CONSIDERACIONES GENERALES.

Cells exposed to 60 and 120 mins of reperfusion, though not those exposed to ischemia alone or to 5 mins reperfusion, showed classical DNA laddering (Fig. 3.2e).

3.3.4 ELECTRON MICROSCOPY

In hearts exposed to ischaemia and ischaemia/reperfusion no morphological features suggestive of necrotic cell death were seen. Typical features of apoptosis, such as diffuse nuclear chromatin condensation and margination, were not seen in endothelial cells or myocytes after ischaemia alone j(Fig.3.2f), but became apparent in endothelial cells after 5 mins reperfusion i(Fig.3.2g). Myocytes with the characteristic apoptotic morphology were observed together with apoptotic endothelial cells after 60 mins reperfusion (Fig.3.3a/b/c).

SECTION 4

3.4.1 DISCUSSION

This study shows that apoptosis following ischaemia/reperfusion proceeds in a cell and time dependent manner.

Ischaemia alone is not sufficient to complete apoptotic death of myocyte and non-myocyte cells assessed by TUNEL and electron microscopy in the isolated perfused rat heart.

After five to 60 minutes of reperfusion, endothelial cells within initially small, but later, larger coronary vessels become TUNEL positive, associated with a perivascular cuff of TUNEL positive myocytes of progressively increasing radius from the vessel with time.

By 2 hours of reperfusion the proportion of TUNEL positive endothelial cells had decreased, presumably reflecting their clearance or secondary necrosis, and the distribution of the positive myocytes had become more homogenous.

Although no TUNEL positive cells were seen after ischaemia alone, some cells, again in the coronary vessels, were stained with an antibody that recognises the cleaved form of caspase-3. This suggests that ischaemia without reperfusion can initiate the molecular pathway of apoptosis, although reperfusion is required to complete the DNA fragmentation and morphological changes characteristic of an end stage apoptotic cell. The colocalisation of cleaved caspase-3 with TUNEL positivity after reperfusion would support this interpretation. This requirement for reperfusion in completing the apoptotic program is in accordance with several previous studies (Gottlieb et al, 1994; Fliss et al, 1996).

The demonstration that endothelial cell apoptosis precedes that of myocytes has two important implications. Firstly, endothelial cell DNA fragmentation following short periods of reperfusion may follow the release into the restored circulation of a

vasoactive mediator(s) generated during ischaemia which is necessary for completion of the apoptotic process. Secondly, the radial distribution of apoptotic myocytes seen 5 and 60 minutes after reperfusion may reflect the diffusion into the myocardium of soluble apoptogenic mediators either released from damaged endothelial cells and/or carried by the blood circulation.

Several candidate mediators for the paracrine apoptosis of myocytes may be postulated, including those that ligate a death receptor, such as tumor necrosis factor a (TNFo) and FasL and those that damage mitochondria, such as free radicals. The potential involvement of soluble factors in cardiac cell apoptosis following ischaemia/reperfusion injury suggests that strategies based on their scavenging or inhibition may both allow endothelial cell rescue and protect the myocardium.

Figure 3.1a - Control heart exhibiting no yellow TUNEL positive cells, orange nuclei stained by propidium iodide (PI). Original magnification: x400.

Figure 3.1b - Heart exposed to ischaemia/reperfusion (5 mins): TUNEL positive endothelial cells appear yellow while non-apoptotic nuclei stained by PI remain red/orange. Original magnification: x200.

Figure 3.1c - Heart exposed to ischaemia/reperfusion (5 mins): TUNEL positive endothelial cells appear yellow while non-apoptotic nuclei stained by PI remain red/orange. Original magnification: x650.

Figure 3.1 d - Longitudinal section of a vessel showing endothelial colocalisation of TUNEL (yellow) and anti-von Willebrand factor (red/orange) (60 mins reperfusion). Original magnification: x650.

Figure 3.1 e - Ischemic-reperfused heart (5 mins): TUNEL positive cardiac myocytes (yellow) surrounding TUNEL positive vessel (yellow), all negative nuclei stained red by propidium iodide. Original magnification: x650.

■-45

I---

H- Control 35*1

[=□ TUNEL EC C=3 TUNEL CM Casp3 EC Casp3 CM

Figure 3.1 f - Percentage o f TUNEL staining and activated caspase-3 expression in endothelial cells and cardiac myocytes at different tim e points.

Figura 3.1g - Heart subjected to ischæmia followed by 60 mine reperfusion:

TUNEL positive vessel (yellow) with cuff of TUNEL positive cardiac myocytes

(yellow), whose numt>er decreases with increasing distance from the lumen.

Negative nuclei are stained red by propidium iodide. Original magnification: x200.

R*perfuaion time pointe

5* pep 60’ Rep 120’ Rep

§ 60

1

i

I

F ig ure 3.2a - Range of (vertical line) and mean (horizontal bar) diameter of TUNEL positive vessels with increasing reperfusion times.

20

I

8

15

i l l .

R#du* from VMM* <mk*on)

100

200

300

400

500

+ 4-

₊

— S'rep 60* r e p j^ p w rep

Figure 3.2b - Distribution of TUNEL positive cardiac myocytes with increasing radius from a vessel.

