Protective effect of heat shock proteins: potential for gene therapy

I. Introduction

amino acid identity with the corresponding yeast proteinIt is now over forty years since Ritossa observed that

and 78% with the Drosophila protein (Rebbe et al, 1987).exposure of the larval salary gland of Drosophila to

The various hsps and their characteristics are listed inelevated temperature resulted in the appearance of new

Table 1. puffs in the giant chromosomes of these cells (Ritossa,

Although originally identified on the basis of their1962). It is now clear that these puffs represent the

induction by elevated temperature and therefore named thetranscriptional induction of specific genes which encode a

heat shock proteins, these proteins are in fact induced by agroup of proteins known as the heat shock proteins (for

wide range of stimuli which are potentially damaging toreview see Lindquist and Craig, 1988; Parsell and

the cell. Such inducers include infections with a wide Lindquist, 1993).

variety of different viruses (Collins and Hightower, 1982;Although originally demonstrated in Drosophila, the

Khandijan and Turler, 1983; La Thangue and Latchman,induction of a small number of heat shock proteins by

1988), treatment with ethanol (Plesset et al, 1982), steroidelevated temperature, is observed in all organisms studied

hormones (Norton and Latchman, 1989), and amino acidranging from prokaryotic bacteria to mammals including

analogues (Li and Laszlo, 1985). Interestingly, hsps are man. Moreover, this evolutionary conservation extends not

also induced by processes which may occur during humanonly to the existence of the heat shock response in

disease, notably exposure of specific cells such as cardiacdifferent organisms but also to the induced proteins

or neuronal cells to ischaemia or elevated levels of free themselves which are very similar to one another in very

radicals (Nowak, 1985; Polla, 1988).different organisms. Thus, the best characterised hsps,

The strong evolutionary conservation of the hsps orhsp90, hsp70, hsp65 and hsp27 (each hsp is named

stress proteins which was discussed above and theiraccording to its mass in kilodaltons) are induced in

induction by a variety of stressful stimuli, indicates thatresponse to heat in all organisms studied from bacteria to

they are likely to have some critical function in the cellular response to stress. Interestingly, however, many of these proteins are also synthesised by normal unstressed cells with their synthesis being further enhanced upon exposure to stress. For example, hsp90 is one of the most abundant proteins in unstressed cells, constituting approximately 1% of the total protein in mammalian cells even prior to exposure to stress. This has led to the idea that the function of the hsps is one which is required in normal cells but is needed to an even greater extent in stressed cells. This idea is in accordance with the detailed functional studies of individual hsps which, as shown in Table 1, have indicated that a number of them have a role in ensuring the correct protein folding of other proteins within the cells, acting as so-called “molecular chaperones” (for review see Ellis, 1990). Thus, for example, hsp90 associates with the steroid receptors, such as the glucocorticoid receptor and keeps them in an inactive form located in the cytoplasm prior to exposure to steroid. Upon steroid treatment, hsp90 dissociates from the receptor which then can move to the nucleus and activate steroid responsive genes.

The idea of the hsps as proteins which are of importance in normal cells but which assume a greater significance in stressed cells, leads logically to the idea that the induction of these proteins by a stressful stimulus is of itself important in assisting the cell to protect itself from stress. In turn, this leads to the idea that the prior induction of the hsps by a mild stress or by some other non-stressful procedure, would be protective against subsequent more severe stress. This idea obviously has considerable medical importance and has therefore been intensively investigated.

Over the years, this work has effectively proceeded in three stages. Firstly, the demonstration that exposure to mildly stressful stimuli which can induce hsp expression, can in turn protect cells against exposure to a more severe stress. Clearly, such findings implicate the hsps as being protective but do not prove this, since the protective effect could be due to some other action of the mildly stressful treatment, other than its ability to induce the hsps. This idea leads directly to the second stage of these investigations, namely, the use of gene constructs to over-express the hsps in cultured cells and then demonstrate a protective effect against subsequent exposure to stress. Finally, more recently, these experiments in cultured cells have been complemented by experiments over-expressing the hsps in an intact animal and again demonstrating a protective effect. In subsequent sections of this review, I will discuss these three stages of work on the protective effect of heat shock proteins, focusing on studies involving neuronal or cardiac cells in culture or in the intact brain and heart, because of the key medical importance of these organs.

II. Protective effect of stimuli which induce hsp synthesis

During the 1980s, a very large number of studies demonstrated that, in cells in culture, stimuli which induced hsp synthesis such as a mild stress resulted in protection against subsequent exposure to a more severe stress. Moreover, it was also demonstrated that the levels of the hsps induced by such mildly stressful procedures, generally correlated with the level of protection which was observed against the subsequent more severe stress (for review see Lindquist and Craig, 1988; Parsell and Lindquist, 1993).

