Browsing by Subject "Fibroblasts"

Now showing 1 - 10 of 23
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    8-aminoquinolines effective against Pneumocystis carinii in vitro and in vivo
    (American Society for Microbiology, 1999-10) Queener, Sherry F.; Bartlett, Marilyn S.; Nasr, Mohamed; Smith, James W.; Pharmacology and Toxicology, School of Medicine
    The activities of 25 8-aminoquinolines were compared in tests assessing the ability of the compounds to inhibit the growth of Pneumocystis carinii in culture. Six compounds were effective at or below 0.03 microM: CDRI 80/53, NSC19894, NSC305805, NSC305812, WR182234, and primaquine. Four others were effective at between 0.2 and 0.03 microM: NSC305835, WR225448, WR238605, and WR242511. Fourteen drugs were also tested in a standard model of P. carinii pneumonia in rats at daily doses of 2 mg/kg of body weight in drinking water. CDRI 80/53, NSC305805, NSC305835, and WR225448 were extremely effective in the animal model. The effectiveness of WR238605, WR242511, and primaquine in the rat model has been reported elsewhere (M. S. Bartlett, S. F. Queener, R. R. Tidwell, W. K. Milhouse, J. D. Berman, W. Y. Ellis, and J. W. Smith, Antimicrob. Agents Chemother. 35:277-282, 1991). The length of the alkyl chain separating the nitrogens in the substituent at position 8 of the quinoline ring was a strong determinant of anti-P. carinii activity.
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    Cancer-associated fibroblast exosomes regulate survival and proliferation of pancreatic cancer cells
    (SpringerNature, 2017-03-30) Richards, Katherine E.; Zeleniak, Ann E.; Fishel, Melissa L.; Wu, Junmin; Littlepage, Laurie E.; Hill, Reginald; Department of Pediatrics, IU School of Medicine
    Cancer associated fibroblasts (CAFs) comprise the majority of the tumor bulk of pancreatic adenocarcinomas (PDACs). Current efforts to eradicate these tumors focus predominantly on targeting the proliferation of rapidly growing cancer epithelial cells. We know that this is largely ineffective with resistance arising in most tumors following exposure to chemotherapy. Despite the long-standing recognition of the prominence of CAFs in PDAC, the effect of chemotherapy on CAFs and how they may contribute to drug resistance in neighboring cancer cells is not well characterized. Here we show that CAFs exposed to chemotherapy play an active role in regulating the survival and proliferation of cancer cells. We found that CAFs are intrinsically resistant to gemcitabine, the chemotherapeutic standard of care for PDAC. Further, CAFs exposed to gemcitabine significantly increase the release of extracellular vesicles called exosomes. These exosomes increased chemoresistance-inducing factor, Snail, in recipient epithelial cells and promote proliferation and drug resistance. Finally, treatment of gemcitabine-exposed CAFs with an inhibitor of exosome release, GW4869, significantly reduces survival in co-cultured epithelial cells, signifying an important role of CAF exosomes in chemotherapeutic drug resistance. Collectively, these findings show the potential for exosome inhibitors as treatment options alongside chemotherapy for overcoming PDAC chemoresistance.
