PEPTIDE MODULATORS OF THE INTERACTION BETWEEN HUMAN C-PEPTIDE AND HUMAN ELASTIN RECEPTOR FOR THERAPEUTIC USE

Abstract

The present disclosure shows that inflammation in metabolic syndrome is augmented by and hitherto overlooked lock- and-key activation of the elastin receptor, a protein involved in vascular (blood vessel) inflammation and elastin repair, with the C-peptide, a small protein that is produced in a 1:1 ratio alongside with widely known insulin. The elastin receptor is the lock that is activated by a key motif of amino acids (PG-domain) found in C-peptide and in breakdown products (PG-domain-fragments) thereof. Until now, no one has ever discovered this lock-and-key interaction between the two, now providing novel development of novel peptides for treatment of metabolic syndrome, exploiting the finding that not only the normal keys of the elastin receptor (elastin peptides), but also the C-peptide, a peptide we produce together with insulin every time glucose rises in our blood after a meal, interacts in a lock-and-key mode with the elastin receptor.

Claims

1. An isolated or synthetic peptide for use in treatment of human disease, wherein the peptide comprises: at least one peptide motif able to modulate binding of human C-peptide to a human elastin receptor.

2. The peptide according to claim 1, wherein the peptide has at least one human elastin receptor binding motif GxxPG and has at least one amino acid Q, wherein G represents a one-letter code for the amino acid glycine, P for the amino acid proline, Q for the amino acid glutamine and x for any amino acid, and wherein the peptide consists of 5-30 amino acids.

3. A peptide according to claim 2 consisting of 5-20 amino acids.

4. A peptide according to claim 2 consisting of 5-15 amino acids.

5. A peptide according to claim 2 consisting of 5-12 amino acids.

6. A peptide according to claim 2 consisting of 5-9 amino acids.

7. A method of treating a subject for inflammation, the method comprising: administering to the subject the peptide according to claim 2 to treat inflammation.

8. A method of treating a subject for type 1 diabetes and/or end-stage type 2 diabetes, the method comprising: administering to the subject the peptide according to claim 2 to treat type 1 diabetes and/or end-stage type 2 diabetes.

9. A method of treating a subject for micro-vascular complications, the method comprising: administering to the subject the peptide according to claim 2 for treatment of micro-vascular complications.

10. A method of treating a subject for micro-vascular complications in type 1 diabetes and/or end-stage type 2 diabetes, the method comprising: administering to the subject the peptide according to claim 2 for treatment of micro-vascular complications in type 1 diabetes and/or end-stage type 2 diabetes.

11. A peptide according to claim 2 able to combine with a human elastin receptor on a cell and initiating the same physiological activity typically produced by the binding of human C-peptide to the human elastin receptor.

12. The peptide according to claim 1 having at least a motif QDEA (SEQ ID NO:31), wherein the peptide inhibits the binding of human C-peptide to a human elastin receptor and reduces the physiological activity of human C-peptide, and wherein the peptide consists of 4-40 amino acids.

13. A peptide according to claim 12 consisting of 4-20 amino acids.

14. A peptide according to claim 12 consisting of 4-15 amino acids.

15. A peptide according to claim 12 consisting of 4-12 amino acids.

16. A peptide according to claim 12 consisting of 4-9 amino acids.

17. A method of treating a subject for human insulin resistance, the method comprising: administering the peptide according to claim 12 for use in to the subject to treat human insulin resistance.

18. A method of treating a subject for human dyslipidemia, the method comprising: administering the peptide according to claim 12 to the subject to treat human dyslipidemia.

19. A method of treating a subject for human hypertension, the method comprising: administering the peptide according to claim 12 to the subject to treat human hypertension.

20. A method of treating a subject for human macrovascular complications, the method comprising: administering to the subject the peptide according to claim 12 to treat human macrovascular complications.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] FIG. 1: Ido et al. (Science, 1997 Jul. 25;277(5325):563-6) show human C-peptide's mid-portion SEQ ID NO:8 (GGGPGAG) to normalize glucose-induced vascular dysfunction. However, finding reverse (retro) and all-D-amino acid (enantio or inverso) C-peptides equipotent to native C-peptide, they conclude the activity of SEQ ID NO:8 (GGGPGAG) to be not mediated by a receptor, thereby teaching away from a receptor for C-peptide. This specification shows that the opposite is a case. All peptides with the mid-portion motif GxxP (SEQ ID NO:38 (GGGP) in human and rat C-peptide, SEQ ID NO:39 (GAGP) in reverse C-peptide and stereochemically equivalent PGAG in D-form C-peptide) normalize vascular dysfunction while none of the peptides without that motif do. Thus, the EBP-binding motif GxxP in SEQ ID NO:8 (GGGPGAG) is both necessary and sufficient to normalize vascular dysfunction. Hence, human C-peptide can be considered a ligand of the EBP that modulates vascular repair via the ERC. Efficacy is expressed as an average percent of the effect of 100 nM human C-peptide. Significantly different for 30 mM glucose: *P<0.05 GxxP motifs in peptides bind to the elastin receptor when allowing for a close to a type VIII beta-turn confirmation, a condition considered always to be met by the motif xGxxPG. All peptides having the GxxP motif (SEQ ID NO:38 (GGGP) or SEQ ID NO:39 (GAGP), or all-D PGAG which is stereometrically equivalent to all-L-SEQ ID NO:39 (GAGP)) show significant normalization of vascular dysfunction while none of the peptides without the motif show significant effects, illustrating that the elastin receptor binding motif GxxP is both necessary and sufficient to elicit the biological activity of C-peptide. Figure adapted from Ido Y, et al. Prevention of vascular and neural dysfunction in diabetic rats by C-peptide. Science 1997; 277: 563-66.

[0108] FIG. 2: Summarized description of pathological vascular effects of GxxP-peptide deficiency versus GxxP-excess

[0109] FIG. 3: Various GxxP hexa-peptides were docked in the peptide-binding site of the elastin binding protein (EBP) using Vina/Autodock and PyMOL (1, 2, 3). The binding conformation of each peptide was chosen from the top 20 best scoring poses. A homology model of EBP (4) was used as receptor in the docking procedure. Peptides tested were:

TABLE-US-00001 SEQ ID NO: 41 (VGVAPG) (prototype GxxP-peptide ligand of EBP (4)) SEQ ID NO: 34 (LGGGPG) (selected from human C-peptide (5)) SEQ ID NO: 43 (QGQLPG) (immunomodulatory peptide provided herein) SEQ ID NO: 44 (PGAYPG) (selected from human Galectin-3 (6)) SEQ ID NO: 45 (QGVLPA) (selected from loop 2 of human beta-hCG (7))

[0110] References Trott, O & Olson, A J (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, J Comp Chem 31: 455-461

[0111] 2 Seeliger, D & de Groot, B L (2010) Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput-Aided Mol Des 24 :417-422.

[0112] 3 www.pymol.org

[0113] 4 Blanchevoye, C et al. (2013) Interaction between the elastin peptide VGVAPG and human elastin binding protein, J Biol Chem 288:1317-28.

