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Visualization of the binding, endocytosis, and transcytosis of low- density lipoprotein in the arterial endothelium in situ
Journal of Cell Biology (JCB)
Abstract
We investigated the interaction and transport of low-density lipoprotein (LDL) through the arterial endothelium in rat aorta and coronary artery, by perfusing in situ native, untagged human, and rat LDL. The latter was rendered electron-opaque after it interacted with the endothelial cell and was subsequently fixed within tissue. We achieved LDL electron-opacity by an improved fixation procedure using 3,3'-diaminobenzidine, and mordanting with tannic acid. The unequivocal identification of LDL was implemented by reacting immunocytochemically the perfused LDL with anti LDL-horseradish peroxidase conjugate. Results indicate that LDL is taken up and internalized through two parallel compartmented routes. (a) A relatively small amount of LDL is taken up by endocytosis via: (i) a receptor-mediated process (adsorptive endocytosis) that involved coated pits/vesicles, and endosomes, and, probably, (ii) a receptor-independent process (fluid endocytosis) carried out by a fraction of plasmalemmal vesicles. Both mechanisms bringing LDL to lysosomes supply cholesterol to the endothelial cell itself. (b) Most circulating LDL is transported across the endothelial cell by transcytosis via plasmalemmal vesicles which deliver LDL to the other cells of the vessel wall. Endocytosis is not enhanced by increasing LDL concentration, but the receptor-mediated internalization decreases at low temperature. Transcytosis is less modified by low temperature but is remarkably augmented at high concentration of LDL. While the endocytosis of homologous (rat) LDL is markedly more pronounced than that of heterologous (human) LDL, both types of LDL are similarly transported by transcytosis. These results indicate that the arterial endothelium possesses a dual mechanism for handling circulating LDL: by a high affinity process, endocytosis secures the endothelial cells' need for cholesterol; by a low-affinity nonsaturable uptake process, transcytosis supplies cholesterol to the other cells of the vascular wall, and can monitor an excessive accumulation of plasma LDL. Since in most of our experiments we used LDL concentrations above those found in normal rats, we presume that at low LDL concentrations saturable high-affinity uptake would be enhanced in relation to nonsaturable pathways.
... Indeed, Cav-1 may affect levels of cholesterol at various levels. Firstly, Cav-1 may affect cholesterol homeostasis by mediating LDLs endocytosis: once internalized by endothelial cells, LDL particles can be used to ensure cellular cholesterol requirements by releasing free cholesterol through the lysosomal degradation of LDLs [33]. When intracellular cholesterol levels increase, Cav-1 mediates the transport of cholesterol from the endoplasmic reticulum to the cell surface [34,35]. ...
... It is well known that, once internalized by endothelial cells, LDL particles can follow two pathways: endocytosis to respond to cellular cholesterol requirements or transcytosis across endothelial cells [33]. Beyond internalization, Cav-1 also contributes to the transcytosis of LDLs in the sub-endothelial space, where they accumulate starting the formation of atheroma. ...
... One of the first studies regarding LDLR saturation reported that the increased concentration of LDL did not result in enhanced endocytosis [33]. In order to quantify the activity of the LDL receptor, in 1986 Meddings et al. provided an equation that defines the relationship between the production of LDLs, numbers of LDLRs, and LDLs concentration in plasma [58]. ...
Caveolae are 50–100 nm cell surface plasma membrane invaginations observed in terminally differentiated cells. They are characterized by the presence of the protein marker caveolin-1. Caveolae and caveolin-1 are involved in regulating several signal transduction pathways and processes. It is well recognized that they have a central role as regulators of atherosclerosis. Caveolin-1 and caveolae are present in most of the cells involved in the development of atherosclerosis, including endothelial cells, macrophages, and smooth muscle cells, with evidence of either pro- or anti-atherogenic functions depending on the cell type examined. Here, we focused on the role of caveolin-1 in the regulation of the LDLs’ fate in endothelial cells.
... Since macrophages were not involved in the antibody transfer, we next focused on the potential role of LECs in the uptake of antibodies. Certain macro molecules can be shuttled from one surface of the cell to the other by transcellular vesicular transport, i.e., transcytosis (15,(25)(26)(27)(28)(29)(30). We found that the extremely attenuated (often <200 nm in thick ness) cytoplasmic projections of sinusoidal LECs are very rich in heterogeneous cytoplasmic vesicles resembling flaskshaped cav eolae, round intracytoplasmic vesicles, and elongated tubulovacu olar structures ( Figure 7A). ...
... To mechanistically study the endocytosis and transcytosis of antibodies in the floor LECs, we functionally interfered with these pathways using chemical inhibitors and, when available, genedeficient mice. Transcytosis in BECs is largely mediated by caveolae (15,(26)(27)(28)(29). We therefore studied antibody transcytosis in caveolin1-deficient mice (Cav1 -/mice), which lack all caveolae and hence caveolindependent endocytosis and transcytosis (32). ...
Lymph nodes (LNs) filter lymph to mount effective immune responses. Small soluble lymph-borne molecules from the periphery enter the draining LNs via a reticular conduit system. Intact antibodies and other larger molecules, in contrast, are physically unable to enter the conduits, and they are thought to be transported to the LNs only within migratory DCs after proteolytic degradation. Here, we discovered that lymph-borne antibodies and other large biomolecules enter within seconds into the parenchyma of the draining LN in an intact form. Mechanistically, we found that the uptake of large molecules is a receptor-independent, fluid-phase process that takes place by dynamin-dependent vesicular transcytosis through the lymphatic endothelial cells in the subcapsular sinus of the LN. Physiologically, this pathway mediates a very fast transfer of large protein antigens from the periphery to LN-resident DCs and macrophages. We show that exploitation of the transcytosis system allows enhanced whole-organ imaging and spatially controlled lymphocyte activation by s.c. administered antibodies in vivo. Transcytosis through the floor of the subcapsular sinus thus represents what we believe to be a new physiological and targetable mode of lymph filtering.
... LDL particles are approximately 20-30 nm in diameter, much larger than the adjacent intercellular gap in the endothelium (3-6 nm), and the LDL receptor (LDLR)-mediated pathway is downregulated upon high LDL concentrations. Therefore, LDL particle transport in the endothelium is largely LDLR-independent and requires caveolae-mediated transcytosis (Fig. 2) [14,91]. Autophagic regulation of Cav1 can directly affect lipoprotein transcytosis via Cav1 degradation, and Cav1 regulation of autophagy can indirectly affect AS by modulating inflammation and endothelial cell function. ...
Atherosclerosis (AS) is a chronic vascular disease characterized by lipid accumulation and the activation of the inflammatory response; it remains the leading nation-wide cause of death. Early in the progression of AS, stimulation by pro-inflammatory agonists (TNF-α, LPS, and others), oxidized lipoproteins (ox-LDL), and biomechanical stimuli (low shear stress) lead to endothelial cell activation and dysfunction. Consequently, it is crucial to investigate how endothelial cells respond to different stressors and ways to alter endothelial cell activation in AS development, as they are the earliest cells to respond. Caveolin-1 (Cav1) is a 21-24-kDa membrane protein located in caveolae and highly expressed in endothelial cells, which plays a vital role in regulating lipid transport, inflammatory responses, and various cellular signaling pathways and has atherogenic effects. This review summarizes recent studies on the structure and physiological functions of Cav1 and outlines the potential mechanisms it mediates in AS development. Included are the roles of Cav1 in the regulation of endothelial cell autophagy, response to shear stress, modulation of the eNOS/NO axis, and transduction of inflammatory signaling pathways. This review provides a rationale for proposing Cav1 as a novel target for the prevention of AS, as well as new ideas for therapeutic strategies for early AS.
... In addition to receptor-mediated transcytosis, there may be a role for fluid-phase internalization [49], a process by which proteins or macromolecules are endocytosed via nonspecific binding to the apical membrane [50]. The study of LDL transcytosis using electron microscopy suggested both receptor-and non-receptor-mediated endocytosis mechanisms [51]. In cultured coronary artery endothelial cells, the presence of excess unlabeled LDL only inhibited about two-thirds of transcytosis [13••], suggesting a potential contribution from non-receptor-mediated internalization [52]. ...
Purpose of Review: The accumulation of LDL in the arterial intima is an initiating event in atherosclerosis. After decades of controversy, it is now clear that transcytosis of LDL across an intact endothelial monolayer contributes to its intimal deposition. We review recent observations in this field and address the question of whether LDL transcytosis can be manipulated therapeutically.
Recent Findings: The development of a live-cell imaging method for studying transcytosis using total internal reflection fluorescence (TIRF) microscopy has catalyzed recent discoveries. LDL transcytosis is mediated by SR-BI and ALK1. Estrogen down-regulates SR-BI and inhibits LDL transcytosis, while the nuclear structural protein HMGB1 promotes LDL transcytosis. LDL transcytosis by ALK1 is independent of the receptor's kinase activity and is antagonized by BMP9, ALK1's canonical ligand. Inflammation stimulates LDL transcytosis.
Summary: Identifying the function and mechanisms of LDL transcytosis may ultimately permit its therapeutic manipulation.
... For example, the concentration of labelled LDL was often far below physiological levels, which would have favoured high-affinity routes. It is unlikely that a receptor exists for quantum dots, so fluid phase transcytosis seems the most plausible mechanism at least for this tracer, and perhaps also for LDL itself (Vasile et al., 1983). ...
A striking feature of atherosclerosis is its patchy distribution within the vascular system; certain arteries and certain locations within each artery are preferentially affected. Identifying the local risk factors underlying this phenomenon may lead to new therapeutic strategies. The large variation in lesion prevalence in areas of curvature and branching has motivated a search for haemodynamic triggers, particular those related to wall shear stress (WSS). The fact that lesions are rich in blood-derived lipids has motivated studies of local endothelial permeability. However, the location of lesions, the underlying haemodynamic triggers, the role of permeability, the routes by which lipids cross the endothelium, and the mechanisms by which WSS affects permeability have all been areas of controversy. This review presents evidence for and against the current consensus that lesions are triggered by low and/or oscillatory WSS and that this type of shear profile leads to elevated entry of low density lipoprotein (LDL) into the wall via widened intercellular junctions; it also evaluates more recent evidence that lesion location changes with age, that multidirectional shear stress plays a key role, that LDL dominantly crosses the endothelium by transcytosis, and that the link between flow and permeability results from hitherto unrecognised shear-sensitive mediators.
... The fatty streak formation represents a precocious sign of atherosclerosis and depends on the accumulation of lowdensity lipoproteins (LDL) beneath the arterial intima thickness [22,23]. In particular, the glycocalyx of the innermost layer of artery walls regulates LDL transport into the intima, which can cross the endothelium via transcytosis through vesicles [24,25]. Once activated, endothelial cells express on their surface some adhesion molecules, such as the intracellular adhesion molecule -1 and vascular adhesion molecule (VCAM)-1, P-selectin, and E-selectin for the binding of incoming leukocytes to occur [26]. ...
Background:
Foam cells, mainly derived from monocytes-macrophages, contain in their cytoplasm lipid droplets essentially composed of cholesterol. They infiltrate the intima of arteries, contributing to the formation of atherosclerotic plaques.
Pathogenesis:
Foam cells damage the arterial cell wall via the release of proinflammatory cytokines, free radicals, and matrix metalloproteinases, enhancing the plaque size up to its rupture.
Therapy:
A correct dietary regimen seems to be the most appropriate therapeutic approach to minimize obesity, which is associated with the formation of foam cells. At the same time, different types of antioxidants have been evaluated to arrest the formation of foam cells, even if the results are still contradictory. In any case, a combination of antioxidants seems to be more efficient in the prevention of atherosclerosis.
