Lifan Liang

Long-term cardiac-targeted RNA interference for the treatment of heart failure restores cardiac function and reduces pathological hypertrophy.

Background— RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. Here, we report on targeted RNAi for the treatment of heart failure, an important disorder in humans that results from multiple causes. Successful treatment of heart failure is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban, a key regulator of cardiac Ca2+ homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy uses regulatory RNAs to achieve its effect. Methods and Results— We describe structural requirements to obtain high RNAi activity from adenoviral and adeno-associated virus (AAV9) vectors and show that an adenoviral short hairpin RNA vector (AdV-shRNA) silenced phospholamban in cardiomyocytes (primary neonatal rat cardiomyocytes) and improved hemodynamics in heart-failure rats 1 month after aortic root injection. For simplified long-term therapy, we developed a dimeric cardiotropic adeno-associated virus vector (rAAV9-shPLB) to deliver RNAi activity to the heart via intravenous injection. Cardiac phospholamban protein was reduced to 25%, and suppression of sacroplasmic reticulum Ca2+ ATPase in the HF groups was rescued. In contrast to traditional vectors, rAAV9 showed high affinity for myocardium but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (left ventricular end-diastolic pressure, dp/dtmin, and &tgr;) and systolic (fractional shortening) functional parameters to normal ranges. The massive cardiac dilation was normalized, and cardiac hypertrophy, cardiomyocyte diameter, and cardiac fibrosis were reduced significantly. Importantly, no evidence was found of microRNA deregulation or hepatotoxicity during these RNAi therapies. Conclusions— Our data show for the first time the high efficacy of an RNAi therapeutic strategy in a cardiac disease.

SERCA2a Gene Transfer Decreases Sarcoplasmic Reticulum Calcium Leak and Reduces Ventricular Arrhythmias in a Model of Chronic Heart Failure

Background—Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca2+) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. Methods and Results—We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca2+ homeostasis and spontaneous SR Ca2+ leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca2+ leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca2+ load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca2+ leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. Conclusions—SERCA2a gene therapy stabilizes SR Ca2+ load, reduces ryanodine receptor phosphorylation and decreases SR Ca2+ leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

JNK modulates FOXO3a for the expression of the mitochondrial death and mitophagy marker BNIP3 in pathological hypertrophy and in heart failure

Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) is a mitochondrial death and mitophagy marker, which is involved in inducing cardiac remodeling post myocardial infarction. In this study, we show that BNIP3 expression increases in stressed cardiomyocytes in vitro and in response to pressure overload in vivo, and that its transcription is directly related to JNK activity. BNIP3 expression gradually increased in the first weeks after pressure overload and peaked at the heart failure stage. Ultrastructurally, the mitochondrial area was inversely proportional to BNIP3 expression. Both JNK and AKT activities increased with pressure overload; however, JNK signaling dominated over AKT signaling for the activation of the transcription factor FOXO3a and for the transcription of its effector, BNIP3. 3-methyladenine attenuated JNK signaling and significantly decreased BNIP3 expression and reversed cardiac remodeling in heart failure. Ultrastructurally, the mitochondrial area was significantly increased in the 3-methyladenine group compared with placebo. Moreover, adenoviral gene delivery of dominant negative JNK in a rat model of pressure overload hypertrophy abolished the increase in BNIP3 expression in response to pressure overload. These results suggest that JNK signaling is a critical modulator of the transcription factor FOXO3a driving the expression of its effector, BNIP3, in heart failure and that JNK, through BNIP3, induces mitochondrial apoptosis and mitophagy.

SERCA2a Gene Transfer Decreases SR Calcium Leak and Reduces Ventricular Arrhythmias in a Model of Chronic Heart Failure

Background—Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca2+) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. Methods and Results—We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca2+ homeostasis and spontaneous SR Ca2+ leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca2+ leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca2+ load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca2+ leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. Conclusions—SERCA2a gene therapy stabilizes SR Ca2+ load, reduces ryanodine receptor phosphorylation and decreases SR Ca2+ leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

Effects of CXCR4 Gene Transfer on Cardiac Function After Ischemia-Reperfusion Injury

