Erythropoietin Amplifies Stroke-Induced Oligodendrogenesis in the Rat

Background

Erythropoietin (EPO), a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia.

Methodology and Principal Findings

Recombinant human EPO (rhEPO) at a dose of 5,000 U/kg (n = 18) or saline (n = 18) was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs) measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ), but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase) positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ) neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU) for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation.

Conclusions

Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.     

Stabilizing HDAC11 with SAHA to assay slow-binding benzamide inhibitors

Among 18 human histone deacetylases (HDAC), HDAC11 is least studied. MS275, a benzamide HDAC inhibitor (HDACi), was stereotypically considered to selectively target Class I HDACs. We verified this slow-binding inhibitor also targeted HDAC11. In a traditional enzyme based assay, MS275 at low concentrations surprisingly behaved as an agonist. This was attributed to the poor stability of HDAC11 which lost 40% activity in 3 h at 37 °C. By adding 0.2 μM SAHA, HDAC11 activity was stabilized during the 3-h assay period. Since 0.2 μM SAHA inhibited 50% HDAC11 activity, the apparent IC₅₀′ of MS275 was adjusted to the true IC₅₀ = 0.65 μM. Finally, the new method demonstrated its superiority in one-dose-screening assays by decreasing false negative results. This work highlighted an optimized strategy to assay slow-binding inhibitors of unstable proteins with known fast-binding inhibitors. It should be especially useful in a hit-discovery stage to find moderate potent compounds.     

Novel HDAC6 isoform selective chiral small molecule histone deacetylase inhibitors

In an effort to identify HDAC isoform selective inhibitors, we designed and synthesized novel, chiral 3,4-dihydroquinoxalin-2(1H)-one and piperazine-2,5-dione aryl hydroxamates showing selectivity (up to 40-fold) for human HDAC6 over other class I/IIa HDACs. The observed selectivity and potency (IC₅₀ values 10–200 nM against HDAC6) is markedly dependent on the absolute configuration of the chiral moiety, and suggests new possibilities for use of chiral compounds in selective HDAC isoform inhibition.     

Lactam based 7-amino suberoylamide hydroxamic acids as potent HDAC inhibitors

A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7 of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data compared with the corresponding linear products, without substituent in position 7. In general, this modification provided an effective reinforcement of in vitro activity. While the lactam size or the CO/NH group orientation did not strongly influence the inhibition, the contemporary modification of the suberoylamide fragment gave vary active variants in the lactam series, with compound 28 (ST8078AA1) that showed IC₅₀ values between 2 and 10 nM against all Class I HDAC isoforms, demonstrating it to be a large spectrum pan-inhibitor. This strong affinity with HDAC was also confirmed by the value of IC₅₀ = 0.5 μM against H460 cells, ranking 28 as one of the most potent HDAC inhibitors described so far.     

Discovery of novel 9H-purin derivatives as dual inhibitors of HDAC1 and CDK2

HDAC and CDK inhibitors have been demonstrated to be synergistically in suppressing cancer cell proliferation and inducing apoptosis. In this work, we incorporated the pharmacophore groups of HDACs and CDKs inhibitors into one molecule to design and synthesize a series of purin derivatives as HDAC/CDK dual inhibitors. The lead compound 6d, showing good HDAC1 and CDK2 inhibitory activity with IC₅₀ values of 5.8 and 56 nM, respectively, exhibited attractive potency against several cancer cell lines in vitro. This work may lead to the discovery of a novel scaffold and potential dual HDAC/CDK inhibitors.     

SelSA,selenium analogs of SAHA as potent histone deacetylase inhibitors

Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase (HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular, HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy. Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In particular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the literature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors; SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC₅₀ values of 196 nM. These results provided novel selenium-containing potent HDAC inhibitors.     

