Poly-l-Lysine inhibits VEGF and c-Myc mediated tumor-angiogenesis and induces apoptosis in 2D and 3D tumor microenvironment of both MDA-MB-231 and B16F10 induced mice model.

Cancer is a widespread disease that has shown promising mortality worldwide. Our previous study has been shown the efficacy of Poly-l-lysine (PLL) as a promising cytotoxic effect against cancer cells. However, exact-mechanism of PLL in 3D physiological relevant tumor-microenvironment and against tumor-angiogenesis has never been analysed. In this study, we have investigated apoptotic efficacy of PLL, if any in opposition to proliferative aggressive cancer cell MDA-MB-231 both 2D and-3D cell culture conditions. Furthermore, PLL was administered in B16F10 murine melanoma cells induced BALB/c mice model. The study has been designed through transcription and translation level of PLL-induced tumor-angiogenesis and apoptotic gene-expression modulation level and various relevant histological studies in comparison with untreated control. Studies have shown anti-proliferative and anti-tumor angiogenic efficacy of PLL better in in-vitro 3D tumor-microenvironment against MDA-MB-231 breast cancer cells. Furthermore, in-vivo model, PLL was found to suppress tumorigenesis process at minimum dose. PLL found to induce apoptosis through-upregulation of cytosolic-cytochrome-C, caspase-3 and PARP activations when administered in B16F10 induced in-vivo tumor. In blocking proliferation and tumor-angiogenesis, PLL was found to be effective as it significantly downregulated activity of VEGF, VEGFR2, Ki-67 and c-Myc expression. As PLL blocked tumor progression and induced DNA-break, also upregulated apoptotic process and recovered tissue architecture as revealed from histological study in comparison with untreated control. Overall PLL was found to be a promising anti-tumor angiogenic and anti-proliferative drug that was effective both in in-vitro breast cancer 3D tumor-microenvironment and in-vivo metastatic-mice-model.

In-vitro and in-vivo evaluation of polymeric microsphere formulation for colon targeted delivery of 5-fluorouracil using biocompatible natural gum katira.

The aim was to develop oral site-specific rate-controlled anticancer drug delivery to pacify systemic side-effects and offer effective and safe therapy for colon cancer with compressed dose and duration of treatment. The double emulsion solvent evaporation method was employed. To check functionality, DAPI-staining and in-vivo anticancer study of Ehrlich Ascites Carcinoma bearing mice was tested. Histopathology of liver and kidney and Cell morphology of EAC cell was also performed. Formulated and optimized polymeric microsphere of 5-FU showed excellent physicochemical features. In-vitro, DAPI results pointed drug-treated groups displayed the prominent feature of apoptosis. The percentage of apoptotic of entrapped drug played in a dose-dependent manner. Significant decreases in EAC liquid tumors and increased life span of treated mice were observed. Rate of variation of cell morphology was more in 5-FU loaded microsphere than 5-FU injection. Hematological and biochemical parameter's and Histopathology of liver and kidney resulted that due to control released formulation have slow release rate, that gives less trace on liver and kidney function. Finally, we foresee that polymeric microsphere of 5-FU applying natural gum katira could be an assuring micro-carrier for active colon targeting delivery tool with augmented chemotherapeutic efficacy and lowering side effect against colon cancer.

Polymeric Microsphere Formulation for Colon Targeted Delivery of 5-Fluorouracil Using Biocompatible Natural Gum Katira.

