Vitreous microparticles contain apoptotic signals suggesting a diabetic vitreopathy

INTRODUCTION

Markers used in this study include Annexin-V, CD144, and 4’,6-diamidino-2-phenylindole (DAPI). Annexin-V binds phosphatidyl-serine which is typically found on the inner leaflet of a lipid bilayer. When cells undergo apoptosis,there is a reversal to the polarity of the lipid bilayer and the phosphatidyl serine residues are externalized. Once externalized, Annexin-V can bind and identify a microparticle as Annexin-V-positive. CD144 (V-cadherin) is an endothelial cell marker. DAPI is a membrane impermeable molecule and can pass through a lipid bilayer if it has been compromised.DAPI positive MPs identified by flow cytometry indicate that the MP membrane has been compromised and DAPI has entered the MP. Once inside the MP, DAPI binds nucleic acids whether DNA or RNA.

Microparticles (MPs) are small vesicles bound by a lipid bilayer that pinch off the surface of a cell membrane. The size of MPs range from 0.03-1.0 μm. Exosomes, or membranebound vesicles released by a cell, are formed in the Golgi apparatus or lysosomes, and range in size from 0.03-0.1 μm in diameter; while apoptotic bodies tend to be larger, measuring at the 0.05‐2.0 μm range. MPs consist of fluid and carry a variety of lipid, protein, and nucleic acid cargo including mRNA and micro‐RNAs (miRNA) which can have downstream effects at remote target locations. Baruttadescribed differences in serum miRNAs within extracellular vesicles between patients with diabetes, and found that patients with significantly lower levels of miR-126 within serum extracellular vesicles (EVs)were found to have microvascular complications including diabetic nephropathy. The function of MPs both systemically and within the eye remains an area of intense investigation.There is clear evidence that the pathogenesis of diabetic macular edema and diabetic retinopathy stem from pericyte dysfunction, vascular leakage, and a violation of the bloodretinal-barrier. Extravasation of serum contents into the retina and vitreous may alter the microenvironment of the posterior segment which may lead to exacerbation of retinopathy. Other measures of systemic hyperpermeability such as urinary albumin excretion rate was found to be directly correlated to optical coherence tomography (OCT) macular thickness in patients with diabetes. As such, the presence of serum contents in the posterior segment as a result of retinal vascular hyperpermeability may play a role in disease pathogenesis.

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Further clinical and basic-science investigations are evaluating the role of the vitreous in various diseases. The Diabetic Retinopathy Clinical Research (DRCR) Network Protocol D evaluated the role of primary vitrectomy in the treatment of diabetic macular edema. In an era when 20-gauge vitrectomy was commonplace and without a consensus regarding the peeling of macular membranes, the outcomes for Protocol D demonstrated that even in patients that had failed conventional therapy for macular edema, up to half of study participants had some short-term improvement in visual acuity and in OCT thickness. This study, however, was a small cohort study that was not designed to address the role of the vitreous in diabetic disease. Protocol AB, which compares prompt vitrectomy to anti-VEGF treatment, will evaluate the role of small-gauge vitrectomy as a primary treatment modality for proliferative diabetic retinopathy. Follow-up for the study is currently underway. The DRIVE-UK study evaluated outcomes following vitrectomy in patients with diabetic eye disease. Patients with mild vitreous hemorrhage treated with vitrectomy had better outcomes than those treated for tractional retinal detachments supporting the concept that the removal of a diseased vitreous may improve future visual outcomes.Vitreous MPs have been evaluated in a few case series.Guptahave evaluated extracellular vesicles using a non-formalin fixative in combination with various imaging techniques including nanoparticle tracking analysis and confocal microscopy. Another group has shown greater concentrations of vitreous MPs and proinflammatory cytokines in patients with rhegmatogenous retinal detachments compared to controls. Exosomes are abundantly expressed in the vitreous. The presence of sub-cellular membrane-derived vesicles in the vitreous with different markers suggests that these vesicles play a role in vitreoretinal pathophysiology. The aim of the current study was two-fold: to optimize the protocol for isolating and identifying human vitreous MPs by flow cytometric analysis, and to identify differences in these MPs between patients with and without diabetes.

The vitreous body fills the posterior segment of the eye and is composed primarily of collagen and hyaluronic acid. These extracellular matrix-molecules are synthesized by hyalocytes, the resident cells of the vitreous. Many disease processes will manifest in the vitreous; for example,uveitis may present secondarily in the form of vitreous cells,or proliferative diabetic retinopathy may present in the form of vitreous hemorrhage or tractional retinal detachment.The vitreous itself, however, has not been evaluated as a primary source for disease pathogenesis. To begin elucidating the role of the vitreous in various diseases, membranebound extracellular vesicles released by hyalocytes have been examined to explore their potential role in disease pathogenesis.

SUBJECTS AND METHODS

This study was approved by the Human Research Protections Office (HRPO) at the Washington University Institutional Review Board (IRB) and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all subjects.

There is convincing evidence that vitreous components play a role in disease pathogenesis relevant to therapy. Physicians use anti-vascular endothelial growth factor (VEGF) agents to treat diverse retinal vascular diseases including diabetic retinopathy and age-related macular degenerationto target elevated levels of VEGF in the posterior segment of the eye. The ability of an intravitreal injection to improve visual outcomes in these patients has revolutionized the way we treat these diseases.As such, the vitreous can serve as a reservoir for diseaseassociated or causal molecules that could potentially be future therapeutic targets or biomarkers.

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Inclusion criteria included eyes undergoing primary vitrectomy for any disease process except uveitis. A chart review was performed to acquire historical data such as presence and absence of diabetes mellitus and the reason for surgery. The patients underwent standard 3-port trans-conjunctival vitrectomy with sterile precautions. Three 25-gauge trocars were inserted into the eye and the vitrector was inserted into the mid-vitreous cavity. An assistant would aspirate the vitreous into a 3 mL syringe to an appropriate volume prior to the visualization of choroidals or ocular collapse. For the initial 9 patients, cut rates varied between 1000 cuts per minute (cpm) to 7500 cpm. Subsequent to this,future specimens were cut at 1000 cpm. Specimens were transferred into freezer-appropriate 3 mL vials and immediately placed on dry ice and later placed into a -80°C freezer.

In order to optimize MP signal for flow cytometry, it was important to titrate parameters to attain the best possible signal. This signal is the ability for the cytometer to identify a single MP, as opposed to two MPs(doublets) that may be stuck together. When a cytometer reads a doublet, the amount of fluorescence emitted by that particle is therefore an aberrant reading since two MPs would have more fluorescence together than a single MP alone and can alter specimen interpretation. Singlets were defined as a population of particles within a 20K-unit difference in sidescatter-width that may span a range of side-scatter-area-levels,while doublets and other multiplets were particles with greater side-scatter-width values. Vitrectomy cut rates were titrated between 1000 and 7500 cpm to identify cut rates with optimal singlet yield.

由于各高校實際情況的不同，實驗班體制的設定也有很大差異。根據具體高校的教育資源和師資力量的不同，實驗班的開展主要采取具體的實體班級制度或虛擬班級制度。以湖南大學為例，實體班級制度與前文的實驗班的開展略有差別，由學生所在院系和合作院系共同管理，與前文實驗班的管理模式差別不大不作贅述；其中虛擬班級制度則是在學生入校之初便按照所報專業作為主專業進入院系學習，由主專業所在院系教學。學生的課程學習則是按照個人傾向的第二專業，由第二專業所在的院系負責課程方案的設定。……