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Am J Physiol Heart Circ Physiol 287: H1104-H1114, 2004. First published April 15, 2004; doi:10.1152/ajpheart.00102.2004
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Heat shock treatment suppresses angiotensin II-induced activation of NF-{kappa}B pathway and heart inflammation: a role for IKK depletion by heat shock?

Yu Chen,1 André-Patrick Arrigo,2 and R. William Currie1,2

1Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5, and 2Laboratoire Stress Oxidant, Chaperons et Apoptose, Centre de Génétique Moleculaire et Cellulaire, Centre National de la Recherche Scientifique UMR-5534, Université Claude Bernard Lyon-1, 69622 Villeurbanne, France

Submitted 3 February 2004 ; accepted in final form 7 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Heat shock (HS) proteins (Hsps) function in tissue protection through their chaperone activity and by interacting with cell signaling pathways to suppress apoptosis. Here, we investigated the effect of HS treatment on the nuclear factor (NF)-{kappa}B signaling pathway in the angiotensin II (ANG II) model of inflammation. Male Sprague-Dawley rats were divided into sham and HS-, ANG II-, and HS + ANG II-treated groups. HS treatment was administered 24 h before the initiation of ANG II infusion. HS treatment (42°C for 15 min) decreased 7-day ANG II-induced hypertension from 191 ± 4 to 147 ± 3 mmHg (P < 0.01). Histological staining of hearts showed that HS treatment reduced ANG II-induced leukocyte infiltration, perivascular and interstitial inflammation, and fibrosis. Heart NF-{kappa}B nuclear translocation and activity, examined by Western blot analysis and electrophoretic mobility shift assay, was suppressed by HS treatment. HS treatment depleted I{kappa}B kinase-{alpha} (IKK-{alpha}) and phosphorylated IKK-{alpha} and suppressed the depletion of I{kappa}B-{alpha} and the accumulation of phosphorylated I{kappa}B-{alpha}. HS treatment blocked ANG II induced expression of IL-6 and ICAM-1 in the heart. ANG II and HS treatment induced high-level expression of Hsp27 and Hsp70 and their phosphorylation. Phosphorylated isoforms of Hsp27 and Hsp70 may play an important role in protecting the heart against ANG II-induced inflammation.

heat shock proteins; I{kappa}B-{alpha}; I{kappa}B kinase-{alpha}; nuclear factor-{kappa}B; protein phosphorylation; hypertension

ANGIOTENSIN (ANG) II, the primary active component of the renin-angiotensin system, plays a key role in the development of hypertension and regulation of body fluid homeostasis. ANG II also induces cell growth, proliferation, and inflammatory responses in the arterial wall (22, 29) and in the heart (12, 62) and kidney (44, 60). ANG II activates NADPH oxidase and induces the production of vascular superoxide and other oxygen free radicals (34, 56). ANG II has also been associated with activation and regulation of the proinflammatory transcription factor nuclear factor (NF)-{kappa}B (29, 60, 61).

NF-{kappa}B, a ubiquitous inducible transcription factor, is involved in the inflammatory process, and promotes transcription of multiple inflammatory factors and cytokines. NF-{kappa}B is a homo- or heterodimer consisting of various combinations of the Rel/NF-{kappa}B family members, including NF-{kappa}B1 (p50 and precursor p105), c-Rel, p65 (RelA), NF-{kappa}B2 (p52 and precursor p100), and RelB (20). Usually, NF-{kappa}B, mainly the p50/p65 heterodimer, resides in the cytoplasm in an inactive state bound to its inhibitor, I{kappa}B. The NF-{kappa}B/I{kappa}B complex is activated by reactive oxygen intermediates, UV light, lipopolysaccharide, TNF-{alpha}, and numerous other stimuli (20). The dissociation of the NF-{kappa}B/I{kappa}B complex is mediated by increased activity of the I{kappa}B kinase (IKK) complex. When organisms receive appropriate cues, the IKK complex becomes active, and it causes the phosphorylation of I{kappa}B. This phosphorylation leads to the ubiquitination and degradation of I{kappa}B, the release of NF-{kappa}B from its inhibitor, and its translocation from cytoplasm to the nucleus. There are three known subunits in the IKK complex: two protein kinases (IKK-{alpha} and IKK-{beta}) and a structural/regulatory subunit (NEMO/IKK-{gamma}) (21). IKK-{alpha} appears to be critical for NF-{kappa}B activation in response to proinflammatory cytokines by specifically phosphorylating I{kappa}B-{alpha} on both Ser32 and Ser36. These modifications are required for targeted degradation of I{kappa}B-{alpha} via the ubiquitin proteasome pathway (14, 58). Several recent studies have reported that NF-{kappa}B inhibition suppresses tissue injury induced by ANG II. Aspirin, enalapril, pioglitazone, rosiglitazone, 3-hydroxy-3-methylglutaryl coenzyme A, and cyclosporine A are all protective against ANG II-induced inflammation and tissue injury by suppressing NF-{kappa}B activity (12, 15, 44, 47, 50).

