General Discussion

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General discussion

Cardiovascular disease is a leading cause of morbidity and mortality in the western world. Acute

myocardial infarction (AMI) is an important contributor to this cause since it induces apoptosis, necrosis

and inflammation in the myocardium, resulting in a loss of cardiomyocytes.[1,2] As spontaneous

regeneration of cardiomyocytes is limited, injured myocardium is replaced by non-contractile scar tissue,

which leads to adverse remodelling of the left ventricle and can result in heart failure.[2,3] Therapy for

AMI focuses on restoration of reperfusion and on prevention of remodeling and secondary

cardiovascular events via specific drug therapy.[4,5] Although these strategies significantly reduce

mortality,[6,7] they do not replace lost cardiomyocytes. The ideal AMI therapy would therefore be to

stimulate revascularization of the ischemic region, to minimize the loss of cardiomyocytes, thus limiting

scar tissue formation, and to replace lost cardiomyocytes.[5] New therapies for AMI, focusing on

inflammatory inhibitors and stem cell therapy using adipose derived stem cells, are the subject of this

thesis.

Inflammatory inhibitors

The major goal of current AMI treatment is restoration of reperfusion in the ischemic myocardium.

Reperfusion, however, results in the induction of inflammation in the heart (figure 1). Through the

binding of inflammatory mediators on injured and dead cardiomyocytes, inflammatory cells, especially

neutrophilic granulocytes and macrophages are attracted, subsequently clearing injured cardiomyocytes

from the heart. However, this inflammatory reaction induces additional damage to the myocardium.[8-

10] After AMI, also non-lethal damaged cardiomyocytes lose their membrane phospholipid

asymmetry.[11] As a result, the anionic phospholipids phosphatidylserine and phosphatidyl-

ethanolamine, which are normally kept within the inner plasma membrane leaflet, are then exposed in

the outer membrane leaflet, a process known as `flip-flop’.[12] It has shown that this flip-flop can be a

reversible phenomenon.[13,14] However, during reperfusion also these flip-flopped/reversibly damaged

cells are targeted by inflammatory mediators, resulting in cell death of these cardiomyocytes, thereby

increasing infarct size in time. A crucial mediator herein is the acute phase protein type IIA secretory-

type phospholipase A2 (sPLA2-IIA), which was shown to bind to and to induce cell death of these

reversibly damaged cardiomyocytes.[13,15,16] As depicted in figure 1, sPLA2-IIA can not only induce cell

death via CRP and complement binding/activation, but also via a direct cytotoxic effect, independent of

other inflammatory mediators.[13] Inhibition of this inflammation cascade might therefore form a

potent therapy to reduce cell death of cardiomyocytes after AMI. To this purpose we analyzed the effect

of the sPLA2-IIA inhibitor PX-18 on cell death after AMI.We then found in a rat model of AMI, that

treatment with PX-18 significantly reduced infarct size and mortality, without impairing wound

healing.[17] The latest is of utmost importance since the inflammation response itself is also essential for

the healing process of the heart after myocardial infarction.[15] When using PX-18, CRP may still bind to

necrotic cardiomyocytes independent of sPLA2-IIA, meaning that necrotic tissue can be cleared via the

CRP-complement dependent cascade (Figure 1).[9,13] Interestingly, we also found in vitro that PX-18

reduced membrane flip-flop and apoptosis in cardiomyocytes subjected to metabolic inhibition in the

Chapter 8

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absence of sPLA2-IIA, suggesting an additional cell protective effect of PX-18 independent of sPLA2-

IIA.[17] Finally we found that after treatment with PX-18, the numbers of so called late type

macrophages in the heart were significantly increased after AMI.[17] The function of macrophages in

wound healing after AMI is quite diverse, including digestion of damaged tissue, but also inhibition of

inflammation and promotion of regeneration.[18,19] It has been shown that early type macrophages

