Primary Cultures Of Human Cells Research Articles

Vitamin D and interferon-γ co-operate to increase the ACE-2 receptor expression in primary cultures of human thyroid cells.

BackgroundA more severe course of COVID-19 was associated with low levels of Vitamin D (VitD). Moreover in vitro data showed that VitD up-regulates the mRNA of the Angiotensin Converting Enzyme 2 (ACE-2), the SARS-COV-2 receptor in different type of cells. ACE-2 is expressed in several type of tissues including thyroid cells, on which its mRNA was shown to be up-regulated by interferon-gamma (IFN-γ). The aim of the present study was to investigate if treatment with VitD alone or in combination with IFN-γ would increase ACE-2 both at mRNA and protein levels in primary cultures of human thyrocytes.Materials and methodsPrimary thyroid cell cultures were treated with VitD and IFN-γ alone or in combination for 24 h. ACE-2 mRNA levels were measured by Real-time Polymerase Chain Reaction (RT-PCR). The presence of ACE-2 on thyroid cell membrane was assessed by immunocytochemistry basally and after the previous mentioned treatments.ResultsACE-2 mRNA levels increased after treatment with VitD and IFN-γ alone. The combination treatment (VitD + IFN-γ) showed an additive increase of ACE-2-mRNA. Immunocytochemistry experiments showed ACE-2 protein on thyroid cells membrane. ACE-2 expression increased after treatment with VitD and IFN-γ alone and further increased by the combination treatment with VitD + IFN-γ.ConclusionsVitD would defend the body by SARS-COV2 both by regulating the host immune defense and by up-regulating of the expression of the ACE-2 receptor. The existence of a co-operation between VitD and IFN-γ demonstrated in other systems is supported also for ACE-2 up-regulation. These observations lead to an increased interest for the potential therapeutic benefits of VitD supplementation in COVID-19.

Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells.

ABSTRACTHuman coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies.IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.

Suppression of EZH2 inhibits TGF-β1-induced EMT in human retinal pigment epithelial cells

Epithelial–mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is critically involved in the occurrence of subretinal fibrosis. This study aimed to investigate the role of enhancer of zeste homolog 2 (EZH2) in EMT of human primary RPE cells and the underlying mechanisms of the anti-fibrotic effect of EZH2 suppression. Primary cultures of human RPE cells were treated with TGF-β1 for EMT induction. EZH2 was silenced by siRNA to assess the expression levels of epithelial and fibrotic markers using qRT-PCR, Western blot, and immunofluorescence staining assay. Furthermore, the cellular migration, proliferation and barrier function of RPE cells were evaluated. RNA-sequencing analyses were performed to investigate the underlying mechanisms of EZH2 inhibition. Herein, EZH2 silencing up-regulated epithelial marker ZO-1 and downregulated fibrotic ones including α-SMA, fibronectin, and collagen 1, alleviating EMT induced by TGF-β1 in RPE cells. Moreover, silencing EZH2 inhibited cellular migration and proliferation, but didn't affect cell apoptosis. Additionally, EZH2 suppression contributed to improved barrier functions after TGF-β1 stimulation. The results from RNA sequencing suggested that the anti-fibrotic effect of EZH2 inhibition was associated with the MAPK signaling pathway, cytokine-cytokine receptor interaction, and the TGF-beta signaling pathway. Our findings provide evidence that the suppression of EZH2 might reverse EMT and maintain the functions of RPE cells. EZH2 could be a potential therapeutic avenue for subretinal fibrosis.

Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis.

In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer's recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA).The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm2. Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients' peripheral blood can be used to develop personalized tissue-engineered constructs.

Inflammasome Activation in Retinal Pigment Epithelium from Human Donors with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. One of the risk factors associated with developing AMD is the single nucleotide polymorphism (SNP) found within the gene encoding complement factor H (CFH). Part of the innate immune system, CFH inhibits alternative complement pathway activation. Multi-protein complexes called inflammasomes also play a role in the innate immune response. Previous studies reported that inflammasome activation may contribute to AMD pathology. In this study, we used primary human adult RPE cell cultures from multiple donors, with and without AMD, that were genotyped for the Y402H CFH risk allele. We found complement and inflammasome-related genes and proteins at basal levels in RPE tissue and cell cultures. Additionally, treatment with rotenone, bafilomycin A, and ATP led to inflammasome activation. Overall, the response to priming and activation was similar, irrespective of disease state or CFH genotype. While these data show that the inflammasome is present and active in RPE, our results suggest that inflammasome activation may not contribute to early AMD pathology.

