The effect of intravitreal anti-vascular endothelial growth factor injections on corneal endothelium in patients with diabetic macular oedema.

PURPOSE: To evaluate the characteristics of corneal parameters in patients with diabetic macular oedema (DME) treated with intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections. METHODS: Participants in this study were 36 patients with DME, treated with either intravitreal ranibizumab (n = 16) or aflibercept (n = 20). All participants underwent best-corrected visual acuity (BCVA) measurement, optical coherence tomography and non-contact specular microscopy to evaluate corneal endothelium parameters (endothelial cell density-ECD, hexagonality, coefficient of variation of the cell size and central corneal thickness-CCT), at baseline and at months 6 and 12 after the first intravitreal injection. Comparisons between baseline and months 6 and 12 were performed. RESULTS: There was no statistically significant difference regarding ECD, hexagonality, coefficient of variation of the cell size and CCT at month 6 and 12 post initial injection compared to baseline in patients with DME. BCVA improved significantly at month 6 and 12 compared to baseline (p < 0.001 for both comparisons). Central retinal thickness was significantly reduced at month 6 and 12 compared to baseline (p < 0.001 for both comparisons). CONCLUSION: Intravitreal anti-VEGF injections in patients with DME were found not to affect corneal parameters, namely ECD, hexagonality, coefficient of variation of the cell size and CCT at the long-term follow-up of 12 months.

The Effect of Diabetes Mellitus on Corneal Endothelial Cells and Central Corneal Thickness: A Case-Control Study.

PURPOSE: The aim of this study was to evaluate the characteristics of corneal endothelial cells and central corneal thickness (CCT) in patients with diabetes mellitus (DM), comparing them with those of healthy subjects (controls) and to determine potential factors affecting the corneal parameters in patients with DM. METHODS: Participants in this study were 72 patients with DM and 88 healthy controls. Diabetic patients were further classified into groups depending on the severity of diabetic retinopathy (no retinopathy, mild, moderate, severe non-proliferative diabetic retinopathy, and proliferative diabetic retinopathy). All participants underwent non-contact specular microscopy to evaluate corneal endothelium parameters and CCT, while factors affecting endothelial cell density and CCT in patients with DM were also analyzed. RESULTS: Patients with DM presented significantly decreased endothelial cell density compared to controls (2,297.9 ± 311.3 and 2,518.3 ± 243.7 cells/mm2, respectively; p < 0.001), while the two groups did not differ significantly in any other measured corneal parameter. In the diabetic group, the multivariate analysis showed a significant association between decreased endothelial cell density and increased HbA1c (p < 0.001), longer DM duration (p = 0.003), and more severe diabetic retinopathy status (p = 0.008). CONCLUSION: DM seems to affect the corneal endothelium, since endothelial cell density was decreased in the diabetic group, while duration of disease, HbA1c levels, and severity of retinopathy were significantly associated with changes in endothelial cell density and should be taken into account.

VEGFR-2 reduces while combined VEGFR-2 and -3 signaling increases inflammation in apical periodontitis.

BACKGROUND: In apical periodontitis, oral pathogens provoke an inflammatory response in the apical area that induces bone resorptive lesions. In inflammation, angio- and lymphangiogenesis take place. Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in these processes and are expressed in immune cells and endothelial cells in the lesions. OBJECTIVE: We aimed at testing the role of VEGFR-2 and -3 in periapical lesion development and investigated their role in lymphangiogenesis in the draining lymph nodes. DESIGN: We induced lesions by pulp exposure in the lower first molars of C57BL/6 mice. The mice received IgG injections or blocking antibodies against VEGFR-2 (anti-R2), VEGFR-3 (anti-R3), or combined VEGFR-2 and -3, starting on day 0 until day 10 or 21 post-exposure. RESULTS: Lesions developed faster in the anti-R2 and anti-R3 group than in the control and anti-R2/R3 groups. In the anti-R2 group, a strong inflammatory response was found expressed as increased number of neutrophils and osteoclasts. A decreased level of pro-inflammatory cytokines was found in the anti-R2/R3 group. Lymphangiogenesis in the draining lymph nodes was inhibited after blocking of VEGFR-2 and/or -3, while the largest lymph node size was seen after anti-R2 treatment. CONCLUSIONS: We demonstrate an anti-inflammatory effect of VEGFR-2 signaling in periapical lesions which seems to involve neutrophil regulation and is independent of angiogenesis. Combined signaling of VEGFR-2 and -3 has a pro-inflammatory effect. Lymph node lymphangiogenesis is promoted through activation of VEGFR-2 and/or VEGFR-3.

