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Center for Neuroscience and Department of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan, Republic of China (F.C.H.L., S.H.H.C., A.Y.W.C.); and Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, Republic of China (J.Y.H.C.)
Received February 1, 2005; accepted April 12, 2005
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Abstract |
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; Welch, 1992; Morimoto and Santoro, 1998). Because of their critical roles in intracellular processing, synthesis, transportation, and degradation of proteins, HSPs have been termed molecular chaperones. The cellular protective mechanisms of HSPs are believed to be related to these chaperone functions, which lead to the prevention of protein denaturation and promotion of refolding of damaged proteins after stress. In addition, HSP chaperones may sustain proteins in the productive folding pathway or maintain newly synthesized proteins in an unfolded conformation suitable for translocation across membranes (Welch, 1992; Morimoto and Santoro, 1998).
A majority of studies on the in vivo neuroprotective role of HSPs centers on protection against cerebral ischemia during heatstroke (Yang and Lin, 1999). HSP70 induced by a brief hyperthermic heat shock also confers cardiovascular protection during heatstroke via potentiation of baroreceptor reflex response (Li et al., 2001) by up-regulation of glutamatergic neurotransmission (Chan et al., 2002a) at the nucleus tractus solitarii. Other investigators reported (Yenari et al., 1998) that HSP70 enhances neuronal survival during transient focal cerebral ischemia or excitotoxin-induced seizures. On the other hand, the best-studied cellular action of HSPs by far is protection against apoptosis, based primarily on studies using cell lines under nonpathological conditions. Within the HSP family, HSP70 and HSP90 are two members that are often reported to be antiapoptotic (Meriin et al., 1998; Robertson et al., 1999; Lee et al., 2001).
Cardiovascular depression during sepsis remains a significant cause of morbidity and mortality (Parrillo, 1993). Our laboratory (Chan et al., 2001a,b) demonstrated previously that, by eliciting a reduction in sympathetic vasomotor outflow and arterial pressure, overproduction of nitric oxide (NO) by NO synthase II (NOS II or iNOS) and formation of peroxynitrite by reacting NO with superoxide anion (Chan et al., 2002b, 2005) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located (Ross et al., 1984), plays a pivotal role in the fatal cardiovascular depression associated with endotoxemia. Based on this rat model of experimental endotoxemia, which mimics clinically the systemic inflammatory response syndrome, the present study was undertaken to assess the hypothesis that HSP70 and HSP90 in the RVLM confers neuroprotection against fatal endotoxemia. This hypothesis was partially validated based on combined physiological, pharmacological, and biochemical results. We demonstrated that whereas an augmented expression of HSP70 resulting from de novo synthesis in the RVLM plays a neuroprotective role in fatal endotoxemia; HSP90 is essentially not involved. We further showed that the cellular mechanisms that underlie this neuroprotective action of HSP70 include prevention of cardiovascular depression by an enhancement of the NOS I (or neuronal NOS)/protein kinase G (PKG) signaling pathway and an inhibition of the NOS II/peroxynitrite cascade in the RVLM.
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Materials and Methods |
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Recording and Power Spectral Analysis of Systemic Arterial Pressure Signals. Animals received cannulation of both femoral artery and vein and tracheal intubation under an initial pentobarbital sodium anesthesia (50 mg/kg, i.p.). They received thereafter intravenous infusion of propofol (Zeneca Pharmaceuticals plc, Macclesfield, UK) at 20 to 25 mg/kg/h. This scheme provided satisfactory anesthetic maintenance and preserved the capacity of central cardiovascular regulation (Yang et al., 1995). Systemic arterial pressure (SAP) signals recorded from the femoral artery were simultaneously subject to on-line power spectral analysis (Chan et al., 2001a,b, 2002b, 2004, 2005; Li et al., 2001; Chang et al., 2003). We were particularly interested in the very low-frequency (VLF; 0–0.25 Hz) and low-frequency (LF; 0.25–0.8 Hz) components of SAP signals. Our laboratory demonstrated previously (Kuo et al., 1997) that these spectral components of SAP signals take origin from the RVLM, and their power density reflects the prevailing sympathetic neurogenic vasomotor tone. Heart rate (HR) was derived instantaneously from SAP signals. The SAP spectra and power density of the two spectral components were displayed during the experiment, alongside pulsatile SAP, mean SAP (MSAP), and HR, in an on-line and real-time manner. During the recording session, body temperature of the animals was maintained at 37°C with a heating pad, and animals were allowed to breathe spontaneously with room air via the intubated trachea.