(type 1) digest damaged tissue, while late type macrophages (type 2) promote healing.[19,20] Therefore

the finding that these late type macrophages were increased after treatment with PX-18 suggests

another mechanism through which PX-18 exerts a protective effect (Figure 1). Taken together, PX-18 can

exert a cardiac protective effect on at least 4 different levels: (1) PX-18 inhibits membrane flip-flop, and

thus prevents the pro-inflammatory state of the plasma membrane of cardiomyocytes. (2) PX-18 inhibits

sPLA2-IIA activity, and thus prohibits sPLA2-IIA mediated cell death of at that moment reversibly

damaged cardiomyocytes. (3) PX-18 directly inhibits apoptosis of cardiomyocytes. (4) PX-18 attracts late

type macrophages which improve wound healing. (Figure 1)

Another inflammatory inhibitor we have studied in this thesis was clusterin. Clusterin is not only

an inhibitor of complement, but also protects cardiomyocytes after ischemia in vitro, independent of

complement.[21] We have now shown in a rat model of AMI, that treatment with clusterin significantly

reduced infarct size and mortality, as compared to untreated rats, again without impairing wound

healing.[22] The exact mechanism through which clusterin protects cardiomyocytes after AMI, next to

complement inhibition, is however not known. It has been shown that during oxidative stress clusterin

exerts a protective effect in H9c2 cells (a cardiomyoblast cell-line) via the Akt/GSK-3β signaling

pathway.[28] It also has been suggested that clusterin plays a role in membrane recycling of

cardiomyocytes in response to ischemia,[21,23-25] and that it functions as a secreted heat-shock protein

or chaperone molecule. As such it has an active role in the removal of damaged cells or toxic molecules

derived from (cardiac) cells.[25-27] The cytoprotective effect of clusterin has been related to its receptor

megalin.[29,30] Although we have shown that megalin is expressed in cardiomyocytes of the human

heart, the cell protective effect of clusterin was independent of megalin, at least in vitro after metabolic

inhibition, which is in line with findings of Jun et al.[22,28] We now have also shown that after treatment

with clusterin, the number of late type macrophages was significantly increased after AMI. Taken

together, also clusterin forms a potential therapeutic agent in the treatment of AMI.

Figure 1. In vivo mechanisms of cell death after AMI: the effect of PX-18 and clusterin hereon. Ischemia induces

reversible and irreversible membrane flip-flop in cardiomyocytes. During the subsequent inflammatory reaction, sPLA2-IIA

binds to flip-flopped membranes and induces cell-death, resulting in necrosis of both reversibly as irreversibly damaged

cardiomyocytes. PX-18 (red) can prevent cell death of reversibly flip-flopped cardiomyocytes by inhibiting flip-flop, by

inhibiting direct cytotoxic effects of sPLA2-IIA, and also by inhibiting sPLA2-IIA-facilitated binding of CRP and complement.

Importantly, irreversibly damaged cardiomyocytes can still be cleared due to binding of CRP to these cells independent of

sPLA2-IIA. Clusterin (blue) prevents cell death by inhibition of complement, and by inhibition of flip-flop independent of

the clusterin receptor megalin. In addition both PX-18 and clusterin induce macrophage infiltration, therewith improving

cardiac remodeling. C: complement, CRP: C-reactive protein, †: cell death, ⊥: inhibition, o: hydrophobic phospholipids

(phosphatidylcholine) that in normal cells composes most of the outer leaflet of plasma membranes, •: anionic

phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) that are normally kept within the inner leaflet

of the plasma membrane.

General Discussion

135

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Chapter 8

136

Next, an important topic is the optimal timeframe to apply these inflammatory inhibitors. We have

found that application of a single bolus of clusterin as well as PX-18 directly after reperfusion did not

result in a significant reduction of infarct size. Reduction was only found when the inflammatory

inhibitors were administered repeatedly during the first 3 days after infarct induction.[17,22] These

results therefore indicate that the acute inflammation period after AMI plays a major role in cell death

induction after AMI, in addition to ischaemia itself. In conclusion, inhibition of the inflammatory

response post AMI, by administration of PX-18 and clusterin, forms a new promising therapeutic

approach to decrease cell death after AMI, thus preventing the development of heart failure.

