GTMB 7 - Gene Therapy & Molecular Biology

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Latchman: Protective effect of heat shock proteinsproduce a protective effect against subsequent exposure toischaemia/reperfusion. These results have subsequentlybeen extended both by examining other parameters ofheart function and by using other species such as the rabbit(Karmazyn et al, 1990; Yellon et al, 1992) (for review seeYellon and Latchman, 1992).These studies demonstrating a protective effect in theheart on a Langerdorff perfusion apparatus following priorexposure to elevated temperature in vivo, lead naturally tothe question of whether a similar protective effect wouldbe observed in hearts exposed to myocardial ischaemiawithin the intact animal following a prior exposure to heatshock.Donnelly et al, (1992) demonstrated that this wasindeed the case with an effective reduction of infarct sizebeing observed when rat hearts were exposed to 35minutes of left coronary artery occlusion in the intactanimal following exposure to heat shock. Moreover, thisprotective effect is not confined to the use of heat shockitself to induce the hsps. Thus, Marber et al, (1993) wereable to demonstrate that four brief periods (5 minuteseach) of cardiac ischaemia were able to induce hspsynthesis and were also able to reduce infarct size whenthe hearts were subsequently exposed to 30 minutes ofischaemia in the intact animal.Hence, stimuli which result in hsp induction in theintact heart in vivo can produce a protective effect againstsubsequent exposure of the heart to ischaemia/reperfusioneither on a perfusion apparatus or within the intact animal.In addition, a number of studies have demonstrated thatthe protective effect correlates with the amount of heatshock protein which is induced. Thus, for example,Marber et al, (1994) showed a correlation between theamount of hsp70 produced by heat stress of papillarymuscle and the muscle’s ability to recover functionfollowing a period of hypoxia. Similarly, Hutter et al,(1994) demonstrated a similar correlation between theamount of hsp70 and the ability to limit infarct sizefollowing exposure of the heart to ischaemia andsubsequent reperfusion.These studies indicate therefore, that neuronal andcardiac cells can be protected by prior exposure to a mildstress sufficient to induce hsp over-expression. Moreover,the correlation between the amount of hsp induced and thedegree of protection observed, suggests that it is theinduction of the hsp rather than some other effect of themild stress, which produces the protective effect againstthe more severe stress. However, such studies areessentially only correlative and to prove that hsps can havea protective effect, it is necessary to over-expressindividual hsps in neuronal or cardiac cells. Such studiesare discussed in the next section.III. Protective effect of individual hspsin neuronal and cardiac cells in vitroIn a number of cases, it has been possible to showthat over-expression of an individual hsp can provide aprotective effect against damaging stimuli, in the samemanner as a mild hsp-inducing stress. Thus, for example,dorsal root ganglion neurones can be protected againstthermal or ischaemic stress by over-expression of eitherhsp70 or hsp90 (Uney et al, 1993; Amin et al, 1996; Wyattet al, 1996) and a similar effect of hsp70 and hsp90 hasbeen observed in the ND7 neuronal cell line (Mailhos etal, 1994). Similarly, Fink et al, (1997) were able to protectcultured hippocampal neurones against subsequent heatshock using a herpes simplex virus (HSV)-derivedamplicon vector expressing hsp70, indicating that thiseffect applies to neurones derived from both the centraland the peripheral nervous systems.Similar studies on the protective effect of the hsps incardiac cells, initially focused on hsp70 and utilised theH9c2 cell line which was derived initially from the ratheart. In 1994, two groups reported the results ofexperiments in which stable transfection was used toproduce clonal cell lines derived from H9c2 whichconstitutively over-expressed hsp70 (Heads et al, 1994;Mestril et al, 1994). These cells were shown to beprotected against subsequent exposure to thermal orischaemic stress compared to control cells which did notover-express hsp70. These studies were subsequentlyextended by Cumming et al, (1996b) who demonstratedthat similar protective effects against heat stress orsimulated ischaemia could be observed when hsp70 wasover-expressed by transfection of primary rat cardiacmyocyte cultures, demonstrating that this protective effectcould be observed both in primary cardiac cells and in celllines derived from them. A similar protective effect wasalso