ABSTRACT
Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4-moesin-talin-ß1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to ß1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5ß1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.
Subject(s)
Cell Movement , Endothelial Cells/cytology , Endothelial Cells/metabolism , Integrins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Motifs , Animals , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Shape/drug effects , Endothelial Cells/drug effects , Epistasis, Genetic , Focal Adhesions/metabolism , Humans , Integrin alpha1/drug effects , Integrin alpha1/metabolism , Integrin beta1/chemistry , Integrin beta1/drug effects , Integrin beta1/metabolism , Integrins/drug effects , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Neovascularization, Pathologic , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Talin/chemistry , Talin/metabolismABSTRACT
Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 µM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbß3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbß3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.
Subject(s)
Blood Platelets/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/drug effects , Stilbenes/metabolism , Animals , Blood Coagulation/drug effects , Blood Platelets/drug effects , Clot Retraction/drug effects , Collagen , Fibrinogen/metabolism , Hemostasis/drug effects , Humans , Integrin alpha2/drug effects , Integrin alpha2/metabolism , Integrin beta3/drug effects , Integrin beta3/metabolism , Integrins/drug effects , Integrins/metabolism , Mice , P-Selectin/metabolism , Phosphorylation , Platelet Activation/drug effects , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Signal Transduction/drug effects , Stilbenes/pharmacology , Thrombosis/metabolismABSTRACT
OBJECTIVES: This article reports a study of cell mechanics in patient-derived (primary) B-cell acute lymphocytic leukemia (ALL) cells treated with antibodies against integrins. Leukemia cell adhesion to stromal cells mediates chemotherapeutic drug resistance, also known as cell adhesion-mediated chemotherapeutic drug resistance. We have previously shown that antibodies against integrin α4 and α6 adhesion molecules can de-adhere ALL cells from stromal cells or counter-receptors. Because drug-resistant cells are more deformable, as evaluated by single-beam acoustic tweezers, we hypothesized that changes in cell mechanics might contribute to the de-adhesive effect of integrin-targeting antibodies. METHODS: In this study, the deformability of primary pre-B ALL cells was evaluated by single-beam acoustic tweezers after treatments with the de-adhering antibody Tysabri or P5G10 against integrin α4 and α6 adhesion molecules. RESULTS: We demonstrated that primary ALL cells treated with P5G10 expressed decreased deformability compared with immunoglobulin G1 -treated control cells (P < .05). Tysabri did not show an effect on deformability (P > .05). CONCLUSIONS: These results suggest that decreased deformability is associated with an integrin α6 blockade. Further assessments of the functional roles of deformability and integrin blockades in B-ALL cell drug resistance and deformability, respectively, are necessary.
Subject(s)
Cell Adhesion/drug effects , Immunologic Factors/therapeutic use , Integrins/drug effects , Natalizumab/therapeutic use , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnostic imaging , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Acoustics , Cells, Cultured , Humans , Immunoglobulin G/administration & dosage , Ultrasonography/methodsABSTRACT
In systemic sclerosis (SSc), a common and aetiologically mysterious form of scleroderma (defined as pathological fibrosis of the skin), previously healthy adults acquire fibrosis of the skin and viscera in association with autoantibodies. Familial recurrence is extremely rare and causal genes have not been identified. Although the onset of fibrosis in SSc typically correlates with the production of autoantibodies, whether they contribute to disease pathogenesis or simply serve as a marker of disease remains controversial and the mechanism for their induction is largely unknown. The study of SSc is hindered by a lack of animal models that recapitulate the aetiology of this complex disease. To gain a foothold in the pathogenesis of pathological skin fibrosis, we studied stiff skin syndrome (SSS), a rare but tractable Mendelian disorder leading to childhood onset of diffuse skin fibrosis with autosomal dominant inheritance and complete penetrance. We showed previously that SSS is caused by heterozygous missense mutations in the gene (FBN1) encoding fibrillin-1, the main constituent of extracellular microfibrils. SSS mutations all localize to the only domain in fibrillin-1 that harbours an Arg-Gly-Asp (RGD) motif needed to mediate cell-matrix interactions by binding to cell-surface integrins. Here we show that mouse lines harbouring analogous amino acid substitutions in fibrillin-1 recapitulate aggressive skin fibrosis that is prevented by integrin-modulating therapies and reversed by antagonism of the pro-fibrotic cytokine transforming growth factor ß (TGF-ß). Mutant mice show skin infiltration of pro-inflammatory immune cells including plasmacytoid dendritic cells, T helper cells and plasma cells, and also autoantibody production; these findings are normalized by integrin-modulating therapies or TGF-ß antagonism. These results show that alterations in cell-matrix interactions are sufficient to initiate and sustain inflammatory and pro-fibrotic programmes and highlight new therapeutic strategies.
