Bone marrow derived cells in collateral formation : studies toward therapeutic arteriogenesis
Hellingman, A.A.
Citation
Hellingman, A. A. (2011, September 15). Bone marrow derived cells in collateral formation : studies toward therapeutic arteriogenesis. Retrieved from https://hdl.handle.net/1887/17838
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Chapter 1
General Introduction
BACKGROUND
Peripheral artery disease (PAD) is a major health issue of the Western world and is associated with symptoms ranging from intermittent claudication to rest pain, ulcers and gangrene1. Advanced PAD leads to significant decreased quality of life, morbidity and mortality. Despite a variety of treatment options like percutaneous transluminal angioplasty (PTA), stenting or bypass surgery, major amputation is reported in up to one third of patients with severe PAD2. In case of extended PAD with multi-level vascular oc- clusions and particularly low quality run-off crural vessels with minor outflow, treatment options become limited. These “no-option” patients underscore the urgent need for new therapeutic interventions. Exploring new strategies for biological revascularization of ischemic limbs is therefore of great importance.
TWO BASIC PRINCIPLES OF NEOVASCULARIZATION: ANGIOGENESIS AND ARTERIOGENESIS
Neovascularization mechanisms include capillary growth (angiogenesis) and the de- velopment of collateral arteries (arteriogenesis). Angiogenesis involves the sprouting of small endothelial tubes occurring from existing capillaries in response to hypoxia3–5. Hypoxia is the strong stimulus and upregulates the hypoxia-inducible factor-1_ (HIF- 1_), which induces a number of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and stromal derived factor-1 (SDF-1). A series of sequential events can then be distinguished, consisting of activation of the endothelial cells, an increase in permeability and a disruption of the basement membrane and interstitial matrix via proteolytic degradation 6. Endothelial cell migration and sprouting into the extra- vascular space towards the angiogenic stimuli is mediated by the interaction of protrud- ing filipodia with the extracellular matrix surrounding the cells7, 8. Cells then elongate and align to form vascular sprouts and these sprouts will form a lumen and connect with neighboring vessels. A recent report showed that monocytic cells play a chaperone function in the fusion of sprouting capillaries forming vascular anastomoses adding new circuits to the existing vessel network9. The newly formed capillaries are small, with a diameter of about 10–20 +m. Subsequently, angiogenesis alone cannot compensate occluded arteries sufficient in the prevention of tissue damage due to ischemia.
Arteriogenesis is the formation of larger collateral arteries to bypass occluded arter- ies. It was defined as the development of adult collateral arteries from the enlargement of pre-existing collateral arterioles10. The driving force to initiate arteriogenesis is the increase of shear stress against the vessel wall induced by the narrowing or occlusion of the artery11–13. Changes of shear stress regulate the expression of intracellular adhesion
12 Chapter 1
molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)11. The upregula- tion of these adhesion molecules and the upregulation of monocyte chemoattractant protein-1 (MCP-1) are potentially important for the adhesion to the endothelium and subsequent invasion of monocytes into the vessel wall14, 15. Monocytes can promote arteriogenesis by the secretion of different chemokines and growth factors16–19. A strong inflammatory reaction then occurs with the extravasation of different leukocytes like CD4+ T cells20–22, CD8+ T cells23 and Natural Killer (NK) cells21 around the growing collat- eral arteries. The invading cells migrate to the perivascular space and activate proteases in the pericollateral space, which degrade matrix components of the connective tissue surrounding the arteriole, creating space for collateral wall expansion24. In addition, these leukocytes can produce various factors leading to growth of pre-existing arteri- oles by proliferation and migration of smooth muscle cells and endothelial cells20.
THERAPEUTIC NEOVASCULARIZATION
The two above described concepts of vascular formation play both a role in adult neovascularization and usually occur simultaneously but at different anatomical levels.
Therefore, to improve oxygenation status of ischemic tissue, stimulation of both arterio- genic collateral arteries and angiogenic capillaries are crucial for sufficient blood flow.
Clinical trials with angiogenic growth factors
Therapeutic angiogenesis is a strategy for the treatment of ischemic vascular disease that uses angiogenic growth factors or genes encoding these proteins to induce neo- vascularization. Evaluation of recombinant growth factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) was performed in preclinical studies in models of peripheral artery disease25, 26. However, the use of recombinant growth factor proteins seemed to be restricted by its limited tissue half-life. Accord- ingly, subsequent experiments studied the potential use of gene therapy. Gene therapy is a promising therapeutic tool that overcomes the inherent instability of angiogenic growth and facilitates sustained and local production of angiogenic growth factors27, 28. In 1996 Isner et al.29 published the first case report of administration of phVEGF (plasmid DNA vector encoding human VEGF) via balloon angioplasty for the treatment of PAD.
