Endothelial and Smooth Muscle Cell Development
Our laboratory is interested in how endothelial cells and smooth muscle cells (SMC) develop during embryogenesis to form a mature, functional vasculature. In addition, changes in the function of vascular cells occur in vascular disease, and we are interested in the control of disease-related phenotypic alterations. Our current focus is the function of the Notch signaling pathway in these processes.
Role of Notch Signaling in Vascular Development
We have developed both gain-of-function and loss-of-function mouse transgenic lines to understand Notch function during vascular development. Unbalanced Notch signaling generates defective vascular development, with phenotypes ranging from mid-gestation lethality to neonatal lethality. One example is the transgenic expression of a constitutively active Notch1 intracellular domain (ICD) construct. We characterized this Notch1ICD Cre-activated allele in cellular lineages expressing Tie2 to target endothelial cells (Fig 1). Transgenic N1ICDxTie2-Cre embryos die in mid-gestation with vascular remodeling defects. We showed that endothelial cells were able to differentiate, but failed to remodel into mature vascular networks. In addition, the endothelial cells present were unable to recruit mesenchymal cells to form a SMC invested embryonic vessel. Mechanistically, we found that endothelial cells with activated Notch1 signaling change their pattern of cytokine secretion, and in co-culture with SMC, suppress SMC proliferation. Therefore, these models will allow us to continue study of endothelial cell/SMC interactions with and without Notch signaling. See Venkatesh et al, 2008 for more details (). Similarly, activation of Notch signaling in developing SMC prohibits vascular maturation and leads to loss of vascular integrity. Ongoing studies are addressing the molecular basis for these phenotypes.
Figure 1. Activation of Notch1 leads to defective embryonic cardiovasculature and lethality. Embryos from Tie2CrexNotch1ICD crosses were collected at E9.5. Shown are a wild type embryo (A, C) and a N1ICD+ activated littermate (B, E). N1ICD+ embryos were smaller than controls, and arteries were not blood-filled. The extensive pericardial edema in N1ICD+ embryos is highlighted in B (arrows). PECAM-1 immunostaining of control (C) and N1ICD+ embryos (E) highlighted the vasculature. Areas of the head and the dorsal somites that are shown in higher magnification in right panels are boxed in (C). Angiogenic sprouts of intersomitic vessels (ISV) were present in N1ICD+ embryos (E, lower inset), although they failed to have proper vascular patterning. This corresponded to increased TUNEL labeling of somites in N1ICD+ embryos (F) compared to controls (D). Normal (G, I) or n1ICD+ (H, J) E9.5 embryos were sectioned and stained with anti-PECAM-1 (G-H, insets in I-J) or TUNEL labeled (I-J). N1ICD+ embryos had smaller dorsal aortae (da) and neural tubes (nt), with lack of blood vessels within the neural tube (H) compared to controls (G). Control neural tubes had minimal TUNEl positive cells (I), corresponding to robust vascularization (inset, PECAM staining), while high rates of apoptosis were observed in N1ICD+ embryos (J), consistent with lack of vascularization (inset, PECAM staining).
Interaction of Notch Signaling with TGFβ/Smad Superfamily Pathways
Jagged-1/Notch signaling is a strong differentiation signal for human primary SMC (Fig 2). To study this mechanism in more detail, we focused on transcriptional regulation of the smooth muscle alpha actin (SM actin) gene. Transcriptional complexes containing NotchICD and CBF1 recognize CBF1 consensus binding sites on the SM actin promoter to increase transcription. Although HRT proteins are Notch targets considered Notch effector proteins, we identified a novel negative feedback loop involving HRT suppression of NotchICD/CBF1 binding to the SM actin promoter. This work led to the important concepts that 1) Notch signaling can induce its own feedback inhibitor when activated in SMC, 2) this is a mechanism for actively repressing SMC Notch target genes, and 3) mechanism of HRT activity is to inhibit binding of the CBF-1 activation complex to the SM actin promoter. See Tang et al, 2008 for more details ()
Figure 2. Notch signaling in primary human smooth muscle cells promotes a highly contractile phenotype. Smooth muscle cells following Notch activation were stained to detect calponin (green) and actin (red).
