Molecular mechanisms of the regulatory action of high-density lipoproteins on the endothelial function.

Endothelial dysfunction underlies the pathogenesis of many diseases, primarily cardiovascular diseases. Epidemiological studies have shown an inverse dependence between the plasma level of high-density lipoproteins (HDL) and cardiovascular diseases. The results of experimental studies indicate that the antiatherogenic effect of HDL is associated not only with their participation in the reverse transport of excess cholesterol, but also with their regulatory effect on the functions of cells of various organs and tissues, including endothelial cells. The purpose of this review is to consider recent data on the participation of plasma receptors and related intracellular signaling pathways in the mechanism of protective effect of HDL on endothelial cell functions. Understanding the mechanisms of cell function regulation under the influence of HDL is an important step for the development of new ways of pharmacological correction of impaired endothelial functions and creation of effective endothelial protection drugs.

A negatively charged cluster in the disordered acidic domain of GPIHBP1 provides selectivity in the interaction with lipoprotein lipase.

GPIHBP1 is a membrane protein of endothelial cells that transports lipoprotein lipase (LPL), the key enzyme in plasma triglyceride metabolism, from the interstitial space to its site of action on the capillary lumen. An intrinsically disordered highly negatively charged N-terminal domain of GPIHBP1 contributes to the interaction with LPL. In this work, we investigated whether the plethora of heparin-binding proteins with positively charged regions found in human plasma affect this interaction. We also wanted to know whether the role of the N-terminal domain is purely non-specific and supportive for the interaction between LPL and full-length GPIHBP1, or whether it participates in the specific recognition mechanism. Using surface plasmon resonance, affinity chromatography, and FRET, we were unable to identify any plasma component, besides LPL, that bound the N-terminus with detectable affinity or affected its interaction with LPL. By examining different synthetic peptides, we show that the high affinity of the LPL/N-terminal domain interaction is ensured by at least ten negatively charged residues, among which at least six must sequentially arranged. We conclude that the association of LPL with the N-terminal domain of GPIHBP1 is highly specific and human plasma does not contain components that significantly affect this complex.

Etiology and emerging treatments for familial chylomicronemia syndrome.

INTRODUCTION: Familial chylomicronemia syndrome (FCS) is a rare autosomal recessive condition. Effective treatment is important as patients are at risk for severe and potentially fatal acute pancreatitis. We review recent developments in pharmacologic treatment for FCS, namely biological inhibitors of apolipoprotein (apo) C-III and angiopoietin-like protein 3 (ANGPTL3). AREAS COVERED: FCS follows a biallelic inheritance pattern in which an individual inherits two pathogenic loss-of-function alleles of one of the five causal genes - LPL (in 60-80% of patients), GPIHBP1, APOA5, APOC2, and LMF1 - leading to the absence of lipolytic activity. Patients present from childhood with severely elevated triglyceride (TG) levels >10 mmol/L. Most patients with severe hypertriglyceridemia do not have FCS. A strict low-fat diet is the current first-line treatment, and existing lipid-lowering therapies are minimally effective in FCS. Apo C-III inhibitors are emerging TG-lowering therapies shown to be efficacious and safe in clinical trials. ANGPTL3 inhibitors, another class of emerging TG-lowering therapies, have been found to require at least partial lipoprotein lipase activity to lower plasma TG in clinical trials. ANGPTL3 inhibitors reduce plasma TG in patients with multifactorial chylomicronemia but not in patients with FCS who completely lack lipoprotein lipase activity. EXPERT OPINION: Apo C-III inhibitors currently in development are promising treatments for FCS.

Expression and Localization Profiles of Tight Junction Proteins in Immune Cells Depend on Their Activation Status.

The ability of the immune system to combat pathogens relies on processes like antigen sampling by dendritic cells and macrophages migrating through endo- and epithelia or penetrating them with their dendrites. In addition, other immune cell subtypes also migrate through the epithelium after activation. For paracellular migration, interactions with tight junctions (TJs) are necessary, and previous studies reported TJ protein expression in several immune cells. Our investigation aimed to characterize, in more detail, the expression profiles of TJ proteins in different immune cells in both naïve and activated states. The mRNA expression analysis revealed distinct expression patterns for TJ proteins, with notable changes, mainly increases, upon activation. At the protein level, LSR appeared predominant, being constitutively present in naïve cell membranes, suggesting roles as a crucial interaction partner. Binding experiments suggested the presence of claudins in the membrane only after stimulation, and claudin-8 translocation to the membrane occurred after stimulation. Our findings suggest a dynamic TJ protein expression in immune cells, implicating diverse functions in response to stimulation, like interaction with TJ proteins or regulatory roles. While further analysis is needed to elucidate the precise roles of TJ proteins, our findings indicate important non-canonical functions of TJ proteins in immune response.

