elastin and valyl-glycyl-valyl-alanyl-prolyl-glycine

Elastic fibers are an important component of the extracellular matrix, providing elasticity and resilience to tissues that require the ability to deform repetitively and reversibly. Among the elastin-derived peptides, the Val-Gly-Val-Ala-Pro-Gly (VGVAPG) hexapeptide is known for its chemotactic activity and metalloproteinases upregulation properties. As other elastin-derived peptides, having homologous similar sequences, do not exhibit any biological activity, the following question arises: Does the peptide-receptor interaction need a specific active conformation? Previous experimental studies including NMR and CD spectroscopies did not clearly identify the conformations adopted by the VGVAPG peptide in solution. However, structural predictions made on VGVAPG and related XGXXPG peptides suggested a folded beta-turn conformation. So we undertook a theoretical and experimental study of the VGVAPG peptide. The work presented here, which gives an overall structural description of VGVAPG behavior in water, also provides an additional insight into its structure-activity relationship. Both theoretical and experimental results suggest the existence of an ensemble of rather extended and folded conformations in solution. All the folded structures obtained exhibit a type VIII beta-turn spanning the GVAP sequence. In the lack of any structural information concerning the elastin receptor, these results suggest that such a conformation could be relevant for the peptide-receptor interaction and thus for biological activity.

Topics: Elastin; Models, Molecular; Oligopeptides; Protein Conformation; Protein Structure, Secondary

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor from the family of basic helix-loop-helix transcription factors. Several studies have indicated an important role of AhR signaling pathways in senescence, aging, and neurodegenerative diseases. During aging, elastin is degraded and elastin-derived peptides (EDPs) are formed. EDPs have been detected in human blood, serum, and cerebrospinal fluid. Literature data suggest a role of EDPs in the development of neurodegenerative diseases. However, the impact of EDPs on the AhR signaling pathway has never been investigated. Therefore, the aim of our paper was to study the role of AhR in the mechanism of action of the VGVAPG peptide (one of the EDPs) in mouse primary astrocytes in vitro. Our experiments have shown that AhR plays an important role in the EDP mechanism of action in a model of mouse primary astrocytes. Moreover, due to the involvement of Sirt3, Pparγ, AhR, Glb1, Nf-κb1, Ece1, Ide, and Nepr genes and the production and release of neurosteroids, VGVAPG can accelerate the development of neurodegenerative diseases in which the proper metabolism of astrocytes is crucial. Furthermore, our studies have proved that AhR is likely involved in the co-control of the Sirt1, Glb1, Nf-κb1, Ece1, and Nepr expression in astrocytes.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Elastin; Humans; Mice; Neurodegenerative Diseases; Peptides; Receptors, Aryl Hydrocarbon

Autophagy is a self-degradative process important for balancing the sources of energy and involved in the development of Alzheimer's disease (AD). To date, a number of papers have shown that elastin-derived peptides (EDPs) affect the expression and activation of peroxisome proliferator-activated receptor gamma (PPARγ), which is crucial for the development of AD and autophagy initiation. Therefore, the aim of the present study was to determine whether EDPs with a Val-Gly-Val-Ala-Pro-Gly (VGVAPG) amino acid sequence activate the autophagic process in undifferentiated SH-SY5Y human neuroblastoma cells. Our study is the first to show that EDPs with the VGVAPG sequence initiate the autophagy process in the undifferentiated SH-SY5Y cell line exhibiting a number of features of normal neuroblasts. In particular, we observed in our study that VGAVPG peptide increased ULK1, AKT, PPARγ, and LC3B protein expression. Moreover, our experiments with the agonist (rosiglitazone) and antagonist (GW9662) of PPARγ confirm that the studied EDP acts through the PPARγ pathway affecting mTOR and finally autophagy. Some studies have shown that autophagy disturbances are involved in the development of AD. Therefore, we believe that our study will provide new evidence of the possible involvement of EDPs (especially VGVAPG) in the development of AD.

Topics: Autophagy; Elastin; Humans; Neuroblastoma; Peptides; PPAR gamma

Tissues are subjected to dynamic communication between cells and the extracellular matrix (ECM), resulting in ECM remodeling. One of the ECM components is elastin, which releases elastin-derived peptides (EDPs) during the aging process. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG hexapeptide and elastin-like peptide VVGPGA (control) on certain metabolism parameters in human breast adenocarcinoma (MCF-7) and human lung carcinoma (A549) cell lines. The results did not show a significant effect of the peptides on metabolic activity and caspase-3 activity. However, more specific analysis revealed that VGVAPG and VVGPGA were able to increase KI67 protein expression in both tested cell lines after 24-h treatment. Moreover, the same correlation was observed at the KI67 gene level. VGVAPG also increased the P53, ATM and SHH gene expression in the A549 cells up to 19.08%, 20.74%, and 28.77%, respectively. Interestingly, the VGVAPG peptide exerted an effect on the expression of antioxidant enzymes SOD2 and CAT in the A549 and MCF-7 cells, especially after the 24-h treatment. Lastly, both peptides influenced the CAV1 and CLTC1 expression. Our results show that the tested EDPs have an effect on both A549 and MCF-7 cells at the cellular level. This may be correlated with the multidrug-resistance (MDR) phenotype in these cancer cells, which is an emerging problem in the current anticancer treatment. However, more research is needed in this field.

