Distinct strategies for intravascular triglyceride metabolism in hearts of mammals and lower vertebrate species.

Lipoprotein lipase (LPL) and multiple regulators of LPL activity (e.g., APOC2 and ANGPTL4) are present in all vertebrates, but GPIHBP1-the endothelial cell (EC) protein that captures LPL within the subendothelial spaces and transports it to its site of action in the capillary lumen-is present in mammals but in not chickens or other lower vertebrates. In mammals, GPIHBP1 deficiency causes severe hypertriglyceridemia, but chickens maintain low triglyceride levels despite the absence of GPIHBP1. To understand intravascular lipolysis in lower vertebrates, we examined LPL expression in mouse and chicken hearts. In both species, LPL was abundant on capillaries, but the distribution of Lpl transcripts was strikingly different. In mouse hearts, Lpl transcripts were extremely abundant in cardiomyocytes but were barely detectable in capillary ECs. In chicken hearts, Lpl transcripts were absent in cardiomyocytes but abundant in capillary ECs. In zebrafish hearts, lpl transcripts were also in capillary ECs but not cardiomyocytes. In both mouse and chicken hearts, LPL was present, as judged by immunogold electron microscopy, in the glycocalyx of capillary ECs. Thus, mammals produce LPL in cardiomyocytes and rely on GPIHBP1 to transport the LPL into capillaries, whereas lower vertebrates produce LPL directly in capillary ECs, rendering an LPL transporter unnecessary.

APOA5 deficiency causes hypertriglyceridemia by reducing amounts of lipoprotein lipase in capillaries.

Apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia in mice and humans. For years, the cause remained a mystery, but the mechanisms have now come into focus. Here, we review progress in defining APOA5's function in plasma triglyceride metabolism. Biochemical studies revealed that APOA5 binds to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppresses its ability to inhibit the activity of lipoprotein lipase (LPL). Thus, APOA5 deficiency is accompanied by increased ANGPTL3/8 activity and lower levels of LPL activity. APOA5 deficiency also reduces amounts of LPL in capillaries of oxidative tissues (e.g., heart, brown adipose tissue). Cell culture experiments revealed the likely explanation: ANGPTL3/8 detaches LPL from its binding sites on the surface of cells, and that effect is blocked by APOA5. Both the low intracapillary LPL levels and the high plasma triglyceride levels in Apoa5-/- mice are normalized by recombinant APOA5. Carboxyl-terminal sequences in APOA5 are crucial for its function; a mutant APOA5 lacking 40-carboxyl-terminal residues cannot bind to ANGPTL3/8 and lacks the ability to change intracapillary LPL levels or plasma triglyceride levels in Apoa5-/- mice. Also, an antibody against the last 26 amino acids of APOA5 reduces intracapillary LPL levels and increases plasma triglyceride levels in wild-type mice. An inhibitory ANGPTL3/8-specific antibody functions as an APOA5-mimetic reagent, increasing intracapillary LPL levels and lowering plasma triglyceride levels in both Apoa5-/- and wild-type mice. That antibody is a potentially attractive strategy for treating elevated plasma lipid levels in human patients.

Carboxyl-terminal sequences in APOA5 are important for suppressing ANGPTL3/8 activity.

Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.

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.

CL-11 circulates in serum as functionally distinct isoforms.

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway capable of interacting with collectin-10 (CL-10) and the MASPs to activate the complement cascade. Alternative splicing of the COLEC11 gene gives rise to two different isoforms found in serum (A and D). These isoforms vary in the length of their collagen-like region, which is involved in the stabilization of the trimeric subunit and the interaction with the MASPs. Here we aim at elucidating the biological differences of naturally occurring CL-11 isoforms A and D. We produced recombinant CL-11 as independent isoforms (CL-11A and CL-11D) and together with CL-10 (CL-10/11A, CL-10/11D). Both CL-11 isoforms associated with CL-10, but CL-11D did so to a lesser extent. CL-10/11 heterocomplexes were composed of trimeric subunits of CL-10 and CL-11, as opposed to CL-10 and CL-11 homotrimers. Heterocomplexes were more stable and migrated with higher apparent molecular weights. Immunoprecipitation of serum CL-11 and subsequent mass spectrometry analysis confirmed that native CL-11 circulates in the form of CL-10/11 heterocomplexes that associate with MASP-1, and MASP-3, but not necessarily MASP-2. Despite a shorter collagen region, CL-11D was capable to bind to the MASPs, suggesting that the missing exon 4 is not required for MASP association CL-11D had a reduced ligand binding compared to full-length CL-11A. Based on its reduced ability to oligomerize, form CL-10/11 heterocomplexes, and bind to ligands, we hypothesize that CL-11D may have a limited complement activation potential compared to full-length CL-11A.

