Endothelial contribution to COVID-19: an update on mechanisms and therapeutic implications.

The global propagation of SARS-CoV-2 leads to an unprecedented public health emergency. Despite that the lungs are the primary organ targeted by COVID-19, systemic endothelial inflammation and dysfunction is observed particularly in patients with severe COVID-19, manifested by elevated endothelial injury markers, endotheliitis, and coagulopathy. Here, we review the clinical characteristics of COVID-19 associated endothelial dysfunction; and the likely pathological mechanisms underlying the disease including direct cell entry or indirect immune overreactions after SARS-CoV-2 infection. In addition, we discuss potential biomarkers that might indicate the disease severity, particularly related to the abnormal development of thrombosis that is a fatal vascular complication of severe COVID-19. Furthermore, we summarize clinical trials targeting the direct and indirect pathological pathways after SARS-CoV-2 infection to prevent or inhibit the virus induced endothelial disorders.

Characterization of SR-B2a and SR-B2b genes and their ability to promote GCRV infection in grass carp (Ctenopharyngodon idellus).

Scavenger receptor class B type 2 (SR-B2) is a pattern recognition receptor involved in innate immunity in mammals; however, the immunological function of SR-Bs in fish remains unclear. In this study, the full-length cDNA sequences of SR-B2a and SR-B2b from grass carp (Ctenopharyngodon idellus) were cloned and designated as CiSR-B2a and CiSR-B2b. Multiple alignments and phylogenetic analyses deduced that CiSR-B2a and CiSR-B2b had the highest evolutionary conservation and were closely related to the zebrafish (Danio rerio) homologs, DrSR-B2a and DrSR-B2b, respectively. Both CiSR-B2a and CiSR-B2b were expressed in all the tested tissues, with the highest expression levels found in the hepatopancreas. In Ctenopharyngodon idellus kidney cells (CIK), CiSR-B2a and CiSR-B2b were mainly located in the cytoplasm, and a small amount located on the plasma membrane. After challenge with Grass Carp Reovirus (GCRV), the expression of CiSR-B2a and CiSR-B2b were significantly upregulated in the spleen (about 10.27 and 27.19 times higher than that at 0 day, p < 0.01). With CiSR-B2a or CiSR-B2b overexpressed in CIK, the relative copy number of GCRV in the cells was both significantly increased compared to that in the control group, indicating that CiSR-B2a and CiSR-B2b may be important proteins during the infection processes of GCRV.

Binding of liposomes composed of phosphatidylcholine to scavenger receptor class B type 1 and its modulation by phosphatidic acid in HEK293T cells.

In this study, we developed a method to analyze liposomal binding to a cell membrane receptor using fluorescence-labeled liposomes and demonstrated that scavenger class B type 1 (SR-B1) plays a crucial role in binding of liposomes containing phosphatidylcholine (PC) to HEK293T cell membrane and phosphatidic acid (PA) can modulate it. Site-directed mutagenesis of SR-B1 revealed that S112F and T175A mutations in its ectodomain abrogated binding and endocytosis of PC liposomes in HEK293T cells. K151A and K156A mutations attenuated their binding and endocytosis too. Although the effects of mutations on binding and endocytosis were similar between PC liposomes and PC/PA and PA liposomes, SR-B1 dependency appeared to be PC > PC/PA > PA liposomes. Our data indicate that (i) nanoparticles including high-density lipoprotein (HDL), silica, and liposomes bind to a common or close site of SR-B1, and (ii) PC/PA and PA liposomes bind not only to SR-B1 but also other receptor(s) in HEK293T cells. In addition, PC/PA liposomes induced lipid droplet (LD) formation in HEK293T cells more than PC liposomes. Treatment of HEK293T cells with SR-B1 siRNA suppressed PC/PA liposome-induced LD formation. Taken together, our results demonstrate that SR-B1 plays an essential role in binding PC-containing liposomes and the subsequent induction of cellular responses, while PA can modulate them.

SR-BI (Scavenger Receptor BI), Not LDL (Low-Density Lipoprotein) Receptor, Mediates Adrenal Stress Response-Brief Report.