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    Cavin‐2 promotes fibroblast‐to‐myofibroblast trans‐differentiation and aggravates cardiac fibrosis
    (Wiley, 2024) Higuchi, Yusuke; Ogata, Takehiro; Nakanishi, Naohiko; Nishi, Masahiro; Tsuji, Yumika; Tomita, Shinya; Conway, Simon J.; Matoba, Satoaki; Pediatrics, School of Medicine
    Aims: Transforming growth factor β (TGF-β) signalling is one of the critical pathways in fibroblast activation, and several drugs targeting the TGF-β/Smad signalling pathway in heart failure with cardiac fibrosis are being tested in clinical trials. Some caveolins and cavins, which are components of caveolae on the plasma membrane, are known for their association with the regulation of TGF-β signalling. Cavin-2 is particularly abundant in fibroblasts; however, the detailed association between Cavin-2 and cardiac fibrosis is still unclear. We tried to clarify the involvement and role of Cavin-2 in fibroblasts and cardiac fibrosis. Methods and results: To clarify the role of Cavin-2 in cardiac fibrosis, we performed transverse aortic constriction (TAC) operations on four types of mice: wild-type (WT), Cavin-2 null (Cavin-2 KO), Cavin-2flox/flox , and activated fibroblast-specific Cavin-2 conditional knockout (Postn-Cre/Cavin-2flox/flox , Cavin-2 cKO) mice. We collected mouse embryonic fibroblasts (MEFs) from WT and Cavin-2 KO mice and investigated the effect of Cavin-2 in fibroblast trans-differentiation into myofibroblasts and associated TGF-β signalling. Four weeks after TAC, cardiac fibrotic areas in both the Cavin-2 KO and the Cavin-2 cKO mice were significantly decreased compared with each control group (WT 8.04 ± 1.58% vs. Cavin-2 KO 0.40 ± 0.03%, P < 0.01; Cavin-2flox/flox , 7.19 ± 0.50% vs. Cavin-2 cKO 0.88 ± 0.44%, P < 0.01). Fibrosis-associated mRNA expression (Col1a1, Ctgf, and Col3) was significantly attenuated in the Cavin-2 KO mice after TAC. α1 type I collagen deposition and non-vascular αSMA-positive cells (WT 43.5 ± 2.4% vs. Cavin-2 KO 25.4 ± 3.2%, P < 0.01) were reduced in the heart of the Cavin-2 cKO mice after TAC operation. The levels of αSMA protein (0.36-fold, P < 0.05) and fibrosis-associated mRNA expression (Col1a1, 0.69-fold, P < 0.01; Ctgf, 0.27-fold, P < 0.01; Col3, 0.60-fold, P < 0.01) were decreased in the Cavin-2 KO MEFs compared with the WT MEFs. On the other hand, αSMA protein levels were higher in the Cavin-2 overexpressed MEFs compared with the control MEFs (2.40-fold, P < 0.01). TGF-β1-induced Smad2 phosphorylation was attenuated in the Cavin-2 KO MEFs compared with WT MEFs (0.60-fold, P < 0.01). Heat shock protein 90 protein levels were significantly reduced in the Cavin-2 KO MEFs compared with the WT MEFs (0.69-fold, P < 0.01). Conclusions: Cavin-2 loss suppressed fibroblast trans-differentiation into myofibroblasts through the TGF-β/Smad signalling. The loss of Cavin-2 in cardiac fibroblasts suppresses cardiac fibrosis and may maintain cardiac function.
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    Effects of platelet‐rich fibrin on human gingival and periodontal ligament fibroblast proliferation from chronic periodontitis versus periodontally healthy subjects
    (Wiley, 2021-08) Goel, Apoorv; Windsor, L. Jack; Gregory, Richard L.; Blanchard, Steven B.; Hamada, Yusuke; Periodontology, School of Dentistry
    Background: Platelet-rich fibrin (PRF), an autogenous blood concentrate, contains multiple growth factors and is used as an adjunct in the periodontal regeneration and implant site development procedures to stimulate wound healing. Patient-related factors such as chronic periodontitis may affect the quality of PRF. Objectives: This study aimed to investigate and compare PRF's effects from patients diagnosed with generalized moderate or severe chronic periodontitis to patients who presented with intact periodontium on human gingival fibroblast (HGF) and human periodontal ligament fibroblast (HPLF) proliferation. Materials and methods: A total of 33 ml of whole intravenous blood was collected from each subject and centrifuged at 2700 rpm for 12 min in three 10 ml tubes, and 3 ml of blood was used for Complete Blood Count analysis. Three PRF clots were compressed to produce the membranes and liquid exudate. PRF membrane and 10% liquid exudate were exposed to 20,000 HPLFs/well or 25,000 HGFs/well in triplets from each subject in a 48 cell well plate. After 72 h of incubation, the conditioned media were evaluated by Water Soluble Tetrazolium-1 assays to determine fibroblast proliferation. Controls included cells alone and media without cells. Complete blood counts were measured. Results: Subjects in both groups were age and gender-matched (intact 46.7 ± 11.4 years and periodontitis 54.8 ± 10.4 years, p-value = 0.1344). Body Mass Index and White Blood Corpuscles in the periodontitis group was significantly higher than the intact group (p = 0.0176 and p = 0.0038) whereas no differences were seen for Red Blood Corpuscles (p = 0.2020), Hemoglobin (p = 0.2290) and Platelets (p = 4,094). There were no significant differences in the HGF and HPLF proliferation with PRF exudates and membranes between intact periodontium and periodontitis groups (all p > 0.05). However, PRF exudates in both groups induced significant more cell proliferation when compared to PRF membranes. Conclusions: PRF exudates induced significant proliferation of fibroblasts and can play a vital role in wound healing. The current study concluded that PRF membranes, in combination with PRF exudates, can be utilized for their therapeutic and wound healing potential, not affected by the periodontal condition of the patient.
