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. Author manuscript; available in PMC: 2010 Aug 30.
Published in final edited form as: Dev Dyn. 2009 Jan;238(1):162–170. doi: 10.1002/dvdy.21811

Expression of Active Notch1 in Avian Coronary Development

Ke Yang 1, Yong-Qiu Doughman 1, Ganga Karunamuni 1, Shi Gu 2, Yu-Chung Yang 2, David M Bader 3, Michiko Watanabe 1,*
PMCID: PMC2929638  NIHMSID: NIHMS225975  PMID: 19097050

Abstract

Notch1 is an important regulator of intercellular interactions in cardiovascular development. We show that the nuclear-localized, cleaved and active form of Notch1, the Notch1 intracellular domain (N1ICD), appeared in mesothelial cells of the pro-epicardium during epicardial formation at looped heart stages. N1ICD was also present in mesothelial cells and mesenchymal cells specifically within the epicardium at sulcus regions. N1ICD-positive endothelial cells were detected within the nascent vessel plexus at the atrio-ventricular junction and within the compact myocardium (HH25-30). The endothelial cells expressing N1ICD were surrounded by N1ICD positive smooth muscle cells after coronary orifice formation (HH32-35), while N1ICD expression was absent in the mesenchymal and mesothelial cells surrounding mature coronary vessels. We propose that differential activation of the hypoxia/HIF1-VEGF-Notch pathway may play a role in epicardial cell interactions that promote epicardial EMT and coronary progenitor cell differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic sulcus regions.

Keywords: chicken embryo, heart development, coronary development, Notch1ICD, pro-epicardial organ, epicardium-derived cells

INTRODUCTION

The progenitors of the coronary vasculature originate from the epicardium, the outer-most layer of the developing heart [reviewed in (Reese et al., 2002)]. The epicardium develops from the pro-epicardial organ (PEO) or serosa that attaches and spreads over the surface of the myocardium. A subpopulation of mesothelial cells undergoes an epithelial/mesenchymal transition (EMT) and gives rise to coronary vascular smooth muscle cells, perivascular and intermyocardial fibroblasts, and presumably vascular endothelial cells (van Tuyn et al., 2007). Interestingly, the first lumenized coronary vessels are found at the atrioventricular sulcus (Gittenberger-de Groot et al., 1998; Wada et al., 2003b). The AV junction and other sulcus regions have a thicker epicardium, accumulate relatively more epicardial derived cells (EPDCs), and are the site of the largest diameter coronary vessels compared with other cardiac regions (Perez-Pomares et al., 1998; Li et al., 2002; Mu et al., 2005). The cellular and molecular mechanisms of the region-dependent formation of the largest coronaries have not been identified.

It was suggested that early coronary vessel formation is driven by limited oxygen diffusion within the myocardium, causing a gradient of hypoxia from the myocardium to the epicardium (Tomanek et al., 1999). In the sulcus regions, the base of OFT, IVS and AVJ, the myocardium and epicardium have been shown to be the most hypoxic cardiac tissues in chicken (Ivnitski-Steele et al., 2004; Sugishita et al., 2004; Ivnitski-Steele et al., 2005; Nanka et al., 2006; Wikenheiser et al., 2006) and mouse (Xu et al., 2007) embryos. These same regions have also been shown to have nuclear localized HIF1a and the highest levels of expression of VEGF, a HIF1a regulated gene (Tomanek et al., 1999; Cox and Poole, 2000; Morabito et al., 2001). The epicardial EMT, preferentially begins and may be more active at these intersegmental grooves (Manner et al., 2001; Wessels and Perez-Pomares, 2004; Lie-Venema et al., 2005). Those findings are consistent with the evidence that the subepicardial extracellular matrix (ECM) is rich in Fibroblast growth factors (FGF-1,FGF-2,FGF-7) and vascular endothelial growth factor A (VEGFA) secreted from the hypoxic myocardium and epicardium during coronary vasculogenesis (Tomanek et al., 1999; Cox and Poole, 2000; Morabito et al., 2001). These data suggest that hypoxia-induced HIF-1-mediated transcription and subsequent VEGFA signaling may play a role in designating the formation of large coronary vessels at particularly hypoxic sulcus regions.