Human papillomavirus targets the YAP1-LATS2 feedback loop to drive cervical cancer development.

Human papillomavirus (HPV) infection is very common in sexually active women, but cervical cancer only develops in a small fraction of HPV-infected women, suggesting that unknown intrinsic factors associated with the unique genetic/genomic background of the high-risk population play a critical role in cervical carcinogenesis. Although our previous studies have identified the hyperactivated YAP1 oncogene as a critical contributor to cervical cancer, the molecular mechanism by which YAP1 drives cervical cancer is unknown. In the present study, we found that although the hyperactivated YAP1 caused a malignant transformation of immortalized cervical epithelial cells, it induced cellular senescence in cultures of primary human cervical epithelial cells (HCvECs). However, the hyperactivated YAP1 induced malignant transformation of HCvECs in the presence of high-risk HPV E6/E7 proteins, suggesting that the hyperactivated YAP1 synergizes with HPV to initiate cervical cancer development. Our mechanistic studies demonstrate that YAP1, via up-regulating LATS2, formed a YAP1-LATS2 negative feedback loop in cervical epithelial cells to maintain homeostasis of cervical tissue. Intriguingly, we found that high-risk HPV targets LATS2 to disrupt the feedback loop leading to the malignant transformation of cervical epithelial cells. Finally, we report that mitomycin C, an FDA-approved drug that could upregulate LATS2 and drive cellular senescence in vitro and in vivo, induced a regression of cervical cancer in a pre-clinial animal model. Thus, high-risk HPV targeting the YAP1-LATS2 feedback loop represents a new mechanism of cervical cancer development.

Effects of ethanol and deferoxamine on rat primary glial cell cultures, in regard with ischemia induced by traumatic spinal cord injury

Although they have been regarded, in the past, as passive support cells, many experimental data have shown that glial cells play a critical role in the development and functioning of the nervous system. Despite the advances that have been made in understanding astrocytes' role in the nervous system's development and function, our knowledge of their interactions with other cells is still limited, albeit neurons are dependent on the trophic support provided by astrocytes release. Materials and Methods. The use of the McCarthy and de Vellis methods for isolating glial cells has been regarded as an essential tool for studying their function. This study aims to evaluate the effects of ethanol and deferoxamine on primary rat glial cell cultures and try to explain, as far as possible, the relevance of such effects for patients with chronic alcoholism and traumatic spinal cord injuries. Discussion. Because glial cells are very important in the functioning of the central nervous system and experiments cannot be performed on human primary nerve cell cultures, we performed an experiment on glial cells harvested from the newborn rat, analyzing the dynamics of IL-6 and TNF alpha on models of suffering in spinal cord injury (hypoxia and thermally stress). Conclusion. Inhibition of TNF alpha synthesis was more important at 7 days posttraumatic in cells with prolonged ethanolic exposure, even if protein levels of IL-6 were elevated (under similar experimental conditions). Thus, we can say that long-term exposure to ethanol of nerve cells can ensure a favorable evolution of medical recovery (by increasing TNF alpha), even if the inflammatory process remains active (shown by elevated IL-6 values). Keywords: ethyl alcohol, deferoxamine, primary glial cells cultures, traumatic Spinal Cord Injury

Effects of Triiodothyronine on Human Osteoblast-Like Cells: Novel Insights From a Global Transcriptome Analysis.

Background: Thyroid hormones play a significant role in bone development and maintenance, with triiodothyronine (T3) particularly being an important modulator of osteoblast differentiation, proliferation, and maintenance. However, details of the biological processes (BPs) and molecular pathways affected by T3 in osteoblasts remain unclear. Methods: To address this issue, primary cultures of human adipose-derived mesenchymal stem cells were subjected to our previously established osteoinduction protocol, and the resultant osteoblast-like cells were treated with 1 nm or 10 nm T3 for 72 h. RNA sequencing (RNA-Seq) was performed using the Illumina platform, and differentially expressed genes (DEGs) were identified from the raw data using Kallisto and DESeq2. Enrichment analysis of DEGs was performed against the Gene Ontology Consortium database for BP terms using the R package clusterProfiler and protein network analysis by STRING. Results: Approximately 16,300 genes were analyzed by RNA-Seq, with 343 DEGs regulated in the 1 nm T3 group and 467 upregulated in the 10 nm T3 group. Several independent BP terms related to bone metabolism were significantly enriched, with a number of genes shared among them (FGFR2, WNT5A, WNT3, ROR2, VEGFA, FBLN1, S1PR1, PRKCZ, TGFB3, and OSR1 for 1nM T3; and FZD1, SMAD6, NOG, NEO1, and ENG for 10 nm T3). An osteoblast-related search in the literature regarding this set of genes suggests that both T3 doses are unfavorable for osteoblast development, mainly hindering BMP and canonical and non-canonical WNT signaling. Conclusions: Therefore, this study provides new directions toward the elucidation of the mechanisms of T3 action on osteoblast metabolism, with potential future implications for the treatment of endocrine-related bone pathologies.

