Differential Cellular Effects of Electroporation and Electrochemotherapy in Monolayers of Human Microvascular Endothelial Cells

In vivo electroporation of tumours shows disruption of blood flow and creates a vascular effect with an initial rapid and transient vasoconstriction phase and a much longer lasting phase with changed microvascular endothelium. These changes are not well understood but are presumed to involve the cytoskeleton. The paper presents for the first time differential in vitro effects describing cytoskeleton changes and monolayer integrity changes by both electroporation and electrochemotherapy of monolayers of human microvascular endothelial cells (HMEC-1). After the application of electric field pulses, the morphology of cells, and both the F-actin and Beta-tubulin cytoskeleton proteins were affected. During both electroporation and electrochemotherapy, the initial phase of cellular damage was noticed at 10 min as swollen cells and honeycomb-like actin bundles. The electroporation-induced cellular effects, observed from electric pulses >150 V, were voltage-dependent and within 24 hrs partly recoverable. The electrochemotherapy-induced cellular effects developed at 2 hrs in spindle-like cells, and more densely packed F-actin and Beta-tubulin were observed, which were dependent on the amount of bleomycin and the voltages applied (>50 V). In addition, for electrochemotherapy with electric pulses >150 V cellular changes were not recoverable within 24 hrs. The effects on monolayer integrity were reflected in the enhanced monolayer permeability, with the electrochemotherapy showing an earlier onset and synergy. We conclude that electrochemotherapy as compared to electroporation leads within 24 hrs to a quicker and more pronounced monolayer integrity damage and endothelial cell death, which together provide further insight into the cellular changes of the vascular disruption of electrochemotherapy.     

Shear stress effects on human embryonic kidney cells in Vitro

Human embryonic kidney cells grown as an attached, confluent monolayer on a flat substrate were subjected to steady, uniform laminar flow of medium in a specially designed chamber in which flow patterns and shear stress are accurately defined and controlled. Experiments were performed for shear stress levels ranging from 0.2 to 6.0 N/m(2) with times of exposure to the shear stress ranging from 2 to 24 h. The influence of the shear field was slight at low shear stress (0.26 N/m(2)). Higher stress levels (0.65 N/m(2) and higher) had significant effects on cell morphology, and on the post-shear release of urokinase enzyme. Still higher stress levels (2.6 N/m(2) and higher) caused marked reduction in cell viability. These results may be of interest in addressing practical problems in developing commercial biosynthesis reactors.     

Morphological analysis of tumor cell/endothelial cell interactions under shear flow

In the process of hematogenous cancer metastasis, tumor cells (TCs) must shed into the blood stream, survive in the blood circulation, migrate through the vascular endothelium (extravasation) and proliferate in the target organs. However, the precise mechanisms by which TCs penetrate the endothelial cell (EC) junctions remain one of the least understood aspects of TC extravasation. This question has generally been addressed under static conditions, despite the important role of flow induced mechanical stress on the circulating cell-endothelium interactions. Moreover, flow studies were generally focused on transient or firm adhesion steps of TC-EC interactions and did not consider TCs spreading or extravasation. In this paper, we used a parallel-plate flow chamber to investigate TC-EC interactions under flow conditions. An EC monolayer was cultured on the lower plate of the flow chamber to model the endothelial barrier. Circulating TCs were introduced into the flow channel under a well-defined flow field and TC cell shape changes on the EC monolayer were followed in vitro with live phase contrast and fluorescence microscopy. Two spreading patterns were observed: radial spreading which corresponds to TC extravasation, and axial spreading where TCs formed a mosaic TC-EC monolayer. By investigating the changes in area and minor/major aspect ratio, we have established a simple quantitative basis for comparing spreading modes under various shear stresses. Contrary to radial spreading, the extent of axial spreading was increased by shear stress.     

Adhesion of nonmetastatic and highly metastatic breast cancer cells to endothelial cells exposed to shear stress

The mechanical stimulus of shear stress has to date been neglected when studying the adhesion of cancer cells to the endothelium. Confluent monolayers of endothelial cells were subjected to either 4 or 15 hours of arterial shear stress. Adhesion of nonmetastatic (MCF-7) and highly metastatic (MDA-MB-435) human breast cancer cells was then quantified using a detachment assay carried out inside the parallel plate flow chamber. Four hours of shear stress exposure had no effect on adhesion. However, 15 hours of shear stress exposure led to marked changes in the ability of the endothelial monolayer to bind human breast cancer cells. An increase in adhesive strength was observed for nonmetastatic MCF-7 cells, while a decrease in adhesive strength was observed for highly metastatic MDA-MB-435 cells. Hence, endothelial shear stress stimulation does influence the adhesion of cancer cells to the endothelium and can have different effects on the adhesion of cancer cells with different metastatic potentials. Furthermore, adhesion of nonmetastatic and highly metastatic human breast cancer cells may be controlled by two different endothelial cell adhesion molecules that are differentially regulated by shear stress. Immunohistochemistry confirmed that shear stress did in fact differentially regulate endothelial cell adhesion molecule expression.     

