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We investigated the anorectal musclulature in normal children and anorectal malformations (ARM) to evaluate its role in bowel control mechanism. Pelves of 50 neonates died of ARM-unrelated diseases and 16 patients with anorectal malformations (8 high, 5 intermediate, and 3 low ARMs) were dissected and analyzed. Normal anorectal musculature was divided into three muscular tubes: the internal sphincter tube (IAST), longitudinal muscle tube (LMT) and transverse muscle tube (TMT). The LMT came from the outer longitudinal smooth muscle fiber of the rectum and the striated muscle fiber of the levator ani, and the TMT composed of the puborectalis and the external anal sphincter. However, in ARM, the IAST was absent and the LMT, the center of the sphincter muscle complex, was only from the levator ani and could be divided into the pelvic portion and the perineal portion. The former, from the upper rim of the puborectalis to the bulbar urethral, became narrowed and dislocated anteriorly near to the posterior urethra in high ARM and rectal pouch in intermediate ARM. The latter, below the bulbar urethra to the anal dimple, was fused to a column both in high and intermediate ARM. The columnar perineal LMT run downwards and then split, penetrated the superficial part of EAS and terminated at the deep aspect of the skin, to form the anal dimple, which represents the center of the perineal LMT from the perineal aspect. The length of the LMT was longer in high and intermediate ARM than the normal neonate. The columnar perineal LMT and narrowed pelvic LMT could be possibly identified by laparoscopic and perineal approaches retrospectively and widened to allow the passage of the rectum through. The anorectal musculature in ARM is composed of agenesic LMT and TMT and the narrowed LMT gives anatomical evidence of the center, where the neorectum should pull through.

Failure of hemodialysis access is caused mostly by venous intimal hyperplasia, a fibro-muscular thickening of the vessel wall. The pathogenesis of venous neointimal hyperplasia in primary arteriovenous fistulae consists of processes that have been identified as upstream and downstream events. Upstream events are the initial events producing injury of the endothelial layer (surgical trauma, hemodynamic shear stress, vessel wall injury due to needle punctures, etc.). Downstream events are the responses of the vascular wall at the endothelial injury that consist of a cascade of processes including leukocyte adhesion, migration of smooth muscle cells from the media to the intimal layer, and proliferation. In arteriovenous fistulae, the stenoses occur in specific sites, consistently related to the local hemodynamics determined by the vessel geometry and blood flow pattern. Recent findings that the localization of these sites matches areas of disturbed flow may add new insights into the pathogenesis of neointimal hyperplasia in the venous side of vascular access after the creation of the anastomosis. The detailed study of fluid flow motion acting on the vascular wall in anastomosed vessels and in the arm vasculature at the patient-specific level may help to elucidate the role of hemodynamics in vascular remodeling and neointimal hyperplasia formation. These computational approaches may also help in surgical planning for the amelioration of clinical outcome. This review aims to discuss the role of the disturbed flow condition in acting as upstream event in the pathogenesis of venous intimal hyperplasia and in producing subsequent local vascular remodeling in autogenous arteriovenous fistulae used for hemodialysis access. The potential use of blood flow analysis in the management of vascular access is also discussed.

Aims Alzheimer's disease (AD) is characterized by vascular dysfunction, in addition to memory impairment. Previously we found that β-sheet breaker peptides (βSBPs) improved memory impairment induced by amyloid β-peptide Aβ40. In this study we investigated βSBP effects on vascular responses in a rat model of AD. Main methods AD model was induced by bilateral injection of aged Aβ40 (3 nmol) into the amygdala. βSBPs 15–22, 16–23 and 17–24 (30 nmol) were injected into the amygdala 8 days after Aβ40. The Aβ40 deposits were examined immunohistochemically in cerebral vessels and thoracic aorta. The effects on high-K + contractility, phenylephrine (PE) contractility, acetylcholine (ACh) relaxation and sodium nitroprusside (SNP) relaxation were investigated in isolated thoracic aorta. Nitric oxide (NO) level in serum was investigated 14 days after Aβ40. Key findings Aβ40 was localized and it induced vascular damage in minute and small perforating cerebral vascular endothelium, and tunica intima (endothelial) and media (smooth muscle cells) of the thoracic aorta. In intact aorta, ACh-relaxation was decreased by Aβ40, an effect reduced by βSBPs 15–22 and 16–23. In denuded aorta, Aβ40 decreased PE-contractility. βSBP15–22 increased ACh-relaxation, whereas βSBP17–24 increased K + -contraction. Aβ40 decreased NO, an effect inhibited by the βSBP15–22. Significance These results provide evidence that Aβ40-perverted endothelium-dependent relaxation and decreased serum NO in AD rats were improved differentially by the βSBP15–22. These results show the ability of Aβ40 to alter vascular responses. βSBPs appear to be promising candidate for prevention of these consequences and therapy of AD.

