The impact of hyperglycemia on adhesion between endothelial and cancer cells revealed by single‐cell force spectroscopy

The impact of hyperglycemia on adhesion between lung carcinoma cells (A549) and pulmonary human aorta endothelial cells (PHAEC) was studied using the single‐cell force spectroscopy. Cancer cells were immobilized on a tipless Atomic Force Microscopy (AFM) cantilever and a single layer of endothelial cells was prepared on a glass slide. The measured force‐distance curves provided information about the detachment force and about the frequency of specific ligand‐receptor rupture events. Measurements were performed for different times of short term (up to 2 h) and prolonged hyperglycemia (3 h ‐ 24 h). Single‐cell force results were correlated with the expression of cell adhesion molecules (intercellular adhesion molecule, P‐selectin) and with the length and density of the PHAECs glycocalyx layer, which were measured by AFM nanoindentation. For short‐term hyperglycemia, we observed a statistically significant increase of the adhesion parameters that was accompanied by an increase of the glycocalyx length and expression of P‐selectin. Removal of hyaluronic acid from PHAECs glycocalyx significantly decreased the adhesion parameters, which indicates that hyaluronic acid has a strong impact on adhesion in A549/PHAEC system in short term of hyperglycemia. For prolonged hyperglycemia, the most significant increase of adhesion parameters was observed for 24 hours and this phenomenon correlated with the expression of adhesion molecules and a decrease of the glycocalyx length. Taking together, presented data indicate that both mechanical and structural properties of the endothelial glycocalyx strongly modulate the adhesion in the A549/PHAEC system.     

Spatial phenotyping of the endocardial endothelium as a function of intracardiac hemodynamic shear stress

Despite substantial evidence for the central role of hemodynamic shear stress in the functional integrity of vascular endothelial cells, hemodynamic and molecular regulation of the endocardial endothelium lining the heart chambers remains understudied. We propose that regional differences in intracardiac hemodynamics influence differential endocardial gene expression leading to phenotypic heterogeneity of this cell layer. Measurement of intracardiac hemodynamics was performed using 4-dimensional flow MRI in healthy humans (n=8) and pigs (n=5). Local wall shear stress (WSS) and oscillatory shear indices (OSI) were calculated in three distinct regions of the LV – base, mid-ventricle (midV), and apex. In both the humans and pigs, WSS values were significantly lower in the apex and midV relative to the base. Additionally, both the apex and midV had greater oscillatory shear indices (OSI) than the base. To investigate regional phenotype, endocardial endothelial cells (EEC) were isolated from an additional 8 pigs and RNA sequencing was performed. A false discovery rate of 0.10 identified 1051 differentially expressed genes between the base and apex, and 321 between base and midV. Pathway analyses revealed apical upregulation of genes associated with translation initiation. Furthermore, tissue factor pathway inhibitor (TFPI; mean 50-fold) and prostacyclin synthase (PTGIS; 5-fold), genes prominently associated with antithrombotic protection, were consistently upregulated in LV apex. These spatio-temporal WSS values in defined regions of the left ventricle link local hemodynamics to regional heterogeneity in endocardial gene expression.     

与转染Endostatin基因的角朊细胞共培养时内皮细胞增殖特性变化

构建含Endostatin基因的腺病毒载体,将Endostatin基因导入培养的角朊细胞,并采用套皿法共培养角朊细胞与内皮细胞,测定培养液中Endostatin含量、内皮细胞增殖周期各时相比例、内皮细胞凋亡及细胞抑制率。结果表明转染Endostatin基因的角朊细胞可有效表达并分泌Endostatin,连续培养3天后,培养液中Endostatin含量可达226ng/ml;与转基因角朊细胞共培养的内皮细胞凋亡百分数与抑制率分别为(32.7±7.1)%、(60.5±8.3)%,均显著高于对照组[(7.3±2.0)%,(13.8±1.6)%],且G0/G1期比例明显高于对照组,而S期、G2/M期比例及增殖指数显著低于对照组。因此,转染Endostatin基因角朊细胞与内皮细胞共培养时,角朊细胞可通过分泌Endostatin促进内皮细胞凋亡,并抑制其增殖。     

