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Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model

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ABSTRACT

Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV.

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Infiltration with inflammatory cells.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, subjected to 24 h CIT, and reperfused for 4 weeks: (a,e,i) Allogeneic non-treated with CIT (group I, n = 10), (b,f,j) allogeneic BH4 – treated with CIT (group II, n = 9), (c,g,k) allogeneic non-treated without CIT (group III, n = 9), (d,h,l) syngeneic control group with CIT (group IV, n = 5). Sections were stained for CD68 (a–d), CD4 (e–h) and CD8 (i–l) (brown; black arrows in the allogeneic groups indicating stained inflammatory cells) and for methylene green (a–d; nuclei, green) and for haematoxylin (e–l; nuclei, blue). Representative images of antibody validation for immunohistochemical staining can be found as Supplementary Fig. S3. (m,n,o) Bar graphs showing the quantitative analysis of inflammatory cell infiltration. Results are expressed as mean ± SEM. ns = not significant.
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f4: Infiltration with inflammatory cells.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, subjected to 24 h CIT, and reperfused for 4 weeks: (a,e,i) Allogeneic non-treated with CIT (group I, n = 10), (b,f,j) allogeneic BH4 – treated with CIT (group II, n = 9), (c,g,k) allogeneic non-treated without CIT (group III, n = 9), (d,h,l) syngeneic control group with CIT (group IV, n = 5). Sections were stained for CD68 (a–d), CD4 (e–h) and CD8 (i–l) (brown; black arrows in the allogeneic groups indicating stained inflammatory cells) and for methylene green (a–d; nuclei, green) and for haematoxylin (e–l; nuclei, blue). Representative images of antibody validation for immunohistochemical staining can be found as Supplementary Fig. S3. (m,n,o) Bar graphs showing the quantitative analysis of inflammatory cell infiltration. Results are expressed as mean ± SEM. ns = not significant.

Mentions: CD4 and CD8 positive inflammatory cells as well as CD68 positive macrophages are crucial players in TV development3031. All allogeneic groups (group I, n = 10, group II, n = 9, and group III, n = 9) showed inflammatory cell infiltration of CD4, CD8 and CD68 positive cells 4 weeks after transplantation, whereas only in the syngeneic controls (group IV, n = 5) no infiltration was detected (Fig. 4).


Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model
Infiltration with inflammatory cells.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, subjected to 24 h CIT, and reperfused for 4 weeks: (a,e,i) Allogeneic non-treated with CIT (group I, n = 10), (b,f,j) allogeneic BH4 – treated with CIT (group II, n = 9), (c,g,k) allogeneic non-treated without CIT (group III, n = 9), (d,h,l) syngeneic control group with CIT (group IV, n = 5). Sections were stained for CD68 (a–d), CD4 (e–h) and CD8 (i–l) (brown; black arrows in the allogeneic groups indicating stained inflammatory cells) and for methylene green (a–d; nuclei, green) and for haematoxylin (e–l; nuclei, blue). Representative images of antibody validation for immunohistochemical staining can be found as Supplementary Fig. S3. (m,n,o) Bar graphs showing the quantitative analysis of inflammatory cell infiltration. Results are expressed as mean ± SEM. ns = not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Infiltration with inflammatory cells.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, subjected to 24 h CIT, and reperfused for 4 weeks: (a,e,i) Allogeneic non-treated with CIT (group I, n = 10), (b,f,j) allogeneic BH4 – treated with CIT (group II, n = 9), (c,g,k) allogeneic non-treated without CIT (group III, n = 9), (d,h,l) syngeneic control group with CIT (group IV, n = 5). Sections were stained for CD68 (a–d), CD4 (e–h) and CD8 (i–l) (brown; black arrows in the allogeneic groups indicating stained inflammatory cells) and for methylene green (a–d; nuclei, green) and for haematoxylin (e–l; nuclei, blue). Representative images of antibody validation for immunohistochemical staining can be found as Supplementary Fig. S3. (m,n,o) Bar graphs showing the quantitative analysis of inflammatory cell infiltration. Results are expressed as mean ± SEM. ns = not significant.
Mentions: CD4 and CD8 positive inflammatory cells as well as CD68 positive macrophages are crucial players in TV development3031. All allogeneic groups (group I, n = 10, group II, n = 9, and group III, n = 9) showed inflammatory cell infiltration of CD4, CD8 and CD68 positive cells 4 weeks after transplantation, whereas only in the syngeneic controls (group IV, n = 5) no infiltration was detected (Fig. 4).

View Article: PubMed Central - PubMed

ABSTRACT

Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV.

No MeSH data available.


Related in: MedlinePlus