Inhibition of a novel Dickkopf-1-LDL receptor-related proteins 5 and 6 axis prevents diabetic cardiomyopathy in mice.

BACKGROUND AND AIMS: Anti-hypertensive agents are one of the most frequently used drugs worldwide. However, no blood pressure-lowering strategy is superior to placebo with respect to survival in diabetic hypertensive patients. Previous findings show that Wnt co-receptors LDL receptor-related proteins 5 and 6 (LRP5/6) can directly bind to several G protein-coupled receptors (GPCRs). Because angiotensin II type 1 receptor (AT1R) is the most important GPCR in regulating hypertension, this study examines the possible mechanistic association between LRP5/6 and their binding protein Dickkopf-1 (DKK1) and activation of the AT1R and further hypothesizes that the LRP5/6-GPCR interaction may affect hypertension and potentiate cardiac impairment in the setting of diabetes. METHODS: The roles of serum DKK1 and DKK1-LRP5/6 signalling in diabetic injuries were investigated in human and diabetic mice. RESULTS: Blood pressure up-regulation positively correlated with serum DKK1 elevations in humans. Notably, LRP5/6 physically and functionally interacted with AT1R. The loss of membrane LRP5/6 caused by injection of a recombinant DKK1 protein or conditional LRP5/6 deletions resulted in AT1R activation and hypertension, as well as ß-arrestin1 activation and cardiac impairment, possibly because of multiple GPCR alterations. Importantly, unlike commonly used anti-hypertensive agents, administration of the anti-DKK1 neutralizing antibody effectively prevented diabetic cardiac impairment in mice. CONCLUSIONS: These findings establish a novel DKK1-LRP5/6-GPCR pathway in inducing diabetic injuries and may resolve the long-standing conundrum as to why elevated blood DKK1 has deleterious effects. Thus, monitoring and therapeutic elimination of blood DKK1 may be a promising strategy to attenuate diabetic injuries.

Identification of a PCSK9-LDLR disruptor peptide with in vivo function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 1013in vitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., 13PCSK9i). In mice, 13PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. 13PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.

Nodakenin represses obesity and its complications via the inhibition of the VLDLR signalling pathway in vivo and in vitro.

OBJECTIVES: Nodakenin (NK) is a coumarin glucoside that is found in the roots of Angelicae gigas. A limited number of studies have been conducted on the pharmacological activities of NK. Although NK is an important natural resource having anti-inflammatory and antioxidant effects, no investigation has been conducted to examine the effects of NK on obesity and obesity-induced inflammation. MATERIALS AND METHODS: The present study investigated the therapeutic effects of NK treatment on obesity and its complications, and its mechanism of action using differentiated 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese mice. Oil red O staining, western blot assay, qRT-PCR assay, siRNA transfection, enzyme-linked immunosorbent assay, H&E staining, immunohistochemistry, molecular docking and immunofluorescence staining were utilized. RESULTS: Treatment with NK demonstrated anti-adipogenesis effects via the regulation of adipogenic transcription factors and genes associated with triglyceride synthesis in differentiated 3T3-L1 adipocytes. Compared with the control group, the group administered NK showed a suppression in weight gain, dyslipidaemia and the development of fatty liver in HFD-induced obese mice. In addition, NK administration inhibited adipogenic differentiation and obesity-induced inflammation and oxidative stress via the suppression of the VLDLR and MEK/ERK1/2 pathways. This is the first study that has documented the interaction between NK and VLDLR structure. CONCLUSION: These results demonstrate the potential of NK as a natural product-based therapeutic candidate for the treatment of obesity and its complications by targeting adipogenesis and adipose tissue inflammation-associated markers.

Himalayan Nettle Girardinia diversifolia as a Candidate Ingredient for Pharmaceutical and Nutraceutical Applications-Phytochemical Analysis and In Vitro Bioassays.

