Stacy Robertson

MicroRNA-24 Is a Novel Regulator of Aldosterone and Cortisol Production in the Human Adrenal Cortex

Dysregulation of aldosterone or cortisol production can predispose to hypertension, as seen in aldosterone-producing adenoma, a form of primary aldosteronism. We investigated the role of microRNA (miRNA) in their production, with particular emphasis on the CYP11B1 (11&bgr;-hydroxylase) and CYP11B2 (aldosterone synthase) genes, which produce the enzymes responsible for the final stages of cortisol and aldosterone biosynthesis, respectively. Knockdown of Dicer1, a key enzyme in miRNA maturation, significantly altered CYP11B1 and CYP11B2 expression in a human adrenocortical cell line. Screening of nondiseased human adrenal and aldosterone-producing adenoma samples yielded reproducible but distinctive miRNA expression signatures for each tissue type, with levels of certain miRNA, including microRNA-24 (miR-24), differing significantly between the 2. Bioinformatic analysis identified putative binding sites for several miRNA, including miR-24, in the 3′ untranslated region of CYP11B1 and CYP11B2 mRNAs. In vitro manipulation of miR-24 confirmed its ability to modulate CYP11B1 and CYP11B2 expression, as well as cortisol and aldosterone production. This study demonstrates that Dicer-dependent miRNA, including miR-24, can post-transcriptionally regulate expression of the CYP11B1 and CYP11B2 genes. Normal adrenal tissue and aldosterone-producing adenoma differ significantly and reproducibly in their miRNA expression profiles, with miR-24 significantly downregulated in the latter. Adrenal miRNA may, therefore, be a novel and valid target for the therapeutic manipulation of corticosteroid biosynthesis.

Colchicine Acutely Suppresses Local Cardiac Production of Inflammatory Cytokines in Patients With an Acute Coronary Syndrome

Background Interleukin (IL)-1β, IL-18, and downstream IL-6 are key inflammatory cytokines in the pathogenesis of coronary artery disease. Colchicine is believed to block the NLRP3 inflammasome, a cytosolic complex responsible for the production of IL-1β and IL-18. In vivo effects of colchicine on cardiac cytokine release have not been previously studied. This study aimed to (1) assess the local cardiac production of inflammatory cytokines in patients with acute coronary syndromes (ACS), stable coronary artery disease and in controls; and (2) determine whether acute administration of colchicine inhibits their production. Methods and Results Forty ACS patients, 33 with stable coronary artery disease, and 10 controls, were included. ACS and stable coronary artery disease patients were randomized to oral colchicine treatment (1 mg followed by 0.5 mg 1 hour later) or no colchicine, 6 to 24 hours prior to cardiac catheterization. Blood samples from the coronary sinus, aortic root (arterial), and lower right atrium (venous) were collected and tested for IL-1β, IL-18, and IL-6 using ELISA. In ACS patients, coronary sinus levels of IL-1β, IL-18, and IL-6 were significantly higher than arterial and venous levels (P=0.017, <0.001 and <0.001, respectively). Transcoronary (coronary sinus-arterial) gradients for IL-1β, IL-18, and IL-6 were highest in ACS patients and lowest in controls (P=0.077, 0.033, and 0.014, respectively). Colchicine administration significantly reduced transcoronary gradients of all 3 cytokines in ACS patients by 40% to 88% (P=0.028, 0.032, and 0.032, for IL-1β, IL-18, and IL-6, respectively). Conclusions ACS patients exhibit increased local cardiac production of inflammatory cytokines. Short-term colchicine administration rapidly and significantly reduces levels of these cytokines.