Heat shock (HS) proteins (Hsps) are well known for their cytoprotective functions under stress (9, 48). The protective role of Hsps has been ascribed to their ability to reversibly interact as molecular chaperones with damage-prone proteins and facilitate the refolding, assembly, and stabilization of denatured proteins (23, 31, 46). Transgenic overexpression of Hsp70 protects the heart from ischemic injury (39, 53) and ameliorates the effects of polyglutamine expansion in a model of neurological disease (1). Recently, several studies indicate a role for the phosphorylation of Hsps in cytoprotection. Phosphorylation of {alpha}-crystallin on Ser59 is necessary and sufficient to confer caspase 3 inhibition and protection of cardiac myocytes against hyperosmotic or hypoxic stress (45). Phosphorylation may be necessary to regulate the post-heat stress molecular chaperone activity of Hsp30 (16). Phosphorylation of Hsp27 also plays a role in the resistance of the cells to oxidative damage (19, 25, 64). Interestingly, Hsp70 is also phosphorylated in response to HS (28) and energy deprivation (32).

In this study, we tested the hypothesis that HS treatment suppresses ANG II-induced inflammation in the heart through the IKK/NF-{kappa}B pathway, and we suggest that the phosphorylation of Hsps may play a role in ANG II-induced heart inflammation and the HS-induced protection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models. Male Sprague-Dawley rats (Charles River Laboratories; Québec, Canada) weighing 280–310 g were cared for in accordance with the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care and were housed in a climate-controlled room with a 12:12-h light-dark cycle and had free access to water and food. Rats were randomly assigned to either the ANG II treatment group (ANG group, n = 12), HS and ANG II treatment group (HS + ANG group, n = 12), HS and sham surgery group (HS group, n = 9), or sham group (n = 9). For HS treatment, animals were anesthetized with ketamine (80 mg/kg ip) and xylazine (10 mg/kg ip) and then placed on a heating pad (50°C) until their rectal temperature reached 42°C. Core body temperature was maintained between 42 and 42.5°C for 15 min. Animals in the other groups were anesthetized with the same drug but not heated. Twenty-four hours after HS or non-HS treatment, an osmotic mini-pump (model 2002, Alzet) containing ANG II dissolved in 0.9% NaCl was implanted subcutaneously between scapulae. The ANG II infusion rate was 0.7 mg·kg–1·day–1. Animals in the sham and HS groups underwent an identical surgical procedure with implantation of an empty osmotic pump. Systolic blood pressures were measured in conscious rats by tail-cuff plethysmography (model 29, IITC; Woodland Hills, CA). Rats were overdosed with pentobarbital sodium 3 and 7 days after ANG II infusion or after sham surgery, and hearts were collected for analysis, frozen in liquid nitrogen, and then stored at –70°C until analyzed.

Preparation of protein extracts. Heart ventricular tissue was thawed and rinsed with PBS and then homogenized on ice in 1 ml of buffer containing 50 mM HEPES (pH 7.5), 5 mM EDTA, 50 mM NaCl, 1 mM PMSF, 10 µg/ml leupetin, and 10 µg/ml aprotinin. The homogenates were stored at –70°C as whole cell extracts.

For the isolation of nuclear protein extracts, a modified method (55) was used. Briefly, heart tissue was rinsed with ice-cold PBS and homogenized on ice in 1 ml of ice-cold buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin]. After a 10-min incubation on ice, the homogenates were centrifuged at 850 g for 10 min at 4°C. The supernatants were discarded, and the pellets were suspended in 150 µl of ice-cold buffer A with 0.1% Triton X-100, incubated on ice for 10 min, and then centrifuged as above. The supernatants were stored as cytoplasmic extracts at –70°C. The pellets were resuspended in 750 µl of buffer A and then centrifuged at 850 g for 10 min. The supernatants were removed, and the purified nuclear pellets were resuspended in 90 µl of buffer B [20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin]. The suspension was incubated for 30 min on ice. After centrifugation at 16,000 g for 15 min at 4°C, the supernatants were transferred in aliquots to new tubes and stored at –70°C until analyzed. Protein concentrations were determined by the method of Lowry et al. (38).

