Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression.

Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.

An observational study protocol to explore loneliness and systemic inflammation in an older adult population with chronic venous leg ulcers.

BACKGROUND: Chronic venous leg ulcers (CVLUs) are the most common type of lower extremity wound. Even when treated with evidenced-based care, 30-50% of CVLUs fail to heal. A specific gap exists about the association between psychosocial stressors, particularly loneliness, and biomarkers of inflammation and immunity. Loneliness is highly prevalent in persons with CVLUs, has damaging effects on health, and contributes to the development of multiple chronic conditions, promotes aberrant inflammation, and diminishes healing. However, the confluence of loneliness, inflammation and the wound healing trajectory has not been elucidated; specifically whether loneliness substantially mediates systemic inflammation and alters healing over time. This study seeks to address whether there is a specific biomarker profile associated with loneliness, CVLUs, and wound healing that is different from non-lonely persons with CVLUs. METHODS: An observational prospective study will identify, characterize and explore associations among psychosocial stressors, symptoms and biomarkers between 2 CVLU groups, with loneliness+ (n = 28) and without loneliness- (n = 28) during 4 weeks of wound treatment, measured at 3 time points. We will examine psychosocial stressors and symptoms using psychometrically-sound measures include PROMIS® and other questionnaires for loneliness, social isolation, depression, anxiety, stigma, sleep, fatigue, pain, quality of life, cognition, and function. Demographics data including health history, sex, age, wound type and size, wound age, and treatment will be recorded from the electronic health record. We will characterize a biomarker panel of inflammatory genes including chemotaxic and growth factors, vascular damage, and immune regulators that express in response to loneliness to loneliness and CVLUs using well-established RNA sequence and PCR methods for whole blood samples. In an exploratory aim we will explore whether age and sex/psychological stressors and symptoms indicate potential moderation/mediation of the effect of loneliness on the biomarker profile over the study period. DISCUSSION: This study will provide insight into the influence of psychosocial stressors, symptoms, and biological mechanisms on wound healing, towards advancing a future healing prediction model and interventions to address these stressors and symptoms experienced by persons with CVLUs.

TAK1/Map3k7 enhances differentiation of cardiogenic endoderm from mouse embryonic stem cells.

Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFß-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFß target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer.

BACKGROUND: Eukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy. METHODS: Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1. RESULTS: Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes. CONCLUSIONS: These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

Sub-clinical dose of bone morphogenetic protein-2 does not precipitate rampant, sustained inflammatory response in bone wound healing.

Large bone injuries, defects, and chronic wounds present a major problem for medicine. Several therapeutic strategies are used clinically to precipitate bone including a combination therapy delivering osteoinductive bone morphogenetic protein 2 (rhBMP-2) via an osteoconductive scaffold (absorbable collagen sponge [ACS], i.e., INFUSE). Adverse side effects reportedly associated with rhBMP2 administration include rampant inflammation and clinical failures. Although acute inflammation is necessary for proper healing in bone, inflammatory cascade dysregulation can result in sustained tissue damage and poor healing. We hypothesized that a subclinical dose of rhBMP2 modeled in the murine calvarial defect would not precipitate alterations to inflammatory markers during acute phases of bone wound healing. We utilized the 5 mm critical size calvarial defect in C57BL6 wild-type mice which were subsequently treated with ACS and a subclinical dose of rhBMP2 shown to be optimal for healing. Three and 7-day postoperative time points were used to assess the role that rhBMP-2 plays in modulating inflammation vs. ACS alone by cytokine array and histological interrogation. Data revealed that rhBMP-2 delivery resulted in substantial modulation of several markers associated with inflammation, most of which decreased to levels similar to control by the 7-day time point. Additionally, while rhBMP-2 administration increased macrophage response, this peptide had a little noticeable effect on traditional markers of macrophage polarization (M1-iNOS, M2-Arg1). These results suggest that rhBMP-2 delivered at a lower dose does not precipitate rampant inflammation. Thus, an assessment of dosing for rhBMP-2 therapies may lead to better healing outcomes and less surgical failure.