[0114] 5 Ido, Y et al. (1997) Prevention of vascular and neural dysfunction in diabetic rats by C-peptide Science 277:563-6

[0115] 6 De Boer, R et al. (2011) Plasma Galectin-3 Is Associated with Near-Term Rehospitalization in Heart Failure: A Meta-Analysis Journal of Cardiac Failure Vol 17, Issue 8, S93

[0116] 7 Khan, NA et al. (2010) Mitigation of septic shock in mice and rhesus monkeys by human chorionic gonadotrophin-related peptides, Clin Exp Immunol 160:465-478

[0117] Similarly, All-D-amino acid peptide GPGAG fits in the model of EBP designed for docking prototype elastin peptide SEQ ID NO:41 (VGVAPG) as well. Also, L-amino acid peptides SEQ ID NO:40 (GGGPG) and SEQ ID NO:46 (GAGPG) fit the model as well. EBP-associated bioactivity is considered to depend on whether the GXXP-peptide can adapt to a type VIII beta-turn confirmation at the proline (P).

[0118] FIG. 4: Overview of pathophysiological pathways in metabolic syndrome, atherosclerosis and diabetes

[0119] FIG. 5: This new perspective sheds new light on diseases that are associated with atherosclerosis and insulin resistance (e.g., cardiovascular disease, stroke, peripheral arterial disease, dementia, chronic kidney disease and beta cell failure leading to diabetes). It not only ties together sugar and smoking but also various other co-existing risk factors that result from diet (excess intake of refined starches or of processed meat products with excess elastin), or lifestyle (exposure to smog or lack of exercise). The disclosure puts elastin receptor activation by C-peptide forward as cause of insulin resistance, hypertension and chronic-low inflammation or blood vessel over repair in metabolic syndrome and ties this syndrome together with other conditions of insulin resistance, such as COPD due to smoking and exposure to fine particular matter, where elastin-derived peptides may activate the elastin receptor to cause insulin resistance over-repair.

DETAILED DESCRIPTION

[0120] Human C-peptide is found a ligand of the human elastin receptor. [0121] Elastin receptor shall mean a chemical group or molecule on the cell surface or in the cell interior that has an affinity for a peptide having an amino acid motif GxxP, wherein G represents the one-letter code for the amino acid glycine, P for the amino acid proline and x for any amino acid, the amino acid following P preferably allowing for a type VIII-beta turn, a condition that is met when P is C-terminally followed by a G, the elastin receptor typically represented in humans by the elastin binding protein known in the publicly accessible database Uniprot as GLB1—isoform 2 under identifier: PI6278-2. [0122] C-peptide shall mean a peptide typically produced by beta-cells in the pancreas together with insulin, the C-peptide represented in humans by the peptide known in the publicly accessible database Uniprot as INS-isoform —1 under identifier: P01308-1, position 57-87.

[0123] Human C-peptide, connecting immature insulin chains A and B and secreted in a 1:1 ratio with mature insulin into the portal circulation, has traditionally been considered inert, despite ever increasing evidence of its biological activity. I show that in dietary excess, excess serum C-peptide leads to chronic-low grade inflammation, insulin resistance and hypertension and is causal to metabolic syndrome. I show C-peptide carrying a hitherto unrecognized xGxxPG motif specific for binding of elastin peptides to the elastin receptor, the receptor fulfilling various roles in tissue inflammation and tissue repair. Recent findings show this receptor to promote insulin resistance, dislipidaemia, hypertension and atherogenesis, all characteristic of metabolic syndrome. This finding takes C-peptide into the limelight, tying in metabolic syndrome with other conditions of insulin resistance, such as COPD, when circulating elastin-derived peptides may combine with C-peptide to stimulate elastin receptor-mediated insulin resistance and inflammation.

Insulin Resistance

[0124] Insulin resistance (IR) is central to metabolic syndrome.sup.1,2. It occupies a crucial place in the aetiology of chronic inflammatory, lifestyle-, diet- or age-related, conditions as atherosclerotic cardiovascular disease and diabetes type 2. Hallmarks of metabolic syndrome are IR, hypertension, dyslipidemia, hyperinsulinemia, and impaired glucose tolerance. Uncertainties exist to the cause of IR. Simplified, the main view.sup.1,3 holds chronic-low grade inflammation to drive IR and subsequent hyperinsulinemia; a seemingly opposed view.sup.2 holds increased hyperinsulinemia to drive IR and subsequent inflammation.

[0125] In humans in dietary excess, excess serum C-peptide causes chronic-low grade inflammation as well as IR and hypertension leading to metabolic syndrome, C-peptide being hitherto unrecognized as a ligand for the elastin receptor.

The Elastin Receptor

[0126] The human elastin receptor.sup.4-6 is involved in chemotaxis of leukocytes and activation of matrix-metallo-proteinases, in endothelial cell migration and angiogenesis and in proliferation of fibroblasts and vascular smooth-muscle cells. The receptor is activated by (proteolytic) fragments of extracellular matrix in granulating tissue after tissue injury or inflammation, fulfilling handyman jobs toward tissue repair.

[0127] The receptor consists of an alternatively spliced variant of human beta-galactosidase. It binds to a hexapeptide x-Gly-x-x-Pro-Gly (xGxxPG) motif in (proteolytic fragments of) extracellular matrix proteins such as elastin and fibrillin-1.sup.4. The best-known representative of the motif is hexapeptide SEQ ID NO:41 (VGVAPG) found in (tropo)elastin, but many other biologically active peptides conforming to the signature sequence xGxxPG, generally called elastin peptides, have been reported as agonist.sup.4,5. A minimally essential sequence for biological activity is GxxP, with the peptide at P adopting a type VIII beta-turn.sup.5. V14 peptide (SEQ ID NO:131 (VVGSPSAQDEASPL)) corresponding to the binding site of the receptor, is used to antagonize elastin peptide binding.sup.6.

[0128] The elastin receptor forms a complex with neuraminidase (Neu-1) and protective protein-cathepsin A (PPCA) on the cell surface.sup.4. After binding to its ligand, the complex internalizes to endosomal compartments in the cell and triggers numerous cellular responses. In mice, elastin peptides potentiate atherosclerosis through Neu-1.sup.7 and regulate IR.sup.8 due to an interaction between Neu-1 and the insulin receptor. Moreover, in mice, PPCA is required for assembly of elastic fibers and inactivation of endothelin-1, impaired activation of endothelin-1 resulting in hypertension.sup.9.

C-Peptide

[0129] Herein recognized, human C-peptide (.sub.1SEQ ID NO:1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ)31) contains the xGxxPG motif (underlined), surprisingly identifying it as a ligand for the elastin receptor. The far reaching implications of that find are discussed below. Classically, C-peptide connects the A- and B-chain of insulin in the pre-proinsulin produced in pancreatic beta-cells from the insulin gene and facilitates folding and binding of chains A and B. After processing, mature insulin and C-peptide are secreted into the portal circulation. Be it under dietary frugality or excess, insulin and C-peptide are produced and secreted in equimolar concentrations. However, C-peptide's plasma half-life of 30 min versus insulin's half-life of ˜4 min.sup.10 causes dietary excess to maintain persistently higher levels of circulating C-peptide than of insulin. The traditional view holds circulating C-peptide essentially inert and, because of its longer half-life, particularly useful as a surrogate marker of insulin release. However, accumulating evidence points at biological functions for C-peptide.sup.11-14. Excess C-peptide in mice experimentally elicits inflammatory effects in vasculature and around glomeruli and C-peptide is found deposited in atherosclerotic lesions of patients.sup.15. Fasting serum C-peptide levels significantly relate to hazards of cardiovascular and overall death in non-diabetic adults.sup.16. These recent findings establish pathophysiological importance to C-peptide in its own right.