Topics: A549 Cells; Elastin; Humans; Ki-67 Antigen; Lung; MCF-7 Cells; Neoplasms; Oligopeptides; Peptides

Elastin-derived peptides (EDPs) contain replications of the Val-Gly-Val-Ala-Pro-Gly (VGVAPG) hexapeptide. It has been described that the VGVAPG peptide induces reactive oxygen species (ROS) production in murine monocytes and astrocytes, human fibroblasts, and the human neuroblastoma (SH-SY5Y) cell line. To date, there is growing evidence that calcium channel blockers (CCBs) reduce oxidative stress and development of inflammation in the nervous system. Therefore, the aim of the present study was to evaluate the impact of such CCBs as Nifedipine, Verapamil, and MK-801 on the expression of peroxisome proliferator-activated receptor (Pparγ), i.e. ROS-related and inflammation-related proteins, in mouse astrocytes exposed in vitro to the VGVAPG peptide. The experiments showed that Nifedipine or MK-801 used in co-treatment with the VGVAPG peptide potentiated the effect of this peptide on the Pparγ level after the 24-h and 48-h treatment. Moreover, all studied compounds decreased the VGVAPG-induced caspase-1 activity in both time intervals. The data also showed that the VGVAPG peptide decreased the interleukin 1 beta (IL-1β) level in both studied time intervals. Upon a short-time exposure, the use of CCBs intensified the decrease in IL-1β stimulated by the VGVAPG peptide, opposite to the longer treatment. Moreover, the VGVAPG peptide decreased the IL-1βR1 level in both studied time intervals. After 24 h, Nifedipine and Verapamil potentiated the effect of the VGVAPG peptide. The VGVAPG peptide decreased the catalase (Cat) protein expression only after 24 h, whereas CCBs did not affect the expression of Cat induced by the VGVAPG peptide. The VGVAPG peptide increased the expression of the superoxide dismutase 1 (Sod1) protein. After 24 h of exposure, Nifedipine and Verapamil potentiated the increase in the Sod1 protein expression. Finally, our data showed that VGVAPG did not change the level of estradiol (E

Topics: Animals; Astrocytes; Calcium Channel Blockers; Dizocilpine Maleate; Elastin; Humans; Inflammation; Mice; Neuroblastoma; Nifedipine; Oligopeptides; Peptides; PPAR gamma; Reactive Oxygen Species; Superoxide Dismutase-1; Verapamil

During vascular aging or in pathological conditions in humans, elastin is degraded and its by-products, the elastin-derived peptides (EDPs), enter the blood circulation. EDPs may be detected in the serum of healthy subjects or people who suffered a stroke. Moreover, recent evidence suggests a potential role of inflammatory mechanisms in neurological conditions, which are usually not categorized as inflammatory. Therefore, the present in vitro study was conducted to investigate the impact of the VGVAPG peptide on the activation of inflammatory process in mouse primary astrocytes, which were maintained in phenol red-free DMEM/F12 supplemented with 10% fetal bovine serum. The cells were exposed to VGVAPG or VVGPGA peptides for 24 and 48 h; this was followed by the determination of the activity of caspase-1 and levels of SOD, CAT, PPARγ, NF-κB, IL-1β, and IL-1βR1. Furthermore, rosiglitazone-a PPARγ agonist-was applied. Our study pioneered the finding that the VGVAPG peptide increases caspase-1 activity in astrocytes in vitro. The VGVAPG peptide simultaneously decreases the release of IL-1β into the cell-culture medium from astrocytes. The ELISA method revealed that the VGVAPG peptide increases the protein expression of SOD1 whereas it decreases the expression of IL-1βR1, CAT, and NF-κB. Therefore, the available data suggest that the VGVAPG peptide (concentration 10 nM) synergistically acts with agonists of PPARγ in mouse astrocytes. However, given the lack of sufficient data to explain the molecular mechanism of action of the VGVAPG peptide in the nervous system, more studies in this area are necessary.

Topics: Animals; Astrocytes; Elastin; Gene Expression; Inflammation; Inflammation Mediators; Mice; Oligopeptides; Peptides; Primary Cell Culture; Rosiglitazone

During aging and ischemic and hemorrhagic stroke, elastin molecules are degraded and elastin-derived peptides are released into the brain microenvironment. Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a repeating hexapeptide in the elastin molecule. It is well documented that the peptide sequence binds with high affinity to elastin-binding protein (EBP) located on the cell surface, thereby transducing a molecular signal into the cell. The aim of our study was to investigate whether EBP, aryl hydrocarbon receptor (Ahr), and peroxisome proliferator-activated receptor gamma (Pparγ) are involved in VGVAPG-stimulated proliferation. Primary astrocytes were maintained in DMEM/F12 medium without phenol red, supplemented with 10 or 1% charcoal/dextran-treated fetal bovine serum (FBS). The cells were exposed to increasing concentrations of VGVAPG peptide, and resazurin reduction was measured. In addition, Glb1, Pparγ, and Ahr genes were silenced. After 48 h of exposure to 10 nM and 1 µM of VGVAPG peptide, the level of estradiol (E

