Reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and intimal hyperplasia.
BACKGROUND
fat derived microvascular endothelial cells (MVEC) seeded on prosthetic vascular grafts, improve patency in animals. Results in humans were disappointing, due to thrombogenicity and progressive intimal hyperplasia. Also in animals intimal hyperplasia was found. We postulate that contaminating cells present in the transplant are involved in the intimal hyperplasia. We developed a method to further purify human MVEC from 40-90%. Here we tested the effects of enrichment upon thrombogenicity and seeding-related intimal hyperplasia.


METHODS
liposuction fat was enzymatically digested and centrifuged. To enrich MVEC, contaminating macrophages and fibroblasts were removed with dynabeads coated with macrophage- and fibroblast-specific antibodies. Thrombogenicity was assessed by measuring tissue factor and thrombomodulin activity, presence of endothelial nitric oxide synthase and via perfusion of the cells with whole blood. To investigate seeding-related intimal hyperplasia, PTFE grafts were seeded with the cells and cultured for 3 weeks.


RESULTS
tissue factor activity of purified cells was reduced compared to nonpurified cells. Purified cells showed thrombomodulin activity and eNOS expression. Fragment 1+2 and Fibrinopeptide A generation after perfusion of purified cells were significantly lower than after perfusion of nonpurified cells, and only nonpurified cells were covered with platelets and fibrin. Prostheses seeded with nonpurified cells showed an EC monolayer above a multilayer of myofibroblasts, prostheses seeded with purified cells only showed a single EC monolayer. Mixing experiments with human umbilical cord EC (HUVEC) and fibroblasts showed that when more than 25% HUVEC were present a confluent EC layer was formed. When the amount of fibroblasts was 25% or less, no development of a subendothelial multilayer of myofibroblasts was found within 3 weeks.


CONCLUSIONS
reduction of non-endothelial cell contamination of microvascular endothelial cell seeded grafts decreases thrombogenicity and might prevent seeding-related intimal hyperplasia.
Arts
C H P
CH
Department of Vascular and Transplantation Surgery (G04.228), University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands.
Blankensteijn
J D
JD
Heijnen-Snyder
G J
GJ
Verhagen
H J M
HJ
Hedeman Joosten
P Ph A
PP
Sixma
J J
JJ
Eikelboom
B C
BC
de Groot
P G
PG
D002469:Cell Separation; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation*; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D006965:Hyperplasia / Q000378:metabolism / Q000601:surgery; D007150:Immunohistochemistry; D008859:Microscopy, Polarization; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III; D011138:Polytetrafluoroethylene / Q000627:therapeutic use; D018180:Thrombomodulin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism; D013927:Thrombosis / Q000209:etiology*; D016378:Tissue Transplantation; D016896:Treatment Outcome; D017539:Tunica Intima / Q000378:metabolism / Q000473:pathology* / Q000637:transplantation*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism / Q000637:transplantation
D018180:Thrombomodulin; D011138:Polytetrafluoroethylene; D013925:Thromboplastin; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III