Quantification of lipoprotein lipase in mouse plasma with a sandwich enzyme-linked immunosorbent assay.

To support in vivo and in vitro studies of intravascular triglyceride metabolism in mice, we created rat monoclonal antibodies (mAbs) against mouse LPL. Two mAbs, mAbs 23A1 and 31A5, were used to develop a sandwich ELISA for mouse LPL. The detection of mouse LPL by the ELISA was linear in concentrations ranging from 0.31 ng/ml to 20 ng/ml. The sensitivity of the ELISA made it possible to quantify LPL in serum and in both pre-heparin and post-heparin plasma samples (including in grossly lipemic samples). LPL mass and activity levels in the post-heparin plasma were lower in Gpihbp1-/- mice than in wild-type mice. In both groups of mice, LPL mass and activity levels were positively correlated. Our mAb-based sandwich ELISA for mouse LPL will be useful for any investigator who uses mouse models to study LPL-mediated intravascular lipolysis.

Familial chylomicronemia syndrome: case reports of siblings with deletions of the GPIHBP1 gene.

BACKGROUND: Familial chylomicronemia syndrome (FCS) is a rare monogenic form of severe hypertriglyceridemia, caused by mutations in genes involved in triglyceride metabolism. Herein, we report the case of a Korean family with familial chylomicronemia syndrome caused by compound heterozygous deletions of glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1). CASE PRESENTATION: A 4-year-old boy was referred for the evaluation of severe hypertriglyceridemia (3734 mg/dL) that was incidentally detected 4 months prior. His elder brother also demonstrated an elevated triglyceride level of 2133 mg/dL at the age of 9. Lipoprotein electrophoresis revealed the presence of chylomicrons, an increase in the proportion of pre-beta lipoproteins, and low serum lipoprotein lipase levels. The patient's parents and first elder brother had stable lipid profiles. For suspected FCS, genetic testing was performed using the next-generation sequencing-based analysis of 31 lipid metabolism-associated genes, which revealed no pathogenic variants. However, copy number variant screening using sequencing depth information suggested large heterozygous deletion encompassing all the coding exons of GPIHBP1. A real-time quantitative polymerase chain reaction was performed to validate the deletion site. The results showed that the siblings had two heterozygous copy number variants consisting of the whole gene and an exon 4 deletion, each inherited from their parents. During the follow-up period of 17 months, the patient did not develop pancreatitis, following dietary intervention. CONCLUSION: These siblings' case of familial chylomicronemia syndrome caused by rare GPIHBP1 deletions highlight the implementation of copy number variants-beyond next-generation sequencing-as an important consideration in diagnosis. Accurate genetic diagnosis is necessary to establish the etiology of severe hypertriglyceridemia, which increases the risk of pancreatitis.

Molecular genetic testing and measurement of levels of GPIHBP1 autoantibodies in patients with severe hypertriglyceridemia: The importance of identifying the underlying cause of hypertriglyceridemia.

BACKGROUND: Severe hypertriglyceridemia can be caused by pathogenic variants in genes encoding proteins involved in the metabolism of triglyceride-rich lipoproteins. A key protein in this respect is lipoprotein lipase (LPL) which hydrolyzes triglycerides in these lipoproteins. Another important protein is glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) which transports LPL to the luminal side of the endothelial cells. OBJECTIVE: Our objective was to identify a genetic cause of hypertriglyceridemia in 459 consecutive unrelated subjects with levels of serum triglycerides ≥20 mmol/l. These patients had been referred for molecular genetic testing from 1998 to 2021. In addition, we wanted to study whether GPIHBP1 autoantibodies also were a cause of hypertriglyceridemia. METHODS: Molecular genetic analyses of the genes encoding LPL, GPIHBP1, apolipoprotein C2, lipase maturation factor 1 and apolipoprotein A5 as well as apolipoprotein E genotyping, were performed in all 459 patients. Serum was obtained from 132 of the patients for measurement of GPIHBP1 autoantibodies approximately nine years after molecular genetic testing was performed. RESULTS: A monogenic cause was found in four of the 459 (0.9%) patients, and nine (2.0%) patients had dyslipoproteinemia due to homozygosity for apolipoprotein E2. One of the 132 (0.8%) patients had GPIHBP1 autoantibody syndrome. CONCLUSION: Only 0.9% of the patients had monogenic hypertriglyceridemia, and only 0.8% had GPIHBP1 autoantibody syndrome. The latter figure is most likely an underestimate because serum samples were obtained approximately nine years after hypertriglyceridemia was first identified. There is a need to implement measurement of GPIHBP1 autoantibodies in clinical medicine to secure that proper therapeutic actions are taken.

