Philip Raake

An angiogenic role for the human peptide antibiotic LL-37/hCAP-18

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

G Protein–Coupled Receptor Kinase 2 Ablation in Cardiac Myocytes Before or After Myocardial Infarction Prevents Heart Failure

Myocardial G protein-coupled receptor kinase (GRK)2 is a critical regulator of cardiac &bgr;-adrenergic receptor (&bgr;AR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated &bgr;AR with a GRK2-derived peptide that binds G&bgr;&ggr; (&bgr;ARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and &bgr;ARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (&agr;MHC-Cre×GRK2 fl/fl) or only after administration of tamoxifen (&agr;MHC-MerCreMer×GRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved &bgr;AR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring &bgr;AR signaling by other means to improve outcomes in heart failure.

Uncovering G protein-coupled receptor kinase-5 as a histone deacetylase kinase in the nucleus of cardiomyocytes

G protein-coupled receptor (GPCR) kinases (GRKs) are critical regulators of cellular signaling and function. In cardiomyocytes, GRK2 and GRK5 are two GRKs important for myocardial regulation, and both have been shown to be up-regulated in the dysfunctional heart. We report that increased levels and activity of GRK5 in failing myocardium may have unique significance due to its nuclear localization, a property not shared by GRK2. We find that transgenic mice with elevated cardiac GRK5 levels have exaggerated hypertrophy and early heart failure compared with control mice after pressure overload. This pathology is not present in cardiac GRK2-overexpressing mice or in mice with overexpression of a mutant GRK5 that is excluded from the nucleus. Nuclear accumulation of GRK5 is enhanced in myocytes after aortic banding in vivo and in vitro in myocytes after increased Gαq activity, the trigger for pressure-overload hypertrophy. GRK5 enhances activation of MEF2 in concert with Gq signals, demonstrating that nuclear localized GRK5 regulates gene transcription via a pathway critically linked to myocardial hypertrophy. Mechanistically, we show that this is due to GRK5 acting, in a non-GPCR manner, as a class II histone deacetylase (HDAC) kinase because it can associate with and phosphorylate the myocyte enhancer factor-2 repressor, HDAC5. Moreover, significant HDAC activity can be found with GRK5 in the heart. Our data show that GRK5 is a nuclear HDAC kinase that plays a key role in maladaptive cardiac hypertrophy apparently independent of any action directly on GPCRs.

Cardiac AAV9-S100A1 Gene Therapy Rescues Post-Ischemic Heart Failure in a Preclinical Large Animal Model

A protein that regulates cellular calcium, delivered percutaneously to the heart by gene therapy, improves cardiac function in pigs with heart failure and may also be effective in humans. Paving the Way for a Gene Therapy Trial for Heart Failure Heart failure, also known as congestive heart failure, results when, for any number of reasons, the heart no longer pumps enough blood to keep organs perfused and oxygenated. Common in the Western world, heart failure’s first-line treatment is diuretics, which help to remove the excess fluid that pools in the body during heart failure, and β-adrenergic receptor–blocking drugs to interfere with the deleterious effects of the excess catecholamines that accompany this disease. These treatments are effective but do not restore normal heart function; more than half of patients with heart failure die within 5 years. The authors of Pleger et al. now present evidence that a gene therapy approach to augmenting the failing heart’s damaged ability to handle intracellular calcium produces marked improvements in heart function in pigs with heart failure. These results provide enough evidence, the authors say, to justify a clinical trial to see whether this approach could improve patient’s quality of life and survival when added to current treatments. The investigators injected an adenovirus gene therapy vector (AAV9) carrying the gene for S100A1 into pigs experiencing heart failure, induced by the experimental occlusion of a coronary artery. They chose to deliver S100A1 to the failing heart because this calcium-binding protein becomes depleted as the heart fails and is needed for proper regulation of the calcium dynamics within myocardial cells. Two weeks after infarction, the vector (under control of a cardiac-specific promoter) was delivered to non-infarcted regions of the heart (which would eventually fail without treatment). Twelve weeks later, S100A1 protein expression increased and heart function improved. By several measures, calcium handling within the cardiomyocytes was improved, as were markers of mitochondrial energy production. The authors saw no toxic effects of the therapy and verified the cardiac-specific expression of the vector. Although similar gene therapy tests were shown previously to be effective in mice, a test in a large animal model was especially important to verify that this approach is likely to be safe and effective in patients. The volume of successfully transduced tissue in a pig heart is similar to that required for humans. Unlike the rodent, the pig’s heart rate, sarcomeric proteins, and cardiomyocyte calcium handling are all similar to human, and therapeutic vector delivery through a percutaneous catheter, which will be required in patients, could be mimicked in pigs. If the value of this therapy is confirmed in clinical trials, it would be available for patients already suffering heart failure. It would likely prove most useful as an adjunct therapy to currently existing drugs where it could augment the strength of the heart’s contraction. As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)–S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca2+) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)–S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca2+ cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.

