Sparse Cultures Research Articles

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Abstract P1001: IGFBP2 Overcomes Cell Contact-mediated Proliferation Inhibition In Human IPSC-derived Cardiomyocytes

Introduction and Hypothesis: Human induced pluripotent stem cells (hiPSCs) and their derivatives provide a promising cellular source for regenerative cardiac therapies. However, generating large-scale hiPSC-derived cardiomyocytes (hiPSC-CMs) remains a major hurdle, caused, in part, by cell contact-driven proliferation inhibition in dense culture conditions. We hypothesize that differentially-secreted factors in sparsely vs densely-cultured hiPSC-CMs are responsible for the increased proliferation rate in sparsely cultured hiPSC-CMs. Methods: We performed a high-throughput screening (HTS) assay to identify growth factors (GFs) released in the supernatant of sparsely- and densely-cultured hiPSC-CMs. Differential expression of candidate GFs in dense vs sparse culture conditions was confirmed by single cell RNA sequencing (scRNAseq), Western Blot (WB), and Immunocytochemistry (ICC) analyses. The confirmed GF hits were further validated by measuring the expression of the cell cycle marker Ki67 in hiPSC-CMs after GF treatment. The GF treatment effects on cell-cell contact was further explored mechanistically using siRNA-based N-cadherin (CDH2) knockdown (KD) model in vitro. Results: Our HTS assay identified IGFBP2, IGFBP6, PDGF-AA as top candidate GFs in the sparse culture supernatant. Subsequent WB, ICC, scRNAseq analyses confirmed 1.5, 3.5, 2.0-fold higher IGFBP2 expression in the sparse condition compared to the dense condition. Supplementation of recombinant IGFBP2 to densely cultured hiPSC-CMs showed a dosage-dependent increase in Ki67 expression level from 13.0 +/- 1.4% in control to 44.1 +/- 8.4% after 3nM IGFBP2 treatment, a level comparable to that achieved in sparse culture. Mechanistically, IGFBP2 treatment resulted in a shift of CDH2 localization from cell-cell junction to the cytoplasm. Reducing CDH2 expression by CDH2-KD to mimic loss of cell-cell contact promoted hiPSC-CM proliferation. Conclusion: We discovered an unexpected role of IGFBP2 to overcome cell contact-mediated inhibition of hiPSC-CM proliferation which provides a promising new approach to engineer in situ large-scale 3D cardiac tissues for regenerative applications.

Yes-associated protein activation potentiates glycogen synthase kinase-3 inhibitor-induced proliferation of neonatal cardiomyocytes and iPS cell-derived cardiomyocytes.

Because mammalian cardiomyocytes largely cease to proliferate immediately after birth, the regenerative activity of the heart is limited. To date, much effort has been made to clarify the regulatory mechanism of cardiomyocyte proliferation because the amplification of cardiomyocytes could be a promising strategy for heart regenerative therapy. Recently, it was reported that the inhibition of glycogen synthase kinase (GSK)‐3 promotes the proliferation of neonatal rat cardiomyocytes (NRCMs) and human iPS cell‐derived cardiomyocytes (hiPSC‐CMs). Additionally, Yes‐associated protein (YAP) induces cardiomyocyte proliferation. The purpose of this study was to address the importance of YAP activity in cardiomyocyte proliferation induced by GSK‐3 inhibitors (GSK‐3Is) to develop a novel strategy for cardiomyocyte amplification. Immunofluorescent microscopic analysis using an anti‐Ki‐67 antibody demonstrated that the treatment of NRCMs with GSK‐3Is, such as BIO and CHIR99021, increased the ratio of proliferative cardiomyocytes. YAP was localized in the nuclei of more than 95% of cardiomyocytes, either in the presence or absence of GSK‐3Is, indicating that YAP was endogenously activated. GSK‐3Is increased the expression of β‐catenin and promoted its translocation into the nucleus without influencing YAP activity. The knockdown of YAP using siRNA or pharmacological inhibition of YAP using verteporfin or CIL56 dramatically reduced GSK‐3I‐induced cardiomyocyte proliferation without suppressing β‐catenin activation. Interestingly, the inhibition of GSK‐3 also induced the proliferation of hiPSC‐CMs under sparse culture conditions, where YAP was constitutively activated. In contrast, under dense culture conditions, in which YAP activity was suppressed, the proliferative effects of GSK‐3Is on hiPSC‐CMs were not detected. Importantly, the activation of YAP by the knockdown of α‐catenin restored the proproliferative activity of GSK‐3Is. Collectively, YAP activation potentiates the GSK‐3I‐induced proliferation of cardiomyocytes. The blockade of GSK‐3 in combination with YAP activation resulted in remarkable amplification of cardiomyocytes.

