Laurent Debelle

Conformational Dependence of Collagenase (Matrix Metalloproteinase-1) Up-regulation by Elastin Peptides in Cultured Fibroblasts

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a “principal” VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII β-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.

The structures of elastins and their function

Elastin structures and their significance towards elastic recoil properties have been reviewed. Starting from the initial hypothesis that elastin conformation is conditioned by that of its monomer, the structure of tropoelastin was first described using theoretical and experimental methods and a beta class folding type was evidenced for the isolated unbound tropoelastin molecules. The structure of elastin in the solid state was consistent with that of its monomer and consequently, fibrous elastin appeared constituted of globular tropoelastin molecules. Finally, theoretical and experimental considerations have led us to the conclusion that the functional form of the elastomer, water swollen elastin, could be a triphasic system comprising the protein chains, hydration water and solvent water. Following this description, the dynamic structural equilibria occurring within elastin hydrophobic domains and the plasticizing effect of water could explain elastin elasticity, in keeping with a classical entropic mechanism.

BOVINE ELASTIN AND KAPPA -ELASTIN SECONDARY STRUCTURE DETERMINATION BY OPTICAL SPECTROSCOPIES

Elastin is the macromolecular polymer of tropoelastin molecules responsible for the elastic properties of tissues. The understanding of its specific elasticity is uncertain because its structure is still unknown. Here, we report the first experimental quantitative determination of bovine elastin secondary structures as well as those of its corresponding soluble κ-elastin. Using circular dichroism and Fourier transform infrared and near infrared Fourier transform Raman spectroscopic data, we estimated the secondary structure contents of elastin to be ∼10% α-helices, ∼45% β-sheets, and ∼45% undefined conformations. These values were very close to those we had previously determined for the free monomeric tropoelastin molecule, suggesting thus that elastin would be constituted of a closely packed assembly of globular β structural class tropoelastin molecules cross-linked to form the elastic network (liquid drop model of elastin architecture). The presence of a strong hydration shell is demonstrated for elastin, and its possible contribution to elasticity is discussed.

Protein carbamylation is a hallmark of aging

Significance Human longevity is increasing worldwide because of the advances in scientific knowledge and patient care, which leads to the frequent development of age-related pathologies. Aging remains an elusive process associated with genetic and environmental features, and better understanding would promote sustained wellbeing. We show here for the first time, to our knowledge, that carbamylation, a nonenzymatic posttranslational modification of proteins characterized by the spontaneous binding of isocyanic acid mainly derived from urea, is highly associated with aging and life expectancy in three mammalian species. Carbamylation promotes molecular aging through alteration of protein functions, especially long-lived extracellular matrix proteins. Tissue accumulation of carbamylated proteins may be considered a general hallmark of aging, enabling us to establish a link between cumulative metabolic alterations and age-related complications. Aging is a progressive process determined by genetic and acquired factors. Among the latter are the chemical reactions referred to as nonenzymatic posttranslational modifications (NEPTMs), such as glycoxidation, which are responsible for protein molecular aging. Carbamylation is a more recently described NEPTM that is caused by the nonenzymatic binding of isocyanate derived from urea dissociation or myeloperoxidase-mediated catabolism of thiocyanate to free amino groups of proteins. This modification is considered an adverse reaction, because it induces alterations of protein and cell properties. It has been shown that carbamylated proteins increase in plasma and tissues during chronic kidney disease and are associated with deleterious clinical outcomes, but nothing is known to date about tissue protein carbamylation during aging. To address this issue, we evaluated homocitrulline rate, the most characteristic carbamylation-derived product (CDP), over time in skin of mammalian species with different life expectancies. Our results show that carbamylation occurs throughout the whole lifespan and leads to tissue accumulation of carbamylated proteins. Because of their remarkably long half-life, matrix proteins, like type I collagen and elastin, are preferential targets. Interestingly, the accumulation rate of CDPs is inversely correlated with longevity, suggesting the occurrence of still unidentified protective mechanisms. In addition, homocitrulline accumulates more intensely than carboxymethyl-lysine, one of the major advanced glycation end products, suggesting the prominent role of carbamylation over glycoxidation reactions in age-related tissue alterations. Thus, protein carbamylation may be considered a hallmark of aging in mammalian species that may significantly contribute in the structural and functional tissue damages encountered during aging.

Elastin fragmentation and atherosclerosis progression: The elastokine concept

Atherosclerosis is a progressive multifaceted inflammatory disease affecting large- and medium-sized arteries. Typical feature of this disease is the formation and build-up of atherosclerotic plaques characterized by vascular extracellular matrix degradation and remodeling. Many studies have documented degradation of native elastin, the main extracellular matrix protein responsible for resilience and elasticity of arteries, by local release of elastases, leading to the production of elastin-derived peptides (EDP). These peptides have been proposed to actively participate in the progression of the disease by accelerating different biological processes, such as LDL oxidation and calcification of the vascular wall. These pathophysiological effects are mediated by the binding of EDP on a peculiar heterotrimeric receptor named elastin receptor complex (ERC). In this article, we review the contribution of elastin in biological processes involved in atherosclerosis progression from its initial elastase-driven degradation to its ultimate cellular effects. Finally, we discuss the ERC and its derived signaling pathways as promising therapeutic targets.