Adipose derived stem cells

Although the above described inflammatory inhibitors significantly reduced infarct size, they do not

replace lost cardiomyocytes. Stem cells, however, can replace lost cardiomyocytes, although results of

early clinical trials using stem cell therapy in AMI are disappointing. As such, stem cell therapy still needs

to be further optimized. This we have studied in the second part of this thesis, using the relatively new

and promising adipose derived stem cells (ASCs). ASCs are derived from the stromal vascular fraction

(SVF) of adipose tissue, and are suggested to have a high potential for the treatment of AMI.[18,32-34]

ASCs can be easily harvested and have properties similar to bone marrow mesenchymal stem cells

including the capacity to differentiate towards several cell types, amongst which cardiomyocytes.[18,32-

35] Even more, when compared to bone marrow, adipose tissue provides up to 100 times more

mesenchymal stem cells per gram tissue.[33,35] Meanwhile, several experimental studies have been

performed analyzing the role of ASCs in AMI in both small and large animal models (table 1). Also the

first clinical trial using ASCs to treat AMI has started recently in the Netherlands.[36]

Although the results of these animal studies generally are promising, it was also found that due

to the harsh ischemic and inflammatory environment in which the cells are transplanted after AMI,

massive cell death of stem cells occurs, lowering the effectiveness of this therapy.[2,37] Several factors

play an important role herein, including the time point of administration, the environment of the

infarcted heart, the passage of ASCs used, and its administration route (figure 2).

Time point of administration

In most animal studies investigators applied stem cells during the same operational procedure as the

infarct induction itself (table 1). However, it is known that the environment in the heart changes

dramatically after AMI. Massive myocardial necrosis and leukocyte infiltration might then harm survival

of implanted stem cells, and therewith reduce the effectiveness of stem cell therapy.[2,38,39] Therefore,

the time point of administration might significantly affect stem cell survival after AMI. Several studies

have additionally investigated the time frame to apply stem cell therapy post AMI, although this was not

studied directly using ASCs.[38,40-42] These in vitro and in vivo studies suggested that the optimal timing

for stem cell therapy is subsequent to the acute inflammatory period (>3 days post AMI), but before two

weeks post AMI when scar tissue is formed and regeneration capacity is limited.[42] Interestingly we

found for ASCs that adhesion, proliferation and differentiation in vitro was improved by the extracellular

General Discussion

137

Table 1: studies on ASC transplantation after AMI in coronary ligation models

Route: I.M.=intramyocardial, I.V.=intravenous, I.C.=intra-coronary, T.E.=trans-endocardial, LVEF= left ventricle ejection

fraction, NS= non significant, ?=unknown/not investigated, ==no difference, * p<0.05, ** p<0.01, *** p<0.001. In studies

that investigated different groups of ASCs also with additives only groups without additives have been included in this

table.

Study Animal model Reperfusion Time after AMI passage route Infarctsize LVEF capillaries

Rat

Schenke-Layland 2009 [45] Lewis yes <1 h SVF I.M. ↓ ns ↑ ** ↑ **

Li 2011 [46] Rat (a-thymic) <1 h p25-30 I.M. ↓ * ↑ * ↑ *

Li 2007 [47] Sprague-Dawley <1 h p3 I.M. ? ↑ ** ↑ **

Yamada 2006 [48] Sprague-Dawley <1 h ? I.M. ↓ ns ↑ ** ?