observed when hsp70 was over-expressed bytransfection in coronary endothelial cells (Suzuki et al,1998) indicating that hsp70 can protect these cells as wellas cardiac myocytes. This is of particular interest since ithas been shown that when the heart is exposed to elevatedtemperature in vivo, hsp70 induction occurs primarily inendothelial cells rather than in cardiac myocytes (Amraniet al, 1998; Leger et al, 2000).To extend these experiments to other hsps,transfection methods were used to over-express hsp90,hsp65, or hsp56 either in the H9c2 cell line (Heads et al,1995) or in cultured primary cardiac cells (Cumming et al,1996a,b). In these experiments, hsp90 over-expression wasable to protect the cells against subsequent thermal stressbut not against subsequent simulated ischaemia whereashsp65 or hsp56 had no protective effect. Since hsp70 overexpressionprotected against both thermal or simulatedischaemic stress in these experiments, these studiesindicate that different hsps can have different protectiveeffects and need to be tested individually for theirprotective effect in any specific situation.This idea is reinforced by findings in neuronal cellswhere the over-expression of an individual hsp does notalways reproduce the protective effect of a mild hspinducingstress. Thus, over-expression of hsp70 or hsp90in ND7 cells (Mailhos et al, 1994) or DRG neurones(Wyatt et al, 1996) does not reproduce the protectiveeffect of mild heat shock against subsequent apoptoticstimuli. Similarly, in the experiments of Fink et al, (1997)over-expression of hsp70 with an hsp vector did notprotect hippocampal neurones against glutamate toxicitywhich may act by inducing apoptosis (Kure et al, 1991),despite the fact that previous studies demonstrated a clear248
Gene Therapy and Molecular Biology Vol 7, page 249protective effect of mild heat stress against subsequentexposure to glutamate (Lowenstein et al, 1991; Rordorf etal, 1991).However, in further experiments we were able toshow that the protective effect of a mild heat stress againstapoptotic stimuli in neuronal cells could be reproduced byover-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 cellsand DRG neurones against apoptosis induced bywithdrawal of serum or nerve growth factor, whereas suchprotection was not observed when hsp70 was overexpressedwith a similar vector (Wagstaff et al, 1999)(Figure 1a). As expected, over-expression of either hsp27or hsp70 by this means was able to protect the neuronalcells against subsequent exposure to heat shock orischaemia, paralleling the results obtained with plasmidconstructs for hsp70 and extending this to hsp27 (Wagstaffet al, 1999).Interestingly, when these experiments with HSVvectors over-expressing individual hsps were used todetermine their protective effect in primary cardiac cells(Brar et al, 1999), we confirmed our earlier results thathsp70 over-expression can protect cardiac cells againstsimulated ischaemia or thermal stress, whereas overexpressionof hsp56 has no such protective effect.Moreover, we were able to extend these studies byshowing firstly, that hsp70 can protect against theinduction of apoptosis (programmed cell death) in cardiaccells by exposure to ceramide, whereas hsp56 has noprotective effect and secondly, to demonstrate that overexpressionof hsp27 (which we had not previously tested)similarly protects cardiac cells against subsequentexposure to thermal or ischaemic stress or to ceramide(Figure 1b). Hence, in cardiac cells both hsp70 and hsp27can protect against apoptosis whereas in neuronal cellsonly hsp27 has this protective effect. This reinforces theneed to study individual hsps for their protective effectagainst specific stimuli and in specific cell types.Figure 1. (A) Number of DRG neurones undergoing apoptosis (as assayed by TUNEL staining) after NGF withdrawal following priorinfection with the indicated virus. Values are the mean of three determinations whose standard error is shown by the bars. Significantenhancement of survival (p < 0.05) was observed only with hsp27-expressing virus. (B) Percentage of apoptotic cells (as assayed byTUNEL staining) in cardiomyocytes pre-infected with the indicated viruses or left untreated (C) and then 24 hours after infection eitherleft untreated or treated for six hours with 25µM ceramide. The data represent the means of two independent experiments whosestandard error is indicated by the bars. Both hsp27 and hsp70-expressing viruses significantly reduced the number of apoptotic cellscompared to uninfected cells (C) or cells infected with a control virus expressing green fluorescent protein (GFP) (p < 0.05).249
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