Topics: Animals; Astrocytes; Cell Proliferation; Cells, Cultured; Elastin; Estradiol; Female; Ki-67 Antigen; Mice; Oligopeptides; Oxazines; PPAR gamma; Pregnancy; Receptors, Aryl Hydrocarbon; Receptors, Cell Surface; RNA Interference; RNA, Small Interfering; S100 Calcium Binding Protein beta Subunit; Xanthenes

Elastin is a highly elastic protein present in connective tissue. As a result of protease activity, elastin hydrolysis occurs, and during this process, elastin-derived peptides (EDPs) are released. One of the constitutively repeating elastin and EDP building sequences is the hexapeptide VGVAPG. Therefore, the aim of our research was to define the effect of VGVAPG peptide on adipogenesis in a mouse 3T3-L1 cell line. 3T3-L1 cells were differentiated according to a previously described protocol and exposed to increasing concentrations of VGVAPG or VVGPGA peptide. The obtained results showed that VGVAPG peptide does not stimulate reactive oxygen species (ROS) production, caspase-1 activation, and 3T3-L1 cell proliferation. In the second part of the experiments, it was proved that VGVAPG peptide decreased lipid accumulation as measured by oil red O staining, which was confirmed by the profile of increased expression markers of undifferentiated preadipocytes. In our experiments, 10 nM VGVAPG added for differentiating to adipocytes increased the expression of Pref-1, serpin E1, and adiponectin as compared to rosiglitazone (PPARγ agonist)-treated group and simultaneously decreased the expression of VEGF and resistin as compared to the rosiglitazone-treated group. The obtained results show that VGVAPG peptide sustains 3T3 cells in undifferentiated state.. DMSO: dimethyl sulphoxide; EBP: elastin-binding protein; EDPs: elastin-derived peptides; FBS: foetal bovine serum;

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Adipokines; Animals; Cell Differentiation; Elastin; Lipid Metabolism; Mice; Oligopeptides; Proteome; Reactive Oxygen Species

Under physiological and pathological conditions, elastin is degraded to produce elastin-derived peptides (EDPs). EDPs are detected in the healthy human brain, and its concentration significantly increases after ischemic stroke. Both elastin and EDPs contains replications of the soluble VGVAPG hexapeptide, which has a broad range of biological activities. Effects of VGVAPG action are mainly mediated by elastin-binding protein (EBP), which is alternatively spliced, enzymatically inactive form of the GLB1 gene. This study was conducted to elucidate the activation and role of the N-methyl-D-aspartate receptor (NMDAR) in elastin-derived VGVAPG peptide-dependent calcium homeostasis in mouse cortical astrocytes in vitro. Cells were exposed to 10 nM VGVAPG peptide and co-treated with MK-801, nifedipine, verapamil, or Src kinase inhibitor I. After cell stimulation, we measured Ca

Topics: Animals; Astrocytes; Biomarkers; Calcium; Cells, Cultured; Cerebral Cortex; Elastin; Gene Silencing; Homeostasis; Mice; Oligopeptides; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; RNA, Small Interfering

The process of degradation of the elastin-rich extracellular matrix produces elastin-derived peptides (EDPs). Different types of EDPs are detectable in the cerebrospinal fluid in healthy individuals and in patients after ischemic stroke. To date, it has been demonstrated that EDPs can regulate the development of insulin resistance in mice in a peroxisome proliferator-activated receptor gamma (Pparγ)-dependent manner. Therefore, the aim of this study was to investigate the impact of the elastin-derived valine-glycine-valine-alanine-proline-glycine (VGVAPG) peptide on Pparγ and beta-galactosidase (β-Gal) expression in mouse cortical astrocytes in vitro. Primary astrocytes were maintained in DMEM/F12 without phenol red supplemented with 10% fetal bovine serum. The cells were exposed to 50 nM, 1 and 50 μM of the VGVAPG peptide. After 3 and 6 h (for mRNA) and 24 and 48 h (for the protein) of exposition to the peptide, the expression of Pparγ and β-Gal was measured. Moreover, the siRNA gene knockdown method was applied. Our study showed, for the first time, that the VGVAPG peptide affected β-Gal and Pparγ mRNA and protein expression in mouse astrocytes in vitro. Furthermore, we suggested a bidirectional interaction between Pparγ and β-Gal. Both pioglitazone and rosiglitazone increased β-Gal and Pparγ protein expression in mouse astrocytes in vitro, and this effect was reduced by the VGVAPG peptide. However, due to the lack of sufficient data explaining the molecular mechanism of action of the VGVAPG peptide in the nervous system, more studies are necessary in this field.

Topics: Animals; Astrocytes; beta-Galactosidase; Cells, Cultured; Elastin; Female; Mice; Oligopeptides; PPAR gamma; RNA, Messenger; RNA, Small Interfering

The development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.