Subject(s)
Autoimmunity/drug effects , Contracture/drug therapy , Contracture/pathology , Integrins/drug effects , Integrins/metabolism , Scleroderma, Systemic/drug therapy , Scleroderma, Systemic/pathology , Skin Diseases, Genetic/drug therapy , Skin Diseases, Genetic/pathology , Amino Acid Motifs/genetics , Amino Acid Substitution/genetics , Animals , Antibodies, Antinuclear/immunology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , Autoimmunity/immunology , Contracture/immunology , Contracture/prevention & control , Dendritic Cells/drug effects , Female , Fibrillin-1 , Fibrillins , Fibrosis/drug therapy , Fibrosis/pathology , Fibrosis/prevention & control , Male , Mice , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Mutation, Missense/genetics , Plasma Cells/drug effects , Scleroderma, Systemic/immunology , Scleroderma, Systemic/prevention & control , Skin Diseases, Genetic/immunology , Skin Diseases, Genetic/prevention & control , T-Lymphocytes, Helper-Inducer/drug effects , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/immunologyABSTRACT
In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O']-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (Ë93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4ß7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101's affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.
Subject(s)
Ethylenes/therapeutic use , Integrins/antagonists & inhibitors , Leishmania donovani/enzymology , Leishmaniasis, Visceral/drug therapy , NADH, NADPH Oxidoreductases/drug effects , Animals , Cells, Cultured , Cricetinae , Disease Models, Animal , Ethylenes/pharmacology , Female , Host-Parasite Interactions/drug effects , Integrins/drug effects , Leishmania donovani/drug effects , Leishmania donovani/metabolism , Leishmaniasis, Visceral/metabolism , Leishmaniasis, Visceral/pathology , Male , Mice , Mice, Inbred BALB C , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction/drug effects , Phosphatidylinositol 3-Kinases/drug effects , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/drug effects , Signal Transduction/drug effectsABSTRACT
Steroid refractory acute graft-versus-host-disease of the gut is a serious complication associated with high mortality after allogeneic stem cell transplantation. Treatment options are limited and not predictably effective. We describe the treatment of steroid-refractory acute graft-versus-host-disease with vedolizumab, an antibody directed against integrin α4ß7, in 6 patients. All patients responded, and 4 of 6 patients are alive with a median follow-up of 10 months.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Graft vs Host Disease/drug therapy , Integrins/drug effects , Intestinal Diseases/drug therapy , Adult , Female , Gastrointestinal Agents/therapeutic use , Graft vs Host Disease/pathology , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Male , Middle Aged , Salvage Therapy/methods , Steroids/therapeutic use , Transplantation, Homologous , Treatment OutcomeABSTRACT
Cadmium (Cd) is a carcinogenic heavy metal which is implicated in breast cancer development. While the mechanisms of Cd-induced breast cancer initiation and promotion have been studied, the molecular processes involved in breast cancer progression remain to be investigated. The purpose of the present study was to evaluate the influence of Cd on metastasis-associated phenotypes, such as cell adhesion, migration, and invasion in triple-negative breast cancer cells. Treatment of MDA-MB-231 cells with 1µM Cd increased cell spreading and cell migration. This was associated with the activation of integrin ß1, FAK, Src, and Rac1. Treatment with Cd also inhibited GSK3ß activity and induced T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription, indicating the involvement of ß-catenin signaling. Furthermore, treatment with 3µM Cd for 4weeks increased the expression of ß-catenin and enhanced TCF/LEF-mediated transcription. Furthermore, enhanced expressions of integrins α5 and ß1, paxillin, and vimentin indicated that prolonged Cd treatment reorganized the cytoskeleton, which aided malignancy, as evidenced by enhanced matrix metalloprotease 2/9 (MMP2/9) secretion and cell invasion. Prolonged Cd treatment also caused an increase in cell growth. Together, these results indicate that Cd alters key signaling processes involved in the regulation of cytoskeleton to enhance cancer cell migration, invasion, adhesion, and proliferation.
