In vitro evaluation of platelet extracellular vesicles (PEVs) for corneal endothelial regeneration.

Corneal endothelial cells (CECs) slowly decrease in number with increasing age, which is a clinical issue as these cells have very limited regenerative ability. Therapeutic platelet biomaterials are increasingly used in regenerative medicine and cell therapy because of their safety, cost-effective manufacture, and global availability from collected platelet concentrates (PCs). Platelet extracellular vesicles (PEVs) are a complex mixture of potent bioactive vesicles rich in molecules believed to be instrumental in tissue repair and regeneration. In this study we investigated the feasibility of using a PEVs preparation as an innovative regenerative biotherapy for corneal endothelial dysfunction. The PEVs were isolated from clinical-grade human PC supernatants by 20,000 × g ultracentrifugation and resuspension. PEVs exhibited a regular, fairly rounded shape, with an average size of <200 nm and were present at a concentration of approximately 1011 /mL. PEVs expressed cluster of differentiation 41 (CD41) and CD61, characteristic platelets membrane markers, and CD9 and CD63. ELISA and LC-MS/MS proteomic analyses revealed that the PEVs contained mixtures of growth factors and multiple other trophic factors, as well as proteins related to extracellular exosomes with functional activities associated with cell cadherin and adherens pathways. CECs treated with PEVs showed increased viability, an enhanced wound-healing rate, stronger proliferation markers, and an improved adhesion rate. PEVs did not exert cellular toxicity as evidenced by the maintenance of cellular morphology and preservation of corneal endothelial proteins. These findings clearly support further investigations of PEV biomaterials in animal models for translation as a new CEC regeneration biotherapy.

A purified human platelet pellet lysate rich in neurotrophic factors and antioxidants repairs and protects corneal endothelial cells from oxidative stress.

Human platelet lysate (HPL) is a complex mixture of potent bioactive molecules instrumental in tissue repair and regeneration. Due to their remarkable safety, cost-effective production, and availability at global level from collected platelet concentrates, HPLs can become a powerful biotherapy for various therapeutic applications, if standardized and carefully validated through pre-clinical and clinical studies. In this work, the possibility to use a tailor-made HPL as a corneal transplant alternative to treat the gradual decrease in the number of corneal endothelial cells (CECs) associated with aging, was evaluated. The HPL preparation was thoroughly characterized using various proteomics tools that revealed a remarkable richness in multiple growth factors and antioxidants. Treatment of B4G12 and BCE C/D-1b CECs with the HPL increased their viability, enhanced the wound closure rate, and maintained cell growth and typical hexagonal morphology. Besides, this HPL significantly protected against tert-butyl hydroperoxide (TBHP)-induced oxidative stress as evidenced by increasing CEC viability, decreased cell death and reactive oxygen species formation, and enhanced antioxidant capacity. Proteomics analysis of treated CECs confirmed that HPL treatment triggered the corneal healing pathway and enhanced oxidative stress. These data strongly support further pre-clinical evaluation of this tailor-made HPL as a novel CEC regeneration biotherapy. HPL treatment may eventually represent a pragmatic and cost-effective alternative to corneal transplant to treat damages of the corneal endothelium which is a major cause of blindness worldwide.

Extensive characterization of the composition and functional activities of five preparations of human platelet lysates for dedicated clinical uses.

Human platelet lysates (HPLs), rich in various growth factors and cell growth-promoting molecules, encompass a new range of blood products that are being used for regenerative medicine, cell therapies, and tissue engineering. Well-characterized dedicated preparations, tailor-made to best fit specific therapeutic applications, are needed for optimal clinical efficacy and safety. Here, five types of HPL were prepared from the same platelet concentrates and extensively characterized to determine and compare their proteins, growth factors, cytokines, biochemical profiles, thrombin-generating capacities, thrombin-associated proteolytic activities, phospholipid-associated procoagulant potential, contents of extracellular vesicles expressing phosphatidylserine and tissue factor, and antioxidative properties. Our results revealed that all five HPL preparations contained detectable supraphysiological levels, in the ca. 0.1 ~ 350-ng/ml range, of all growth factors assessed, except insulin-like growth factor-1 detected only in HPL containing plasma. There were significant differences observed among these HPLs in total protein content, fibrinogen, complement components C3 and C4, albumin, and immunoglobulin G, and, most importantly, in their functional coagulant and procoagulant activities and antioxidative capacities. Our data revealed that the biochemical and functional properties of HPL preparations greatly vary depending upon their mode of production, with potential impacts on the safety and efficacy for certain clinical indications. Modes of preparation of HPLs should be carefully designed, and the product properties carefully evaluated based on the intended therapeutic use to ensure optimal clinical outcomes.

Estimated Resting Metabolic Rate and Body Composition Measures Are Strongly Associated With Diabetic Retinopathy in Indonesian Adults With Type 2 Diabetes.

