KLF15-Wnt-Dependent Cardiac Reprogramming Up-Regulates SHISA3 in the Mammalian Heart.

BACKGROUND: The combination of cardiomyocyte (CM) and vascular cell (VC) fetal reprogramming upon stress culminates in end-stage heart failure (HF) by mechanisms that are not fully understood. Previous studies suggest KLF15 as a key regulator of CM hypertrophy. OBJECTIVES: This study aimed to characterize the impact of KLF15-dependent cardiac transcriptional networks leading to HF progression, amenable to therapeutic intervention in the adult heart. METHODS: Transcriptomic bioinformatics, phenotyping of Klf15 knockout mice, Wnt-signaling-modulated hearts, and pressure overload and myocardial ischemia models were applied. Human KLF15 knockout embryonic stem cells and engineered human myocardium, and human samples were used to validate the relevance of the identified mechanisms. RESULTS: The authors identified a sequential, postnatal transcriptional repression mediated by KLF15 of pathways implicated in pathological tissue remodeling, including distinct Wnt-pathways that control CM fetal reprogramming and VC remodeling. The authors further uncovered a vascular program induced by a cellular crosstalk initiated by CM, characterized by a reduction of KLF15 and a concomitant activation of Wnt-dependent transcriptional signaling. Within this program, a so-far uncharacterized cardiac player, SHISA3, primarily expressed in VCs in fetal hearts and pathological remodeling was identified. Importantly, the KLF15 and Wnt codependent SHISA3 regulation was demonstrated to be conserved in mouse and human models. CONCLUSIONS: The authors unraveled a network interplay defined by KLF15-Wnt dynamics controlling CM and VC homeostasis in the postnatal heart and demonstrated its potential as a cardiac-specific therapeutic target in HF. Within this network, they identified SHISA3 as a novel, evolutionarily conserved VC marker involved in pathological remodeling in HF.

A context-specific cardiac ß-catenin and GATA4 interaction influences TCF7L2 occupancy and remodels chromatin driving disease progression in the adult heart.

Chromatin remodelling precedes transcriptional and structural changes in heart failure. A body of work suggests roles for the developmental Wnt signalling pathway in cardiac remodelling. Hitherto, there is no evidence supporting a direct role of Wnt nuclear components in regulating chromatin landscapes in this process. We show that transcriptionally active, nuclear, phosphorylated(p)Ser675-ß-catenin and TCF7L2 are upregulated in diseased murine and human cardiac ventricles. We report that inducible cardiomyocytes (CM)-specific pSer675-ß-catenin accumulation mimics the disease situation by triggering TCF7L2 expression. This enhances active chromatin, characterized by increased H3K27ac and TCF7L2 occupancies to cardiac developmental and remodelling genes in vivo. Accordingly, transcriptomic analysis of ß-catenin stabilized hearts shows a strong recapitulation of cardiac developmental processes like cell cycling and cytoskeletal remodelling. Mechanistically, TCF7L2 co-occupies distal genomic regions with cardiac transcription factors NKX2-5 and GATA4 in stabilized-ß-catenin hearts. Validation assays revealed a previously unrecognized function of GATA4 as a cardiac repressor of the TCF7L2/ß-catenin complex in vivo, thereby defining a transcriptional switch controlling disease progression. Conversely, preventing ß-catenin activation post-pressure-overload results in a downregulation of these novel TCF7L2-targets and rescues cardiac function. Thus, we present a novel role for TCF7L2/ß-catenin in CMs-specific chromatin modulation, which could be exploited for manipulating the ubiquitous Wnt pathway.

Erythropoietin responsive cardiomyogenic cells contribute to heart repair post myocardial infarction.

The role of erythropoietin (Epo) in myocardial repair after infarction remains inconclusive. We observed high Epo receptor (EPOR) expression in cardiac progenitor cells (CPCs). Therefore, we aimed to characterize these cells and elucidate their contribution to myocardial regeneration on Epo stimulation. High EPOR expression was detected during murine embryonic heart development followed by a marked decrease until adulthood. EPOR-positive cells in the adult heart were identified in a CPC-enriched cell population and showed coexpression of stem, mesenchymal, endothelial, and cardiomyogenic cell markers. We focused on the population coexpressing early (TBX5, NKX2.5) and definitive (myosin heavy chain [MHC], cardiac Troponin T [cTNT]) cardiomyocyte markers. Epo increased their proliferation and thus were designated as Epo-responsive MHC expressing cells (EMCs). In vitro, EMCs proliferated and partially differentiated toward cardiomyocyte-like cells. Repetitive Epo administration in mice with myocardial infarction (cumulative dose 4 IU/g) resulted in an increase in cardiac EMCs and cTNT-positive cells in the infarcted area. This was further accompanied by a significant preservation of cardiac function when compared with control mice. Our study characterized an EPO-responsive MHC-expressing cell population in the adult heart. Repetitive, moderate-dose Epo treatment enhanced the proliferation of EMCs resulting in preservation of post-ischemic cardiac function.