Subject(s)
Cadmium/toxicity , Integrins/drug effects , Neoplasm Metastasis/pathology , Signal Transduction/drug effects , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology , beta Catenin/drug effects , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cytoskeleton/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Phenotype , Wound Healing/drug effectsABSTRACT
BACKGROUND AND PURPOSE: Recombinant osteopontin (rOPN) has been reported to be neuroprotective in stroke animal models. The purpose of this study is to investigate a potential role and mechanism of nasal administration of rOPN on preserving the vascular smooth muscle phenotype in early brain injury after subarachnoid hemorrhage (SAH). METHODS: One hundred and ninety-two male adult Sprague-Dawley rats were used. The SAH model was induced by endovascular perforation. Integrin-linked kinase small interfering RNA was intracerebroventricularly injected 48 hours before SAH. The integrin receptor antagonist fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), focal adhesion kinase inhibitor Fib-14, and Rac-1 inhibitor NSC23766 were administered 1 hour before SAH induction. rOPN was administered via the intracerebroventricular and nasal route after SAH. SAH grade, neurological scores, brain water content, brain swelling, hematoxylin and eosin staining, India ink angiography, Western blots, and immunofluorescence were used to study the mechanisms of rOPN on the vascular smooth muscle phenotypic transformation. RESULTS: The marker proteins of vascular smooth muscle phenotypic transformation α-smooth muscle actin decreased and embryonic smooth muscle myosin heavy chain (SMemb) increased significantly at 24 and 72 hours in the cerebral arteries after SAH. rOPN prevented the changes of α-smooth muscle actin and SMemb and significantly alleviated neurobehavioral dysfunction, increased the cross-sectional area and the lumen diameter of the cerebral arteries, reduced the brain water content and brain swelling, and improved the wall thickness of cerebral arteries. These effects of rOPN were abolished by GRGDSP, integrin-linked kinase small interfering RNA, and NSC23766. Intranasal application of rOPN at 3 hours after SAH also reduced neurological deficits. CONCLUSIONS: rOPN prevented the vascular smooth muscle phenotypic transformation and improved the neurological outcome, which was possibly mediated by the integrin receptor/integrin-linked kinase/Rac-1 pathway.