Pentapeptide .SUB.27 .SEQ ID NO:6 (EGSLQ) .SUB.31

[0130] A first concerns is the pentapeptide .sub.27 SEQ ID NO:6 (EGSLQ) .sub.31, corresponding to the C-terminal five residues of human C-peptide that mimics several effects of the full-length peptide. The pentapeptide displaces cell membrane-bound C-peptide, increases intracellular Ca(2+) and stimulates MAP kinase signaling pathways and Na(+),K(+)-ATPase.sup.8. Of note, the glutamate at position 27 was shown essential to activation of alpha-enolase by C-peptide.sup.14, hinting that the C-terminal pentapeptide site may be involved in interaction of C-peptide with alpha-enolase.

Midportion .SUB.13 .SEQ ID NO:8 (GGGPGAG) .SUB.19

[0131] A second site, and main focus of this disclosure, the mid-portion of human C-peptide 13 SEQ ID NO:8 (GGGPGAG) .sub.19, was detected when structural features of C-peptide critical for mediating its effects on vascular dysfunction were investigated in a skin chamber granulation tissue model in rats .sup.14. .sub.13 SEQ ID NO:8 (GGGPGAG) .sub.19 was shown to be central to C-peptide's biological activity. However, as synthetic reverse sequence (retro) and all-D-amino acid (enantio) C-peptides were found equipotent to native C-peptide, it was concluded then .sup.14 that the effects of this mid-portion must rely on non-chiral interactions, thereby teaching away from any possible stereospecific receptor binding to .sub.13 SEQ ID NO:8 (GGGPGAG) .sub.19. This teaching has since then dominated the literature on C-peptide. However, I here conclude that an xGxxPG elastin receptor binding motif is overlapping with C-peptide's bioactive mid-portion (iSEQ ID NO:1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ).sub.31), distinctly associated with effects on vascular function in granulation tissue, identifying C-peptide as a biologically active ligand of the elastin receptor.

C-Peptide Receptor

[0132] Until now, a distinct C-peptide receptor is unknown. A binding study .sup.12 of C-peptide to human cell membranes indicates the existence of at least two C-peptide/receptor complexes, one with high affinity and low mobility and one with low affinity and high mobility and recent studies suggest alpha-enolase .sup.17, a cell surface receptor of plasminogen, or GPR146 .sup.18, associated with dyslipidemia .sup.19, as possible receptor candidates for C-peptide. Biologically active sites in C-peptide. At least two biologically active sites have been identified in the C-peptide.

Non-chirality is revoked

[0133] Surprisingly, studying reference 14 anew, the xGxxPG motif is also present in the biologically active retro C-peptide (.sub.1 SEQ ID NO:136 (QLSGELALPQLSGAGPGGGLEVQGVQLDEAE) 31). Also, the biologically active enantio C-peptide (D--.sub.1EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ.sub.31) carries the motif, being hidden as the retro-enantio sequence D--GPGAGS; retro-enantio peptides being stereometrically nearly identical as their parent peptides, maintaining overall side-chain topology albeit for different N-terminal and C-terminal endings .sup.20. These observations revoke the teaching .sup.14 of non-chirality and instead allow for stereospecific binding of these peptides to a receptor recognizing the motif: the elastin receptor.

C-peptide is a species of the genus of elastin peptides.

[0134] Moreover, additional examples of fragments of the human C-peptide are provided herein and .sup.14, all bearing a mid-portion hexapeptide .sub.12 SEQ ID NO:34 (LGGGPG) .sub.17, that all prevented vascular dysfunction whereas other human C-peptide fragments, wherein the hexapeptide mid-portion was disrupted, were found not active. Rat C-peptide, comprising a hexapeptide (.sub.12 SEQ ID NO:134 (LGGGPE) .sub.17) GxxP motif (the P allowing a type VIII beta-turn required for biological activity .sup.5) was found active as well, whereas pig C-peptide (mid-portion .sub.12 SEQ ID NO:135 (LGGGLG) .sub.17) not containing the essential P in the elastin binding motif, was found inactive. Of note, all 11 C-peptide(fragments) with the GxxP motif prevented vascular dysfunction, whereas all 5 without the motif did not, showing that even fragments of circulating C-peptide may contribute to elastin receptor activation, as long as the GxxP motif and the type VIII beta-turn is present. Human C-peptide and its xGxxPG containing fragments may thus be considered an unexpected species of the genus of a larger class of peptides: elastin peptides capable of elastin receptor activation, whereby excess C-peptide may be meddling with elastin receptor mediated tissue repair, modulating chronic-low grade inflammation, IR and hypertension. Insulin resistance extends beyond metabolic syndrome. Based on the above, I pose that in humans in dietary excess and prone to develop metabolic syndrome excess C-peptide binds to the elastin receptor, eliciting at least three effects, chronic-low grade inflammation (rather to be seen as excess vascular repair activity), insulin resistance and hypertension. The finding ties together conditions seen with metabolic syndrome with conditions possibly caused by circulating elastin degradation products, such as COPD, caused by smoking or by exposure to fine particulate matter (smog), or by physiological conditions, such as pregnancy and growth; allowing for a cumulative pathology when both C-peptide and elastin-derived peptides are increased. This provides a substantial jump in our understanding of the causes of metabolic syndrome and other lifestyle- or age-related conditions of IR. Elastin peptide/elastin receptor binding has been demonstrated for synthetic peptides such as SEQ ID NO:41 (VGVAPG) and inhibited by antagonist V14 peptide .sup.4-6. It is provided to do these tests with synthetic human C-peptide variants or fragments, provided with or without the sequence GxxP, to study binding, including classical elastin receptor antagonist V14 peptide to study inhibition of binding. Similarly, one can do the C-peptide tests in a skin chamber granulation tissue model of vascular function .sup.14, or test synthetic, inducibly or constitutively expressed C-peptide in established models of atherosclerosis, Neu-1 mediated IR or PPCA mediated hypertension .sup.7-9. For example, in a classical Boyden chamber experiment, a 100% increase of migration of CD4+ immune cells in 1% serum medium was demonstrated in vitro by C-peptide at 10 nM which CD4-migration was then inhibited, antagonized and diminished by >50% by V14-peptide at 1.3 microM, specifically demonstrating reduction of C-peptide-specific biological activity by elastin receptor antagonist V14 peptide.