Actinidin,a protease from kiwifruit,induces changes in morphology and adhesion of T84 intestinal epithelial cells

Actinidin belongs to the papain-like family of cysteine proteases and is a major kiwifruit allergen. In this study, the effect of actinidin on cellular morphology and adhesion of T84 intestinal cells was investigated. Both rounding and detachment of T84 cells were observed upon actinidin treatment. The morphological changes and cell desquamation was protease-dependent, as well as time- and concentration-dependent. Changes of intercellular adhesion and adhesion of epithelial cells to collagen upon actinidin treatment could be responsible for the cell rounding and give rise to discontinuous breaches in the epithelial monolayer observed in this study. Actinidin’s action on cell morphology, adhesion and monolayer integrity were not due to compromised viability of T84 epithelial cells, as confirmed by MTT assay and flow cytometric analysis of the cell cycle. Damage to the epithelial monolayer of the intestine induced by actinidin should be further evaluated as an important factor in the development of kiwifruit allergy and other intestinal disorders.     

Volume changes of an endothelial cell monolayer on exposure to anisotonic media

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120-630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.     

Apical Junctional Fluctuations Lead to Cell Flow while Maintaining Epithelial Integrity

Epithelial sheet integrity is robustly maintained during morphogenesis, which is essential to shape organs and embryos. While maintaining the planar monolayer in three-dimensional space, cells dynamically flow via rearranging their connections between each other. However, little is known about how cells maintain the plane sheet integrity in three-dimensional space and provide cell flow in the in-plane sheet. In this study, using a three-dimensional vertex model, we demonstrate that apical junctional fluctuations allow stable cell rearrangements while ensuring monolayer integrity. In addition to the fluctuations, direction-dependent contraction on the apical cell boundaries, which corresponds to forces from adherens junctions, induces cell flow in a definite direction. We compared the kinematic behaviors of this apical-force-driven cell flow with those of typical cell flow that is driven by forces generated on basal regions and revealed the characteristic differences between them. These differences can be used to distinguish the mechanism of epithelial cell flow observed in experiments, i.e., whether it is apical- or basal-force-driven. Our numerical simulations suggest that cells actively generate fluctuations and use them to regulate both epithelial integrity and plasticity during morphogenesis.     

Study of local hydrodynamic environment in cell-substrate adhesion using side-view μPIV technology

Tumor cell adhesion to the endothelium under shear flow conditions is a critical step that results in circulation-mediated tumor metastasis. This study presents experimental and computational techniques for studying the local hydrodynamic environment around adherent cells and how local shear conditions affect cell-cell interactions on the endothelium in tumor cell adhesion. To study the local hydrodynamic profile around heterotypic adherent cells, a side-view flow chamber assay coupled with micro particle imaging velocimetry (μPIV) technique was developed, where interactions between leukocytes and tumor cells in the near-endothelial wall region and the local shear flow environment were characterized. Computational fluid dynamics (CFD) simulations were also used to obtain quantitative flow properties around those adherent cells. Results showed that cell dimension and relative cell-cell positions had strong influence on local shear rates. The velocity profile above leukocytes and tumor cells displayed very different patterns. Larger cell deformations led to less disturbance to the flow. Local shear rates above smaller cells were observed to be more affected by relative positions between two cells.     

