Hemodynamic regulation of metalloendopeptidases EC3.4; 24.15 and EC3.4; 24.16: expression and function in the vascular endothelium

Hemodynamic forces, namely shear stress and cyclic strain, have been well characterised as modulators of vascular endothelial function, and have been assigned an important role in the maintainence of vascular tone, haemostasis, and regulation of vascular growth and health. They exert their influence in part by effecting changes in the production and release of vasoactive compounds by the endothelium, and by effecting changes in the levels and activity of various enzymes. Thimet oligopeptidase (EC3.4.24.15, EP24.15) and neurolysin (EC3.4.24.16, EP24.15) are closely related zinc metalloendopeptidases that have been shown to be expressed and active in the vascular endothelium. Their substrates include the vasoactive peptides bradykinin and angiotensin I, which have been identified as important regulators of both blood pressure and angiogenic processes. Other related peptidases, namely endothelin converting enzyme (ECE) and angiotensin converting enzyme (ACE), have been shown to be regulated by hemodynamic forces in the vascular endothelium. As such EP24.15 and EP24.16 are likely candidates for regulation by hemodynamic forces. In this regard we have investigated the effect of cyclic strain on the expression and activty of EP24.15 and EP24.16 in cultured bovine aortic endothelial cells (BAECs). We have shown that exposure to cyclic strain significantly increases the mRNA expression as well as both the cellular and secreted activity of both enzymes. We have demonstrated that up-regulation of both enzymes is dependent on Gi-protein mediated signalling, although with varying Gia/G(3y subunit specificity for either enzyme. Using immunocytochemistry, we have also demonstrated a strain-dependent increase in EP24.15 protein expression within the nucleus and cytoplasm in parallel with an increase in membrane associated EP 24.15 The effects of strain on the ability of BAECs in culture to cleave both Ang I, and BK in an EC24.15/EC24.16 specific manner was also studied. We observed that exposure to cyclic strain induces a significant increase in the EP24.15 specific hydrolysis of both exogenously added BK and Ang I. The potential of the observed effects of cyclic strain on EP24.15 to effect changes in endothelial cell function were also examined. Use of the dual EP24.15/EP24.16 inhibitor, cFP-AAF-pAB, and the EP24.15 specific antisense, FLIP, was seen to significantly attenuate cyclic strain-induced endothelial cell tubule formation and migration. We also found that the effects of FLIP transfection on cyclic strain-induced endothelial cell tubule formation could be largely reversed by addition of exogenous Ang-(l-7). Taken together these results suggest that strain-induced endothelial cell angiogenesis and migration putatively involves EP24.15 cleavage of vasoactive peptide substrates

Is PPARγ a Prospective Player in HIV-1-Associated Bone Disease?

Currently infection with the human immunodeficiency virus-1 (HIV-1) is in most instances a chronic disease that can be controlled by effective antiretroviral therapy (ART). However, chronic use of ART has been associated with a number of toxicities; including significant reductions in bone mineral density (BMD) and disorders of the fat metabolism. The peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor is vital for the development and maintenance of mature and developing adipocytes. Alterations in PPARγ expression have been implicated as a factor in the mechanism of HIV-1-associated lipodystrophy. Both reduced BMD and lipodystrophy have been well described as complications of HIV-1 infection and treatment, and a question remains as to their interdependence. Interestingly, both adipocytes and osteoblasts are derived from a common precursor cell type; the mesenchymal stem cell. The possibility that dysregulation of PPARγ (and the subsequent effect on both osteoblastogenesis and adipogenesis) is a contributory factor in the lipid- and bone-abnormalities observed in HIV-1 infection and treatment has also been investigated. This review deals with the hypothesis that dysregulation of PPARγ may underpin the bone abnormalities associated with HIV-1 infection, and treats the current knowledge and prospective developments, in our understanding of PPARγ involvement in HIV-1-associated bone disease

Rapid depletion of dissolved oxygen in 96 well microtitre plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration

Biofilm-related research using 96-well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96-well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96-well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96-well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model.Science Foundation Irelan

Oxygen-mediated regulation of biofilm development is controlled by the alternative sigma factor sigma(B) in Staphylococcus epidermidis