Electrophoretic mobility shift assay. NF-{kappa}B consensus oligonucleotide sequence (5'-AGTGAGGGACTTTCCCAGGC-3', Promega; Madison, WI) was end labeled with [{gamma}-32P]ATP (Amersham Pharmacia; Piscataway, NJ) using T4 polynucleotide kinase (Promega) and purified in G-25 Sephadex columns (Amersham Pharmacia). Nuclear extracts (20 µg) were incubated with 2 µl of binding buffer [20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris·HCl (pH 7.5), and 0.25 mg/ml poly(dI-dC)·poly(dI-dC)], and 1 µl of labeled probe (0.035 pmol) was then added and incubated for 20 min at room temperature. Negative controls contained no nuclear extracts, and HeLa cell nuclear extracts were used as a positive control. To establish the specificity of the reaction, competition assays with a 50x excess of unlabeled NF-{kappa}B and SP-1 oligonucleotides (5'-ATTCGATCGGGGCGGGGCGAGC-3', Promega) were performed by adding unlabeled probes 10 min before the addition of the labeled probe. For supershift assays, 2 µg of anti-p65 and anti-p50 antibodies (Santa Cruz Biotechnology; Santa Cruz, CA) were added to nuclear protein extracts and incubated for 1 h after the addition of the labeled probe. The reaction was stopped by adding gel loading buffer [250 mM Tris·HCl (pH 7.5), 0.2% bromophenol blue, and 40% glycerol]. The protein-DNA complexes were separated on a nondenaturing 4% acrylamide gel in Tris-borate. Gels were dried onto Whatman 3MM paper and exposed to X-ray film with an intensify screen at –70°C overnight.

Western blot analysis. Protein samples (20 µg) were boiled for 10 min in sample buffer [250 mM Tris·HCl (pH 6.8), 4% SDS, 10% glycerol, 2% {beta}-mercaptoethanol, and 0.003% bromophenol blue], separated on denaturing 10% SDS-polyacrylamide gels, and then transferred onto a polyvinylidene difluoride membrane (Millipore; Billercia, MA). After being blocked with 5% dry nonfat milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T) for 1 h at room temperature, membranes were washed three times for 5 min in TBS-T and then incubated at 4°C overnight with either rabbit polyclonal anti-NF-{kappa}B p50 (1:1,000, Santa Cruz Biotechnology), rabbit polyclonal anti-IKK-{alpha}, anti-phospho IKK-{alpha} (1:1,000, Cell Signaling Technology; Beverly, MA), rabbit polyclonal anti-I{kappa}B-{alpha}, anti-phospho IKK-{alpha} (1:1,000, Cell Signaling Technology) goat polyclonal anti-IL-6 (1:1,000, R&D Systems; Minneapolis, MN), rabbit polyclonal anti-Hsp27 antibody (1:5,000, StressGen; Victoria, Canada), or mouse monoclonal anti-Hsp70 (1:1,000, StressGen), each in TBS-T containing 5% BSA. Membranes were washed three times in TBS-T for 5 min and incubated with appropriate peroxidase-conjugated secondary antibodies in TBS. After another three washes with TBS-T for 5 min, membranes were reacted with the enhanced chemiluminescence system (Amersham Pharmacia) according to the manufacturer's protocol and then exposed to films. Protein levels were quantified by scanning densitometry using image-analysis systems (Scion; Frederick, MD). The membranes were stained with amido black to ensure that approximately equal amounts of protein were loaded on each lane.

Two-dimensional electrophoresis and immunoblotting. Two-dimensional electrophoresis was performed as previously described (27, 30). In brief, 1 mg of whole cell protein extracts was subjected to isoelectric focusing in the first dimension using ampholines of pI range 3–10 (Bio-Rad; Hercules, CA). Proteins in isoelectric gels were separated in the second dimension in 10% SDS-polyacrylamide gels and transferred onto a polyvinylidene difluoride membrane. The membranes were then immunoreacted with anti-Hsp27 and anti-Hsp70 antibodies as described above.

Alkaline phosphatase treatment. For protein dephosphorylation, a subset of whole cell protein extracts (1 mg) were precipitated with 20% trichloroacetic acid, washed with ethanol and then water, and resuspended in a buffer containing 60 mmol/l Tris·HCl (pH 7.5), 1 mmol/l DTT, 1 µg/ml aprotinin, and 100 mmol/l NaCl. Samples were treated with 100 units of alkaline phosphatase (New England Biolabs; Beverly, MA) for 120 min at 30°C (43). At the end of the reaction, the proteins were subjected to two-dimensional electrophoresis followed by Western blot analysis for Hsp27 and Hsp70.

Tissue processing and staining. The frozen heart tissues were routinely processed and cut by cryostat (14 µm thick). Sections were stained with hematoxylin and eosin for histopathological analysis and collagen-specific Masson's trichrome staining for determination of myocardial interstitial fibrosis.

Immunohistochemistry. Frozen tissue sections were thawed and hydrated in 0.1 M PBS for 30 min at room temperature. Sections were fixed in 2% paraformaldehyde for 10 min at room temperature. The tissue sections were washed with 0.1 M PBS and blocked with goat serum (1:1,000, Sigma-Aldrich Canada; Oakville, Ontario, Canada) for 30 min. Depending on the experiment, rabbit polyclonal anti-NF-{kappa}B p50 (1:500, Santa Cruz Biotechnology) or anti-ICAM-1 (1:500, Santa Cruz Biotechnology) was added to tissue sections at 4°C and kept in a humidified chamber overnight. The primary antibodies were discarded, and sections were washed with PBS. P50 and ICAM-1 positive staining was detected after incubation with fluorescein-conjugated IgG secondary antibody (Molecular Probes; Eugene, OR) at room temperature for 1 h.