Experimental Endotoxemia. Escherichia coli lipopolysaccharide (LPS; serotype 0111:B4; Sigma-Aldrich, St. Louis, MO) was administrated intravenously over 1 to 2 min at 30 mg/kg (Chan et al., 2001a). Temporal changes in MSAP, HR, or power density of LF or VLF component of the SAP signals were routinely followed for 240 min, or until the animal succumbed to endotoxemia. The survival rate within 240 min was also recorded.
Collection of Ventrolateral Medullary Tissue Samples. As we reported previously (Chan et al., 2001a, 2004), the sequence of cardiovascular events during LPS-induced endotoxemia can be divided into three phases. At the peak of each of these phases of experimental endotoxemia (LPS group) or 30, 150, or 240 min after intravenous injection of saline (saline group), rats were perfused intracardiacally with 100 ml of warm (37°C) saline that contains heparin (100 U/ml). The brain was removed rapidly and placed on dry ice. Tissues on both sides of the ventrolateral part of medulla oblongata, at the level of RVLM (0.5–2.5 mm rostral to the obex), were collected and processed (Chan et al., 2001a,b, 2002b, 2004, 2005; Chang et al., 2003) for subsequent proteomic or Western blot analysis. Tissues obtained from animals that were anesthetized and received preparatory surgery served as our sham controls. Protein concentration was determined by the BCA protein assay (Pierce Biotechnology, Rockford, IL).
Proteomic Analysis. Proteomic analysis of proteins extracted from the ventrolateral medulla was carried out as detailed previously (Huang et al., 2002). The silver-stained two-dimensional electrophoresis gels in the domain of pI = 3 to 10 and Mr = 14.4 to 94 were scanned by an ImageScanner (Amersham Biosciences AB, Uppsala, Sweden). Protein spots were quantified and numbered using the ImageMaster 2D Elite software (Amersham Biosciences Inc.) and were checked manually to eliminate artifacts caused by gel distortion, abnormal silver staining, or poorly detectable spots. The protein level of each spot was expressed as a percentage of total spot volume in the two-dimensional electrophoresis gel. Protein spots of interests excised from corresponding Coomassie Brilliant Blue-stained gels were further subject to in-gel digestion and analyzed by MALDI-TOF mass spectrometry (Voyager-ED PRO; Applied Biosystems, Foster City, CA). To identify proteins, the measured monoisotopic masses of peptides were routinely analyzed using both MS-Fit (Protein Prospector; University of California San Francisco Mass Spectrometry Facility, San Francisco, CA) and MASCOT (Matrix Science, Boston, MA) search programs.
Western Blot Analysis. Western blot analysis (Chan et al., 2002a,b, 2004, 2005; Chang et al., 2003) was carried out on proteins extracted from the ventrolateral medulla for HSP70, HSP90, NOS I, NOS II, PKG, nitrotyrosine, an experimental index for peroxynitrite activity (Chan et al., 2002b; 2005), or 
-actin. The primary antisera used included mouse monoclonal antisera against HSP70, HSP90 (both from StressGen Biotechnologies, Victoria, BC, Canada), nitrotyrosine (Upstate Biotechnology, Lake Placid, NY), or -actin (Chemicon International, Temecula, CA); or rabbit polyclonal antisera against NOS I, NOS II (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA), or PKG (Calbiochem, San Diego, CA). The secondary antisera used included horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) for HSP70, HSP 90, nitrotyrosine, or -actin; or donkey anti-rabbit IgG (Amersham Biosciences UK, Ltd.) for NOS I, NOS II, or PKG. Specific antibody-antigen complex was detected by an enhanced chemiluminescence Western blot detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). The amount of protein was quantified by the ImageMaster software (Amersham Biosciences UK, Ltd.), and was expressed as the ratio relative to -actin protein.
