ARHGAP18 Protects Against Thoracic Aortic Aneurysm Formation by Mitigating the Synthetic and Proinflammatory Smooth Muscle Cell Phenotype.

RATIONALE: Thoracic aortic aneurysm (TAA) is a potentially lethal condition, which can affect individuals of all ages. TAA may be complicated by the sudden onset of life-threatening dissection or rupture. The underlying mechanisms leading to TAA formation, particularly in the nonsyndromal idiopathic group of patients, are not well understood. Thus, identification of new genes and targets that are involved in TAA pathogenesis are required to help prevent and reverse the disease phenotype. OBJECTIVE: Here we explore the role of ARHGAP18, a novel Rho GAP expressed by smooth muscle cells (SMCs), in the pathogenesis of TAA. METHODS AND RESULTS: Using human and mouse aortic samples, we report that ARHGAP18 levels were significantly reduced in the SMC layer of aortic aneurysms. Arhgap18 global knockout (Arhgap18-/-) mice exhibited a highly synthetic, proteolytic, and proinflammatory smooth muscle phenotype under basal conditions and when challenged with angiotensin II, developed TAA with increased frequency and severity compared with littermate controls. Chromatin immunoprecipitation studies revealed this phenotype is partly associated with strong enrichment of H3K4me3 and depletion of H3K27me3 at the MMP2 and TNF-α promoters in Arhgap18-deficient SMC. We further show that TAA formation in the Arhgap18-/- mice is associated with loss of Akt activation. The abnormal SMC phenotype observed in the Arhgap18-/- mice can be partially rescued by pharmacological treatment with the mTORC1 inhibitor rapamycin, which reduces the synthetic and proinflammatory phenotype of Arhgap18-deficient SMC. CONCLUSION: We have identified ARHGAP18 as a novel protective gene against TAA formation and define an additional target for the future development of treatments to limit TAA pathogenesis.

Connective tissue growth factor/CCN-2 is upregulated in epididymal and subcutaneous fat depots in a dietary-induced obesity model.

Connective tissue growth factor (CTGF), also known as CCN-2, is a cysteine-rich secreted protein that is involved in a range of biological processes, including regulation of cell growth and differentiation. Our previous in vitro studies have shown that CCN-2 inhibits adipocyte differentiation, although whether CCN-2 is regulated in vivo in adipogenesis is undetermined and was investigated in this study. C57BL/6 male mice were fed either standard laboratory chow (ND) or a diet high in fat (HFD; 45% fat) for 15 or 24 wk. HFD animals that gained >5 g in weight (termed HFD-fat) were insulin resistant and were compared with HFD-fed animals, which failed to gain weight (termed HFD-lean). HFD-fat mice had significantly increased CCN-2 mRNA levels in both the subcutaneous and epididymal fat pads, whereas CCN-2 mRNA was not induced in the epididymal site in HFD-lean mice. Also in HFD-fed animals, epididymal CCN-2 mRNA correlated positively with key genes involved in adipocyte differentiation, adiponectin and PPARγ (P < 0.001 and P < 0.002, respectively). Additionally, epididymal CCN-2 mRNA correlated positively with two markers of tissue turnover, PAI-1 in HFD-fat mice only and TIMP-1, but only in the HFD-lean mice. Collectively, these findings suggest that CCN-2 plays a role in adipocyte differentiation in vivo and thus in the pathogenesis of obesity linked with insulin resistance.

Diabetes is a progression factor for hepatic fibrosis in a high fat fed mouse obesity model of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: While type 2 diabetes is an independent risk factor for worsening of human non-alcoholic steatohepatitis (NASH) in clinical studies, it has not been systematically reported in any model whether diabetes exacerbates NASH. The study aim was to determine if diabetes causes NASH progression in a mouse model of diet induced obesity. METHODS: C57BL/6 mice were fed a high fat diet (HFD: 45% kcal fat) or standard chow (CHOW: 12% kcal fat) for 20 weeks and some animals (HFD+DM or CHOW+DM) were also rendered diabetic by low dose streptozotocin for the final 5 weeks, to model type 2 diabetes. Serum assays included circulating insulin, triglyceride, ALT and AST, glucose, and ultrasensitive CRP and results of insulin tolerance tests. Intrahepatic lipid, triglyceride, macrophage infiltration, and fibrosis were determined. Fibrosis markers collagen-I, collagen-III, CTGF, TIMP-1, and FAP were assessed by qPCR and CTGF and collagen-I by immunostaining. RESULTS: HFD mice were obese, insulin resistant and hyperinsulinaemic, with NASH features of elevated intrahepatic lipid and macrophages, but without fibrosis. In contrast, the HFD+DM mice exhibited fibrosis in addition to these NASH features. By ANOVA, Sirius red staining at perisinusoidal, portal tract and central vein sites, collagen-I, collagen-III, FAP, and TIMP-1 transcripts and collagen-I and CTGF protein were each significantly increased in HFD+DM, compared with CHOW alone. In a further experiment, insulin treatment protected against fibrosis and CRP increases in HFD+DM, showing that diabetes, not streptozotocin, causes the fibrosis. CONCLUSIONS: This novel model indicates that diet-induced NASH fibrosis is exacerbated by diabetes and attenuated by insulin therapy.

The anti-inflammatory agent Propolis improves wound healing in a rodent model of experimental diabetes.

