S7111 Sigma-AldrichApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit

Biomaterials that can drive stem cells to an appropriate differentiation level and decrease apoptosis of transplanted cells are needed in regenerative medicine. Nanomaterials are promising novel materials for such applications. Here we reported that carboxylated multiwalled carbon nanotube (MWCNT 1) promotes myogenic differentiation of mouse myoblast cells and inhibits cell apoptosis under the differentiation conditions by regulating basic helix-loop-helix transcription factors. MWCNT 1 attenuates bone morphogenetic protein receptor (BMPR) signaling activity by binding to BMPR2 and attenuating the phosphorylation of BMPR1. This molecular understanding allowed us to tune stem cell differentiation to various levels by chemical modifications, demonstrating human control of biological activities of nanoparticles and opening an avenue for potential applications of nanomaterials in regenerative medicine.

Myocardin belongs to the SAF-A/B, Acinus, PIAS (SAP) domain family of transcription factors and is specifically expressed in cardiac and smooth muscle. Myocardin functions as a transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. We have previously found that myocardin induces the acetylation of nucleosomal histones surrounding SRF-binding sites in the control regions of cardiac and smooth muscle genes through recruiting chromatin-modifying enzyme p300, yet no studies have determined whether myocardin itself is similarly modified. In this study, we show that myocardin is a direct target for p300-mediated acetylation. p300 acetylates lysine residues at the N terminus of the myocardin protein. Interestingly, a direct interaction between p300 and myocardin, which is mediated by the C terminus of myocardin, is required for the acetylation event. Acetylation of myocardin by p300 enhances the association of myocardin and SRF as well as the formation of the myocardin-SRF-CArG box ternary complex. Conversely, acetylation of myocardin decreases the binding of histone deacetylase 5 (HDAC5) to myocardin. Acetylation of myocardin is required for myocardin to activate smooth muscle genes. Our study demonstrates that acetylation plays a key role in modulating myocardin function in controlling cardiac and smooth muscle gene expression.

Maxillofacial dysmorphogenesis is found in 5% of the population. To begin to understand the mechanisms required for maxillofacial morphogenesis, we employed the inhibitors of the differentiation 2 (Id2) knock-out mouse model, in which Id proteins, members of the regulator of basic helix-loop-helix (bHLH) transcription factors, modulate cell proliferation, apoptosis, and differentiation. We now report that spatially-restricted growth defects are localized at the skull base of Id2 KO mice. Curiously, at birth, neither the mutant Id2 KO nor wild-type (WT) mice differed, based upon cephalometric and histological analyses of cranial base synchondroses. In postnatal week 2, a narrower hypertrophic zone and an inhibited proliferative zone in presphenoid synchondrosis (PSS) and spheno-occipital synchondrosis (SOS) with maxillary hypoplasia were identified in the Id2 mutant mice. Complementary studies revealed that exogenous bone morphogenetic proteins (BMPs) enhanced cartilage growth, matrix deposition, and chondrocyte proliferation in the WT but not in the mutant model. Id2-deficient chondrocytes expressed more Smad7 transcripts. Based on our results, we assert that Id2 plays an essential role, acting downstream of BMP signaling, to regulate cartilage formation at the postnatal stage by enhancing BMP signals through inhibiting Smad7 expression. As a consequence, abnormal endochondral ossification was observed in cranial base synchondroses during the postnatal growth period, resulting in the clinical phenotype of maxillofacial dysmorphogenesis.

Intraventricular ependymal infection by adenoviruses expressing brain-derived neurotrophic factor (BDNF) and noggin is sufficient to induce the heterotopic recruitment of new medium spiny neurons to the adult neostriatum, from endogenous subependymal neural progenitor cells. This approach was found to slow disease progression and extend survival in an R6/2 mouse model of Huntington's disease (HD). However, the practical therapeutic value of this strategy is limited by the transient expression and immunogenicity of adenoviral vectors. In addition, it has been unclear whether sustained overexpression of BDNF and noggin would yield similarly sustained neuronal production and striatal recruitment, or whether progenitor depletion or tachyphylaxis might supervene to limit the therapeutic potential of this approach. To address these issues, we used adeno-associated virus serotype 4 (AAV4), an ependymotrophic vector that is neither immunogenic nor neurotoxic, to achieve sustained BDNF and noggin expression. Using AAV4, we found that BDNF and noggin achieved levels sufficient to initiate and maintain, for at least 4 months, ongoing neuronal addition to the neostriatum and olfactory bulb. Over this period, we noted no diminution of treatment-associated neuronal recruitment from resident progenitors. AAV4:BDNF and noggin-induced neuronal addition may thus provide a means to provide longlasting and persistent striatal neuronal replacement in conditions of striatal neuronal loss, such as HD.

Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.

We previously described the death of vascular cells (vascular rhexis) following persistent coronary occlusion. The present study was designed to determine whether non-sustained ischemia can initiate vascular rhexis and if so, whether relatively brief ischemic insults are sufficient. C57BL6 mice were subjected to coronary ligation for 15 min or 3 h followed by reperfusion. Soluble fractions of left ventricular (LV) homogenates were obtained 48 h after the onset of transitory coronary occlusion. They were assayed by Western blotting for quantification of alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) that we have shown reflect vascular rhexis delineated immunohistochemically. Non-sustained coronary occlusion for 3 h initiated vascular rhexis evident 45 h after reperfusion, but not earlier, as judged from Western blotting of α-SMA and SM-MHC. The number of small- and medium-sized vessels in the previously ischemic zones was reduced at 45 h after reperfusion as well. Thus, vascular rhexis occurs after ischemia as brief as 3 h but evolves slowly and is not evident for 45 h. The delayed disintegration of the vasculature makes it likely that it can be ameliorated by interventions initiated after non-sustained ischemia, rendering it an attractive target for diminution of phenomena such as late negative LV remodeling, and \'no reflow.\'

Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: β-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event.

Background and aims: Acute liver failure (ALF) is frequently complicated by cerebral oedema, systemic inflammation and multiorgan dysfunction. Vascular endothelial growth factor (VEGF) may stimulate liver regeneration but it can also be pro-inflammatory, activating endothelial cells and increasing permeability, actions mediated through Src kinase signalling. We therefore examined whether a Src inhibitor could have therapeutic potential in ALF. Methods: Murine ALF was induced with azoxymethane. Liver pathology was graded by a blinded examiner and apoptosis quantified by immunohistochemistry. Cerebral VEGF expression was imaged using VEGF-green fluorescent protein transgenic mice. Circulating and macrophage-secreted VEGF levels were measured. Experimental animals received a Src inhibitor or vehicle controls. Results: VEGF was undetectable in normal plasma but reached a mean of 835 pg/ml at grade III encephalopathy (P<0.001). Ammonia, lipopolysaccharide and interferon-gamma acted synergistically to enhance VEGF secretion by macrophages. Production of VEGF by cerebral cortical astrocytes increased with disease progression. Late treatment with inhibitors of Src or VEGF did not improve liver histology, encephalopathy or survival. However, early use of a Src kinase inhibitor significantly reduced hepatic injury, delayed encephalopathy and allowed 25% of mice to survive an otherwise lethal insult. Conclusion: Systemic and cerebral VEGF levels are significantly elevated during experimental ALF and may be exacerbated by hyperammonemia and macrophage activation. Early use of a Src inhibitor reduced hepatocellular injury and enabled survival, indicating such agents may have some promise in the treatment of ALF.© 2011 John Wiley & Sons A/S.

The primary aim of this study was to compare the effects of pentoxifylline (PTX) versus placebo on the histological features of nonalcoholic steatohepatitis (NASH). In all, 55 adults with biopsy-confirmed NASH were randomized to receive PTX at a dose of 400 mg three times a day (n = 26) or placebo (n = 29) over 1 year. The primary efficacy endpoint was defined as improvement in histological features of NASH through reduction in steatosis, lobular inflammation, and/or hepatocellular ballooning as reflected by a decrease of ≥2 points in the nonalcoholic fatty liver disease (NAFLD) activity score (NAS). After 1 year, intention-to-treat analysis showed a decrease of ≥2 points in the NAS in 38.5% of patients on PTX versus 13.8% of those on placebo (P = 0.036). Per protocol analysis, a decrease of ≥2 points in the NAS from baseline was observed in 50% of the patients on PTX versus 15.4% of those on placebo (P = 0.01). The mean change in NAS score from baseline was -1.6 in the PTX group, versus -0.1 in the placebo group (P < 0.001). PTX significantly improved steatosis (mean change in score -0.9 versus -0.04 with placebo, P < 0.001) and lobular inflammation (median change -1 versus 0 with placebo, P = 0.02). No significant effects in hepatocellular ballooning were observed. PTX also improved liver fibrosis (mean change in fibrosis score was -0.2 among those on PTX versus +0.4 among those on placebo, P = 0.038). Although not statistically significant (P = 0.17), improvement in fibrosis was observed in a greater proportion (35%) of patients in the PTX group compared to placebo (15%). Adverse effects were similar in both groups. Conclusion: PTX improved histological features of NASH compared to placebo. PTX was well tolerated in patients with NASH (ClinicalTrials.gov number NCT00590161). (HEPATOLOGY 2011).