Design,synthesis and biological evaluation of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as novel histone deacetylase inhibitors

A series of 4-anilinothieno[2,3-d]pyrimidine-based hydroxamic acid derivatives as novel HDACs inhibitors were designed, synthesized and evaluated. Most of these compounds displayed good to excellent inhibitory activities against HDAC1, 3, 6. The IC₅₀ values of compound 10r against HDAC1, HDAC3, HDAC6 was 1.14 ± 0.03 nM, 3.56 ± 0.08 nM, 11.43 ± 0.12 nM. Compound 10r noticeably up-regulated the level of histone H3 acetylation compared to the SAHA. Most of the compounds showed the strong anti-proliferative activity against human cancer cell lines including RMPI8226 and HCT-116. The IC₅₀ values of Compounds 10r and 10t against RPMI8226 was 2.39 ± 0.20 μM, 1.41 ± 0.44 μM, respectively, and the HCT-116 was sensitive to the compounds 10h, 10m, 10r, 10w with the IC₅₀ values <1.9 μM.     

Erythropoietin promotes oligodendrogenesis and myelin repair following lysolecithin-induced injury in spinal cord slice culture

Here, we sought to delineate the effect of EPO on the remyelination processes using an in vitro model of demyelination. We report that lysolecithin-induced demyelination elevated EPO receptor (EpoR) expression in oligodendrocyte progenitor cells (OPCs), facilitating the beneficial effect of EPO on the formation of oligodendrocytes (oligodendrogenesis). In the absence of EPO, the resultant remyelination was insufficient, possibly due to a limiting number of oligodendrocytes rather than their progenitors, which proliferate in response to lysolecithin-induced injury. By EPO treatment, lysolecithin-induced proliferation of OPCs was accelerated and the number of myelinating oligodendrocytes and myelin recovery was increased. EPO also enhanced the differentiation of neural progenitor cells expressing EpoR at high level toward the oligodendrocyte-lineage cells through activation of cyclin E and Janus kinase 2 pathways. Induction of myelin-forming oligodendrocytes by high dose of EPO implies that EPO might be the key factor influencing the final differentiation of OPCs. Taken together, our data suggest that EPO treatment could be an effective way to enhance remyelination by promoting oligodendrogenesis in association with elevated EpoR expression in spinal cord slice culture after lysolecithin-induced demyelination.     

Design,synthesis and biological evaluation of β-boswellic acid based HDAC inhibitors as inducers of cancer cell death

The synthesis and bio-evaluation of naturally occurring boswellic acids (BAs) as an alternate CAP for the design of new HDAC inhibitors is described. All the compounds were screened against a panel of human cancer cell lines to identify leads, which were subsequently examined for their potential to inhibit HDACs. The identified lead compound showed IC₅₀ value of 6 μm for HDACs, found to induce G₁ cell cycle arrest at significantly low concentration (1 μM) and caused significant loss in mitochondrial membrane potential at 5 and 10 μM. Furthermore, specific interactions of the lead molecule inside the catalytic domain were also studied through in silico molecular modeling.     

Design,synthesis and biological evaluation of indeno[1,2-d]thiazole derivatives as potent histone deacetylase inhibitors

Novel indeno[1,2-d]thiazole hydroxamic acids were designed, synthesized, and evaluated for histone deacetylases (HDACs) inhibition and antiproliferative activities on tumor cell lines. Most of the tested compounds exhibited HDAC inhibition and antiproliferative activity against both MCF7 and HCT116 cells with GI₅₀ values in the sub-micromolar range. Among them, compound 6o showed good inhibitory activity against pan-HDAC with IC₅₀ value of 0.14 μM and significant growth inhibition on MCF7 and HCT116 cells with GI₅₀ values of 0.869 and 0.535 μM, respectively.     

Design,synthesis and preliminary activity assay of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as novel Histone deacetylases (HDACs) inhibitors

Histone deacetylases (HDACs) are enzymes involved in tumor genesis and development. Herein, we report a novel series of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives as HDACs inhibitors. The preliminary biological screening showed that most of our compounds exhibited potent inhibitory activity against HDACs. Within this series, five compounds, 13a (IC₅₀ = 0.58 ± 0.10 μM), 7d (IC₅₀ = 1.00 ± 0.16 μM), 8l (IC₅₀ = 1.06 ± 0.14 μM), 7i (IC₅₀ = 1.17 ± 0.19 μM) and 7a (IC₅₀ = 1.29 ± 0.15 μM) possessed better HDACs inhibitory activity than Vorinostat (IC₅₀ = 1.48 ± 0.20 μM). So these five compounds could be used as novel lead compounds for further design of HDACs inhibitors. The anti-proliferative activities of a few compounds and the structure–activity relationships are also briefly discussed.     

