BACKGROUND:Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. METHODS:EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1β activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. RESULTS:Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. CONCLUSIONS:Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.

Acute respiratory distress syndrome is characterized by spatial heterogeneity, with severely injured lung regions adjacent to relatively normal areas. This makes targeting treatment to the injured regions difficult. Here we use thoracic ultrasound and intravenous microbubbles (USMBs) to direct gene transfection specifically to injured lung regions. Transfection of the tight junction protein claudin-5 improved oxygenation and decreased vascular leakage without impairing innate immunity. These findings suggest that USMB is a novel treatment for ARDS.

Atypical chemokine receptor-1 (ACKR1), previously known as the Duffy antigen receptor for chemokines, is a widely conserved cell surface protein that is expressed on erythrocytes and the endothelium of post-capillary venules. In addition to being the receptor for the parasite causing malaria, ACKR1 has been postulated to regulate innate immunity by displaying and trafficking chemokines. Intriguingly, a common mutation in its promoter leads to loss of the erythrocyte protein but leaves endothelial expression unaffected. Study of endothelial ACKR1 has been limited by the rapid down-regulation of both transcript and protein when endothelial cells are extracted and cultured from tissue. Thus, to date the study of endothelial ACKR1 has been limited to heterologous over-expression models or the use of transgenic mice. Here we report that exposure to whole blood induces ACKR1 mRNA and protein expression in cultured primary human lung microvascular endothelial cells. We found that contact with neutrophils is required for this effect. We show that NF-κB regulates ACKR1 expression and that upon removal of blood, the protein is rapidly secreted by extracellular vesicles. Finally, we confirm that endogenous ACKR1 does not signal upon stimulation with IL-8 or CXCL1. Our observations define a simple method for inducing endogenous endothelial ACKR1 protein that will facilitate further functional studies.

