Role of HSP70 protein in human periodontal ligament cell function and physiology.

OBJECTIVE: Heat pre-treatment of mechanically loaded human periodontal ligament cells (hPDL) dampens the inflammatory cellular response, as evidenced by a reduced expression of pro-inflammatory cytokines, inhibition of monocyte adhesion and osteoclastic differentiation. These findings imply heat shock proteins (HSP) as cell protective molecules acting in the PDL that are up-regulated upon ischemia caused by mechanical loading. HSP70 and its inhibition by VER155008 as the active agent in several pharmaceuticals are established targets and strategies, respectively, in the treatment of neoproliferative diseases. However, the effect of both players on periodontal remodeling in unknown. Therefore, we analyzed the role of HSP70 and its frequently used inhibitor VER155008 in the regulation of physiological hPDL cell functions and immune cell interaction. MATERIALS AND METHODS: Fifth passage hPDL cells were cultured in the presence of 25µm HSP70 inactivating agent VER155008. At harvest, HSP70 expression, cell proliferation, and parameters of cell interaction, colony formation and wound healing were analyzed by means of real-time PCR, immunohistochemistry, Western blot, biochemical MTS assay, microscopy, and functional assays for monocyte adhesion and differentiation. RESULTS: Basal HSP70 expression and hPDL cell morphology were not affected by HSP70 inhibitor VER155008. In contrast, cell proliferation, tissue defect healing, and colony formation were reduced significantly following HSP70 inhibition, whereas apoptosis and necrosis, monocyte adhesion and osteoclastic differentiation were markedly increased. CONCLUSIONS: The present data indicate a regulatory role for HSP70 protein in hPDL cell biology. CLINICAL RELEVANCE: These findings identify HSP70 as a promising target in the attempt to modify periodontal remodeling and point to potential periodontal side effects of HSP70 pharmaceutical usage.

Potencial rol protector del óxido nítrico y Hsp70 asociado a alimentos funcionales en la aterosclerosis / Potential protective role of nitric oxide and Hsp70 linked to functional foods in the atherosclerosis

En la actualidad se encuentran en constante revisión los mecanismos determinantes primarios y las posibles terapéuticas de una de las principales entidades patológicas considerada epidémica y constituida como problema de salud pública mundial: la aterosclerosis. En tal sentido, pacientes que la padecen presentan como común denominador disfunción mitocondrial, estrés oxidativo e inflamación. De especial interés, el óxido nítrico, un conocido gas mensajero vasoactivo, ha sido estrechamente relacionado con el proceso inflamatorio, oxidativo y disfuncional mitocondrial propio de la aterosclerosis. Por otro lado, muy recientemente se ha demostrado que alteraciones en la biodisponibilidad del óxido nítrico inducirían la expresión de proteínas de shock térmico. Este mecanismo sería inducido también por el uso de los denominados alimentos funcionales como estrategia para prevenir el envejecimiento vascular así como el desarrollo de aterosclerosis. Finalmente, el mayor conocimiento de los mecanismos implicados en el desarrollo de la aterosclerosis nos permitirá proponer nuevas y posibles intervenciones higiénicas, sanitarias y terapéuticas

Atherosclerosis, one of the main pathologic entities considered epidemic and a worldwide public health problem, is currently under constant review as regards its basic determining mechanisms and therapeutic possibilities. In this regard, all patients afflicted with the disease exhibit mitochondrial dysfunction, oxidative stress and inflammation. Interestingly, nitric oxide - a known vasoactive messenger gas - has been closely related to the inflammatory, oxidative and mitochondrial dysfunctional process that characterizes atherosclerosis. In addition, it has recently been demonstrated that alterations in the bioavailability of nitric oxide would induce the expression of heat shock proteins. This agrees with the use of functional foods as a strategy to prevent both vascular aging and the development of atherosclerosis. Finally, a greater knowledge regarding the mechanisms implied in the development of atherosclerosis will enable proposing new and possible hygiene, health and therapeutic interventions

Detection of experimental myocardium infarction in rats by MRI using heat shock protein 70 conjugated superparamagnetic iron oxide nanoparticle.