Abstract 199: A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments

Abstract Mantle cell lymphomas (MCL) are rare B-cell neoplasms belonging to the non-Hodgkin’s lymphomas and often show an aggressive clinical course. MCL respond relatively briefly to conventional chemotherapy and are characterized by frequent relapses (Dreyling et al., Ann Oncol, 2017). Therapy for MCL remains a challenge also due to limited cultivation time of primary cells. In vitro suspension culture of primary human MCL cells is not easily feasible (Bryant et al., Lab Invest, 2000) and cells undergo spontaneous apoptosis ex vivo (Zhang et al., Blood, 2012; Medina et al., Heamatologica, 2012). Useful in vitro MCL models that allow long-term culture of primary MCL cells could be helpful in the biological and therapeutic study of MCL. For this purpose, we established a 3D hydrogel culture model for cultivation of primary MCL cells that were first tested with cell lines. After 3 days of culture, the cells cultured in the hydrogel can successfully be analysed for cell viability (live-dead confocal laser scanning microscope-based assay, flow cytometry), metabolism (pH, glucose, lactate), and biomarker expression by immunohistochemistry. The 3D hydrogel cultivation model allows a variety of analytical methods, also because it is reversible and the cells can easily be separated as single cell suspension from hydrogel for further analytical purposes, such as flow cytometric analyses. Next, we cultured primary MCL cells in the alginate hydrogel 3D culture system. MCL cells were isolated from blood of different patients and separated by Ficoll centrifugation. The isolated cell population was identified as MCL cells by double-staining of anti-CD20 and anti-CD5 followed by flow cytometric analysis. Cells were further cultured in medium as conventional suspension culture or in alginate hydrogel as 3D culture. Cell death was assessed with AnnexinV by staining phospholipids of apoptotic cells, allowing flow cytometric quantification. All samples had less than 6% AnnexinV positive (+) cells at day 0 before the experiment started. After 2 days cultivation, primary MCL cells showed a strong induction of cell death in suspension culture with 65.5% (±4.2%) AnnexinV+ cells versus 21.9% (±0.6%) AnnexinV+ cells in 3D hydrogel culture. Subsequently after 3 days cultivation, 74.2% (±12.2%) AnnexinV+ cells in suspension versus 33.5% (±1.3%) AnnexinV+ cells in the hydrogel 3D culture were determined. Therefore, we conclude that our novel 3D culture system might be a more suitable cultivation method for primary MCL cells than conventional suspension culture in cell culture medium. We have developed a 3D cultivation system with alginate hydrogel that allows nearly tripling the in vitro cultivation time of primary MCL. This could pave the way for new long-term treatments in research in the future and serve a better understanding of the cells. Citation Format: Kathrin Böpple, Annette M. Staiger, Heike Horn, German Ott, Walter E. Aulitzky, Meng Dong. A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 199.

GDF-11 promotes human trophoblast cell invasion by increasing ID2-mediated MMP2 expression

