Ischemic heart diseases: Current treatments and future

Journal of Controlled Release 140 (2009) 194–202

Contents lists available at ScienceDirect

Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Review

Ischemic heart diseases: Current treatments and future Donghoon Choi a, Ki-Chul Hwang a, Kuen-Yong Lee b, Yong-Hee Kim b,⁎ a b

Division of Cardiology, Yonsei Cardiovascular Hospital, Yonsei Cardiovascular Research Institute, Yonsei University, College of Medicine, Seoul, 120-749, Republic of Korea Department of Bioengineering, Hanyang University, 17, Haengdang-dong, Seongdong-gu, Seoul, 133-791, Republic of Korea

a r t i c l e

i n f o

Article history: Received 15 February 2009 Accepted 20 June 2009 Available online 27 June 2009 Keywords: Ischemic heart disease Percutaneous coronary intervention Protein therapy Cell and gene therapy Polymeric drug delivery system

a b s t r a c t Ischemic heart disease is a rapidly increasing common cause of death in the world. This disease is the insufficient status of oxygen within the cardiac muscles due to an imbalance between oxygen supply and demand, and a cardiac disease that occurs as a result of coronary artery stenosis. Conventional surgery-based therapy for the treatment of ischemic heart diseases has been advanced with biopharmaceutical-based therapy, such as protein, gene and cell therapy. The conventional medical therapy focuses on the use of drug eluting stents, coronary-artery bypass-graft surgery and anti-thrombosis. Biopharmaceutical-based therapies including recombinant protein therapy, gene therapy and cell transplantation are recognized as promising approaches in inducing neovascularization and improving collateral blood flow in the ischemic heart. This review explores the current status and future of the treatment of ischemic heart diseases with conventional medical therapy, biopharmaceutical-based therapy focused on the proteins and polymeric hydrogels for delivery of therapeutic proteins. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . Conventional medical treatments . . . . . . . . . . . 2.1. Percutaneous coronary intervention (PCI) . . . . 2.2. Coronary-artery bypass-graft surgery (CABG) . . 2.3. Anti-thrombotic therapy . . . . . . . . . . . . 3. Protein, cell and gene therapy . . . . . . . . . . . . . 3.1. Protein therapy . . . . . . . . . . . . . . . . 3.1.1. Heat shock protein 27 . . . . . . . . . 3.1.2. Angiogenic growth factors . . . . . . . 3.1.3. Anti-apoptotic fusion proteins . . . . . 3.2. Cell therapy . . . . . . . . . . . . . . . . . . 3.2.1. Bone marrow derived cells . . . . . . . 3.2.2. Adipose tissue derived cells . . . . . . 3.2.3. Umbilical cord derived cells . . . . . . 3.2.4. Embryonic stem cells (ESC) . . . . . . 3.3. Gene therapy . . . . . . . . . . . . . . . . . 4. Polymeric hydrogels for delivery of therapeutic proteins 4.1. Natural polymer-based hydrogels . . . . . . . . 4.2. Synthetic polymer-based hydrogels . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

⁎ Corresponding author. Tel.: +82 2 2220 2345. E-mail address: [email protected] (Y.-H. Kim). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.06.016

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D. Choi et al. / Journal of Controlled Release 140 (2009) 194–202

1. Introduction Ischemic heart disease characterized by reduced blood supply to the heart muscle is the most common cause of death in most western countries. It results from the blockage in coronary arteries by atherosclerosis and thrombus. Ischemic heart disease presents with symptoms such as temporary pain (angina), irregular heart beat (arrhythmia), permanent heart muscle damage (myocardial infarction) and loss of muscle activity (heart failure). Cardiac remodeling is the global and cellular change in the ventricular shape and function following chamber dilation and interstitial and perivascular fibrosis. The remodeling leads to the chronic heart failure. The remodeling includes neurohormonal responses, cytokine activation, loss of cardiomyocytes due to necrosis or apoptosis, cardiomyocyte hypertrophy, disruption of extracellular matrix (ECM) and collagen accumulation followed by scar formation [1]. The critical cause of heart failure is myocardial ischemia, resulting in dysfunction and death of cardiomyocytes. The main cardiac response to myocardial infarction (MI) can be seen as cardiomyocyte hypertrophy, apoptotic myocyte loss, progressive collagen replacement, and enlargement of the left ventricle [2]. 2. Conventional medical treatments There have been enormous advances in percutaneous coronary intervention (PCI) [previously called angioplasty, percutaneous transluminal coronary angioplasty (PTCA) or balloon angioplasty] techniques, devices and medications since Andreas Grutzig first performed in 1977. In particular, drug-eluting stent (DES) has been popularized as an effective intervention tool inhibiting overgrowth of scar tissue that can renarrow the artery and block blood flow to the heart, a complication called restenosis. However, it should be emphasized that although the coronary artery intervention has remarkably reduced restenosis, the incidence of myocardial infarction and heart-related deaths was not notably decreased. This fact suggests that it is important to deter the disease progression and prevent the recurrence with the appropriate drug treatment providing protective effects at the cellular level of myocardium [3]. 2.1. Percutaneous coronary intervention (PCI) PCI is a medical procedure which involves the inflation of a balloon within the blocked coronary artery to crush the plaque into the walls of the artery and restores normal blood flow to the heart muscle. While balloon angioplasty has been performed as a part of nearly all percutaneous coronary interventions, sometimes a stent, recently drug eluting stent is often introduced into the blood vessel or artery. Stenting is an alternative to heart surgery for some forms of non-severe coronary artery disease. This procedure is effective in mostly acute heart attack and reduces mortality of coronary artery disease compared with a common medical treatment administrating thrombolytic agent [4]. Other procedures that are done during a percutaneous coronary intervention include rotational or laser atherectomy, brachytherapy (use of radioactive source to inhibit restenosis) in addition to balloon angioplasty and implantation of stents. Percutaneous coronary angioplasty has been advanced with the development of drug eluting stent (DES) into coronary artery, demonstrating remarkable effect in reducing the restenosis incidence [5]. Unlike the era when the conventional balloon dilatation was used, the type and structure of blood vessels for which coronary angioplasty could be possible, the function of left ventricle and the systemic condition rather than the number of blood vessels with a stenosis are crucial criteria for determining the optimal DES procedure. The use of DES has been reported to reduce the incidence of cardiovascular events to a significant extent compared to that of general stents. Besides, studies have shown that the use of DES (Cypher) could obtain