High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I

Disordered neovascularization and impaired wound healing are important contributors to diabetic vascular complications. We recently showed that high-density lipoproteins (HDLs) enhance ischemia-mediated neovascularization, and mounting evidence suggests HDL have antidiabetic properties. We therefore hypothesized that HDL rescue diabetes-impaired neovascularization. Streptozotocin-induced diabetic mice had reduced blood flow recovery and neovessel formation in a hindlimb ischemia model compared with nondiabetic mice. Reconstituted HDL (rHDL) infusions in diabetic mice restored blood flow recovery and capillary density to nondiabetic levels. Topical rHDL application rescued diabetes-impaired wound closure, wound angiogenesis, and capillary density. In vitro, rHDL increased key mediators involved in hypoxia-inducible factor-1α (HIF-1α) stabilization, including the phosphoinositide 3-kinase/Akt pathway, Siah1, and Siah2, and suppressed the prolyl hydroxylases (PHD) 2 and PHD3. rHDL rescued high glucose–induced impairment of tubulogenesis and vascular endothelial growth factor (VEGF) A protein production, a finding associated with enhanced phosphorylation of proangiogenic mediators VEGF receptor 2 and endothelial nitric oxide synthase. Siah1/2 small interfering RNA knockdown confirmed the importance of HIF-1α stability in mediating rHDL action. Lentiviral short hairpin RNA knockdown of scavenger receptor class B type I (SR-BI) in vitro and SR-BI−/− diabetic mice in vivo attenuated rHDL rescue of diabetes-impaired angiogenesis, indicating a key role for SR-BI. These findings provide a greater understanding of the vascular biological effects of HDL, with potential therapeutic implications for diabetic vascular complications.

Neutrophil-derived microparticles are released into the coronary circulation following percutaneous coronary intervention in acute coronary syndrome patients

To evaluate (i) local coronary and systemic levels of microparticles (MP) in acute coronary syndrome (ACS) and stable angina pectoris (SAP) patients and (ii) their release after plaque disruption with percutaneous coronary intervention (PCI). MP are small vesicles originating from plasma membranes of cells after activation or apoptosis and are implicated in the pathogenesis of atherosclerosis. Neutrophils play a role in plaque destabilization and shed neutrophil-derived MP that have the potential to drive significant proinflammatory and thrombotic downstream effects. Eight ACS and eight SAP patients were included. Coronary sinus (CS) samples pre-intervention (CS1), 45 s following balloon angioplasty (CS2) and at 45 s intervals following stent deployment (CS3, CS4 and CS5), together with peripheral vein samples, pre- and post-PCI were analysed for neutrophil-derived (CD66b+), endothelial-derived (CD144+), platelet-derived (CD41a+), monocyte-derived (CD14+) and apoptotic (Annexin V+) MP. ELISA for interleukin (IL)-6, myeloperoxidase (MPO) and P-selectin was also performed. CD66b+ MP levels were similar in both groups pre-intervention. Post-PCI, CS levels rose significantly in ACS but not SAP patients (ACS area under the curve (AUC): 549 ± 83, SAP AUC: 24 ± 29, P<0.01). CS CD41a+, CD144+, CD14+ and Annexin V+ MP levels did not differ between groups. Acute neutrophil-derived MP release post-PCI occurs in ACS compared with stable patients, likely to be reflective of plaque MP content in vulnerable lesions.

Regulation of Corticosteroidogenic Genes by MicroRNAs

The loss of normal regulation of corticosteroid secretion is important in the development of cardiovascular disease. We previously showed that microRNAs regulate the terminal stages of corticosteroid biosynthesis. Here, we assess microRNA regulation across the whole corticosteroid pathway. Knockdown of microRNA using Dicer1 siRNA in H295R adrenocortical cells increased levels of CYP11A1, CYP21A1, and CYP17A1 mRNA and the secretion of cortisol, corticosterone, 11-deoxycorticosterone, 18-hydroxycorticosterone, and aldosterone. Bioinformatic analysis of genes involved in corticosteroid biosynthesis or metabolism identified many putative microRNA-binding sites, and some were selected for further study. Manipulation of individual microRNA levels demonstrated a direct effect of miR-125a-5p and miR-125b-5p on CYP11B2 and of miR-320a-3p levels on CYP11A1 and CYP17A1 mRNA. Finally, comparison of microRNA expression profiles from human aldosterone-producing adenoma and normal adrenal tissue showed levels of various microRNAs, including miR-125a-5p to be significantly different. This study demonstrates that corticosteroidogenesis is regulated at multiple points by several microRNAs and that certain of these microRNAs are differentially expressed in tumorous adrenal tissue, which may contribute to dysregulation of corticosteroid secretion. These findings provide new insights into the regulation of corticosteroid production and have implications for understanding the pathology of disease states where abnormal hormone secretion is a feature.