Figure 3.2c - Ischemic-reperfused heart: the two central cardiac myocytes show colocalisation of TUNEL (yellow) and activated caspase-3 staining (bright red), all the other cardiac myocytes stained only for propidium iodide (orange). Original magnification: x650.

C

l/R

CaspSa

Figure 3.2d - W estern blotting of activated caspase-3 protein level in cardiac tissues from control hearts, which were buffer perfused for 60 minutes, and hearts exposed to 35 minutes ischaemia and 1 hour reperfusion. Actin was used as internal control.

Figure 3.2e - Ethidium bromide stained agarose gels of DNA from control hearts (lane 2), hearts exposed to 35 mins ischemia (lane 3), and hearts exposed to 5, 60 and 120 mins reperfusion (lanes 4-6 respectively). Lane 1 is a molecular weight marker and Lane 7 is DNA from dexam ethasone treated thymocytes (positive control).

... Æ #

: - ' V i v ' * t '

% r '%

Fig ure 3.2 - f Electron microscopy of endothelial cell from heart exposed to ischaemia alone and (g) ischaemia/reperfusion (5 mins), the latter showing chromatin condensation and margination. Original magnification; xIOOOO.

a

b

c

Figure 3.3 - Cardiac myocyte showing normal ultrastructure (a). Cardiac cell exhibiting nuclear chromatin condensation

and margination, morphological hallmarks of apoptotic cell death (b). Endothelial cell and cardiac myocytes showing

ultrastructural features of apoptosls. The nuclear alterations occurring in the endothelial cell appear to be more

advanced, suggesting that in this cell type the apoptotic process had an earlier start, compared to cardiac myocytes (c).

RESULTS

ENDOTHELIAL

CELLS

AND

CARDIAC

MYOCYTES

UNDERGO

APOPTOSIS

USING

DIFFERENT

SIGNALING

PATHWAYS

DURING

ISCHAEMIA/REPERFUSION INJURY

SECTION 1

4.1 INTRODUCTION

The apoptotic cascade can be elicited by activation of two main distinct signaling pathways: the first being death receptor-mediated, the other following mitochondrial damage (see 1.4).

In the death receptor-initiated pathway, apoptosis is triggered by the interaction between receptors like TNFRl, FAS, DR-3, DR-4 or DR-5 with their cognate

ligands. In the FAS-mediated apoptosis, for instance, the binding event leads to trimerization of the receptor and subsequent interaction between the death domain (DD) of the cytoplasmic region of the FAS receptor and the DD of the adaptor protein FADD (FAS-associated death domain). FADD, in its turn, by means of its death effector domain (DED), interacts with DED of procaspase 8, which results in caspase- 8 activation (Boldin et al, 1996). The assembled FAS-FADD-caspase-8 complex is referred as to death-inducing signaling complex (DISC). Once activated, caspase-8 cleaves caspase-3 and caspase-7, after which caspase-3 cleaves the additional downstream caspases-6 and -2.

In the mitochondria-initiated pathway, upon activation via a death signal leading to mitochondrial dysfunction or damage, cytochrome c is released from the mitochondrial intermembrane space to the cytoplasm. Once relocated, cytochrome c, in presence of ATP or dATP, binds to APAF-1 (apoptosis protease-activating factor 1), forming a multimeric complex called the apoptosome. Upon assembling, this complex recruits and activates pro-caspase 9 through the interaction between the CARD

domain of APAF-1 and the CARD domain of procaspase 9 (Li et al, 1997). Active caspase-9 propagates the caspase cascade cleaving caspase-3 and caspase-7.

These two apoptotic pathways are unilaterally cross-linked by BID (Bcl2-binding protein), a BH3 only protein, member of the Bcl2 family. When cleaved by caspase-8, tBID, the truncated form of BID, relocates from the cytoplasm to the mitochondria, where it induces leakage of cytochrome c and subsequent caspase-8-mediated caspase-9 activation (Luo et al, 1998).

In our previous studies with primary cultures of neonatal and adult cardiac myocytes, we showed that ischaemia alone leads mainly to processing of caspase-9 but not of caspase-8; conversely, exposure of cardiac myocytes to ischaemia/reperfusion resulted in cleavage of both caspase-9 and caspase-8 (Stephanou et al, 2001).

Additionally, in cardiac myocytes exposed to simulated ischaemia alone, the pretreatment with a specific caspase-9 inhibitor, reduced the number of annexin V

positive cells, whilst the C8 inhibitor was ineffective. In contrast, when cardiac myocytes were exposed to simulated ischaemia/reperfusion, both inhibitors, added separately, reduced apoptotic death and the reduction was more consistent when both inhibitors were added together. (Stephanou et al, 2001)

This study, carried out in the isolated rat heart, attempts to address 3 unsolved issues:

1. The level of activation of each apoptotic pathway during ischaemia and during reperfusion

2. The relative contribution of the two apoptotic pathways in inducing apoptosis both in endothelial cells and cardiac myocytes

SECTION 2