Targeted imaging of uPAR expression in vivo with cyclic AE105 variants.

A comprehensive literature reports on the correlation between elevated levels of urokinase-type plasminogen activator receptor (uPAR) and the severity of diseases with chronic inflammation including solid cancers. Molecular imaging is widely used as a non-invasive method to locate disease dissemination via full body scans and to stratify patients for targeted treatment. To date, the only imaging probe targeting uPAR that has reached clinical phase-II testing relies on a high-affinity 9-mer peptide (AE105), and several studies by positron emission tomography (PET) scanning or near-infra red (NIR) fluorescence imaging have validated its utility and specificity in vivo. While our previous studies focused on applying various reporter groups, the current study aims to improve uPAR-targeting properties of AE105. We successfully stabilized the small uPAR-targeting core of AE105 by constraining its conformational landscape by disulfide-mediated cyclization. Importantly, this modification mitigated the penalty on uPAR-affinity typically observed after conjugation to macrocyclic chelators. Cyclization did not impair tumor targeting efficiency of AE105 in vivo as assessed by PET imaging and a trend towards increased tracer uptake was observed. In future studies, we predict that this knowledge will aid development of new fluorescent AE105 derivatives with a view to optical imaging of uPAR to assist precision guided cancer surgery.

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.

Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase.

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPL's α/ß-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPL's active site.

Intracapillary LPL levels in brown adipose tissue, visualized with an antibody-based approach, are regulated by ANGPTL4 at thermoneutral temperatures.

Lipoprotein lipase (LPL) is secreted into the interstitial spaces by parenchymal cells and then transported into capillaries by GPIHBP1. LPL carries out the lipolytic processing of triglyceride (TG)-rich lipoproteins (TRLs), but the tissue-specific regulation of LPL is incompletely understood. Plasma levels of TG hydrolase activity after heparin injection are often used to draw inferences about intravascular LPL levels, but the validity of these inferences is unclear. Moreover, plasma TG hydrolase activity levels are not helpful for understanding LPL regulation in specific tissues. Here, we sought to elucidate LPL regulation under thermoneutral conditions (30 °C). To pursue this objective, we developed an antibody-based method to quantify (in a direct fashion) LPL levels inside capillaries. At 30 °C, intracapillary LPL levels fell sharply in brown adipose tissue (BAT) but not heart. The reduced intracapillary LPL levels were accompanied by reduced margination of TRLs along capillaries. ANGPTL4 expression in BAT increased fourfold at 30 °C, suggesting a potential explanation for the lower intracapillary LPL levels. Consistent with that idea, Angptl4 deficiency normalized both LPL levels and TRL margination in BAT at 30 °C. In Gpihbp1-/- mice housed at 30 °C, we observed an ANGPTL4-dependent decrease in LPL levels within the interstitial spaces of BAT, providing in vivo proof that ANGPTL4 regulates LPL levels before LPL transport into capillaries. In conclusion, our studies have illuminated intracapillary LPL regulation under thermoneutral conditions. Our approaches will be useful for defining the impact of genetic variation and metabolic disease on intracapillary LPL levels and TRL processing.

A protein of capillary endothelial cells, GPIHBP1, is crucial for plasma triglyceride metabolism.

GPIHBP1, a protein of capillary endothelial cells (ECs), is a crucial partner for lipoprotein lipase (LPL) in the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1, which contains a three-fingered cysteine-rich LU (Ly6/uPAR) domain and an intrinsically disordered acidic domain (AD), captures LPL from within the interstitial spaces (where it is secreted by parenchymal cells) and shuttles it across ECs to the capillary lumen. Without GPIHBP1, LPL remains stranded within the interstitial spaces, causing severe hypertriglyceridemia (chylomicronemia). Biophysical studies revealed that GPIHBP1 stabilizes LPL structure and preserves LPL activity. That discovery was the key to crystallizing the GPIHBP1-LPL complex. The crystal structure revealed that GPIHBP1's LU domain binds, largely by hydrophobic contacts, to LPL's C-terminal lipid-binding domain and that the AD is positioned to project across and interact, by electrostatic forces, with a large basic patch spanning LPL's lipid-binding and catalytic domains. We uncovered three functions for GPIHBP1's AD. First, it accelerates the kinetics of LPL binding. Second, it preserves LPL activity by inhibiting unfolding of LPL's catalytic domain. Third, by sheathing LPL's basic patch, the AD makes it possible for LPL to move across ECs to the capillary lumen. Without the AD, GPIHBP1-bound LPL is trapped by persistent interactions between LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the abluminal surface of ECs. The AD interrupts the HSPG interactions, freeing LPL-GPIHBP1 complexes to move across ECs to the capillary lumen. GPIHBP1 is medically important; GPIHBP1 mutations cause lifelong chylomicronemia, and GPIHBP1 autoantibodies cause some acquired cases of chylomicronemia.