Retargeting FX binding-ablated HAdV-5 to vascular cells by inclusion of the RGD-4C peptide in hexon hypervariable region 7 and the HI loop

Recent studies have generated interest in the function of human adenovirus serotype 5 (HAdV-5) hexon:  factor X (FX) binding and subsequent hepatocyte transduction and interaction with the immune system. Here, we retargeted adenovirus serotype 5 vectors, ablated for FX interaction, by replacing amino acids in hexon HVR7 with RGD-4C or inserting the peptide into the fibre HI loop. These genetic modifications in the capsid were compatible with virus assembly, and could efficiently retarget transduction of the vector via the αvβ3/5 integrin-mediated pathway, but did not alter immune recognition by pre-existing human neutralizing anti-HAdV-5 antibodies or by natural antibodies in mouse serum. Thus, FX-binding-ablated HAdV-5 can be retargeted but remain sensitive to immune-mediated attack. These findings further refine HAdV-5-based vectors for human gene therapy and inform future vector development.

VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia.

High‐density lipoproteins augment hypoxia‐induced angiogenesis by inducing the key angiogenic vascular endothelial growth factor A (VEGFA) and total protein levels of its receptor 2 (VEGFR2). The activation/ phosphorylation of VEGFR2 is critical for mediating downstream, angiogenic signaling events. This study aimed to determine whether reconstituted high‐density lipoprotein (rHDL) activates VEGFR2 phosphorylation and the downstream signaling events and the importance of VEGFR2 in the proangiogenic effects of rHDL in hypoxia. In vitro, rHDL increased VEGFR2 activation and enhanced phosphorylation of downstream, angiogenic signaling proteins ERK1/2 and p38 MAPK in hypoxia. Incubation with a VEGFR2‐neutralizing antibody attenuated rHDL‐induced phosphorylation of VEGFR2, ERK1/2, p38 MAPK, and tubule formation. In a murine model of ischemia‐driven neovascularization, rHDL infusions enhanced blood perfusion and augmented capillary and arteriolar density. Infusion of a VEGFR2‐neutralizing antibody ablated those proangiogenic effects of rHDL. Circulating Sca1+/CXCR4+ angiogenic progenitor cell levels, important for neovascularization in response to ischemia, were higher in rHDL‐infused mice 3 d after ischemic induction, but that did not occur in mice that also received the VEGFR2‐neutralizing antibody. In summary, VEGFR2 has a key role in the proangiogenic effects of rHDL in hypoxia/ischemia. These findings have therapeutic implications for angiogenic diseases associated with an impaired response to tissue ischemia.—Cannizzo, C. M., Adonopulos, A. A., Solly, E. L., Ridiandries, A., Vanags, L. Z., Mulangala, J., Yuen, S. C. G., Tsatralis, T., Henriquez, R., Robertson, S., Nicholls, S. J., Di Bartolo, B. A., Ng, M. K. C., Lam, Y. T., Bursill, C. A., Tan, J. T. M. VEGFR2 is activated by high‐density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia. FASEB J. 32, 2911–2922 (2018). www.fasebj.org

NEUTROPHIL-DERIVED MICROPARTICLES ARE RELEASED INTO THE CORONARY CIRCULATION FOLLOWING CORONARY INTERVENTION IN ACUTE CORONARY SYNDROME PATIENTS

