Immunoglobulin E signal inhibition during allergen ingestion leads to reversal of established food allergy and induction of regulatory T cells.

Immunoglobulin E (IgE) antibodies are known for triggering immediate hypersensitivity reactions such as food anaphylaxis. In this study, we tested whether they might additionally function to amplify nascent antibody and T helper 2 (Th2) cell-mediated responses to ingested proteins and whether blocking IgE would modify sensitization. By using mice harboring a disinhibited form of the IL-4 receptor, we developed an adjuvant-free model of peanut allergy. Mast cells and IgE were required for induction of antibody and Th2-cell-mediated responses to peanut ingestion and they impaired regulatory T (Treg) cell induction. Mast-cell-targeted genetic deletion of the FcεRI signaling kinase Syk or Syk blockade also prevented peanut sensitization. In mice with established allergy, Syk blockade facilitated desensitization and induction of Treg cells, which suppressed allergy when transferred to naive recipients. Our study suggests a key role for IgE in driving Th2 cell and IgE responses while suppressing Treg cells in food allergy.

Inhibition of αvß5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvß5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvß5 in a rat model of renal IRI. Pretreatment with this anti-αvß5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvß5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvß5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvß5 antibody treatment. Immunostaining for αvß5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvß5 in modulating vascular leak. Additional studies showed αvß5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvß5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvß5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvß5 inhibition as a promising therapeutic strategy for AKI.

Genes related to emphysema are enriched for ubiquitination pathways.

BACKGROUND: Increased small airway resistance and decreased lung elasticity contribute to the airflow limitation in chronic obstructive pulmonary disease (COPD). The lesion that corresponds to loss of lung elasticity is emphysema; the small airway obstruction is due to inflammatory narrowing and obliteration. Despite their convergence in altered physiology, different mechanisms contribute to these processes. The relationships between gene expression and these specific phenotypes may be more revealing than comparison with lung function. METHODS: We measured the ratio of alveolar surface area to lung volume (SA/V) in lung tissue from 43 smokers. Two samples from 21 subjects, in which SA/V differed by >49 cm2/mL were profiled to select genes whose expression correlated with SA/V. Significant genes were tested for replication in the 22 remaining subjects. RESULTS: The level of expression of 181 transcripts was related to SA/V ( p < 0.05). When these genes were tested in the 22 remaining subjects as a replication, thirty of the 181 genes remained significantly associated with SA/V (P < 0.05) and the direction of association was the same in 164/181. Pathway and network analysis revealed enrichment of genes involved in protein ubiquitination, and western blotting showed altered expression of genes involved in protein ubiquitination in obstructed individuals. CONCLUSION: This study implicates modified protein ubiquitination and degradation as a potentially important pathway in the pathogenesis of emphysema.

Inhibition of spleen tyrosine kinase attenuates allergen-mediated airway constriction.

Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers.

Common inflammatome gene signatures as well as disease-specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co-expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue-specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response-related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non-drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.

Gene expression profiling following NRF2 and KEAP1 siRNA knockdown in human lung fibroblasts identifies CCL11/Eotaxin-1 as a novel NRF2 regulated gene.

BACKGROUND: Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system. METHODS: Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools. RESULTS: An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts. CONCLUSIONS: These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.

Advances toward structure-based drug discovery for inflammasome targets.

Inflammasome proteins play an important role in many diseases of high unmet need, making them attractive drug targets. However, drug discovery for inflammasome proteins has been challenging in part due to the difficulty in solving high-resolution structures using cryo-EM or crystallography. Recent advances in the structural biology of NLRP3 and NLRP1 have provided the first set of data that proves a promise for structure-based drug design for this important family of targets.

Pharmacological characterization of MK-7246, a potent and selective CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells) antagonist.

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor that has been reported to modulate inflammatory responses in various rodent models of asthma, allergic rhinitis and atopic dermatitis. In this study, we describe the biological and pharmacological properties of {(7R)-7-[[(4-fluorophenyl)sulfonyl](methyl)amino]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}acetic acid (MK-7246), a novel synthetic CRTH2 antagonist. We show that MK-7246 1) has high affinity for the human, monkey, dog, rat, and mouse CRTH2, 2) interacts with CRTH2 in a reversible manner, 3) exhibits high selectivity over all prostanoid receptors as well as 157 other receptors and enzymes, 4) acts as a full antagonist on recombinant and endogenously expressed CRTH2, 5) demonstrates good oral bioavailability and metabolic stability in various animal species, 6) yields ex vivo blockade of CRTH2 on eosinophils in monkeys and sheep, and 7) significantly blocks antigen-induced late-phase bronchoconstriction and airway hyper-responsiveness in sheep. MK-7246 represents a potent and selective tool to further investigate the in vivo function of CRTH2.

