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Item Cytokine-induced F-actin reorganization in endothelial cells involves RhoA activation(2009-03) Campos, Silvia B; Ashworth, Sharon L; Wean, Sarah; Hosford, Melanie; Sandoval, Ruben M; Hallett, Mark A; Atkinson, Simon J; Molitoris, Bruce AAcute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1α, IL-6, and TNF-α orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P ≤ 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 ± 15%, P ≤ 0.05) and RhoA activation (up to 227 ± 26% increase, P ≤ 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.Item Endothelial colony-forming cells and pro-angiogenic cells: clarifying definitions and their potential role in mitigating acute kidney injury(Wiley, 2017) Basile, David P.; Collett, Jason A.; Yoder, Mervin C.; Department of Cellular & Integrative Physiology, IU School of MedicineAcute kidney injury (AKI) represents a significant clinical concern that is associated with high mortality rates and also represents a significant risk factor for the development of chronic kidney disease (CKD). This article will consider alterations in renal endothelial function in the setting of AKI that may underlie impairment in renal perfusion and how inefficient vascular repair may manifest post-AKI and contribute to the potential transition to CKD. We provide updated terminology for cells previously classified as ‘endothelial progenitor’ that may mediate vascular repair such as pro-angiogenic cells and endothelial colony-forming cells. We consider how endothelial repair may be mediated by these different cell types following vascular injury, particularly in models of AKI. We further summarize the potential ability of these different cells to mitigate the severity of AKI, improve perfusion and maintain vascular structure in pre-clinical studies.Item Hydrogen Sulfide: A Potential Novel Therapy for the Treatment of Ischemia(Wolters Kluwer, 2017-11) Jensen, Amanda R.; Drucker, Natalie A.; Khaneki, Sina; Surgery, School of MedicineHydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental importance in cellular function as a regulator of apoptosis, inflammation, and perfusion. Mechanisms of endogenous H2S signaling are poorly understood; however, signal transmission is thought to occur via persulfidation at reactive cysteine residues on proteins. Although much has been discovered about how H2S is synthesized in the body, less is known about how it is metabolized. Recent studies have discovered a multitude of different targets for H2S therapy, including those related to protein modification, intracellular signaling, and ion channel depolarization. The most difficult part of studying hydrogen sulfide has been finding a way to accurately and reproducibly measure it. The purpose of this review is to: elaborate on the biosynthesis and catabolism of H2S in the human body, review current knowledge of the mechanisms of action of this gas in relation to ischemic injury, define strategies for physiological measurement of H2S in biological systems, and review potential novel therapies that use H2S for treatment.Item Incidence of seizures following initial ischemic stroke in a community-based cohort: The Framingham Heart Study(Elsevier, 2017-04) Stefanidou, Maria; Das, Rohit R.; Beiser, Alexa S.; Sundar, Banu; Kelly-Hayes, Margaret; Kase, Carlos S.; Devinsky, Orrin; Seshadri, Sudha; Freidman, Daniel; Department of Neurology, IU School of MedicinePurpose We examined the incidence of seizures following ischemic stroke in a community-based sample. Methods All subjects with incident ischemic strokes in the Framingham Original and Offspring cohorts between 1982 and 2003 were identified and followed for up to 20 years to determine incidence of seizures. Seizure-type was based on the 2010 International League Against Epilepsy (ILAE) classification. Disability was stratified into mild/none, moderate and severe, based on post-stroke neurological deficit documentation according to the Framingham Heart Study (FHS) protocol and functional status was determined using the Barthel Index. Results An initial ischemic stroke occurred in 469 subjects in the cohort and seizures occurred in 25 (5.3%) of these subjects. Seizure incidence was similar in both large artery atherosclerosis (LAA) (6.8%) and cardio-embolic (CE) (6.2%) strokes. No seizures occurred following lacunar strokes. The predominant seizure type was focal seizure with or without evolution to bilateral convulsive seizure. One third of participants had seizures within the first 24 h from stroke onset and half of all seizures occurred within the first 30 days. On multivariate analysis, moderate and severe disability following stroke was associated with increased risk of incident seizure. Conclusions Seizures occurred in approximately 5% of subjects after an ischemic stroke. One third of these seizures occurred in the first 24 h after stroke and none followed lacunar strokes. Focal seizures with or without evolution in bilateral convulsive seizures were the most common seizure type. Moderate and severe disability was predictive of incident seizures.Item Neuroprotective Mechanisms of Lycium barbarum Polysaccharides Against Ischemic Insults by Regulating NR2B and NR2A Containing NMDA Receptor Signaling Pathways(Frontiers, 