Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/25—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• This invention relates to vascular remodeling and to the treatment of vascular diseases, to methods and pharmaceutical compositions for treating a cardiovascular disease, methods and pharmaceutical compositions for increasing the number of circulating angiogenic cells (CAC) and/or improving the function of CAC, and methods and pharmaceutical compositions for improving vascular remodelling and/or neovascularisation.
• CAC circulating angiogenic cells
• Ghrelin is a 28 amino acid peptide, purified and identified from rat stomach, and characterized by the presence of an n-octanoyl modification on the Ser 3 residue (Ref. 1 ).
• AG is the endogenous ligand of the growth hormone secretagogue receptor (GHSR) (Refs. 2, 3) and in addition to growth-hormone-releasing properties, AG is also detected in the cardiovascular system including in the heart, vasculature and endothelial cells of large vessels, indicating that it may also influence vascular biology, vascular physiology, and atherogenesis (Refs. 4, 5, 6).
• GHSR growth hormone secretagogue receptor
• Des-acyl ghrelin (or unacylated ghrelin, UAG), the unacylated form of ghrelin, whose concentration in plasma and tissue is higher, compared to AG, fails to bind GHSR-Ia and is devoid of any central activity (Ref. 7).
• UAG shares with AG many biological activities and common binding sites on several peripheral tissues.
• AG and UAG exhibit similar GHS-R independent biological activities, including a cytoprotective effect (Ref. 9) and an effect on adipogenesis in vivo (Ref. 10).
• GHSR-I a is not expressed, suggesting that such pleiotropic activities shared with AG may be mediated by a yet unidentified receptor distinct from GHSR-Ia.
• UAG is a biologically active peptide, particularly at the metabolic level, having notably been shown to exert anti-diabetogenic effects as described in U.S. Patent 7,485,620, in U.S. patent application publication number U.S. 20080159991 , in U.S. patent application publication number U.S. 20080312133 and in WO/2008/145749.
• Accelerated vascular disease is the major cause of death and disability in patients with diabetes. Endothelial injury is thought to represent a crucial step in initiation and progression of atherosclerotic vascular disease in diabetes setting (Ref. 1 1 ).
• AGEs advanced glycated end products
• ROS reactive oxygen species
• vascular remodeling does not rely exclusively on proliferation of resident endothelial cells but also involves circulating endothelial progenitor cells (EPC).
• EPC endothelial progenitor cells
• EPC circulating angiogenic cells
• Compelling evidence indicates that as the cardiovascular risk factor profile increases, CAC number decreases and CAC functional activity is impaired, thus limiting CAC delivery to, for example, sites of ischemia where angiogenesis could be required.
• Treatment with certain cytokines induces bone-marrow (BM) mobilization of CAC which, in turn, likely protects against cardiovascular risk (Refs. 17, 18).
• BM bone-marrow
• AGEs advanced glycated end products
• One solution is to increase CAC cell number and/or improve CAC functionality, which can be achieved by, improving their mobilization from the bone marrow, decreasing ROS production induced by AGEs, decreasing CAC senescence or apoptosis rate, and by enhancing CAC capacity to differentiate into an arterial or a venous phenotype (i.e., to form vessels in vivo).
• the earlier observations that AG may have an effect on vascular dysfunction and cardio- protection led to evaluate the in vitro and in vivo effect of UAG on same, as well as to evaluate the effect of UAG on CAC biology, which notably, has not been demonstrated before.
• the present invention relates to a method for treating a cardiovascular disease in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a fragment or analog thereof having the biological activity of SEQ ID NO: 1.
• the present invention relates to a method for increasing the number of circulating angiogenic cells (CAC) and/or improving the function of CAC in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1.
• CAC circulating angiogenic cells
• the present invention relates to a method for improving vascular remodeling and/or neovascularization in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide comprising the sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1.
• the present invention relates to a pharmaceutical composition for use in the treatment of a cardiovascular disease, comprising a therapeutically effective amount of a polypeptide, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 , together with a pharmaceutically acceptable carrier.
• the present invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 , for increasing the number of circulating angiogenic cells (CAC) and/or improving the function of CAC in a subject.
• CAC circulating angiogenic cells
• the present invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof, for improving vascular remodeling and/or neovascularization in a subject.
• the present invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof, for improving wound healing in a subject.
• the present invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof, for improving engraftment associated with transplantation.
• the present invention relates to an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof, for use in tissue engineering.
• the present invention relates to the use of a therapeutically effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 , in the preparation of a medicament for the treatment of a cardiovascular disease in a subject.
• the present invention relates to the use of a therapeutically effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 for increasing the number of circulating angiogenic cells (CAC) and/or improving the function of CAC in a subject.
• a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 for increasing the number of circulating angiogenic cells (CAC) and/or improving the function of CAC in a subject.
• CAC circulating angiogenic cells
• the present invention relates to the use of a therapeutically effective amount of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 in the preparation of a medicament for increasing the number of circulating angiogenic cells (CAC) and/or improving the function of CAC in a subject.
• a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 in the preparation of a medicament for increasing the number of circulating angiogenic cells (CAC) and/or improving the function of CAC in a subject.