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the equivalent treatment outcome in patients who had equivalent lesions to the surgical group. 2.2. Coronary-artery bypass-graft surgery (CABG) Coronary artery bypass graft surgery is a general surgical procedure performed to reduce the risk of death from coronary artery disease. In most cases, coronary bypass surgery requires a general anesthesia and median sternotomy. Arteries or veins originated from the patient's body are grafted to the coronary arteries to bypass atherosclerotic narrowings and increase the blood supply to the coronary circulation supplying the myocardium. The representative blood vessels that are used for coronary bypass surgery include saphenous vein and internal thoracic artery. Saphenous vein has been reported to have some disadvantages including a time-dependent restenosis risk [6]. The administration of anti-platelet drugs or lipid-lowering agents prevented the progression of arthrosclerosis and reduced the progression of restenosis. When the saphenous vein was used, the long-term follow-up results were not satisfactory. In contrast, the short-term and long-term follow-up results with the artery were superior to those with the vein. The bypass graft with the internal thoracic artery positively functioned in more than 90% of patients. Accordingly, left internal thoracic artery is frequently used for coronary bypass surgery. Recently, right internal thoracic artery and radial artery are becoming popular. Trials with the superficial gastric artery showed promising mid-term and short-term follow-up results. 2.3. Anti-thrombotic therapy Anti-thrombotic therapy is one of the important treatment modalities for patients with acute coronary syndrome (ACS) such as unstable angina (UA) and non-ST-elevation myocardial infarction (NSTEMI) [7]. Anti-thrombotic drugs suppress the formation of thrombin, which prevent the formation of blood clots. Goals for antithrombotic therapy are to prevent development and progression of thrombosis, promote dissolution or stabilization of acute and residual mural thrombus, and reduce death, myocardial infarction, stroke, thromboembolism, and, in patients undergoing PCI, reduce acute thrombosis and need for urgent revascularization. The use of antithrombotic therapy in ACS has reduced the incidence of death and myocardial infarctions dramatically in recent years. To date, unfractionated heparin (UFH) has been widely used as the standard treatment regimen as its efficacy has been documented in several large, randomized trials [8]. UFH is a heterogeneous mixture of polysaccharide chains of molecular weights that range from 5000 to 30,000 Da and that have varying anticoagulant activity. More recent studies indicate that low molecular weight heparin (LMWH) is also effective in the reduction of end points such as myocardial infarction or death. More recently, LMWH when used in combination with acetyl salicylic acid, has been reported to produce more efficient treatment outcome than UFH. The LMWHs are obtained through chemical or enzymatic depolymerization of the polysaccharide chains of heparin to provide chains with different molecular-weight distributions. LMWH binds to plasma protein and endothelial cells to a lesser extent, and subsequently has a long halflife. A subcutaneous injection of LMWH once or twice a day is able to maintain its anti-coagulative effects. LMWH has the disadvantage of cost-ineffectiveness despite the advantage that a laboratory monitoring is not essential. Factor Xa inhibitor (fondaparinux) is an anti-thrombogenic agent acting through the coagulation cascade to inhibit the multiplier effects of the downstream reactions, thereby suppressing thrombin generation [9]. Factor Xa inhibitor significantly lowers the death, myocardial infarction and stroke compared to LMWH. It has the advantage of a lower incidence of hemorrhage. Based on the effectiveness and safety, Factor Xa inhibitor is the most beneficial drug. It is disadvantageous, however, in that it cannot be used as the single agent during the PCI.

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Direct thrombin inhibitor (DTI) suppresses the activity of thrombin (factor IIa) and thereby inhibits the conversion from fibrinogen to fibrin with the mediation of thrombin. A single use of DTI in patients with acute coronary artery syndrome produced a lower incidence of hemorrhage. To perform PCI in these patients, a concomitant use of thienopyridine or GP IIb/IIIa inhibitors is mandatory. 3. Protein, cell and gene therapy 3.1. Protein therapy Proteins with other bio-macromolecules such as polysaccharides and nucleic acids are essential composition of organisms and participate in crucial process in cells. Proteins are potent hormones, antibodies, enzymes that catalyze biochemical reactions, structural or mechanical functions, involved in cell signaling, immune responses, and cell cycling, and energy source. Various human diseases are closely related to the malfunctioning of particular proteins. A variety of therapeutically important human proteins can be produced with relative ease by means of the recombinant DNA technology in mammalian cells, yeast or bacteria. Novel protein expression systems, including phage technology process, permit biologically active, properly folded, engineered proteins to be manufactured with high yield at a low cost. Therapeutic proteins can be used for a wide array of diseases in which the proteins are either lacking or defective, or to inhibit biological processes. Recently, protein transduction domains (PTD) or cell penetrating peptides (CPP) including HIV-1 Tat protein, homeodomain protein (penetratin), Drosophila Antennapedia and oligo/poly arginines have been identified to be efficient cargo carriers for proteins, genes, siRNAs, and particles so that water soluble macromolecules such as proteins are able to penetrate cells. For the ischemic heart disease treatment, protein-based angio-therapy approach based on dual delivery of arteriogenic and angiogenic factors showed significantly improved myocardial collateral growth, blood perfusion, and cardiac function in animal ischemic myocardial model (Table 1). 3.1.1. Heat shock protein 27 Heat shock proteins (HSPs), highly conserved molecular chaperones, including HSP90, HSP70, HSP27 and HSP22 classified by their molecular weight, have the ability to protect other proteins from aggregation, fold nascent proteins or refold damaged proteins and lead to degradation of severely damaged proteins [10]. Aging and hyperlipidemia have been known to diminish the expression of the cardio protective HSPs in response to heat-shock stress and hypoxia, resulting in the decreased function of the heart in protecting itself under ischemic condition [11,12]. HSPs play important roles in protecting against stresses such as high temperature, hypoxia/ischemia and oxidative stress in all mammalian cell types. HSP27, one of important HSPs, is highly induced and overexpressed in response to a stress or cytotoxic stimuli. The cardio protective effect of HSP27 under apoptotic stimuli might be associated with a direct inhibition of caspase cascade. Recently, it was reported that enhanced intracellular delivery of HSP27 by a TAT PTD could preserve cytoprotective effect after ischemic insult which induces cardiac cell death in the cardiomyocytes and rat left anterior descending (LAD) coronary artery ligation model, demonstrating that HSP27 protein is considered as a promising protein therapeutic to protect cardiomyocytes against ischemic insults [13]. 3.1.2. Angiogenic growth factors Despite of the complexity of angiogenic process, angiogenesismediated treatment of the ischemic heart disease focuses on an induction of neovascularization in the ischemic heart by introducing angiogenic growth factors, such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). Angiogenesis, a formation of new capillaries from the

