Focal Adhesion Assembly in Myofibroblasts Fosters a Microenvironment that Promotes Tumor Growth

Cells within the tumor microenvironment influence tumor growth through multiple mechanisms. Pericytes such as hepatic stellate cells are an important cell within the tumor microenvironment; their transformation into highly motile myofibroblasts leads to angiogenesis, stromal cell recruitment, matrix deposition, and ensuing tumor growth. Thus, a better understanding of mechanisms that regulate motility of pericytes is required. Focal adhesions (FAs) form a physical link between the extracellular environment and the actin cytoskeleton, a requisite step for cell motility. FAs contain a collection of proteins including the Ena/VASP family member, vasodilator-stimulated phosphoprotein (VASP); however, a role for VASP in FA development has been elusive. Using a comprehensive siRNA knockdown approach and a variety of VASP mutants coupled with complementary cell imaging methodologies, we demonstrate a requirement of VASP for optimal development of FAs and cell spreading in LX2 liver myofibroblasts, which express high levels of endogenous VASP. Rac1, a binding partner of VASP, acts in tandem with VASP to regulate FAs. In vivo, perturbation of Ena/VASP function in tumor myofibroblast precursor cells significantly reduces pericyte recruitment to tumor vasculature, myofibroblastic transformation, tumor angiogenesis, and tumor growth, providing in vivo pathobiologic relevance to these findings. Taken together, our results identify Ena/VASP as a significant modifier of tumor growth through regulation of FA dynamics and ensuing pericyte/myofibroblast function within the tumor microenvironment.Tumor stroma importantly influences tumor growth and progression with pericytes representing an important stromal cell owing to their importance in angiogenesis and their transformation into highly motile tumor myofibroblasts.¹ Focal adhesions (FAs) are specialized structures that connect extracellular environment to actin cytoskeleton thus facilitating the signal transduction and actin remodeling requisite for the process of pericyte migration during angiogenesis.^2,3 Thus, mechanisms that govern FA assembly and pericyte motility may present potential targets for anti-cancer therapy but are not yet fully defined.vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that regulate actin cytoskeleton and cell migration.⁴ It contains distinct subdomains that facilitate specific protein interactions and actin binding characteristics that culminate in an anticapping/branching function within the cytoskeleton.^4,5 Although VASP resides within FAs, its precise role in FA dynamics has not been completely defined, probably due in part to cell-type specific VASP functions.^6–8Because the role of VASP in pericyte function and ensuing effects within the tumor microenvironment remain unexplored, we investigated the role of VASP in FA development in human hepatic stellate cells (HSC), liver pericytes that express a high level of VASP and which on transformation into myofibroblasts, develop a highly motile state that has been postulated to influence tumor growth. Our data reveal a requirement of VASP in FA dynamics and pericyte motility, which requires cooperation of each distinct VASP subdomain. Indeed, overexpression of the EVH2 domain alone results in a dominant negative function that inhibits FA assembly, cell motility, and angiogenesis. We also identify a role of Rac1 as a key binding partner of VASP that promotes its ability to regulate FA and cell motility. Perturbation of Ena/VASP function in tumor myofibroblast precursor cells significantly reduced tumor growth in mice demonstrating an important role for these cell biological mechanisms in microenvironmental regulation of tumor growth. Taken together, our results identify VASP as a regulator of FA development, pericyte motility, and tumor growth and identify several important mechanisms by which this regulation is conferred.     

New flow cytometric technique for the evaluation of circulating endothelial progenitor cell levels in various disease groups

Circulating endothelial progenitor cells (EPC) localise to sites of ischaemia and play a role in vascular repair and re-endothelialisation of injured blood vessels. Low levels of EPCs are associated with cardiovascular disease (CVD) in the general population. It is not clear at present whether and how the numbers of circulating EPCs vary in diseases other than CVD. We have enumerated EPCs by the flow cytometric analysis of whole blood by using a novel cocktail of monoclonal antibodies. This consisted of CD2FITC, CD13FITC and CD22FITC to eliminate non-progenitor cells and VEGFR2PE and CD133-streptavidin-PeCy7 to include only EPCs. We analysed 250 patients with varying stages of uraemia, 36 patients with inflammatory bowel disease (IBD) and 9 patients with acute respiratory distress syndrome and compared this to 74 healthy controls. Using flow cytometry we were able to measure the circulating levels of EPCs, with a result available within hours of the sample being obtained. Circulating EPC numbers vary in different patient groups and healthy controls. In uraemic patients, irrespective of disease severity, there are lower numbers of circulating EPC numbers compared to normal controls (46.6+/-3.7 vs. 66.1+/-4.7; p=0.03). This new technique provides a means of monitoring patients and shows a reduction in circulating EPCs in uraemic patients; this abnormality may be a target of novel therapies.     

