Smooth Muscle alpha-actin is a direct target of Notch/CSL

Intercellular signaling mediated by Notch receptors is essential for proper cardiovascular development and homeostasis. Notch regulates cell fate decisions that affect proliferation, survival, and differentiation of endothelial and smooth muscle cells. It has been reported that Jagged1-Notch interactions may participate in endocardial cushion formation by inducing endothelial-to-mesenchymal transformation. Here, we show that Notch directly regulates expression of the mesenchymal and smooth muscle cell marker smooth muscle alpha-actin (SMA) in endothelial and vascular smooth muscle cells via activation of its major effector, CSL. Notch/CSL activation induces SMA expression during endothelial-to-mesenchymal transformation, and Notch activation is required for expression of SMA in vascular smooth muscle cells. CSL directly binds a conserved cis element in the SMA promoter, and this consensus sequence is required for Notch-mediated SMA induction. This is the first evidence of the requirement for Notch activation in the regulation of SMA expression.     

Tumor necrosis factor-alpha and endothelial cells modulate Notch signaling in the bone marrow microenvironment during inflammation

OBJECTIVE: Homeostasis of the hematopoietic compartment is challenged and maintained during conditions of stress by mechanisms that are poorly defined. To understand how the bone marrow (BM) microenvironment influences hematopoiesis, we explored the role of Notch signaling and BM endothelial cells in providing microenvironmental cues to hematopoietic cells in the presence of inflammatory stimuli. MATERIALS AND METHODS: The human BM endothelial cell line (BMEC) and primary human BM endothelial cells were analyzed for expression of Notch ligands and the ability to expand hematopoietic progenitors in an in vitro coculture system. In vivo experiments were carried out to identify modulation of Notch signaling in BM endothelial and hematopoietic cells in mice challenged with tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS), or in Tie2-tmTNF-alpha transgenic mice characterized by constitutive TNF-alpha activation. RESULTS: BM endothelial cells were found to express Jagged ligands and to greatly support progenitor's colony-forming ability. This effect was markedly decreased by Notch antagonists and augmented by increasing levels of Jagged2. Physiologic upregulation of Jagged2 expression on BMEC was observed upon TNF-alpha activation. Injection of TNF-alpha or LPS upregulated three- to fourfold Jagged2 expression on murine BM endothelial cells in vivo and resulted in increased Notch activation on murine hematopoietic stem/progenitor cells. Similarly, constitutive activation of endothelial cells in Tie2-tmTNF-alpha mice was characterized by increased expression of Jagged2 and by augmented Notch activation on hematopoietic stem/progenitor cells. CONCLUSIONS: Our results provide the first evidence that BM endothelial cells promote expansion of hematopoietic progenitor cells by a Notch-dependent mechanism and that TNF-alpha and LPS can modulate the levels of Notch ligand expression and Notch activation in the BM microenvironment in vivo.     

Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa

Epithelial-mesenchymal transformation is a critical developmental process reiterated in multiple organs throughout embryogenesis. Formation of endocardial cushions, primordia of valves and septa, is a classic example of epithelial-mesenchymal transformation. Several gene mutations are known to affect cardiac valve formation. Sox9 is activated when endocardial endothelial cells undergo mesenchymal transformation and migrate into an extracellular matrix, called cardiac jelly, to form endocardial cushions. In Sox9-null mutants, endocardial cushions are markedly hypoplastic. In these mutants, Nfatc1 is ectopically expressed and no longer restricted to endothelial cells. Further, Sox9-deficient endocardial mesenchymal cells fail to express ErbB3, which is required for endocardial cushion cell differentiation and proliferation. Our results reveal a succession of molecular steps in the pathway of endocardial cushion development. We propose that loss of Sox9 inhibits epithelial-mesenchymal transformation after delamination and initial migration, but before definitive mesenchymal transformation.     

Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.     

