PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Comparisons of α2-Adrenergic Agents, Medetomidine and Xylazine, with Pentobarbital for Anesthesia: Important Pitfalls in Diabetic and Nondiabetic Rats.

Purpose: Anesthesia is necessary to conduct rodent electroretinograms (ERGs). We evaluated utility of the α2-agonist medetomidine versus xylazine for ERG studies in nondiabetic and diabetic rats. Pentobarbital was included as a comparator. Methods: Male Sprague-Dawley rats, with and without streptozotocin (STZ)-induced diabetes, were anesthetized with medetomidine (1 mg/kg), xylazine (10 mg/kg) (both with ketamine 75 mg/kg), or pentobarbital (70 mg/kg). The depth of anesthesia was assessed, and if adequate, scotopic ERGs were recorded. Blood glucose was monitored. Results: In nondiabetic rats, all three agents induced satisfactory anesthesia, but with differing durations: medetomidine > pentobarbital > xylazine. ERG responses were similar under medetomidine and xylazine, but relatively reduced under pentobarbital. Both α2-agonists (but not pentobarbital) elicited marked hyperglycemia (peak values 316.1 ± 42.6 and 300.3 ± 29.5 mg/dL, respectively), persisting for 12 h. In diabetic rats, elevated blood glucose concentrations were not affected by any of the agents, but the depth of anesthesia under medetomidine and xylazine was inadequate for ERG recording. Conclusions: In nondiabetic rats, medetomidine and xylazine elicited comparable effects on ERGs that differ from pentobarbital, but both perturbed glucose metabolism, potentially confounding experimental outcomes. In STZ-diabetic rats, neither α2-agent provided adequate anesthesia, while pentobarbital did so. Problems with α2-anesthetic agents, including medetomidine, must be recognized to ensure meaningful interpretation of experimental results.

Modelling preeclampsia: a comparative analysis of the common human trophoblast cell lines.

Preeclampsia remains a challenge without an effective therapy. Evidence supports targetability of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), which are released excessively from the placenta under ischemic and hypoxic stresses. We compared four trophoblast cell lines, BeWo, Jar, Jeg-3, and HTR-8/SVneo, in order to identify a suitable model for drug screening. Cultured trophoblasts were exposed to 1% oxygen vs. normoxia for 24-48 hr; human umbilical vein and aortic endothelial cells were included for comparison. Supernatant sFlt-1 and sEng concentrations were measured by ELISA, and sFlt-1 mRNA expression determined by RT-PCR. Cellular responses to experimental therapeutics were explored. All four trophoblast lines secreted sEng, which did not increase by hypoxia. BeWo, Jar, and Jeg-3 exhibited significantly enhanced expression of sFlt-1 i13 and e15a mRNA in response to hypoxia; however, only BeWo released a detectable level of sFlt-1 protein, which was doubled by hypoxia. In contrast, hypoxia decreased sFlt-1 mRNA expression and protein release in HTR-8/SVneo, similarly to endothelial cells. The cellular mechanism involved HIFα. BeWo responded to representative agents similarly to human primary placental tissues in the literature. These data support that the BeWo-hypoxia model mimics a key pathogenic mechanism of preeclampsia and has potential value for translational drug discovery.

Effects of Modified Low-Density Lipoproteins and Fenofibrate on an Outer Blood-Retina Barrier Model: Implications for Diabetic Retinopathy.

Purpose: There is a lack of treatment for early diabetic retinopathy (DR), including blood-retina barrier (BRB) breakdown. The robust clinical benefit of fenofibrate in DR provides an opportunity to explore disease mechanisms and therapeutic targets. We have previously found that modified lipoproteins contribute to DR and that fenofibrate protects the inner BRB. We now investigate (1) whether modified lipoproteins elicit outer BRB injury and (2) whether fenofibrate may alleviate such damage. Methods: Human retinal pigment epithelium ARPE-19 cells were cultured in semipermeable transwells to establish a monolayer barrier and then exposed to heavily oxidized, glycated low-density lipoprotein (HOG-LDL, 25-300 mg/L, up to 24 h) versus native (N)-LDL. Transepithelial electric resistance (TEER) and FITC-dextran permeability were measured. The effects of fenofibrate, its active metabolite fenofibric acid, and other peroxisome proliferator-activated receptor (PPARα) agonists (gemfibrozil, bezafibrate, and WY14643) were evaluated, with and without the PPARα antagonist GW6471 or the adenosine monophosphate-activated protein kinase (AMPK) inhibitor Compound C. Results: HOG-LDL induced concentration- and time-dependent barrier impairment, decreasing TEER and increasing dextran leakage, effects that were amplified by high glucose. Fenofibric acid, but not fenofibrate, gemfibrozil, bezafibrate, or WY14643, attenuated barrier impairment. This effect was reversed significantly by Compound C, but not by GW6471. Conclusions: Modified lipoproteins elicited outer BRB injury in an experimental model, which was reduced by fenofibric acid through a PPARα-independent, AMPK-mediated mechanism. These findings suggest a protective role of fenofibric acid on the outer BRB in diabetic retina.