Acute coronary occlusion is the leading cause of death in the Western world. There is an unmet need for the development of treatments to limit the extent of myocardial infarction (MI) during the acute phase of occlusion. Recently, investigators have focused on the use of a chemokine, CXCL12, the only identified ligand for CXCR4, as a new therapeutic modality to recruit stem cells to individuals suffering from MI. Here, we examined the effects of overexpression of CXCR4 by gene transfer on MI. Adenoviruses carrying the CXCR4 gene were injected into the rat heart one week before ligation of the left anterior descending coronary artery followed by 24 hours reperfusion. Cardiac function was assessed by echocardiography couple with 2,3,5-Triphenyltetrazolium chloride staining to measure MI size. In comparison with control groups, rats receiving Ad-CXCR4 displayed an increase in infarct area (13.5% +/- 4.1%) and decreased fractional shortening (38% +/- 5%). Histological analysis revealed a significant increase in CXCL12 and tumor necrosis factor-alpha expression in ischemic area of CXCR4 overexpressed hearts. CXCR4 overexpression was associated with increased influx of inflammatory cells and enhanced cardiomyocyte apoptosis in the infarcted heart. These data suggest that in our model overexpressing CXCR4 appears to enhance ischemia/reperfusion injury possibly due to enhanced recruitment of inflammatory cells, increased tumor necrosis factor-alpha production, and activation of cell death/apoptotic pathways.

Therapeutic Efficacy of AAV1.SERCA2a in Monocrotaline-Induced Pulmonary Arterial Hypertension

Background— Pulmonary arterial hypertension (PAH) is characterized by dysregulated proliferation of pulmonary artery smooth muscle cells leading to (mal)adaptive vascular remodeling. In the systemic circulation, vascular injury is associated with downregulation of sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and alterations in Ca2+ homeostasis in vascular smooth muscle cells that stimulate proliferation. We, therefore, hypothesized that downregulation of SERCA2a is permissive for pulmonary vascular remodeling and the development of PAH. Methods and Results— SERCA2a expression was decreased significantly in remodeled pulmonary arteries from patients with PAH and the rat monocrotaline model of PAH in comparison with controls. In human pulmonary artery smooth muscle cells in vitro, SERCA2a overexpression by gene transfer decreased proliferation and migration significantly by inhibiting NFAT/STAT3. Overexpresion of SERCA2a in human pulmonary artery endothelial cells in vitro increased endothelial nitric oxide synthase expression and activation. In monocrotaline rats with established PAH, gene transfer of SERCA2a via intratracheal delivery of aerosolized adeno-associated virus serotype 1 (AAV1) carrying the human SERCA2a gene (AAV1.SERCA2a) decreased pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and fibrosis in comparison with monocrotaline-PAH rats treated with a control AAV1 carrying &bgr;-galactosidase or saline. In a prevention protocol, aerosolized AAV1.SERCA2a delivered at the time of monocrotaline administration limited adverse hemodynamic profiles and indices of pulmonary and cardiac remodeling in comparison with rats administered AAV1 carrying &bgr;-galactosidase or saline. Conclusions— Downregulation of SERCA2a plays a critical role in modulating the vascular and right ventricular pathophenotype associated with PAH. Selective pulmonary SERCA2a gene transfer may offer benefit as a therapeutic intervention in PAH.

SERCA2a controls the mode of agonist-induced intracellular Ca2+ signal, transcription factor NFAT and proliferation in human vascular smooth muscle cells

In blood vessels, tone is maintained by agonist-induced cytosolic Ca(2+) oscillations of quiescent/contractile vascular smooth muscle cells (VSMCs). However, in synthetic/proliferative VSMCs, Gq/phosphoinositide receptor-coupled agonists trigger a steady-state increase in cytosolic Ca(2+) followed by a Store Operated Calcium Entry (SOCE) which translates into activation of the proliferation-associated transcription factor NFAT. Here, we report that in human coronary artery smooth muscle cells (hCASMCs), the sarco/endoplasmic reticulum calcium ATPase type 2a (SERCA2a) expressed in the contractile form of the hCASMCs, controls the nature of the agonist-induced Ca(2+) transient and the resulting down-stream signaling pathway. Indeed, restoring SERCA2a expression by gene transfer in synthetic hCASMCs 1) increased Ca(2+) storage capacity; 2) modified agonist-induced IP(3)R Ca(2+) release from steady-state to oscillatory mode (the frequency of agonist-induced IP(3)R Ca(2+) signal was 11.66 ± 1.40/100 s in SERCA2a-expressing cells (n=39) vs 1.37 ± 0.20/100 s in control cells (n=45), p<0.01); 3) suppressed SOCE by preventing interactions between SR calcium sensor STIM1 and pore forming unit ORAI1; 4) inhibited calcium regulated transcription factor NFAT and its down-stream physiological function such as proliferation and migration. This study provides evidence for the first time that oscillatory and steady-state patterns of Ca(2+) transients have different effects on calcium-dependent physiological functions in smooth muscle cells.