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[0155] Again, when we eat too much, we provide the beta-cells of our pancreas with continued glucose signaling to produce insulin, to harbor the ever excess glucose derived from our food in peripheral liver, muscle, and fat cells. When eating in excess, we demand ever increasing insulin production from our beta-cells, and therewith demand ever increasing production of C-peptide from our beta-cells, C-peptide and insulin being produced and excreted in equal amounts. As insulin has a typical half-life of about 3-4 minutes, conditions of excess insulin may be easily coped with. However C-peptide has a much longer half-life, typically of >30 minutes, and depositions of excess C-peptide (and of partly or whole unprocessed pro-insulin) will be formed around the rim of the beta-cells and the islets of Langerhans, and also in the vascular wall of our blood-vessels. Pericytes, smooth muscle cells, fibroblasts, adipose tissue cells, pancreatic stellate cells, and others, together with endothelial cells, and possibly circulating leucocytes, respond to binding of EBP to GxxPG bearing C-peptide, thereby causing matrix-metallo-proteinase (MMP) induced hydrolysis accompanied by interleukin-1-beta mediated proliferation and subsequent low-grade inflammatory activation, in and around beta-cells in the islets of Langerhans. IL-1-beta thus drives tissue inflammation that impacts on both beta-cells functional mass and subsequently may also drive insulin sensitivity in type-2 diabetes. Binding of EBP to C-peptide's GxxPG sequences may further facilitate shedding of EBP from cellular surfaces and increased presentation of the interleukin-I receptor, allowing for a continued interleukin-1-beta mediated proliferation and inflammatory activation wherever C-peptide deposits are present, again driving insulin sensitivity in type-2 diabetes. In a patient thus developing diabetes type-2 or metabolic syndrome damage to and destruction of the beta-cells in the pancreas is following excess C-peptide production by those cells. Phenomena commonly seen as insulin resistance are then often secondary to initial events in the pancreatic beta-cells and arise out of interaction of fibroblasts, smooth muscle cells pericytes and leucocytes with vascular or peripheral C-peptide overload.

[0156] C-peptide exerts chemotactic and bioactive effects via interaction of its GXXPG and XGXPG motif with the elastin-binding protein. It is taught in this disclosure that C-peptide exerts chemotactic and bioactive influence on monocytes, pericytes, smooth muscle cells, fibroblasts, and other cells via interaction with the elastin-binding protein (EBP) (Privitera et al., J. Biol. Chem. 1998; 273:6319-6326). This receptor recognizes Gly-X-X-Pro-Gly (XGXXPG) or X-Gly-X-Pro-Gly (XGXPG) motifs found in C-peptides, wherein X can be any amino acid, and preferably a hydrophobic amino acid. The identity of this receptor protein, commonly called the elastin binding protein (EBP), has been established as an enzymatically inactive, alternatively spliced variant of beta-galactosidase. EBP forms a complex with protective protein/cathepsin A (PPCA) and lysosomal sialidase (neuraminidase-1, Neu-1). As C-peptide is released in equimolar concentrations together with insulin, but has a much longer half-life, increased insulin excretion as result of increased food-intake will result in even higher C-peptide levels. This evokes an oversupply of C-peptide and deposits of the C-peptide are observed in the periphery of beta-cells and even in the (micro)vasculature where these C-peptide deposits evoke the low-grade inflammation so typical of what is commonly called insulin-resistance. GXXPG and XGXPG motif binding to the EBP induces interleukin-I beta mediated proliferation of vascular and connective tissue cells.

[0157] Pericytes, smooth muscle cells, fibroblasts, adipose tissue cells, pancreatic stellate cells, and others, together with endothelial cells, and circulating leucocytes, respond to binding of EBP to GxxPG or xGxPG bearing proteins and peptides by interleukin-1-beta mediated proliferation and low-grade inflammatory activation. Analysis of the human proteome shows that proteins with multiple GxxPG or xGxPG motifs are highly related to the extracellular matrix (ECM). Matrix proteins with multiple GxxPG or xGxPG sites include fibrillin-1, -2, and -3, elastin, fibronectin, laminin, and several tenascins and collagens.

[0158] Recent studies have shown that the Neu-1 component of the EBP complex is responsible for triggering cellular activation. EBP is present on many cell types, including various types of leukocytes, mesenchymal cells, vascular smooth muscle cells, and skin fibroblasts. Whereas the hexapeptide SEQ ID NO:41 (VGVAPG), a commonly repeated sequence in human elastin, is the most well-recognized ligand for this receptor, C-peptide, galectin-3, the amino acid sequence SEQ ID NO:25 (FRAAPLQGMLPGLLAPLRT) in human collagen 6 A3 (COL6A3, Uniprot identifier P1211) and the beta-2 loop of human choriogonadotropin (hCG) are now herein also recognized as also capable of binding to the EBP. In addition to SEQ ID NO:41 (VGVAPG), (all elastin-derived) peptides that follow the motif GXXPG or XGXPG (where X is a hydrophobic amino acid) display chemotaxis for monocytes in vitro (Bisaccia F, et al., Int. J. Pept. Protein Res. 1994 ;44:332-341, Castiglione Morelli M A, et al., J. Pept. Res. 1997 ;49:492-499). This is noteworthy, albeit not having been observed before, because primate C-peptide sequences do not contain the SEQ ID NO:41 (VGVAPG) sequence; however, primate C-peptide contain significant quantities of both GXXP, GXXPG and XGXPG motifs that show similar activities. C-peptide's GxxP, GxxPG and xGxPG interactions explain IL-1-beta involvement. C-peptide's GxxP, GxxPG and xGxPG interactions have until now been overlooked by those skilled in the art of diabetes or metabolic disorder research as well as by those skilled in the art of elastin peptide and extracellular matrix (ECM) research. This earlier unobserved fact explains the macrophage-predominant, IL-1-beta mediated chronic inflammatory disease process as seen in, for example, adipose tissue in patients suffering from diabetes type-2, it explains the intima thickening and smooth muscle cell proliferation seen in vessels of patients suffering from atherosclerosis, the direct insulitis and peri-islet inflammation around beta cells in the pancreas as seen in the early phases of diabetes, and many other disease manifestations of metabolic syndrome wherein the patients suffer from C-peptide overproduction and C-peptide deposits, likely as a consequence of over-eating. C-peptide's GxxP, GxxPG and xGxPG interactions also explain leucocyte involvement. In addition, IL-1-beta signaling results in the production of pro-inflammatory mediators that act in a feed-forward autocrine/paracrine manner in beta-cells and local innate immune cells to amplify these effects, amplified by the fact that circulating leucocytes show strong chemotaxis to GxxPG or xGxPG bearing proteins and peptides; again C-peptide will thus attract those cells to wherever C-peptide is present, and in situations of C-peptide overload or even C-peptide deposits, this will exacerbate disease. As indicated herein, the concept that C-peptide and degradation products thereof can drive a macrophage-predominant, chronic inflammatory disease process via its GxxPG and xGxPG motif is now elucidating the etiology of diabetes of all types and is applicable to all diseases that occur in vasculature-rich organs and tissues, including coronary artery disease, peripheral vascular disease, and aortic aneurysm.