postcapillary venules, is stimulated in hypoxia via activation of the hypoxia inducible factor (HIF-1α) to accelerate transcription of growth factors and their receptors [14]. FGFs, mitogenic and angiogenic factors stimulating proliferation, migration, and differentiation of vascular cells, have been validated for its therapeutic effect on ischemic diseases [15]. FGF-1 and FGF-2, wellknown angiogenic cardioprotective members among FGF family, have binding sites on various cells such as endothelial cells, smooth muscle cells, and myoblasts, resulting in stimulation of the proliferation of the respective cell types [16]. The first neovascularization of myocardium was studied with FGF-1 [17]. The administration of FGF-1 induced the neoangiogenesis in the ischemic rat and human heart with the development of new vascular structures through the stimulation of angiogenesis, granulation, and tissue formation [18,19]. Although, the mechanism of action of FGF family on the cardioprotective effect is controversial, the effect was reported to associate with enhanced postischemic hemodynamic recovery and decreased lactate dehydrogenase release during reperfusion, and the cardiac functional recovery was through the FGF receptors, protein kinase C (PKC), and tyrosine kinase (TK) [20]. Angiogenesis independent mechanism has been postulated, which follows cardioprotection at the level of the cardiac myocyte and at least partially mediated via activation of the mitogen activated protein kinase (MAP) ERK-1 and -2 [21]. Human trials demonstrated that local intramyocardial injection of FGF-1 increased the capillary density by 3 times [22]. Similar to FGF-1, the intracoronary administration of FGF-2 to the ischemic myocardium in canine and porcine models resulted in a significantly increased transmural collateral flow for 4 weeks [23]. The administration of FGF-2 to myocardial perfusion in patients with advanced coronary disease resulted in an attenuation of stress-induced ischemia and an improvement in resting myocardial perfusion [24]. FGF stimulates VEGF expression and VEGF also stimulates FGF expression in endothelial cells. The VEGF inducedangiogenesis is inhibited by neutralization of FGF-2 activity and vice versa [25,26,27]. The most extensively studied angiogenic growth factor is VEGF and its expression is stimulated by hypoxia-dependent and -independent mechanisms [28]. The functions of VEGF include enhanced migration, increased permeability, enhanced survival and the production of plasminogen activators in endothelial cells, all of which are related to angiogenesis [29]. VEGF plays a key role in angiogenesis in fetal myocardium, ischemic hind limb, wound healing, and coronary collateral development. The development of coronary flow, a fourfold increase in capillary density of the collateral-dependent myocardium and a decrease in the ischemic area were observed with the administration of VEGF perivascularly over 4 weeks in ischemic swine model [30,31]. The perivascular was regarded as one of administration routes to effectively deliver the angiogenic proteins compared to other administration routes, such as intravenous, intracoronary, intrapericardial, and catheter-based intramyocardial [32]. Although several administration routes are available in growth factor treatment, intracoronary administration of VEGF showed slight effect in preclinical studies [33,34]. The popular phenomenon in ischemic heart diseases is endothelial dysfunction and deficits in

Table 1 Therapeutic proteins for the ischemic heart diseases. Protein

Injection route

Model

TAT-FNK

Intramyocardial

TAT-BH4

Perfusion

Isolated heart Decrease in myocardial [39] ischemia infarction, recovery of myocardial function Isolated heart Inhibition of Caspase-3 [41,42] ischemia Heart Decrease in myocardial [43] ischemia infarction