Controlled release delivery system of chemotherapeutic agents at the site of colon endorses modern drug-entrapped delivery tools, which release the entrappedagents at a controlled rate for anextended period providing patient compliance and additional protection from the degradinggastric environment. Thus, the present study was aimed to develop and optimize a novel polymeric microsphere of 5-fluorouracil (5-FU) using natural gum katira to obtain an optimal therapeutic response at the colon. Due course of experimentation, in-vivo safety profile of the gum katira in an animal model was established. Modified solvent extraction/evaporation technique wasemployed to encapsulate 5-FU in the natural polymeric microsphere and was characterized using in-vitro studies to investigate particle size, morphology, encapsulation efficiency and release of the drug from developed formulation. Formulated and optimized polymeric microsphere of 5-FU using gum katira polymer own optimal physicochemical characteristics with a fine spherical particle with size ranged from 210.37±7.50 to 314.45±7.80 µm.Targeted microsphere exhibited good cytotoxicity and also has high drug entrapment efficiency, and satisfactory release pattern of the drug within a time frame of 12 h. Finally, we foresee that the optimized polymeric gum katiramicrosphere of 5-FU could be a promising micro-carrier for efficient colon drug targeting delivery tool with improved chemotherapeutic efficacy against colon cancer.

Induction of apoptosis, anti-proliferation, tumor-angiogenic suppression and down-regulation of Dalton's Ascitic Lymphoma (DAL) induced tumorigenesis by poly-l-lysine: A mechanistic study.

PURPOSE: The present study, attempts to validate the molecular mechanism(s) of Poly-l-lysine (PLL) induced apoptosis, anti-proliferative and anti-tumorigenic properties in in-vitro HUVECs cells and Dalton's Ascitic Lymphoma (DAL) and in in-vivo DAL cell bearing BALB/c mice model. MATERIALS AND METHODS: The cell proliferation assay and morphological assay was carried out using the MTT assay and Giemsa staining method. The antitumor activity of PLL was evaluated in BALB/c mice at 20 and 40 mg/kg/b.w doses for 21 days for DAL solid tumor model. Several tumor evaluation endpoints, hematological and biochemical parameters were estimated. Additionally, the tumor apoptosis, anti-proliferative and anti-tumor angiogenesis effects were assessed using western blots and immunohistochemistry. RESULTS: PLL significantly decreased cell proliferation in in-vitro HUVECs and DAL cells without significant effects on normal cell growth. PLL also induced alteration in cellular morphology in DAL cells. Therafter, in the BALB/c mouse model, PLL had noticeable inhibition in DAL-induced tumorigenesis. This inhibition was evident through reduced solid tumor volume and weight versus the control group. However, PLL promoted tumor apoptosis and suppressed cell-proliferation and tumor-angiogenesis. PLL also increased hematological markers significantly compared to 5-flurouracil (5-FU). The amount of TdT in the nuclei of DAL cells in mice treated with PLL was significantly increased while in contrast decreases of anti-apoptotic protein Bcl-2 expression were observed. PLL also significantly upregulated the pro-apoptotic protein Bax and activated caspase-3. Measurable decreases of cyclin-D1 were observed through PLL treatments, an indicator of cell-cycle arrest. These studies also indicate PLL's induction and anti-proliferative effects through suppression of the c-Myc and Ki-67 proliferation-indices. Additionally, PLL inhibited tumor-angiogenesis through suppression of VEGF and CD34 protein expression levels and reduction ofmicrovesseldensityversus similar parameters in tumors from control mice. CONCLUSION: The present study offers opportunities and hopes for possible anti-tumortherapies with PLL in the near future and warrants further formulation developments.

Poly-L-Lysine Inhibits Tumor Angiogenesis and Induces Apoptosis in Ehrlich Ascites Carcinoma and in Sarcoma S-180 Tumor

