Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.     

In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.     

Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.     

Cross-tolerance in the tidepool sculpin: the role of heat shock proteins

Cross-tolerance, or the ability of one stressor to transiently increase tolerance to a second heterologous stressor, is thought to involve the induction of heat shock proteins (Hsp). We thus investigated the boundaries of cross-tolerance in tidepool sculpins (Oligocottus maculosus) and their relationship to Hsp70 levels. Survival of sculpins exposed to severe osmotic (90 ppt, 2 h) and hypoxic (0.33 mg O(2)/L, 2 h) stressors increased from 68% to 96%, and from 47% to 76%, respectively, following a +12 degrees C heat shock. The magnitude of this heat shock was critical for protection. A +10 degrees C heat shock did not confer cross-tolerance, while a +15 degrees C heat shock was deleterious. Sculpins required between 8 and 48 h of recovery following the +12 degrees C heat shock to develop cross-tolerance. There was no association between Hsp70 levels before the onset of the secondary stressor and cross-tolerance. However, branchial Hsp70 levels following osmotic shock were highly correlated with the time frame of cross-tolerance. Thus, Hsp70 induction by the priming stressor may be less important than the ability of the cell to mount an Hsp response to subsequent stressors. The time frame of cross-tolerance is similar to the interval between low tides, suggesting the possible relevance of this response in nature.     

Glutamine's protection against sepsis and lung injury is dependent on heat shock protein 70 expression

Glutamine (GLN) has been shown to protect against inflammatory injury and illness in experimental and clinical settings. The mechanism of this protection is unknown; however, laboratory and clinical trial data have indicated a relationship between GLN-mediated protection and enhanced heat shock protein 70 (HSP70) expression. The aim of this study was to examine the hypothesis that GLN's beneficial effect on survival, tissue injury, and inflammatory response after inflammatory injury is dependent on HSP70 expression. Mice with a specific deletion of the HSP70 gene underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 1 h post-CLP. Lung tissue NF-kappaB activation, inflammatory cytokine response, and lung injury were assessed post-CLP. Survival was assessed for 5 days post-CLP. Our results indicate that GLN administration improved survival in Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN; however, GLN exerted no survival benefit in Hsp70(-/-) mice. This was accompanied by a significant decrease in lung injury, attenuation of NF-kappaB activation, and proinflammatory cytokine expression in GLN-treated Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN. In the Hsp70(-/-) mice, GLN's attenuation of lung injury, NF-kappaB activation, and proinflammatory cytokine expression was lost. These results confirm our hypothesis that HSP70 expression is required for GLN's effects on survival, tissue injury, and the inflammatory response after global inflammatory injury.     

Heat shock response and acute lung injury

All cells respond to stress through the activation of primitive, evolutionarily conserved genetic programs that maintain homeostasis and assure cell survival. Stress adaptation, which is known in the literature by a myriad of terms, including tolerance, desensitization, conditioning, and reprogramming, is a common paradigm found throughout nature, in which a primary exposure of a cell or organism to a stressful stimulus (e.g., heat) results in an adaptive response by which a second exposure to the same stimulus produces a minimal response. More interesting is the phenomenon of cross-tolerance, by which a primary exposure to a stressful stimulus results in an adaptive response whereby the cell or organism is resistant to a subsequent stress that is different from the initial stress (i.e., exposure to heat stress leading to resistance to oxidant stress). The heat shock response is one of the more commonly described examples of stress adaptation and is characterized by the rapid expression of a unique group of proteins collectively known as heat shock proteins (also commonly referred to as stress proteins). The expression of heat shock proteins is well described in both whole lungs and in specific lung cells from a variety of species and in response to a variety of stressors. More importantly, in vitro data, as well as data from various animal models of acute lung injury, demonstrate that heat shock proteins, especially Hsp27, Hsp32, Hsp60, and Hsp70 have an important cytoprotective role during lung inflammation and injury.     

Exogenous heat shock protein 70 mediates sepsis manifestations and decreases the mortality rate in rats

Mammalian responses to bacterial lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria can lead to an uncontrolled inflammatory reaction that can be deadly for the host. We checked whether heat shock protein 70 (Hsp70) protein is able to protect animals from the deleterious effects of bacterial LPS by monitoring the effect of exogenous Hsp70 injections before and after LPS administration. Our research with rats demonstrates for the first time that administration of exogeneous Hsp70 before and after LPS challenges can reduce mortality rates and modify several parameters of hemostasis and hemodynamics. Hsp70 isolated from bovine muscles showed significant protective effects against the impaired coagulation and fibrinolytic systems caused by LPS, and reduced the mortality caused by Escherichia coli and Salmonella typhimurium LPS injections significantly. Characteristically, Hsp70 preparations used in the experiments result in different effects when administered before and after an LPS challenge, and the effects of Hsp70 injections also differ significantly depending on the origin of the LPS (E coli vs S typhimurium). Based on our data, mammalian Hsp70 appears to be an attractive target in therapeutic strategies designed to stimulate endogenous protective mechanisms against many deleterious consequences of septic shock by accelerating the functional recovery of susceptible organs in humans.     

Loss of Hsp70 in Drosophila is pleiotropic, with effects on thermotolerance, recovery from heat shock and neurodegeneration

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.     

Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death

Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In this system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Hsp70 overexpression inhibits aminoglycoside-induced hair cell death in vitro. In this study, we utilized Hsp70-overexpressing mice to determine whether Hsp70 is protective in vivo. Both Hsp70-overexpressing mice and their wild-type littermates were treated with systemic kanamycin (700 mg/kg body weight) twice daily for 14 days. While kanamycin treatment resulted in significant hearing loss and hair cell death in wild-type mice, Hsp70-overexpressing mice were significantly protected against aminoglycoside-induced hearing loss and hair cell death. These data indicate that Hsp70 is protective against aminoglycoside-induced ototoxicity in vivo.