Editorial

Mending the broken heart: ischemic preconditioning-stimulatedmyocardial angiogenesis

The phenomenon of ischemic preconditioning (IP), inwhich a period of sub-lethal ischemia can profoundly protectthe cell from subsequent lethal ischemic insult, has withoutdoubt been responsible for an enormous amount of researchover the last 15 years. Since the original publication [1], therehave been in excess of 2000 publications directly addressingthis phenomenon and virtually all the publications demon-strate the ability of sub-lethal ischemia to protect the myo-cardium from a subsequent ischemia-induced injury, thusattesting to the power of the phenomenon of IP. This earlyform of protection has become known as “classic or earlypreconditioning”. This was followed by the first descriptionof the “second window of protection” or delayed precondi-tioning [2] in which a second period of protection was ob-served 24 h after a sub-lethal ischemic insult. That a myocar-dial cell has an innate ability to protect itself from severelethal ischemia based upon what it can “remember” from aprior sub-lethal ischemic insult, has caught the imaginationof both basic and clinical scientists. What is believed to be sounique about this phenomenon is that it provides a window tocertain mechanisms by which myocardial tissue attempts tosurvive a severe ischemic insult. It is therefore important todiscern the myriad of pathways that are associated with thissurvival mechanism. From the initial receptor that triggers orinitiates the complex and elaborate signaling pathways to theincompletely understood end-effectors that finally producethe profound protection known as IP, all attest to the com-plexity of inter-related biological pathways. There is nodoubt that since the initial discovery of IP we have learned agreat deal with regard to the cellular physiology/molecularbiology that was not appreciated before. We are gaining a realunderstanding of how a cell requires to protect itself frominjury and to orchestrate repair. What is amazing, however, isthat despite having observed the phenomenon of IP in greatdetail, we still are comparatively ignorant of the fundamentalmechanism as to how IP actually protects the heart. Since thediscovery that the mitochondrial KATP channel is pivotal toIP, controversy has existed as to whether this channel is atrigger, a mediator, or the end-effector of IP and this issue hasrecently been discussed in a detailed review [3].Additionally,the importance and roles of apoptosis [4], intracellular signaltransduction modulated-gap junction-mediated intercellularcommunication [5], protein kinase C (PKC) signaling andinnate immune systems [6] in the setting of IP have beendescribed. Furthermore, the ability of adenosine receptor

agonists to activate complex protein kinase signaling cas-cades with subsequent activation of gene transcription andpost-translational regulation of several proteins includingmitochondrial manganese superoxide dismutase and the27 kDa heat shock protein has also been reported as media-tors of IP [7–9]. Finally, Laude et al. [10] examined theimportant but poorly understood area of preconditioning andthe vasculature. They discussed this under investigated areain relation to both early and delayed preconditioning and theyhighlighted the potentially important role of the endothe-lium. They also described mediators of endothelial protec-tion such as adenosine, bradykinin, nitric oxide, and freeradicals under IP conditions.

In spite of these documented mechanisms of IP-mediatedcardio-protection, neo-vascularization (neo-angiogenesis) inresponse to preconditioning may represent an additional, yeta potent mechanism leading to the survival of ischemic tissueduring infarction. Unfortunately, this possibility has not beensufficiently studied. Information regarding the effect of IP onthe augmentation of angiogenic signals, both in terms ofangiogenic gene upregulation and resultant collateral vesseldevelopment leading to cardio-protection during myocardialischemia, remains scant. In this regard the study by Fukuda etal. on pp. 547–559 of the last April issue of the Journal ofMolecular and Cellular Cardiology, Ref. [11], showing anaugmentation of neo-vascularization following precondi-tioning in a chronic myocardial infarction rat model, at-tempts to fill in the void in this area of investigation and pavethe way for future research.