An intriguing candidate for coronary development regulation downstream of VEGF is the signaling molecule: Notch. This transmembrane protein has been show to act downstream of the VEGFA pathway in arterial endothelial cells (Liu et al., 2003) and interacts with HIF1 to determine the arterial cell fate of endothelial progenitor cells (Diez et al., 2007). It has been implicated in many aspects of cardiovascular development, including endocardial cushion EMT during development of the heart valves, in arterial-venous differentiation, endothelial tip cell differentiation and in remodeling of the primitive vascular plexus (Gridley, 2007; Niessen and Karsan, 2007). Notch signaling requires two steps of ligand-induced receptor cleavage to generate the active Notch intracellular domain (NICD). In this study, we observed intense staining for active Notch1ICD in the epicardium, myocardial vessels, presumptive cardiac valves, and ventricular and atrial endocardium, in addition to the previously reported expression in the nascent endocardium at E14.5 mouse heart (Del Monte et al., 2007). It was previously proposed that not only is Notch1/CSL-dependent induction of SMA involved in endocardial cushion EMT (Noseda et al., 2004), but it is also required for SMA expression in SMC (Noseda et al., 2006). Thus, Notch signaling may also be required for maturation and stabilization of the coronary vasculature.

While Notch and its ligands have been detected in cardiac tissues (Del Monte et al., 2007), the cleaved and active form of Notch (NICD) has not been evaluated in detail in terms of its roles in coronary development. The molecular mechanisms linking the hypoxic microenvironment and activated Notch to epicardial EMT and coronary progenitor cell fate in coronary development have yet to be clarified. We hypothesized that a uniquely hypoxic environment in sulcus regions induces HIF-1 and VEGF signaling and activates the Notch pathway that promotes epicardial EMT and determines arterial cell fate at these regions during epicardial development and coronary vascularization. Furthermore, Notch signaling may be required for maturation and stabilization of the coronary vasculature. In this study, we followed in detail the timing and cell type specific expression pattern of Notch1ICD protein in normal chicken and quail epicardial development and coronary vasculogenesis. Our findings support a role for Notch 1 signaling in the early steps of coronary vessel development and its preferential activity at sulcus regions during coronary vasculogenesis.

RESULTS AND DISCUSSION

N1ICD Expression during Epicardial Formation

An N1ICD-specific antibody against the conserved N-terminal V1744 amino acid of N1ICD was used for this study (Del Monte et al., 2007). By Western blot analysis, we detected a specific N1ICD band (110-kDa, arrow) in chicken and quail embryonic (HH30) hearts (Fig.1 A) and an additional lower molecular weight band (asterisk) in adult rat epicardial/mesothelial cells (ARMECs) (Eid et al., 1992; Wada et al., 2003a) (Fig.1 B and C). Western blotting analysis showed that N1ICD levels were cell density dependent (Fig.1 C) and decreased in ARMECs treated with γ-secretase inhibitor (L685,485 at 10 μm, for 48h) compared with DMSO vehicle exposed negative controls (Fig.1 B). The secretase inhibitor prevents Notch cleavage and therefore inhibits the formation of N1ICD.

Fig. 1.

Fig. 1

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Immunoblot analysis of Notch1 intracellular domain (N1ICD) levels in adult rat epicardial/mesothelial cells (ARMECs), quail (HH30) and chicken (HH30) heart tissue. A: The N1ICD antibody recognized a 110 kDa protein band (arrow) in quail (HH30) and chicken (HH30) whole heart lysates. B: In whole cell lysates of ARMECs, a 110 kDa band (arrow) and a smaller molecular weight band (asterisk, *) was detected by N1ICD antibody. Both protein bands decreased in intensity after treatment with γ-secretase inhibitor L-685,458 (10 μm, 48h) compared with that treated by DMSO vehicle control (DMSO). C: N1ICD levels were elevated with increased cell density (25%, 50% and 100% confluence of ARMECs).D: Hypoxia (1% O2 incubation for 24h) and ectopic HIF1 expression [infection with adenovirus encoding constitutively active HIF1α (100MOI)) increased Notch1ICD (lower panel) levels correlated with the induction of HIF1α expression showed in upper panel. 50 μg proteins per lane were loaded and β-actin was used as the loading control.

