Use of VEGF-C or VEGF-D in Reconstructive Surgery
The present invention provides materials and methods for repairing tissue and using vascular endothelial growth factor C (VEGF-C) genes and/or proteins. Methods and materials related to the use of VEGF-C for the reduction of edema and improvement of skin perfusion is provided. Also provided is are materials and methods for using VEGF-C before, during, and after reconstructive surgery.
1 . A method of improving the healing of a skin graft or skin flap to underlying tissue of a mammalian subject, comprising:
contacting skin graft or skin flap tissue or underlying tissue with a composition comprising a healing agent that is a Vascular Endothelial Growth Factor C (VEGF-C) polynucleotide that comprises a nucleotide sequence that encodes a VEGF-C polypeptide,
wherein the healing agent is present in said composition in an amount effective to improve the healing of the skin graft or skin flap.
2 . A method according to claim 1 wherein said mammalian subject is human.
3 . A method according to claim 2 , further comprising a step of attaching the skin graft or skin flap tissue to the underlying tissue.
4 . A method according to claim 3 wherein the contacting precedes the attaching.
5 . A method according to claim 3 wherein the contacting is subsequent to the attaching.
6 . A method according to claim 3 wherein the underlying tissue is breast tissue.
7 . A method according to claim 6 wherein the skin graft or skin flap is attached in a breast augmentation, breast reduction, mastopexy, or gynecomastia procedure.
8 . A method according to claim 3 wherein the skin graft or skin flap is attached in a cosmetic surgery procedure.
9 . A method according to claim 8 , wherein the procedure is a facial cosmetic procedure selected from the group consisting of rhytidectomy, browlift, otoplasty, blepharoplasty, rhinoplasty, facial implant, and hair replacement therapy.
10 . A method according to claim 3 , wherein the skin graft or skin flap is attached in an abdominoplasty (abdominal lipectomy) or liposuction procedure.
11 . A method according to claim 3 , wherein the skin graft or skin flap is attached in a reconstructive surgery.
12 . A method according to claim 11 , wherein the reconstructive surgery corrects a congenital defect selected from the group consisting of birthmark, cleft palate, cleft lip, syndactyly, urogenital and anorectal malformations, craniofacial birth defects, ear and nasal deformities, and vaginal agenesis.
13 . A method according to claim 11 , wherein the reconstructive surgery corrects a defect from an injury, infection, or disease.
14 . A method according to claim 13 , wherein the injury is a burn.
15 . A method according to claim 13 , wherein the disease is skin cancer.
16 . A method according to claim 13 , wherein the reconstructive surgery is breast reconstruction following mastectomy or injury.
17 . A method according to claim 3 , wherein the skin graft is selected from the group consisting of a split thickness, a full thickness, of and a composite graft.
18 . A method according to claim 3 , wherein the skin flap is selected from the group consisting of a local flap, a regional flap, a musculocutaneous flap, an osteomyocutaneous flap and a soft tissue flap.
19 . A method according to claim 1 , wherein the contacting step comprises injecting the composition intradermally or subdermally.
20 . A method according to claim 19 , wherein the contacting comprises injection into the dermis of the skin graft or skin flap.
21 . A method according to claim 1 , wherein the contacting step comprises topical application of the composition to the skin graft or skin flap.
22 . (canceled)
23 . A method according to claim 1 wherein said VEGF-C polynucleotide further encodes a heparin-binding domain in frame with the VEGF-C polypeptide.
24 . A method according to claim 1 , wherein said polynucleotide further comprises a nucleotide sequence encoding a secretory signal peptide, wherein the sequence encoding the secretor signal peptide is connected in-frame with the sequence that encodes the VEGF-C polypeptide.
25 . A method according to claim 24 , wherein the polynucleotide further comprises a promoter sequence operably connected to the sequence that encodes the secretory signal sequence and VEGF-C polypeptide, wherein the promoter sequence promotes transcription of the sequence that encodes the secretory signal sequence and the VEGF-C polypeptide in cells of the mammalian subject.
26 . A method according to claim 25 wherein the promoter sequence comprises a skin-specific promoter.
27 . A method according to claim 26 wherein the promoter is selected from the group consisting of K14, K5, K6, K16 and alpha 1(I) collagen promoter.
28 . A method according to claim 25 wherein the polynucleotide further comprises a polyadenylation sequence operably connected to the sequence that encodes the VEGF-C polypeptide.
