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Background: Endothelial progenitor cells (EPCs) are present in lower numbers in kidney disease patients who are dialysis-dependent and can be used to predict adverse cardiovascular events. The function of EPCs can be measured using colony forming unit (CFU) assays and the appearance of late outgrowth endothelial cells (OECs) in vitro. Specific clinical parameters can affect EPC function, yet less is known about the relationship between clinical observations and OEC function. Therefore the aim of this study was to determine if the appearance of OECs derived from dialysis-dependent patients was influenced by their clinical history. Method(s): Dialysis-dependent patients (n=20) were recruited to this study; and their age, time on dialysis, blood pressure (BP), erythropoietin (EPO), statin use and smoking status was collected as these parameters have previously demonstrated to affect circulating EPC levels. Blood (10mls) was obtained prior to a single dialysis session and peripheral blood mononuclear cells isolated and cultured. After 7 days CFU wasassessed, then cells were further cultured for 21 days or until OECs appeared (identified by cobblestone morphology). Result(s): The patient cohort had a mean age of 64.2(+/-15.5) years and 80% were male. The mean time on hemodialysis was 46 months (+/-69.6) and blood pressures of 139.1(+/-27.1)/75.2 (+/-18.9). Half of the patients received EPO, 30% were administered statins and 65% had a history of or were current smokers. Circulating %EPC of patients receiving EPO was significantly lower then patients who were not (mean diff. 1.487+/-0.538, 95% CI 0.3-2.7. p=0.0184) and high systolic blood pressure was negatively correlated with %EPC (r=-0.59, p=0.033). We observed a 45% conversion of EPCs to OECs, which was not dependent on starting %EPC (p=0.19). Both systolic (mean diff. 24.95mmHg, 95% CI 1.7-48.1, p=0.0365) and diastolic (mean diff. 18.9, 95%CI 3.2-34.6, p=0.0208) blood pressures were significantly lower in patients whe

Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation.Copyright © 2019 Huuskes, DeBuque, Kerr, Samuel and Ricardo.

Objective To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. Methods Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4 mg/d, s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10 days post-surgery, mice commenced treatment with anakinra (75 mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11 days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. Results By 10 days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3 +/- 2.4 mmHg) compared to control mice (121.7 +/- 2.7 mmHg; n = 18, P < 0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by ~20 mmHg (n = 16, P < 0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced (~30%) renal expression of some (CCL5, CCL2; n = 7-8; P < 0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n = 7-8, P > 0.05). Anakinra reduced renal collagen content (n = 6, P < 0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n = 8-9, P < 0.001) that accompanied 1K/DOCA/salt-induced hypertension. Conclusion Despite its anti-hypertensive and renal anti-fibrotic actions, anakinra had minimal effects on inflammation and leukocyte infiltration in mice with 1K/DOCA/salt-induced hypertension. Future studies will assess whether the anti-hypertensive actions of anakinra are mediated by protective actions in other BP-regulating or salt-handling organs such as the arteries, skin and brain.Copyright © 2016 The Authors

Background and Purpose Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1beta and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1beta and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. Experimental Approach Wild-type and inflammasome-deficient ASC-/- mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. Key Results 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1beta, as well as protein levels of active caspase-1 and mature IL-1beta. Following treatment with 1K/DOCA/salt, ASC-/- mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. Conclusions and Implications Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1beta pathway as a potential therapeutic target in hypertension.Copyright © 2015 The British Pharmacological Society.

