Summary β‐site amyloid precursor protein cleaving enzyme‐1 (BACE1) research has historically focused on its actions as the β‐secretase responsible for the production of β‐amyloid beta, observed in Alzheimer's disease. Although the greatest expression of BACE1 is found in the brain, BACE1 mRNA and protein is also found in many cell types including pancreatic β‐cells, adipocytes, hepatocytes, and vascular cells. Pathologically elevated BACE1 expression in these cells has been implicated in the development of metabolic diseases, including type 2 diabetes, obesity, and cardiovascular disease. In this review, we examine key questions surrounding the BACE1 literature, including how is BACE1 regulated and how dysregulation may occur in disease, and understand how BACE1 regulates metabolism via cleavage of a myriad of substrates. The phenotype of the BACE1 knockout mice models, including reduced weight gain, increased energy expenditure, and enhanced leptin signaling, proposes a physiological role of BACE1 in regulating energy metabolism and homeostasis. Taken together with the weight loss observed with BACE1 inhibitors in clinical trials, these data highlight a novel role for BACE1 in regulation of metabolic physiology. Finally, this review aims to examine the possibility that BACE1 inhibitors could provide a innovative treatment for obesity and its comorbidities.

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The β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer’s disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aβ) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aβ concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD.

Background The beta-amyloid precursor protein cleaving enzyme 1 (BACE1) is well known for its role in the development of Alzheimers disease via the generation of beta-amyloid. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic inflammatory diseases, including type 2 diabetes and cardiovascular disease. However, to date there has been no studies looking into the role of BACE1 in the autoimmune condition Systemic Sclerosis (SSc). The aim of this study was to investigate the role of BACE1 in SSc tissue fibrosis. Methods Patient fibroblasts were obtained from full thickness forearm skin biopsies from healthy and early diffuse SSc patients. BACE1 was inhibited with the small molecule inhibitors M-3 and AZD3839 and siRNA specific to BACE1. Morphogen signalling was activated with recombinant TGF-B, Wnt-3a or the smoothened agonist SAG. Xenotransplant mouse model using patient pDC was used to interrogate in vivo expression of BACE1 in fibrosis. Results Here we show that BACE1 protein levels are elevated in SSc patient skin biopsies, as well as dermal fibroblasts and in mouse skin during fibrosis. Inhibition of BACE1 with small molecule inhibitors or siRNA blocked SSc and morphogen mediated fibroblast pro-fibrotic activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on the B-catenin and Notch signalling pathway activation. Conclusions The ability of BACE1 to regulate SSc fibroblast activation reveals an exciting new therapeutic target in SSc. Several BACE1 inhibitors, including AZD3839, have been shown to be safe in clinical trials for Alzheimers disease.

The insulin receptor (IR) and insulin like growth factor-1 receptor (IGF-1R) are heterodimers consisting of 2 extracellular α-subunits and 2 transmembrane β-subunits. IR α/β and IGF-1R α/β hemi-receptors can heterodimerize to form hybrids composed of one IR α/β and one IGF-1R α/β. Widely distributed in mammalian tissues, in contrast to IR and IGF-1R the physiological function of hybrids is unclear. To identify tool compounds that inhibit hybrid formation we performed a high-throughput small molecule screen based on a homology model of hybrid structure. Our studies unveil a first in class quinoline-containing heterocyclic small molecule that reduces hybrids by >50% in human umbilical vein endothelial cells (HUVECs) with no effect on IR or IGF-1R. Downstream of IR and IGF-1R our small molecule led to reduced expression of the negative regulatory p85α subunit of phosphatidylinositol 3-kinase, an increase in phosphorylation of its downstream target Akt and enhanced insulin and shear-induced phosphorylation of Akt. We show that hybrids have a role in human EC physiology distinct from IR and IGF1R.