Subject(s)
Cerebral Arteries/drug effects , Integrins/drug effects , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Neuroprotective Agents/pharmacology , Osteopontin/pharmacology , Protein Serine-Threonine Kinases/drug effects , Subarachnoid Hemorrhage/drug therapy , rac1 GTP-Binding Protein/drug effects , Animals , Disease Models, Animal , Male , Neuroprotective Agents/administration & dosage , Osteopontin/administration & dosage , Phenotype , Rats , Rats, Sprague-Dawley , Recombinant ProteinsABSTRACT
OBJECTIVE: Hydrogen sulfide (H(2)S)-releasing NSAIDs exert potent anti-inflammatory effects beyond classical cyclooxygenase inhibition. Here, we compared the platelet inhibitory effects of the H(2)S-releasing aspirin derivative ACS14 with its mother compound aspirin to analyze additional effects on platelets. METHODS AND RESULTS: In platelets of mice fed with ACS14 for 6 days (50 mg/kg per day), not only arachidonic acid-induced platelet aggregation but also ADP-dependent aggregation was decreased, an effect that was not observed with an equimolar dose of aspirin (23 mg/kg per day). ACS14 led to a significantly longer arterial occlusion time after light-dye-induced endothelial injury as well as decreased thrombus formation after ferric chloride-induced injury in the carotid artery. Bleeding time was not prolonged compared with animals treated with equimolar doses of aspirin. In vitro, in human whole blood, ACS14 (25-500 µmol/L) inhibited arachidonic acid-induced platelet aggregation, but compared with aspirin additionally reduced thrombin receptor-activating peptide-, ADP-, and collagen-dependent aggregation. In washed human platelets, ACS14 (500 µmol/L) attenuated αIIbß3 integrin activation and fibrinogen binding and increased intracellular cAMP levels and cAMP-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation. CONCLUSIONS: The H(2)S-releasing aspirin derivative ACS14 exerts strong antiaggregatory effects by impairing the activation of the fibrinogen receptor by mechanisms involving increased intracellular cyclic nucleotides. These additional antithrombotic properties result in a more efficient inhibition of thrombus formation in vivo as achieved with aspirin alone.
Subject(s)
Aspirin/metabolism , Aspirin/pharmacology , Blood Platelets/drug effects , Fibrinolytic Agents/pharmacology , Hydrogen Sulfide/metabolism , Platelet Aggregation/drug effects , Animals , Aspirin/analogs & derivatives , Bleeding Time , Blood Platelets/metabolism , Cyclic AMP/metabolism , Disulfides/pharmacology , Humans , In Vitro Techniques , Integrins/drug effects , Integrins/metabolism , Mice , Mice, Inbred C57BL , Models, Animal , Platelet Activation/drug effects , Platelet Activation/physiology , Platelet Aggregation/physiology , Prostaglandin-Endoperoxide Synthases/drug effects , Prostaglandin-Endoperoxide Synthases/metabolism , Thrombosis/metabolism , Thrombosis/prevention & controlABSTRACT
The integrity of transplanted mesenchymal stem cells (MSCs) for cardiac regeneration is dependent on cell-cell or cell-matrix adhesion, which is inhibited by reactive oxygen species (ROS) generated in ischemic surroundings after myocardial infarction. Intracellular ROS play a key role in the regulation of cell adhesion, migration, and proliferation. This study was designed to investigate the role of ROS on MSC adhesion. In H(2)O(2) treated MSCs, adhesion and spreading were inhibited and detachment was increased in a dose-dependent manner, and these effects were significantly rescued by co-treatment with the free radical scavenger, N-acetyl-L-cysteine (NAC, 1 mM). A similar pattern was observed on plates coated with different matrices such as fibronectin and cardiogel. Hydrogen peroxide treatment resulted in a marked decrease in the level of focal adhesion-related molecules, such as phospho-FAK and p-Src in MSCs. We also observed a significant decrease in the integrin-related adhesion molecules, alpha V and beta1, in H(2)O(2) treated MSCs. When injected into infarcted hearts, the adhesion of MSCs co-injected with NAC to the border region was significantly improved. Consequently, we observed that fibrosis and infarct size were reduced in MSC and NAC-injected rat hearts compared to in MSC-only injected hearts. These results indicate that ROS inhibit cellular adhesion of engrafted MSCs and provide evidence that the elimination of ROS might be a novel strategy for improving the survival of engrafted MSCs.