Hong 2011 [49] Rat (a-thymic) 1 h p3-4 I.M. ↓ *** ↑ *** ↑ **

Cai 2009 [50] Rat (a-thymic) 1 h p3 I.M. ↓ * ↑ * ↑ *

Shi 2011 [51] Lewis 1 h p3 I.M. = = ?

van Dijk 2011 [52] Wistar yes 1 day SVF I.V. ↓ ns ↑ ns =

Danoviz 2010 [53] Lewis yes 1 day p3-4 T.E. ? ↑ * ↑ *

Zhu 2009 [54] Sprague-Dawl 1 day p5 I.V. ↓ * ↑ * ↑ *

van Dijk 2011 [52] Wistar yes 7 days SVF I.V. ↓ * ↑ ns ↑ ns

van Dijk 2011 [52] Wistar yes 7 days p3 I.V. ↓ * ↑ ns ↑ ns

Wang 2009 [55] Lewis 7 days p3-5 I.M. ↓ * ↑ * ↑ *

Bayes-Genis 2010 [56] Rat (SCID) 7 days p2-5 I.M. ↓ ** ↑ * ↑ **

Hwangbo 2010 [57] Sprague-Dawl 7 days p3-5 I.M. ? ↑ * ↑ ns

Berardi 2011 [58] Wistar 8 days P5 I.M. ? = ↑ ns

Mouse

Yu 2010 [59] C57BL/6 <1 h p2-3 I.M. ↓ ** ↑ ** ↑ *

Tokunaga 2010 [60] C57BL/6 <1 h p3-5 I.M. ↓ ns ? ↑ ns

van der Bogt 2009 [61] FVB <1 h ? I.M. = ↑ ns ?

Leobon 2009 [62] C57BL/6 <1 h ? I.M. ? ↑ * ↑ **

Bayes-Genis 2010 [56] NIH-Foxn1rnu <1 h p2-5 I.M. ↓ * ? ↑ **

Bai 2010 [63] Mouse (SCID) <1 h SVF I.M. ? ↑ ** ↑ **

Bai 2010 [63] Mouse (SCID) <1 h p3 I.M. ? ↑ ** ↑ **

pig

Alt 2010 [64] Pig yes <1 h SVF I.C. ↓ * ↑ * ↑ *

Valina 2007 [65] Pig yes <1 h p3 I.C. ↓ ns ↑ * ↑ *

De Siena 2010 [66] Pig <1 h p5-8 I.M. ↓ * ↑ * ↑ ns

Rigol 2010 [67] Pig yes 7 days p3 T.E. = = ↑ *

Chapter 8

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matrix molecules fibronectin and laminin, which are upregulated in the infarcted area after AMI

(fibronectin from 12 hours on and laminin from 3 days on). This indicates that in case ASCs are

transplanted subsequent to the acute inflammatory period, thus when fibronectin and laminin

depositions are high, these proteins might stimulate ASC attachment and differentiation.[43,44]

As depicted in table 1 infarct size reduction in rats indeed was more often non-significant after

early ASC injection (within 24 hours), compared with injection after the acute inflammatory period (1

week). We have now shown that application of stem cells 7 days post AMI, leads to a significant

reduction in scar size, which was not found when cells were injected at day 1 post AMI.[52] To the best

of our knowledge, no other studies have compared the effect of injection of ASCs at different timepoints

within one study. Taken together, after the acute inflammation period after AMI (>3 days), the

environment in the heart is more favourable for stem cell transplantation. In future studies we will

address whether this inflammatory environment can be modified, to improve stem cell therapy, for

example by adding inflammatory inhibitors prior to stem cell therapy.

Culturing of ASCs before transplantation

Another factor that might influence the fate of stem cells after AMI is the process of culturing these cells

before transplantation, which is usually done to achieve a high number of stem cells. Culturing, however,

is time consuming, expensive, increases cell size, and, probably most important, affects the functional

characteristics of cells.[33,35,52,68] As such it has been shown that already during early culture ASCs

loose stem cell markers like CD34.[69] Additionally, we have now found that ASCs express the multi drug

resistance protein BCRP, that protects ASCs against ischemia. Also the expression of this protective

factor decreases during culture. These data suggest that using the uncultured stromal vascular fraction

cells (SVF), which contains ASCs, is more favourable for clinical practice than using long cultured ASCs.