OBJECTIVE: Adrenal gland secretes stress-induced glucocorticoids (iGCs) to coping with stress. Previous study showed that SR-BI (scavenger receptor BI) null (SR-BI-/-) mice failed to generate iGC in stress conditions, suggesting that SR-BI-mediated cholesterol uptake from HDL (high-density lipoprotein) is a key regulator for iGC production. However, the LDL (low-density lipoprotein)/LDLr (LDL receptor) pathway can also provide cholesterol for iGC synthesis, but rodents have limited LDL levels in circulation. Here, we generated SR-BI-/-ApoBtg (apolipoprotein B transgenic) mice with normal LDL levels in circulation to determine the relative contribution of the HDL/SR-BI and LDL/LDLr pathways to iGC production in stress conditions. Approach and Results: To obtain mouse models with normal LDL levels, SR-BI-/- mice were bred to ApoBtg mice. Then, the F1 SR-BI±ApoBtg mice were backcrossed to SR-BI-/- to obtain SR-BI-/-ApoBtg, SR-BI-/-ApoBwt (apolipoprotein B wild type), and SR-BI+/+ApoBtg mice. We first examined the lipoprotein profile, which shows a 6.5-fold increase in LDL levels in SR-BI-/-ApoBtg mice compared with SR-BI-/-ApoBwt mice. Then, we induced stress with adrenocorticotropic hormone and cecal ligation and puncture. One hour after adrenocorticotropic hormone stimulation, SR-BI+/+ApoBtg control mice produced iGC (14.9-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg showed no iGC production (P<0.001). Three hours after cecal ligation and puncture treatment, SR-BI+/+ApoBtg control mice showed iGC production (6.4-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg mice showed no iGC production (P<0.001). CONCLUSIONS: SR-BI-/-ApoBtg mice fail to produce iGC in stress conditions even though with restored LDL levels in circulation. These findings clarify that the HDL/SR-BI, not LDL/LDLr, pathway is responsible for iGC production in stress conditions.

Identification of scavenger receptor B1 as the airway microfold cell receptor for Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, we show that M cell transcytosis depends on the Mtb Type VII secretion machine and its major virulence factor EsxA. We identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, our data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.

The high-density lipoprotein receptor Scarb1 is required for normal bone differentiation in vivo and in vitro.

We examined bone formation and turnover in high-density lipoprotein (HDL) receptor, scavenger receptor type I (Scarb1), knockout animals relative to wild-type (WT) controls. Scarb1-/- animals have elevated serum adrenocorticotropic hormone (ACTH) due to the role of Scarb1 in glucocorticoid production, which might cause increased bone mass. However, this was not observed: Scarb1-/- mice, with ACTH, over 1000 pg/ml relative to wild-type ACTH ~ 25 pg/ml, bone of the knockout animals was osteopenic relative to the wild type at 16 weeks, including bone volume/total volume and trabecular thickness. Other serum parameters of WT and Scarb1-/- animals in cortisol or calcium were unaffected, although Scarb1-/- animals had significantly elevated PTH and decreased phosphate. Osteoblast and osteoclast-related mRNAs extracted from bone were greatly decreased at 8 or 16 weeks. Importantly, in normal ACTH, osteogenic differentiation in vitro from mesenchymal stem cells showed reduced alkaline phosphatase and mineralization. In Scarb1-/- cells relative to WT, mRNAs for RunX2, alkaline phosphatase, type I collagen, and osteocalcin were reduced 40-90%, all p < 0.01, indicating a role of Scarb1 in osteoblast differentiation independent of ACTH. Additionally, in vitro osteoblast differentiation at variable ACTH in WT cells confirmed ACTH increasing bone differentiation, mineralization, alkaline phosphatase, and osteocalcin mRNA at 0-10 nM ACTH, but reduced bone differentiation at 100-1000 nM ACTH. Overall Scarb1-/- animals show inhibited bone formation with age. This may be a mixed effect on direct bone formation and of very high ACTH. Further, this work shows that both ACTH concentration and the HDL receptor Scarb1 play important independent roles in osteoblast differentiation.