Superparamagnetic iron-oxide based contrast agents can provide important diagnostic information regarding the assessment of cardiac inflammatory diseases. The aim of the study was to analyze whether nanoparticles conjugated to recombinant 70-kDa heat shock protein (Hsp70-SPION) can be applied for the detection of acute myocardium infarct by MRI. Cellular experiments demonstrated increased CD40-mediated uptake of Hsp70-SPIONs in comparison to non-conjugated SPIONs. Following induction of an acute infarct in rats by ligation of the left anterior descending artery SPIONs and Hsp70-SPION conjugates were injected intravenously on day 4. The animals underwent sequential MRI that showed the presence of the particles in the infarcted zone. Subsequent biodistribution analyses with the help of method on non-linear magnetic response indicated the preferential accumulation of the Hsp70-SPIONs in the heart tissue that was further confirmed with histological analyses. The study demonstrated that an acute infarct can be visualized by MRI using Hsp70-functionalized SPION conjugates. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxides nanoparticles (SPIONs) have been studied extensively as a contrast agent for MRI. Their tissue specificity can be further enhanced by conjugation with various ligands. In this study, the authors conjugated superparamagnetic nanoparticles to 70-kDa heat shock protein (Hsp70-SPION) to investigate the feasibility for the detection of acute myocardium infarct. The positive findings would suggest that this approach might be used clinically in the future.

Hsp70 plays an important role in high-fat diet induced gestational hyperglycemia in mice

Gestational diabetes mellitus (GDM) has emerged as an epidemic disease during the last decade, affecting about 2 to 5 % pregnant women. Even among women who have gestational hyperglycemia may also be positively related to adverse outcomes as GDM. Since heat shock protein (Hsp) 70 has been reported to be associated with diabetes and insulin resistance and its expression was reported to be negatively regulated by the membrane-permeable Hsp70 inhibitor MAL3-101 while positively regulated by the Hsp70 activator BGP-15, we investigated whether Hsp70 played a role in a gestational hyperglycemia mouse model. Mice were divided into non-pregnant and pregnant groups, and each comprised three subgroups: control, high-fat diet (HFD) + MAL3-101, and HFD + BGP-15. We examined the serum levels of triglycerides, total cholesterol, glucose, and insulin, as well as conducted thermal detection of brown adipose tissue (BAT). The role of Hsp70 in BAT apoptosis was also investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and caspase-3 staining. Higher serum level of Hsp70 was associated with increased bodyweight gain after pregnancy in mice fed HFD. Circulating Hsp70 was elevated in control pregnant mice compared to control non-pregnant mice. BGP-induced serum Hsp70 expression reduced triglycerides, total cholesterol, glucose, and insulin levels in the serum. Additionally, thermal detection of BAT, TUNEL, and caspase-3 staining revealed relationship correlation between Hsp70 and BAT functions. Hsp70 level is associated with hyperglycemia during pregnancy. Our results support the role of Hsp70 in facilitating BAT activities and protecting BAT cells from apoptosis via caspase-3 pathway

Oxidative stress protection by exogenous delivery of rhHsp70 chaperone to the retinal pigment epithelium (RPE), a possible therapeutic strategy against RPE degeneration.