IFN-α Negatively Regulates the Expression of Endothelial Nitric Oxide Synthase and Nitric Oxide Production: Implications for Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a known risk factor for endothelial dysfunction. Murine and human lupus studies revealed a role for IFN-α in vascular abnormalities associated with impaired blood vessel dilation. However, the impact of IFN-α on mediators that induce vasodilation and modulate inflammation, including endothelial NO synthase (eNOS) and NO bioavailability, are unknown. The objectives of this study were to determine how IFN-α promotes endothelial dysfunction in SLE, focusing on its regulation of eNOS and NO production in endothelial cells. We demonstrate that IFN-α promotes an endothelial dysfunction signature in HUVECs that is characterized by transcription suppression and mRNA instability of eNOS complemented by upregulation of MCP1 and VCAM1 These changes are associated with IFN-inducible gene expression. IFN-α impairs insulin-mediated NO production, and altered gene expression resulted from eNOS instability, possibly due to enhanced miR-155 expression. IFN-α significantly impaired NO production in insulin-stimulated HUVECs. IFN-α treatment also led to enhanced neutrophil adhesion. Our study introduces a novel pathway by which IFN-α serves as a proatherogenic mediator through repression of eNOS-dependent pathways. This could promote the development of endothelial dysfunction and cardiovascular disease in SLE.

HUNK Phosphorylates Rubicon to Support Autophagy.

BACKGROUND: Autophagy is a catabolic cellular recycling pathway that is essential for maintaining intracellular homeostasis. Autophagosome formation is achieved via the coordination of the Beclin-1 protein complex. Rubicon is a Beclin-1 associated protein that suppresses autophagy by impairing the activity of the class III PI3K, Vps34. However, very little is known about the molecular mechanisms that regulate Rubicon function. METHODS: In this study, co-immunoprecipitation and kinase assays were used to investigate the ability of Hormonally Upregulated Neu-associated Kinase (HUNK) to bind to and phosphorylate Rubicon. LC3B was monitored by immunofluorescence and immunoblotting to determine whether phosphorylation of Rubicon by HUNK controls the autophagy suppressive function of Rubicon. RESULTS: Findings from this study identify Rubicon as a novel substrate of HUNK and show that phosphorylation of Rubicon inhibits its function, promoting autophagy.

Optimizing bone wound healing using BMP2 with absorbable collagen sponge and Talymed nanofiber scaffold.

BACKGROUND: Bone is a highly vascularized and resilient organ with innate healing abilities, however some bone injuries overwhelm these attributes and require intervention, such as bone tissue engineering strategies. Combining biomaterials and growth factors, such as bone morphogenetic protein 2 (BMP2), is one of the most commonly used tissue engineering strategies. However, use of BMP2 has been correlated with negative clinical outcomes including aberrant inflammatory response, poor quality bone, and ectopic bone. METHODS: In the present study, a novel poly-n-acetyl glucosamine (pGlcNAc, trade name Talymed) scaffold was utilized in addition to the commonly used acellular collagen sponge (ACS) BMP2 delivery system in a murine calvarial defect model to investigate whether the innate properties of Talymed can reduce the noted negative bone phenotypes associated with BMP2 treatment. RESULTS: Comparison of murine calvarial defect healing between ACS with and without Talymed revealed that there was no measurable healing benefit for the combined treatment. Healing was most effective utilizing the traditional acellular collagen sponge with a reduced dose of BMP2. CONCLUSIONS: The results of this investigation lead to the conclusion that excessive dosing of BMP2 may be responsible for the negative clinical side effects observed with this bone tissue engineering strategy. Rather than augmenting the currently used ACS BMP2 bone wound healing strategy with an additional anti-inflammatory scaffold, reducing the dose of BMP2 used in the traditional delivery system results in optimal healing without the published negative side effects of BMP2 treatment.

Involvement of calvarial stem cells in healing: A regional analysis of large cranial defects.