... Caveolae are thought to play a significant role in ensuring transcytosis ( Figure 3) [303,317,318]. Endothelial transcytosis depends on the function of caveolin-1 [319]. ...
Atherosclerosis is one of the most important problems in modern medicine. Its high prevalence and social significance determine the need for a better understanding of the mechanisms of the disease's development and progression. Lipid metabolism and its disorders are one of the key links in the pathogenesis of atherosclerosis. Lipids are involved in many processes, including those related to the mechanoreception of endothelial cells. The multifaceted role of lipids in endothelial mechanobiology and mechanisms of atherogenesis are discussed in this review. Endothelium is involved in ensuring adequate vascular hemodynamics, and changes in blood flow characteristics are detected by endothelial cells and affect their structure and function.
... It has been reported that AP to BL LDL transport occurs in vivo across arterial endothelial cells [99]. The findings from this report suggest that this transport is probably not through LDLR, as lysosome inhibition ( Figure 5) significantly promoted Dil-LDL accumulation with or without TNFɑ. ...
Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFɑ) in modulating the low-density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFɑ promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFɑ could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFɑ-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. The effects of TNFɑ included an LDLR cell surface increase of 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFɑ-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFɑ on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFɑ did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFɑ promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.
... 158 Electron microscopic studies of rat aorta perfused with LDL clearly show its internalization into cellular vesicles and LDL targeting to the basolateral membrane. 159 Support for a specific aortic EC LDL transporter is provided by the atheroprotective effect of Cav-1 deletion in APO E knockout mice, 133 with its rescue in ECs being sufficient to promote lesion progression and LDL infiltration of the aortic wall. 134,160 These findings strongly suggest that cholesterol-carrying lipoproteins cross the aortic endothelium via a transcytotic route. ...
Lipid uptake and metabolism are central to the function of organs such as heart, skeletal muscle, and adipose tissue. Although most heart energy derives from fatty acids (FAs), excess lipid accumulation can cause cardiomyopathy. Similarly, high delivery of cholesterol can initiate coronary artery atherosclerosis. Hearts and arteries—unlike liver and adrenals—have nonfenestrated capillaries and lipid accumulation in both health and disease requires lipid movement from the circulation across the endothelial barrier. This review summarizes recent in vitro and in vivo findings on the importance of endothelial cell receptors and uptake pathways in regulating FAs and cholesterol uptake in normal physiology and cardiovascular disease. We highlight clinical and experimental data on the roles of ECs in lipid supply to tissues, heart, and arterial wall in particular, and how this affects organ metabolism and function. Models of FA uptake into ECs suggest that receptor-mediated uptake predominates at low FA concentrations, such as during fasting, whereas FA uptake during lipolysis of chylomicrons may involve paracellular movement. Similarly, in the setting of an intact arterial endothelial layer, recent and historic data support a role for receptor-mediated processes in the movement of lipoproteins into the subarterial space. We conclude with thoughts on the need to better understand endothelial lipid transfer for fuller comprehension of the pathophysiology of hyperlipidemia, and lipotoxic diseases such as some forms of cardiomyopathy and atherosclerosis.
... The classical LDL receptor (LDLR) pathway, which mediates the uptake of LDLs for internalization and subsequent degradation in the lysosomes, does not operate in transcytosis and, actually, does not explain the accumulation of LDLs in the subendothelium of systemic circulation (Dehouck et al., 1997). In addition, the LDLR-mediated pathway is down-regulated at high concentrations of LDLs, while LDLR-independent pathways are enhanced in conditions of hypercholesterolemia (Vasile, Simionescu, & Simionescu, 1983). Thus, transcytosis in endothelial cells is LDLR-independent and, importantly, requires the presence of caveolae ( Figure 1). ...
Oxidized LDLs (oxLDLs) and oxysterols play a key role in endothelial dysfunction and the development of atherosclerosis. The loss of vascular endothelium function negatively impacts vasomotion, cell growth, adhesiveness and barrier functions. While for some of these disturbances, a reasonable explanation can be provided from a mechanistic standpoint, for many others, the molecular mediators that are involved are unknown. Caveolae, specific plasma membrane domains, have recently emerged as targets and mediators of oxLDL‐induced endothelial dysfunction. Caveolae and their associated protein caveolin‐1 (Cav‐1) are involved in oxLDLs/LDLs transcytosis, mainly through the scavenger receptor class B type 1 (SR‐B1 or SCARB1). In contrast, oxLDLs endocytosis is mediated by the lectin‐like oxidized LDL receptor 1 (LOX‐1), whose activity depends on an intact caveolae system. In addition, LOX‐1 regulates the expression of Cav‐1 and vice versa. On the other hand, oxLDLs may affect cholesterol plasma membrane content/distribution thus influencing caveolae architecture, Cav‐1 localization and the associated signalling. Overall, the evidence indicate that caveolae have both active and passive roles in oxLDL‐induced endothelial cell dysfunction. First, as mediators of lipid uptake and transfer in the subendothelial space and, later, as targets of changes in composition/dynamics of plasma membrane lipids resulting from increased levels of circulating oxLDLs. Gaining a better understanding of how oxLDLs interact with endothelial cells and modulate caveolae‐mediated signalling pathways, leading to endothelial dysfunction, is crucial to find new targets for intervention to tackle atherosclerosis and the related clinical entities.
LINKED ARTICLES
This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc
... Reducing the thickness of the glycocalyx, or decreasing its barrier function would both favour the access of LDL to the endothelial surface, expose endothelial receptors and enhance binding of monocytes, which lead to enhanced accumulation of LDL into the intima [45,46]. Once through the glycocalyx, LDL can cross the endothelium via transcytosis, a process that occurs through vesicles, which transports lipoproteins from the apical to the basolateral aspect of ECs [47]. Such transcytosis of LDL raises the intimal LDL content and also leads to their enhanced persistence in the lesion. ...
Polydatin (PD) is a monocrystalline metabolite from the underground parts of Polygonum cuspidatum Sieb. et Zucc., a member of the Polygonaceae family, which has been traditionally used in Asian countries as both foodstuffs and medicine. PD, also reckoned as pieceid, 3,4′,5-trihydroxystilbene-3-β-D-glucoside, (E)-piceid, (E)-polydatin, and trans-polydatin. It possesses potent biological activities i.e. analgesic, anti-inflammatory, antidiabetic, anticancer, and anti-atherosclerotic properties. The initial part of this report specifically explains distinct sequential mechanisms underlying the initiation and development of atherosclerotic plaques and later part deals with the pharmacological efficacy of PD in the management of major cardiac event i.e. atherosclerotic cardiovascular diseases (ASCVD) via modulation of a set of molecular mechanisms i.e. antioxidant potential, lipid and lipoprotein metabolism including total cholesterol (TC) and low density lipoprotein (LDL) levels, β-hydroxy-β-methyl-glutaryl-CoA reductase (HMG-R) expression and functionality, SIRT signalling, LDL-receptor (LDL-R), LDL oxidation status (Ox-LDL), effects on endothelial cells (ECs), smooth muscle cells (SMCs), macrophage, foam cell formation and plaque stabilization, inflammatory signalling pathways and hypertension. In contrast, one of the major insight into the potential cardioprotective molecular mechanism is the PD-mediated targeting of proprotein convertase subtilisin/kexin type-9 (PCSK-9) and LDL-R pathway, both at transcriptional and protein functional level, which makes it a better alternative therapeutic medicinal candidate to treat hypercholesterolemia, especially for the patients facing inadequate lipid lowering with classical HMG-R inhibitors (statins) and statin intolerance. Finally, to sum up the whole, we concluded that PD may be promoted from alternative to mainstream medicine in targeting risk factors mediated ASCVD.
... The selection of appropriate controls poses further challenges. For example, fluorescently labelled low-density lipoprotein and transferrin can be used as markers for clathrin-mediated endocytosis [205,206], dextran as a fluid phase marker for phagocytosis and for the CLEE/GEEC pathway [193], and LacCer (C5-lactosylceramide) for cholesterol-dependent uptake [133,207]. However, while cholera toxin and SV40 were previously used as markers of caveolae-mediated endocytosis [95], they have been found to enter cells using preferentially other routes and thus should not be used anymore as markers for this pathway [208][209][210]. ...
Nano-sized materials have great potential as drug carriers for nanomedicine applications. Thanks to their size, they can exploit the cellular machinery to enter cells and be trafficked intracellularly, thus they can be used to overcome some of the cellular barriers to drug delivery. Nano-sized drug carriers of very different properties can be prepared, and their surface can be modified by the addition of targeting moieties to recognize specific cells. However, it is still difficult to understand how the material properties affect the subsequent interactions and outcomes at cellular level. As a consequence of this, designing targeted drugs remains a major challenge in drug delivery. Within this context, we discuss the current understanding of the initial steps in the interactions of nano-sized materials with cells in relation to nanomedicine applications. In particular, we focus on the difficult interplay between the initial adhesion of nano-sized materials to the cell surface, the potential recognition by cell receptors, and the subsequent mechanisms cells use to internalize them. The factors affecting these initial events are discussed. Then, we briefly describe the different pathways of endocytosis in cells and illustrate with some examples the challenges in understanding how nanomaterial properties, such as size, charge, and shape, affect the mechanisms cells use for their internalization. Technical difficulties in characterizing these mechanisms are presented. A better understanding of the first interactions of nano-sized materials with cells will help to design nanomedicines with improved targeting.
... In general, the metabolism of extracellular macromolecules in cells is firstly recognized by the corresponding receptor on the cell membrane and dissociated from plasma membrane through a coated vesicle [4,5]; the coated vesicle fuses with early endosomes (EEs) and release the cargos [6]; the cargos are sorted in the endosome, and are then transported to lysosome for degradation [7]. low-density-lipoprotein receptor (LDL-R) system has become recognized as a prototype of the process ...
Endocytosis is an important pathway to regulate the metabolism of low-density lipoprotein (LDL) in cells. At the same time, engineering nanoparticles (ENPs) enter the cell through endocytosis in biomedical applications. Therefore, a crucial question is whether the nanoparticles involved in endocytosis could impact the natural metabolism of LDL in cells. In this study, we fabricated a series of gold nanoparticles (AuNPs) (13.00 ± 0.69 nm) with varied surface charge densities. The internalized AuNPs with high-surface negative-charge densities (HSNCD) significantly reduced LDL uptake in HepG-2, HeLa, and SMMC-7721 cells compared with those cells in control group. Notably, the significant reduction of LDL uptake in cells correlates with the reduction of LDL receptors (LDL-R) on the cell surface, but there is no change in protein and mRNA of LDL-Rs. The cyclic utilization of LDL-R in cells is a crucial pathway to maintain the homoeostasis of LDL uptake. The release of LDL-Rs from LDL/LDL-R complexes in endosomes depended on reduction of the pH in the lumen. AuNPs with HSNCD hampered vacuolar-type H+-ATPase V1 (ATPaseV1) and ATPaseV0 binding on the endosome membrane, blocking protons to enter the endosome by the pump. Hence, fewer freed LDL-Rs were transported into recycling endosomes (REs) to be returned to cell surface for reuse, reducing the LDL uptake of cells by receptor-mediated endocytosis. The restrained LDL-Rs in the LDL/LDL-R complex were degraded in lysosomes.
Atherosclerosis begins with the infiltration of cholesterol-containing lipoproteins into the arterial wall. White blood cell (WBC)-associated inflammation follows. Despite decades of research using genetic and pharmacologic methods to alter WBC function, in humans, the most effective method to prevent the initiation and progression of disease remains low-density lipoprotein (LDL) reduction. However, additional approaches to reducing cardiovascular disease would be useful as residual risk of events continues even with currently effective LDL-reducing treatments. Some of this residual risk may be due to vascular toxicity of triglyceride-rich lipoproteins (TRLs). Another option is that LDL transcytosis continues, albeit at reduced rates due to lower circulating levels of this lipoprotein. This review will address these two topics. The evidence that TRLs promote atherosclerosis and the processes that allow LDL and TRLs to be taken up by endothelial cells leading to their accumulation with the subendothelial space.