Subject(s)
Cell Adhesion Molecules/metabolism , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , Myocardial Ischemia/metabolism , Myocardial Ischemia/surgery , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Animals , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Adhesion Molecules/drug effects , Cell Differentiation/physiology , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Focal Adhesion Kinase 1/drug effects , Focal Adhesion Kinase 1/metabolism , Focal Adhesions/drug effects , Focal Adhesions/metabolism , Free Radical Scavengers/pharmacology , Gene Knock-In Techniques , Graft Survival/physiology , Hydrogen Peroxide/pharmacology , Integrins/drug effects , Integrins/metabolism , Male , Myocardial Ischemia/physiopathology , Oxidants/pharmacology , Oxidative Stress/drug effects , Oxidative Stress/physiology , Rats , Rats, Sprague-Dawley , Regeneration/physiology , src-Family Kinases/drug effects , src-Family Kinases/metabolismABSTRACT
The formation of a suitable extracellular matrix (ECM) that promotes cell adhesion, organization, and proliferation is essential within biomaterial scaffolds for tissue engineering applications. In this work, short elastin mimetic peptide sequences, EM-19 and EM-23, were engineered to mimic the active motifs of human elastin in hopes that they can stimulate ECM development in synthetic polymer scaffolds. Each peptide was incubated with human aortic smooth muscle cells (SMCs) and elastin and desmosine production were quantified after 48 h. EM-19 inhibited elastin production through competitive binding phenomena with the elastin binding protein (EBP), whereas EM-23, which contains an RGDS domain, induces recovery of elastin production at higher concentrations, alluding to a higher binding affinity for the integrins than for the EBP and the involvement of integrins in elastin production. Colocalization of each peptide with the elastin matrix was confirmed using immunofluorescent techniques. Our data suggest that with appropriate cell-binding motifs, we can simulate the cross-linking of tropoelastin into the developing elastin matrix using short peptide sequences. The potential for increased cell adhesion and the incorporation of elastin chains into tissue engineering scaffolds make these peptides attractive bioactive moieties that can easily be incorporated into synthetic biomaterials to induce ECM formation.
Subject(s)
Elastic Tissue/metabolism , Elastin/metabolism , Extracellular Matrix/metabolism , Integrins/metabolism , Oligopeptides/metabolism , Receptors, Cell Surface/metabolism , Cell Adhesion/drug effects , Cells, Cultured , Elastic Tissue/chemistry , Elastic Tissue/drug effects , Elastin/antagonists & inhibitors , Elastin/chemistry , Extracellular Matrix/chemistry , Extracellular Matrix/drug effects , Humans , Hydrogels/chemistry , Hydrogels/metabolism , Integrins/chemistry , Integrins/drug effects , Models, Biological , Muscle, Smooth/cytology , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Oligopeptides/chemistry , Oligopeptides/pharmacology , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/drug effects , Tissue EngineeringABSTRACT
Pharmacological agents directed against the integrins alpha(v)beta(3) and alpha(v)beta(5) have been reported to inhibit angiogenesis. However, genetic ablations of the genes encoding these integrins fail to block angiogenesis and in some cases even enhance it. This apparent paradox suggests the hypotheses that these integrins are negative regulators of angiogenesis and that the drugs targeting them may be acting as agonists rather than antagonists.
Subject(s)
Integrins/drug effects , Integrins/metabolism , Neovascularization, Pathologic/drug therapy , Neovascularization, Physiologic/physiology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Apoptosis/physiology , Autoantigens/pharmacology , Collagen Type IV/pharmacology , Humans , Integrins/genetics , Neovascularization, Physiologic/drug effects , Receptors, Vitronectin/drug effects , Receptors, Vitronectin/genetics , Receptors, Vitronectin/metabolismABSTRACT