Statistical analysis. Data are expressed as means ± SE. The significance of differences was determined by ANOVA and post hoc multiple-comparison test using SPSS 10.0 software (Chicago, IL). P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Blood pressure. ANG II infusion significantly increased systolic blood pressure from day 1 and throughout the study (P < 0.01; Fig. 1). HS treatment suppressed the ANG II-induced elevation of blood pressure. This apparent protective role of HS treatment was significant from 3 to 7 days of ANG II infusion (P < 0.01). However, this protection was only partial in that at 7 days HS treatment decreased ANG II-induced hypertension from 191 ± 4 to 147 ± 3 mmHg (P < 0.01) and both were significantly (P < 0.01) elevated compared with the HS (90 ± 2 mmHg) or sham (92 ± 4 mmHg) groups.



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  		Fig. 1. Effect of heat shock (HS) treatment on ANG II-induced hypertension in rats. HS was administered 24 h before the initiation of ANG II infusion. Systolic blood pressure was measured at 0, 1, 3, 5, and 7 days (d) of ANG II infusion. Data points represent means ± SE; n = 9. *P < 0.01 vs. sham; #P < 0.01 vs. ANG.

 
Myocardial histopathology. In sham hearts, with the use of hematoxylin and eosin staining, few leukocytes were seen around coronary vessels or between cardiomyocytes (Fig. 2A). After 7 days of ANG II infusion, perivascular leukocyte infiltration was evident and was associated with mild to moderate thickening of the tunica media and disorganization of coronary vessels (Fig. 2B). Leukocytes were also found adhered in clusters to the endothelium of coronary arteries. Cardiomyocyte degeneration was sporadically observed in some animals, in association with severe interstitial leukocyte infiltration (Fig. 2C). Hearts from HS + ANG rats (Fig. 2D) were histologically similar to those of sham animals and had few leukocytes around coronary vessels or between cardiomyocytes. Low levels of interstitial collagen, as revealed by Masson's trichrome staining, were detected between cardiomyocytes and around blood vessels of sham hearts (Fig. 3A). After 7 days of ANG II infusion, high levels of interstitial collagen were detectable in between cardiomyocytes and around blood vessels (Fig. 3B). In contrast, HS treatment (HS + ANG) markedly suppressed the myocardial interstitial fibrosis (Fig. 3C) induced by ANG II.



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  		Fig. 2. Cardiac histology (hematoxylin and eosin). A: histological section from a sham rat. B and C: 7 days of ANG II infusion caused myocardial perivascular inflammatory lesions indicated by leukocyte infiltration around blood vessels (B) and sporadic cardiomyocyte degeneration and severe interstitial leukocyte infiltration (C). D: myocardium of animals that received HS treatment 24 h before ANG II infusion showed histology similar to that of sham animals. Scale bar = 50 µm in all.

 


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  		Fig. 3. Cardiac histology (Masson's trichrome stain). A: histological section from a sham rat. B: 7 days of ANG II infusion caused myocardial fibrosis indicated by abundant interstitial collagen (blue). C: myocardium of animals that received HS treatment 24 h before ANG II infusion showed histology similar to the sham animals. Scale bar = 100 µm in all.

 
Activation of the IKK/NF-{kappa}B pathway. ANG II increased the activation of the transcription factor NF-{kappa}B (Fig. 4A). HS treatment suppressed the activation of NF-{kappa}B induced by ANG II, whereas HS itself had no effect on NF-{kappa}B activity. The specificity of NF-{kappa}B complexes was examined using a 50x concentration of unlabeled NF-{kappa}B or SP-1 oligonucleotides (Fig. 4B). Antibodies against the p65 and p50 subunits of NF-{kappa}B shifted the migration characteristic of the complexes and showed that these complexes were specific to NF-{kappa}B p50/p65 heterodimers (Fig. 4B). By Western blot analysis, in sham hearts NF-{kappa}B p50 was abundant in the cytoplasmic fraction and was weakly detected in the nuclear fraction (Fig. 5). After the ANG II infusion, the p50 subunit was detected at significantly increased levels in cytoplasmic and nuclear fractions. In the nuclear fractions, HS reduced the level of p50 induced by ANG II, and this was significant after 7 days of ANG II infusion (Fig. 5).



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  		Fig. 4. Effect of HS treatment on ANG II-induced nuclear factor (NF)-{kappa}B activation in the heart. A: EMSA of NF-{kappa}B DNA binding activity showing that HS treatment suppressed ANG II-induced activation of NF-{kappa}B at 3 and 7 days. B: specific NF-{kappa}B binding activity and supershift assay showing identification of p50 and p65 subunits in the NF-{kappa}B complex. Data are representative of 3 separate experiments.