High-density lipoproteins induce miR-223-3p biogenesis and export from myeloid cells: Role of scavenger receptor BI-mediated lipid transfer.

BACKGROUND AND AIMS: We recently showed that miR-223-3p on high-density lipoproteins (HDL) is exported to endothelial cells, where it inhibits inflammation. However, the origin of miR-223-3p on HDL is unknown. We hypothesize that HDL-associated miR-223-3p originates in myeloid cells and is exported to HDL in a scavenger receptor BI (SR-BI)-dependent manner. METHODS: Polymorphonuclear neutrophils (PMNs) and human monocyte derived macrophages (HMDMs) were incubated with native HDL (nHDL) or discoidal reconstituted HDL (rHDL). Total RNA was isolated before and after incubation. Mature and primary miR-223-3p (pri-mir-223-3p) levels were quantified by real-time PCR. RESULTS: Incubation with nHDL and rHDL increased miR-223-3p export from PMNs and HMDMs. In PMNs, nHDL but not rHDL, increased mature and pri-mir-223-3p. Incubation with HDL also increased Dicer mRNA, a critical regulator of miRNA biogenesis. Incubation of HMDMs with nHDL did not increase cellular levels of mature miR-223-3p, but significantly increased pri-mir-223 levels. Incubation with rHDL had no effect on either mature or pri-mir-223-3p levels. Activated PMNs increased miR-223-3p export to HDL and the production of reactive oxygen species and activated protein kinase C. Blocking HDL binding to SR-BI increased miR-223-3p export to HDL in both PMNs and HMDMs, but did not affect mature and primary miR-223-3p levels. Chemical inhibition of cholesterol flux by Block Lipid Transport (BLT)-1 inhibited HDL-induced pri-mir-223 expression in PMNs. CONCLUSIONS: HDL-associated miR-223-3p originates in PMNs and macrophages. HDL stimulates miR-223-3p biogenesis in PMNs in a process that is regulated by SR-BI-mediated lipid flux.

Involvement of cutaneous SR-B1 in skin lipid homeostasis.

BACKGROUND: The main functions of the skin are to protect against environmental insults and prevent water loss, which are performed by the complex lipid- and protein matrix present in the outermost layers of the epithelium. The lipidome of these outer layers is mainly composed of ceramides, fatty acids, and cholesterol, which regulates keratinocyte differentiation and skin barrier function. SR-B1 is a multifunctional scavenger receptor that is best known for facilitating uptake of cholesterol from HDL particles in the liver, but it is also expressed in the skin. OBJECTIVE: To determine the role of SR-B1 in keratinocyte differentiation. METHODS: We investigated the relationship between SR-B1 and keratinocyte differentiation using a physiologically relevant model, organotypic skin equivalents (SEs), wherein SR-B1 was knocked down via siRNA transfection. To assess effects of SR-B1 knockdown on keratinocyte differentiation, we performed hematoxylin/eosin staining, RT-PCR, western blotting, and immunohistochemistry. We also examined the effect of SR-B1 knockdown on lipid production by performing Oil Red O staining and thin layer chromatography. RESULTS: SR-B1 knockdown resulted in decreased lipid levels in SEs, specifically ceramides, and in decreased transcript levels of LDLR, PPAR-α and PPAR-γ, which are factors involved in regulating ceramide synthesis. In addition, filaggrin levels increased in SR-B1 KD tissues, but neither keratin 14 nor keratin 10 were affected. CONCLUSION: We conclude that one of the main functions of SR-B1 in the skin is to regulate ceramide levels and thereby maintain the barrier function of the skin, resulting in the protection of cutaneous tissues from outdoor insults.

Rivaroxaban Suppresses the Progression of Ischemic Cardiomyopathy in a Murine Model of Diet-Induced Myocardial Infarction.