Discovery of MK-7246, a selective CRTH2 antagonist for the treatment of respiratory diseases.

In this manuscript we wish to report the discovery of MK-7246 (4), a potent and selective CRTH2 (DP2) antagonist. SAR studies leading to MK-7246 along with two synthetic sequences enabling the preparation of this novel class of CRTH2 antagonist are reported. Finally, the pharmacokinetic and metabolic profile of MK-7246 is disclosed.

Angiotensin-converting enzyme 2 protects from severe acute lung failure.

Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is a devastating clinical syndrome with a high mortality rate (30-60%) (refs 1-3). Predisposing factors for ARDS are diverse and include sepsis, aspiration, pneumonias and infections with the severe acute respiratory syndrome (SARS) coronavirus. At present, there are no effective drugs for improving the clinical outcome of ARDS. Angiotensin-converting enzyme (ACE) and ACE2 are homologues with different key functions in the renin-angiotensin system. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 inactivates angiotensin II and is a negative regulator of the system. ACE2 has also recently been identified as a potential SARS virus receptor and is expressed in lungs. Here we report that ACE2 and the angiotensin II type 2 receptor (AT2) protect mice from severe acute lung injury induced by acid aspiration or sepsis. However, other components of the renin-angiotensin system, including ACE, angiotensin II and the angiotensin II type 1a receptor (AT1a), promote disease pathogenesis, induce lung oedemas and impair lung function. We show that mice deficient for Ace show markedly improved disease, and also that recombinant ACE2 can protect mice from severe acute lung injury. Our data identify a critical function for ACE2 in acute lung injury, pointing to a possible therapy for a syndrome affecting millions of people worldwide every year.

Identification of distinct plasma biomarker signatures in patients with rapid and slow declining forms of COPD.

Chronic obstructive pulmonary disease (COPD) is a prevalent pulmonary disease characterized by a progressive decline in lung function. The identification of biomarkers capable of predicting the rate of lung function decline or capable of giving an early read on drug efficacy in clinical trials would be very useful. The aim of this study was to identify plasma biomarkers capable of accurately distinguishing patients with COPD from healthy controls. Eighty-nine plasma markers in 40 COPD patients and 20 healthy smoker controls were analyzed. The COPD patients were divided into two subgroups, rapid and slow decliners based on their rate of lung function decline measured over 15 years. Univariate analysis revealed that 25 plasma markers were statistically different between rapid decliners and controls, 4 markers were different between slow decliners and controls, and 10 markers were different between rapid and slow decliners (p < 0.05). Multivariate analysis led to the identification of groups of plasma markers capable of distinguishing rapid decliners from controls (signature 1), slow decliners from controls (signature 2) and rapid from slow decliners (signature 3) with over 90% classification accuracy. Importantly, signature 1 was shown to be longitudinally stable using plasma samples taken a year later from a subset of patients. This study describes a novel set of plasma markers differentiating slow from rapid decline of lung function in COPD. If validated in distinct and larger cohorts, the signatures identified will have important implications in both disease diagnosis, as well as the clinical evaluation of new therapies.

Mice with a targeted mutation of patched2 are viable but develop alopecia and epidermal hyperplasia.

Hedgehog (Hh) signaling plays pivotal roles in tissue patterning and development in Drosophila melanogaster and vertebrates. The Patched1 (Ptc1) gene, encoding the Hh receptor, is mutated in nevoid basal cell carcinoma syndrome, a human genetic disorder associated with developmental abnormalities and increased incidences of basal cell carcinoma (BCC) and medulloblastoma (MB). Ptc1 mutations also occur in sporadic forms of BCC and MB. Mutational studies with mice have verified that Ptc1 is a tumor suppressor. We previously identified a second mammalian Patched gene, Ptc2, and demonstrated its distinct expression pattern during embryogenesis, suggesting a unique role in development. Most notably, Ptc2 is expressed in an overlapping pattern with Shh in the epidermal compartment of developing hair follicles and is highly expressed in the developing limb bud, cerebellum, and testis. Here, we describe the generation and phenotypic analysis of Ptc2(tm1/tm1) mice. Our molecular analysis suggests that Ptc2(tm1) likely represents a hypomorphic allele. Despite the dynamic expression of Ptc2 during embryogenesis, Ptc2(tm1/tm1) mice are viable, fertile, and apparently normal. Interestingly, adult Ptc2(tm1/tm1) male animals develop skin lesions consisting of alopecia, ulceration, and epidermal hyperplasia. While functional compensation by Ptc1 might account for the lack of a strong mutant phenotype in Ptc2-deficient mice, our results suggest that normal Ptc2 function is required for adult skin homeostasis.

