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Purpose: Although regular water is composed of two hydrogens and one oxygen, referred to as H 2 O, a small amount of water on this planet contains alternative forms of elements with different molecular weights because of the addition of neutrons. The present study was dedicated to studying the effect of heavy water (D 2 O), in which the two hydrogens become substituted by deuterium, on the cell physiology of different human cells with particular focus on malignant melanoma cells., Methods: Cells were cultured in regular medium in which the content of H 2 O was gradually substituted by D 2 O or deuterium-depleted water (DDW). Following this, the changes of basic cellular parameters, such as morphology, migration, proliferation, cell cycle, apoptosis and microtubule integrity were examined., Results: It was found that raising the D 2 O content above the standard levels led to a concentration-dependent decrease in proliferation. Lowering the D 2 O levels below this level had no effect. Likewise, elevated D 2 O levels hampered migration. Moreover, cell-cycle analysis showed an increase of sub-G1 cells. Corroboratively, markers for apoptosis were induced (histone-associated DNA fragments, Bax, and PARP). In regard to microtubule integrity, only very high levels of D 2 O (75%) caused partial filament condensation., Conclusion: D 2 O, although chemically identical with H 2 O, shows proapoptotic and antiproliferative effects on melanoma cells. These findings give a closer look of this interesting compound., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Kleemann et al.)

Background: Adipose-derived stem cells (ASC) are known to transdifferentiate into a wide range of different cell species in vitro including along the epidermal lineage. This property makes them a promising tool for regenerative medicine to restore the epidermal barrier., Objective: This study is dedicated to identify in vitro conditions enabling transdifferentiation to a keratinocyte-like phenotype. In particular, the impact of different culture conditions (media compositions, 2D, 3D cultures) and extracellular matrix (ECM) molecules was evaluated., Methods: Adipose-derived stem cells derived from subcutaneous abdominal fat were characterized by stemness-associated markers and subjected to different media. Epithelial differentiation in 2D cultures was monitored by pan-cytokeratin expression using flow cytometry and immunocytochemistry. To evaluate the impact of different ECM molecules on epidermal stratification, 3D cultures were produced, lifted to the air-liquid interface (ALI) and examined by histological analysis and quantitative real-time RT-PCR., Results: We identified a medium composition containing retinoic acid, hydrocortisone, ascorbic acid and BMP-4 enabling maximum pan-cytokeratin expression in 2D cultures. Moreover, adhesion to type IV collagen further promotes the pan-cytokeratin expression. When cultures were lifted to the ALI, significant stratification was observed, particularly in supports coated with type IV collagen or fibronectin. Moreover, epidermal differentiation markers (involucrin, cytokeratin 1 and 14) become induced., Conclusion: Conditions with hampered wound healing such as non-healing ulcers demand new treatment regimes. The here introduced optimized protocols for transdifferentiation of ASC into keratinocyte-like cells may help to establish more effective treatment procedures., (© 2018 European Academy of Dermatology and Venereology.)

Cathepsin B has been shown to be important in angiogenesis; therefore, understanding its regulation in endothelial cells should provide fundamental information that will aid in the development of new treatment options. Peroxisome proliferator-activated receptors (PPARs) have been shown to have anti-inflammatory, anti-angiogenic and anti-tumorigenic properties. We explored the influence of a PPARα agonist on cathepsin B expression in human endothelial cells. The PPARα agonist, Wy14643, was found to inhibit cathepsin B protein expression. Further studies demonstrated the Wy14643-dependent but PPARα-independent suppression of cathepsin B. This has been previously described for other PPAR agonists. Wy14643 suppressed the accumulation of cathepsin B mRNA, which was accompanied by the selective suppression of a 5'-alternative splice variant. Consistent with these results, luciferase promoter assays and electrophoretic mobility shift analysis demonstrated that the suppression was facilitated by reduced binding of the transcription factors USF1/2 to an E-box within the cathepsin B promoter. Additionally, Wy14643 treatment resulted in a reduction in cathepsin B half-life, suggesting a posttranslational regulatory mechanism. Overall, our results suggest that the PPARα-dependent anti-angiogenic action of Wy14643 seems to be mediated, in part, by Wy14643-dependent but PPARα-independent regulation of cathepsin B expression.