The functional importance of spontaneously developingcollateral vessels in supplementing perfusion of the myocar-dium rendered ischemic by vascular obstruction was recog-nized many years ago, prompting attempts, both experimen-tal and clinical, to enhance collateral development. However,it was not until potent angiogenesis factors such as basicfibroblast growth factor (bFGF) [12] and vascular endothelialgrowth factor (VEGF) [13], proteins that could actuallystimulate collateral flow, were identified, purified, and pro-duced in sufficient quantities, that the field began its rapiddevelopment. In recent years, enhancement of ischemic an-giogenesis with angiogenic growth factors has been attract-ing increasing attention as a promising new way to treatischemic cardiovascular diseases [14,15]. Triggered by thetherapeutic potential of modifying angiogenesis in diseaseprocesses, interest and research in this area have exploded in

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recent years, and led to rapidly changing views on the mecha-nisms and regulation of angiogenesis. Tissue hypoxia alsoupregulates expression of VEGF and bFGF and their recep-tors, with temporal and spatial kinetics indicating the role ofthese factors in ischemic angiogenesis [16]. Consequently,several investigators have explored the therapeutic potentialof local administration of exogenous angiogenic growthfactors—either as proteins, naked cDNA, or plasmids—intothe ischemic tissues to enhance collateral vessel formationand restore blood flow [14,17–20].Although such attempts torevascularize ischemic tissues have often been successful inanimals, early studies have also uncovered certain potentialshortcomings. One of the complications of the use of VEGFis its ability to increase vascular permeability, resulting intissue edema [21,22], whereas one of the complicationsof bFGF is its mitogenic effects on cells other thanvascular—that is, tumor cells that may lead to the spread ofcancer [23]. In fact, both bFGF and VEGF have been impli-cated in tumor growth and neoplastic angiogenesis [23].Moreover, additional studies also suggested that the durationof exposure of the vessels to angiogenesis factors was criti-cal, and that the administration of proteins, with their rela-tively brief half-lives, may pose important practical limita-tions. It could be argued that gene therapy that improvescollateral function, presents one of the solutions to the co-nundrum, since gene therapy can be considered a sophisti-cated form of a sustained delivery system. Consistent withthis notion, clinical trials that involved administration ofsingle angiogenesis agent showed promising results in PhaseI trials; however, some Phase II studies demonstrated notreatment effect on the primary end-point [24]. It may, there-fore, be relevant to consider that the molecular mechanismsresponsible for angiogenesis are extraordinarily complex,and an optimal angiogenesis intervention may require a“multiple factor” strategy. Adverse effects of gene therapysuch as neo-vascularization of non-targeted tissues and tu-mor growth, though not excessively reported using singleagent, also need to be considered.

An alternate strategy to augment vascularization in theischemic myocardium may be to stimulate “intrinsic” angio-genesis potential in the targeted tissue. The current study byFukuda et al. [11] proposes IP as a mean to achieve this goal.The study describes several important and interesting find-ings. The first important finding of this study is that leftanterior descending coronary artery (LAD) occlusion fol-lowing IP resulted in enhanced neo-vascularization, as evi-dent from significant increases in capillary and arterioledensities. This enhanced vascularization in turn improvedregional blood flow, preserved left ventricular functionalreserve, reduced infarct size, and inhibited myocyte andendothelial cell apoptosis via an upregulation of anti-apoptotic genes Bcl-2 and survivin, in a rat model of chronicmyocardial infarction. These findings suggest that increasedangiogenesis by IP may have an “added” effect in chronicstage over the known “direct effect” of IP on myocyte sur-vival observed in early stages. Augmentation in neo-

vascularization by IP has also been reported in a separatestudy which also showed PKC-mediated increase in neo-vascularization and VEGF upregulation in the setting ofchronic infarction [25]. Nevertheless, this concept remainsextremely important and warrants further research.