Since the initial clinical trial of gene therapy, various groups have evaluated the use of VEGF-A, VEGF-C, FGF-1, FGF-4 and hepatic growth factor (HGF) for treatment of PAD30. Early preclinical studies and phase I clinical studies achieved promising results, however phase III clinical trials have failed to demonstrate unambiguous success in that angio- genic agents are beneficial in patients studies. To illustrate this, local gene therapy using non-viral plasmid DNA encoding acidic FGF demonstrates in a phase II trial statistical
improvement in ulcer healing and a significantly reduced risk of all amputations31. How- ever, in the large multicenter clinical trial (TAMARIS phase III trial) enrolling 525 patients with limb ischemia, the results did not demonstrate any benefit of the treatment with this FGF gene therapy on amputation rate or wound healing32.
Cell therapy for therapeutic neovascularization
The concept of arteriogenesis strengthens the importance of various different bone mar- row (BM) derived (precursor) cells and inflammatory cells. The recruitment of different BM-derived cells is a central event in arteriogenesis and leads to the fundamental idea of the application of these cells for the development of new therapeutic approaches. Asa- hara et al. 33 discovered in 1997 a subset of bone marrow derived cells called endothelial progenitor cells (EPCs). These cells were able to differentiate into endothelium and pro- mote new blood vessel formation 34 in vitro and in vivo. Despite the fact that there are more than 800 articles35 published about these EPCs, there is still controversy about the nature and description of the human EPC36. Because of the lack of standardized criteria or clear definition and the abundance of different EPC characteristics reported such as EPC colony formation or EPC number in peripheral blood, comparing between different studies has become very complex37. Bone marrow derived mononuclear cells (BM-MNCs) or monocytic cells are also highlighted as possible candidates for a cell therapy. Tateishi- Yuyama et al. 38 reported for the first time about the use of BM-derived mononuclear cells (MNCs) (including EPCs) in the treatment of limb ischemia in patients with PAD. In their randomized controlled trial 22 patients with bilateral limb ischemia were randomly treated with either autologous BM-MNCs or with peripheral blood mononuclear cells as a control. Ankle brachial index (ABI) improved in limbs injected with BM-MNCs in 13 out of 20 patients. Pain at rest in limbs treated with BM-MNCs was abolished in 16 out of 20 patients and the pain free walking distance was also improved. The publication of Tateishi-Yuyama’s TACT (Therapeutic Angiogenesis using Cell Transplantation) study raised considerable interest and led to many clinical trials using stem cells or BM-derived cells in patients with PAD. About 700 PAD patients without revascularization options were treated in these trials39. Considering all these trials in a recent meta-analysis, the hard objective endpoints like ulcer healing and amputation rate were significantly improved in patients treated with cell therapy. Subjective endpoints such as pain free walking distance and pain scale showed also significant improvement, but are more susceptible to placebo effect39, 40. Unfortunately, various limitations of these trials like small number of patients included, variations in degree of ischemia throughout study groups and the lack of a control group, make these studies not easy to interpret.
14 Chapter 1
MOUSE MODEL FOR TESTING THERAPEUTIC APPROACHES
The development of new therapeutic approaches requires careful preclinical investiga- tion involving the use of animal models of human disease. Many research groups use the hind limb ischemia mouse model to mimic PAD. In this model, unilateral hind limb ischemia is operatively induced by electro-coagulation of the artery. First, good vessel exposure is obtained by dissecting the artery from the vein and nerve. Subsequently, hind limb ischemia is induced by electro-coagulation of the artery at a standardized anatomical level. In this model, the contra-lateral leg functions as a control. This model is characterized by post-operative blood flow reduction and subsequent endogenous blood flow restoration in the hind limb. Blood flow restoration is demonstrated with Laser Doppler Perfusion Imaging (LDPI). Repetitive LDPI measurements can follow post-ischemic blood flow recovery within time. Post-ischemic neovascularization, which consists of angiogenesis and arteriogenesis, can be studied at the tissue level with immunohistochemistry. Capillaries in the post-ischemic calf muscle and collaterals in the post-ischemic adductor muscle can be visualized and quantified using antibodies recognizing CD31 on endothelial cells or _-smooth muscle actin in smooth muscle cell layers of collaterals respectively. Furthermore, angiography and micro-computed tomography (micro-CT) are used to visualize post-ischemic collateral artery and capil- lary formation. The differences between animal models and patients with PAD cannot be understated. The animals used in preclinical studies are usually young and healthy, whereas PAD patients are typically older with various comorbidities. Furthermore, PAD in patients develops chronically, whereas ischemia in the mouse model is induced by acute electro-coagulation of the artery. Additionally, PAD is a multi-level occlusion dis- ease and mainly the ischemia in the mouse model is performed at one anatomical level.
