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Background: Genomic classification of breast cancer has advanced breast cancer diagnosis and outcomes. However, extensive heterogeneity still exists beyond their DNA or RNA profiles. Newer classifications based on protein profiling are being developed to investigate the molecular oncology of breast cancers at the level where most drugs act. Using a recently-developed technology, we performed global proteomic profiling of 300 breast cancer specimens linked to outcome data. Methods: Sections of 75 samples from each PAM50 intrinsic subtype (Luminal A, Luminal B, Her2-enriched, Basal-like; n = 300) were macrodissected and analyzed using the Single-Pot Solid-Phase enhanced Sample Preparation Clinical Tissue Proteomics, a highly sensitive 11-sample multiplex massspectrometry protocol applicable to formalin-fixed, paraffin embedded (FFPE) specimens. This methodology enables comprehensive quantification of protein expression for classifier and biomarker discovery. Patients were diagnosed during 2008-2013 (n = 178, dataset I) and 1986-1992 (n = 122, dataset II). Results: In-depth proteomic analysis measured 9088 proteins in total, including 4214 proteins quantified in every sample. Consensus clustering of 174 evaluable cases in dataset I identified four distinct groups based on expression values for 1054 highly variant proteins. Cluster 3 (n = 47, mostly basal-like with HER2-Enriched) displayed the most favorable recurrence free survival (RFS) when compared to other clusters (HR = 0.22, 95%CI [0.08-0.63], p = 0.005). This cluster was enriched for immune related pathways including antigen processing and presentation and type I & II interferon signaling, and displayed high tumor infiltrating lymphocyte counts, characterizing this cluster as “immune hot”. In contrast, cluster 2 (n = 50, mostly basal-like) exhibited the poorest RFS (HR = 2.88, 95%CI [1.45-5.70], p = 0.002) and was enriched for proteins related to stromal and extracellular matrix with few immune related peptides. Cluster 1 (n = 34, luminal B and HER2-Enriched) was associated with lipid metabolism, whereas cluster 4 (n = 43, mostly HER2-Enriched with luminal A and luminal B) had a profile enriched for extracellular matrix, blood coagulation and complement activation. Conclusions: Global proteomic analysis on FFPE specimens can characterize the heterogeneity of breast cancer in a reliable and clinically-applicable high throughput manner. Our methodology identifies protein candidates that potentially serve as therapeutic targets and could be adapted to archived clinical specimens from other tumors. Citation Format: Karama Asleh, Gian Luca Negri, Sandra E. Spencer Miko, Shane Colborne, Christopher S. Hughes, Xiu Q. Wang, Dongxia Gao, C. Blake Gilks, Stephen K.L. Chia, Torsten O. Nielsen, Gregg B. Morin. Proteomic analysis of breast cancer formalin-fixed paraffin-embedded clinical specimens identifies biologically-important subtypes with distinct clinical outcomes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-06.

The aim of this study was to determine the effects of dietary supplementation of palygorskite instead of zinc oxide (ZnO) on growth performance, apparent nutrient digestibility and zinc contents in serum and feces in weaned piglets. One hundred and sixty-five piglets (28 days of age, 7.10 ± 0.86 kg) were allotted to five treatments on the basis of weight and sex. Each treatment included three replicates of 11 piglets. The piglets were fed the same basal diet supplemented with 0 mg/kg (control group), 1800 mg/kg, 2400 mg/kg, or 3000 mg/kg palygorskite or 2500 mg/kg ZnO for 28 days. There was no significant difference for incidence of diarrhea among the four treatments during the 0–14-day feeding period. Compared with the control group, piglets offered diets supplemented with 1800 mg/kg palygorskite had higher (P