Integrins are critically involved in many tumour-promoting activities. The development of inhibitors against integrins may suppress tumour growth by inhibiting tumour angiogenic signalling. In this study, we investigated the effects of two novel peptides containing the integrin binding arginine-glycine-aspartic acid-motif on inhibiting diverse cell behaviours, including cell adhesion, motility, invasion, tube formation and cell cytoskeleton. Cell adhesion and motility assays demonstrated that cyclopeptides c-Gly and c-Lys might inhibit the adhesive and motile activity at the concentration of 25 µM. There was no significant effect on cell invasion, indicating the importance of extracellular matrix degradation in modulating the anti-invasive effect of human umbilical vein endothelial cells (HUVECs). More importantly, the tubular network formation of HUVECs was significantly inhibited by cyclopeptide c-Lys besides causing a remarkable inhibition of cytoskeletal organization, disrupting the focal adhesion and actin stress fibres formation. In conclusion, this study results indicated that the novel peptide c-Lys has the ability to inhibit diverse cell behaviours of HUVECs, and the effects may be mediated at different levels of the tumour growth. Therefore, c-Lys is perhaps proposed to be a potent anti-angiogenic drug candidate.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Integrins/drug effects , Peptides, Cyclic/pharmacology , Actins/drug effects , Amino Acid Motifs , Angiogenesis Inhibitors/chemistry , Binding Sites , Cell Adhesion/drug effects , Cell Line , Cell Migration Assays , Cell Movement/drug effects , Cytoskeleton/drug effects , Endothelial Cells/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Extracellular Matrix/drug effects , Focal Adhesions/drug effects , Humans , Integrins/metabolism , Neoplasm Invasiveness , Neovascularization, Pathologic/drug therapy , Peptides, Cyclic/chemistry , Stress Fibers/drug effects , Umbilical Veins/cytology , Umbilical Veins/metabolismABSTRACT
Opioids play a pivotal role in pain transmission but are also able to modulate immune cell functions. In the last decades a connection between opioids and integrins-adhesion molecules involved, among many other processes, in leukocyte recruitment at inflamed site-has been established. To study immune cell integrin-mediated adhesion, cell adhesion assay is a simple, reproducible, and valuable tool capable of unraveling concentration-dependent effects of a test candidate on integrin-mediated cell adhesion.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Adhesion/drug effects , Immunohistochemistry/methods , Analgesics, Opioid/metabolism , Analgesics, Opioid/pharmacology , Animals , Cell Adhesion/physiology , Cell Adhesion Molecules/drug effects , Humans , Immunologic Factors/pharmacology , Inflammation/metabolism , Integrins/drug effects , Integrins/metabolism , Jurkat Cells , Leukocytes/metabolism , U937 CellsABSTRACT
T7 peptide is considered as an antiangiogenic polypeptide. The presents study aimed to further detect the antiangiogenic mechanisms of T7 peptide and determine whether combining T7 peptide and meloxicam (COX-2/PGE2 specific inhibitor) could offer a better therapy to combat hepatocellular carcinoma (HCC). T7 peptide suppressed the proliferation, migration, tube formation, and promoted the apoptosis of endothelial cells under both normoxic and hypoxic conditions via integrin α3ß1 and αvß3 pathways. Cell proliferation, migration, apoptosis, or tube formation ability were detected, and the expression of integrin-associated regulatory proteins was detected. The anti-tumor activity of T7 peptide, meloxicam, and their combination were evaluated in HCC tumor models established in mice. T7 peptide suppressed the proliferation, migration, tube formation, and promoted the apoptosis of endothelial cells under both normoxic and hypoxic conditions via integrin α3ß1 and αvß3 pathways. Meloxicam enhanced the activity of T7 peptide under hypoxic condition. T7 peptide partly inhibited COX-2 expression via integrin α3ß1 not αvß3-dependent pathways under hypoxic condition. T7 peptide regulated apoptosis associated protein through MAPK-dependent and -independent pathways under hypoxic condition. The MAPK pathway was activated by the COX-2/PGE2 axis under hypoxic condition. The combination of T7 and meloxicam showed a stronger anti-tumor effect against HCC tumors in mice. The data highlight that meloxicam enhanced the antiangiogenic activity of T7 peptide in vitro and in vivo.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Collagen Type IV/pharmacology , Cyclooxygenase 2 Inhibitors/pharmacology , Dinoprostone/antagonists & inhibitors , Liver Neoplasms/drug therapy , Meloxicam/pharmacology , Peptide Fragments/pharmacology , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation , Cyclooxygenase 2/metabolism , Drug Combinations , Endothelial Cells/drug effects , Humans , Hypoxia/pathology , Integrins/drug effects , Mice , Neovascularization, Pathologic/drug therapy , RNA, Small Interfering/metabolism , Random Allocation , Xenograft Model Antitumor AssaysABSTRACT