To evaluate (i) local coronary and systemic levels of microparticles (MP) in acute coronary syndrome (ACS) and stable angina pectoris (SAP) patients and (ii) their release after plaque disruption with percutaneous coronary intervention (PCI). MP are small vesicles originating from plasma membranes of cells after activation or apoptosis and are implicated in the pathogenesis of atherosclerosis. Neutrophils play a role in plaque destabilization and shed neutrophil-derived MP that have the potential to drive significant proinflammatory and thrombotic downstream effects. Eight ACS and eight SAP patients were included. Coronary sinus (CS) samples pre-intervention (CS1), 45 s following balloon angioplasty (CS2) and at 45 s intervals following stent deployment (CS3, CS4 and CS5), together with peripheral vein samples, pre- and post-PCI were analysed for neutrophil-derived (CD66b+), endothelial-derived (CD144+), platelet-derived (CD41a+), monocyte-derived (CD14+) and apoptotic (Annexin V+) MP. ELISA for interleukin (IL)-6, myeloperoxidase (MPO) and P-selectin was also performed. CD66b+ MP levels were similar in both groups pre-intervention. Post-PCI, CS levels rose significantly in ACS but not SAP patients (ACS area under the curve (AUC): 549 +− 83, SAP AUC: 24 +− 29, P<0.01). CS CD41a+, CD144+, CD14+ and Annexin V+ MP levels did not differ between groups. Acute neutrophil-derived MP release post-PCI occurs in ACS compared with stable patients, likely to be reflective of plaque MP content in vulnerable lesions.

The regulation of miRNAs by reconstituted high-density lipoproteins in diabetes-impaired angiogenesis

Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. We recently found that reconstituted high-density lipoproteins (rHDL) rescue diabetes-impaired angiogenesis. microRNAs (miRNAs) regulate angiogenesis and are transported within HDL to sites of injury/repair. The role of miRNAs in the rescue of diabetes-impaired angiogenesis by rHDL is unknown. Using a miRNA array, we found that rHDL inhibits hsa-miR-181c-5p expression in vitro and using a hsa-miR-181c-5p mimic and antimiR identify a novel anti-angiogenic role for miR-181c-5p. miRNA expression was tracked over time post-hindlimb ischaemic induction in diabetic mice. Early post-ischaemia when angiogenesis is important, rHDL suppressed hindlimb mmu-miR-181c-5p. mmu-miR-181c-5p was not detected in the plasma or within HDL, suggesting rHDL specifically targets mmu-miR-181c-5p at the ischaemic site. Three known angiogenic miRNAs (mmu-miR-223-3p, mmu-miR-27b-3p, mmu-miR-92a-3p) were elevated in the HDL fraction of diabetic rHDL-infused mice early post-ischaemia. This was accompanied by a decrease in plasma levels. Only mmu-miR-223-3p levels were elevated in the hindlimb 3 days post-ischaemia, indicating that rHDL regulates mmu-miR-223-3p in a time-dependent and site-specific manner. The early regulation of miRNAs, particularly miR-181c-5p, may underpin the rescue of diabetes-impaired angiogenesis by rHDL and has implications for the treatment of diabetes-related vascular complications.

Erratum. High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I. Diabetes 2016;65:3091–3103

Erratum. High-Density Lipoproteins Rescue Diabetes-Impaired Angiogenesis via Scavenger Receptor Class B Type I. Diabetes 2016;65:3091–3103 DOI: 10.2337/db17-er04b Joanne T.M. Tan, Hamish C.G. Prosser, Louise L. Dunn, Laura Z. Vanags, Anisyah Ridiandries, Tania Tsatralis, Laura Leece, Zoë E. Clayton, Sui Ching G. Yuen, Stacy Robertson, Yuen Ting Lam, David S. Celermajer, Martin K.C. Ng, and Christina A. Bursill In the article listed above, Laura Lecce’s name was erroneously listed as Laura Leece. The authors regret the error.