Gene Therapy in Heart Failure

With increasing knowledge of basic molecular mechanisms governing the development of heart failure (HF), the possibility of specifically targeting key pathological players is evolving. Technology allowing for efficient in vivo transduction of myocardial tissue with long-term expression of a transgene enables translation of basic mechanistic knowledge into potential gene therapy approaches. Gene therapy in HF is in its infancy clinically with the predominant amount of experience being from animal models. Nevertheless, this challenging and promising field is gaining momentum as recent preclinical studies in larger animals have been carried out and, importantly, there are 2 newly initiated phase I clinical trials for HF gene therapy. To put it simply, 2 parameters are needed for achieving success with HF gene therapy: (1) clearly identified detrimental/beneficial molecular targets; and (2) the means to manipulate these targets at a molecular level in a sufficient number of cardiac cells. However, several obstacles do exist on our way to efficient and safe gene transfer to human myocardium. Some of these obstacles are discussed in this review; however, it primarily focuses on the molecular target systems that have been subjected to intense investigation over the last decade in an attempt to make gene therapy for human HF a reality.

Level of G protein–Coupled Receptor Kinase-2 Determines Myocardial Ischemia/Reperfusion Injury via Pro- and Anti-Apoptotic Mechanisms

Rationale: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein–coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. Objective: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac &bgr;-adrenergic receptor (&bgr;AR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the &bgr;ARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. Methods and Results: We demonstrate, using cardiac-specific GRK2 and &bgr;ARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with &bgr;ARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in &bgr;ARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via &bgr;ARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of &bgr;2ARs abolished &bgr;ARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. Conclusion: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the &bgr;ARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

AAV6.βARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model

AIMS G protein-coupled receptor kinase 2 (GRK2), which is markedly upregulated in failing human myocardium, has been implicated as a contributing factor or consequence of heart failure (HF). Importantly, cardiac-specific GRK2 knockout mice have recently proved the pathological nature of GRK2 in HF. Targeted inhibition of GRK2 is possible using a peptide inhibitor known as the βARKct, which has rescued several disparate small animal HF models. This study was designed to evaluate long-term βARKct expression in a clinically relevant large animal HF model, using stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). METHODS AND RESULTS A porcine model of HF subsequent to left ventricular (LV) myocardial infarction (MI) was used to study the effects of retrograde injection into the anterior interventricular vein of either AAV6.βARKct or AAV6.luciferase as a control 2 weeks after MI. Echocardiography and LV hemodynamics were performed before and 6 weeks after gene transfer. Robust and long-term βARKct expression was found after AAV6-mediated delivery, leading to significant amelioration of LV haemodynamics and contractile function in HF pigs compared with AAV6.luciferase-treated control animals that showed a continued decline in cardiac function. Interestingly, the neurohormonal axis was virtually normalized in AVV6.βARKct-treated HF animals, represented by reductions in plasma norepinephrine levels, whereas AAV6.luciferase-treated pigs showed further increases in plasma catecholamine levels. As a result, LV remodelling and foetal gene expression was reversed by AVV6.βARKct gene therapy. CONCLUSION These data--showing sustained amelioration of cardiac function in a post-MI pig HF model--demonstrate the therapeutic potential of βARKct gene therapy for HF.

Heat Shock Protein 90 Transfection Reduces Ischemia-Reperfusion–Induced Myocardial Dysfunction via Reciprocal Endothelial NO Synthase Serine 1177 Phosphorylation and Threonine 495 Dephosphorylation