Integrins are essential in the complex multistep process of angiogenesis and are thus attractive targets for the development of antiangiogenic therapies. Integrins are antagonized by disintegrins and C-type lectin-like proteins, two protein families from snake venom. Here, we report that CC-PLA2-1 and CC-PLA2-2, two novel secreted phospholipases A(2) (PLA(2)) isolated from Cerastes cerastes venom, also showed anti-integrin activity. Indeed, both PLA(2)s efficiently inhibited human brain microvascular endothelial cell adhesion and migration to fibrinogen and fibronectin in a dose-dependent manner. Interestingly, we show that this anti-adhesive effect was mediated by alpha5beta1 and alphav-containing integrins. CC-PLA2s also impaired in vitro human brain microvascular endothelial cell tubulogenesis on Matrigel and showed antiangiogenic activity in vivo in chicken chorioallantoic membrane assay. The complete PLA(2) cDNAs were cloned from a venom gland cDNA library. Mature CC-PLA2-1 and CC-PLA2-2 contain 121 and 120 amino acids, respectively, including 14 cysteines each and showed 83% identity. Tertiary model structures of CC-PLA2-1 and CC-PLA2-2 were generated by homology modeling. This is thus the first study describing an antiangiogenic effect for snake venom PLA(2)s and reporting first clues to their mechanism of action on endothelial cells.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Group I Phospholipases A2/pharmacology , Group II Phospholipases A2/pharmacology , Integrins/drug effects , Viper Venoms/enzymology , Animals , Chorioallantoic Membrane/drug effects , Endothelial Cells , Focal Adhesions/drug effects , Group I Phospholipases A2/chemistry , Group II Phospholipases A2/chemistry , Humans , In Vitro Techniques , Models, Structural , Static Electricity , Viper Venoms/chemistryABSTRACT
Active targeting strategies are currently being extensively investigated in order to enhance the selectivity of photodynamic therapy. The aim of the present research was to evaluate whether the external decoration of nanopolymeric carriers with targeting peptides could add more value to a photosensitizer formulation and increase antitumor therapeutic efficacy and selectivity. To this end, we assessed PLGA-PLA-PEG nanoparticles (NPs) covalently attached to a hydrophilic photosensitizer 5-[4-azidophenyl]-10,15,20-tri-(N-methyl-4-pyridinium)porphyrinato zinc (II) trichloride (ZnTriMPyP) and also to c(RGDfK) peptides, in order to target αv ß3 integrin-expressing cells. In vitro phototoxicity investigations showed that the ZnTriMPyP-PLGA-PLA-PEG-c(RGDfK) nanosystem is effective at submicromolar concentrations, is devoid of dark toxicity, successfully targets αv ß3 integrin-expressing cells and is 10-fold more potent than related nanosystems where the PS is occluded instead of covalently bound.
Subject(s)
Drug Carriers , Nanoparticles , Neoplasms/drug therapy , Oligopeptides/chemistry , Photochemotherapy , Photosensitizing Agents/pharmacology , Polymers/chemistry , Cell Line, Tumor , Humans , Integrins/drug effects , Kinetics , Photosensitizing Agents/therapeutic use , Singlet Oxygen/chemistry , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
Multiple spontaneous transient elevations of cytosolic-free calcium ([Ca2+]i) are observed in single human neutrophils during adherence. The interrelation between adherence and spontaneous [Ca2+]i transients was analyzed by simultaneous monitoring of [Ca2+]i and cell morphology. Fluorescent images of fura 2-loaded neutrophils attached to albumin-coated glass were recorded with a high sensitivity CCD camera while [Ca2+]i was assessed with a dual excitation microfluorimetry. The majority of the initially round cells studied showed changes in shape which started either before or at the same time as the onset of the [Ca2+]i transients. These data suggested that a rise in [Ca2+]i is not a prerequisite for shape change. This conclusion was confirmed by observation of movement and spreading in cells whose [Ca2+]i transients were abolished by chelation of extracellular Ca2+. Instead, our data suggest that spreading or adhesion itself initiates the [Ca2+]i activity. In keeping with this hypothesis, cytochalasin B, which prevents both cell movement and adhesion, completely inhibited generation of [Ca2+]i transients. To determine if the movement alone or adhesion alone is responsible for [Ca2+]i activity, we treated cells with antibodies against the beta chain (CD18, beta 2) or the alpha subunit (CD11b, alpha m) of the dominant leukocyte integrin (CR3). Antibody-treated cells showed normal extension of pseudopods but impaired ability to adhere. Inhibition of adhesion in this way inhibited [Ca2+]i activity. Taken together these results suggest that following sequence of events after contact of neutrophils with surfaces: (a) cell movement and shape change lead to enhanced contact of integrins with the surface; and (b) integrins-mediated adhesion generates multiple [Ca2+]i transients. The [Ca2+]i transients may then control exocytic events associated with movement and may provide a link between adherence and activation or priming of neutrophils to other stimuli.
