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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.

No MeSH data available.


Protein oxidation, eNOS monomer formation and biopterin levels.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, at different time points (at the time of graft procurement ≙ “recovery”, following 24 h CIT ≙ “CIT”, following 24 h CIT plus 45 min WIT ≙ “WIT”, and following 24 h CIT plus 45 min WIT and 10 h of graft reperfusion ≙ “10 h rep”) and assessed for protein oxidation, eNOS dimerisation as well as biopterin content. (a) Bar graph showing the ratio between densitometric values of the Oxyblot® bands and those stained with Ponceau red in order to quantify the level of protein oxidation at distinct time points (n = 5/group for each time point). Representative images of protein oxidation and Ponceau red staining blots can be found as Supplementary Fig. S4. (b) Quantification of eNOS dimers and monomers in aortas at distinct time points (n = 5/group for each time point). (c) Representative blot for determination of eNOS dimers and monomers by western blot at distinct time points in native (non-boiled) aortas. Boiled and native (non-boiled) mouse heart tissue samples served as controls. D = dimer, M = monomer. (d) Bar graph showing BH4-to-total biopterin ratios in aortas at distinct time points (n = 5/group for each time point). Results are expressed as mean ± SEM. ns = not significant.
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f5: Protein oxidation, eNOS monomer formation and biopterin levels.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, at different time points (at the time of graft procurement ≙ “recovery”, following 24 h CIT ≙ “CIT”, following 24 h CIT plus 45 min WIT ≙ “WIT”, and following 24 h CIT plus 45 min WIT and 10 h of graft reperfusion ≙ “10 h rep”) and assessed for protein oxidation, eNOS dimerisation as well as biopterin content. (a) Bar graph showing the ratio between densitometric values of the Oxyblot® bands and those stained with Ponceau red in order to quantify the level of protein oxidation at distinct time points (n = 5/group for each time point). Representative images of protein oxidation and Ponceau red staining blots can be found as Supplementary Fig. S4. (b) Quantification of eNOS dimers and monomers in aortas at distinct time points (n = 5/group for each time point). (c) Representative blot for determination of eNOS dimers and monomers by western blot at distinct time points in native (non-boiled) aortas. Boiled and native (non-boiled) mouse heart tissue samples served as controls. D = dimer, M = monomer. (d) Bar graph showing BH4-to-total biopterin ratios in aortas at distinct time points (n = 5/group for each time point). Results are expressed as mean ± SEM. ns = not significant.

Mentions: BH4 is known to be essential for stabilising the active, NO producing, dimeric form of eNOS16. In contrast, in its monomeric form eNOS generates radical oxygen species promoting oxidative stress32. We therefore assessed protein oxidation status and the amount of eNOS monomers. Oxyblot® analysis revealed that already at the time of graft recovery, aortic grafts from non-treated animals showed significantly higher protein oxidation compared to grafts from BH4-treated donor animals (n = 5/group; p = 0.003; Fig. 5a). After 24 h CIT differences in protein oxidation were still significant (n =  = 5/group; p = 0.02; Fig. 5a), whereas after additional 45 min of warm ischemia time (WIT) observed differences in protein oxidation did not reach statistical significance anymore (n = 5/group; p = ns; Fig. 5a).


Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model
Protein oxidation, eNOS monomer formation and biopterin levels.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, at different time points (at the time of graft procurement ≙ “recovery”, following 24 h CIT ≙ “CIT”, following 24 h CIT plus 45 min WIT ≙ “WIT”, and following 24 h CIT plus 45 min WIT and 10 h of graft reperfusion ≙ “10 h rep”) and assessed for protein oxidation, eNOS dimerisation as well as biopterin content. (a) Bar graph showing the ratio between densitometric values of the Oxyblot® bands and those stained with Ponceau red in order to quantify the level of protein oxidation at distinct time points (n = 5/group for each time point). Representative images of protein oxidation and Ponceau red staining blots can be found as Supplementary Fig. S4. (b) Quantification of eNOS dimers and monomers in aortas at distinct time points (n = 5/group for each time point). (c) Representative blot for determination of eNOS dimers and monomers by western blot at distinct time points in native (non-boiled) aortas. Boiled and native (non-boiled) mouse heart tissue samples served as controls. D = dimer, M = monomer. (d) Bar graph showing BH4-to-total biopterin ratios in aortas at distinct time points (n = 5/group for each time point). Results are expressed as mean ± SEM. ns = not significant.
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f5: Protein oxidation, eNOS monomer formation and biopterin levels.Aortic grafts were taken from tetrahydrobiopterin (BH4) - treated or non-treated donors, at different time points (at the time of graft procurement ≙ “recovery”, following 24 h CIT ≙ “CIT”, following 24 h CIT plus 45 min WIT ≙ “WIT”, and following 24 h CIT plus 45 min WIT and 10 h of graft reperfusion ≙ “10 h rep”) and assessed for protein oxidation, eNOS dimerisation as well as biopterin content. (a) Bar graph showing the ratio between densitometric values of the Oxyblot® bands and those stained with Ponceau red in order to quantify the level of protein oxidation at distinct time points (n = 5/group for each time point). Representative images of protein oxidation and Ponceau red staining blots can be found as Supplementary Fig. S4. (b) Quantification of eNOS dimers and monomers in aortas at distinct time points (n = 5/group for each time point). (c) Representative blot for determination of eNOS dimers and monomers by western blot at distinct time points in native (non-boiled) aortas. Boiled and native (non-boiled) mouse heart tissue samples served as controls. D = dimer, M = monomer. (d) Bar graph showing BH4-to-total biopterin ratios in aortas at distinct time points (n = 5/group for each time point). Results are expressed as mean ± SEM. ns = not significant.
Mentions: BH4 is known to be essential for stabilising the active, NO producing, dimeric form of eNOS16. In contrast, in its monomeric form eNOS generates radical oxygen species promoting oxidative stress32. We therefore assessed protein oxidation status and the amount of eNOS monomers. Oxyblot® analysis revealed that already at the time of graft recovery, aortic grafts from non-treated animals showed significantly higher protein oxidation compared to grafts from BH4-treated donor animals (n = 5/group; p = 0.003; Fig. 5a). After 24 h CIT differences in protein oxidation were still significant (n =  = 5/group; p = 0.02; Fig. 5a), whereas after additional 45 min of warm ischemia time (WIT) observed differences in protein oxidation did not reach statistical significance anymore (n = 5/group; p = ns; Fig. 5a).

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.