Increasing fungal infections in cardiovascular ICUs

2005

Increased mechanical stretch of alveolar type II (ATII) cells occurs during mechanical ventilation. The effects of three patterns of stretching rat ATII cells (frequency [min -1 ]-⌬surface area [%]: S40-13, S60-13, S40-30) were compared with those in static cultures at 12, 18, and 24 h. Cell viability and expression of cyclooxygenase-2, 5-lipoxygenase, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) were characterized. Supernatants were analyzed for eicosanoids, nitrite, cytokines, and stimulatory effects on rat lymphocytes. S40-13 simulates normal breathing; the other patterns increased amplitude and frequency. There were no significant differences between S40-13 and static cultures. S60-13 only significantly increased the supernatant nitrite (11.2 Ϯ 1.6 versus 3.9 Ϯ 0.4 M at 24 h). S40-30 significantly reduced the number of trypan blue-excluding cells, increased the supernatant concentration of TXB2 (4.1 Ϯ 0.61 versus 2.2 Ϯ 0.36 pg/ml), 6-keto-PGF1␣ (8.7 Ϯ 1.0 versus 6.7 Ϯ 0.52 pg/ml), cysteinyl-LT (12.2 Ϯ 2.0 versus 6.1 Ϯ 0.75 pg/ml) and nitrite (7.2 Ϯ 1.7 versus 3.9 Ϯ 0.4 M). S40-30 did not alter the release of tumor necrosis factor-␣ and monocyte chemotactic protein-1, but significantly reduced the concentration of the anti-inflammatory interleukin-10 (20.8 Ϯ 13.3 versus 130 Ϯ 21.5 pg/ml). Expression of cyclooxygenase-2/5-lipoxygenase was increased/decreased; expression of iNOS/eNOS was unchanged by high-amplitude stretch. Supernatants from S40-30 experiments caused lymphocyte activation measured by CD71 and CD54 surface expression. Continuing mechanical distension of ATII cells contributes to an inflammatory response by a shift in the balance of proand anti-inflammatory mediators.

Cells Tissues Organs, 2010

The interest of scientists in the effects of mechanical stresses on cells is growing, in order to reproduce and understand cell behaviour in an environment closely reproducing physiological conditions. There have been many studies showing that mechanical stimulations are involved in regulating the proliferation, apoptosis and synthesis of proteins and cell morphology. In this study, we have considered the effects of a 20% stretching mechanical stress on MRC5 lung fibroblast cells in order to verify the role of survival/apoptotic pathways. As a survival pathway, the activation of Akt has been studied in association with pro-apoptotic or anti-apoptotic signals such as the Bax/Bcl-2 ratio and cleavage of caspases 3 and 9. Findings have shown the effects of overstressed cellular stretching to be a balance of a cause-and-effect reaction between survival and apoptosis.

Critical Care, 2006

Objective To examine the effects of short-term cyclic stretch on apoptosis in alveolar type II cells (A549). To study in vitro the direct influence of alveolar type II cells on mechanical stretch. Methods A549 were treated with different doses of lipopolysaccharide (LPS), 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, and then A549 were lengthened 5%, 15%, 30% using a FLEXCELL tension unit 4000, a vacuum-driven device that applies strain to cells, which were cultured in six-well plates coated with collagen-I, and 12 cycles/min for 4 hours. Apoptosis was measured using the flow cytometry method that measures annexin V and propidium iodide (PI) staining. The morphological changes of apoptotic cells were observed by transmission electron microscope. Results Apoptosis could be induced in alveolar type II cells (A549) by mechanical stretch. The percentage of annexin V + PI cells increased after being treated with cyclic stretch for 4 hours by 5%, 15%, 30% in all groups. The morphological features of apoptotic cells demonstrated by transmission electron microscope were as follows: shrinkage of the cell, chromatin condensation and aggregation under the nuclear membrane as a crescent or lump, membrane-encapsulated nuclear fragment or cell organ formed by invagination of the cell membrane, and apoptotic body formation followed by vacuolization. Conclusion Apoptosis induced by mechanical stretch and LPS is dose dependent. Mechanical stretch aggravates apoptosis especially in cells treated with LPS. Annexin V and PI double staining is a specific, sensitive, and quantitative method for analyzing apoptotic cells. It is also helpful to clarify the protective mechanism of low-volume ventilation in ARDS.