Large craniofacial defects present a substantial clinical challenge that often requires the use of osteoconductive matrices and osteoinductive cues (i.e., bone morphogenetic proteins [BMP2]) to augment healing. While these methods have improved clinical outcomes, a better understanding of how the osteogenic fronts surrounding the defect, the underlying dura mater, and the cranial suture area contribute to healing may lead to more targeted therapies to enhance bone regeneration. We hypothesized that healing within a large bone defect will be precipitated from cells within the remaining or available suture mesenchyme abutting the edges of a murine critical sized defect. To investigate this hypothesis, 39 adult, wild-type mice were randomly arranged into groups (9 or 10 per group) by time (4 and 8 weeks) and treatment (control, acellular collagen sponge alone, or acellular collagen sponge loaded with a clinically relevant scaled dosage of BMP2). The skulls were then subjected to microcomputed tomography and histological analysis to assess bone regeneration in regions of interest within the defect area. A regional assessment of healing indicated that BMP2 drives greater healing than control and that healing emanates from the surgical margin, particularly from the margin associated with undisrupted suture mesenchyme. Though BMP2 treatment drove an increase in cell presence within the healing defect, there was no regional orientation of craniofacial stem cells or vascularity. Overall, these data reinforce that osteoconductive matrices in conjunction with osteoinductive peptides result in better healing of large calvarial defects. This healing is characterized as emanating from the surgical margin where there is an abundant supply of vasculature and progenitor cells.

AKT3 controls mitochondrial biogenesis and autophagy via regulation of the major nuclear export protein CRM-1.

Our previous work has shown that Akt3 is required for mitochondrial biogenesis in primary human endothelial cells (ECs) and in Akt3-null mice; Akt3 affects subcellular localization of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α), the master regulator of mitochondrial biogenesis. The purpose of this study is to determine the mechanism by which Akt3 controls the subcellular distribution of PGC-1α and to explore the effect on mitochondrial biogenesis and turnover during angiogenesis. Here we use standard biochemical analyses and Akt3-knockdown strategies to show that Akt3 controls the stabilization of chromosome maintenance region-1 (CRM-1), the major nuclear export receptor. Site-directed mutagenesis and association analyses show that PGC-1α nuclear export is CRM-1 dependent. Akt3 knockdown and CRM-1 overexpression cause 3-fold reductions in PGC-1α target gene expression, compared to control levels. Akt3 inhibition causes autophagy, as measured by autophagosome formation, in a CRM-1-dependent, Akt1/mTOR-independent pathway. In vivo, Akt3-null and heterozygous mice show dose-dependent decreases in angiogenesis compared to wild-type littermates (~5- and 2.5-fold decreases, respectively), as assessed by Matrigel plug assays. This correlates with an ~1.5-fold decrease in mitochondrial Cox IV expression. Our studies suggest that Akt3 is a regulator of mitochondrial dynamics in the vasculature via regulation of CRM-1-dependent nuclear export.

VEGF-mediated phosphorylation of eNOS regulates angioblast and embryonic endothelial cell proliferation.

To evaluate potential roles of nitric oxide (NO) in the regulation of the endothelial lineage and neovascular processes (vasculogenesis and angiogenesis) we evaluated endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) expression in 7.2-8.5 days post-coitum (dpc) mouse embryos. Analysis revealed that p-eNOS((S1177)) but not P-eNOS((S617)) or P-eNOS((T495)) was expressed in a subpopulation of angioblasts (TAL-1(+)/Flk-1(+)/CD31(-)/CD34(-)/VE-Cadherin(-)) at 7.2 dpc. A role of the VEGF/Akt1/eNOS signaling pathway in the regulation of the endothelial cell (EC) lineage was suggested by the strong correlation observed between cell division and p-eNOS((S1177)) expression in both angioblasts and embryonic endothelial cells (EECs, TAL-1(+)/Flk-1(+)/CD31(+)/CD34(+)/VE-Cadherin(+)). Our studies using Akt1 null mouse embryos show a reduction in p-eNOS((S1177)) expression in angioblast and EECs that is correlated with a decrease in endothelial cell proliferation and results in changes in VEGF-induced vascular patterning. Further, we show that VEGF-mediated cell proliferation in Flk-1(+) cells in allantoic cultures is decreased by pharmacological inhibitors of the VEGF/Akt1/eNOS signaling pathways. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation on Ser1177 regulates angioblast and EEC division, which underlies the formation of blood vessels and vascular networks.