PI3K-AKT, JAK2-STAT3 pathways and cell\u2013cell contact regulate maspin subcellular localization

BackgroundMaspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell–cell contact in this process.MethodsMCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. Proteomic and interactome analyses were conducted to identify maspin-binding proteins in EGF-treated cells only. To investigate the role of cell–cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence.ResultsWe found that PI3K-Akt and JAK2-STAT3, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. We observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell–cell adhesion.ConclusionsMaspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-Akt and JAK2-STAT3 pathways) and cell–cell contact.DeaMyif71VSQHA76EZpJtKVideo

Cell Density-Dependent Fibroblast Growth Factor-2 Signaling Regulates Syndecan-4 Expression in Cultured Vascular Endothelial Cells.

Syndecan-4 is a member of the syndecan family of transmembrane heparan sulfate proteoglycans, and is involved in cell protection, proliferation, and the blood coagulation-fibrinolytic system in vascular endothelial cells. Heparan sulfate chains enable fibroblast growth factor-2 (FGF-2) to form a complex with its receptor and to transduce the cell growth signal. In the present study, bovine aortic endothelial cells were cultured, and the intracellular signal pathways that mediate the regulation of syndecan-4 expression in dense and sparse cultures by FGF-2 were analyzed. We demonstrated the cell density-dependent differential regulation of syndecan-4 expression. Specifically, we found that FGF-2 upregulated the synthesis of syndecan-4 in vascular endothelial cells via the MEK1/2-ERK1/2 pathway in dense cell cultures, with only a transcriptional induction of syndecan-4 at a low cell density via the Akt pathway. This study highlights a critical mechanism underlying the regulation of endothelial cell functions by proteoglycans.

A revalidation and critique of assumptions about urinary sample collection methods, specimen quality and contamination

Introduction and hypothesisMidstream urine (MSU) is key in assessing lower urinary tract syndrome (LUTS), but contingent on some assumptions. The aim of this study was to compare the occurrence of contamination and the quality of substrates obtained from four different collections: MSU, catheter specimen urine (CSU), a commercial MSU collecting device (Peezy) and a natural void. Contamination was quantified by differential, uroplakin-positive, urothelial cell counts.MethodsThis was a single blind, crossover study conducted in two phases. First, we compared the MSU with CSU using urine culture, pyuria counts and differential counting of epithelial cells after immunofluorescence staining for uroplakin III (UP3). Second, we compared the three non-invasive (MSU, Peezy MSU™, natural void) methods using UP3 antibody staining only.ResultsThe natural void was best at collecting bladder urinary sediment, with the majority of epithelial cells present derived from the urinary tract. CSU sampling missed much of the urinary sediment and showed sparse culture results. Finally, the MSU collection methods did not capture much of the bladder sediment.ConclusionWe found little evidence for contamination with the four methods. Natural void was the best method for harvesting shed urothelial cells and white blood cells. It provides a richer sample of the inflammatory exudate, including parasitised urothelial cells and the microbial substrate. However, if the midstream sample is believed to be important, the MSU collection device is advantageous.

