Epithelial-mesenchymal transdifferentiation in pediatric lens epithelial cells

PURPOSE. Posterior capsule opacification (PCO) is a complication after cataract surgery, particularly in children. Epithelial-mesenchymal transition (EMT) of lens epithelial cells, mediated by transforming growth factor beta (TGF beta), contributes to PCO. However, its pathogenesis in children is poorly understood. We correlated cell growth in culture with patient characteristics, studied gene expression of pediatric lens epithelial cells (pLEC), and examined the effects of TGF beta-2 on these cells in vitro. METHODS. Clinical characteristics of children with cataracts correlated with growth behavior of pLEC in vitro. mRNA expression of epithelial (alpha B-crystallin, connexin-43) and mesenchymal (alpha(V)-integrin, alpha-smooth muscle actin, collagen-I alpha 2, fibronectin-1) markers was quantified in pLEC and in cell line HLE-B3 in the presence and absence of TGF beta-2. RESULTS. Fifty-four anterior lens capsules from 40 children aged 1 to 180 months were obtained. Cell outgrowth occurred in 44% of the capsules from patients <= 12 months and in 33% of capsules from children aged 13 to 60 months, but in only 6% of capsules from children over 60 months. TGF beta-2 significantly upregulated expression of alpha B-crystallin (HLE-B3), alpha(V)-integrin (HLE-B3), collagen-I alpha 2, and fibronectin-1 (in pLEC and HLE-B3 cells). CONCLUSIONS. Patient characteristics correlated with growth behavior of pLEC in vitro, paralleling a higher clinical incidence of PCO in younger children. Gene expression profiles of pLEC and HLE-B3 suggest that upregulation of alpha(V)-integrin, collagen-I alpha 2, and fibronectin-1 are involved in EMT

Modulation of TTX-sensitive voltage-dependent Na+ channels by β-bungarotoxin in rat cerebellar neurons

Background The modulation of voltage-dependent Na+ channels by lipid metabolites such as arachidonic acid or eicosanoids plays a role in physiological functions as well as in degenerative diseases. So far TTX-resistant channels were found mainly to be regulated by lipid metabolites. Results We investigated the lipid-dependent modulation of TTX-sensitive (TTX-s) Na+ channels using beta-bungarotoxin (beta-BuTX, 10 pM), which has an intrinsic phospholipase-A2 activity, and indomethacin (10 muM), which blocks cyclooxygenase activity in primary cerebellar neurons. To investigate TTX-s Na+ channels, whole-currents were measured under K+-free conditions and blocked by 10 nM TTX. The currents resulting from calculating the difference of currents measured in the presence and the absence of TTX were used for further analysis. Application of indomethacin mainly changed the current kinetics but has only minor effects on voltage-dependence. In contrast beta-BuTX increased the maximal current amplitude and shifted the voltage-dependent activation towards more negative potentials. The effects of beta-BuTX were blocked by indomethacin. Analysis of lipid metabolites which accumulate by treatment with beta-BuTX using MALDI-TOF MS showed an increase of cyclooxygenase reaction products in relation to arachidonic acid. Conclusions In summary, we conclude that TTX-sensitive Na+ channels can be directly modulated by cyclooxygenase reaction products leading to higher activity at less depolarized potentials and subsequent higher excitability of neurons. Since activation of cyclooxygenase is also involved in pathways leading to apoptotic cells death this could play a role in degenerative diseases of the CNS and highlights a possible protective effect of cyclooxygenase inhibition

Intended healthcare utilisation in cases of severe COVID-19 and inflammatory gastrointestinal disease: results of a population survey with vignettes

Objectives: To examine variations in intended healthcare utilisation in severe cases of COVID-19 and inflammatory gastrointestinal disease (IGD).Design Representative cross-sectional telephone survey. Setting and participants 1207 randomly drawn adults of the city of Hamburg, Germany, between November 2020 and January 2021. Outcome measures: Different vignettes with severe symptoms were presented varying in sex, age (child, middle-aged person, older person), daytime (Tuesday morning or Tuesday evening) and disease (COVID-19 or IGD), while the degree of urgency was equivalent for all cases. The respondents were asked for the intended healthcare utilisation resulting in three different alternatives: general practitioner (GP)/paediatrician, medical on-call service ('116117') and emergency care (accident and emergency department, emergency practice, rescue service). In multivariate analyses, associations of characteristics of the vignettes and participants (sex, age, education, migration background) with intended healthcare utilisation were tested. In a further step, analyses were conducted separately for IGD and COVID-19. Results: Regarding the vignettes' characteristics, intended utilisation of GP/paediatrician is associated with female sex, higher age, daytime (morning) and COVID-19 symptoms, the medical on-call service with male sex, daytime (evening) and COVID-19 symptoms and the emergency medicine with younger age, daytime (evening) and IGD. Women chose more often the GP/paediatrician, men preferred emergency medicine. Only in case of IGD, higher educated persons more often chose the medical on-call service while people with a migration background decided less often for medical on-call service and emergency medicine. Conclusions: Despite comparable urgency, the findings suggest variations of intended healthcare utilisation depending on various characteristics of the vignettes and respondents. Depending on the type of disease inequalities vary. Overall, information about healthcare alternatives in severe cases has to be improved and clear pathways to facilitate healthcare utilisation has to be further developed.Data are available on reasonable request