The four and a half LIM-domain 2 controls early cardiac cell commitment and expansion via regulating ß-catenin-dependent transcription.

The multiphasic regulation of the Wnt/ß-catenin canonical pathway is essential for cardiogenesis in vivo and in vitro. To achieve tight regulation of the Wnt/ß-catenin signaling, tissue- and cell-specific coactivators and repressors need to be recruited. The identification of such factors may help to elucidate mechanisms leading to enhanced cardiac differentiation efficiency in vitro as well as promote regeneration in vivo. Using a yeast-two-hybrid screen, we identified four-and-a-half-LIM-domain 2 (FHL2) as a cardiac-specific ß-catenin interaction partner and activator of Wnt/ß-catenin-dependent transcription. We analyzed the role of this interaction for early cardiogenesis in an in vitro model by making use of embryoid body cultures from mouse embryonic stem cells (ESCs). In this model, stable FHL2 gain-of-function promoted mesodermal cell formation and cell proliferation while arresting cardiac differentiation in an early cardiogenic mesodermal progenitor state. Mechanistically, FHL2 overexpression enhanced nuclear accumulation of ß-catenin and activated Wnt/ß-catenin-dependent transcription leading to sustained upregulation of the early cardiogenic gene Igfbp5. In an alternative P19 cell model, transient FHL2 overexpression led to early activation of Wnt/ß-catenin-dependent transcription, but not sustained high-level of Igfbp5 expression. This resulted in enhanced cardiogenesis. We propose that early Wnt/ß-catenin-dependent transcriptional activation mediated by FHL2 is important for the transition to and expansion of early cardiogenic mesodermal cells. Collectively, our findings offer mechanistic insight into the early cardiogenic code and may be further exploited to enhance cardiac progenitor cell activity in vitro and in vivo.

Krueppel-like factor 15 regulates Wnt/ß-catenin transcription and controls cardiac progenitor cell fate in the postnatal heart.

Wnt/ß-catenin signalling controls adult heart remodelling in part via regulation of cardiac progenitor cell (CPC) differentiation. An enhanced understanding of mechanisms controlling CPC biology might facilitate the development of new therapeutic strategies in heart failure. We identified and characterized a novel cardiac interaction between Krueppel-like factor 15 and components of the Wnt/ß-catenin pathway leading to inhibition of transcription. In vitro mutation, reporter assays and co-localization analyses revealed that KLF15 requires both the C-terminus, necessary for nuclear localization, and a minimal N-terminal regulatory region to inhibit transcription. In line with this, functional Klf15 knock-out mice exhibited cardiac ß-catenin transcriptional activation along with functional cardiac deterioration in normal homeostasis and upon hypertrophy. We further provide in vivo and in vitro evidences for preferential endothelial lineage differentiation of CPCs upon KLF15 deletion. Via inhibition of ß-catenin transcription, KLF15 controls CPC homeostasis in the adult heart similar to embryonic cardiogenesis. This knowledge may provide a tool for reactivation of this apparently dormant CPC population in the adult heart and thus be an attractive approach to enhance endogenous cardiac repair.

Hepoxilin A(3) protects ß-cells from apoptosis in contrast to its precursor, 12-hydroperoxyeicosatetraenoic acid.

Pancreatic ß-cells have a deficit of scavenging enzymes such as catalase (Cat) and glutathione peroxidase (GPx) and therefore are susceptible to oxidative stress and apoptosis. Our previous work showed that, in the absence of cytosolic GPx in insulinoma RINm5F cells, an intrinsic activity of 12 lipoxygenase (12(S)-LOX) converts 12S-hydroperoxyeicosatetraenoic acid (12(S)-HpETE) to the bioactive epoxide hepoxilin A(3) (HXA(3)). The aim of the present study was to investigate the effect of HXA(3) on apoptosis as compared to its precursor 12(S)-HpETE and shed light upon the underlying pathways. In contrast to 12(S)-HpETE, which induced apoptosis via the extrinsic pathway, we found HXA(3) not only to prevent it but also to promote cell proliferation. In particular, HXA(3) suppressed the pro-apoptotic BAX and upregulated the anti-apoptotic Bcl-2. Moreover, HXA(3) induced the anti-apoptotic 12(S)-LOX by recruiting heat shock protein 90 (HSP90), another anti-apoptotic protein. Finally, a co-chaperone protein of HSP90, protein phosphatase 5 (PP5), was upregulated by HXA(3), which counteracted oxidative stress-induced apoptosis by dephosphorylating and thus inactivating apoptosis signal-regulating kinase 1 (ASK1). Taken together, these findings suggest that HXA(3) protects insulinoma cells from oxidative stress and, via multiple signaling pathways, prevents them from undergoing apoptosis.