Girardinia diversifolia, also known as Himalayan nettle, is a perennial herb used in Nepal to make fiber as well as in traditional medicine for the treatment of several diseases. To date, phytochemical studies and biological assays on this plant are scarce. Thus, in the present work, the G. diversifolia extracts have been evaluated for their potential pharmaceutical, cosmetic and nutraceutical uses. For this purpose, detailed phytochemical analyses were performed, evidencing the presence of phytosterols, fatty acids, carotenoids, polyphenols and saponins. The most abundant secondary metabolites were ß- and γ-sitosterol (11 and 9% dw, respectively), and trans syringin (0.5 mg/g) was the most abundant phenolic. Fatty acids with an abundant portion of unsaturated derivatives (linoleic and linolenic acid at 22.0 and 9.7 mg/g respectively), vitamin C (2.9 mg/g) and vitamin B2 (0.12 mg/g) were also present. The antioxidant activity was moderate while a significant ability to inhibit acetylcholinesterase (AChE), butyrilcholinesterase (BuChE), tyrosinase, α-amylase and α-glucosidase was observed. A cytotoxic effect was observed on human ovarian, pancreatic and hepatic cancer cell lines. The effect in hepatocarcinoma cells was associated to a downregulation of the low-density lipoprotein receptor (LDLR), a pivotal regulator of cellular cholesterol homeostasis. These data show the potential usefulness of this species for possible applications in pharmaceuticals, nutraceuticals and cosmetics.

A small molecule inhibitor of PCSK9 that antagonizes LDL receptor binding via interaction with a cryptic PCSK9 binding groove.

Proprotein convertase (PC) subtilisin kexin type 9 (PCSK9) inhibits the clearance of low density lipoprotein (LDL) cholesterol from plasma by directly interacting with the LDL receptor (LDLR). As the interaction promotes elevated plasma LDL cholesterol levels and a predisposition to cardiovascular disease (CVD), it has attracted much interest as a therapeutic target. While anti-PCSK9 monoclonal antibodies have been successful in the treatment of hypercholesteremia by decreasing CVD risk, their high cost and a requirement for injection have prohibited widespread use. The advent of an orally bioavailable small molecule inhibitor of the PCSK9-LDLR interaction is an attractive alternative, however efforts have been tempered as the binding interface is unfavourable for binding by small organic molecules. Despite its challenging nature, we report herein the discovery of compound 3f as a small molecule inhibitor of PCSK9. The kinase inhibitor nilotinib emerged from a computational screen that was applied to identify compounds that may bind to a cryptic groove within PCSK9 and proximal to the LDLR-binding interface. A subsequent in vitro PCSK9-LDLR binding assay established that nilotinib was a bona fide but modest inhibitor of the interaction (IC50 = 9.8 µM). Through multiple rounds of medicinal chemistry, 3f emerged as a lead-like molecule by demonstrating disruption of the PCSK9-LDLR interaction at nanomolar levels in vitro (IC50 = 537 nM) with no inhibitory activity (IC50 > 10 µM) against a small panel of kinases. Compound 3f restored LDL uptake by liver cells at sub-micromolar levels and demonstrated excellent bioavailability when delivered subcutaneously in mice. Most significantly, compound 3f lowered total cholesterol levels in the plasma of wild-type mice, thereby providing proof-of-concept that the notion of a small molecule inhibitor against PCSK9 is therapeutically viable.

Wilms tumor 1 regulates lipid accumulation in human endometrial stromal cells during decidualization.

We previously reported that the transcription factor Wilms tumor 1 (WT1) regulates the expression of insulin-like growth factor-binding protein-1 (IGFBP-1) and prolactin (PRL) during decidualization of human endometrial stromal cells (ESCs). However, other roles of WT1 in decidualization remain to be fully clarified. Here, we investigated how WT1 regulates the physiological functions of human ESCs during decidualization. We incubated ESCs isolated from proliferative-phase endometrium with cAMP to induce decidualization, knocked down WT1 with siRNA, and generated three types of treatments (nontreated cells, cAMP-treated cells, and cAMP-treated + WT1-knockdown cells). To identify WT1-regulated genes, we used gene microarrays and compared the transcriptome data obtained among these three treatments. We observed that WT1 up-regulates 121 genes during decidualization, including several genes involved in lipid transport. The WT1 knockdown inhibited lipid accumulation (LA) in the cAMP-induced ESCs. To examine the mechanisms by which WT1 regulates LA, we focused on very low-density lipoprotein receptor (VLDLR), which is involved in lipoprotein uptake. We found that cAMP up-regulates VLDLR and that the WT1 knockdown inhibits it. Results of ChIP assays revealed that cAMP increases the recruitment of WT1 to the promoter region of the VLDLR gene, indicating that WT1 regulates VLDLR expression. Moreover, VLDLR knockdown inhibited cAMP-induced LA, and VLDLR overexpression reverted the suppression of LA caused by the WT1 knockdown. Taken together, our results indicate that WT1 enhances lipid storage by up-regulating VLDLR expression in human ESCs during decidualization.