Secondly, preconditioning induced upregulation of VEGFexpression, an observation which authors propose, may con-tribute to enhanced neo-vascularization in the infracted myo-cardium. While the contribution of VEGF in neo-angiogenesis is not debatable, certain questions remain to bevalidated. Is enhanced angiogenesis by IP solely attributableto VEGF or rather it represents the involvement of otherangiogenic growth factors like bFGF and PDGF as well?This possibility has not been explored in this study and theinclusion of VEGF-blocking experiments could have shedsome light on this issue. If IP does in fact increase theexpression of other angiogenic growth factors, then IP mayrepresent a “multi-factorial” angiogenesis approach, whichmay potentially have advantage over the single factor ap-proach achieved by gene therapy. Ever since our laboratoryreported the existence of endothelial progenitor cells (EPCs)in the adult circulation [26], it has now become apparentthat EPCs clearly contribute to the process of neo-vascularization. Therefore, another important question thatremains to be answered is whether or not enhanced neo-vascularization by IP modulates the kinetics of EPC bymobilizing these precursors from the bone marrow.

Another interesting but not entirely surprising finding inthis study [11] is the observed IP-induced upregulation ofcertain transcription factors including that of AP1, SP1, andNFjB. In this regard, a previous study by Strohm et al. [27]demonstrated that systemic infusion of transcription inhibi-tor actinomycin D, completely cancelled the IP-inducedcardio-protection, indicating that cardio-protective effects ofIP are mediated by the modulations in gene transcription.While it is true that transcription factors AP1, SP1, andNFjB are known to regulate angiogenesis and VEGF expres-sion, the link between observed modulation of these trans-factors to the VEGF expression and neo-angiogenesis in thecurrent study [11] is at best correlative and should be inter-preted with caution. Since both AP1 and NFjB are known toregulate the expression of a wide variety of genes, includingthat of pro-inflammatory markers and cytokines it is conceiv-able that an upregulation of these trans-factors merely repre-sents ensuing inflammation that follows myocardial injury.Similarly an upregulation of stat-3 might represent the induc-tion of IL-10 signaling, an anti-inflammatory cytokine thathas been linked to the healing of infracted myocardium [28].In the absence of a direct “cause-effect” data in the currentstudy, the interpretation that modulations in the activation ofthese trans-factors leads to the enhanced angiogenesis viamodulations in VEGF, Bcl-2, and survivin expression mayrequire further functional validation.

Finally, the authors [11] show that enhanced VEGF up-regulation by preconditioning did not increase vascular per-meability which leads to edema and myocardial damage.

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Authors provide reasonable data showing that the suppres-sion of vascular permeability despite increased VEGF ex-pression is mediated by a simultaneous inhibition of Srcprotein. Since VEGF is known to promote vascular perme-ability, this observation is significant in its clinical implica-tion [29].

In summary, data shown by Fukuda et al. [11] provideexperimental evidence justifying an optimistic outlook relat-ing to our ability to augment collateral flow to ischemicmyocardium in a clinical setting by stimulating “innate”angiogenic signals via ischemic/hypoxic preconditioning.However, we are not there yet, and identification of theoptimal angiogenesis strategy is still unclear. Additional ex-perimental work, in parallel with large, carefully controlledanimal studies and clinical trials are needed to continue theexciting advances in the field, and to achieve the goal ofproviding patients with alternative potent therapies to im-prove collateral flow, and thereby to alleviate their symptomsand perhaps to prolong their lives.

References

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[29] Weis S, Shintani S, Weber A, Kirchmair R, Wood M, Cravens A, et al.Src blockade stabilizes a Flk/cadherin complex, reducing edema andtissue injury following myocardial infarction. J Clin Invest 2004;113:885–94.

Raj KishoreDouglas W. Losordo

Division of Cardiovascular Research, St. Elizabeth’sMedical Center, Tufts University School of Medicine,736 Cambridge Street, Boston, MA 02135 2997, USA

E-mail address: douglas.losordo@tufts.edu(D.W. Losordo).

Received and accepted 6April 2004

Available online 08 June 2004

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