This is possible since high numbers of SVF-cells/ASCs can be harvested from a relatively small amount of

adipose tissue.[33,35]

The question then arises whether there is a difference in therapeutic effect between uncultured

SVF-cells and cultured ASCs. We therefore compared the potential of uncultured SVF-cells and passage 3

ASCs in a rat model of AMI with reperfusion. We have found that uncultured SVF cells reduced infarct

size and improved cardiac outcome comparable to cultured ASCs. However, injection with cultured ASCs

(p3) occasionally resulted in severe pulmonary complications (thrombo-embolism), which was not found

with SVF cells. Since the results in cardiac outcome between uncultured SVF cells and cultured ASCs were

comparable, the use of uncultured SVF cells theoretically would be clinically more favourable.

Administration route

The route of delivery of stem cells is another important determinant for the fate of the stem cells after

AMI. Commonly used administration routes are intra-myocardial (IM) injection (directly, trans-coronary

or trans-endocardial) and intravascular delivery (intravenously, or intra-coronary). Several studies have

compared the outcome for these different administration routes. Generally it was found that

independent of the administration route the majority of the cells is lost, but slightly more cells can be

General Discussion

139

retraced in the heart after IM injection, compared with intravascular delivery.[70-73] However, IM

injections have the disadvantage that they can induce severe complications like for instance wall

rupture, arrhythmia and calcifications at the injection site.[74-77]

In the clinical setting intra-coronary injection is usually performed during percutaneous coronary

intervention.[74] This method, however, is not optimal since the time point of the percutaneous

coronary intervention usually is just before or during the acute inflammation phase of AMI (see above).

Other disadvantages of intra-coronary injection are the risk of in-stent restenosis and embolisation of

microvessels.[74,75] In contrast, intravenous injection has the advantage that it can be applied at any

timepoint desired after AMI. It is even possible to inject stem cells several times intravenously, instead of

the application of one single bolus. However, a major disadvantage of intravenous stem cell therapy is

the low number of stem cells that reaches the infarcted area, as the majority of the injected stem cells

are found to be trapped in the lungs.[78-80] This so called pulmonary first pass effect is at least partly

related to the size of the cells injected as stated above.[79] Uncultured cells are smaller compared to

cultured ASCs, as culturing of ASCs increases cell size. Therefore, complications after intravenous

injection might be reduced by using uncultured SVF cells, as we have also demonstrated.[52]

Figure 2. Factors that we have shown to influence the faith of ASCs after transplantation for AMI.

Faith of ASCs is influenced by the environment in the heart, and the time point of transplantation. Also the route of

transplantation and culture of ASCs influences stem cell therapy. + improves stem cell therapy, - reduces chance of

successful ASC therapy

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In future studies we will further optimize the intravenous delivery method of ASCs/SVF cells, by

specifically targeting the cells to the infarction area using microbubbles. Microbubbles are gas-filled

bubbles with a diameter of 2-5µm, that originally were used as ultrasound contrast agents.[81] These

microbubbles will be labelled with an antibody against ASCs (within the SVF fraction) as well as against a

specific target in the infarction area. As such we not only intend to increase the retention time of ASCs

within the infarcted area, but also intend to facilitate the binding of this complex to the endothelium by

pushing the ASC-microbubble complexes to the vessel wall using the acoustic radiation force of

ultrasound.

