Michelle B. Hookham

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-β3, 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.

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‐β1 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.

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.

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.

Response to Comment on Kelly et al. Subclinical First Trimester Renal Abnormalities Are Associated With Preeclampsia in Normoalbuminuric Women With Type 1 Diabetes. Diabetes Care 2018;41:120–127

We thank Foussard et al. (1) for their interest in our study (2). We agree that caution is required when identifying predictive markers of clinical interest in pregnant women with diabetes with regard to preeclampsia (PE). Accurate estimation of renal function is critical for the care of pregnant patients. Alper et al. (3) highlighted the limitations of currently available formulas for accurately predicting the glomerular filtration rate (GFR) in patients with PE. Our study focused on markers of renal function early in pregnancy prior to the clinical onset of PE. We studied a population with type 1 diabetes, thus at high risk of …

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

BackgroundEndothelial 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.MethodsIn 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.ResultsIn 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.ConclusionsThis 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.

52 Endothelial progenitor cell responses to hypoxia: time-course dependent changes in Akt signalling and gene expression

The aim of this work was to examine endothelial progenitor cells (EPCs) and their potential role as a cell based autologous therapy to revascularise the ischaemic tissue of the diabetic retina. Our group have isolated and characterised a distinctive human blood derived EPC with high proliferative capacity known as outgrowth endothelial cells (OECs). To mimic low O2 concentrations in the ischaemic retina, we have exposed OECs to 1% O2 for varying times. Hypoxia induces reduced cell proliferation, cell senescence and increased cell size in OECs. Changes in Akt activity, a key regulator of cell proliferation were noted after 30 min exposure to hypoxia which then returned to basal levels by 1 h. Hypoxia also induced considerable alterations in the phosphorylation status of several proteins involved in mTOR signalling, a crucial regulator of cell size. Acute exposure (30 min) to 1% O2 triggered p70S6 K phosphorylation in OECs. In contrast chronic exposure to hypoxia resulted in a considerable decrease in p70S6 K which correlated closely with an increase in both AMPK and Raptor phosphorylation. We have also identified that BMP pathway activation is a feature of OEC responses to hypoxia, with rapid induction of Smad1/5/8 phosphorylation observed in these cells within 60 min of hypoxia. The effect of recombinant BMPs on OEC tube formation and other angiogenic markers is currently being examined. These data provide novel insights into OEC responses in the hypoxic/ischaemic milieu, and have important implications for micro and macrovascular damage seen in diabetes.