BackgroundGrowth differentiation factor-11 (GDF-11), also known as bone morphogenetic protein-11, belongs to the transforming growth factor-beta superfamily. GDF-11 was first identified as an important regulator during embryonic development. Increasing evidence has demonstrated that GDF-11 regulates the development of various organs and its aberrant expressions are associated with the risk of cardiovascular diseases and cancers. Extravillous trophoblast (EVT) cells invasion is a critical event for placenta development and needs to be finely regulated. However, to date, the biological function of GDF-11 in the human EVT cells remains unknown.MethodsHTR-8/SVneo, a human EVT cell line, and primary cultures of human EVT cells were used to examine the effect of GDF-11 on matrix metalloproteinase 2 (MMP2) expression. Matrigel-coated transwell invasion assay was used to examine cell invasiveness. A series of in vitro experiments were applied to explore the underlying mechanisms that mediate the effect of GDF-11 on MMP2 expression and cell invasion.ResultsTreatment with GDF-11 stimulates MMP2 expression, in the HTR-8/SVneo and primary human EVT cells. Using a pharmacological inhibitor and siRNA-mediated knockdown approaches, our results demonstrated that the stimulatory effect of GDF-11 on MMP2 expression was mediated by the ALK4/5-SMAD2/3 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 2 (ID2) was upregulated by GDF-11 and that was required for the GDF-11-stimulated MMP2 expression and EVT cell invasion.ConclusionsThese findings discover a new biological function and underlying molecular mechanisms of GDF-11 in the regulation of human EVT cell invasion.Ede8tEoyGya1QPPVVUV7JYVideo

Models and Techniques to Study Aortic Valve Calcification in Vitro, ex Vivo and in Vivo. An Overview.

Aortic valve stenosis secondary to aortic valve calcification is the most common valve disease in the Western world. Calcification is a result of pathological proliferation and osteogenic differentiation of resident valve interstitial cells. To develop non-surgical treatments, the molecular and cellular mechanisms of pathological calcification must be revealed. In the current overview, we present methods for evaluation of calcification in different ex vivo, in vitro and in vivo situations including imaging in patients. The latter include echocardiography, scanning with computed tomography and magnetic resonance imaging. Particular emphasis is on translational studies of calcific aortic valve stenosis with a special focus on cell culture using human primary cell cultures. Such models are widely used and suitable for screening of drugs against calcification. Animal models are presented, but there is no animal model that faithfully mimics human calcific aortic valve disease. A model of experimentally induced calcification in whole porcine aortic valve leaflets ex vivo is also included. Finally, miscellaneous methods and aspects of aortic valve calcification, such as, for instance, biomarkers are presented.

Hydroxyapatite/tricalcium phosphate (HA/beta-TCP) scaffold combined with bone and endothelial cells as a potential candidate for oral and maxillofacial bone regeneration.

Cell-based scaffolds have been proposed as useful for tissue repair and bone regeneration in oral and maxillofacial tissue engineering. However, vascularization, a key player in bone regeneration, is often inadequate. To improve this, the use of biphasic calcium phosphate biomaterial (30% hydroxyapatite (HA)-70% tricalcium phosphate (TCP) mixture) HA/beta-TCP-scaffold with defined micron-channel sizes and pore sizes capable of driving angiogenesis, may prove helpful. Therefore, our study aimed to combine the HA/beta-TCP scaffold with in vitro differentiated cells, especially endothelial cells, well-known players in angiogenesis. The HA/beta-TCP-scaffold was combined with primary cultures of human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs). Cells were co-cultured with HA/beta-TCP-scaffold in normoxia (20% oxygen tension) for 14 days. Then, we evaluated: cell adhesion/morphology (Scanning Electron Microscopy and Confocal Microscopy); cell viability (Calcein AM/Propidium Iodide Assay); the presence of multinucleated hOCs (Tartrate Resistant- Acid Phosphatase assay); expression of specific markers of both osteo- and vasculogenic differentiation (Immunohistochemistry). All cell types were viable and well adhered to the entire scaffold surface. Interestingly, bone matrix formation and newly formed vessel-like structures were both detected. Although further investigation is needed, our data proved that differentiated cells combined in vitro with HA/beta-TCP-scaffold prior to implantation could represent a useful strategy for future applications in bone regeneration. Especially, the use of customized micro-vascularized cell-based scaffold would fall within the innovative "personalized medicine” approach.

Human Dental Pulp Cells form Spheroids in the Presence of Serum When Seeded on a Low-Attachment Cultural Surface