TAT-c-Jun binding Intradomain (JBD) peritoneal

Effect

References

D. Choi et al. / Journal of Controlled Release 140 (2009) 194–202

endothelial function, both of which have a common risk factor, nitric oxide (NO)-associated oxidative stress. Recent studies, which showed relationship between VEGF and NO production, demonstrated that VEGF and the stimulation of NO production are important factor in coronary collateral growth [35]. Furthermore, animal studies showed that the administration of both VEGF and FGF protein to ischemic heart led to increased collateral blood flow in the ischemic myocardium in animal model [36] (Table 2). 3.1.3. Anti-apoptotic fusion proteins The crucial roles of Bcl-2 protein family in anti-apoptosis and proapoptosis have been widely investigated. Bcl-2 and Bcl-xL, inhibitors of apoptosis, are anti-apoptotic protein members, while Bax, Bak and Bid, inducers of apoptosis, are pro-apoptotic protein members. The balance between anti-apoptotic proteins and pro-apoptotic protein inside cell is considered to determine survival or death following an apoptotic stimulus. FNK which is a Bcl-xL derivative is constructed from a structural remodeling of Bcl-xL and have the powerful cytoprotective activity against various death stimuli in ischemia/ reperfusion injuries of the brain, liver and heart [37]. The protective function of TAT-FNK fusion protein has been demonstrated in apoptotic hepatic cells and hearts infarcted by ischemia/reperfusion. Levels of a mediator of apoptosis, caspase-3 and the vacuolized area that represents cytoplasmic degeneration were reduced by ~ 80% and ~ 50% after TAT-FNK treatment, respectively [38]. TAT-FNK treatment also showed temporally recovered function of left ventricular, inhibition of procaspase-3 cleavage and reduced number of stained cells in TUNEL assay, suggesting the reduction of myocardial infarction area through the suppression of myocardial apoptosis following ischemia/reperfusion [39]. BH domains, which inhibit apoptosis by a regulation of MPT, are highly conserved regions in anti-apoptotic members of Bcl-2 family [40]. Sugioka et al. evaluated the anti-apoptotic ability of BH4 in the ischemia/reperfusion-induced heart failure with a pre-treatment of TAT-BH4, and a considerable suppression in heart injury was observed [41]. Bcl-2 and Bcl-xL are localized in mitochondria as well as nuclear envelop and cytosol, and mainly prevent the release of apoptotic factors, including cytocrome c and Smac/DIABLO, from mitochondria. The Bcl-2 family regulate permeability transition pore (PT pore) by opening or closing of the voltage-dependent anion channel (VDAC, also called porin), a major protein of the mitochondrial outer membrane, controlled by the anti-apoptotic or pro-apoptotic members. BH4 domain plays an essential role to inhibit VDAC activity [37]. As a result of the relationship between BH4 domain and mitochondria, mitochondria respiration was markedly recovered in TAT-BH4 treated group, and TAT-BH4 decreased expression of caspase-3 and TUNEL-positive cells, demonstrating that the myocardial ischemia/reperfusion injury was induced through mitochondrial apoptosis [42]. In spite of advantageous ability to inhibit the apoptosis over Bcl-2 and Bcl-xL, BH4 domains can only prevent some types of apoptosis [41].

Table 2 The clinical trials of therapeutic growth factors. Growth factor

Injection route

Trial

Effect

References

VEGF VEGF

Intracoronary Intravenous

Phase I Phase I

[44] [45]

VEGF VEGF

Intracoronary Intracoronary/ intravenous Intracoronary/ intravenous

Phase I VIVA trial Phase I

Improvement in perfusion Improvement in SPECT, collaterals Improvement in perfusion Improvement in angina class

Intracoronary

Phase I

FGF-2

FGF-2

Improvement in perfusion, attenuation of stress-induced ischemia Improvement in QOL

[46] [47] [24]

[48]

SPECT — single-positron emission-computed tomography; QOL — quality of life.

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3.2. Cell therapy Cell therapy refers to the transplantation of human or animal cells to replace or repair damaged tissues or cells. Cell therapy presents new opportunities to enhance cardiac performance through transdifferentiation of the injected cells into cardiomyocytes. Although early studies opened the plausibility of cell therapy to regenerate cardiomyocytes, subsequent studies failed to advance these initial observations. The mechanistic underpinnings of cardiac cell therapy appear to be far more complex. It has been reported that cell therapy may promote vascularization by physical fusion of the injected cells into new capillaries or in perivascular cells. Cell transplantation is considered to be a novel and hopeful therapeutic alternative to support endogenous regenerative mechanisms in ischemic heart disease and heart failure. In the past decade, bone marrow derived cells, skeletal myoblasts, embryonic stem cells, adipose tissue derived resident cardiac progenitor cells and umbilical cord derived cells have been tested for cardiac repair [49]. Bone marrow stem cells (BMCs) and hematopoietic progenitor cells (HPCs) have been widely challenged in cell transplantation after ischemic heart disease. However, due to the heterogeneous nature of the used cell populations, the most effective cell type and ordered cell dose have not been clearly determined, and recent clinical trials have represented conflicting results [1]. Bone marrow incorporates several cell populations that have the capacity to proliferate, migrate and differentiate into various mature cell types. Surrounded by these are haematopoietic stem cells (HSC), endothelial progenitor cells (EPC), mesenchymal stem cells (MSC), multipotent adult progenitor cells (MAPC), and side population cells. HSC have been used spaciously for cell transplantation in haematological disorders because these cells can differentiate into all types of blood cells. The recent studies, however, could not replicate the promising in vivo transdifferentiation data. EPC dwells from the bone marrow and moves can stimulate to the location of circumferential circulation, home to sites of ischemia and stimulate neovascularization. These cells can be isolated from bone marrow or from peripheral blood following mobilization with cytokine controls. MSCs have been identified as potential candidates for cell therapy. In the animal models, a close normalization of ventricular function after myocardial infarction was observed after transplantation of MSCs. Therefore, MSCs may be used as a novel therapy showing regenerative and protective effects against injured myocardium, although the mechanism remains obscure. However, a combination of these above factors may provide to myocardial repair. More attention should focus on explaining the mechanisms. This will help describe the optimal conditions for therapeutic application [1]. 3.2.1. Bone marrow derived cells HSCs are confirmed by the expression of the hematopoietic marker proteins CD34 or CD133. These cells have been used commonly for cell transplantation in the world, because these cells can give rise to various types of blood cells [49]. In 2001, Orlic et al. suggested that a bone marrow cell transplanted in the infarcted heart. The transplanted cells generated substantial amounts of new myocardium, comprising multiplication myocytes as well as vascular structures [50]. Recent studies could not reproduce the expecting in vivo data. HSC transplantation prevented left ventricular (LV) dilatation and improved LV function, proposing that endothelial progenitor cell mechanisms other than differentiation promoted functional recovery [49]. EPCs existent in the bone marrow and have the capacity to migrate to the peripheral circulation, home to sites of ischemia and stimulate neovascularisation. These cells were able to isolate from bone marrow or from peripheral blood following mobilization. EPC were confirmed by presentation of the hematopoietic markers CD133, the co-expression of vascular endothelial growth factor (VEGF), and the ability to differentiate into endothelial cells [49]. Some researchers reported that plate expanded these cells also contained CD14+/CD34-cell population, which promotes neovascularisation by secreting pro-