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 42, No. 1Angiopoietin-2: An Emerging Tie to Pathological Vessel Enlargement Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessResearch ArticlePDF/EPUBAngiopoietin-2: An Emerging Tie to Pathological Vessel Enlargement Negar Khosraviani, Ruilin Wu and Jason E. Fish Negar KhosravianiNegar Khosraviani Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada (N.K., R.W., J.E.F.). Toronto General Hospital Research Institute (N.K., R.W., J.E.F.), University Health Network, Ontario, Canada. , Ruilin WuRuilin Wu Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada (N.K., R.W., J.E.F.). Toronto General Hospital Research Institute (N.K., R.W., J.E.F.), University Health Network, Ontario, Canada. and Jason E. FishJason E. Fish Correspondence to: Jason E. Fish, PhD, Toronto General Hospital Research Institute, University Health Network, 101 College St, 3-309 Princess Margaret Cancer Research Tower, Toronto, Ontario, Canada, M5G 1L7. Email E-mail Address: [email protected] https://orcid.org/0000-0003-0640-7277 Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada (N.K., R.W., J.E.F.). Toronto General Hospital Research Institute (N.K., R.W., J.E.F.), University Health Network, Ontario, Canada. Peter Munk Cardiac Centre (J.E.F.), University Health Network, Ontario, Canada. Originally published11 Nov 2021https://doi.org/10.1161/ATVBAHA.121.317102Arteriosclerosis, Thrombosis, and Vascular Biology. 2022;42:3–5is related toEndothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood VesselsOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: November 11, 2021: Ahead of Print Dynamic hierarchical vascular networks are requisite for tissue homeostasis and are formed and maintained by highly interconnected signaling pathways. A wide range of vascular malformations—including arteriovenous, cavernous, venous, lymphatic, and capillary malformations—occur due to mutations in genes encoding signaling components that act to control endothelial cell (EC) behavior. In the case of sporadic disease, these are typically the result of somatic mutations within ECs. The clonal expansion of mutated cells drives altered vessel morphology, including lumen enlargement—a characteristic feature of malformed vessels. By identifying mutant genes and characterizing downstream pathways, novel therapeutic approaches can be discovered for these vascular lesions, which are notoriously difficult to treat. In this issue of ATVB, Huang et al1 identify ANGPT2 (angiopoietin-2) as a regulator of vessel enlargement in a mouse xenograft model of capillary malformation (CM) in which endothelial colony forming cells express GNAQ p.R183Q, the most common somatic mutation in CM. This exciting discovery opens up new possibilities for therapeutic intervention for vascular malformations.See accompanying article on page e27Cutaneous CM, also known as port-wine stain, affects 0.3% of infants and is associated with Sturge-Weber syndrome, a rare neurocutaneous disorder. CM is a slow-flow vascular anomaly characterized by clusters of abnormal capillaries and venules that are enlarged, have enhanced sprouting, and abnormal pericyte coverage.2 The occurrence of cutaneous and brain CMs has been linked primarily to somatic missense mutation in GNAQ (p. R183Q), an alpha subunit of the heterotrimeric G protein complex, Gαq. This gain-of-function mutation occurs in the conserved GTP-binding pocket that mediates the hydrolysis of GTP to GDP, resulting in constitutive activity.3 This somatic mutation occurs predominantly in ECs of brain and skin CMs at a low allele frequency but is also present in some surrounding stromal cells.4,5By characterizing pathways downstream of activated GNAQ, Huang et al reveal a critical role for phospholipase C β3 and identify a bifurcation of signaling pathways that result in activation of PKC-NFκB-ANGPT2 and calcineurin-NFATc1-DSCR1.41 (Figure). The increased expression of ANGPT2 and DSCR1.4 were further confirmed in mouse xenograft studies and human CM tissues. Importantly, GNAQ p.R183Q expression in endothelial colony forming cells was sufficient to induce the formation of enlarged CM-like vessels, and ANGPT2 inhibition prevented pathological vessel enlargement, positioning ANGPT2 as a promising therapeutic target.Download figureDownload PowerPointFigure. GNAQ mutations drive ANGPT2-dependent vessel enlargement. Left, Schematic of the molecular pathway identified by Huang et al. Activating mutations in GNAQ (p.R183Q) drive PLCβ3 (phospholipase C β3) signaling and conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol phosphate (IP3) and diacylglyceride (DAG). Calcineurin activation leads to the nuclear translocation of NFATc1 (nuclear factor of activated T cells-1) and induction of Down Syndrome critical region gene 1.4 (DSCR1.4). PKC (protein kinase C) signaling leads to the nuclear translation of NFκB (nuclear factor κ-light chain enhancer of activated B-cells) and induction of Angiopoietin-2 (ANGPT2). Right, Secretion of ANGPT2 drives pathological vessel enlargement. The figure was created with Biorender.com.Angiopoietins regulate multiple aspects of vascular biology through their interaction with the TIE2 receptor on ECs. ANGPT1 is highly expressed in adult tissue and promotes vascular maturation and maintains vascular stability.6,7 Conversely, ANGPT2 destabilizes vessels and promotes angiogenesis through antagonism of ANGPT1.6 However, ANGPT2 action is highly context dependent and treatment with ANGPT2 mimic can also activate TIE2 to mediate vessel enlargement.8 Notably, ANGPT2 has been implicated in the pathogenesis of several distinct vascular malformations, where the aberrant elevation of ANGPT2 contributes to enlargement of vessels and morphological changes. For example, ANGPT2 is elevated in patient tissue from brain arteriovenous malformations.9 In cerebral cavernous malformation, loss of CCM3 (cerebral cavernous malformation 3) results in exocytosis of ANGPT2, contributing to disrupted EC junctions, enlargement of lumens, and defective pericyte recruitment.10 Consistent with these observations, EC-specific SMAD4-deficient mice, modeling hereditary hemorrhagic telangiectasia, also have robust ANGPT2 elevation.11 Inhibition of ANGPT2 function through knockdown or the use of inhibitors, restored normal EC shape and size and reduced vessel diameter.11 Collectively, these studies reveal that ANGPT2 induction is a unifying characteristic of vascular malformations with distinct causes, and that ANGPT2 is a potent mediator of vessel enlargement.Aberrant lumen expansion plays an important role in the progression of malformations as it significantly alters blood flow and local hemodynamics. Yet, the underlying molecular mechanisms responsible for the development of enlarged vessels remains an open question. Expanded lumens can be due to increases in the number or size/morphology of ECs. It is unclear which of these processes is involved in lumen expansion in the xenograft model used by Huang et al, although the decrease in vascular density in the xenograft model may suggest the latter. Interestingly, in the setting of hemorrhagic telangiectasia, vessel enlargement involves ECs becoming larger and not elongating properly in response to laminar flow.12,13 