Perlecan Maintains microvessel integrity in vivo and modulates their formation in vitro

Perlecan is a heparan sulfate proteoglycan assembled into the vascular basement membranes (BMs) during vasculogenesis. In the present study we have investigated vessel formation in mice, teratomas and embryoid bodies (EBs) in the absence of perlecan. We found that perlecan was dispensable for blood vessel formation and maturation until embryonic day (E) 12.5. At later stages of development 40% of mutant embryos showed dilated microvessels in brain and skin, which ruptured and led to severe bleedings. Surprisingly, teratomas derived from perlecan-null ES cells showed efficient contribution of perlecan-deficient endothelial cells to an apparently normal tumor vasculature. However, in perlecan-deficient EBs the area occupied by an endothelial network and the number of vessel branches were significantly diminished. Addition of FGF-2 but not VEGF165 rescued the in vitro deficiency of the mutant ES cells. Furthermore, in the absence of perlecan in the EB matrix lower levels of FGFs are bound, stored and available for cell surface presentation. Altogether these findings suggest that perlecan supports the maintenance of brain and skin subendothelial BMs and promotes vasculo- and angiogenesis by modulating FGF-2 function

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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

Polymerized-Type I Collagen Induces a High Quality Cartilage Repair in a Rat Model of Osteoarthritis

Background: Osteoarthritis (OA) is a chronic, degenerative and inflammatory disease. It is characterized by progressive deterioration of articular cartilage. It is the most common disabling rheumatic pathology in adults older than 45 years, and there is no specific treatment. Objectives: Based on the rationale that in vitro polymerized-type I collagen induces chondrocytes proliferation, up-regulates the cartilage extracellular matrix proteins and down-regulates proinflammatory cytokines, we decided to evaluate its effect on cartilage repair in a rat model of OA. Methods: Thirty Wistar male rats with partial meniscectomy were subjected to daily high impact exercise during 3 weeks. Rats were randomly allocated into 5 groups a) training control, b)sham/operated control, c) toxicity control, d) OA treated with 4 intraarticular (IA) injections of placebo, and e) OA treated with 4 IA injections of polymerized-type I collagen. Weight, temperature and thickness of the knee were measured. Histological and radiological analysis was also performed. Type I and II collagen as well as, MMP13 expression was determined by immunofluorescence. Results: Clinimorphometric analysis showed a higher temperature and thickness of the knee in OA/placebo vs. OA/polymerized-type I collagen treated rats. Radiological and histological analysis demonstrated that polymerized-type I collagen but not placebo preserved joint cavity structure and proteoglycans content and induced an increase of 2 to 4 fold type II collagenexpressing chondrocytes whereas it inhibited type I collagen and MMP13 producing chondrocytes. Conclusion: The results suggest that polymerized-type I collagen is safe and effective chondroprotective biodrug with disease modifying effects. It induces high quality cartilage repair