AIM: A direct oral anti-coagulant, FXa inhibitor, has been applied to the clinical treatment of myocardial infarction (MI). Experimental studies in mice indicated that FXa inhibitors reduced atherosclerosis and prevented cardiac dysfunction after coronary ligation. These studies suggested that protease-activated receptor (PAR) 2, a major receptor of activated FX, may play an important role in atherosclerosis and cardiac remodeling. METHODS: The effects of a FXa inhibitor, rivaroxaban, were investigated in a new murine model of ischemic cardiomyopathy (ICM) using SR-BI KO/ApoeR61h/h mice (Hypo E mice) that developed MI by high-fat diet loading. RESULTS: Hypo E mice were fed rivaroxaban-containing (n=49) or control chow diets (n=126) after the induction of MI. The survival curve of the rivaroxaban-treated group 2 weeks after the induction of MI was improved significantly as compared with the non-treatment group (survival rate: 75.5% vs. 47.4%, respectively, p=0.0012). Echocardiography and the expression of BNP showed that rivaroxaban attenuated heart failure. Histological analyses revealed that rivaroxaban reduced aortic atherosclerosis and coronary occlusion, and markedly attenuated cardiac fibrosis. Rivaroxaban treatment decreased cardiac PAR2 levels and pro-inflammatory genes. In vitro, rivaroxaban application demonstrated the increase of cell viability against hypoxia in cardiac myocytes and the reduction of hypoxia-induced inflammation and fibrosis-related molecules in cardiac fibroblasts. The effects of the PAR2 antagonist against hypoxia-induced inflammation were comparable to rivaroxaban in cardiac fibroblasts. CONCLUSIONS: Rivaroxaban treatment just after MI in Hypo E mice prevented the progression of ICM by attenuating cardiac remodeling, partially through the suppression of the PAR2-mediated inflammatory pathway.

SR-B1 involvement in keratinocytes in vitro wound closure.

Skin represents the most extended organ of human body, having as main function the protection of our body from outdoor stressors. Its protective ability is compromised when the skin is disrupted as a consequence of mechanical insults. For this purpose, cutaneous tissue is equipped with an efficient and fine mechanism involved in repairing the wounded area. Among the numerous players that take part in the wound healing process, SR-B1 has been recently shown to have a role in keratinocyte re-epithelialization. SR-B1 is a mediator of cholesterol uptake from HDLs, whereas it is implicated in other cellular processes such as vitamins absorption, vesicle trafficking or pathogen identification. The aim of this study was to investigate the mechanisms involved in SR-B1 role in skin wound closure. Our in vitro data demonstrated that SR-B1 influenced keratinocyte proliferation and migration through a downregulation of nuclear cyclin D1 levels and active MMP9 expression respectively possibly in an NF-kB-dependent mechanism. In addition, SR-B1 was also able to modulate keratinocyte morphology into a pro-migratory cytoskeleton rearrangement. The present in vitro study suggests a new role of SRB1 as a possible new key player in cutaneous wound healing mechanism.

The function of high-density lipoprotein and low-density lipoprotein in the maintenance of mouse ovarian steroid balance.

The membrane proteins, low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SR-BI, gene name Scarb1), are lipoprotein receptors that play central roles in lipoprotein metabolism. Cholesterol bound in high-density lipoprotein (HDL) and LDL is transported into cells mainly by SR-BI and LDLR. The relative contribution of LDL and HDL to the steroidogenic cholesterol pool varies among species and may vary among tissues within one species. To investigate which of these pathways is more important in the supply of cholesterol in mouse ovary, we utilized immunohistochemistry, western blotting, RNAi, and RT-PCR as well as Ldlr-/- mice to explore the uptake of HDL and LDL in the ovary. Our data demonstrate that both SR-BI and LDLR are present in the interstitial cells, thecal cells, and corpora lutea (CLs), and their expression fluctuates with the development of follicles and CLs. The intracellular cholesterol concentration was significantly decreased when Ldlr or Scarb1 was silenced in luteal cells. Furthermore, Ldlr-/- mice had lower progesterone and estrogen levels compared to wild-type mice, and when Ldlr-/- mice were treated with the inhibitor of de novo cholesterol synthesis, lovastatin, serum progesterone, and estrogen concentrations were further reduced. These results demonstrate that both LDLR and SR-BI play important roles in importing cholesterol and that both HDL and LDL are crucial in steroidogenesis in mouse ovaries.