OUTLINE OF THE THESIS
The first part of this thesis contains two important pillars of this thesis: inflammatory key players in arteriogenesis and the hind limb ischemia mouse model in which we study different cells and their role in arteriogenesis. Wolfgang Schaper41 started studies on cel- lular and molecular mechanisms of collateral artery growth already in 1966. A detailed overview of leukocytes involved in arteriogenesis is provided in Chapter 2. In particular, the role of monocytes, NK cells, T cells and various subpopulations of T cells will be ad- dressed extensively. A good understanding of the mechanisms how different leukocytes are involved in arteriogenesis is important for the design of effective strategies for the treatment of patients with PAD.
Experimental mouse models have been developed to study the complex arteriogenic process. Today, a broad spectrum of pro-arteriogenic cells and growth factors has been shown to be able to stimulate collateral artery formation in hind limb ischemia models in mice. Nevertheless, a large variety of surgical procedures to induce hind limb isch- emia complicate the interpretation and comparison of these results. Therefore, chapter 3 provides an overview of different surgical approaches to induce hind limb ischemia in mice and study differences in outcomes of various surgical variations. An alternative model of double electro-coagulation of both the femoral artery and iliac artery for test- ing new therapeutic approaches is studied, since the extremely fast blood flow recovery in the original hind limb ischemia mouse model makes it difficult to test the potential stimulating effects of different cells in collateral artery formation.
The second part of this thesis studies different bone marrow derived cells and inflam- matory cells in the hind limb ischemia mouse model. Bone marrow derived mononuclear cells (BM-MNCs) gain interest in stimulating collateral artery formation. Promising results are reported from initial clinical trials using autologous BM-MNC transplantation in PAD patients. But the exact cellular kinetics of these cells is still unclear. Chapter 4 studies MNC-behavior by monitoring the presence of MNCs after transplantation into a hind limb ischemia mouse model with molecular imaging. Next to the fact that we want to answer critical questions regarding MNC survival and homing to optimize clinical use, functional consequences of different transplantation routes (intramuscular injections versus intravenous transfusion) or dosage of cells needs to be elucidated. Moreover, it is still unknown what exact cell type is required for the induction of arteriogenesis and how these cells need to be (pre-) treated. In the mononuclear cell fraction, monocytes comprise a highly plastic and divergent cell population that may differentiate into pro- genitor cells, paracrine pro-arteriogenic factor releasing cells, capillary bridging tip cells etc. The beneficial effect of monocytes transfusion on arteriogenesis was illustrated by several recent mouse studies42, 43. Monocytes play a significant role in arteriogenesis, but the exact mechanism is not fully understood yet. It seems that these cells need a differentiation along a pro-arteriogenic-supporting pathway to support this process.
Recently, Beem et al.44 reported that co-culture of monocytes with activated CD4+ T cells was indispensable for colony-forming-unit (CFU)-Hill colonies, which were cor- related to vascular reactivity. Chapter 5 studies pre-stimulation of human monocytes with soluble factors derived from activated CD4+ T cells and the effects of these pre- stimulated monocytes on arteriogenesis. An additional interesting role for monocytes was observed in the arteriogenesis studies in protease-activated receptor (PAR)-1-/- mice and PAR-2-/- mice as described in chapter 6. The activation status of monocytes differs between these mice and results in differences in arteriogenesis.
Our research group reported previously that T cells are involved in the regulation of arteriogenesis21. Furthermore, impaired blood flow recovery was observed after hind
16 Chapter 1
limb ischemia in CD4-/- mice22 and CD8-/- mice23. The contribution of a specific cell of the immune system, regulatory T cells (CD4+CD25+FoxP3+ T cell) and their role in arte- riogenesis is studied in chapter 7. Here, the contribution of regulatory T cells in the arteriogenic response is studied by major modulations in regulatory T cell number in vivo.
The last part of this thesis studies a new approach to stimulate post-ischemic neovas- cularization. Chapter 8 describes a new approach to stimulate hypoxia inducible factor (HIF)-dependent growth factors using the silencing of the HIF-1_ degrading enzyme prolyl hydroxylases (PHD2). Silencing was achieved using the overexpression of a specific shRNA45 targeting PHD2 by a novel gene transfer vector, a so-called minicircle vector.