Subject(s)
Calcium/blood , Integrins/physiology , Neutrophils/physiology , Antibodies, Monoclonal , Cell Adhesion/drug effects , Cell Membrane/physiology , Cytochalasin B/pharmacology , Cytosol/metabolism , Egtazic Acid/pharmacology , Fura-2 , Humans , In Vitro Techniques , Integrins/drug effects , Kinetics , Microscopy, Fluorescence , Neutrophils/cytology , Neutrophils/drug effects , SoftwareABSTRACT
The capability of the integrin VLA-3 to function as a receptor for collagen (Coll), laminin (Lm), and fibronectin (Fn) was addressed using both whole cell adhesion assays and ligand affinity columns. Analysis of VLA-3-mediated cell adhesion was facilitated by the use of a small cell lung carcinoma line (NCI-H69), which expresses VLA-3 but few other integrins. While VLA-3 interaction with Fn was often low or undetectable in cells having both VLA-3 and VLA-5, NCI-H69 cells readily attached to Fn in a VLA-3-dependent manner. Both Arg-Gly-Asp (RGD) peptide inhibition studies, and Fn fragment affinity columns suggested that VLA-3, like VLA-5, may bind to the RGD site in human Fn. However, unlike Fn, both Coll and Lm supported VLA-3-mediated adhesion that was not inhibited by RGD peptide, and was totally unaffected by the presence of VLA-5. In addition, VLA-3-mediated binding to Fn was low in the presence of Ca++, but was increased 6.6-fold with Mg++, and 30-fold in the presence of Mn++. In contrast, binding to Coll was increased only 1.2-fold with Mg++, and 1.7-fold in Mn++, as compared to the level seen with Ca++. Together, these experiments indicate that VLA-3 can bind Coll, Lm, and Fn, and also show that (a) VLA-3 can recognize both RGD-dependent and RGD-independent ligands, and (b) different VLA-3 ligands have distinctly dissimilar divalent cation sensitivities.
Subject(s)
Cell Adhesion/physiology , Collagen/metabolism , Fibronectins/metabolism , Integrins/metabolism , Laminin/metabolism , Amino Acid Sequence , Binding, Competitive , Cations, Divalent/pharmacology , Cell Adhesion/drug effects , Chromatography, Affinity , Extracellular Matrix/metabolism , Humans , Integrins/drug effects , Integrins/physiology , Molecular Sequence Data , Oligopeptides/pharmacology , Tumor Cells, CulturedABSTRACT
Platelet activation in vivo can be part of the hemostatic response to injury or a pathological response to disease. In either setting, platelets adhere to the vessel wall and to each other, forming a closely packed mass interspersed with fibrin. Recent studies have identified new molecules on the platelet surface and within platelets that support and regulate thrombus growth and stability, ensuring that platelet accumulation after injury is sufficient to stop bleeding, but not so exuberant that vascular occlusion occurs. An understanding of how this balance is achieved helps to illuminate the events of platelet activation and, at the same time, provides potential targets for new classes of antiplatelet agents.