Source tracking of fecal pollution is an emerging component in water quality monitoring. It may be implemented in a tiered approach involving Escherichia coli and/or Enterococcus spp. as the standard fecal indicator bacteria (FIB) and the 16S rRNA gene markers of Bacteroidales as source identifiers. The relative population dynamics of the source identifiers and the FIB may strongly influence the implementation of such approach. Currently, the relative performance of DNA and RNA as detection targets of Bacteroidales markers in the tiered approach is not known. We compared the decay of the DNA and RNA of the total (AllBac) and ruminant specific (CF128) Bacteroidales markers with those of the FIB in seawater spiked with cattle feces. Four treatments of light and oxygen availability simulating the subtropical seawater of Hong Kong were tested. All Bacteroidales markers decayed significantly slower than the FIB in all treatments. Nonetheless, the concentrations of the DNA and RNA markers and E. coli correlated significantly in normoxic seawater independent of light availability, and in hypoxic seawater only under light. In hypoxic seawater without light, the concentrations of RNA but not DNA markers correlated with that of E. coli. Generally, the correlations between Enterococcus spp. and Bacteroidales were insignificant. These results suggest that either DNA or RNA markers may complement E. coli in the tiered approach for normoxic or hypoxic seawater under light. When light is absent, either DNA or RNA markers may serve for normoxic seawater, but only the RNA markers are suitable for hypoxic seawater.

Macrophages accumulate in blood vessels during hypertension. However, their contribution to vessel remodeling is unknown. In the present study, we examined the polarization state of macrophages (M1/M2) in aortas of mice during hypertension and investigated whether antagonism of chemokine receptors involved in macrophage accumulation reduces vessel remodeling and blood pressure (BP). Mice treated with ANG II (0.7 mg.kg-1.day-1, 14 days) had elevated systolic BP (158 +/- 3 mmHg) compared with saline-treated animals (122 +/- 3 mmHg). Flow cytometry revealed that ANG II infusion increased numbers of CD45+CD11b+Ly6Chi monocytes and CD45+CD11b+F4/80+ macrophages by 10- and 2-fold, respectively. The majority of macrophages were positive for the M2 marker CD206 but negative for the M1 marker inducible nitric oxide synthase. Expression of other M2 genes (arginase-1, Fc receptor-like S scavenger receptor, and receptor-1) was elevated in aortas from ANG II-treated mice, whereas M1 genes [TNF and chemokine (C-X-C motif) ligand 2] were unaltered. A PCR array to identify chemokine receptor targets for intervention revealed chemokine (C-C motif) receptor 2 (CCR2) to be upregulated in aortas from ANG II-treated mice, while flow cytometry identified Ly6Chi monocytes as the main CCR2-expressing cell type. Intervention with a CCR2 antagonist (INCB3344; 30 mg.kg-1.day-1), 7 days after the commencement of ANG II infusion, reduced aortic macrophage numbers. INCB334 also reduced aortic collagen deposition, elastin loss, and BP in ANG II-treated mice. Thus, ANG II-dependent hypertension in mice is associated with Ly6Chi monocyte and M2 macrophage accumulation in the aorta. Inhibition of macrophage accumulation with a CCR2 antagonist prevents ANG II-induced vessel fibrosis and elevated BP, highlighting this as a promising approach for the future treatment of vessel remodeling/stiffening in hypertension.Copyright © 2015 the American Physiological Society.

Clinical hypertension is associated with raised serum IgG antibodies. However, whether antibodies are causative agents in hypertension remains unknown. We investigated whether hypertension in mice is associated with B-cell activation and IgG production and moreover whether B-cell/IgG deficiency affords protection against hypertension and vascular remodeling. Angiotensin II (Ang II) infusion (0.7 mg/kg per day; 28 days) was associated with (1) a 25% increase in the proportion of splenic B cells expressing the activation marker CD86, (2) an 80% increase in splenic plasma cell numbers, (3) a 500% increase in circulating IgG, and (4) marked IgG accumulation in the aortic adventitia. In B-cell-activating factor receptor-deficient (BAFF-R-/-) mice, which lack mature B cells, there was no evidence of Ang II-induced increases in serum IgG. Furthermore, the hypertensive response to Ang II was attenuated in BAFF-R-/- (DELTA30+/-4 mm Hg) relative to wild-type (DELTA41+/-5 mm Hg) mice, and this response was rescued by B-cell transfer. BAFF-R-/- mice displayed reduced IgG accumulation in the aorta, which was associated with 80% fewer aortic macrophages and a 70% reduction in transforming growth factor-beta expression. BAFF-R-/- mice were also protected from Ang II-induced collagen deposition and aortic stiffening (assessed by pulse wave velocity analysis). Finally, like BAFF-R deficiency, pharmacological depletion of B cells with an anti-CD20 antibody attenuated Ang II-induced hypertension by =35%. Hence, these studies demonstrate that B cells/IgGs are crucial for the development of Ang II-induced hypertension and vessel remodeling in mice. Thus, B-cell-targeted therapies - currently used for autoimmune diseases - may hold promise as future treatments for hypertension.Copyright © 2015 American Heart Association, Inc.