Survival of corneal endothelium following exposure to a vitrification solution

Corneal endothelium, a monolayer of cells lining the inner surface of the cornea, is particularly susceptible to freezing injury. Ice formation damages the structural and functional integrity of the endothelium, and this results in a loss of corneal transparency. Instead of freezing, an alternative method of cryopreservation is vitrification, which avoids damage associated with ice formation. Vitrification at practicable cooling rates, however, requires exposure of tissues to very high concentrations of cryoprotectants, and this can cause damage through chemical toxicity and osmotic stress. The effects of a vitrification solution (VS1) containing 2.62 mol/liter (20.5%, w/v) dimethyl sulfoxide, 2.62 mol/liter (15.5%, w/v) acetamide, 1.32 mol/liter (10%, w/v) propane-1,2-diol, and 6% (w/v) polyethylene glycol were studied on corneal endothelium. Endothelial function was assessed by monitoring corneal thickness during 6 hr of perfusion at 35 degrees C with a Ringer solution supplemented with glutathione and adenosine. Various dilutions of the vitrification solution were introduced and removed in a stepwise manner to mitigate osmotic stress. Survival of endothelium after exposure to VS1 or a solution containing 90% of the cryoprotectant concentrations in VS1 (90% VS1) was dependent on the duration of exposure, the temperature of exposure, and the dilution protocol. The basic dilution protocol was performed at 25 degrees C: corneas were transferred from 90% VS1 or VS1 into 50% VS1 for 15 min, followed by 25% VS1 for 15 min and finally into isosmotic Ringer solution. Using this protocol, corneal endothelium survived exposure to 90% VS1 for 15 min at -5 degrees C, but 5 min in VS1 at -5 degrees C was harmful and resulted in some very large and misshapen endothelial cells. This damage was not ameliorated by using a sucrose dilution technique; but endothelial function was improved when the temperature of exposure to VS1 was reduced from -5 to -10 degrees C. Exposure to VS1 for 5 min at -5 degrees C was well tolerated, however, when the temperature of the first dilution step into 50% VS1 was reduced from 25 to 0 degree C. The large, misshapen cells were not observed under these conditions nor after exposure to VS1 at -10 degrees C. These results suggested that damage was the result of cryoprotectant toxicity rather than osmotic stress. Thus, corneal endothelium survived exposure to two solutions of cryoprotectants, namely, 90% VS1 and VS1, that were sufficiently concentrated to vitrify. Whether corneas can be cooled fast enough in these solutions to achieve vitrification and warmed fast enough to avoid devitrification remains to be determined.     

Hydrodynamic effects on cells in agitated tissue culture reactors

Tissue cells are known to be sensitive to mechanical stresses imposed on them by agitation in bioreactors. The amount of agitation provided in a microcarrier or suspension bioreactor should be only enough to provide an effective homogeneity. Three distinct flow regions can be identified in the reactor: bulk turbulent flow, bulk laminar flow, and boundary-layer flows. Possible mechanisms of cell damage are examined by analyzing the motion of microcarriers or free cells relative to the surrounding fluid, to each other, and to moving or stationary solid surfaces. The primary mechanisms of cell damage appear to result from (a) direct interaction between microcarriers and turbulent eddies, (b) collisions between microcarriers in turbulent flow, and (c) collisions against the impeller or other stationary surfaces. If the smallest eddies of turbulent flow are of the same size as the microcarrier beads, they may cause high shear stresses on the cells. Eddies the size of the average interbead spacing may cause bead-bead collisions which damage cells. The severity of the collisions increases when the eddies are also of the same size as the beads. Bead size and the interbead distance are virtually equal in typical microcarrier suspensions. Impeller collisions occur when the beads cannot avoid the impeller leading edge as it advances through the liquid. The implications of the results of this analysis on the design and operation of tissue culture bioreactors are also discussed.     

Visualization of flow-dependent concentration polarization of macromolecules at the surface of a cultured endothelial cell monolayer by means of fluorescence microscopy

To substantiate the occurrence of flow-dependent concentration or depletion of atherogenic lipoproteins, which has been theoretically predicted to take place at a blood/endothelium boundary, we have studied the effects of perfusion pressure and wall shear rate on the accumulation and uptake of microspheres by cultured vascular endothelial cells in a monolayer. The study was carried out by flowing a cell culture medium containing fetal calf serum and fluorescent microspheres through a parallel-plate flow chamber having a cultured bovine aortic endothelial cell (BAEC) monolayer on one wall of the chamber. The microspheres had a nominal diameter of 19 nm, approximately the same as that of low-density lipoproteins, and thus served as models and tracers of plasma proteins and lipoproteins. Experiments were carried out in steady flow in the physiological range of wall shear rate and water filtration velocity at the monolayer, while monitoring the intensity of fluorescence of the spheres accumulated at and taken up by the endothelial cells. It was found that in a perfusate containing only fluorescent microspheres, due to increased phagocytic activity of the endothelial cells, the intensity of fluorescence which reflected the number of the microspheres taken up by the endothelial cells, increased almost linearly with time and independently of wall shear rate. However, with perfusates containing fetal calf serum, this abnormal phenomenon did not occur, and the intensity of fluorescence increased with increasing perfusion pressure and decreasing wall shear rate. It was also found that the number of fluorescent microspheres accumulated at and taken up by the BAEC monolayer was shear-dependent only at low wall shear rates, and increased sharply when the flow rate was reduced to zero. These results provided solid experimental evidence that flow-dependent concentration or depletion of macromolecules occurs at the luminal surface of the endothelium at physiological wall shear rates and water filtration velocities, and strongly supports the hypothesis that flow-dependent concentration polarization of lipoproteins plays an important role in the localization of atherosclerosis and intimal hyperplasia in man by facilitating the uptake of atherogenic lipoproteins by endothelial cells.     