[0159] By “peptide” the inventor includes not only molecules in which amino acid residues are joined by peptide (—CO—NH—) linkages, but also functionally equivalent molecules in which the peptide bond is reversed. Retro-inverse peptides are composed of D-amino acids assembled in a reverse order from that of the parent L-sequence, thus maintaining the overall topology of the native sequence. Such retro-inverso peptidomimetics may be made using methods known in the art, for example, such as those described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, and Carver et al. (1997) Biopolymers. 1997 Apr. 15; 41(5):569-90, incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al. and Carver et al. (1997) show that these pseudopeptides are useful. Retro-inverse peptides are much more resistant to proteolysis. Retro-inversion is a way of protecting peptide substances against proteolysis. It entails retro-inverting those peptide bonds most susceptible to enzymatic hydrolysis by inverting the direction of the peptide bonds. The “retro-inverso peptides” are structural isomers of the reference peptides and as such preserve their biological activity while being more resistant to enzymatic hydrolysis. A peptidomimetic is a small protein-like chain designed to mimic a peptide. They typically arise from modification of an existing peptide in order to alter the molecule's properties. For example, they may arise from modifications to change the molecule's stability or biological activity (only useful when the peptide's biological activity is chiral). Chemically synthesized peptides generally have free N- and C-termini. N-terminal acetylation and C-terminal amidation reduce the overall charge of a peptide; therefore, its overall solubility might decrease. However, the stability of the peptide could also be increased because the terminal acetylation/amidation generates a closer mimic of the native protein. These modifications might increase the biological activity of a peptide and are herein also provided.

Peptide Synthesis

[0160] Synthetic PG-domain or GxxP-type peptides such as SEQ ID NO:41 (VGVAPG), SEQ ID NO:138 (GVAPGV), SEQ ID NO:139 (VAPGVG), SEQ ID NO:140 (APGVGV), SEQ ID NO:141 (PGVGVA), SEQ ID NO:142 (GVGVAP), SEQ ID NO:60 (PGAIPG), SEQ ID NO:137 (LGTIPG), SEQ ID NO:32 (LGGGPGAG), SEQ ID NO:8 (GGGPGAG), SEQ ID NO:49 (GGGPGA), SEQ ID NO:38 (GGGP), SEQ ID NO:40 (GGGPG), SEQ ID NO:46 (GAGPG), SEQ ID NO:50 (GGGPE), SEQ ID NO:51 (GAIPG), SEQ ID NO:52 (GGVPG), SEQ ID NO:53 (GVAPG), SEQ ID NO:54 (YTTGKLPYGYGPGG), SEQ ID NO:55 (YGARPGVGVGIP), SEQ ID NO:56 (PGFGAVPGA), SEQ ID NO:57 (GVYPG), SEQ ID NO:58 (GFGPG), SEQ ID NO:59 (GVLPG), SEQ ID NO:51 (GAIPG), SEQ ID NO:60 (PGAIPG), SEQ ID NO:61 (PGAVGP), SEQ ID NO:62 (VGAMPG), SEQ ID NO:63 (VGSLPG), SEQ ID NO:64 (VGMAPG), SEQ ID NO:65 (VPGVG), SEQ ID NO:66 (IPGVG), SEQ ID NO:63 (VGSLPG), SEQ ID NO:41 (VGVAPG), SEQ ID NO:67 (VGVPG), SEQ ID NO:68 (AGAIPG), SEQ ID NO:69 (VPGV), SEQ ID NO:70 (LGITPG), SEQ ID NO:71 (GDNP), SEQ ID NO:72 (GAIP), SEQ ID NO:73 (GKVP), SEQ ID NO:74 (GVQY), SEQ ID NO:75 (GVLP), SEQ ID NO:76 (GVGP), SEQ ID NO:77 (GFGP), SEQ ID NO:78 (GGIP), SEQ ID NO:79 (GVAP), SEQ ID NO:80 (GIGP), SEQ ID NO:39 (GAGP), SEQ ID NO:81 (GGIPP), SEQ ID NO:82 (GQFP), SEQ ID NO:83 (GLSP), SEQ ID NO:84 (GPQP), SEQ ID NO:85 (GGPQP), SEQ ID NO:86 (GPQPG), SEQ ID NO:87 (GGPQPG), SEQ ID NO:88 (GIPP), SEQ ID NO:81 (GGIPP), SEQ ID NO:89 (GIPPA), SEQ ID NO:90 (GGIPPA), or retro-inverso variants thereof are synthesized according to classical solid phase synthesis. V14 peptide, a peptide reproducing the sequence of S-Gal interacting with elastin peptides bearing the PG-domain, in particular, the motif GxxP, is obtained from Neosystem (Strasbourg, France). Alternatively, V14 peptide and variants thereof are synthesised as described herein. Purity of the peptides is confirmed by high performance liquid chromatography and by fast atom bombardment mass spectrometry.

[0161] Traditionally, peptides are defined as molecules that consist of between 2 and 50 amino acids, whereas proteins are made up of 50 or more amino acids. In addition, peptides tend to be less well defined in structure than proteins and can adopt complex conformations known as secondary, tertiary, and quaternary structures. Functional distinctions may also be made between peptides and proteins. Peptides, however, may be subdivided into peptides that have few amino acids (e.g., 2 to 30-50), and polypeptides that have many amino acids (>50). Proteins are formed from one or more polypeptides joined together. Hence, proteins essentially are very large peptides. In fact, most researchers, as well as this disclosure, use the term peptide to refer specifically to peptides, or otherwise relatively short amino acid chains, with the term polypeptide being used to describe proteins, or chains of >50 or much more amino acids.

[0162] Treatment of cultured cells with C-peptide or fragments thereof.

[0163] Cells may be plated at a density of 450 per mm.sup.2 in a 24-well microplate or 32 mm diameter Petri dish and cultured for 2-4 days or in cultures as described above. On day 2 in culture, cells are treated with various C-peptides (preferably selected from Table 1) or peptide fragments thereof for 1 or 2 days. In an experiment, cells were treated with a combination of C-peptide (1 micro-M) or polyclonal anti-67 kDa elastin receptor antibody (anti-S-Gal antibody) (10 ng per ml) for 2 d. At the end of the treatment, cells may be trypsinized (0.25%) and the cell number determined with a Coulter counter. For determination of thymidine incorporation, cells are labeled with 50 micro-Ci of [methyl-.sup.3H] thymidine (3.2 TBq per mmol; Amersham) for the final 18 h of the treatment. Incorporated thymidine is determined as trichloroacetic acid-precipitable counts with a liquid scintillation spectrometer (Beckman LS9800). Binding may be antagonized by adding V32-peptide or V32-peptide fragments or V14 peptide or V14-peptide fragments.

[0164] Detection of the 67 kDa elastin receptor. To select for or confirm the presence of the 67 kDa elastin receptor in cells, reverse transcriptionpolymerase chain reaction is performed using cellular RNA and synthetic oligoprimers corresponding to the beta-galactosidase cDNA sequences upstream and downstream spanning the region between exons 2 and 5. The reaction is run for 40 cycles with denaturation at 90° C. for 1 min, annealing at 50° C. for 2 min, and extension at 72° C. for 5 min in a DNA Thermal Cycler (Perkin-Elmer Cetus). The polymerase chain reaction products are preferably analyzed on 1% agarose gel.