Background: This study focuses on the role of Poly-L-lysine (PLL), an essential amino acid, on molecular changes of tumor angiogenesis suppression, pro-apoptotic and anti-apoptotic gene expression after treatment on Ehrlich ascites carcinoma (EAC) and solid sarcoma-180 tumor cells bearing mice. Materials and Methods: The cell viability was carried out using MTT assay. The antitumor activity was evaluated by treatment with PLL at 20 and 40mg/kg/b.w doses for 14 days in EAC ascites tumor and 21 days for Sarcoma-180 solid tumor model. Several tumor evaluation studies, haematological and biochemical parameters were estimated. Importantly, the tumor cell apoptosis was assessed using microscopic observations, DNA fragmentation assay, Flow cytometric analysis, cell-cycle and electron-microscopic study, following which, the expression of several signal proteins related to pro-apoptosis, anti-apoptosis and tumor angiogenesis were quantified using western blotting and immunohistochemistry study. Results: Precisely, PLL had cytotoxic effect on K562; A549; U937 and B16F10 cancer cells. Significant decreases in liquid and solid tumors and increased life span of treated mice were observed (P<0.05). Typical morphological changes, apoptosis bleb phenomenon and sub-G1 cell cycle arrests revealed that PLL promoted apoptotic cell death. Western blot and immunohistochemistry confirms, PLL activated apoptotic signalling cascades through down regulation of Bcl-2 and CD31 protein and upregulation of Bax and p53 proteins. The anti-angiogenic effects were also accompanied with decreased VEGF expression and reduced peritoneal-angiogenesis and microvessel density. Conclusions: The antitumor and antitumor-angiogenic activity of PLL was confirmed from all the results via up and down regulation of relevant signal proteins reported in this publication.

A novel role for the Wnt inhibitor APCDD1 in adipocyte differentiation: Implications for diet-induced obesity.

Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and ß-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3'-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBPα, and PPARγ compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.

Inositol hexa phosphoric acid (phytic acid), a nutraceuticals, attenuates iron-induced oxidative stress and alleviates liver injury in iron overloaded mice.

Inositol hexa phosphoric acid (IP6) or Phytic acid, a natural antioxidant of some leguminous plants, known to act as a protective agent for seed storage in plants by suppressing iron catalyzed oxidative process. Following the same mechanism, we have tested the effect of IP6 on iron overloaded in vitro oxidative stress, and studied it's in vivo hepatoprotective ability in iron-dextran (injection)-induced iron overloaded liver injury in mice (intraperitoneal). Our results showed that IP6 had in vitro iron chelation (IC50 38.4µg/ml) activity, with the inhibition of iron-induced lipid peroxidation (IC50 552µg/ml), and deoxyribose sugar degrading hydroxyl radicals (IC50 448.6µg/ml). Oral administration of IP6 (0-200mg/kg) revealed significant decrease in biochemical markers such as serum iron, total iron binding, serum ferritin and serum enzymes. Histopathology of liver stained with hematoxylin-eosin and Prussian blue showed reduced hepatocellular necrosis, ballooning and inflammation, indicating the restoration of normal cellular integrity. Interestingly, the IP6 was found to down-regulate the mRNA expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1ß, and IL-6 in iron overloaded liver tissues. Thus, we provide an insight that IP6, a natural food component, can serve as an iron chelator against iron overload diseases like Thalassemia, and also as a dietary hepatoprotective supplement.

Pharmacological and toxicological investigations of etodolac loaded gum katira microspheres prepared by W1/O/W2 emulsion solvent evaporation technique in rats

ABSTRACT Etodolac is a non-steroidal anti-inflammatory drug (NSAID) and approved by USFDA as a COX2 inhibitor. Although etodolac therapy provides clinical benefits, it is associated with upper gastrointestinal (GI) tract complications also. Etodolac loaded gum Katira microsphere (ELGKM) was prepared by W1/O/W2 emulsion solvent evaporation technique. The gastric irritation properties of orally administered pure etodolac, ELGKM and blank microspheres (without etodolac) were evaluated in experimental rats treated for 6 days. The stomach examination and biochemical investigation of stomach tissue of treated rats indicated that ELGKM formulation remarkably reduced ulcerogenecity as compared to pure etodolac. The anti-inflammatory activities of pure etodolac and ELGKMs were ascertained by the implantation of cotton pellets in rats for 6 days. Based on the results, ELGKMs showed significant anti-inflammatory activities (P<0.01) as compared to control group. The cotton pellets test suggested that ELGKM formulation retained more anti-inflammatory properties among the groups. The hematological changes, biochemical analysis and histopathological studies of subacute toxicity in rats revealed that ELGKM were the effective sustained release formulation in the treatment of chronic pain and inflammation. In conclusion, the physicochemical characterization, pharmacological and toxicological studies suggest that ELGKMs may represent as a potential candidate for sustained drug delivery (10-12 hours) in chronic joint pain related diseases with remarkably diminished gastrointestinal side effects.

Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis.

BACKGROUND: High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). METHODS AND RESULTS: A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC(-/-) mice. In RBCs from HFD-fed wild-type and DARC(-/-) mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. CONCLUSIONS: RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

Transplanted perivascular adipose tissue accelerates injury-induced neointimal hyperplasia: role of monocyte chemoattractant protein-1.

OBJECTIVE: Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. APPROACH AND RESULTS: We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet-fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. CONCLUSIONS: These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1-dependent mechanisms.

Proinflammatory phenotype of perivascular adipocytes.

Perivascular adipose tissue (PVAT) directly abuts the lamina adventitia of conduit arteries and actively communicates with the vessel wall to regulate vascular function and inflammation. Mounting evidence suggests that the biological activities of PVAT are governed by perivascular adipocytes, a unique class of adipocyte with distinct molecular and phenotypic characteristics. Perivascular adipocytes surrounding human coronary arteries (pericoronary perivascular adipocytes) exhibit a reduced state of adipogenic differentiation and a heightened proinflammatory state, secreting ≤50-fold higher levels of the proinflammatory cytokine monocyte chemoattractant peptide-1 compared with adipocytes from other regional depots. Thus, perivascular adipocytes may contribute to upregulated inflammation of PVAT observed in atherosclerotic human blood vessels. However, perivascular adipocytes also secrete anti-inflammatory molecules such as adiponectin, and elimination of PVAT in rodent models has been shown to augment vascular disease, suggesting that some amount of PVAT is required to maintain vascular homeostasis. Evidence in animal models and humans suggests that inflammation of PVAT may be modulated by environmental factors, such as high-fat diet and tobacco smoke, which are relevant to atherosclerosis. These findings suggest that the inflammatory phenotype of PVAT is diverse depending on species, anatomic location, and environmental factors and that these differences are fundamentally important in determining a pathogenic versus protective role of PVAT in vascular disease. Additional research into the mechanisms that regulate the inflammatory balance of perivascular adipocytes may yield new insight into, and treatment strategies for, cardiovascular disease.

Inhibition of stearoyl-coA desaturase selectively eliminates tumorigenic Nanog-positive cells: improving the safety of iPS cell transplantation to myocardium.

Induced pluripotent stem cells (iPS) can differentiate into cardiomyocytes (CM) and represent a promising form of cellular therapy for heart regeneration. However, residual undifferentiated iPS derivates (iPSD), which are not fully eliminated by cell differentiation or purification protocols, may form tumors after transplantation, thus compromising therapeutic application. Inhibition of stearoyl-coA desaturase (SCD) has recently been reported to eliminate undifferentiated human embryonic stem cells, which share many features with iPSD. Here, we tested the effects of PluriSin#1, a small-molecule inhibitor of SCD, on iPS-derived CM. We found that plurisin#1 treatment significantly decreased the mRNA and protein level of Nanog, a marker for both cell pluripotency and tumor progression; importantly, we provide evidence that PluriSin#1 treatment at 20 µM for 1 day significantly induces the apoptosis of Nanog-positive iPSD. In addition, PluriSin#1 treatment at 20 µM for 4 days diminished Nanog-positive stem cells in cultured iPSD while not increasing apoptosis of iPS-derived CM. To investigate whether PluriSin#1 treatment prevents tumorigenicity of iPSD after cell transplantation, we intramyocardially injected PluriSin#1- or DMSO-treated iPSD in a mouse model of myocardial infarction (MI). DMSO-treated iPSD readily formed Nanog-expressing tumors 2 weeks after injection, which was prevented by treatment with PluriSin#1. Moreover, treatment with PluriSin#1 did not change the expression of cTnI, α-MHC, or MLC-2v, markers of cardiac differentiation (P>0.05, n = 4). Importantly, pluriSin#1-treated iPS-derived CM exhibited the ability to engraft and survive in the infarcted myocardium. We conclude that inhibition of SCD holds the potential to enhance the safety of therapeutic application of iPS cells for heart regeneration.