At a stage when the tubular chicken heart is looped and beginning chamber differentiation, N1ICD was not expressed in any cell types in the pro-epicardial organ (Fig.2 B,PEO), . while intense staining was present in endothelial cells of the endocardium of the atrial ventricular canal (AVC) (Fig.2. B arrow) in a pattern similar to that described for E8.5-10.5 mouse hearts (Schroeter et al., 1998; Serneels et al., 2005; Del Monte et al., 2007). The mesothelial cells on the surface of PEO were identified as epicardial cells by immunostaining for the epicardial marker, Wilms tumor suppressor gene1 (Wt1) protein, in the immediately adjacent section (Fig2 C arrow). After the PEO cells attached to the myocardium and during epicardial monolayer formation at HH21, N1ICD immunostaining appeared in clusters of mesothelial cells that were covering the myocardium from the dorsal to the ventral surfaces (Fig.2 E, arrows). These cells were Wt1 positive in the immediately adjacent section (Fig.2 F, arrow). At this time point, the elongated mesothelial protrusions extending from the PEO at the sinus venosus and adhering to the myocardium was detected by Wt1 immunostaining (Fig.2. I). Some mesothelial cells on the PEO extension showed N1ICD staining (Fig.2 G,H arrows) that was comparable in intensity to the signal in the endocardium (Fig.2.E arrow head). Lateral induction of Notch signaling was reported in cardiac valve development (Gittenberger-de Groot et al., 1998) and in other systems such as wing margin boundary formation in flies (Timmerman et al., 2004), induction of proneural domains in the ear in vertebrates (Panin et al., 1997), limb bud margin formation (Daudet and Lewis, 2005), and somite boundary formation (Irvine and Vogt, 1997). The formation of a contiguous cell layer may stimulate the lateral induction mechanism among mesothelial cells of the epicardium and induce the N1ICD expression.

Fig. 2.

Fig. 2

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Expression of Notch 1ICD, Wt1 and VEGFA in HH17 and HH21 chicken hearts during epicardial formation. A: A semi-sagittal section of HH17 chicken heart. B,C Higher magnifications of the boxed area in panel A. B: Nuclear staining for N1ICD (green) was absent in the cells of the pro-epicardial organ (PEO) at HH17 but was intense in endothelial cells of the endocardium at the atrial ventricular canal (AVC; solid arrowhead). C: An immediately adjacent 3μm serial section was stained with Wt1, a marker for epicardial cells in this setting (red). Cardiomyocytes did not express Notch1ICD in the myocardial wall (empty arrow head). D: A sagittal section of a HH21 heart. E,F: Higher magnifications of the boxed area in panel D. A few epicardial mesothelial cells covering the ventricular myocardium had nuclear staining for N1ICD (E, arrows) co-localized with Wt1 in a consecutive sections (F, arrows).G,H,I: Higher magnifications of the boxed area in panel E and F showed the N1ICD (G, arrows) co-localized with Wt1 in a consecutive sections (I, arrows). E and F Were immediately adjacent sections. Nuclei were visualized by DAPI staining (blue; G,H). Scale bars=500 μm in A (applies to D), 50 μm in B (applies to C,E,F), 5 μm in G (applies to H).

N1ICD Expression during Epicardial EMT and EPDC Differentiation

In order to identify endothelial cell type specific N1ICD expression, the quail endothelial cell marker, QH1, was utilized for co-staining in embryonic quail hearts. At HH17 and HH21, N1ICD was not co-localized with QH1 positive cells in the pro-epicardial organ (data not shown). From HH17 (the earliest stage we studied), the expression of N1ICD was always present in the endothelial cells lining the endocardium of the ventricles, atria and AV cushion , but it was never observed in cardiomyocytes in the compact myocardium. By HH25 when the epicardial coverage over the ventricular myocardium was complete, a subset of Wt1 positive epicardial mesothelial cells (Fig.3. B, empty arrow heads) and epicardial derived mesenchymal cells were N1ICD positive (Fig.3. C arrow heads). Some QH1 positive endothelial cells at the AVJ (Fig.3.C arrows) and some endothelial cells in myocardial vessels (Fig.3. B arrows) showed intense N1ICD expression. The N1ICD immunostaining in mesothelial cells at the ventricular epicardium (Fig.3. B, empty arrow heads) was less intense compared with N1ICD immunostaining of endothelial cells suggesting an increase in N1ICD levels with EMT. Similarly, at the AVJ: endothelial cells and subepicardial mesenchymal cells (Fig.3. C, arrow head) adjacent to the endothelial cells (Fig.3. C, arrows) showed more intense staining than mesothelial cells (Fig.3. C, empty arrow head).