[0165] In describing protein or peptide composition, structure and function herein, reference is made to amino acids. In the present specification, amino acid residues are expressed by using the following abbreviations. Also, unless explicitly otherwise indicated, the amino acid sequences of peptides and proteins are identified from N-terminal to C-terminal, left terminal to right terminal, the N-terminal being identified as a first residue. Ala: alanine residue; Asp: aspartate residue; Glu: glutamate residue; Phe: phenylalanine residue; Gly: glycine residue; His: histidine residue; Ile: isoleucine residue; Lys: lysine residue; Leu: leucine residue; Met: methionine residue; Asn: asparagine residue; Pro: proline residue; Gln: glutamine residue; Arg: arginine residue; Ser: serine residue; Thr: threonine residue; Val: valine residue; Trp: tryptophan residue; Tyr: tyrosine residue; Cys: cysteine residue. The amino acids may also be referred to by their conventional one-letter code abbreviations; A=Ala; T=Thr; V=Val; C=Cys; L=Leu; Y=Tyr; I=Ile; N=Asn; P=Pro; Q=Gln; F=Phe; D=Asp; W=Trp; E=Glu; M=Met; K=Lys; G=Gly; R=Arg; S=Ser; and H=His.

Overview 1. Elastin Degradation and Elastin Peptides with a Gxxp Motif are Associated with Vascular Disease

Elastin-Derived Peptides and Elastin Receptor Complex (ERC) Mediated Vascular Disease

[0166] Activation of ERC by proteolytically degraded elastin peptides is associated with vascular disease.

[0167] Matrix ageing and vascular impacts: focus on elastin fragmentation. Duca L, et al; Cardiovasc Res. 2016 Jun. 1;110(3):298-308.

[0168] Hellenthal F A, Buurman W A, Wodzig W K, Schurink G W. Biomarkers of AAA progression. Part 1: extracellular matrix degeneration. Nat Rev Cardiol 2009;6: 464-474.

[0169] Monocyte chemotactic activity in human abdominal aortic aneurysms: role of elastin degradation-peptides and the 67-kD cell surface elastin receptor. Hance K A, et al; J Vasc Surg 2002;35:254-261.

[0170] Elastin degradation is associated with progressive aortic stiffening and all-cause mortality in predialysis chronic kidney disease. Smith E R, et al; Hypertension. 2012 May;59(5):973-8

Prototype Synthetic Elastin Peptide SEQ ID NO:41 (VGVAPG)

[0171] Evidence that interaction of SEQ ID NO:41 (VGVAPG) with ERC may cause atherosclerosis and is involved in macrophage chemotaxis and angiogenesis.

[0172] Elastin-derived peptides potentiate atherosclerosis through the immune Neul-PI3Kγ pathway. Gayral S, et al; Cardiovasc Res. 2014 Apr. 1;102(1):118-27.

[0173] Induction of macrophage chemotaxis by aortic extracts from patients with Marfan syndrome is related to elastin binding protein. Guo G, et al; PLoS One. 2011;6(5): e20138.

[0174] Elastin-derived peptides enhance angiogenesis by promoting endothelial cell migration and tubulogenesis through upregulation of MT1-MMP. Robinet A, et al; J Cell Sci. 2005 Jan. 15;118 (Pt 2):343-56.

[0175] Proteolytically degraded elastin peptides SEQ ID NO:143 (VPGVGISPEA) and SEQ ID NO:144 (GVAPGIGPGG)

[0176] Evidence that SEQ ID NO:143 (VPGVGISPEA) and SEQ ID NO:144 (GVAPGIGPGG) localize in human atherosclerotic lesions and that serum levels of SEQ ID NO:144 (GVAPGIGPGG) associate with acute myocardial infarction. Note: None of the below authors recognize the GxxP motif in SEQ ID NO:143 (VPGVGISPEA) and SEQ ID NO:144 (GVAPGIGPGG)

[0177] Acute Myocardial Infarction and Pulmonary Diseases Result in Two Different Degradation Profiles of Elastin as Quantified by Two Novel ELISAs. Skjøt-Arkil H, et al; PLoS One. 2013 Jun. 21;8(6):e60936.

[0178] Additional elastin derived peptides that interact with ERC and have biological activity are extensively discussed in:

[0179] Degradation of tropoelastin by matrix metalloproteinases—cleavage site specificities and release of matrikines. Heinz A, et al; FEBS J. 2010 Apr;277(8):1939-56.

Overview 2. Non-Elastin Peptides That Have a GxxP-Motif and are Associated with Vascular Disease

[0180] C-peptide with midportion SEQ ID NO:8 (GGGPGAG)

[0181] Evidence that C-peptide localizes in human atherosclerotic lesions, induces macrophage chemotaxis and angiogenesis, that C-peptide may cause atherosclerosis and that serum levels of C-peptide associate with overall, cardiovascular and diabetes mortality. Typical degradation products of C-peptide are SEQ ID NO:145 (VELGGGPGAGSLQP), SEQ ID NO:146 (LGGGPGAGSLQP) and SEQ ID NO:147 (LGGGPGAGS). Note: None of the below authors recognize the GxxP motif in C-peptide.

[0182] C-peptide co-localizes with macrophages in early arteriosclerotic lesions of diabetic subjects and induces monocyte chemotaxis in vitro. Marx N, et al; Arterioscler Thromb Vasc Biol. 2004 Mar;24(3):540-5.

[0183] Proinsulin C-peptide prevents impaired wound healing by activating angiogenesis in diabetes. Lim Y C, et al; J Invest Dermatol. 2015 Jan;135(1):269-78.

[0184] C-peptide promotes lesion development in a mouse model of arteriosclerosis. Vasic D, et al; J Cell Mol Med. 2012 Apr;16(4):927-35.

[0185] Fasting serum C-peptide levels predict cardiovascular and overall death in nondiabetic adults. Patel N, et al; J Am Heart Assoc. 2012 Dec;1(6): e003152.

[0186] C-peptide levels are associated with mortality and cardiovascular mortality in patients undergoing angiography: the LURIC study. Marx N, et al; Diabetes Care. 2013 Mar;36(3):708-14.

[0187] Serum C-peptide levels and risk of death among adults without diabetes mellitus. Min J Y, Min K B.

[0188] CMAJ. 2013 Jun. 11;185(9):E402-8.

[0189] Serum C-peptide levels as an independent predictor of diabetes mellitus mortality in non-diabetic individuals. Min J Y, Min K B. Eur J Epidemiol. 2013 Sep;28(9):771-4.

Galectin-3 with N-terminal “collagen-like-stretch” SEQ ID NO:148 (AGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSG)

[0190] Evidence that galectin-3 plasma levels associate with heart failure. Note: None of the below authors recognize the GxxP motif in galectin-3.

[0191] Galectin-3, a novel marker of macrophage activity, predicts outcome in patients with stable chronic heart failure. Van der Lok, D, et al; J Am Coll Cardiol 2007 49Suppl. A 98A [Abstract]

[0192] Predictive value of plasma galectin-3 levels in heart failure with reduced and preserved ejection fraction. de Boer RA, et al; Ann Med. 2011 Feb;43(1):60-8.

Fibrillinin-1 with motif SEQ ID NO:149 (EGFEPG)

[0193] Evidence that interaction of SEQ ID NO:149 (EGFEPG) with ERC is involved in macrophage chemotaxis.

[0194] Induction of macrophage chemotaxis by aortic extracts of the mgR Marfan mouse model and a GxxPG-containing fibrillin-1 fragment. Guo G, et al; Circulation 2006; 114:1855-1862.

