Enzymatic Activity Is Not Required for Phospholipase D Mediated TNF-α Regulation and Myocardial Healing

Phospholipase D1 is a regulator of tumor necrosis factor-α expression and release upon LPS-induced sepsis and following myocardial infarction (MI). Lack of PLD1 leads to a reduced TNF-α mediated inflammatory response and to enhanced infarct size with declined cardiac function 21 days after ischemia reperfusion (I/R) injury. Deficiency of both PLD isoforms PLD1 and PLD2 as well as pharmacological inhibition of the enzymatic activity of PLD with the PLD inhibitor FIPI protected mice from arterial thrombosis and ischemic brain infarction. Here we treated mice with the PLD inhibitor FIPI to analyze if pharmacological inhibition of PLD after myocardial ischemia protects mice from cardiac damage. Inhibition of PLD with FIPI leads to reduced migration of inflammatory cells into the infarct border zone 24 h after experimental MI in mice, providing first evidence for immune cell migration to be dependent on the enzymatic activity of PLD. In contrast to PLD1 deficient mice, TNF-α plasma level was not altered after FIPI treatment of mice. Consequently, infarct size and left ventricular (LV) function were comparable between FIPI-treated and control mice 21 days post MI. Moreover, cell survival 24 h post I/R was not altered upon FIPI treatment. Our results indicate that the enzymatic activity of PLD is not responsible for PLD mediated TNF-α signaling and myocardial healing after I/R injury in mice. Furthermore, reduced TNF-α plasma levels in PLD1 deficient mice might be responsible for increased infarct size and impaired cardiac function 21 days post MI

Enhanced Integrin Activation of PLD2-Deficient Platelets Accelerates Inflammation after Myocardial Infarction

Background: Phospholipase (PL)D1 is crucial for integrin αIIbβ3 activation of platelets in arterial thrombosis and TNF-α-mediated inflammation and TGF-β-mediated collagen scar formation after myocardial infarction (MI) in mice. Enzymatic activity of PLD is not responsible for PLD-mediated TNF-α signaling and myocardial healing. The impact of PLD2 in ischemia reperfusion injury is unknown. Methods: PLD2-deficient mice underwent myocardial ischemia and reperfusion (I/R). Results: Enhanced integrin αIIbβ3 activation of platelets resulted in elevated interleukin (IL)-6 release from endothelial cells in vitro and enhanced IL-6 plasma levels after MI in PLD2-deficient mice. This was accompanied by enhanced migration of inflammatory cells into the infarct border zone and reduced TGF-β plasma levels after 72 h that might account for enhanced inflammation in PLD2-deficient mice. In contrast to PLD1, TNF-α signaling, infarct size and cardiac function 24 h after I/R were not altered when PLD2 was deleted. Furthermore, TGF-β plasma levels, scar formation and heart function were comparable between PLD2-deficient and control mice 21 days post MI. Conclusions: The present study contributes to our understanding about the role of PLD isoforms and altered platelet signaling in the process of myocardial I/R injury

Establishment of a New Murine Elastase-Induced Aneurysm Model Combined with Transplantation

Introduction: The aim of our study was to develop a reproducible murine model of elastase-induced aneurysm formation combined with aortic transplantation. Methods: Adult male mice (n = 6-9 per group) underwent infrarenal, orthotopic transplantation of the aorta treated with elastase or left untreated. Subsequently, both groups of mice were monitored by ultrasound until 7 weeks after grafting. Results: Mice receiving an elastase-pretreated aorta developed aneurysms and exhibited a significantly increased diastolic vessel diameter compared to control grafted mice at 7 week after surgery (1.11 +/- 0.10 mm vs. 0.75 +/- 0.03 mm; p <= 0.001). Histopathological examination revealed disruption of medial elastin, an increase in collagen content and smooth muscle cells, and neointima formation in aneurysm grafts. Conclusions: We developed a reproducible murine model of elastase-induced aneurysm combined with aortic transplantation. This model may be suitable to investigate aneurysm-specific inflammatory processes and for use in gene-targeted animals

Representative examples of histological and immunohistochemical cross-section (CHE, MAC2, SMA).

<p>A–D) Elastase treated aortae after 7 weeks: (A) Cholinesterase (CHE) staining (x100) shows absence of neutrophils, (B) MAC2 (x100) staining shows absence of macrophages, (C) alpha-Smooth Muscle Actin (SMA) staining (x100) shows abundant content of smooth muscle cells, (D) DAPI staining. E–H) Control grafted aortae after 7 weeks: (E) Cholinesterase (CHE) staining (x100) shows absence of neutrophils (F), MAC2 (x100) staining shows no accumulation of macrophages, (G) alpha-Smooth Muscle Actin (SMA) staining (x100) shows minimal loss of smooth muscle cell actin, (H) DAPI staining.</p

Parameters measured by high resolution ultrasound.

<p>Indicated parameters were measured in control grafted mice and mice transplanted with elastase-pretreated aortae at 4 and 7 weeks after surgery. * p≤0,05; ** p ≤0,01; *** p ≤0,001 <i>versus</i> controls.</p

Representative examples of histological cross-section (EVG, SIR).

<p>A,B) Elastase treated aortae after 7 weeks: (A) Elastic van Gieson (EVG) staining (×100, ×200) reveals destruction of medial elastin layer, neointima formation, but no existence of pseudoaneurysms. (B) Sirius Red Staining (SIR) (x100) shows substantial collagen content. C,D) Control grafted aortae after 7 weeks: (C) Elastic van Gieson (EVG) staining (x100) shows no destruction of the medial elastin layer, no existence of aneurysms or no neoitimal formation, (D) Sirius Red Staining (SIR) (x100) shows only low collagen content.</p

Anterior δa/Dd and posterior δp/Dd wall displacement ratio.

<p>(A) Anterior and (B) posterior wall displacement ratios were measured in control grafted mice and mice transplanted with elastase-pretreated aortae at 4 and 7 weeks after surgery using high resolution ultrasound. *p<0,05, ***p<0,001.</p

Schematic presentation of the main steps of the procedure.

<p>(A, D) Step 1: Mouse jugular catheter introduced through an aortotomy and secured with a silk tie. (B, E) Step 2: Aorta filled with saline containing type I porcine pancreatic elastase. (C, F, G) Step 3: Aortic transplantation using sleeve technique/F: Proximal anastomosis, G: Distal anastomosis/. (H) Aorta after transplantation.</p