Regarding CM, Gαq has been implicated in flow sensing,14,15 but the impact of the R183Q mutation in this context has not been assessed. It will be of interest to determine whether ANGPT2 is involved in regulating EC hemodynamic responses in the setting of CM and other malformations. Notably, ANGPT2 is known to be a flow-responsive gene and ANGPT2 regulates phenotypes that are dependent on hemodynamics.16 Determining whether ANGPT2 influences cell-cell or cell-ECM adhesion, modulates pericyte recruitment or if ANGPT2 secretion affects the phenotype of wild-type cells within the lesion, should also be pursued. Aside from antagonizing ANGPT1/TIE2 signaling, ANGPT2 can bind to integrins to activate signaling through the FAK-RAC1 pathway to modulate cytoskeletal rearrangements, cell migration, and angiogenesis.17,18 Thus, ANGPT2-dependent cytoskeleton changes at the cellular level may lead to altered vascular organization and the development of enlarged vessels in CM. Further investigations are needed to determine whether ANGPT2 acts in a TIE2-dependent or -independent manner in CM.Another intriguing aspect of the Huang et al study is the concurrent activation of both angiogenic and inflammatory pathways downstream of GNAQ p.R183Q.1 ANGPT2 is known to regulate both inflammation and angiogenesis.19 The extent to which angiogenic and inflammatory pathways contribute to CM pathogenesis remains to be uncovered, but inflammation has been postulated to participate in other vascular malformations through the modulation of the microenvironment.20ANGPT2, initially characterized as an antagonistic ligand to ANGPT1, has been shown to be involved in multiple aspects of endothelial biology. It is currently being explored as a therapeutic target in multiple diseases, including sepsis and cancer.21 As such, it is exciting that ANGPT2 inhibition can prevent vessel enlargement in animal models of CM and other vascular malformations—a critical disease phenotype. In future studies, it will be important to determine whether ANGPT2 acts to initiate and maintain CM phenotypes to elucidate whether ANGPT2 antagonism would be an effective treatment in CM patients with established vascular pathologies.Article InformationSources of FundingN. Khosraviani is supported by an Ontario Graduate Studentship. R. Wu is supported by a Canada Graduate Scholarship from the Canadian Institutes of Health Research (CIHR). J.E. Fish is supported by a Canada Research Chair from CIHR and his lab received infrastructure funding from the Canada Foundation for Innovation, the John R. Evans Leaders Fund and the Ontario Research Fund. Vascular malformation research in the Fish laboratory is supported by a Project Grant from CIHR (PJT 155922) and the US Department of Defense (W81XWH-18-1-0351).Disclosures None.Footnotes*N. Khosraviani and R. Wu contributed equally.For Sources of Funding and Disclosures, see page 5.The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Correspondence to: Jason E. Fish, PhD, Toronto General Hospital Research Institute, University Health Network, 101 College St, 3-309 Princess Margaret Cancer Research Tower, Toronto, Ontario, Canada, M5G 1L7. Email jason.[email protected]caReferences1. Huang L, Bichsel C, Norris AL, Thorpe J, Pevsner J, Alexandrescu S, Pinto A, Zurakowski D, Kleiman RJ, Sahin M, et al.. Endothelial GNAQ p.R183Q increases ANGPT2 (angiopoietin-2) and drives formation of enlarged blood vessels.Arterioscler Thromb Vasc Biol. 2022; 42:e27–e43. doi: 10.1161/ATVBAHA.121.316651LinkGoogle Scholar2. Bichsel C, Bischoff J. A somatic missense mutation in GNAQ causes capillary malformation.Curr Opin Hematol. 2019; 26:179–184. doi: 10.1097/MOH.0000000000000500CrossrefMedlineGoogle Scholar3. 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Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.Science. 1997; 277:55–60. doi: 10.1126/science.277.5322.55CrossrefMedlineGoogle Scholar7. Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis.Cell. 1996; 87:1171–1180. doi: 10.1016/s0092-8674(00)81813-9CrossrefMedlineGoogle Scholar8. Kim M, Allen B, Korhonen EA, Nitschké M, Yang HW, Baluk P, Saharinen P, Alitalo K, Daly C, Thurston G, et al.. Opposing actions of angiopoietin-2 on Tie2 signaling and FOXO1 activation.J Clin Invest. 2016; 126:3511–3525. doi: 10.1172/JCI84871CrossrefMedlineGoogle Scholar9. Hashimoto T, Lam T, Boudreau NJ, Bollen AW, Lawton MT, Young WL. Abnormal balance in the angiopoietin-tie2 system in human brain arteriovenous malformations.Circ Res. 2001; 89:111–113. doi: 10.1161/hh1401.094281LinkGoogle Scholar10. Jenny Zhou H, Qin L, Zhang H, Tang W, Ji W, He Y, Liang X, Wang Z, Yuan Q, Vortmeyer A, et al.. Endothelial exocytosis of angiopoietin-2 resulting from CCM3 deficiency contributes to cerebral cavernous malformation.Nat Med. 2016; 22:1033–1042. doi: 10.1038/nm.4169CrossrefMedlineGoogle Scholar11. Crist AM, Zhou X, Garai J, Lee AR, Thoele J, Ullmer C, Klein C, Zabaleta J, Meadows SM. Angiopoietin-2 inhibition rescues arteriovenous malformation in a smad4 hereditary hemorrhagic telangiectasia mouse model.Circulation. 2019; 139:2049–2063. doi: 10.1161/CIRCULATIONAHA.118.036952LinkGoogle Scholar12. Crist AM, Lee AR, Patel NR, Westhoff DE, Meadows SM. Vascular deficiency of Smad4 causes arteriovenous malformations: a mouse model of Hereditary Hemorrhagic Telangiectasia.Angiogenesis. 2018; 21:363–380. doi: 10.1007/s10456-018-9602-0CrossrefMedlineGoogle Scholar13. Sugden WW, Meissner R, Aegerter-Wilmsen T, Tsaryk R, Leonard EV, Bussmann J, Hamm MJ, Herzog W, Jin Y, Jakobsson L, et al.. Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues.Nat Cell Biol. 2017; 19:653–665. doi: 10.1038/ncb3528CrossrefMedlineGoogle Scholar14. Albarrán-Juárez J, Iring A, Wang S, Joseph S, Grimm M, Strilic B, Wettschureck N, Althoff TF, Offermanns S. Piezo1 and Gq/G11 promote endothelial inflammation depending on flow pattern and integrin activation.J Exp Med. 2018; 215:2655–2672. doi: 10.1084/jem.20180483CrossrefMedlineGoogle Scholar15. Dela Paz NG, Melchior B, Frangos JA. Shear stress induces Gαq/11 activation independently of G protein-coupled receptor activation in endothelial cells.Am J Physiol Cell Physiol. 2017; 312:C428–C437. doi: 10.1152/ajpcell.00148.2016CrossrefMedlineGoogle Scholar16. Tressel SL, Huang RP, Tomsen N, Jo H. 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Mouchtouris N, Jabbour PM, Starke RM, Hasan DM, Zanaty M, Theofanis T, Ding D, Tjoumakaris SI, Dumont AS, Ghobrial GM, et al.. Biology of cerebral arteriovenous malformations with a focus on inflammation.J Cereb Blood Flow Metab. 2015; 35:167–175. doi: 10.1038/jcbfm.2014.179CrossrefMedlineGoogle Scholar21. Akwii RG, Sajib MS, Zahra FT, Mikelis CM. Role of Angiopoietin-2 in Vascular Physiology and Pathophysiology.Cells. 2019; 8:E471. doi: 10.3390/cells8050471CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesEndothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood VesselsLan Huang, et al. Arteriosclerosis, Thrombosis, and Vascular Biology. 2022;42:e27-e43 January 2022Vol 42, Issue 1Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/ATVBAHA.121.317102PMID: 34758631 Originally publishedNovember 11, 2021 KeywordsEditorialsangiopoietin-2capillary malformationsomatic mutationGNAQvessel enlargementPDF download Advertisement