The Administration of Semax and HLDF-6 Peptides to Rats Regulates Protein Synthesis Rhythm in Hepatocytes and Corrects Senescent Disturbances

To clarify the organizing effect of Semax and HLDF-6 peptides on the kinetics of protein synthesis in hepatocytes, in addition to an in vitro study (Brodsky et al., 2019), the effects of these peptides in vivo have been studied. The circahoralian (ultradian) rhythm of protein synthesis, that is, a marker of the direct cell-to-cell communication, was investigated in rats of different ages. Peptides were injected intraperitoneally into young (2–3-month-old) or old (1.5–2-year-old) rats at a 50–100-µg/kg dose. Hepatocytes were isolated and sparse or dense cultures were established. In sparse cultures from young rats that received one or another peptide, the rhythm of protein synthesis was observed; in the cultures from rats of the same age that were injected with saline, no rhythm was found. In dense cultures of old rats after the action of the peptide, the amplitudes of the rhythm of protein synthesis did not differ from the rhythms observed in young rats; after saline injection, the rhythm amplitudes were twice as low. Injection of the peptide into adult rats that had previously received dopamine caused a protein synthesis rhythm characteristic for rats of this age; the administration of dopamine abolished the rhythm. The synchronizing effect of the peptides was maintained for at least 2 days after their single administration to the rat. The use of Semax or HLDF-6 is recommended to compensate for the disturbances of the kinetics of protein synthesis in humans in aging and pathology.

Insulin-like growth factor 1 partially rescues early developmental defects caused by SHANK2 knockdown in human neurons.

SHANK2 is a scaffold protein that serves as a protein anchor at the postsynaptic density in neurons. Genetic variants of SHANK2 are strongly associated with synaptic dysfunction and the pathophysiology of autism spectrum disorder. Recent studies indicate that early neuronal developmental defects play a role in the pathogenesis of autism spectrum disorder, and that insulin-like growth factor 1 has a positive effect on neurite development. To investigate the effects of SHANK2 knockdown on early neuronal development, we generated a sparse culture system using human induced pluripotent stem cells, which then differentiated into neural progenitor cells after 3-14 days in culture, and which were dissociated into single neurons. Neurons in the experimental group were infected with shSHANK2 lentivirus carrying a red fluorescent protein reporter (shSHANK2 group). Control neurons were infected with scrambled shControl lentivirus carrying a red fluorescent protein reporter (shControl group). Neuronal somata and neurites were reconstructed based on the lentiviral red fluorescent protein signal. Developmental dendritic and motility changes in VGLUT1+ glutamatergic neurons and TH+ dopaminergic neurons were then evaluated in both groups. Compared with shControl VGLUT1+ neurons, the dendritic length and arborizations of shSHANK2 VGLUT1+ neurons were shorter and fewer, while cell soma speed was higher. Furthermore, dendritic length and arborization were significantly increased after insulin-like growth factor 1 treatment of shSHANK2 neurons, while cell soma speed remained unaffected. These results suggest that insulin-like growth factor 1 can rescue morphological defects, but not the change in neuronal motility. Collectively, our findings demonstrate that SHANK2 deficiency perturbs early neuronal development, and that IGF1 can partially rescue the neuronal defects caused by SHANK2 knockdown. All experimental procedures and protocols were approved by the Laboratory Animal Ethics Committee of Jinan University, China (approval No. 20170228010) on February 28, 2017.

Glycosylation is a novel TGFβ1-independent post-translational modification of Smad2

Smad2 is a crucial component of intracellular signaling by transforming growth factor-β (TGFβ). Here we describe that Smad2 is glycosylated, which is a novel for Smad2 post-translational modification. We showed that the Smad2 glycosylation was inhibited upon treatment of cells with 17β-estradiol, and was enhanced in cells in a dense culture as compared to cells in a sparse culture. The Smad2 glycosylation was not dependent on the C-terminal phosphorylation of Smad2, and was not affected by TGFβ1 treatment of the cells. Smad2 was glycosylated at multiple sites, and one of the predicted sites is Serine110. Thus, Smad2 is glycosylated, and this post-translational modification was modulated by 17β-estradiol but not by TGFβ1.

