Epidermal growth factor prevents oligomeric amyloid-β induced angiogenesis deficits in vitro

Cells, oAβ42 and GFs

hCMEC/D3 cells (BECs) were a kind gift from Pierre-Olivier Couraud (Inserm) and cultured as described previously. ¹⁰ Twenty-four hours before experiments, BECs were serum- and GF-starved. Primary human pericytes and astrocytes (Sciencell) were cultured according to manufacturer protocol between passages 2 and 5. oAβ42 was prepared as previously described ¹¹ and used at the indicated concentrations (0–100 nM). GFs (Shenandoah) were utilized at 100 nM.

Cell viability assays

Cell viability was assessed in hCMEC/D3 cells with oAβ42 ± GFs (24 hr) via the MTT assay ¹² (ThermoFisher).

Tube formation and disruption assays

Immunocytochemistry and imaging analysis

All angiogenesis plates were fixed in 4% paraformaldehyde at t = 24 h. Fluorescent images of BECs in each well were captured at × 1.6 magnification and analyzed using the ImageJ angiogenesis plug-in. ¹³ Immunocytochemistry (ICC) was then performed as described previously ¹⁰ using the Aβ specific antibody MOAB-2 (1:400, Biosensis). To image BECs, astrocytes and pericytes, high magnification images were captured on a Zeiss Axio Imager M1 under identical capture settings at × 5 or × 10 magnification. Aβ and pericyte coverage were assessed using × 5 magnification images in imageJ (Figures 1 and 2).

OPEN IN VIEWER

Figure 1.

oAβ42 inhibits tube formation and induces vessel disruption in BEC cultures, prevention by EGF. (a–e) Treatment Paradigm 1 – Simultaneous. (a) hCMEC/D3 viability in response to oAβ42 at 0, 100 nM, and 5μM with and without GFs, as assessed by MTT assay (****p < 0.0001 by two-way ANOVA, main effect of concentration). (b) Schematic representation of the workflow. BECs were plated and treated with oAβ42 (0–100 nM) alone or in combination with VEGF (100 nM), FGFβ (100 nM), or EGF (100 nM). (c) Total vessel length and number of meshes formed by BECs quantified using × 1.6 magnification images (n = 3 with 2–4 replicates, *p < 0.05, ****p < 0.0001 by standard two-way ANOVA, main effect of treatment group; Φp < 0.05 by Dunnet’s multiple comparison vs. oAβ42 group. #p < 0.05 by ANOVA for both oAβ42 50 and 100 nM vs. VC, $p < 0.05 by ANOVA for oAβ42 100 nM vs. VC). (d) Representative × 10 images of VC, VEGF, and EGF treatments (from left to right, top) and oAβ42, oAβ42 plus VEGF, and oAβ42 plus EGF (from left to right, bottom. All images at 100 nM oAβ42. Green = BECs; red = Aβ42). (e) Percent area occupied by Aβ42 (100 nM, n = 3 with 2–4 replicates). (f–i) Treatment paradigm 2 – spike/protection. (f) Schematic representation of the workflow. In the spike/prevention treatment paradigm, BECs were treated with VEGF (100 nM), FGFβ (100 nM), or EGF (100 nM) and oAβ42 (0–100 nM) spiked into the culture. (g) Quantification of total vessel length and number of meshes formed by BECs quantified using × 1.6 magnification images (n = 4 with 2–4 replicates, ****p < 0.0001 by standard two-way ANOVA, main effect of treatment group; Φp < 0.05, ΦΦΦp < 0.001 by Dunnet’s multiple comparison vs. oAβ42 group. #p < 0.05 by ANOVA for both oAβ42 50 and 100 nM vs. VC). (h) Representative × 10 images of VC, VEGF, and EGF treatments alone (from left to right, top) and oAβ42, oAβ42 plus VEGF, and oAβ42 plus EGF (from left to right, bottom. All images at 100 nM oAβ42. Green = BECs; red = Aβ42). (i) Percent area occupied by Aβ42 (100 nM, n = 4 with 2–4 replicates). (j) Schematic representation of the workflow for the reversal paradigm. (k) Total vessel length and number of meshes formed by BECs quantified using × 1.6 magnification images (n = 5 in triplicate, #p < 0.05 by ANOVA for both oAβ42 50 and 100 nM vs. VC, $p < 0.05 by ANOVA for oAβ42 100 nM vs. VC, + p < 0.05 by Student’s t-test vs. oAβ42 50 nM). (l) Representative × 10 images of oAβ42, and oAβ42 plus EGF (from left to right. All images at 100 nM oAβ42. Green = BECs). All data are expressed as mean ± s.e.m compared to oAβ42 VC. Scale bar represents 100 µm.