Infection with certain strains of Escherichia coli and endotoxemia results in renal glomerular thrombotic microangiopathy (TMA) characterized by endothelial swelling and prominent glomerular microthrombus formation. Nitric oxide (NO) is an endogenous biologic modulator with diverse physiologic functions including vasodilation and inhibition of platelet adhesion and aggregation. NO is synthesized from conversion of L-arginine to L-citrulline by a family of NO synthases (NOS), which include constitutive and inducible isoforms. Indirect evidence supports the hypothesis that TMA is associated with depressed intrarenal NO production. However, the effect of TMA on renal tissue NOS expression has not been fully elucidated. We studied rats with TMA induced by iv bolus injection of high dose (20 mg/kg) E. coli endotoxin. Subgroups of six animals each were sacrificed before or at 30, 90, 180, 360, and 720 minutes after the administration of endotoxin. Renal histology and tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) were examined. Additionally, we examined the effect of endotoxin on glomerular NO production, and eNOS and iNOS protein expression in vitro. Glomerular capillary thrombosis developed by 180 minutes after endotoxin administration in approximately half of the animals. The glomeruli without thrombotic lesions apparent by light microscopy disclosed early signs of TMA characterized by endothelial swelling, platelet accumulation/adhesion, and patchy fibrinogen deposition. These morphologic changes were associated with a marked reduction of renal tissue eNOS expression beyond 180 minutes after the endotoxin administration. The fall in eNOS expression was coupled with a significant rise in iNOS protein abundance, which was expressed largely by glomerular circulating neutrophils and endothelial cells, peritubular vascular endothelium, and collecting ducts of cortex and medulla. In vitro incubation of isolated glomeruli with endotoxin also resulted in a marked reduction in eNOS expression and a significant rise in iNOS content. Administration of E. coli endotoxin leads to a sustained fall in renal eNOS expression both in vivo and in vitro. The associated decline in intrarenal endothelial NO production/availability may result in renal vasoconstriction and a hypercoagulative state, which may contribute to the pathogenesis of endotoxin-induced TMA.

The available data on the effect of chronic renal failure (CRF) on nitric oxide (NO) metabolism are limited and contradictory. We studied rats with CRF 6 wk after a five-sixths nephrectomy and compared the results with those in the sham-operated controls, felodipine-treated CRF, and parathyroidectomized (CRF-PTX) animals. CRF was produced by surgical resection of the upper and lower thirds of the left kidney, followed by contralateral nephrectomy. We chose this model, as opposed to that produced by renal artery branch ligation, because the latter causes exuberant hypertension (HTN), which independently affects NO metabolism. The CRF group exhibited a mild HTN coupled with elevated basal platelet cytosolic Ca2+concentration ([Ca2+]i), blunted hypotensive response tol-arginine, decreased hypertensive response to NO synthase (NOS) inhibitor, NG-monomethyl-l-arginine, and normal hypotensive response to NO donor, sodium nitroprusside. This was associated with a significant reduction in urinary excretion of stable NO metabolites (NOX) and depressed NOS activity, as well as endothelial and inducible NO synthase (eNOS and iNOS, respectively) protein contents of thoracic aorta and the remnant kidney in the CRF animals. Calcium channel blockade and PTX lowered blood pressure, increased urinary NOX, and enhanced vascular NOS activity, as well as eNOS and iNOS protein expressions in the tested tissues. Thus CRF animals exhibited significant reductions in vascular NOS activity and eNOS and iNOS expressions. These abnormalities were reversed by calcium channel blockade and PTX, suggesting the possible causal role of CRF-induced dysregulation of [Ca2+]i.