Molecular beacons (MBs) of DCurrent treatments for human coronary artery disease necessitate the development of the next generations of vascular bioimplants. Recent reports provide evidence that controlling cell orientation and morphology through topographical patterning might be beneficial for bioimplants and tissue engineering scaffolds. However, a concise understanding of cellular events underlying cell-biomaterial interaction remains missing. In this study, applying methods of laser material processing, we aimed to obtain useful markers to guide in the choice of better vascular biomaterials. Our data show that topographically treated human primary vascular smooth muscle cells (VSMC) have a distinct differentiation profile. In particular, cultivation of VSMC on the microgrooved biocompatible polymer E-shell induces VSMC modulation from synthetic to contractile phenotype and directs formation and maintaining of cell-cell communication and adhesion structures. We show that the urokinase receptor (uPAR) interferes with VSMC behavior on microstructured surfaces and serves as a critical regulator of VSMC functional fate. Our findings suggest that microtopography of the E-shell polymer could be important in determining VSMC phenotype and cytoskeleton organization. They further suggest uPAR as a useful target in the development of predictive models for clinical VSMC phenotyping on functional advanced biomaterials.NA and RNA have aroused increasing interest because they allow a continuous readout, excellent spatial and temporal resolution to observe in real time. This kind of dual-labeled oligonucleotide probes can differentiate between bound and unbound DNA/RNA in homogenous hybridization with a high signal-to-background ratio in living cells. This review briefly summarizes the different unnatural sugar backbones of oligonucleotides combined with fluoro-phores that have been employed to sense DNA/RNA. With different probes, we epitomize the fundamental understanding of driving forces and these recognition processes. Moreover, we will introduce a few novel and attractive emerging applications and discuss their advantages and dis-advantages. We also highlight several perspective probes in the application of cancer therapeutics.

Atherosclerosis is one of the major complications of diabetes and involves endothelial dysfunction, matrix alteration, and most importantly migration and proliferation of vascular smooth muscle cells (VSMCs). Although hyperglycemia and hyperinsulinemia are known to contribute to atherosclerosis, little is known about the specific cellular signaling pathways that mediate the detrimental hyperinsulinemic effects in VSMCs. Therefore, we investigated the cellular mechanisms of hyperinsulinemia-induced migration and proliferation of VSMCs. VSMCs were treated with insulin (100 nM) for 6 days and subjected to various physiological and molecular investigations. VSMCs subjected to hyperinsulinemia exhibited increased migration and proliferation, and this is paralleled by oxidative stress [increased NADPH oxidase activity, NADPH oxidase 1 mRNA expression, and reactive oxygen species (ROS) generation], alterations in mitochondrial physiology (membrane depolarization, decreased mitochondrial mass, and increased mitochondrial ROS), changes in mitochondrial biogenesis-related genes (mitofusin 1, mitofusin 2, dynamin-related protein 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, peroxisome proliferator-activated receptor gamma coactivator 1-beta, nuclear respiratory factor 1, and uncoupling protein 2), and increased Akt phosphorylation. Diphenyleneiodonium, a known NADPH oxidase inhibitor significantly inhibited migration and proliferation of VSMCs and normalized all the above physiological and molecular perturbations. This study suggests a plausible crosstalk between mitochondrial dysfunction and oxidative stress under hyperinsulinemia and emphasizes counteracting mitochondrial dysfunction and oxidative stress as a novel therapeutic strategy for atherosclerosis. � 2012 Springer Science+Business Media New York.

