In this study, we have identified a previously unrecognized, protective role for Bmper in the setting of atherosclerosis. ApoE−/− mice, who develop an atherosclerotic phenotype when fed a high fat diet, displayed significantly worsened symptoms when they carried only one phenocopy of the Bmper gene (Bmper+/−;ApoE−/− mice). Not only did the decrease in Bmper levels in these mice cause a dramatic increase in lesion size, it also resulted in increased arterial calcification and a heightened induction of endothelial inflammatory adhesion molecules in the aorta in regions subjected to oscillatory and laminar shear stress. Similar results were found in cultured cell studies, solidifying the notion that Bmper is a critical regulator of vascular inflammation and broadening our understanding of the role that Bmper plays in the myriad of events that result from the Bmp signaling pathway.
Previously, our studies revealed the essential roles of Bmp and Bmper signaling in endothelial cell differentiation, migration and angiogenesis13, 14, 16, 17, 23, 24
. With the results from the present study, we can now add vascular inflammation to the growing list of Bmp signaling events that are regulated by Bmper. Bmper+/−
mice demonstrated increased Smad activation and expression of the inflammatory markers ICAM1 and VCAM1 in the endothelium layer of the lesser curvature of the aorta, a region known to be predisposed to atherogenic activity due to Bmp-mediated vascular inflammation brought about by oscillatory shear stress effects on endothelial cells (). In addition, decreased levels of Bmper in Bmper+/−
mice led to an increase in the number of macrophages that were recruited and that migrated into the inflamed, atherogenic regions of the aortas (), supporting the notion that Bmper acts as a protective regulator of vascular inflammation. It is worth noting however, that Bmper may also play a role in maintaining general vascular health in addition to its role in inflammatory responses. Our analysis of aortas taken from ApoE+/+
mice that were haploinsufficient for Bmper (Bmper+/−
mice) revealed a significant increase in the area of atherosclerotic lesions in mouse fed a regular diet (). This suggests that Bmper may be important in maintaining vascular health even under basal conditions, a theory that will require further experiments to determine.
As compelling as these in vivo
results are, however, it is important to remember that the decrease in Bmper expression in the Bmper+/−
mouse is not limited to endothelial cells. Since atherosclerosis is a pathological condition that results from the dysfunction of multiple cell types and involves different cellular events, it is not possible, from these in vivo
studies, to be able to localize the protective effect of Bmper to endothelial cells alone. Indeed, published reports demonstrate that Bmps enhance smooth muscle cell migration and induce proinflammatory factors such as inducible nitric oxide synthase (iNOS) and TNF in macrophages25, 26
. The inhibition of Bmp activity by specific inhibitors and antagonist decreases vascular calcification, suggesting important roles of Bmp in vascular calcification as well as early vascular injury10, 11
. Therefore, it is entirely possible that Bmper, a secreted extracellular Bmp modulator, may also be able to influence Bmp activity not only in endothelial cells but in additional cell types such as smooth muscle cells and macrophages. Therefore, the contribution of Bmper to protection against atherogenic processes and vascular inflammation will need further investigation.
As mentioned above, a number of published reports have detailed the role of Bmper/Bmp signaling in various aspects of endothelial cell function13, 14, 16, 17, 23, 24
. However, the ability of Bmper to inhibit endothelial inflammatory responses has not been investigated directly. In order to examine this aspect of endothelial Bmp signaling, we cultured HUVECs and subjected them to fluid shear stress to induce an inflammatory response similar to what occurs within atherogenic areas of the aorta. We found that Bmper inhibits the inflammatory response usually elicited from endothelial cells in response to oscillatory shear stress ( and ). This result is consistent with other reports demonstrating that the expression of Bmper and other Bmp antagonists is increased by oscillatory shear stress compared to laminar shear stress8
. Surprisingly, however, we also found that Bmper also exerts an anti-inflammatory effect on endothelial cells exposed to laminar shear stress. Laminar shear stress promotes endothelial survival and integrity by activating MAP kinases such as ERK/BMK1 and endothelial NOS (eNOS) signaling4
. Our data have demonstrated that Bmper is also involved in protecting endothelial cells exposed to laminar shear stress, partly due to increases in activity of eNOS and decreases ICAM1 and VCAM1 expression () that have recently been attributed to Bmper activity in endothelial cells19
. The exact molecular mechanisms through which Bmper modulates eNOS activity and other signaling pathways of laminar shear stress need further investigation.
Bmper has been identified as a critical regulator of Bmp signaling activity, important in both vascular development and in hypoxia-induced retinal neovascularization13, 14
. Previously we reported a gradient effect of Bmper's ability to influence Bmp signaling, whereby superstoichiometric concentrations of Bmper compared to Bmp inhibit Bmp signaling and substoichiometric concentrations of Bmper compared to Bmp activate Bmp signaling13
. In this study, we observed 61% and 15% higher Bmp4 and Bmper expression in oscillatory shear stress conditions, resulting in a significantly lower Bmper to Bmp4 ratio compared to laminar shear stress conditions, an expression pattern consistent with an anti-inflammatory role for Bmper (). This observation further suggests that the fine-tuning of Bmp activity by Bmper is essential for modulating Bmp-mediated cellular functions. Collectively, the data presented in this report demonstrates that the regulation of Bmp activity by Bmper is essential for the maintenance of normal vascular homeostasis and its disruption increases the risk of inflammatory vascular diseases such as atherosclerosis.