AAV-mediated hepatic LPL expression ameliorates severe hypertriglyceridemia and acute pancreatitis in Gpihbp1 deficient mice and rats.

GPIHBP1 plays an important role in the hydrolysis of triglyceride (TG) lipoproteins by lipoprotein lipases (LPLs). However, Gpihbp1 knockout mice did not develop hypertriglyceridemia (HTG) during the suckling period but developed severe HTG after weaning on a chow diet. It has been postulated that LPL expression in the liver of suckling mice may be involved. To determine whether hepatic LPL expression could correct severe HTG in Gpihbp1 deficiency, liver-targeted LPL expression was achieved via intravenous administration of the adeno-associated virus (AAV)-human LPL gene, and the effects of AAV-LPL on HTG and HTG-related acute pancreatitis (HTG-AP) were observed. Suckling Gpihbp1-/- mice with high hepatic LPL expression did not develop HTG, whereas Gpihbp1-/- rat pups without hepatic LPL expression developed severe HTG. AAV-mediated liver-targeted LPL expression dose-dependently decreased plasma TG levels in Gpihbp1-/- mice and rats, increased post-heparin plasma LPL mass and activity, decreased mortality in Gpihbp1-/- rat pups, and reduced the susceptibility and severity of both Gpihbp1-/- animals to HTG-AP. However, the muscle expression of AAV-LPL had no significant effect on HTG. Targeted expression of LPL in the liver showed no obvious adverse reactions. Thus, liver-targeted LPL expression may be a new therapeutic approach for HTG-AP caused by GPIHBP1 deficiency.

Tumor cell-expressed lipolysis-stimulated lipoprotein receptor negatively regulates T-cell function.

Lipolysis-stimulated lipoprotein receptor (LSR) is known as a lipoprotein receptor. LSR is expressed in various solid tumors, including epithelial ovarian, gastric, and colon cancers. High LSR expression is significantly associated with poor prognosis, but its role in cancer has not been fully elucidated. LSR belongs to the Ig protein superfamily, which is conserved in B7 family. Here, we assessed LSR as a novel immune checkpoint molecule. We developed a novel anti-LSR antibody (#27-6 mF-18) that defects antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. The #27-6 mF-18 cross-reacts with both human and mouse LSR. We found that LSR was expressed on 4T1 murine breast cancer cell line. The #27-6 mF-18 exhibited antitumor effects against the 4T1 syngeneic tumor model, a poor immunogenic model refractory to treatment with anti-PD-1 or anti-CTLA-4 antibodies. Compared with control antibody-treated mice, mice treated with #27-6 mF-18 showed significantly increased numbers of CD8+ T cells and a ratio of activated CD8+ T cells infiltrated in the tumor tissue. This antitumor effect was abrogated by CD8+ T-cell depletion through anti-CD8 antibody treatment, indicating that LSR negatively regulates tumor immunity by repressing CD8+ T cells. These findings show that LSR negatively regulates T-cell immune activity. LSR targeting could provide immune checkpoint inhibitors for cancer immunotherapy.

Hypertriglyceridemia in Apoa5-/- mice results from reduced amounts of lipoprotein lipase in the capillary lumen.

Why apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia has remained unclear, but we have suspected that the underlying cause is reduced amounts of lipoprotein lipase (LPL) in capillaries. By routine immunohistochemistry, we observed reduced LPL staining of heart and brown adipose tissue (BAT) capillaries in Apoa5-/- mice. Also, after an intravenous injection of LPL-, CD31-, and GPIHBP1-specific mAbs, the binding of LPL Abs to heart and BAT capillaries (relative to CD31 or GPIHBP1 Abs) was reduced in Apoa5-/- mice. LPL levels in the postheparin plasma were also lower in Apoa5-/- mice. We suspected that a recent biochemical observation - that APOA5 binds to the ANGPTL3/8 complex and suppresses its capacity to inhibit LPL catalytic activity - could be related to the low intracapillary LPL levels in Apoa5-/- mice. We showed that an ANGPTL3/8-specific mAb (IBA490) and APOA5 normalized plasma triglyceride (TG) levels and intracapillary LPL levels in Apoa5-/- mice. We also showed that ANGPTL3/8 detached LPL from heparan sulfate proteoglycans and GPIHBP1 on the surface of cells and that the LPL detachment was blocked by IBA490 and APOA5. Our studies explain the hypertriglyceridemia in Apoa5-/- mice and further illuminate the molecular mechanisms that regulate plasma TG metabolism.