We have demonstrated that bladder wall tissue in spinal cord injury (SCI) rats at 10 days post-injury is more compliant and accompanied by changes in material class from orthotropic to isotropic as compared to normal tissue. The present study examined the long-term effects (3-, 6-, and 10-weeks) post-SCI on the mechanical properties of bladder wall tissues, along with quantitative changes in smooth muscle orientation and collagen and elastin content. Bladder wall compliance (defined as det(F) - 1 under an equi-biaxial stress state of 100 kPa, where F is the deformation gradient tensor) was found to be significantly greater at 3- and 6-weeks (0.873 +/- 0.092 and 0.864 +/- 0.112, respectively) when compared to the normal bladders (0.260 +/- 0.028), but at 10 weeks the compliance reduced (0.389 +/- 0.061) to near that of normal bladders. This trend in mechanical compliance closely paralleled the collagen/elastin ratio. Moreover, changes in material class, assessed using a graphical technique, correlated closely with quantitative changes in smooth muscle fiber orientation. The results of the present study provide the first evidence that, while similarities exist between acute and chronic responses of the urinary bladder wall tissue to SCI, the overall alterations are distinct, result in profound and complex time dependent changes in bladder wall structure, and will lay the basis for simulations of the bladder wall disease process.

Purpose: Acute and/or chronic radiation enteritis can develop after radiotherapy for pelvic cancers. Experimental and clinical observations have provided evidence of a role played by acute mucosal disruption in the appearance of late effects. The therapeutic potential of acute administration of glucagon-like peptide-2 (GLP-2) against acute and chronic intestinal injury was investigated in this study. Methods and Materials: Intestinal segments were surgically exteriorized and exposed to 16.7 or 19 Gy X-rays. The rats were treated once daily with vehicle or a protease-resistant GLP-2 derivative for 14 days before irradiation, with or without 7 days of GLP-2 after treatment. Macroscopic and microscopic observations were made 2 and 15 weeks after radiation exposure. Results: In the control animals, GLP-2 induced an increase in intestinal mucosal mass, along with an increase in villus height and crypt depth. GLP-2 administration before and after irradiation completely prevented the acute radiation-induced mucosal ulcerations observed after exposure to 16.7 Gy. GLP-2 treatment strikingly reduced the late radiation damage observed after 19 Gy irradiation. Microscopic observations revealed an improved organization of the intestinal wall and an efficient wound healing process, especially in the smooth muscle layers. Conclusion: GLP-2 has a clear therapeutic potential against both acute and chronic radiation enteritis. This therapeutic effect is mediated through an increased mucosal mass before tissue injury and the stimulation of still unknown mechanisms of tissue response to radiation damage. Although these preliminary results still need to be confirmed, GLP-2 might be a way to limit patient discomfort during radiotherapy and reduce the risk of consequential late effects. © 2007 Elsevier Inc. All rights reserved.

Propionyl-l-carnitine (PLC) has been introduced among the therapeutic approaches of peripheral arterial disease, and more recently, an increase of intimal cell apoptosis has been demonstrated to contribute to its effectiveness in rabbit carotid postinjury myointimal hyperplasia prevention. How PLC mediates these effects on vascular smooth muscle cells (SMCs) remains poorly understood. We investigated the role of NF-kappaB in PLC-induced arterial remodeling. In vivo, daily PLC treatment 15 days after injury resulted in a reduction of relative rat aortic intimal volume, an increase of apoptosis, Bax up-regulation without changing the Bcl-2 level, and a reduction of NF-kappaB, vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and survivin in myointimal thickening compared with controls. In the presence of 10% serum, a reduced G(1) --S phase progression preceded PLC-induced intimal cell apoptosis; in 0.1% serum cultures, in a dose-dependent manner, PLC rapidly induced intimal cell apoptosis and reduced p65, p50, IAP-1, and IAP-2 expression. Inhibiting NF-kappaB activation through SN50 increased apoptotic rate and Bax expression in intimal but not in medial SMCs, and successive PLC treatment failed to induce a further increase in apoptotic rate. Bax antisense oligodeoxynucleotide reduced PLC-induced intimal cell apoptosis and cytochrome c release. The PLC-induced attenuation of NF-kappaB activity in intimal cells was also due to the increase of IkappaB-alpha bioavailability, as the result of a parallel induction of IkappaB-alpha synthesis and reduction of phosphorylation and degradation. Collectively, these findings document that NF-kappaB activity inhibition contributes to PLC-induced proliferative arrest and Bax-related apoptosis of intimal SMCs.