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angiogenic factors. Other researchers proposed that a subpopulation of circulating myeloid cells could also promote endothelial cell production by releasing angiogenic factors [51]. Forcing evidence to agree that EPC transplantation may preserve LV function after myocardial infarction through promoting neovascularisation. The effects' mechanism for this therapy was primarily considered to be combination, differentiation and proliferation of EPC for new blood vessel formation. Recent studies suggested that EPC also have the possible to differentiate into cardiomyocytes in vivo. These discoverings have led to a new concept that EPC may improve both vasculogenesis and cardiomyogenesis, making this cell type a promising candidate for cardiac cell therapy. In spite of these confidence in experimental data there is growing information that EPC numbers and their ability to promote neovascularisation are impaired in patients with ischemic heart diseases, which may limit the therapeutic effectiveness in the clinical setting. MSCs indicate a group of distinct stem cell populations found in the adult bone marrow non-hematopoietic tissues. MSCs can be isolated and expanded from adult bone marrow in vitro without any apparent modification in phenotype or loss of pluripotent function. Purified and culture-expanded MSCs have the ability to differentiate into multiple phenotypes various types of both in vitro and in vivo [52]. The first use of MSC as a cell for cardiomyoplasty was reported in 1999 by Tomita et al. in a rat model. Five weeks after transplantation, bone marrow cells were established in all animals and expressed muscle specific proteins not present before cell implantation. Furthermore, improved systolic and diastolic function was observed in animals receiving cells pretreated with specific drugs or some chemicals, which has been reported to enhance differentiation of cells. Several experimental studies suggested that MSC have the potential to differentiate in functional cardiomyocytes both in vivo and in vitro. In addition, cell transplantation certainly improved LV function and beneficially affected LV remodeling [49]. Another advantage of MSC is plausibility of an allogenic setting since they have low immunogenicity. Allogenic MSCs are not rejected in vivo studies showed that by the recipient host, we expect that the future use of allogenic MSC has significant clinical potential. It was reported that when transplanted into lethally irradiated mice, they were able to home to areas of damage in ischemic mouse hearts and differentiate into vascular endothelial cells and cardiac myocytes [53]. These so called side population cells are characterized by their natural capacity to efflux Hoechst 33342, a member of the ATP binding cassette transporter family. Side population cells have also can be isolation from extra-hematopoietic tissues, including skeletal muscle and adult cardiac tissue. Pfister et al. established a cardiac side population, which was efficient of differentiating into mature cardiomyocytes through a process mediated by cellular linking with adult cardiomyocytes [54]. Therefore, the additional study which investigates the possible of side population cells to repair ischemic myocardium in vivo may be of interest which is considerable. 3.2.2. Adipose tissue derived cells Adipose tissue is derived from the embryonic mesenchyme. Recently studies demonstrated that adipose tissue derived cells could differentiate into cardiomyocytes with morphological, molecular and functional properties. In a mouse model, adipose tissue derived cells had the potential to transdifferentiate into an endothelial phenotype and promote tissue vascularisation [55]. The facilitation of access to fat and its abundance in humans make adipose tissue derived cells an appealing source for cell based cardiac repair [49]. 3.2.3. Umbilical cord derived cells Umbilical cord blood contains a number of progenitor cell populations, including HSC, MSC and unrestricted somatic stem cells (USSC). USSC have a high proliferation potential and have been suggested to be capable of differentiating into several cell lineages, including cardio-

myocytes. Human USSC transplantation in a myocardial infarction model prevented LV dilatation and enhanced LV function. The implanted cells also increased regional perfusion, proposing that USSC may also relate in the formation of new blood vessels [49]. It was reported that intracoronary injection of USSC in a porcine infarction model did not attenuate LV remodeling or ameliorate LV function [56]. 3.2.4. Embryonic stem cells (ESC) Embryonic stem cells have several advantageous features for cardiac cell therapy — they have unlimited proliferation possible and can form cardiomyocytes with a distinct electrophysiologic and contractile phenotype [57]. Researchers demonstrated that transplanted embryonic stem cells could differentiate into cardiomyocytes and improve the contractile function of previously infarcted myocardium through the animal model [58]. Embryonic stem cell transplantation provides an expected framework for cell based cardiac repair, their use in clinical studies is inhibited by unsolved ethical and legal issues, the risk of rejection by the immune system, and concern about potential of tumor formation. Cell therapy is a promising therapeutic modality for patients. Animal model studies supplied evidence that various cell types may beneficially involve myocardial perfusion and contractile performance. However, a number of open questions with regard to basic mechanisms and definition of a favorable therapeutic approach remain, which stimulate additional preclinical research. In particular, forthcoming studies should further explain the precise cellular mechanisms by which the cell therapy can improve cardiac performance [49]. 3.3. Gene therapy Recent achievements in the cardiovascular area have been made with viral and non-viral gene therapies. A variety of catheter or surgical approaches for in vivo cardiac gene transfer showed promising results in animal and clinical studies. Transgene expression would be required only during a period of defined risk, such as remodeling after myocardial infarction. In common with the angiogenic protein therapy, angiogenic gene therapy is getting much interest as an alternative therapy to improve the ischemic heart failure. An animal model with chronic ischemic myocardium showed an increase in collateral blood flow and an improvement of cardiac function by an injection of plasmid VEGF or FGF [59,60]. The angiogenic gene therapy intensively studied in human clinical trials. The administration of plasmid VEGF into human ischemic myocardium through a small left anterior thoracotomy resulted in improved heart responses, demonstrating the therapeutic efficacy of this approach [61]. Various isoforms of VEGF have been delivered to patient in clinical studies by different delivery methods. VEGF165 delivered by myocardial injection and intramyocardial transfection to patient demonstrated the significant improvement of ischemic myocardium area with increased perfusion and the reduced angina, respectively [62,63]. The administration of VEGF in those studies resulted in increased level of VEGF in plasma, providing a direct evidence of the transfection and expression of plasmid VEGF. Viral vectors are frequently used to effectively transfect DNA because the expression level of naked DNA in cells is too low due to its poor transfection efficiency. Adenovirus vector-based injection of VEGF121 led to an improvement of angina in all patients and of exercise time with increased VEGF level in plasma in phase I trial [64]. Similar to VEGF, administration of FGF into epicardial fat demonstrated an improvement of angina symptoms and an increase in myocardial blood flow [65]. Intracoronary administration of FGF-4 using adenovirus vector also showed significantly improved exercise time in treated group compared to placebo group in angiogenic gene therapy (AGENT) trial [66]. The gene therapy has the great potential to the ischemic heart diseases; however, the transfection efficiency, stability and longterm expression of the therapeutic genes should be improved to be practical.

