EMT induces characteristic changes of Rho GTPases and downstream effectors with a mitosis-specific twist.

Epithelial-mesenchymal transition (EMT) is a key cellular transformation for many physiological and pathological processes ranging from cancer over wound healing to embryogenesis. Changes in cell migration, cell morphology and cellular contractility were identified as hallmarks of EMT. These cellular properties are known to be tightly regulated by the actin cytoskeleton. EMT-induced changes of actin-cytoskeletal regulation were demonstrated by previous reports of changes of actin cortex mechanics in conjunction with modifications of cortex-associated f-actin and myosin. However, at the current state, the changes of upstream actomyosin signaling that lead to corresponding mechanical and compositional changes of the cortex are not well understood. In this work, we show in breast epithelial cancer cells MCF-7 that EMT results in characteristic changes of the cortical association of Rho-GTPases Rac1, RhoA and RhoC and downstream actin regulators cofilin, mDia1 and Arp2/3. In the light of our findings, we propose that EMT-induced changes in cortical mechanics rely on two hitherto unappreciated signaling paths-i) an interaction between Rac1 and RhoC and ii) an inhibitory effect of Arp2/3 activity on cortical association of myosin II.

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.

Skin epithelial cells change their mechanics and proliferation upon snail-mediated EMT signalling.

Skin cancer is the most commonly occurring cancer in the USA and Germany, and the fourth most common cancer worldwide. Snail-dependent epithelial-mesenchymal transition (EMT) was shown to initiate and promote skin cancer. Previous studies could show that EMT changes actin cortex regulation and cellular mechanics in epithelial cells of diverse tissue origin. However, in spite of its potentially high significance in the context of skin cancer, the effect of EMT on cellular mechanics, mitotic rounding and proliferation has not been studied in skin epithelial cells so far. In this work, we show that TGF-ß-induced partial EMT results in a transformation of the mechanical phenotype of skin epithelial cells in a cell-cycle dependent manner. Concomitantly, we looked at EMT-induced changes of cell proliferation. While EMT decreases proliferation in 2D culture, we observed an EMT-induced boost of cellular proliferation when culturing cells as mechanically confined aggregates of skin epithelial cells. This proliferation boost was accompanied by enhanced mitotic rounding and composition changes of the actin cortex. We give evidence that observed EMT-induced changes depend on the EMT-upregulated transcription factor snail. Overall, our findings indicate that EMT-induced changes of cellular mechanics might play a currently unappreciated role in EMT-induced promotion of skin tumor proliferation.

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.

EMT changes actin cortex rheology in a cell-cycle-dependent manner.

The actin cortex is a key structure for cellular mechanics and cellular migration. Accordingly, cancer cells were shown to change their actin cytoskeleton and their mechanical properties in correlation with different degrees of malignancy and metastatic potential. Epithelial-mesenchymal transition (EMT) is a cellular transformation associated with cancer progression and malignancy. To date, a detailed study of the effects of EMT on the frequency-dependent viscoelastic mechanics of the actin cortex is still lacking. In this work, we have used an established atomic force microscope-based method of cell confinement to quantify the rheology of the actin cortex of human breast, lung, and prostate epithelial cells before and after EMT in a frequency range of 0.02-2 Hz. Interestingly, we find for all cell lines opposite EMT-induced changes in interphase and mitosis; whereas the actin cortex softens upon EMT in interphase, the cortex stiffens in mitosis. Our rheological data can be accounted for by a rheological model with a characteristic timescale of slowest relaxation. In conclusion, our study discloses a consistent rheological trend induced by EMT in human cells of diverse tissue origin, reflecting major structural changes of the actin cytoskeleton upon EMT.

Impact of omega-3 fatty acids on expression of angiogenic cytokines and angiogenesis by adipose-derived stem cells.