Regular administration of recombinant erythropoietin (EPO) in patients with chronic renal failure (CRF) is frequently complicated by a rise in arterial blood pressure. Clinical studies intended to discern the possible role of endothelin (ET) in the pathogenesis of EPO-induced hypertension have produced contradictory results. Given the limitations of the clinical studies, this placebo-controlled study was carried out in CRF (5/6 nephrectomized) rats treated with either EPO, 150 U/kg intraperitoneally, or the vehicle alone twice weekly for 6 wk. Plasma ET was measured at baseline, and weeks 2, 4, and 6. In addition, plasma arginine vasopressin (AVP) and atrial natriuretic peptide (ANP) were determined at the conclusion of the study period. As expected, blood pressure rose markedly after 1 wk of EPO therapy as compared with the placebo therapy. However, there was no significant difference in plasma ET levels between the EPO- and placebo-treated groups during the study period. Likewise, EPO therapy had no effect on plasma ANP level but depressed plasma AVP concentration. Thus, this placebo-controlled animal study revealed that EPO therapy markedly raised arterial blood pressure but had no effect on plasma ET in the CRF rats. This observation suggests that EPO-induced hypertension in this model is not mediated by an increased circulating ET level. However, the possible effect, if any, of EPO on local vascular tissue ET level is uncertain and awaits further investigation.

BACKGROUND: The effect of reactive oxygen species (ROS) on nitric oxide synthase (NOS) expression remains uncertain. This study explored the effect of increased ROS activity on NOS expression in vitro in human coronary artery endothelial cells (HCAECs) grown in culture and in intact animals. METHODS: Endothelial NOS (eNOS) expression and nuclear factor kappaB (NFkappaB) activation were determined in HCAECs grown in culture and exposed to oxidative stress with xanthine-xanthine oxidase (X-XO) generated superoxide, H(2)O(2), or glutathione depletion with buthionine sulfoximine (BSO) for 24 h. In parallel experiments, cells were treated with a nitric oxide (NO) scavenger (hemoglobin), and with an NO donor S-nitroso-N-acetyl penicillamine (SNAP)]. In addition, eNOS and inducible NOS (iNOS) expressions were determined in rats treated with either BSO or vehicle for 48 h. RESULTS: Increases in ROS activity, achieved by exogenous superoxide and H(2)O(2) or by glutathione depletion, upregulated the expression of eNOS at both transcriptional and translational levels in HCAECs. Similar effects were seen with the non-radical NO scavenger, hemoglobin. The upregulatory action of hemoglobin on eNOS messenger RNA (mRNA) and protein expressions was overcome by the NO donor, SNAP, thereby suggesting that there is a negative feedback regulation of eNOS by NO. Nuclear translocation of NFkappaB (p65) was noted within 5 min of exposure to H(2)O(2) and at least 15 min after exposure to superoxide or BSO. Induction of oxidative stress by glutathione depletion led to upregulation of renal and aorta eNOS and iNOS in live animals. CONCLUSIONS: An increase in ROS activity upregulates NOS expression in vitro in HCAECs grown in culture, and also in vivo in animals. This effect appears to be, in part, mediated by limiting the availability of NO, thereby exerting a negative feedback influence on NOS expression through activation of NFkappaB.

Association of renal injury with nitric oxide deficiency in aged SHR: Prevention by hypertension control with AT 1 blockade. Background Aged spontaneously hypertensive rats (SHR) develop end-stage renal disease resembling that of uncontrolled essential hypertension in humans. Nitric oxide (NO) and angiotensin II (Ang II) play an important role in the regulation of blood pressure and the growth of vascular smooth muscle and renal mesangial cells. The relationship between renal NO system, Ang II activity and renal injury in aged SHR is not fully understood. Methods The 8-week-old SHR were randomized into losartan-treated (30 mg/kg/day for 55 weeks) and vehicle treated groups. The age-matched Wistar-Kyoto rats (WKY) served as controls. Renal histology and tissue expressions of endothelial and inducible NO synthases (eNOS and iNOS) and nitrotyrosine were examined at 63-weeks of age. Results Compared to the WKY group, untreated SHR showed severe hypertension, proteinuria, renal insufficiency, a twofold decrease in renal tissue eNOS and iNOS expressions and massive nitrotyrosine accumulation. This was associated with severe glomerulosclerosis, tubular atrophy and interstitial fibrosis. Losartan therapy normalized blood pressure, prevented proteinuria and renal insufficiency, abrogated the fall in renal eNOS and iNOS protein contents, mitigated renal nitrotyrosine accumulation, and prevented the histological abnormalities found in the untreated SHR. Conclusions Aged SHR exhibit severe renal lesions with acquired NO deficiency that are prevented by hypertension control with AT 1 blockade. These findings point to the possible role of NO deficiency in the pathogenesis of renal lesions in aged SHR.