C. albicans activates cyclooxygenase but not its product prostaglandin E2in HPV 16-stabilized cells.

OBJECTIVE: The selective induction of cyclooxygenase-2 (COX-2) in human cells by Candida albicans was the first report of its role in infectious disease. This led us to question whether recurrent vulvovaginal candidosis in the cancer patient is involved in the formation of malignant tumors of the genital tract. Our speculation coincided with the patients' assessments in our hospital, where few cancer patients had a prior history of Candida infection. We wanted to study the contribution of C. albicans to gynecological cancers. STUDY DESIGN: In the present study, we used the developed vaginal epithelial cells system, having an insertion of HPV 16 viral sequence, as a model system (VK2/E6E7) to investigate the effect of Candida infection on prostaglandin E2 synthesis, which is known to be associated with cancers. We infected VK2/E6E7 cells with wild-type C. albicans and determined its effect on COX-2 and prostaglandin E2 synthesis, and its alteration in dependence on p53, and we analyzed the ubiquitin-proteasome degradation pathways and the involvement of 14-3-3 protein, which is involved in the modulation of the cell cycle. RESULTS: Our work using the cellular model indicates that recurrent Candida infection of the genital tract in patients carrying HPV 16 viral infection blocks the proliferation of host cells, PGE2 synthase expression and thus PGE2 production. CONCLUSION: We found that Candida infection contributes only to cell cycle arrest and does not by itself contribute actively to the development of cancer, although it is associated with patients diagnosed as having cancer of the genital tract induced by HPV 16 virus.

NF-kappaB activation is required for adaptive cardiac hypertrophy.

AIMS: We have previously shown that cardiac-specific inhibition of NF-kappaB attenuates angiotensin II (AngII)-induced left ventricular (LV) hypertrophy in vivo. We now tested whether NF-kappaB inhibition is able to block LV remodelling upon chronic pressure overload and chronic AngII stimulation. METHODS AND RESULTS: Cardiac-restricted NF-kappaB inhibition was achieved by expression of a stabilized IkappaBalpha mutant (IkappaBalphaDeltaN) in cells with an active alpha-myosin heavy chain (alphaMHC) promoter employing the Cre/lox technique. Upon low-gradient trans-aortic constriction (TAC, gradient 21 +/- 3 mmHg), hypertrophy was induced in both male and female control mice after 4 weeks. At this time, LV hypertrophy was blocked in transgenic (TG) male but not female mice with NF-kappaB inhibition. Amelioration of LV hypertrophy was associated with activation of NF-kappaB by dihydrotestosterone in isolated neonatal cardiomyocytes. LV remodelling was not attenuated by NF-kappaB inhibition after 8 weeks TAC, demonstrated by decreased fractional shortening (FS) in both control and TG mice irrespective of gender. Similar results were obtained when TAC was performed with higher gradients (48 +/- 4 mmHg). In TG mice, FS dropped to similar low levels over the same time course [FS sham, 29 +/- 1% (mean +/- SEM); FS control + 14 days TAC, 13 +/- 3%; FS TG + 14 days TAC, 9 +/- 5%]. Similarly, LV remodelling was accelerated by NF-kappaB inhibition in an AngII-dependent genetic heart failure model (AT1-R(alphaMHC)) associated with significantly increased cardiac fibrosis in double AT1-R(alphaMHC)/TG mice. CONCLUSION: NF-kappaB inhibition attenuates cardiac hypertrophy in a gender-specific manner but does not alter the course of stress-induced LV remodelling, indicating NF-kappaB to be required for adaptive cardiac hypertrophy.

Beta-Catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation.