Subclinical First Trimester Renal Abnormalities Are Associated With Preeclampsia in Normoalbuminuric Women With Type 1 Diabetes.

OBJECTIVE: This study was conducted to determine the utility of tubular (urinary/plasma neutrophil gelatinase-associated lipocalin [NGAL] and urinary kidney injury molecule 1 [KIM-1]) and glomerular (estimated glomerular filtration rate [eGFR]) biomarkers in predicting preeclampsia (PE) in pregnant women with type 1 diabetes mellitus (T1DM) who were free of microalbuminuria and hypertension at the first trimester. RESEARCH DESIGN AND METHODS: This was a prospective study of T1DM pregnancy. Maternal urinary and plasma NGAL, urinary KIM-1 (ELISA of frozen samples), and eGFR (Chronic Kidney Disease Epidemiology Collaboration equation) were determined at three study visits (V1: 12.4 ± 1.8; V2: 21.7 ± 1.4; V3: 31.4 ± 1.5 weeks' gestation [mean ± SD]) in 23 women with T1DM with subsequent PE (DM+PE+), 24 who remained normotensive (DM+PE-), and, for reference, in 19 normotensive pregnant women without diabetes (DM-). The groups with diabetes were matched for age, diabetes duration, and parity. All subjects were normotensive and free of microalbuminuria or albuminuria at V1. All study visits preceded the onset of PE. RESULTS: Urinary creatinine-corrected NGAL (uNGALcc, ng/mg) was significantly elevated at V1 in DM+PE+ vs. DM+PE- women (P = 0.01); this remained significant after exclusion of leukocyte-positive samples (5 DM+PE+ and 2 DM+PE-) (P = 0.02). Accounting for BMI, HbA1c, and total daily insulin dose, a doubling of uNGALcc at V1 conferred a sevenfold increase in risk for PE (P = 0.026). In contrast, neither plasma NGAL nor urinary KIM-1 predicted PE. Also at V1, eGFR was elevated in DM+PE+ vs. DM+PE- (P = 0.04). CONCLUSIONS: Early tubular and glomerular dysfunction may predict PE in first trimester women with T1DM, even if free of microalbuminuria. These data suggest that subclinical renal tubular and glomerular injury, if present early in pregnancy, may predispose women with T1DM to PE.

Circulating adipokines are associated with pre-eclampsia in women with type 1 diabetes.

AIMS/HYPOTHESIS: The incidence of pre-eclampsia, a multisystem disorder of pregnancy, is fourfold higher in type 1 diabetic than non-diabetic women; it is also increased in women with features of the metabolic syndrome and insulin resistance. In a prospective study of pregnant women with type 1 diabetes, we measured plasma levels of adipokines known to be associated with insulin resistance: leptin, fatty acid binding protein 4 (FABP4), adiponectin (total and high molecular weight [HMW]; also known as high molecular mass), retinol binding protein 4 (RBP4) and resistin and evaluated associations with the subsequent development of pre-eclampsia. METHODS: From an established prospective cohort of pregnant type 1 diabetic women, we studied 23 who developed pre-eclampsia and 24 who remained normotensive; for reference values we included 19 healthy non-diabetic normotensive pregnant women. Plasma adipokines were measured (by ELISA) in stored samples from three study visits (Visit 1- Visit 3) at different gestational ages (mean ± SD): Visit 1, 12.4 ± 1.8 weeks; Visit 2, 21.7 ± 1.4 weeks; and Visit 3, 31.4 ± 1.5 weeks. All the women were free of microalbuminuria and hypertension at enrolment. All study visits preceded the clinical onset of pre-eclampsia. RESULTS: In all groups, leptin, the ratio of leptin to total or HMW adiponectin, FABP4 concentration, ratio of FABP4 to total or HMW adiponectin and resistin level increased, while total and HMW adiponectin decreased, with gestational age. At Visit 1: (1) in diabetic women with vs without subsequent pre-eclampsia, leptin, ratio of leptin to total or HMW adiponectin, and ratio of FABP4 to total or HMW adiponectin, were increased (p < 0.05), while total adiponectin was decreased (p < 0.05); and (2) in normotensive diabetic vs non-diabetic women, total adiponectin was elevated (p < 0.05). At Visits 2 and 3: (1) the primary findings in the two diabetic groups persisted, and FABP4 also increased in women with subsequent pre-eclampsia (p < 0.05); and (2) there were no differences between the two normotensive groups. By logistic regression analyses after covariate adjustment (HbA1c, insulin kg-1 day-1 and gestational age), the best predictive models for pre-eclampsia were as follows: Visit 1, doubling of leptin, OR 9.0 (p < 0.01); Visit 2, doubling of the leptin:total adiponectin ratio, OR 3.7 (p < 0.05); and Visit 3, doubling of FABP4 concentration, OR 25.1 (p < 0.01). The associations were independent of BMI. CONCLUSIONS/INTERPRETATION: As early as the first trimester in type 1 diabetic women, adipokine profiles that suggest insulin resistance are associated with subsequent pre-eclampsia, possibly reflecting maternal characteristics that precede pregnancy. These associations persist in the second and third trimesters, and are independent of BMI. Insulin resistance may predispose women with type 1 diabetes to pre-eclampsia.