The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing.

Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.

LSR targets YAP to modulate intestinal Paneth cell differentiation.

Lipolysis-stimulated lipoprotein receptor (LSR) is a multi-functional protein that is best known for its roles in assembly of epithelial tricellular tight junctions and hepatic clearance of lipoproteins. Here, we investigated whether LSR contributes to intestinal epithelium homeostasis and pathogenesis of intestinal disease. By using multiple conditional deletion mouse models and ex vivo cultured organoids, we find that LSR elimination in intestinal stem cells results in the disappearance of Paneth cells without affecting the differentiation of other cell lineages. Mechanistic studies reveal that LSR deficiency increases abundance of YAP by modulating its phosphorylation and proteasomal degradation. Using gain- and loss-of-function studies, we show that LSR protects against necrotizing enterocolitis through enhancement of Paneth cell differentiation in small-intestinal epithelium. Thus, this study identifies LSR as an upstream negative regulator of YAP activity, an essential factor for Paneth cell differentiation, and a potential therapeutic target for necrotizing enterocolitis.

Luteolin in the Qi Bi Anshen decoction improves propionic acid-induced autism-like behavior in rats by inhibiting LRP1/MMP9.

BACKGROUND: A neurodevelopmental illness with a high frequency and unidentified pathophysiology is known as autism spectrum disorder (ASD). A research hotspot in this field is the identification of disease-specific biomarkers and drug intervention targets. Traditional Chinese medicine (TCM) can eliminate the symptoms of autism by precisely regulating human physiology. The Qi Bi Anshen decoction (QAT) is a commonly used TCM clinical drug commonly-used to treat for treating ASD. However, the primary active ingredients and underlying mechanisms of action of this decoction remain unknown. PURPOSE: This study aimed to investigate the active ingredients and pharmacodynamics of QAT in the treatment of ASD using a Sprague-Dawley rat model that resembled autism. METHODS: Autism-like rat models were established through intracerebroventricular injections of propionic acid (PPA). Subsequently, the rats were treated with QAT, and their efficacy was evaluated using the three-chamber method to analyze social interactions and grooming behavior. Additionally, open-field tests, elevated cross-maze tests, hematoxylin and eosin staining, Nissl staining, and enzyme-linked immunosorbent assays were performed; Western blot analysis was employed to determine the expression of synaptic plasticity-related proteins. Utilizing ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS), the effectiveness of active QAT components was assessed, and potential QAT targets were screened through molecular docking, surface plasmon resonance, and thermal migration experiments. To better understand the precise processes involved in treating ASD with active QAT components, in vivo and in vitro knockdown tests were also performed. RESULTS: QATexhibited a significant improvement in autism-like behavior and a notable increase in the production of proteins associated with synaptic plasticity. Furthermore, luteolin (LUT), identified as a potentially important active ingredient in QAT for treating ASD, reduced matrix metallopeptidase-9 (MMP9) expression. However, this effect was attenuated by the knockdown of low-density lipoprotein receptor-associated protein 1 (LRP1), which is the target binding site for LUT. CONCLUSIONS: LUT emerges as a potentially crucial active component of QAT in the treatment of ASD, with the ability to antagonize LRP1 and subsequently reduce MMP9 expression.

An LRP1-binding motif in cellular prion protein replicates cell-signaling activities of the full-length protein.

Low-density lipoprotein receptor-related protein-1 (LRP1) functions as a receptor for nonpathogenic cellular prion protein (PrPC), which is released from cells by ADAM (a disintegrin and metalloproteinase domain) proteases or in extracellular vesicles. This interaction activates cell signaling and attenuates inflammatory responses. We screened 14-mer PrPC-derived peptides and identified a putative LRP1 recognition motif in the PrPC sequence spanning residues 98-111. A synthetic peptide (P3) corresponding to this region replicated the cell-signaling and biological activities of full-length shed PrPC. P3 blocked LPS-elicited cytokine expression in macrophages and microglia and rescued the heightened sensitivity to LPS in mice in which the PrPC gene (Prnp) had been deleted. P3 activated ERK1/2 and induced neurite outgrowth in PC12 cells. The response to P3 required LRP1 and the NMDA receptor and was blocked by the PrPC-specific antibody, POM2. P3 has Lys residues, which are typically necessary for LRP1 binding. Converting Lys100 and Lys103 into Ala eliminated the activity of P3, suggesting that these residues are essential in the LRP1-binding motif. A P3 derivative in which Lys105 and Lys109 were converted into Ala retained activity. We conclude that the biological activities of shed PrPC, attributed to interaction with LRP1, are retained in synthetic peptides, which may be templates for therapeutics development.