Aim: The remodelled asthmatic airway has increased airway smooth muscle cell (ASMC) growth, expanded vasculature, and altered extracellular matrix (ECM). The ECM is the external cellular microenvironment which regulates cell behaviour. Under proliferative, inflammatory, or fibrotic conditions, the asthmaticASM cells (AASMCs) deposit altered patterns of ECM proteins. However, it is unclear whether this is the case under basal, unstimulated conditions. This study aims to examine the composition of the ECM deposited by unstimulated NA (nonasthmatic)- and AASMCs, and biological potential for modulating blood vessel formation. Methods: Primary ASMCs from asthmatic and non-asthmatic individuals were quiesced in 0.1% BSA media after 72 hr of growth. After 24 hr ASMCs were lysed and the ECM either collected in lysate buffer and quantified using BCA, or kept intact and examined for collagen and fibronectin using Picrosirius Red and ELISA, respectively. Angiogenic potential was determined by examining Human Umbilical Vein Endothelial Cell (HUVEC) proliferation on (MTT and Cyquant assays), and attachment to ASMC-derived ECM. Unpaired t-tests were utilised to identify differences between NAASMC and AASMC-derived ECM. Results: Although the total amount of ECM deposited did not change (AASM N = 4 and NAASM N = 3 respectively), AASMCs deposited more collagen (N = 5, P

Aim: The remodelled asthmatic airway has increased airway smooth muscle cell (ASMC) growth, expanded vasculature, and altered extracellular matrix (ECM). The ECM is the external cellular microenvironment which regulates cell behaviour. Under proliferative, inflammatory, or fibrotic conditions, the asthmaticASM cells (AASMCs) deposit altered patterns of ECM proteins. However, it is unclear whether this is the case under basal, unstimulated conditions. This study aims to examine the composition of the ECM deposited by unstimulated NA (nonasthmatic)- and AASMCs, and biological potential for modulating blood vessel formation. Methods: Primary ASMCs from asthmatic and non-asthmatic individuals were quiesced in 0.1% BSA media after 72 hr of growth. After 24 hr ASMCs were lysed and the ECM either collected in lysate buffer and quantified using BCA, or kept intact and examined for collagen and fibronectin using Picrosirius Red and ELISA, respectively. Angiogenic potential was determined by examining Human Umbilical Vein Endothelial Cell (HUVEC) proliferation on (MTT and Cyquant assays), and attachment to ASMC-derived ECM. Unpaired t-tests were utilised to identify differences between NAASMC and AASMC-derived ECM. Results: Although the total amount of ECM deposited did not change (AASM N = 4 and NAASM N = 3 respectively), AASMCs deposited more collagen (N = 5, P

To evaluate the effect of local anesthesia on the skin vasodilation induced by the iontophoresis of sodium nitroprusside and heating.Skin vascular reactivity, in response to iontophoresis of sodium nitroprusside (SNP), was evaluated at the forearm and foot in 13 neuropathic diabetic (DN) and 11 nonneuropathic diabetic (D) patients and 9 healthy, nondiabetic subjects who served as controls (C). The direct (DI) and nerve axon reflex-related (N-V) vasodilation were measured by using two single-point laser Doppler probes. The vasodilation in response to local warming was also assessed. A topical anesthetic was applied on the contralateral forearm and foot and all the measurements were repeated.Dermal anesthesia resulted in a reduction of the direct vasodilation to SNP at the forearm [C: 58.1 +/- 16, D: 60.6 +/- 11%, and DN: 48.3 +/- 37% (postanesthesia percentage of reduction; mean +/- SEM), P0.01] and at the foot in all three groups (D: 38.5 +/- 12%, P0.01; C: 27.2 +/- 14% and DN: 11.3 +/- 17.5%, P=NS). The N-V related vasodilation was very low before and did not change after local anesthesia. The postanesthesia hyperemic response to warming was significantly reduced at low temperatures but did not change at 44 degrees C.The sodium nitroprusside-related vasodilation is reduced after local anesthesia in a similar way in healthy subjects and diabetic patients with and without neuropathy. The response to heating is also reduced at low temperatures. This indicates a stabilizing effect of local anesthesia on the smooth muscle cell.

Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.

Rationale Airway hyperresponsiveness is a principle feature of asthma, explained in part by remodeling of airway smooth muscle (ASM), including muscle thickening and increased extracellular matrix (ECM) protein production by the ASM. Current therapies are largely successful in targeting the inflammatory component of asthma, but lack the necessary means to reverse or prevent the remodeling process. β-catenin is a potential therapeutic target in this regard, as we have shown previously its critical role in the regulation of ASM proliferation and ECM production. Accordingly we have tested several small-molecule signaling modulators that attenuate the nuclear functions of β-catenin. Methods Three small-molecule compounds were employed in this study that each selectively target different components of β-catenin signaling: ICG-001 (inhibits β-catenin/CBP interaction), IQ-1 (prevents β-catenin/p300 interaction) and XAV-939 (a WNT-specific β-catenin antagonist that inhibits Tankyrase 1 and 2 to stabilize Axin). Immortalized human bronchial smooth muscle cell lines were treated with Fetal Bovine Serum (FBS) or Transforming Growth Factor-β1 (TGF-β1) in combination with increasing concentrations of ICG-001, IQ-1 or XAV-939. We assessed cell proliferation using an Alamar Blue conversion assay and Western blotting. ECM production was determined via RT-qPCR and Western blotting. Results ICG-001 dose dependently (0,1-10 μM) inhibited FBS-induced airway smooth muscle proliferation (58% at 3 μM, p

