TY - JOUR
T1 - Vitreous microparticles contain apoptotic signals suggesting a diabetic vitreopathy
AU - Sultan, Harris
AU - Rajagopal, Rithwick
AU - Rao, Prabakar Kumar
AU - Piggott, Kisha Deslee
AU - Paley, Michael A.
AU - Hassman, Lynn Marisa
AU - Li, Albert S.
AU - Marshall, Brigid
AU - Apte, Rajendra Shridhar
N1 - Funding Information:
In conclusion, vitreous MPs reflect changes seen in progressive diabetic retinopathy including those seen with vitreous hemorrhage and proliferative diabetic retinopathy. The presence of blood in the vitreous of patients with diabetes may contribute to a diseased vitreous by evidence of increased Annexin-V+ MPs, a sign of cellular apoptosis. Significant work is needed to further understand the role and function of MPs in the vitreous and how they may contribute to progression of diabetic eye disease. Limitations Limitations to this study include the relatively small sample sizes in group comparisons. This study is also un-masked which may bias the results. Limitation in sample collection includes the variability in aspiration. Standardized aspiration rates have not been established as it is an assistant that collects the specimen in the 3 mL syringe. Despite establishing a vitrectomy cut-rate, person-to-person differences in aspiration rate can also change MP singlet populations which was not controlled in this study. Most of the current analysis was done on vitreous specimens that were frozen. The current microparticle literature demonstrates that freezing MPs may alter MP integrity and artifactually increase the number of MPs seen by flow cytometry known as MP-fracture[27]. Due to the labor intensiveness of processing and evaluating samples the same day as sample collection, the majority of samples were still evaluated with a single freeze-thaw cycle, and thus the results should be interpreted with caution. A significant amount of clinical MP literature, however, supports a protocol where specimens have undergone a single freeze-thaw cycle. The BD LSR II flow cytometer has a lower limit of detection at 0.488 µm, the wavelength of its forward scatter laser. This study only evaluates MPs that are between detectable range through 2.0 µm but not those that are smaller, in the 0.03 µm range. This study may bias its results towards MPs that are larger and neglecting smaller MPs that are beyond the lower detection-limit of the cytometer. Sample size calculations were not done to make the conclusions seen in the study and thus must be considered exploratory. ACKNOWLEDGEMENTS Foundation: Supported by an unrestricted grant to Washington University by Research to Prevent Blindness Inc., New York, NY. Conflicts of Interest: Sultan H, None; Rajagopal R, None; Rao PK, None; Piggott KD, None; Paley MA, None; Hassman LM, None; Li AS, None; Marshall B, None; Apte RS, None.
Publisher Copyright:
© 2022 International Journal of Ophthalmology (c/o Editorial Office). All rights reserved.
PY - 2022
Y1 - 2022
N2 - AIM: To evaluate differences in microparticle profiles in vitreous samples between diabetic and non-diabetic eyes undergoing vitrectomy. METHODS: Un-masked cross-sectional series of 34 eyes undergoing vitrectomy. Vitreous specimens were collected and processed to evaluate for membrane integrity (DAPI), apoptosis (Annexin-V), and endothelial-cell origin (V-Cadherin). A BD LSR II flow cytometer was used for analysis and standardized sub-micron-sized beads were used for size comparison. RESULTS: Thirty-four specimens underwent analysis. Greater levels of Annexin-V were found on microparticles from specimens in which blood had entered the vitreous (n=12) compared to those without blood (n=22; 52.3%±30.7% vs 19.6%±27.2%, P=0.002). Patients with diabetes having surgery with hemorrhage (n=7) had greater expression of Annexin-V than those without hemorrhage (n=8; 62.1%±31.7% vs 18.9%±20.9%, P=0.009). However, in patients with non-diabetic vitreous hemorrhage, the level of Annexin-V expression was not significantly different compared to other disease processes (38.6%±25.7%, n=5 vs 20.0%±30.9%, n=14, P=0.087). CONCLUSION: Increased expression of the apoptotic marker, Annexin-V is detected on vitreous microparticles in diabetes-related vitreous hemorrhage. When evaluating vitreous hemorrhage in patients without diabetes, the apoptotic signal is not significantly different. Vitrectomy in patients with diabetes, and improvement in visual outcomes, may be related to the removal of a serum-derived, pro-apoptotic vitreous. Further investigation is warranted in order to identify the molecular characteristics of microparticles that regulate disease.
AB - AIM: To evaluate differences in microparticle profiles in vitreous samples between diabetic and non-diabetic eyes undergoing vitrectomy. METHODS: Un-masked cross-sectional series of 34 eyes undergoing vitrectomy. Vitreous specimens were collected and processed to evaluate for membrane integrity (DAPI), apoptosis (Annexin-V), and endothelial-cell origin (V-Cadherin). A BD LSR II flow cytometer was used for analysis and standardized sub-micron-sized beads were used for size comparison. RESULTS: Thirty-four specimens underwent analysis. Greater levels of Annexin-V were found on microparticles from specimens in which blood had entered the vitreous (n=12) compared to those without blood (n=22; 52.3%±30.7% vs 19.6%±27.2%, P=0.002). Patients with diabetes having surgery with hemorrhage (n=7) had greater expression of Annexin-V than those without hemorrhage (n=8; 62.1%±31.7% vs 18.9%±20.9%, P=0.009). However, in patients with non-diabetic vitreous hemorrhage, the level of Annexin-V expression was not significantly different compared to other disease processes (38.6%±25.7%, n=5 vs 20.0%±30.9%, n=14, P=0.087). CONCLUSION: Increased expression of the apoptotic marker, Annexin-V is detected on vitreous microparticles in diabetes-related vitreous hemorrhage. When evaluating vitreous hemorrhage in patients without diabetes, the apoptotic signal is not significantly different. Vitrectomy in patients with diabetes, and improvement in visual outcomes, may be related to the removal of a serum-derived, pro-apoptotic vitreous. Further investigation is warranted in order to identify the molecular characteristics of microparticles that regulate disease.
KW - Apoptosis
KW - Cell-derived microparticles
KW - Diabetes mellitus
KW - Vitrectomy
KW - Vitreous
UR - http://www.scopus.com/inward/record.url?scp=85123061979&partnerID=8YFLogxK
U2 - 10.18240/ijo.2022.01.14
DO - 10.18240/ijo.2022.01.14
M3 - Article
C2 - 35047362
AN - SCOPUS:85123061979
VL - 15
SP - 89
EP - 97
JO - International Journal of Ophthalmology
JF - International Journal of Ophthalmology
SN - 2222-3959
IS - 1
ER -