Critical Care, 2006

Objective To examine the effects of short-term cyclic stretch on apoptosis in alveolar type II cells (A549). To study in vitro the direct influence of alveolar type II cells on mechanical stretch. Methods A549 were treated with different doses of lipopolysaccharide (LPS), 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, and then A549 were lengthened 5%, 15%, 30% using a FLEXCELL tension unit 4000, a vacuum-driven device that applies strain to cells, which were cultured in six-well plates coated with collagen-I, and 12 cycles/min for 4 hours. Apoptosis was measured using the flow cytometry method that measures annexin V and propidium iodide (PI) staining. The morphological changes of apoptotic cells were observed by transmission electron microscope. Results Apoptosis could be induced in alveolar type II cells (A549) by mechanical stretch. The percentage of annexin V + PI cells increased after being treated with cyclic stretch for 4 hours by 5%, 15%, 30% in all groups. The morphological features of apoptotic cells demonstrated by transmission electron microscope were as follows: shrinkage of the cell, chromatin condensation and aggregation under the nuclear membrane as a crescent or lump, membrane-encapsulated nuclear fragment or cell organ formed by invagination of the cell membrane, and apoptotic body formation followed by vacuolization. Conclusion Apoptosis induced by mechanical stretch and LPS is dose dependent. Mechanical stretch aggravates apoptosis especially in cells treated with LPS. Annexin V and PI double staining is a specific, sensitive, and quantitative method for analyzing apoptotic cells. It is also helpful to clarify the protective mechanism of low-volume ventilation in ARDS. Acknowledgement The study was funded by the 'One Hundred People' project of Shanghai Sanitary Bureau (03-77-20).

2006

Objective To examine the effects of short-term cyclic stretch on apoptosis in alveolar type II cells (A549). To study in vitro the direct influence of alveolar type II cells on mechanical stretch. Methods A549 were treated with different doses of lipopolysaccharide (LPS), 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, and then A549 were lengthened 5%, 15%, 30% using a FLEXCELL tension unit 4000, a vacuum-driven device that applies strain to cells, which were cultured in six-well plates coated with collagen-I, and 12 cycles/min for 4 hours. Apoptosis was measured using the flow cytometry method that measures annexin V and propidium iodide (PI) staining. The morphological changes of apoptotic cells were observed by transmission electron microscope. Results Apoptosis could be induced in alveolar type II cells (A549) by mechanical stretch. The percentage of annexin V + PI cells increased after being treated with cyclic stretch for 4 hours by 5%, 15%, 30% in all groups. The morphological features of apoptotic cells demonstrated by transmission electron microscope were as follows: shrinkage of the cell, chromatin condensation and aggregation under the nuclear membrane as a crescent or lump, membrane-encapsulated nuclear fragment or cell organ formed by invagination of the cell membrane, and apoptotic body formation followed by vacuolization. Conclusion Apoptosis induced by mechanical stretch and LPS is dose dependent. Mechanical stretch aggravates apoptosis especially in cells treated with LPS. Annexin V and PI double staining is a specific, sensitive, and quantitative method for analyzing apoptotic cells. It is also helpful to clarify the protective mechanism of low-volume ventilation in ARDS.

Critical Care Clinics, 2011

Objective To examine the effects of short-term cyclic stretch on apoptosis in alveolar type II cells (A549). To study in vitro the direct influence of alveolar type II cells on mechanical stretch. Methods A549 were treated with different doses of lipopolysaccharide (LPS), 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, and then A549 were lengthened 5%, 15%, 30% using a FLEXCELL tension unit 4000, a vacuum-driven device that applies strain to cells, which were cultured in six-well plates coated with collagen-I, and 12 cycles/min for 4 hours. Apoptosis was measured using the flow cytometry method that measures annexin V and propidium iodide (PI) staining. The morphological changes of apoptotic cells were observed by transmission electron microscope. Results Apoptosis could be induced in alveolar type II cells (A549) by mechanical stretch. The percentage of annexin V + PI cells increased after being treated with cyclic stretch for 4 hours by 5%, 15%, 30% in all groups. The morphological features of apoptotic cells demonstrated by transmission electron microscope were as follows: shrinkage of the cell, chromatin condensation and aggregation under the nuclear membrane as a crescent or lump, membrane-encapsulated nuclear fragment or cell organ formed by invagination of the cell membrane, and apoptotic body formation followed by vacuolization. Conclusion Apoptosis induced by mechanical stretch and LPS is dose dependent. Mechanical stretch aggravates apoptosis especially in cells treated with LPS. Annexin V and PI double staining is a specific, sensitive, and quantitative method for analyzing apoptotic cells. It is also helpful to clarify the protective mechanism of low-volume ventilation in ARDS. PaO 2 /FiO 2 430 [421; 440] # 380 [349; 397] 165 [68; 289] # C (ml/cmH 2 O) 28 [24; 32]* 18 [16; 21]* 12 [8; 17]* R i (cmH 2 O/l/s) 4.1 [3.9; 4.5] 4.5 [4.3; 5.1] 5.1 [3.7; 7.9] # P < 0.05 control vs 24-hour peritonitis, *P < 0.05 control vs 12-hour and 24-hour peritonitis.