Background: Extracellular vesicle (EV)-derived microcalcifications formed in collagen-poor fibrous caps contribute to plaque rupture. Collagen accumulation and calcification are major determinants of plaque stability, although the mechanisms linking fibrotic and calcific responses are unknown. The collagen receptor discoidin domain receptor-1 (DDR-1) regulates plaque calcification in vivo; however, its role in the release of calcifying EVs remains unclear. We hypothesize that DDR-1 regulates the processes of fibrosis and EV-induced calcification in atherosclerotic plaques. Methods and results: Smooth muscle cells (SMC) from the carotid arteries of wild type and DDR-1 knockout (DDR-1-/-) mice (n=5 per group) were cultured in control or calcifying media. At days 14 and 21, cells were harvested and EVs isolated for analysis. Compared to wild type cells, DDR-1-/- SMCs exhibited a 3.5-fold increase in EV release (p

Rationale Airway hyperresponsiveness is a principle feature of asthma, explained in part by remodeling of airway smooth muscle (ASM), including muscle thickening and increased extracellular matrix (ECM) protein production by the ASM. Current therapies are largely successful in targeting the inflammatory component of asthma, but lack the necessary means to reverse or prevent the remodeling process. β-catenin is a potential therapeutic target in this regard, as we have shown previously its critical role in the regulation of ASM proliferation and ECM production. Accordingly we have tested several small-molecule signaling modulators that attenuate the nuclear functions of β-catenin. Methods Three small-molecule compounds were employed in this study that each selectively target different components of β-catenin signaling: ICG-001 (inhibits β-catenin/CBP interaction), IQ-1 (prevents β-catenin/p300 interaction) and XAV-939 (a WNT-specific β-catenin antagonist that inhibits Tankyrase 1 and 2 to stabilize Axin). Immortalized human bronchial smooth muscle cell lines were treated with Fetal Bovine Serum (FBS) or Transforming Growth Factor-β1 (TGF-β1) in combination with increasing concentrations of ICG-001, IQ-1 or XAV-939. We assessed cell proliferation using an Alamar Blue conversion assay and Western blotting. ECM production was determined via RT-qPCR and Western blotting. Results ICG-001 dose dependently (0,1-10 μM) inhibited FBS-induced airway smooth muscle proliferation (58% at 3 μM, p

Protein-disulphide isomerase family (PDI) are an ER-stress protein that controls TF-procoagulant activity but its role in HVSMC migration and coronary artery disease remains to be elucidated. We aimed to investigate whether in human coronary smooth muscle cells (HVSMC) the ER-stress protein-disulphide isomerase family A member 2 (PDIA2) regulates tissue factor (TF) polarisation during migration and atherosclerotic remodeling. PDIA2 and TF were analysed by confocal microscopy, silenced by small interfering RNAs (siRNA) and their function analysed by transwell and migration assays in vitro and in vivo. PDIA2and TF co-localise in the front edge of motile HVSMC. Silencing PDIA2, as well as silencing TF, reduces migration. PDIA2 silenced cells show increased TF-rich microparticle shedding. In vivo cell-loaded plug implants in nude mice of PDIA2 silenced HVSMC together with microvascular endothelial cells showed a significant impairment in mature microvessel formation. PDIA2 and TF are found in remodelled atherosclerotic plaques but not in healthy coronaries. In conclusion, we demonstrate that TF is chaperoned by PDIA2 to the HVSMC membrane and to the cell migratory front. Absence of PDIA2 impairs TF intracellular trafficking to its membrane docking favoring its uncontrolled release in microparticles. TF-regulated HVSMC migration and microvessel formation is under the control of the ER-protein PDIA2. © 2015 Schattauer.