Foot ulcers and poor wound healing are problematic for patients with diabetes. The beehive protectant Propolis can improve wound healing but whether it can improve healing in diabetic wounds has not been investigated. In this study, the effect of a single application of Propolis on epithelial closure, wound morphology, cellular infiltrate, and blood vessel density were investigated. Diabetes was induced in rats using streptozocin. After 6 weeks, diabetic and control animals were wounded and the wounds were treated with Propolis or saline as control. At days 6 and 12 animals were sacrificed and wounds were excised. Compared with controls, diabetes decreased epithelial closure and reepithelialization but had no effect on wound contraction. These delays were prevented by Propolis. At day 12, the impaired macrophage infiltration (C:1.49+/-0.09 vs. D:0.25+/-0.14), persistent neutrophil infiltration (C:0.22+/-0.19 vs. D:1.33+/-0.81), and increased myeloperoxidase activity (fourfold) in diabetic wounds were prevented by Propolis. Diabetes had no effect on wound volume, vessel number, or branch points. These novel data indicate that Propolis can accelerate wound healing in diabetes. As neutrophil infiltration is normalized, its mechanism of action may be through anti-inflammatory pathways. This result and the established safety profile of Propolis provide a rationale for studying topical application of this agent in a clinical setting.

A Web-based e-learning course: integration of pathophysiology into pharmacology.

The Internet is becoming the preferred place to find information. Millions of people go online in search of health and medical information. Likewise, the demand for Web-based courses is growing. This paper presents the development, utilization, and evaluation of a Web-based e-learning course for nursing students, entitled Integration of Pathophysiology into Pharmacology. The pathophysiology component included cardiovascular, respiratory, central nervous and immune system diseases, while the pharmacology component was developed based on 150 commonly used drugs. One hundred and nineteen Year 1 nursing students took part in the course. The Web-based e-learning course materials were uploaded to a WebCT for students' self-directed learning and attempts to pass two scheduled online quizzes. At the end of the semester, students were given a questionnaire to measure the e-learning experience. Their experience in the e-learning course was a positive one. Students stated that they were able to understand rather than memorize the subject content, and develop their problem solving and critical thinking abilities. Online quizzes yielded satisfactory results. In the focus group interview, students indicated that they appreciated the time flexibility and convenience associated with Web-based learning, and also made good suggestions for enhancing Web-based learning. The Web-based approach is promising for teaching and learning pathophysiology and pharmacology for nurses and other healthcare professionals.

The use of health technology and information: e-learning technological approach.

The Internet is becoming the preferred place for finding information. Millions of people go online in search of health and medical information. Likewise, health care professionals must be able to retrieve and manage information in an efficient, effective manner. The development of health technology and informatics (HTI) has been rapid, yet little is known about the knowledge and attitudes of nursing students with regard to using HTI. Therefore, the objective of the present study was to explore nursing students' knowledge of and attitudes toward using HTI and to use an e-learning workshop to enhance the effective use of HTI. In Phase I, 115 first-year full-time nursing students were invited to complete a 13-item questionnaire regarding knowledge of and attitudes toward computer and Internet use, knowledge of HTI, and attitudes toward the future development of HTI. Participants perceived themselves to be knowledgeable in computer and Internet use, while knowledge of HTI was perceived to be low and attitudes toward the future development of HTI were generally positive. Phase II was an e-learning HTI workshop; topics included an introduction to HTI, examples of HTI in use, and the application and implementation of various HTI projects. There were 30 participants in the E-learning workshop, and their feedback was positive. Nurses and all health care professionals must be prepared to participate in the development of health technology and informatics. Teaching and learning activities should be geared toward mastery of HTI in the nursing education curriculum in order to enhance health care quality and safety.

Connective tissue growth factor inhibits adipocyte differentiation.

Adipocyte differentiation is a key process implicated in the pathogenesis of obesity and insulin resistance. Its regulation is triggered by a cascade of transcription factors, including the CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-gamma (PPARgamma). Growth factors such as transforming growth factor-beta1 (TGF-beta1) are known to inhibit adipocyte differentiation in vitro, via the C/EBP pathway, and in vivo, but whether a downstream mediator of TGF-beta1, connective tissue growth factor (CTGF), also known as CCN2, has a similar role is unknown. Mouse 3T3-L1 cells were differentiated into adipocytes by using standard methods, and effects and regulation of CTGF were studied. Intervention with recombinant human CTGF during differing stages of differentiation caused an inhibition in the development of the adipocyte phenotype, according to the gene expression of the differentiation markers adiponectin and PPARgamma, as well as suppression of lipid accumulation and expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. Whereas CTGF gene expression promptly fell by 90% as 3T3-L1 preadipocytes differentiated into mature adipocytes, CTGF mRNA expression was induced by added TGF-beta1. CTGF applied to cells early in the course of differentiation inhibited total cell protein levels and nuclear localization of the beta-isoform of C/EBP (C/EBP-beta) and, subsequently, total cell C/EBP-alpha levels. CTGF also inhibited the adipocyte differentiation program in primary cultures of mouse preadipocytes. Expression of CTGF mRNA was twofold higher in the central fat depots of mice compared with subcutaneous fat, suggesting a potential role for CTGF in vivo. In summary, these data show that CTGF inhibits the adipocyte differentiation program.

2-Methoxy-2,4-diphenyl-3(2H)-furanone-labeled gelatin zymography and reverse zymography: a rapid real-time method for quantification of matrix metalloproteinases-2 and -9 and tissue inhibitors of metalloproteinases.

Measurement of matrix metalloproteinases (MMPs) and their specific tissue inhibitors of metalloproteinases (TIMPs) by the techniques of zymography and reverse zymography provide useful information regarding the status of matrix accumulation or breakdown. This report describes the use of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), a fluorescent compound which can be used to label gelatin as a substrate for detection of the gelatin degrading MMP-2 and -9 by zymography. In addition, a modification of the zymographic technique by addition of excess MMPs enables the use of the MDPF-labeled gelatin substrate for the identification and quantification of TIMPs by reverse zymography. Both systems are real-time sensitive reliable quantification techniques, easily used for measurement of these MMPs and TIMPs in clinical, biological, and tissue culture samples.