• CAC circulating angiogenic cells
• the present invention relates to the use of a therapeutically effective amount of a polypeptide comprising the sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 for improving vascular remodeling and/or neovascularization in a subject.
• the present invention relates to the use of a therapeutically effective amount of a polypeptide comprising the sequence set forth in SEQ ID NO: 1 , or a fragment or analog thereof having the biological activity of SEQ ID NO: 1 in the preparation of a medicament for improving vascular remodeling and/or neovascularization in a subject.
• Figures 1A to 1 F illustrate the protective effect of UAG on CAC from diabetes-associated oxidative stress.
• the percentage of CAC in S phase was evaluated by FACS analysis.
• ROS production from CAC was measured over the indicated periods of time.
• Figures 1 E and 1 F illustrate that UAG (6-13), a fragment of UAG, also has a protective effect on CAC from diabetes-associated oxidative stress.
• FIGs 2A to 2E illustrate that UAG prevents CAC senescence.
• the senescence was evaluated by acidic ⁇ -gal activity on cultured CAC with AGE, UAG and AGE+UAG.
• AGE-, UAG- and AGE+UAG-cultured CAC were assayed by Western blotting using the indicated antibodies.
• CAC were transfected with p53 siRNA or with the scrambled sequence.
• FIG 2D CAC from age- and sex- matched diabetic patients treated with saline or UAG were evaluated for acidic ⁇ -gal activity.
• CAC recovered from diabetic patients treated as above were assayed by Western blotting using the indicated antibodies.
• FIGs 3A to 3D illustrate induction by UAG of CAC mobilization and arterial specification.
• FIG 3A a representative FACS analysis of CAC recovered from type 2 diabetic patients analyzed for the expression of CD45, CD14, CD146 and CD105.
• Figure 3B CAC recovered after saline or UAG or AG treatment from healthy donors (N) and diabetic patients (D) were counted.
• Figure 3C the percentage of cells in the S phase was evaluated on CAC from diabetic patients (D) or healthy subjects (N), treated with UAG, AG or with saline.
• Q-RT-PCR was performed on CAC cells from type 2 diabetic patients or healthy donors cultured as above. The indicated arterial and venous markers were evaluated.
• FIGs 4A to 4F illustrate that UAG induces CAC mobilization in diabetic mice and CAC exposed to UAG act as angiogenic cells.
• a representative FACS analysis of CAC recovered from diabetic mice (NOD/SCI D and ob/ob) or from wt mice analyzed for the expression of CD45, CD31 , CD33 and KDR is reported.
• Figure 4B blood, drawn from NOD/SCI D, ob/ob and wt mice, after UAG or saline treatment was analyzed.
• Figure 4C the percentage of cells in S phase was evaluated by FACS analysis on CAC obtained from different mouse models, treated or not with UAG.
• FIG 4D a Q-RT- PCR was performed on CAC from mouse models cultured as above. The indicated arterial and venous markers were evaluated.
• Figure 4E an immunohistochemical analysis of Matrigel plugs containing IL-3 and labelled CAC, recovered after implantation into SCID mice. Panel a (left) corresponds to CAC recovered after saline treatment, panel b (right) after UAG treatment. The ability of CAC to form functional vessels is reported in panel a. Black arrows indicate positive CAC cells.
• Matrigel plugs recovered indicate that the majority of vessels are lined by human HLA Class I positive cells. The number of human or host derived vessels is reported in the histogram.
• FIGS 5A and 5B illustrate that UAG (UAG (1-28)) and UAG (6-13) fragment have binding sites on CAC membranes.
• the present invention relates to the unexpected discovery of the effects of UAG on CAC. More particularly, it relates to the unexpected discovery that UAG mobilizes CAC, protects CAC from oxidative stress or from diabetes-associated oxidative stress, reduces CAC accelerated onset of senescence and restores CAC functional activity, increases CAC number, prevents ROS production in CAC both in physiological conditions and in diabetes setting, binds to CAC and/or rescues functional impairment of CAC.
• UAG can be used as a novel therapeutic strategy to improve or ameliorate impaired vascular remodeling in a subject or patient with cardiovascular risk, or to treat a subject or a patient with a cardiovascular disease or an ischemic disease, to increase CAC number and improve CAC function, to improve engraftment, to improve engraftment associated with or following transplantation of, for example, an organ or part thereof, to be used in or to facilitate in vitro or ex vivo tissue engineering, such as, but not limited to, blood vessels engineering, to improve wound healing, to improve wound healing in diabetic patients, such as diabetic patients suffering from diabetic ulcers.
• ghrelin and “acylated ghrelin” or “AG” are used interchangeably and have the same meaning.
• unacylated ghrelin or "UAG” is intended to mean peptides that have the amino acid sequence specified in SEQ ID NO: 1 (1-NH 2 Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His- Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-28; SEQ ID NO: 1 ).
• UAG may also be referred to as UAG (1-28).
• Naturally-occurring variations of unacylated ghrelin include peptides that contain substitutions, additions or deletions of one or more amino acids which result due to discrete changes in the nucleotide sequence of the encoding ghrelin gene or alleles thereof or due to alternative splicing of the transcribed RNA. It is understood that the said changes do not substantially affect the properties, pharmacological and biological characteristics of unacylated ghrelin variants.