Esophageal adenocarcinomas are believed to follow the intestinal metaplasia–dysplasia–carcinoma pathway. The metaplastic glandular epithelium is lined by a layer of periglandular myofibroblastic cells which regulate many aspects of the epithelial cells like in the intestine (refer to the intestine section for more detailed discussion) [4]. Due to the presence of the periglandular myofibroblastic sheath, benign intestinal epithelial proliferations have a characteristic serrated appearance. Except for the rare serrated adenocarcinomas in the colon which possess other easily discernible characteristic features, genuine serration is rarely seen in gastrointestinal epithelial malignancies. This important feature can be very useful in the diagnosis of well-differentiated carcinomas where invasion is insidious and desmoplasia is not evident.

High Throughput Screening (HTS) now plays an important role in the discovery of new lead compounds for novel therapeutic targets. The advantage of HTS over the conventional method, now termed as Low Throughput Screening (LTS), is that valuable compounds can be selected rapidly from a large number of samples with minimal human involvement. In spite of the growing awareness of HTS, the importance of the LTS in the drug discovery and development is still not changed. Advances in pharmacogenomics will also provide us many pharmacological targets, and thus increase the number of compounds that should be assayed by HTS and LTS. In this review, we will first describe the outline of HTS. We will next describe new approaches to develop and brush up the LTS: 1) screening method of drugs acting on ion channels by voltage-sensitive fluorescent dye, 2) functional assay method using reconstituted smooth muscle fiber, and 3) organ culture method as a useful model of vascular proliferative disease. These approaches, which work cooperatively with HTS, will contribute greatly to the development of new drugs.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition is a potential novel strategy for treatment of CVD. Alirocumab is a fully human PCSK9 monoclonal antibody in phase 3 clinical development. We evaluated the antiatherogenic potential of alirocumab in APOE∗3Leiden. CETP mice. Mice received a Western-type diet and were treated with alirocumab (3 or 10 mg/kg, weekly subcutaneous dosing) alone and in combination with atorvastatin (3.6 mg/kg/d) for 18 weeks. Alirocumab alone dose-dependently decreased total cholesterol (-37%; -46%, P < 0.001) and TGs (-36%; -39%, P < 0.001) and further decreased cholesterol in combination with atorvastatin (-48%; -58%, P < 0.001). Alirocumab increased hepatic LDL receptor protein levels but did not affect hepatic cholesterol and TG content. Fecal output of bile acids and neutral sterols was not changed. Alirocumab dose-dependently decreased atherosclerotic lesion size (-71%; -88%, P < 0.001) and severity and enhanced these effects when added to atorvastatin (-89%; -98%, P < 0.001). Alirocumab reduced monocyte recruitment and improved the lesion composition by increasing the smooth muscle cell and collagen content and decreasing the macrophage and necrotic core content. Alirocumab dose-dependently decreases plasma lipids and, as a result, atherosclerosis development, and it enhances the beneficial effects of atorvastatin in APOE∗3Leiden.CETP mice. In addition, alirocumab improves plaque morphology. Chemicals/CAS: alirocumab, 1245916-14-6; atorvastatin, 134523-00-5, 134523-03-8; cholesterol, 57-88-5; collagen, 9007-34-5

Rationale Airway hyperresponsiveness is a common feature of asthma explained in part by an excessive contractile response of the airway smooth muscle (ASM). The underlying mechanisms are complex and in need of study. WNT-5A and WNT-5B, two members of the WNT signaling pathway, may be of significance, as we have previously shown an active role for these ligands in cultured ASM and an increased WNT-5A expression in ASM isolated from asthmatics compared to healthy controls. Methods Using immortalized human bronchial smooth muscle cell lines, we investigated the role of WNT-5A and WNT-5B in ASM. Adopting a gain-of-function approach, we treated cells with recombinant WNT-5A or WNT-5B (100-1000 ng/ml) up to 96 hours. Furthermore, bovine tracheal smooth muscle (BTSM) strips were isolated and treated with recombinant WNT-5A or WNT-5B to determine isometric contraction. Results An initial microarray study indicated a modulatory role for WNT-5A, highlighting a wide subset of genes involved in mitochondrial function. Subsequent studies confirmed this role, indicating that treatment with recombinant WNT-5A and WNT-5B induced a strong depolarization of the mitochondrial membrane (Δψm), as was assessed with the fluorescent probe JC-1. Depolarization occurred dose dependently where the strongest effect was observed 48 hours following stimulation (32% and 22% for WNT-5A and WNT-5B respectively). Changes in Δψm were not due to initiation of apoptosis, as we could not find any difference in intracellular ATP levels nor in expression of mitochondrial mediated caspase-9 initiation. In addition, an Alamar Blue conversion assay indicated increased cellular viability following WNT treatment. Due to their crucial role in calcium buffering, we hypothesized that the observed mitochondrial membrane depolarization was calcium dependent. Using the calcium-sensitive ratiometric fluorescent dye fura-2, the involvement of calcium was corroborated as we observed a strong increase in resting cytosolic calcium levels following WNT-5A or WNT-5B 48 hour pre-treatment. Interestingly, direct WNT stimulation did not induce intracellular calcium release. To extent our findings in a functional setting, we pre-treated isolated BTSM strips with WNT-5A or WNT-5B for 48 hours. WNT-5A increased maximal contractility (25%) and sensitivity to histamine in isolated BTSM strips, whereas WNT-5B had no effect. Conclusions Our findings indicate a role for WNT-5A and WNT-5B in the regulation of calcium levels in ASM cells that likely includes a previously unidentified mitochondrial component. These findings may be of relevance to increased ASM contractility in asthma.

