title: Adventitial transplantation of blood outgrowth endothelial cells in porcine haemodialysis grafts alleviates hypoxia and decreases neointimal proliferation through a matrix metalloproteinase-9-mediated pathway--a pilot study.
abstract: Purpose. We hypothesized that adventitial transplantation of blood outgrowth endothelial cells (BOEC) to the vein-to-graft anastomosis of polytetrafluoroethylene grafts will reduce neointimal hyperplasia by reducing hypoxia inducible factor-1alpha (HIF-1alpha), by increasing angiogenesis in a porcine model of chronic renal insufficiency with haemodialysis polytetrafluoroethylene grafts. Because matrix metalloproteinases (MMPs) have been shown to be involved with angiogenesis, the expression of MMPs and their inhibitors was determined.
  
  
  METHODS
  Chronic renal insufficiency was created by subtotal renal infarction and 28 days later, arteriovenous PTFE grafts were placed bilaterally from the carotid artery to the jugular vein. Autologous blood outgrowth endothelial cells labeled with Lac Z were transplanted to the adventitia of the vein-to-graft anastomosis using polyglycolic acid scaffolding and scaffolding only to other side (control). Animals were killed 14 days later and vessels were explanted from the vein-to-graft anastomosis of both sides and underwent immunohistochemical analysis, western blotting and zymography for HIF-1alpha, MMP-2, MMP-9, TIMP-1 and TIMP-2. BOEC were also made hypoxic and normoxic for 12, 24 and 48 h to determine protein expression for MMPs and TIMPs.
  
  
  RESULTS
  Under hypoxia, BOEC significantly increased the expression of pro MMP-2 by 12 h and TIMP-2 by 24 h when compared to normoxic cells (P < 0.05). Transplantation of BOEC resulted in a significant decrease in both HIF-1alpha and intima-to-media ratio with a significant increase in both pro and active MMP-9 when compared to control vessels (P < 0.05). MMP-9 activity was localized to the neointima of the transplanted vessels by immunohistochemistry. There was increased CD31 density with engraftment of BOEC cells into the neointima of both the transplanted vessels compared to controls (P = NS).
  
