Building a Vessel Wall with Notch Signaling (original) (raw)

. Author manuscript; available in PMC: 2010 Feb 27.

Blood vessel walls are built and maintained by two interdependent cell types, endothelial cells and mural cells. Signals exchanged between these two cell types control the formation, maturation, remodeling and function of microvascular networks. Mural cells are either pericytes in the microvasculature or smooth muscle cells (SMCs) in larger vessels. Interactions between endothelial cells and mural cells serve to stabilize nascent capillary vessels, provide endothelial cell survival factors, inhibit endothelial cell proliferation, promote mural cell differentiation and guide vascular network remodeling.1,2 Yet precisely how endothelial cells recruit mural cells to form a functional vessel wall is still incompletely understood.

Mural Cell Investment

The initial steps in this process of vessel wall formation, called investment, involve the establishment of cell-cell contacts between endothelium and incoming mural cells, which triggers a self-reinforcing mechanism to maintain the contacts and initiate the next phase of vessel wall maturation. N-cadherin appears to be important for heterotypic cell-cell adhesion between endothelial cells and nascent SMCs in the embryonic day 12.5 (E12.5) dorsal aorta.3 As vascular development proceeds, an iterative investment sequence is set into motion, as is evident by the multilayered structure of the tunica media that is produced in large vessels. In the microvasculature, communication between endothelial cells and mural cells prevents vascular regression and promotes maturation processes in both cell types.2 Investment of microvessels with pericytes is a reversible process that is highly responsive to changes in the rate and direction of blood flow.4 Although these basic steps of vessel wall investment have been known for many years, the molecular mechanisms that are responsible for morphogenesis of this structure are still not well understood.

Endothelial Cell-Mural Cell Contact Induces Notch3 in Mural Cells

In this issue of Circulation Research, a report by Liu et al begins to identify some of the key early players in this process.5 In particular, the authors showed that Notch3 is one of the genes most highly induced when mural cells are cocultured with endothelial cells under conditions that promote cell-cell contact. Human umbilical artery and coronary artery SMCs, bovine retinal pericytes, and human neonatal dermal fibroblasts (HNDFs, used to emulate natural mural progenitor cells) all greatly enhanced vascular network formation by human umbilical vein endothelial cells (HUVEC) in three-dimensional collagen gel in vitro coculture assays. HUVEC-mural cell coculture stimulated Notch3 expression in the mural cell partners. By contrast, no induction of Notch3 expression was found in HepG2 liver cells cocultured with HUVEC under identical conditions. Tang et al also reported that notch signaling was activated in human aortic SMCs that were cocultured with human aortic endothelial cells.6 Canonical notch signaling is initiated when ligand binding induces cleavage of the notch receptor by a membrane-associated γ-secretase complex, thus releasing an intracellular domain of the receptor (NotchICD). Upregulation of Notch3 expression in mural cells was accompanied by activation of a NotchICD-dependent signaling pathway and increased expression of known Notch target genes, including Hes1 and HEYL/HRT3. Moreover, the authors showed that inhibiting the Notch pathway strongly attenuated the upregulation of Notch3, suggesting that activated Notch3 promotes its own expression through auto-activation. The upregulation of Notch3 expression in mural cells was dependent upon mural cell contact with endothelial cells expressing Jagged1.

Endothelial Cell Contact Promotes Mural Cell Maturation

Of interest were the observations made by Liu et al5 that increased expression of Notch3 in mural cells was accompanied by upregulation of expression of SMC differentiation marker genes SMα-actin, calponin and SM-myosin heavy chain (SM-MHC). The increases in SMC marker gene expression were dependent upon Notch3 expression in mural cells, since siRNA knockdown of Notch3 in these cells abolished the upregulation. Increases in SMC differentiation marker gene expression in mural cell-endothelial cell cocultures were not uniform, however, and were greatest in those mural cell types that were already expressing SMC marker genes at readily detectable levels prior to coculture. This suggests that some degree of SMC differentiation in the mural cell partner may be required in order for robust SMC maturation responses to result from contact with an endothelial cell partner.

Activated Notch3 Induces Jagged1 Expression in Mural Cells

Upon activation of Notch signaling in mural cells, the authors observed upregulation not only of Notch3 receptor, but also found parallel increases in expression of the Notch ligand Jagged1 in the same cells.5 This is an intriguing finding that suggests a self-reinforcing, feed-forward mechanism has potentially been triggered. Such a mechanism could promote a second layer of mural cell investment by the same rationale applied above to stabilize endothelial cell-mural cell interactions in the first place. Alternatively, it may serve to further amplify the initial endothelial cell-mural cell contact as part of a mechanism to “fix” the initial interaction and to maintain endothelial cell-mural cell contacts over an extended period.