Mechanisms through which ASCs can improve cardiac outcome

Although many studies now have shown that ASCs form a promising new therapy in myocardial

infarction, the mechanisms through which ASCs improve cardiac outcome after AMI are not fully

elucidated. Studies have postulated different mechanisms for this cardiac improvement, mainly via an

effect of ASCs on differentiation or via paracrine effects (figure 3). In vitro and in vivo studies have shown

differentiation of ASCs towards cardiomyocytes and endothelial cells, suggesting that ASCs could

improve cardiac outcome by differentiation into these cells.[43,52,63,65,82-88] However, the number of

differentiated endothelial cells and cardiomyocytes that could be retrieved in the heart in animal studies

was relatively low, suggesting that the improvement of cardiac outcome was not only related to

differentiation effects of ASCs.[45,50,52,63] In line with this, several in vivo studies have now shown that

after treatment with ASCs the number of vessels in the peri-infarction area was increased, albeit without

evidence of differentiation of ASCs into endothelial cells.[45,50,52,55]

It was therefore suggested that ASCs might also improve cardiac outcome by so called paracrine

effects, indicating that factors excreted by stem cells affect the surrounding cells. Growth factors,

cytokines and signalling molecules produced by the infused stem cells can improve vascularisation,

reduce inflammation, and favour the viability of cardiomyocytes by inhibition of apoptosis.[89] As such,

it has been shown that ASCs secreted significant amounts of vascular endothelial growth factor (VEGF),

insulin-like growth factor (IGF)-1, hepatic growth factor and transforming growth factor-β (TGF-

β).[90,91] Both the secreted VEGF and IGF-1 have an anti-apoptotic effect on cardiomyocytes subjected

to hypoxia in vitro,[91] while VEGF also promotes endothelial cell growth and reduced endothelial

apoptosis in vitro.[90] Finally, it has been suggested that ASCs might also improve cardiac outcome by

reducing inflammation or attract resident stem cells to the infarcted area, but this has not been

confirmed yet.[92] Also, we did not find a significant effect of ASC therapy on the number of

macrophages in the heart after AMI.[52] Taken together, ASCs can improve cardiac outcome after AMI

through both induction of differentiation into cardiomyocytes and endothelial cells and through

paracrine effects (figure 3).

General Discussion

141

Figure 3. Mechanisms through which ASCs can improve outcome after AMI. ASCs can directly improve cardiac outcome

after AMI through differentiation into cardiomyocytes or endothelial cells. In addition, ASCs secrete factors that promote

survival of ischaemic cardiomyocytes and that induce angiogenesis, thereby improving perfusion around the ischaemic

area. In addition, it has also been suggested that ASCs might improve clinical outcome after AMI in a paracrine manner by

reducing inflammation through modulating protease activity and scar formation and by recruitment of resident cardiac

stem cells. HGF= hepatocyte growth factor; IGF= insulin-like growth factor; VEGF= vascular endothelial growth factor.

Conclusions of this thesis

The aim of this thesis was to develop and improve new putative therapies for myocardial infarction. We

now have shown that related to the inflammation, the first three days post AMI play a major role in the

induction of additional damage to cardiomyocytes, next to ischaemia itself. Specific inflammatory

inhibitors, clusterin and PX-18, indeed significantly reduced infarct size, without impairing normal cardiac

wound healing, when applied during these first three days post AMI. Therefore these inflammation

inhibitors form a promising new therapy in patients with AMI, especially since they not only inhibit

inflammatory mediators, but also reduce the pro-inflammatory state of the cardiomyocyte membrane.

To replace lost cardiomyocytes post AMI human ASCs in the SVF fraction of adipose tissue form

a potent therapy. These cells are abundantly available and show a high potential of endothelial and

cardiomyocyte differentiation. We have now shown that uncultured SVF cells are more favourable over

cultured ASCs for therapy after AMI. Furthermore, our studies indicate that SVF-cells/ASCs should be

transplanted after the acute inflammation period (>3 days), as the acute inflammatory environment

after infarction is a harmful environment also for stem cells. Finally, we have shown that intravenous

injection is a save transplantation route for stem cells that can be used at any desired timepoint post

AMI. In conclusion, inflammatory inhibitors and ASCs or their combination, can further improve clinical

outcome after AMI, by preventing development of heart failure later on.

Chapter 8

142

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