Analysis of ADAMTS Effects on Cell Adhesion and Migration.

As extracellular enzymes that interact extensively with extracellular matrix (ECM) components, several ADAMTS enzymes are understood to influence aspects of cell adhesion to the ECM and the ability of cells to migrate. A standard approach to investigate the involvement of an ADAMTS in these aspects of mammalian cell behavior involves siRNA-mediated knockdown of the expression of the gene of interest in cell culture, followed by methods for quantification of migratory or adhesive behavior. We describe here two methods for cell migration quantification: a time-lapse videomicroscopy method suitable for measuring single cell migration in sparse cultures that allows for determination of migration speed and directionality (persistence), and scratch wound assays for directional migration in confluent cell monolayers. We also present assays to quantify total adhesion to ECM components, as well as more detailed visualization and quantification of focal adhesion structures.

Lipophilic statins inhibit YAP nuclear localization, co-activator activity and colony formation in pancreatic cancer cells and prevent the initial stages of pancreatic ductal adenocarcinoma in KrasG12D mice.

We examined the impact of statins on Yes-associated Protein (YAP) localization, phosphorylation and transcriptional activity in human and mouse pancreatic ductal adenocarcinoma (PDAC) cells. Exposure of sparse cultures of PANC-1 and MiaPaCa-2 cells to cerivastatin or simvastatin induced a striking re-localization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEAD-regulated genes Connective Tissue Growth Factor (CTGF) and Cysteine-rich angiogenic inducer 61 (CYR61). Statins also prevented YAP nuclear import and expression of CTGF and CYR61 stimulated by the mitogenic combination of insulin and neurotensin in dense culture of these PDAC cells. Cerivastatin, simvastatin, atorvastatin and fluvastatin also inhibited colony formation by PANC-1 and MiaPaCa-2 cells in a dose-dependent manner. In contrast, the hydrophilic statin pravastatin did not exert any inhibitory effect even at a high concentration (10 μM). Mechanistically, cerivastatin did not alter the phosphorylation of YAP at Ser127 in either PANC-1 or MiaPaCa-2 cells incubated without or with neurotensin and insulin but blunted the assembly of actin stress fiber in these cells. We extended these findings with human PDAC cells using primary KC and KPC cells, (expressing KrasG12D or both KrasG12D and mutant p53, respectively) isolated from KC or KPC mice. Using cultures of these murine cells, we show that lipophilic statins induced striking YAP translocation from the nucleus to the cytoplasm, inhibited the expression of Ctgf, Cyr61 and Birc5 and profoundly inhibited colony formation of these cells. Administration of simvastatin to KC mice subjected to diet-induced obesity prevented early pancreatic acini depletion and PanIN formation. Collectively, our results show that lipophilic statins restrain YAP activity and proliferation in pancreatic cancer cell models in vitro and attenuates early lesions leading to PDAC in vivo.

山形県における水稲「はえぬき」の疎植栽培について

山形県における水稲「はえぬき」の疎植栽培について

Properties of Dental Pulp–derived Mesenchymal Stem Cells and the Effects of Culture Conditions

Dental pulp mesenchymal stem cells (DPMSCs) highly express mesenchymal stem cell markers and possess the potential to differentiate into neural cells, osteoblasts, adipocytes, and chondrocytes. Thus, DPMSCs are considered suitable for tissue regeneration.The colony isolation method has commonly been used to collect relatively large amounts of heterogeneous DPMSCs. Homogenous DPMSCs can be isolated by fluorescence-activated cell sorting using antibodies against mesenchymal stem cell markers, although this method yields a limited number of cells. Both quality and quantity of DPMSCs are critical to regenerative therapy, and cell culture methods need to be improved. We thus investigated the properties of DPMSCs cultured with different methods.DPMSCs in a three-dimensional spheroid culture system, which is similar to the hanging drop culture for differentiation of embryonic stem cells, showed upregulation of odonto-/osteoblastic markers and mineralized nodule formation. This suggests that this three-dimensional spheroid culturing system for DPMSCs may be suitable for inducing hard tissues. We further examined the effect of cell culture density on the properties of DPMSCs because the properties of stem cells can be altered depending on the cell density. DPMSCs cultured under the confluent cell density condition showed slight downregulation of some mesenchymal stem cell markers compared with those under the sparse condition.The ability of DPMSCs to differentiate into hard tissue–forming cells was found to be enhanced in the confluent condition, suggesting that the confluent culture condition may not be suitable for maintaining the stemness of DPMSCs. When DPMSCs are to be used for hard tissue regeneration, dense followed by sparse cell culture conditions may be a better alternative strategy.

