Porcine aortic organ culture: A model to study the cellular response to vascular injury

Summary Organ cultures of porcine thoracic aorta were studied to define the characteristics of this system as a model to study the reaction of endothelial cells (ECs) and the underlying smooth muscle cells (SMCs) to injury. Both nonwounded and wounded cultures the latter having had part of the endothelial surface gently denuded with a scalpel blade, were studied over a 7 d period by scanning and transmission electron microscopy. The results showed that the nonwounded ECs underwent a shape change from elongated to polygonal within 24 h in culture. In both nonwounded and wounded explants there was cell proliferation beneath the nondenuded endothelium so that by 7 d several layers of cells were present showing features of the secretory type of SMCs. This proliferation, however, did not occur if the endothelium was totally removed from the aorta. There was also evidence of gaps between the surface ECs, and by 7 d lamellipodia of cells beneath the surface were present in these gaps. Occasionally, elongated cells were seen to be present on the surface of the endothelium. In the wounded organ culture, cell migration and proliferation occurred extending from the wound edge and producing a covering of cells on the denuded area. There were also multilayered cells beneath the surface similar to the nonwounded area. Occasional foam cells were seen in the depth of the multilayered proliferating cells. The results indicate that organ culture of porcine thoracic aorta is a good model to study the reaction of ECs and underlying SMCs to injury. This work was supported by a grant from the Ontario Heart Foundation.     

Plasma membrane vesiculation: a cellular response to injury.

The shedding of plasma membrane vesicles has been shown to result from exposure of monolayer cell cultures to formaldehyde and other sulfhydryl blocking agents. Incubation of cells in concentrations of these agents as low as 5 to 10 mM for intervals as brief as fifteen minutes is effective (Scott, 1976). Plasma membrane vesiculation has been shown to be an energy-dependent process that requires Ca++ and physiological temperature. Following plasma membrane vesiculation, cell monolayers appear intact by phase microscopy and show only slight evidence of cell injury by electron microscopy. In view of these observations, the question has been raised whether plasma membrane vesiculation is compatible with continued cell growth and metabolism. The experiments described in this paper were designed to answer these questions. We pulse exposed 3T3 mouse embryo cells to concentrations of formaldehyde, between 2.5 and 250 mM, for intervals 15, 30 or 60 min. Cell momolayers were then washed in a variety of different media in an attempt to reverse the effect of formaldehyde on cells. Cell monolayers were thereafter assayed for the shedding of plasma membrane vesicles and for their ability to transport 2-deoxy-D-glucose. Cells were also replated in serum-containing medium and their ability to grow was assayed over a seven day interval. The results show an inverse relationship between the shedding of plasma membrane vesicles and the ability of the cells to transport nutrients and to grow. We interpret these data to suggest that the process of plasma membrane vesiculation results from a form of cell injury which blocks cellular metabolism and growth.     

Design of a sterile organ culture system for the ex vivo study of aortic heart valves

The biological response of valves to mechanical forces is not well understood. The aim of this study was to design a pulsatile system to enable the ex vivo study of aortic valves when subjected to various hemodynamic conditions. A bioreactor was designed to subject porcine aortic valves to physiological and pathophysiological pressure and flow conditions, while maintaining viability and sterility. Pressure and flow rate could be independently controlled to produce clinically relevant mechanical conditions. The oxygen transfer rate was characterized and sterile operation was achieved over 96 hours. The oxygenation capabilities ensure sufficient oxygen transport to valves, allowing operation for extended periods.     

Angiotensin II-induced growth effects in vascular smooth muscle in cell culture and in the aortic tunica media in organ culture

Several different studies have investigated the growth effects of angiotensin II on vascular smooth muscle cells in culture. However, smooth muscle cells change their phenotype when placed in culture. The objective of the present study was to investigate the effects of angiotensin II on (3)H-thymidine and (3)H-proline incorporation in vascular smooth muscle cells in culture and in the tunica media of blood vessels perfused at normal physiological pressures in organ culture, thus avoiding the phenotypic changes observed in cell culture. The perfusion system consisted of a peristaltic pump and a closed circuit of plastic tubing connected to a culture media bottle where thoracic rat aortae were placed. Angiotensin II induced an increase in (3)H-thymidine and (3)H-proline incorporation in both culture systems. The results suggest that angiotensin II may play a role in mediating cell growth in vascular smooth muscle cells in their 'contractile' as well as in their 'synthetic' phenotype.     

