characterizing-extracellular-vesicles-from-biological-fluids

Characterizing Extracellular Vesicles from Biological Fluids

Extracellular vesicles (EVs) are structures that are produced from cells and participate in intercellular communication by transporting biomolecules from ...

University of North Texas Health Science Center

Extracellular vesicles (EVs) are lipid-bound vesicles that originate from the endosomal system or budded off from the plasma membrane. EVs are involved in cell-cell communication via transporting DNA, RNA, and proteins from one cell to another. Tear EVs (tEVs) have been reported in dry eye, Sjӧgren's Syndrome, and primary open-angle glaucoma. In this study, we sought to investigate the presence of tEVs in relation to keratoconus (KC). Tears were passively collected from the lateral meniscus from 10 healthy (5 males and 5 females) and 9 KC (4 males and 5 females) subjects. Tear samples were processed and analyzed using the ExoView™ R100. Statistical analysis was performed using a Mann-Whitney U non-parametric Student's t-test. All tEVs, in both Healthy and KC subjects, showed a CD9 dominant tEV cohort independent of sex. A significant decrease in CD63+/CD9+ and CD63+/CD81+/CD9+ was found in the male KC tEVs (p < 0.05), but not in females compared to their healthy counterparts. Neither Healthy nor KC tEVs showed differences in the total number of tEVs, however significant differences were identified between the sexes (p < 0.05), with males having a higher number of tEVs. tEVs diameters ranged from 50 to 200 nm, in both Healthy and KC cohorts, with the majority in the 50-80 nm range suggesting exosome-dominant cohorts. To our knowledge, this is the first time, to date, that tEVs have been isolated and characterized in KCs. While further studies are warranted, the tEVs differences between KC and Healthy subjects suggest a potential role for tEVs in KC pathogenesis.

Revealing the presence of tear extracellular vesicles in Keratoconus.

This study investigated the interplay between transforming growth factor beta (TGF-β1/T1 and TGF-β3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 10 cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα) and beta (ERβ), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERα, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERα, ERβ, and GnRHR. T3 caused significant upregulation in expressions PR, ERα, ERβ, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERα, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-β isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.

Novel Correlation between TGF-β1/-β3 and Hormone Receptors in the Human Corneal Stroma.

Diabetes mellitus (DM) is a common metabolic disease associated with severe macrovascular and microvascular complications that influence nearly every tissue in the body, including the anterior and posterior segments of the eye. In the cornea, DM is associated with recurrent epithelial erosion and reduced wound-healing capacity, which increases the risk of corneal scarring. We previously developed a co-culture model of the cornea consisting of immortalized human corneal epithelial cells (hCE-TJ) overlaying a self-assembled stromal layer generated by human corneal fibroblasts (hCFs) over a 4-week period. In this study, we investigated epithelial-stromal constructs generated from hCFs derived from subjects with Type 1 (T1DM) or 2 diabetes (T2DM) compared to controls. We found that T2DM constructs exhibited a disrupted epithelium and a thicker, stratified stromal layer compared to controls or T1DM. Both T1DM and T2DM stromal constructs expressed lower expression of thrombospondin-1 in isolated extracellular vesicles (EVs) compared to controls with no significant difference observed in the presence of epithelial cells, suggesting that reduced provisional matrix secretion in the corneal stroma may be a factor that promotes delayed corneal wound healing in diabetes. The tetraspanins are established extracellular vesicle (EV) markers and include CD63, CD81, and CD9, and were highly expressed by EVs in all three cell types. Control corneal stromal fibroblasts produced more and larger EVs when compared to T1DM and T2DM hCF-derived EVs, supporting a role for altered cell-cell communication in the context of DM. Further characterization of EVs and their cargo is expected to aid in the development of targeted treatments to improve corneal wound healing.

Corneal epithelial-stromal constructs to study differences associated with diabetes mellitus.

Keratoconus (KC) is the most prevalent corneal ectasia in the world and its pathogenesis is influenced by both ocular and systemic factors. This review explores the multifaceted associations between keratoconus and systemic health conditions, ocular characteristics, and various other environmental/exogenous factors, aiming to illuminate how these relationships influence the pathophysiology of the disease.

Brenna S. Hefley

North Texas Eye Research Institute,
Department of Pharmaceutical Sciences,
College of Pharmacy,
Department of Pharmaceutical Sciences, College of Pharmacy

University of North Texas Health Science Center

Characterizing Extracellular Vesicles from Biological Fluids

Brenna S. Hefley

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}North Texas Eye Research Institute,Department of Pharmaceutical Sciences,College of Pharmacy,Department of Pharmaceutical Sciences, College of Pharmacy

University of North Texas Health Science Center

Characterizing Extracellular Vesicles from Biological Fluids

North Texas Eye Research Institute,
Department of Pharmaceutical Sciences,
College of Pharmacy,
Department of Pharmaceutical Sciences, College of Pharmacy