OPEN IN VIEWER

Figure 2.

oAβ42 induces vessel disruption in MVU cultures, prevention by EGF. (a) Schematic representation of the workflow for the treatment paradigm in MVU cultures, treated with oAβ42 (0–100 nM) alone or in combination with VEGF (100 nM), FGFβ (100 nM), or EGF (100 nM). (b) Total vessel length and number of meshes formed by BECs quantified using × 1.6 magnification images (n = 3 in triplicate, **p < 0.01, ****p < 0.001 by standard two–way ANOVA, main effect of treatment group; ΦΦΦp < 0.001, ΦΦΦΦΦΦΦp < 0.0001 by Dunnet’s multiple comparison vs. oAβ42 group. $p < 0.05 by ANOVA for oAβ42 100 nM vs. VC). (c) Representative × 10 images of VC, VEGF, and EGF treatments alone (from left to right, top) and oAβ42, oAβ42 plus VEGF, and oAβ42 plus EGF (from left to right, bottom. All images at 100 nM oAβ42. Green = BECs; red = astrocytes; blue = pericytes). (d) Quantification of pericyte count and percent area occupied (n = 3 in triplicate, &p < 0.05 compared to oAβ42 VC; %p < 0.05 compared to oAβ42 100 nM by Student’s t-test). All data are expressed as mean ± s.e.m compared to oAβ42 VC. Scale bar represents 100 µm.

Statistical analysis

All data are presented as mean +/− s.e.m and were analyzed using two-way AVOVA, one-way ANOVA, or Student’s t-test, as specified, in GraphPad Prism version 6 (Table 1).

OPEN IN VIEWER

Table 1.

Summary of statistics from all paradigms.

Paradigm	Measure	p: treatment effect (Two-way ANOVA)	p: oAβ42 vs VEGF (Dunnet’s)	p: oAβ42 vs FGFβ (Dunnet’s)	p: oAβ42 vs EGF (Dunnet’s)
Paradigm 1-simultaneous	Total length [3, 144]	p < 0.0001****	p = 0.4750	p = 0.0965	p = 0.0195Φ
	Number of meshes [3, 144]	p = 0.0154*	p = 0.9762	p = 0.09891	p = 0.0466Φ
Paradigm 2-spike	Total length [3, 144]	p < 0.0001****	p = 0.9811	p = 0.0006ΦΦΦ	p = 0.0249Φ
	Number of meshes [3,144]	p < 0.0001****	p = 0.4336	p = 0.0161Φ	p = 0.0003ΦΦΦ
Paradigm 3-reversal	Total length [3, 224]	p = 0.1370	p = 0.1456 (DNS)	p = 0.9847 (DNS)	p = 0.3575
	Number of meshes [3, 224]	p = 0.6259	p = 0.6714 (DNS)	p = 0.9847 (DNS)	p = 0.9996
MVU paradigm T1-spike	Total length [3, 128]	p = 0.0013**	p = 0.5260	p = 0.6041	p = 0.0006ΦΦΦ
	Number of meshes [3, 128]	p < 0.0001****	p = 0.0747	p = 0.3438	p < 0.0001ΦΦΦΦ

EGF prevents direct oAβ42-induced disruption of tube formation in single BEC cultures (paradigm 1)

Initially, the effect of non-toxic oAβ42 concentrations (identified by MTT assay, Figure 1(a)) on tube formation was assessed in single cultures of immortalized human BECs (hCMEC/D3 cells) by simultaneously adding cells +/−oAβ42 (VC, 25 nM, 50 nM, 100 nM) for 24 h to a Matrigel® matrix (Figure 1(b)). At non-toxic doses, oAβ42 dose-dependently disrupted tube formation (Figure 1(c)). Compared to the VC, total vessel length was 20% and 16% lower with 50 and 100 nM oAβ42, respectively. Further, the number of meshes was 10% (50 nM) and 40% (100 nM) lower with oAβ42. Next, the effect of simultaneous oAβ42 (0–100 nM) and GF treatment was determined (Figure 1(b)). EGF (100 nM), but not VEGF, FGFβ, or IGF (data not shown) ameliorated oAβ42-induced disruption of tube formation (Figure 1(c) and (d)). For example, compared to the VC, total tube length was 110% for combined oAβ42-EGF treatment (Figure 1(c)). Interestingly, EGF-induced protection was not due to changes in oAβ42 accumulation in the BECs (Figure 1(e)). These data support that direct oAβ42 signaling prevents tube formation and EGF ameliorates this effect.

