Tissue engineering of endothelial cells and the immune response

Abstract

Background: While tissue engineering offers promise for organ and tissue transplantation, it can also be used to examine transplant and immune biology. Endothelial cells engrafted within 3-dimensional matrices create stable units that produce all of the factors of a functional quiescent endothelium. Perivascular implantation of tissue engineered endothelial cell constructs provides long-term control of vascular repair after injury. This control is established without restoration of the natural luminal:mural endothelium, and most intriguingly, without engendering host allo- and xenogeneic immune responses. We examined how endothelial immunogenicity is controlled by interaction with 3-dimensional matrices.

Materials and methods: Human aortic endothelial cells (HAE) were either grown to confluence on polystyrene tissue culture plates or within 3-dimensional collagen-based matrices. Major histocompatibility complex (MHC) class II, integrin, interferon (IFN)-gamma receptor expression, and signaling were analyzed via confocal microscopy, flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), and microarray. Splenocyte proliferation was assayed by thymidine incorporation.

Results: Despite similar expression levels of IFN-gamma receptors, matrix-embedded HAE elicited far less STAT-1 phosphorylation upon IFN-gamma stimulation, and expressed 2-fold less MHC II than HAE grown to confluence on culture plates (P < .001). This effect correlated with reduced expression of integrin alpha(v) and beta(3) (P < .002), and muted proliferation of porcine splenocytes (P < .001).

Conclusions: Matrix architecture is critical for modulation of endothelial immunogenicity. Embedding HAE within a physiologic 3-dimensional environment affects activity of intracellular signaling pathways, MHC II expression, and subsequent activation of immune cells. These findings might offer novel insights into our understanding of endothelial-mediated diseases and might enhance our ability to leverage the potential for cell-based therapies.

Figure 1. Matrix embedding attenuates IFN-γ induced endothelial MHC class II expression

A 10⁴ endothelial cells were analyzed for surface expression of MHC class II expression via flow cytometry. B RT-PCR analysis revealed reduced mRNA transcript levels of HLA-DRα in matrix-embedded HAE. C Spearman correlation of HLA-DRα mRNA transcript levels and MHC class II surface expression on HAE. Area of the density ellipse represents the 95% confidence interval.

Figure 2. Though expression of IFN-γ receptors was similar in HAE grown on TCPS or cultured within a three-dimensional matrix, matrix-embedding attenuates intracellular signaling pathways downstream of IFN-γ binding to its receptor

A IFN-γ receptor subunit II analyzed by immunofluorescence confocal microscopy (100X). B Matrix-embedding has no influence on endothelial expression of IFN-γ as revealed via flow cytometry for IFN-γ receptor chain 1 and 2. C Western blot demonstrated attenuated phosphorylation of STAT-1 after IFN-γ stimulation for indicated time periods in matrix-embedded HAE when compared to HAE grown to confluence on tissue culture polystyrene plates.

Figure 3. Matrix-embedded endothelial cells exhibit an altered integrin expression

A Microarray analysis (A) and RT-PCR (B) revealed significant differences in integrin expression between matrix-embedded endothelial cells and endothelial cells grown to confluence on tissue culture polystyrene plates.

Figure 4

Unstimulated HAE did not evoke xenogeneic splenocyte proliferation. HAE-stimulation with IFN-γ resulted in a significant proliferative response of splenocytes, which was significantly greater against HAE grown to confluence on tissue culture plates compared to matrix embedded cells. The presence of MHC II antibody blocked splenocyte proliferation in response to IFN-γ treated HAE. Each value represents mean±SD.