Fig. 3.

Fig. 3

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Expression of Notch 1ICD, QH1 and VEGFA in sections of HH25 quail heart. A: A semi-sagittal section of a HH25 quail heart. N1ICD immunostaining was intensely positive in the endothelial cells lining the endocardium of the ventricles, atria and AV cushions as well as on some mesenchymal cells within the AV cushions B,C: Higher magnifications of the interventricular sulcus (B) and AVJ (C) regions indicated by boxed areas in panel A. QH1 positive endothelial cells in endocardium were N1ICD positive (B Solid arrow heads). Notch1ICD was expressed in epicardial mesothelial (B,C: empty arrow heads) and mesenchymal cells (C, solid arrow head) and co-localized with Wt1 at the atrial-ventricular junction (AVJ). Some QH1 positive (red) endothelial cells in the myocardium (B arrows) and nascent vessels (C arrows) within the epicardium at the AVJ showed intense N1ICD staining. D. Alternate sections immunostained for N1ICD, Wt1 and VEGFA revealed staining for all three in epicardial mesothelial cells and subepicardial mesenchymal cells at the AVJ. The dotted line indicates the boundary of the epicardium (left) and myocardium (right). Nuclei were labeled by DAPI staining (dark blue). Scale bars=500 μm in A, 50 μm in B (applies to C,D,E,F).

At HH25, staining of alternative sections showed that VEGFA immunostaining coincided with N1ICD expression in a subset of the epicardial mesothelial cells and subepicardial mesenchymal cells at the AVJ and less intense VEGFA staining was also observed in the myocardium adjacent to the epicardium (Fig.3.F, arrow heads). Notch1 was proposed as a downstream gene of VEGF in arterial endothelial cells (Timmerman et al., 2004). VEGF stimulation was shown to upregulate Notch1 and Dll4 expression exclusively in arterial endothelial cells (Lawson et al., 2002). Whereas VEGFA could not rescue arterial marker gene expression in Notch deficient zebrafish embryos, the expression of an activated Notch1 in VEGFA-deficient embryos could rescue the expression of arterial markers. Furthermore, the spatiotemporal VEGF-distribution as well as specificity of the isoforms are important for proper coronary vascular development (van den Akker et al., 2007a). It is likely that epicardium-derived and myocardium-derived VEGFA may have an additive effect to induce N1ICD expression in epicardial and subepicardial mesenchymal cells during epicardial EMT and endothelial cell differentiation.

In the atrio-ventricular sulcus, some of the epicardially derived mesenchymal cells coalesced to form channels within the extracellular matrix and formed the endothelium of the coronary vessels at HH30 quail heart (Fig.4.B,F). N1ICD was present in endothelial cells in these nascent vessels in the AVJ epicardium (Fig 4, F arrows) and within the compact ventricular myocardium (Fig.4D-G). At HH30, no smooth muscle actin-positive cells were detected (data not shown). At this stage, there is no blood flow and hence no blood pressure because the coronary arteries do not connect to the aorta until HH32. This is consistent with previous findings that the timing of coronary smooth muscle cell differentiation occurs after the formation of ostia when the blood flow may stimulate SMC formation (Lawson et al., 2002).

Fig. 4.

Fig. 4

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N1ICD immunostaining in the HH30 quail heart. A: Double immunofluorescence staining for N1ICD (green) and Qh1 (red) at lower magnification of a frontal section of HH30 quail heart. B: At the AVJ, endothelial cells in nascent capillary vessels are Notch1ICD positive. C: An immediately adjacent section to that shown in panel B, was exposed to N1ICD antibody blocked with N1ICD peptide (1:4) for the negative control (Fig. 4 C). D,E: Higher magnification of regions from panel A showed N1ICD stained vessels in the myocardium of the right ventricle and IVS. F: Higher magnification of the boxed region in B showed that N1ICD and Qh1 were co-localized in endothelial cells of the nascent coronary plexus (arrows). G: N1ICD co-localized with Qh1 in endothelial cells of myocardial vessels (arrows), G is an enlargement of the region within the white box in D. Nuclei were stained with DAPI (blue). Scale bars=500 μm in A, 50 μm in B (applies to C,D,E), 10 μm in F (applies to G).