Microinjection of Test Agents. Microinjection bilaterally of test agents into the RVLM was carried out stereotaxically and sequentially at a volume of 50 nl. The coordinates used were 4.5 to 5 mm posterior to lambda, 1.8 to 2.1 mm lateral to midline, and 8.1 to 8.4 mm below the dorsal surface of cerebellum (Chan et al., 2001a,b, 2002b, 2004, 2005; Chang et al., 2003). Test agents used included a transcription inhibitor (Fernando et al., 2000; Chang et al., 2004), actinomycin D (Tocris Cookson Inc., Bristol, UK); a translation inhibitor (Fernando et al., 2000; Chang et al., 2004), cycloheximide (Tocris Cookson); normal mouse serum (Sigma-Aldrich), mouse monoclonal antiserum against HSP70 or HSP90 (StressGen Biotechnologies); or sense, antisense, or scrambled oligonucleotide (Genemed Synthesis, South San Francisco, CA) against hsp70 (Robertson et al., 1999; Chan et al., 2004) or hsp90 (Zucchi et al., 2002) gene (Table 1). The dose and treatment regimen were adopted from previous reports (Fernando et al., 2000; Li et al., 2001; Zucchi et al., 2002; Chan et al., 2004; Chang et al., 2004) that used those test agents for the same purpose as in this study. Actinomycin D was prepared with 0.1% DMSO, and cycloheximide with 50% EtOH. We added 0.02% Triton X-100 (Sigma-Aldrich) to anti-HSP70 or -HSP90 antiserum to facilitate its transport across the cell membrane (Li et al., 2001; Chan et al., 2002a). All oligonucleotides were phosphorothioated in all positions and were diluted in artificial cerebrospinal fluid (aCSF) at pH 7.4. Microinjection of 0.1% DMSO, 50% EtOH, normal mouse serum plus Triton X-100 or aCSF served as our vehicle and volume control.
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Statistical Analysis. All values are expressed as mean ± S.E. The averaged value of MSAP or HR calculated every 20 min after administration of test agents or vehicle, the sum total of power density for LF or VLF component in the SAP spectra over 20 min, or the protein expression level in the ventrolateral medulla during each phase of experimental endotoxemia was used for statistical analysis. One-way or two-way analysis of variance with repeated measures was used, as appropriate, to assess group means, followed by the Scheffé multiple range test for post hoc assessment of individual means. p < 0.05 was considered statistically significant.
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Results |
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Three Phases of Cardiovascular Responses Are Present during Endotoxemia. As reported previously (Chan et al., 2001a), based on the decrease, increase, and a secondary decrease in the power density of the LF or VLF component in the SAP spectrum, the sequence of cardiovascular responses induced by intravenous administration of LPS (30 mg/kg) can be divided into three phases (Figs. 3 and 4). Both SAP and HR underwent typically a significant decrease and a rebound during phase I, to be followed by progressive hypotension or bradycardia during phases II and III endotoxemia.
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), the HSP70 expression in the ventrolateral medulla underwent a significant and progressive elevation during phases I and II endotoxemia, followed by a return to baseline during phase III (Fig. 5E). On the other hand, HSP90 expression remained relatively constant at all phases of experimental endotoxemia (Fig. 5F). The level of HSP70 or HSP90 in the RVLM of saline controls was stable and was comparable with sham controls. Western blot analysis using an antiserum that recognizes selectively the inducible form of HSP70, but not HSC70, or an anti-HSP90 antiserum that recognizes both HSP86 and HSP84, essentially confirmed these observations (Fig. 6). There was a progressive and significant augmentation of HSP70 expression in the ventrolateral medulla during phase I and phase II endotoxemia, followed by a decline toward baseline during phase III. The level of HSP90 again remained constant during the entire course of experimental endotoxemia.