HDAC9 knockout mice are protected from adipose tissue dysfunction and systemic metabolic disease during high-fat feeding.

During chronic caloric excess, adipose tissue expands primarily by enlargement of individual adipocytes, which become stressed with lipid overloading, thereby contributing to obesity-related disease. Although adipose tissue contains numerous preadipocytes, differentiation into functionally competent adipocytes is insufficient to accommodate the chronic caloric excess and prevent adipocyte overloading. We report for the first time that a chronic high-fat diet (HFD) impairs adipogenic differentiation, leading to accumulation of inefficiently differentiated adipocytes with blunted expression of adipogenic differentiation-specific genes. Preadipocytes from these mice likewise exhibit impaired adipogenic differentiation, and this phenotype persists during in vitro cell culture. HFD-induced impaired adipogenic differentiation is associated with elevated expression of histone deacetylase 9 (HDAC9), an endogenous negative regulator of adipogenic differentiation. Genetic ablation of HDAC9 improves adipogenic differentiation and systemic metabolic state during an HFD, resulting in diminished weight gain, improved glucose tolerance and insulin sensitivity, and reduced hepatosteatosis. Moreover, compared with wild-type mice, HDAC9 knockout mice exhibit upregulated expression of beige adipocyte marker genes, particularly during an HFD, in association with increased energy expenditure and adaptive thermogenesis. These results suggest that targeting HDAC9 may be an effective strategy for combating obesity-related metabolic disease.

Evaluation of the wound healing activity of Shorea robusta, an Indian ethnomedicine, and its isolated constituent(s) in topical formulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Indian ethnomedicinal plant Shorea robusta is traditionally used for several ailments including wounds and burn by different tribal groups, since ages. Here we have validated, for the first time, the effectiveness and the possible mechanism of action of young leaf extracts of Shorea robusta, used by two distinct tribes of India, and its isolated compounds as a topical formulation in three wound models in rats. MATERIALS AND METHODS: Bioactivity-guided study of the active extract resulted in the isolation of two known compounds. The prepared ointment containing extracts (2.5 and 5%, w/w), fractions (5% w/w) and isolated compounds (0.25% w/w) were evaluated on excision, incision and dead space wound models in rats by the rate of wound closure, period of epithelialisation, tensile strength, granulation tissue weight, hydroxyproline content and histopathology. RESULTS: The animals treated with the extracts and fractions (5%) showed significant reduction in wound area 96.55 and 96.41% with faster epithelialisation (17.50 and 17.86), while the isolated compounds bergenin and ursolic acid heal the wound faster, but complete epithelialisation with 100% wound contraction was evident with 5% povidone-iodine group on 18th post-wounding day. Moreover, the tensile strength of incision wound, granuloma tissue weight, and hydroxyproline content was significantly increased in both the extract and compound(s) treated animals. Furthermore, the tissue histology of animals treated with the isolated compound(s) showed complete epithelialisation with increased collagenation, similar to povidone-iodine group. CONCLUSION: Thus, our results validated the traditional use of Shorea robusta young leaves in wound management.

Allele-specific expression of angiotensinogen in human subcutaneous adipose tissue.

The angiotensinogen gene is genetically linked with hypertension, but the mechanistic basis for association of sequence variants in the promoter and coding region of the gene remains unclear. An E-box at position -20 has been hypothesized to control the level of angiotensinogen expression, but its mechanistic importance for angiotensinogen expression in human tissues is uncertain. We developed an allele-specific polymerase chain reaction-based assay to distinguish between angiotensinogen mRNA derived from variants at the -20 position (rs5050) in the angiotensinogen promoter in adipose tissues obtained during surgery. The assay takes advantage of linkage disequilibrium between the rs5050 (located in the promoter) and rs4762 (located in the coding region) single nucleotide polymorphisms. This strategy allowed us to assess the level of allele-specific expression in A-20C heterozygous subjects comparing the relative proportion of each allele with the total, thus eliminating the problem of variability in the level of total angiotensinogen mRNA among subjects. We show that angiotensinogen mRNA derived from the -20C allele is expressed significantly higher than that derived from the -20A allele in subcutaneous adipose tissue, and increased expression correlates with enriched chromatin binding of upstream stimulatory factor-2 to the -20C E-box compared with -20A. This may be depot selective because we were unable to detect these differences in omental adipose. This provides the first data directly comparing expression of angiotensinogen mRNA and differential transcription factor binding derived from 2 variant alleles in human tissue where the ratio of expression of one allele to another can be accurately determined.