Two sources of coronary smooth muscle cells were proposed, the neural crest derivatives and epicardial cells. It was determined that the proximal coronary stems contain some neural crest-derived SMCs for a short distance from their origin at the aortic root, using a neural crest-specific Wnt1-Cre recombinase system for lineage mapping (van den Akker et al., 2007b). As was described for aortic arch arteries in chick-quail chimeras (Hood et al., 1992; Gittenberger-de Groot et al., 1998), mouse neural crest cells in the coronary stems exhibit sharp boundaries and little or no intermixing with PEO-derived SMCs (Jiang et al., 2000). SMCs of coronary arteries originate mainly from the epicardium in avian systems. Fate mapping studies using retroviral labeling (Mikawa and Gourdie, 1996) provided evidence that proepicardial cells may be already committed to distinct endothelial or SMC fates prior to contact with the heart. We chose HH35 to study the role of N1ICD in SMC recruitment to the nascent plexus. Double immunofluorescence staining of N1ICD showed clear co-localization with SMA. Notch 1ICD was expressed in SMCs lining coronary vessels at the AVJ in HH35 quail heart (Fig.5, B arrows). The endothelial cells lining the vessel lumen were N1ICD positive (Fig.5, E, arrow heads) as were SMCs surrounding vessels within the myocardial compact layer (Fig.5, D box,). Interestingly, the N1ICD staining of epicardial mesothelial cells and subepicardial mesenchyme around the vessels was absent by stage HH35. These cells appeared to have lost N1ICD expression after epicardial EMT and differentiation after the initiation of coronary circulation. Notch-mediated lateral inhibition of N1ICD was demonstrated in several developmental events such as neuroblast segregation in Drosophila (Daudet and Lewis, 2005), vertebrate early neurogenesis (Skeath and Thor, 2003); and sensory hair cell formation in the vertebrate inner ear (Chitnis, 1995) and may be playing a role in down-regulating N1ICD expression at this late stage. Alternatively hypoxia and VEGF levels may be lower at these sites because of the initiation of coronary circulation.

Fig.5.

Fig.5

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Expression of Notch 1ICD and SMA in HH35 quail heart. A: Co-immunofluorescence staining for N1ICD (nuclear, green) and SMA (cytoplasmic, red) of a frontal section of the HH35 quail heart. B: At the right AVJ, several smooth muscle cells (red) enwrapping coronary vessels had Notch1ICD positive nuclei (green). C: The coronary vessels at the left AVJ were also covered by N1ICD positive SMCs (arrows). D: The SMCs (red) adjacent to vessels within the myocardial compact layer of the ventricles were stained by N1ICD (green). E: Higher magnification of the boxed region in panel C showed nuclear staining of N1ICD (green) co-localized (arrows) with SMA staining (red) around a vessel containing a nucleated (blue) red blood cell (negative for N1ICD, asterisk). A few endothelial cells lining the vessel lumen were N1ICD positive (arrow heads).F: A higher magnification of a region from panel D showed N1ICD staining (green) within the left ventricular wall. N1ICD (green) co-localized with SMA (red) in SMCs of myocardial vessels (arrows). Nuclei were stained by DAPI (blue). Scale bars=500 μm in A, 50 μm in B (applies to C,D), 10 μm in E (applies to F).

As summarized in Figure 6, N1ICD appears in mesothelial cells of the pro-epicardium during epicardial formation at looped heart stages. After epicardial EMT, a subset of N1ICD positive EPDCs differentiate and link to form a nascent plexus that induces “local” N1ICD positive mesenchymal cells to become smooth muscle cells. And the “local” mesenchymal cells were most likely the EPDCs migrating with the endothelial cells. Once these EPDCs commit to the endothelial cell and SMC / pericyte lineage, NICD expression is lost in the neighboring EPDCs and mesothelial cells. We propose that Notch1 signaling may play essential roles in coronary progenitor cell fate determination. Recently, evidence was obtained that epicardial cells may also serve as precursors for a subset of cardiomyocytes at sulcus regions (Cai et al., 2008; Zhou et al., 2008). It will be interesting to look at the role of Notch 1 during development of this special set of cardiomyocytes.