Combined loss of Hey1 and HeyL causes congenital heart defects because of impaired epithelial to mesenchymal transition

Congenital heart defects affect almost 1% of human newborns. Recently, mutations in Notch ligands and receptors have been found to cause a variety of heart defects in rodents and humans. However, the molecular effects downstream of Notch are still poorly understood. Here we report that combined inactivation of Hey1 and HeyL, two primary target genes of Notch, causes severe heart malformations, including membranous ventricular septal defects and dysplastic atrioventricular and pulmonary valves. These defects lead to congestive cardiac failure with high lethality. We found both genes to be coexpressed with Notch1, Notch2 and the Notch ligand Jagged1 in the endocardium of the atrioventricular canal, representing the primary source of mesenchymal cells forming membraneous septum and valves. Atrioventricular explants from Hey1/HeyL deficient mice exhibited impaired epithelial to mesenchymal transition. Although epithelial to mesenchymal transition was initiated regularly, full transformation into mesenchymal cells failed. This was accompanied by reduced levels of matrix metalloproteinase-2 expression and reduced cell density in endocardial cushions in vivo. We further show that loss of Hey2 leads to very similar deficiencies, whereas a Notch1 null mutation completely abolishes epithelial to mesenchymal transition. Thus, the Hey gene family shows overlap in controlling Notch induced endocardial epithelial to mesenchymal transition, a process critical for valve and septum formation.     

Kindlin-2 is an essential component of intercalated discs and is required for vertebrate cardiac structure and function

Notch signaling is critical for the development and maintenance of the cardiovasculature, with loss-of-function studies defining roles of Notch1 in the endothelial/hematopoietic lineages. No in vivo studies have addressed complementary gain-of-function strategies within these tissues to define consequences of Notch activation. We developed a transgenic model of Cre recombinase-mediated activation of a constitutively active mouse Notch1 allele (N1ICD(+)) and studied transgene activation in Tie2-expressing lineages. The in vivo phenotype was compared to effects of Notch1 activation on endothelial tubulogenesis, paracrine regulation of smooth muscle cell proliferation, and hematopoiesis. N1ICD(+) embryos showed midgestation lethality with defects in angiogenic remodeling of embryonic and yolk sac vasculature, cardiac development, smooth muscle cell investment of vessels, and hematopoietic differentiation. Angiogenic defects corresponded with impaired endothelial tubulogenesis in vitro following Notch1 activation and paracrine inhibition of smooth muscle cells when grown with Notch1-activated endothelial cells. Flow cytometric analysis of hematopoietic and endothelial precursor populations demonstrated a significant loss of CD71(+)/Ter119(+) populations with an active N1ICD(+) allele and a corresponding increase in c-Kit(+)/CD71 and Flk1(+) populations, suggesting a developmental block during the transition between c-Kit- and Ter119-expressing erythroblasts. Cardiovascular lineages are sensitive to an imbalance in Notch signaling, with aberrant activation reflecting a vascular phenotype comparable to a loss-of-function Notch1 mutation.     

Notch promotes vascular maturation by inducing integrin-mediated smooth muscle cell adhesion to the endothelial basement membrane

Vascular development and angiogenesis initially depend on endothelial tip cell invasion, which is followed by a series of maturation steps, including lumen formation and recruitment of perivascular cells. Notch ligands expressed on the endothelium and their cognate receptors expressed on perivascular cells are involved in blood vessel maturation, though little is known regarding the Notch-dependent effectors that facilitate perivascular coverage of nascent vessels. Here, we report that vascular smooth muscle cell (VSMC) recognition of the Notch ligand Jagged1 on endothelial cells leads to expression of integrin αvβ3 on VSMCs. Once expressed, integrin αvβ3 facilitates VSMC adhesion to VWF in the endothelial basement membrane of developing retinal arteries, leading to vessel maturation. Genetic or pharmacologic disruption of Jagged1, Notch, αvβ3, or VWF suppresses VSMC coverage of nascent vessels and arterial maturation during vascular development. Therefore, we define a Notch-mediated interaction between the developing endothelium and VSMCs leading to adhesion of VSMCs to the endothelial basement membrane and arterial maturation.     