 


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  		Fig. 5. Western blot analysis showing that HS treatment suppressed NF-{kappa}B p50 subunit nuclear translocation induced by ANG II infusion. c, Cytoplasmic fraction; n, nuclear fraction. **P < 0.01 vs. respective cytoplasmic fraction or nuclear fraction sham; ##P < 0.01, HS + ANG vs. ANG. Data are means ± SE and are representative of 3 separate experiments.

 
Localization of the NF-{kappa}B p50 subunit was in the cytoplasm and after ANG II infusion also in the nucleus (Fig. 6). No immunofluorescences was detected when the primary antibody was omitted in the staining reaction (Fig. 6A). Immunofluorescences showed cytoplasmic localization of NF-{kappa}B p50 in the myocardium of sham rats (Fig. 6B). After 7 days of ANG II infusion, localization of p50 in cardiomyocytes and interstitial cells appeared to be mainly cytoplasmic and nuclear (Fig. 6, C and D). NF-{kappa}B p50 nuclear localization in interstitial cells and leukocytes around coronary vessels indicated activation of the vascular inflammatory response (Fig. 6D). HS treatment 24 h before ANG II infusion suppressed the nuclear localization of p50 (Fig. 6E); the immunofluorescence histology was similar to that of sham animals.



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  		Fig. 6. Immunofluorescences for p50 in the myocardium. A: no immunofluorescence was detected after the omission of the primary antibody against the NF-{kappa}B p50 subunit in the staining reaction. B: low-level immunofluorescence is localized throughout the myocardium from a sham rat. C and D: ANG II infusion for 7 days caused localization of p50 in the cytoplasm and nuclei of cardiomyocytes (C) and clustered interstitial cells found around vessels (D). The nuclear localization of p50 in perivascular and interstitial myocardial structures corresponds to the areas of inflammation in Fig. 2. E: myocardium of animals that received HS treatment 24 h before ANG II infusion showed immunofluorescent histology similar to that of sham animals, i.e., minimal nuclear localization of p50. Scale bar = 50 µm in all.

 
The relative cytoplasmic levels of IKK-{alpha} and I{kappa}B-{alpha} were examined by Western blot analysis (Fig. 7). HS treatment significantly depleted IKK-{alpha} and ANG II-induced phosphorylated IKK-{alpha} and significantly suppressed the depletion of I{kappa}B-{alpha} and the accumulation of phosphorylated I{kappa}B-{alpha}. ANG II significantly decreased the abundance of I{kappa}B-{alpha} in a steplike fashion at 3 and 7 days of infusion, whereas the abundance of the phosphorylated I{kappa}B-{alpha} was significantly increased. HS treatment reversed the effect of ANG II on I{kappa}B-{alpha} and phosphorylated I{kappa}B-{alpha}.



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  		Fig. 7. Western blot analysis of I{kappa}B kinase (IKK)-{alpha} and I{kappa}B-{alpha}. HS treatment depleted IKK-{alpha} and blocked the ANG II-induced phosphorylation of IKK-{alpha} (pIKK-{alpha}). HS restored the ANG II-suppressed expression of I{kappa}B-{alpha}, and HS suppressed the ANG II-induced expression of phosphorylated I{kappa}B-{alpha} (pI{kappa}B). For IKK-{alpha}: *P < 0.05 vs. sham; **P < 0.01 vs. sham. For pIKK-{alpha}: **P < 0.01 vs. sham; ##P < 0.01, HS + ANG vs. ANG. For I{kappa}B-{alpha}: **P < 0.01 vs. sham; ##P < 0.01, HS + ANG vs. ANG. For pI{kappa}B-{alpha}: **P < 0.01 vs. sham; ##P < 0.01, HS + ANG vs. ANG. Data are means ± SE and are representative of 3 separate experiments.

 
Expression of IL-6 and ICAM-1. By Western blot analysis, the relative level of IL-6 was elevated between 2 and 3.8 times that of the sham animals at 3 and 7 days, respectively, post-ANG II infusion (Fig. 8, A and B). HS per se had no effect on the abundance of IL-6; however, HS suppressed the relative abundance of ANG II-induced IL-6. ICAM-1 localization was examined using immunofluorescence (Fig. 8, C–E). ICAM-1 immunoreactivity was increased in vessels after ANG II infusion, and the staining appeared to be localized in endothelial cells and in perivascular cells. HS treatment suppressed the ANG II-induced ICAM-1 immunoreactivity.