Following such early studies primarily carried out in cell lines of fibroblast origin, this work was extended also by carrying out similar studies both in cell lines of neuronal origin and in primary neuronal cells. Thus, for example, primary neuronal cells cultured in vitro are protected by exposure to mild heat or ischaemic stress from a subsequent more severe heat or ischaemic stress or exposure to the excitotoxin glutamate (Lowenstein et al, 1991; Rordorf et al, 1991; Amin et al, 1995). Indeed, in our own studies, the degree of protection afforded by an initial mild stress correlated with the amount of hsp induced, rather than the nature of the subsequent stress. Thus, a mild heat stress produced a better protective effect against subsequent severe heat stress or severe ischaemia, than was observed for a mild ischaemic stress correlating with the greater degree of hsp induction produced by the mild heat stress (Amin et al, 1995).

These in vitro studies examining the protective effect of a mild stress against a more severe stress, have also been supplemented by examining the protective effect of such mild stresses against exposure to stimuli which induce apoptosis (programmed cell death). Thus, it has been shown that prior mild heat shock can protected the ND7 neuronal cell line against apoptosis induced by serum withdrawal and addition of retinoic acid (Mailhos et al, 1993). Similarly, primary neonatal dorsal root ganglion cells are protected by mild heat shock against subsequent withdrawal of nerve growth factor, which induces apoptosis in these neurones (Mailhos et al, 1994).

These studies demonstrating a protective effect in the heart on a Langerdorff perfusion apparatus following prior exposure to elevated temperature in vivo, lead naturally to the question of whether a similar protective effect would be observed in hearts exposed to myocardial ischaemia within the intact animal following a prior exposure to heat shock.

Donnelly et al, (1992) demonstrated that this was indeed the case with an effective reduction of infarct size being observed when rat hearts were exposed to 35 minutes of left coronary artery occlusion in the intact animal following exposure to heat shock. Moreover, this protective effect is not confined to the use of heat shock itself to induce the hsps. Thus, Marber et al, (1993) were able to demonstrate that four brief periods (5 minutes each) of cardiac ischaemia were able to induce hsp synthesis and were also able to reduce infarct size when the hearts were subsequently exposed to 30 minutes of ischaemia in the intact animal.

Hence, stimuli which result in hsp induction in the intact heart in vivo can produce a protective effect against subsequent exposure of the heart to ischaemia/reperfusion either on a perfusion apparatus or within the intact animal. In addition, a number of studies have demonstrated that the protective effect correlates with the amount of heat shock protein which is induced. Thus, for example, Marber et al, (1994) showed a correlation between the amount of hsp70 produced by heat stress of papillary muscle and the muscle’s ability to recover function following a period of hypoxia. Similarly, Hutter et al, (1994) demonstrated a similar correlation between the amount of hsp70 and the ability to limit infarct size following exposure of the heart to ischaemia and subsequent reperfusion.

These studies indicate therefore, that neuronal and cardiac cells can be protected by prior exposure to a mild stress sufficient to induce hsp over-expression. Moreover, the correlation between the amount of hsp induced and the degree of protection observed, suggests that it is the induction of the hsp rather than some other effect of the mild stress, which produces the protective effect against the more severe stress. However, such studies are essentially only correlative and to prove that hsps can have a protective effect, it is necessary to over-express individual hsps in neuronal or cardiac cells. Such studies are discussed in the next section.

III. Protective effect of individual hsps in neuronal and cardiac cells in vitro

In a number of cases, it has been possible to show that over-expression of an individual hsp can provide a protective effect against damaging stimuli, in the same manner as a mild hsp-inducing stress. Thus, for example, dorsal root ganglion neurones can be protected against thermal or ischaemic stress by over-expression of either hsp70 or hsp90 (Uney et al, 1993; Amin et al, 1996; Wyatt et al, 1996) and a similar effect of hsp70 and hsp90 has been observed in the ND7 neuronal cell line (Mailhos et al, 1994). Similarly, Fink et al, (1997) were able to protect cultured hippocampal neurones against subsequent heat shock using a herpes simplex virus (HSV)-derived amplicon vector expressing hsp70, indicating that this effect applies to neurones derived from both the central and the peripheral nervous systems.