Topics: Animals; Cells, Cultured; Elastin; Human Umbilical Vein Endothelial Cells; Humans; Hydrogels; Male; Mice; Mice, Inbred C57BL; Microvessels; Neovascularization, Physiologic; Oligopeptides; Peptides; Polymers; Recombinant Proteins; Wound Healing

Throughout the lifetime of humans, the amount of stem cells and the rate of cell proliferation continue to decrease. Reactive oxygen species (ROS) are one among the many factors that promote stem cell aging. Both a decrease in the level of stem cells and increase in ROS production can lead to the development of different neurodegenerative diseases. This study was conducted to determine how the VGVAPG peptide, liberated from elastin during the aging process and under pathological conditions, affects ROS production and activities of antioxidant enzymes in undifferentiated, proliferating SH-SY5Y cells. SH-SY5Y cells were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After treating the SH-SY5Y cells with VGVAPG peptide, we measured ROS production; cell metabolism, proliferation, and expression; and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). We demonstrated that the VGVAPG peptide increases GPx expression and activity, whereas it decreases CAT expression in SH-SY5Y cells. Silencing of the GLB1 gene prevents changes in GPx activity. Despite the fact that the VGVAPG peptide increases GPx expression, it increases the ROS level. Moreover, the VGVAPG peptide decreases SH-SY5Y proliferation, which is prevented by the ROS scavenger N-acetyl-L-cysteine. Our data suggest that ROS production and decreased proliferation of SH-SY5Y cells are the results of excitotoxicity meditated through close unrecognized molecular pathways. More research is needed to elucidate the unknown mechanism of action of VGVAPG peptide in the nervous system.

Topics: Catalase; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Elastin; Enzyme-Linked Immunosorbent Assay; Glutathione Peroxidase; Humans; Ki-67 Antigen; Neuroblastoma; Oligopeptides; PPAR gamma; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Superoxide Dismutase-1

During melanoma tumour growth, cancerous cells are exposed to the immediate surrounding the micro- and macro environment, which is largely modified through the degradation of the extracellular matrix by fibroblast-derived metalloproteinases. Among the degradation products, (VGVAPG)3, an elastin peptide is known to stimulate the proliferation of both fibroblasts and cancerous cells by binding to the elastin-binding receptor and activating the MEK/ERK signal transduction pathway. As this process strongly modifies mRNA synthesis, we investigated its effect on the relative three-dimensional organisation of the major partners of the mRNA splicing machinery: promyelocytic nuclear bodies (PML-NBs ) and splicing component 35 speckles (SC35) of normal fibroblasts and melanoma SK-MEL-28 cells. SC35 and PML-NBs proteins were immunolabeled and imaged by confocal microscopy within these cells cultured with (VGVAPG)3. Three-dimensional reconstruction was performed to elucidate the organisation of PML-NBs and SC35 speckles and their spatial relationship. In G0 cells, SC35 speckles were sequestered in PML-NBs. Shortly after (VGVAPG)3 stimulation, the three-dimensional organisation of PML-NBs and SC35 speckles changed markedly. In particular, SC35 speckles gradually enlarged and adopted a heterogeneous organisation, intermingled with PML-NBs. Conversely, inhibition of the elastin-binding protein or MEK/ERK pathway induced a remarkable early sequestration of condensed SC35 speckles in PML-NBs, the hallmark of splicing inhibition. The 3D architecture of speckles/PML-NBs highlights the modulation in their spatial relationship, the multiple roles of PML-NBs in activation, inhibition and sequestration, and provides the first demonstration of the dependence of PML-NBs and SC35 speckles on the elastin peptide for these functions.

Topics: Adult; Cell Line; Cell Nucleus; Cell Proliferation; Elastin; Fibroblasts; Humans; Imaging, Three-Dimensional; Melanoma; Oligopeptides; RNA Splicing; Structure-Activity Relationship

Elastin is the major structural extracellular matrix component of the arterial wall that provides the elastic recoil properties and resilience essential for proper vascular function. Elastin-derived peptides (EDP) originating from elastin fragmentation during vascular remodeling have been shown to play an important role in cell physiology and development of cardiovascular diseases. However, their involvement in thrombosis has been unexplored to date. In this study, we investigated the effects of EDP on (1) platelet aggregation and related signaling and (2) thrombus formation. We also characterized the mechanism by which EDP regulate thrombosis.. We show that EDP, derived from organo-alkaline hydrolysate of bovine insoluble elastin (kappa-elastin), decrease human platelet aggregation in whole blood induced by weak and strong agonists, such as ADP, epinephrine, arachidonic acid, collagen, TRAP, and U46619. In a mouse whole blood perfusion assay over a collagen matrix, kappa-elastin and VGVAPG, the canonical peptide recognizing the elastin receptor complex, significantly decrease thrombus formation under arterial shear conditions. We confirmed these results in vivo by demonstrating that both kappa-elastin and VGVAPG significantly prolonged the time for complete arteriole occlusion in a mouse model of thrombosis and increased tail bleeding times. Finally, we demonstrate that the regulatory role of EDP on thrombosis relies on platelets that express a functional elastin receptor complex and on the ability of EDP to disrupt plasma von Willebrand factor interaction with collagen.. These results highlight the complex nature of the mechanisms governing thrombus formation and reveal an unsuspected regulatory role for circulating EDP in thrombosis.