Metal-based compounds represent promising anticancer therapeutic agents. In this study, the mechanism of action of a novel metal-based drug, a palladium(II) (Pd) complex ([PdCl(terpy)](sac)2H(2)O, terpy=2,2':6',2 ''-terpyridine and sac=saccharinate), was elucidated. The tested compound induced cytotoxicity in nine different human cancer cell lines that originated from various organs, suggesting a broad spectrum of activity. The IC50 values were significantly higher for noncancerous cells when compared with cancer cells. We found that cells treated with the Pd(II) complex exhibited increased caspase 3/7 activities and condensed/fragmented nuclei, as demonstrated by nuclear staining and DNA laddering. Morphological features, such as cellular shrinkage and blebbing, were also observed, indicating that apoptosis was the primary mechanism of cell death. Pd(II) treatment induced DNA double-stranded breaks both in vitro and in vivo, potentially accounting for the source of stress in these cells. Although caspase 3/7 activities were elevated after Pd(II) treatment, silencing or using inhibitors of caspase 3 did not block apoptosis. Other molecules that could potentially play a role in Pd(II)-induced apoptosis, such as p53 and Bax, were also tested using silencing technology. However, none of these proteins were essential for cell death, indicating either that these molecules do not participate in Pd(II)-induced apoptosis or that other pathways were activated in their absence. Hence, this new molecule might represent a promising anticancer agent that exhibits cytotoxicity in p53-mutant, Bax-mutant, and/or caspase 3-mutant cancer cells. (C) 2013 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Increased airway smooth muscle (ASM) mass is a common pathological feature associated with chronic airway diseases, notably asthma. The mechanisms, however, remain poorly understood. The WNT (Wingless/integrase-1) signaling pathway has been implicated in various proliferative diseases, including lung cancer. In addition, the phosphorylation of GSK-3b, a key regulator of the WNT pathway, correlates with ASM hypertrophy. The WNT pathway may therefore be of particular significance in understanding ASM remodeling. Using immortalized human bronchial smooth muscle cell lines, we investigated the role of the WNT pathway in ASM remodeling. Our approach was twofold: first, to establish which WNT ligands are expressed in ASM and determine their ability to induce a mitogenic response and second, to characterize the potential mechanisms of action. An initial screen for all known WNT ligands showed WNT-5A, WNT-5B and WNT-16 to be strongly expressed in serum-deprived ASM cells, while displaying an additional increase after stimulation with platelet derived growth factor (PDGF) or fetal bovine serum (FBS). The increase in gene expression appeared to be concentration-dependent. Interestingly, recombinant WNT-5A exhibited a strong mitogenic effect on ASM cells, using [3H]thymidine incorporation and Alamar blue conversion assays, whereas WNT-5B and WNT-16 had no effect. Moreover, WNT-5A knockdown attenuated PDGF induced proliferation. A subsequent microarray study confirmed the modulatory role of WNT-5A, highlighting a wide subset of genes involved in cell-cycle progression and proliferation. Together, these findings underline a previously unidentified role for WNT-5A in the regulation of airway smooth muscle proliferation in vitro.

