The perlecan fragment LG3 is a novel regulator of obliterative remodeling associated with allograft vascular rejection

RATIONALE: Endothelial apoptosis is increased in association with acute and chronic vascular rejection (VR) of solid allografts. Apoptotic endothelial cells (EC) release LG3, a C-terminal fragment of perlecan of potential importance in vascular remodeling and neointima formation. OBJECTIVE: Our 2 goals were to determine whether circulating levels of LG3 are increased in association with acute VR of renal allografts and to evaluate the impact of LG3 on vascular remodeling. METHODS AND RESULTS: We conducted a case-control study to compare serum LG3 levels in human renal transplant patients with acute VR, tubulo-interstitial rejection (ATIR) and normal graft function. Aorta transplantation between fully MHC-mismatched mice in association with intravenous LG3 injection was used to characterize the impact of LG3 on vascular remodeling. Scratch assays evaluated the promigratory activity of LG3 on vascular smooth muscle cells (VSMC) in vitro. Serum LG3 levels were significantly elevated in human renal transplant patients with acute VR (n = 16) compared to ATIR (n = 16) and normal graft function (n = 32, P = 0.004). In patients with acute VR, graft loss was associated with elevated LG3 levels. Increasing LG3 serum levels in aortic allograft recipients significantly increased neointima formation. LG3 injection fostered accumulation of \u3b1-smooth muscle actin-positive cells and decreased the number of CD31 positive EC. LG3 increased the migration of VSMC through extracellular signal-regulated kinases 1/2-dependent pathways. CONCLUSION: These results indicate that LG3 is a novel regulator of obliterative vascular remodeling during rejection.Peer reviewed: YesNRC publication: Ye

Guidelines for the use and interpretation of assays for monitoring autophagy

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