Anti-LDLR modified TPZ@Ce6-PEG complexes for tumor hypoxia-targeting chemo-/radio-/photodynamic/photothermal therapy.

Hypoxia, a state of low oxygen tension in solid tumors, is not only closely correlated with resistance to both radiotherapy and chemotherapy, but also associated with poor prognosis of tumors and regional lymph node status. Herein, based on the analysis of cell samples from tumor patients, low-density lipoprotein receptor (LDLR) was found to be overexpressed on the surface of hypoxic tumor cell membranes, and confirmed to be an effective hypoxia marker through specific binding with anti-LDLR antibody in solid tumors. In addition, using the special therapeutic microenvironment of hypoxia, tirapazamine (TPZ, which can be used as both a hypoxia-activated chemotherapy prodrug and radiotherapy sensitizer) was integrated with PEGylated photosensitizer chlorin e6 (Ce6-PEG) by self-assembly, and anti-LDLR was then modified on the surface to form tumor hypoxia-targeting multifunctional nanoparticles (CPTA). CPTA possesses a multimodal antitumor effect via a simultaneous photothermal therapy (PTT)/photodynamic therapy (PDT) effect generated by Ce6, and chemotherapy/radiotherapy actions sensitized by TPZ. It is noteworthy that tumor oxygen was consumed in the process of PDT and the hypoxia was subsequently exacerbated, which can greatly increase the TPZ-sensitized chemotherapy and lead to a synergistic antitumor effect. Both in vitro and in vivo experiments demonstrated that CPTA possesses an excellent therapeutic effect through PTT, PDT, and TPZ sensitized radiotherapy and chemotherapy. This hypoxic tumor targeting synergetic therapeutic strategy has great potential for future clinical transformation.

Bone morphogenetic protein 1 cleaves the linker region between ligand-binding repeats 4 and 5 of the LDL receptor and makes the LDL receptor non-functional.

The cell-surface low-density lipoprotein receptor (LDLR) internalizes low-density lipoprotein (LDL) by receptor-mediated endocytosis and plays a key role in the regulation of plasma cholesterol levels. The ligand-binding domain of the LDLR contains seven ligand-binding repeats of approximately 40 residues each. Between ligand-binding repeats 4 and 5, there is a 10-residue linker region that is subject to enzymatic cleavage. The cleaved LDLR is unable to bind LDL. In this study, we have screened a series of enzyme inhibitors in order to identify the enzyme that cleaves the linker region. These studies have identified bone morphogenetic protein 1 (BMP1) as being the cleavage enzyme. This conclusion is based upon the use of the specific BMP1 inhibitor UK 383367, silencing of the BMP1 gene by the use of siRNA or CRISPR/Cas9 technology and overexpression of wild-type BMP1 or the loss-of-function mutant E214A-BMP1. We have also shown that the propeptide of BMP1 has to be cleaved at RSRR120↓ by furin-like proprotein convertases for BMP1 to have an activity towards the LDLR. Targeting BMP1 could represent a novel strategy to increase the number of functioning LDLRs in order to lower plasma LDL cholesterol levels. However, a concern by using BMP1 inhibitors as cholesterol-lowering drugs could be the risk of side effects based on the important role of BMP1 in collagen assembly.

Molecular dynamics insights on the role ß-augmentation of the peptide N-terminus with binding site ß-hairpin of proprotein convertase subtilisin/kexin 9.

