T-helper 2 cells are essential for modulation of vascular repair by allogeneic endothelial cells

Abstract

Background: Endothelial cells (ECs) embedded within 3-dimensional matrices (MEEC) control lumenal inflammation and intimal hyperplasia when placed in the vascular adventitia. Matrix embedding alters endothelial immunogenicity in vitro. T-helper (Th) cell-driven host immunity is an impediment of allogeneic grafts. We aimed to identify if modulation of Th balance would affect immune compatibility and endothelial regulation of vascular repair in vivo.

Methods: Pigs (n = 4/group) underwent carotid artery balloon injury and were left untreated (Group 1) or received perivascular porcine MEEC implants (Group 2), 12 days of cyclosporine A (CsA; Group 3), or MEEC and CsA (Group 4). Host immune reactivity was analyzed after 28 and 90 days.

Results: MEEC treatment induced formation of EC-specific immunoglobulin (Ig) G(1) antibodies (41 +/- 6 mean fluorescence intensity [MFI]) and differentiation of host splenocytes into Th2, but not Th1, cytokine-producing cells (interleukin [IL]-4, 242 +/- 102; IL-10, 273 +/- 114 number of spots). Concomitant CsA therapy reduced IgG(1) antibody frequency (25 +/- 2 MFI; p < 0.02) and Th2-cytokine producing splenocytes upon MEEC treatment (IL-4, 157 +/- 19; IL-10, 124 +/- 26 number of spots; p < 0.05). MEECs inhibited luminal occlusion 28 and 90 days after balloon injury (12 +/- 7%) vs untreated controls (68 +/- 14%; p < 0.001) but to a lesser extent with concomitant CsA treatment (34 +/- 13%; p < 0.02 vs Group 2).

Conclusions: MEECs do not induce a significant Th1-driven immune response but do enhance differentiation of splenocytes into cells producing Th2 cytokine. Reduction in this Th2 response reduces the vasoregulatory effects of allogeneic ECs after injury.

Figure 1. Cyclosporine A is without effect on cytokine-induced endothelial expression of adhesion and costimulatory molecules. Matrix-embedded EC reveal a significant reduced expression of proinflammatory molecules

*p<0.05 PAE on TCPS vs. MEEC ^†p<0.02 PAE on TCPS vs. MEEC

Figure 2. Circulating PAE-specific IgG in pigs from the four treatment groups (n=4/group day 0–28, n=2/group day 29–90). Formation of PAE-specific IgG₁ antibodies by perivascular implantation of allogeneic matrix-embedded PAE (MEEC) is limited by concomitant cyclosporine A treatment (CsA)

A circulating PAE-specific IgM B circulating PAE-specific IgG_2a C circulating PAE-specific IgG₁ ^*p<0.005 vs. group 1 and group 3 ^†p<0.05 vs. group 4 ^‡p<0.02 vs. group 1 and group 3

Figure 2. Circulating PAE-specific IgG in pigs from the four treatment groups (n=4/group day 0–28, n=2/group day 29–90). Formation of PAE-specific IgG₁ antibodies by perivascular implantation of allogeneic matrix-embedded PAE (MEEC) is limited by concomitant cyclosporine A treatment (CsA)

A circulating PAE-specific IgM B circulating PAE-specific IgG_2a C circulating PAE-specific IgG₁ ^*p<0.005 vs. group 1 and group 3 ^†p<0.05 vs. group 4 ^‡p<0.02 vs. group 1 and group 3

Figure 2. Circulating PAE-specific IgG in pigs from the four treatment groups (n=4/group day 0–28, n=2/group day 29–90). Formation of PAE-specific IgG₁ antibodies by perivascular implantation of allogeneic matrix-embedded PAE (MEEC) is limited by concomitant cyclosporine A treatment (CsA)

A circulating PAE-specific IgM B circulating PAE-specific IgG_2a C circulating PAE-specific IgG₁ ^*p<0.005 vs. group 1 and group 3 ^†p<0.05 vs. group 4 ^‡p<0.02 vs. group 1 and group 3

Figure 3. Induction of Th2-polarized splenocytes is reduced in pigs after vascular injury and treatment with perivascular implants of allogeneic matrix-embedded PAE (MEEC) with concomitant cyclosporine A treatment (CsA). ELISPOT assay was performed 28 (A) and 90 days (B) after balloon injury

^*p<0.002 vs. group 1 and group 3 ^†p<0.05 vs. group 2 ^‡p<0.02 vs. group 1 and group 3

Figure 3. Induction of Th2-polarized splenocytes is reduced in pigs after vascular injury and treatment with perivascular implants of allogeneic matrix-embedded PAE (MEEC) with concomitant cyclosporine A treatment (CsA). ELISPOT assay was performed 28 (A) and 90 days (B) after balloon injury

^*p<0.002 vs. group 1 and group 3 ^†p<0.05 vs. group 2 ^‡p<0.02 vs. group 1 and group 3

Figure 4. Splenocytes isolated from the four different treatment groups display no significant difference in in vitro lysis of allogeneic PAE. 10⁴ PAE were labeled with calcein and incubated with 5×10⁵ splenocytes isolated after 28 and 90 days, respectively

MEEC: matrix-embedded PAE, CsA: cyclosporine A

Figure 5. Concomitant cyclosporine A treatment (CsA) modulates the ability of perivascular implanted matrix embedded PAE (MEEC) to influence vascular repair after balloon injury

A Representative angiograms of carotid arteries before (left) and 90 days (right) after balloon injury in the four treatment groups (sham; matrix-embedded PAE, MEEC; cyclosporine A alone, CsA; matrix-embedded PAE with CsA, MEEC+CsA). ^*carotid artery ^#endotracheal tube B Degree of stenosis (%) after balloon injury of both carotid arteries in the four pig treatment groups. Symbols: individual data, dashed lines: mean±SD ^*p<0.001 vs. group 1 and group 3 ^†p<0.02 vs. group 2 ^‡p<0.005 vs. group 1 and group 3

Figure 5. Concomitant cyclosporine A treatment (CsA) modulates the ability of perivascular implanted matrix embedded PAE (MEEC) to influence vascular repair after balloon injury

A Representative angiograms of carotid arteries before (left) and 90 days (right) after balloon injury in the four treatment groups (sham; matrix-embedded PAE, MEEC; cyclosporine A alone, CsA; matrix-embedded PAE with CsA, MEEC+CsA). ^*carotid artery ^#endotracheal tube B Degree of stenosis (%) after balloon injury of both carotid arteries in the four pig treatment groups. Symbols: individual data, dashed lines: mean±SD ^*p<0.001 vs. group 1 and group 3 ^†p<0.02 vs. group 2 ^‡p<0.005 vs. group 1 and group 3