Electrostatic sheathing of lipoprotein lipase is essential for its movement across capillary endothelial cells.

GPIHBP1, an endothelial cell (EC) protein, captures lipoprotein lipase (LPL) within the interstitial spaces (where it is secreted by myocytes and adipocytes) and transports it across ECs to its site of action in the capillary lumen. GPIHBP1's 3-fingered LU domain is required for LPL binding, but the function of its acidic domain (AD) has remained unclear. We created mutant mice lacking the AD and found severe hypertriglyceridemia. As expected, the mutant GPIHBP1 retained the capacity to bind LPL. Unexpectedly, however, most of the GPIHBP1 and LPL in the mutant mice was located on the abluminal surface of ECs (explaining the hypertriglyceridemia). The GPIHBP1-bound LPL was trapped on the abluminal surface of ECs by electrostatic interactions between the large basic patch on the surface of LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the surface of ECs. GPIHBP1 trafficking across ECs in the mutant mice was normalized by disrupting LPL-HSPG electrostatic interactions with either heparin or an AD peptide. Thus, GPIHBP1's AD plays a crucial function in plasma triglyceride metabolism; it sheathes LPL's basic patch on the abluminal surface of ECs, thereby preventing LPL-HSPG interactions and freeing GPIHBP1-LPL complexes to move across ECs to the capillary lumen.

ANGPTL4: a new mode in the regulation of intravascular lipolysis.

PURPOSE OF REVIEW: Lipoprotein lipase (LPL) is the rate-limiting enzyme for intravascular processing of circulating triglyceride-rich lipoproteins (TRLs). One emerging strategy for therapeutic lowering of plasma triglyceride levels aims at increasing the longevity of LPL activity by attenuating its inhibition from angiopoietin-like proteins (ANGPTL) 3, 4 and 8. This mini-review focuses on recent insights into the molecular mechanisms underpinning the regulation of LPL activity in the intravascular unit by ANGPTLs with special emphasis on ANGPTL4. RECENT FINDINGS: Our knowledge on the molecular interplays between LPL, its endothelial transporter GPIHBP1, and its inhibitor(s) ANGPTL4, ANGPTL3 and ANGPTL8 have advanced considerably in the last 2 years and provides an outlined on how these proteins regulate the activity and compartmentalization of LPL. A decisive determinant instigating this control is the inherent protein instability of LPL at normal body temperature, a property that is reciprocally impacted by the binding of GPIHBP1 and ANGPTLs. Additional layers in this complex LPL regulation is provided by the different modulation of ANGPTL4 and ANGPTL3 activities by ANGPTL8 and the inhibition of ANGPTL3/8 complexes by apolipoprotein A5 (APOA5). SUMMARY: Posttranslational regulation of LPL activity in the intravascular space is essential for the differential partitioning of TRLs across tissues and their lipolytic processing in response to nutritional cues.

The Urokinase Receptor (uPAR) as a "Trojan Horse" in Targeted Cancer Therapy: Challenges and Opportunities.

One of the largest challenges to the implementation of precision oncology is identifying and validating selective tumor-driving targets to enhance the therapeutic efficacy while limiting off-target toxicity. In this context, the urokinase-type plasminogen activator receptor (uPAR) has progressively emerged as a promising therapeutic target in the management of aggressive malignancies. By focalizing the plasminogen activation cascade and subsequent extracellular proteolysis on the cell surface of migrating cells, uPAR endows malignant cells with a high proteolytic and migratory potential to dissolve the restraining extracellular matrix (ECM) barriers and metastasize to distant sites. uPAR is also assumed to choreograph multiple other neoplastic stages via a complex molecular interplay with distinct cancer-associated signaling pathways. Accordingly, high uPAR expression is observed in virtually all human cancers and is frequently associated with poor patient prognosis and survival. The promising therapeutic potential unveiled by the pleiotropic nature of this receptor has prompted the development of distinct targeted intervention strategies. The present review will focus on recently emerged cytotoxic approaches emphasizing the novel technologies and related limits hindering their application in the clinical setting. Finally, future research directions and emerging opportunities in the field of uPAR targeting are also discussed.