2017-09-27) Shi, Zhongshan; Zhu, Lihui; Li, Tingting; Tang, Xiaoya; Xiang, Yonghui; Han, Xinjia; Xia, Luoxing; Zeng, Ling; Nie, Junhua; Huang, Yongxia; Tsang, Chi Kwan; Wang, Ying; Lei, Zhigang; Xu, Zaocheng; So, Kwok-fai; Ruan, Yiwen; Anatomy and Cell Biology, School of MedicineGlutamate excitotoxicity plays an important role in neuronal death after ischemia. However, all clinical trials using glutamate receptor inhibitors have failed. This may be related to the evidence that activation of different subunit of NMDA receptor will induce different effects. Many studies have shown that activation of the intrasynaptic NR2A subunit will stimulate survival signaling pathways, whereas upregulation of extrasynaptic NR2B will trigger apoptotic pathways. A Lycium barbarum polysaccharide (LBP) is a mixed compound extracted from Lycium barbarum fruit. Recent studies have shown that LBP protects neurons against ischemic injury by anti-oxidative effects. Here we first reported that the effect of LBP against ischemic injury can be achieved by regulating NR2B and NR2A signaling pathways. By in vivo study, we found LBP substantially reduced CA1 neurons from death after transient global ischemia and ameliorated memory deficit in ischemic rats. By in vitro study, we further confirmed that LBP increased the viability of primary cultured cortical neurons when exposed to oxygen-glucose deprivation (OGD) for 4 h. Importantly, we found that LBP antagonized increase in expression of major proteins in the NR2B signal pathway including NR2B, nNOS, Bcl-2-associated death promoter (BAD), cytochrome C (cytC) and cleaved caspase-3, and also reduced ROS level, calcium influx and mitochondrial permeability after 4 h OGD. In addition, LBP prevented the downregulation in the expression of NR2A, pAkt and pCREB, which are important cell survival pathway components. Furthermore, LBP attenuated the effects of a NR2B co-agonist and NR2A inhibitor on cell mortality under OGD conditions. Taken together, our results demonstrated that LBP is neuroprotective against ischemic injury by its dual roles in activation of NR2A and inhibition of NR2B signaling pathways, which suggests that LBP may be a superior therapeutic candidate for targeting glutamate excitotoxicity for the treatment of ischemic stroke.Item Neutrophil Diversity in the Pathogenesis of Ischemic Acute Kidney Injury(2020-09) Winfree, Seth; El-Achkar, Tarek M.; Dagher, Pierre C.; Day, Richard N.; Williams, James C., Jr.Acute kidney injury (AKI) affects millions of patients worldwide yet has few treatment options. There is a critical need to identify novel interventions for AKI, especially approaches targeting cell types that are central to the disease, such as neutrophils. Neutrophils are professional phagocytic cells that respond early to tissue injury. In rodent models of severe ischemic-reperfusion-injury AKI, neutrophils transiently infiltrate the injured kidney, appearing within 6 hours, and are gone by 72 hours. These infiltrating neutrophils are considered proinflammatory and harmful to tissue repair and recovery of kidney function. However, neutrophils can exhibit atypical activity such as antigen presentation and have a central role in recovery from myocardial ischemic injury. Furthermore, little is known of neutrophil polarization, atypical activity, or neutrophil diversity in AKI. Lastly, the kidney generated and renal-protective immunomodulatory protein uromodulin (Tamm-Horsfall Protein, THP) regulates granulopoiesis. In the absence of uromodulin, there is a systemic increase in neutrophils and mouse kidneys are sensitive to injury in AKI. To elucidate neutrophil diversity in AKI and their sensitivity to uromodulin, I performed a series of single-cell sequencing experiments to generate transcriptional profiles of neutrophils from the blood and kidneys of wild-type and THPknockout mice after renal ischemic-reperfusion-injury (IRI). Neutrophil diversity was detected following IRI of the mouse kidney in the blood and kidney. The distribution of subpopulations was sensitive to the kidney milieu. Within the kidney, this diversity and the transcriptional programs of neutrophil subpopulations was sensitive to the severity of ischemic injury. Lastly, Cxcl3 was uniquely upregulated in specific neutrophils after severe ischemic injury. Using single-cell sequencing of uromodulin knock-out mice, I detected the upregulation of toll-like receptor pathways and complement cascades across neutrophil subpopulations in a THP sensitive manner. Furthermore, CXCR2 ligand expression was a combination of moderate and severe injury in wild-type mice. This confirmed previously reported cytokine dysregulation in the uromodulin knock-out mouse after IRI and uncovers a novel role for Cxcl3. Thus, upon revisiting the well-studied neutrophil, I have uncovered novel neutrophil diversity that correlates with recovery of kidney function in AKI and suggests new roles for an old player.Item Obesity Alters Molecular and Functional Cardiac Responses to Ischemia-Reperfusion and Glucagon-Like Peptide-1 Receptor Agonism(Springer, 2016-07) Sassoon, Daniel J.; Goodwill, Adam G.; Noblet, Jillian N.; Conteh, Abass M.; Herring, B. Paul; McClintick, Jeanette N.; Tune, Johnathan D.; Mather, Kieren J.; Department of Cellular & Integrative Physiology, Indiana University School of MedicineThis study tested the hypothesis that obesity alters the cardiac response to