CD14 directs adventitial macrophage precursor recruitment: role in early abdominal aortic aneurysm formation.

BACKGROUND: Recruitment of macrophage precursors to the adventitia plays a key role in the pathogenesis of abdominal aortic aneurysms (AAAs), but molecular mechanisms remain undefined. The innate immune signaling molecule CD14 was reported to be upregulated in adventitial macrophages in a murine model of AAA and in monocytes cocultured with aortic adventitial fibroblasts (AoAf) in vitro, concurrent with increased interleukin-6 (IL-6) expression. We hypothesized that CD14 plays a crucial role in adventitial macrophage precursor recruitment early during AAA formation. METHODS AND RESULTS: CD14(-/-) mice were resistant to AAA formation induced by 2 different AAA induction models: aortic elastase infusion and systemic angiotensin II (AngII) infusion. CD14 gene deletion led to reduced aortic macrophage infiltration and diminished elastin degradation. Adventitial monocyte binding to AngII-infused aorta in vitro was dependent on CD14, and incubation of human acute monocytic leukemia cell line-1 (THP-1) monocytes with IL-6 or conditioned medium from perivascular adipose tissue (PVAT) upregulated CD14 expression. Conditioned medium from AoAf and PVAT induced CD14-dependent monocyte chemotaxis, which was potentiated by IL-6. CD14 expression in aorta and plasma CD14 levels were increased in AAA patients compared with controls. CONCLUSIONS: These findings link CD14 innate immune signaling via a novel IL-6 amplification loop to adventitial macrophage precursor recruitment in the pathogenesis of AAA.

Apolipoprotein E2 accentuates postprandial inflammation and diet-induced obesity to promote hyperinsulinemia in mice.

Genetic studies have revealed the association between the ε2 allele of the apolipoprotein E (apoE) gene and greater risk of metabolic diseases. This study compared C57BL/6 mice in which the endogenous mouse gene has been replaced by the human APOE2 or APOE3 gene (APOE2 and APOE3 mice) to identify the mechanism underlying the relationship between ε2 and obesity and diabetes. In comparison with APOE3 mice, the APOE2 mice had elevated fasting plasma lipid and insulin levels and displayed prolonged postprandial hyperlipidemia accompanied by increased granulocyte number and inflammation 2 h after being fed a lipid-rich meal. In comparison with APOE3 mice, the APOE2 mice also showed increased adiposity when maintained on a Western-type, high-fat, high-cholesterol diet. Adipose tissue dysfunction with increased macrophage infiltration, abundant crown-like structures, and inflammation were also observed in adipose tissues of APOE2 mice. The severe adipocyte dysfunction and tissue inflammation corresponded with the robust hyperinsulinemia observed in APOE2 mice after being fed the Western-type diet. Taken together, these data showed that impaired plasma clearance of apoE2-containing, triglyceride-rich lipoproteins promotes lipid redistribution to neutrophils and adipocytes to accentuate inflammation and adiposity, thereby accelerating the development of hyperinsulinemia that will ultimately lead to advanced metabolic diseases.

Anti-herpes virus activities of Achyranthes aspera: an indian ethnomedicine, and its triterpene acid.