Relationship between serum Betatrophin, GPIHBP1, and LDL subfractions in patients with gestational diabetes mellitus.

OBJECTIVES: Gestational diabetes mellitus (GDM) leads to changes in the lipid metabolism. In this study, we aimed to compare serum levels of LDL subfractions, betatrophin, and glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) between patients with GDM and healthy pregnant women. DESIGN AND METHODS: We designed a prospective case-control study with 41 pregnant women. Subjects were divided into two groups: GDM and control. Betatrophin and GPIHBP1 levels were measured by ELISA method. Lipoprint LDL subfraction kit was used to perform LDL subfraction analysis electrophoretically. RESULTS: Serum levels of LDL6 subfraction, betatrophin, and GPIHBP1 were found to be higher in GDM group compared to the controls (p < 0.001). The mean LDL size were also found larger in GDM group. A positive correlation was found between betatrophin and GPIHBP1 levels (rho = 0.96, p < 0.001). CONCLUSIONS: Our findings suggest that betatrophin, and GPIHBP1 levels were found to be increased in GDM. This maybe the result of adaptive mechanisms in response to insulin resistance, but also this relationship should be evaluated for their effects on impaired lipid metabolism and lipoprotein lipase metabolism. There is a need for further prospective studies with larger samples to fully elucidate the mechanisms of this relationship both in pregnant patients and the other patient groups.

Andrographolide exerts a neuroprotective effect by regulating the LRP1-mediated PPARγ/NF-κB pathway.

Low-density lipoprotein receptor-associated protein 1 (LRP1) is widely expressed in neurons, microglia and astrocytes. Studies have revealed that the suppression of LRP1 expression in the brain significantly exacerbates Alzheimer's disease (AD)-related neuropathology. Andrographolide (Andro) has been demonstrated to possess neuroprotective properties, although its underlying mechanisms remain largely unknown. This study aims to investigate whether Andro can inhibit neuroinflammation in AD by modulating the LRP1-mediated PPARγ/NF-κB pathway. In Aß-induced BV-2 cells, Andro was found to increase cell viability and enhance the expression of LRP1, while decreasing the expression of p-NF-κB (p65) and NF-κB(p65), as well as IL-1ß, IL-6 and TNF-α levels. In addition, when Aß was cotreatment with Andro to BV2 cells with either LRP1 or PPARγ knockdown, increased mRNA and protein expression of p-NF-κB(p65) and NF-κB(p65), NF-κB DNA binding activity as well as IL-1ß, IL-6 and TNF-α levels were observed. These findings suggested that Andro could attenuate Aß induced cytotoxicity by reducing neuroinflammation which may be partly attributed to its effects on this LRP1 mediated PPARγ/NF-κB pathway.

Severe hypertriglyceridemia caused by Gpihbp1 deficiency facilitates vascular remodeling through increasing endothelial activation and oxidative stress.

Hypertriglyceridemia (HTG) is an independent risk factor for atherosclerosis. However, its impact on non-atherosclerotic cardiovascular diseases remains largely unknown. Glycosylphosphatidylinositol anchored high-density lipoprotein binding protein 1 (GPIHBP1) is essential for the hydrolysis of circulating triglycerides and loss of functional GPIHBP1 causes severe HTG. In this study, we used Gpihbp1 knockout (GKO) mice to investigate the potential effects of HTG on non-atherosclerotic vascular remodeling. We compared the aortic morphology and gene expressions between three-month-old and ten-month-old GKO mice and their age-matched wild-type controls. We also conducted similar comparisons between GKO mice and wild-type controls in an angiotensin II (AngII)-induced vascular remodeling model. Our data showed that the intima-media wall of ten-month-old GKO mice but not three-month-olds was significantly thickened compared to wild-type controls. Moreover, ten-month-old GKO mice but not three-month-olds had increased aortic macrophage infiltration and perivascular fibrosis, along with increased endothelial activation and oxidative stress. Similarly, the AngII-induced vascular remodeling, as well as endothelial activation and oxidative stress, were also exacerbated in the GKO mice compared to wild-type controls. In conclusion, we demonstrated that severe HTG caused by Gpihbp1 deficiency could facilitate the onset and progression of non-atherosclerotic vascular remodeling through endothelial activation and oxidative stress in mice.