Table 1.
Temporal artery implant (TAI) numbers and treatment.
Fig 1.
Aortic Window Xenograft / TAI Model.
(A) Schematic of human temporal artery biopsy implant into the aorta of SCID mice. (B) Left panel—surgical window in the anterior abdominal aortic wall, Right panel—TA xenopatch covering the surgical window. Arrowhead—surgical window in recipient mouse, Short black arrows—donor human TAI graft. Longer black arrows—recipient mouse aorta. (C) Cross section of TAI biopsy graft in SCID mouse aorta. Blue line outlines TA graft (HA—human artery). Black line outlines mouse artery (MA). (D) Mouse aortic cells display excellent viability after storage in OCT at -80°C for 1–7 days. I—intima). M—media, A—adventitial, VC—vena cava, S—suture.
Fig 2.
Immunostained human cells in spleen and temporal artery (TA) xenograft sections demonstrate persistent human PBMC colonization at 28 days.
(A) MHC-1 positive staining. (B) Mitochondria positive staining. (C) CD3+ positive staining. Brown stained cells (arrow) are positively stained for selected human antigens (n = 11. MHC-I—major histocompatibility complex-I-I. Magnification: 400X for spleen, 200X for TA grafts and 800X for insets.
Fig 3.
GCApos TAI grafts have increased vascular inflammatory lesion (VIL) thickness when compared to GCAneg TAI; Serpin treatment reduces intimal thickness.
H & E stained cross sections of TAI engrafted SCID mouse aorta at 4 weeks follow up; (A) GCAneg TAI graft saline treated, (B) GCApos TAI saline treated, (C) GCAneg section, Serp-1 treated, (D) GCApos section, Serp-1 treated. Invading mononuclear cells in TAI engrafted aortic cross sections in mice after PBMC infusion at 4 weeks: (E) GCAneg cross section, Saline treated, (F) GCApos section, Saline treated, (G) GCAneg, Serp-1 treated. (H) GCApos Serp-1 treated. (I) Bar graphs of mean intimal/ medial (I/M) thickness ratios (± SE) for TAI grafts. GCApos TAI with Saline treatment have increased VIL. I/M are significantly reduced with Serp-1 (p = 0.005). (J) Inflammatory cell counts are significantly reduced with Serp-1 treatment in GCApos implants after PBMC transfusion (p ≤ 0.041; n = 3 each for GCAneg and pos). (K) When the GCApos and GCAneg data are combined, mean I/M thickness ratios are significantly reduced in Serp-1 treated mice when compared to Saline and PBMC/Saline groups (P ≤ 0.011 and 0.039, respectively; n = 5–10). (L) The mean mononuclear cell counts in combined GCApos and GCAneg data was significantly reduced in mice treated with Serp-1 after PBMC transfusion but not without PBMC, when compared to controls (p ≤ 0.046; n = 5–10). (I—intima, M—media, A—adventitia, S—suture. Arrow—inflammatory cell. Mag-100–200X).
Fig 4.
Immunohistochemical analysis demonstrates significantly reduced CD11b positive cell counts with Serp-1 treatment in TAI grafts in SCID mice at 4 weeks (n = 5–10).
(A) CD11b+ staining of GCApos sections in Saline + PBMC treated mice. Brown stained areas are positively stained cells (marked by arrows) (B) CD11b+ staining of GCApos sections in TAI grafts with Serp-1 + PBMC treatment. (C) GCApos or GCAneg engrafted sections without PBMC did not show a significant change for CD3, CD11b, CCR6 nor CD83 stained cell counts (n = 3 each for GCApos or GCAneg). (D) CD11b+ cells were significantly reduced by Serp-1 treatment after PBMC transfusion when combining data for GCApos and GCAneg. No difference in CD3+, CCR6+, and CD83+ cell counts were detected for combined data (n = 5–10). Magnification- 400X.
Fig 5.
Flow cytometry illustrates decreased inflammatory cell numbers after Serp-1 treatment in splenocytes from TAI engrafted SCID mice.
A, B, C and D are representative flow cytometry results for CD4+IL-17+ cells treated by Saline, Serp-1, Saline+PBMC and Serp-1+PBMC respectively. E, F and G show the increased Th1 (CD4+ IFN-γ+), Th17 (CD4+IL-17+) and Treg (CD4+FoxP3+) cells in PBMC transfused TAI engrafted SCID mice when compared to Saline treated mice (P ≤ 0.01), respectively. Serp-1 with PBMC transfusion decreased the cell numbers when compared to PBMC transfusion alone (P ≤ 0.02, P ≤ 0.01, P ≤ 0.01). For Th1 cells, the absolute positive cell count was low, but still a trend can be noted. To increase cell numbers, spleen cells were pooled from the same group.
Fig 6.
Gene expression changes in splenocytes isolated from mice with TAI after Saline and Serp-1 treatments.
(A) Real-time PCR analysis demonstrates significantly increased gene expressions for IL-1β, IL-17, and CD25, in spleen isolates from Saline+PBMC treated GCApos TA implanted mice (P ≤ 0.0317, 0.0281, and 0.0354, respectively). fII and IL-1β gene expression in GCApos engrafted mice are significantly reduced by Serp-1+ PBMC treatment (P ≤ 0.002; n = 3 for GCApos and GCAneg) (B) In combined data of GCApos and GCAneg engrafted mice, PCR array analyses demonstrate significantly increased expression of thrombotic and thrombolytic proteases, serpins and receptors, inflammatory cell markers and cytokines/chemokines in TAI engrafted mice transfused with PBMCs. Serp-1+PBMC treatment significantly reduced expression when compared to Saline + PBMC. (* P ≤ 0.05, ** P ≤ 0.01; N = 4 for Saline + PBMC, N = 5 for Serp-1 + PBMC).