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4. Polymeric hydrogels for delivery of therapeutic proteins Despite recent progress in production of various therapeutic proteins, it is still challenging to regulate enzymatic susceptibility, stability during storage, and efficacy of the proteins in the body [67]. Many studies have been reported to overcome these limitations and to prolong the biological activity of proteins in the body. One potential approach includes a use of delivery systems to achieve the controlled protein release, systemically or locally, over an extended time period. These delivery systems can protect protein drugs from enzymatic degradation and antibody neutralization, allowing a prolonged retention of the protein drugs in the body. Polymeric hydrogels have been of special interest as a delivery vehicle of various drug molecules including peptides and proteins [68,69]. Hydrogels are three-dimensionallystructured networks of hydrophilic polymers containing a large amount of water. Hydrogels can be formed through chemical or physical crosslinking of polymers, and have structural similarity to the macromolecular-based components in the body. In addition, hydrogels can be injected into the body via minimally invasive administration, which may reduce the pain of patients [70,71]. In this review, potential and currently used biocompatible polymer-based hydrogels for protein delivery are summarized. 4.1. Natural polymer-based hydrogels Collagen is a main component of the extracellular matrices (ECMs) of tissues in the body and forms thermally reversible gels. Chemically cross-linked collagen gels were also reported to improve the physical properties [72,73] and used for delivery of various growth factors [74]. A bioactive glass (Bioglass® 45S5) loaded into collagen gels showed proangiogenic potential over a limited range of concentrations, which induced endothelial cell proliferation, tubule formation in co-culture, and upregulated vascular endothelial growth factor (VEGF) production [75]. Gelatin is a denatured form of collagen and composed of singlestrand molecules unlike the triplex helix structure of collagen. Gelatin is biocompatible and easily forms gels by temperature changes. Similar to collagen, gelatin has been frequently used for protein delivery [76]. Myocardial infarction-induced heart failure was carried out on pigs. The bFGF-loaded gelatin gels were injected into the left ventricular wall of the pig heart, and an improvement in left ventricular function was observed [77]. Site-specific intra-arterial delivery of bFGF-releasing gelatin gels was useful to augment functional collateral vessels in ischemic hindlimb of rabbits. After 28 days of intra-arterial administration, remarkable collateral vessel improvement was observed in the hindlimbs [78]. Fibrin plays an important role in natural wound healing, and has been frequently used as surgery sealant and adhesive [79]. A fibrin gel is formed by enzymatic polymerization of fibrinogen in the presence of thrombin. Fibrin gels can be used as an autologous carrier for protein delivery, as they can be produced from the patient's own blood. Fibrin gels have been used to deliver various growth factors [80], and the addition of heparin to a fibrin gel was useful for the sustained release and enhanced activity of angiogenic factors [81]. The bFGF-loaded fibrin gels were injected into mouse ischemic limbs, and a significant increase of the microvessel density in the ischemic site was observed by immunohistological analysis (Table 3). Hyaluronic acid is a glycosaminoglycan (GAG) component of the ECMs in the body and is degradable by hyaluronidase [82]. Hyaluronic acid can form hydrogels by various covalent cross-linking methods [83,84]. Cross-linked hyaluronan gels, loaded with VEGF and keratinocyte growth factor (KGF), were reported to promote new microvessel growth in vivo. Hyaluronan gels containing growth factors were subcutaneously implanted into the ear of mice. Strikingly, intact microvessel beds with well-defined borders and the greatest angiogenic response were generated in case of using gels containing both VEGF and