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), died 160 to 170 min after the induction of endotoxemia; along with an antagonism of the augmented HSP70 expression in the ventrolateral medulla during phases I and II endotoxemia (Fig. 9). On the other hand, pretreatments with normal mouse serum (1:20) or sense or scrambled hsp70 oligonucleotide (50 pmol) were ineffective (Fig. 8A). Likewise, immunoneutralization using the same anti-HSP90 antiserum used in Western blot analysis, or gene knockdown using an antisense hsp90 oligonucleotide that acts selectively on HSP84 (Zucchi et al., 2002), in the RVLM, similar to normal mouse serum (1:20), or sense or scrambled hsp90 oligonucleotide (50 pmol), did not affect the LPS-induced mortality rate (Fig. 8B).
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The Augmented HSP70 Expression in the Ventrolateral Medulla Confers Cardiovascular Protection during Endotoxemia. We recently reported (Chan et al., 2004) that HSP70 induced in the RVLM by a prior hyperthermic heat shock confers cardiovascular protection during experimental endotoxemia. It thus is of interest to delineate whether the augmented HSP70 expression in the ventrolateral medulla during endotoxemia may also antagonize the LPS-induced cardiovascular depression, leading to our observed neuroprotection against fatality. Based again on a loss-of-function approach, we observed that pretreatment by microinjection into the bilateral RVLM of an anti-HSP70 antiserum (1:20; Fig. 3, left column) or an antisense hsp70 oligonucleotide (50 pmol; Fig. 3, right column) significantly potentiated the elicited hypotension or bradycardia and blunted the increase in power density of LF or VLF component of the SAP signals during the significantly shortened phase II endotoxemia. It is intriguing to note that, comparable with survival rate (Fig. 8), all these animals succumbed to fatal endotoxemia 140 to 160 min after LPS administration. Again, pretreatments with normal mouse serum (1:20; Fig. 3, left column) or sense or scrambled hsp70 oligonucleotide (50 pmol; Fig. 3, right column) were ineffective. Immunoneutralization or gene knockdown of HSP90 in the RVLM also did not affect the LPS-induced cardiovascular depressions (Fig. 4).
The Augmented HSP70 Expression in the Ventrolateral Medulla Enhances NOS I/PKG Signaling Pathway during Endotoxemia. We reported previously that the tonically active NOS I in the RVLM is responsible for the sympathoexcitatory cardiovascular actions of the endogenous NO at this medullary site (Chan et al., 2001b) and confers cardiovascular protection possibly via activation of the soluble guanylyl cyclase/cGMP/PKG cascade (Chan et al., 2005). It is thus intriguing to note that whereas the HSP70 level in the ventrolateral medulla underwent a progressive augmentation during phases I and II, and a decline toward baseline during phase III endotoxemia (Figs. 5 and 6), NOS I expression in the RVLM was found (Chan et al., 2001a) to be maintained until its significant reduction during phase III endotoxemia. Whether these temporally correlated changes in HSP70 and NOS I in the ventrolateral medulla are causally related was evaluated in the present study (Fig. 9). We first ascertained that the temporal changes of HSP70 and NOS I expression in the same sample of ventrolateral medulla indeed took place during experimental endotoxemia. Western blot analysis revealed that animals that received pretreatment via microinjection of aCSF bilaterally into the RVLM exhibited a progressive augmentation of HSP70 expression, alongside maintained NOS I or PKG level in the ventrolateral medulla during phases I and II, to be followed by a significant reduction in HSP70, NOS I, or PKG expression during phase III endotoxemia. Pretreating animals by microinjection bilaterally into the RVLM of an antisense hsp70 oligonucleotide (50 pmol) blunted significantly the LPS-induced surge in HSP70 level at the ventrolateral medulla. It is intriguing that this pretreatment also elicited a significant and progressive down-regulation of NOS I or PKG in the ventrolateral medulla that commenced at phase I endotoxemia (Fig. 9). With the exception of a significantly reduced baseline expression, pretreatment with an antisense hsp90 oligonucleotide (50 pmol) exerted no discernible effects on the temporal changes in HSP70, NOS I, or PKG levels in the ventrolateral medulla during endotoxemia. Likewise, pretreatments with sense or scrambled hsp70 or hsp90 oligonucleotide (50 pmol) were ineffective against NOS I or PKG level (data not shown), and antisense hsp70 oligonucleotide (50 pmol) pretreatment did not affect HSP90 expression (Fig. 9).