These minicircles are devoid of bacterial DNA sequence and therefore are thought to be a more effective vector than traditional vectors. The inhibition of PHD2 should prevent the ubiquitinilation of HIF-1_ which consequently should result in an upregulation of a series of pro-arteriogenic and pro-angiogenic factors stimulating neovascularization.
Finally, the studies described in this thesis are discussed and placed in a broader perspective in chapter 9, the general discussion.
REFERENCE LIST
1. Hiatt WR, Goldstone J, Smith SC, Jr., McDermott M, Moneta G, Oka R, et al. Atherosclerotic Pe- ripheral Vascular Disease Symposium II: nomenclature for vascular diseases. Circulation 2008 Dec 16;118(25):2826-9.
2. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG, et al. Inter-Society Consen- sus for the Management of Peripheral Arterial Disease (TASC II). Eur J Vasc Endovasc Surg 2007;33 Suppl 1:S1-75.
3. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003 Jun;3(6):401-10.
4. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996 Aug 9;86(3):353-64.
5. Carmeliet P. Angiogenesis in health and disease. Nat Med 2003 Jun;9(6):653-60.
6. Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes memorial Award lecture. Cancer Res 1986 Feb;46(2):467-73.
7. Gross JL, Moscatelli D, Rifkin DB. Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro. Proc Natl Acad Sci U S A 1983 May;80(9):2623-7.
8. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000 Apr;6(4):389-95.
9. Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q, Prykhozhij S, et al. Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood 2010 Aug 5;116(5):829-40.
10. van Royen N, Piek JJ, Buschmann I, Hoefer I, Voskuil M, Schaper W. Stimulation of arteriogenesis; a new concept for the treatment of arterial occlusive disease. Cardiovasc Res 2001 Feb 16;49(3):543- 53.
11. Hoefer IE, van Royen N, Rectenwald JE, Deindl E, Hua J, Jost M, et al. Arteriogenesis proceeds via ICAM-1/Mac-1- mediated mechanisms. Circ Res 2004 May 14;94(9):1179-85.
12. Deindl E, Buschmann I, Hoefer IE, Podzuweit T, Boengler K, Vogel S, et al. Role of ischemia and of hypoxia-inducible genes in arteriogenesis after femoral artery occlusion in the rabbit. Circ Res 2001 Oct 26;89(9):779-86.
13. Hershey JC, Baskin EP, Glass JD, Hartman HA, Gilberto DB, Rogers IT, et al. Revascularization in the rabbit hindlimb: dissociation between capillary sprouting and arteriogenesis. Cardiovasc Res 2001 Feb 16;49(3):618-25.
14. Heil M, Schaper W. Influence of mechanical, cellular, and molecular factors on collateral artery growth (arteriogenesis). Circ Res 2004 Sep 3;95(5):449-58.
15. Herold J, Pipp F, Fernandez B, Xing Z, Heil M, Tillmanns H, et al. Transplantation of monocytes:
a novel strategy for in vivo augmentation of collateral vessel growth. Hum Gene Ther 2004 Jan;15(1):1-12.
16. Kusch A, Tkachuk S, Haller H, Dietz R, Gulba DC, Lipp M, et al. Urokinase stimulates human vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk2 interaction. J Biol Chem 2000 Dec 15;275(50):39466-73.
17. Menshikov M, Elizarova E, Plakida K, Timofeeva A, Khaspekov G, Beabealashvilli R, et al. Urokinase upregulates matrix metalloproteinase-9 expression in THP-1 monocytes via gene transcription and protein synthesis. Biochem J 2002 Nov 1;367(Pt 3):833-9.
18. Khan KM, Falcone DJ. Role of laminin in matrix induction of macrophage urokinase-type plasmin- ogen activator and 92-kDa metalloproteinase expression. J Biol Chem 1997 Mar 28;272(13):8270- 5.
18 Chapter 1
19. Schirmer SH, van Nooijen FC, Piek JJ, van Royen N. Stimulation of collateral artery growth: travel- ling further down the road to clinical application. Heart 2009 Mar;95(3):191-7.
20. Schaper W, Scholz D. Factors regulating arteriogenesis. Arterioscler Thromb Vasc Biol 2003 Jul 1;23(7):1143-51.
21. van Weel V, Toes RE, Seghers L, Deckers MM, de Vries MR, Eilers PH, et al. Natural killer cells and CD4+ T-cells modulate collateral artery development. Arterioscler Thromb Vasc Biol 2007 Nov;27(11):2310-8.
22. Stabile E, Burnett MS, Watkins C, Kinnaird T, Bachis A, la Sala A, et al. Impaired arteriogenic re- sponse to acute hindlimb ischemia in CD4-knockout mice. Circulation 2003 Jul 15;108(2):205-10.