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KGF [85]. A covalently bound heparin provided a cross-linkable heparan sulfate analog, which forms a gel that can release basic fibroblast growth factor (bFGF) in a sustained manner, and neovascularization increased dramatically when the gels were transplanted into mice [86]. Alginate is obtained from brown seaweeds, and has been frequently used for many biomedical applications due to its biocompatibility, low toxicity, and simple gelation behavior with divalent cations (e.g., calcium ions) [87]. Alginate has been widely used as an injectable of cells and proteins [88,89]. Alginate gels were used as a localized delivery vehicle of angiogenic molecules, and the ability to promote new blood vessel formation was tested in vivo. The in vitro release rates of VEGF and bFGF were similar. However, VEGF was more appropriate for creating a dense bed of new blood vessels in vivo [90]. Local and sequential delivery of VEGF followed by platelet-derived growth factor-BB (PDGF-BB) using alginate hydrogels induced mature vessels and improved cardiac function more than single factors after myocardial infarction in vivo [91]. Strikingly, sequential delivery of VEGF and PDGF-BB increased the systolic velocity-time integral, which is a marker of myocardial function. No significant difference in the left ventricular end-systolic dimension (LVDd) and ejection fraction was observed. Chitosan is prepared by N-deacetylation of chitin, usually obtained from shrimp and crab shells. Chitosan has shown excellent biocompatibility, low toxicity, structural similarity to natural GAGs and controlled degradation by enzymes [92]. Chitosan forms hydrogels by physical cross-linking [93] or chemical cross-linking [94]. The bFGFincorporated chitosan gels were immobilized to the ischemic myocardium surface of rabbits with chronic myocardial infarctions. The extent of fibrosis was significantly reduced in infracted area of rabbits treated with bFGF-releasing chitosan hydrogels. The controlled release of bFGF from the gels induced angiogenesis and promoted collateral circulation in the ischemic myocardium [95]. Temperature/ pH-sensitive gels were formed from quaternized chitosan and glycerophosphate, and used as an intelligent carrier. The solution formed a hydrogel once the surrounding temperature reached the body temperature, allowing the sustained release of model drugs in vitro. This system was considered useful for delivery of living cells, proteins, and enzymes [96]. 4.2. Synthetic polymer-based hydrogels Poly(ethylene oxide) (PEO) is one representative biopolymer that has been approved by the FDA for many medical applications due to its biocompatibility and low toxicity. Branched [97] or bi-functional poly (ethylene glycol) (PEG) [98] were used to form gels and were demonstrated to be useful for protein delivery. A heparin-binding peptide was conjugated to four-arm PEG, and the bFGF release from

Table 3 Natural polymer-based hydrogels for the delivery of angiogenic growth factors. Polymer

Therapeutic protein

Injection route

Effect

References

Gelatin

bFGF

Left ventricular wall

[68]

Gelatin

bFGF

Intra-artery

Fibrin

bFGF

Ischemic limbs

Improved Left ventricular function Improved collateral vessels Increased microvessel density Microvessel growth

Hyaluronic VEGF/KGF acid Alginate VEGF/ PDGF-BB Chitosan

bFGF

Subcutaneous implant Local and sequential Immobilization on ischemic myocardium surface

Improved cardiac function Reduced fibrosis/ improved collateral blood flow

[69] [72] [76]

[86]

KGF — keratinocyte growth factor; PDGF-BB — platelet-derived growth factor-BB.

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the resultant gel was mainly dependent on an erosion-controlled process [99]. Enzymatically cross-linked PEG gels were prepared from functionalized PEG and a lysine-containing polypeptide through the action of transglutaminase [100]. Gelation was occurred under mild conditions and diffusion coefficients of small proteins and albumin in the gel were comparable to those in free solution. Various PEO derivatives have also been synthesized and used for protein delivery. Heparin-modified star-shape PEG copolymers were used to form bioactive hydrogels via peptide–saccharide interactions. The sustained release of bFGF from the gel was achieved by gel erosion [101]. The bFGF-releasing nitrocinnamate-derived PEG gels were not cytotoxic to human neonatal fibroblasts, and the released growth factor maintained its activity and induced cell proliferation in vitro. In addition, the effect of the released bFGF on collagen production was determined by quantifying the amount of hydroxyproline in the medium using a liquid scintillation counter. Collagen production was directly proportional to the amount of bFGF released into the medium [102]. Various PEO-based copolymers have been synthesized and used for protein delivery applications [103]. A triblock copolymer of PEO and poly(propylene oxide) (PEO-b-PPO-b-PEO), commercially available as Pluronic or Poloxamer, forms thermally reversible gels, and has been frequently used for drug delivery applications [104]. Degradable PLA-b-PEO-b-PLA gels were prepared, and were injected through a trochar for delivery of proteins [105]. Multi-block copolymers of PEO and PLA were also synthesized and the gels exhibited temperaturedependent reversible sol-gel transitions near the body temperature [106], which was considered useful for delivery of proteins and cells in a minimally invasive manner. Polyacrylamide and its various derivatives were synthesized and cross-linked with native proteins [107], oligodeoxyribonucleotides [108], or engineered coiled-coil proteins [109] to form hydrogels. Poly (N-isopropylacrylamide) (PNIPAAm) is attractive for protein delivery applications, as it undergoes phase transition near the body temperature [110]. Therefore, one can easily prepare a mixed solution of a protein drug and the polymer at room temperature and inject the mixture into the body in a minimally invasive manner using a syringe or endoscope. The mixed solution eventually solidifies and forms a gel in the body. PNIPAAm was useful to deliver VEGF to human vascular endothelial cells over an extended time period [111]. Biodegradable poly(aspartic acid) (PASP) hydrogels and PASP/ gelatin complexes were prepared as pH-sensitive matrices for controlled delivery of myoglobin (Mb), and the pulsatile release of Mb from the matrices was achieved [112]. The pH-sensitive release of bFGF from a biodegradable semi-interpenetrating network composed of poly(γ-glutamic acid) (γ-PGA) and sulfonated γ-PGA was reported. The bFGF released from the gel in vitro maintained its biological activity without denaturation [113]. The release behavior of FGF-2 from gelatin-polylysine (gelatin-PLL) and gelatin-poly(glutamic acid) (gelatin-PLG) hydrogels was sustained over 4 weeks in vitro. FGF-2 releasing gelatin-PLG hydrogels induced marked reperfusion in animals. In contrast, the bolus injection of FGF-2 produced an initial angiogenic response that lasted for 4 weeks. Total limb loss was observed within 1 week in groups without FGF-2. However, groups with gelatin-PLL and gelatin-PLG gels demonstrated no significant change on Laser Doppler perfusion imaging (LPDI) ratios over 8 weeks. Significant capillary regrowth was also observed after 8 weeks, although the LPDI ratios had decreased substantially [114]. 5. Conclusion The coronary artery intervention has been accepted as the most popular regimen for the treatment of coronary artery diseases in conventional medical therapy. In particular, the development of DES dramatically reduced the incidence of restenosis, which is the disadvantage of the conventional bare metal stent (BMS). Inter-disciplinary