Topics: Animals; Blood Platelets; Cathepsin A; Cattle; Collagen; Elastin; Humans; Mice; Neuraminidase; Oligopeptides; Peptide Fragments; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Proteolysis; Receptors, Cell Surface; Signal Transduction; Thrombosis; Vascular Remodeling; von Willebrand Factor

To determine the role of multinucleated giant cells (MGCs) in cardiovascular diseases.. MGCs are a hallmark of giant cell arteritis. They are also described in atherosclerotic plaques from aortic aneurysms and carotid and coronary arteries. Herein, we demonstrate that the cholate-containing Paigen diet yields many MGCs in atherosclerotic plaques of apolipoprotein E-/- mice. These mice revealed a 4-fold increase in MGC numbers when compared with mice on a Western or Paigen diet without cholate. Most of the MGCs stained intensively for cathepsin K and were located at fibrous caps and close to damaged elastic laminae, with associated medial smooth muscle cell depletion. During in vitro experiments, MGCs demonstrated a 6-fold increase in elastolytic activity when compared with macrophages and facilitated transmigration of smooth muscle cells through a collagen-elastin matrix. An elastin-derived hexapeptide (Val-Gly-Val-Ala-Pro-Gly [VGVAPG]) significantly increased the rate of macrophage fusion, providing a possible mechanism of in vivo MGC formation. Comparable to the mouse model, human specimens from carotid arteries and aortic aneurysms contained cathepsin K-positive MGCs.. Apolipoprotein E-/- mice fed a Paigen diet provide a model to analyze the tissue-destructive role of MGCs in vascular diseases.

Topics: Animals; Antigens, Differentiation; Aortic Aneurysm; Apolipoproteins E; Atherosclerosis; Carotid Artery Diseases; Cathepsin K; Cell Fusion; Cell Movement; Cells, Cultured; Cholates; Collagen; Dietary Fats; Disease Models, Animal; Elastin; Endotoxins; Giant Cells, Foreign-Body; Humans; Immunohistochemistry; Interleukin-4; Macrophages, Peritoneal; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oligopeptides; Time Factors; Toll-Like Receptor 4

In the most common primary brain tumors, malignant glioma cells invade the extracellular matrix (ECM) and proliferate rapidly in the cerebral tissue, which is mainly composed of hyaluronan (HA) along with the elastin present in the basement membrane of blood vessels. To determine the role of ECM components in the invasive capacity of glioma cell lines, we developed a 3-D cell-culture system, based on a hydrogel in which HA can be coreticulated with kappa-elastin (HA-kappaE). Using this system, the invasiveness of cells from four glioma cell lines was dramatically increased by the presence of kappaE and a related, specific peptide (VGVAPG)(3). In addition, MMP-2 secretion increased and MMP-12 synthesis occurred. Extracellular injections of kappaE or (VGVAPG)(3) provoked a pronounced and dose-dependent increase in [Ca(2+)](i). kappaE significantly enhanced the expression of the genes encoding elastin-receptor and tropoelastin. We propose the existence of a positive feedback loop in which degradation of elastin generates fragments that stimulate synthesis of tropoelastin followed by further degradation as well as migration and proliferation of the very cells responsible for degradation. All steps in this ECM-based loop could be blocked by the addition of either of the EBP antagonists, lactose, and V-14 peptide, suggesting that the loop itself should be considered as a new therapeutic target.

Topics: Calcium; Cell Adhesion; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Elastin; Extracellular Matrix; Glioblastoma; Humans; Matrix Metalloproteinase 12; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Oligopeptides; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Tropoelastin

Melanomas containing more elastin are associated with higher stages of the disease. The interaction between elastin-derived peptides and melanoma cells appears to play an important role in the progression of melanomas. The effects of the elastin-derived peptides VGVAPG and VAPG have been investigated on the migration, invasion, adhesion and angiogenesis of human melanoma cells of different invasive potential. Elastin, tropoelastin and VGVAPG peptide were demonstrated at the invasion site of melanoma using histochemistry and immunohistochemistry. Not only the VGVAPG elastin-derived peptide, which exhibits the XGXXPG consensus sequence in its primary structure, but also the shorter VAPG bind directly to 3 cell surface receptors: galectin-3, integrin alpha v beta 3 and elastin-binding protein. Our results suggest that the increased levels of elastin-derived peptides facilitate the invasion of melanoma cells: (i) VGVAPG and VAPG elastin-derived peptides are chemotactic for melanoma cells; (ii) they can increase the migration of melanoma cells and the expression of CXCR-4 and CXCL-12 chemokines; (iii) they enhance the expression of the elastin-degrading MMP-2 and MMP-3; (iv) they increase the attachment of melanoma cells and the expression of different adhesion molecules; (v) they increase the expression of the lymphangiogenic VEGF-C and (vi) the galectin-3 receptor can mediate all these effects. Clinical and therapeutic aspects are also discussed.

Topics: Antigens, Neoplasm; Biomarkers, Tumor; Carrier Proteins; Cell Adhesion; Cell Adhesion Molecules; Cell Movement; Chemokine CXCL12; Chemotaxis; Disease Progression; Elastin; Flow Cytometry; Galectin 3; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Immunohistochemistry; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Melanoma; Neoplasm Invasiveness; Neovascularization, Pathologic; Oligopeptides; Polymerase Chain Reaction; Receptors, CXCR4; Skin Neoplasms; Tropoelastin; Vascular Endothelial Growth Factor C

