PB1296 - Elevated NET-Formation and Citrullinated-Fibrinogen are Found in a Porcine Model of Acute Limb Ischemia Reperfusion Injury

Tuesday, July 12, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Valentina Zollet

PhD student
University of Bern, Switzerland
fribourg, Fribourg, Switzerland

Co-Author(s)

IA

Isabel Arenas

PhD student
DBMR
bern, Bern, Switzerland
SH

Stefanie Hirsiger

Surgeant
Inselspital
Bern, Bern, Switzerland
BB

Bilal Ben Brahim

student
DBMR
bern, Bern, Switzerland
MP

Mariafrancesca Petrucci

PhD student
DBMR
bern, Bern, Switzerland
JM

Josip Mikulic

ex postdoc
DBMR
bern, Bern, Switzerland
DC

Daniela Casoni

anaesthesiologist
DBMR
bern, Bern, Switzerland
JW

Junhua Wang

research associate
DBMR
bern, Bern, Switzerland
OB

Olgica Beslac

scrub technician
dbmr
bern, Bern, Switzerland
KN

Kay Nettelbeck

nurse
DBMR
bern, Bern, Switzerland
LG

Luisana Garcia

anaestheiologist
DBMR
bern, Bern, Switzerland
lF

lena Fuest

surgeant
Inselspital
bern, Bern, Switzerland
EV

Esther Vögelin

surgeant
Inselpspital
bern, Bern, Switzerland
MC

Mihai Constantinescu

surgeant
Inselspital
bern, Bern, Switzerland
RR

Robert Rieben

Prof. Dr.
Department for biomedical research (DBMR), university of Bern, Switzerland
Bern, Bern, Switzerland
NS

Nicoletta Sorvillo

Dr.
Department for biomedical research (DBMR), university of Bern, Switzerland
Bern, Bern, Switzerland

Background: Ischemia/reperfusion injury (IRI) is a pathological inflammatory process in which activated infiltrating neutrophils release neutrophil extracellular traps (NETs), a decondensed chromatin meshwork. During NET-formation, the intracellular enzyme peptidyl-arginine deiminase 4 (PAD4) is discharged to the extracellular milieu, where it citrullinates proteins leading to loss of protein function. While NET-derived PAD4 was found to citrullinate plasma proteins in several diseases, its role in IRI is unknown.

Aims: Here, we aim to determine the effect of NETs and PAD4-mediated protein citrullination in a model of acute limb IRI.

Methods: Amputated pig forelimbs were exposed to 1h or 9h of ischemia and then reperfused in vivo for 12h. Limb weight and compartmental pressure were measured, and skeletal muscle was analyzed by immunofluorescence (TUNEL assay and dystrophin staining) to evaluate tissue damage. Myeloperoxidase and citrullinated histone 4 were used to detect neutrophils and NETs, respectively. Candidate citrullinated proteins were immuno-precipitated from plasma samples using an anti-citrulline probe and identified by Western blot.

Results: Limb weight and compartmental pressure were increased (0.3- and 3.4-fold, respectively) in limbs that underwent 9h ischemia compared to 1h. Reperfused limbs after 9h ischemia also displayed higher infiltration of neutrophils and NETs. Interestingly, immunohistochemical analysis revealed that the extent of disruption of skeletal muscle architecture correlated with the presence of NETs, suggesting a role for NETs in tissue damage. Next, we looked for citrullinated plasma fibrinogen and antithrombin, which could play a critical role in IRI. We found a 0.5-fold increase of citrullinated fibrinogen in plasma of pigs which were grafted a limb after 9h ischemia. Citrullinated antithrombin did not rise during IRI.

Conclusion(s): Our data reveal that NETs and levels of citrullinated fibrinogen correlated with tissue damage in IRI. Given its established role in the formation of plasmin-resistant thrombi, this suggests that citrullinated fibrinogen contributes to the thrombo-inflammatory events in IRI.