Expression and one-step purification of active LPL contemplated by biophysical considerations.

LPL is essential for intravascular lipid metabolism and is of high medical relevance. Since LPL is notoriously unstable, there is an unmet need for a robust expression system producing high quantities of active and pure recombinant human LPL (hLPL). We showed previously that bovine LPL purified from milk is unstable at body temperature (Tm is 34.8°C), but in the presence of the endothelial transporter glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), LPL is stabile (Tm increases to 57.6°C). Building on this information, we now designed an expression system for hLPL using Drosophila Schneider 2 cells grown in suspension at high cell density and at an advantageous temperature of 25°C. We cotransfected Schneider 2 cells with hLPL, lipase maturation factor 1, and soluble GPIHBP1 to provide an efficient chaperoning and stabilization of LPL in all compartments during synthesis and after secretion into the conditioned medium. For LPL purification, we used heparin-Sepharose affinity chromatography, which disrupted LPL-GPIHBP1 complexes causing GPIHBP1 to elute with the flow-through of the conditioned media. This one-step purification procedure yielded high quantities of pure and active LPL (4-28 mg/l). Purification of several hLPL variants (furin cleavage-resistant mutant R297A, active-site mutant S132A, and lipid-binding-deficient mutant W390A-W393A-W394A) as well as murine LPL underscores the versatility and robustness of this protocol. Notably, we were able to produce and purify LPL containing the cognate furin cleavage site. This method provides an efficient and cost-effective approach to produce large quantities of LPL for biophysical and large-scale drug discovery studies.

Targeting the Urokinase-Type Plasminogen Activator Receptor (uPAR) in Human Diseases With a View to Non-invasive Imaging and Therapeutic Intervention.

The interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) focalizes plasminogen activation to cell surfaces, thereby regulating extravascular fibrinolysis, cell adhesion, and migration. uPAR belongs to the Ly6/uPAR (LU) gene superfamily and the high-affinity binding site for uPA is assembled by a dynamic association of its three consecutive LU domains. In most human solid cancers, uPAR is expressed at the invasive areas of the tumor-stromal microenvironment. High levels of uPAR in resected tumors or shed to the plasma of cancer patients are robustly associated with poor prognosis and increased risk of relapse and metastasis. Over the years, a plethora of different strategies to inhibit uPA and uPAR function have been designed and investigated in vitro and in vivo in mouse models, but so far none have been implemented in the clinics. In recent years, uPAR-targeting with the intent of cytotoxic eradication of uPAR-expressing cells have nonetheless gained increasing momentum. Another avenue that is currently being explored is non-invasive imaging with specific uPAR-targeted reporter-molecules containing positron emitting radionuclides or near-infrared (NIR) florescence probes with the overarching aim of being able to: (i) localize disease dissemination using positron emission tomography (PET) and (ii) assist fluorescence guided surgery using optical imaging. In this review, we will discuss these advancements with special emphasis on applications using a small 9-mer peptide antagonist that targets uPAR with high affinity.

GPIHBP1 and ANGPTL4 Utilize Protein Disorder to Orchestrate Order in Plasma Triglyceride Metabolism and Regulate Compartmentalization of LPL Activity.