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  		Fig. 8. Effect of HS treatment on ANG II-induced IL-6 and ICAM-1 expression in the heart. A and B: HS treatment suppressed ANG II-induced IL-6 expression. *P < 0.05 vs. sham; **P < 0.01 vs. sham; #P < 0.05, HS + ANG vs. ANG; ##P < 0.01, HS + ANG vs. ANG. Data are representative of 3 separate experiments. C–E: immunofluorescences for ICAM-1 in the myocardium. C: ICAM-1 immunofluorescent section from a sham rat. D: after 7 days of ANG II infusion, ICAM-1 was localized at elevated levels in endothelial perivascular cells. E: HS treatment suppressed the ANG II-induced ICAM-1 expression. Scale bar = 50 µm in all.

 
Hsp expression. After 7 days of ANG II infusion, the relative abundance of Hsp70 in the heart did not appear different from sham rats (Fig. 9). As expected, HS treatment increased Hsp70 levels significantly (Fig. 9). Hsp27 was increased by 3 days and at 7 days of ANG II infusion, and the HS treatment further increased the level of Hsp27.



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  		Fig. 9. Western blot analysis of HS protein (Hsp)70 and Hsp27 in rat hearts after HS treatment and ANG II infusion. *P < 0.05 and **P < 0.01 vs. sham. Data are means ± SE and are representative of 3 separate experiments.

 
Finally, we then investigated the phosphorylation of Hsp70 and Hsp27 in our model of ANG II-induced inflammation. After 7 days of ANG II infusion, various isoforms of Hsp70 and Hsp27 were abundant (Fig. 10, A and A'), as revealed by two-dimensional gel electrophoresis and Western blot analysis. When a sample of the same aliquot was treated with alkaline phosphatase, the more acidic isoforms of Hsp70 and Hsp27 were diminished (Fig. 10, B and B'). Compared with the sham group, 3 and 7 days of ANG II infusion caused an increase of both nonphosphorylated and phosphorylated (acidic) isoforms of Hsp27 and Hsp70 in the heart (Fig. 10C). HS treatment followed by 3 days of ANG II infusion also caused an increase of both phosphorylated and nonphosphorylated Hsp27 and Hsp70 in the heart (Fig. 10C). HS treatment followed by 7 days of recovery (no ANG II infusion) caused an increase of mainly phosphorylated isoforms of Hsp27 and both phosphorylated and nonphosphorylated isoforms of Hsp70 in the heart (Fig. 10C).



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  		Fig. 10. Two-dimensional Western blot analysis of Hsp70 and Hsp27 phosphorylated isoforms. A and B: the acidic side of the gels is on the right. Approximately 1 mg of heart protein samples from a rat infused with ANG II for 7 days was either nontreated (A) or treated with alkaline phosphatase (B). Western blot analysis revealed one phosphorylated isoform of Hsp70 (PP) and two phosphorylated isoforms of Hsp27 (PP1 and PP2). Nonphosphorylated isoforms of Hsp70 (NP) and Hsp27 (NP1 and NP2) are indicated. A' and B': membranes were counterstained with amido black to show other proteins and confirm approximately equal loading of protein. C: comparison of the expression of Hsp70 and Hsp27 isoforms in hearts from sham rats and from rats after 3 and 7 days ANG II infusion, after HS treatment and 3 days of ANG II infusion, and after HS treatment and 7 days of recovery (no ANG II). Representative results from 3 different experiments are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In a previous study (8), we found that HS treatment suppressed the ANG II-induced hypertension, and this was mediated through the suppression of NF-{kappa}B activation. Here, we confirm these findings and further explore the in vivo inhibition of the IKK/NF-{kappa}B pathway by HS treatment in the ANG II-induced model of hypertension and inflammation. HS treatment suppressed ANG II-induced histological changes indicative of inflammation in the heart. At the molecular level, HS depleted IKK-{alpha} and blocked the expression of phosphorylated IKK-{alpha}. HS blocked the ANG II-induced depletion of I{kappa}B-{alpha} and suppressed the ANG II-induced expression of phosphorylated I{kappa}B-{alpha}. In addition to HS treatment suppressing NF-{kappa}B activation at 3 and 7 days of ANG II infusion, the expression of downstream genes regulated by NF-{kappa}B, IL-6 and ICAM-1, were suppressed by HS. The HS protection was correlated with the higher expression of Hsp27 and Hsp70 and their phosphorylated isoforms.

ANG II is a multifunction hormone and plays a central role in the pathophysiology of cardiovascular and renal diseases and in the etiology of hypertension by causing peripheral vascular constriction, aldosterone release, and renal sodium reabsorption. ANG II also appears to have a role in the inflammatory response. ANG II causes significant T helper cell (CD4+), cytotoxic T cell (CD8+), and monocyte/macrophage infiltration into the heart (47). ANG II induces inflammation by triggering immediate-early genes and tyrosine receptor-coupled transcription factors such as NF-{kappa}B, activator protein-1, ERK, p38MAPK, and signal transducers and activators of transcription (40, 60, 62). The activation of these proinflammatory pathways may relate to ANG II-induced activation of NADH/NADPH oxidase and the production of reactive oxygen species as shown in vascular smooth muscle cells (34, 56, 63). In the present study, ANG II infusion caused inflammatory cell infiltration around blood vessels and in the interstitium (Fig. 2, B and C). The cardiomyocytes and infiltrating inflammatory cells exhibited NF-{kappa}B p50 subunit nuclear localization (Fig. 6, C and D). After 7 days of ANG II infusion, there was an apparent myocardial fibrosis (Fig. 3B). These histopathological changes induced by ANG II infusion were all ameliorated by HS treatment.

