +/- 0.4 (P <or= 0.02). The mean DAS was reduced from 6.0 +/- 0.5 to 3.6 +/- 0.6 (P <or= 0.02). We found some fluctuations in DAS response as compared with the change in PASI, with the latter exhibiting a steady decrease over time. After 4 weeks the cell infiltrate was reduced in both skin and synovium. There was a significant reduction in the number of blood vessels in dermis and synovium at week 4. A significant reduction in the expression of alphavbeta3 integrin, a marker of neovascularization, was also found in both skin and synovium at week 4. In addition, a significant reduction in the expression of adhesion molecules was observed in both skin and synovium at week 4. We also observed a trend toward reduced expression of vascular endothelial growth factor in both skin and synovium. In conclusion, low-dose infliximab treatment leads to decreased neoangiogenesis and deactivation of the endothelium, resulting in decreased cell infiltration and clinical improvement in psoriasis and psoriatic arthritis.

Adult↗
PubMed2006

Adenoviral-encoded antigens are presented efficiently by a subset of dendritic cells expressing high levels of alpha(v)beta3 integrins.

Dendritic cells (DC) play a central role in antigen presentation and are often targeted by adenoviral (Ad)-based gene therapy. However, DC lack the coxsackie-Ad receptor, and little is known about the process by which they acquire and present Ad-encoded antigens. We examined the expression of alpha(v)beta3 integrins (CD51/CD61) on mouse bone marrow-derived DC (BM-DC) and their susceptibility to transduction by Ad vectors. Less than 10% of BM-DC precursors expressed CD51, but expression increased over time in culture with granulocyte macrophage-colony stimulating factor (GM-CSF)/interleukin (IL)-4. After 7 days, 28 +/- 1.7% of CD11c+ DC expressed high levels of CD51 (CD51(hi)), and the remaining DC expressed low levels of CD51 (CD51(lo)). CD51(hi) CD express higher major histocompatibility complex type 1 (MHC I); however, both of the DC subsets expressed similar levels of MHC II and costimulatory molecules. When exposed to a first-generation Ad vector, transgene expression was restricted to the CD51(hi) DC subset and blocked by soluble peptides expressing an arginine, glycine, aspartic acid (RGD) sequence, confirming the role of integrins in viral entry. Consistent with this, a modified Ad expressing an RGD-binding sequence in its fiber knob (Ad-RGD) transduced the CD51(hi) DC subset with significantly higher efficiency. When BM-DC were transduced with an Ad-expressing ovalbumin (Ad-OVA), the CD51(hi) subset proved superior in activating OT-I (T cell receptor-OVA) T cells. Similar to in vitro effects, systemic administration of GM-CSF/IL-4 increased the expression of CD51 on splenic DC and rendered these cells susceptible to Ad transduction. These results suggest that a limited subset of DC expressing high levels of alpha(v)beta3 integrins is preferentially transduced by Ad vectors and activates CD8+ T cell responses against Ad-encoded antigens.

Adenoviridae↗
PubMed2006

A tumor vasculature targeted liposome delivery system for combretastatin A4: design, characterization, and in vitro evaluation.

The objective of this study was to develop an efficient tumor vasculature targeted liposome delivery system for combretastatin A4, a novel antivascular agent. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG), and DSPE-PEG-maleimide were prepared by the lipid film hydration and extrusion process. Cyclic RGD (Arg-Gly-Asp) peptides with affinity for alphavbeta3-integrins expressed on tumor vascular endothelial cells were coupled to the distal end of PEG on the liposomes sterically stabilized with PEG (long circulating liposomes, LCL). The liposome delivery system was characterized in terms of size, lamellarity, ligand density, drug loading, and leakage properties. Targeting nature of the delivery system was evaluated in vitro using cultured human umbilical vein endothelial cells (HUVEC). Electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of approximately 120 nm in diameter. High performance liquid chromatography determination of ligand coupling to the liposome surface indicated that more than 99% of the RGD peptides were reacted with maleimide groups on the liposome surface. Up to 3 mg/mL of stable liposomal combretastatin A4 loading was achieved with approximately 80% of this being entrapped within the liposomes. In the in vitro cell culture studies, targeted liposomes showed significantly higher binding to their target cells than nontargeted liposomes, presumably through specific interaction of the RGD with its receptors on the cell surface. It was concluded that the targeting properties of the prepared delivery system would potentially improve the therapeutic benefits of combretastatin A4 compared with nontargeted liposomes or solution dosage forms.

Angiogenesis Inhibitors↗