Evaluation of Freezing Effects on Human Microvascular-Endothelial Cells (hMEC)

2001 ◽  
Author(s):  
Marwane S. Berrada ◽  
John C. Bischof
Keyword(s):  
Endothelial Cells ◽  
Predictive Ability ◽  
Cell Types ◽  
Cell System ◽  
Ice Formation ◽  
Microvascular Endothelial Cells ◽  
Cooling Rates ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Microvascular Endothelial

Abstract There is mounting evidence that the endothelium may play an important role in traditional cryosurgical treatments by acting to locally foster thrombi in the microvasculature of various tissues after freezing. Therefore, this study was designed to investigate, at the cellular level in human microvascular endothelial cells (hMEC), the various biophysical changes that occur during freezing and compare them with post-freeze viability. The hMECs were loaded on a cryomicroscope stage and freezing experiments at 5, 10, 15, 25, 100 and 130°C/min were performed to experimentally evaluate dehydration (water transport) as well as intracellular ice formation (IIF) within this cell system. The dehydration kinetics were found to be governed by a membrane permeability Lpg and activation energy ELp of 0.05 (μm/min.atm) and 14.8 (kcal/mole) respectively [R2 = 0.94]. These parameters were then tested for predictive ability against the experimentally measured behavior at 15°C/min with a good agreement [R2 = 0.98]. Intracellular Ice Formation (IIF) was found to occur at lower temperatures than many cell types (i.e. TIIF 50% ∼ −18°C) and at cooling rates greater than or equal to 25°C/min. At cooling rates above 50°C/min, two types of IIF, cell darkening and twitching, were both observed and quantified and were assumed to be governed by Surface Catalyzed Nucleation (SCN). IIF parameters Ωo, and κo were found to be 6.8 × 10−8 (m2.s)−1 and 8.3 × 10−9 (K5) [R2 = 0.94] respectively. Viability results suggest an inverted U-shape curve between 1 and 50°C/min (with a maximum at 10°C/min). But viability appears to increase again at cooling rates > 50°C/min (i.e. it does not continue to drop) which suggests that the traditional two factor hypothesis may not completely describe viability in this system. Additional cellular destruction was found by lowering the end-temperature to −30°C or below. At this temperature the majority of the cell population was destroyed regardless of the cooling rate.

A Determination of Biophysical Parameters Related to Freezing of an ELT-3 Cell Line

2000 ◽  
Author(s):  
Marwane S. Berrada ◽  
John C. Bischof
Keyword(s):  
Cell Line ◽  
Volume Fraction ◽  
Cell Types ◽  
Cellular Level ◽  
Ice Formation ◽  
Biophysical Parameters ◽  
Cooling Rates ◽  
Intracellular Ice Formation ◽  
Biophysical Study ◽  
Intracellular Ice

Abstract This study investigates two destructive biophysical mechanisms during freezing (extensive dehydration and intracellular ice formation) at the cellular level in the rodent ELT-3 uterine fibroid cell-line. The osmotically inactive volume fraction (Vb) of ELT-3 cells was approximated to 0.35 of the initial isotonic cell volume (Vo). The water transport characteristics of this cell-line are such that ELT-3 cells are highly permeable with a strong ability to lose water even at low subzero temperatures. The hydraulic reference permeability, Lpg and activation energy, Elp associated with Lp were found to be 0.13 (μm/min.atm) and 19.0 (kcal/mole) [R2 = 0.86] respectively. Intracellular Ice Formation (IIF) occurs at lower temperatures than many cell-types (i.e. TIIF 50% below −15°C) at cooling rates > 25 °C/min. Darkening IIF, which was assumed to occur by Surface Catalyzed Nucleation (SCN), is governed by kinetic Ωo and thermodynamic κo biophysical parameters, which were found to be 6.1×108(m2.s)−1 and 5.3×109(K5) [R2 = 0.94] respectively. At a cooling rate of 100°C/min, twitching IIF (non-darkening IIF) was observed. Viability data from a separate study (Bischof et. al., 2000) indicated that at cooling rates ≤ 1°C/min and ≥ 50°C/min with an end-temperature of −20°C, extensive damage to cells was observed. The current biophysical study shows that extensive dehydration occurs at 1°C/min while substantial IIF (77%) occurs at 50 °C/min. This data suggests that while biophysics can explain some of the destruction occurring at the investigated temperatures, other effects or mechanisms may be playing a role at lower end-temperatures.