• Those peptides may be in the form of salts. Particularly the acidic functions of the molecule may be replaced by a salt derivative thereof such as, but not limited to, a trifluoroacetate salt.
• peptide By “peptide”, “polypeptide” or “protein” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation), or chemical modification, or those containing unnatural or unusual amino acids such as D- Tyr, ornithine, amino-adipic acid.
• post-translational modification e.g., glycosylation or phosphorylation
• chemical modification e.g., amino acids that is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation), or chemical modification, or those containing unnatural or unusual amino acids such as D- Tyr, ornithine, amino-adipic acid.
• D- Tyr e.g., D- Tyr, ornithine, amino-adipic acid
• fragments refers to amino acid fragments of a peptide such as unacylated ghrelin. Fragments of unacylated ghrelin are shorter than 28 amino acid residues. Fragments of unacylated ghrelin may therefore be 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5 or 4 amino acid residues in length.
• fragments of UAG may have residues 1-14 of SEQ ID NO: 1 as shown in SEQ ID NO: 2 ((UAG 1-14); Gly-Ser-Ser-Phe-
• Val-Gln or residues 8-12 of SEQ ID NO: 1 as shown in SEQ ID NO: 8 ((UAG 8-12); Glu-His-Gln-Arg- VaI) or analogs thereof.
• the polypeptides are used in a form that is “purified”, “isolated” or “substantially pure”.
• the polypeptides are “purified”, “isolated” or “substantially pure” when they are separated from the components that naturally accompany them.
• a compound is substantially pure when it is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, by weight, of the total material in a sample.
• analog of unacylated ghrelin refers to both structural and functional analogs of unacylated ghrelin or fragments thereof which are, inter alia, capable of replacing UAG in the biological function of UAG as described in the present application, such as, but not limited to, in the prevention and treatment of cardiovascular diseases, in protecting CAC cells from oxidative stress or from diabetes-associated oxidative stress, promoting vascular remodeling and neovascularization, increasing CAC number, improving CAC function, protecting CAC from oxidative stress, preventing ROS production in CAC both in physiological conditions and in diabetes setting, protecting against oxidative stress associated with inhibition of the accelerated onset of CAC senescence, binding to CAC membranes, and increase mobilization of CAC and/or rescue functional impairment of CAC, as described in the present application.
• structural and functional analogues will be useful for realizing therapeutic benefits in medical conditions as described
• Simple structural analogs comprise peptides showing homology with unacylated ghrelin as set forth in SEQ ID NO: 1 or homology with any fragment thereof.
• An example of an analog of ghrelin is an isoform of ghrelin-28 (SEQ ID NO: 1 ), des Gln-14 Ghrelin (a 27 amino acid peptide possessing serine 3 modification by n-octanoic acid) which is shown to be present in stomach. It is functionally identical to ghrelin in that it binds to GHSR-Ia with similar binding affinity, elicits Ca 2+ fluxes in cloned cells and induces GH secretion with similar potency as Ghrelin-28. It is expected that UAG also has a des Gln-14 UAG that is functionally identical to UAG.
• Preferred analogs of UAG and preferred analogs of fragments of UAG are those that vary from the native UAG sequence or from the native UAG fragment sequence by conservative amino acid substitutions; i.e., those that substitute a residue with another of like characteristics.
• Typical substitutions include those among Ala, VaI, Leu and lie; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; among the basic residues Lys and Arg; and among the aromatic residues Phe and Tyr.
• Particularly preferred are analogs in which several, for example, but not limited to, 5-10, 1- 5, or 1-2 amino acids are substituted, deleted, or added in any combination.
• the analogs of UAG may differ in sequence from UAG by 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions (preferably conservative substitutions), deletions, or additions, or combinations thereof.
• analogs of the peptides of the invention that have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with the amino acid sequences described herein over its full length, and sharing at least one of the metabolic effects or biological activity of UAG.
• a person skilled in the art would readily identify an analog sequence of unacylated ghrelin or an analog sequence of a fragment of unacylated ghrelin.
• Analogs of UAG or analogs of fragments thereof are, for example, analogs obtained by alanine scans, by substitution with D-amino acids or with synthetic amino acids or by cyclization of the peptide.
• Analogs of UAG or fragments thereof may comprise a non- naturally encoded amino acid, wherein the non-naturally encoding amino acid refers to an amino acid that is not one of the common amino acids or pyrrolysine or selenocysteine, or an amino acid that occur by modification (e.g.
• Naturally encoded amino acid including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine
• naturally encoded amino acid including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine
• non-naturally-occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N- acetylglucosaminyl-L-threonine and O-phosphotyrosine.
• modified refers to any changes made to a given polypeptide, such as changes to the length of the polypeptide, the amino acid sequence, chemical structure, co-translational modification, or post-translational modification of a polypeptide.
• post-translational modification refers to any modification of a natural or non- natural amino acid that occurs to such an amino acid after it has been incorporated into a polypeptide chain.
• the term encompasses, by way of example only, co-translational in vivo modifications, co-translational in vitro modifications (such as in cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.
• Examples of post-translational modifications are, but are not limited to, glycosylation, acetylation, acylation, amidation, carboxylation, phosphorylation, addition of salts, amides or esters, in particular C-terminal esters, and N-acyl derivatives of the peptides of the invention.
