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.
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.
Hughes
Deborah
D
Division of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.
Fu
Alex A
AA
Puggioni
Alessandra
A
Glockner
James F
JF
Anwer
Bilal
B
McGuire
Antonio M
AM
Mukhopadhyay
Debabrata
D
Misra
Sanjay
S
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
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
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