Inhibition of Ca 2+ channel surface expression by mutant bestrophin‐1 in RPE cells

The BEST1 gene product bestrophin-1, a Ca2+-dependent anion channel, interacts with CaV1.3 Ca2+ channels in the retinal pigment epithelium (RPE). BEST1 mutations lead to Best vitelliform macular dystrophy. A common functional defect of these mutations is reduced trafficking of bestrophin-1 into the plasma membrane. We hypothesized that this defect affects the interaction partner CaV1.3 channel affecting Ca2+ signaling and altered RPE function. Thus, we investigated the protein interaction between CaV1.3 channels and bestrophin-1 by immunoprecipitation, CaV1.3 activity in the presence of mutant bestrophin-1 and intracellular trafficking of the interaction partners in confluent RPE monolayers. We selected four BEST1 mutations, each representing one mutational hotspot of the disease: T6P, F80L, R218C, and F305S. Heterologously expressed L-type channels and mutant bestrophin-1 showed reduced interaction, reduced CaV1.3 channel activity, and changes in surface expression. Transfection of polarized RPE (porcine primary cells, iPSC-RPE) that endogenously express CaV1.3 and wild-type bestrophin-1, with mutant bestrophin-1 confirmed reduction of CaV1.3 surface expression. For the four selected BEST1 mutations, presence of mutant bestrophin-1 led to reduced CaV1.3 activity by modulating pore-function or decreasing surface expression. Reduced CaV1.3 activity might open new ways to understand symptoms of Best vitelliform macular dystrophy such as reduced electro-oculogram, lipofuscin accumulation, and vision impairment

Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mouse

Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11–0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis

Lack of netrin-4 modulates pathologic neovascularization in the eye

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4−/− mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4−/− mice. In OIR, Ntn-4−/− mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4−/− mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4−/− mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization

Lack of netrin-4 modulates pathologic neovascularization in the eye

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4−/− mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4−/− mice. In OIR, Ntn-4−/− mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4−/− mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4−/− mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration

Background: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. Methods: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1 beta and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. Results: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1 beta to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. Conclusion: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function

Effects of empagliflozin and target-organ damage in a novel rodent model of heart failure induced by combined hypertension and diabetes

Type 2 diabetes mellitus and hypertension are two major risk factors leading to heart failure and cardiovascular damage. Lowering blood sugar by the sodium-glucose co-transporter 2 inhibitor empagliflozin provides cardiac protection. We established a new rat model that develops both inducible diabetes and genetic hypertension and investigated the effect of empagliflozin treatment to test the hypothesis if empagliflozin will be protective in a heart failure model which is not based on a primary vascular event. The transgenic Tet29 rat model for inducible diabetes was crossed with the mRen27 hypertensive rat to create a novel model for heart failure with two stressors. The diabetic, hypertensive heart failure rat (mRen27/tetO-shIR) were treated with empagliflozin (10 mg/kg/d) or vehicle for 4 weeks. Cardiovascular alterations were monitored by advanced speckle tracking echocardiography, gene expression analysis and immunohistological staining. The novel model with increased blood pressure und higher blood sugar levels had a reduced survival compared to controls. The rats develop heart failure with reduced ejection fraction. Empagliflozin lowered blood sugar levels compared to vehicle treated animals (182.3 ± 10.4 mg/dl vs. 359.4 ± 35.8 mg/dl) but not blood pressure (135.7 ± 10.3 mmHg vs. 128.2 ± 3.8 mmHg). The cardiac function was improved in all three global strains (global longitudinal strain − 8.5 ± 0.5% vs. − 5.5 ± 0.6%, global radial strain 20.4 ± 2.7% vs. 8.8 ± 1.1%, global circumferential strain − 11.0 ± 0.7% vs. − 7.6 ± 0.8%) and by increased ejection fraction (42.8 ± 4.0% vs. 28.2 ± 3.0%). In addition, infiltration of macrophages was decreased by treatment (22.4 ± 1.7 vs. 32.3 ± 2.3 per field of view), despite mortality was not improved. Empagliflozin showed beneficial effects on cardiovascular dysfunction. In this novel rat model of combined hypertension and diabetes, the improvement in systolic and diastolic function was not secondary to a reduction in left ventricular mass or through modulation of the afterload, since blood pressure was not changed. The mRen27/tetO-shIR strain should provide utility in separating blood sugar from blood pressure-related treatment effects