PURPOSE: To measure the cytoprotective effects of rhHsp70 against oxidative stress and study its cellular uptake, intracellular and intraocular distribution in the retinal pigment epithelium. METHODS: Human retinal pigment epithelial cells (ARPE-19) were pre-treated with rhHsp70 for 24 h, 48 h, and 72 h before being exposed to 1.25 mM hydrogen peroxide. Non-treated cells served as control. We analysed interleukin 6 secretion, cell viability, and cytolysis. Uptake and intracellular distribution of fluorescently labelled rhHsp70 were investigated with flow cytometry and confocal microscopy, respectively. Ocular distribution of radioactively labelled rhHsp70 was followed ex vivo in porcine eyes by micro SPECT/CT. RESULTS: After exposure to hydrogen peroxide, IL-6 secretion decreased by 35-39% when ARPE-19 cells were pre-treated with rhHsp70. Cell viability increased by 17-32%, and cell lysis, measured by the release of lactate dehydrogenase, decreased by 6-43%. ARPE-19 cells endocytosed rhHsp70 added to the culture medium and the protein was localized in late endosomes and lysosomes. Following intravitreal injection into isolated porcine eyes, we found 20% rhHsp70 in the RPE. CONCLUSIONS: Recombinant hHsp70 protein offers protection against oxidative stress. RPE cells take up the exogenously delivered rhHsp70 and localize it in late endosomes and lysosomes. This work provides the basis for a therapeutic strategy to target aggregate-associated neurodegeneration in AMD.

[Biodistribution of the recombinant heat shock protein rhHsp70 in intracranial C6 glioma models in Wistar rats and subcutaneos B16/F10 melanoma in C57BL/6 mice].

For the first time, the biodistribution of recombinant heat shock protein in rhHsp70 rats with grafted intracranial C6 glioma was evaluated. It was assessed using the fluorescent antibody accumulation chaperone rhHsp70 conjugated with fluorochrome Alexa Fluor 555 in tumor cells by intratumoral or intravenous administration. Assessment of the distribution and accumulation of labeled protein was carried out on the model of subcutaneous B16/F10 melanoma in C57BL/6 mice with the use of single-photon emission computer tomography. After 60 minutes after intravenous administration rhHsp70-I123 (20 MBq, 5 mg chaperone) accumulation of the drug mainly in the liver and tumor tissue was showed. The coefficient of the differential accumulation of the labeled protein KDN(tumor/background) was 3.14. It was turned out that comparing the level of fixation of rhHsp70-I123 in the liver and the tumor KDN(tumor/ liver) = 0.76. After 24 hours from the time of injection of rhHsp70-I123 it was observed increase the level of fixation of the labeled protein in the liver and melanoma: KDN(tumor/background) = 3.43; KDN(tumor/liver = 0.78.

Heat shock protein derivatives for delivery of antigens to antigen presenting cells.

Delivery of antigens to antigen presenting cells (APCs) is a key issue for developing effective cancer vaccines. Controlling the tissue distribution of antigens can increase antigen-specific immune responses, including the induction of cytotoxic T lymphocytes (CTL). Heat shock protein 70 (Hsp70) forms complexes with a variety of tumor-related antigens via its polypeptide-binding domain. Because Hsp70 is taken up by APCs through recognition by Hsp receptors, such as CD91 and LOX-1, its application to antigen delivery systems has been examined both in experimental and clinical settings. A tissue distribution study revealed that Hsp70 is mainly taken up by the liver, especially by hepatocytes, after intravenous injection in mice. A significant amount of Hsp70 was also delivered to regional lymph nodes when it was injected subcutaneously, supporting the hypothesis that Hsp70 is a natural targeting system for APCs. Model antigens were complexed with or conjugated to Hsp70, resulting in greater antigen-specific immune responses. Cytoplasmic delivery of Hsp70-antigen further increased the efficacy of the Hsp70-based vaccines. These findings indicate that effective cancer therapy can be achieved by developing Hsp70-based anticancer vaccines when their tissue and intracellular distribution is properly controlled.

[Development of heat shock proteins with controlled distribution properties and their application to vaccine delivery].