Laminin with Motif SEQ ID NO:137 (LGTIPG)

[0195] Evidence that laminin interacts via motif SEQ ID NO:137 (LGTIPG) with ERC and induces fibroblast and tumor cell chemotaxis.

[0196] The elastin receptor shows structural and functional similarities to the 67-kDa tumor cell laminin receptor. Mecham RP et al; J Biol Chem. 1989 Oct 5;264(28):16652-7.

TABLE-US-00002 TABLE 1  C-peptide, interspecies comparisons and alignments Species Uniprot identifier C-peptide amino acid sequence Human >sp|P01308|57-87 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) human variant rs121908279 SEQ ID NO: 150 (EAEDLQVGQVEMGGGPGAGSLQPLALEGSLQ) human variant rs121908274 SEQ ID NO: 151 (EAEDLQVGQVELGGGPGAGSLQPLALERSLQ) chimpanzee >sp|P30410|57-87 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) Gorilla >sp|Q6YK33|57-87 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) orangutan >sp|Q8HXV2|57-87 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) Gibbon G1RSS5 SEQ ID NO: 152 (EAEDPQVGQVELGGGPGAGSLQPLALEGSLQ) macaque >sp|P30406|57-87 SEQ ID NO: 152 (EAEDPQVGQVELGGGPGAGSLQPLALEGSLQ) green monkey >sp|P30407|57-87 SEQ ID NO: 152 (EAEDPQVGQVELGGGPGAGSLQPLALEGSLQ) mouse insulin 2  >sp|P01326|57-87 SEQ ID NO: 153 (EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ) mouse insulin 1  >sp|P01325|57-85 SEQ ID NO: 167 (EVEDPQVEQLELGGSPGDLQTLALEVARQ) rat insulin 2 >sp|P01323|57-87 SEQ ID NO: 154 EVEDPQVAQLELGGGPGAGDLQTLALEVARQ) rat insulin 1 >sp|P01322|57-87 SEQ ID NO: 155 (EVEDPQVPQLELGGGPEAGDLQTLALEVARQ) Horse F6QQU6 SEQ ID NO: 156 (EAEDPQVGQEELGGGPGLGGLQPLALAGPQQ) Horse >sp|P01310|33-63 SEQ ID NO: 157 (EAEDPQVGEVELGGGPGLGGLQPLALAGPQQ) Horse Most horses SEQ ID NO: 158 (EAEDPQVGQVELGGGPGLGGLQPLALAGPQQ) chinchilla >sp|P01327|33-63 SEQ ID NO: 159 (ELEDPQVGQADPGVVPEAGRLQPLALEMTLQ) Guinea pig >sp|P01329|57-87 SEQ ID NO: 160 (ELEDPQVEQTELGMGLGAGGLQPLALEMALQ) Rabbit >sp|P01311|57-87 SEQ ID NO: 161 (EVEELQVGQAELGGGPGAGGLQPSALELALQ) Bovine >sp|P01317|57-82 SEQ ID NO: 164 (EVEGPQVGALELAGGPGAGGLEGPPQ) Bovine Fleckvieh variant SEQ ID NO: 165 (EVEGPQVGALELAGGLGAGGLEGPPQ) Sheep >sp|P01318|57-82 SEQ ID NO: 164 (EVEGPQVGALELAGGPGAGGLEGPPQ) Pig >sp|P01315|57-85 SEQ ID NO: 166 (EAENPQAGAVELGGGLGGLQALALEGPPQ) Dog >sp|P01321|57-87 SEQ ID NO: 162 (EVEDLQVRDVELAGAPGEGGLQPLALEGALQ) Cat >sp|P06306|57-87 SEQ ID NO: 163 (EAEDLQGKDAELGEAPGAGGLQPSALEAPLQ)

[0197] Table 2 The presence of the elastin receptor binding motif GxxP (underlined) in vascular matrix proteins elastin and fibrillin and in C-peptides. Peptides are shown with their respective identifiers and amino acids are numbered as shown in the database Uniprot.

TABLE-US-00003 TABLE 2 A, elastic fiber proteins Elastin, P15502, H. .sub.501 SEQ ID NO: 170 sapiens (GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPG) .sub.541 Fibrillin-1, P35555, .sub.411 SEQ ID NO: 168 (PVLPVPPGFPPGPQIPVPRP) .sub.430 - H. sapiens .sub.2191 SEQ ID NO: 169 (TCEEGFEPGPM) .sub.2201 Fibrillin-2, P35556, .sub.421 SEQ ID NO: 171 (LPMGGIPGSAGSRPGGTGGN) .sub.440 - H. sapiens .sub.2237 SEQ ID NO: 172 (NCNEGFEPGPM) .sub.2247 B, C-peptides P01308, H. sapiens .sub.57 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) .sub.87 P30410, P. troglodytes .sub.57 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) .sub.87 Q6YK33, G. gorilla .sub.57 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) .sub.87 Q8HXV2, P. pygmaeus .sub.57 SEQ ID NO: 1 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ) .sub.87 P01325, M. .sub.57 SEQ ID NO: 167 (EVEDPQVEQLELGGSPGDLQTLALEVARQ) .sub.85 musculus;(Ins-1) P01326, M. .sub.57 SEQ ID NO: 153 (EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ) .sub.87 musculus;(Ins-2) P01322, R. .sub.57 SEQ ID NO: 155 (EVEDPQVPQLELGGGPEAGDLQTLALEVARQ) .sub.87 norvegicus;(Ins-1) P01323, R. .sub.57 SEQ ID NO: 154 (EVEDPQVAQLELGGGPGAGDLQTLALEVARQ) .sub.87 norvegicus;(Ins-2) Q62587, P. obesus .sub.57 SEQ ID NO: 173 (GVDDPQMPQLELGGSPGAGDLRALALEVARQ) .sub.87 G5C2F2, H. glaber .sub.57 SEQ ID NO: 174 (ELENLQVGQAEPGMGLEAGGLQPLAQELALQ) .sub.87 P01315, S. scrofa .sub.57 SEQ ID NO: 166 (EAENPQAGAVELGGGLGGLQALALEGPPQ) .sub.85

Further Identification of ERC-Docking Sites

[0198] The elastin-receptor-complex (ERC) is thought to cause human vascular disease by binding excess peptide ligands derived from proteolysis of extra-cellular-matrix (ECM) after aging or smoking. Novel ERC-ligands were identified, notably in well-known biomarkers of vascular disease C-peptide (induced with insulin by high blood-glucose) and NTproBNP (induced in cardiomyocyte stress). It is proposed that A) to investigate accumulation of ERC-ligands as central etiology of human vascular disease, B) to early detect vascular disease risk by testing for ERC-ligands arising from accumulated risks diet, lifestyle and aging, that may all result in human vascular disease.