• the types of post-translational modifications are well known.
• Certain peptides according to the present invention may also be in cyclized form, such that the N- or C-termini are linked head-to-tail either directly, or through the insertion of a linker moiety, such moiety itself generally comprises one or more amino acid residues as required to join the backbone in such a manner as to avoid altering the three-dimensional structure of the peptide with respect to the non-cyclized form.
• Such peptide derivatives may have improved stability and bioavailability relative to the non-cyclized peptides.
• cyclising peptides are well known in the art and for example may be accomplished by disulfide bond formation between two side chain functional groups, amide or ester bond formation between one side chain functional group and the backbone ⁇ -amino or carboxyl function, amide or ester bond formation between two side chain functional groups, or amide bond formation between the backbone ⁇ -amino and carboxyl functions.
• These cyclisation reactions have been traditionally carried out at high dilution in solution. Cyclisation is commonly accomplished while the peptide is attached to the resin.
• One of the most common ways of synthesising cyclic peptides on a solid support is by attaching the side chain of an amino acid to the resin.
• the C-and N-termini can be selectively deprotected and cyclised on the resin after chain assembly.
• This strategy is widely used, and is compatible with either tert- butyloxycarbonyl (Boc) or 9- fluorenylmethoxycarbonyl (Fmoc) protocols.
• Boc tert- butyloxycarbonyl
• Fmoc 9- fluorenylmethoxycarbonyl
• a number of approaches may be used to achieve efficient synthesis of cyclic peptides.
• One procedure for synthesising cyclic peptides is based on cyclisation with simultaneous cleavage from the resin.
• the deprotected amino group can react with its anchoring active linkage to produce protected cyclic peptides. In general, a final deprotection step is required to yield the target cyclic peptide.
• Lactamazation a form of cyclisation, may be performed to form a lactam bridge using Fmoc synthesis, amino acids with different protecting groups at the lateral chains may be introduced, such as, but not limited to, aspartic acid (or glutamic) protected with allyl ester at the beta ester (or gamma ester for glutamic acid) and lysine protected with allyloxy carbamate at the N- ⁇ .
• the allyl and alloc protecting groups of aspartic acid and lysine may be deprotected with, for example, palladium (0) followed by cyclization using PyAOP (7-Azabenzotriazol-1- yloxytris(pyrrolidino) phosphonium-hexafluorophosphate) to produce the lactam bridge.
• an amino acid named herein refers to the L-form.
• Levoratory amino acids L-amino acids or L or L-form
• dextrorotary amino acids D- amino acids or D or D-form
• Alanine Al or A
• Arginine Arg or R
• Asparagine Asn or N
• Aspartic acid Asp or D
• Cysteine Cys or C
• Glutamic acid GIu or E
• Glutamine GIn or Q
• Glycine GIy or G
• Histidine Histidine
• lsoleucine lie or I
• Leucine Leu or L
• Lysine Lysine
• Methionine Met or M
• Phenylalanine Phe or F
• Proline Pro or P
• Serine Serine
• Ser or S Threonine
• Thr or T Tryptophan
• Trp or W Tyrosine
• L-amino acid residue within the native peptide sequence may be altered to any one of the 20 L-amino acids commonly found in proteins or any one of the corresponding D-amino acids, rare amino acids, such as, but not limited to, 4- hydroxyproline or hydroxylysine, or a non-protein amino acid, such as P-alanine or homoserine.
• UAG peptides may also be part of a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro- sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
• homology refers to sequence similarity between two peptides while retaining an equivalent biological activity. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences so that a "homologous sequence” refers to a sequence sharing homology and an equivalent function or biological activity. Assessment of percent homology is known by those of skill in the art. Methods to determine homology, identity and similarity of peptides are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, BLASTP, BLASTN, and FASTA.
• the BLAST X program is publicly available from NCBI and other sources.
• the well known Smith Waterman algorithm may also be used to determine identity.
• Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. MoI Biol. 48: 443-453 (1970);
• polypeptides of the invention may be prepared in any suitable manner as known in the art.
• Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means and methods for preparing such polypeptides are well known in the art.
• UAG polynucleotides include isolated polynucleotides which encode the UAG polypeptides, fragments and analogs defined in the application.
• polynucleotide refers to a molecule comprised of a plurality of deoxyribonucleotides or nucleoside subunits.
• the linkage between the nucleoside subunits can be provided by phosphates, phosphonates, phosphoramidates, phosphorothioates, or the like, or by nonphosphate groups as are known in the art, such as peptoid-type linkages utilized in peptide nucleic acids (PNAs).
• PNAs peptide nucleic acids
• the linking groups can be chiral or achiral.
• the oligonucleotides or polynucleotides can range in length from 2 nucleoside subunits to hundreds or thousands of nucleoside subunits.
• oligonucleotides are preferably 5 to 100 subunits in length, and more preferably, 5 to 60 subunits in length, the length of polynucleotides can be much greater (e.g., up to 100).
• the polynucleotide may be any of DNA and RNA.
• the DNA may be in any form of genomic DNA, a genomic DNA library, cDNA derived from a cell or tissue, and synthetic DNA.
• the present invention may, in certain aspects, use vectors which include bacteriophage, plasmid, cosmid, or phagemid.