Aims
The entry of lipoproteins from blood into the arterial wall is a rate-limiting step in atherosclerosis. It is controversial whether this happens by filtration or regulated transendothelial transport.
Because sphingosine-1-phosphate (S1P) preserves the endothelial barrier, we investigated in vivo and in vitro, whether S1P and its cognate S1P receptor 3 (S1P3) regulate the transendothelial transport of lipoproteins.
Methods and Results
Compared to apoE-haploinsufficient mice (CTRL), apoE-haploinsufficient mice with additional endothelium specific knock-in of S1P3 (S1P3-iECKI) showed decreased transport of LDL and Evan’s Blue but increased transport of HDL from blood into the peritoneal cave. After 30 weeks of high-fat diet feeding, S1P3-iECKI mice had lower levels of non-HDL-cholesterol and less atherosclerosis than CTRL mice. In vitro, stimulation with an S1P3 agonist increased the transport of 125I-HDL but decreased the transport of 125I-LDL through human aortic endothelial cells (HAECs). Conversely, inhibition or knock-down of S1P3 decreased the transport of 125I-HDL but increased the transport of 125I-LDL. Silencing of SCARB1 encoding scavenger receptor B1 (SR-BI) abrogated the stimulation of 125I-HDL transport by the S1P3 agonist. The transendothelial transport of 125I-LDL was decreased by silencing of SCARB1 or ACVLR1 encoding activin-like kinase 1 but not by interference with LDLR. None of the three knock-downs prevented the stimulatory effect of S1P3 inhibition on transendothelial 125I-LDL transport.
Conclusion
S1P3 regulates the transendothelial transport of HDL and LDL oppositely by SR-BI-dependent and SR-BI-independent mechanisms, respectively. This divergence supports a contention that lipoproteins pass the endothelial barrier by specifically regulated mechanisms rather than passive filtration.
Cardiovascular diseases (CVD) are currently the leading cause of death worldwide. Atherosclerosis is the main mechanism underlying CVD and is often symptomless for decades. Over the past decades, cholesterol crystals (CCs) have emerged as a major component and driver of atherogenesis affecting multiple cell types known to play crucial roles during atherogenesis. One important cell type that is often overlooked are endothelial cells (ECs), the most inner lining of all blood vessels. Recent work has suggested that ECs are capable of CC production. However, only little is known about the impact of CCs on EC function and dysfunction in atherogenesis, as most studies focus on other cell types such as macrophages and smooth muscle cells. In this book chapter, we aimed to achieve two main goals. First, we will summarize signaling mechanisms shown to be important for EC-specific signaling pathways activated by CCs. Second, we will elaborate on the mechanisms and pathways we believe should be of focus for future investigations in an endothelial-specific manner to better understand the underlying mechanisms of endothelial derived CCs as well as the impact CCs have on the vasculature. We will focus in particular on the potential importance of lysosomes and their transcription factor EB (TFEB). Lastly, we will describe the need for the development of targeted interventions potentially preventing EC dysfunction in atherosclerosis.
BACKGROUND
Inflammation contributes to the pathogenesis of atherosclerosis. But little is known about the potential benefits of inflammatory cells to atherosclerosis. The aim of this study was to investigate the function of inflammatory cells/endothelium axis and determine whether and how inflammatory cell–derived MYDGF (myeloid-derived growth factor) inhibited endothelial LDL (low-density lipoprotein) transcytosis.
METHODS
In in vivo experiments, both loss- and gain-of-function strategies were used to evaluate the effect of inflammatory cell–derived MYDGF on LDL transcytosis. We generated monocyte/macrophage-targeted MYDGF-null mice on an Ldlr (LDL receptor) −/− background in the loss-of-function strategy and restored the inflammatory cell–derived MYDGF by bone marrow transplantation and inflammatory cell–specific overexpression of MYDGF mice model in the gain-of-function strategy. In in vitro experiments, coculture experiments between primary mouse aortic endothelial cells and macrophages and mouse aortic endothelial cells supplemented with or without recombinant MYDGF were conducted.
RESULTS
Inflammatory cell–derived MYDGF deficiency aggravated endothelial LDL transcytosis, drove LDL uptake by artery wall, and thus exacerbated atherosclerosis in vivo. Inflammatory cell–derived MYDGF restoration by bone marrow transplantation and inflammatory cell MYDGF overexpression alleviated LDL transport across the endothelium, prevented LDL accumulation in the subendothelial space, and subsequently ameliorated atherosclerosis in vivo. Furthermore, in the in vitro study, macrophages isolated from MYDGF +/+ mice and recombinant MYDGF attenuated LDL transcytosis and uptake in mouse aortic endothelial cells. Mechanistically, MYDGF inhibited MAP4K4 (mitogen-activated protein kinase kinase kinase kinase isoform 4) phosphorylation, enhanced activation of Akt (protein kinase B)-1, and diminished the FoxO (forkhead box O) 3a signaling cascade to exert protective effects of MYDGF on LDL transcytosis and atherosclerosis.
CONCLUSIONS
The findings support a role for inflammatory cell–derived MYDGF served as a cross talk factor between inflammatory cells and endothelial cells that inhibits LDL transcytosis across endothelium. MYDGF may become a novel therapeutic drug for atherosclerosis, and the beneficial effects of inflammatory cell in atherosclerosis deserve further attention.
Real-time fluorescence sensing can provide insight into biodynamics. However, few fluorescent tools are available to overcome the tissue scattering and autofluorescence interference for high-contrast in vivo sensing with high spatiotemporal resolution. Here, we develop a molecular-based FRET nanosensor (MFN) capable of producing a dynamic ratiometric NIR-IIb (1500-1700 nm) fluorescence signal under a frequency-modulated dual-wavelength excitation bioimaging system. The MFN provides reliable signals in highly scattering tissues and enables in vivo real-time imaging at micrometer-scale spatial resolution and millisecond-scale temporal resolution. As a proof of concept, a physiological pH-responsive nanosensor (MFNpH) was designed as a nanoreporter for intravital real-time monitoring of the endocytosis dynamics of nanoparticles in the tumor microenvironment. We also show that MFNpH allows the accurate quantification of pH changes in a solid tumor through video-rate ratiometric imaging. Our study offers a powerful approach for noninvasive imaging and sensing of biodynamics with micrometer-scale spatial resolution and millisecond-scale temporal resolution.
The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.
We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo–scanning electron microscopy (SEM), and cryo–focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media–lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media–lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.
The caveolae-mediated transport across polarized epithelial cell barriers has been largely deciphered in the last decades and is considered the second essential intracellular transfer mechanism, after the clathrin-dependent endocytosis. The basic cell biology knowledge was supplemented recently, with the molecular mechanisms beyond caveolae generation implying the key contribution of the lipid-binding proteins (the structural protein Caveolin and the adapter protein Cavin), along with the bulb coat stabilizing molecules PACSIN-2 and Eps15 homology domain protein-2. The current attention is focused also on caveolae architecture (such as the bulb coat, the neck, the membrane funnel inside the bulb, and the associated receptors), and their specific tasks during the intracellular transport of various cargoes. Here, we resume the present understanding of the assembly, detachment, and internalization of caveolae from the plasma membrane lipid raft domains, and give an updated view on transcytosis and endocytosis, the two itineraries of cargoes transport via caveolae. The review adds novel data on the signalling molecules regulating caveolae intracellular routes and on the transport dysregulation in diseases. The therapeutic possibilities offered by exploitation of Caveolin-1 expression and caveolae trafficking, and the urgent issues to be uncovered conclude the review.
Obesity has reached epidemic proportions and is a major contributor to insulin resistance (IR) and type 2 diabetes (T2D). Importantly, IR and T2D substantially increase the risk of cardiovascular (CV) disease. Although there are successful approaches to maintain glycemic control, there continue to be increased CV morbidity and mortality associated with metabolic disease. Therefore, there is an urgent need to understand the cellular and molecular processes that underlie cardiometabolic changes that occur during obesity so that optimal medical therapies can be designed to attenuate or prevent the sequelae of this disease. The vascular endothelium is in constant contact with the circulating milieu; thus, it is not surprising that obesity-driven elevations in lipids, glucose, and proinflammatory mediators induce endothelial dysfunction, vascular inflammation, and vascular remodeling in all segments of the vasculature. As cardiometabolic disease progresses, so do pathological changes in the entire vascular network, which can feed forward to exacerbate disease progression. Recent cellular and molecular data have implicated the vasculature as an initiating and instigating factor in the development of several cardiometabolic diseases. This Review discusses these findings in the context of atherosclerosis, IR and T2D, and heart failure with preserved ejection fraction. In addition, novel strategies to therapeutically target the vasculature to lessen cardiometabolic disease burden are introduced.
Atherosclerosis is characterized by focal accumulations of lipid within the arterial wall, thought to arise from effects of hemodynamic wall shear stress (WSS) on endothelial permeability. Identifying pathways that mediate the effects of shear on permeability could therefore provide new therapeutic opportunities. Here, we consider whether the sphingosine-1-phosphate (S1P) pathway could constitute such a route. We review effects of S1P in endothelial barrier function, the influence of WSS on S1P production and signaling, the results of trials investigating S1P in experimental atherosclerosis in mice, and associations between S1P levels and cardiovascular disease in humans. Although it seems clear that S1P reduces endothelial permeability and responds to WSS, the evidence that it influences atherosclerosis is equivocal. The effects of specifically pro- and anti-atherosclerotic WSS profiles on the S1P pathway require investigation, as do influences of S1P on the vesicular pathways likely to dominate low-density lipoprotein transport across endothelium.
Background and aims
When endothelium is cultured in wells swirled on an orbital shaker, cells at the well centre experience putatively pro-atherogenic flow whereas those near the edge experience putatively atheroprotective flow. Transcellular transport is decreased equally in both regions, consistent with it being reduced by a mediator released from cells in one part of the well and mixed in the swirling medium. Similar effects have been reported for pro-inflammatory changes. Here we identify the mediator and the flow characteristics that stimulate its release.
Methods and results
Medium conditioned by cells swirled at the edge, but not by cells swirled at the centre or cultured under static conditions, significantly reduced transendothelial transport of a low density lipoprotein (LDL)-sized tracer and tumor necrosis factor α (TNF-α)-induced vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression, activation of nuclear factor κB (NF-κB), and monocyte adhesion. An inhibitor of transcytosis similarly decreased tracer transport. Mass spectrometry identified follistatin-like 1 (FSTL1) as a candidate mediator. Cells from the swirled edge produced significantly more FSTL1 than cells from the swirled centre or from static wells. Exogenous FSTL1 reduced transendothelial transport of the LDL-sized tracer and of LDL itself, as well as TNF-α-induced VCAM-1 and ICAM-1 expression. Bone morphogenetic protein (4BMP4) increased transendothelial transport of the LDL-sized tracer and expression of VCAM-1 and ICAM-1; these effects were abolished by FSTL1.
Conclusions
Putatively atheroprotective flow stimulates production of FSTL1 from cultured endothelial cells. FSTL1 reduces transcellular transport of LDL-sized particles and of LDL itself, and inhibits endothelial activation. If this also occurs in vivo, it may account for the atheroprotective nature of such flow.
