Inhibition of Trophoblast-Induced Spiral Artery Remodeling Reduces Placental Perfusion in Rat Pregnancy.

Rats harboring the human angiotensinogen and human renin genes develop preeclamptic features in pregnancy. The preeclamptic rats exhibit a deeper trophoblast invasion associated with a reduced resistance index by uterine Doppler. Doxycycline inhibits matrix metalloproteinase activity. We tested the hypothesis that matrix metalloproteinase inhibition reduces trophoblast invasion with subsequent changes in placental perfusion. Preeclamptic and pregnant control Sprague-Dawley rats were treated with doxycycline (30 mg/kg of body weight orally) from gestational day 12 until day 18. Placental perfusion was assessed using a micromarker contrast agent. The animals were euthanized on day 18 of pregnancy; biometric data were acquired, and trophoblast invasion was analyzed. Doxycycline resulted in intrauterine growth retardation and lighter placentas in both groups. Maternal body weight was not affected. As shown earlier, preeclamptic rats exhibited a deeper endovascular trophoblast invasion. However, doxycycline treatment reduced trophoblast invasion in the preeclamptic rats. The physiological spiral artery remodeling, as assessed by the deposition of fibrinoid and α-actin in the spiral artery contour, was significantly reduced by doxycycline. The vascularity index, as assessed by perfusion measurement of the placenta, was reduced after doxycycline treatment in preeclamptic rats. Thus, matrix metalloproteinase inhibition with doxycycline leads to reduced trophoblast invasion and associated reduced placental perfusion. These studies are the first to show that reducing trophoblast-induced vascular remodeling decreases subsequent placental perfusion. Our model allows the study of dysregulated trophoblast invasion and vascular remodeling in vivo to gain important insights into preeclampsia-related mechanisms

Tetracycline Actions Relevant to Rosacea Treatment

Until today, the pathogenesis of rosacea is not known in detail. Yet in recent years evidence has been accumulating that rosacea with its common symptoms such as inflammatory lesions, erythema, telangiectasia, phymatous changes, and ocular symptoms is of inflammatory nature. Tetracycline derivatives like doxycycline successfully used in the treatment of skin diseases like acne and rosacea seem to inhibit different inflammatory pathways involved in the pathogenesis by various modes of action. Although data for skin diseases are relatively scanty, the following modes of action of tetracyclines seem to be most relevant for an effective treatment of acne and rosacea: inhibition of matrix metalloproteinases, downmodulation of cytokines, inhibition of cell movement and proliferation, inhibition of granuloma formation, inhibition of reactive oxygen species, nitric oxide, and angiogenesis, whereas inhibition of phospholipase A2 seems to be of lower importance. The role of the saprophytic mite Demodex folliculorum remains to be clarified. Additional studies are necessary to further elucidate how tetracyclines work in rosacea treatment. Copyright (C) 2009 S. Karger AG, Base

Non-viral Smad7 gene delivery and attenuation of postoperative peritoneal adhesion in an experimental model

Background: Postoperative intra-abdominal adhesion is associated with high morbidity and mortality. Smad7, a protein that occupies a strategic position in fibrogenesis, inhibits the transforming growth factor (TGF) β/Smad signalling pathway. In this study the therapeutic potential of exogenous Smad7 in preventing fibrogenesis in postoperative intra-abdominal adhesion was investigated. Methods: Intra-abdominal adhesion was induced in a rodent model by peritoneal abrasion. Smad7 was delivered into the peritoneal cavity by a non-viral ultrasound-microbubble-mediated naked gene transfection system. The effect of Smad7 transgene on adhesion formation was studied by measuring changes in TGF-β, fibrogenic factors, α-SMA and Smad2/3 activation in the anterior abdominal wall. Results: Four weeks after surgical abrasion, all rats developed significant peritoneal adhesion with enhanced TGF-β expression, increased levels of extracellular matrix components and activated myofibroblasts, accompanied by decreased Smad7 expression and increased Smad2/3 activation. In rats treated with the Smad7 transgene, the incidence and severity of peritoneal adhesion were significantly reduced, with biochemical downregulation of fibrogenic factors and inhibition of Smad2/3 activation. Serial quantitation using magnetic resonance imaging revealed a significant reduction in adhesion areas from day 14 onwards. Conclusion: Ultrasound-microbubble-mediated gene transfection provides timely targeted gene delivery for the treatment of postoperative peritoneal adhesions. Copyright © 2009 British Journal of Surgery Society Ltd. Published by John Wiley & Sons Ltd.postprin

Bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fibrosis.

