The two major splice variants of scavenger receptor BI differ by their interactions with lipoproteins and cellular localization in endothelial cells.

The scavenger receptor SR-BI facilitates the transport of both HDL and LDL through endothelial cells. Its two splice variants, SR-BIvar1 and SR-BIvar2, differ in their carboxyterminal domains. Only the one of SR-BIvar1 contains the putative binding sites for the adapter proteins PDZK1 and DOCK4, which limit the cell surface abundance and internalization of the receptor. To investigate the cellular localization of the SR-BI variants and their interaction with lipoproteins in endothelial cells, EA.hy926 cells were stably transfected with vectors encoding untagged, GFP- or mCherry-tagged constructs of the two SR-BI variants. Additionally, the cells were transfected with shRNAs against PDZK1 or DOCK4. Microscopy investigation showed that SR-BIvar1 was predominantly localized on the cell surface together with clathrin whereas SR-BIvar2 was absent from the cell surface but retrieved in endosomes and lysosomes. Accordingly, only SR-BIvar1 increased lipoprotein binding to endothelial while HDL and LDL uptake were enhanced by both variants. Silencing of PDZK1 or DOCK3 only reduced HDL association in SR-BIvar2 overexpressing cells while LDL association was reduced both in wild type and SR-BIvar2 overexpressing cells. In conclusion, either SR-BI variant facilitates the uptake of HDL and LDL into endothelial cells, however by different mechanisms and trafficking routes. This dual role may explain why the loss of DOCK4 or PDZK1 differently affects the uptake of HDL and LDL in different endothelial cells.

Human fertilization in vivo and in vitro requires the CatSper channel to initiate sperm hyperactivation.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

Sphingosine-1-phosphate receptor 3 regulates the transendothelial transport of high-density lipoproteins and low-density lipoproteins in opposite ways.

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.

Mapping the dynamic high-density lipoprotein synapse.

BACKGROUND AND AIMS: Heterogeneous high-density lipoprotein (HDL) particles, which can contain hundreds of proteins, affect human health and disease through dynamic molecular interactions with cell surface proteins. How HDL mediates its long-range signaling functions and interactions with various cell types is largely unknown. Due to the complexity of HDL, we hypothesize that multiple receptors engage with HDL particles resulting in condition-dependent receptor-HDL interaction clusters at the cell surface. METHODS: Here we used the mass spectrometry-based and light-controlled proximity labeling strategy LUX-MS in a discovery-driven manner to decode HDL-receptor interactions. RESULTS: Surfaceome nanoscale organization analysis of hepatocytes and endothelial cells using LUX-MS revealed that the previously known HDL-binding protein scavenger receptor B1 (SCRB1) is embedded in a cell surface protein community, which we term HDL synapse. Modulating the endothelial HDL synapse, composed of 60 proteins, by silencing individual members, showed that the HDL synapse can be assembled in the absence of SCRB1 and that the members are interlinked. The aminopeptidase N (AMPN) (also known as CD13) was identified as an HDL synapse member that directly influences HDL uptake into the primary human aortic endothelial cells (HAECs). CONCLUSIONS: Our data indicate that preformed cell surface residing protein complexes modulate HDL function and suggest new theragnostic opportunities.

Apolipoprotein M and Sphingosine-1-Phosphate Receptor 1 Promote the Transendothelial Transport of High-Density Lipoprotein.

Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.