Purpose of Review
Scavenger receptor class B type I (SR-BI) serves a key role in the reverse cholesterol transport in the liver as the high-affinity receptor for HDL. SR-BI is abundantly expressed in endothelium, and earlier works indicate that the receptor mediates anti-atherogenic actions of HDL. However, more recent studies uncovered novel functions of endothelial SR-BI as a lipoprotein transporter, which regulates transcellular transport process of both LDL and HDL. This brief review focuses on the unique functions of endothelial SR-BI and how they influence atherogenesis.
Recent Findings
Earlier studies indicate that SR-BI facilitates anti-atherogenic actions of HDL through modulation of intracellular signaling to stimulate endothelial nitric oxide synthase. In vivo studies in global SR-BI knockout mice also showed a strong atheroprotective role of the receptor; however, a contribution of endothelial SR-BI to atherosclerosis process in vivo has not been fully appreciated. Recent studies using cultured endothelial cells and in mice with endothelial-specific deletion of the receptor revealed previously unappreciated pro-atherogenic actions of SR-BI, which relates to its ability to deliver LDL into arteries. On the other hand, SR-BI has also been implicated in transport of HDL to the sub-intimal space as a part of reverse cholesterol transport.
Summary
SR-BI mediates internalization and transcellular transport of both HDL and LDL, and the cellular and molecular mechanism of the process has just begun to emerge. Harnessing these dual transport functions of the endothelial SR-BI may provide a novel, effective intervention to atherosclerosis.
Endothelial cell insulin receptors mediate the transcytosis of insulin from luminal to abluminal cell surface. We have investigated the kinetics of insulin receptor translocation by immunoprecipitation of radiolabeled receptors at various times before and after trypsin treatment of intact endothelial cells. Insulin receptors were constitutively internalized with t½ = 18 ± 2 min and were recycled to the cell surface. Insulin stimulated receptor internalization and externalization rates 2.6- and 2.4-fold, respectively. Changes in cell-surface binding of ¹²⁵I-insulin were consistent with the receptor translocation rates observed in surface-labeling experiments. Phorbol myristate acetate (PMA) treatment increased the rate of insulin-stimulated receptor externalization 1.7-fold. PMA treatment increased the constitutive externalization rate 3.5-fold without affecting the constitutive internalization rate, suggesting that recycling might occur via a mobilization of receptors from intracellular sites in a manner independent of internalization rate. Analysis of the intracellular distribution of receptors by ¹²⁵I-insulin binding and immunogold electron microscopy revealed that less than one-third of the total insulin receptor pool resided on the cell surface. In summary, endothelial cell insulin receptors are constitutively recycled, and internalization and externalization rates are increased by receptor occupancy and PMA treatment.
In humans, smooth muscle cells (SMCs) are the main cell type in the artery medial layer, in pre-atherosclerotic diffuse thickening of the intima, and in all stages of atherosclerotic lesion development. SMCs secrete the proteoglycans responsible for the initial binding and retention of atherogenic lipoproteins in the artery intima, with this retention driving foam cell formation and subsequent stages of atherosclerosis. In this chapter we review current knowledge of the extracellular matrix generated by SMCs in medial and intimal arterial layers, their relationship to atherosclerotic lesion development and stabilization, how these findings correlate with mouse models of atherosclerosis, and potential therapies aimed at targeting the SMC matrix-lipoprotein interaction for atherosclerosis prevention.
Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis, and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a non-degradative manner. Here we examine the cross talk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9 mediated internalization of ALK-1, BMP-9 dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9 induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL mediated ALK-1 internalization. Interestingly, BMP-9 mediated ALK-1 internalization strongly reduces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.
The accumulation of low-density lipoproteins (LDL) in the arterial wall plays a pivotal role in the initiation and pathogenesis of atherosclerosis. Conversely, the removal of cholesterol from the intima by cholesterol efflux to high density lipoproteins (HDL) and subsequent reverse cholesterol transport shall confer protection against atherosclerosis. To reach the subendothelial space, both LDL and HDL must cross the intact endothelium. Traditionally, this transit is explained by passive filtration. This dogma has been challenged by the identification of several rate-limiting factors namely scavenger receptor SR-BI, activin like kinase 1, and caveolin-1 for LDL as well as SR-BI, ATP binding cassette transporter G1, and endothelial lipase for HDL. In addition, estradiol, vascular endothelial growth factor, interleukins 6 and 17, purinergic signals, and sphingosine-1-phosphate were found to regulate transendothelial transport of either LDL or HDL. Thorough understanding of transendothelial lipoprotein transport is expected to elucidate new therapeutic targets for the treatment or prevention of atherosclerotic cardiovascular disease and the development of strategies for the local delivery of drugs or diagnostic tracers into diseased tissues including atherosclerotic lesions.
Cells, much like mammals, possess an internal skeleton. This cellular skeleton (called the cytoskeleton) provides structure to cells, enables their movement within the environment and promotes the internalization of extracellular cargo (endocytosis). Many pathogens have devised strategies to hijack the cytoskeleton and other crucial sub-cellular processes for their disease processes. The bacterium Listeria monocytogenes (L. monocytogenes) utilizes the clathrin endocytic machinery to invade cells, and later, the actin polymerization machinery to generate actin-rich comet/rocket tails to move within and amongst host cells. Salmonella enterica serovar Typhimurium (S. Typhimurium) and Shigella flexneri (S. flexneri) generate actin-rich membrane ruffles at the cell surface to enter cells. Once inside, S. Typhimurium occupies a long-lived vacuole, whereas S. flexneri generates comet/rocket tails. Enteropathogenic Escherichia coli (EPEC) on the other hand remain extracellular and co-opt clathrin and actin to form motile pedestals directly beneath the site of bacterial adherence. In this thesis, I explored the involvement of several host actin- and/or endocytic-associated proteins during bacterial infections and simultaneously used these infections to gain insight into novel roles of the proteins studied. In chapter 2, I discovered that L. monocytogenes co-opts the actin-associated protein palladin during its entry and intracellular motility. Importantly, I revealed that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility. In chapter 3, I uncovered that the internalization strategy used by L. monocytogenes to transfer between host cells exploits caveolin-mediated endocytosis. In chapter 4, I investigated the host enzyme cyclophilin A (CypA) and found that it is crucial for maintaining the structural integrity of L. monocytogenes membrane protrusions generated during bacterial dissemination events. In chapter 5, I determined that CypA restricts S. Typhimurium invasion but is dispensable for EPEC pedestal formation. Finally, in chapters 6 and 7, I examined the receptor of CypA, CD147, and found that this membrane protein, like CypA, is crucial for the proper formation and function of L. monocytogenes membrane protrusions. In conclusion, my research has 2 major implications: 1) I have uncovered new insight into the mechanisms behind how actin-hijacking pathogens cause disease and 2) I have demonstrated novel cellular functions for host actin-associated proteins.
Blood-brain barrier (BBB) disruption is one of the critical mechanisms of brain injury induced by subarachnoid hemorrhage (SAH). Past studies have often focused on the tight junctions of endothelial cells. However, low transcellular transport levels also play an important role in the normal functioning of the BBB. Major facilitator superfamily domain-containing 2a (Mfsd2a) has been demonstrated to be essential for the maintenance of the normal BBB. Our present study aimed to explore the roles and mechanisms of Mfsd2a in BBB disruption after SAH. In this study, a prechiasmatic cistern single-injection model was used to produce experimental SAH in Sprague-Dawley rats. Specific small-interfering RNA and plasmids were used to downregulate and upregulate the expression of Mfsd2a prior to assessments in our SAH model. Omega-3 fatty acid deficiency diet was used to reduce DHA in rat brain. The expression level of Mfsd2a decreased significantly after SAH and reached its lowest level at 72 h post-SAH, which then gradually recovered. At 72 h after SAH, BBB function was disrupted; upregulation of Mfsd2a reversed this damage, whereas downregulation of Mfsd2a exacerbated this damage. These effects were primarily mediated through transcellular transport, especially for changes in caveolae compared to those of tight junctions. After stopping the supply of omega-3 fatty acids, the effect of Mfsd2a on inhibition of caveolae and protection of the blood-brain barrier was eliminated. Taken together, Mfsd2a inhibits caveolae-based transcellular transport by transporting omega-3 fatty acids to protect the BBB after SAH.
Human microvascular ECs from the neonatal foreskin of two donors purchased from one distributor were used in an angiogenesis assay under the same culture conditions. Different angiogenic potency was apparent in these two batches (ECang and ECnon-ang). During the cultivation period of three weeks, ECang ran through all stages of angiogenesis starting from proliferation to migration up to the formation of three-dimensional capillary-like structures. Despite of expression of endothelial markers, ECnon-ang showed excessive intracellular storage of lipids in form of multilamellar bodies and decreased angiogenic potency in contrast to its counterpart, ECang. Results indicate that lipid metabolism differs in ECang versus ECnon-ang. This study points up that these differences are based on the different donors and presents a novel and valuable model for the study of mechanisms of atherosclerosis in endothelial cells in vitro.
Purpose of review:
High-fat diets contribute to hyperlipidemia and dysregulated metabolism underlying insulin resistant states and cardiovascular diseases. Neointimal hyperplasia is a significant resulting morbidity. Increased fatty acid (FA) levels lead to dysfunctional endothelium, defined as activated, proinflammatory and prothrombotic. The purpose of this review is to assess the recent literature on the emerging concept that uptake of FA into many tissues is regulated at the endothelial level, and this in turn contributes to endothelial dysfunction, an initiating factor in insulin resistant states, atherosclerosis and neointimal hyperplasia.
Recent findings:
Studies support the role of endothelial FA uptake proteins as an additional level of regulation in tissue FA uptake. These proteins include CD36, FA transport proteins, FA-binding proteins and caveolin-1. In many cases, inappropriate expression of these proteins can result in a change in FA and glucose uptake, storage and utilization. Accumulation of plasma FA is one mechanism by which alterations in expression of FA uptake proteins can lead to endothelial dysfunction; changes in tissue substrate metabolism leading to inflammation are also implicated.
Summary:
Identification of the critical players and regulators can lead to therapeutic targeting to reduce endothelial dysfunction and sequela such as insulin resistance and neointimal hyperplasia.
Aim: In this work we illustrate limits and challenges associated with the use of pharmacological inhibitors to study how nanomedicines enter cells and show how such limits can be overcome. Materials & methods: We selected a panel of six common pharmacological inhibitors and a model nanoparticle-cell system. We tested eventual toxicity by measuring cell viability. We confirmed drug efficacy by measuring the uptake of control markers for the pathways involved by flow cytometry and fluorescence microscopy. Results & conclusion: We show how to optimize the use of pharmacological inhibitors and interpret the results generated. Furthermore, we demonstrate that some inhibitors cannot be used for nanomedicine studies because they lose their efficacy when serum is added, as required for nanoparticle exposure to cells.
The entry of remnants of cholesterol-rich lipoproteins, such as low-density lipoprotein (LDL), from the blood stream into the intima of large arteries initiates and then perpetuates atherosclerosis. A study published in Nature sheds new light on this important process by identifying scavenger receptor BI (SR-BI) as a major receptor that mediates LDL delivery across the endothelium into arteries.
Elevated plasma low density lipoprotein (LDL) is an established risk factor for cardiovascular disease. In addition to being able to cross the endothelial barrier to become accumulated in subendothelial space and thereby initiate atherosclerosis, LDL may exert a direct effect on vascular endothelial cells through activation of LDL receptor and its downstream signaling. Whether LDL can modulate the signaling for autophagy in endothelial cells is not clear. The present study firstly demonstrated that LDL can suppress endothelial autophagy through activation of the PI3K/Akt/mTOR signaling pathway and can promote glucose uptake by translocating glucose transporter 1 (GLUT1) from cytoplasm to cell membrane, actions similar to those of insulin. A co-immunoprecipitation assay found that LDL receptor (LDLR) and insulin receptor (IR) formed a complex in HUVECs. Knock down of the insulin receptor by small interfering RNA blocked the suppression of autophagy by LDL, as well as the signaling pathway involved. We conclude that LDL may mimic the action of insulin in endothelial cells, which might partly explain the increased incidence of diabetes in patients receiving some LDL-lowering therapy.