Gene expression profiling in patients with chronic obstructive pulmonary disease and lung cancer.

RATIONALE: Chronic obstructive lung disease (COPD) is a common and disabling lung disease for which there are few therapeutic options. OBJECTIVES: We reasoned that gene expression profiling of COPD lungs could reveal previously unidentified disease pathways. METHODS: Forty-eight human lung samples were obtained from tissue resected from five nonsmokers, 21 GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage 0, 9 GOLD stage 1, 10 GOLD stage 2, and 3 GOLD stage 3 patients. mRNA from the specimens was profiled using Agilent's Functional ID v2.0 array (Agilent, Santa Clara, CA) containing 23,720 sequences. MEASUREMENTS AND MAIN RESULTS: The gene expression pattern was influenced by the percentage of the sample made up of parenchyma. Gene expression was related to forced expiratory flow between 25 and 75% of forced expiratory volume (FEF(25-75%) % predicted) revealing a signature gene set of 203 transcripts. Genes involved in extracellular matrix synthesis/degradation and apoptosis were among the up-regulated genes, whereas genes that participate in antiinflammatory responses were down-regulated. Immunohistochemistry confirmed expression of urokinase plasminogen activator (PLAU), urokinase plasminogen activator receptor (PLAUR), and thrombospondin (THBS1) by alveolar macrophages and airway epithelial cells. Genes in this pathway have been shown to be involved in the activation of transforming growth factor (TGF)-beta1 and matrix metalloproteinases and are subject to inhibition by SERPINE2. Interestingly, both TGF-beta1 and SERPINE2 have been identified as candidate genes in COPD genetic linkage and association studies. CONCLUSIONS: The results provide evidence that genes involved in tissue remodeling and repair are differentially regulated in the lungs of obstructed smokers and suggest that they are potential therapeutic targets. Data deposited in GEO at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE8500.

Loss of PTEN attenuates the development of pathological hypertrophy and heart failure in response to biomechanical stress.

AIMS: The maladaptive response to biomechanical stress is a fundamental response in heart disease. Loss of the 3'-lipid phosphatase, phosphatase and tensin homolog deleted on chromosome ten (PTEN), is associated with increased phosphorylation of Akt/protein kinase B and glycogen synthase kinase-beta. We hypothesize that these key changes will halt the development of pathological hypertrophy and the progression to heart failure in response to pressure overload. METHODS AND RESULTS: In mice, muscle-specific knockout of PTEN, mckCRE-PTEN(flox/flox) (PTEN KO), resulted in basal hypertrophy and mild reduction in left ventricular (LV) systolic function. Male mice were subjected to aortic banding (AB) or sham operation. In contrast to mckCRE-PTEN(+/+) control mice, pressure overload in PTEN KO mice resulted in reduced pathological hypertrophy, less interstitial fibrosis, and reduced apoptosis with a marked preservation of LV function. Western blot analysis of mitogen-activated protein kinase (MAPK) signalling showed equivalent phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 with markedly reduced phosphorylation of jun N-terminal kinase (JNK)1 and JNK2, and p38 in PTEN KO mice subjected to AB. Loss of PTEN was associated with increased expression of the proangiogenic factors, vascular endothelial growth factor-A and angiopoietin-2, with preservation of the myocardial capillary density in response to pressure overload. Moreover, banded PTEN KO mice maintained the expression of several key metabolic genes that are known to be dysregulated in heart failure. In contrast, a subpressor dose of the G protein-coupled receptor (GPCR) agonist angiotensin II (Ang II) leads to increased pathological hypertrophy and MAPK activation in PTEN KO mice. CONCLUSION: Loss of PTEN prevents the development of maladaptive ventricular remodelling with preservation of angiogenesis and metabolic gene expression in response to pressure overload but not in response to the GPCR agonist, Ang II. Inhibition of PTEN signalling in the heart may represent a novel approach to slow the progression of heart failure in response to pathological biomechanical stress.

The use of strictly standardized mean difference for hit selection in primary RNA interference high-throughput screening experiments.