American Journal of Respiratory Cell and Molecular Biology, 2005

Increased mechanical stretch of alveolar type II (ATII) cells occurs during mechanical ventilation. The effects of three patterns of stretching rat ATII cells (frequency [min -1 ]-⌬surface area [%]: S40-13, S60-13, S40-30) were compared with those in static cultures at 12, 18, and 24 h. Cell viability and expression of cyclooxygenase-2, 5-lipoxygenase, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) were characterized. Supernatants were analyzed for eicosanoids, nitrite, cytokines, and stimulatory effects on rat lymphocytes. S40-13 simulates normal breathing; the other patterns increased amplitude and frequency. There were no significant differences between S40-13 and static cultures. S60-13 only significantly increased the supernatant nitrite (11.2 Ϯ 1.6 versus 3.9 Ϯ 0.4 M at 24 h). S40-30 significantly reduced the number of trypan blue-excluding cells, increased the supernatant concentration of TXB2 (4.1 Ϯ 0.61 versus 2.2 Ϯ 0.36 pg/ml), 6-keto-PGF1␣ (8.7 Ϯ 1.0 versus 6.7 Ϯ 0.52 pg/ml), cysteinyl-LT (12.2 Ϯ 2.0 versus 6.1 Ϯ 0.75 pg/ml) and nitrite (7.2 Ϯ 1.7 versus 3.9 Ϯ 0.4 M). S40-30 did not alter the release of tumor necrosis factor-␣ and monocyte chemotactic protein-1, but significantly reduced the concentration of the anti-inflammatory interleukin-10 (20.8 Ϯ 13.3 versus 130 Ϯ 21.5 pg/ml). Expression of cyclooxygenase-2/5-lipoxygenase was increased/decreased; expression of iNOS/eNOS was unchanged by high-amplitude stretch. Supernatants from S40-30 experiments caused lymphocyte activation measured by CD71 and CD54 surface expression. Continuing mechanical distension of ATII cells contributes to an inflammatory response by a shift in the balance of proand anti-inflammatory mediators.

Critical Care, 2006

Objective To examine the effects of short-term cyclic stretch on apoptosis in alveolar type II cells (A549). To study in vitro the direct influence of alveolar type II cells on mechanical stretch. Methods A549 were treated with different doses of lipopolysaccharide (LPS), 0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml, and then A549 were lengthened 5%, 15%, 30% using a FLEXCELL tension unit 4000, a vacuum-driven device that applies strain to cells, which were cultured in six-well plates coated with collagen-I, and 12 cycles/min for 4 hours. Apoptosis was measured using the flow cytometry method that measures annexin V and propidium iodide (PI) staining. The morphological changes of apoptotic cells were observed by transmission electron microscope. Results Apoptosis could be induced in alveolar type II cells (A549) by mechanical stretch. The percentage of annexin V + PI cells increased after being treated with cyclic stretch for 4 hours by 5%, 15%, 30% in all groups. The morphological features of apoptotic cells demonstrated by transmission electron microscope were as follows: shrinkage of the cell, chromatin condensation and aggregation under the nuclear membrane as a crescent or lump, membrane-encapsulated nuclear fragment or cell organ formed by invagination of the cell membrane, and apoptotic body formation followed by vacuolization. Conclusion Apoptosis induced by mechanical stretch and LPS is dose dependent. Mechanical stretch aggravates apoptosis especially in cells treated with LPS. Annexin V and PI double staining is a specific, sensitive, and quantitative method for analyzing apoptotic cells. It is also helpful to clarify the protective mechanism of low-volume ventilation in ARDS. Acknowledgement The study was funded by the 'One Hundred People' project of Shanghai Sanitary Bureau (03-77-20).