Studies on the modification of the cellular response to injury

Extracellular acidosis (pH 6.5) was found to significantly retard the response of Ehrlich ascites tumor cells (EATC) to direct plasma membrane injury with the non-penetrating organic mercurial compound, p-chloromercuribenzene sulfonic acid (PCMBS). Treatment of cells with 1 mM PCMBS resulted in loss of viability of all cells by 45 minutes at pH 7.4, and by 90 minutes at pH 6.5. Pregression of cellular changes through the various stages of cell injury at the ultrastructural level was correspondingly slower at pH 6.5. The results support the concept that stage 3 of cell injury, associated with condensed mitochondria, dilated ER and swollen cell sap is compatible with cell survival, while stage 5 with high amplitude swelling of mitochondria, fragmentation of membrane systems, and beginning of karyolysis is characteristic of irreversible injury. All cells entered stage 3 at 7.5 minutes at pH 7.4, while essentially all cells entered stage 5 by 45 minutes. At pH 6.5, stage 3 was maintained for 45 minutes and 100% of the cells entered stage 5 by 90 minutes. Although the mechanism of the protection against PCMBS-induced injury is not known, the present electron microscopical results are compatible with the hypothesis that the extracellular acidosis acts to partially stabilize plasma membrane, perhaps by interaction with sulfhydryl (SH) groups.     

Organ culture: a new model for vascular endothelium dysfunction

Background  

Endothelium dysfunction is believed to play a role in the development of cardiovascular disease. The aim of the present study was to evaluate the suitability of organ culture as a model for endothelium dysfunction.     

Chronic effect of doxorubicin on vascular endothelium assessed by organ culture study.

We have attempted to determine the chronic effects of doxorubicin, a commonly used anticancer agent, on vascular endothelium using an organ culture system. In rabbit mesenteric arteries treated with 0.3 microM doxorubicin for 7 days, rounding and concentrated nuclei and TUNEL-positive staining were observed in endothelial cells, indicating DNA damage and the induction of apoptosis. However, the endothelium-dependent relaxation induced by substance P and the expression of mRNA encoding endothelial NO synthase (eNOS) did not differ from those in control arteries. In arteries treated with a higher concentration (1 microM) of doxorubicin, apoptosis and damage to nuclei occurred in the endothelial cells at the third day of treatment, and the detachment and excoriation of endothelium from the tunica interna of the vascular wall were also observed. The impairment of endothelium-dependent relaxation was observed at the fifth day of the treatment with 1 microM doxorubicin. Additionally, apoptotic change in the smooth muscle layer was observed at this concentration of doxorubicin. Apoptotic phenomena were further confirmed by DNA fragmentation using isolated bovine aortic endothelial cells (BAECs) and A7r5 vascular smooth muscle cells, and it was revealed that BAECs are more sensitive than A7r5 to the apoptotic effect of doxorubicin. These results suggest that chronic treatment with doxorubicin at therapeutic concentrations induces apoptosis and excoriation of endothelial cells, which diminishes endothelium-dependent relaxation.     

Stiffening response of a cellular tensegrity model

Living cells exhibit, as most biological tissues, a stiffening (strain-hardening) response which reflects the nonlinearity of the stress-strain relationship. Tensegrity structures have been proposed as a comprehensive model of such a cell's mechanical response. Based on a theoretical model of a 30-element tensegrity structure, we propose a quantitative analysis of its nonlinear mechanical behavior under static conditions and large deformations. This study provides theoretical foundation to the passage from large-scale tensegrity models to microscale living cells, as well as the comparison between results obtained in biological specimens of different sizes. We found two non-dimensional parameters (L*-normalized element length and T*-normalized elastic tension) which govern the mechanical response of the structure for three types of loading tested (extension, compression and shear). The linear strain-hardening is uniquely observed for extension but differed for the two other types of loading tested. The stiffening response of the theoretical model was compared and discussed with the living cells stiffening response observed by different methods (shear flow experiments, micromanipulation and magnetocytometry).     

Endothelial cells in culture: An experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [³H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor- and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selectin And vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also rn rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.Abbreviations AGEs advanced glycated end-products - ICAM intracellular adhesion molecule - IL-1 interleukin-1 - IFN- interferon- - MECIF monocyte-derived endothelial cell inhibitory factor - NO nitric oxide - PECAM-1 platelet-endothelial cell adhesion molecule-1 - RAGE receptor for advanced glycated end-products - RBCs red blood cells - TNF- tumor necrosis factor- - VCAM vascular adhesion molecule     

Adult neurogenesis as a cellular model to study schizophrenia

Comment on: Kim JY, et al. Neuron 2009; 63:761-73.     

