IU Indianapolis ScholarWorks :: Browsing by Author "Witzmann, Frank A."

Browsing by Author "Witzmann, Frank A."

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Bioinformatic Analysis of Differential Protein Expression in Calu-3 Cells Exposed to Carbon Nanotubes
(MDPI, 2013-10-24) Li, Pin; Lai, Xianyin; Witzmann, Frank A.; Blazer-Yost, Bonnie L.; Biology, School of Science
Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 hexposure to 10 μg/mL and 100 ng/mLof two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformaticanalysis of proteins identified in LFQMS. Interestingly, after exposure to ahigh concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins in common to both nanotubes occurred within the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The majority of the protein changes represent a decrease in amount suggesting a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure.
Cancer and Chemotherapy Contribute to Muscle Loss by Activating Common Signaling Pathways
(Frontiers, 2016) Barreto, Rafael; Mandili, Giorgia; Witzmann, Frank A.; Novelli, Francesco; Zimmers, Teresa A.; Bonetto, Andrea; Department of Surgery, IU School of Medicine
Cachexia represents one of the primary complications of colorectal cancer due to its effects on depletion of muscle and fat. Evidence suggests that chemotherapeutic regimens, such as Folfiri, contribute to cachexia-related symptoms. The purpose of the present study was to investigate the cachexia signature in different conditions associated with severe muscle wasting, namely Colon-26 (C26) and Folfiri-associated cachexia. Using a quantitative LC-MS/MS approach, we identified significant changes in 386 proteins in the quadriceps muscle of Folfiri-treated mice, and 269 proteins differentially expressed in the C26 hosts (p < 0.05; -1.5 ≥ fold change ≥ +1.5). Comparative analysis isolated 240 proteins that were modulated in common, with a large majority (218) that were down-regulated in both experimental settings. Interestingly, metabolic (47.08%) and structural (21.25%) proteins were the most represented. Pathway analysis revealed mitochondrial dysfunctions in both experimental conditions, also consistent with reduced expression of mediators of mitochondrial fusion (OPA-1, mitofusin-2), fission (DRP-1) and biogenesis (Cytochrome C, PGC-1α). Alterations of oxidative phosphorylation within the TCA cycle, fatty acid metabolism, and Ca(2+) signaling were also detected. Overall, the proteomic signature in the presence of both chemotherapy and cancer suggests the activation of mechanisms associated with movement disorders, necrosis, muscle cell death, muscle weakness and muscle damage. Conversely, this is consistent with the inhibition of pathways that regulate nucleotide and fatty acid metabolism, synthesis of ATP, muscle and heart function, as well as ROS scavenging. Interestingly, strong up-regulation of pro-inflammatory acute-phase proteins and a more coordinated modulation of mitochondrial and lipidic metabolisms were observed in the muscle of the C26 hosts that were different from the Folfiri-treated animals. In conclusion, our results suggest that both cancer and chemotherapy contribute to muscle loss by activating common signaling pathways. These data support the undertaking of combination strategies that aim to both counteract tumor growth and reduce chemotherapy side effects.
Circulating Uromodulin inhibits systemic oxidative stress by inactivating the TRPM2 channel
(American Association for the Advancement of Science, 2019-10) LaFavers, Kaice A.; Macedo, Etienne; Garimella, Pranav S.; Lima, Camila; Khan, Shehnaz; Myslinski, Jered; McClintick, Jeanette; Witzmann, Frank A.; Winfree, Seth; Phillips, Carrie; Hato, Takashi; Dagher, Pierre; Wu, Xue-Ru; El-Achkar, Tarek M.; Micanovic, Radmila; Medicine, School of Medicine
High serum concentrations of kidney-derived protein uromodulin (Tamm-Horsfall protein or THP) have recently been shown to be independently associated with low mortality in both older adults and cardiac patients, but the underlying mechanism remains unclear. Here, we show that THP inhibits the generation of reactive oxygen species (ROS) both in the kidney and systemically. Consistent with this experimental data, the concentration of circulating THP in patients with surgery-induced acute kidney injury (AKI) correlated with systemic oxidative damage. THP in the serum dropped after AKI, and was associated with an increase in systemic ROS. The increase in oxidant injury correlated with post-surgical mortality and need for dialysis. Mechanistically, THP inhibited the activation of the transient receptor potential cation channel, subfamily M, member 2 (TRPM2) channel. Furthermore, inhibition of TRPM2 in vivo in a mouse model, mitigated the systemic increase in ROS during AKI and THP deficiency. Our results suggest that THP is a key regulator of systemic oxidative stress by suppressing TRPM2 activity and our findings might help to explain how circulating THP deficiency is linked with poor outcomes and increased mortality.
Comparative Proteomics and Biological Effects of Functionalized Carbon Nanotubes in Intestinal Cell Co-culture
(Office of the Vice Chancellor for Research, 2011-04-08) Lai, Xianyin; Fang, Meixian; Fears, Sharry; Mitra, Somenath; Ringham, Heather; Witzmann, Frank A.