EGF prevents oAβ42-induced tube disruption in single BEC cultures (paradigm 2)

To determine whether oAβ42 causes tube degeneration, BECs were incubated for 4 h to form vessel networks, and oAβ42 was spiked into the media for a further 20 h (Figure 1(f)). oAβ42 reduced total vessel length by 20% and the number of meshes by ∼40% at both 50 and 100 nM (Figure 1(g)). EGF prevented the oAβ42-induced vessel disruption to BECs (Figure 1(g) and (h)). Indeed, compared to 50 and 100 nM oAβ42, total vessel length values were 20% and 35% higher with combined oAβ42-EGF treatment, respectively. Further, with combined EGF and oAβ42 treatment, the number of meshes were 88% (50 nM oAβ42) and 61% (100 nM oAβ42) higher compared to oAβ42 alone (Figure 1(g)). As for tube formation, the beneficial effects of EGF were not related to alterations in oAβ42 uptake or degradation (Figure 1(i)). For other GFs, there was a non-significant trend for VEGF to prevent oAβ42-induced tube disruption and interestingly, FGFβ in combination with oAβ42-induced more robust vessel disruption compared to oAβ42 alone (Figure 1(g)).

EGF does not reverse oAβ42-induced disruption of tube formation in single BEC cultures (paradigm 3)

To determine whether EGF could reverse oAβ42-induced tube formation disruption, BECs were incubated ± oAβ42 from 0–4 h and GFs spiked into the media for a further 20 h (Figure 1(j)). As in paradigm 1, oAβ42 (0–100 nM) disrupted tube formation (Figure 1(k)). However, with the exception that EGF appeared to reverse oAβ42-induced disruption in total vessel length at 50 nM oAβ42, EGF was largely unable to reverse oAβ42-induced tube disruption in this paradigm (Figure 1(k) and (l)).

EGF prevents oAβ42-induced tube disruption in MVU cultures

Although our data demonstrates that oAβ42 induces detrimental effects on tube formation directly in BECs, it is important to conduct complementary analysis using the main cell types of the MVU, which can respond to oAβ42 to collectively determine vessel length. Therefore, we next investigated the effects of oAβ42 and GFs in cultures containing BECs, primary human pericytes and primary human astrocytes. We focused on the oAβ42-induced effects on preformed vessels to mimic the in vivo scenario of oAβ42 accumulation with an established CV (Figure 2(a)).

oAβ42 treatment resulted in tube disruption only at the highest dose (100 nM) causing a 20% reduction in number of meshes and 13% reduction in total tube length (Figure 2(b)). As 50 nM oAβ42 caused disruption in single BEC cultures, these data imply that BECs are more resistant to insult within the MVU (proposed in Itoh et al. ¹⁴ ). At high magnification, pericyte coverage at tube junctions and tube projections appeared lower in oAβ42-treated triple cultures (Figure 2(c)). Indeed, when quantified, 100 nM oAβ42 reduced the percent area occupied by pericytes by 49% compared to VC (Figure 2(d)).

As observed in BEC cultures, EGF protected against 100 nM oAβ42-induced tube disruption, with a 20% higher total tube length and a 50% greater number of meshes compared to oAβ42 treatment alone (Figure 2(b)). Interestingly, treatment with 25 and 50 nM oAβ42 in combination with EGF resulted in higher in total length and number of meshes, despite oAβ42 treatment alone at the same concentrations not causing any direct disruption. Further, EGF prevented the 100 nM oAβ42-induced decrease in the number of and percent area covered by pericytes, while VEGF and FGFβ had no effect (Figure 2(d)). These data suggest a direct link between pericyte health/coverage and the ability of BECs to form viable networks that is disrupted in the presence of oAβ42. Mirroring data in single BECs, VEGF trended to protect against the oAβ42-induced disruption at 100 nM.