Altered Functionality of Lipoprotein(a) Impacts on Angiogenesis in Diabetic Retinopathy.

Purpose: Diabetic retinopathy (DR) is a complication of type 2 diabetes mellitus (T2DM). Lipoprotein(a) (Lp(a)) contributes to the progression of DR, but how is unclear. In homeostasis of the retinal microvasculature, myeloid-derived pro-angiogenic cells (PACs) also play a pivotal role, and fail to function properly in diabetic conditions. Here, we explored the putative contribution of Lp(a) from patients with T2DM with/without DR and healthy controls on inflammation and angiogenesis of retinal endothelial cells (RECs), and on PAC differentiation. Subsequently, we compared the lipid composition of Lp(a) from patients to that from healthy controls. Methods: Lp(a)/LDL obtained from patients and healthy controls were added to TNF-alpha-activated RECs. Expression of VCAM-1/ICAM-1 was measured using flowcytometry. Angiogenesis was determined in REC-pericyte co-cultures stimulated by pro-angiogenic growth factors. PAC differentiation from peripheral blood mononuclear cells was determined by measuring expression of PAC markers. The lipoprotein lipid composition was quantified using detailed lipidomics analysis. Results: Lp(a) from patients with DR (DR-Lp(a)) failed to block TNF-alpha-induced expression of VCAM-1/ICAM-1 in REC whereas Lp(a) from healthy controls (healthy control [HC]-Lp(a)) did. DR-Lp(a) increased REC angiogenesis more than HC-Lp(a) did. Lp(a) from patients without DR showed intermediate profiles. HC-Lp(a) reduced the expression of CD16 and CD105 in PAC, but T2DM-Lp(a) did not. Phosphatidylethanolamine content was lower in T2DM-Lp(a) than in HC-Lp(a). Conclusions: DR-Lp(a) does not show the anti-inflammatory capacity seen with HC-Lp(a), but increases REC angiogenesis, and affects PAC differentiation less than HC-Lp(a). These functional differences in Lp(a) in T2DM-related retinopathy are associated with alterations in the lipid composition as compared to healthy conditions.

Aberration in myeloid-derived pro-angiogenic cells in type-2 diabetes mellitus; implication for diabetic retinopathy?

Purpose: Diabetic retinopathy (DR) is a major microvascular complication of type 2 diabetes mellitus (T2DM). Myelomonocytic proangiogenic cells (PAC) have been implicated in DR pathogenesis, but their functional and developmental abnormalities are unclear. In this study we assessed PAC characteristics from healthy controls, T2DM patients with DR (DR) and without (NoDR) in order to determine the consequence of the diabetic condition on PAC phenotype and function, and whether these differ between DR and NoDR patients. Methods: PAC were generated by culturing PBMC on fibronectin coating and then immunophenotyped using flow cytometry. Furthermore, cells were sorted based on CD14, CD105, and CD133 expression and added to an in vitro 3-D endothelial tubule formation assay, containing GFP-expressing human retinal endothelial cells (REC), pericytes, and pro-angiogenic growth factors. Tubule formation was quantified by fluorescence microscopy and image analysis. Moreover, sorted populations were analyzed for angiogenic mediator production using a multiplex assay. Results: The expression of CD16, CD105 and CD31, but not CD133, was lower in PAC from T2DM patients with or without DR. Myeloid and non-myeloid T2DM-derived sorted populations increased REC angiogenesis in vitro as compared to control cultures. They also showed increased S100A8 secretion, decreased VEGF-A secretion, and similar levels of IL-8, HGF, and IL-3 as compared to healthy control (HC)-derived cell populations. Conclusion: T2DM PAC are phenotypically and functionally altered compared to PAC from HC. Differences between DR and NoDR PAC are limited. We propose that impaired T2DM PAC provide inadequate vascular support and promote compensatory, albeit pathological, retinal neovascularization.

Three-dimensional tubule formation assay as therapeutic screening model for ocular microvascular disorders.

PURPOSE: This study is aimed to adapt a three-dimensional (3-D) in vitro angiogenesis model to the ophthalmology field using retinal endothelial cells (REC). This system is applied to assess the angiogenic capacity of aqueous humor (AH) from patients with ocular disorders, and to test the effect of VEGF inhibitor (aflibercept) on induced angiogenesis. METHODS: Human REC and umbilical vein endothelial cells (HUVEC) and pericytes were co-cultured in a gel matrix with 25-200 ng/ml pro-angiogenic growth factors (GF). AH from patients with cataract, glaucoma or proliferative diabetic retinopathy (PDR) was tested in the REC-pericyte co-culture. Aflibercept was then introduced to the co-culture containing PDR AH. The surface area and total tubule length were measured using Image J. RESULTS: Optimal GF concentrations at 200 ng/ml induced angiogenesis by REC as well as HUVEC, while vessel formation by both cell types was strongly reduced using 25-50 ng/ml GF. Addition of AH from the PDR patient triggered tubule formation by REC at low GF concentration. Aflibercept, however, significantly inhibited angiogenesis induced by PDR AH, but showed no significant influence on other conditions. CONCLUSION: REC can be applied efficiently in the 3-D in vitro angiogenesis model as a diagnostic tool to assess the AH angiogenic status and to validate new anti-angiogenic therapeutic compounds prior to clinical trial.

Evaluation of Cardiac Size in the Common Kestrel ( Falco tinnunculus ) Based on Radiographic Measurements.

The common kestrel ( Falco tinnunculus ) is a common raptor found worldwide. To establish reference intervals for cardiac size by radiography, 14 adult common kestrels were evaluated clinically and with radiographs (lateral and ventrodorsal views). The cardiac width, thoracic width, distance between third and fourth ribs, synsacrum width, coracoid width, and distance between clavicle bones were measured on ventrodorsal radiographs. The ratio between cardiac width and the other 5 indices was calculated. Results showed a significant, positive correlation between the cardiac width and thoracic width and between cardiac width and coracoid width. The values and ratios of this study can be used as reference intervals for cardiac size evaluated radiographically in the common kestrel for diagnosis of the cardiomegaly in this bird.

A survey on intestinal parasites of golden hamster (Mesocricetus auratus) in the northeast of Iran.

The aims of this study were to provide baseline knowledge about gastrointestinal parasites in golden hamster (Mesocricetus auratus) that inhabit the Mashhad area, and to analyze possible independent variable from October 2011-August 2012. To determine the prevalence of intestinal parasites in golden hamster, faecal samples were tested specifically for nematode eggs, protozoan oocysts and sporocysts utilizing a combined sedimentation-flotation technique. In addition, all fecal samples were examined to detect oocysts of Cryptosporidium spp. using modified Ziehl-Neelsen staining. Of 100 golden hamsters, 52 % were females and 48 % males. Of all examined fecal samples of golden hamsters, 44 % (95 % CI: 34.3-53.7 %) were found to harbor at least one parasite species. The following parasites were detected (with their respective prevalence): undetermined Trichurata (42 %, 95 % CI: 29.5-48.5 %), Syphacia spp. (4.3 %, 95 % CI: 1-7.3 %). Cryptosporidium and protozoan oocysts were not found in these animals. There was no significant difference (p > 0.05) between age, sex, litter, breeding place, breeding style and anthelminthic treatment with individual helminth infection in faecal examination. This is the first record of the gastrointestinal parasites of golden hamster in Iran. Considering that hamster and other rodents are pets in many homes, the likelihood of cross-infections, particularly involving children and mainly due to unhygienic habits, should be determined.