29 . A method according to claim 1 , wherein the composition comprises a gene therapy vector that comprises the VEGF-C polynucleotide.
30 . A method according to claim 29 , wherein the gene therapy vector is an adenoviral or adeno-associated viral vector.
31 . A method according to claim 29 wherein said vector comprises a replication-deficient adenovirus, said adenovirus comprising the polynucleotide operably connected to a promoter and flanked by adenoviral polynucleotide sequences.
32 . A method according to claim 31 wherein the adenoviral vector is present in the composition at a titer of 10 7 -10 13 viral particles.
33 . (canceled)
34 . A method according to claim 1 wherein said VEGF-C polypeptide comprises the formula X-B-Z or Z-B-X,
wherein X binds Vascular Endothelial Growth Factor Receptor 3 (VEGFR-3) and comprises an amino acid sequence at least 90% identical to a VEGFR-3 ligand selected from the group consisting of:
(a) the prepro-VEGF-C amino acid sequence set forth in SEQ ID NO: 2; and
(b) fragments of (a) that bind VEGFR-3;
wherein Z comprises a heparin-binding amino acid sequence; and
wherein B comprises a covalent attachment linking X to Z.
35 . A method according to any 1 , wherein said VEGF-C polypeptide comprises a mammalian VEGF-C polypeptide.
36 . A method according to claim 35 , wherein said VEGF-C polypeptide comprises a human VEGF-C polypeptide.
37 . A method according to claim 35 , wherein said VEGF-C polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof that binds to VEGFR-3.
38 . A method according to claim 35 , wherein said VEGF-C polypeptide comprises an amino acid sequence comprising a continuous portion of SEQ ID NO: 2, said continuous portion having, as its amino terminus, an amino acid selected from the group consisting of positions 32 to 111 of SEQ ID NO: 2, and having, as its carboxyl terminus, an amino acid selected from the group consisting of positions 228 to 419 of SEQ ID NO: 2.
39 . A method according to claim 35 , wherein the VEGF-C polypeptide selectively binds VEGFR-3.
40 . A method according to claim 39 , wherein the VEGF-C polypeptide comprises a VEGF-C156X polypeptide,
wherein the cysteine residue at position 156 of SEQ ID NO: 8 has been deleted or replaced by an amino acid other than cysteine; and
wherein the VEGF-C156X polypeptide binds human VEGFR-3 and has reduced human VEGFR-2 binding affinity relative to the prepro-VEGF-C polypeptide or a fragment thereof
41 . A method according to claim 35 , wherein the attaching step includes surgical connection of blood vessels between the underlying tissue and the skin graft or skin flap.
42 . A method according to claim 35 , wherein the contacting and attaching are performed without use of an angiogenic polypeptide that binds VEGFR-1 or VEGFR-2.
43 . A method according to claim 1 , further comprising contacting the skin graft or skin flap with an angiogenic growth factor.
44 . A method according to claim 43 , wherein the angiogenic growth factor is substantially free of vascular permeability increasing activity.
45 . A method according to claim 2 , wherein the composition further comprises a pharmaceutically acceptable carrier.
46 . A method according to claim 2 , wherein said administering comprises at least one intravascular injection of said composition.
47 . A method according to claim 2 wherein said administering comprises a patch- or dressing-mediated transfer of said composition to the skin graft or skin flap.
48 . A method according to claim 2 , wherein the mammalian subject is diabetic.
49 . A patch comprising a pad material having an upper surface and lower surface, an adhesive on the lower surface, and a therapeutic composition,
wherein the composition comprises a healing agent selected from the group consisting of Vascular Endothelial Growth Factor C (VEGF-C) polynucleotides, VEGF-C polypeptides, Vascular Endothelial Growth Factor D (VEGF-D) polynucleotides, and VEGF-D polypeptides.
50 . (canceled)
51 . A method according to claim 35 wherein said VEGF-C polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof, and binds to VEGFR-3.
52 . A method according to claim 1 wherein the healing agent comprises a VEGF-D polynucleotide that encodes a VEGF-D polypeptide.
53 . A method according to claim 52 wherein said VEGF-D polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, or a fragment thereof, and binds to VEGFR-3.
54 . A method according to claim 1 wherein the healing agent comprises a VEGF-D polypeptide.
55 . A method according to claim 54 wherein said VEGF-D polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4 or a fragment thereof that binds VEGFR-3.