The elastin peptides-mediated induction of pro-collagenase-1 production by human fibroblasts involves activation of MEK/ERK pathway via PKA- and PI3K-dependent signaling

Elastin peptides, such as κ‐elastin (kE), bind to the elastin receptor at the cell surface of human dermal fibroblasts and stimulate collagenase‐1 expression at the gene and protein levels. Using specific inhibitors and phosphospecific antibodies, we show here that the binding of elastin peptides to their receptor activates the extracellular signal‐regulated kinase (ERK) pathway; this activation is essential for the induction of pro‐collagenase‐1 production. Moreover, protein kinase A (PKA) and phosphatidylinositol 3‐kinase (PI3K) signaling were found to participate in ERK activation. Concomitantly, we demonstrate that stimulation by elastin peptides leads to enhanced DNA binding of activator protein‐1 (AP‐1). Our data indicate that the up‐regulation of collagenase‐1 following treatment of fibroblasts with elastin peptides results from a cross‐talk between PKA, PI3K and the ERK signaling pathways and that this regulation is accompanied by activation of AP‐1 transcription factors.

Elastin-Derived Peptides Are New Regulators of Insulin Resistance Development in Mice

Although it has long been established that the extracellular matrix acts as a mechanical support, its degradation products, which mainly accumulate during aging, have also been demonstrated to play an important role in cell physiology and the development of cardiovascular and metabolic diseases. In the current study, we show that elastin-derived peptides (EDPs) may be involved in the development of insulin resistance (IRES) in mice. In chow-fed mice, acute or chronic intravenous injections of EDPs induced hyperglycemic effects associated with glucose uptake reduction and IRES in skeletal muscle, liver, and adipose tissue. Based on in vivo, in vitro, and in silico approaches, we propose that this IRES is due to interaction between the insulin receptor (IR) and the neuraminidase-1 subunit of the elastin receptor complex triggered by EDPs. This interplay was correlated with decreased sialic acid levels on the β-chain of the IR and reduction of IR signaling. In conclusion, this is the first study to demonstrate that EDPs, which mainly accumulate with aging, may be involved in the insidious development of IRES.

Elastin-derived peptides potentiate atherosclerosis through the immune Neu1-PI3Kγ pathway.

AIMS Elastin is degraded during vascular ageing and its products, elastin-derived peptides (EP), are present in the human blood circulation. EP binds to the elastin receptor complex (ERC) at the cell surface, composed of elastin-binding protein (EBP), a cathepsin A and a neuraminidase 1. Some in vitro functions have clearly been attributed to this binding, but the in vivo implications for arterial diseases have never been clearly investigated. METHODS AND RESULTS Here, we demonstrate that chronic doses of EP injected into mouse models of atherosclerosis increase atherosclerotic plaque size formation. Similar effects were observed following an injection of a VGVAPG peptide, suggesting that the ERC mediates these effects. The absence of phosphoinositide 3-kinase γ (PI3Kγ) in bone marrow-derived cells prevented EP-induced atherosclerosis development, demonstrating that PI3Kγ drive EP-induced arterial lesions. Accordingly, in vitro studies showed that PI3Kγ was required for EP-induced monocyte migration and ROS production and that this effect was dependent upon neuraminidase activity. Finally, we showed that degradation of elastic lamellae in LDLR(-/-) mice fed an atherogenic diet correlated with atherosclerotic plaque formation. At the same time, the absence of the cathepsin A-neuraminidase 1 complex in cells of the haematopoietic lineage abolished atheroma plaque size progression and decreased leucocytes infiltration, clearly demonstrating the role of this complex in atherogenesis and suggesting the involvement of endogenous EP. CONCLUSION Altogether, this work identifies EP as an enhancer of atherogenesis and defines the Neuraminidase 1/PI3Kγ signalling pathway as a key mediator of this function in vitro and in vivo.

Cumulative influence of elastin peptides and plasminogen on matrix metalloproteinase activation and type I collagen invasion by HT-1080 fibrosarcoma cells

HT-1080 fibrosarcoma cells express at their plasma membrane the elastin-binding protein (EBP). Occupancy of EBP by elastin fragments, tropoelastin or XGVAPG peptides was found to trigger procollagenase-1 (proMMP-1) overproduction by HT-1080 cells at the protein and enzyme levels. RT-PCR analysis indicated that elastin peptides did not modify the MMP-1 mRNA steady state levels, suggesting the involvement of a post-transcriptional mechanism. We previously reported that binding of elastin peptides to EBP induced other matrix metalloproteinases (MMP-2 and MT1-MMP) expression [20]. Since those peptides were here found to also accelerate the secretion of urokinase from HT-1080 cells, culture medium was supplemented with plasminogen together with elastin peptides at aims to induce or potentiate MMPs activation cascades. In such conditions, plasmin activity was generated and exacerbate proMMP-1 and proMMP-2 activation. As a consequence, elastin peptides and plasminogen-treated HT-1080 cells displayed a significant type I collagen matrix invasive capacity.

Matrix ageing and vascular impacts: focus on elastin fragmentation

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and represent a major problem of public health. Over the years, life expectancy has considerably increased throughout the world, and the prevalence of CVD is inevitably rising with the growing ageing of the population. The normal process of ageing is associated with progressive deterioration in structure and function of the vasculature, commonly called vascular ageing. At the vascular level, extracellular matrix (ECM) ageing leads to molecular alterations in long half-life proteins, such as elastin and collagen, and have critical effects on vascular diseases. This review highlights ECM alterations occurring during vascular ageing with a specific focus on elastin fragmentation and also the contribution of elastin-derived peptides (EDP) in age-related vascular complications. Moreover, current and new pharmacological strategies aiming at minimizing elastin degradation, EDP generation, and associated biological effects are discussed. These strategies may be of major relevance for preventing and/or delaying vascular ageing and its complications.