Antigen delivery to antigen-presenting cells (APCs) is a key issue in developing effective cancer vaccines. Controlling the tissue distribution of antigens, which are administered in a peptide/protein or DNA form, can increase antigen-specific immune responses, including the induction of cytotoxic T lymphocytes. Heat-shock protein 70 (Hsp70), a member of a highly conserved family of molecular chaperones, forms complexes with a variety of tumor-related antigens via its polypeptide binding domain. Because Hsp70 is taken up by APCs through the recognition by Hsp receptors, such as CD91 and LOX-1, its application to antigen delivery systems has been examined both in experimental and clinical settings. A tissue distribution study revealed that Hsp70 is mainly taken up by the liver, especially by hepatocytes, after intravenous injection in mice. A significant amount of Hsp70 was also delivered to regional lymph nodes when it was injected subcutaneously, supporting the hypothesis that Hsp70 is a natural targeting system to APCs. Model antigens were complexed with or conjugated to Hsp70, by which greater antigen-specific immune responses were achieved. Cytoplasmic delivery of Hsp70-antigen further increased the efficacy of the Hsp70-based vaccines. These findings indicate that effective cancer therapy can be achieved by developing Hsp70-based anticancer vaccines when their tissue and intracellular distribution is properly controlled.

Histogram analysis of pharmacokinetic parameters by bootstrap resampling from one-point sampling data in animal experiments.

A bootstrap method is proposed for assessing statistical histograms of pharmacokinetic parameters (AUC, MRT, CL and V(ss)) from one-point sampling data in animal experiments. A computer program, MOMENT(BS), written in Visual Basic on Microsoft Excel, was developed for the bootstrap calculation and the construction of histograms. MOMENT(BS) was applied to one-point sampling data of the blood concentration of three physiologically active proteins ((111)In labeled Hsp70, Suc(20)-BSA and Suc(40)-BSA) administered in different doses to mice. The histograms of AUC, MRT, CL and V(ss) were close to a normal (Gaussian) distribution with the bootstrap resampling number (200), or more, considering the skewness and kurtosis of the histograms. A good agreement of means and SD was obtained between the bootstrap and Bailer's approaches. The hypothesis test based on the normal distribution clearly demonstrated that the disposition of (111)In-Hsp70 and Suc(20)-BSA was almost independent of dose, whereas that of (111)In-Suc(40)-BSA was definitely dose-dependent. In conclusion, the bootstrap method was found to be an efficient method for assessing the histogram of pharmacokinetic parameters of blood or tissue disposition data by one-point sampling.

Heat shock protein 70 delays gentamicin-induced vestibular hair cell death.

In this study, geranylgeranylacetone (GGA) was shown to induce heat shock protein (HSP)70 in the vestibular end organs of the guinea pig and to alleviate gentamicin (GM) ototoxicity. This was accomplished without thermal preconditioning. In isolated guinea pig vestibular end organs we demonstrated possible prophylactic (preventive) effects of GGA on GM ototoxicity by actively inducing HSP70. When HSP70 was pre-incubated with GGA, its content in sensory cell cytoplasm and transitional dark cells was increased. Pre-incubation of vestibular end organs with GGA gave sensory cells partial protection from GM toxicity. These findings show that administration of GGA can protect vestibular sensory cells from GM ototoxicity and suggest that induction of HSP70 by GGA may be a useful adjunct for the treatment of vestibular disorders.

Pharmacokinetic and tissue distribution mechanism of mouse recombinant heat shock protein 70 in mice.

PURPOSE: To investigate the in vivo pharmacokinetics and uptake mechanisms of recombinant mouse heat shock protein 70 (Hsp70) by hepatocytes in mice. METHODS: The tissue distribution and intrahepatic localization of Hsp70 were determined after an intravenous injection of 111In-Hsp70 (111In-Hsp70) into mice. Ligands of CD91 or scavenger receptors were injected prior to Hsp70 to examine the involvement of these molecules on the distribution of 111In-Hsp70. The uptake of 111In-Hsp70 by primary mouse hepatocytes was also examined. RESULTS: After intravenous injection, 111In-Hsp70 was rapidly eliminated from the circulation and taken up mainly by the liver. The hepatic uptake was significantly inhibited by preinjection of ligands for CD91 or scavenger receptors. The separation of liver-constituting cells revealed a major contribution of hepatocytes to the overall hepatic uptake of 111In-Hsp70. The uptake of 111In-Hsp70 by cultured hepatocytes was inhibited by a CD91 ligand or anti-CD91 anibody. In addition, after subcutaneous injection, 111In-Hsp70 gradually disappeared from the injection site and accumulated in primary lymph nodes. CONCLUSIONS: These results indicate for the first time that intravenous Hsp70 is, at least partially, recognized by CD91 and eliminated by hepatocytes, whereas subcutaneous Hsp70 is efficiently delivered to regional lymph nodes.