BackgroundThe beta-amyloid precursor protein cleaving enzyme 1 (BACE1) is well known for its role in the development of Alzheimer’s disease via the generation of β-amyloid. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic inflammatory diseases, including type 2 diabetes and cardiovascular disease. However, to date there has been no studies looking into the role of BACE1 in the autoimmune condition Systemic Sclerosis (SSc).ObjectivesThe aim of this study was to assess the expression profile of BACE1 in SSc patient samples and investigate the effects of BACE1 inhibitors and siRNA on SSc fibroblast activation.MethodsPatient fibroblasts were obtained from full thickness forearm skin biopsies from healthy and early diffuse SSc patients. BACE1 was inhibited with 2 specific small molecule inhibitors and siRNA specific to BACE1. Morphogen signalling was activated with recombinant TGF-β, Wnt-3a or the smoothened agonist SAG. A xenotransplant bleomycin mouse model using patient pDC was used to interrogatein vivoexpression of BACE1 in fibrosis.ResultsHere we show that BACE1 protein levels are elevated in SSc patient skin biopsies. In particular BACE1 was increased in the fibroblasts and endothelial cells of the SSc skin. BACE1 was elevated in isolated dermal fibroblasts grown in culture (2.3 fold increase, N=4). BACE1 protein levels were elevated in the bleomycin skin fibrosis model. Interestingly BACE1 mRNA levels were unaffected in cultured SSc fibroblasts, suggesting a post-translational modification led to the elevated protein levels.Inhibition of BACE1 with small molecule inhibitors (that have been proven safe in phase 1 clinical trials for Alzheimer’s) or siRNA blocked pro-fibrotic gene (alpha SMA, Collagen Type 1 and CTGF) expression in SSc fibroblasts. In addition overexpression of BACE1 in healthy fibroblasts resulted in myofibroblast activation (2-fold increase in alpha SMA protein expression). Interestingly overexpression of a BACE1 mutant construct which disrupts the secretase activity of the protein, was unable to induce fibroblast activation.Disruption of BACE1 (with both the inhibitors and siRNA) blocked morphogen mediated fibroblasts activation. The BACE1 inhibitors and siRNA blocked TGF-β, Wnt-3a and Hedgehog mediated alpha-SMA expression in healthy fibroblasts. Furthermore, we show that BACE1 regulation of dermal fibroblast activation was dependent on the β-catenin and Notch signalling pathways. BACE1 ability to regulate non-canonical Wnt receptors led to elevated β-catenin expression which in turn activated the Notch signalling pathway.ConclusionThis is the first evidence that BACE1 and in particular its secretase activity, plays a role in SSc and fibrosis in general. The ability of BACE1 to regulate SSc fibroblast activation reveals an exciting new therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in phase 1 clinical trials for Alzheimer’s disease. Future work includes investigating the role of BACE1 in vascular/endothelial cell dysfunction in SSc.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation British Microcirculation and Vascular Biology Society Background β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease notorious for its contribution to amyloid plaque formation in the pathophysiology of Alzheimer’s disease (AD) [1]. Further research has suggested a role for BACE1 in vascular homeostasis [2,3] and has shown that it proteolytically cleaves various angiogenic signalling factors including VEGF receptor 1 (VEGFR1) [2], NOTCH ligands [4] and the insulin receptor [5]. Similar to AD, BACE1 activity is elevated in models of type 2 diabetes [5], suggesting a potential role for its contribution to aberrant vessel growth characteristic of diabetes-related complications. Purpose Type 2 diabetes dramatically increases an individual's risk of developing microvascular complications and consequent lower limb amputations [6]. Therefore, identifying novel roles for BACE1 in angiogenic dysregulation will aid progression of future biomedical interventions in this field. Methods Retinal staining and the fibrin gel angiogenesis assay were used to identify a role for BACE1 in vessel growth in vivo and in vitro, respectively. Endothelium of the developing retinal vasculature in BACE1-/- and wild type (WT) mice was stained with IsolectinB4-Alexa488 and imaged using confocal microscopy. Sprout formation was further analysed using the fibrin gel angiogenesis assay with human umbilical vein endothelial cells (HUVECs) treated with or without a highly specific BACE1 inhibitor or transfected to over-express BACE1. Primary isolated pulmonary endothelial cells (PECs) were isolated from BACE1-/- and wild type control mice prior to Western blots, and real-time PCR. Results BACE1-/- retinas had increased branch points, vasculature area and quantity of filopodia compared to WT mice. Moreover, BACE1-/- PECs had reduced NOTCH1 signalling (26.73% ± 14.15, P=0.05) and soluble Jagged-1 protein (28.48% ± 14.61, P=<0.05). HUVECs treated with a BACE1 inhibitor had increased sprouting (18.70%± 5.92, P=<0.05) as well as increased phosphorylation of eNOS (83% ± 22, P=<0.05) and Akt (85.5% ± 9.24, P=NS) compared to untreated cells. Moreover, HUVECs transfected to over-express BACE1 had decreased sprouting (35.22% ± 7.34, P=<0.01) and increased NOTCH1 signalling (23.4% ± 2.42, P=0.01). Conclusion Our findings indicate a role of BACE1 in negatively regulating angiogenesis, possibly via NOTCH1 or Akt/eNOS/NO signalling. This provides a potential therapeutic purpose for BACE1 inhibitors, previously trialled to treat AD, in normalising BACE1 levels in individuals with type 2 diabetes and preventing associated microvascular complications.

Sirtuin 1 (SIRT1) is an NAD+ dependent deacetylase with vasculoprotective properties, which maintains cardiometabolic homeostasis under oxidative stress and prevents ischaemiareperfusion injury. Patients at high risk of atherothrombosis, such as diabetics and obese, display reduced SIRT1 levels, which were associated with enhanced thrombus formation in a murine model of arterial thrombosis. However, the importance of SIRT1 in regulating platelet function has not been evaluated. The aim of this study was to investigate the effect of the SIRT1 activators on platelet function and assess their potential use as a novel antithrombotic therapy.Unless stated otherwise, all experiments were performed with platelets from healthy donors and SIRT1 was activated with SRT1720 10 μM.The expression of SIRT1 was confirmed in platelets by Western blotting. Using PBA, it was demonstrated that SIRT1 activation with SRT1720 attenuates platelet aggregation induced with collagen and TRAP-6. Further evaluation of the role of SIRT1 in platelet activation showed that SRT1720 causes a 50% decrease in fibrinogen binding and a 30% reduction in a-granule release. The effect of SIRT1 activation in cytoskeletal rearrangement during platelet activation was also explored. Incubation of platelets with SRT1720 significantly reduced actin polymerization after platelet stimulation. Activation of SIRT1 with SRT1720 inhibited platelet adhesion and spreading on collagen and fibrinogen, but it did not alter agonist-induced coiling of the tubulin ring, indicating that SIRT1 is involved in platelet actin cytoskeleton reorganization. This was further supported by clot retraction assays, which demonstrated that SIRT1 inhibition with EX 527 caused a dramatic reduction in clot retraction, represented by an increase in the clot weight and the area occupied by the thrombus at different time points.These results suggest that SIRT1 regulates platelet integrin aIIbβ3 activation and the subsequent cytoskeletal rearrangement. SIRT1 may therefore provide a novel therapeutic target to limit platelet activation and accelerate reperfusion after a thrombotic obstruction in a vessel while protecting against oxidative damage.