Nucleoside and nucleobase transporters of primary human cardiac microvascular endothelial cells: characterization of a novel nucleobase transporter

2007 ◽  
Vol 293 (6) ◽  
pp. H3325-H3332 ◽  
Author(s):  
Derek B. J. Bone ◽  
James R. Hammond
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
Nucleoside Transporter ◽  
Active Metabolites ◽  
Equilibrative Nucleoside Transporter ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells ◽  
Nucleobase Transport

Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated 2-[3H]chloroadenosine via the nitrobenzylmercaptopurine riboside-sensitive equilibrative nucleoside transporter 1 (ENT1) at a Vmaxof 3.4 ± 1 pmol·μl−1·s−1, with no contribution from the nitrobenzylmercaptopurine riboside-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves, which are also compatible with minimal ENT2 expression. The nucleobase [3H]hypoxanthine was accumulated within hMVECs ( Km= 96 ± 37 μM; Vmax= 1.6 ± 0.3 pmol·μl−1·s−1) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole-insensitive but could be inhibited by adenine ( Ki= 19 ± 7 μM) and other purine nucleobases, including chemotherapeutic analogs. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter-deficient PK( 15 ) cell line (PK15NTD). Further characterization of [3H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na+or H+. PK15NTD cells expressing human ENT2 accumulated 4.5-fold more [3H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD cells or hMVECs, suggesting trapping of ENT2-permeable metabolites. Understanding the nucleoside and nucleobase transporter profiles in the vasculature will allow for further study into their roles in pathophysiological conditions such as hypoxia or ischemia.

scholarly journals Rickettsia rickettsii Infection of Human Macrovascular and Microvascular Endothelial Cells Reveals Activation of Both Common and Cell Type-Specific Host Response Mechanisms

2010 ◽  
Vol 78 (6) ◽  
pp. 2599-2606 ◽  
Author(s):  
Elena Rydkina ◽  
Loel C. Turpin ◽  
Sanjeev K. Sahni
Keyword(s):  
Endothelial Cells ◽  
Host Response ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
Cell Type ◽  
Rickettsia Rickettsii ◽  
Specific Host ◽  
Cell Type Specific ◽  
Cox 2 ◽  
Microvascular Endothelial

ABSTRACT Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-κB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-κB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E2 after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

Clot Lysis Mediated by Cultured Human Microvascular Endothelial Cells

1988 ◽  
Vol 60 (03) ◽  
pp. 463-467 ◽  
Author(s):  
Wolfgang Speiser ◽  
Elisabeth Anders ◽  
Bernd R Binder ◽  
Gert Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Shape Change ◽  
Umbilical Vein ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
Clot Lysis ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Fibrin Clots

SummaryThe lysis of fibrin clots on the surface of cultured human omental tissue microvascular endothelial cells (HOTMEC) and cultured human umbilical vein endothelial cells (HUVEC) was studied. Fibrin clots were made by mixing fibrinogen, plasminogen and thrombin on the surface of both cell types. Clot lysis was seen only on the surface of HOTMEC, which were found to synthesize about 100-fold more tissue plasminogen activator (tPA) antigen than HUVEC. Clot lysis of HOTMEC could be blocked by anti-tPA IgG but was not affected by the incorporation of exogenous plasminogen activator (PAI) into the clot in concentrations (75 arbitrary units) exceeding the tPA activity (21 ± 2.5 IU) of the cells. Thus, it is likely that tPA secreted by HOTMEC is protected from inhibition by PAI in the presence of fibrin and endothelial cells. The stimulation of EC to release an excess of tPA over PAI, in contrast to the secretion of an excess of PAI over tPA found in unstimulated cells in the absence of fibrin, is obviously no prerequisite for the initiation of fibrinolysis on the surface of HOTMEC. As thrombin was used for clot formation, its influence on tPA and PAI synthesis of both cell types was investigated. In contrast to HOTMEC, which were not affected by Α-thrombin, HUVEC revealed a dose-dependent increase in tPA and PAI synthesis upon incubation with the enzyme. This increase in tPA production by HUVEC was not sufficient to lyse the clots within 48 hours. Furthermore, HUVEC. behaved differently towards thrombin as these cells in contrast to HOTMEC revealed the typical shape change reaction upon incubation with the enzyme

scholarly journals Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 505-515 ◽  
Author(s):  
Shinsuke Seki ◽  
Keisuke Edashige ◽  
Sakiko Wada ◽  
Peter Mazur
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Types ◽  
Ice Nucleation ◽  
Nucleation Temperature ◽  
Ice Formation ◽  
Mouse Oocytes ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Mature Mouse

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to −7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to −70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were −34, −40, −35, and −25 °C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10–15 °C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-strandedAqp3RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

Difference in proangiogenic potential of systemic and pulmonary endothelium: role of CXCR2

