Adventitial transplantation of blood outgrowth endothelial cells in porcine haemodialysis grafts alleviates hypoxia and decreases neointimal proliferation through a matrix metalloproteinase-9-mediated pathway--a pilot study

Abstract

Purpose. We hypothesized that adventitial transplantation of blood outgrowth endothelial cells (BOEC) to the vein-to-graft anastomosis of polytetrafluoroethylene grafts will reduce neointimal hyperplasia by reducing hypoxia inducible factor-1alpha (HIF-1alpha), by increasing angiogenesis in a porcine model of chronic renal insufficiency with haemodialysis polytetrafluoroethylene grafts. Because matrix metalloproteinases (MMPs) have been shown to be involved with angiogenesis, the expression of MMPs and their inhibitors was determined.

Methods: Chronic renal insufficiency was created by subtotal renal infarction and 28 days later, arteriovenous PTFE grafts were placed bilaterally from the carotid artery to the jugular vein. Autologous blood outgrowth endothelial cells labeled with Lac Z were transplanted to the adventitia of the vein-to-graft anastomosis using polyglycolic acid scaffolding and scaffolding only to other side (control). Animals were killed 14 days later and vessels were explanted from the vein-to-graft anastomosis of both sides and underwent immunohistochemical analysis, western blotting and zymography for HIF-1alpha, MMP-2, MMP-9, TIMP-1 and TIMP-2. BOEC were also made hypoxic and normoxic for 12, 24 and 48 h to determine protein expression for MMPs and TIMPs.

Results: Under hypoxia, BOEC significantly increased the expression of pro MMP-2 by 12 h and TIMP-2 by 24 h when compared to normoxic cells (P < 0.05). Transplantation of BOEC resulted in a significant decrease in both HIF-1alpha and intima-to-media ratio with a significant increase in both pro and active MMP-9 when compared to control vessels (P < 0.05). MMP-9 activity was localized to the neointima of the transplanted vessels by immunohistochemistry. There was increased CD31 density with engraftment of BOEC cells into the neointima of both the transplanted vessels compared to controls (P = NS).

Conclusion: Transplantation of BOEC resulted in a significant decrease in intimal hyperplasia and HIF-1alpha with a significant increase in both pro and active MMP-9 that was localized to the neointima of transplanted vessels. The increase in MMP-9 offers a possible mechanism for angiogenesis and the reduced intima-to-media ratio. Furthermore, we observed that BOEC had homed to the neointima of the contralateral vessels that had increased levels of HIF-1alpha, suggesting that hypoxia may be an important stimulus for BOEC migration.

Fig. 1

Placement of polytetrafluoroethylene haemodialysis graft and representative MRI and PC MRA of venous stenoses. (A) Placement of polytetrafluoroethylene haemodialysis grafts. (B) MRI and PC MRA were performed in a Day 14 animal with BOEC treatment on the right (white arrow) and control on the left (yellow arrow). (C) Schematic showing the location of the vein-to-graft anastomosis used for histology (V1) and for protein analysis (V2). PTFE = polytetrafluoroethylene, VS = venous stenosis, GA = grafted artery, CA = control artery, CV = control vein.

Fig. 2

HIF-1α expression as a function of time in the BOEC cells during hypoxia at different time points. (A) Graph (upper) representing appropriate protein band of HIF-1α from Western blot analysis and graph (lower) representing appropriate band for IgG from Western blot for protein loading. N is normoxia and H is hypoxia. (B) Semiquantitative analysis for HIF-1α performed at 12, 24 and 48 h. The normalized density of HIF-1α was significantly higher in hypoxic specimens when compared to normoxic specimens at all time points. *A significantly higher value (P < 0.05). Data are mean ± SD.

Fig. 3

Pro MMP-2 expression by BOEC under hypoxic conditions at different time points. (A) Graph (upper) representing appropriate protein band of pro MMP-2 from zymographic analysis. N is normoxia and H is hypoxia. (B) Semiquantitative analysis for HIF-1α performed at 12, 24 and 48 h. The normalized density of pro MMP-2 was significantly higher in hypoxic specimens when compared to normoxic specimens at 12 h. ^*A significantly higher value (P < 0.05). Data are mean ± SD.