researches advanced biopharmaceutical therapy, such as protein, cell and gene therapy, due to their potential advantages over conventional medical therapy for the ischemic diseases. Angiogenesis-based therapy using angiogenic proteins or genes showed positive effects on the ischemic heart in pre-clinical and/or clinical studies, and anti-apoptotic proteins also demonstrated the ability to prevent the apoptosis-induced cell death by inhibiting apoptotic pathway in ischemic heart. The intracellular delivery of those proteins and genes had to be improved in order to maximize their therapeutic efficacy because the target region of the proteins and genes is intracellular organ. PTDs, positively charged peptides including TAT and oligo-arginine, have shown efficient delivery of large molecules into various cells. For the protein and gene delivery, the long-term stability, bioactivity, low side effects and intracellular delivery efficiency should be guaranteed in future in vivo trials. One potential approach to improve their possible limitations of in vivo applications is to use of polymeric drug delivery systems, including hydrogels. Hydrogels with structural similarity to the macromolecularbased components in the body can entrap therapeutic proteins and genes with high loading efficiency, and provide sustained release of biomacromolecules. Among them, stimuli-sensitive systems (i.e., intelligent or smart systems) could be useful for biomacromolecule delivery, as these systems can release targets in response to environmental changes [115]. Overall, combination therapies of the conventional medical therapy and the biopharmaceutical therapy taking advantage of polymeric drug delivery system have great potential and should be further examined for the treatment of ischemic heart diseases in the future. Acknowledgments This work was partially supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST, F104AA010005-08A0101-00510, R01-2007-000-20602-0 (2008)) and Seoul R&BD program (CR070027). References [1] X.J. Wang, Q.P. Li, The roles of mesenchymal stem cells (MSCs) therapy in ischemic heart diseases, Biochem. Biophys. Res. Commun. 359 (2007) 189–193. [2] C. Templin, D. Kotlarz, J. Faulhaber, S. Schnabel, K. Grote, G. Salguero, M. Luchtefeld, K.H. Hiller, P. Jakob, H.Y. Naim, B. Schieffer, D. Hilfiker-Kleiner, U. Landmesser, F.P. Limbourg, H. Drexler, Ex vivo expanded hematopoietic progenitor cells improve cardiac function after myocardial infarction: role of beta-catenin transduction and cell dose, J. Mol. Cell. Cardiol. 45 (2008) 394–403. [3] D.R. Holmes Jr, M.B. Leon, J.W. Moses, J.J. Popma, D. Cutlip, P.J. Fitzgerald, C. Brown, T. Fischell, S.C. Wong, M. Midei, D. Snead, R.E. Kuntz, Analysis of 1-year clinical outcomes in the SIRIUS trial: a randomized trial of a sirolimus-eluting stent versus a standard stent in patients at high risk for coronary restenosis, Circulation 109 (2004) 634–640. [4] A.D. Michaels, K. Chatterjee, Angioplasty versus bypass surgery for coronary artery disease, Circulation 106 (2002) e187. [5] Y.M. Yang, I. Moussa, Percutaneous coronary intervention and drug-eluting stents, Can. Med. Assoc. J. 172 (2005) 323–325. [6] E. Van Belle, K. Abolmaali, C. Bauters, E.P. McFadden, J.M. Lablanche, M.E. Bertrand, Restenosis, late vessel occlusion and left ventricular function six months after balloon angioplasty in diabetic patients, J. Am. Coll. Cardiol. 34 (1999) 476–485. [7] R. Bhatheja, D. Mukherjee, Acute coronary syndromes: unstable angina/non-ST elevation myocardial infarction, Crit. Care Clin. 23 (2007) 709–735. [8] J. Hirsh, Low-molecular-weight heparin: a review of the results of recent studies of the treatment of venous thromboembolism and unstable angina, Circulation 98 (1998) 1575–1582. [9] J.L. Anderson, C.D. Adams, E.M. Antman, C.R. Bridges, R.M. Califf, D.E. Casey, W.E. Chavey, F.M. Fesmire, J.S. Hochman, T.N. Levin, A.M. Lincoff, E.D. Peterson, P. Theroux, N.K. Wenger, R.S. Wright, S.C. Smith, A.K. Jacobs, C.D. Adams, J.L. Anderson, E.M. Antman, J.L. Halperin, S.A. Hunt, H.M. Krumholz, F.G. Kushner, B.W. Lytle, R. Nishimura, J.P. Ornato, R.L. Page, B. Riegel, ACC/AHA 2007 guidelines for the management of patients with unstable angina/non-ST-elevation myocardial infarction—executive summary, J. Am. Coll. Cardiol. 50 (2007) 652–726. [10] C. Sõti, E. Nagy, Z. Giricz, L. Vígh, P. Csermely, P. Ferdinandy, Heat shock proteins as emerging therapeutic targets, Br. J. Pharmacol. 146 (2005) 769–780. [11] P. Ferdinandy, Z. Szilvassy, G.F. Baxter, Adaptation to myocardial stress in disease states: is preconditioning a healthy heart phenomenon, Trends Pharmacol. Sci. 19 (1998) 223–229. [12] P. Ferdinandy, Myocardial ischaemia/reperfusion injury and preconditioning: effects of hypercholesterolaemia/hyperlipidaemia, Br. J. Pharmacol. 138 (2003) 283–285.

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