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE-/-) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE-/- versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE-/- mice. To examine the impact of a further reduction in NOX1 activity, APOE-/- mice were crossed with NOX1-/y mice to generate NOX1-/y/APOE-/- double-knockouts. NOX1 deficiency in APOE-/- mice was associated with 30-50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1-/y/APOE-/- versus APOE-/- mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1-/y/APOE-/- and APOE-/- mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE-/- mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE-/- mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.Copyright © 2015 Informa UK, Ltd.

Chronic liver injury and inflammation lead to hepatic fibrosis, cirrhosis, and liver failure. Embryonic and mesenchymal stem cells have been shown to reduce experimental liver fibrosis but have potential limitations, including the formation of dysplastic precursors, tumors, and profibrogenic cells. Other stem-like cells may reduce hepatic inflammation and fibrosis without tumor and profibrogenic cell formation. To test this hypothesis we transplanted human amnion epithelial cells (hAEC), isolated from term delivered placenta, into immunocompetent C57/BL6 mice at week 2 of a 4-week regimen of carbon tetrachloride (CCl4) exposure to induce liver fibrosis. Two weeks following hAEC infusion, intact cells expressing the human-specific markers inner mitochondrial membrane protein and human leukocyte antigen-G were found in mouse liver without evidence of host rejection of the transplanted cells. Human albumin, known to be produced by hAEC, was detected in sera of hAEC-treated mice. Human DNA was detected in mouse liver and also spleen, lungs, and heart of some animals. Following hAEC transplantation, CCl4-treated animals showed decreased serum ALT levels and reduced hepatocyte apoptosis, compared to controls. hAEC-treated mouse liver had lower TNF-alpha and IL-6 protein levels and higher IL-10 compared to animals given CCl4 alone. Compared to CCl4 controls, hAEC-treated mice showed fewer activated collagen-producing hepatic stellate cells and less fibrosis area and collagen content. Reduced hepatic TGF-beta levels in conjunction with a twofold increase in the active form of the collagen-degrading enzyme matrix metalloproteinase-2 in hAEC-treated mice compared to CCl4 controls may account for the reduction in fibrosis. hAEC transplantation into immunocompetent mice leads to cell engraftment, reduced hepatocyte apoptosis, and decreased hepatic inflammation and fibrosis. All rights reserved. Copyright © 2010 Cognizant Comm. Corp.

Acute respiratory distress syndrome is characterized by loss of lung tissue as a result of inflammation and fibrosis. Augmenting tissue repair by the use of mesenchymal stem cells may be an important advance in treating this condition. We evaluated the role of term human umbilical cord cells derived from Wharton's jelly with a phenotype consistent with mesenchymal stem cells (uMSCs) in the treatment of a bleomycininduced mouse model of lung injury. uMSCs were administered systemically, and lungs were harvested at 7, 14, and 28 days post-bleomycin. Injected uMSCs were located in the lung 2 weeks later only in areas of inflammation and fibrosis but not in healthy lung tissue. The administration of uMSCs reduced inflammation and inhibited the expression of transforming growth factor-beta, interferon-gamma, and the proinflammatory cytokines macrophage migratory inhibitory factor and tumor necrosis factor-alpha. Collagen concentration in the lung was significantly reduced by uMSC treatment, which may have been a consequence of the simultaneous reduction in Smad2 phosphorylation (transforming growth factor-beta activity). uMSCs also increased matrix metalloproteinase-2 levels and reduced their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases, favoring a pro-degradative milieu following collagen deposition. Notably, injected human lung fibroblasts did not influence either collagen or matrix metalloproteinase levels in the lung. The results of this study suggest that uMSCs have antifibrotic properties and may augment lung repair if used to treat acute respiratory distress syndrome. Copyright © American Society for Investigative Pathology.