The spike protein of severe acute respira-tory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses mediates host cell entry and is S-acyl-ated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes have been reported to post-translationally modify spike proteins; however, strategies to exploit this modification are lacking. Using resin-assisted capture MS, we demonstrate that the spike protein is S-acyl-ated in SARS-CoV-2-infected human and monkey epithelial cells. We further show that increased abundance of the acyltransferase ZDHHC5 associates with increased S-acylation of the spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also found that the S-acylation of the spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also labelled the protein. Yet, we observed that ZDHHC5 was only modified when incubated with alkyne-palmitate, suggesting it has specificity for this acyl-CoA, and that other ZDHHC enzymes may use additional fatty acids to modify the spike protein. Since multiple ZDHHC isoforms may modify the spike protein, we also examined the ability of the FASN inhibitor TVB-3166 to prevent S-acylation of the spike proteins of SARS-CoV-2 and human CoV-229E. We show that treating cells with TVB-3166 inhibited S-acylation of expressed spike proteins and attenuated the ability of SARS-CoV-2 and human CoV-229E to spread in vitro. Our find-ings further substantiate the necessity of CoV spike protein S-acylation and demonstrate that de novo fatty acid synthesis is critical for the proper S-acyla-tion of the spike protein.

Brain arteriovenous malformations (AVMs) are a disorder wherein abnormal, enlarged blood vessels connect arteries directly to veins, without an intervening capillary bed. AVMs are one of the leading causes of hemorrhagic stroke in children and young adults. Most human sporadic brain AVMs are associated with genetic activating mutations in the KRAS gene. Our goal was to develop an in vitro model that would allow for simultaneous morphological and functional phenotypic data capture in real time during AVM disease progression. By generating human endothelial cells harboring a clinically relevant mutation found in most human patients (activating mutations within the small GTPase KRAS) and seeding them in a dynamic microfluidic cell culture system that enables vessel formation and perfusion, we demonstrate that vessels formed by KRAS4AG12V mutant endothelial cells (ECs) were significantly wider and more leaky than vascular beds formed by wild-type ECs, recapitulating key structural and functional hallmarks of human AVM pathogenesis. Immunofluorescence staining revealed a breakdown of adherens junctions in mutant KRAS vessels, leading to increased vascular permeability, a hallmark of hemorrhagic stroke. Finally, pharmacological blockade of MEK kinase activity, but not PI3K inhibition, improved endothelial barrier function (decreased permeability) without affecting vessel diameter. Collectively, our studies describe the creation of human KRAS-dependent AVM-like vessels in vitro in a self-assembling microvessel platform that is amenable to phenotypic observation and drug delivery.