Immunogenicity is preferentially induced in sparse dendritic cell cultures

We have previously shown that human monocyte-derived dendritic cells (DCs) acquired different characteristics in dense or sparse cell cultures. Sparsity promoted the development of IL-12 producing migratory DCs, whereas dense cultures increased IL-10 production. Here we analysed whether the density-dependent endogenous breaks could modulate DC-based vaccines. Using murine bone marrow-derived DC models we show that sparse cultures were essential to achieve several key functions required for immunogenic DC vaccines, including mobility to draining lymph nodes, recruitment and massive proliferation of antigen-specific CD4+ T cells, in addition to their TH1 polarization. Transcription analyses confirmed higher commitment in sparse cultures towards T cell activation, whereas DCs obtained from dense cultures up-regulated immunosuppressive pathway components and genes suggesting higher differentiation plasticity towards osteoclasts. Interestingly, we detected a striking up-regulation of fatty acid and cholesterol biosynthesis pathways in sparse cultures, suggesting an important link between DC immunogenicity and lipid homeostasis regulation.

Tropicalizing Philip Quaque: On African Atlantic Literatures

Whereas slave narratives are classics of eighteenth-century studies, there has been scant attention to African eighteenth-century writing written by parties implicated in the slave trade. That absence reduces the diversity of African statements about the slave trade and African participation in the pre-abolition Atlantic world. Using the letters of Philip Quaque, as a case study, I argue that attention to the sparse eighteenth-century West African literary culture complicates the repertoire of African representations of the slave-trading Atlantic world.

CXCL11 Expression by Keratinocytes Occurs Transiently Between Reaching Confluence and Cellular Compaction.

Objective: To investigate whether differentiation or cellular confluence is responsible for CXCL11 expression patterns in re-epithelialization. Approach:In vitro model systems of re-epithelialization using the HaCaT keratinocyte cell line were utilized in monitoring expression of differentiation markers, including desmoplakin and various cytokeratins while evaluating for an association with chemokine CXCL11 expression. Results: CXCL11 expression was elevated in sparse culture with peak expression near the time of confluence. This somewhat followed the accumulation of desmoplakin in detergent-insoluble pool of proteins. However, in postconfluent, despite continued accumulation of desmoplakin within cells, CXCL11 expression decreased to baseline levels. This biphasic pattern was also seen in low calcium culture, an environment that inhibits keratinocyte differentiation and accumulation of desmosomal proteins. Highest CXCL11-expressing areas best correlated with newly confluent areas within culture expressing basal keratin 14, but also activated keratin 6. Innovation: Achievement of a threshold cellular density induces cell signaling cascade through CXCR3 that, in addition to other undiscovered pathways, can progress cutaneous wounds from the proliferative into the remodeling phases of cutaneous wound healing. Conclusion: These results suggest that the achievement of confluence with increased cellular density by migrating keratinocytes at the wound edge triggers expression of CXCL11. Since CXCR3 stimulation in endothelial cells results in apoptosis and causes neovascular pruning, whereas stimulation of CXCR3 in fibroblasts results decreased motility and cellular contraction, we speculate that CXCL11 expression by epidermal cells upon achieving cellular confluence could be the source of CXCR3 stimulation in the dermis ushering a transition from proliferative to remodeling phases of wound healing.