PCSK9, a member of the proprotein convertase family, is a key negative regulator of hepatic low-density lipoprotein receptor (LDLR) concentrations in the blood plasma and is associated with the risk of coronary artery disease (CAD). Peptide inhibitors designed to block PCSK9-LDLR interactions could reduce the risk of CAD. We present a study of the interaction of a PCSK9 bound peptide and its design through modification by phosphorylation using molecular dynamics simulations. Extensive explicit solvent simulations of PCSK9 and its mutant (Asp374 â†’ Tyr374) with designed peptides provide insights into the mechanism of peptide binding at the protein interface. We establish that ß-augmentation is the key mechanism of peptide association with PCSK9. Position-specific phosphorylation of threonine residues is observed to have noticeable effect in modulating protein-peptide association. This study provides a handle to explore and improve the design of peptides bound to PCSK9 by incorporating knowledge-derived functional motifs into designing potent binders.

Novel strategies to target proprotein convertase subtilisin kexin 9: beyond monoclonal antibodies.

Since the discovery of the role of proprotein convertase subtilisin kexin 9 (PCSK9) in the regulation of low-density lipoprotein cholesterol (LDL-C) in 2003, a paradigm shift in the treatment of hypercholesterolaemia has occurred. The PCSK9 secreted into the circulation is a major downregulator of the low-density lipoprotein receptor (LDLR) protein, as it chaperones it to endosomes/lysosomes for degradation. Humans with loss-of-function of PCSK9 exhibit exceedingly low levels of LDL-C and are protected from atherosclerosis. As a consequence, innovative strategies to modulate the levels of PCSK9 have been developed. Since 2015 inhibitory monoclonal antibodies (evolocumab and alirocumab) are commercially available. When subcutaneously injected every 2-4 weeks, they trigger a ∼60% LDL-C lowering and a 15% reduction in the risk of cardiovascular events. Another promising approach consists of a liver-targetable specific PCSK9 siRNA which results in ∼50-60% LDL-C lowering that lasts up to 6 months (Phases II-III clinical trials). Other strategies under consideration include: (i) antibodies targeting the C-terminal domain of PCSK9, thereby inhibiting the trafficking of PCSK9-LDLR to lysosomes; (ii) small molecules that either prevent PCSK9 binding to the LDLR, its trafficking to lysosomes or its secretion from cells; (iii) complete silencing of PCSK9 by CRISPR-Cas9 strategies; (iv) PCSK9 vaccines that inhibit the activity of circulating PCSK9. Time will tell whether other strategies can be as potent and safe as monoclonal antibodies to lower LDL-C levels.

An Innovation for Treating Orthotopic Pancreatic Cancer by Preoperative Screening and Imaging-Guided Surgery.

PURPOSE: Pancreatic cancer is still associated with a poor outcome and low patient quality of life, which are mainly attributed to the late detection and requirement of distal pancreatectomy with extended resection of pancreatic tumors. Therefore, novel strategies for early screening and precise tumor resection are urgently needed. In this study, we evaluated the feasibility of a low-density lipoprotein receptor (LDLR)-targeted small-molecule contrast agent (peptide-22-Cy7) for early screening with photoacoustic tomography and near-infrared (NIR) imaging as guided surgical navigation to achieve precise resection. PROCEDURE: Normal pancreatic cells (HPDE6-C7) and cancer cells (PANC-1) were respectively used in the in vitro targeting evaluations. The ability of peptide-22-Cy7 for preoperative in vivo pancreatic tumor detection was investigated in a mouse orthotopic pancreatic cancer model (n = 10) using photoacoustic tomography; 18 tumor-bearing mice were further divided into three groups for different treatments. After intravenous injection of peptide-22-Cy7, surgical navigation was conducted through laparotomy. Histopathological analysis was used to further confirm the tumor area and the state of surgical margins. RESULTS: Flow cytometry demonstrated that peptide-22 is highly specific to pancreatic cancer cells, with a fluorescence intensity of approximately 87.3 %. Orthotopic pancreatic tumors with a size of 4 mm could be accurately detected by photoacoustic tomography. Surgical navigation effectively achieved R0 resection and minimized the range of resection, which led to increased body weight of the mice following surgery. CONCLUSION: Overall, our newly developed targeted contrast agent facilitated the accurate positioning and resection of pancreatic tumors. Photoacoustic tomography and optical imaging-guided surgical navigation may be a novel direction for improving the survival, quality of life, and disease management of pancreatic cancer patients.