• biological activity or “biological property”, or the term “activity” in reference to the polypeptides of the present invention, are used interchangeably herein and refer to the pharmacological, biological or cellular abilities of the polypeptides of the invention and include, but are not limited to, the capacity of replacing UAG in the biological functions of UAG such as but not limited to, in the prevention and/or the treatment of cardiovascular diseases, in protecting CAC cells from oxidative stress or from diabetes-associated oxidative stress, in promoting vascular remodeling and neovascularization, in increasing CAC number, in improving CAC function, in protecting CAC from oxidative stress, in preventing ROS production in CAC both in physiological conditions and in diabetes setting, in protecting against oxidative stress associated with inhibition of the accelerated onset of CAC senescence, in binding to CAC membranes, and in increasing mobilization of CAC and/or rescuing functional impairment of CAC.
• the present invention provides a method for treating subjects such as patients at risk of having or having a cardiovascular disease or an ischemic disease.
• the present invention also provides a method for improving vascular remodeling and/or neovascularization in a subject.
• the present invention provides a method for increasing the number of CAC and improving the function of CAC in a subject.
• the present invention also provides for a method to improve engraftment, more particularly to improve engraftment associated with or following a transplantation of, for example, a tissue or an organ or any part thereof.
• the present invention also provides for a method to improve wound healing, to improve engraftment and/or to facilitate tissue engineering.
• treatment refers to both therapeutic treatments as well as to prophylactic and preventative measures. Those in need of treatment include those already with the disease or disorder or condition as well as those in which the disease, disorder or condition is to be prevented. Those in need of treatment are also those in which the disorder, disease or condition has occurred and left after-effects or scars. Treatment also refers to administering a therapeutic substance effective to improve or ameliorate symptoms associated with a disease, a disorder or a condition to lessen the severity of or cure the disease, disorder or condition, or to prevent the disease, disorder or condition from occurring.
• the method of the invention includes the step of administering to a subject a therapeutically effective amount of a polypeptide defined herein which shares the same potential therapeutic indication as UAG itself.
• a polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or any fragments or any analogs thereof as described above, such as, but not limited to, UAG (6-13), UAG (8-12) and UAG (8-13) fragments.
• Subjects that would benefit from being administered with the polypeptides of the invention include, but are not limited to, those that are at risk of suffering or are suffering or have suffered from a cardiovascular disease or an ischemic disease. Such subjects may for example be subjects suffering from type 1 or type 2 diabetes and/or may be subjects suffering from obesity.
• cardiovascular disease refers to diseases that involve the heart or blood vessels (arteries and veins). The term refers to any disease that affects the cardiovascular system. It also refers to diseases related to atherosclerosis (arterial disease). Cardiovascular diseases include, but are not limited to, aneurysm, angina, arrhythmia, cardiomyopathy, cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease, coronary artery disease, dilated cardiomyopathy, diastolic dysfunction, endocarditis, high blood pressure (hypertension), hypertrophic cardiomyopathy, mitral valve prolapse, myocardial infarction (heart attack), venous thromboembolism, ischemia and wound healing.
• aneurysm angina, arrhythmia, cardiomyopathy, cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease, coronary artery disease, dilated cardio
• any subject suffering from or being at risk of suffering from vascular complications, cardiovascular diseases associated with the metabolic syndrome or syndrome X or obesity, atherosclerosis, primary artherosclerotic vascular degeneration such as central and peripheral arteriopathy, or any subject in need of vascular remodeling or in need of neovascularization would benefit from being administered with the polypeptides of the invention.
• the expression syndrome X refers to a combination of medical disorders that includes the risk of developing cardiovascular diseases.
• oxidative stress refers to an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damages.
• Any subject or patient that would benefit from an increase in the CAC number and/or an improvement in CAC function would benefit from administration of the polypeptides of the invention.
• CAC number refers to the amount or concentration of CAC that home from the bone marrow into sites of vascular remodeling and/or sites of noeovascularization.
• CAC function refers to the property of CAC to mobilize to a site of endothelization (CAC mobilization), differentiate into endothelial cells and participate in endothelization. Improvement of CAC function refers to bringing into a more desirable condition CAC function, such as for example, improving CAC mobilization refers to bringing into a more desirable condition CAC mobilization. Any subject in need of vascular remodeling or vascular restoration and/or in need of noeovascularization would also benefit from administration of the polypeptides of the invention.
• vascular remodeling refers to any lasting changes in the diameter, thickness, or structure of a mature blood vessel.
• vascular remodeling also includes collateral vessel formation.
• atherosclerosis vascular remodeling acts as a compensatory mechanism to preserve blood flow in the face of plaque growth, which tends to cause stenosis or narrowing of the artery.
• neovascularization refers to the formation of functional microvascular networks with red blood cell perfusion.
• Improvement of vascular remodeling and/or neovascularization refers to bringing into a more desirable condition vascular remodeling and/or neovascularization.
• polypeptides of the invention are also useful to promote engraftment of, for example, cells, tissues and/or organs or parts thereof, in a transplant recipient such as an animal or a human.
• engraftment refers to incorporation of grafted tissue into the body of a host.
