Pre-eclampsia (PE) is related to an impaired endothelial function. Endothelial dysfunction accounts for altered vascular reactivity, activation of the coagulation cascade and loss of vascular integrity. Impaired endothelial function originates from production of inflammatory and cytotoxic factors by the ischemic placenta and results in systemic oxidative stress (OS) and an altered bioavailability of nitric oxide (•NO). The free radical •NO, is an endogenous endothelium-derived relaxing factor influencing endothelial function. In placental circulation, endothelial release of •NO dilates the fetal placental vascular bed, ensuring feto-maternal exchange. The Endopreg study was designed to evaluate in vivo endothelial function and to quantify in vitro OS in normal and pre-eclamptic pregnancies. The study is divided into two arms, a prospective longitudinal study and a matched case control study. In the longitudinal study, pregnant patients ≥18 years old with a singleton pregnancy will be followed throughout pregnancy and until 6 months post-partum. In the case control study, cases with PE will be compared to matched normotensive pregnant women. Maternal blood concentration of superoxide (O2•) and placental concentration of •NO will be determined using EPR (electron paramagnetic resonance). Endothelial function and arterial stiffness will be evaluated using respectively Peripheral Arterial Tonometry (PAT), Flow-Mediated Dilatation (FMD) and applanation tonometry. Placental expression of eNOS (endothelial NOS) will be determined using immune-histochemical staining. Target recruitment will be 110 patients for the longitudinal study and 90 patients in the case-control study. The results of Endopreg will provide longitudinal information on in vivo endothelial function and in vitro OS during normal pregnancy and PE. Adoption of these vascular tests in clinical practice potentially predicts patients at risk to develop cardiovascular events later in life after PE pregnancies. •NO, O2•− and eNOS measurements provide further inside in the pathophysiology of PE. This trial has been registered on clinicaltrials.gov. ClinicalTrials.gov Identifier: NCT02603913 . Registered October 2015.

OBJECTIVES Pulmonary ischaemia–reperfusion injury (IRI) is associated with several life-threatening pulmonary disorders, and may severely compromise the outcome of lung transplantation. Highly reactive molecules such as superoxide, nitric oxide (NO) and peroxynitrite (ONOO⁻) are presumed to contribute to IRI pathogenesis, but this assumption is based on indirect measurements. We use electron spin resonance (ESR) to directly quantify free radical formation after pulmonary ischaemia and reperfusion. METHODS Five groups of 10 Swiss mice were subjected to left pulmonary hilum clamping for 1 h of ischaemia followed by 0, 1, 4 and 24 h of reperfusion or to sham thoracotomy alone as control procedure. In five mice per group, ESR was used to measure iron-diethyldithio-carbamate trihydrate-trapped NO in the lung. In the other group of 5, reactive oxygen species generation in the lung and in blood was quantified with ESR by detection of ascorbyl radical and CMH spin probe, respectively. Pulmonary ONOO⁻ was monitored with nitrotyrosine Western blotting. RESULTS After 1 h of reperfusion, a pulmonary NO peak (14.69 ± 0.91 × 10⁴ Arbitrary Units (A.U.). vs 1.84 ± 0.75 × 10⁴ A.U. in sham; P < 0.001) coincided with a significant increase in nitrosated proteins (0.105 ± 0.015 A.U.) compared with sham (0.047 ± 0.006 A.U.); P < 0.005). Peripheral blood showed a significant free radical burst after 1 h of ischaemia (11 774 ± 728 A.U. vs 6660 ± 833 A.U. in sham; P < 0.001). CONCLUSIONS Longitudinal quantification of free radicals during IRI reveals the occurrence of two major radical bursts. The radical peak in peripheral blood after ischaemia may be related to systemic hypoxia. After 1 h of reperfusion, the lung tissue shows a significant increase of superoxide, NO and their reaction products, which are probably involved in IRI pathogenesis.