Pharmacology of PCSK9 Inhibitors: Current Status and Future Perspectives.

Protein Convertase Subtilisin/Kexin type 9 (PCSK9) is a serine protease primarily expressed in the liver, which represents the main source of the plasma enzyme. The best characterized function of PCSK9 relates to the binding to Low-Density Lipoprotein Receptor (LDL-R) in hepatocytes, increasing its endosomal and lysosomal degradation. This results in the inhibition of LDL-R recycling to the cell surface and therefore the reduction of the hepatic uptake of LDL, leading to the increase in plasma levels of LDL-cholesterol, a major risk factor of Cardiovascular Diseases (CVD). Therefore, PCSK9 is an important therapeutic target to reduce LDLcholesterol levels. PCSK9 inhibition can occur at the level of its interaction with LDL-R as well as at several sites across the pathway of its intracellular synthesis and secretion. Two fully human mAbs, Alirocumab and Evolocumab, that selectively bind to PCSK9 and prevent its interaction with the LDL-R, are currently used in the clinical practice. These mAbs are the most potent cholesterol-lowering agents available today and can decrease LDLcholesterol levels up to 73% while they also reduce the risk of atherosclerotic CVD. Ongoing research has led to the development of new PCSK9 inhibitors through genome editing technology (CRISPR-Cas9), siRNA or antisense oligonucleotide silencing agents, vaccines, mimetic peptides, adnectins, and inhibitors of PCSK9 secretion. The above inhibitors have been studied in vitro, in animal models in vivo, as well as in phase I and II trials and have demonstrated an important efficacy profile. Future studies with these agents will demonstrate their possible clinical value and will further enlighten the various targets and activities of PCSK9 intracellularly and extracellularly, the underlying mechanisms, as well as the clinical significance of these actions beyond the inhibition of LDL-R recycling.

Multimodal Treatment of Homozygous Familial Hypercholesterolemia.

BACKGROUND: Familial Hypercholesterolemia (FH) is an autosomal-dominant genetic disease, associated with premature atherosclerotic Cardiovascular Disease (CVD), especially in its homozygous type (HoFH). OBJECTIVE: The aim of this review is to discuss the safety and efficacy of combination treatments (procedures and drugs) for HoFH. RESULTS: Historically, liver transplantation was used first; however, it is currently considered only as a last resort for some patients. In the mid 70's, LDL aphaeresis was introduced and remains up today the treatment of choice for patients of any age, despite its significant cost. The use of Ezetimibe results in additive 15-20% reductions in LDL-C regardless of the therapeutic approach, while statins are modestly effective in patients with class 4 or 5 mutations, in which LDL Receptors (LDLR) are present. One of the novel drugs for HoFH is Lomitapide, which is a highly effective oral agent, but is also exceedingly expensive ($350, 000/year). Mipomersen is administered every week subcutaneously, is also effective but has been approved only in the US mainly due to injection site reactions up to 80%. Both Lomitapide (mainly) and Mipomersen have been found to promote fat accumulation in the liver, resulting in subsequent serum transaminases elevations. PCSK9 inhibitors are effective in those with partial LDLR presence and function by reducing frequency of LDL apheresis, improve cost effectiveness of treatment. CONCLUSION: Pediatric and adult HoFH treatment needs combination of procedures and drugs. The main treatment is LDL-C apheresis aided by ezetimibe and PCSK9 inhibitors. Lomitapide needs caution, and liver transplantation is an alternative as the last resort.

New Horizons in the Pathogenesis, Pathophysiology and Treatment of Familial Hypercholesterolaemia.