GRP94 is an IGF-1R chaperone and regulates beta cell death in diabetes.

High workload-induced cellular stress can cause pancreatic islet ß cell death and dysfunction, or ß cell failure, a hallmark of type 2 diabetes mellitus. Thus, activation of molecular chaperones and other stress-response genes prevents ß cell failure. To this end, we have shown that deletion of the glucose-regulated protein 94 (GRP94) in Pdx1+ pancreatic progenitor cells led to pancreas hypoplasia and reduced ß cell mass during pancreas development in mice. Here, we show that GRP94 was involved in ß cell adaption and compensation (or failure) in islets from leptin receptor-deficient (db/db) mice in an age-dependent manner. GRP94-deficient cells were more susceptible to cell death induced by various diabetogenic stress conditions. We also identified a new client of GRP94, insulin-like growth factor-1 receptor (IGF-1R), a critical factor for ß cell survival and function that may mediate the effect of GRP94 in the pathogenesis of diabetes. This study has identified essential functions of GRP94 in ß cell failure related to diabetes.

Akt3 controls vascular endothelial growth factor secretion and angiogenesis in ovarian cancer cells.

The PI3 kinase/Akt pathway is commonly deregulated in human cancers, functioning in such processes as proliferation, glucose metabolism, survival and motility. We have previously described a novel function for one of the Akt isoforms (Akt3) in primary endothelial cells: the control of VEGF-induced mitochondrial biogenesis. We sought to determine if Akt3 played a similar role in carcinoma cells. Because the PI3 kinase/Akt pathway has been strongly implicated as a key regulator in ovarian carcinoma, we tested the role of Akt3 in this tumor type. Silencing of Akt3 by shRNA did not cause an overt reduction in mitochondrial gene expression in a series of PTEN positive ovarian cancer cells. Rather, we find that blockade of Akt3, results in smaller, less vascularized tumors in a xenograft mouse model that is correlated with a reduction in VEGF expression. We find that blockade of Akt3, but not Akt1, results in a reduction in VEGF secretion and retention of VEGF protein in the endoplasmic reticulum (ER). The reduction in secretion under conditions of Akt3 blockade is, at least in part, due to the down regulation of the resident golgi protein and reported tumor cell marker, RCAS1. Conversely, over-expression of Akt3 results in an increase in RCAS1 expression and in VEGF secretion. Silencing of RCAS1 using siRNA inhibits VEGF secretion. These findings suggest an important role for Akt3 in the regulation of RCAS1 and VEGF secretion in ovarian cancer cells.

Integrin-dependent Akt1 activation regulates PGC-1 expression and fatty acid oxidation.

BACKGROUND: Poly-N-acetyl glucosamine nanofibers derived from a marine diatom have been used to increase cutaneous wound healing. These nanofibers exert their activity by specifically activating integrins, which makes them a useful tool for dissecting integrin-mediated pathways. We have shown that short-fiber poly-N-acetyl glucosamine nanofiber (sNAG) treatment of endothelial cells results in increased cell motility and metabolic rate in the absence of increased cell proliferation. RESULTS: Using a Seahorse Bioanalyzer to measure oxygen consumption in real time, we show that sNAG treatment increases oxygen consumption rates, correlated with an integrin-dependent activation of Akt1. Akt1 activation leads to an increase in the expression of the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). This is not due to increased mitochondrial biogenesis, but is associated with an increase in the expression of pyruvate dehydrogenase kinase 4 (PDK4), suggesting regulation of fatty acid oxidation. Blockade of fatty acid oxidation with etomoxir, an O-carnitine palmitoyltransferase-1 inhibitor, blocks the sNAG-dependent increased oxygen consumption. (3)H-palmitate uptake experiments indicate a PDK4-dependent increase in fatty acid oxidation, which is required for nanofiber-induced cell motility. CONCLUSIONS: Our findings imply a linear pathway whereby an integrin-dependent activation of Akt1 leads to increased PGC-1α and PDK4 expression resulting in increased energy production by fatty acid oxidation.

Cellular and Molecular Mechanisms of MEK1 Inhibitor-Induced Cardiotoxicity.