The aim of this study was to evaluate the antiviral potential of methanolic extract (ME) of Achyranthes aspera, an Indian folk medicine and one of its pure compound oleanolic acid (OA) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The ME possessed weak anti-herpes virus activity (EC50 64.4µg/ml for HSV-1 and 72.8µg/ml for HSV-2). While OA exhibited potent antiherpesvirus activity against both HSV-1 (EC50 6.8µg/ml) and HSV-2 (EC50 7.8µg/ml). The time response study revealed that the antiviral activity of ME and OA is highest at 2-6h post infection. The infected and drug-treated peritoneal macrophage at specific time showed increased level of pro-inflammatory cytokines (IL6 and IL12). Further, the PCR of DNA from infected cultures treated with ME and OA, at various time intervals, failed to show amplification at 48-72h, similar to that of HSV infected cells treated with acyclovir, indicating that the ME and OA probably inhibit the early stage of multiplication (post infection of 2-6h). Thus, our study demonstrated that ME and OA have good anti-HSV activity, with SI values of 12, suggesting the potential use of this plant.

Effect of potent ethyl acetate fraction of Stereospermum suaveolens extract in streptozotocin-induced diabetic rats.

To evaluate the antihyperglycemic effect of ethyl acetate fraction of ethanol extract of Stereospermum suaveolens in streptozotocin-(STZ-) induced diabetic rats by acute and subacute models. In this paper, various fractions of ethanol extract of Stereospermum suaveolens were prepared and their effects on blood glucose levels in STZ-induced diabetic rats were studied after a single oral administration (200 mg/kg). Administration of the ethyl acetate fraction at 200 mg/kg once daily for 14 days to STZ-induced diabetic rats was also carried out. The parameters such as the fasting blood glucose, hepatic glycogen content, and pancreatic antioxidant levels were monitored. In the acute study, the ethyl acetate fraction is the most potent in reducing the fasting serum glucose levels of the STZ-induced diabetic rats. The 14-day repeated oral administration of the ethyl acetate fraction significantly reduced the fasting blood glucose and pancreatic TBARS level and significantly increased the liver glycogen, pancreatic superoxide dismutase, and catalase activities as well as reduced glutathione levels. The histopathological studies during the subacute treatment have been shown to ameliorate the STZ-induced histological damage of pancreas. This paper concludes that the ethyl acetate fraction from ethanol extract of Stereospermum suaveolens possesses potent antihyperglycemic and antioxidant properties, thereby substantiating the use of plant in the indigenous system of medicine.

A novel mechanism involving coordinated regulation of nuclear levels and acetylation of NF-YA and Bcl6 activates RGS4 transcription.

Neuronally enriched RGS4 plays a critical role attenuating G protein signaling in brain, although the mechanisms regulating RGS4 expression are unknown. Here we describe a novel mechanism for transcriptional activation of RGS4 in neuron-like PC6 cells, where RGS4 is markedly induced during confluence-induced growth arrest. Transcriptional activation of RGS4 in confluent PC6 cells was accompanied by impaired G(i/o)-dependent MAPK activation. In the human RGS4 gene promoter, we identified three phylogenetically conserved cis-elements: an inverted CCAAT box element (ICE), a cAMP response element, and a B-cell lymphoma 6 (Bcl6)-binding site. The ICE and the cAMP response element mediate activation, and the Bcl6 site mediates repression of RGS4 transcription. Activation of RGS4 transcription in confluent PC6 cells is accompanied by increases in NF-YA and C/EBPß and decreases in Bcl6 levels in the nucleus. Increases in NF-YA and C/EBPß lead to their increased binding to the RGS4 promoter in vivo, and dominant negative forms of these proteins repressed RGS4 promoter activity. Acetylation of NF-YA and Bcl6 were increased in postconfluent cells. Trichostatin A stimulation of RGS4 promoter activity, accompanied by increased binding of NF-YA and decreased binding of Bcl6 to the promoter, was abolished by mutation of the ICE and enhanced by mutation of the Bcl6 site. These findings demonstrate a dynamic and coordinated regulation of nuclear levels and acetylation status of trans-acting factors critical in determining the off/on state of the RGS4 promoter.