However, in further experiments we were able to show that the protective effect of a mild heat stress against apoptotic stimuli in neuronal cells could be reproduced by over-expressing the small heat shock protein hsp27. Thus, over-expression of hsp27 using an herpes simplex virus (HSV-)based vector was able to protect both ND7 cells and DRG neurones against apoptosis induced by withdrawal of serum or nerve growth factor, whereas such protection was not observed when hsp70 was over-expressed with a similar vector (Wagstaff et al, 1999) (Figure 1a). As expected, over-expression of either hsp27 or hsp70 by this means was able to protect the neuronal cells against subsequent exposure to heat shock or ischaemia, paralleling the results obtained with plasmid constructs for hsp70 and extending this to hsp27 (Wagstaff et al, 1999).

These findings also suggest that hsp27 may be as protective as hsp70 in cardiac cells whilst potentially being more protective in neuronal cells. Similar results were also obtained by Martin et al, (1997; 1999) who used an adenovirus vector to over-express hsp27 or the related protein αB-crystallin in cardiac cells. They were able to demonstrate that both these proteins were able to protect cardiac myocytes from the effect of simulated ischaemia and that decreasing the level of endogenous hsp27 using an antisense approach enhanced the damaging effects of a subsequent ischaemic stimulus.

Taken together therefore, these results demonstrate that several hsps can play an important role in protecting cells in culture from the effects of damaging stimuli. They therefore raise the possibility that the ultimate use of hsps in therapeutic procedures (see below) may be optimised by stimulating the over-expression of more than one hsp to produce an optimal protective effect. This is reinforced by the findings of Lau et al (1997) who were unable to demonstrate any protective effect of over-expressing hsp60 or hsp10 individually in cultured cardiac cells but did observe a protective effect when both proteins were over-expressed together.

Hence, these studies do clearly demonstrate that over-expression of individual hsps can protect cultured neuronal or cardiac cells against different death-inducing stimuli, extending the results with prior mild hsp-inducing stresses. Moreover, they demonstrate that the protective effect of individual hsps may vary with the cell type being investigated and the nature of the stress being used and also, provide the first suggestion that hsp27 may have a more potent protective effect than hsp70 in the nervous system. These findings reinforce the need to extend these in vitro studies on over-expression of the hsps to their over-expression in the intact animal. Such studies are discussed in the next section.

IV. Protective effect of hsps in vivo

In keeping with the strong focus in the hsp field on the major inducible hsp, hsp70, transgenic animals over-expressing this hsp were reported by several groups in 1995-96 (Marber et al, 1995; Plumier et al, 1995; Radford et al, 1996). In their initial analysis of these mice, all these groups focused primarily on the potential protective effect of hsp70 in the heart. In all cases, they were able to demonstrate that such over-expression of hsp70 was able to protect the heart against the damaging effects of ischaemia using a variety of assays such as infarct size, creatine kinase release, recovery of high-energy phosphate stores and correction of metabolic acidosis. Moreover, in a subsequent study, it was demonstrated that such a protective effect could also be observed against myocardial dysfunction caused by a brief ischaemia which was insufficient to produce an infarct (Trost et al, 1998).

These studies thus establish for the first time, that the over-expression of a single hsp in vivo in the intact animal is sufficient to protect a specific organ, namely the heart, against the damaging effects of a stressful stimulus. In view of the clear evidence demonstrating that there is also a protective effect for the hsps in neuronal cells in vitro (see above), it is not surprising that these hsp70 transgenic animals were rapidly used in attempts to demonstrate that over-expression of hsp70 would also protect the brain of the intact animal from specific stresses. In general, however, the results of these studies have been far more equivocal than the corresponding studies in the heart, with protection being observed against some insults but not against others (Plumier et al, 1997; Rajdev et al, 2000; Lee et al, 2001). Thus, for example, Lee et al, (2001) found that one of the hsp70 transgenic mouse strains showed no reduction of infarct size or enhanced survival of neuronal cells following cerebral ischaemia and similar results were also obtained using a different strain by Plumier et al, (1997) in terms of infarct size and striatal neurone survival, although they did observe enhanced survival of hippocampal neurones.

Hence, hsp27 over-expression has a clear potent protective effect in the nervous system in terms of kainate toxicity, both at the level of whole animal seizure activity and survival and at the level of vulnerable neurones within the hippocampus (Akbar et al, 2003). It will evidently be of considerable interest to extend this study by determining whether hsp27 can have protective effects against other damaging stimuli in the nervous system including, for example, cerebral ischaemia. It will also be of interest to conduct a detailed study of the hsp27 and hsp70 over-expressing transgenic animals, to compare the potency of these different heat shock proteins against various damaging stimuli in both the heart and the brain and to confirm or deny the suggestion that hsp27 may be more potent than hsp70 in protecting the nervous system. It is already clear however, that hsp27 does have a clear protective effect in the nervous system in vivo as well as in vitro. This reinforces the need to conduct studies using different individual hsps and investigating their protective effects, rather than simply focusing on the major heat shock protein hsp70.

