We showed that nitric oxide (NO) signaling is decreased in the pulmonary vasculature before the development of endothelial dysfunction in a lamb model of congenital heart disease and increased pulmonary blood flow (Shunt). The elucidation of the molecular mechanism by which this occurs was the purpose of this study. Here, we demonstrate that concentrations of the endogenous NO synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), are elevated, whereas the NOS cofactor tetrahydrobiopterin (BH(4)) is decreased in Shunt lambs. Our previous studies demonstrated that ADMA decreases heat shock protein-90 (Hsp90) chaperone activity, whereas other studies suggest that guanosine-5'-triphosphate cyclohydrolase 1 (GCH1), the rate-limiting enzyme in the generation of BH(4), may be a client protein for Hsp90. Thus, we determined whether increases in ADMA could alter GCH1 protein and activity. Our data demonstrate that ADMA decreased GCH1 protein, but not mRNA concentrations, in pulmonary arterial endothelial cells (PAECs) because of the ubiquitination and proteasome-dependent degradation of GCH1. We also found that Hsp90-GCH1 interactions were reduced, whereas the association of GCH1 with Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) increased in ADMA-exposed PAECs. The overexpression of CHIP potentiated, whereas a CHIP U-box domain mutant attenuated, ADMA-induced GCH1 degradation and reductions in cellular BH(4) concentrations. We also found in vivo that Hsp90/GCH1 interactions are decreased, whereas GCH1-Hsp70 and GCH1-CHIP interactions and GCH1 ubiquitination are increased. Finally, we found that supplementation with l-arginine restored Hsp90-GCH1 interactions and increased both BH(4) and NO(x) concentrations in Shunt lambs. In conclusion, increased concentrations of ADMA can indirectly alter NO signaling through decreased cellular BH(4) concentrations, secondary to the disruption of Hsp90-GCH1 interactions and the CHIP-dependent proteasomal degradation of GCH1.

Surgical castration of male piglets without analgesia is a painful procedure. This prospective, randomized and double-blinded study aimed to evaluate the analgesic effects of four different local anesthetics for piglet castration during the first week of life. In total, 54 piglets aged 3 to 7 days were distributed into 6 treatment groups: handling (H); castration without pain relief (sodium chloride, NaCl); and castration with a local anesthetic: 4% procaine (P), 2% lidocaine (L), 0.5% bupivacaine (B) or 20 mg/ml mepivacaine (M). By excluding stress and fear as disruptive factors via a minimum anesthesia model, all piglets received individual minimum alveolar concentration (MAC) isoflurane anesthesia. Twenty minutes before castration, all treatment groups except group H received one injection per testis. Then, 0.5 ml of a local anesthetic or NaCl was injected intratesticularly (i.t.), and 0.5 ml was administered subscrotally. Acute physiological responses to noxious stimuli at injection and castration were evaluated by measuring blood pressure (BP), heart rate (HR), cortisol, epinephrine, norepinephrine and chromogranin A (CgA); limb movements were quantified. The results confirm that castration without analgesia is highly painful. Surgical castration without pain relief revealed significant changes in mean arterial blood pressure (MAP) and HR. Local anesthetic administration significantly reduced changes in BP and HR associated with castration. Piglets receiving a preoperative local anesthetic exhibited the fewest limb movements during castration, while the NaCl group exhibited the most. Injection itself was not associated with significant changes in MAP or HR. However, many piglets exhibited limb movements during injection, indicating that the injection itself causes nociceptive pain. No significant differences were found between groups regarding parameters of plasma cortisol, catecholamines and CgA. In conclusion, all four local anesthetics administered are highly effective at reducing signs of nociception during castration under light isoflurane anesthesia. However, injection of a local anesthetic seems to be painful.

Rationale: To evaluate the repurposing of lenvatinib, a multi tyrosine kinase inhibitor, in limiting experimental abdominal aortic aneurysm (AAA) growth.Objective: AAA is a disease with high morbidity and mortality, especially in case of rupture. No pharmacologic treatment is currently available. Novel therapeutic strategies are targeting vascular smooth muscle cells (VSMC) and angiogenesis.Repurposing of clinically available drugs has proven its benefit to extend treatment strategies and may be suited to halt aortic dilatation.Findings: Porcine pancreatic elastase (PPE) was used to induce murine AAAs.Systemic and local lenvatinib forms of treatment were evaluated, and RNA profiling identified myosin heavy chain 11 (Myh11) as the most deregulated transcript. Daily oral treatment significantly reduced aneurysm formation in two independent mouse models. In addition, a novel large animal aneurysm model in hypercholesterolemic low-density lipoprotein receptor knockout (LDLR -/-) Yucatan minipigs was applied to endovascularly deliver lenvatinib via drug-eluting balloons (DEB). Here, a single local endovascular delivery blocked AAA progression successfully compared to a DEBdelivered control treatment when reaching 28 days post aneurysm induction.Reduced VSMC proliferation and a restored contractile phenotype were observed in animal tissues (murine and porcine), as well as AAA patient-derived cells. Apart from increasing MYH11 levels, lenvatinib reduced downstream ERK signaling. Conclusion:Lenvatinib is a promising new therapy to limit aortic aneurysm expansion upon local endovascular delivery. The tyrosine kinase inhibitor was able to positively affect pathways of key relevance to human AAA disease. The novel endovascular local delivery using DEBs offers new treatment strategies for additional therapies capable of slowing down aneurysm expansion.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable‐isotope dimethyl labeling prior to nanoLC‐MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N‐glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

The aim of the present study was to investigate the effect of four local anesthetics on pain relief during surgical castration under standardized conditions in conscious piglets. Therefore, 71 male piglets (three to seven days) were distributed into control groups (handling, castration without anesthesia or analgesia) and local anesthetic trial groups (procaine, lidocaine, bupivacaine, mepivacaine). Then, 20 min prior to castration, animals of the treatment groups, except piglets in the handling group, received an injection of a local anesthetic or sodium chloride of 0.5 mL intratesticularly and 0.5 mL subscrotally. During injection and castration, defensive behavior was evaluated. Locomotor activity, as well as postoperative bleeding, wound healing and average daily weight gain were assessed to detect side effects. The injection caused increased defensive movements, significantly in the bupivacaine group. Lidocaine and mepivacaine significantly reduced defensive movements during castration, and procaine and bupivacaine only during severing of the spermatic cord. Impairments of locomotor activity were found in piglets injected with lidocaine, bupivacaine or sodium chloride. Considering healing, bleeding and weight gain, no negative impacts were observed. In conclusion, lidocaine and mepivacaine were able to achieve significant pain relief during the castration procedure, whereas procaine and bupivacaine only during the severing of the spermatic cord. Moreover, the injection of bupivacaine seemed to be painful itself.