“Carbon nanotubes (CNTs) possess unique electrical, mechanical, and thermal properties, with potential applications in the electronics, catalysts, polymer composites, aerospace, and other industries. CNTs are also being developed for a broad range of applications in biomedicine, including oral drug delivery. Functionalized, water dispersible CNTs (fCNTS) can be expected to enter the digestive tract and exert biological effects on its barrier epithelial cells. To characterize these effects, we developed an in vitro model of the large intestinal tract using a coculture of Caco-2 (75%) and HT29-MTX (25%, mucus secreting) cells, and exposed these cells to functionalized single-walled (SWNT) and multi-walled (MWNT) carbon nanotubes at realistic concentrations (500 pg/mL and 10 µg/mL; 48 h). Protein expression was analyzed using our recently developed label-free quantitative mass spectrometry (LFQMS) platform, IdentiQuantXL™, while typical toxicological endpoint assays were used to characterize various cellular responses. LFQMS identified 5,007 unique protein database entries, from which 4,200 proteins were considered qualified for quantitation. These proteins represented 1,978 protein groups (containing isoforms, splice-variants, etc). Differences in expression were calculated by ANOVA (P<0.001) and post hoc Holm Sidak comparisons (P<0.05). fCNT significantly altered protein expression in a moderate number of proteins, the extent and type of which were fCNT specific. Only 13 proteins were universally altered by all exposures (except 500 pg/mL COOHSWNT which had no effect), and these represent a broad range of cellular functions. Bioinformatic analysis using the Gene Ontology Database and Ingenuity Pathway Analysis revealed statistically significant protein associations with a broad range of functional networks and signaling/metabolic pathways. Again, little overlap between fCNT was observed. None of the exposures was associated with overt toxicity or proinflammatory response. The results suggest that significant biological effects result from fCNT exposure, responses that are specific to CNT-type and dose, but occurring in the absence of toxicity or irritation. Supported by NIEHS RC2ES018810.”
Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations
(American Society for Biochemistry and Molecular Biology, 2016-06) Gilda, Jennifer E.; Lai, Xianyin; Witzmann, Frank A.; Gomes, Aldrin V.; Cellular and Integrative Physiology, School of Medicine
Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism.
Distinctive and pervasive alterations in aqueous humor protein composition following different types of glaucoma surgery
(Molecular Vision, 2015) Rosenfeld, Cyril; Price, Marianne O.; Lai, Xianyin; Witzmann, Frank A.; Price, Francis W.; Department of Biochemistry and Molecular Biology, IU School of Medicine
PURPOSE: To investigate whether specific glaucoma surgeries are associated with differences in aqueous humor protein concentrations compared to eyes without filters. METHODS: In this cross-sectional study, aqueous humor samples were prospectively collected from control subjects who underwent routine cataract surgery (n=14) and from patients who had different glaucoma filters: Baerveldt aqueous shunt (n=6), Ahmed aqueous shunt (n=6), trabeculectomy (n=5), and Ex-Press trabeculectomy (n=3). Total protein concentrations were determined with Bradford assay. Tryptic digests were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and were quantitatively compared with a label-free platform. Relative protein quantities were compared across groups with ANOVA. Post hoc pair-wise comparisons were adjusted for multiple comparisons. RESULTS: Compared to the control eyes, the aqueous humor protein concentration was increased approximately tenfold in the Ahmed and Baerveldt eyes and fivefold in the trabeculectomy and Ex-Press eyes. Overall, 718 unique proteins, splice variants, or isoforms were identified. No differences in the protein concentrations were detected between the Baerveldt and Ahmed groups. Likewise, the trabeculectomy and Ex-Press groups were remarkably similar. Therefore, the aqueous shunt groups were pooled, and the trabeculectomy groups were pooled for a three-way comparison with the controls. More than 500 proteins differed significantly in relative abundance (ANOVA p<0.01) among the control, aqueous shunt, and trabeculectomy groups. Functional analyses suggested these alterations in relative protein abundance affected dozens of signaling pathways. CONCLUSIONS: Different glaucoma surgical procedures were associated with marked increases in the aqueous humor protein concentration and distinctive changes in the relative abundance of numerous proteins involved in multiple signaling pathways.
Excess TGF-β mediates muscle weakness associated with bone metastases in mice
(SpringerNature, 2015-11) Waning, David L.; Mohammad, Khalid S.; Reiken, Steven; Xie, Wenjun; Andersson, Daniel C.; John, Sutha; Chiechi, Antonella; Wright, Laura E.; Umanskaya, Alisa; Niewolna, Maria; Trivedi, Trupti; Charkhzarrin, Sahba; Khatiwada, Pooja; Wronska, Anetta; Haynes, Ashley; Benassi, Maria Serena; Witzmann, Frank A.; Zhen, Gehua; Wang, Xiao; Cao, Xu; Roodman, G. David; Marks, Andrew R.; Guise, Theresa A.; Department of Medicine, IU School of Medicine
Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases-representing breast, lung and prostate cancers, as well as multiple myeloma-exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-β, released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca(2+)) release channel (RyR1). The oxidized RyR1 channels leaked Ca(2+), resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-β signaling, TGF-β release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-β activity. Thus, pathological TGF-β release from bone contributes to muscle weakness by decreasing Ca(2+)-induced muscle force production.
Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection
(American Society of Nephrology, 2018-04) Kolb, Alexander L.; Corridon, Peter R.; Zhang, Shijun; Xu, Weimin; Witzmann, Frank A.; Collett, Jason A.; Rhodes, George J.; Winfree, Seth; Bready, Devin; Pfeffenberger, Zechariah J.; Pomerantz, Jeremy M.; Hato, Takashi; Nagami, Glenn T.; Molitoris, Bruce A.; Basile, David P.; Atkinson, Simon J.; Bacallao, Robert L.; Biology, School of Science
Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identified highly upregulated proteins, including NADP+-dependent isocitrate dehydrogenase 2 (IDH2), and we confirmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney susceptibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P<0.05) observed in controls and increased the mitochondria membrane potential (P<0.05), maximal respiratory capacity (P<0.05), and intracellular ATP levels (P<0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemia-reperfusion injury and mimics ischemic preconditioning.
Functional Effects of Nanoparticle Exposure on Calu-3 Airway Epithelial Cells
(2012) Banga, Amiraj; Witzmann, Frank A.; Petrache, Horia I.; Blazer-Yost, Bonnie
High concentrations of manufactured carbon nanoparticles (CNP) are known to cause oxidative stress, inflammatory responses and granuloma formation in respiratory epithelia. To examine the effects of lower, more physiologically relevant concentrations, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in transepithelial permeability and cellular function of airway epithelia after exposure to environmentally realistic concentrations of carbon nanoparticles. Three common carbon nanoparticles, fullerenes, single- and multi-wall carbon nanotubes (SWCNT, MWCNT) were used in these experiments. Electrophysiological measurements were performed to assay transepithelial electrical resistance (TEER) and epinephrine-stimulated chloride (Cl(-)) ion secretion of epithelial cell monolayers that had been exposed to nanoparticles for three different times (1 h, 24 h and 48 h) and over a 7 log unit range of concentrations. Fullerenes did not have any effect on the TEER or stimulated ion transport. However, the carbon nanotubes (CNT) significantly decreased TEER and inhibited epinephrine-stimulated Cl(-) secretion. The changes were time dependent and at more chronic exposures caused functional effects which were evident at concentrations substantially lower than have been previously examined. The functional changes manifested in response to physiologically relevant exposures would inhibit mucociliary clearance mechanisms and compromise the barrier function of airway epithelia.
Increasing serum pre-adipocyte factor-1 (Pref-1) correlates with decreased body fat, increased free fatty acids, and level of recent alcohol consumption in excessive alcohol drinkers
(Elsevier, 2014-12) Liangpunsakul, Suthat; Bennett, Rachel; Westerhold, Chi; Ross, Ruth A.; Crabb, David W.; Lai, Xianyin; Witzmann, Frank A.; Department of Medicine, IU School of Medicine
Patients with alcoholic liver disease have been reported to have a significantly lower percentage of body fat (%BF) than controls. The mechanism for the reduction in %BF in heavy alcohol users has not been elucidated. In adipose tissue, Pref-1 is specifically expressed in pre-adipocytes but not in adipocytes. Pref-1 inhibits adipogenesis and elevated levels are associated with reduced adipose tissue mass. We investigated the association between serum Pref-1 and %BF, alcohol consumption, and serum free fatty acids (FFA) in a well-characterized cohort of heavy alcohol users compared to controls. One hundred forty-eight subjects were prospectively recruited. The Time Line Follow-Back (TLFB) questionnaire was used to quantify the amount of alcohol consumed over the 30-day period before their enrollment. Anthropometric measurements were performed to calculate %BF. Serum Pref-1 and FFA were measured. Fifty-one subjects (mean age 32 ± 9 years, 88% men) were non-excessive drinkers whereas 97 were excessive drinkers (mean age 41 ± 18 years, 69% men). Compared to non-excessive drinkers, individuals with excessive drinking had significantly higher levels of Pref-1 (p<0.01), FFA (p < 0.001), and lower %BF (p = 0.03). Serum levels of Pref-1 were associated with the amount of alcohol consumed during the previous 30 days. Serum Pref-1 was negatively correlated with %BF, but positively associated with serum FFA. Our data suggest that elevated Pref-1 levels in excessive drinkers might inhibit the expansion of adipose tissue, decreasing %BF in alcoholics. Further work is needed to validate these findings and to better understand the role of Pref-1 and its clinical significance in subjects with heavy alcohol use.

Browsing by Author "Witzmann, Frank A."

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