Spheroid formation is a characteristic feature of stem/progenitor cells. Under a serum-free cultural condition, human dental pulp cells can form spheroids. In the present study, we report that these cells can also form spheroids in the presence of serum when seeded on a low-attachment cultural surface. Dental pulp cells derived from three teeth were seeded with surface densities 103–105/cm2 in wells of low attachment and standard cultural plates. Fibroblasts were also seeded onto a low-attachment surface as a comparison. The growth of single spheroids of pulp cells was observed for 7 days. Pulp cells in spheroids and cells attached to the low-attachment surface were separated and further expanded on standard cultural surface in the monolayer and studied for their viability and osteogenic differentiation comparatively. In all three cultures of primary human dental pulp cells on low attachment surface, spheroids formed one day after seeding and grew in size over the 7 days of observation. The optimal seeding density for spheroids was around 104 cells/cm2 (105 cells/mL). Expanded pulp cells from the spheroids exhibited lower viability but higher osteogenic differentiation potential compared to pulp cells expanded from those attached to the surface of the low attachment plate. Human dental pulp cells have the specific capacity to forms spheroids when seeded on a low-attachment surface and may enable selection of a subpopulation with stronger differentiation potential and may also provide a strategy for culturing these cells in a three-dimensional organization without scaffolds.

CRTH2 Mediates Profibrotic Macrophage Differentiation and Promotes Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a particularly deadly form of pulmonary fibrosis of unknown cause. In patients with IPF, high serum and lung concentrations of CHI3L1 (chitinase 3 like 1) can be detected and are associated with poor survival. However, the roles of CHI3L1 in these diseases have not been fully elucidated. We hypothesize that CHI3L1 interacts with CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells) to stimulate profibrotic macrophage differentiation and the development of pulmonary fibrosis and that circulating blood monocytes from patients with IPF are hyperresponsive to CHI3L1-CRTH2 signaling. We used murine pulmonary fibrosis models to investigate the role of CRTH2 in profibrotic macrophage differentiation and fibrosis development and primary human peripheral blood mononuclear cell culture to detect the difference of monocytes in the responses to CHI3L1 stimulation and CRTH2 inhibition between patients with IPF and normal control subjects. Our results showed that null mutation or small-molecule inhibition of CRTH2 prevents the development of pulmonary fibrosis in murine models. Furthermore, CHI3L1 stimulation induces a greater increase in CD206 expression in IPF monocytes than control monocytes. These results demonstrated that monocytes from patients with IPF appear to be hyperresponsive to CHI3L1 stimulation. These studies support targeting the CHI3L1-CRTH2 pathway as a promising therapeutic approach for IPF and that the sensitivity of blood monocytes to CHI3L1-induced profibrotic differentiation may serve as a biomarker that predicts responsiveness to CHI3L1- or CRTH2-based interventions.

Generic benzalkonium chloride-preserved travoprost eye drops are not identical to the branded polyquarternium-1-preserved travoprost eye drop: Effect on cultured human conjunctival goblet cells and their physicochemical properties.

To investigate the effect of polyquaternium-1 (PQ)-preserved and benzalkonium chloride (BAK)-preserved travoprost eye drops on viability of primary human conjunctival goblet cell (GC) cultures and on secretion of mucin and cytokines. Furthermore, to evaluate the physicochemical properties of the branded travoprost eye drop Travatan® and available generics. The effect of travoprost eye drops was evaluated on GC cultures. Cell viability was assessed through lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin secretion was evaluated by immunohistochemical staining. Secretion of interleukin (IL)-6 and IL-8 was measured using BD Cytometric Bead Arrays. pH, viscosity, droplet mass, osmolality and surface tension were measured for all included eye drops. In the LDH assay, BAK travoprost caused significant GC loss after 2 hrs of incubation compared to the control. PQ travoprost caused no GC loss at any time point. Both PQ- and BAK travoprost caused secretion of mucin to the cytoplasma. No difference in IL-6 and IL-8 secretion was identified compared to controls. The pH values for the generics were lower (pH 6.0) than the pH value for Travatan (pH 6.7; p < 0.0001). The viscosity was lowest for Travatan, while the mean droplet mass was higher for Travatan (35 mg) than the generics (28-30 mg; p ≤ 0.0318). The osmolality and surface tension did not differ between the eye drops investigated. BAK travoprost caused GC loss, indicating that PQ preservation may be preferable in treatment of glaucoma. Furthermore, physicochemical properties of branded and generic travoprost eye drops can not be assumed to be identical.

Glycogene Expression Profile of Human Limbal Epithelial Cells with Distinct Clonogenic Potential.