Cell proliferation and survival in the mating circuit of adult male hamsters: Effects of testosterone and sexual behavior

The transient actions of gonadal steroids on the adult brain facilitate social behaviors, including reproduction. In male rodents, testosterone acts in the posterior medial amygdala (MeP) and medial preoptic area (MPOA) to promote mating. Adult neurogenesis occurs in both regions. The current study determined if testosterone and/or sexual behavior promote cell proliferation and survival in MeP and MPOA. Two experiments were conducted using the thymidine analog BrdU. First, gonad-intact and castrated male hamsters (n = 6/group) were compared 24 h or 7 weeks after BrdU. In MeP, testosterone-stimulated cell proliferation 24 h after BrdU (intact: 22.8 ± 3.9 cells/mm², castrate: 13.2 ± 1.4 cells/mm²). Testosterone did not promote cell proliferation in MPOA. Seven weeks after BrdU, cell survival was sparse in both regions (MeP: 2.5 ± 0.6 and MPOA: 1.7 ± 0.2 cells/mm²), and was not enhanced by testosterone. In Experiment 2, gonad-intact sexually-experienced animals were mated weekly to determine if regular neural activation enhances cell survival 7 weeks after BrdU in MeP and MPOA. Weekly mating failed to increase cell survival in MeP (8.1 ± 1.6 vs. 9.9 ± 3.2 cells/mm²) or MPOA (3.9 ± 0.7 vs. 3.4 ± 0.3 cells/mm²). Furthermore, mating at the time of BrdU injection did not stimulate cell proliferation in MeP (8.9 ± 1.7 vs. 8.1 ± 1.6 cells/mm²) or MPOA (3.6 ± 0.5 vs. 3.9 ± 0.7 cells/mm²). Taken together, our results demonstrate a limited capacity for neurogenesis in the mating circuitry. Specifically, cell proliferation in MeP and MPOA are differentially influenced by testosterone, and the birth and survival of new cells in either region are not enhanced by reproductive activity.     

Constitutive hyperactivity of histone deacetylases enhances radioresistance in Lepidopteran Sf9 insect cells

BackgroundLepidopteran insect cells withstand multifold higher radiation doses and suffer far less DNA damage despite carrying numerous structural/functional homologies with mammalian cells. Since DNA–histone interactions significantly influence radiation-induced DNA damage, we investigated the role of histones in insect cell radioresistance.MethodsModified comet assay was used to assess the γ-radiation-induced DNA damage following serial histone depletion by varied salt concentrations. Acid–Urea–Triton (AUT) gel analysis combined with in silico predictions was used to compare mammalian and insect histones and acetylation status while HDAC activity was assessed/modified for studying the latter's role in radioresistance. Cell death was measured by morphological analysis and flow cytometry.ResultsHigh-salt extraction pattern from Sf9 nuclei suggested stronger DNA–histone affinity as the two core histones H2A/H2B could be extracted at much higher (2 M) concentration as compared to 1.2 M NaCl in mammalian (AA8) cells. Electrophoretic mobility of unirradiated Sf9 cells remained unaltered at all salt concentrations (0.14 M–2 M NaCl), and radiation-induced DNA damage increased only by 2 M-NaCl pre-treatment. In silico analysis confirmed excellent conservation of Lepidopteran H2A/H2B sequence with human histones including comparable N-terminal lysine residues, yet these had ~ 60% lower acetylation. Importantly, insect cells showed ~ 70% higher histone deacetylase activity whose inhibition by Trichostatin-A reversed hypo-acetylation state and caused significant radiosensitization, thereby confirming the protective contribution of reduced acetylation.ConclusionOur study reveals that the hypo-acetylated state of well-conserved core histones, maintained by considerable HDAC activity, contributes significantly in Lepidopteran radioresistance.General SignificanceThis investigation shows constitutively high activity of HDACs as a potential radioprotective mechanism existing in insect cells.     