OBJECTIVE: To investigate the associations of estimated resting metabolic rate (RMR), body fat (BF), subcutaneous fat (SCF), visceral fat (VF), fat-free mass (FFM) percentage, BMI, and waist circumference (WC) with diabetic retinopathy (DR) in Indonesian adults with type 2 diabetes. RESEARCH DESIGN AND METHODS: This was a community-based cross-sectional study of 1,184 subjects with type 2 diabetes. DR was assessed from fundus photography and categorized as mild, moderate nonproliferative DR (NPDR), and vision-threatening DR (VTDR). RMR and body composition parameters were measured using automated body composition scan. Logistic regression with semipartial correlation analysis was used. RESULTS: DR and VTDR were present in 43.1 and 26.3% of participants, respectively. After adjustment for age, sex, diabetes duration, fasting glucose, systolic blood pressure, smoking, diabetic ulcer, and use of combined diabetes treatment, per SD increase in RMR (odds ratio [OR] 2.60 [95% CI 2.19-3.07]; P < 0.001) was associated with DR, while per SD increases in BF (0.66 [95% CI 0.56-0.78]; P < 0.001), FFM (0.69 [0.57-0.84]; P < 0.001), VF (0.77 [0.67-0.88]; P < 0.001), BMI (0.83 [0.73-0.94]; P = 0.004), and WC (0.81 [0.73-0.91]; P < 0.001) were inversely associated with presence of DR. Similar associations were found for VTDR. Among all variables, RMR had the largest contribution to the variance in the DR model (39%). CONCLUSIONS: In this study, RMR and body composition measures were strongly associated with and contributed considerably to the presence and severity of DR. These findings, if confirmed, suggest that RMR and body composition may be strong markers that represent actual metabolic state in the pathophysiology of DR.

Prevalence of Diabetic Retinopathy and Blindness in Indonesian Adults With Type 2 Diabetes.

PURPOSE: To report the prevalence of diabetic retinopathy (DR) and DR-related blindness in an Indonesian population with type 2 diabetes. METHODS: Design: Population-based cross-sectional study. SETTING: Community health centers. STUDY POPULATION: We recruited 1184 people aged older than 30 years with type 2 diabetes residing in Jogjakarta, Indonesia. Multistage, clustered random sampling based on regencies and districts in Jogjakarta was used. OBSERVATION PROCEDURE: Detailed interviews, general and eye examinations, and anthropometric measurement were performed. Disc- and macula-centered retinal photographs were taken to assess DR. The definition of DR followed a modified Airlie House classification system and was categorized into mild, moderate, and vision-threatening DR (VTDR). MAIN OUTCOME MEASURE(S): Prevalence and severity of DR. RESULTS: The median (range) age and diabetes duration of participants was 59 (52-65) and 4 (2-9) years. The prevalence of DR was 43.1% (95% confidence interval 39.6%-46.6%), with mild, moderate, and severe NPDR and PDR to be 9.41%, 7.46%, 11.1%, and 12.1%, respectively. The prevalence of VTDR was 26.3% (23.1%-29.5%). Longer diabetes duration, higher fasting glucose, presence of hypertension, and foot ulcers were associated with DR and VTDR. The prevalence of bilateral blindness was 4% and 7.7% in persons with DR and VTDR. CONCLUSIONS: This study reports a high prevalence of any DR and VTDR among Indonesian adults with type 2 diabetes in urban and rural areas: approximately 1 in 4 adults with diabetes had VTDR and 1 in 12 of those with VTDR was bilaterally blind, suggesting the need for appropriate screening and management of DR among the Indonesian population.

Rationale and Methodology for a Community-Based Study of Diabetic Retinopathy in an Indonesian Population with Type 2 Diabetes Mellitus: The Jogjakarta Eye Diabetic Study in the Community.

PURPOSE: There are no available data about diabetic retinopathy (DR) in the Indonesian population. This report summarizes the rationale and study design of the Jogjakarta Eye Diabetic Study in the Community (JOGED.COM), a community-based study to estimate the prevalence and risk factors of DR in persons with type 2 diabetes in Jogjakarta, Indonesia. METHODS: The JOGED.COM aimed to examine a cross-sectional sample of 1200 persons with type 2 diabetes aged 30 years and older residing in the study area. We identified 121 community health centers (CHCs) in Jogjakarta and listed 35 CHCs with non-communicable diseases facilities. Multi-stage, clustered random sampling was used to select 22 CHCs randomly. We included CHCs with coverage population >30,000, and excluded those classified as 100% rural. Lists of persons with diabetes confirmed by their family physician were provided from each CHC. Examinations procedures included detailed interviews, general and eye examinations, anthropometry and body composition scan, and dilated fundus photography. RESULTS: We collaborated with local health authorities, family physicians, and local health practitioners in the recruitment phase. A total of 1435 invitations were distributed, and 1184 people (82.5%) with type 2 diabetes participated in this study, of whom 1138 (79.3%) had completed data with gradable retinal images. CONCLUSIONS: JOGED.COM is the first epidemiologic study of DR in an Indonesian population. This study will provide key information about the prevalence and risk factors of DR in the community. These data are very important for future health promotion programs to reduce the burden of DR in the population.