Glycans function as valuable markers of stem cells but also regulate the ability of these cells to self-renew and differentiate. Approximately 2% of the human genome encodes for proteins that are involved in the biosynthesis and recognition of glycans. In the present study, we evaluated the expression of a small subset of glycogenes in human limbal epithelial cells with distinct clonogenic potential. Individual clones were classified as abortive or clonogenic, based on the fraction of the terminal colonies produced; clones leading exclusively to terminal colonies were referred to as abortive while those with half or fewer terminal colonies were referred to as clonogenic. An analysis of glycogene expression in clonogenic cultures revealed a high content of transcripts regulating the galactose and mannose metabolic pathways. Abortive clones were characterized by increased levels of GCNT4 and FUCA2, genes that are responsible for the branching of mucin-type O-glycans and the hydrolysis of fucose residues on N-glycans, respectively. The expansion of primary cultures of human limbal epithelial cells for 10 days resulted in stratification and a concomitant increase in MUC16, GCNT4 and FUCA2 expression. These data indicate that the clonogenic potential of human limbal epithelial cells is associated with specific glycosylation pathways. Mucin-type O-glycan branching and increased fucose metabolism are linked to limbal epithelial cell differentiation.

Genetic deletion of endothelial Caveolin‐1 is protective against metabolic disease

In metabolic disease, the endothelium is exposed to a substantial increase in fatty acid concentrations creating a pathological role for endothelial cells (ECs) as lipid metabolizing cells. One mechanism whereby lipids may enter ECs is by caveolin‐1 (Cav1)‐mediated endocytosis. Because both ECs and adipocytes both have abundant Cav1 and both can readily take up lipids, we wanted to differentiate the role of Cav1 between the two cell types. For these reasons, we used an inducible EC‐specific Cav1 knockout mouse (Cav1fl/fl/Cdh5‐CreERT2+). Using these mice, we induced Cav1 genetic deletion from endothelium at 6 weeks, and began a high fat diet (HFD; 60% fat) to induce metabolic disease at 8 weeks. Normal chow fed littermates were used as controls. All mice were examined at 20 weeks (i.e., 12 weeks of HFD). Thus, we had four cohorts: Cre+ HFD, Cre‐ HFD, Cre+ normal chow, and Cre‐ normal chow all injected with tamoxifen. All mice regardless of genotype had comparable weight gain within diet groups, and gonadal fat pad mass. Food consumption was similar between groups, and electron microscopy was used to confirm presence of caveola in adipocytes, but not endothelium. Initial experiments examining plasma lipids found HFD Cre+ mice had both increased serum cholesterol (175mg/dL) and LDL (75mg/dL) compared to HFD Cre‐ controls (cholesterol, 125mg/dL; LDL, 60mg/dL). When these mice were challenged with a bolus of insulin, the blood glucose of Cre+ HFD mice dropped significantly resembling an insulin tolerance curve from normal chow fed controls. Similarly, Cre+ HFD mice were protected from hyperglycemia after bolus glucose injections, resulting in a glucose tolerance phenotype similar to that of a normal chow fed mouse (HFD Cre+ AUC 3000 vs. HFD Cre‐ AUC 60000). There was no difference in Cre+ or Cre‐ mice on normal chow diets with serum lipids or after challenge with insulin or glucose. It isn’t clear why the insulin and glucose protection correspond with significantly increased lipids in the Cre+ HFD mice. For these reasons, we began investigating the possible mechanisms with primary cultures of Human Adipose Microvascular Endothelial Cells (HAMECs) and knocked down Cav1 with siRNA. In monolayers of HAMEC without any other cell type present, we did not observe altered lipid uptake after addition of 50µM of exogenous fatty acids with a 70% knockdown of Cav1 protein. Additionally, HAMECs seahorse assays were used to determine if there were changes in metabolic function when Cav1 was knocked down. This work provides initial evidence for the unique metabolic roles of endothelial Cav1 during periods of high lipid circulation.

Oxidative stress and metformin: An in-vitro study on serum and primary human granulosa cell cultures