Quantitative dispersion microscopy

Refractive index dispersion is an intrinsic optical property and a useful source of contrast in biological imaging studies. In this report, we present the first dispersion phase imaging of living eukaryotic cells. We have developed quantitative dispersion microscopy based on the principle of quantitative phase microscopy. The dual-wavelength quantitative phase microscope makes phase measurements at 310 nm and 400 nm wavelengths to quantify dispersion (refractive index increment ratio) of live cells. The measured dispersion of living HeLa cells is found to be around 1.088, which agrees well with that measured directly for protein solutions using total internal reflection. This technique, together with the dry mass and morphology measurements provided by quantitative phase microscopy, could prove to be a useful tool for distinguishing different types of biomaterials and studying spatial inhomogeneities of biological samples.     

Could metformin be used in patients with diabetes and advanced chronic kidney disease?

Diabetes is an important cause of end stage renal failure worldwide. As renal impairment progresses, managing hyperglycaemia can prove increasingly challenging, as many medications are contra‐indicated in moderate to severe renal impairment. Whilst evidence for tight glycaemic control reducing progression to renal failure in patients with established renal disease is limited, poor glycaemic control is not desirable, and is likely to lead to progressive complications. Metformin is a first‐line therapy in patients with Type 2 diabetes, as it appears to be effective in reducing diabetes related end points and mortality in overweight patients. Cessation of metformin in patients with progressive renal disease may not only lead to deterioration in glucose control, but also to loss of protection from cardiovascular disease in a cohort of patients at particularly high risk. We advocate the need for further study to determine the role of metformin in patients with severe renal disease (chronic kidney disease stage 4‐5), as well as patients on dialysis, or pre‐/peri‐renal transplantation. We explore possible roles of metformin in these circumstances, and suggest potential key areas for further study.     

Components and determinants of therapeutic delay in patients with acute ST-elevation myocardial infarction: A tertiary care hospital-based study

Background

Delayed reperfusion is associated with worse outcomes in ST-segment elevation myocardial infarction (STEMI). This study was conducted to assess the components and determinants of therapeutic delay in STEMI patients of our state.

The Role of Grape Seed Extract in the Treatment of Chemo/Radiotherapy Induced Toxicity: A Systematic Review of Preclinical Studies

Grapes are one of the most consumed fruits in the world and are rich in polyphenols. Grape seed proanthocyanidins (GSP) have demonstrated chemopreventive and/or chemotherapeutic effects in various cancer cell cultures and animal models. The clinical efficacy of chemotherapy is often limited by its adverse effects. Several studies show that reactive oxygen species mediate the cardiotoxicity and neurotoxicity induced by various cancer chemotherapeutic agents. This implies that concomitant administration of antioxidants may prevent these adverse effects. The review was carried out in accordance with the PRISMA guidelines. An electronic search strategy in Medline and Embase databases was conducted. Of the 41 studies reviewed, 27 studied GSP while the remainder (14) studied grape seed or skin extracts (GSE). All the studies were published in English, except 2 in Chinese. A significant percentage (34%) of the studies we reviewed assessed the effect of GSE or GSP on cardiotoxicity induced by chemotherapy. Doxorubicin was the most common chemotherapeutic drug studied followed by cisplatin. Research studies that assessed the effect of GSE or GSP on radiation treatment accounted for 22% of the articles reviewed. GSE/GSP ameliorates some of the cytotoxic effects on normal cells/tissues induced by chemo/radiotherapy.     

Iron supplementation decreases plasma zinc but has no effect on plasma fatty acids in non-anemic women

Limited information is available on the role of iron in fatty acid metabolism in humans. We hypothesized that iron supplementation will increase desaturase activity, and so, the aim of this study was to determine the effect of iron supplementation on fatty acid desaturase activity in young women. Participants were randomly assigned to a control group (CG) or supplementation group (SG) who were provided with 37.4 mg of elemental iron daily for 12 weeks. Forty women completed the trial, n = 19 in CG and n = 21 in SG. The mean ages were 25.2 and 24.6 years, and body mass indices were 21.8 and 21.2 (kg/m²) in CG and SG, respectively. Serum ferritin concentrations increased significantly (P < .01) in subjects assigned to SG but were unchanged in CG. Serum iron concentrations were not significantly changed. Plasma zinc concentrations at the end of the intervention were similar to baseline values for individuals in CG but were decreased significantly (P = .004) in SG. Plasma fatty acids, phospholipid fatty acids, and desaturase activities, expressed as precursor-to-product ratios, were not significantly affected by the intervention, although in SG the concentration of serum ferritin was correlated positively (P < .05) with Δ6-desaturase activity. Supplementing non-anemic women with low dose iron improves iron status but has no significant effect on desaturase activity. The lack of a clear effect on an indirect indicator of desaturase activity may be related to the antagonism between iron and zinc, as illustrated by the decrease in plasma zinc concentrations in women who were supplemented with iron.