Atherosclerosis is a lipid disease characterized by accumulation of low density lipoprotein (LDL) in the artery wall. The transport of LDL across the endothelium of coronary artery is an initiating event of atherosclerosis, whose mechanism remains poorly understood. In the last decade, it has been shown that in caveolin-1 (Cav-1) deficient mice, LDL infiltration in aorta wall is decreased and CD36 expression in aortas is down-regulated, leading to regression of atherosclerotic lesions. In the present study, we show that native LDL endocytosis is decreased in endothelial cells deficient in Cav-1 or CD36. We demonstrate that Cav-1 and CD36 interact in caveolae-rich domains by different biochemical approaches. In addition, confocal microscopy reveals some colocalization of Cav-1 with CD36. These findings indicate that caveolae and CD36 are involved in native LDL endocytosis and suggest that CD36 might be a good candidate for the transport of native LDL across the endothelium, an early event in atherosclerosis.
Seminal studies from Nikolai Anichckov identified the accumulation of cholesterol in the arteries as the initial event that lead to the formation of atherosclerotic plaques. Further studies by Gofman and colleagues demonstrated that high levels of circulating low-density lipoprotein cholesterol (LDL-C) was responsible for the accelerated atherosclerosis observed in humans. These findings were confirmed by numerous epidemiological studies which identified elevated LDL-C levels as a major risk factor for cardiovascular disease. LDL infiltrates in the arterial wall and interacts with the proteoglycan matrix promoting the retention and modification of LDL to a toxic form, which results in endothelial cell (EC) activation and vascular inflammation. Despite the relevance of LDL transport across the endothelium during atherogenesis, the molecular mechanism that control this process is still not fully understood. A number of studies have recently demonstrated that low density lipoprotein (LDL) transcytosis across the endothelium is dependent on the function of caveolae, scavenger receptor B1 (SR-B1), activin receptor-like kinase 1 (ALK1), and LDL receptor (LDLR), whereas high-density lipoproteins (HDL) and its major protein component apolipoprotein AI transcytose ECs through SR-B1, ATP-Binding cassette transporter A1 (ABCA1) and ABCG1. In this review article, we briefly summarize the function of the EC barrier in regulating lipoprotein transport, and its relevance during the progression of atherosclerosis. A better understanding of the mechanisms that mediate lipoprotein transcytosis across ECs will help to develop therapies targeting the early events of atherosclerosis and thus exert potential benefits for treating atherosclerotic vascular disease.
During their metabolism, all lipoproteins undergo endocytosis, either to be degraded intracellularly, for example in hepatocytes or macrophages, or to be re-secreted, for example in the course of transcytosis by endothelial cells. Moreover, there are several examples of internalized lipoproteins sequestered intracellularly, possibly to exert intracellular functions, for example the cytolysis of trypanosoma. Endocytosis and the subsequent intracellular itinerary of lipoproteins hence are key areas for understanding the regulation of plasma lipid levels as well as the biological functions of lipoproteins. Indeed, the identification of the low-density lipoprotein (LDL)-receptor and the unraveling of its transcriptional regulation led to the elucidation of familial hypercholesterolemia as well as to the development of statins, the most successful therapeutics for lowering of cholesterol levels and risk of atherosclerotic cardiovascular diseases. Novel limiting factors of intracellular trafficking of LDL and the LDL receptor continue to be discovered and to provide drug targets such as PCSK9. Surprisingly, the receptors mediating endocytosis of high-density lipoproteins or lipoprotein(a) are still a matter of controversy or even new discovery. Finally, the receptors and mechanisms, which mediate the uptake of lipoproteins into non-degrading intracellular itineraries for re-secretion (transcytosis, retroendocytosis), storage, or execution of intracellular functions, are largely unknown.
Oxidatively modified low-density lipoprotein (oxLDL) is known to be involved in various diseases, including cardiovascular diseases. The presence of oxLDL in the human circulatory system and in atherosclerotic lesions has been demonstrated using monoclonal antibodies. Studies have shown the significance of circulating oxLDL in various systemic diseases, including acute myocardial infarction and diabetic mellitus. Several different enzyme-linked immunosorbent assay (ELISA) procedures to measure oxLDL were utilized. Evidence has been accumulating that reveals changes in oxLDL levels under certain pathological conditions. Since oxLDL concentration tends to correlate with low-density lipoprotein (LDL)-cholesterol, the ratio of oxLDL and LDL rather than oxLDL concentration alone has been focused attention. In addition to circulating plasma, LDL and oxLDL are found in gingival crevicular fluid (GCF), where the ratio of oxLDL to LDL in GCF is much higher than in plasma. LDL and oxLDL levels in GCF show an increase in diabetic patients and periodontal patients, suggesting that GCF might be useful in examining systemic conditions. GCF oxLDL increased when the teeth were affected by periodontitis. It is likely that oxLDL levels in plasma and GCF could reflect oxidative stress and transfer efficacy in circulatory system.
Processing and transport of hormones across vascular endothelial cells may modulate hormone action at subendothelial tissue sites. Insulin was transported across cultured rat capillary and bovine aortic endothelial cells, after a delay of 5-10 min, at a constant rate for 60 min at 37 degrees C. 125I-labeled insulin transport was inhibited by 88 +/- 11% (SE, n = 4) and 75 +/- 18% (SE, n = 4) in the presence of anti-insulin receptor antibody and unlabeled insulin (at 10(-7) M), respectively. Reverse phase high-performance liquid chromatography showed 88% of the 125I-insulin transported over 60 min was indistinguishable from the 125I-insulin added to the cells at 4 degrees C. In aortic endothelial cells preincubated with 2.3 x 10(-9) M of insulin for 24 h, insulin receptor binding was downregulated by 67%, and 125I-insulin transport was decreased by 52 +/- 11%. The proton ionophore monensin (0.05 mM) increased the internalized insulin in bovine aortic endothelial cells by 78%, with a corresponding decrease in 125I-insulin released by 76 +/- 2% (SE, n = 4). 125I-insulin transport across the aortic endothelial cell monolayer was similarly decreased (54 +/- 12%, SE, n = 4) by monensin. In contrast, the lysosomal protease inhibitor leupeptin had no effect. Degradation and transport were similarly dissociated by low temperature. At 15 degrees C, no significant insulin degradation was detected, whereas 125I-insulin release from the cells continued at 30 +/- 3% of the rate at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Early atherosclerosis features functional and structural changes in the endothelial barrier function that affect the traffic of molecules and solutes between the vessel lumen and the vascular wall. Such changes are mechanistically related to the development of atherosclerosis.Proatherogenic stimuli and cardiovascular risk factors, such as dyslipidemias, diabetes, obesity, and smoking, all increase endothelial permeability sharing a common signalling denominator: an imbalance in the production/disposal of reactive oxygen species (ROS), broadly termed oxidative stress. Mostly as a consequence of the activation of enzymatic systems leading to ROS overproduction, proatherogenic factors lead to a pro-inflammatory status that translates in changes in gene expression and functional rearrangements, including changes in the transendothelial transport of molecules, leading to the deposition of low-density lipoproteins (LDL) and the subsequent infiltration of circulating leukocytes in the intima.In this review we focus on such early changes in atherogenesis and on the concept that proatherogenic stimuli and risk factors for cardiovascular disease, by altering the endothelial barrier properties, coordinately trigger the accumulation of LDL in the intima and ultimately plaque formation.
Endothelial cells (EC) play a key role in atherosclerosis. Although EC are in constant contact with low density lipoproteins (LDL), how EC process LDL and whether this influences atherogenesis, is unclear. Here we show that EC take up and metabolize LDL, and when overburdened with intracellular cholesterol, generate cholesterol crystals (CC). The CC are deposited on the basolateral side, and compromise endothelial function. When hyperlipidemic mice are given a high fat diet, CC appear in aortic sinus within 1 week. Treatment with cAMP-enhancing agents, forskolin/rolipram (F/R), mitigates effects of CC on endothelial function by not only improving barrier function, but also inhibiting CC formation both in vitro and in vivo. A proof of principle study using F/R incorporated into liposomes, designed to target inflamed endothelium, shows reduced atherosclerosis and CC formation in ApoE−/− mice. Our findings highlight an important mechanism by which EC contribute to atherogenesis under hyperlipidemic conditions.
Vesicle-mediated transcellular transport or simply “transcytosis” is a cellular process used to shuttle macromolecules such as lipoproteins, antibodies, and albumin from one surface of a polarized cell to the other. This mechanism is in contrast to the transit of small molecules such as anions, cations and amino acids that occur via uptake, diffusion through the cytosol and release and is also distinct from paracellular leak between cells. Importantly, transcytosis has evolved as a process to selectively move macromolecules between two neighboring yet unique microenvironments within a multicellular organism. Examples include the movement of lipoproteins out of the circulatory system and into tissues and the delivery of immunoglobulins to mucosal surfaces. Regardless of whether the transport is conducted by endothelial or epithelial cells the process often involves receptor-mediated uptake of a ligand into an endocytic vesicle, regulated transit of the carrier through the cytoplasm, and release of the cargo via an exocytic event. While transcytosis has been examined in detail in epithelial cells, for both historical and technical reasons, the process is less understood in endothelial cells. Here, we spotlight aspects of epithelial transcytosis including recent findings and review the comparative dearth of knowledge regarding the process in endothelial cells highlighting the opportunity for further study.
Due to the fact that one of the main causes of worldwide deaths are directly related to atherosclerosis, scientists are constantly looking for atherosclerotic factors, in an attempt to reduce prevalence of this disease. The most important known pro-atherosclerotic factors include: elevated levels of LDL, low HDL levels, obesity and overweight, diabetes, family history of coronary heart disease and cigarette smoking. Since finding oxidized forms of cholesterol – oxysterols – in lesion in the arteries, it has also been presumed they possess pro-atherosclerotic properties. The formation of oxysterols in the atherosclerosis lesions, as a result of LDL oxidation due to the inflammatory response of cells to mechanical stress, is confirmed. However, it is still unknown, what exactly oxysterols cause in connection with atherosclerosis, after gaining entry to the human body e.g., with food containing high amounts of cholesterol, after being heated. The in vivo studies should provide data to finally prove or disprove the thesis regarding the pro-atherosclerotic prosperities of oxysterols, yet despite dozens of available in vivo research some studies confirm such properties, other disprove them. In this article we present the current knowledge about the mechanism of formation of atherosclerotic lesions and we summarize available data on in vivo studies, which investigated whether oxysterols have properties to cause the formation and accelerate the progress of the disease. Additionally we will try to discuss why such different results were obtained in all in vivo studies.
Isolated rat livers were perfused for 4 hours in a recirculating system containing washed rat erythrocytes. Biologically screened, radioiodinated low density lipoproteins (1.030 < d < 1.055 g/ml) were added to the perfusate with different amounts of whole serum to supply unlabeled rat low density lipoproteins. Apolipoprotein B contained 90% of the bound (131)I, other apolipoproteins contained 4%, and lipids contained the remainder. The fraction of apolipoprotein mass degraded during the perfusion was quantified by the linear increment of non-protein-bound radioiodine in the perfusate, corrected for the increment observed during recirculation of the perfusate in the absence of a liver. The fractional catabolic rate ranged from 0.3 to 1.7%/hr in seven experiments and was inversely related to the size of perfusate pool of low density apolipoprotein. The catabolic rate of low density apolipoprotein (fractional catabolic rate x pool size) in four livers, in which the concentration of rat low density lipoproteins was 50-100% of that present in intact rats, was 5.3 +/- 2.7 micro g hr(-1) (mean +/- SD). Similar results were obtained with human low density lipoproteins. These rates were compared with catabolic rates for the apoprotein of rat low density lipoproteins in intact animals. Fractional catabolic rate in vivo, obtained by multi-compartmental analysis of the disappearance curve of (131)I-labeled low density apolipoprotein from blood plasma, was 15.2 +/- 3.1% hr(-1) (mean +/- SD). Total catabolic rate in vivo (fractional catabolic rate x intravascular pool of low density apolipoprotein) was 76 +/- 14 micro g hr(-1) (mean +/- SD). The results suggest that only a small fraction of low density apolipoprotein mass in rats is degraded by the liver.
