Effect of vitamin B12 supplementation on retinal lesions in diabetic rats.

Purpose: Diabetic retinopathy (DR) is the most common complication of diabetes involving microvasculature and neuronal alterations in the retina. Previously, we reported that vitamin B12 deficiency could be an independent risk factor for DR in humans. However, the effect of vitamin B12 supplementation in experimental DR is unknown. Thus, in this study, we investigated the impact of dietary supplementation of vitamin B12 on retinal changes in diabetic rats. Methods: Diabetes was induced in 2-month-old Sprague-Dawley rats and maintained for 4 months. One group of diabetic rats were fed normal levels of vitamin B12, and one group double the quantity of vitamin B12 (50 µg/kg diet). Vitamin B12 and homocysteine levels in the plasma were analyzed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC), respectively. At the end of 4 months of experimentation, the eyeballs were collected. Retinal changes were analyzed with hematoxylin and eosin (H&E) staining, immunoblotting, and immunofluorescence methods. Results: Dietary supplementation of vitamin B12 had no effect on food intake, bodyweight, fasting blood glucose, and plasma homocysteine levels in the diabetic rats. However, vitamin B12 supplementation prevented loss of rhodopsin, and overexpression of VEGF, and completely prevented overexpression of HIF1α, GFAP, and endoplasmic reticulum (ER) stress markers (GRP78, ATF6α, XBP1, CHOP, and caspase 12) in the diabetic rat retina. Further, vitamin B12 ameliorated apoptosis in the retina as shown with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and prevented retinal thinning. Conclusions: Vitamin B12 supplementation of diabetic rats appeared to be beneficial by circumventing retinal hypoxia, VEGF overexpression, and ER stress-mediated cell death in the retina. The present study adds another potential therapeutic strategy of vitamin B12 in diabetes.

Circulating proteolytic signatures of chemotherapy-induced cell death in humans discovered by N-terminal labeling.

It is known that many chemotherapeutics induce cellular apoptosis over hours to days. During apoptosis, numerous cellular proteases are activated, most canonically the caspases. We speculated that detection of proteolytic fragments released from apoptotic cells into the peripheral blood may serve as a unique indicator of chemotherapy-induced cell death. Here we used an enzymatic labeling process to positively enrich free peptide α-amines in the plasma of hematologic malignancy patients soon after beginning treatment. This N-terminomic approach largely avoids interference by high-abundance proteins that complicate traditional plasma proteomic analyses. Significantly, by mass spectrometry methods, we found strong biological signatures of apoptosis directly in the postchemotherapy plasma, including numerous caspase-cleaved peptides as well as relevant peptides from apoptotic and cell-stress proteins second mitochondria-derived activator of caspases, HtrA serine peptidase 2, and activating transcription factor 6. We also treated hematologic cancer cell lines with clinically relevant chemotherapeutics and monitored proteolytic fragments released into the media. Remarkably, many of these peptides coincided with those found in patient samples. Overall, we identified 153 proteolytic peptides in postchemotherapy patient plasma as potential indicators of cellular apoptosis. Through targeted quantitative proteomics, we verified that many of these peptides were indeed increased post- vs. prechemotherapy in additional patients. Our findings reveal that numerous proteolytic fragments are released from dying tumor cells. Monitoring posttreatment proteolysis may lead to a novel class of inexpensive, rapid biomarkers of cell death.