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Coronary artery disease is the number one cause of morbidity and mortality in the Western world. It typically occurs when heart muscle receives inadequate blood supply due to rupture of atherosclerotic plaques. During ischemia, up-regulation of hypoxia inducible factor-1 alpha (HIF-1α) transcriptional factor can activate several downstream angiogenic genes. However, HIF-1α is naturally degraded by prolyl hydroxylase-2 (PHD2) protein. Recently, we cloned the mouse PHD2 gene by comparing the homolog gene in human and rat. The best candidate shRNA sequence for inhibiting PHD2 was inserted behind H1 promoter, followed by a separate hypoxia response element (HRE)-incorporated promoter driving a firefly luciferase (Fluc) reporter gene. This construct allowed us to monitor gene expression noninvasively and was used to test the hypothesis that inhibition of PHD2 by short hairpin RNA interference (shRNA) can lead to significant improvement in angiogenesis and contractility as revealed by in vitro and in vivo experiments.
Coronary artery disease (CAD) is the leading cause of morbidity and mortality in the Western world (1). Conventional treatment for CAD consists of medical therapy as the first-line strategy, followed by percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG). However, a significant number of patients will still have refractory angina despite these treatments (2). Over the past decades, a better understanding of the molecular and genetic bases of different diseases has made gene therapy an increasingly viable treatment option (3). With the use of gene transfer techniques, it is now possible to modify somatic cells in ischemic myocardium, to overexpress beneficial or inhibitory pathologic proteins, and to achieve positive therapeutic effects (4). Indeed, several clinical trials evaluating both viral and non-viral delivery of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) have been completed to date (5).
A growing body of evidence suggests that the expression of a single angiogenic factor alone is not sufficient for the functional revascularization of ischemic tissues (6). Newer approaches based on the transcriptional factor HIF-1α may be a more natural choice. HIF-1α is known to control the expression of over 60 genes that affect cell survival and metabolism in adverse conditions, including VEGF (7), insulin-like growth factor (8), erythropoietin (9), nitric oxide synthase (10), among others. However, during normoxia, HIF-1α subunits have an exceptionally short half-life (~3–5 min) and low steady-state levels (11). This is due to hydroxylation of two prolyl residues (Pro402 and Pro564) by a family of prolyl-4-hydroxylases (PHDs) (12). Hydroxylation of HIF-1α allows recognition by the von Hippel-Lindau (VHL) tumor suppressor, which targets HIF-1α for proteosomal destruction (13). In contrast, increasing the severity of hypoxia retards degradation of HIF-1α subunits, allowing nuclear localization, dimerization with HIF-1β subunits, and formation of a stable DNA-binding HIF complex (14). Thus, the short hairpin RNA (shRNA) plasmid for knockdown of PHD2 (shPHD2) can potentially be used as a novel gene therapy for treatment of ischemic heart disease.
To date, the majority of cardiac gene therapy studies have relied on ex vivo quantification of gene expression (e.g., GFP or lacZ) in small animals or indirect markers (e.g., changes in perfusion or contractility) in clinical trials (15, 16). In order to characterize, visualize, and quantify biological processes at the molecular and cellular levels within intact living organisms, in vivo imaging techniques are needed. Over the past 10 years, molecular imaging has been widely used for oncology studies, but applications in cardiology have been a recent development (17). One such example is the use of reporter genes that can be transferred into cells via a delivery vector and regulated by constitutive, inducible, or tissue-specific promoters.
In this protocol, we outline the procedures used to address the two issues mentioned above--better therapeutic gene and more sophisticated tracking method. We show that the inhibition of HIF-1α degradation via shRNA knockdown of PHD2 in the ischemic heart represents a novel angiogenic therapy approach. At the same time, we track the shRNA vector in vivo through novel molecular imaging technology (18).
The authors would like to thank Dr. Robert Robbins and his staff for providing the surgical service. This work was supported in part by grants from the NIH HL095571 (JCW), NIH HL093172 (JCW), and AHA Western Postdoctoral Fellowship (MH).
1We harvest the heart tissue under microscopy to isolate the peri-infarct and infarct area tissues.
2To obtain the optimal quality of plasmids for in vivo injection, we recommend preparing the plasmids by CsCl2-ethidium bromide equilibrium centrifugation.
3To obtain the large yield of shPHD2 plasmid, we select DH5 alpha and fresh E. coli solution to do the isolation.