V. Therapeutic potential of hsps

The experiments described in earlier sections, clearly suggest that procedures which elevate hsp levels in the heart or the brain may be of significant benefit, for example, during reperfusion following a period of ischaemia, or in patients undergoing neuronal cell loss due to neurodegenerative diseases such as Alzheimer’s or Parkinson’s diseases. Similarly, elevation of hsp levels may be beneficial during cardiac bypass or to preserve donor heart function prior to transplantation. Indeed, in view of the use of cold storage during transportation prior to transplantation, it is of particular interest that reduced as well as elevated temperature induces hsp expression in the heart (Laios et al, 1997). Moreover, a mild heat treatment prior to hypothermic storage has been shown to enhance subsequent recovery of the heart (Gowda et al, 1998a).

Such temperature-based manipulations of the hsps may thus have a role to play in cardiac transplantation procedures. Similarly, it has been shown that a protective effect across the whole heart can be obtained by using a thermal probe to produce local heating of the heart (Gouda et al, 1998b) suggesting that a similar procedure could be used therapeutically.

The induction of the hsps by stressful stimuli such as elevated temperature (for review see Morimoto, 1998) or ischaemia (Nishizawa et al, 1999) is mediated by the heat shock transcription factor (HSF-1). Thus following exposure to elevated temperature, the cytoplasmic HSF-1 monomer, forms a trimer and moves to the nucleus where it binds to its target sites (known as heat shock elements) in the regulatory regions of the hsp genes and, following HSF-1 phosphorylation, it induces hsp gene expression.

Interestingly, the induction of the hsps by stressful stimuli diminishes with age in a variety of tissues including the heart and this has been shown to be due to impaired activation of HSF-1 by stress in the aged heart (Locke and Tanguay, 1996). Moreover, this effect is associated with a reduced protective effect of mild heat shock or ischaemia against a subsequent severe ischaemic stress in aged hearts (Locke and Tanguay, 1996; Fenton et al, 2000). As many of the situations where the protective effect of hsps would be valuable involve elderly individuals, this suggest that other procedures not involving stressful stimuli or HSF-1 may be required for the therapeutic induction of the hsps.

Although the hsps were identified on the basis of their induction by stressful procedures, they are also induced naturally by specific non-stressful procedures (for review see Latchman, 1998). For example, the cytokines interleukin-6 (Stephanou et al, 1997) and interleukin-10 (Ripley et al, 1999) can induce hsp gene expression in a non-stressful manner. It has been shown that these inducers do not act via HSF-1 but activate hsp expression via other transcription factors such as NF-IL6 and STAT3 (for review see Stephanou and Latchman, 1999). Based on the use of these inducers in non-cardiac cells, we demonstrated that the interleukin-6-like cytokine cardiotrophin-1 (CT-1) was able to induce hsp synthesis in cultured cardiac cells and that such treatment protects them against subsequent exposure to severe thermal or ischaemic stress (Stephanou et al, 1998). Similar induction of the hsps in cultured cardiac cells and protection against subsequent severe stress is also observed with the tyrosine kinase inhibitor herbimycin A (Morris et al, 1996; Conde et al, 1997).

These protective effects in cardiac cells in culture have also been extended to the intact heart. For example, Vigh et al, (1997) showed that bimoclomol, a novel hydroxylamine derivative was able to induce hsp synthesis in the intact perfused heart ex vivo and to produce a protective effect against a subsequent ischaemia. Similarly, Meng et al, (1996) demonstrated that norepinephrine treatment of an intact rat resulted in hsp induction in the heart and protection against ischaemia when the heart was subsequently perfused ex vivo.

Several compounds thus exist which can induce hsps and produce a protective effect, although it should be note that in no case has this protective effect been directly shown to be due to the ability to induce hsps. Before the protective effect of any of these compounds could be exploited clinically, it is evidently necessary to investigate whether their protective effect in the heart can be achieved without any significant side-effects. For example, CT-1 was originally identified on the basis of its ability to induce cardiac hypertrophy (Pennica et al, 1995) whilst herbimycin as a tyrosine kinase inhibitor is likely to have significant effects on cellular growth and division.

factor, HSF1, which should induce a range of hsps. When

an HSV vector expressing this HSF1 mutant was used to

infect neuronal cells, it induced accumulation of hsp70 but

not of hsp27 and therefore, did not result in protection

against apoptosis, although it was able to produce

protection against heat shock and ischaemia (Wagstaff et

al, 1998). Nonetheless, the clear protective effect of the hsps in

the cardiac and nervous systems offers hope for

therapeutic procedures which enhance endogenous or

exogenous hsp expression.

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