The Augmented HSP70 Expression in the Ventrolateral Medulla Also Inhibits NOS II/Peroxynitrite Cascade during Endotoxemia. We reported previously that experimental endotoxemia is accompanied by a progressive augmentation in both molecular synthesis and functional expression of NOS II (Chan et al., 2001a), followed by the formation of the cytotoxic substance peroxynitrite via a reaction between NO and superoxide anion (Chan et al., 2002b) in the RVLM. Subsequent experiments (Chan et al., 2004) showed that HSP70 induced in the RVLM by a prior heat shock confers cardiovascular protection by down-regulating NOS II expression in this medullary site. It thus is of interest to delineate whether the augmented HSP70 expression in the ventrolateral medulla during endotoxemia may also antagonize the LPS-induced up-regulation of NOS II. Figure 9 demonstrated that, in animals that were pretreated by microinjection of an antisense hsp70 oligonucleotide (50 pmol) into the bilateral RVLM, the progressive elevations of NOS II or nitrotyrosine (marker for peroxynitrite) in the ventrolateral medulla over the course of experimental endotoxemia was significantly potentiated. Again, pretreatment with an antisense hsp90 oligonucleotide (50 pmol) exerted no discernible effects on the temporal changes in HSP70, NOS II, or nitrotyrosine levels in the ventrolateral medulla during experimental endotoxemia. Likewise, pretreatments with sense or scrambled hsp70 or hsp90 oligonucleotide (50 pmol) were ineffective against NOS II or nitrotyrosine level (data not shown), and antisense hsp70 oligonucleotide (50 pmol) pretreatment did not affect HSP90 expression (Fig. 9).
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Discussion |
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One notion derived from the conflicting role of HSP70 or HSP90 in apoptosis (Galea-Lauri et al., 1996; Meriin et al., 1998; Robertson et al., 1999; Wagstaff et al., 1999; Lee et al., 2001; Lopez-Maderuelo et al., 2001) is that whether an HSP family member is pro- or antiapoptotic depends on the cell type or the stressor. The present study provided credence to this notion by demonstrating a differential neuroprotective role for HSP70 and HSP90 in the RVLM against fatal endotoxemia. We found that whereas both HSPs are present in the proteomic map of the ventrolateral medulla, complementary data from proteomic and Western Blot analyses revealed that only HSP70 manifested a progressive augmentation in its expression level during phases I and II endotoxemia. More importantly, despite its short duration (160–180 min), the induced HSP70 expression in the RVLM conferred neuroprotection against fatal endotoxemia. Our results showed that functional blockade of HSP70 in the RVLM by immunoneutralization using an anti-HSP70 antiserum that recognizes selectively the inducible form of HSP70 (Chan et al., 2002a), or prevention of synthesis by gene knockdown using an antisense hsp70 oligonucleotide that acts selectively on the hsp70 gene (Robertson et al., 1999), exacerbated mortality and potentiated the cardiovascular depression during experimental endotoxemia. It is intriguing to note that augmentation of HSP70 expression in the ventrolateral medulla that peaks 24 h after exposing animals to a brief hyperthermic heat shock is also causally and temporally related to antagonism of the circulatory suppression during endotoxemia (Chan et al., 2004). Together, these observations indicated that regardless of the means of induction or the duration of expression, an augmented HSP70 level in the ventrolateral medulla confers neuroprotection against fatal endotoxemia via prevention of cardiovascular depression.