BACKGROUND: Familial Hypercholesterolaemia (FH) is an autosomal-dominant genetic disease and represents the most common genetic disorder: heterozygous 1/250 births, homozygous 1/300, 000 births. FH is characterized by high to very high low-density lipoprotein cholesterol (LDL-C), which is the main cause of increased incidence of premature atherosclerotic Cardiovascular Disease (CVD) or aortic stenosis. OBJECTIVE: The aim of the review was to investigate the pathogenesis and the pathophysiology of FH. RESULTS: The most common (60-80%) FH cause is mutations of the LDL Receptor (LDLR) protein (6 classes with a different number of receptors and functionality). Moreover, mutations in apolipoprotein B (APOB) (<5%) and gain-of-function mutations of proprotein convertase subtilisin/kexin type 9 genes (PCSK9) (<1%) contribute to its pathogenesis. An Autosomal Recessive Hypercholesterolaemia (ARH) is another cause, very rare (1/2.500 births), mainly in Sardinia. The remaining patients with a clinical diagnosis of monogenic hypercholesterolaemia do not present any known genetic cause. Since FH is a significant public health problem, early diagnosis and treatment are of utmost importance. Recent studies demonstrated the influence of the LDLR mutation type in the FH phenotype, associating a more severe clinical phenotype and worse advanced CVD in patients with null mutation than those with receptor-defective mutations. This analysis completes the adequate clinical diagnosis. CONCLUSION: Both homozygous and heterozygous FH are related to mutations of LDLR (mainly), APOB, PCSK9, while other rare forms exist. All aberrations lead to the impaired removal of LDL-C from the blood leading to its accumulation and subsequent CVD earlier than in the general population.

A New Strategy for Rapidly Screening Natural Inhibitors Targeting the PCSK9/LDLR Interaction In Vitro.

The interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and the low-density lipoprotein receptor (LDLR) is a promising target for the treatment of hyperc-holesterolemia. In this study, a new method based on competitive affinity and tag detection was developed, which aimed to evaluate potent natural inhibitors preventing the interaction of PCSK9/LDLR directly. Herein, natural compounds with efficacy in the treatment of hypercholesterolemia were chosen to investigate their inhibitory activities on the PCSK9/LDLR interaction. Two of them, polydatin (1) and tetrahydroxydiphenylethylene-2-O-glucoside (2), were identified as potential inhibitors for the PCSK9/LDLR interaction and were proven to prevent PCSK9-mediated LDLR degradation in HepG2 cells. The results suggested that this strategy could be applied for evaluating potential bioactive compounds inhibiting the interaction of PCSK9/LDLR and this strategy could accelerate the discovery of new drug candidates for the treatment of PCSK9-mediated hypercholesterolemia.

Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol.

Clinical translation of in vivo genome editing to treat human genetic diseases requires thorough preclinical studies in relevant animal models to assess safety and efficacy. A promising approach to treat hypercholesterolemia is inactivating the secreted protein PCSK9, an antagonist of the LDL receptor. Here we show that single infusions in six non-human primates of adeno-associated virus vector expressing an engineered meganuclease targeting PCSK9 results in dose-dependent disruption of PCSK9 in liver, as well as a stable reduction in circulating PCSK9 and serum cholesterol. Animals experienced transient, asymptomatic elevations of serum transaminases owing to the formation of T cells against the transgene product. Vector DNA and meganuclease expression declined rapidly, leaving stable populations of genome-edited hepatocytes. A second-generation PCSK9-specific meganuclease showed reduced off-target cleavage. These studies demonstrate efficient, physiologically relevant in vivo editing in non-human primates, and highlight safety considerations for clinical translation.

Bempedoic Acid (ETC-1002): A Current Review.

Although statins are first-line therapy for low-density lipoprotein cholesterol (LDL-C) reduction, many individuals on maximally tolerated statin therapy have elevated LDL-C. Bempedoic acid (ETC-1002) is a novel once-daily LDL-C-lowering agent in phase 3 clinical trials. In phase 1 and 2 studies, ETC-1002 was efficacious in lowering LDL-C when used as monotherapy and when added to statin and/or ezetimibe and was well tolerated in patients with statin intolerance. ETC-1002 also improved cardiometabolic risk factors. Ongoing phase 3 studies of ETC-1002 are evaluating its long-term efficacy and safety, and effects on cardiovascular events. This article discusses current evidence and future directions for ETC-1002.