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tThis is the final version of the article. Available from Public Library of Science via the DOI in this record.Many common diseases of the gas exchange surface of the lung have no specific treatment but cause serious morbidity and mortality. Idiopathic Pulmonary Fibrosis (IPF) is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma. Keratinocyte Growth Factor (KGF, also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. During repair, the lung not only uses resident cells after injury but also recruits circulating bone marrow-derived cells (BMDC). Several groups have used Mesenchymal Stromal Cells (MSCs) as therapeutic vectors, but little is known about the potential of Hematopoietic Stem cells (HSCs). Using an inducible lentiviral vector (Tet-On) expressing KGF, we were able to efficiently transduce both MSCs and HSCs, and demonstrated that KGF expression is induced in a regulated manner both in vitro and in vivo. We used the in vivo bleomycin-induced lung fibrosis model to assess the potential therapeutic effect of MSCs and HSCs. While both populations reduced the collagen accumulation associated with bleomycin-induced lung fibrosis, only transplantation of transduced HSCs greatly attenuated the histological damage. Using double immunohistochemistry, we show that the reduced lung damage likely occurs through endogenous type II pneumocyte proliferation induced by KGF. Taken together, our data indicates that bone marrow transplantation of lentivirus-transduced HSCs can attenuate lung damage, and shows for the first time the potential of using an inducible Tet-On system for cell based gene therapy in the lung.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. DB is a Cancer Research UK principle Investigator. SJ was an MRC Clinician Scientist. This work was in part funded by a Johnson and Johnson “Focussed Giving Grant” to SJ and partly undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme (SJ) and by internal Cancer Research UK funding (DB)

Future directions for the management of pain in osteoarthritis.

Osteoarthritis (OA) is the predominant form of arthritis worldwide, resulting in a high degree of functional impairment and reduced quality of life owing to chronic pain. To date, there are no treatments that are known to modify disease progression of OA in the long term. Current treatments are largely based on the modulation of pain, including NSAIDs, opiates and, more recently, centrally acting pharmacotherapies to avert pain. This review will focus on the rationale for new avenues in pain modulation, including inhibition with anti-NGF antibodies and centrally acting analgesics. The authors also consider the potential for structure modification in cartilage/bone using growth factors and stem cell therapies. The possible mismatch between structural change and pain perception will also be discussed, introducing recent techniques that may assist in improved patient phenotyping of pain subsets in OA. Such developments could help further stratify subgroups and treatments for people with OA in future

Poly(ADP-ribose) Polymerase-1 (PARP1) in Atherosclerosis: From Molecular Mechanisms to Therapeutic Potential

Poly(ADP-ribosyl)ation reactions, carried out by poly(ADP-ribose) polymerases (PARPs/ARTDs), are reversible posttranslational modifications impacting on numerous cellular processes (e.g., DNA repair, transcription, metabolism, or immune functions). PARP1 (EC 2.4.2.30), the founding member of PARPs, is particularly important for drug development for its role in DNA repair, cell death, and transcription of proinflammatory genes. Recent studies have established a novel concept that PARP1 is critically involved in the formation and destabilization of atherosclerotic plaques in experimental animal models and in humans. Reduction of PARP1 activity by pharmacological or molecular approaches attenuates atherosclerotic plaque development and enhances plaque stability as well as promotes the regression of pre-established atherosclerotic plaques. Mechanistically, PARP1 inhibition significantly reduces monocyte differentiation, macrophage recruitment, Sirtuin 1 (SIRT1) inactivation, endothelial dysfunction, neointima formation, foam cell death, and inflammatory responses within plaques, all of which are central to the pathogenesis of atherosclerosis. This article presents an overview of the multiple roles and underlying mechanisms of PARP1 activation (poly(ADP-ribose) accumulation) in atherosclerosis and emphasizes the therapeutic potential of PARP1 inhibition in preventing or reversing atherosclerosis and its cardiovascular clinical sequalae