ischemia/reperfusion and/or glucagon like peptide-1 (GLP-1) receptor activation, and that these differences are associated with alterations in the obese cardiac proteome and microRNA (miRNA) transcriptome. Ossabaw swine were fed normal chow or obesogenic diet for 6 months. Cardiac function was assessed at baseline, during a 30-minutes coronary occlusion, and during 2 hours of reperfusion in anesthetized swine treated with saline or exendin-4 for 24 hours. Cardiac biopsies were obtained from normal and ischemia/reperfusion territories. Fat-fed animals were heavier, and exhibited hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. Plasma troponin-I concentration (index of myocardial injury) was increased following ischemia/reperfusion and decreased by exendin-4 treatment in both groups. Ischemia/reperfusion produced reductions in systolic pressure and stroke volume in lean swine. These indices were higher in obese hearts at baseline and relatively maintained throughout ischemia/reperfusion. Exendin-4 administration increased systolic pressure in lean swine but did not affect the blood pressure in obese swine. End-diastolic volume was reduced by exendin-4 following ischemia/reperfusion in obese swine. These divergent physiologic responses were associated with obesity-related differences in proteins related to myocardial structure/function (e.g. titin) and calcium handling (e.g. SERCA2a, histidine-rich Ca2+ binding protein). Alterations in expression of cardiac miRs in obese hearts included miR-15, miR-27, miR-130, miR-181, and let-7. Taken together, these observations validate this discovery approach and reveal novel associations that suggest previously undiscovered mechanisms contributing to the effects of obesity on the heart and contributing to the actions of GLP-1 following ischemia/reperfusion.Item Renal C3 complement component: feed forward to diabetic kidney disease(Karger, 2015) Kelly, Katherine J.; Liu, Yunlong; Zhang, Jizhong; Dominguez, Jesus H.; Department of Medicine, IU School of MedicineBACKGROUND: Diabetic nephropathy is the main cause of end-stage renal disease and has reached epidemic proportions. METHODS: Comprehensive genomic profiling (RNAseq) was employed in the ZS (F1 hybrids of Zucker and spontaneously hypertensive heart failure) model of diabetic nephropathy. Controls were lean littermates. RESULTS: Diabetic nephropathy in obese, diabetic ZS was accelerated by a single episode of renal ischemia (DI). This rapid renal decline was accompanied by the activation of the renal complement system in DI, and to a lesser extent in sham-operated diabetic rats (DS). In DI there were significant increases in renal mRNA encoding C3, C4, C5, C6, C8, and C9 over sham-operated lean normal controls (LS). Moreover, mRNAs encoding the receptors for the anaphylatoxins C3a and C5a were also significantly increased in DI compared to LS. The classic complement pathway was activated in diabetic kidneys with significant increases of C1qa, C1qb, and C1qc mRNAs in DI over LS. In addition, critical regulators of complement activation were significantly attenuated in DI and DS. These included mRNAs encoding CD55, decay accelerating factor, and CD59, which inhibit the membrane attack complex. C3, C4, and C9 proteins were demonstrated in renal tubules and glomeruli. The complement RNAseq data were incorporated into a gene network showing interactions among C3-generating renal tubular cells and other immune competent migratory cells. CONCLUSIONS: We conclude that local activation of the complement system mediates renal injury in diabetic nephropathy.Item Renal iron overload in rats with diabetic nephropathy.(APS, 2015-12) Dominguez, Jesus H.; Liu, Yunlong; Kelly, Katherine J.; Department of Medicine, IU School of MedicineDiabetic nephropathy (DN) remains incurable and is the main cause of end‐stage renal disease. We approached the pathophysiology of DN with systems biology, and a comprehensive profile of renal transcripts was obtained with RNA‐Seq in ZS (F1 hybrids of Zucker and spontaneously hypertensive heart failure) rats, a model of diabetic nephropathy. We included sham‐operated lean control rats (LS), sham‐operated diabetic (DS), and diabetic rats with induced renal ischemia (DI). Diabetic nephropathy in DI was accelerated by the single episode of renal ischemia. This progressive renal decline was associated with renal iron accumulation, although serum and urinary iron levels were far lower in DI than in LS. Furthermore, obese/diabetic ZS rats have severe dyslipidemia, a condition that has been linked to hepatic iron overload. Hence, we tested and found that the fatty acids oleic acid and palmitate stimulated iron accumulation in renal tubular cells in vitro. Renal mRNAs encoding several key proteins that promote iron accumulation were increased in DI. Moreover, renal mRNAs encoding the antioxidant proteins superoxide dismutase, catalase, and most of the glutathione synthetic system were suppressed, which would magnify the prooxidant effects of renal iron loads. Substantial renal iron loads occur in obese/diabetic rats. We propose that in diabetes, specific renal gene activation is partly responsible for iron accumulation. This state might be further aggravated by lipid‐stimulated iron uptake. We suggest that progressive renal iron overload may further advance renal injury in obese/diabetic ZS rats.