That HSP90 did not play a neuroprotective role in our model of endotoxemia may not be confounded by the nonselectivity of the anti-HSP90 antiserum or antisense hsp90 oligonucleotide used is supported by two observations. First, both proteomic and Western blot analyses demonstrated that HSP90 expression in the ventrolateral medulla remained constant during the course of experimental endotoxemia. Second, pretreatment with an anti-HSP90 antiserum that recognizes both HSP86 and HSP84, or an antisense hsp90 oligonucleotide that knocks down selectively HSP84 (Zucchi et al., 2002), produced complementary results on cardiovascular depression or mortality during fatal endotoxemia.
Our observations with actinomycin D or cycloheximide pretreatment showed that the elevated HSP70 expression during phases I and II was the result of de novo synthesis. The ability of a cell to rapidly change its gene expression pattern in response to extracellular signals usually involves modulation of the activity of preexisting transcription factors, a major mechanism of which is protein phosphorylation (Hunter, 2000). In this regard, heat shock transcription factor 1 (HSF1) is known to regulate hsp induction in response to stress, and HSP70 expression is attributable to phosphorylation of HSF1 at serine 230 (Holmberg et al., 2001, Pirkkala et al., 2001). Overexpression of HSP70 inhibits phosphorylation of HSF1 in the nucleus at serine residues (Mosser et al., 1993; Pirkkala et al., 2001). This negative feedback mechanism, which was proposed to explain the well established reduction in HSP70 expression after repetitive exposure of cells or animals to heat shock (Mosser et al., 1993), may also account for our observation that HSP70 expression returned to baseline during phase III endotoxemia.
Our laboratory demonstrated recently (Chang et al., 2003) that whereas all three NOS isoforms are expressed in the ventrolateral medulla at both mRNA and protein levels, only NOS I and II are present in RVLM neurons. Furthermore, physiological regulation of sympathetic vasomotor outflow by the endogenous NO at the RVLM is determined by a balance between the tonically active NOS I and NOS II (Chan et al., 2001b). We propose that, under physiological conditions, the prevalence of NOS I over NOS II activity at the RVLM and the associated dominance of sympathoexcitation over sympathoinhibition underlie the maintenance of sympathetic vasomotor outflow and stable SAP by the endogenous NO in the RVLM (Chan et al., 2001b). On the other hand, a tilt toward the progressive augmentation in molecular synthesis and functional expression of NOS II in the RVLM underlies the cardiovascular depression seen during pathological conditions such as endotoxemia (Chan et al., 2001a). It is therefore intriguing that the present study demonstrated that HSP70 may confer neuroprotection by regulating cardiovascular functions during endotoxemia via modulating NOS I or II expression in the RVLM.
Low concentration of NO generated by NOS I in the RVLM increases sympathetic vasomotor outflow (Chan et al., 2001b) by exciting sympathetic premotor neurons in this medullary site via a cGMP/PKG-dependent facilitation of presynaptic glutamate release (Huang et al., 2003). It is therefore interesting to observe that the progressive augmentation of HSP70 expression during phases I and II endotoxemia was associated with maintained NOS I or PKG level in the ventrolateral medulla, alongside an increase in vasomotor tone during phase II. Significant reduction in NOS I or PKG expression took place only during phase III when HSP70 returned to baseline level and when significant and severe cardiovascular depression ensued. Results from antisense hsp70 oligonucleotide pretreatment further confirmed that these temporally correlated cellular (HSP70, NOS I, or PKG expression; Fig. 9) and cardiovascular events (Fig. 3) are causally related. It follows that the up-regulated HSP70 may confer neuroprotection against fatal endotoxemia by preventing cardiovascular depression via enhancement of the NOS I/PKG signaling pathway in the RVLM. The potential candidates whereby HSP70 exerts this regulatory effects on NOS I gene expression include the Oct-2 transcription factor (Deans et al., 1996) and members of the Sp and ZNF families of transcription factors (Saur et al., 2002).