Peroxisome proliferator-activated receptor (PPAR) delta agonists are known to have distinct anti-inflammatory and antitumor effects; though, the knowledge regarding their mode of action has thus far been limited. Different cathepsins have been shown to be upregulated in a broad range of pathological events, such as rheumatoid arthritis, psoriasis, atherosclerosis and diverse tumor entities, for example melanoma. Recent work demonstrated that cathepsin B in particular is an important pro-angiogenic protease in various pathological conditions. We therefore analysed whether cathepsins are a valid target for PPARδ agonists. This study reveals an inhibitory effect of two commonly used PPARδ agonists, GW501516 and L-165,041, on the protein expression and enzyme activity of cathepsin B in human endothelial cells. In contrast, no inhibitory effects were observed on cathepsin L and cathepsin D protein expression after treatment with PPARδ agonists. Furthermore, the results substantiate that PPARδ activators mediate their inhibitory action in a PPARδ-dependent manner and that the underlying regulatory mechanism is not based on a transcriptional but rather on a posttranslational mode of action, via the reduction in the cathepsin B protein half-life. Mechanisms conveying the suppressive effect by 5'-alternative splicing, a 3'-UTR-dependent way or by miRNA could be excluded. The data of this study explore cathepsin B as a new valid target for PPARδ agonists in endothelial cells. The results bolster other studies demonstrating PPARδ agonists as anti-inflammatory and anticarcinogenic agents and thus might have the potential to help to develop new pharmaceutical drugs., (© 2012 John Wiley & Sons A/S.)

The association between angiogenesis and chronic inflammatory diseases, such as psoriasis, seems to be an important phenomenon implicated in the pathogenesis of these medical conditions. Recent studies provide evidence that dimethylfumarate (DMF) has a profound anti-inflammatory as well as anti-tumorigenic action, although the effect of DMF on angiogenesis is unknown. Signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses. Therefore, we explored whether the known anti-inflammatory and anti-tumorigenic properties of DMF might be mediated in part by anti-angiogenic effects through the reduction in VEGFR2 expression. In this study, DMF was found to inhibit endothelial VEGFR2 expression; time- and concentration-dependent inhibition was demonstrated both at the level of protein and mRNA expression. This blockade was coincident with the inhibition of the formation of capillary-like structures. The DMF-dependent inhibition of VEGFR2 transcription was found to be mediated by an element located between base pairs -60 and -37, which contains two adjacent, consensus Sp1 transcription factor-binding sites, and the constitutive formation of complexes containing Sp1 at this site is decreased by DMF treatment. Inhibition of VEGFR-2 is shown to be one critical aspect in DMF-mediated anti-angiogenic effects.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that function mainly in the regulation of glucose and lipid homeostasis. PPAR agonists have been shown to control inflammation by inhibition of distinct proinflammatory genes. Aberrant activation of the epidermal growth factor receptor and/or overexpression of its ligand, transforming growth factor-α (TGFα), are key features of both neoplastic and inflammatory hyperproliferative epithelia. Matrix metalloproteinase 9 (MMP9) belongs to the set of genes that are effectively induced by TGFα in keratinocytes. Induction of MMP9 expression is strongly linked to regenerative skin repair mechanisms, inflammatory skin diseases and tumor metastasis. We explored whether the known anti-inflammatory effects of PPARδ ligands involve inhibiting the TGFα-mediated upregulation of MMP9. The PPARδ agonists potently inhibited TGFα-induced MMP9 expression in human keratinocytes. This inhibition was observed at both the protein and mRNA levels. Transcriptional activation studies with deletion constructs of a reporter gene revealed that PPARδ agonists mediate their inhibitory effects via an AP1-binding site. Electromobility shift assay analysis indicated that MMP9 gene expression is inhibited by repressing site-dependent DNA binding and transactivation by c-fos. In conclusion, our data provide the first evidence that MMP9 expression induced by TGFα is a valid target of PPARδ ligands in keratinocytes., (© 2011 John Wiley & Sons A/S.)