Using a modified rotating-disk reactor to sparge oxygen to Staphylococcus epidermidis cultures, we found that oxygen negatively regulates biofilm development by influencing the activity of {sigma}B. Under anaerobic conditions, increased {sigma}B activity activates icaADBC, which encodes enzymes responsible for polysaccharide intercellular adhesin synthesis, by repressing transcription of the negative regulator icaR.Science Foundation IrelandThe title of the submitted paper is different from the article\u27s. Copyright fine. JG suggests to amend the bibliographic details - AV 24/01/201

Characterisation of a modified rotating disk reactor for the cultivation of Staphylococcus epidermidis biofilm

Aims:  The purpose of this study was to develop a system that would allow biofilms to be cultivated under strictly defined conditions in terms of dissolved oxygen, fluid shear and to assess whether the method was suitable for the detection of respiratory activity stratification in biofilm samples. Methods:  The system is a modified version a commercially available laboratory biofilm reactor and incorporates a number of features such as the provision of defined levels of dissolved oxygen, constant average shear, enhanced gas–liquid mass transfer, aseptic operation and the ability to remove biofilm for ex situ analysis during or after continuous cultivation. Conclusions:  The system was shown to be effective for the characterization of the effects of dissolved oxygen on a pure culture of Staphylococcus epidermidis. The versatility of the system offers the potential for cultivating pure culture biofilm in defined, controlled conditions and facilitates a range of analyses that can be performed ex situ. Significance and Impact of the Study:  The ability to provide strict regulation of environmental conditions and enhanced transfer of oxygen to the biofilm during cultivation are important, first because oxygen is known to regulate biofilm development in several micro-organisms and second because many conventional biofilm cultivation systems may not provide adequate oxygen supply to the biofilm.Science Foundation Irelan

Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation-6

Sence of HIV REV and p55-gag. Untreated, undifferentiated cells were included as controls. Cells were harvested at 3, 6, 9 and 15 days post induction of differentiation, fixed and stained for either calcium deposition (alizarin red) or alkaline phosphatase (BCIP/NBT). Plates were first photographed, then the stain extracted, and the degree of staining quantified spectrophotometrically and normalised to cell number. Fig 3a; alkaline phosphatase (ALP) staining in untreated MSCs (con), diferentiating MSCs (cd), and differentiating MSCs exposed to HIV p55-gag and REV proteins. Fig 3b; ALP staining normalised to cell number, data expressed as percentage of untreated control ± SEM. Fig 3c; calcium staining in untreated MSCs (con), diferentiating MSCs (cd), and differentiating MSCs exposed to HIV p55-gag and REV proteins. Fig 3d; calcium staining normalised to cell number, data expressed as percentage of untreated control ± SEM. All data is the mean of several independent experiments. (<p><b>Copyright information:</b></p><p>Taken from "Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation"</p><p>http://www.biomedcentral.com/1471-2474/9/33</p><p>BMC Musculoskeletal Disorders 2008;9():33-33.</p><p>Published online 13 Mar 2008</p><p>PMCID:PMC2330047.</p><p></p

Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation-1

Sence of HIV REV and p55-gag. Untreated, undifferentiated cells were included as controls. Cells were harvested at 3, 6, 9 and 15 days post induction of differentiation, fixed and stained for either calcium deposition (alizarin red) or alkaline phosphatase (BCIP/NBT). Plates were first photographed, then the stain extracted, and the degree of staining quantified spectrophotometrically and normalised to cell number. Fig 3a; alkaline phosphatase (ALP) staining in untreated MSCs (con), diferentiating MSCs (cd), and differentiating MSCs exposed to HIV p55-gag and REV proteins. Fig 3b; ALP staining normalised to cell number, data expressed as percentage of untreated control ± SEM. Fig 3c; calcium staining in untreated MSCs (con), diferentiating MSCs (cd), and differentiating MSCs exposed to HIV p55-gag and REV proteins. Fig 3d; calcium staining normalised to cell number, data expressed as percentage of untreated control ± SEM. All data is the mean of several independent experiments. (<p><b>Copyright information:</b></p><p>Taken from "Mechanism of HIV protein induced modulation of mesenchymal stem cell osteogenic differentiation"</p><p>http://www.biomedcentral.com/1471-2474/9/33</p><p>BMC Musculoskeletal Disorders 2008;9():33-33.</p><p>Published online 13 Mar 2008</p><p>PMCID:PMC2330047.</p><p></p