Age-related Notch-4 quiescence is associated with altered wall remodeling during vein graft adaptation

Abstract

Background: The link of aging to specific mechanisms of vascular biology is not well understood. We have previously shown that aging is associated with increased vein graft wall thickness and that this process involves the VEGF-Delta/Notch-ephrin/Eph cascade. Therefore, we examined whether Dll-4 or Notch-4 are differentially expressed, according to age, during vein graft adaptation.

Materials and methods: Vein grafts were performed in 6-mo and 24-mo Fischer 344 rats. Gene expression was analyzed by quantitative real-time PCR, and the distribution of Dll-4 and Notch-4 was observed by immunofluorescence.

Results: The expression of Dll-4 and Notch-4 was reduced in vein grafts performed in aged rats compared with the expression in young adult rats. Both Dll-4 and Notch-4 were distributed in vein graft endothelium as well as the outer adventitia, with reduced amounts in the outer adventitia of aged vein grafts. Aged veins had reduced eNOS membrane targeting and colocalization with caveolin-1 as well as reduced eNOS protein expression in comparison to young adult veins. In an exchange model between young and aged animals, heterogeneous vein grafts (Yo(Ag) and Ag(Yo)) showed significantly thicker neointima compared with young (Yo(Yo)) controls, and had Notch-4-positive cells, but not Dll-4-positive cells, diminished in the adventitia. Vein grafts that were air-denuded of endothelium did not show any adaptation to the arterial environment and also lacked both Dll-4 and Notch-4 expression at 3 wk.

Conclusions: During vein graft adaptation to the arterial environment, both Dll-4 and Notch-4 expression are down-regulated in an aged, but not a young, background. Loss of Notch-4 is associated with loss of attenuation of neointima. The delta-Notch signaling pathway may be active during vein graft adaptation.

Figure 1

(A) Real time quantitative RT-PCR shows impaired Dll-4, Notch-4, ephrin-B2, and Eph-B4 gene expression in aged rat vein grafts. Data is number of mRNA transcripts detectable by qPCR and normalized to GAPDH, with the bar graph showing the difference between aged and young adult vein graft expression, in n=4 distinct young adult and aged samples. *: p<0.05 of aged in comparison to young adult number of transcripts. (B) Representative immunofluorescence staining of Notch-4, from n=4 young adult and n=4 aged samples. White arrowheads indicate positive signal in the outer adventitia. I+M: intima and medial layer, Ad: adventitial layer, O: outer adventitia, SM: Smooth muscle. Scale bar: 20 micrometer.

Figure 2

Representative immunofluorescence staining of Dll-4, from n=4 young adult and n=4 aged vein graft samples. I+M: intima and medial layer, Ad: adventitial layer, O: outer adventitia. Scale bar: 20 micrometer.

Figure 3

(A) Representative immunofluorescence images of young adult (upper 2 rows) and aged (lower 2 rows) vein and vein grafts. eNOS, green signal; caveolin-1, red signal; merge, yellow signal. n=3 young adult and aged animals. (B, C) Bar graphs show mean number of transcripts detectable by qPCR at 0, 6 hr, 24 hr, and 3 wks after vein graft implantation, in both young adult and aged animals. Panel (B) shows PDGF-BB, and panel (C) shows VEGF-A, normalized to GAPDH. n=4. *, p<0.05 in comparison of young vs. aged data. Some the data from panel (C) has been previously published.⁵

Figure 4

Representative immunofluorescence images of young adult (upper 2 rows) and aged (lower 2 rows) vein and vein grafts. eNOS, green signal; Golgi, red signal; merge, yellow signal. n=3 young adult and aged animals.

Figure 5

Representative Western blot analysis from n=3 young adult and aged veins. There is reduced density of eNOS in the aged veins.

Figure 6

Morphological analysis of syngeneic exchanged vein grafts between young adult and aged hosts. (A) Representative images of hematoxylin-eosin (H&E) and Masson’s trichrome (MT) staining. Yellow arrowheads indicate neointimal hyperplasia formation. (B) Measurement of rat neointimal thickness as the intima+media layers (I+M) and (C) adventitial thickness. n=4 in each group. *: p<0.05 in comparison to the Yo^Yo group.

Figure 7

(A) Representive immunofluorescence staining images of Dll-4 (upper panels) and Notch-4 (lower panels) in perivascular connective tissue (outer adventitia) of syngeneic switched rat vein grafts. Yellow arrowheads show positive cells. (B) Dll-4 and (C) Notch-4 positively stained cell counts. n=4 in each group. *: p<0.05 in comparison to the Yo^Yo group. Scale bar shows 20 micrometer.

Figure 8

EC denuded vein grafts. (A) Schema of EC denudation in vein grafts by air injury of the preimplantation vein. The external jugular vein was tied at three points: proximal end, distal end, and primary major branches. A pin hole for egress of air was made at the distal end, and a 30G needle was inserted at the primary major branch (red arrows). After air injury, the venous lumen was flushed with saline, and harvested at the blue arrows. Thick black lines indicate tie points. (B) Confirmation of EC denudation. Evans blue was injected 1 hour after vein graft surgery, and sacrificed 30 minutes after injection. Vein grafts were harvested with the proximal and distal carotid artery, and opened for observation of graft en face. Compared to the endothelial-intact artery, the vein graft luminal surface demonstrates blue staining due to EC denudation. Arrowheads show blue staining in artery luminal surface due to EC injury by clamping. CCA: common carotid artery, ICA: internal carotid artery, ECA: external carotid artery, EJV: external jugular vein, VG: vein graft. (C) Representive Masson’s trichrome staining images of control and EC denuded vein grafts. No obvious neointimal hyperplasia was present in either denuded young adult or aged vein grafts. (D) Morphological analysis of EC denuded vein graft. Rat neointimal thickness is measured as the total of the intima + media layers. n=3 in each group. *: p<0.05 in comparison of young adult and aged groups. n. s.: not significant.

Figure 9

Analysis of Dll-4 and Notch-4 positive cells in EC denuded vein grafts. (A) Representative images of Dll-4 immunofluorescence staining. (B) Dll-4 positive cell counts in pre-implantion vein (Vein), control vein graft (Control), and EC-denuded vein grafts (Denuded). (C) Representative images of Notch-4 immunofluorescence staining. (D) Notch-4 positive cell counts in pre-implantion vein, control VG, and EC-denuded VG. Yellow arrowheads indicate positive cells. Scale bar: 20 micrometer. n=3 in each group. *: p<0.05 between control vs. denuded vein graft. §: p<0.05 between young adult vs. aged.