iNOS-inhibitor driven neuroprotection in a porcine retina organ culture model

Nitrite oxide plays an important role in the pathogenesis of various retinal diseases, especially when hypoxic processes are involved. This degeneration can be simulated by incubating porcine retinal explants with CoCl₂. Here, the therapeutic potential of iNOS-inhibitor 1400W was evaluated. Degeneration through CoCl₂ and treatment with the 1400W were applied simultaneously to porcine retinae explants. Three groups were compared: control, CoCl₂, and CoCl₂ + iNOS-inhibitor (1400W). At days 4 and 8, retinal ganglion cells (RGCs), bipolar, and amacrine cells were analysed. Furthermore, the influence on the glia cells and different stress markers were evaluated. Treatment with CoCl₂ resulted in a significant loss of RGCs already after 4 days, which was counteracted by the iNOS-inhibitor. Expression of HIF-1α and its downstream targets confirmed the effective treatment with 1400W. After 8 days, the CoCl₂ group displayed a significant loss in amacrine cells and also a drastic reduction in bipolar cells was observed, which was prevented by 1400W. The decrease in microglia could not be prevented by the inhibitor. CoCl₂ induces strong degeneration in porcine retinae by mimicking hypoxia, damaging certain retinal cell types. Treatment with the iNOS-inhibitor counteracted these effects to some extent, by preventing loss of retinal ganglion and bipolar cells. Hence, this inhibitor seems to be a very promising treatment for retinal diseases.     

An organ culture system to model early degenerative changes of the intervertebral disc

Introduction  

Back pain, a significant source of morbidity in our society, is related to the degenerative changes of the intervertebral disc. At present, the treatment of disc disease consists of therapies that are aimed at symptomatic relief. This shortcoming stems in large part from our lack of understanding of the biochemical and molecular events that drive the disease process. The goal of this study is to develop a model of early disc degeneration using an organ culture. This approach is based on our previous studies that indicate that organ culture closely models molecular events that occur in vivo in an ex vivo setting.     

Development of the response to adenosine during organ culture of young embryonic chick hearts

Young (3 days-old) embryonic chick hearts (i.e., prior to innervation) show little electrophysiological response to adenosine. However, during embryonic development the sensitivity to adenosine greatly increases. In the present experiments, in which the chick hearts were placed into organ culture, the sensitivity to adenosine was found to increase with time in culture. Thus, the ability of adenosine to slow the spontaneous heart rate became greater during the course of organ culture. These results suggest that physiological responses to adenosine continue to develop in organ culture, and that the increased sensitivity to adenosine seen in ovo may be independent from the development of sympathetic or parasympathetic innervation.     

A study of dose response and organ susceptibility of copper toxicity in a rat model

Copper (Cu) in higher concentration is toxic and results in various organ dysfunction. We report Cu concentration in liver, brain and kidney in the rat model following chronic exposure of oral copper sulphate at different subtoxic doses and correlate the tissue Cu concentrations with respective organ dysfunction. Fifty-four male wistar rats divided in 3 groups, the control group received saline water and the experimental group (Group-IIA and IIB) received oral copper sulphate in dose of 100 and 200 mg/kg Body Weight. At the end of 30 days, 60 days and 90 days of exposure, six rats were sacrificed from each group. The maximum peak force in grip strength, latency to fall in rotarod and percentage attention score in Y-maze were significantly reduced in the copper sulphate exposed rats compared to the controls at all time points and these were more marked in Group-IIB compared to Group-IIA. Cu concentration was significantly higher in liver, kidney and brain in the Group-II compared to the Group-I. The Cu concentration was highest in the liver (29 folds) followed by kidney (3 folds) and brain (1.5 folds). Serum ALT, AST and bilirubin correlated with liver Cu, BUN with kidney Cu, and grip strength, rotarod and Y-maze findings correlated with brain Cu level. In rats, chronic oral copper sulphate exposure at subtoxic level results in neurobehavioral abnormality and liver and kidney dysfunctions due to increased Cu concentration in the respective organs. Liver is the most vulnerable organ and copper toxicity increases with increasing dose and duration of exposure.     

A theoretical model to study the effects of cellular stiffening on the damage evolution in deep tissue injury

Pressure induced deep tissue injury (DTI) is a severe form of pressure ulcers that is hard to detect in early stages and difficult to prevent and treat. High prevalence figures are partly due to a lack of understanding of pathological pathways involved in DTI. The aim of this study was to investigate, whether changes in material properties of damaged tissue can play a role in DTI aetiology. A numerical model was developed based on muscle microstructure and tissue engineering experiments. A time dependent damage law was proposed and stiffening of dead cells incorporated. The results obtained in the microstructural investigations were used to include the stiffening information in a pre-existing macroscopic model based on animal experiments, which correlated strains to tissue damage measured in the tibialis anterior muscle in rat limbs. With the modelling approach employed in this paper, the damaged area in the rat limb models increased up to 1.65-fold and the rate of damage progression was up to 2.1 times higher in microstructural simulations when stiffening was included.