• engraftment also refers to the process in which transplanted stem cells or bone marrow cells migrate to the bone marrow and begin producing blood cells.
• tissue engineering also refers to the production of natural or synthetic organs and tissues that can be implanted as fully functional units or may develop to perform necessary functions following implantation.
• tissue engineering also refers to the production of natural or synthetic organs and tissues that can be implanted as fully functional units or may develop to perform necessary functions following implantation.
• the methods of regenerative medecine or tissue engineering are known to those of skill in the art.
• polypeptide defined herein are also useful for improving wound healing.
• the polypeptide defined herein are also useful for improving wound healing in diabetic patients such as improving healing of diabetic ulcers.
• metabolic disorders refers to, but is not limited to, disorders of carbohydrate metabolism, disorders of amino acid metabolism, disorders of organic acid metabolism (organic acidurias), disorders of fatty acid oxidation and mitochondrial metabolism, disorders of porphyrin metabolism, disorders of purine or pyrimidine metabolism, disorders of steroid metabolism, disorders of mitochondrial function, disorders of peroxisomal function and lysosomal storage disorders.
• metabolism syndrome refers to a combination of medical disorders that increase one's risk for cardiovascular disease and diabetes.
• the polypeptides as defined herein may be formulated for, but not limited to, intravenous, subcutaneous, transdermal, topical, oral, buccal, sublingual, nasal, inhalation, pulmonary, or parenteral administration according to conventional methods. Intravenous injection may be by bolus or infusion over a conventional period of time.
• the polypeptides as defined herein may also be administered directly to a target site within a subject e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
• Active ingredients such as the polypeptides defined herein, to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain, but not be limited to, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents.
• these compositions may contain, but are not limited to microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.
• the active ingredients may be administered by way of a controlled-release delivery system.
• Administered by nasal aerosol or inhalation formulations may be prepared, for example, as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, employing fluorocarbons, and/or employing other solubilizing or dispersing agents.
• polypeptides of the invention may be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form.
• the injectable solution or suspension may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, well known in the art.
• the polypeptides of the invention may also be formulated for topical administration.
• topical as used herein includes any route of administration that enables the compounds to line the skin or mucosal tissus. Topical administration of the polypeptides defined herein is useful for example, to improve wound healing or to improve wound treatment or for in situ treatment in a subject.
• the formulation suitable for topical application may be in the form of, for example, cream, lotion, solution, gel, ointment, paste, plaster, paint, bioadhesive, or the like, and/or may be prepared so as to contain liposomes, micelles, microparticles and/or microspheres.
• the formulation may be aqueous, i.e., contain water, or may be nonaqueous and optionally used in combination with an occlusive overlayer so that moisture evaporating from the body surface is maintained within the formulation upon application to the body surface and thereafter.
• Ointments as is well known in the art of pharmaceutical formulation, are semisolid preparations that are typically based on petrolatum or other petroleum derivatives.
• Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. As will be appreciated by those working in the field of pharmaceutical formulation, gels are semisolid, suspension-type systems.
• Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.
• Lotions which are preferred for delivery of cosmetic agents, are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids.
• Pastes are semisolid dosage forms in which the active agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from a single-phase aqueous gels.
• Plasters are comprised of a pasty mixture that is spread on the body, either directly or after being saturated into a base material such as cloth. Formulations of the invention, may be dissolved or dispersed within the plaster to make a medicated plaster.
• Bioadhesives are preparations that adhere to surfaces of body tissues. Polymeric bioadhesive formulations are well known in the art.
• Formulations may also be prepared with liposomes, micelles, microparticles and/or microspheres.
• Liposomes are microscopic vesicles having a lipid wall comprising a lipid bilayer, and can be used as drug delivery systems.
• Micelles are known in the art to be comprised of surfactant molecules arranged so that their polar headgroups form an outer spherical shell, while the hydrophobic, hydrocarbon chains are oriented towards the center of the sphere, forming a core.
• Microparticles are particulate carrier systems in the micron size range, normally prepared with polymers, which can be used as delivery systems for drugs or vaccines that are usually trapped within the particles. Microspheres, similarly, may be incorporated into the present formulations and drug delivery systems.
• microspheres essentially encapsulate a drug or drug- containing formulation.
• Microspheres are generally, although not necessarily, formed from synthetic or naturally occurring biocompatible polymers, but may also be comprised of charged lipids such as phospholipids.
• compositions will comprise at least one of the polypeptides of the invention together with a pharmaceutically acceptable carrier which will be well known to those skilled in the art.
• the compositions may further comprise for example, one or more suitable excipients, diluents, fillers, solubilizers, preservatives, salts, buffering agents and other materials well known in the art depending upon the dosage form utilized. Methods of composition are well known in the art.
• pharmaceutically acceptable carrier is intended to denote any material, which is inert in the sense that it substantially does not have any therapeutic and/or prophylactic effect per se and that are non-toxic.
• a pharmaceutically acceptable carrier may be added to the polypeptides of the invention with the purpose of making it possible to obtain a pharmaceutical composition, which has acceptable technical properties.
• Such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and PEG.
• buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone
• Carriers for topical or gel-based forms of polypeptides include polysaccharides such as sodium carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, and wood wax alcohols.