Hypoxia-induced responses by endothelial colony-forming cells are modulated by placental growth factor.

BACKGROUND: Endothelial colony-forming cells (ECFCs), also termed late outgrowth endothelial cells, are a well-defined circulating endothelial progenitor cell type with an established role in vascular repair. ECFCs have clear potential for cell therapy to treat ischaemic disease, although the precise mechanism(s) underlying their response to hypoxia remains ill-defined. METHODS: In this study, we isolated ECFCs from umbilical cord blood and cultured them on collagen. We defined the response of ECFCs to 1% O2 exposure at acute and chronic time points. RESULTS: In response to low oxygen, changes in ECFC cell shape, proliferation, size and cytoskeleton phenotype were detected. An increase in the number of senescent ECFCs also occurred as a result of long-term culture in 1% O2. Low oxygen exposure altered ECFC migration and tube formation in Matrigel®. Increases in angiogenic factors secreted from ECFCs exposed to hypoxia were also detected, in particular, after treatment with placental growth factor (PlGF). Exposure of cells to agents that stabilise hypoxia-inducible factors such as dimethyloxalylglycine (DMOG) also increased PlGF levels. Conditioned medium from both hypoxia-treated and DMOG-treated cells inhibited ECFC tube formation. This effect was reversed by the addition of PlGF neutralising antibody to the conditioned medium, confirming the direct role of PlGF in this effect. CONCLUSIONS: This study deepens our understanding of the response of ECFCs to hypoxia and also identifies a novel and important role for PlGF in regulating the vasculogenic potential of ECFCs.

Beneficial Effects of Berberine on Oxidized LDL-Induced Cytotoxicity to Human Retinal Müller Cells.

PURPOSE: Limited mechanistic understanding of diabetic retinopathy (DR) has hindered therapeutic advances. Berberine, an isoquinolone alkaloid, has shown favorable effects on glucose and lipid metabolism in animal and human studies, but effects on DR are unknown. We previously demonstrated intraretinal extravasation and modification of LDL in human diabetes, and toxicity of modified LDL to human retinal Müller cells. We now explore pathogenic effects of modified LDL on Müller cells, and the efficacy of berberine in mitigating this cytotoxicity. METHODS: Confluent human Müller cells were exposed to in vitro-modified 'highly oxidized, glycated (HOG-) LDL versus native-LDL (N-LDL; 200 mg protein/L) for 6 or 24 hours, with/without pretreatment with berberine (5 µM, 1 hour) and/or the adenosine monophosphate (AMP)-activated protein kinase (AMPK) inhibitor, Compound C (5 µM, 1 hour). Using techniques including Western blots, reactive oxygen species (ROS) detection assay, and quantitative real-time PCR, the following outcomes were assessed: cell viability (CCK-8 assay), autophagy (LC3, Beclin-1, ATG-5), apoptosis (cleaved caspase 3, cleaved poly-ADP ribose polymerase), oxidative stress (ROS, nuclear factor erythroid 2-related factor 2, glutathione peroxidase 1, NADPH oxidase 4), angiogenesis (VEGF, pigment epithelium-derived factor), inflammation (inducible nitric oxide synthase, intercellular adhesion molecule 1, IL-6, IL-8, TNF-α), and glial cell activation (glial fibrillary acidic protein). RESULTS: Native-LDL had no effect on cultured human Müller cells, but HOG-LDL exhibited marked toxicity, significantly decreasing viability and inducing autophagy, apoptosis, oxidative stress, expression of angiogenic factors, inflammation, and glial cell activation. Berberine attenuated all the effects of HOG-LDL (all P < 0.05), and its effects were mitigated by AMPK inhibition (P < 0.05). CONCLUSIONS: Berberine inhibits modified LDL-induced Müller cell injury by activating the AMPK pathway, and merits further study as an agent for preventing and/or treating DR.