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that are implicated in the regulation of lipid and glucose homeostasis. PPAR agonists have been shown to control inflammatory processes, in part by inhibiting distinct proinflammatory genes (e.g. Il-1β and IFN-γ). IL-8 is a member of the proinflammatory chemokine family that is important for various functions, such as mediating the adhesion of eosinophilic granulocytes onto endothelial cells. The influence of PPARδ activators on the expression of IL-8 in noninduced quiescent endothelial cells is unclear. Therefore, we explored the influence of PPARδ activators on the expression of IL-8 in nonstimulated endothelial cells. PPARδ agonists induce IL-8 expression in human umbilical vein endothelial cells. This induction is demonstrated at the level of both protein and mRNA expression. Transcriptional activation studies using IL-8 reporter gene constructs and DNA binding assays revealed that PPARδ agonists mediated their effects via an NFκB binding site. It is well known that IL-8 is also regulated by mRNA stability. To provide further evidence for this concept, we performed mRNA stability assays and found that PPARδ agonists induce the mRNA stability of IL-8. In addition, we showed that PPARδ agonists induce the phosphorylation of ERK1/2 and p38, which are known to be involved in the increase of mRNA stability. The inhibition of these MAPK signaling pathways resulted in a significant suppression of the induced IL-8 expression and the reduced mRNA stability. Therefore, our data provide the first evidence that PPARδ induces IL-8 expression in nonstimulated endothelial cells via transcriptional as well as posttranscriptional mechanisms.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. This system controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. Recent evidence also established the importance of the proteasome in tumor development, showing antitumor and antiangiogenic actions by using selective inhibitors in vivo. As signaling via the vascular endothelial growth factor receptor 2 (VEGFR2) pathway is critical for angiogenic responses to occur, we explored whether antiangiogenic effects due to proteasome inhibition were partly mediated through decreased endothelial VEGFR2 expression. This study shows that different proteasome inhibitors blocked VEGFR2 expression in a time-dependent and concentration-dependent manner. This blockade was paralleled by the respective inhibition of the formation of capillary-like structures and endothelial cell migration. In contrast, neither tie-2 nor VEGFR1 expression was significantly affected by proteasome inhibitor treatment. The suppressive effects on VEGFR2 expression were not conveyed by increased shedding or a decrease in protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. In line with this conclusion, proteasome inhibition significantly suppressed VEGFR2 mRNA accumulation. In addition, inhibitor treatment considerably decreased the transcriptional activity of 5' deletional VEGFR2 promoter gene constructs. Proteasome inhibition-mediated repression was controlled by a GC-rich region that harbored one consensus Sp1-binding site. Subsequent EMSA analyses showed decreased constitutive Sp1-dependent DNA binding in response to proteasome inhibition. In addition, we could show that proteasome inhibitors reduced VEGFR2 mRNA stability. Therefore, VEGFR2 expression may constitute a critical molecular target of proteasome inhibitors that may mediate their antiangiogenic effects in vivo.

Nitric oxide (NO) is a small gaseous molecule with an odd number of electrons and is rather soluble in hydrophobic phases. It was once known for its toxicity in the environment and for its applications in meat curing. After 1980 its importance was discovered in many physiological fields such as vascular regulation, neuronal communication, cytotoxic action by macrophages in bacterial infections etc. On the other side NO is involved in toxic shock, DNA damage and many pathological conditions. In 1992 the journal Science designated it as "molecule of the year" and in the last years there has been an explosion of publications on the subject. The publications are concerned with the spectroscopic characterisation of NO derivatives, with the reactivity of NO with Myoglobin, Cytochrome and Hemoglobin and in particular with the chemical activities and biological applications of nitric oxide donors and nitric oxide scavengers. All such researches have produced until now many patents. The most famous products are Viagra and nitroglycerine (Trinitrin). Particular attention is given to the applications of NO to cardiovascular and hematological disorders. To this aim the authors examine the physiologic activities of NO and the mechanism of its antiplatelet, vasodilatory and antiproliferative action. Studies in animals and humans are also reported. Another section examines the drugs that increase the endogenous production of NO and modulate its activities. The last part is dedicated to the novel antithrombotic agent Nitroaspirin. Methods for NO detection will also be examined.

Ferrimyoglobin at pH 7.4 binds nitric oxide to yield nitric oxide adducts. In the presence of glutathione (GSH), nitrosoadducts of Mb(III) react with it to give nitrosoglutathione, whose concentration has been determined with an apparatus based on a specific and sensitive solid-state amperometric gas sensor. The reaction constant between the adduct and glutathione, kGSH = (47 +/- 1) M(-1) x s(-1), obtained by UV-Vis spectroscopy kinetic measurements, is about one-eighth of the constant with OH- determined by other authors. We can explain this fact with the higher nucleophilicity of OH- compared to GSH, due to the bulkiness and charge of the species. It is known that the formation of nitrosothiols starting from nitrite or NO (nitrogen monoxide) and glutathione, in the absence of oxygen, is impossible. Thus, from a biological point of view, it is important to point out that GSH reacts with NO in the presence of ferrimyoglobin, even at physiological pH, to form nitrosoglutathione., (Copyright 2001 Academic Press.)