RNA interference (RNAi) high-throughput screening (HTS) has been hailed as the 2nd genomics wave following the 1st genomics wave of gene expression microarrays and single-nucleotide polymorphism discovery platforms. Following an RNAi HTS, the authors are interested in identifying short interfering RNA (siRNA) hits with large inhibition/activation effects. For hit selection, the z-score method and its variants are commonly used in primary RNAi HTS experiments. Recently, strictly standardized mean difference (SSMD) has been proposed to measure the siRNA effect represented by the magnitude of difference between an siRNA and a negative reference group. The links between SSMD and d+-probability offer a clear interpretation of siRNA effects from a probability perspective. Hence, SSMD can be used as a ranking metric for hit selection. In this article, the authors investigated both the SSMD-based testing process and the use of SSMD as a ranking metric for hit selection in 2 primary siRNA HTS experiments. The analysis results showed that, as a ranking metric, SSMD was more stable and reliable than percentage inhibition and led to more robust hit selection results. Using the SSMD -based testing method, the false-negative rate can more readily be obtained. More important, the use of the SSMD-based method can result in a reduction in both the false-negative and false-positive rates. The applications presented in this article demonstrate that the SSMD method addresses scientific questions and fills scientific needs better than both percentage inhibition and the commonly used z-score method for hit selection.

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system.

While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the 'immune fingerprint' of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.

Inhibition of spleen tyrosine kinase attenuates IgE-mediated airway contraction and mediator release in human precision cut lung slices.

BACKGROUND AND PURPOSE: Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways. EXPERIMENTAL APPROACH: Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). High-affinity IgE receptor (FcεRI) activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function. KEY RESULTS: While dexamethasone had little effect on FcεR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi abolished anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells. CONCLUSIONS AND IMPLICATIONS: A selective Syk inhibitor, but not corticosteroids, abolished FcεR-mediated contraction in human small airways ex vivo. The mechanism involved FcεRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.

Phosphoinositide 3-kinase gamma-deficient mice are protected from isoproterenol-induced heart failure.

BACKGROUND: We have recently shown that genetic inactivation of phosphoinositide 3-kinase gamma (PI3Kgamma), the isoform linked to G-protein-coupled receptors, results in increased cardiac contractility with no effect on basal cell size. Signaling via the G-protein-coupled beta-adrenergic receptors has been implicated in cardiac hypertrophy and heart failure, suggesting that PI3Kgamma might play a role in the pathogenesis of heart disease. METHODS AND RESULTS: To determine the role for PI3Kgamma in hypertrophy induced by G-protein-coupled receptors and cardiomyopathy, we infused isoproterenol, a beta-adrenergic receptor agonist, into PI3Kgamma-deficient mice. Compared with controls, isoproterenol infusion in PI3Kgamma-deficient mice resulted in an attenuated cardiac hypertrophic response and markedly reduced interstitial fibrosis. Intriguingly, chronic beta-adrenergic receptor stimulation triggered impaired heart functions in wild-type mice, whereas PI3Kgamma-deficient mice retained their increased heart function and did not develop heart failure. The lack of PI3Kgamma attenuated the activation of Akt/protein kinase B and extracellular signal-regulated kinase 1/2 signaling pathways in cardiac myocytes in response to isoproterenol. beta1- and beta2-adrenergic receptor densities were decreased by similar amounts in PI3Kgamma-deficient and control mice, suggesting that PI3Kgamma isoform plays no role in the downregulation of beta-adrenergic receptors after chronic beta-adrenergic stimulation. CONCLUSIONS: Our data show that PI3Kgamma is critical for the induction of hypertrophy, fibrosis, and cardiac dysfunction function in response to beta-adrenergic receptor stimulation in vivo. Thus, PI3Kgamma may represent a novel therapeutic target for the treatment of decreased cardiac function in heart failure.

A story of two ACEs.

According to the World Health Organization predictions cardiovascular diseases will be the leading cause of death by the year 2020. High blood pressure is a major risk factor for myocardial infarction, cerebrovascular disease, and stroke. Modulation of the renin-angiotensin system, particularly inhibition of the angiotensin-converting enzyme (ACE), has become a prime strategy in the treatment of hypertension and heart failure. Recently the gene of a new ACE, termed ACE2, has been characterized. The ACE2 gene maps to defined quantitative trait loci on the X chromosome in three different rat models of hypertension, suggesting ACE2 as a candidate gene for hypertension. In mice the targeted disruption of ACE2 resulted in increased systemic angiotensin II levels, impaired cardiac contractility, and upregulation of hypoxia-induced genes in the heart. Since mice deficient in both ACE2 and ACE show completely normal heart function, it appears that ACE and ACE2 negatively regulate each other. The mechanisms and physiological significance of the interplay between ACE and ACE2 are not yet elucidated, but it may involve several new peptides and peptide systems. In view of drug development the increasing complexity of the renin-angiotensin system offers both challenge and opportunity to develop new and refined treatment strategies against cardiovascular diseases.