Background: Trametinib is a MEK1 (mitogen-activated extracellular signal-related kinase kinase 1) inhibitor used in the treatment of BRAF (rapid accelerated fibrosarcoma B-type)-mutated metastatic melanoma. Roughly 11% of patients develop cardiomyopathy following long-term trametinib exposure. Although described clinically, the molecular landscape of trametinib cardiotoxicity has not been characterized. Objectives: The aim of this study was to test the hypothesis that trametinib promotes widespread transcriptomic and cellular changes consistent with oxidative stress and impairs cardiac function. Methods: Mice were treated with trametinib (1 mg/kg/d). Echocardiography was performed pre- and post-treatment. Gross, histopathologic, and biochemical assessments were performed to probe for molecular and cellular changes. Human cardiac organoids were used as an in vitro measurement of cardiotoxicity and recovery. Results: Long-term administration of trametinib was associated with significant reductions in survival and left ventricular ejection fraction. Histologic analyses of the heart revealed myocardial vacuolization and calcification in 28% of animals. Bulk RNA sequencing identified 435 differentially expressed genes and 116 differential signaling pathways following trametinib treatment. Upstream gene analysis predicted interleukin-6 as a regulator of 17 relevant differentially expressed genes, suggestive of PI3K/AKT and JAK/STAT activation, which was subsequently validated. Trametinib hearts displayed elevated markers of oxidative stress, myofibrillar degeneration, an 11-fold down-regulation of the apelin receptor, and connexin-43 mislocalization. To confirm the direct cardiotoxic effects of trametinib, human cardiac organoids were treated for 6 days, followed by a 6-day media-only recovery. Trametinib-treated organoids exhibited reductions in diameter and contractility, followed by partial recovery with removal of treatment. Conclusions: These data describe pathologic changes observed in trametinib cardiotoxicity, supporting the exploration of drug holidays and alternative pharmacologic strategies for disease prevention.

Poly-N-acetyl glucosamine fibers activate bone regeneration in a rabbit femur injury model.

BACKGROUND: The purpose of this study was to evaluate the ability of a membrane material, consisting only of short poly-N-acetyl glucosamine (sNAG) nanofibers, to regenerate bone tissue after implantation into circular holes in the rabbit femur. METHODS: Three circular holes were created in the femurs of five male New Zealand white rabbits. The holes were ∼ 2.0 mm in diameter. Three holes in the left femur were implanted with the comparative control substance (Bone Wax; Ethicon, Inc.); three holes in the right femur were implanted with the sNAG membrane test article. Animals were killed 4 weeks after surgery, and macroscopic evaluation of the implant sites was made. Hematoxylin and eosin histology was performed on both control and test sites. RESULTS: All control (bone wax) sites had visible holes (defects) at the 28-day end point of the study and showed no evidence of new bone formation. All the 15 sNAG test sites were found to have new bone tissue present in the bone hole defects. Hematoxylin and eosin histology of the sNAG-treated test sites showed the presence of osteoblasts, osteocytes, and trabecula of new bone formation at the 28-day end point of the study. CONCLUSIONS: The sNAG membrane test material activated the regeneration of new bone tissue in a rabbit femur bone model after 28 days of implantation, whereas the control bone wax material did not.

Thymomegaly, microsplenia, and defective homeostatic proliferation of peripheral lymphocytes in p51-Ets1 isoform-specific null mice.

Ets1 is a member of the Ets transcription factor family. Alternative splicing of exon VII results in two naturally occurring protein isoforms: full-length Ets1 (p51-Ets1) and Ets1(DeltaVII) (p42-Ets1). These isoforms bear key distinctions regarding protein-protein interactions, DNA binding kinetics, and transcriptional target specificity. Disruption of both Ets1 isoforms in mice results in the loss of detectable NK and NKT cell activity and defects in B and T lymphocytes. We generated mice that express only the Ets1(DeltaVII) isoform. Ets1(DeltaVII) homozygous mice express no p51-Ets1 and elevated levels of the p42-Ets1 protein relative to the wild type and display increased perinatal lethality, thymomegaly, and peripheral lymphopenia. Proliferation was increased in both the thymus and the spleen, while apoptosis was decreased in the thymus and increased in the spleen of homozygotes. Significant elevations of CD8(+) and CD8(+)CD4(+) thymocytes were observed. Lymphoid cell (CD19(+), CD4(+), and CD8(+)) reductions were predominantly responsible for diminished spleen cellularity, with fewer memory cells and a failure of homeostatic proliferation to maintain peripheral lymphocytes. Collectively, the Ets1(DeltaVII) mutants demonstrate lymphocyte maturation defects associated with misregulation of p16(Ink4a), p27(Kip1), and CD44. Thus, a balance in the differential regulation of Ets1 isoforms represents a potential mechanism in the control of lymphoid maturation and homeostasis.