Intravascular processing of triglyceride-rich lipoproteins (TRLs) is crucial for delivery of dietary lipids fueling energy metabolism in heart and skeletal muscle and for storage in white adipose tissue. During the last decade, mechanisms underlying focal lipolytic processing of TRLs along the luminal surface of capillaries have been clarified by fresh insights into the functions of lipoprotein lipase (LPL); LPL's dedicated transporter protein, glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1); and its endogenous inhibitors, angiopoietin-like (ANGPTL) proteins 3, 4, and 8. Key discoveries in LPL biology include solving the crystal structure of LPL, showing LPL is catalytically active as a monomer rather than as a homodimer, and that the borderline stability of LPL's hydrolase domain is crucial for the regulation of LPL activity. Another key discovery was understanding how ANGPTL4 regulates LPL activity. The binding of ANGPTL4 to LPL sequences adjacent to the catalytic cavity triggers cooperative and sequential unfolding of LPL's hydrolase domain resulting in irreversible collapse of the catalytic cavity and loss of LPL activity. Recent studies have highlighted the importance of the ANGPTL3-ANGPTL8 complex for endocrine regulation of LPL activity in oxidative organs (e.g., heart, skeletal muscle, brown adipose tissue), but the molecular mechanisms have not been fully defined. New insights have also been gained into LPL-GPIHBP1 interactions and how GPIHBP1 moves LPL to its site of action in the capillary lumen. GPIHBP1 is an atypical member of the LU (Ly6/uPAR) domain protein superfamily, containing an intrinsically disordered and highly acidic N-terminal extension and a disulfide bond-rich three-fingered LU domain. Both the disordered acidic domain and the folded LU domain are crucial for the stability and transport of LPL, and for modulating its susceptibility to ANGPTL4-mediated unfolding. This review focuses on recent advances in the biology and biochemistry of crucial proteins for intravascular lipolysis.

The Importance of Lipoprotein Lipase Regulation in Atherosclerosis.

Lipoprotein lipase (LPL) plays a major role in the lipid homeostasis mainly by mediating the intravascular lipolysis of triglyceride rich lipoproteins. Impaired LPL activity leads to the accumulation of chylomicrons and very low-density lipoproteins (VLDL) in plasma, resulting in hypertriglyceridemia. While low-density lipoprotein cholesterol (LDL-C) is recognized as a primary risk factor for atherosclerosis, hypertriglyceridemia has been shown to be an independent risk factor for cardiovascular disease (CVD) and a residual risk factor in atherosclerosis development. In this review, we focus on the lipolysis machinery and discuss the potential role of triglycerides, remnant particles, and lipolysis mediators in the onset and progression of atherosclerotic cardiovascular disease (ASCVD). This review details a number of important factors involved in the maturation and transportation of LPL to the capillaries, where the triglycerides are hydrolyzed, generating remnant lipoproteins. Moreover, LPL and other factors involved in intravascular lipolysis are also reported to impact the clearance of remnant lipoproteins from plasma and promote lipoprotein retention in capillaries. Apolipoproteins (Apo) and angiopoietin-like proteins (ANGPTLs) play a crucial role in regulating LPL activity and recent insights into LPL regulation may elucidate new pharmacological means to address the challenge of hypertriglyceridemia in atherosclerosis development.

IRDye800CW labeled uPAR-targeting peptide for fluorescence-guided glioblastoma surgery: Preclinical studies in orthotopic xenografts.

Glioblastoma (GBM) is a devastating cancer with basically no curative treatment. Even with aggressive treatment, the median survival is disappointing 14 months. Surgery remains the key treatment and the postoperative survival is determined by the extent of resection. Unfortunately, the invasive growth with irregular infiltrating margins complicates an optimal surgical resection. Precise intraoperative tumor visualization is therefore highly needed and molecular targeted near-infrared (NIR) fluorescence imaging potentially constitutes such a tool. The urokinase-type Plasminogen Activator Receptor (uPAR) is expressed in most solid cancers primarily at the invading front and the adjacent activated peritumoral stroma making it an attractive target for targeted fluorescence imaging. The purpose of this study was to develop and evaluate a new uPAR-targeted optical probe, IRDye800CW-AE344, for fluorescence guided surgery (FGS). Methods: In the present study we characterized the fluorescent probe with regard to binding affinity, optical properties, and plasma stability. Further, in vivo imaging characterization was performed in nude mice with orthotopic human patient derived glioblastoma xenografts, and we performed head-to-head comparison within FGS between our probe and the traditional procedure using 5-ALA. Finally, the blood-brain barrier (BBB) penetration was characterized in a 3D BBB spheroid model. Results: The probe effectively visualized GBM in vivo with a tumor-to-background ratio (TBR) above 4.5 between 1 to 12 h post injection and could be used for FGS of orthotopic human glioblastoma xenografts in mice where it was superior to 5-ALA. The probe showed a favorable safety profile with no evidence of any acute toxicity. Finally, the 3D BBB model showed uptake of the probe into the spheroids indicating that the probe crosses the BBB. Conclusion: IRDye800CW-AE344 is a promising uPAR-targeted optical probe for FGS and a candidate for translation into human use.