Glutamic Acid - Amino Acid, Neurotransmitter, and Drug - Is Responsible for Protein Synthesis Rhythm in Hepatocyte Populations in vitro and in vivo.

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell-cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.

Distinct effects of β1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells.

An aberrant expression of integrin β1 has been implicated in breast cancer progression. Here, we compared the cell behaviors of wild-type (WT), β1 gene deleted (KO), and β1 gene restored (Res) MDA-MB-231 cells. Surprisingly, the expression of β1 exhibited opposite effects on cell proliferation. These effects were dependent on cell densities, and they showed an up-regulation of cell proliferation when cells were cultured under sparse conditions, and a down-regulation of cell growth under dense conditions. By comparison with WT cells, the phosphorylation levels of ERK in KO cells were consistently suppressed under sparse culture conditions, but consistently up-regulated under dense culture conditions. The phosphorylation levels of EGFR were increased in the KO cells. By contrast, the phosphorylation levels of AKT were decreased in the KO cells. The abilities for both colony and tumor formation were significantly suppressed in the KO cells, suggesting that β1 plays an important role in cell survival signaling for tumorigenesis. These aberrant phenotypes in the KO cells were rescued in the Res cells. Taken together, these results clearly showed the distinct roles of β1 in cancer cells: the inhibition of cell growth and the promotion of cell survival, which may shed light on cancer therapies.

Cell coupling mediated by connexin 26 selectively contributes to reduced adhesivity and increased migration.

Gap junction proteins (connexins) have crucial effects on cell motility in many systems, from migration of neural crest cells to promotion of metastatic invasiveness. Here, we show that expression of Cx26 (also known as GJB2) in HeLa cells specifically enhances cell motility in scrape wounding and sparse culture models. This effect is dependent on gap junction channels and is isotype specific [Cx26 enhances motility, whereas Cx43 (also known as GJA1) does not and Cx32 (also known as GJB1) has an intermediate effect]. The increased motility is associated with reduced cell adhesiveness, caused by loss of N-cadherin protein and RNA at the wound edge. This in turn causes a redistribution of N-cadherin-binding proteins (p120 catenin and β-catenin) to the cytosol and nucleus, respectively. The former activates Rac-1, which mediates cytoskeletal rearrangements needed for filopod extension. The latter is associated with increased expression of urokinase plasminogen activating receptor (an activator of extracellular proteases) and secretion of extracellular matrix components like collagen. Although these effects were dependent on Cx26-mediated coupling of the cells, they are not mediated by the same signal (i.e. cAMP) through which Cx26 has been shown to suppress proliferation in the same system.

Serglycin in Quiescent and Proliferating Primary Endothelial Cells.

Proteoglycans are fundamental components of the endothelial barrier, but the functions of the proteoglycan serglycin in endothelium are less described. Our aim was to describe the roles of serglycin in processes relevant for endothelial dysfunction. Primary human umbilical vein endothelial cells (HUVEC) were cultured in vitro and the expression of proteoglycans was investigated. Dense cell cultures representing the quiescent endothelium coating the vasculature was compared to sparse activated cell cultures, relevant for diabetes, cancer and cardiovascular disease. Secretion of 35S- proteoglycans increased in sparse cultures, and we showed that serglycin is a major component of the cell-density sensitive proteoglycan population. In contrast to the other proteoglycans, serglycin expression and secretion was higher in proliferating compared to quiescent HUVEC. RNAi silencing of serglycin inhibited proliferation and wound healing, and serglycin expression and secretion was augmented by hypoxia, mechanical strain and IL-1β induced inflammation. Notably, the secretion of the angiogenic chemokine CCL2 resulting from IL-1β activation, was increased in serglycin knockdown cells, while angiopoietin was not affected. Both serglycin and CCL2 were secreted predominantly to the apical side of polarized HUVEC, and serglycin and CCL2 co-localized both in perinuclear areas and in vesicles. These results suggest functions for serglycin in endothelial cells trough interactions with partner molecules, in biological processes with relevance for diabetic complications, cardiovascular disease and cancer development.