Fig.6.

Fig.6

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Notch1ICD expression pattern during coronary vascular development. A: The N1ICD is not present in cells of the proepicardial organ (PEO) before the PEO attaches to the myocardium. B: N1ICD (Red nuclei) appears in mesothelial cells extending from the pro-epicardium on the looping heart. C,D: After epicardial EMT, a subset of N1ICD positive mesenchymal cells (red nuclei) differentiate into endothelial cells (D, pink cells with red nuclei) and form a nascent vessel plexus within the subepicardial extracellular matrix (SEM) of the atrio-ventricular junction (AVJ) and within the compact layer of myocardium (Myo) (HH25-30). E: The endothelial cells expressing N1ICD in the vessels recruit local N1ICD positive mesenchymal cells and the latter commit to the smooth muscle cell fate(yellow cells with red nuclei) (HH32-35). There is a loss of NICD expression in the neighboring and mesothelial cells (with light blue nuclei) surrounding the mature coronary vessels.

HIF1 and Notch

The regulation of Notch signaling has recently been linked to hypoxia and HIF-1. Hypoxia or ectopically expressed HIF-1 α elevates Notch1 ICD protein levels and increases the Notch downstream response in vitro (Eddison et al., 2000). The crosstalk between hypoxia with the Notch signaling pathway has been shown to be required for hypoxia-mediated reduction of progenitor cell differentiation in neuronal and myogenic differentiation (Gustafsson et al., 2005). The Notch intracellular domain (ICD) interacted with HIF-1α, and HIF-1α was recruited to Notch-responsive promoters upon Notch activation during hypoxic conditions. Hypoxia induced Dll4, Hey1 and Hey2 in various cell types including embryonic endothelial progenitor cells (eEPCs), was mediated by activation of HIF-1α and Notch signaling. Hypoxia might also play essential roles in arterial cell fate determination by activation of the Dll4-Notch-Hey2 signalling cascade with subsequent repression of COUPTFII (Sainson and Harris, 2006). In ARMECs, we also found that hypoxia (1% O2 incubation for 24h) and ectopic stabilized HIF1 expression [infection by constitutively active HIF1α described previously (Kelly et al., 2003)] increased Notch1ICD levels correlated with the induction of HIF1α expression. (Figure 1 D.) This observation is consistent with a similar observation in C2C12 cells (Gustafsson et al., 2005). We hypothesize that N1ICD may be also be stabilized in adult rat epicardial cell EMT in some circumstance, such as under ischemic conditions. Our findings suggest that the hypoxia/HIF1-VEGF-Notch pathway plays a role in epicardial cell interactions that promote epicardial EMT and determines coronary progenitor cell fate and differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic microenvironments at sulcus regions.

EXPERIMENTAL PROCEDURES

Embryos

Fertilized eggs of the White Leghorn chicken (Gallus domesticus) and of the Japanese quail (Coturnix coturnix japonica, Boyd's Bird Company, Pullman, WA) were incubated at 37°C (80% humidity) for 2.5–9 days (HH17-35), harvested, and staged using Hamburger and Hamilton (1951) staging criteria.

Cell culture and Hypoxia treatment or ectopic HIF1 α expression

Adult rat epicardial/mesothelial cells (ARMECs) were obtained from Dr. David M. Bader (Vanderbilt University) and grown in DMEM containing 4.5 mg/mL glucose, 4 mmol/L L-glutamine with 10% FBS, and 10 μg/mL penicillin/streptomycin at 37°C with 5% CO2. ARMECs 2.5 × 106 cells in a 100-mm dish. ARMECs were exposed to 1%O2 filled with in a hypoxia chamber. Normoxic controls were exposed to atmospheric O2 concentrations within a tissue culture incubator. Another set of cells were infected by Adeno-GFP or Adeno- constitutively active HIF1α (caHIF1α,100MOI) (Kelly et al., 2003) for 1 h in 1 ml of media, and then an additional 9 ml of fresh media was added, and infections were allowed to proceed for 24h. Control cells infected by Adeno-GFP.

