Diabetic hyperglycemia-induced glycation regulates tumor vasculature barrier integrity via NF-K b-mediated tumor cell angiogenic cytokine secretion

Introduction::
Diabetes is a complex disease associated with hyperglycemia. Clinical studies suggest diabetes leads to pathogenic tumor vasculature. However, the mechanistic link between diabetes and tumor vasculature function remains unclear. We previously showed that hyperglycemia promotes glycation to induce mechanical signaling by stiffening tumor extracellular matrix (ECM) and chemical signaling through formation of advanced glycation end products (AGEs). As both ECM stiffness and AGE signaling through RAGEs have been implicated in regulation of endothelial barrier integrity, we hypothesize that hyperglycemia-induced glycation regulates tumor vasculature function via ECM stiffening and AGE-RAGE mediated tumor cell angiogenic cytokine secretion.




Materials and Methods::
For in vivo studies, MMTV-PyMT female mice were given a high-fat diet and Streptozotocin to induce hyperglycemia and alagebrium or aminoguanidine to inhibit glycation. Tumors were sectioned and stained for CD31 to quantify vessel density. Vessel permeability was measured by injecting mice with 2 MDa FITC and Evans Blue and measuring the extravasation distance of Evans Blue. For ex ovo studies, collagen gels glycated with ribose, with or without MDA-MB-231 cells (MDAs), were placed onto the chick chorioallantoic membrane (CAM). Neovessels permeability was quantified by co-injecting embryos with 72 kDa TRITC-dextran and 2 MDa FITC-dextran and measuring the normalized signal intensity. The secretome of MDAs on PA gels treated with or without AGEs was assayed using a cytokine array. GM-CSF secretion of MDAs was measured via ELISA. NF-κB activity was quantified by measuring the nuclear NF-κB signal. HUVEC monolayers were treated with conditioned medium from control or NF-κB inhibitor-treated MDAs to study permeability.




Results, Conclusions, and Discussions::
Results and Discussion Hyperglycemia significantly increased vessel permeability, and inhibition of glycation decreased tumor vessel permeability, suggesting that hyperglycemia-mediated glycation impairs endothelial barrier function (Fig. 1A-B). While we previously showed matrix stiffness directly increases vessel permeability, the role of tumor cells in this process remains unclear. To address this issue, we employed the CAM model and found that permeability within glycated on-plants seeded with MDAs was much higher than non-glycated or acellular gels (Fig. 1C), implying that MDAs secrete factors in response to glycation that induce increased tumor vascular phenotype. Thus, we next examined the secretome of MDAs on PA gels with different stiffnesses and AGE treatments. We found that GM-CSF was one of the most significantly upregulated factors in cells exposed to high stiffness and glycation. Inhibiting Rho/ROCK signaling via Y27632 (Y27) lowered GM-CSF secretion, and this effect continued when Y27 was combined with ZM1, an AGE-RAGE inhibitor (Fig. 1D-E). Additionally, we observed that MDAs on stiffer substrates exposed to AGEs had significantly higher NF-κB activation while treating cells with Y27 or ZM1 decreased NF-κB activation (Fig. 1F). Furthermore, cells on stiffer substrates had higher GM-CSF secretion, and NF-κB inhibition significantly decreased GM-CSF secretion (Fig. 1G). Finally, we found that conditioned medium from MDAs treated with an NF-κB inhibitor decreased the permeability of HUVEC monolayers (Fig. 1H). These results suggest that glycation-mediated ECM stiffening and activated AGE-RAGE signaling impair endothelial barrier function via NF-κB-mediated GM-CSF secretion.




Conclusion Our study demonstrates that hyperglycemia regulates tumor vasculature integrity via stiffening tumor ECM and activating AGE-RAGE signaling. Our data describe a novel mechanism by which diabetic hyperglycemia alters tumor vasculature barrier function and highlights a key role for matrix stiffness within this regulation.




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