Endothelial expression of the Notch ligand Jagged1 is required for vascular smooth muscle development

The Notch ligand Jagged1 (Jag1) is essential for vascular remodeling and has been linked to congenital heart disease in humans, but its precise role in various cell types of the cardiovascular system has not been extensively investigated. We show that endothelial-specific deletion of Jag1 results in embryonic lethality and cardiovascular defects, recapitulating the Jag1 null phenotype. These embryos show striking deficits in vascular smooth muscle, whereas endothelial Notch activation and arterial-venous differentiation appear normal. Endothelial Jag1 mutant embryos are phenotypically distinct from embryos in which Notch signaling is inhibited in endothelium. Together, these results imply that the primary role of endothelial Jag1 is to potentiate the development of neighboring vascular smooth muscle.     

The role of the Angiopoietins in vascular morphogenesis

The Angiopoietin/Tie system acts as a vascular specific ligand/receptor system to control endothelial cell survival and vascular maturation. The Angiopoietin family includes four ligands (Angiopoietin-1, Angiopoietin-2 and Angiopoietin-3/4) and two corresponding tyrosine kinase receptors (Tie1 and Tie2). Ang-1 and Ang-2 are specific ligands of Tie2 binding the receptor with similar affinity. Tie2 activation promotes vessel assembly and maturation by mediating survival signals for endothelial cells and regulating the recruitment of mural cells. Ang-1 acts in a paracrine agonistic manner inducing Tie2 phosphorylation and subsequent vessel stabilization. In contrast, Ang-2 is produced by endothelial cells and acts as an autocrine antagonist of Ang-1-mediated Tie2 activation. Ang-2 thereby primes the vascular endothelium to exogenous cytokines and induces vascular destabilization at higher concentrations. Ang-2 is strongly expressed in the vasculature of many tumors and it has been suggested that Ang-2 may act synergistically with other cytokines such as vascular endothelial growth factor to promote tumor-associated angiogenesis and tumor progression. The better mechanistic understanding of the Ang/Tie system is gradually paving the way toward the rationale exploitation of this vascular signaling system as a therapeutic target for neoplastic and non-neoplastic diseases.     

Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking notch signaling in endothelial cells

Angiogenesis is highly sensitive to the composition of the vascular microenvironment, however, our understanding of the structural and matricellular components of the vascular microenvironment that regulate angiogenesis and the molecular mechanisms by which these molecules function remains incomplete. Our previous results described a novel pro-angiogenic activity for Microfibril-Associated Glycoprotein-2 (MAGP-2), but did not address the molecular mechanism(s) by which this is accomplished. We now demonstrate that MAGP-2 promotes angiogenic cell sprouting by antagonizing Notch signaling pathways in endothelial cells. MAGP-2 decreased basal and Jagged1 induced expression from the Notch sensitive Hes-1 promoter in ECs, and blocked Jagged1 stimulated Notch1 receptor processing in transiently transfected 293T cells. Interestingly, inhibition of Notch signaling by MAGP-2 seems to be restricted to ECs since MAGP-2 increased Hes-1 promoter activity and Notch1 receptor processing in heterologous cell types. Importantly, constitutive activation of the Notch signaling pathway blocked the ability of MAGP-2 to promote angiogenic cell sprouting, as well as morphological changes associated with angiogenesis. Collectively, these observations indicate that MAGP-2 promotes angiogenic cell spouting in vitro by antagonizing Notch signaling pathways in ECs.     

Notch-dependent expression of epithelial-mesenchymal transition markers in cholangiocytes after liver transplantation

