Hemodynamic forces have long been postulated to regulate vessel identity, but testing this hypothesis has not been straightforward. The fact that vessel identity is established by a genetic program prior to the presence of significant and diverse hemodynamic forces has made it particularly difficult to discern later contributions of fluid forces on vessel identity, particularly in the case of pathologic flow states such as those in vascular malformations. Most experimental approaches have utilized exogenous, surgical manipulations to dramatically alter blood flow in the developing or mature cardiovascular system to test the effect of hemodynamic forces on vessel identity and cardiovascular organ formation (31
). While these approaches have provided valuable insights, whether they accurately reflect the more gradual responses that occur in the context of congenital or acquired vascular diseases is unclear. In addition, whether hemodynamic forces can permanently reprogram endothelial and vessel identity is not known. In the present study, we show that postnatal vascular remodeling in SLP76-deficient mice exposes normally formed mesenteric lymphatic vessels to flowing blood that reprograms the LECs lining these vessels as blood endothelial cells. Similar changes are observed in LECs exposed to fluid shear ex vivo, suggesting that hemodynamic forces can reprogram lymphatic vessels to blood vessels through transcriptional pathways that establish and maintain endothelial identity.
The key finding in this study is the demonstration that lymphatic vessels exposed to blood flow in vivo after birth are reprogrammed to acquire blood vessel identity. Studies performed more than 30 years ago revealed that endothelial cell turnover in large vessels such as the aorta is low but non-uniform and higher in areas of turbulent blood flow where hemodynamic forces are more varied (34
). Thus, the onset of significant fluid shear forces in the lymphatic vessels of SLP76-deficient mice could stimulate endothelial turnover and the replacement of LECs by either circulating blood endothelial cells or circulating blood endothelial precursor cells believed to contribute to the endothelium of injured or new vessels (36
). Alternatively, the gradual rise in hemodynamic shear forces may alter the gene expression of the LECs lining these vessels and reprogram them to a blood endothelial identity. Our genetic lineage tracing experiments demonstrate that virtually all of the PROX1-negative endothelial cells that line the SVs of surviving SLP76-deficient animals derive from PROX1-expressing cells. These studies therefore provide definitive evidence of molecular reprogramming of endothelial and vessel identity in response to blood flow in vivo.
Our findings provide strong evidence that hemodynamic forces underlie the reprogramming of lymphatic vessels to blood vessels in response to blood flow. Studies using Vav-Cre;Slp76fl/–
mice demonstrate that the endothelial and vessel identity changes observed do not reflect an unexpected role for SLP76 in endothelial cells, but instead arise due to changes in the vascular environment that result from blood-lymphatic vascular connections in the intestine. Radiation chimeras reconstituted with SLP76-deficient hematopoietic cells develop blood-filled mesenteric lymphatics but have little or no flow in those vessels, most likely because these mature, irradiated animals do not remodel their vasculature to create arterio-venous-lymphatic shunts like neonatal animals. These animals demonstrate that the formation of blood-lymphatic connections and contact with blood is not sufficient to alter lymphatic endothelial and vessel identity. In contrast, LECs exposed to fluid shear forces in the absence of blood ex vivo exhibit rapid downregulation of PROX1. Loss of PROX1 in LECs has been shown to result in the loss of other lymphatic identity markers such as LYVE1 and PDPN (17
), and genetic deletion of Prox1
has recently been shown to be sufficient to convert LECs to blood endothelial cells in vivo (18
). Thus, our studies support the concept that expression of the PROX1 transcription factor is required to maintain lymphatic endothelial and vessel identity (18
) and that loss of PROX1 expression in LECs exposed to fluid shear forces associated with blood flow is a likely mechanism by which lymphatic vessels are reprogrammed to blood vessels in SLP76-deficient mice.
What are the molecular pathways through which hemodynamic forces negatively regulate PROX1 and endothelial lymphatic identity? We found that HEY1 and HEY2 are strongly upregulated in coordination with downregulation of PROX1 in LECs exposed to fluid shear forces (Supplemental Figure 2), and HEY1/2 or NOTCH expression was recently demonstrated to negatively regulate lymphatic endothelial identity in cultured LECs by reducing expression of PROX1 (40
). To test the role of HEY1/2 expression and loss of lymphatic endothelial identity in SLP76-deficient mice in vivo, we have examined Slp76–/–;Hey2LacZ/+
mice, in which LacZ is expressed in place of Hey2
). However, we did not detect LacZ expression in the endothelium of mesenteric arteries or SVs, suggesting that the NOTCH/HEY signaling is not the basis for this endothelial reprogramming event in vivo. It is possible that flow-dependent activation of HEY1/2 signaling plays a transient role in this process that was not detected by these studies, but it seems more likely that ex vivo studies of cultured endothelial cells performed over hours do not fully model molecular changes that take place over weeks in vivo. Flow chamber experiments have identified large numbers of endothelial genes that are upregulated and downregulated by fluid flow, including many involved in endothelial identity (31
), but there is no definitive means of testing whether and to what extent these genes mediate endothelial responses to hemodynamic forces in vivo. Transiently blocking blood flow in very young embryos can distinguish between programmed endothelial identity gene expression and gene expression driven by fluid forces (44
), but this approach is not feasible when hemodynamic changes arise more gradually, as they do in patients with congenital vascular and cardiac defects. Thus, the identity of the molecular signals that downregulate PROX1 expression in response to fluid flow in vivo is not yet defined.
An important implication of this study is that vessel identity remains plastic after vascular development is complete and may be radically altered by hemodynamic forces later in life. The healthy mature vasculature is thought to be very quiescent, but many congenital and acquired human cardiovascular diseases are associated with persistent changes in blood flow and fluid shear forces, e.g., the left-to-right shunting of blood to the low-pressure pulmonary vasculature from the high-pressure arterial system in congenital heart disease. Molecular changes in endothelial and vessel identity are very likely to accompany the hemodynamic alterations in these diseases. Defining these molecular changes is expected to provide new insight into the pathogenesis and treatment of human cardiovascular diseases such as vascular malformations.