Interestingly, in animal models of arterial pressure overload, histopathological changes such as hypertrophy, myocardial fibrosis, and infiltration of inflammatory cells are characteristic (33). While we have not completely ruled out the possibility that HS treatment is directly suppressing blood pressure and hence the inflammatory response, for several reasons we think that the HS protection against ANG II-induced inflammation is independent of blood pressure (8). First, while HS treatment suppressed the hypertension induced by ANG II, the systolic pressures were still higher than normal. Second, norepinephrine infusion increased blood pressure similar to ANG II infusion, but norepinephrine did not activate NF-{kappa}B (8). Interestingly, while ANG II-induced hypertension is associated with an increase in vascular superoxide production and endothelial regulation of vasomotion, norepinephrine is not (56), suggesting that ANG II has unique vascular effects not shared by other forms of hypertension. Third, because HS treatment also suppresses the norephrine-induced increase in blood pressure (8), it may be that the vascular cells responsible for the increased blood pressure may be in a unresponsive state. After HS treatment, these vascular cells may not be responding to the signaling stimuli involved in establishing hypertension. Interestingly, Hsp70 and Hsp27 block apoptotic signaling pathways (17, 46, 52), so it seems reasonable to consider that Hsps may block other signaling pathways, such as the IKK/NF-{kappa}B pathway.

In this study, we demonstrated that HS suppressed ANG II-induced IKK/NF-{kappa}B activation. NF-{kappa}B is a proinflammatory transcription factor that is required for maximal transcription of many cytokines, including IL-6, IL-8, TNF-{alpha}, and ICAM-1 (5). IL-6 is a multifunctional cytokine that mediates the acute-phase response and lymphocyte proliferation/activation (22, 36). ICAM-1 has been shown to mediate rolling and attachment of activated monocytes to the endothelium, which is critical in the process of cell migration into the perivascular interstitium in inflamed tissues (37). We have shown that HS suppressed not only the ANG II-induced IKK/NF-{kappa}B activation but also the ANG II-induced expression of IL-6 and ICAM-1.

The IKK complex includes two catalytic subunits, IKK-{alpha} and IKK-{beta}. Both IKK-{alpha} and IKK-{beta} have a high degree of amino acid homology and a similar domain structure that facilitates their dimerization (13). Autophosphorylated IKK-{alpha} specifically phosphorylates I{kappa}B-{alpha} on both Ser32 and Ser36, which then causes the degradation of I{kappa}B-{alpha} (14, 58). In addition to this conventional function, IKK-{alpha} may also shuttle from the cytoplasm to the nucleus and have a nuclear role in phosphorylation of histones (3, 66). Interestingly, IKK-{alpha} is predominant in alveolar macrophages (57) and IKK-{beta} is predominant in monocytes (49), suggesting that IKK activation may be cell specific. In the present study, the apparent depletion of IKK-{alpha} after HS and the suppression of ANG II-induced expression of pIKK-{alpha} after HS may be the key regulatory step in the suppression of NF-{kappa}B activation after HS. While the mechanism of IKK-{alpha} depletion is currently unknown, at least two possibilities exist. First, the HS treatment may have interrupted the synthesis of IKK-{alpha} or caused its denaturation or degradation. Second, Hsp70, or more likely Hsp27, may interact with IKK-{alpha} (51), prevent the activation of IKK-{alpha}, and possibly alter its stability.

The infusion of ANG II induced an increase in the expression of Hsp27 and no apparent expression of Hsp70 when examined by one-dimensional Western blot analysis (Fig. 9). However, after the ANG II infusion, low-level expression of Hsp70 was detectable by two-dimensional Western blot analysis (Fig. 10). After HS treatment, Hsp70 protein levels in the heart are maximal by 24 h and then slowly decline, but are still above background levels after 8 days of recovery (27). Curiously, after HS treatment (no ANG II), most of Hsp27 appears to be phosphorylated (Fig. 10C), suggesting that HS and ANG II treatments promote the accumulation of different isoforms of Hsp27. In a previous study (8), ANG II induced high-level expression of Hsp27 and Hsp70 in the aorta. After ANG II treatment, Hsps are expressed in smooth muscle cell cultures and in the aorta and kidney of live rats (2, 26, 43, 65) and are suggested to have a protective role against ANG II-induced end organ injury. Interestingly, Ishizaka et al. (26) showed increased expression of Hsp27 and Hsp70 in the kidney after ANG II treatment and also showed increased expression of Hsp27 in the heart but minimal or no increase in the expression of Hsp70. These findings suggest that the expression of Hsps is tissue specific and may depend on the local cellular environment.