23. Stabile E, Kinnaird T, la Sala A, Hanson SK, Watkins C, Campia U, et al. CD8+ T lymphocytes regulate the arteriogenic response to ischemia by infiltrating the site of collateral vessel development and recruiting CD4+ mononuclear cells through the expression of interleukin-16. Circulation 2006 Jan 3;113(1):118-24.
24. Kinnaird T, Stabile E, Burnett MS, Epstein SE. Bone-marrow-derived cells for enhancing collat- eral development: mechanisms, animal data, and initial clinical experiences. Circ Res 2004 Aug 20;95(4):354-63.
25. Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor.
J Vasc Surg 1992 Aug;16(2):181-91.
26. Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest 1994 Feb;93(2):662-70.
27. Giordano FJ, Ping P, McKirnan MD, Nozaki S, DeMaria AN, Dillmann WH, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nat Med 1996 May;2(5):534-9.
28. Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, et al. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral develop- ment and tissue perfusion. Circulation 1996 Dec 15;94(12):3281-90.
29. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, et al. Clinical evidence of angiogen- esis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet 1996 Aug 10;348(9024):370-4.
30. Gupta R, Tongers J, Losordo DW. Human studies of angiogenic gene therapy. Circ Res 2009 Oct 9;105(8):724-36.
31. Baumgartner I, Chronos N, Comerota A, Henry T, Pasquet JP, Finiels F, et al. Local gene transfer and expression following intramuscular administration of FGF-1 plasmid DNA in patients with critical limb ischemia. Mol Ther 2009 May;17(5):914-21.
32. Hiatt WR, Baumgartner I, Van Belle E. NV1FGF Gene Therapy on Amputation-Free Survival in Critical Limb Ischemia – Phase 3 Randomized Double-Blind Placebo-Controlled Trial. Circulation 2010(122)
33. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progeni- tor endothelial cells for angiogenesis. Science 1997 Feb 14;275(5302):964-7.
34. Fadini GP, Agostini C, Avogaro A. Endothelial progenitor cells in cerebrovascular disease. Stroke 2005 Jun;36(6):1112-3.
35. Timmermans F, Plum J, Yoder MC, Ingram DA, Vandekerckhove B, Case J. Endothelial progenitor cells: identity defined? J Cell Mol Med 2009 Jan;13(1):87-102.
36. Timmermans F, Van Hauwermeiren F, De Smedt M, Raedt R, Plasschaert F, De Buyzere ML, et al.
Endothelial outgrowth cells are not derived from CD133+ cells or CD45+ hematopoietic precur- sors. Arterioscler Thromb Vasc Biol 2007 Jul;27(7):1572-9.
37. Everaert BR, Van Craenenbroeck EM, Hoymans VY, Haine SE, Van Nassauw L, Conraads VM, et al.
Current perspective of pathophysiological and interventional effects on endothelial progenitor cell biology: focus on PI3K/AKT/eNOS pathway. Int J Cardiol 2010 Oct 29;144(3):350-66.
38. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002 Aug 10;360(9331):427-35.
39. Fadini GP, Agostini C, Avogaro A. Autologous stem cell therapy for peripheral arterial disease meta-analysis and systematic review of the literature. Atherosclerosis 2010 Mar;209(1):10-7.
40. Lawall H, Bramlage P, Amann B. Stem cell and progenitor cell therapy in peripheral artery disease.
A critical appraisal. Thromb Haemost 2010 Apr;103(4):696-709.
41. Schaper W. Influence of physical factors on the radial growth of collateral vessels in coronary circulation. Verh Dtsch Ges Kreislaufforsch 1966;32:282-6.
42. Herold J, Pipp F, Fernandez B, Xing Z, Heil M, Tillmanns H, et al. Transplantation of monocytes:
a novel strategy for in vivo augmentation of collateral vessel growth. Hum Gene Ther 2004 Jan;15(1):1-12.
43. Urbich C, Heeschen C, Aicher A, Dernbach E, Zeiher AM, Dimmeler S. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation 2003 Nov 18;108(20):2511-6.
44. van Beem RT, Noort WA, Voermans C, Kleijer M, ten Brinke A, van Ham SM, et al. The presence of activated CD4(+) T cells is essential for the formation of colony-forming unit-endothelial cells by CD14(+) cells. J Immunol 2008 Apr 1;180(7):5141-8.
45. Huang M, Chan DA, Jia F, Xie X, Li Z, Hoyt G, et al. Short hairpin RNA interference therapy for ischemic heart disease. Circulation 2008 Sep 30;118(14 Suppl):S226-S233.