Plasma interleukin-1β concentration is associated with stroke in sickle cell disease

The pathogenesis of sickle cell disease (HbSS), which has numerous complications including stroke, involves inflammation resulting in alteration of plasma inflammatory protein concentration. We investigated HbSS children with abnormal cerebral blood flow detected by trans-cranial Doppler ultrasound (TCD) who participated in the multi-center stroke prevention (STOP) study, to determine if plasma inflammatory protein concentration is associated with the outcome of stroke. Thirty-nine plasma samples from HbSS participants with elevated TCD who had no stroke, HbSS-NS (n = 13) or had stroke, HbSS-S (n = 13), HbSS steady-state controls (n = 7) and controls with normal hemoglobin, HbAA (n = 6), were analyzed simultaneously for 27 circulating inflammatory proteins. Logistic regression and receiver operating characteristics curve analysis of stroke on plasma inflammatory mediator concentration, adjusted for age and gender, demonstrated that interleukin-1β (IL-1β) was protective against stroke development (HbSS-NS = 19, 17–23, HbSS-S = 17, 16–19 pg/mL, median and 25th–75th percentile; odds ratio = 0.59, C.I. = 0.36–0.96) and was a good predictor of stroke (area under curve = 0.852). This result demonstrates a strong association of systemic inflammation with stroke development in HbSS via moderately increased plasma IL-1β concentration, which is furthermore associated with a decreased likelihood of stroke in HbSS.     

Synergistic induction of miR-126 by hypoxia and HDAC inhibitors in cardiac myocytes

HDAC inhibitors are under clinical development for the treatment of hypertrophic cardiomyopathy and heart failure although the mechanisms of protection are incompletely understood. Micro-RNA 126, an endothelium-specific miR has been assigned essential developmental roles in the heart by activating survival kinases ERK1/2 and Akt and increasing pro-angiogenic signaling. Here we provide the first evidence that hypoxia and HDAC inhibitors selectively and synergistically stimulate expression of miR-126 in cardiac myocytes. MiR-126 expression was increased 1.7-fold (p < 0.05) after 1 h of hypoxic exposure and this was further enhanced to 3.0-fold (p < 0.01) by simultaneously blocking HDAC with the pan-HDAC inhibitor Tricostatin A (TSA). TSA alone did not increase miR-126. In parallel, hypoxia and TSA synergistically increased p-ERK and p-Akt without effecting VEGF-A level. Knockdown of miR-126 with si-RNA eliminated inductions of p-ERK and p-Akt by hypoxia, whereas miR-126 overexpression mimicked hypoxia and amplified p-ERK and p-Akt in parallel with miR-126. The results suggest that miR-126 is a hypoxia-inducible target of HAT/HDAC and its activation in cardiac myocytes may contribute to cardioprotection by activating cell survival and pro-angiogenic pathways selectively during ischemia.     

Thromboxane A receptor-mediated cell proliferation, survival and gene expression in oligodendrocytes

Thromboxane A(2) receptors (TP) were previously localized to discrete regions in the rat brain on myelinated fiber tracts and oligodendrocytes (OLGs). The present studies extended these findings and investigated the effects of TP signaling on cell proliferation, survival, and gene expression in OLG progenitor cells (OPCs) and OLGs. It was found that the TP agonist, U46619 stimulated the proliferation of OPCs and promoted the survival of mature OLGs. Examination of the early gene expression events involved in OPC proliferation, revealed that c-fos expression was substantially increased by U46619 stimulation. Treatment of OPCs or OLGs with U46619 caused activation of the mitogen-activated protein kinases (MAPK) ERK 1/2. In OPCs this activation was blocked by inhibition of src. However, in OLGs this phosphorylation was not only blocked by inhibition of src but also by inhibition of protein kinase C (PKC). Furthermore, U46619 was found to increase CREB phosphorylation in both OPCs and OLGs. Similar to ERK 1/2 activation, there was a divergence in the mechanism of the TP-mediated CREB response for each cell type. Specifically, U46619 activation was attenuated by src and protein kinase A (PKA) inhibition in OPCs, whereas in OLGs this effect was blocked by inhibition of src, PKA as well as by inhibition of PKC. Collectively, these results provide the first demonstration that TP-activated nuclear signaling events are involved in the proliferation of OPCs, the survival of mature OLGs, and the stimulation of gene expression.