2005 ◽  
Vol 288 (6) ◽  
pp. L1117-L1123 ◽  
Author(s):  
Aigul Moldobaeva ◽  
Elizabeth M. Wagner
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Cell Surface Expression ◽  
Endothelial Cell Proliferation ◽  
Microvascular Endothelial Cells ◽  
Pulmonary Vasculature ◽  
Cathepsin S ◽  
Thymidine Uptake ◽  
Pulmonary Endothelium ◽  
Microvascular Endothelial

The systemic vasculature in and surrounding the lung is proangiogenic, whereas the pulmonary vasculature rarely participates in neovascularization. We studied the effects of the proangiogenic ELR+ CXC chemokine MIP-2 (macrophage inflammatory protein-2) on endothelial cell proliferation and chemotaxis. Mouse aortic, pulmonary arterial, and lung microvascular endothelial cells were isolated and subcultured. Proliferation ([3H]thymidine uptake) and migration (Transwell chemotaxis) were evaluated in each cell type at baseline and upon exposure to MIP-2 (1–100 ng/ml) without and with exposure to hypoxia (24 h)-reoxygenation. Baseline proliferation did not vary among cell types, and all cells showed increased proliferation after MIP-2. Aortic cell chemotaxis increased markedly upon exposure to MIP-2; however, neither pulmonary artery nor lung microvascular endothelial cells responded to this chemokine. Assessment of CXCR2, the G protein-coupled receptor through which MIP-2 signals, displayed no baseline difference in mRNA, protein, or cell surface expression among cell types. Exposure to hypoxia increased expression of CXCR2 of aortic endothelial cells only. Additionally, aortic cells, compared with pulmonary cells, showed significantly greater protein and activity of cathepsin S, a proteolytic enzyme important for cell motility. Thus the combined effects of increased cathepsin S activity, providing increased motility and enhanced CXCR2 expression after hypoxia, both contribute to the proangiogenic phenotype of systemic arterial endothelial cells.

scholarly journals Differences in coagulant and fibrinolytic activities of cultured human endothelial cells derived from omental tissue microvessels and umbilical veins

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 964-967 ◽  
Author(s):  
W Speiser ◽  
E Anders ◽  
KT Preissner ◽  
O Wagner ◽  
G Muller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Von Willebrand Factor ◽  
Protein C ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microvascular Endothelial ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Large vessel and microvascular endothelial cells were compared in their capacity to synthesize and secrete coagulant and fibrinolytic factors. Human omental tissue microvascular endothelial cells (HOTMEC) and human umbilical vein endothelial cells (HUVEC) were isolated, grown to confluency under identical conditions, and studied in primary cultures. After an incubation period of 12 hours in serum-free medium, the conditioned medium of confluent HOTMEC contained 100-fold higher levels of tissue plasminogen activator (tPA) antigen than that of HUVEC. The conditioned media as well as the lysates of both cell types did not contain any free tPA activity, but the free plasminogen activator inhibitor capacity was found intracellularly as well as extracellularly. Although von Willebrand factor was detected in both cell types by immunofluorescence, measurable amounts were only found in HUVEC using an enzyme-linked immunosorbent assay. The kinetics of protein C activation by thrombin on the surface of once-passaged cells were identical for HOTMEC and HUVEC. The present study indicates that cultivated HOTMEC produce larger quantities of tPA than HUVEC do, possess smaller amounts of von Willebrand factor than HUVEC do, and express thrombomodulin for protein C activation as effectively as HUVEC.

A Study of Intracellular Ice Formation in Jurkat Cells

2008 ◽  
Author(s):  
Tathagata Acharya ◽  
Ram V. Devireddy
Keyword(s):  
Cooling Rate ◽  
Jurkat Cells ◽  
Freezing Rate ◽  
Ice Formation ◽  
Substantial Evidence ◽  
Cooling Rates ◽  
Cryoprotective Agents ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
Phosphate Buffered Saline

The objective of this study was to characterize the IIF behavior of Jurkat cells in isotonic conditions in the absence of any cryoprotective agents. The Jurkat cells were collected from culture and then washed and re-suspended in Dulbecco’s Phosphate Buffered Saline (PBS). The freezing experiments were carried out at defined freezing protocols and at various freezing rates of 5, 20, 30 and 50 °C/min. The results suggest there was no substantial evidence of intracellular ice formation at lower cooling rates of 5, 20 and 30° C/min. The first conspicuous indication of intracellular ice formation (IIF) was observed at a freezing rate of 50 °C/min. At this cooling rate, unlike the usual sudden blackening of cells, the cells suddenly grew and exploded suggesting the formation of intracellular ice, which was reminiscent of a prior observed phenomenon for IIF.

Sign in / Sign up

Export Citation Format

Share Document