Cholesterol synthesis in actively growing bovine vascular endothelial cells is regulated by low density lipoprotein (LDL) at a step prior to mevalonate formation, in a manner comparable to that found in aortic smooth muscle cells. LDL uptake by these cells is associated with induction of cholesterol esterification, an increase in total cell cholesterol, and an inhibition of endogenous sterol synthesis. In contrast, cholesterol metabolism in confluent contact-inhibited endothelial cultures was not significantly affected by LDL even though the cells bind the lipoprotein at high affinity receptor sites. Lysosomal degradation and subsequent regulatory effects on cellular cholesterol metabolism, however, were observed in contact-inhibited endothelial cells incubated with cationized rather than native LDL. Cationized LDL enter the cells independently of the high affinity sites. Therefore, the primary regulation of cholesterol metabolism in these cells is neither through the appropriate intracellular enzymes nor through the high affinity surface receptors, but via an inhibition of LDL internalization. It is suggested that this inhibition is due to a strict contact-inhibited morphology which enables the endothelium of the larger arteries to function as a selective barrier to the high circulating levels of plasma LDL.
Pharmacologic doses of 17α-ethinyl estradiol have been reported to cause a marked lowering of plasma lipoprotein levels in the rat. The drop in plasma low density lipoprotein (LDL) is associated with enhanced uptake of LDL by the liver. In the current studies, we show that membranes prepared from livers of ethinyl estradiol-treated rats exhibit a 3- to 10-fold increase in saturable binding sites for human 125I-LDL. These binding sites resembled the LDL receptors previously described in extrahepatic human, mouse, and bovine cells in that they: 1) showed a marked preference for human LDL as opposed to human high density lipoprotein (HDL); 2) required calcium; 3) failed to bind LDL in which the lysine residues had been acetylated or methylated in vitro; and 4) were destroyed by pronase. Hepatic uptake of intravenously administered human 125I-HDL was 12-fold higher in estradiol-treated rats as compared with controls. Uptake of 125I-LDL was competitively inhibited by unlabeled human LDL, but not by human HDL. Moreover, methylated human 125I-HDL, which did not bind to the LDL site on membranes, was taken up by the liver in vivo at a 10-fold lower rate than 125I-LDL. These data suggest that the 125I-LDL membrane binding site that was detected in vitro mediated the uptake of 125I-LDL in vivo. Livers of untreated and ethinyl estradiol-treated rats also exhibited a saturable binding site for human 125I-HDL. This site bound human HDL much more avidly than human LDL. HDL binding was not increased by ethinyl estradiol treatment, did not require calcium, and was not destroyed by pronase. Whether the 125I-LDL and 125I-HDL binding sites function as hepatic lipoprotein receptors in untreated rats is not yet known.
The destruction of large pinosomes was examined with phase-contrast microscopy in cultured mouse fibroblasts. In areas of rapid pinosome breakdown, lysosomes were observed to repeatedly collide with pinosomes without fusing, tearing off small pieces until the pinosomes became smaller and denser. This segmentation of pinosomes by lysosomal collision has been named "piranhalysis."
Gallotannin, consisting mainly of low molecular weight esters such as penta- and hexagalloylglucoses (commercially available as tannic acid produced from Turkish nutgall), can be used for increasing and diversifying tissue contrast in electron microscopy. When applied on tissue specimens previously fixed by conventional methods (aldehydes and OsO4), the low molecular weight galloylglucoses (LMGG) penetrate satisfactorily the cells and induce general high contrast with fine delineation of extra- and intracellular structures, especially membranes. In some features, additional details of their intimate configuration are revealed. Various experimental conditions tested indicate that the LMGG display a complex effect on fixed tissues: they act primarily as a mordant between osmium-treated structures and lead, and concomitantly stabilize some tissue components against extraction incurred during dehydration and subsequent processing. Experiments with aldehyde blocking reagents (sodium borohydride and glycine) suggested that the LMGG mordanting effect is not dependent on residual aldehydes groups in tissues.
Two improved procedures were developed for activating ferritin so that the ferritin could be covalently linked to antibodies. One procedure involved use of a water-soluble carbodiimide and N-hydroxysuccinimide to prepare ferritin-containing activated esters. The other involved activation of the ferritin with excess glutaraldehyde. The ferritin-antibody conjugates prepared with the two procedures were shown to have a number of properties which made them suitable for locating antigenic components in cells.
A method has been developed for the separation of serum or plasma lipoproteins by electrophoresis in an agarose-agar gel mixture. The gel is applied to the surface of a thin polyester photographic film strip. With minor alterations in technique either single samples on individual strips or many samples on one large sheet may be processed. After fixation and dehydration the transparent film is stained with Sudan Black B and washed with water. The finished electrophoretogram can be obtained in 5 hr and consists of widely separated bands of lipoprotein fractions on a colorless transparent background, ideally suited for scanning with a densitometer.
Plasma samples from different subjects show pre-β lipoproteins of different mobilities. An effect of gel concentration on the extent of lipoprotein migration is demonstrated. The clear-cut separation of lipoproteins by this method will facilitate the classification of hyperlipoproteinemias and improve quantitative estimates of lipoprotein distribution.
The density distribution of lipoproteins in rats fed chow or chow containing 1% cholesterol and 10% olive oil was studied. Lipoprotein fractions were prepared in the ultracentrifuge between narrow density bands within the density range of 1.006–1.21 and were analyzed by chemical, electrophoretic, and immunological methods. In serum from normal rats there were three major lipoprotein fractions, with densities less than 1.006, 1.030–1.063, and 1.063–1.21. Almost no lipoprotein was found between d 1.006 and 1.030. Most of the low density lipoprotein appeared between a density of 1.04 and 1.05. In the density range 1.05–1.07, small amounts of both low density and high density lipoprotein were found.
Feeding a diet high in cholesterol resulted in a marked increase in the concentration of lipoproteins of density less than 1.006, and a new lipoprotein fraction appeared between d 1.006 and 1.030; this fraction contained immunologically demonstrable low density and high density lipoproteins. In addition, there was a decrease in the high density lipoprotein fraction between d 1.070 and 1.21.
The plasma lipoproteins of the Zucker fatty rat were characterized with respect to lipid and apoprotein composition, and results were compared with those obtained from lean controls. Information on apoproteins was obtained from gel filtration experiments and electrophoresis on polyacrylamide gels. Very low density lipoproteins (VLDL) were increased several-fold in fatties, and 78% of their mass was triglycerides compared with 60% in the controls. Low density (LDL) and high density (HDL) lipoproteins were increased by a factor of 2, although their compositions were similar to those of the controls. Levels of apoVLDL, apoLDL, and apoHDL were five, two and two times higher, respectively, in the fatties, and the two most rapidly moving subunit peptides on polyacrylamide gels were disproportionately elevated in the apoproteins. The slower of these two bands was present in relatively greater amounts than the faster one in fatties. If the slower peptide is an activator of lipoprotein lipase, analogous to the comparable subunit peptides of human apolipoproteins, plasmas of fatties could contain up to 10 times more lipase activator activity than control plasma. This finding, and the fact that adipose tissue lipoprotein lipase activity of fatties was about 150% of controls, suggests that fatties have increased capacities for VLDL catabolism. We have previously shown that hepatic VLDL secretory rates are higher than normal in these animals. The increased capacity for catabolism may be a response to the altered secretory rates.
Recent years have seen accelerating progress in our understanding of lipoprotein structure and metabolism. The system grows ever more complex and all of us are striving to find the simplest possible scheme within which to understand and account for all of the observed phenomena. This is as it should be. Our goal in science is to find the broadest general principles that can be applied. The danger, however, is that in seeking simplification and generalization we may be tempted to overlook exceptions that are possibly awkward (sometimes even irritating!) but nevertheless important. The purpose of these opening remarks, then, is to sound a cautionary note to all of us who are students of lipoprotein structure and metabolism. I will cite three general areas in which the early bloom of enthusiasm for simplicity and generality has faded with the accumulation of new information.
Endocytosis, exocytosis, and the related traffic of membranes have several common features shared by all eukaryotic cells. In addition, these processes have some characteristics which reflect the special functions fulfilled by various cells. In this respect, the endothelial cell is a revealing example. Interposed between two fluid compartments — plasma, and interstitial fluid — the vascular endothelium is highly differentiated to mediate and monitor the bidirectional exchange of substances between these two adjoining milieux. The endothelial cell contains the complete set of components usually implicated in endocytosis and exocytosis. In order to accomplish its transport function, this cell has developed the capability of coupling extensively and efficiently endocytosis and exocytosis in a special process named “transcytosis” (Fig. 1) (N. Simionescu 1979).
The lipids in human aortic intima occur in two chemically and morphologically distinct forms. In typical fatty streaks the lipid is within fat-filled cells and the cholesterol ester fatty acids are characterized by a very high proportion of oleic (18 : 1) acid, suggesting that the cholesterol has been esterified by the cells in situ. In normal intima and early fibrous lesions the lipid is in the form of fine, extracellular droplets orientated along collagen and elastic fibres, with a cholesterol ester fatty acid pattern in which linoleic (18 : 2) is the predominant fatty acid. This closely resembles the cholesterol ester in serum low-density lipoprotein, and is probably derived directly from it.The atheroma lipid pool underlying large plaques contains the linoleic acid rich, low-density lipoprotein type of cholesterol ester, and it is concluded that most of the lipid in large human plaques is derived directly from plasma lipoprotein.The amount of immunologically intact lipoprotein in the intima has been measured by electrophoresis directly from the minced tissue into an antibody-containing gel. In normal intinia the concentration of low-dmsity lipoprotein is highly correlated with the serum cholesterol level during the week before death (r= 0.965; P< 0.001). The volume of the patient's own serum in the intima is independent of cholesterol level, but is increased in hypertension. In the ‘gelatinous’ precursors of plaques and at the edges of developing plaques the lipoprotein concentrations are respectively 2 and 3 1/2 times greater than normal. There is a particularly marked increase in lipoprotein concentration in the deep layers, where in young plaques it exceeds the concentration in the superficial layers, thus providing a substantial pool of lipoprotein from which lipid could be split.
Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.
Aortae of various mammals were shown to utilize free fatty acid and choline for the synthesis of complex lipids, such as phospholipids, triglycerides and cholesterol esters. The synthesis of the major aortic phospholipid, lecithin, also proceeds through acylation of lysolecithin, which in itself can serve as a precursor. With the help of radioautography these reactions have been localized to aortic smooth muscle cells. In addition to determining synthetic pathways, we have characterized aortic enzymes active in the catabolism of phospholipids and have shown that their activity changes with the age of the individual. It was postulated that the rise in aortic phospholipase Az and the fall or lack of change in sphingomyelinase are responsible for the rise in sphingomyelin: lecithin ratio encountered in the ageing artery.
