Inhibition of Chemokine-Glycosaminoglycan Interactions in Donor Tissue Reduces Mouse Allograft Vasculopathy and Transplant Rejection

Abstract

BackgroundBinding of chemokines to glycosaminoglycans (GAGs) is classically described as initiating inflammatory cell migration and creating tissue chemokine gradients that direct local leukocyte chemotaxis into damaged or transplanted tissues. While chemokine-receptor binding has been extensively studied during allograft transplantation, effects of glycosaminoglycan (GAG) interactions with chemokines on transplant longevity are less well known. Here we examine the impact of interrupting chemokine-GAG interactions and chemokine-receptor interactions, both locally and systemically, on vascular disease in allografts.Methodology/Principal FindingsAnalysis of GAG or CC chemokine receptor 2 (CCR2) deficiency were coupled with the infusion of viral chemokine modulating proteins (CMPs) in mouse aortic allograft transplants (n = 239 mice). Inflammatory cell invasion and neointimal hyperplasia were significantly reduced in N-deacetylase-N-sulfotransferase-1 (Ndst1 f/f TekCre +) heparan sulfate (GAG)-deficient (Ndst1−/−, p<0.044) and CCR2-deficient (Ccr2−/−, p<0.04) donor transplants. Donor tissue GAG or CCR2 deficiency markedly reduced inflammation and vasculopathy, whereas recipient deficiencies did not. Treatment with three CMPs was also investigated; Poxviral M-T1 blocks CC chemokine receptor binding, M-T7 blocks C, CC, and CXC GAG binding, and herpesviral M3 binds receptor and GAG binding for all classes. M-T7 reduced intimal hyperplasia in wild type (WT) (Ccr2+/+, p≤0.003 and Ccr2−/−, p≤0.027) aortic allografts, but not in Ndst1−/− aortic allografts (p = 0.933). M-T1 and M3 inhibited WT (Ccr2+/+ and Ndst1 +/+, p≤0.006) allograft vasculopathy, but did not block vasculopathy in Ccr2−/− (p = 0.61). M-T7 treatment alone, even without immunosuppressive drugs, also significantly prolonged survival of renal allograft transplants (p≤0.001).Conclusions/SignificanceInterruption of chemokine-GAG interactions, even in the absence of chemokine-receptor blockade, is a highly effective approach to reduction of allograft rejection, reducing vascular inflammation and prolonging allograft survival. Although chemokines direct both local and systemic cell migration, interruption of inherent chemokine responses in the donor tissue unexpectedly had a greater therapeutic impact on allograft vasculopathy.

Glycosaminoglycans (GAGs) in Cardiovascular Disease: Searching for the Sweet Spot

“…Modified GAGs and metabolizing enzymes, heparanase and chondroitinase, are also variably reported to modify acute kidney damage. We have also found that changes in Ndst1 expression in mouse aortic transplants can reduce plaque growth [11] and more recent work has demonstrated a correlation between changes in HS GAG disaccharide content and reductions in acute rejection in Ndst1-/-donor allograft transplants in mice (Manuscript submitted, unpublished data). What is both fascinating and also supremely frustrating is the fact that one cannot fully predict the functions of HS and other GAGs, such as chondroitin and dermatan sulfate the main connective tissue and glycocalyx GAGs, by a direct correlation to gene expression.…”

Comparative Analysis of Plaque Growth after Arterial Stent Implant with Anti-Inflammatory Chemokine and Serine Protease Inhibitors Treatment

“…Harvested arterial sections were processed and stained with hematoxylin and eosin (H & E) or Masson's trichrome stains as previously described [25][26][27][28][29][30][31]33]. Plaque area, was measured by morphometric analysis using an Olympus CCD color video camera attached to an Olympus microscope, and the ImagePro application program calibrated to the microscope objective [26][27][28][29][30][31]33].…”

“…Plaque area, was measured by morphometric analysis using an Olympus CCD color video camera attached to an Olympus microscope, and the ImagePro application program calibrated to the microscope objective [26][27][28][29][30][31]33]. Both the mean total cross-sectional intimal area and the mean intimal thickness after normalized to the medial thickness were calculated for each arterial section.…”

“…These inflammatory moderators function at femptomolar to picomolar concentrations when secreted by the originating viruses [23,24]. Two such proteins, Serp-1 that inhibits uPA/uPAR [23][24][25][26][27][28] and M-T7 [26,[29][30][31] that blocks the C terminal GAG binding domain of C, CC and CXC class chemokines [26,[29][30][31] have been purified individually and studied as anti-inflammatory reagents in a wide range of animal models with demonstrated reduction in inflammatory cell invasion and plaque growth. One protein, Serp-1 has been successfully tested in clinical trial for treatment of patients with unstable coronary syndromes (Unstable angina and NSTEMI) who have received stent implants [26,32].…”

“…M-T1 from vMyxlacT1gpt infected BGMK cell cultures as previously described [26,29,31]. Concentrated supernatants were fractionated by Mono QHR5/5 anion exchange chromatography followed by gel filtration chromatography using a HiLoadSuperdex 200 column (AmershamPahrmacia Biotech Inc.).…”