• the polypeptides used for in vivo administration must be sterile. This may be accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The polypeptidesordinarily will be stored in lyophilized form or in solution.
• Therapeutic polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
• the invention also provides an article of manufacture or a commercial package or kit, comprising: a container, a label on the container, and a composition comprising the polypeptides of the invention as an active agent within the container when used at the indicated level, wherein the composition is effective for, inter alia, the treatment of a cardiovascular disease and/or for improving vascular remodeling or neovascularization and/or increasing CAC number and improving
• the label on the container indicates for what the composition can be used.
• a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
• a therapeutically effective amount of the peptides noted herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
• a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting the onset of a condition related to insulin levels and/or activity. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.
• a therapeutically effective amount or effective dose of the peptides of the invention is an amount sufficient to improve or ameliorate impaired vascular remodeling in a subject or patient with cardiovascular risk, or to treat a subject or a patient with a cardiovascular disease or an ischemic disease, to improve engraftment, to improve engraftment associated with or following transplantation of an organ or a part thereof, to be used in or to improve or facilitate in vitro or ex vivo tissue engineering, such as, but not limited to blood vessels engineering, to improve wound healing, to improve wound healing in diabetic patients, such as diabetic patients suffering from diabetic ulcers.
• the methods and/or assays for measuring such parameters are known to those of ordinary skill in the art.
• the therapeutically effective amount of the invention will generally vary from about 0.001 ⁇ g/kg to about 10 mg/kg, more particularly from about 0.01 ⁇ g/kg to about 10 mg/kg, and even more particularly from about 1 ⁇ g/kg to about 1 mg/kg. Therapeutically effective amounts or effective doses that are outside this range but that have the desired therapeutic effect are also encompassed by the present invention.
• the subject noted above is a mammal, in a further aspect, a human.
• UAG protects CAC from oxidative stress
• Oxidative stress plays a major role in tissue damage (Ref. 21 ) and endothelial injury associated with diabetes mainly depends on the production of reactive oxygen species (ROS).
• ROS reactive oxygen species
• type 2 diabetic patients were subjected to UAG treatment. After 12 hours of treatment, CAC were recovered from UAG treated and untreated patients and cultured. As shown in Figures 2D and 2E, when administered in vivo, UAG reduced the number of senescent cells and prevented p53 accumulation, phosphorylation of Rb and p21 expression. These data further confirm that a p53-mediated signalling pathway contributes to the impaired CAC function in a diabetic setting and that UAG can prevent these events.
• UAG affects CAC mobilization
• CAC physiologically populate in BM and mobilize into circulation in response to microenvironment changes. Impairment in CAC mobilization has been reported in patients with cardiovascular risk factors (Refs. 19, 31 ).
• NOD/SCID mice and ob/ob mice were treated for different time intervals with saline or UAG.
• the person skilled in the art will be familiar with the NOD/SCID mice and ob/ob mice models.
• FACS analysis demonstrates that, as with the human counterpart, cells recovered from UAG-treated ob/ob mice expressed the CD45 marker and CD31 , KDR, but not CD33.
• Black lines refer to preimmune IgG used as negative control and grey lines refer to surface marker expression.
• Similar results were obtained when CAC from untreated diabetic mice as well as untreated or treated wild-type mice were analyzed (data not shown).
• UAG challenge was able to increase the percentage of recovered cells ( Figure 4B).
• UAG (3.0 ⁇ g/kg/h iv. as infusion for 12 h from 0 to 12 hours); isotonic saline (infusion from 0 to 12 hours).
• Reagents - M199 medium (endotoxin tested), bovine serum albumin, fetal bovine serum (FBS), glycated human albumin (AGE) were from Sigma-Aldrich (St Louis, MO).
• Bovine calf serum (endotoxin-tested) was obtained from HyClone (Logan, UT). Trypsin was purchased from Difco.
• Nitrocellulose filters, horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG, molecular weight markers, and chemiluminescence reagents (ECL) were from Amersham Biosciences.
• the acidic ⁇ -galactosidase staining kit was from Invitrogen.
• Peroxide hydrogen was obtained from Carlo Erba reagents. The presence of endotoxin contamination was tested by the Limulus amebocyte assay (concentration was ⁇ 0.1 ng/ml). Human IL-3 was a gift from Sandoz Pharma Ltd (Basel, Switzerland). Human UAG was purchased from Phoenix Europe GmbH (Karlsruhe, Germany).
• PB-MNC Peripheral-blood mononuclear cells isolated by Fycoll Histopaque 1077 (Sigma) were resuspended in 20% FBS 199 Medium and plated on fibronectin-coated dishes (Biocoat, Becton Dickinson Labware) as described by Hill et al. (Ref. 19). Human UAG studies were performed on CAC recovered from normal donors and diabetic patients. The purity of sorted cells was assessed by FACS analysis (Ref. 20).
• the isolated CAC were cultured under 5% CO 2 at 37°C for 4 days on 20 ⁇ g/ml fibronectin-coated dishes in EGM-2 medium containing 10% FBS, hydrocortisone, human Fibroblast Growth Factor, Vascular Endothelial Growth Factor, Insulin Growth Factor 1 , ascorbic acid, human Epidermal Growth Factor, gentamicin and amphotericin-B (Cambrex, Walkersville, MD, USA) alone or in combination with 1 ⁇ M UAG and 1.2 mg/ml AGE.
