Blood and lymphatic endothelial cell-specific differentiation programs are stringently controlled by the tissue environment

Abstract

The discovery of marker proteins of human blood (BECs) and lymphatic endothelial cells (LECs) has allowed researchers to isolate these cells. So far, efforts to unravel their transcriptional and functional programs made use of cultured cells only. Hence, it is unknown to which extent previously identified LEC-and BEC-specific programs are representative of the in vivo situation. Here, we define the human BEC-and LEC-specific in vivo transcriptomes by comparative genomewide expression profiling of freshly isolated cutaneous EC subsets and of non-EC skin cells (fibroblasts, mast cells, dendritic cells, epithelial cells). Interestingly, the expression of most of the newly identified EC subset-discriminating genes depends strictly on the in vivo tissue environment as revealed by comparative analyses of freshly isolated and cultured EC subsets. The identified environment-dependent, EC subsetrestricted gene expression regulates lineage fidelity, fluid exchange, and MHC class II-dependent antigen presentation.As an example for a BEC-restricted in vivo function, we show that non-activated BECs in situ, but not in vitro, assemble and display MHC class II protein complexes loaded with self-peptides. Thus, our data demonstrate the key importance of using precisely defined native IntroductionThe microvasculature is actively involved in metabolism and immune cell trafficking. Although the supply with oxygen and nutrients as well as the attraction of leukocytes is accomplished by blood vessels, the removal of extracellular fluid from the tissues and the guidance of immune cells to lymph nodes are carried out by lymphatic vessels. At the molecular level, these different functional capabilities are likely encoded by the transcriptional repertoires of blood vessel endothelial cells (BECs) and lymphatic ECs (LECs). Recently, the discovery of the LEC-specific marker proteins podoplanin (PDPN) and LYVE-1 allowed researchers to isolate and propagate BECs and LECs in vitro. 1,2 Accordingly, several techniques, including genomewide expression profiling coupled to bioinformatic approaches, have been used to identify the spectrum of genes that are expressed in cultured LECs and BECs. [3][4][5][6] However, probably as a result of technical limitations, genomewide analyses were not performed on freshly isolated ECs. Thus, it is possible that ECs, in their tissue-resident state, have a transcriptional and, thus, functional repertoire that differs from that of cultured ECs. In fact, the specialized ECs of high endothelial venules rapidly lose EC subset-defining antigen expression and morphology when these cells were placed in culture. 7 This supports that active signal exchange between ECs and the local tissue environment can also be of critical importance for the maintenance of differentiation and function of LECs and BECs.To address whether and, if so, to which extent tissue-resident LECs or BECs resemble their cultured counterparts, we established for the first time the complete transcriptomes of freshly isolated cutaneous LECs, BECs, a...

Phospholipase Cγ2 is necessary for separation of blood and lymphatic vasculature in mice

“…This expression pattern may be due to altered gene expression caused by changes in the EC microenvironment (Amatschek et al, 2007). These results suggest that PLCγ2 may not function in differentiated LECs that have become separated from blood vessels; however, we cannot exclude the possibility that transient expression of PLCγ2 in EC-lineage cells in a temporal and spatial manner is involved in the separation process.…”

D6 and the atypical chemokine receptor family: Novel regulators of immune and inflammatory processes

“…This is in contrast to DARC, which displays a reciprocal expression pattern with no expression on LEC but high expression on blood endothelial cells [23]. Interestingly, a number of array analyses comparing the transcriptional profiles of lymphatic and blood endothelial cells, cultured in vitro, have failed to pick up D6 as a significantly differentially expressed transcript [70][71][72]. This suggests that there is some context-specific regulation of D6 expression, which is lost upon in vitro culture.…”

Fluid Flow Regulates Stromal Cell Organization and CCL21 Expression in a Tissue-Engineered Lymph Node Microenvironment

“…These findings highlight the notion that naturally occurring biophysical conditions are necessary for these cells to be maintained in an activated and functional state. The partial dedifferentiation seen with TRCs cultured in 2D is reminiscent of observations with other cells isolated ex vivo, e.g., high endothelial venule cells from LNs and cutaneous endothelial cells (58,59). Thus, the chemokine environment in the LN is likely to be cooperatively regulated and perhaps fine tuned by the combination of biophysical as well as biochemical signals.…”