A study was made of the effectiveness of the fixatives most commonly used for lipoproteins after electrophoresis on agarose gel. The results prove that acetic acid in water or in alcohol does not fix, but allows the removal of the albumin together with the middle part of the alpha lipoprotein band during drying. A double alpha band, therefore, arises artificially. After fixation with methanol or ethanol, not only the alpha, but also the greater part of the beta lipoprotein group remains soluble.Both trichloroacetic acid + formaldehyde, and picric acid, proposed earlier in the literature for the fixation of serum proteins, proved to be effective for lipoproteins, but uranyl acetate was found to be by far the best of all the fixatives yet investigated.
Since hyperlipoproteinemia is associated with an increased risk of atherosclerosis we have evaluated the effects of sera of different hyperlipoproteinemic clinical patterns on human endothelial cells in vitro. Cultured human umbilical vein endothelial cells were treated with sera from 2 patients homozygous for familial hypercholesterolemia and 4 patients heterozygous for that disorder. Familial hypercholesterolemic sera inhibited endothelial cell migration by 50% during a 72 hour incubation (p <0.0001) compared to normal pooled human serum or single donor AB serum when measured by an agarose gel technique. The inhibition of migration was not observed when cells were treated with familial combined hyperlipidemic sera (4 patients) or familial hypertriglyceridemic sera (5 patients). Endothelial cell detachment in vitro was not induced by any of the classical patterns of hyperlipoproteinemic sera tested. The development of atherosclerosis in familial hypercholesterolemia may be in part related to an impairment of endothelial repair.
Atherosclerosis is generally considered to be a multifactorial disease; as such, the prevention or cure of the disease may be approached at different levels, control of serum cholesterol or β-lipoprotein being an important one. However, the ultimate manifestation of the disease is in the arterial wall, and any prospective drug will have to be effective in producing the beneficial action at the arterial level. From this point of view it is of great importance to be able to monitor the effect of a drug on the relevant processes at the arterial wall. For the present discussion the arterial permeability to serum proteins, low density lipoprotein (LDL) in particular, will be taken as the relevant process.
The original Lowry method of protein determination has been modified by the addition of sodium dodecyl sulfate in the alkali reagent and an increase in the amount of copper tartrate reagent. These alterations allowed the method to be used with membrane and lipoprotein preparations without prior solubilization or lipid extraction and with samples containing 200 mm sucrose or 2.5 mm EDTA.
The plasma lipoproteins are complex structures composed of proteins and lipids and involved with the transport of lipids in the circulation. The various lipoproteins are customarily separated into several groups following their flotation properties in salt solutions. Lipoproteins thus obtained differ in size, particle weight, lipid and protein composition, electrophoretic mobility and other physical and chemical properties. Yet, the lipoprotein system resembles more a continuous spectrum of particles of changing composition than families of distinct and discernible character. This is especially true for the lower density lipoproteins — chylomicrons, VLDL and LDL. Clearly, the classification of lipoproteins is somewhat arbitrary and a functional approach towards the lipoproteins is needed.
The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.
Monolayers of normal human fibroblasts were observed to bind ferritin-labeled low density lipoprotein (LDL-ferritin) at specific receptor sites on the cell surface membrane. When fibroblasts were incubated with LDL-ferritin at 4 degrees, more than 70% of the surface-bound ferritin cores were localized by electron microscopy to short segments of the plasma membrane where the membrane appeared indented and coated on both of its sides by a fuzzy material. These membrane segments corresponded to "coated regions" previously described in other cell types. Unver the conditions of these experiments, an average of 55 LDL-ferritin particles were bound to each millimeter of plasma membrane in normal cells. In the presence of a 15-fold excess of native LDL, the number of bound ferritin cores was reduced by 75%, suggesting that the LDL-ferritin was binding to specific LDL receptor sites. Although fibroblasts from a patient with the homozygous form of familial hypercholesterolemia contained the same number of indented, coated membrane regions per millimeter of cell surface as did normal cells, no LDL-ferritin was observed to bind to the cell membrane in these mutant cells. The present ultrastructural data are consistent with previous biochemical and genetic evidence indicating that LDL exerts its regulatory action on cellular cholesterol metabolism in fibroblasts through an interaction with a specific cell surface receptor and that this receptor is defective in homozygous familial hypercholesterolemia fibroblasts. Moreover, the data suggest that the LDL receptor is localized to indented, coated regions of the plasma membrane that appear to participate in the adsorptive endocytosis of proteins.
Previous studies from our laboratory (1, 2) have indicated that the lipoproteins contained in the human atherosclerotic plaque are extractable into saline and have biochemical properties which are similar to those of the serum lipoproteins except for very low density lipoproteins (VLDL). The major lipid component of arterial VLDL appears to be cholesterol ester as opposed to triglycerides for serum VLDL. It also was found that the saline extractable content of VLDL and LDL (low density lipoproteins) in atherosclerotic vessels was much higher than that of the normal intima and increased with increasing severity of atherosclerosis (Table 1). On the other hand, the high density lipoprotein (HDL) content of the arterial intima was small and did not appear to change with the progression of atherosclerosis. Of the total lipids contained in the diseased intima about 35 to 40% appeared to be extractable into saline (1.65 M NaCl) and recoverable as lipoproteins.
1. (1) The long-term effect of the administration of a diet containing olive oil and cholesterol on the chemical composition and structure of plasma lipoproteins was studied in 3 groups of rats receiving respectively 2% olive oil plus 2% cholesterol (group 1), 2% olive oil (group II) and normal laboratory chow (group III). 2. (2) The administration of 2% olive oil for 6 weeks caused an increased concentration of VLDL and LDL1, whereas the addition of 2% cholesterol to this diet led to a further increase of both VLDL and LDL1 and a reduction of HDL2. 3. (3) In the rats of groups I and II, VLDL, LDL1 and LDL2 contained more hydrophobic lipids (cholesteryl esters and triglycerides) and less protein than the normal counterparts. 4. (4) Agarose gel chromatography showed that LDL2 of both groups I and II contained 3 different subfractions. Subfractions A and B were rich in cholesteryl esters and had a low protein content, whereas subfraction C had a composition fairly similar to that of normal LDL2. 5. (5) The plasma of the rats of both groups I and II contained large polymorphous globules (30.0-350.0 nm in diameter) which showed a low affinity for the negative stain and differed from the chylomicrons and VLDL in that their shape was irregular and they had a strong tendency to coalesce into larger complexes. 6. (6) These polymorphous globules were present together with normal particles in all lipoprotein fractions of the rats of both groups I and II, but they were more numerous in the lipoprotein classes of density less than 1.050 g/ml. 7. (7) The structure of the polymorphous globules was greatly affected by the different negative staining procedures we used which did not alter the structure of the normal lipoprotein particles. 8. (8) The ultrastructure of the polymorphous globules strongly resembled that of an artificially prepared emulsion of fat having a high content of neutral lipids.
Transmural concentration profiles of 125I-labeled low-density lipoproteins (LDL) within the descending thoracic aorta were determined as a function of time following intravenous injection into normal conscious rabbits. The animals were sacrificed after 10 min to 67 h, and the descending thoracic aorta was immediately excised, opened longitudinally, rinsed and frozen. Samples of frozen aorta were sectioned parallel to the intimal surface and washed with trichloroacetic acid (TCA) prior to counting. Up to 4 h, transmural concentration profiles of TCA-precipitable radioactivity had steep gradients near the intimal surface, moderate gradients near the medial-adventitial border, and were relatively flat in the middle of the media. After 24 h, the steep intimal gradient had disappeared. Concentration levels were otherwise comparable to those at 4 h. All gradients disappeared and concentration levels were lower after 67 h. The rate of accumulation of TCA-precipitable radioactivity was initially rapid (measurable concentrations were found throughout the media after only 10 min), although it was less than that of [125I] albumin observed in a previous study. The results are consistent with entry of [125I]LDL into the media from both the luminal and adventitial sides and with gradual degradation of the labeled LDL within the aortic wall. Approximate calculations indicate that the LDL mass transfer resistance associated with the intimal endothelium is about an order of magnitude greater than that associated with the media.
Bovine vascular endothelial cells during logarithmic growth bind, internalize, and degrade low density lipoprotein (LDL) via a receptor-mediated pathway. However, contact-inhibited (confluent) monolayers bind but do not internalize LDL. This is in contrast to aortic smooth muscle cells or endothelial cells that have lost the property of contact inhibition. These cells internalize and degrade LDL at both high and low cell densities. The LDL receptors of smooth muscle and sparse endothelial cells down-regulate in response to LDL. In contrast, normal endothelial cells at confluency show little response. When contact inhibition in endothelial monolayers was locally released by wounding, and LDL was present, only cells released from contact inhibition accumulated LDL cholesterol. In smooth muscle cells under the same conditions, the entire culture interiorized lipid. It thus appears that in endothelial cells, unlike smooth muscle cells, contact inhibition is the major factor regulating cellular uptake of LDL cholesteryl ester. Reversal of contact inhibition by wounding provides a mechanism by which the endothelium could be the primary initiator of the atherosclerotic plaque.
Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18-24 h incubation in the presence of LDL at 100 microgram LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 microgram LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 microgram LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 microgram/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.
The primary purpose of this paper is to review some of the recent developments in our understanding of how lipoproteins are metabolized by peripheral cells. Studies utilizing cultured mammalian cells promise to enhance considerably our insights into factors regulating steady-state levels of lipoproteins in the plasma compartment and, at least potentially, our insights into the cellular basis for atherogenesis. Quite possibly we may see the development of new modalities of pharmacologic intervention based on a better understanding of how lipoproteins are degraded by or modified by interactions with peripheral cells. Research in this area is still at an early stage of development but progress is being made rapidly. Those of us interested in the role of lipids in atherogenesis and in the possibilities for preventive intervention should be aware of the opportunities to capitalize on the new findings as they come along. Before turning to the cellular level, however, we should establish the context by briefly reviewing current concepts of lipoprotein metabolism in
vivo.
Using a quantitative EM autoradiographic technique, we have visualized the membrane binding and receptor-mediated uptake of low density lipoprotein (LDL) in human fibroblasts. The initial binding was restricted to the plasma membrane (2 h of incubation at 4 °C) and approx. 62% of the grains could be localized to coated pits in the plasma membrane. When the incubations were carried out at 37 °C, 125I radioactivity was found both on the membrane and within the cell and predominantly localized on or within lysosomes. In cells from the patient J. D., a familial hypercholesterolemic homozygote with an internalization defect, initial binding of 125I-LDL was restricted to the plasma membrane but not preferentially localized to coated segments of the plasma membrane. After incubation for 30 min at 37 °C, the membrane bound 125I-LDL in J. D. cells was not internalized. These data confirm results obtained with ferritin-labeled LDL and illustrate the complementary application of two different morphologic probes, each of which offers special advantages for special problems.
Proteins and peptides can enter cells by receptor-mediated endocytosis, a coupled process by which selected extracellular proteins or peptides are first bound to specific cell surface receptors and then rapidly internalised by the cell. Internalisation follows clustering of the receptors in specialised regions of the cell surface called coated pits that invaginate to form intracellular coated vesicles. It is now recognised that receptor-mediated endocytosis has a fundamental role in the growth, nutrition and differentiation of animal cells.
Cultured human endothelial cells preincubated with the infranatant of human serum increased their content of cholesterol when subsequently exposed to low density lipoproteins (LDL) as compared to control cultures further incubated in the presence of infranatant only. Replacing LDL with high density lipoproteins (HDL) resulted in no change in the cellular cholesterol content compared to the control. The addition of HDL did not influence the increase in cellular cholesterol content mediated by LDL. HDL stimulated the efflux of endogenously synthesized 14C-labelled sterols compared to the infranatant fraction, whereas LDL had only a slight effect. Cells preincubated with whole serum did not change their cholesterol content when subsequently exposed to LDL, compared to cultures further incubated in presence of whole serum. Replacing whole serum (during the final incubation) with infranatant, resulted in a decrease of the cellular cholesterol content, which was not influenced by further addition of HDL.