Objective: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)—a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)—a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis. Conclusions: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.

The Duffy antigen receptor for chemokines (DARC, also known as atypical chemokine receptor 1) is a blood group antigen associated with susceptibility to the parasite causing malaria. Despite homology to other chemokine receptors, DARC lacks the intracellular DRY motif present on most G‐protein‐coupled receptors that is required for signaling. Human biopsy and animal studies have indicated the presence of DARC on the endothelium of post‐capillary venules where its function and regulation are unclear. The lack of literature on endothelial DARC is attributed in part to the fact that its expression disappears in cultured endothelial cells within hours of isolation from tissue. Thus, almost all studies on endothelial DARC have required in vivo approaches. However, while the in vivo work is intriguing, the ability to study endogenous endothelial DARC in vitro will be necessary to elucidate its regulation and true physiologic function. We have discovered that incubation with human whole blood for 24 hours leads to induction of DARC on cultured primary human pulmonary microvascular endothelial cells (HPMEC). DARC induced in this manner is appropriately targeted to the cell surface, as it renders endothelial cells sensitive to an extracellular leukocidin. Induction of DARC on the endothelium was sensitive to cycloheximide, implicating de novo protein synthesis, and chromatin immunoprecipitation (ChIP) assays for RNA polymerase II also confirm transcription of the gene. While exposure to whole blood for 24 hours induced endogenous endothelial DARC expression, exposure to plasma had no effect. Transwell experiments indicated that physical contact with cellular elements in blood was necessary for induction of DARC. After induction of DARC and removal of blood, we observed rapid loss of DARC protein and mRNA within 24 hours. Our preliminary experiments suggest that endothelial DARC is rapidly degraded at least in part by matrix metalloproteinases. In conclusion, DARC expression on endothelial cells is regulated by cues from the microenvironment. DARC may play an important role in the immune system and its regulation is likely to have implications for our understanding of how cultured cells maintain or lose their in situ tissue phenotype.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant to W.L.L., and N.K. was supported by the Canadian Graduate Scholarship ‐ Master’s Program (CGS‐M).

To act on tissues, circulating insulin must perfuse the relevant organ and then leave the bloodstream by crossing the endothelium-a process known as insulin delivery. It has been postulated that the continuous endothelium is a rate-limiting barrier to insulin delivery but existing data are contradictory. This conflict is in part due to the limitations of current models, including the inability to maintain a constant blood pressure in animals and the absence of shear stress in cultured cells. We developed a murine cardiac ex vivo perfusion model that delivers insulin to the heart in situ at a constant flow. We hypothesized that if the endothelial barrier were rate-limiting to insulin delivery, increasing endothelial permeability would accelerate insulin action. The kinetics of myocardial insulin action were determined in the presence or absence of agents that increased endothelial permeability. Permeability was measured using Evans Blue, which binds with high affinity to albumin. During our experiments, the myocardium remained sensitive to insulin and the vasculature retained barrier integrity. Perfusion with insulin induced Akt phosphorylation in myocytes but not in the endothelium. Infusion of platelet-activating factor or vascular endothelial growth factor significantly increased permeability to albumin without altering insulin action. Amiloride, an inhibitor of fluid-phase uptake, also did not alter insulin action. These data suggest that the endothelial barrier is not rate limiting to insulin's action in the heart; its passage out of the coronary circulation is consistent with diffusion or convection. Modulation of transendothelial transport to overcome insulin resistance is unlikely to be a viable therapeutic strategy.

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase ( FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog ( PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.

In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.