Critical Care Medicine, 2006

epsis is a major public health problem both in the United States and worldwide. It often precipitates lung injury, accounting for 25% to 40% of cases of acute respiratory distress syndrome (ARDS) (1, 2). Although several disease processes can lead to ARDS, the common pathologic finding is diffuse alveolar damage. This is characterized by neutrophil accumulation and endothelial damage. The net result is an increase in permeability, transudation of protein into the interstitial and alveolar spaces, and a loss of pulmonary epithelial cells, primarily type I pneumocytes (3). Clinically, this is reflected in decreased lung compliance, hypoxemia, atelectasis, and respiratory failure (4-6). One consequence of respiratory failure is the need to administer positive pressure mechanical ventilation. This may lead to barotrauma or volutrauma to alveoli (7-14). In a seminal randomized, prospective trial comparing low vs. traditional tidal volumes, the ARDSnet investigators demonstrated improved outcome from ARDS (15). This and other studies (16, 17) have stimulated investigations to determine the mechanism by which over-distension or increased pressure might damage alveoli. Recent laboratory investigations demonstrate that stretch can alter the behavior of isolated pneumocytes. Specifically, cell deformation has been shown to activate calcium-sensitive ion channels and stimulate mechanosensitive receptors. These changes led to the activation of the transcription factors nuclear factor (NF)-B and c-fos, which in turn enhanced cytokine expression (18-22). In addition, stretch of healthy alveolar cells, both in vivo and in vitro, has been shown to precipitate structural and cytosolic changes. Moreover, larger deformations increased paracellular permeability and caused cell death (23-26). Although it is clear that stretch alone can lead to cellular injury, in most cases mechanical ventilation does not precipitate clinically relevant lung injury. However, this may not be true in the presence of preexisting sepsis. Therefore, we tested the hypothesis that lung injury secondary to sepsis increases the vulnerability of pulmonary epithelial cells to stretch injury. We also hypothesized that increas-Objective: Previous in vitro models have shown that cellular deformation causes dose-dependent injury and death in healthy rat alveolar epithelial cells (AECs). We compared the viability of AECs from septic rats with those from nonseptic rats after 1 hr of cyclic equibiaxial stretch. We hypothesized that sepsis would increase stretch-induced cell death. Design: Laboratory investigation. Setting: University research laboratory. Subjects: Thirty-seven male Sprague-Dawley rats weighing 240-260 g. Interventions: Anesthetized rats were subjected to cecal ligation and double puncture (2CLP) or sham laparotomy without cecal ligation or puncture (sham). After 24 or 48 hrs, AECs were isolated, seeded in custom wells, and maintained in culture for 48 hrs before study. AECs were stretched cyclically (15/min) to a 0%, 12%, 25%, or 37% change in surface area (⌬SA) for 1 hr. Cell viability, phenotypic markers, and nuclear factor-B intracellular localization were assessed using fluorescent immunocytochemistry. Measurements and Main Results: Phase and fluorescent images were evaluated for all studies. Response to stretch was the same at 24 and 48 hrs after 2CLP. Relative to sham, 2CLP significantly increased cell death at 25 and 37% ⌬SA (p < .003, analysis of variance). Relative to sham, 2CLP did not alter expression of type I or type II phenotypic markers. Nuclear factor-B within the nuclear compartment was observed after 2CLP in unstretched cells and after 1 hr of cyclic stretch at 37% ⌬SA. In sham, nuclear factor-B within the nuclear compartment was seen only after stretch. Conclusions: AECs isolated from septic rats are more vulnerable to mechanical deformation injury than AECs from nonseptic animals.

American journal of physiology. Heart and circulatory physiology, 2002

We studied the response of porcine vascular smooth muscle cells (PVSMCs) to cyclic sinusoidal stretch at a frequency of 1 Hz. Cyclic stretch with an area change of 25% caused an increase in PVSMC apoptosis, which was accompanied by sustained activation of c-Jun NH(2)-terminal kinases (JNK) and the mitogen-activated protein kinase p38. Cyclic stretch with an area change of 7% had no such effect. Infection of PVSMCs with recombinant adenoviruses expressing constitutively active forms of upstream molecules that activate JNK and p38 also led to apoptosis. The simultaneous blockade of both JNK and p38 pathways with adenovirus-mediated expression of dominant-negative mutants of c-Jun and p38 caused a significant decrease (to 1/2) of the apoptosis induced by 25% cyclic stretch. The 25% stretch also caused sustained clustering of tumor necrosis factor-alpha (TNF-alpha) receptor-1 and its association with TNF-alpha receptor-associated factor-2 (TRAF-2). Overexpressing the wild-type TRAF-2 in...