  
  CONCLUSIONS
  Transplantation of BOEC resulted in a significant decrease in intimal hyperplasia and HIF-1alpha with a significant increase in both pro and active MMP-9 that was localized to the neointima of transplanted vessels. The increase in MMP-9 offers a possible mechanism for angiogenesis and the reduced intima-to-media ratio. Furthermore, we observed that BOEC had homed to the neointima of the contralateral vessels that had increased levels of HIF-1alpha, suggesting that hypoxia may be an important stimulus for BOEC migration.
authors.affiliation: Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Deborah
authors.initials: D
authors.lastname: Hughes
authors.forename: Alex A
authors.initials: AA
authors.lastname: Fu
authors.forename: Alessandra
authors.initials: A
authors.lastname: Puggioni
authors.forename: James F
authors.initials: JF
authors.lastname: Glockner
authors.forename: Bilal
authors.initials: B
authors.lastname: Anwer
authors.forename: Antonio M
authors.initials: AM
authors.lastname: McGuire
authors.forename: Debabrata
authors.initials: D
authors.lastname: Mukhopadhyay
authors.forename: Sanjay
authors.initials: S
authors.lastname: Misra
mesh_terms: D000818:Animals; D001166:Arteriovenous Shunt, Surgical / Q000379:methods*; D001807:Blood Vessel Prosthesis; D002408:Catheters, Indwelling; D015687:Cell Hypoxia / Q000502:physiology; D003238:Connective Tissue / Q000601:surgery; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism* / Q000637:transplantation*; D004792:Enzyme Precursors / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D017930:Genes, Reporter; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007763:Lac Operon; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D018919:Neovascularization, Physiologic; D010865:Pilot Projects; D011138:Polytetrafluoroethylene; D006435:Renal Dialysis; D051436:Renal Insufficiency, Chronic / Q000628:therapy; D034421:Sus scrofa; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D054457:Tissue Scaffolds; D014182:Transplantation, Autologous; D017539:Tunica Intima / Q000473:pathology
chemical_list: D004792:Enzyme Precursors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019716:Tissue Inhibitor of Metalloproteinase-2; D011138:Polytetrafluoroethylene; D018093:Gelatinases; C069860:progelatinase; D020780:Matrix Metalloproteinase 9
references.citation: Arterioscler Thromb Vasc Biol. 2004 Apr;24(4):650-7
references.pmid: 14764423
references.citation: J Surg Res. 2006 Oct;135(2):370-9
references.pmid: 16815448
references.citation: Radiology. 1993 Mar;186(3):867-72
references.pmid: 8430200
references.citation: J Clin Invest. 2000 Jan;105(1):71-7
references.pmid: 10619863
references.citation: Circulation. 2003 Sep 23;108(12):1520-6
references.pmid: 12952850
references.citation: J Vasc Interv Radiol. 2008 Feb;19(2 Pt 1):252-9
references.pmid: 18341958
references.citation: J Biol Chem. 2003 Aug 29;278(35):32994-9
references.pmid: 12816956
references.citation: Am J Physiol Renal Physiol. 2007 Apr;292(4):F1132-40
references.pmid: 17200161
references.citation: J Clin Invest. 2003 Jul;112(2):160-9
references.pmid: 12865405
references.citation: Nephrol Dial Transplant. 2008 Feb;23(2):525-33
references.pmid: 18037619
references.citation: Kidney Int. 2005 Dec;68(6):2890-900
references.pmid: 16316367
references.citation: Cardiovasc Res. 2008 Jun 1;78(3):440-8
references.pmid: 18218684
references.citation: Cardiovasc Intervent Radiol. 2006 Jul-Aug;29(4):624-9
references.pmid: 16729233
references.citation: Circ Res. 1994 Jul;75(1):181-9
references.pmid: 8013077
references.citation: Nat Rev Cancer. 2003 Oct;3(10):721-32
references.pmid: 13130303
references.citation: Am J Kidney Dis. 2003 Dec;42(6 Suppl 5):A5-7, S1-230
references.pmid: 14655179
references.citation: Kidney Int. 1999 May;55(5):2078-90
references.pmid: 10231476
references.citation: Nat Med. 2004 Aug;10(8):858-64
references.pmid: 15235597
references.citation: Am J Physiol. 1998 Sep;275(3):H1103-14
references.pmid: 9724319
references.citation: Breast Cancer Res. 2008;10(3):R44
references.pmid: 18474097
references.citation: Kidney Int. 2001 Jun;59(6):2325-34
references.pmid: 11380837
references.citation: Nat Cell Biol. 2000 Oct;2(10):737-44
references.pmid: 11025665
references.citation: Nature. 2001 Apr 5;410(6829):701-5
references.pmid: 11287958
references.citation: Science. 1997 Feb 14;275(5302):964-7
references.pmid: 9020076
references.citation: Biomaterials. 2004 Jun;25(14):2799-805
references.pmid: 14962558
references.citation: Cell. 1996 Aug 9;86(3):353-64
references.pmid: 8756718
references.citation: Clin Sci (Lond). 2005 Jul;109(1):27-37
references.pmid: 15966869
references.citation: Circ Res. 2004 Feb 6;94(2):262-8
references.pmid: 14670843
references.citation: J Cell Biol. 2005 May 23;169(4):681-91
references.pmid: 15911882
references.citation: J Vasc Interv Radiol. 2008 Apr;19(4):587-94
references.pmid: 18375305
references.citation: Am J Physiol Heart Circ Physiol. 2008 May;294(5):H2219-30
references.pmid: 18326810
references.citation: Anat Rec A Discov Mol Cell Evol Biol. 2003 Jun;272(2):526-37
references.pmid: 12740947
references.citation: Lancet. 2000 Jul 8;356(9224):147-52
references.pmid: 10963260
references.citation: J Vasc Interv Radiol. 2008 Feb;19(2 Pt 1):260-5
references.pmid: 18341959
references.citation: Biochem J. 1993 Dec 15;296 ( Pt 3):803-9
references.pmid: 8280080
references.citation: Int J Biochem Cell Biol. 1997 Oct;29(10):1167-77
references.pmid: 9438380
references.citation: J Vasc Surg. 2004 Feb;39(2):432-9
references.pmid: 14743149
references.citation: Circulation. 2005 Sep 13;112(11):1618-27
references.pmid: 16145003
references.citation: J Cell Mol Med. 2005 Apr-Jun;9(2):267-85
references.pmid: 15963249