Integration of Biochemical and Biomechanical Signals Regulating Endothelial Barrier Function

Endothelial barrier function is critical for tissue homeostasis throughout the body. Disruption of the endothelial monolayer leads to edema, vascular diseases and even cancer metastasis among other pathological conditions. Breakdown of the endothelial barrier integrity triggered by cytokines (e.g.IL-8,IL-1β) and growth factors (e.g.VEGF) is well documented. However, endothelial cells are subject to major biomechanical forces that affect their behavior. Due to their unique location at the interface between circulating blood and surrounding tissues, endothelial cells experience shear stress, strain and contraction forces. More than three decades ago, it was already appreciated that shear flow caused endothelial cells alignment in the direction of the flow. After that observation, it took around 20 years to begin to uncover some of the mechanisms used by the cells for mechanotransduction. In this review, we describe mechanosensors on the endothelium identified to date and the associated signaling pathways that integrate biochemical and biomechanical inputs into biological responses and how they modulate the integrity of the endothelial barrier.     

Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me₂SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me₂SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Induction of Endothelial Cell Apoptosis by Solid Tumor Cells

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performedin vivoandin vitrodemonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.     

The effects of Clostridium perfringens enterotoxin on morphology, viability, and macromolecular synthesis in Vero cells.

Vero (African green monkey kidney) cells grown in tissue culture monolayer were sensitive to Clostridium perfringens enterotoxin. Within 30 minutes of exposure to the enterotoxin gross morphological damage was observed and within 40 minutes approximately 75% of the cells had detached. Nearly half of the cells were nonviable following 35 to 40 minutes incubation with the enterotoxin. Doses as low as 0.1 ng caused small but detectable inhibition of plating efficiency of the cells while more than 100 ng caused the inhibition to approach 100%. Total inhibition of DNA, RNA, and protein synthesis occurred within 30 minutes exposure to enterotoxin. Heat inactivated enterotoxin had no apparent effects upon cellular morphology, detachment, viability, plating efficiency, or incorporation. We propose that the enterotoxin induces structural damage to the cytoplasmic membrane which results in loss of electrolytes and other essential substances from the cells. The outcome of this process is shut down of macromolecular synthesis, gross morphological damage, and eventual death of the cell.     

Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1.     

Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm².h and 0.385 μg/cm².h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.     

Mathematical modeling of vascular endothelial layer maintenance: the role of endothelial cell division, progenitor cell homing, and telomere shortening

Maintenance of the endothelial cell (EC) layer of the vessel wall is essential for proper functioning of the vessel and prevention of vascular disorders. Replacement of damaged ECs could occur through division of surrounding ECs. Furthermore, EC progenitor cells (EPCs), derived from the bone marrow and circulating in the bloodstream, can differentiate into ECs. Therefore, these cells might also play a role in maintenance of the endothelial layer in the vascular system. The proliferative potential of both cell types is limited by shortening of telomeric DNA. Accelerated telomere shortening might lead to senescent vascular wall cells and eventually to the inability of the endothelium to maintain a continuous monolayer. The aim of this study was to describe the dynamics of EC damage and repair and telomere shortening by a mathematical model. In the model, ECs were integrated in a two-dimensional structure resembling the endothelium in a large artery. Telomere shortening was described as a stochastic process with oxidative damage as the main cause of attrition. Simulating the model illustrated that increased cellular turnover or elevated levels of oxidative stress could lead to critical telomere shortening and senescence at an age of 65 yr. The model predicted that under those conditions the EC layer could display defects, which could initiate severe vascular wall damage in reality. Furthermore, simulations showed that 5% progenitor cell homing/yr can significantly delay the EC layer defects. This stresses the potential importance of EPC number and function to the maintenance of vascular wall integrity during the human life span.