Oxidative and nitrosative stress are an umbrella term for pathophysiological processes that involve free radical generation during inflammation. In this review, the involvement of reactive oxygen and nitrogen species is evaluated during lung ischemia-reperfusion injury (LIRI) from a surgical point of view. The main biochemical and cellular mechanisms behind free radical generation are discussed, together with surgical procedures that may cause reperfusion injury. Finally, different therapeutic strategies are further explored. A literature search was performed, searching for “lung ischemia reperfusion injury”, “reperfusion injury”, “large animal model” and different search terms for each section: “surgery”, “treatment”, “cellular mechanism”, or “enzyme”. Although reperfusion injury is not an uncommon entity and there is a lot of evidence concerning myocardial ischemia-reperfusion injury, in the lung this phenomenon is less extensively described and studies in large animals are not easy to come by. With increasing number of patients on waiting lists for lung transplant, awareness for this entity should all but rise.

OBJECTIVES: Ischaemia–reperfusion injury is a necessary part of organ transplantation and a key determinant of both acute and chronic graft failure. We have assessed the contribution of endothelial nitric oxide synthase (eNOS) and eNOS uncoupling to oxidative and nitrosative stress formation during lung ischaemia–reperfusion injury dependent on ischaemia time. METHODS: Forty eNOS wild-type mice (eNOS^+/+) and 40 eNOS knock-out mice (eNOS^−/−) received either a sham thoracotomy or 60 or 90 min of ischaemia, followed by 0, 1 or 24 h of reperfusion. Lung tissue was analysed with electron spin resonance for NO production and reactive oxygen species content. Protein nitrosation, eNOS and eNOS uncoupling were determined using western blotting. In peripheral blood, arterial blood gases were taken and reactive oxygen species content was determined. RESULTS: eNOS^+/+mice had lower reactive oxygen species production in their peripheral circulation but worse blood gas values after 1 h of reperfusion. Lung tissue of eNOS^−/− mice showed lower reactive oxygen species and NO production and lower protein nitrosation compared with wild-type mice. Longer ischaemia times result in more elaborate oxidative and nitrosative stress dependent on eNOS genotype. Structural eNOS uncoupling was present after 60 min of ischaemia but diminished after 90 min of ischaemia. CONCLUSIONS: eNOS uncoupling may contribute to lung ischaemia–reperfusion injury and inflammation. This ultimately leads to worse clinical outcome. Stabilizing eNOS may therefore be a new approach to extend pulmonary graft survival.

Pulmonary ossifications have often been regarded as rare, post-mortem findings without any clinical significance. We have investigated the occurrence of pulmonary ossifications in patients undergoing thoracic procedures, and how this may affect the differential diagnosis of solitary pulmonary nodules. In addition, we have performed a literature search on the occurrence and possible pathogenesis of these ossifications. From January 2008 until August 2019, we identified pulmonary ossifications in 34 patients who underwent elective pulmonary surgery. Pre-operative imaging was unable to differentiate these ossifications from solid tumors. A definitive diagnosis was made by an experienced pathologist (VS, ML). The PubMed database was researched in December 2019 with the search terms “pulmonary ossifications”; “heterotopic ossifications”; and “solitary pulmonary nodule”. In total, 27 patients were male, with a mean age of 63 ± 12 years (age 41 to 82 on diagnosis). All lesions were identified on thoracic CT and marked for resection by a multidisciplinary team. A total of 17 patients were diagnosed with malignancy concurrent with ossifications. There was a clear predilection for the right lower lobe (12 cases, 35.3%) and most ossifications had a nodular form (70.6%). We could not identify a clear association with any other pathology, either cancerous or non-cancerous in origin. Oncologic or pulmonary comorbidities did not influence patient survival. Pulmonary ossifications are not as seldom as thought and are not just a curiosity finding by pathologists. These formations may be mistaken for a malignant space-occupying lesion, both pre-and perioperatively, as they are indistinguishable in imaging. We propose these ossifications as an underestimated addition to the differential diagnosis of a solitary pulmonary nodule.