Aim: Epithelial–mesenchymal transition (EMT) has been identified in chronic cholestatic liver diseases, which are characterized by biliary proliferation and fibrosis. Activation of Notch signaling mediates EMT in a variety of epithelial cell types. In the present study, we investigated the role of Notch signaling in the regulation of EMT marker expression in cholangiocytes after liver transplantation. Methods: Orthotopic liver transplantation was performed in Sprague–Dawley rats. Liver tissues and isolated cholangiocytes were collected 1 week after transplantation. The expression of mesenchymal and biliary epithelial markers was evaluated by immunohistochemistry, quantitative polymerase chain reaction (PCR) and western blotting in liver sections and isolated cholangiocytes. Quantitative real‐time PCR and western blotting for Jagged1 and HES1 were utilized to evaluate the activation of Notch signaling. Proliferation and migration of cholangiocytes were assessed by 5‐bromodeoxyuridine and transwell assays, respectively. Cholangiocyte proliferation, migration and expression of EMT markers were also evaluated following the inhibition of Notch signaling with N,(N‐[3,5‐difluorophenacetyl]‐L‐alanyl)‐S‐phenylglycine t‐butylester (γ‐secretase inhibitor) and a Jagged1‐neutralizing antibody. Results: Expression of EMT markers by cholangiocytes was observed in liver grafts and isolated cholangiocytes obtained 1 week after transplantation. Inhibition of Notch signaling prevented the expression of EMT markers in bile ducts of liver sections and isolated cholangiocytes. Cholangiocyte proliferative and migratory capacities were also suppressed by the inhibition of Notch signaling. Conclusion: Activation of Notch signaling promotes cholangiocyte proliferation and expression of EMT markers after liver transplantation.     

Notch ligand Delta-like 1 is essential for postnatal arteriogenesis

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.     

Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells

Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.     

Critical role of endothelial Notch1 signaling in postnatal angiogenesis

Notch receptors are important mediators of cell fate during embryogenesis, but their role in adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in haploinsufficient global or endothelial-specific Notch1(+/-) mice, but not Notch4(-/-) mice, were impaired compared with wild-type mice. The expression of vascular endothelial growth factor (VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice, suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with VEGF increases presenilin proteolytic processing, gamma-secretase activity, Notch1 cleavage, and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of gamma-secretase activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell proliferation, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued the inhibitory effects of gamma-secretase inhibitors on VEGF-induced angiogenesis. These findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates gamma-secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for improving angiogenic response and blood flow recovery in ischemic limbs.     

Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration

The Notch/Jagged signaling pathway is important for cellular differentiation and proliferation. Its dysfunction is associated with human pathologies in several tissues including liver. Point mutations in Jagged-1 gene are the cause for Alagille syndrome, associated with paucity of intrahepatic bile ducts. To determine the putative role of the trans-membrane receptor Notch and its ligand Jagged-1 in liver regeneration, we investigated the expression of Notch and Jagged-1 in rat liver following 2/3 partial hepatectomy. Immunohistochemical staining of normal rat liver showed that Notch was expressed in hepatocytes, bile duct cells and endothelial cells, whereas Jagged-1 was expressed in bile duct cells and hepatocytes. Both Notch-1 and Jagged-1 proteins were upregulated in hepatocytes after partial hepatectomy up to day 4. After partial hepatectomy, nuclear translocation of the intracellular cytoplasmic domain of Notch (NICD) increased and peaked within 15 minutes, indicating the activation of Notch. Expression of the Notch-dependent target gene (HES-1) expression increased within 30-60 minutes. Addition of recombinant Jagged-1 protein to primary cultures of hepatocytes stimulated hepatocyte DNA synthesis. Furthermore, injection of silencing RNA for Notch and Jagged-1 to livers 2 days before partial hepatectomy significantly suppressed proliferation of hepatocytes at days 2 to 4 of the regenerative response. In conclusion, Notch/Jagged signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting cell growth and differentiation.     

Jagged1-induced Notch signaling drives proliferation of multiple myeloma cells

Notch receptors expressed on hematopoietic stem cells interact with their ligands on bone marrow stromal cells and thereby control cell fate decisions and survival. We recently demonstrated that Notch signaling is involved in proliferation and survival of B cell-derived tumor cells of classic Hodgkin disease and described a novel mechanism for the oncogenic capacity of Notch. In this study we investigated whether Notch signaling is involved in the tight interactions between neoplastic plasma cells and their bone marrow microenvironment, which are essential for tumor cell growth in multiple myeloma (MM). Here we demonstrate that Notch receptors and their ligand Jagged1 are highly expressed in cultured and primary MM cells, whereas nonneoplastic counterparts show low to undetectable levels of Notch. Functional data indicate that ligand-induced Notch signaling is a growth factor for MM cells and suggest that these interactions contribute to myelomagenesis in vivo.