Maria-Patapia Zafiriou

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

We analyzed the effect of conditional, αMHC-dependent genetic β-catenin depletion and stabilization on cardiac remodeling following experimental infarct. β-Catenin depletion significantly improved 4-week survival and left ventricular (LV) function (fractional shortening: CTΔex3–6: 24 ± 1.9%; β-catΔex3–6: 30.2 ± 1.6%, P < 0.001). β-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: β-catenin-depleted mice showed a marked subendocardial and subepicardial layer of small cTnTpos 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 αMHC-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 (αMHC, GATA4, Tbx5) but not cardiac Troponin T (cTnT). The cell population overlaps in part with both the previously described c-kitpos and stem cell antigen-1 (Sca-1)pos precursor cell population but not with the Islet-1pos precursor cell pool. An in vitro coculture assay of highly enriched (>95%) Sca-1pos cardiac precursor cells from β-catenin-depleted mice compared to cells isolated from control littermate demonstrated increased differentiation toward α-actinpos and cTnTpos cardiomyocytes after 10 days (CTΔex3–6: 38.0 ± 1.0% α-actinpos; β-catΔex3–6: 49.9 ± 2.4% α-actinpos, P < 0.001). We conclude that β-catenin depletion attenuates postinfarct LV remodeling in part through increased differentiation of GATA4pos/Sca-1pos resident cardiac progenitor cells.

NF-κB 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.

Structure, biochemistry and biology of hepoxilins

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 (HXA3) synthase activity. Site‐directed mutagenesis studies on rat 12S‐LOX showed that the HXA3 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 HXA3 formation is dependent on the cellular overall peroxide tone. Cellular glutathione peroxidases (cGPxs) compete with HXA3 synthase for 12S‐HpETE as substrate either to reduce to 12S‐HETE or to convert to HXA3, respectively. Therefore, RINm5F cells, which are devoid of GPxs, are capable of converting AA or 12S‐HpETE to HXA3 under basal conditions, whereas cells overexpressing cGPx are unable to do so. HXA3 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 HXA3–G‐protein‐coupled receptors explains many of the extracellular effects of HXA3, including AA‐ and diacylglycerol (DAG) release in human neutrophils, insulin secretion in rat pancreatic β‐cells or islets, and synaptic actions in the brain. The availability of stable analogs of HXA3, 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 HXA3 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. HXA3 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), HXA3 also stimulated insulin secretion in RINm5F cells through the activation of IRE1α, an endoplasmic reticulum‐resident kinase. The latter regulates the protein folding for insulin biosynthesis. In conclusion, HXA3‐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 protects β-cells from apoptosis in contrast to its precursor, 12-hydroperoxyeicosatetraenoic acid

Pancreatic β-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.

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‐HXA3 by hepoxilin A3 (HXA3) synthase (12‐lipoxygenase), which exhibits oxygenase activity. We also presented a novel form of ichthyosis in a patient, termed hepoxilin A3 synthase‐linked ichthyosis (HXALI), whose scales expressed high levels of 12R‐LOX, but were deficient of HXA3 synthase.