Surface binding and uptake of heat shock protein 70 by antigen-presenting cells require all 3 domains of the molecule.

The surprisingly efficient uptake of peptide-loaded heat shock proteins (Hsps) by antigen-presenting cells (APCs) has been recently associated with a specific receptor-ligand-based mechanism, and the identity of at least 1 receptor has been determined. In this study, we tested how the domain composition of the stress protein affected its surface association and internalization by APCs, and this was facilitated by the availability of the 70-kDa human heat shock protein (Hsp70) and its various deletion mutants. We show that both these processes strictly depend on the presence of all 3 domains of Hsp70. We propose that the previously described interdomain interactions as a determinant of a favorable conformational status might also govern a sterical adaptation of Hsps to components of the internalization machinery.

Mycobacterium paratuberculosis heat shock protein 70 as a tool in control of paratuberculosis.

Paratuberculosis in cattle is a chronic intestinal disease in which a distinctive cellular reactivity of a Th1-type preceeds the phase in which antibody titers are easily detectable and the animal becomes clinically ill. During infection with Mycobacterium avium ssp. paratuberculosis (M.a.p), a decrease in CD4 T-helper cells has been observed in the clinical phase. Our ultimate aim is to elicit a cytotoxic reaction against infected macrophages, using recombinant Hsp70 (rHsp70) of M.a.p. as a tool to shuttle antigen into the MHC class I antigen presentation pathway. To investigate the mechanism of rHsp70 as a carrier for antigen into the cell, we studied the interaction between APC and Fitc-labelled rHsp70, using FACS analysis and confocal microscopy. Interaction of rHsp70 with the cell surface of bovine APC, presumably via a receptor, was shown on monocytes, monocyte derived macrophages and dendritic cell (DC). The interaction is detectable on the complete population of freshly derived monocytes, although peak intensity of fluorescence is lower on these cells than on macrophages and DCs. DCs show interaction on a high percentage of the cells, with high intensity, while in the case of macrophages only a subpopulation interacts with rHsp70. Efficient uptake of rHsp70 as compared to OVA is shown. Preincubation of DC with unlabelled rHsp70 leads to a decreased interaction with rHsp70-FITC. DC interacting with rHsp70 in addition showed high expression of MHC I, MHC II, Myd-1 (CD172a) and CD40. Further research will focus on loading of the rHsp70 with M.a.p. antigen for presentation in MHC class I.

In vitro studies show that Hsp70 can be released by glia and that exogenous Hsp70 can enhance neuronal stress tolerance.

Glial cells release a variety of molecules that support neuronal function. Because heat shock proteins (Hsps) are important in the survival of neurons subjected to metabolic stress, the possibility that glia can release the inducible form of the 70 kDa Hsp (Hsp70) was examined. Additionally, the ability of neuronal cells to show increased stress tolerance by taking up a mixture of constitutive and inducible forms of Hsp70 (Hsc/Hsp70) added to the extracellular fluid was tested. Human T98G glioma cells and differentiated LA-N-5 neuroblastoma cells were used as model glia and neurons to investigate these points. Hsp70 was analyzed using affinity chromatography, Western blotting, and immunofluorescence microscopy. The glioma cells were shown to export Hsp70 into the culture medium whether under normal conditions or subjected to heat shock. The amount of glial Hsp70 released ranged from 5 to 15 pg per 10(6) cells per day, being greater following heat shock. Neuroblastoma cells took up biotinylated Hsc/Hsp70 within 1 h after it was added to the culture medium and it made them more resistant to heat shock (44 degrees C) and to staurosporine-induced apoptosis. This increased stress tolerance was especially important in neuroblastoma cells induced to differentiate with phorbol ester because those 'mature neurons' showed a 10-fold decline in endogenous Hsp70, which was accompanied by increased susceptibility to heat shock and staurosporine-induced apoptosis. These results suggest that extracellular Hsp70 may provide a means by which glia can affect neuronal function, perhaps enhancing neuronal stress tolerance.

