Varying levels of 6-keto-prostaglandin F1α and thromboxane B2 in serum and endothelialization and hyperplasia in small-diameter grafts seeded with CD34+ bone marrow cells in canines

Abstract

The aim of the present study was to investigate the serum levels of 6-keto-prostaglandin (PG)F1α and thromboxane (TX)B₂, as well as the endothelialization and hyperplasia of polytetrafluoroethylene (PTFE) and Dacron prostheses seeded with CD34+ cells in medium-term observation. A total of 24 crossbred dogs were randomly distributed into PTFE or Dacron groups. CD34+ cells were isolated from bone marrow aspirate and collected using an immunomagnetic bead-based system. The PTFE or Dacron prostheses were implanted into the abdominal aortic artery and inferior vena cava of the dogs. In each group, 8 dogs were implanted with prostheses that had been seeded with CD34+ cells, while 4 dogs were implanted with prostheses that had been seeded with autogenous blood as a control. Serum concentrations of 6-keto-PGF1α and TXB₂ were determined at days 0, 10, 30 and 60 following surgery. The grafts were removed and examined at days 10, 30, 60 and 100 following surgery. Finally, CD34 factor staining was used to identify endothelial cells, while light and electron microscopy were applied to examine endothelialization and patency. The results revealed that confluent endothelial cells appeared on the neointima of prostheses seeded with CD34+ cells at day 30 following surgery. In the control groups compared with the experimental groups, there were fewer endothelial cells and the neointima was significantly thicker in the arterial (PTFE, 174±1.41 vs. 117±2.83 μm, respectively; P=0.001; Dacron, 187.5±3.5 vs. 100±1.41 μm, respectively; P<0.001) and venous (PTFE, 230.5±6.36 vs. 135±5.66 μm, respectively; P=0.001; Dacron, 249±2.83 vs. 121.5±3.54 μm, respectively; P<0.001) prostheses. In the experimental groups, intimal hyperplasia in the venous prostheses (PTFE, 135±5.66 μm; Dacron, 121.5±3.54 μm) was more severe compared with that in the arterial prostheses (PTFE, 117±2.83 μm; Dacron, 100±1.41 μm) at day 60. Compared with the 6-keto-PGF1α concentrations in the experimental groups, those in the control groups were significantly lower on day 10 (PTFE, 135±6.01 vs. 80.5±4.35 pg/l, respectively; P=0.001; Dacron, 145±6.54 vs. 81.2±5.10 pg/l, respectively; P<0.001) and were then maintained at a lower level. By contrast, the TXB₂ concentration, following marked increases on day 10 in the experimental and control groups (PTFE, 635±32.8 vs. 1,256±63.5 pg/l, respectively; P<0.001; Dacron, 652±30.9 vs. 1,136±53.2 pg/l, respectively; P=0.001), remained at a high level in the control groups. Therefore, the results of the present study indicate that it is possible to achieve rapid endothelialization in PTFE or Dacron prostheses by implanting CD34+ cells. Endothelialization inhibited the reduction in the concentration of 6-keto-PGF1α and the increase in the concentration of TXB₂. In addition, endothelialization inhibited excessive intimal hyperplasia and thrombosis. Thus, CD34+ cell seeding provides a theoretical basis for the clinical application of artificial vessel endothelialization.

Keywords: 6-keto-prostaglandin F1α; CD34+ cells; endothelialization; grafts; thromboxane B2.

Figure 1

(A) Grafts were patent following surgery. (B) Grafts without significant deformation adjoined with the surrounding tissue. (C) In the test groups, grafts had smooth and translucent surfaces with no apparent thrombosis. (D) Venous grafts were occlusive in the control groups at day 100 following surgery.

Figure 2

(A) CD34+ cells were identified by flow cytometry. (B) Immunocytochemical staining with FVIII/vWF and CD34 antibodies resulted in staining of endothelial cells. (C) With the PTFE test grafts, there was a layer of neointima consisting of a single layer of endothelial cells on the surface, shown to be positive with H&E staining. The subintima was largely composed of smooth muscle cells, fibroblasts and collagen. (D) With the Dacron test grafts, the subintima was largely composed of smooth muscle cells, fibroblasts and collagen. There were no osteocytes, osteoblasts or microcalcification in the seeded grafts. PTFE, polytetrafluoroethylene; H&E, hematoxylin and eosin; FVIII, factor VIII; vWF, von Willebrand factor.

Figure 3

Endometrial thickness measurements of (A) the test groups and (B) the test and control groups at day 60. Serum concentration of (C) 6-keto-PGF1α and (D) TXB₂. PG, prostaglandin; TX, thromboxane; PTFE, polytetrafluoroethylene.

Figure 4

Scanning and transmission electron microscopy images of the (A) Dacron test group at day 10 (magnification, ×1,000), (B) PTFE test group at day 10 (magnification, ×2,000), (C) Dacron test group at day 30 (magnification, ×1,000) and (D) PTFE test group at day 30 (magnification, ×2,000). PTFE, polytetrafluoroethylene.

Figure 5

Scanning and transmission electron microscopy images of the (A) Dacron test group at day 60 (magnification, ×1,000), (B) PTFE test group at day 60 (magnification, ×1,000), (C) Dacron test group at day 60 (magnification, ×2,000) and (D) PTFE test group at day 60 (magnification, ×1,500). PTFE, polytetrafluoroethylene.

Figure 6

Scanning and transmission electron microscopy images of (A) Dacron test group at day 100 (magnification, ×500), (B) PTFE test group at day 100 (magnification, ×2,000), (C) Dacron control group at day 60 (magnification, ×500) and (D) PTFE control group at day 60 (magnification, ×2,000). PTFE, polytetrafluoroethylene.