The feed-forward nature of the model proposed by Liu et al5 has the attractive feature of providing a mechanism for how interactions between endothelial cells and mural cells become stable and mediate maturation responses in both cell types. It may turn out to explain at least part of the iterative sequence underlying tunica media formation seen in the multilayered structure of large artery walls. However, there are several issues that remain to be considered before it becomes clear when and where to apply the interesting results reported by Liu et al5 to the intact vascular system.

Remaining Questions

  1. A critical role for Notch3 expression in endothelial cell-mural cell interactions must be reconciled by the finding that _Notch3_-deficient mice are viable and phenotypically normal.7 Large elastic and muscular arteries exhibit no differences from wild type in their overall structure or function. Resting blood pressures are normal in _Notch3_−/− mice, and vasopressor responses to phenylephrine or angiotensin II do not differ from wild type. Therefore a great deal of vessel wall building goes on normally in the absence of Notch3 expression by mural cells. _Notch3_-deficient mice do, however, exhibit significant structural and functional defects in small arteries and arterioles, particularly in the cerebral circulation.7 These vessels exhibit a disorganized and hypoplastic medial layer with pronounced defects in pressure-induced myogenic contractile responses that correspond with reduced RhoA activation and myosin light chain phosphorylation.8
  2. A role for Notch3 signaling in promoting SMC maturation, while consistent with a number of previous studies9–11, must be weighed against reports that activation of the canonical Notch pathway can inhibit SRF/myocardin-dependent expression of SMC differentiation marker genes.6,12–14
  3. Endothelial cell-pericyte interactions are readily reversible.4,15 Loss of mural cell interactions can lead to microvascular regression.16–18 Therefore feed-forward pathways of the type presented here have overriding controls that can interrupt the self-reinforcing signaling in order to promote vascular regression. It will be important to identify signaling inputs that govern whether or not self-reinforcing, notch-dependent pathways are induced or maintained in mural cells.
  4. Mural cells are recruited to endothelial cells during the investment process by two distinct pathways. One is by de novo recruitment from perivascular mesenchymal progenitors that surround capillary-like vessels, and the other is by division of preexisting SMCs and downstream migration of these cells along capillary basement membranes.19,20 Whether or not Notch3-Jagged1 interactions apply equally well to both types of investment processes will need to be determined.
  5. Pericytes are not equally distributed over the surface of microvessels. Rather, they are most frequently found localized over endothelial cell-cell junctions and at microvascular branch points.15 Moreover, pericyte density varies considerably among different vascular beds, reflecting the functional specialization of particular microvascular beds.2 It will be of interest to determine if the pattern of Jagged1 expression in microvascular endothelial cells correlates with the relative density and distribution of pericytes in the microvasculature.

Summary

Notch signaling is well known for context-dependent effects on the outcome of signal pathway activation.21,22 Therefore, the experimental conditions, type of vessel, origin of cells, and physiological context of the experimental model under study all need to be carefully defined and considered as important determinants of the results obtained. With this in mind, the findings of Liu et al5 offer an exciting glimpse into critical signaling pathways that are activated upon contact between endothelial cells and mural cells that both stabilize interactions between these two cell types and stimulate maturation responses in mural cells that may promote subsequent steps in formation of a vessel wall.

Figure 1.

Figure 1

Mural cells (pericytes) are closely associated with endothelial cells in the microvasculature. Contact between mural cells and endothelial cells that are expressing the Notch ligand Jagged1 induces expression of both the Notch3 receptor and Jagged1 in mural cells. Upregulation of notch signaling in mural cells is associated with increased expression of SMC differentiation marker genes indicative of a maturation response by the mural cell. The predicted outcome of this interaction is to stabilize the contact between mural cell and endothelial cell, and to promote the formation of more robust microvascular networks in angiogenesis and in vascular network remodeling.

Acknowledgments

Sources of Funding

This work was supported by National Institutes of Health grant HL-19424 (to M.W.M), by American Heart Association Fellowship grant 0715320U to J.N.R, and by the Carolina Cardiovascular Biology Center.

Footnotes

References