High concentration of NO generated by NOS II decreases sympathetic vasomotor outflow (Chan et al., 2001b) by inhibiting RVLM neurons via a peroxynitrite-mediated reduction of presynaptic glutamate release (Huang et al., 2004). It is intriguing that the present study showed that whereas the HSP70 level returned to baseline during phase III endotoxemia, the NOS II and nitrotyrosine levels were further enhanced, alongside significant reduction in SAP, HR, or sympathetic vasomotor outflow. Results from our antisense hsp70 oligonucleotide pretreatment again confirmed that these temporally correlated cellular (HSP70, NOS II, or nitrotyrosine expression; Fig. 9) and cardiovascular events (Fig. 3) are causally related. HSP70 inhibits NOS II gene expression by transcriptional mechanisms that involve the nuclear factor-
B/inhibitory B pathway (Feinstein et al., 1996; Chan et al., 2004). HSP70 also protects macrophages against peroxynitrite cytotoxicity (Hirvonen et al., 1996; Szabo et al., 1996). It follows that the up-regulated HSP70 may also confer neuroprotection by preventing cardiovascular depression via holding NOS II expression and peroxynitrite formation at the RVLM in check. The return of HSP70 level to baseline, the reduction in NOS I/PKG expression and augmentation of the NOS II/peroxynitrite level in the RVLM, the significant circulatory depression and the ensuing fatality during phase III endotoxemia, together with the exacerbated mortality and potentiated cardiovascular depression by loss-of-function manipulations of HSP70, provided ample credence to this notion.
Brain death is associated with the permanent termination of essentially all brain functions, particularly the autonomic cardiovascular regulatory mechanisms in the brain stem (Anonymous, 1981). It is thus intriguing that the neuroprotective action of HSP70 against fatal endotoxemia was elicited on the RVLM, a neural substrate that is intimately related to sympathetic vasomotor tone (Ross et al., 1984) and whose neuronal activity is closely associated with a "life-and-death" signal (Kuo et al., 1997) that is drastically reduced or lost before patients succumbed to systemic inflammatory response syndrome (Yien et al., 1997). Because death represents the end of existence for an individual organism, we propose that multiple "pro-life" and "pro-death" programs must be engaged during the progression toward brain stem death. The present study provided novel findings to indicate that HSP70 in the RVLM may be one of those pro-life programs. We demonstrated that de novo synthesis of HSP70 in the RVLM during experimental endotoxemia confers neuroprotection against fatality by preventing cardiovascular depression via enhancing the NOS I/PKG signaling pathway and inhibiting the NOS II/peroxynitrite cascade in this crucial neural substrate. We also showed that, although present in the RVLM, HSP90 does not seem to play a protective role in endotoxemia. This information should provide further insights on the etiology of brain stem death, and offer new directions for the development of therapeutic strategy against sepsis.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: HSP, heat shock protein; HSP70, 70-kDa heat shock protein; HSP90, 90-kDa heat shock protein; NOS, nitric-oxide synthase; RVLM, rostral ventrolateral medulla; PKG, protein kinase G; SAP, systemic arterial pressure; VLF, very low-frequency; LF, low-frequency; HR, heart rate; MSAP, mean systemic arterial pressure; LPS, lipopolysaccharide; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; DMSO, dimethyl sulfoxide; aCSF, artificial cerebrospinal fluid; HSF1, heat shock transcription factor 1.
Address correspondence to: Dr. Alice Y. W. Chang, Center for Neuroscience, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, Republic of China. E-mail: achang{at}mail.nsysu.edu.tw
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