Membrane-type I matrix metalloproteinase (MT1-MMP) has been previously reported to be up-regulated in human microvascular endothelial cell-1 line (HMEC) by elastin-derived peptides (elastokines). The aim of the present study was to identify the signaling pathways responsible for this effect. We showed that elastokines such as (VGVAPG)(3) peptide and kappa elastin induced nitric oxide (NO) production in a time-, concentration- and receptor-dependent manner as it could be abolished by lactose and a receptor-derived competitive peptide. As evidenced by the use of NO synthase inhibitors, elastokine-mediated up-regulation of MT1-MMP and pseudotube formation on Matrigel required NO production through activation of the PI(3)-kinase/Akt/NO synthase and NO/cGMP/Erk1/2 pathways. Elastokines induced both PI(3)-kinase p110gamma sub-unit, Akt and Erk1/2 activation, as shown by a transient increase in phospho-Akt and phospho-Erk1/2, reaching a maximum after 5 and 15 min incubation, respectively. Inhibitors of PI(3)-kinase and MEK1/2 suppressed elastokine-mediated MT1-MMP expression at both the mRNA and protein levels, and decreased the ability of elastokines to accelerate pseudotube formation. Besides, elastokines mediated a time- and concentration-dependent increase of cGMP, suggesting a link between NO and MT1-MMP expression. This was validated by the use of a guanylyl cyclase inhibitor, a NO donor and a cGMP analog. The guanylyl cyclase inhibitor abolished the stimulatory effect of elastokines on MT1-MMP expression. Inversely, the cGMP analog, mimicked the effect of both elastokines and NO donor in a concentration- and time-dependent manner. Overall, our results demonstrated that such elastokine properties through NO and MT1-MMP may be of importance in the context of tumour progression.

Topics: Cell Line; Chromones; Elastin; Endothelial Cells; Humans; Matrix Metalloproteinase 14; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Nitric Oxide; Nitric Oxide Synthase; Oligopeptides; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Up-Regulation

In congenital or acquired dermal melanocytosis, attachment of melanocyte with elastic fiber was shown in electron microscopy of unknown biological meaning. We hypothesize elastin-derived peptide may play a role in activating dermal melanocyte precursors.. This study was designed to determine: (i) whether melanocyte precursors express elastin binding protein (EBP); (ii) ontogenic expression of EBP and elastin in murine embryonic skin; (iii) the effects of elastin-derived peptide (VGVAPG) on melanocyte precursors.. Using immunohistochemistry or Western blot to identify EBP on murine embryonic sections, neural crest cell (NCC) primary culture explants, or two melanocyte precursor cell lines, NCCmelb4 and NCCmelan5. NCC explants or cells were treated with VGVAPG to compare its effect on proliferation, dendrite formation, melanosome maturation and tyrosinase mRNA expression of melanocyte precursors.. EBP was immunostained on c-kit+ melanocyte precursors. 67kDa EBP protein was immunoblotted on NCCmelb4 and NCCmelan5 cells. EBP was expressed early at embryonic day (E) 9.5, but elastin appeared later at E12.5 skin. VGVAPG increased DOPA-positive cell number and enhanced their dendrite formation in NCC explants. Electron microscopy showed advanced melanosome maturation in NCC explants or cells treated with VGVAPG. VGVAPG enhanced tyrosinase mRNA expression in NCCmelan5 cells.. Melanocyte precursors expressed EBP. VGVAPG stimulated their melanogenesis and dendrite formation. In the developmental journey interaction between elastin and EBP-expressed melanocyte precursors in embryos happened mainly since the stage of tertiary melanoblasts. These findings first provide biological evidences for the interaction between melanocyte and elastic fiber.

Topics: Animals; Base Sequence; Cell Line; Cells, Cultured; Dendrites; Dihydroxyphenylalanine; DNA Primers; Elastin; Embryonic Stem Cells; Female; Gene Expression Regulation, Developmental; Immunohistochemistry; Melanocytes; Melanosomes; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Monophenol Monooxygenase; Oligopeptides; Pregnancy; Receptors, Cell Surface; RNA, Messenger

A polyclonal antibody to elastin-derived hexapeptide repeat, H-(Val-Gly-Val-Ala-Pro-Gly)(3)-NH(2), was prepared in order to investigate the differences between elastin fibres in intimal hyperplasia and media in human atheroscleroic lesions. The hexapeptide repeat and alpha-elastin were recognized by this polyclonal antibody in enzyme-linked immunosorbent assay (ELISA), but other elastin-derived peptides such as tetrapeptide repeat, pentapeptide repeat and nonapeptide were not. In the series of hexapeptide repeats, H-(VGVAPG)(n)-NH(2) where n is 1-7, the polyclonal antibody reacted strongly with oligomers (n = 3-7) and weakly with dimer (n = 2), but not with monomer (n = 1). CD measurements suggested that the beta-turn structure is important for recognition by the polyclonal antibody. In an immunohistochemical study, elastin was stained more strongly in intimal hyperplasia than in media, suggesting that newly synthesized elastin in intimal hyperplasia is morphologically distinct from that in media.

Topics: Amino Acid Sequence; Antibodies; Atherosclerosis; Circular Dichroism; Dimerization; Elastic Tissue; Elastin; Humans; Hyperplasia; Immunochemistry; Immunohistochemistry; Microscopy; Molecular Sequence Data; Oligopeptides; Tunica Intima

In normal and pathological tissues, polymorphonuclear leukocyte proteases (elastase, cathepsin G and proteinase 3) may generate soluble peptides through limited proteolysis of elastin, the main component of mature elastic fibres. Elastin-derived peptides display diverse biological activities including cell migration, differentiation, proliferation, chemotaxis, tumor progression and up-regulation of metalloproteinases. To be biologically active, their structures must adopt a beta-turn conformation which accommodates to the cell surface-located elastin binding protein. In this study, we established that human elastin exon 24-derived peptides are hydrolysed by leukocyte elastase, when the active site is fully occupied (from S(5) to S'(3)). As shown by mass spectrometry analyses, a major cleavage site was demonstrated at a Val-Ala bond and a minor one at Gly-Val bond. For longer peptides, the hydrolysed fragments could themselves be re-hydrolysed. If the shortest fragments do not contain the GxxPG sequence known to stimulate cellular effects, some of the intermediates together with hydrolysis fragments generated by other proteases such as proteinase 3, may possess this motif.