Determination of lymph flow in murine oral mucosa using depot clearance of near-infrared-labeled albumin.

The lymphatic vessels are playing an important role in inflammation since they return extravasated fluid, proteins, and cells back into the circulation and regulate immune cell trafficking. The oral mucosa, including gingiva, is well supplied with lymphatic vessels and is frequently challenged with inflammatory insults. Lymphatic vessels in gingiva protect against periodontal disease development, but quantification of lymph flow in this area has so far never been performed, due to lack of reliable methods. Mice of FVB strain (n=17) were anesthetized with isoflurane and placed on a jaw retraction board allowing the mouth to be kept open and stable. Albumin conjugated with Alexa680-fluorochrome (with or without LPS from Porphyromonas gingivalis) was injected superficially in oral mucosa mesio-buccal to the left first molar in each mouse. 60 min post-injection the mouse was transferred to an OptixMX3 optical imager where the total fluorescence was measured in the posterior facial area. The measurements continued further every 60 min for 7h for each mouse. The mice were awake and active between measurements. The in vivo washout of Alexa680-albumin was calculated using the natural logarithm of the relative values creating a negative slope for each mouse. Statistical analysis of variance was performed. The injection and distribution site for tracer was verified with India ink and shown to be in the interstitium below the oral mucosal epithelium, in an area well supplied with initial lymphatic vessels. Washout of the tracer Alexa680-albumin was log-linear, and the basal lymph flow calculated from depot clearance averaged -0.28 ± 0.08%/min (n=8). The clearance was significantly faster (-0.30 ± 0.08%/min, n=9) in acutely inflamed oral mucosa (p=0.0326). We developed a method that can successfully quantify the lymph flow in oral mucosa in steady state conditions and under acute perturbation. By use of this method, new information about the lymphatic function in oral mucosa during physiological and pathological conditions can be achieved.

Leukocyte transmigration into tissue-engineered constructs is influenced by endothelial cells through Toll-like receptor signaling.

INTRODUCTION: Inflammation plays a crucial role in tissue regeneration, wound healing, and the success of tissue-engineered constructs. The aim of this study was to investigate the influence of human umbilical vein endothelial cells (ECs) on leukocyte transmigration when co-cultured with primary human bone marrow-derived multipotent stromal cells (MSCs). METHODS: MSCs with and without ECs were cultured in poly (L-lactide-co-1, 5-dioxepan-2-one) (poly (LLA-co-DXO)) scaffolds for 1 week in vitro in a bioreactor system, after which they were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. After 1 and 3 weeks, scaffolds were retrieved, and the mRNA expression of interleukin 1-beta (IL-1ß), IL-6, IL-10, hypoxia-inducible factor 1-alpha (HIF-1α), HIF-1ß, and mammalian target of rapamycin was examined by real-time reverse transcription-polymerase chain reaction. Furthermore, immunofluorescent staining was performed for IL-1ß, IL-6, neutrophils, and CD11b. In addition, Western blotting was done for IL-1ß and IL-6. Leukocyte transmigration genes and genes in Toll-like receptor pathways, expressed by MSCs cultured in vitro with or without ECs, were further investigated with a microarray dataset. RESULTS: In vitro, genes involved in leukocyte transmigration and Toll-like receptor pathways were clearly influenced by the addition of ECs. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) and cadherin-5 (CDH5), both genes involved in leukocyte transmigration, were expressed significantly higher in the MSC/EC group. CONCLUSIONS: The recruitment of leukocytes into tissue-engineered constructs with MSCs is strongly influenced by the addition of ECs via activation of leukocyte transmigration and Toll-like receptor pathways.