Poly-N-acetyl glucosamine nanofibers: a new bioactive material to enhance diabetic wound healing by cell migration and angiogenesis.

INTRODUCTION: In several fields of surgery, the treatment of complicated tissue defects is an unsolved clinical problem. In particular, the use of tissue scaffolds has been limited by poor revascularization and integration. In this study, we developed a polymer, poly-N-acetyl-glucosamine (sNAG), with bioactive properties that may be useful to overcome these limitations. OBJECTIVE: To develop a scaffold-like membrane with bioactive properties and test the biologic effects in vitro and in vivo in diabetic wound healing. METHODS: In vitro, cells-nanofibers interactions were tested by cell metabolism and migration assays. In vivo, full thickness wounds in diabetic mice (n = 15 per group) were treated either with sNAG scaffolds, with a cellulosic control material, or were left untreated. Wound healing kinetics, including wound reepithelialization and wound contraction as well as microscopic metrics such as tissue growth, cell proliferation (Ki67), angiogenesis (PECAM-1), cell migration (MAP-Kinase), and keratinocyte migration (p 63) were monitored over a period of 28 days. Messenger RNA levels related to migration (uPAR), angiogenesis (VEGF), inflammatory response (IL-1beta), and extracellular matrix remodeling (MMP3 and 9) were measured in wound tissues. RESULTS: sNAG fibers stimulated cell metabolism and the in vitro migratory activity of endothelial cells and fibroblasts. sNAG membranes profoundly accelerated wound closure mainly by reepithelialization and increased keratinocyte migration (7.5-fold), granulation tissue formation (2.8-fold), cell proliferation (4-fold), and vascularization (2.7-fold) compared with control wounds. Expression of markers of angiogenesis (VEGF), cell migration (uPAR) and ECM remodeling (MMP3, MMP9) were up-regulated in sNAG treated wounds compared with controls. CONCLUSIONS: The key mechanism of the bioactive membranes is the cell-nanofiber stimulatory interaction. Engineering of bioactive materials may represent the clinical solution for a number of complex tissue defects.

VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase.

The growth factor, vascular endothelial growth factor (VEGF), induces angiogenesis and promotes endothelial cell (EC) proliferation. Affymetrix gene array analyses show that VEGF stimulates the expression of a cluster of nuclear-encoded mitochondrial genes, suggesting a role for VEGF in the regulation of mitochondrial biogenesis. We show that the serine threonine kinase Akt3 specifically links VEGF to mitochondrial biogenesis. A direct comparison of Akt1 vs. Akt3 gene silencing was performed in ECs and has uncovered a discrete role for Akt3 in the control of mitochondrial biogenesis. Silencing of Akt3, but not Akt1, results in a decrease in mitochondrial gene expression and mtDNA content. Nuclear-encoded mitochondrial gene transcripts are also found to decrease when Akt3 expression is silenced. Concurrent with these changes in mitochondrial gene expression, lower O(2) consumption was observed. VEGF stimulation of the major mitochondrial import protein TOM70 is also blocked by Akt3 inhibition. In support of a role for Akt3 in the regulation of mitochondrial biogenesis, Akt3 silencing results in the cytoplasmic accumulation of the master regulator of mitochondrial biogenesis, PGC-1alpha, and a reduction in known PGC-1alpha target genes. Finally, a subtle but significant, abnormal mitochondrial phenotype is observed in the brain tissue of AKT3 knockout mice. These results suggest that Akt3 is important in coordinating mitochondrial biogenesis with growth factor-induced increases in cellular energy demands.