Metabolism of low density lipoproteins (LDL) was studied in cultures of endothelial cells derived from bovine aorta or heart and from human umbilical veins. At low LDL concentrations nonconfluent cultures of bovine endothelial cells catabolized more LDL protein than contact-inhibited confluent cultures but this difference was reduced at high LDL concentrations. Nonconfluent human endothelial cells displayed also a higher rate of LDL degradation than their contact-inhibited counterparts, but this difference was less pronounced than in the bovine cells. Bovine endothelial cells grown in the presence of fibroblast growth factor metabolized less LDL than those cultured without fibroblast growth factor (FGF), but this difference was not consistent in the human endothelial cells. The data presented provide evidence that contact-inhibited confluent human endothelial cells are capable of catabolizing LDL when exposed to physiological concentrations of this lipoprotein.
Cultured human endothelial cells derived from umbilical cord veins were injured when exposed to low density lipoproteins (LDL). Addition of high density lipoproteins (HDL), together with LDL, inhibited the cellular injury induced by LDL as demonstrated by lowered 51Cr release and prevention of morphological changes. Serum albumin had a similar, but far weaker effect. Preincubation of the cells with HDL did not reduce injury inflicted during a subsequent incubation with LDL, while preincubation with LDL aggravated later damage. The protective effect of HDL could be overcome by increasing the DLD concentration.
We tested the hypothesis that loss of endothelium results in increased transport of lipoprotein into the arterial wall, favors accumulation of lipid, and thus predisposes to atherosclerosis. In rabbits initially fed a diet low in lipid, the aortas were de-endothlialized with an intraarterial balloon catheter; 28 days later, the animals were divided into two groups. Group I animals were continued on a diet low in lipid and sacrificed at 8, 11, 13, and 15 weeks after de-endothelialization. Group II animals were fed the same diet supplemented with 0.5% cholesterol and sacrificed at comparable intervals. Aortas of group I animals revealed proliferative fibromuscular intimal thickening in both de-endothelialized and re-endothelialized areas, with little or no fatty change in the intima. In contrast, aortas of group II animals revealed slight to marked fatty change in the intima, characterized by accumulation of oil red O-positive material with anisotropic lipid inclusions. The greatest quantity of lipid was present in intimal thickening beneath regenerated endothelium, and not in adjacent intimal thickening lacking an endothelial lining. These results do not support the hypothesis that the absence of endothelium favors accumulation of lipid and predisposes to atherosclerosis. The experiments indicate that lipid accumulates preferentially in areas of intimal thickening covered by regenerated endothelium.
Apoliporotein B (apoB) was measured in buffer-extracted homogenates of grossly normal and artherosclerotic human aortic intima by means of an electroimmunoassay procedure. The apoB values which were expressed as microgram per mg tissue dry weight, varied widely, ranging from 0.34 to 18.45 in normal intima and from 0.8 to 12.5 in fatty fibrous plaques. No consistent differences in apoB content were found between normal intimas from thoracic and abdominal aortic regions. There was a statistically significant positive correlation between the quantity of buffer-extractable apoB in normal regions and the plasma cholesterol and triglyceride concentration. Buffer-extractable apoB values were significantly higher in fatty fibrous plaques than in ulcerated lesions from the same vessel. However, fatty fibrous plaque apoB values were significantly lower than those from grossly normal regions from the same aorta, although the topographical distribution of apoB was more widespread in plaques than in normal regions, as shown by immunofluorescence studies. This apparent discrepancy reflected the incomplete extraction of apoB from plaques as contrasted to normal regions. The relatively loosely bound apoB, extractable by standard buffers, may represent intact low density lipoprotein (LDL) and/or very low density lipoprotein (VLDL), while the tightly bound fraction may represent insoluble complexes of intact lipoproteins within the plaque or delipidated apoB.
This article reviews recent findings and current views concerning the structural aspects of microvascular permeability. The vascular endothelium is considered as a simple squamous epithelium which has acquired a remarkably high permeability to water and water soluble solutes (including macromolecules) through a characteristic process of differentiation of its cells. In terms of cellular structures, this differentiation involves an unusually large population of plasmalemmal vesicles. The evidence so far obtained indicates that these vesicles function as (1) mass-carriers of fluid and solutes across the endothelium and as (2) generators of transendothelial channels by concomitant fusion (followed by fission) with both domains (luminal and tissular) of the plasmalemma. The endothelial fenestrae of visceral capillaries are initially transendothelial channels subsequently collapsed to minimal length. The intercellular junctions of the endothelium are not detectably permeable to tracers of diam. greater than or equal to 18--20 A in capillaries, but are focally open to probes of 50--60 A diam. in postcapillary (pericytic) venules. A correlation is attempted between transendothelial channels (and fenestrae) and the pore systems postulated by the pore theory of capillary permeability. The channels appear to function as either small or large pores depending on the porosity of their associated diaphragms and on the size of local strictures along their pathway. Two main components are recognized in the analysis of capillary permeability: 1) a basic component comparable to that of other simple epithelia and involving transport across the plasmalemma and probably along the intercellular junctions (for molecules of diam. greater than or equal to 10 A); and a differentiated component which involves plasmalemmal vesicles and their derivatives (transendothelial channels and fenestrae). The postulated pores of the capillary endothelium are part of this differentiated component. The special situation found in postcapillary venules (focally open junctions) seems to be related to the role played by these vessels in inflammatory reactions.
The ultrastructural localization of apolipoprotein B (apo B), the major protein in human plasma low (LDL) and very low density lipoproteins (VLDL), was determined in advanced atherosclerotic plaques from human coronary arteries and aorta using an immunoperoxidase procedure. Positive regions were most predominant in the lipid core at the base of the plaque. Apo B was localized on the surface of circular structures, presumably spheres, ranging in size from 250 to 2000 Å diameters. These spheres were found either free, attached to collagen and elastic fibers, or bound to extracellular lipid droplets and crystals. Smaller apo B-positive spheres (250 to 1000 Å diameters) frequently surrounded the larger spheres (1000 to 2000 Å). Saline extracts as well as homogenates of plaques contained particles of 250 to 800 Å diameters after negative staining. It is suggested that the smaller spheres are intact LDL and VLDL, whereas the larger ones may either represent lipid-free apo B attached to cell debris, or the ultrastructural morphology of denatured lipoproteins.
A comparison is made of the concentration and chemical composition of serum lipoproteins of normal rats and rats deficient in essential fatty acids. The concentration of very low density lipoproteins (VLDL) and of low density lipoproteins (LDL) in serum of deficient rats is about half that found in normal rats, but the concentration of high density lipoproteins (HDL) is higher than normal and they contain an increased amount of cholesterol esters. The proportion of cholesterol that is esterified is much greater than normal in the serum of deficient rats. The deficiency of essential fatty acids also appears to result in compensating changes occurring in the composition of serum lipoproteins. In both VLDL and LDL of deficient rats the proportion of protein is raised and that of phospholipid lowered compared to normal, while the proportions of trigly ceride and cholesterol esters are unchanged.
The transport of 125I-labeled serum lipoproteins through the aortic endothelium was studied by radioautography. Rat aorta and heart was perfused in vitro with a medium containing human very low density (VLDL), low density (LDL), high density lipoprotein (HDL), delipidated HDL apolipoprotein or rat HDL. In all lipoproteins more than 95% of the radioactivity was TCA precipitable and lipid radioactivity was from 2–4% in HDL, 4–6% in LDL, 7–15% in VLDL. After 18–60 min of perfusion and wash with unlabeled medium, most of the aortic radioactivity was TCA precipitable and the percent of lipid counts was similar to that in the original lipoprotein. Following perfusion with VLDL, LDL, or HDL the radioautographic reaction was seen over the endothelium, the subendothelial space and the inner media, and was separated by an unlabeled zone from the reaction present over the adventitia. Uniform labeling of the entire wall was found after perfusion with HDL apolipoprotein. The presence of silver grains over endothelial cells in regions rich in plasmalemmal vesicles suggested that these organelles participate in the transport of the labeled lipoprotein, as was shown for lactoperoxidase (Stein and Stein, 1972). The present data indicate that cholesterol may enter the aortic wall as a constituent of lipoprotein particles. Since an HDL particle carries less than 1/20 of the cholesterol present in a LDL particle, it seems that the lower susceptibility of the female to atheromatosis might be related to the higher ratio of HDL to LDL particles in the female serum.
A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with sodium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immunoglobulin retained its immunologic as well as enzymatic activities.
Male and female rabbits from 3 to 36 months of age were fed tracer amounts of labeled cholesterol for 2 days. Incorporation of plasma cholesterol into cardiac ventricular and skeletal muscle as well as into aorta was measured. In addition, the cholesterol specific activities of liver, lung, heart, skeletal muscle, skin, adipose tissue and aorta were compared to that of unesterified cholesterol in plasma.Liver was the only tissue that contained relatively large amounts of labeled esterified cholesterol. Aorta and skeletal muscle contained small amounts of labeled cholesterol ester and studies with 51Cr-labeled red blood cells showed that nearly all of this was due to contamination of these tissues with small amounts of labeled blood. The incorporation of plasma unesterified cholesterol per gram of aorta was smaller for the older than for the younger animals. In females this difference, which was less pronounced than in the males, was statistically only marginally significant. Expressed as intimal “clearance” of unesterified cholesterol (μl of plasma per cm2 per day) no age difference for aorta was seen in either sex.In female rabbits the specific activities of aorta, skeletal muscle, skin and adipose tissue, after 2 days of labeled cholesterol feeding, varied from 3 to 15 % of the plasma cholesterol specific activity, whereas the relative values for cardiac muscle, lung and liver were 40, 50 and 100 % respectively. The relative specific activities of cholesterol in adipose tissue were about four times higher in the very young than in the older animals.Plasma cholesterol concentrations in male and female abbits at 7 weeks of age varied between 100 and 150 mg/100 ml. With increasing age, the cholesterol concentrations in the females decreased somewhat, but in the males the levels decreased to about one-fourth of the earlier values.
A low-viscosity embedding medium based on ERL-4206 is recommended for use in electron microscopy. The composition is: ERL-4206 (vinyl cyclohexene dioxide) 10 g, D.E.R. 736 (diglycidyl ether of polypropylene glycol) 6 g, NSA (nonenyl succinic anhydride) 26 g, and S-1 (dimethylaminoethanol or DMAE) 0.4 g. The medium is easily and rapidly prepared by dispensing the components, in turn by weight, into a single flask. The relatively low viscosity of the medium (60 cP) permits rapid mixing by shaking and swirling. The medium is infiltrated into specimens after the use of any one of several dehydrating fluids, such as ethanol, acetone, dioxan, hexylene glycol, isopropyl alcohol, propylene oxide, and tert.-butyl alcohol. It is compatible with each of these in all proportions. After infiltration the castings are polymerized at 70°C in 8 hours. Longer curing does not adversely affect the physical properties of the castings. Curing time can be reduced by increasing the temperature or the accelerator, S-1, or both; and the hardness of the castings is controlled by changes in the D.E.R. 736 flexibilizer. The medium has a long pot life of several days and infiltrates readily because of its low viscosity. The castings have good trimming and sectioning qualities. The embedding matrix of the sections is very resistant to oxidation by KMnO4 and Ba(MnO4)2, compared with resins containing NADIC methyl anhydride. Sections are tough under the electron beam and may be used without a supporting membrane on the grids. The background plastic in the sections shows no perceptible substructure at magnifications commonly used for biological materials. The medium has been used successfully with a wide range of specimens, including endosperms with a high lipid content, tissues with hard, lignified cell walls, and highly vacuolated parenchymatous tissues of ripe fruits.