Objective: To observe the effect of metformin on the oxidative stress (OS) levels and SIRT1 expression in primary human granulosa cell cultures from infertile females. Methods: This cross-sectional study was conducted from August 2017-July 2019 on ten infertile patients. After written informed consent, the follicular fluid of these subjects was collected for establishing primary human granulosa cell cultures (HGCs), to test the capacity of metformin to reduce OS. The cultured HGCs were divided into (i) control: HGCs without any treatment, (ii) test1: HGCs induced with H2O2 (metformin absent), and (iii) test2: HGCs treated with metformin after H2O2 induction. Baseline OS (control group) was estimated, it was induced and measured by an increase in optical density (OD) (test1 & test 2). Finally, test2 was incubated with metformin (1 ml of 100 mmol/l concentration) and it effect on the levels of OS were determined by Mishra OS assay. Effect of metformin modulating SIRT1 expression in OS-induced cells was analyzed using quantitative-PCR. Results: Age of female subjects was 32.04±2.29years and BMI was 27.61±2.15 kg/m2. Test (OS induced) samples gave an OD of 0.28 (0.16-0.40) while control HGC samples gave an OD; 0.153 (0.09-0.23). Test cells showed significant reduction in ODs after metformin treatment. The relative expression of SIRT1 in metformin untreated and treated cells was found to be 61.5% and 80%, respectively. Conclusion: Metformin was found to suppress OS in HGCs and increases the expression of SIRT1 in OS induced environment of primary cell cultures, suggesting a relationship between metformin, SIRT1 expression, and reduction of OS.

Topical Nanoemulsion of a Runt-related Transcription Factor 1 Inhibitor for the Treatment of Pathologic Ocular Angiogenesis.

Purpose:To test the efficacy of runt-related transcription factor 1 (RUNX1) inhibition with topical nanoemulsion containing Ro5-3335 (eNano-Ro5) in experimental ocular neovascularization.Design:Preclinical experimental study.Participants:In vitro primary culture human retinal endothelial cell (HREC) culture. C57BL/6J 6- to 10-week-old male and female mice.Methods:We evaluated the effect of eNano-Ro5 in cell proliferation, cell toxicity, and migration of HRECs. We used an alkali burn model of corneal neovascularization and a laser-induced model of choroidal neovascularization to test in vivo efficacy of eNano-Ro5 in pathologic angiogenesis in mice. We used mass spectrometry to measure penetration of Ro5-3335 released from the nanoemulsion in ocular tissues.Main Outcome Measures:Neovascular area.Results:RUNX1 inhibition reduced cell proliferation and migration in vitro. Mass spectrometry analysis revealed detectable levels of the active RUNX1 small-molecule inhibitor Ro5-3335 in the anterior and posterior segment of the mice eyes. Topical treatment with eNano-Ro5 significantly reduced corneal neovascularization and improved corneal wound healing after alkali burn. Choroidal neovascularization lesion size and leakage were significantly reduced after treatment with topical eNano-Ro5.Conclusions:Topical treatment with eNano-Ro5 is an effective and viable platform to deliver a small-molecule RUNX1 inhibitor. This route of administration offers advantages that could improve the management and outcomes of these sight-threatening conditions. Topical noninvasive delivery of RUNX1 inhibitor could be beneficial for many patients with pathologic ocular neovascularization.

Molecular Genetics and Cytotoxic Responses to Titanium Diboride and Zinc Borate Nanoparticles on Cultured Human Primary Alveolar Epithelial Cells

Titanium diboride (TiB2) and zinc borate (Zn3BO6) have been utilized in wide spectrum industrial areas because of their favorable properties such as a high melting point, good wear resistance, high hardness and thermal conductivity. On the other hand, the biomedical potentials of TiB2 and Zn3BO6 are still unknown because there is no comprehensive analysis that uncovers their biocompatibility features. Thus, the toxicogenomic properties of TiB2 and Zn3BO6 nanoparticles (NPs) were investigated on human primary alveolar epithelial cell cultures (HPAEpiC) by using different cell viability assays and microarray analyses. Protein-Protein Interaction Networks Functional Enrichment Analysis (STRING) was used to associate differentially expressed gene probes. According to the results, up to 10 mg/L concentration of TiB2 and Zn3BO6 NPs application did not stimulate a cytotoxic effect on the HPAEpiC cell cultures. Microarray analysis revealed that TiB2 NPs exposure enhances cellular adhesion molecules, proteases and carrier protein expression. Furthermore, Zn3BO6 NPs caused differential gene expressions in the cell cycle, cell division and extracellular matrix regulators. Finally, STRING analyses put forth that inflammation, cell regeneration and tissue repair-related gene interactions were affected by TiB2 NPs application. Zn3BO6 NPs exposure significantly altered inflammation, lipid metabolism and infection response activator-related gene interactions. These investigations illustrated that TiB2 and Zn3BO6 NPs exposure may affect different aspects of cellular machineries such as immunogenic responses, tissue regeneration and cell survival. Thus, these types of cellular mechanisms should be taken into account before the use of the related NPs in further biomedical applications.