Development of the vascular system is a complex process that begins with the formation of simple tubes. In vasculogenesis, endothelial precursors called angioblasts associate to form vessel tubes. Major axial vessels such as the aorta, and the vascular plexus of endodermal organs such as the spleen, are formed by vasculogenesis (8
). In angiogenesis, vessels form by sprouting from pre-existing vessels. Vascularization of neural and mesodermal tissues, like the brain and limb buds, occurs by angiogenesis (15
). In both cases, the primitive vessel tubes then remodel, forming the more complex architecture of the adult vasculature. Developmental studies suggest that the EC in these nascent tubes may govern the acquisition of additional vessel layers (23
). Knowledge of the mechanism(s) by which EC recruit mural cell precursors and enable their differentiation remains unknown, and would significantly add to the understanding not only of blood vessel development, but of pathological conditions such as atherosclerosis or vascular malformations, in which these basic processes appear to be deregulated.
Our studies demonstrate that EC do indeed recruit undifferentiated mesenchymal cells. Furthermore, EC-derived PDGF-BB mediates the mesenchymal movement. We found that the ability of BAE as well as bovine or rodent capillary EC to recruit 10T1/2 cells (presumptive mural cell-precursors) was totally blocked by a neutralizing antibody to PDGF-B. PDGF has been shown in previous in vitro studies to act as a mitogen and chemoattractant for mesenchymal cells (67
). Neutralizing antibodies to PDGF-A or to bFGF had no effect on EC recruitment of 10T1/2 cells, even though these factors have been shown to be mitogenic and chemotactic for SMC and fibroblasts. These findings suggest that EC-derived PDGF-B specifically exerts a paracrine effect on undifferentiated mesenchymal cells during vascular development in vivo. Observations by Holmgren et al. (24
) on the expression of PDGF ligand and receptors in forming blood vessels of human placenta, support this concept. They found that EC of developing blood vessels express the mRNA and protein for PDGF-B, but not the PDGF-β receptor, whereas PDGF-β receptor mRNA was detectable in fibroblast-like cells and SMC surrounding intermediate and large blood vessels.
The phenotypes of mice deficient for components of the PDGF system lend further support to our hypothesis. Patch
mice that have a spontaneous mutation deleting the PDGF-α receptor are embryonic lethal, and have cardiovascular defects characterized by reduced numbers of SMC (56
). Mice lacking PDGF-B also exhibited severe cardiovascular and renal abnormalities (31
). The lack of mesangial cells in the kidneys of PDGF-B null mice is of particular interest, since mesangial cells are considered to be specialized pericytes and developmentally related to SMC (57
). Indeed, more recently, PDGF-B null mice have been reported to lack pericytes (32
Our studies show that once undifferentiated mesenchymal cells are recruited to EC, they are induced to become SMC-like. Mature SMC and pericytes in intact vessels express a series of proteins that define the SMC lineage (for review see reference 58
). These include, but are not limited to, αSM-actin (46
), SM-myosin (29
), calponin (3
), SM22α (14
), h-caldesmon (59
), and desmin (39
). A variety of cells including astrocytes (20
) and myofibroblasts (11
) have been reported to express αSM-actin in culture. Therefore, induction of αSM-actin in an otherwise undifferentiated cell is not evidence for differentiation towards an SMC lineage. Furthermore, a few of these proteins expressed specifically in adult SMC are also transiently expressed in other cell types (e.g., cardiac muscle) during embryogenesis (36
). However, simultaneous expression of multiple SM-specific proteins is indicative of a SM phenotype. Hence, simultaneous expression of αSM-actin, SM-myosin, SM22α, and calponin in 10T1/2 cells in culture with EC, along with a dramatic change in cell shape, provides strong evidence that these mesenchymal cells are truly induced toward the smooth muscle lineage. Certainly it is possible that this coculture system will have limitations and will not reproduce all aspects of differentiated SMC function. However, the biologically relevant induction of SM-specific protein expression in 10T1/2 cells, which can be easily genetically manipulated, make this an ideal system in which to investigate the molecular aspects of SM-specific gene expression.
We demonstrated that induction of 10T1/2 to an SMC phenotype occurs only after the mesenchymal cells come into close proximity with the EC. This sequence of events has been shown to occur during development of the aorta in the quail. Hungerford et al. found that mesodermal cells become associated with the developing aorta, beginning at the ventral surface, and that they express αSM-actin only after associating with the EC tube (26
). By recreating aspects of vessel assembly in vitro, we have begun to investigate directly the mechanisms involved in this EC–mesenchymal interaction. We have found that the induction of the SMC phenotype in this model is mediated at least in part by TGF-β. In addition to showing that direct treatment of 10T1/2 cells with TGF-β1 induces expression of SM-specific markers, we have demonstrated that neutralizing antibodies against TGF-β blocks induction of SM-specific markers in the cocultures.