Involvement of scavenger receptor class B type 1 and low-density lipoprotein receptor in the internalization of liposomes into HepG2 cells.

In this study, HepG2 cells, an in vitro model system for human hepatocytes, were used to evaluate the interaction of lipoprotein receptors with liposomes carrying fluorescently labeled cholesterol and their subsequent intracellular uptake. In these experiments, two lipoprotein receptors, scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR), accounted for approximately 20% and 10%, respectively, of the intracellular uptake of the labeled liposomes. These findings indicate that additional mechanisms contributed to liposomal internalization. Liposomes modified with both apolipoproteins A-I and E were internalized in HepG2 cells in FBS-depleted culture medium at the same levels as unmodified liposomes in FBS-containing culture medium, which indicates that apolipoproteins A-I and E were the major serum components involved in liposomal binding to SR-B1 or LDLR (or both). These results increase our understanding of the disposition of liposomes, processes that can directly affect the efficacy and safety of drug products.

Scavenger receptor class B member 1 (SCARB1) variants modulate hepatitis C virus replication cycle and viral load.

BACKGROUND & AIMS: There are numerous coding and non-coding variants in the SCARB1 gene that encodes scavenger receptor class B member 1 (SR-BI), a key receptor for both high density lipoproteins and hepatitis C virus (HCV). Many have been linked to clinical phenotypes, yet their impact on the HCV replication cycle is incompletely understood. The aim of this study was to analyze the impact of these variants on the molecular biology and clinical course of HCV. METHODS: We analyzed key coding non-synonymous as well as non-coding SCARB1 variants using virological in vitro and human genetics approaches. RESULTS: Non-synonymous variants: S112F and T175A have greatly reduced HCV receptor function. When present on the cell surface, these variants are impaired in their ability to interact with HCV E2. Non-coding variants: The G allele in rs3782287 is associated with decreased viral load. Haplotype analysis confirmed these findings and identified haplotype rs3782287 A/rs5888 C as a risk allele associated with increased viral load. We also detected a trend towards lower hepatic SR-BI expression in individuals with the rs3782287 GG genotype associated with low viral load suggesting a potential underlying mechanism. CONCLUSION: Coding and non-coding genetic SCARB1 variants modulate the HCV replication cycle and possibly clinical features of hepatitis C. These findings underscore the relevance of SR-BI as an HCV receptor and contribute to our understanding of inter-individual variation in HCV infection. LAY SUMMARY: The cell surface receptor SR-BI (scavenger receptor class B member 1), is essential for hepatitis C virus (HCV) entry into hepatocytes. Variations in the gene coding this receptor influence infectivity and viral load. We analyzed these variations to gain a better understanding of inter-individual differences over the course of HCV infection.

Scavenger Receptor Class B Type 1 Deletion Led to Coronary Atherosclerosis and Ischemic Heart Disease in Low-density Lipoprotein Receptor Knockout Mice on Modified Western-type Diet.

AIM: Atherosclerosis-prone apolipoprotein E (apoE) or low-density lipoprotein receptor (LDL-R) knockout (KO) mice are generally resistant to developing coronary atherosclerosis (CA) and ischemic heart disease (IHD). However, studies have demonstrated the occurrence of spontaneous CA and IHD in scavenger receptor class B type 1 (SR-BI)/apoE double KO (dKO) mice, which suggests that SR-BI could be a potential target for the prevention and therapy of CA and IHD. This possibility was later investigated in SR-BI/LDL-R dKO mice, but no signs of CA or IHD was identified when mice were fed a normal western-type diet. Here we explored whether SR-BI deletion could result in CA and IHD in LDL-R KO mice when fed a modified western-type diet containing higher (0.5%) cholesterol. METHODS: Cardiac functions were detected by electrocardiography, single photon emission computed tomography (SPECT), echocardiography (Echo) and 2,3,5-triphenyltetrazolium chloride staining. CA was visualized by hematoxylin-eosin staining. RESULTS: After 12 weeks on the modified diet, SR-BI/LDL-R dKO mice developed cardiac ischemia/infarction, together with systolic dysfunction and left ventricular dilatation. CA was most severe at the aortic sinus level to an extent that no dKO mice survived to 20 weeks on the modified diet. None of control mice, however, developed CA or IHD. CONCLUSIONS: SR-BI deletion led to CA and IHD in LDL-R KO mice when fed the modified western-type diet. We established SR-BI/LDL-R dKO mice as a diet-induced murine model of human IHD and developed detection methods, using a combination of SPECT and Echo, for effective in vivo evaluation of cardiac functions.