Increased airway smooth muscle (ASM) mass is a common pathological feature associated with chronic airway diseases, notably asthma. The mechanisms, however, remain poorly understood. The WNT (Wingless/integrase-1) signaling pathway has been implicated in various proliferative diseases, including lung cancer. In addition, the phosphorylation of GSK-3b, a key regulator of the WNT pathway, correlates with ASM hypertrophy. The WNT pathway may therefore be of particular significance in understanding ASM remodeling. Using immortalized human bronchial smooth muscle cell lines, we investigated the role of the WNT pathway in ASM remodeling. Our approach was twofold: first, to establish which WNT ligands are expressed in ASM and determine their ability to induce a mitogenic response and second, to characterize the potential mechanisms of action. An initial screen for all known WNT ligands showed WNT-5A, WNT-5B and WNT-16 to be strongly expressed in serum-deprived ASM cells, while displaying an additional increase after stimulation with platelet derived growth factor (PDGF) or fetal bovine serum (FBS). The increase in gene expression appeared to be concentration-dependent. Interestingly, recombinant WNT-5A exhibited a strong mitogenic effect on ASM cells, using [3H]thymidine incorporation and Alamar blue conversion assays, whereas WNT-5B and WNT-16 had no effect. Moreover, WNT-5A knockdown attenuated PDGF induced proliferation. A subsequent microarray study confirmed the modulatory role of WNT-5A, highlighting a wide subset of genes involved in cell-cycle progression and proliferation. Together, these findings underline a previously unidentified role for WNT-5A in the regulation of airway smooth muscle proliferation in vitro.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:54Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:37:27Z : No. of bitstreams: 1 2-s2.0-84875899455.pdf: 3515438 bytes, checksum: bcbb303312508bf21ce057f018240a02 (MD5) Made available in DSpace on 2014-05-27T11:28:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-09 Background: Cimetidine, histamine H2 receptors antagonist, has caused adverse effects on the male hormones and reproductive tract due to its antiandrogenic effect. In the testes, peritubular myoid cells and muscle vascular cells death has been associated to seminiferous tubules and testicular microvascularization damages, respectively. Either androgen or histamine H2 receptors have been detected in the mucosa and smooth muscular layer of vas deferens. Thus, the effect of cimetidine on this androgen and histamine-dependent muscular duct was morphologically evaluated.Methods: The animals from cimetidine group (CMTG; n=5) received intraperitoneal injections of 100 mg/kg b.w. of cimetidine for 50 days; the control group (CG) received saline solution. The distal portions of vas deferens were fixed in formaldehyde and embedded in paraffin. Massońs trichrome-stained sections were subjected to morphological and the following morphometrical analyzes: epithelial perimeter and area of the smooth muscular layer. TUNEL (Terminal deoxynucleotidyl-transferase mediated dUTP Nick End Labeling) method, NF-kB (nuclear factor kappa B) and AR (androgen receptors) immunohistochemical detection were also carried out. The birefringent collagen of the muscular layer was quantified in picrosirius red-stained sections under polarized light. The muscular layer was also evaluated under Transmission Electron Microscopy (TEM).Results: In CMTG, the mucosa of vas deferens was intensely folded; the epithelial cells showed numerous pyknotic nuclei and the epithelial perimeter and the area of the muscular layer decreased significantly. Numerous TUNEL-labeled nuclei were found either in the epithelial cells, mainly basal cells, or in the smooth muscle cells which also showed typical features of apoptosis under TEM. While an enhanced NF-kB immunoexpression was found in the cytoplasm of muscle cells, a weak AR immunolabeling was detected in these cells. In CMTG, no significant difference was observed in the birefringent collagen content of the muscular layer in comparison to CG.Conclusions: Cimetidine induces significant damages in the epithelium; a possible antiandrogenic effect on the basal cells turnover should be considered. The cimetidine-induced muscle cells apoptosis confirms the susceptibility of these cells to this drug. The parallelism between enhanced cytoplasmic NF-kB immunolabeling in the damaged muscular tissue and muscle cell apoptosis suggests that this drug may avoid the translocation of NF-kB to the nucleus and interfere in the control of NF-kB-mediated smooth muscle cell apoptosis. The decreased immunoexpression of ARs verified in the damaged muscular tissue reinforces this possibility. © 2013 Koshimizu et al.; licensee BioMed Central Ltd. Department of Morphology Laboratory of Histology and Embryology Araraquara Dental School-UNESP Univ. Estadual Paulista Department of Morphology and Genetics Federal University of São Paulo (UNIFESP), São Paulo/SP Department of Morphology Laboratory of Histology and Embryology Araraquara Dental School-UNESP Univ. Estadual Paulista