Stem Cell Factor Gene Transfer Promotes Cardiac Repair After Myocardial Infarction via In Situ Recruitment and Expansion of c-kit+ Cells

Rationale: There is growing evidence that the myocardium responds to injury by recruiting c-kit+ cardiac progenitor cells to the damage tissue. Even though the ability of exogenously introducing c-kit+ cells to injured myocardium has been established, the capability of recruiting these cells through modulation of local signaling pathways by gene transfer has not been tested. Objective: To determine whether stem cell factor gene transfer mediates cardiac regeneration in a rat myocardial infarction model, through survival and recruitment of c-kit+ progenitors and cell-cycle activation in cardiomyocytes, and explore the mechanisms involved. Methods and Results: Infarct size, cardiac function, cardiac progenitor cells recruitment, fibrosis, and cardiomyocyte cell-cycle activation were measured at different time points in controls (n=10) and upon stem cell factor gene transfer (n=13) after myocardial infarction. We found a regenerative response because of stem cell factor overexpression characterized by an enhancement in cardiac hemodynamic function: an improvement in survival; a reduction in fibrosis, infarct size and apoptosis; an increase in cardiac c-kit+ progenitor cells recruitment to the injured area; an increase in cardiomyocyte cell-cycle activation; and Wnt/&bgr;-catenin pathway induction. Conclusions: Stem cell factor gene transfer induces c-kit+ stem/progenitor cell expansion in situ and cardiomyocyte proliferation, which may represent a new therapeutic strategy to reverse adverse remodeling after myocardial infarction.

Aortic Implantation of Mesenchymal Stem Cells after Aneurysm Injury in a Porcine Model

BACKGROUND Cell-based therapies are being evaluated in the setting of degenerative pathophysiologic conditions. The search for the ideal method of delivery and improvement in cell engraftment continue to pose a challenge. This study explores the feasibility of introducing mesenchymal stem cells (MSC) following aortic injury in a porcine model. METHODS Bone marrow-derived MSC were obtained from eight pigs, characterized for the MSC markers CD13 and CD 29, labeled with green fluorescent protein (GFP), and collected for autologous injection in a porcine model of abdominal aortic aneurysm (AAA). The pigs were euthanized (1-7 d) after the procedure to assess the histologic characteristics and presence of MSC in the aortic tissue. Negative controls included noninjured aorta. Tracking of the MSC was conducted by the identification of the GFP-labeled cells using immunofluorescence. RESULTS AAA sections stained with hematoxylin and eosin showed disorganization of the aortic tissue; collagen-muscle-elastin stain demonstrated fragmentation of elastin fibers. The presence of the implanted MSC in the aortic wall was evidenced by fluorescent microscopy showing GFP labeled cells. Engraftment of MSC up to 7 d after introduction was observed. CONCLUSION Autologous implantation of bone marrow-derived MSC following aortic injury in a porcine model may be successfully accomplished. The long-term impact and therapeutic value of such cell-based therapy will require further investigation.

Assessment of Cardiovascular Fibrosis Using Novel Fluorescent Probes

Cardiovascular fibrosis resulted from pressure overload or ischemia could alter myocardial stiffness and lead to ventricular dysfunction. Fluorescently labeled collagen-binding protein CNA 35, derived from the surface component of Staphylococcus aureus, and a novel synthetic biphenylalanine containing peptide are applied to stain fibrosis associated collagen and myocytes, respectively. Detailed pathological characteristics of cardiovascular fibrosis could be identified clearly in 2 hours. This staining pair requires only simple staining and brief washing, generating less than 10 ml of waste. The image information collected by this novel fluorescent staining pair is compatible with it collected by the traditional Massons Trichrome and Picrosirius Red staining which are widely used to stain cardiovascular fibrosis and isolated cells.