Topics: Amino Acid Sequence; Elastin; Exons; Humans; Hydrolysis; Leukocyte Elastase; Mass Spectrometry; Molecular Sequence Data; Oligopeptides; Peptide Fragments

Elastin is one of the most significant components of the extracellular matrix, which supports the stretchiness of the blood vessels via its helical structure and cross-links. Enzymatic decomposition of this protein could induce chemotactic responses of cell populations in the surrounding tissues by several peptide sequences, e.g. XGXXPG. In our present work the VGVAPG variant and its oligomers were studied. The objective of the experiments was to learn (i) whether the chemotactic effect of these peptides is general in different levels of phylogeny: (ii) whether increasing the number of monomer units influences the chemotactic behaviour of the cell? The trimer had the strongest chemoattractant effect in a wide concentration range (10(-12) - 10(-7) M), while the monomer and the pentamer were chemorepellent. All tri-, tetra-, penta- and hexamers could chemotactically select subpopulations with a high chemotactic responsiveness to the identical peptide, in the long term. With regard to its repellent effect, the pentamer had a negative effect on phagocytosis. All six oligomers had a growth-promoter effect in Tetrahymena. The characteristic cell-physiological effects of VGVAPG oligomers signal that molecules of the extracellular matrix can induce identical responses even in lower levels of phylogeny, e.g. in the Ciliates.

Topics: Animals; Cell Proliferation; Chemotactic Factors; Chemotaxis; Circular Dichroism; Elastin; Models, Biological; Oligopeptides; Phagocytosis; Tetrahymena pyriformis

Chronic inflammation is a characteristic feature of abdominal aortic aneurysms (AAAs), but the molecular signals responsible for recruiting monocytes into the outer aortic wall are unresolved. The purpose of this study was to examine whether AAA tissues elaborate chemotactic activity for mononuclear phagocytes and to determine whether this activity is attributable to interactions between elastin degradation peptides (EDPs) and their cell surface receptor, the 67-kD elastin binding protein (EBP).. Soluble proteins were extracted from human AAA tissues, and chemotactic activity for differentiated U937 mononuclear phagocytes was measured by use of a modified Boyden chamber. Chemotactic activity induced by N -formyl-Met-Leu-Phe was used as a positive control and checkerboard analysis was used to distinguish chemotaxis from chemokinesis. Inhibition of chemotaxis was tested by peptide competition, blocking antibodies and galactosugar-mediated dissociation of the 67-kD EBP.. AAA extracts stimulated a concentration-dependent increase in monocyte migration that reached up to 24% of the maximal effect induced by N -formyl-Met-Leu-Phe. Checkerboard analysis demonstrated that AAA extracts stimulated chemotaxis without a chemokinetic effect. AAA-derived chemotactic activity was eliminated by competition with Val-Gly-Val-Arg-Pro-Gly (VGVAPG), a repetitive peptide found in human elastin that binds to cellular elastin receptors, and decreased nearly 40% in the presence of BA-4, an antielastin monoclonal antibody that can block EDP-mediated chemotactic activity. Monocyte chemotaxis in response to both VGVAPG and AAA extracts was abolished in the presence of lactose, a galactosugar that specifically dissociates the 67-kD EBP, but it was unaffected by either glucose, fructose, or mannose.. These findings indicate that soluble EDPs released within human AAA tissue can subsequently attract mononuclear phagocytes through ligand-receptor interactions with the 67-kD EBP, thereby providing a plausible molecular mechanism to explain the inflammatory response that accompanies aneurysmal degeneration. Better understanding of factors regulating inflammatory cell recruitment may lead to novel forms of therapy for early stages of aneurysmal degeneration.

Topics: Antibodies; Aortic Aneurysm, Abdominal; Chemotaxis, Leukocyte; Elastin; Humans; Immunoglobulin G; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Oligopeptides; Receptors, Cell Surface

Vessels remodel to compensate for increases in blood flow/pressure. The chronic exposure of blood vessels to increased flow and circulatory redox-homocysteine may injure vascular endothelium and disrupt elastic laminae. In order to understand the role of extracellular matrix (ECM) degradation in vascular structure and function, we isolated human vascular smooth muscle cells (VSMC) from normal and injured coronary arteries. The apparently normal vessels were isolated from explanted human hearts. The vessels were injured by inserting a blade into the lumen of the vessel, which damages the inner elastic laminae in the vessel wall and polarizes the VSMC by producing a pseudopodial phenotypic shift in VSMC. This shift is characteristic of migratory, invasive, and contractile nature of VSMC. We measured extracellular matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA), tissue inhibitor of metalloproteinase (TIMP), and collagen I expression in VSMC by specific substrate zymography and Northern blot analyses. The injured and elastin peptide, val-gly-val-ala-pro-gly, treated VSMC synthesized active MMPs and reduced expression of TIMP. The level of tPA and collagen type I was induced in the injured, invasive VSMC and in the val-gly-val-ala-pro-gly treated cells. To demonstrate the angiogenic role of elastin peptide to VSMC we performed in vitro organ culture with rings from normal coronary artery. After 3 days in culture the vascular rings in the collagen gel containing elastin peptide elaborated MMP activity and sprouted and grew. The results suggest that val-gly-val-ala-pro-gly peptide generated at the site of proteolysis during vascular injury may have angiogenic activity.