[0199] Background: ERC is a complex of elastin binding protein (EBP), protective protein/cathepsin A and neuraminidase-1, found on leucocytes, fibroblasts and smooth muscle cells. ERC-ligands confirm to binding motifs xGxxPG or xxGxPG (G being glycine, P proline, x any amino acid), or xGxxPx if adapted to a type VIII beta-turn. Prototype ERC-ligand SEQ ID NO:41 (VGVAPG) and others, such as SEQ ID NO:197 (YGYGPG), SEQ ID NO:198 (YGARPG), SEQ ID NO:199 (FGAVPG), are derived by proteolysis from repeat areas in elastin. Others are SEQ ID NO:149 (EGFEPG) (fibrilin) and SEQ ID NO:137 (LGTIPG) (laminin). EBP separately binds galactosides. ERC-ligand binding to EBP is antagonized by V14 peptide. Circulating levels of ERC-ligands, generated from elastin proteolysis in aging or by smoking, have been associated with atherosclerosis, arterial stiffness, abdominal aortic aneurysms and myocardial infarction in humans, providing ample basis to explore early diagnosis, prevention and treatment of ERC-mediated vascular disease. A composite in silico model is available to dock ERC-ligands in EBP for structural analyses and candidate drug-development. In vitro, ERC-ligand/EBP structure-function relationship may be studied in human cells by testing leukocyte chemotaxis, and proliferation of smooth muscle cells. ERC-ligands induce atherosclerosis and resistance to insulin in mice allowing in vivo study of ERC-mediated vascular disease.

Identification of ERC-Ligand Motifs Derived by Proteolysis from Non-ECM Proteins

[0200] A first find is C-peptide, a peptide derived by prohormone convertase cleavage (PC) from the pre-proinsulin gene and excreted in equimolar amounts with insulin. C-peptide carries the ERC-ligand motif SEQ ID NO:34 (LGGGPG). Ido et al how C-peptide fragments with core motif SEQ ID NO:8 (GGGPGAG) to mitigate glucose-induced vascular dysfunction in rats but do not recognize the ERC-ligand motif. C-peptide has been found atherogenic in mice and an independent marker of human vascular disease. Thus, finding a putative ERC-ligand SEQ ID NO:34 (LGGGPG) in C-peptide acutely links ERC-mediated vascular disease to high-circulating C-peptide levels. It surprisingly provides a common etiology of vascular disease after smoking as well as after diets high in glucose or starch, wherein both etiologies are causally linked to circulating ligands of ERC.

[0201] A second find is galectin-3 that has an N-terminal domain, susceptible to proteolysis, with putative ERC-ligand repeat motifs SEQ ID NO:44 (PGAYPG). Galectin-3 is an independent marker of human vascular disease as well as obesity that underlies vascular disease. As galectin-3 and EBP both bind galactosides and are causal to insulin resistance in mice, it is suggested that a second relationship of galectin-3 to EBP next to putative ERC-ligand-receptor interaction.

[0202] A third find is ERC-ligand peptide motif SEQ ID NO:45 (QGVLPA) in loop 2 of beta-chorionic gonadotropin (beta-hCG), expressed during pregnancy, which loop is nicked by proteolysis from beta-hCG and involved in immunomodulation and angiogenesis.

[0203] We docked newly found SEQ ID NO:34 (LGGGPG), SEQ ID NO:44 (PGAYPG) and SEQ ID NO:45 (QGVLPA), and prototype SEQ ID NO:41 (VGVAPG), in the in-silico model of EBP. All fit this composite model. Also, preliminary in-vitro results show inhibition of bioactivity of C-peptide by ERC-antagonists V14 peptide.

[0204] We then performed a further search for proteins with xGxxPG or xxGxPG motifs closely flanked by PC cleavage sites, to identify ERC-ligands in refulatory model elements rf fragments thereof that may derive from pro-proteins. SEQ ID NO:200 (GVGAPG), SEQ ID NO:186 (PLGSPG), SEQ ID NO:201 (DGAKPG), SEQ ID NO:202 (QGMLPG), and SEQ ID NO:196 (AGGAPG) were found in procalcitonin (PCT), amino-terminal pro-brain natriuretic peptide (NTproBNP), pro-opiomelanacortin (POMC), collagen 6A3 (COL6A3), and pyrin, respectively. PCT and NTproBNP each correlate with heart failure. POMC relates to regulation of feeding behavior and COL6A3 relates to adipocyte function in obesity and insulin resistance. Pyrin relates to innate immunity.

TABLE-US-00004 TABLE 3 Biomarkers of vascular disease that carry the elastin receptor binding motif. Table 3 Biomarkers of vascular disease that carry the elastin  relevant in silico receptor binding motif name hexa-peptide fit in EBP Multiple occurrences of docking motif SEQ ID NO: 216 Elastin SEQ ID + (VGVAPGVGVAPGVGVAPGVGL NO: 41 APGVGVAPGVGVAPGVGVAPG) (VGVAPG) SEQ ID NO: 203 (FGLVPGVGVA) SEQ ID NO: 214 (FGLVPG) SEQ ID NO: 144 (GVAPGIGPGG) Elastin after SEQ ID MMP9/12 NO: 215 (PGIGPG) SEQ ID NO: 205 Galectin-3 SEQ ID + (PPGAYPGQAPPGAYPGAPGAYP NO : 44 GAPAPG) (PGAYPG) Single occurrence of docking motif SEQ ID NO: 206 (TCEEGFEPGP) Fibrillin-1 SEQ ID NO: 149 (EGFEPG) SEQ ID NO: 207 (NPLGTIPGGN) Laminin beta-1 SEQ ID NO: 137 (LGTIPG) Single occurrence of docking motif regulatory model element peptide SEQ ID NO: 208 proinsulin C- SEQ ID + (RREAEDLQVGQVELGGGPGAGS peptide NO: 34 LQPLALEGSLQKR) (LGGGPG) SEQ ID NO: 209 beta-hCG loop 2 SEQ ID + (RVLQGVLPALPQVVCNYR) NO: 45 (QGVLPA) SEQ ID NO: 210 Procalcitonin SEQ ID (KRCGNLSTCMLGTYTQDFNKFH NO: 200 TFPQTAIGVGAPGKKR) (GVGAPG) SEQ ID NO: 211 NT-proBNP SEQ ID (RSHPLGSPGSASDLETSGLQEQR) NO: 186 (PLGSPG) SEQ ID NO: 212 Pro- SEQ ID (KREDVSAGEDCGPLPEGGPEPRS opiomelanacortin NO: 201 DGAKPGPREGKR) (DGAKPG) SEQ ID NO: 213 Collagen 6A3 SEQ ID (RAAPLQGMLPGLLAPLR) NO: 202 (QGMLPG) SEQ ID NO: 192 Pyrin SEQ ID (RRNASSAGRLQGLAGGAPGQKE NO: 196 CR) (AGGAPG)

[0205] We found that three well-known circulating biomarkers of vascular disease, C-peptide, amino-terminal pro-B-type natriuretic peptide (NT-proBNP) and galectin-3, and others, share a little-known docking site with circulating elastin-derived-peptides (EDP). Through this docking site, EDP activate the elastin-receptor-complex (ERC) that is expressed on cells throughout the human arterial system. ERC contributes to elastin degradation and arterial wall remodeling. Experimental activation of ERC by EDP induces insulin resistance and atherosclerosis in mice. Excess EDP/ERC docking causes chemotaxis of human leukocytes and proliferation of human smooth muscle cells (SMC) and is associated with loss of arterial elasticity, atherosclerosis, increased arterial stiffness, abdominal aortic aneurysms and myocardial infarction in humans.