Retinal uptake of intravitreally injected Hsc/Hsp70 and its effect on susceptibility to light damage.

PURPOSE: To evaluate the uptake by the rat retina of an intravitreally injected mixture of the constitutive and inducible forms of the 70 kD heat shock protein (Hsc/Hsp70) and test its potential to protect photoreceptors from light damage. METHODS: Hsc/Hsp70 and actin (control protein) were labeled with fluorescein (referred to as fl-Hsc/Hsp70 and fl-actin). The labeled proteins were microinjected intravitreally into the normal or light damaged rat eye and each eye collected at three intervals after the injections. Retinal uptake of Hsc/Hsp70 or actin was studied in frozen sections using epifluorescence microscopy and in western blots of retinal homogenates using an anti-fluorescein antibody. Additionally, the cytoprotective effects of Hsc/Hsp70 were tested in rats that first were exposed to bright light (170 ft-c) for 24 h and then given an intravitreal injection of the protein immediately thereafter. Ten days later, photoreceptor damage was evaluated by measuring the area of the outer nuclear layer at fixed locations along the circumference of the retina. RESULTS: The fluorescein-labeled proteins were detected in the retina one h after administration and were retained there for more than 6 h. They were diffusely distributed, primarily in the nerve fiber layer, ganglion cell layer, and plexiform layers. Fl-Hsc/Hsp70 was also found in the outer nuclear layer (ONL) at 6 h after injection. At 24 h post-injection, the proteins were undetectable by epifluorescence microscopy of retinal sections, but could still be detected in western blots of retinal homogenates. The pattern of protein uptake was similar in light-damaged retinas. Ten days after light damage, the retinas in those eyes that received injections of Hsc/Hsp70 had greater ONL areas compared to either the light-damaged retinas of uninjected eyes or those that had received actin. The difference was statistically significant (p<0.05). CONCLUSIONS: Intravitreally injected Hsc/Hsp70 is taken up by retinal cells and, when administered after an acute injury like light damage, increased the number of surviving photoreceptors.

HSP70 peptidembearing and peptide-negative preparations act as chaperokines.

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-kappaB activity and subsequent augmentation of proinflammatory cytokine TNF-alpha, IL-1beta, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

Kinetics of induction and protective effect of heat-shock proteins after cardioplegic arrest.

BACKGROUND: Heat-shock proteins are known to enhance cardiac resistance to ischemia. METHODS: To evaluate the kinetics of heat-shock protein 70 in relation to its effect on postischemic recovery of cardiac mechanical (cardiac output) and endothelial function (as percentage increase of coronary flow in response to 5-hydroxytryptamine), isolated rat hearts were subjected to prolonged hypothermic cardioplegic arrest at different intervals ranging from 12 to 96 hours after heat stress (n = 6 in each interval). RESULTS: Immunoblotting showed the maximal level of heat-shock protein 70, 0.65 +/- 0.10 (arbitrary units +/- standard error of the mean), at 24 hours after heat shock and similar values at 26 and 30 hours (p = not significant). Postischemic recovery of cardiac output and endothelial function (percentage of preischemic value +/- standard error of the mean) observed at 24 hours was 74.0 +/- 2.4 and 58.3 +/- 7.2, respectively. Similar values were observed at 26 and 30 hours (p = not significant). CONCLUSIONS: In a protocol mimicking conditions for cardiac transplantation, postischemic recovery of cardiac output and endothelial function was improved when the interval between heat stress and ischemia ranged from 24 to 30 hours. This correlated with an apparently critical amount of heat-shock protein 70.