Mechanisms of xenogeneic baboon platelet aggregation and phagocytosis by porcine liver sinusoidal endothelial cells

Abstract

Background: Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies.

Methods: Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-α(M)β(2) integrin Ab) were tested for the ability to inhibit phagocytosis.

Results: None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01).

Conclusions: Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis.

Figure 1. Cell culture and identification.

Porcine aortic endothelial cells (a), hepatocytes (b), and liver sinusoidal endothelial cells (c) were in culture. The primary cells where then tested by anti- CD31 antibody(d), anti-hepatocyte specific antigen antibody (OCH1E5) (e), and Dil-Ac-LDL up-take(f) and examined under fluorescence microscope.To understand MAC-1 receptor expressed in liver sinusoidal endothelial cells,flow cytometry test for MAC-1 and CD14 in cells was done(g). The expression rate of Mac-1 in cells was 14.2%. In another hand, CD14 expression in cells was negative. It meaned that all the cells were not Kupffer cells.

Figure 2. Pig platelets aggregation in different pig cells.

(a) Pig or (b) baboon platelets (2×10⁸) were stimulated by collagen (0.5 ug/ml) and showed strong aggregation. When pig platelets (2×10⁸) were mixed with (c) pig hepatocytes, (d) pig aortic endothelial cells and (e) pig liver endothelial cells separately (2×10⁶), no platelet aggregation was observed.

Figure 3. Baboon platelets aggregation in different pig cells.

Baboon platelets (2×10⁸) were mixed with (a) pig hepatocytes, (b) pig aortic endothelial cells and (c) pig liver endothelial cells separately (2×10⁶).Mild platelet aggregation was observed, corresponding to 10.8±0.7%, 10.5±0.4%, and 10.4±0.4%. There was no significant difference between these various groups (P>0.05).

Figure 4. Platelets phagocytosis in different cells.

In (A) CFSE labeled porcine platelets (green) were mixed with pig hepatocytes, aortic endothelial cells and liver endothelial cells (stained by CellTracker™ Blue CMAC, blue), co-cultured for 1 h at 37°C. No phagocytosis was observed (confocal microscopy×400).In (B), CFSE labeled baboon platelets (green) were mixed with pig hepatocytes, aortic endothelial cells and liver endothelial cells (stained by CellTracker™ Blue CMAC, blue), co-cultured for 1 h at 37°C. Platelet internalization by hepatocytes, aortic endothelial cells and liver endothelial cells was absent, mild and strong, respectively (confocal microscopy ×400). In (C), baboon platelets (labeled by CFSE, green) are rapidly internalized by pig liver endothelial cells (stained by CellTracker™7-amino-4-chloromethylcoumarin, blue) (confocal microscopy ×400). The left panel shows the sequential section analysis of a single endothelial cell that has internalized platelets, the sections go from top to bottom. The right panel shows the analysis of one section with simultaneous indication of location of platelets within the endothelial cell, indicating that these are clearly internalized and not on the cell surface.

Figure 5. Platelets phagocytosis blocked by different drugs.

Phagocytosis index of baboon platelets and pig cells (phagocytosis index = number of platelets internalized per 100 cells and expressed as means ± SD of values from three independent experiments. In (A), no drug was added to the pig cell- baboon platelet combinations (#is statistically significant p<0.01). In (B), various drugs (aspirin, aurintricarboxylic acid, eptifibatide, or ICRF44 were added to the pig liver endothelial cell- baboon platelet combinations, (#is significantly lower when compared with no drug, P<0.01).