Although the cells in these cocultures make contact, it is not clear if physical contact between the cells is necessary for the inductive event. Speculation regarding the source of the TGF-β in the cocultures is based on previous studies from our lab and others, showing that both EC and mural cells, when grown separately, produce a latent form of TGF-β that is activated in EC-mural cell cocultures (2
). The demonstration that local activation of TGF-β may be involved in directing undifferentiated mesenchymal cells to an SMC lineage in the vessel wall is consistent with previous observations of elevated TGF-β in mesenchymal remodeling, and at sites of epithelial–mesenchymal interactions (22
An increasing body of evidence indicates roles for members of the TGF-β family in regulating muscle differentiation in general. TGF-β has been implicated in the regulation of both cardiac and skeletal muscle differentiation (for review see references 42
). The epithelial–mesenchymal transformation in embryonic heart can be mimicked by TGF-β (49
), and antisense against TGF-β3 blocks epithelial–mesenchymal transformation of cardiac endothelial cells (49
). Expression of a dominant-negative form of the type II TGF-β receptor suppressed myogenic differentiation in a culture model (16
). Furthermore, data from gene disruption studies suggest that a new member of the TGF-β family, growth/differentiation factor-8, acts as a negative regulator of skeletal muscle growth (35
Whether all of the effects of TGF-β on SM-specific gene expression in this model are direct remains to be determined. To date, regulatory regions identified as conferring responsiveness to TGF-β have been found within the 5′ flanking sequences of αSM-actin (21
). The 5′ and 3′ regulatory regions of other SMC-specific genes, including SM-myosin and SM22α, are under investigation (27
). In addition, some of the effects of TGF-β on vascular wall cell differentiation may be due to TGF-β–induced changes in the extracellular matrix (34
). It is well known that the matrix can influence cell growth, polarity, and organization as well as differentiation (1
). In fact, the interaction of presumptive SMC and pericytes with the abluminal EC surface are temporally associated with deposition of a basement membrane (9
). In this regard, it is interesting to speculate that TGF-β, activated when mural cells associate with EC, may serve several functions, all aimed at establishing a mature vessel. These include inhibition of endothelial proliferation (45
) and migration (54
), stimulation of SMC/pericyte differentiation (our present observations), and induction of basement membrane assembly (34
Observations of mice with a targeted disruption of TGF-β support this concept. Fifty percent of TGF-β1 null mice die in utero from a defect in yolk sac vasculogenesis, which is thought to be due to improper interactions between epithelial cells and mesenchymal cells, and may be the result of altered cell–matrix interactions (12
). This observation also suggests that local activation of TGF-β, resulting from such cell–cell interactions, may occur widely throughout development. Whether TGF-β activation is a constitutive event at the site of EC–mural cell contact is not known. The reversibility of the SMC phenotype (4
) and the concept that differentiation may require constant signaling, at least under some circumstances (5
), leads us to suspect that local activation of TGF-β is an ongoing process. In fact, it is well-documented that the endothelium makes frequent contacts with SMC and pericytes throughout the vasculature (51
Although we have shown that 10T1/2 cells are capable of becoming SMC-like in vitro in response to EC, we wanted to confirm that 10T1/2 cells have the capacity to become incorporated into developing vessels in vivo. Therefore, undifferentiated 10T1/2 cells were permanently labeled with a fluorescent dye, and were placed in the proximity of developing vessels in collagen matrices subcutaneously in the mouse. Not only did these cells become incorporated into the medial layers of the newly forming vessels (Fig. ), but once associated with the vessel, simultaneously expressed αSM-actin, SM-myosin, and calponin, reflective of a SM phenotype. The resolution was not sufficient to comment on the role of contact in the induction of the SMC phenotype in these vessels. However, the 10T1/2 cells expressing SM markers usually comprised the innermost layer of cells in the vessel wall.
In summary, we have developed coculture systems to identify potential regulators of vessel formation, and to elucidate their relative contributions. A complimentary in vivo system was established, and observations of developing vessels in the model corroborate our tissue culture observations. We believe that the information gained from this system regarding the cellular and molecular regulation of vessel formation will be important in understanding not only developmental regulation, but also pathophysiological processes such as atherosclerosis and vascular malformations, where there appear to be defects in the normal control mechanisms.