We recently characterized the interaction between Notch signaling and another SMC differentiation signal, TGFβ1, in human SMC (Fig 3). We found that Notch and TGFβ/Smad have parallel transcriptional pathways regulating SMC marker genes. These pathways were also interactive, because Notch activation led to enhancement of pSmad-compex binding to Smad consensus sites in SMC marker genes. Transcriptional activity from a TGFβ response element was synergistically activated by TGFβ/Notch signaling. We confirmed a mechanism by which Notch activation increased Smad-complex binding to the SM actin, calponin1, and SM22α gene promoters. See Tang et al, 2010 for more details ().
Figure 3. Molecular interaction of Notch and TGFβ1 signaling. A) Human SMC expressing Notch1ICD, CBF1, or treated with TGFβ1 were lysed and immunoprecipitated with antibodies against V5 (N1ICD epitope tag), CBF1, or pSmad2/3. Immunoprecipitates were separated and immunoblotted with anti-pSmad2/3. B) Luciferase promoter transactivation assays were performed with the TGFβ1 responsive CAGA12-luc reporter construct. SMC were transduced and stimulated with 2ng/ml TGFβ1 for 24h before quantification of luciferase activity. C) Promoter sequences were evaluated 2kb upstream of the transcriptional start site. Indicated are consensus binding sites for Smad and CBF1. D) SMC were transduced with GFP or N1ICD, and stimulated with TGFβ1 for 1h. Cells were collected for chromatin immunoprecipitation assays. Quantitative RT-PCR was performed to amplify the Smad binding sites of SM actin, calponin1, and the three regions in the SM22α promoter that contain Smad sites.
We are also studying the interaction of Notch signaling with members of the TGFβ/BMP family in endothelial cells. In our mouse N1ICD model, we found that short-term activation of Notch signaling was sufficient to induce a senescent phenotype in large vessels in vivo. We utilized human endothelial cells to study this mechanism. Constitutive activation of Notch or ligand activation by Dll4 led to premature cellular senescence in low passage cells, associated with increased p53 and p21 expression. In addition, Notch signaling activated Rho kinase, which led to changes in endothelial cell adhesion molecule expression, including suppression of VE-cadherin. Functionally, Notch activation was assocated with decreased tubulogenesis and loss of barrier function in an intact monolayer. These findings identified Notch as an upstream regulator of Rho kinase and endothelial permeability and senescence. For more details, see Venkatesh et al. (). Because some BMPs appear to function as vascular quiescence factors, our studies are extending to address how BMP and Notch may interact in endothelial cells. Ongoing studies are geared towards how these pathways initiate vascular proliferation or quiescence (Fig 4).
Figure 4. Proliferation of human endothelial cells in vitro. Incorporation of DNA was detected with anti-BrdU labeling (red) to quantify proliferating cells. Endothelial cells under different conditions and signaling activators are analyzed for changes in cell growth.
Notch regulation of vascular microRNAs
Recent studies show that microRNAs (miR) including miR-143/145 regulate VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Our recent studies determined that Jagged-1/Notch signaling transcriptionally activates miR-143/145 in human SMC, and this induction is required for Jagged-1/Notch induced VSMC differentiation. This transcriptional effect induced by Jagged-1/Notch signaling is independent of SRF activity, but the combination of SRF and Notch activity are required for acquisition of the full SMC contractile phenotype (Fig 5). For more details, see Boucher et al, 2011 ().
Figure 5. Model of parallel miR-143/145 regulatory pathways in SMC. Activation of Notch receptors by Jag-1 leads to proteolytic cleavage of the NICD, translocation to the nucleus, and complex formation with CBF1 to transcriptionally activate miR-143/145. Jag-1/Notch mediated activation is CBF1-dependent, but SRF-independent. Notch-responsive CBF1 sites flank the CArG box within the previously defined cardiovascular enhancer. We propose that while the Notch and SRF pathways independently activate miR-143/145, potential interaction of these pathways may occur to increase miR143/145 expression. miR-143/145 is required for the reduced cell proliferation and increased acquisition of contractile phenotype by Jag-1/Notch signaling. VSMC differentiation is further promoted by Notch and SRF/myocardin via direct transcriptional activation of VSMC contractile genes by both pathways.