Fig. 4

TIMP-2 expression by BOEC under hypoxic conditions at different time points. (A) Graph (upper) representing appropriate protein band of TIMP-2 from Western blot analysis and graph (lower) representing appropriate band for IgG from Western blot for protein loading. N is normoxia and H is hypoxia. (B) Semiquantitative analysis for TIMP-2 performed at 12, 24 and 48 h. The normalized density of TIMP-2 was significantly higher in hypoxic specimens when compared to normoxic specimens at 12 h. ^*A significantly higher value (P < 0.05). Data are mean ± SD.

Fig. 5

Verhoeff's van Giesen staining was performed at the venous stenosis (section V1, see Figure 1C) from the cushioning region of the BOEC-transplanted (A and C) and contralateral non-transplanted (control) veins (B and D). A and B are 5× and C and D are 40× magnification. The lower panel (E) shows that there was a 50% decrease in the intima-to-media ratio in the BOEC-transplanted samples when compared to controls (P < 0.05). Data are mean ± SD.

Fig. 6

Confocal triple immunostaining of the vein-to-graft anastomosis with transplanted BOEC from a Day 14 animal (A,B). For comparison, (C) is a normal vein. Blue denotes cell nuclei, green α-SMA and red CD31-positive cells. A shows that CD31-positive cells are lining the adventitial microvessels (^*) surrounded by α-SMA-positive cells. (B) The neointima is composed primarily of α-SMA-positive cells. Ad is the adventitia, M is the media, N is the neointima and L is the lumen. For comparison, normal vein is shown in C. L is the lumen, I is the intima and M is the media. (D) A confocal double immunostaining of eNOS-positive cells (red) from BOEC-transplanted vessels. (E) A normal vein for comparison.

Fig. 7

Indirect immunofluorescence for CD31-positive cells was performed at the venous stenosis from BOEC grafted (A) and control specimens (B). Ad is adventitia, M is the media and L is the lumen, and the yellow arrow shows microvessel formation. Lower panel shows quantitative assessment from the venous stenosis from BOEC and control grafts. There was increased amount of CD31-positive cells in the BOEC samples when compared to the controls.

Fig. 8

Staining for β-gal was performed at the vein-to-graft anastomosis removed from the BOEC and contralateral non-transplanted (control) specimens. In (A), L is the lumen, N is the neointima, M is a microvessel and A is adventitia. B is a high magnification of the adventitia. (A) Engraftment of Lac Z-positive labeled BOEC cells (blue) into the neointima (N). In (B), there is engraftment of Lac Z-positive cells into the microvessel (yellow arrows) in the adventitia. Lower panel shows quantitative analysis of the Lac Z-positive BOEC cells into the neointima and intima of both the transplanted and non-transplanted contralateral (control) vein-to-graft anastomosis. There was an increase in the engraftment of Lac Z-positive cells into the neointima of the control vein-to-graft anastomosis when compared to the BOEC.

Fig. 9

Graph representing appropriate protein band HIF-1α (A) from western blot analysis and pro and active MMP-9 from zymography (B) with blood outgrowth endothelial cell (BOEC) delivery compared to control (C). According to semiquantitative analysis, by Day 14, the normalized density of HIF-1α was significantly less in the BOEC samples when compared to control (P < 0.05) while the normalized density of both pro and active MMP-9 was significantly higher than the control vessels (P < 0.05). (D), (E) Confocal double immunostaining of MMP-9-positive cells (red) from BOEC-transplanted vessels and (F) the contralateral non-transplanted (control) vessel. Ad is the adventitia, M is the media, N is the neointima and L is the lumen. ^*An adventitial microvessel surrounded by cells expressing MMP-9. More intense immunostaining was observed in the neointima of the transplanted vessels when compared to non-transplanted vessels.

Fig. 10

Three-dimensional microscopic computed tomographic evaluation of the carotid artery and jugular vein. (A) A pig carotid artery cross section showing the lumen and the origin of a vasa vasorum externa with microfilm (box). Yellow arrow is shown in (B). (B) The arterial vasa vasorum (red) and the venous vasa vasorum (blue) with the yellow arrow on the vasa vasorum from (A). (C) The vasa vasorum surrounding the vein that communicates with the arterial vasa vasorum.