LDL receptor blockade reduces mortality in a mouse model of ischaemic stroke without improving tissue-type plasminogen activator-induced brain haemorrhage: towards pre-clinical simulation of symptomatic ICH.

BACKGROUND: Symptomatic intracerebral haemorrhage (sICH) following tissue-type plasminogen activator (rt-PA) administration is the most feared and lethal complication of thrombolytic therapy for ischaemic stroke, creating a significant obstacle for a broader uptake of this beneficial treatment. rt-PA also undermines cerebral vasculature stability in a multimodal process which involves engagement with LDL receptor-related protein 1 (LRP-1), potentially underlying the development of sICH. AIMS AND METHODS: We aimed to simulate rt-PA-induced haemorrhagic transformation (HT) in a mouse model of stroke and to assess if it drives symptomatic neurological deterioration and whether it is attenuated by LDL receptor blockade. rt-PA (10 mg/kg) or its vehicle, with or without the LDL receptor antagonist, receptor-associated protein (RAP; 2 mg/kg), were intravenously injected at reperfusion after 0.5 or 4 h of middle cerebral artery occlusion (MCAo). Albumin and haemoglobin content were measured in the perfused mouse brains 24 h post MCAo as indications of blood-brain barrier (BBB) compromise and HT, respectively. RESULTS: rt-PA did not elevate brain albumin and haemoglobin levels in sham mice or in mice subjected to 0.5 h MCAo. In contrast, administration of rt-PA after prolonged MCAo (4 h) caused a marked increase in HT (but similar changes in brain albumin) compared to vehicle, mimicking the clinical shift from a safe to detrimental intervention. Interestingly, this HT did not correlate with functional deficit severity at 24 h, suggesting that it does not play a symptomatic role in our mouse stroke model. Co-administration of RAP with or without rt-PA reduced mortality and neurological scores but did not effectively decrease brain albumin and haemoglobin levels. CONCLUSION: Despite the proven causative relationship between severe HT and neurological deterioration in human stroke, rt-PA-triggered HT in mouse MCAo does not contribute to neurological deficit or simulate sICH. Model limitations, such as the long duration of occlusion required, the type of HT achieved and the timing of deficit assessment may account for this mismatch. Our results further suggest that blockade of LDL receptors improves stroke outcome irrespective of rt-PA, blood-brain barrier breakdown and HT.

Shedding of membrane-associated LDL receptor-related protein-1 from microglia amplifies and sustains neuroinflammation.

In the CNS, microglia are activated in response to injury or infection and in neurodegenerative diseases. The endocytic and cell signaling receptor, LDL receptor-related protein-1 (LRP1), is reported to suppress innate immunity in macrophages and oppose microglial activation. The goal of this study was to identify novel mechanisms by which LRP1 may regulate microglial activation. Using primary cultures of microglia isolated from mouse brains, we demonstrated that LRP1 gene silencing increases expression of proinflammatory mediators; however, the observed response was modest. By contrast, the LRP1 ligand, receptor-associated protein (RAP), robustly activated microglia, and its activity was attenuated in LRP1-deficient cells. An important element of the mechanism by which RAP activated microglia was its ability to cause LRP1 shedding from the plasma membrane. This process eliminated cellular LRP1, which is anti-inflammatory, and generated a soluble product, shed LRP1 (sLRP1), which is potently proinflammatory. Purified sLRP1 induced expression of multiple proinflammatory cytokines and the mRNA encoding inducible nitric-oxide synthase in both LRP1-expressing and -deficient microglia. LPS also stimulated LRP1 shedding, as did the heat-shock protein and LRP1 ligand, calreticulin. Other LRP1 ligands, including α2-macroglobulin and tissue-type plasminogen activator, failed to cause LRP1 shedding. Treatment of microglia with a metalloproteinase inhibitor inhibited LRP1 shedding and significantly attenuated RAP-induced cytokine expression. RAP and sLRP1 both caused neuroinflammation in vivo when administered by stereotaxic injection into mouse spinal cords. Collectively, these results suggest that LRP1 shedding from microglia may amplify and sustain neuroinflammation in response to proinflammatory stimuli.