We analyzed the effect of conditional, alphaMHC-dependent genetic beta-catenin depletion and stabilization on cardiac remodeling following experimental infarct. beta-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CT(Deltaex3-6): 24 +/- 1.9%; beta-cat(Deltaex3-6): 30.2 +/- 1.6%, P < 0.001). beta-Catenin stabilization had opposite effects. No significant changes in adult cardiomyocyte survival or hypertrophy were observed in either transgenic line. Associated with the functional improvement, LV scar cellularity was altered: beta-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnT(pos) cardiomyocytes associated with increased expression of cardiac lineage markers Tbx5 and GATA4. Using a Cre-dependent lacZ reporter gene, we identified a noncardiomyocyte cell population affected by alphaMHC-driven gene recombination localized to these tissue compartments at baseline. These cells were found to be cardiac progenitor cells since they coexpressed markers of proliferation (Ki67) and the cardiomyocyte lineage (alphaMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kit(pos) and stem cell antigen-1 (Sca-1)(pos) precursor cell population but not with the Islet-1(pos) precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1(pos) cardiac precursor cells from beta-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward alpha-actin(pos) and cTnT(pos) cardiomyocytes after 10 days (CT(Deltaex3-6): 38.0 +/- 1.0% alpha-actin(pos); beta-cat(Deltaex3-6): 49.9 +/- 2.4% alpha-actin(pos), P < 0.001). We conclude that beta-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4(pos)/Sca-1(pos) resident cardiac progenitor cells.

Hepoxilin A3 (HXA3) synthase deficiency is causative of a novel ichthyosis form.

Non-bullous congenital ichthyosis erythroderma (NCIE) and lamellar ichthyosis (LI) are characterized by mutations in 12R-lipoxygenase (12R-LOX) and/or epidermal lipoxygenase 3 (eLOX3) enzymes. The eLOX3 lacks oxygenase activity, but is capable of forming hepoxilin-type products from arachidonic acid-derived hydroperoxide from 12R-LOX, termed 12R-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12R-HpETE). Mutations in either of two enzymes lead to NCIE or LI. Moreover, 12R-LOX-deficient mice exhibit severe phenotypic water barrier dysfunctions. Here, we demonstrate that 12R-HpETE can also be transformed to 8R-HXA(3) by hepoxilin A(3) (HXA(3)) synthase (12-lipoxygenase), which exhibits oxygenase activity. We also presented a novel form of ichthyosis in a patient, termed hepoxilin A(3) synthase-linked ichthyosis (HXALI), whose scales expressed high levels of 12R-LOX, but were deficient of HXA(3) synthase.

Structure, biochemistry and biology of hepoxilins: an update.

Hepoxilins are biologically relevant epoxy-hydroxy eicosanoids synthesized through the 12S-lipoxygenase (12S-LOX) pathway of the arachidonic acid (AA) metabolism. The pathway is bifurcated at the level of 12S-hydroperoxy-eicosatetraenoic acid (12S-HpETE), which can either be reduced to 12S-hydro-eicosatetraenoic acid (12S-HETE) or converted to hepoxilins. The present review gives an update on the biochemistry, biology and clinical aspects of hepoxilin-based drug development. The isolation, cloning and characterization of a rat leukocyte-type 12S-LOX from rat insulinoma RINm5F cells revealed a 12S-LOX possessing an intrinsic 8S/R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA(3)) synthase activity. Site-directed mutagenesis studies on rat 12S-LOX showed that the HXA(3) synthase activity was impaired when the positional specificity of AA was altered. Interestingly, amino acid Leu353, and not conventional sequence determinants Met419 and Ile418, was found to be a crucial sequence determinant for AA oxygenation. The regulation of HXA(3) formation is dependent on the cellular overall peroxide tone. Cellular glutathione peroxidases (cGPxs) compete with HXA(3) synthase for 12S-HpETE as substrate either to reduce to 12S-HETE or to convert to HXA(3), respectively. Therefore, RINm5F cells, which are devoid of GPxs, are capable of converting AA or 12S-HpETE to HXA(3) under basal conditions, whereas cells overexpressing cGPx are unable to do so. HXA(3) exhibits a myriad of biological effects, most of which are associated with the stimulation of intracellular calcium or the transport of calcium across the membrane. The activation of HXA(3)-G-protein-coupled receptors explains many of the extracellular effects of HXA(3), including AA- and diacylglycerol (DAG) release in human neutrophils, insulin secretion in rat pancreatic beta-cells or islets, and synaptic actions in the brain. The availability of stable analogs of HXA(3), termed 10-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid derivatives (PBTs), recently made several animal studies possible and explored the role of HXA(3) as a therapeutic in treatment of diseases. Thus, PBT-3 induced apoptosis in K562 tumour cells and inhibited growth of K562 CML solid tumours in nude mice. HXA(3) inhibited bleomycin-evoked lung fibrosis and inflammation in mice and the raised insulin level in the circulation of rats. At low glucose concentrations (0-3 mm), HXA(3) also stimulated insulin secretion in RINm5F cells through the activation of IRE1alpha, an endoplasmic reticulum-resident kinase. The latter regulates the protein folding for insulin biosynthesis. In conclusion, HXA(3)-mediated signaling may be involved in normal physiological functions, and hepoxilin-based drugs may serve as therapeutics in diseases such as type II diabetes and idiopathic lung fibrosis.