Imaging gene expression in the brain with peptide nucleic acid (PNA) antisense radiopharmaceuticals and drug targeting technology

Antisense oligomers are potential pharmaceutical and radiopharmaceutical agents that can be used to modulate and image gene expression. Progress with in vivogene targeting using antisense-based therapeutics has been slower than expected during the last decade, owing to poor trans-cellular delivery of antisense agents. This chapter suggests that if antisense pharmacology is merged with drug targeting technology, then membrane barriers can be circumvented and antisense agents can be delivered to tissues in vivo. Without the application of drug targeting, the likelihood of success for an antisense drug development program is low, particularly for the brain which is protected by the blood-brain barrier (BBB). Among the different classes of antisense agents, peptide nucleic acids (PNA) present advantages for in vivoapplications over conventional and modified oligodeoxynucleotides (ODN), including phosphorothioates (PS)-ODN. Some advantages of PNAs include their electrically neutral backbone, low toxicity to neural cells, resistance to nucleases and peptidases, and lack of binding to plasma proteins. PNAs are poorly transported through cellular membranes, however, including the BBB and the brain cell membrane (BCM). Because the mRNA target for the antisense agent lies within the cytosol of the target cell, the BBB and the BCM must be circumvented in vivo, which ispossible with the use of chimeric peptide drug targeting technology. Chimeric peptides are formed by conjugation of a non-transportable drug, such as a PNA, to a drug delivery vector. The vector undergoes receptor-mediated transcytosis (RMT) through the BBB and receptor-mediated endocytosis through the BCM in vivo. When labeled with a radioisotope (e.g., ¹²⁵I or ¹¹¹In), the antisense chimeric peptide provides imaging of gene expressionin the brain in vivoin a sequence-specific manner. Further development of antisense radiopharmaceutical agents may allow for in vivoimaging of genes in pathological states, and may provide tools for the analysis of novel genes with functional genomics.     

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O₂, 5% CO₂, 93% N₂) for 24 h followed by reoxygenation (21% O₂, 5% CO₂, 74% N₂) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ～30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.     

Cell cycle dynamics and complement expression distinguishes mature haematopoietic subsets arising from hemogenic endothelium

The emergence of haematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium results in the formation of sizeable HSPC clusters attached to the vascular wall. We evaluate the cell cycle and proliferation of HSPCs involved in cluster formation, as well as the molecular signatures from their initial appearance to the point when cluster cells are capable of adult engraftment (definitive HSCs). We uncover a non-clonal origin of HSPC clusters with differing cell cycle, migration, and cell signaling attributes. In addition, we find that the complement cascade is highly enriched in mature HSPC clusters, possibly delineating a new role for this pathway in engraftment.     

Vitamin D and cancer: Deciphering the truth

Vitamin D is a hormone-like micronutrient involved not only in calcium metabolism but also in a variety of biological activities (e.g., cell proliferation, apoptosis, angiogenesis, inflammation) that makes it a candidate anticancer agent. Preclinical studies support the therapeutic potential of vitamin D both alone and in combination with other therapeutics. Overall, epidemiological data suggest the existence of a link between vitamin D and cancer risk, whereas the results of clinical trials are quite conflicting.     

A pH-regulated dimeric bouquet in the structure of von Willebrand factor

At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal ～1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.     

Discrete Notch signaling requirements in the specification of hematopoietic stem cells

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.     

Scanning electron microscope evidence of telocytes in vasculature

Here, we here present scanning electron microscope data for the existent telocytes (TCs) on the endothelial surface of the wall of pig coronary arteries, internal thoracic arteries and carotid arteries. These cells have a small (8.39 ± 1.97 μm/4.95 ± 0.91 μm) cell body of different shapes (from round to triangular, depending on the number of cellular prolongations) with very long (of about 30 μm) and thin cellular processes called telopodes (Tps), which have uneven calibre. The number of Tps ranges between 2 and 6. Tps typically present the alternation of podoms and podomers, and also have a dichotomic branching pattern. These data could influence the current attempts for elucidating the role(s) of TCs.