| Popis: |
Hepatic endothelial cells constitute 25% of all liver cells and exhibit unique properties, including coagulation factor synthesis, participation in host immune responses, paracrine cell signaling, etc. In short-term assays, we recently showed that intraperitoneal transplantation of healthy LSEC in hemophilia A mice restored FVIII activity with phenotypic correction and intraportally transplanted LSEC could be targeted to mouse liver. To determine the potential of genetically-manipulated LSEC, we isolated LSEC by collagenase perfusion of mouse liver followed by Percoll-gradient separation. LSEC were 75% CD31+ and 25% were CD45+, 10% of which were F4/80+. Isolated LSEC were efficiently transduced by lentiviral vectors (LV) and expressed the GFP in culture conditions under an ubiquitous PGK promoter and several endothelial-specific promoters. Further analysis of these endothelial promoters, Tie2, Flk-1, VE-cadherin and ICAM-2, in LSEC and hepatocytes showed that the promoters retained activity in LSEC but were largely inactive in hepatocytes. Moreover, LV-modified primary LSEC survived in the liver of recipients. In parallel studies, to further address whether LSEC will engraft, proliferate and function in the liver of intact animals, we isolated LSEC from transgenic FVB/N Tie-2-GFP mice. Cells were transplanted intraportally into syngeneic FVB/N mice followed by analysis of recipient livers at multiple intervals. Transplanted LSEC expressing the endothelially-restricted Tie2-GFP were observed in the liver of FVB/N mice for the entire 3 month-duration of the studies using cyclophosphamide to inhibit host responses against GFP. EM studies established that transplanted LSEC were incorporated in the sinusoidal endothelial lining of the liver. Immunostaining studies verified that GFP-expressing transplanted LSEC were distinct from Kupffer cells, stained with the F4/80 marker. To further determine whether engraftment and proliferation in transplanted LSEC could be induced by prior endothelial injury in recipients, we used monocrotaline (MCT), a pyrollizidine alkaloid, which damages the vascular endothelium. In MCT-conditioned mice, engraftment of transplanted Tie2-GFP- LSEC significantly improved (e.g., 5-fold after 1 week). Moreover, transplanted LSEC proliferated in mice preconditioned with MCT, such that FACS analysis of Tie-2-GFP LSEC recovered from liver of FVB/N recipients over a 3-month period indicated that 25% of the LSEC compartment was reconstituted. The identity of recovered Tie-2-GFP LSEC was verified by CD31 expression. Also, transplanted Tie-2-GFP LSEC incorporated Dil-Ac-LDL in vivo, which indicated their functional integrity. Conclusions: Primary LSEC were amenable to LV-mediated gene transfer and correctly regulated endothelial promoters. Transplanted LSEC survived and functioned indefinitely in the liver. The ability to reconstitute the liver with transgenic LSEC will offer new ways to investigate biological mechanisms and to pursue cell and gene therapy in hemophilia A and in other suitable disorders. |