• isolated CAC were cultured in EBM-2 medium (Cambrex, Walkersville, MD, USA) supplemented with 10 ng/ml of IL-3.
• FACS was used to analyze their phenotype (anti-CD45, anti-CD31 , anti-CD105, anti-CD14 antibodies were used)(Ref. 22).
• Mobilization assay - CAC recovered from healthy subjects and diabetic patients, treated with UAG or saline for 6 h and 12 h, were evaluated. Total number of MNC was counted by 3 independent investigators. The percentage of CAC was calculated by comparing CAC obtained from diabetic patients and healthy donors, treated for 6 h and 12 h with UAG, AG or saline, to CAC recovered at time 0.
• siRNA Small Interfering RNAs
• CAC Senescence was evaluated by acidic ⁇ -gal activity on CAC recovered from normal subjects cultured in EGM2 medium for 4 days with UAG or AGE+UAG and saline- or UAG-treated diabetic patients (Ref. 22).
• SA- ⁇ -gal stain solution 1 mg/ml 5-bromo-4-chloro-3-indolyl ⁇ -D-galactopyranoside (X-gal), 5 mM/liter potassium ferrocyanide, 5 mM/liter ferricyanide, 150 mM/liter NaCI, 2 mM/liter MgCI 2 , 0.01% sodium deoxycholate, and 0.02% Nonidet P-40.
• SA- ⁇ -gal-positive cells were counted manually by 3 independent investigators.
• RNA isolation and quantitative real-time PCR - CAC from diabetic patients, healthy subjects and mice treated with UAG or saline were cultured in EGM-2 medium. Human arterial and venous cells were used as a positive controls. mRNA quantification was performed by Q-RT-PCR, as previously described (Ref. 24). The relative expression of
• Ephrin B2 Notchi , Notch4 and EphB4 were calculated by using comparative threshold cycle methods.
• Diabetic and control mice - Mice were divided in four groups. The plasma glucose and insulin determination for each group are reported. Sixteen 6 weeks old ob/ob mice (blood glucose, 600 ⁇ 45 mg/dL, insulin, 5.5 ⁇ 0.9 ng/ml); sixteen C56BL6/J wild type mice (blood glucose, 150 ⁇ 18.2 mg/dL, insulin, 1 ⁇ 0.05 ng/ml); ten 7 weeks old NOD/SCID mice (blood glucose, 164 ⁇ 12.9 mg/dL, insulin 1.2 ⁇ .0.12 ng/ml); ten 17 weeks old NOD/SCID mice (blood glucose, 536 ⁇ 36.3 mg/dL, insulin 4.85 ⁇ 0.74 ng/ml).
• Blood glucose and serum insulin determinations Blood glucose was measured with a One Touch Il glucose meter (Lifescane, Mountain View, CA). Serum insulin was measured with a mouse insulin radioimmumoassay kit (Linco Research immunoassay, St. Charles, MO), following the manufacturer's instructions.
• the isolated CAC were cultured in EBM-2 medium supplemented with 10 ng/ml of IL-3.
• Matrigel-plug assay For murine angiogenesis assay, untreated or UAG-treated CAC, obtained from type 2 diabetic patients, were counted and resuspended in DMEM (4 x 10 6 in 250 ⁇ L DMEM). Cells were chilled in ice, labelled with fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CSFE, Molecular Probes), added to 250 ⁇ L Matrigel containing of IL-3 (100 ng/ml) at 4°C, and injected sc. in the abdominal paramedian region of 7 week old NOD/SCID mice (5 mice per group).
• CSFE carboxyfluorescein diacetate succinimidyl ester
• mice were killed and Matrigel plugs were recovered, fixed in 10% buffered formalin and embedded in paraffin for immunohistochemistry.
• Immunohistochemistry and immunofluorescence For immunohistochemistry, sections from paraffin-embedded blocks of Matrigel plugs were collected onto poly-lysine-coated slides. Endogenous peroxidase activity was blocked with 6% H 2 O 2 for 8 minutes at room temperature. To detect cells labelled with fluorescent dye CSFE, anti-Fluorescein/Oregon Green polyclonal Abs (Molecular Probes) were applied to slides overnight at 4°C.
• Horseradish peroxidase-labeled antirabbit Envision polymer (DakoCytomation, Carpinteria, CA) was incubated for 30 minutes. The reaction product was developed using 3,3-diaminobenzidine. Omission of the primary Ab or substitution with an unrelated rabbit serum IgG used as negative control. The percentage of positive cells was counted in 4 non-sequential sections for each experiment at x40 magnification. For immunofluorescence, the samples were processed using anti-human HLA I and anti- mouse MHC Il antibodies as previously described (Ref. 28). The number of MHC Il or HLA I positive vessels was determined by counting 10 randomly selected fields in three different samples.
• Delhanty PJ van der Eerden BC, van der Velde M, Gauna C, Pols HA, Jahr H, Chiba H, van der LeIy AJ, van Leeuwen J; Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-RI a. J Endocrinol. 188:37-47 (2006). 10.
• MAPK mitogen-activated protein kinase
• PI3K phosphoinositide 3-kinase

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