Characterization of Sirt2 using conditional RNAi in mice

Within the past eight years, RNA interference (RNAi) has emerged as a powerful experimental tool for gene function analysis in mice. Reversible control of shRNA mediated RNAi has been achieved by using a tetracycline (tet)-inducible promoter. In the presence of the inductor doxycycline (dox), shRNA mediated gene silencing is initiated, whereas RNAi mechanism is blocked in the absence of dox. To achieve spatially and temporally regulated RNAi, the tet inducible system was combined with a Cre/loxP based strategy for tissue specific activation of shRNA constructs. To this end, a loxP-flanked "promoter inhibitory element" (PIE) was placed between the proximal (PSE) and distal sequence element (DSE) of a dox inducible promoter such that promoter function is completely blocked. Re-activation can be achieved through Cre mediated excision of PIE. To allow for gene silencing in a selected tissue, Cre expression can be regulated by a tissue-specific promoter. In mouse ES cells, the system mediated tight regulation of shRNA expression upon Cre mediated activation and dox administration, reaching knockdown efficiencies of >80%. Unexpectedly, the system showed a limited activity in transgenic mice when applied for conditional silencing of two different targets, LacZ and Sirt2. Sirt2 is a member of the sirtuin family which has considerably gained attention in vitro for its possible role in many physiological processes, including adipogenesis and neurodegenerative diseases. To investigate the function of Sirt2 in vivo, the unmodified dox-responsive and tet-inducible promoter was further used for conditional RNAi in transgenic mice. Inducible shRNA expression resulted in efficient silencing of Sirt2 (>90%) in all tissues which have been analyzed. Suppression of Sirt2 during embryogenesis resulted in offspring consisting of equal ratios of wild type and transgenic pups, indicating that Sirt2 is not indispensable for development. In adult animals, glucose metabolism, insulin sensitivity and energy balance appeared to be unaffected by Sirt2 deficiency. Likewise, expression of PPARγ, a downstream target of Sirt2, was not found to be altered upon Sirt2 inhibition. Finally, Sirt2 silencing was induced in an experimental model of Parkinson disease (PD). Data from Rotarod performances to study motor behaviour did not provide any evidence for a role of Sirt2 in PD pathogenesis as suggested by previous in vitro studies. Taken together, conditional Sirt2 silencing in vivo does not support speculation concerning a central role of Sirt2 in physiological processes, embryogenesis and in a mouse model of Parkinson disease

Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway

We previously established that DNA methyltransferase 3b (Dnmt3b) is the sole Dnmt responsive to fracture repair and that Dnmt3b expression is induced in progenitor cells during fracture repair. In the current study, we confirmed that Dnmt3b ablation in mesenchymal progenitor cells (MPCs) resulted in impaired endochondral ossification, delayed fracture repair, and reduced mechanical strength of the newly formed bone in Prx1-Cre;Dnmt3bf/f (Dnmt3bPrx1) mice. Mechanistically, deletion of Dnmt3b in MPCs led to reduced chondrogenic and osteogenic differentiation in vitro. We further identified Rbpjκ as a downstream target of Dnmt3b in MPCs. In fact, we located 2 Dnmt3b binding sites in the murine proximal Rbpjκ promoter and gene body and confirmed Dnmt3b interaction with the 2 binding sites by ChIP assays. Luciferase assays showed functional utilization of the Dnmt3b binding sites in murine C3H10T1/2 cells. Importantly, we showed that the MPC differentiation defect observed in Dnmt3b deficiency cells was due to the upregulation of Rbpjκ, evident by restored MPC differentiation upon Rbpjκ inhibition. Consistent with in vitro findings, Rbpjκ blockage via dual antiplatelet therapy reversed the differentiation defect and accelerated fracture repair in Dnmt3bPrx1 mice. Collectively, our data suggest that Dnmt3b suppresses Notch signaling during MPC differentiation and is necessary for normal fracture repair

The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration.

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices