[Show abstract][Hide abstract] ABSTRACT: Background
Basement matrices such as MatrigelTM and GeltrexTM are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 ¿l/cm2) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations.ResultsGeltrexTM basement matrix at 5 ¿l/cm2 in 24-well (10 ¿l) or 96-well (2 ¿l) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix.Conclusions
We present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.
[Show abstract][Hide abstract] ABSTRACT: Chronic kidney disease (CKD) is a common medical condition of ageing cats. In most cases the underlying aetiology is unknown, but the most frequently reported pathological diagnosis is renal tubulointerstitial fibrosis. Renal fibrosis, characterised by extensive accumulation of extra-cellular matrix within the interstitium, is thought to be the final common pathway for all kidney diseases and is the pathological lesion best correlated with function in both humans and cats. As a convergent pathway, renal fibrosis provides an ideal target for the treatment of CKD and knowledge of the underlying fibrotic process is essential for the future development of novel therapies. There are many mediators and mechanisms of renal fibrosis reported in the literature, of which only a few have been investigated in the cat. This article reviews the process of renal fibrosis and discusses the most commonly cited mediators and mechanisms of progressive renal injury, with particular focus on the potential significance to feline CKD.
The Veterinary Journal 10/2014; 203(1). DOI:10.1016/j.tvjl.2014.10.009 · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50-100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25-100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.
PLoS ONE 10/2014; 9(10):e109375. DOI:10.1371/journal.pone.0109375 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Redox regulation of angiogenesis is an important and emerging topic in vascular sciences; reactive oxygen species (ROS) modulate vascular endothelial cell (EC) proliferation and migration, but the molecular mechanism underlying this phenomenon remains largely unclear. We have shown micromolar concentrations of the pro-angiogenic metabolite deoxyribose-1-phosphate (dRP) detected by mass spectrometry (MS) and nuclear magnetic resonance (NMR) in the supernatants of collagen- and thrombin-stimulated platelets. The genetic silencing of dRP-generating enzymes uridine phosphorylase (UP) and thymidine phosphorylase (TP) in mouse platelets significantly reduced the levels of dRP in platelet supernatants and impaired the ability of protein-free platelet supernatants to enhance human umbilical vein endothelial cell (HUVEC) motility. Chick chorioallantoic membrane (CAM) vascularisation assays performed with protein-free supernatants suggested that the release of dRP by platelets stimulates angiogenesis in vivo.
To understand the pro-angiogenic effect of dRP, we investigated further the molecular mechanism of action of dRP and whether this effect was observed in other nucleoside derivatives deoxyribose-5-phospate (dR5P), ribose-1-phosphate (R1P) and deoxyribose (dR). Using live cell real-time confocal imaging and fluorescence assays, HUVEC treatment with dRP significantly increased ROS generation. Similarly, dR5P and R1P also induced ROS accumulation in HUVECs, whereas dR did not. The protein expression of the redox stress marker heme oxygenase-1 (HO-1) and Integrin β3 were shown to be upregulated by dRP and its phosphorylated derivatives (except dR) by immunoblot experiments. Vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), interleukin 8 (IL-8), and stromal cell-derived factor 1α (SDF-1α) showed a similar effect. In parallel, only dRP and its phosphorylated derivatives increased HUVEC cell motility in scratch-healing assays and capillary tube formation using Matrigel™ assays. The causative link between integrin β3 upregulation and increased HUVEC motility/capillary tube formation was proved using integrin-specific inhibitory antibodies'. Furthermore the angiogenic potential of dRP was confirmed in vivo.
In summary, dRP and its phosphorylated derivatives were shown to play a relevant role in the paracrine regulation of EC motility and pro-angiogenic activity by up-regulating integrin β3 in a ROS-dependent manner possibly signaling via VEGFR2. dRP is likely to play a role in the stimulation of postnatal angiogenesis and tissue regeneration.
Cardiovascular Research 07/2014; 103(suppl 1):S97. DOI:10.1093/cvr/cvu093.2 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Obesity and saturated fatty acid (SFA) treatment are both associated with skeletal muscle insulin resistance (IR) and increased macrophage infiltration. However, the relative effects of SFA and unsaturated fatty acid (UFA)-activated macrophages on muscle are unknown. Here, macrophages were treated with palmitic acid, palmitoleic acid or both and the effects of the conditioned medium (CM) on C2C12 myotubes investigated. CM from palmitic acid-treated J774s (palm-mac-CM) impaired insulin signalling and insulin-stimulated glycogen synthesis, reduced Inhibitor κBα and increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in myotubes. p38 MAPK inhibition or siRNA partially ameliorated these defects, as did addition of tumour necrosis factor-α blocking antibody to the CM. Macrophages incubated with both FAs generated CM that did not induce IR, while palmitoleic acid-mac-CM alone was insulin sensitising. Thus UFAs may improve muscle insulin sensitivity and counteract SFA-mediated IR through an effect on macrophage activation.
[Show abstract][Hide abstract] ABSTRACT: Protease activated receptors (PARs) form a distinct group of G-protein coupled receptors with key roles in several pathophysiological processes including inflammation and atherothrombotic disease. This study aimed to investigate the effects of oxidative stress on PAR biology in human endothelial cells. EA.hy926 cells were investigated as an endothelial cell model and subsequently the effect of tert-butylhydroperoxide (tert-BHP) as an oxidative stressor was investigated in terms of PAR expression and activation.
[Show abstract][Hide abstract] ABSTRACT: Dogs with congenital portosystemic shunts (CPSS) have liver hypoplasia and hepatic insufficiency. Surgical CPSS attenuation results in liver growth associated with clinical improvement. The mechanism of this hepatic response is unknown, although liver regeneration is suspected. This study investigated whether markers of liver regeneration were associated with CPSS attenuation. Dogs treated with CPSS attenuation were prospectively recruited. Residual liver tissue was collected for gene expression analysis (seven genes) from 24 CPSS dogs that tolerated complete attenuation, 25 dogs that tolerated partial attenuation and seven control dogs. Relative gene expression was measured using quantitative polymerase chain reaction (qPCR). Blood samples were collected before, 24 h and 48 h post-surgery from 36 CPSS dogs and from 10 control dogs. Serum hepatocyte growth factor (HGF) concentration was measured using a canine specific enzyme-linked immunosorbent assay (ELISA).
HGF mRNA expression was significantly decreased in CPSS compared to control dogs (P = 0.046). There were significant increases in HGF (P = 0.050) and methionine adenosyltransferase 2 A (MAT2A; P = 0.002) mRNA expression following partial CPSS attenuation. Dogs with complete attenuation had significantly greater MAT2A (P = 0.024) mRNA expression compared to dogs with partial attenuation. Serum HGF concentration significantly increased 24 h following CPSS attenuation (P < 0.001). Hepatic mRNA expression of two markers of hepatocyte proliferation (HGF and MAT2A) was associated with the response to surgery in dogs with CPSS, and serum HGF significantly increased following surgery, suggesting hepatocyte proliferation. These findings support the concept that hepatic regeneration is important in the hepatic response to CPSS surgery.
The Veterinary Journal 05/2014; 200(2). DOI:10.1016/j.tvjl.2014.02.027 · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytochrome P450s (CYPs) represent a highly divergent class of enzymes involved in the oxidation of organic compounds. A subgroup of CYPs metabolize ω3-arachidonic and linoleic acids and ω6-docosahexaenoic and eicosapentaenoic polyunsaturated fatty acids (PUFAs) into a series of related biologically active mediators. Over the past 20 years, increasing evidence has emerged for a role of these PUFA-derived mediators in physiological and pathophysiological processes in the vasculature, during inflammation, and in the regulation of metabolism. With recent technological advances and increased availability of lipid mass spectroscopy, we are now starting to discern the patterns of these CYP-PUFA products in health and disease. These analyses are revealing not only the diverse spectrum of lipid nutrients regulated by CYPs, but also clearly indicate that the balance of these mediators changes with dietary intake of different PUFA classes. These findings suggest that we are only just beginning to understand all of the relevant lipid species produced by CYP pathways. Moreover, we are still a long way from understanding the nature and presence of their receptors, their tissue expression, and the pathophysiological processes they regulate. This review highlights these future issues in the context of lipid-metabolizing CYP enzymes, focusing particularly on the CYP450 family of epoxygenases and the lipid mediators they produce. Expected final online publication date for the Annual Review of Nutrition Volume 34 is July 17, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
[Show abstract][Hide abstract] ABSTRACT: The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders.
Cell and Tissue Research 12/2013; 355(2). DOI:10.1007/s00441-013-1763-y · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adenosine is released from the myocar- dium, endothelial cells, and skeletal muscle in ischemia and is an important regulator of coronary blood flow. We have already shown that acute (2 min) activation of A2a purinoceptors stimulates NO production in human fetal umbilical vein endothelial cells (1) and now report a key role for p42/p44 mitogen-activated protein ki- nases (p42/p44MAPK) in the regulation of the L-argi- nine-nitric oxide (NO) signaling pathway. Expression of mRNA for the A2a-, A2b-, and A3-adenosine receptor subtypes was abundant whereas A1-adenosine receptor mRNA levels were negligible. Activation of A2a purino- ceptors by adenosine (10 M) or the A2a receptor agonist CGS21680 (100 nM) resulted in an increase in L-arginine transport and NO release that was not medi- ated by changes in intracellular Ca2, pH, or cAMP. Stimulation of endothelial cells with adenosine was associated with a membrane hyperpolarization and phosphorylation of p42/p44MAPK. L-NAME abolished the adenosine-induced hyperpolarization and stimula- tion of L-arginine transport whereas sodium nitroprus- side activated an outward potassium current. Genistein (10 M) and PD98059 (10 M), an inhibitor of MAPK kinase 1/2 (MEK1/2), inhibited adenosine-stimulated L-arginine transport, NO production, and phosphoryla- tion of p42/p44MAPK. We found no evidence for acti-
[Show abstract][Hide abstract] ABSTRACT: Blood levels of triglyceride-rich lipoproteins (TRL), increase postprandially, and a delay in their clearance results in postprandial hyperlipidemia, an important risk factor in atherosclerosis development. Atherosclerosis is a multifactorial inflammatory disease, and its initiation involves endothelial dysfunction, invasion of the artery wall by leukocytes and subsequent formation of foam cells.. TRL are implicated in several of these inflammatory processes, including the formation of damaging free radicals, leukocyte activation, endothelial dysfunction and foam cell formation. Recent studies have provided insights into the mechanisms of uptake and the signal transduction pathways mediating the interactions of TRL with leukocytes and vascular cells, and how they are modified by dietary lipids. Multiple receptor and non-receptor mediated pathways function in macrophage uptake of TRL by macrophages. TRL also induce expression of adhesion molecules, cyclooxygenase-2 and heme-oxygenase-1 in endothelial cells, and activate intracellular signalling pathways involving mitogen-activated protein kinases, NF-κB and Nrf2. Many of these effects are strongly influenced by dietary components carried in TRL. There is extensive evidence indicating that raised postprandial TRL levels are a risk factor for atherosclerosis, but the molecular mechanisms involved are only now becoming appreciated. Here, we review current understanding of the mechanisms by which TRL influence vascular cell function.
Progress in lipid research 06/2013; 52(4). DOI:10.1016/j.plipres.2013.06.001 · 12.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2; VE-cadherin; CD31) which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labelling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro co-culture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behaviour and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC co-cultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signalling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF(164) . Thus, endoglin-expressing islet MSCs influence EC ALK5 signalling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-β signalling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation.
[Show abstract][Hide abstract] ABSTRACT: C-type natriuretic peptide (CNP/Nppc) is expressed at high levels in the anterior pituitary of rats and mice and activates guanylyl cyclase B receptors (GC-B/Npr2) to regulate hormone secretion. Mutations in NPR2/Npr2 can cause achondroplasia, GH deficiency, and female infertility, yet the normal expression profile within the anterior pituitary remains to be established in humans. The current study examined the expression profile and transcriptional regulation of NPR2 and GC-B protein in normal human fetal pituitaries, normal adult pituitaries, and human pituitary adenomas using RT-PCR and immunohistochemistry. Transcriptional regulation of human NPR2 promoter constructs was characterized in anterior pituitary cell lines of gonadotroph, somatolactotroph, and corticotroph origin. NPR2 was detected in all human fetal and adult pituitary samples regardless of age or sex, as well as in all adenoma samples examined regardless of tumor origin. GC-B immunoreactivity was variable in normal pituitary, gonadotrophinomas, and somatotrophinomas. Maximal transcriptional regulation of the NPR2 promoter mapped to a region within -214 bp upstream of the start site in all anterior pituitary cell lines examined. Electrophoretic mobility shift assays revealed that this region contains Sp1/Sp3 response elements. These data are the first to show NPR2 expression in normal human fetal and adult pituitaries and adenomatous pituitary tissue and suggest a role for these receptors in both pituitary development and oncogenesis, introducing a new target to manipulate these processes in pituitary adenomas.
Endocrine Related Cancer 05/2012; 19(4):497-508. DOI:10.1530/ERC-12-0129 · 4.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atherosclerosis is known to be an inflammatory disease and there is increasing evidence that chylomicron remnants (CMR), the lipoproteins which carry dietary fats in the blood, cause macrophage foam cell formation and inflammation. In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is increased, and clearance of CMR from blood may be delayed, however, whether CMR contribute directly to monocyte activation and subsequent egress into the arterial wall has not been established. Here, the contribution of CMR to activation of monocyte pro-inflammatory pathways was assessed using an in vitro model.
Primary human monocytes and CMR-like particles (CRLP) were used to measure several endpoints of monocyte activation. Treatment with CRLP caused rapid and prolonged generation of reactive oxygen species by monocytes. The pro-inflammatory chemokines MCP-1 and IL-8 were secreted in nanogram quantities by the cells in the absence of CRLP. IL-8 secretion was transiently increased after CRLP treatment, and CRLP maintained secretion in the presence of pharmacological inhibitors of IL-8 production. In contrast, exposure to CRLP significantly reduced MCP-1 secretion. Chemotaxis towards MCP-1 was increased in monocytes pre-exposed to CRLP and was reversed by addition of exogenous MCP-1.
Our findings indicate that CRLP activate human monocytes and augment their migration in vitro by reducing cellular MCP-1 expression. Our data support the current hypothesis that CMR contribute to the inflammatory milieu of the arterial wall in early atherosclerosis, and suggest that this may reflect direct interaction with circulating blood monocytes.
[Show abstract][Hide abstract] ABSTRACT: Surgical attenuation of a congenital portosystemic shunt (CPSS) results in increased liver mass, development of intrahepatic portal vasculature and improved liver function. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The aim of this study was to investigate the role of VEGF and its receptor in the hepatic response to CPSS surgery. The study included 99 dogs with CPSS treated with either partial or complete suture attenuation. Forty-four dogs with partial attenuation underwent a second surgery for complete attenuation. The expression of VEGF and VEGF receptor 2 (VEGFR2) in biopsy samples of liver was assessed by immunohistochemistry with rabbit anti-human VEGF polyclonal antibody and mouse anti-human VEGFR2 monoclonal antibody. Expression of these molecules was graded. The proportion of samples expressing VEGF was significantly greater in samples from dogs with CPSS compared with control samples (P=0.04) and the proportion of samples expressing VEGFR2 was significantly greater in control samples compared with samples from dogs with CPSS (P=0.04). VEGF labelling grade decreased significantly (P=0.038) and VEGFR2 increased significantly (P=0.046) between first and second surgery. The decrease in VEGF may reflect transient expression, preferential expression of other factors, reperfusion of existing vessels and/or increased angiogenesis before surgery in the form of arterialization and subsequent reduction due to improved portal blood flow. Partial suture attenuation was associated with a degree of 'normalization' of VEGF and VEGFR2 expression when compared with the control samples. Further investigation is needed to provide more information on the hepatic response to CPSS surgery.