Western blotting

Quail or chick embryo hearts (HH30) were homogenized in RIPA buffer (Sigma) containing protease inhibitors cock tail (Roche).The ARMECs were harvested by RIPA buffer at different cell densities. For γ-secretase inhibitor studies, confluent ARMECs were harvested after treatment with γ-secretase inhibitor (10 μm) (L-685,458, Calbiochem) for 48h. After the first 24h, the media were replaced by fresh γ-secretase inhibitor (10 μm). DMSO vehicle treated cells were the negative controls. Protein content in the homogenate was determined by DC protein assay kit (Biorad). Proteins were separated on 8% polyacrylamide gels and transferred to a PVDF membrane (Millipore, Bedford, MA) using a semidry transfer cell (Bio-Rad trans-Blot SD). The blots were probed anti-cleaved Notch1 antibody (V1744 antibody, Cell Signaling) (1:500) followed with the appropriate secondary antibody conjugated with horse radish peroxidase (HRP) and were visualized with a HRP detection protocol (Pierce Chemical Co., Rockford, IL).

Immunohistochemistry

Embryonic chicken and quail hearts were dissected and fixed for 1-2 hr in 4% paraformaldehyde at 4°C, cryopreserved using a graded sucrose series (12%,15%,20%), and embedded in OCT (source of OCT). Serial 3-5μm sections were collected on pre-treated slides (Superfrost Plus, Fisher Scientific). For antigen retrieval, the sections were boiled for 30 min in sodium citrate (10 mM, pH 6.0, allowed to cool to room temperature and washed in phosphate buffered saline (PBS, 5 min). After blocking for 1hr in TNT solution anti-cleaved Notch1 antibody (V1744 antibody, Cell Signaling; 1:200; 4°C, overnight) was added, followed by washing with TNT buffer (3 washes, 5 min each). Slides were then incubated with biotinylated anti-rabbit IgG antibody (1:200, 1 hr, room temperature). Finally, the signal was amplified with tyramide signal amplification (TSA)-System (Perkin Elmer). Slides were mounted and nuclei counterstained by DAPI containing mounting media (Vector Labs). For co-staining of N1ICD and QH1 or SMA, the protocol described above for N1ICD was followed by incubation with an anti-smooth muscle actin (SMA) antibody (clone1A4, Sigma) conjugated with a Cy-3 fluorochrome (1:200) or anti-QH1 antibody (1:2000). The endothelial precursor marker Qh-1 was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA.. For Wt1 staining, alternative sections were stained with anti-Wt1 (Santa Cruz, sc-19). Wt1 and QH1 were detected with an appropriate secondary antibody conjugated with Alexa Fluor 594 (Molecular Probes, Invitrogen) at 1:200 dilution. Immunohistochemical staining of VEGFA on sections were performed using the Vectastain ABC staining kit. The endogenous peroxidase activity was quenched by incubation for 15 min in 0.3% H2O2 in PBS. Sections were incubated overnight with the anti-VEGFA (Santa Cruz) and incubated with a secondary biotin-labeled antibody were then incubated with Vectastain ABC staining kit for 1 hr. Slides were rinsed with PBS and Tris/Maleate buffer (pH 7.6). 3-3′-diaminobenzidine tetrahydrochloride (DAB) was used as chromogen. Because the anti-N1ICD, anti-Wt1 and anti-VEGFA antibodies were all generated in rabbit, consecutive sections (3μm) were stained with the three antibodies separately in order to identify the type of cells that express N1ICD. To further confirm the specificity of the signal obtained with the V1744 antibody, we stained sections of HH30 chicken embryos by N1ICD antibody pre-mixed with control N1ICD peptide (1:4). This section immediately adjacent to the one stained with N1ICD antibody showed no staining (Fig. 4 C). Stained sections were observed with a fluorescence microscope (Nikon, DIAPHOT200), and images were captured with Capture pro. Digital images were adjusted with Adobe Photoshop 7.0. Negative control sections were photographed at the same exposure as the experimental sections and digital images adjusted in parallel.

ACKNOWLEDGMENTS

The authors thank Dr. Steven A. Fisher with cardiovascular institute at University Hospitals in Cleveland, Ohio, for his comments.

This study was supported by NIH grants HL65314. HL075436. ES013607. GK was supported by AHA grant TRN104519.

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