A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I.

While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties. IMPORTANCE: Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability.

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

AIMS: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. METHODS AND RESULTS: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. CONCLUSION: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.

Scavenger receptor class B, type I-mediated uptake of A1AT by pulmonary endothelial cells.

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30-50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.

A genetic dissection of intestinal fat-soluble vitamin and carotenoid absorption.

Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency. Surprisingly, responses to dietary supplementation with these compounds are quite variable between individuals. Genome-wide studies have associated common genetic polymorphisms in the BCO1 gene with this variability. The BCO1 gene encodes an enzyme that is expressed in the intestine and converts provitamin A carotenoids to vitamin A-aldehyde. However, it is not clear how this enzyme can impact the bioavailability and metabolism of other carotenoids such as xanthophyll. We here provide evidence that BCO1 is a key component of a regulatory network that controls the absorption of carotenoids and fat-soluble vitamins. In this process, conversion of ß-carotene to vitamin A by BCO1 induces via retinoid signaling the expression of the intestinal homeobox transcription factor ISX. Subsequently, ISX binds to conserved DNA-binding motifs upstream of the BCO1 and SCARB1 genes. SCARB1 encodes a membrane protein that facilitates absorption of fat-soluble vitamins and carotenoids. In keeping with its role as a transcriptional repressor, SCARB1 protein levels are significantly increased in the intestine of ISX-deficient mice. This increase results in augmented absorption and tissue accumulation of xanthophyll carotenoids and tocopherols. Our study shows that fat-soluble vitamin and carotenoid absorption is controlled by a BCO1-dependent negative feedback regulation. Thus, our findings provide a molecular framework for the controversial relationship between genetics and fat-soluble vitamin status in the human population.

Excess cholesterol induces mouse egg activation and may cause female infertility.

The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-ß-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.

Intestinal scavenger receptors are involved in vitamin K1 absorption.

Vitamin K1 (phylloquinone) intestinal absorption is thought to be mediated by a carrier protein that still remains to be identified. Apical transport of vitamin K1 was examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium and in transfected HEK cells. Phylloquinone uptake was then measured ex vivo using mouse intestinal explants. Finally, vitamin K1 absorption was compared between wild-type mice and mice overexpressing scavenger receptor class B type I (SR-BI) in the intestine and mice deficient in cluster determinant 36 (CD36). Phylloquinone uptake by Caco-2 cells was saturable and was significantly impaired by co-incubation with α-tocopherol (and vice versa). Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 85% of vitamin K1 uptake. BLT1 also decreased phylloquinone apical efflux by ∼80%. Transfection of HEK cells with SR-BI and CD36 significantly enhanced vitamin K1 uptake, which was subsequently decreased by the addition of BLT1 or sulfo-N-succinimidyl oleate (CD36 inhibitor), respectively. Similar results were obtained in mouse intestinal explants. In vivo, the phylloquinone postprandial response was significantly higher, and the proximal intestine mucosa phylloquinone content 4 h after gavage was increased in mice overexpressing SR-BI compared with controls. Phylloquinone postprandial response was also significantly increased in CD36-deficient mice compared with wild-type mice, but their vitamin K1 intestinal content remained unchanged. Overall, the present data demonstrate for the first time that intestinal scavenger receptors participate in the absorption of dietary phylloquinone.