The importance of Prox1 in initializing differentiation was shown to be a result of its ability to reprogram blood endothelial cells to a lymphatic endothelial cell profile. This molecular switch shown in vitro suggested that Prox1 initiates the transcriptional machinery necessary for lymphangiogenesis [8, 11]. While the Prox1 knockout study as well as a tissue specific deletion of Prox1 in the endothelium  point to the necessity of Prox1 in lymphatic development, it does not provide a complete extension on the molecular players that confer the ability to reprogram the blood vasculature as suggested from in vitro studies [7, 8]. To this end, one way to address this involves the specific overexpression of Prox1 in the endothelial cell compartment. In the present study, we examine the importance of vascular reprogramming and fate specificity in vivo with a tie1/prox1 bigenic mouse model that specifically expresses Prox1 in early vascular endothelial cells.
Early expression of Prox1 in vascular endothelial cells result in no overt embryonic phenotype, however as development progresses Prox1 double transgenic embryos appear growth delayed and display more overt bleeding. Of the most serious cases, DT embryos are anemic and edematous. This correlated with the alteration of a number of molecular targets associated with vascular reprogramming such as an increase in VEGFR-3, Neuropilin-2 and STAT6 and a decrease in Neuropilin-1, VEGFR-2, Tie-2 and CyclinE2 [7, 8, 12]. Examination of the most severely affected DT embryos show a general degradation of the embryonic structure, separation of the epidermis and enlarged lymph sacs when compared to their wild type counterparts. Furthermore, vascular structures such as the jugular vein, which normally express low levels of VEGFR-3 and Podoplanin are now highly positive for these lymphatic markers. In conjunction with previous data demonstrating the importance of Prox1 in BEC differentiation to LECs, we present data that provides further in vivo evidence of the molecular players involved in the reprogramming of BECs to LECs early in lymphatic vascular development. Consistent with our data, it was recently observed that Sox18 is upstream of Prox1; in vitro overexpression of Sox18 in embryonic stem cells and vascular endothelial cells results in the upregulation of Prox1 and an increase in lymphatic markers such as Podoplanin, indirectly confirming our data presented here . In addition, the conditional deletion of Prox1 results in the dedifferentiation of lymphatic endothelial cells to that of a more vascular endothelial cell-like phenotype . This further suggests that Prox1 is critical for the maintenance of the lymphatic endothelial cell fate, and that endothelial cells are genetically malleable. Our in vivo study further confirms the importance of Prox1 in generating a lymphatic profile by altering the gene signature of vascular endothelial cells.
One consequence that results from the overexpression of Prox1 in the vascular compartment of the developing embryo is edema. At the histological level, we observe the separation of the dermal layer from the epidermis as a result of fluid leakage, characteristic of an improper lymphatic drainage system. Furthermore, a prominent feature of the most severely affected embryos is the enlargement of the lymph sacs. It is unclear as to the mechanism of the increased size, whether it is from an increase in the number of differentiating early lymphatic endothelial cells budding from the jugular vein, or from an increase in proliferation of committed lymphatic endothelial cells that are in the process of or have already formed the lymph sac. Interestingly, early analysis of E10.5 DT embryos show an increase in Prox1 positive cells in the periphery; this increase in population may play a contributing factor to the increase in lymph sac size. The potential for an increase in proliferation due to Prox1 overexpression is consistent with previously published work that identifies the upregulation of a number of cell cycle targets such as CyclinE2 and p57kip .
The data suggests that the overexpression of Prox1 during early embryogenesis results in the reprogramming of vascular endothelial cells to a more lymphatic cell fate resulting in edema, hemorrhaging and death. While it demonstrates the importance of tightly regulating Prox1 in development, it is unclear as to why the hemorrhaging occurs. In our model, one can hypothesize that the overexpression of Prox1 may influence junctional proteins involved in maintaining the integrity of cell-cell contacts; alterations in these same complexes may result in changes in vascular permeability leading to a more permeable, lymphatic-like state . Indeed, upon Prox1 overepression changes in junctional proteins are observed away from the normal molecular profile associated with control embryos. Previous studies have shown that increases as well as decreases in junction protein levels have been shown to be associated with an increase in permeability [16, 17], suggesting that a balance is required in order to maintain the integrity of the cell-cell interface. Recent data has found that the overexpression of Prox1 in colon epithelial cells result in dysplasia and transformation that correlates with changes in cellular adhesion . Furthermore, conditional deletion of Prox1 results in aberrant junctional complex formation and abnormal pericyte association . These lines of evidence point to the importance of cell-cell integrity in the regulation of vascular permeability. Moreover, the controlled expression of Prox1 is critical for normal lymphatic development in the embryo.
It is clear that the measured expression of Prox1 as well as other lymphangiogenic factors during embryonic development is essential for the proper formation of the lymphatic vasculature. Not surprisingly, compromising this regulated expression has been found to result in negative clinical consequences. For example, the growth of lymphatic endothelium in tumors is a result of the presence of VEGF-C, which acts on lymph vessels to enhance metastasis . Other events include the transformation and cellular reprogramming of spindle-like lesional cells by Karposi sarcoma herpesvirus (KHSV)/human herpesvirus-8 (HHV8). Interestingly, gene profiling of KHSV infected lesional cells produce a signature similar to that derived from a lymphatic endothelial cell; targets that include Prox1, LYVE-1 and VEGFR-3. This suggests that Karposi sarcoma genetically mimics an expression pattern similar to a differentiated lymphatic endothelial cell [19–21].