BACKGROUND AND AIMS: Human adipose-tissue derived stem cells (ADSC) are interesting novel targets in tissue engineering and regenerative medicine with pronounced angiogenic capacities. Furthermore, omega-3 fatty acids have been described to mediate cardioprotective effects, but their role in angiogenesis and vascular regeneration is not well-understood. Here, we analyzed the impact of different omega-3 fatty acids on angiogenesis by ADSCs. METHODS: Stem cells were cultured as monolayers or in 3D models, in spheroids embedded in collagen matrix or in co-cultures with human umbilical vein endothelial cells (HUVECs) in the Matrigel™ assay. The angiogenic properties of ADSCs were assessed by their sprouting and paracrine activities, gene expression by RT-PCR, Western blot, and enzyme immunoassay. RESULTS: Stimulation of undifferentiated ADSCs with docosahexaenoic acid (DHA) strongly upregulated angiopoietin-1 mRNA levels up to 4.6 ± 0.3 fold. Furthermore, Il-6 and Il-8 mRNAs were increased 4.2 ± 0.5 fold and 7.1 ± 1.1 fold, respectively. On the other hand, addition of DHA significantly decreased the cumulative sprout length by 2.7 ± 0.8 fold and reduced the total number of sprouts by 2.3 ± 0.9 fold in the in vitro angiogenesis assay. Moreover, excretion of IL-8 into the medium rapidly increased up to 1.7 ± 0.3 fold in response to treatment of ADSCs with DHA. Finally, protein kinase C inhibitor RO-31-8220 abrogated DHA-mediated up-regulation of angiopoietin-1 without significantly affecting ADSCs cell viability. CONCLUSION: In conclusion, ADSCs might regulate the formation and function of microvascular networks.

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-11ß-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.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice.

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db/db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db/db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg/d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db/db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db/db mice.

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.

Stress responses of human retinal pigment epithelial cells to glyoxal.

PURPOSE: Intracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells. METHODS: Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of Nε-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal. The percentage of apoptotic cells was further quantified by flow cytometry after staining of fixed cells with propidium iodide to determine cells with a subdiploid (fragmented) DNA content. Apoptosis related activation of caspase 3 was determined by Western blotting. Glyoxal induced changes in VEGF-A165a mRNA expression and protein production were determined by real-time PCR and by flow cytometry after immunocytochemical staining. RESULTS: Increasing glyoxal concentrations resulted in enhanced formation of AGEs, such as CML modifications of proteins. This was associated with elevated levels of intracellular reactive oxygen species, a depolarized MMP, and a decreased intracellular pH, resulting in an increased number of apoptotic cells. Apoptosis related caspase 3 activation increased in a dose dependent manner after glyoxal incubation. In consequence, the cells activated compensatory mechanisms and increased the levels of the anti-oxidative and stress-related proteins heme oxygenase-1, osteopontin, heat shock protein 27, copper/zinc superoxide dismutase, manganese superoxide dismutase, and cathepsin D. Furthermore, VEGF-A165a mRNA expression and VEGF-A protein production were significantly increased after incubation with glyoxal in ARPE-19 cells. CONCLUSIONS: The glyoxal-induced oxidative stress and apoptosis in ARPE-19 cells may provide a suitable in vitro model for studying RPE cellular reactions to AGEs that occur in AMD or in DR.

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.Trial Registration:ClinicalTrials.gov NCT02168790

Hand coordination following capsular stroke.

Motor outcome following stroke of the internal capsule is variable and its determinants are poorly understood. While many patients fully regain their abilities, recovery of motor functions remains incomplete in others. We analysed functional motor tasks of the upper limb to determine the pattern of focal disability after a small infarct of the internal capsule ('pure motor stroke') in the chronic stage (mean 2.4 years after stroke) with kinematic recordings of a reaching-to-grasp movement, with a quantitative analysis of the precision grip, and with clinical rating scales. The location of the lesions within the posterior limb of the internal capsule (PLIC) in 18 patients was determined from neuroimages obtained in the acute stage (5-20 days after the insult). Involvement of the PLIC was assessed at the level of the basal ganglia, approximately 8 mm above the anterior commissure-posterior commissure level. The distance between the posterior edge of the internal capsule and the centre of gravity of the lesion was determined. Chronic disabilities affected dextrous movements, while paresis was mild and sensitivity for light touch or passive finger flexion was almost normal. For both the reaching-to-grasp movement and the precision grip paradigm, the slowness of movement or force development was confined to the phases when grip formation and stabilization occur, while the onset of hand transport and of the vertical lifting force were not delayed. Grip forces were increased. We observed a close correlation between posterior location within the PLIC and the altered measures of timing and precision grip force. The more posterior the acute lesion was located within the PLIC, the more pronounced were the chronic motor deficits, as seen both in the quantitative measures and in the rating scales. The present study demonstrates for the first time that the amount and quality of chronic motor deficits of dextrous movements are related to a simple measure drawn from routine neuroimaging in the acute stage in patients with capsular stroke. The poor motor outcome in lesions involving the most posterior parts of the PLIC could be due to the condensed organization of corticofugal projections and the density of pyramidal fibres from the primary motor cortex in this subsector. Even small infarcts of this strategic area can disrupt many of the projections from the motor cortices and could thereby limit recovery strategies between homolateral motor representations.