Role of AMP-protein kinase (AMPK) in regulation of perivascular adipose tissue (PVAT) function

This thesis, entitled: ‘Role of AMP-protein kinase (AMPK) in regulation of perivascular adipose tissue (PVAT) function’, has been submitted by author Tarek Ali Mohamed Almabrouk for a degree of Doctor of Philosophy (PhD) in the College of Medical, Veterinary and Life Sciences at the University of Glasgow, October 2016. Apart from the cerebral circulation, all vasculature is surrounded by layers of adipose tissue known as perivascular adipose tissue (PVAT). In health, PVAT can function as an endocrine organ to produce a wide range of adipocytokines which can attenuate vascular contraction. The exact mechanism of this anti-contractile effect is still ill-defined, although much evidence suggests that PVAT-released adipocytokines may activate K+ channels on VSMCs or eNOS on endothelial layer possibly via AMP-activated protein kinase (AMPK). However, obesity results in oxidative stress and inflammation of the PVAT leading to abnormal adipocytokine release and PVAT dysfunction. AMPK is a serine/threonine kinase with many potential physiological functions, including regulation of energy heamostasis. AMPK is expressed in the three layers of the blood vessel: smooth muscle (VSM), the endothelium and PVAT and it is known that activation of AMPK leads to vascular dilatation via both endothelium- and non-endothelium-dependent mechanisms. Although it is known that AMPK can modulate VSM and endothelial function, it is unknown whether AMPK can influence the anti-contractile activity of PVAT. Therefore, this project aimed to investigate the mechanism of the anticontractile effect of PVAT by determining the functions of AMPK within adipocytes, as well as to assess the importance of vascular AMPK to the PVAT anti-contractile function. Experiments were conducted using wild type (WT) and global AMPKα1 knockout (KO) mice aortae. The phenotypic features of the PVAT were assessed by both histological, immunohistochemical and immunofluorescent methods. Secretory function of the PVAT was tested using an immunoblotting array and ELISA, whereas the anti-contractile effect of PVAT was studied using wire myography. Immunoblotting methods were used to test AMPK activity in the PVAT and VSMCs. Aortic rings from WT and KO mice were denuded of endothelium and mounted on a wire myograph in the presence and absence of PVAT. The responses to an AMPK activator (AICAR) and the AMPK-independent vasodilator cromakalim were subsequently assessed. Relaxation responses to AICAR or cromakalim in the Sv129 (wild type) mouse were significantly enhanced in the presence of endogenous attached or unattached PVAT, an effect that was absent in vessels from KO mice. Furthermore, enhanced relaxation was observed in vessels from KO mice incubated with PVAT from Sv129 mice, whereas PVAT from KO mice had no effect on relaxation of vessels from Sv129 mice. Furthermore, conditioned medium (CM) transfer experiments demonstrated the presence of an anticontractile factor released from PVAT that was absent in KO mice. Adiponectin secretion was reduced in PVAT from KO mice and PVAT-enhanced relaxation was attenuated in the presence of adiponectin blocking peptide. Adipokine array and ELISA demonstrated that adiponectin release is significantly reduced in the KO conditioned media in comparison with wild type CM. Globular adiponectin restores the relaxation response in both wild type aortae without PVAT and in KO aortae with and without PVAT. High fat diet (HFD) fed mice showed a reduction in the relaxation response to cromakalim in wild type vessels with intact PVAT in comparison with animals fed a normal chow diet (ND). HFD animals had increased inflammatory infiltrates in the PVAT which were associated with reduced AMPK activity and adiponectin release in comparison with ND fed WT mice. In KO mice, AMPK activity was also reduced and increased inflammatory infiltration was observed in both ND and HFD mice. In conclusion, the current project demonstrates that AMPKα1 has a critical role in maintaining PVAT’s anti-contractile effect; likely mediated through altered adiponectin secretion or sensitivity, and through protection of PVAT against inflammation. Marked reduction in AMPK activity in WT PVAT, accompanied with the reduction in the release of adiponectin in HFD and KO animal may explain the impaired vascular function observed in obesity

Deletion of AMPKα1 attenuates the anticontractile effect of perivascular adipose tissue (PVAT) and reduces adiponectin release

Background and Purpose: Perivascular adipose tissue (PVAT) surrounds most blood vessels and secretes numerous active substances, including adiponectin, which produce a net anticontractile effect in healthy individuals. AMPK is a key mediator of cellular energy balance and may mediate the vascular effects of adiponectin. In this study, we investigated the role of AMPK within PVAT in mediating the anticontractile effect of PVAT. Experimental Approach: Endothelium-denuded aortic rings from wild-type (WT; Sv129) and α1AMPK knockout (KO) mice were mounted on a wire myograph. Dose–response curves to the AMPK-independent vasodilator cromakalim were studied in vessels with and without PVAT, and effect of pre-incubation with conditioned media and adiponectin on relaxation was also studied. The effect of AMPKα1 KO on the secretory profile of PVAT was assessed by elisa. Key Results: Thoracic aortic PVAT from KO mice was morphologically indistinct from that of WT and primarily composed of brown adipose tissue. PVAT augmented relaxation to cromakalim in WT but not KO aortic rings. Addition of WT PVAT augmented relaxation in KO aortic rings but KO PVAT had no effect in WT rings. PVAT from KO mice secreted significantly less adiponectin and addition of adiponectin to either KO or WT aortic rings without PVAT augmented relaxation to cromakalim. An adiponectin blocking peptide significantly attenuated relaxation in WT rings with PVAT but not in KO rings. Conclusions and Implications: AMPKα1 has a critical role in maintaining the anticontractile actions of PVAT; an effect independent of the endothelium but likely mediated through altered adiponectin secretion or sensitivity

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030