Insulin receptor substrate-2 is expressed in kidney epithelium and up-regulated in diabetic nephropathy.

Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.

FKBPL and peptide derivatives: novel biological agents that inhibit angiogenesis by a CD44-dependent mechanism.

PURPOSE: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. EXPERIMENTAL DESIGN: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. RESULTS: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. CONCLUSION: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

Nox2 NADPH oxidase promotes pathologic cardiac remodeling associated with Doxorubicin chemotherapy.

Doxorubicin is a highly effective cancer treatment whose use is severely limited by dose-dependent cardiotoxicity. It is well established that doxorubicin increases reactive oxygen species (ROS) production. In this study, we investigated contributions to doxorubicin cardiotoxicity from Nox2 NADPH oxidase, an important ROS source in cardiac cells, which is known to modulate several key processes underlying the myocardial response to injury. Nox2-deficient mice (Nox2-/-) and wild-type (WT) controls were injected with doxorubicin (12 mg/kg) or vehicle and studied 8 weeks later. Echocardiography indicated that doxorubicin-induced contractile dysfunction was attenuated in Nox2-/- versus WT mice (fractional shortening: 29.5±1.4 versus 25.7±1.0%; P<0.05). Similarly, in vivo pressure-volume analysis revealed that systolic and diastolic function was preserved in doxorubicin-treated Nox2-/- versus WT mice (ejection fraction: 52.6±2.5 versus 28.5±2.3%, LVdP/dtmin: -8,379±416 versus -5,198±527 mmHg s(-1); end-diastolic pressure-volume relation: 0.051±0.009 versus 0.114±0.012; P<0.001). Furthermore, in response to doxorubicin, Nox2-/- mice exhibited less myocardial atrophy, cardiomyocyte apoptosis, and interstitial fibrosis, together with reduced increases in profibrotic gene expression (procollagen IIIαI, transforming growth factor-ß3, and connective tissue growth factor) and matrix metalloproteinase-9 activity, versus WT controls. These alterations were associated with beneficial changes in NADPH oxidase activity, oxidative/nitrosative stress, and inflammatory cell infiltration. We found that adverse effects of doxorubicin were attenuated by acute or chronic treatment with the AT1 receptor antagonist losartan, which is commonly used to reduce blood pressure. Our findings suggest that ROS specifically derived from Nox2 NADPH oxidase make a substantial contribution to several key processes underlying development of cardiac contractile dysfunction and remodeling associated with doxorubicin chemotherapy.

The suppressors of cytokine signalling E3 ligases behave as tumour suppressors.

Many studies have suggested that E3 ubiquitin ligases can behave as either oncogenes or tumour suppressor genes and, recently, it has become clear that the SOCS (suppressor of cytokine signalling) E3 ligases fit this mould. While most cancer-associated E3s regulate the cell cycle or DNA repair, the SOCS proteins inhibit growth factor responses by degrading signalling intermediates such as JAKs (Janus kinases) via the SOCS-box-associated ECS (Elongin-Cullin-SOCS) E3 ligase. Clinical studies have found that (epi)genetic (mutation or methylation) phenomena can occur in many solid tumours and a growing number of clinical findings reveal post-translational modifications that disrupt SOCS function in haematological malignancy. In the present review, we provide a summary of the functions of the SOCS E3s and propose the potential use of members of this family as diagnostic markers and therapeutic targets in cancer.

The myeloproliferative disorder-associated JAK2 V617F mutant escapes negative regulation by suppressor of cytokine signaling 3.

The somatic JAK2 valine-to-phenylalanine (V617F) mutation has been detected in up to 90% of patients with polycythemia and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis. Suppressor of cytokine signaling 3 (SOCS3) is known to be a strong negative regulator of erythropoietin (EPO) signaling through interaction with both the EPO receptor (EPOR) and JAK2. We report here that JAK2 V617F cannot be regulated and that its activation is actually potentiated in the presence of SOCS3. Instead of acting as a suppressor, SOCS3 enhanced the proliferation of cells expressing both JAK2 V617F and EPOR. Additionally, although SOCS1 and SOCS2 are degraded in the presence of JAK2 V617F, turnover of SOCS3 is inhibited by the JAK2 mutant kinase and this correlated with marked tyrosine phosphorylation of SOCS3 protein. We also observed constitutive tyrosine phosphorylation of SOCS3 in peripheral blood mononuclear cells (PBMCs) derived from patients homozygous for the JAK2 V617F mutant. These findings suggest that the JAK2 V617F has overcome normal SOCS regulation by hyperphosphorylating SOCS3, rendering it unable to inhibit the mutant kinase. Thus, JAK2 V617F may even exploit SOCS3 to potentiate its myeloproliferative capacity.