Sutureless fixation of amniotic membrane for therapy of ocular surface disorders

Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders

Induction of vascular endothelial growth factor-A165a in human retinal and endothelial cells in response to glyoxal

Low-density lipoprotein (LDL) apheresis is effective and safe for patients with diabetes, proteinuria, and dyslipidemia. Diabetes mellitus is accompanied by ocular microvascular complications like retinal neovascularization or diabetic macular edema. These are leading causes of blindness and can be mediated by abnormal vessel growth and increased vascular permeability due to elevated levels of vascular endothelial growth factor (VEGF) in diabetic patients. In this study, we established methods to study the expression of different VEGF isoforms in human retinal and endothelial cells. The VEGF-A165a isoform is much higher expressed in retinal cells, compared to endothelial cells. Stimulation with glyoxal as a model of oxidative stress under diabetic conditions lead to a pronounced induction of VEGF-A165a in human retinal and endothelial cells. These data suggest that diabetes and oxidative stress induce VEGF-A isoforms which could be relevant in regulating the ingrowths of novel blood vessels into the retina in diabetic patients

Sutureless Fixation of Amniotic Membrane for Therapy of Ocular Surface Disorders

Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders

Elevated Steroid Hormone Production in the db/db Mouse Model of Obesity and Type 2 Diabetes

Obesity and type 2 diabetes have become a major public health problem worldwide. Steroid hormone dysfunction appears to be linked to development of obesity and type 2 diabetes and correction of steroid abnormalities may offer new approaches to therapy. We therefore analyzed plasma steroids in 15-16 week old obese and diabetic db/db mice using liquid chromatography-tandem mass spectrometry. Lean db/+ served as controls. Db/db mice developed obesity, hyperglycemia, hyperleptinemia, and hyperlipidemia. Hepatic triglyceride storage was increased and adiponectin and pancreatic insulin were lowered. Aldosterone, corticosterone, 11-deoxycorticosterone, and progesterone were respectively increased by 3.6-, 2.9-, 3.4, and 1.7-fold in db/db mice compared to controls. Ratios of aldosterone-to-progesterone and corticosterone-to-progesterone were respectively 2.0- and 1.5-fold higher in db/db mice. Genes associated with steroidogenesis were quantified in the adrenal glands and gonadal adipose tissues. In adrenals, Cyp11b2, Cyp11b1, Cyp21a1, Hsd3b1, Cyp11a1, and StAR were all significantly increased in db/db mice compared with db/+ controls. In adipose tissue, no Cyp11b2 or Cyp11b1 transcripts were detected and no differences in Cyp21a1, Hsd3b1, Cyp11a1, or StAR expression were found between db/+ and db/db mice. In conclusion, the present study showed an elevated steroid hormone production and adrenal steroidogenesis in the db/db model of obesity and type 2 diabetes

Impact of Dietary Sodium Reduction on the Development of Obesity and Type 2 Diabetes in db/db Mice

The impact of dietary sodium reduction on mouse models of type 2 diabetes is not well understood. Therefore, we analyzed the effect of a low-salt diet on obesity and parameters of type 2 diabetes in db/db mice. Five-week-old male db/db and lean db/m mice were fed a normal salt (0.19% Na+, NS) or a low-salt diet (<0.03% Na+, LS) for 5 weeks. Body and organ weight and parameters of glucose and insulin tolerance were analyzed. Plasma levels of steroids were determined by liquid chromatography tandem mass spectrometry. Body weight, glucose, and insulin tolerance were not affected by LS. The amount of gonadal adipose tissue showed a trend to be increased by LS whereas liver, pancreas, kidney, heart, and adrenal weight remained unaffected. LS reduced urinary sodium-to-creatinine ratio but did not affect plasma Na+ levels in both genotypes. Plasma and urinary potassium-to-creatinine ratio did not differ in all groups of mice. Aldosterone as a major determinant of changes in dietary sodium remained unaffected by LS in db/db mice as well as further investigated steroid hormones. The present study showed reduced sodium-to-creatinine ratio, but no additional effects of dietary sodium reduction on major metabolic parameters and steroid levels in obese and hyper-glycemic db/db mice

Impact of Aldosterone Synthase Inhibitor FAD286 on Steroid Hormone Profile in Human Adrenocortical Cells

Inhibition of aldosterone synthase (CYP11B2) is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone effects. FAD286 is the best characterized aldosterone synthase inhibitor. However, to date, no study has used sensitive liquid chromatography-tandem mass spectrometry to characterize in detail the effect of FAD286 on the secreted steroid hormone profile of adrenocortical cells. Basal aldosterone production in NCI-H295R cells was detectable and 9-fold elevated after stimulation with angiotensin II. FAD286 inhibited this increase, showing a maximal effect at 10 nmol/l. Higher concentrations of FAD286 did not further reduce aldosterone concentrations, but showed a parallel reduction in corticosterone, cortisol and cortisone levels, reflecting additional inhibition of steroid-11β-hydroxylase (CYP11B1). Pregnenolone, progesterone and 17-OH-progesterone levels remained unaffected. In conclusion, the aldosterone synthase inhibitor FAD286 lowers angiotensin II-induced aldosterone concentrations in adrenocortical cells but the relative lack of selectivity over CYP11B1 is evident at higher FAD286 concentrations

Growth factor expression in AMs at different time points during processing and use.

<p>A) Concentration of RNA from amniotic membranes directly after cryopreservation (AM), AMs after mechanical handling before transplantation (AM-transplant), or AMs after being worn on the ocular surface for one week (AM-explant) (n≥3). B) mRNA of vascular endothelial growth factor-A (isoform VEGF-A165a), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF-2) and pigment epithelium-derived factor (PEDF) in amniotic membranes immediately after thawing (AM), or AMs after mechanical handling before transplantation (AM-transplant). Bars represent mean ± SEM of three or more measurements. *p<0.05 vs. AM.</p

Components of AmnioClip and mounting device.

<p>A) AmnioClip, disassembled, left: outer silicone ring, right: inner steel ring; B) AmnioClip, assembled without AM, view from the inner (ocular) side; C) AmnioClip, assembled with AM, view from the inner (ocular) side, D) mounting device, open position allowing placement of the rings and AM; E) mounting device, closed “mounting” position.</p

Sutureless application of AM for treating refractory epithelial erosion (case 1, patient #6).

<p>A) Slit lamp examination and B) fluorescein demonstration of the corneal erosion before sutureless AM application, arrow indicates the lesioned site; C,D) slit lamp control of AM fit at days 3 and 5 of wearing, arrow in D points on AmnioClip; E) slit lamp control and F) fluorescein demonstration of the corneal surface after removal of the AM at day 7, the arrow indicates the reduced lesion.</p

Primers used for gene expression analysis by real-time PCR.

<p>Primers used for gene expression analysis by real-time PCR.</p