Topics: Amino Acid Sequence; Chemotaxis; Collagen; Coronary Vessels; Elastin; Extracellular Matrix; Humans; In Vitro Techniques; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Muscle, Smooth, Vascular; Neovascularization, Pathologic; Oligopeptides; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-1; Tissue Plasminogen Activator

Infusion of the abdominal aorta with pancreatic elastase induces aneurysms in a rat model (Anidjar/Dobrin). Because elastolysis liberates elastin degradation products (EDPs), the present experiment was carried out to test the hypothesis that an EDP alone could induce features of aneurysm disease.. The EDP val/gly/val/ala/pro/gly (VGVAPG), elastase, or saline solution was infused into infrarenal aorta (n = 4/group). After 1 week aortic diameters were measured, and the tissues were prepared for histologic examination. Adventitial capillaries (vessels per high-power field) were counted over a standardized preparation of aorta. Wall thickness was measured by means of computer-aided planimetry.. There was an increase of greater than 100-fold in mean vessels per high-power field in aortas receiving VGVAPG or elastase versus saline controls (4.10 +/- 0.68 SEM or 4.48 +/- 0.49 SEM versus 0.03 +/- 0.03 SEM, respectively, p < 0.05). The VGVAPG-perfused group had a 26% +/- 4% SEM increase in diameter from baseline that was statistically significant (p < 0.01), but the aortas did not reach aneurysmal dimensions.. Although no aneurysms occurred at 1 week after the infusion of EDP, the results demonstrate that the EDP VGVAPG can induce a characteristic feature of aneurysm disease. The model permits study of the earliest stages of experimental aneurysm formation and raises interesting questions regarding the role of the vasa vasorum in this pathologic process.

Topics: Animals; Aortic Aneurysm, Abdominal; Disease Models, Animal; Elastin; Muscle, Smooth, Vascular; Neovascularization, Pathologic; Oligopeptides; Rats; Rats, Wistar

Topics: Animals; Aortic Aneurysm, Abdominal; Disease Models, Animal; Elastin; Neovascularization, Pathologic; Oligopeptides; Rats; Rats, Wistar; Vasa Vasorum

Synthetic elastin peptides, VPGVG or its polymer (VPGVG)n, enhanced the proliferation of smooth muscle cells 1.5-fold during 48 h treatment at the concentrations over 10(-6) M or 1.0 microgram/ml, respectively. Monomeric and polymeric VPGVG sequences reduced elastin synthesis and its mRNA level to one-third and one-half of control respectively under the conditions in which the proliferation of cells were enhanced, but did not change collagen synthesis as measured by bacterial collagenase digestion. The elastin-specific autoregulation by elastin fragments may reflect the feedback regulation of elastin expression which may play an essential role in elastin metabolism under the normal and diseased conditions.

Topics: Amino Acid Sequence; Animals; Cell Division; Cells, Cultured; Chickens; Collagen; Elastin; Homeostasis; Molecular Sequence Data; Muscle, Smooth, Vascular; Oligopeptides; RNA, Messenger

Extracellular matrix proteins and their proteolytic products have been shown to modulate cell motility. We have found that certain tumor cells display a chemotactic response to degradation products of the matrix protein elastin, and to an elastin-derived peptide, VGVAPG. The hexapeptide VGVAPG is a particularly potent chemotaxin for lung-colonizing Lewis lung carcinoma cells (line M27), with 5 nM VGVAPG eliciting maximal chemotactic response when assayed in 48-microwell chemotaxis chambers. Binding of the elastin-derived peptide to M27 cells was studied using a tyrosinated analog (Y-VGVAPG) to allow iodination. Scatchard analysis of [125I]Y-VGVAPG binding to viable M27 tumor cells at both 37 and 4 degrees C indicates the presence of a single class of high affinity binding sites. The dissociation constant obtained from these studies (2.7 X 10(-9) M) is equivalent to the concentration of VGVAPG required for chemotactic activity. The receptor molecule was identified as an Mr 59,000 species by covalent cross-linking of the radiolabeled ligand to the M27 tumor cell surface and subsequent analysis of the cross-linked material by electrophoresis and size-exclusion high performance liquid chromatography. These results suggest that M27 tumor cell chemotaxis to VGVAPG is initiated by high affinity binding of the peptide to a distinct cell surface receptor.

Topics: Animals; Chemotactic Factors; Cross-Linking Reagents; Elastin; Lung Neoplasms; Molecular Weight; Neoplasm Proteins; Oligopeptides; Receptors, Cell Surface; Tumor Cells, Cultured

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.

Topics: Animals; Cattle; Chemotactic Factors; Chemotaxis; Elastin; Fetus; Fibroblasts; Humans; Indicators and Reagents; Ligaments; Monocytes; Oligopeptides