Induction of chronic oxidative stress by glutathione (GSH) depletion has been shown to cause hypertension in normal rats. This was accompanied by and perhaps in part due to inactivation and sequestration of NO by reactive oxygen species (ROS), leading to diminished NO bioavailability. This study was designed to examine renal histology, nitric oxide synthase (NOS) isotype expression, and nitrotyrosine distribution in this model. Sprague-Dawley rats were subjected to oxidative stress by administration of the GSH synthase inhibitor buthionine sulfoximine (BSO; 30 mM/l in drinking water) for 2 weeks. The controls were given tap water. Blood pressure, renal histology, tissue expression of endothelial and inducible NOS (eNOS and iNOS) and nitrotyrosine, tissue GSH content, and urinary excretion of NO metabolites (NOx) were examined. The BSO-treated group showed a 3-fold decrease in tissue GSH content, a marked elevation in blood pressure, and a significant reduction in the urinary excretion of NOx. Histological examination of kidneys revealed no significant abnormalities in either group. In addition, no significant differences were observed in either intensities or localizations of eNOS and iNOS in the kidney. However, the BSO-treated group exhibited intense accumulation in the renal tissue of nitrotyrosine, which is the footprint of NO oxidation by ROS. These observations suggest that oxidative stress-induced hypertension is not caused by either structural abnormality of or depressed NOS expression by the kidney in this model. Instead, it is associated with and perhaps partially related to enhanced renal NO inactivation by ROS and diminished NO bioavailability.

Chronic iron (Fe) overload is associated with a marked increase in renal tissue iron content and injury. It is estimated that 10% of the American population carry the gene for hemochromatosis and 1% actually suffer from iron overload. The mechanism of iron overload-associated renal damage has not been fully elucidated. Iron can accelerate lipid peroxidation leading to organelle membrane dysfunction and subsequent cell injury/death. Iron-catalyzed generation of reactive oxygen species (ROS) is responsible for initiating the peroxidatic reaction. We investigated the possible association of oxidative stress and its impact on nitric oxide (NO) metabolism in iron-overload-associated renal injury. Rats were randomized into Fe-loaded (given 0.5 g elemental iron/kg body weight as iron dextran; i.v.), Fe-depleted (given an iron-free diet for 20 weeks), and control groups. Renal histology, tissue expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS), renal tissue expression of nitrotyrosine, plasma, and renal tissue lipid peroxidation product, malondialdehyde (MDA), and plasma and urinary NO metabolites (NOx) were examined. Iron overload was associated with mild proteinuria, tissue iron deposition together with significant glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Rare focal glomerulosclerosis and tubulointerstitial changes were noted in normal controls. No renal lesions were observed in Fe-depleted rats. Iron deposits were seen in glomeruli, proximal tubules, and interstitium. The iron staining in the distal tubules was negligible. Both plasma and renal tissue MDA and renal tissue nitrotyrosine were increased significantly in Fe-loaded rats compared with control rats. In contrast, Fe-depleted animals showed a marked reduction in plasma and renal tissue MDA and nitrotyrosine together with significant elevation of urinary NOx excretion. In addition, iron-overload was associated with up-regulation of renal eNOS and iNOS expressions when compared with the control and Fe-depleted rats that showed comparable values. In conclusion, chronic iron overload resulted in iron deposition in the glomeruli and proximal tubules with various renal lesions and evidence of increased ROS activity, enhanced ROS-mediated inactivation, and sequestration of NO and compensatory up-regulation of renal eNOS and iNOS expressions. However, iron depletion was associated with reduced MDA and tissue nitrotyrosine abundance, increased urinary NOx excretion, normal nitric oxide synthase (NOS) expression, and absence of renal injury. These findings point to the possible role of ROS in chronic iron overload-induced renal injury.

