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.

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.

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.

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.