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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
- Gaengel, K., et al.
(författare)
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The Sphingosine-1-Phosphate Receptor S1PR1 Restricts Sprouting Angiogenesis by Regulating the Interplay between VE-Cadherin and VEGFR2
- 2012
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Ingår i: Developmental Cell. - : Elsevier BV. - 1534-5807 .- 1878-1551. ; 23:3, s. 587-599
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Tidskriftsartikel (refereegranskat)abstract
- Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S1PR1, a receptor for the blood-borne bioactive lipid sphingosine-1-phosphate (S1P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S1PR1 leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S1PR1 signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S1PR1 signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses.
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| 3. |
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| 4. |
- Shang, Ming-Mei, et al.
(författare)
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Lim domain binding 2 : a key driver of transendothelial migration of leukocytes and atherosclerosis
- 2014
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 34:9, s. 2068-2077
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Using a multi-tissue, genome-wide gene expression approach, we recently identified a gene module linked to the extent of human atherosclerosis. This atherosclerosis module was enriched with inherited risk for coronary and carotid artery disease (CAD) and overlapped with genes in the transendothelial migration of leukocyte (TEML) pathway. Among the atherosclerosis module genes, the transcription cofactor Lim domain binding 2 (LDB2) was the most connected in a CAD vascular wall regulatory gene network. Here, we used human genomics and atherosclerosis-prone mice to evaluate the possible role of LDB2 in TEML and atherosclerosis.APPROACH AND RESULTS: mRNA profiles generated from blood macrophages in patients with CAD were used to infer transcription factor regulatory gene networks; Ldlr(-/-)Apob(100/100) mice were used to study the effects of Ldb2 deficiency on TEML activity and atherogenesis. LDB2 was the most connected gene in a transcription factor regulatory network inferred from TEML and atherosclerosis module genes in CAD macrophages. In Ldlr(-/-)Apob(100/100) mice, loss of Ldb2 increased atherosclerotic lesion size ≈2-fold and decreased plaque stability. The exacerbated atherosclerosis was caused by increased TEML activity, as demonstrated in air-pouch and retinal vasculature models in vivo, by ex vivo perfusion of primary leukocytes, and by leukocyte migration in vitro. In THP1 cells, migration was increased by overexpression and decreased by small interfering RNA inhibition of LDB2. A functional LDB2 variant (rs10939673) was associated with the risk and extent of CAD across several cohorts.CONCLUSIONS: As a key driver of the TEML pathway in CAD macrophages, LDB2 is a novel candidate to target CAD by inhibiting the overall activity of TEML.
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