Abstract —We have recently demonstrated that long-term consumption of a high-fat, refined-carbohydrate (HFS) diet induces hypertension (HTN) in normal rats compared with a low-fat, complex-carbohydrate (LFCC) diet. Limited evidence suggests that high-fat or high-sugar diets cause enhanced generation of reactive oxygen species (ROS). We therefore hypothesized that by inducing oxidative stress, the HFS diet may promote nitric oxide (NO) inactivation and HTN. To test this hypothesis, female Fischer rats were placed on either the HFS or the LFCC diet starting at 2 months of age. Blood pressure, urinary NO metabolites (NO x ), and total renal NO synthase activity were monitored, and the tissue abundance of nitrotyrosine (NT), which is the stable “footprint” of NO oxidation by ROS, was determined. The HFS diet group exhibited a gradual rise in arterial blood pressure and were hypertensive by 18 months. This trend was accompanied by a marked accumulation of NT in all tested tissues, an initial rise and a subsequent fall in NO synthase activity, and a fall in urinary NO x excretion. The HFS diet–fed animals had a blunted blood pressure response to the NO synthase inhibitor N ω -nitro- l -arginine methyl ester ( l -NAME) compared with the LFCC diet group, which showed a marked hypertensive response to l -NAME. l -NAME–induced HTN was reversible with l -arginine in the LFCC diet group; however, HTN was not corrected by l -arginine supplementation in the HFS diet group. These findings point to enhanced ROS-mediated inactivation and sequestration of NO, which may contribute to the reduction of bioactive NO and HTN in the HFS diet–fed animals.

Abstract —Earlier studies have demonstrated that nitric oxide (NO) exerts a fast-acting inhibitory influence on endothelial NO synthase (eNOS) enzymatic activity in isolated vascular tissue preparations. The present study was designed to examine the possible effect of NO on eNOS protein expression in cultured endothelial cells and intact animals. Human coronary endothelial cells were incubated with S -nitroso- N -acetyl-penicillamine (SNAP, an NO donor), oxyhemoglobin (HGB, an NO trapping agent), SNAP plus HGB, or inactive vehicle (control). In other experiments, cells were treated with 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), 1 H -[1,2,4]oxadiazolo-[4,3–2]quinoxalin-1-one (ODQ, a guanylate cyclase inhibitor), SNAP plus ODQ, 8-bromo-cGMP (8-Br-cGMP, a cell-permeable cGMP compound), 8-Br-cGMP plus HGB, or inactive vehicle in order to discern the effect of cGMP. The incubations were conducted for 24 hours, and total nitrate plus nitrite production and eNOS protein abundance (Western analysis) were measured. To determine the effect of NO on eNOS expression in vivo, rats were treated with either the NO donor isosorbide dinitrate or placebo by gastric gavage for 48 hours, and aortic eNOS protein expression was examined. The NO donor SNAP markedly depressed, whereas the NO scavenger HGB significantly raised, eNOS protein expression. The downregulatory action of SNAP was completely abrogated by HGB. Phosphodiesterase inhibitor and 8-Br-cGMP downregulated, whereas the guanylate cyclase inhibitor ODQ upregulated eNOS protein expression. The downregulatory action of SNAP was completely overcome by the guanylate cyclase inhibitor ODQ, and the upregulatory action of the NO scavenger HGB was abrogated by 8-Br-cGMP. Administration of NO donor resulted in a marked downregulation of aortic eNOS protein expression in intact animals, thus confirming the in vitro findings. NO serves as a negative-feedback regulator of eNOS expression via a cGMP-mediated process.