In the present study, ANG II (as well as HS treatment) induced an increase in the phophorylated isoforms of both Hsp27 and Hsp70 in the heart. Human Hsp27 can be phosphorylated at serine residues 15, 78, and 82 (35), whereas rodent Hsp27 can be phosphorylated at serine residues 15 and 86 (18). The importance of phosphorylation of Hsp27 is indicated by the increased resistance of the cells to oxidative damage (19, 25, 64). In contrast, the phosphorylation of Hsp27 impedes the formation of Hsp27 large oligomers that are important for both chaperone action and resistance against oxidative stress (54, 59). Whereas nonphosphorylated small oligomers and monomeric Hsp27 bind to F-actin as a capping protein and block actin polymerization, Hsp27 phosphoisoforms enhance actin remodeling, which is essential for vascular smooth muscle cell migration and contraction (43). In addition to phosphorylation playing a role in the chaperone activity of Hsp27, phosphorylation of Hsp27 also has effects on cell death/survival pathways. Phosphorylated Hsp27 interacts with Daxx to inhibit Fas-induced caspase-independent apoptosis (6). Hsp27 is phosphorylated in injured adult sensory and motor neurons, and expression knockdown of Hsp27 in vitro and in vivo in injured sensory neurons results in apoptosis (4). The delivery of phosphorylatable human Hsp27 is required for the survival of injured sensory and motor neurons, whereas the delivery of nonphosphorylatable mutant Hsp27 failed to rescue injured sensory and motor neurons (4). Moreover, the expression of Hsp27 as well as its phosphorylation and formation of larger oligomers is observed during early differentiation in many cell types. Inhibition of this expression results in apoptosis (41, 42) or in aberrant differentiation (7, 11). Whether Hsp27 expression in ANG II-treated cells reflects a cell differentiation phenomenon is currently unknown. More recently, it has been reported that Hsp27 interacts directly with IKK-{alpha} and IKK-{beta} in downregulating TNF-{alpha}-induced NF-{kappa}B activation, and the association of Hsp27 with IKK is mediated by its p38-MK2 phosphorylation as demonstrated using Hsp27 phosphorylation mutants and the p38 specific inhibitor SB203580 (51). While we have not tested this possibility in vivo, it is possible that phosphorylated isoforms of Hsp27 are interacting directly with IKK-{alpha} to prevent its activation and cause its depletion. While Hsp70 phosphorylation is currently less intensively studied, isoforms of Hsp70 have been reported (10, 24, 27, 32).

ANG II infusion appears to induce expression of Hsp27 and Hsp70 (Fig. 10C), and it is clear that HS treatment does so also. Other proteins that have altered expression after HS may also regulate the ANG II-induced hypertension and inflammation. The HS treatment almost certainly only delays the hypertension and other inflammatory changes induced by ANG II (8). As shown here, HS treatment suppressed the ANG II-induced hypertension to 7 days. After HS treatment, with 11 and 14 days of ANG II infusion, blood pressure begins to rise more steeply (8). Coincident with this, in the aorta, after HS treatment with 11 and 14 days of ANG II infusion, the activation of NF-{kappa}B is no longer suppressed and is similar to ANG II (no HS treatment).

In conclusion, the present study demonstrates that HS protects against ANG II-induced hypertension and inflammation by suppression of the IKK/NF-{kappa}B pathway. The HS-induced depletion of IKK-{alpha} appears to be the critical step in the pathway. As part of the HS response, elevated levels of phosphorylated Hsp27 are present. Interestingly, phosphorylated Hsp27 interacts with IKK-{alpha} (51), and, although we have not tested this possibility in vivo, it is possible that phosphorylated isoforms of Hsp27 are interacting directly with IKK-{alpha} to prevent its activation and cause its depletion.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded by grants from the Heart and Stroke Foundation of New Brunswick and the Canadian Stroke Network. Y. Chen was supported by scholarships from the Killam Memorial Trust and the Nova Scotia Health Research Foundation. R. W. Currie was a visiting scientist with A. P. Arrigo and was supported by an International Scientific Exchange Scholarship from the Canadian Institutes of Health Research and Centre National de la Recherche Scientifique.


   ACKNOWLEDGMENTS
 
The authors thank Brenda Ross, Xiaoying Jia, Nan Cheng, and Pat Colp for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. W. Currie, Dept. of Anatomy and Neurobiology, Dalhousie Univ., Halifax, Nova Scotia, Canada B3H 1X5 (E-mail: wcurrie{at}dal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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