Hepoxilin A3 synthase.

Hepoxilins constitute a group of 12S-hydroperoxyeicosatetraenoic acid (12S-HpETE)-derived epoxy-hydroxy fatty acids that have been detected in various cell types and tissues. Although hepoxilin A3 (HXA3) exhibits a myriad of biological activities, its biosynthetic mechanism was not investigated in detail. Here we review the isolation, cloning, and characterization of a leukocyte-type 12S-lipoxygenase (12S-LOX) from rat insulinoma cells RINm5F, which exhibits an intrinsic hepoxilin A3 synthase activity. Confirmation for this observation was achieved by coimmunoprecipitation of HXA3 synthase activity with an anti-leukocyte 12S-LOX antibody, preparation of recombinant rat 12S-LOX enzyme from RINm5F cells, and assay of HXA3 synthase activity therein. Site-directed mutagenesis studies performed on rat 12S-LOX showed that 12-lipoxygenating enzyme species exhibit a strong HXA3 synthase activity that is impaired when the positional specificity of arachidonic acid is altered in favor of 15-lipoxygenation. Inasmuch as cellular glutathione peroxidases (cGPx and PHGPx) and HXA3 synthase compete for the same substrate 12S-HpETE, it can be proposed that the overall activity of glutathione peroxidases, representing the overall peroxide tone, finely tunes the rate of HXA3 formation.

Oxygenation by COX-2 (cyclo-oxygenase-2) of 3-HETE (3-hydroxyeicosatetraenoic acid), a fungal mimetic of arachidonic acid, produces a cascade of novel bioactive 3-hydroxyeicosanoids.

Cyclo-oxygenases-1/2 (COX-1/2) catalyse the oxygenation of AA (arachidonic acid) and related polyunsaturated fatty acids to endoperoxide precursors of prostanoids. COX-1 is referred to as a constitutive enzyme involved in haemostasis, whereas COX-2 is an inducible enzyme expressed in inflammatory diseases and cancer. The fungus Dipodascopsis uninucleata has been shown by us to convert exogenous AA into 3(R)-HETE [3(R)-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid]. 3R-HETE is stereochemically identical with AA, except that a hydroxy group is attached at its C-3 position. Molecular modelling studies with 3-HETE and COX-1/2 revealed a similar enzyme-substrate structure as reported for AA and COX-1/2. Here, we report that 3-HETE is an appropriate substrate for COX-1 and -2, albeit with a lower activity of oxygenation than AA. Oxygenation of 3-HETE by COX-2 produced a novel cascade of 3-hydroxyeicosanoids, as identified with EI (electron impact)-GC-MS, LC-MS-ES (electrospray) and LC-MS-API (atmospheric pressure ionization) methods. Evidence for in vitro production of 3-hydroxy-PGE2 (3-hydroxy-prostaglandin E2) was obtained upon infection of HeLa cells with Candida albicans at an MOI (multiplicity of infection) of 100. Analogous to interaction of AA and aspirin-treated COX-2, 3-HETE was transformed by acetylated COX-2 to 3,15-di-HETE (3,15-dihydroxy-HETE), whereby C-15 showed the (R)-stereochemistry. 3-Hydroxy-PGs are potent biologically active compounds. Thus 3-hydroxy-PGE2 induced interleukin-6 gene expression via the EP3 receptor (PGE2 receptor 3) in A549 cells, and raised cAMP levels via the EP4 receptor in Jurkat cells. Moreover, 3R,15S-di-HETE triggered the opening of the K+ channel in HTM (human trabecular meshwork) cells, as measured by the patch-clamp technique. Since many fatty acid disorders are associated with an 'escape' of 3-hydroxy fatty acids from the b-oxidation cycle, the production of 3-hydroxyeicosanoids may be critical in modulation of effects of endogenously produced eicosanoids.