Objectives—The interaction of the heat shock protein 90 (Hsp90) with the endothelial NO synthase (eNOS) has been shown to account for a sustained production of NO in vitro. Here, we examined whether overexpression of Hsp90 in a pig model of cardiac infarct could preserve the myocardium from the deleterious effects of ischemia–reperfusion. Methods and Results—Percutaneous liposome-based gene transfer was performed by retroinfusion of the anterior interventricular vein before left anterior descending occlusion and reperfusion. We found that recombinant Hsp90 expression in the ischemic region of the heart led to a 33% reduction in infarct size and prevented the increase in postischemic left ventricular end diastolic pressure observed in mock-transfected animals. Regional myocardial function, assessed by subendocardial segment shortening in the infarct region, was increased in Hsp90-transfected animals at baseline and after pacing. All these effects were completely abrogated by administration of the NOS inhibitor NG-nitro-l-arginine methyl ester. We further documented in vivo and in cultured endothelial cells that the cardioprotective effects of Hsp90 were associated to its capacity to act as an adaptor for both the kinase Akt and the phosphatase calcineurin, thereby promoting eNOS serine 1177 phosphorylation and threonine 495 dephosphorylation, respectively. Conclusions—Hsp90 is a promising target to enhance NO formation in vivo, which may efficiently reduce myocardial reperfusion injury.

Cardio-specific long-term gene expression in a porcine model after selective pressure-regulated retroinfusion of adeno-associated viral (AAV) vectors

Cornerstone for an efficient cardiac gene therapy is the need for a vector system, which enables selective and long-term expression of the gene of interest. In rodent animal models adeno-associated viral (AAV) vectors like AAV-6 have been shown to efficiently transduce cardiomyocytes. However, since significant species-dependent differences in transduction characteristics exist, large animal models are of imminent need for preclinical evaluations. We compared gene transfer efficiencies of AAV-6 and heparin binding site-deleted AAV-2 vectors in a porcine model. Application of the AAVs was performed by pressure-regulated retroinfusion of the anterior interventricular cardiac vein, which has been previously shown to efficiently deliver genes to the myocardium (3.5 × 1010 viral genomes per animal; n=5 animals per group). All vectors harbored a luciferase reporter gene under control of a cytomegalovirus (CMV)-enhanced 1.5 kb rat myosin light chain promoter (CMV-MLC2v). Expression levels were evaluated 4 weeks after gene transfer by determining luciferase activities. To rule out a systemic spillover peripheral tissue was analyzed by PCR for the presence of vector genomes. Selective retroinfusion of AAV serotype 6 vectors into the anterior cardiac vein substantially increased reporter gene expression in the targeted distal left anterior descending (LAD) territory (65 943±31 122 vs control territory 294±69, P<0.05). Retroinfusion of AAV-2 vectors showed lower transgene expression, which could be increased with coadministration of recombinant human vascular endothelial growth factor (1365±707 no vascular endothelial growth factor (VEGF) vs 38 760±2448 with VEGF, P<0.05). Significant transgene expression was not detected in other organs than the heart, although vector genomes were detected also in the lung and liver. Thus, selective retroinfusion of AAV-6 into the coronary vein led to efficient long-term myocardial reporter gene expression in the targeted LAD area of the porcine heart. Coapplication of VEGF significantly increased transduction efficiency of AAV-2.

Targeted Inhibition of Cardiomyocyte Gi Signaling Enhances Susceptibility to Apoptotic Cell Death in Response to Ischemic Stress

Background— A salient characteristic of dysfunctional myocardium progressing to heart failure is an upregulation of the adenylyl cyclase inhibitory guanine nucleotide (G) protein &agr; subunit, G&agr;i2. It has not been determined conclusively whether increased Gi activity in the heart is beneficial or deleterious in vivo. Gi signaling has been implicated in the mechanism of cardioprotective agents; however, no in vivo evidence exists that any of the G&agr; subunits are cardioprotective. We have created a novel molecular tool to specifically address the role of Gi proteins in normal and dysfunctional myocardium. Methods and Results— We have developed a class-specific Gi inhibitor peptide, GiCT, composed of the region of G&agr;i2 that interacts specifically with G protein–coupled receptors. GiCT inhibits Gi signals specifically in vitro and in vivo, whereas Gs and Gq signals are not affected. In vivo expression of GiCT in transgenic mice effectively causes a “functional knockout” of cardiac G&agr;i2 signaling. Inducible, cardiac-specific GiCT transgenic mice display a baseline phenotype consistent with nontransgenic mice. However, when subjected to ischemia/reperfusion injury, GiCT transgenic mice demonstrate a significant increase in infarct size compared with nontransgenic mice (from 36.9±2.5% to 50.9±4.3%). Mechanistically, this post-ischemia/reperfusion phenotype includes increased myocardial apoptosis and resultant decreased contractile performance. Conclusions— Overall, our results demonstrate the in vivo utility of GiCT to dissect specific mechanisms attributed to Gi signaling in stressed myocardium. Our results with GiCT indicate that upregulation of G&agr;i2 is an adaptive protective response after ischemia to shield myocytes from apoptosis.