Abstract —We have recently shown that erythropoietin (EPO)-induced hypertension is unrelated to the rise in hematocrit and is marked by elevated cytosolic [Ca +2 ] and nitric oxide (NO) resistance. The present study was done to determine the effect of EPO on NO production and endothelial NO synthase (eNOS) expression by endothelial cells. Human coronary artery endothelial cells were cultured to subconfluence and then were incubated for 24 hours in the presence of either EPO (0, 5, and 20 U/mL) alone or EPO plus the calcium channel blocker felodipine. The experiments were carried out with quiescent (0.5% FCS) and proliferating (5% FCS) cells. Total nitrate and nitrite, eNOS protein, DNA synthesis (thymidine incorporation), and cell proliferation (cell count) were determined. In addition, NO production in response to acetylcholine stimulation was tested. EPO resulted in a dose-dependent inhibition of basal and acetylcholine-stimulated NO production and eNOS protein expression and also led to a significant dose-dependent stimulation of DNA synthesis in endothelial cells. The inhibitory effects of EPO on NO production and eNOS expression were reversed by felodipine. Thus, EPO downregulates basal and acetylcholine-stimulated NO production, depresses eNOS expression, and stimulates proliferation in isolated human endothelial cells. The suppressive effects of EPO on NO production and on eNOS expression are reversed by calcium channel blockade.

Abstract —Long-term administration of erythropoietin (EPO) frequently causes hypertension in humans and animals with chronic renal failure (CRF). We recently demonstrated that EPO-induced hypertension is hematocrit independent and accompanied by elevated cytosolic [Ca 2+ ] i and nitric oxide (NO) resistance. This study was undertaken to examine the effects of therapy with EPO alone or together with calcium channel blockade on NO metabolism. Urinary excretion of NO metabolites (NOx) and thoracic aorta and kidney endothelial and inducible NO synthases (eNOS and iNOS) were studied in 4 groups of 6 nephrectomized rats treated with either placebo, EPO, the calcium channel blocker felodipine, or EPO plus felodipine for 6 weeks. A group of sham-operated placebo-treated animals served as control. The placebo-treated CRF group exhibited moderate hypertension, elevated basal and depressed stimulated platelet [Ca 2+ ] i , reduced urinary NOx excretion, and diminished vascular and renal eNOS and iNOS proteins. EPO therapy further raised blood pressure and increased resting and stimulated [Ca 2+ ] i but did not change NOx excretion or NOS proteins. Concurrent administration of felodipine abrogated EPO-induced hypertension, normalized resting and stimulated [Ca 2+ ] i , and increased NOx excretion and eNOS and iNOS proteins. Thus, EPO therapy leads to marked increases in blood pressure and resting and stimulated [Ca 2+ ] i . These abnormalities are ameliorated by calcium channel blockade, which restores [Ca 2+ ] i to normal and increases vascular and renal NOS expression.

59 Several recent studies have shown that certain forms of genetic and acquired hypertension (HTN) are associated with oxidative stress and respond favorably to antioxidant therapy. We hypothesize that oxidative stress, per se, may cause HTN via (among other mechanisms) enhanced oxidation and inactivation of nitric oxide (NO). To test this hypothesis, Sprague-Dawley rats were subjected to oxidative stress by glutathione (GSH) depletion using GSH synthase inhibitor, buthionine sulfoximine (BSO, 30 mmol/L in drinking water), for two weeks. The control group was given drug-free drinking water. In parallel experiments, subgroups of animals were provided vitamin E-fortified chow and vitamin C-supplemented drinking water. Arterial blood pressure, urinary excretion of NO metabolites (NO 2 +NO 3 , NOx), and liver tissue GSH were measured. In addition, plasma, kidney, heart, liver and aorta were tested by Western blot analysis for nitrotyrosine, which is the footprint of NO inactivation by reactive oxygen species (ROS). The BSO-treated group showed a three-fold fall in hepatic tissue GSH content (0.99±0.3 vs 3.4±0.3 μmol/g wet tissue, P