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    Fanconi anemia proteins function in mitophagy and immunity
    (Elsevier, 2016-05-05) Sumpter Jr., Rhea; Sirasanagandla, Shyam; Fernández, Álvaro F.; Wei, Yongjie; Dong, Xiaonan; Franco, Luis; Zou, Zhongju; Marchal, Christophe; Lee, Ming Yeh; Clapp, D. Wade; Hanenberg, Helmut; Levine, Beth; Department of Pediatrics, IU School of Medicine
    Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes.
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    The Fanconi anemia signaling network regulates the mitotic spindle assembly checkpoint
    (2014) Enzor, Rikki S.; Clapp, D. Wade; Broxmeyer, Hal E.; Haneline, Laura S.; Srour, Edward F.
    Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.
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    Human fibroblast and stem cell resource from the Dominantly Inherited Alzheimer Network
    (BMC, 2018-07-25) Karch, Celeste M.; Hernández, Damián Hernández; Wang, Jen-Chyong; Marsh, Jacob; Hewit, Alex W.; Hsu, Simon; Norton, Joanne; Levitch, Denise; Donahue, Tamara; Sigurdson, Wendy; Ghetti, Bernardino; Farlow, Martin; Chhatwal, Jasmeer; Berman, Sarah; Cruchaga, Carlos; Morris, John C.; Bateman, Randall J.; Dominantly Inherited Alzheimer Network (DIAN); Pébay, Alice; Goate, Alison M.; Pathology and Laboratory Medicine, School of Medicine
    BACKGROUND: Mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) cause autosomal dominant forms of Alzheimer disease (ADAD). More than 280 pathogenic mutations have been reported in APP, PSEN1, and PSEN2. However, understanding of the basic biological mechanisms that drive the disease are limited. The Dominantly Inherited Alzheimer Network (DIAN) is an international observational study of APP, PSEN1, and PSEN2 mutation carriers with the goal of determining the sequence of changes in presymptomatic mutation carriers who are destined to develop Alzheimer disease. RESULTS: We generated a library of 98 dermal fibroblast lines from 42 ADAD families enrolled in DIAN. We have reprogrammed a subset of the DIAN fibroblast lines into patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized for pluripotency markers. CONCLUSIONS: This library represents a comprehensive resource that can be used for disease modeling and the development of novel therapeutics.
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    Idiopathic gastroparesis is associated with specific transcriptional changes in the gastric muscularis externa
    (Wiley, 2018-04) Herring, B. Paul; Hoggatt, April M.; Gupta, Anita; Griffith, Sarah; Nakeeb, Attila; Choi, Jennifer N.; Idrees, Muhammad T.; Nowak, Thomas; Morris, David L.; Wo, John M.; Cellular and Integrative Physiology, School of Medicine
    BACKGROUND: The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach. METHODS: Full-thickness stomach biopsy samples were collected from non-diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. KEY RESULTS: Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA-encoding platelet-derived growth factor receptor α (PDGFRα) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRα showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder-symptom severity index scores. CONCLUSIONS AND INFERENCES: Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRα+ cells without a significant change in ICCs.
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    Nanotransfection-based vasculogenic cell reprogramming drives functional recovery in a mouse model of ischemic stroke
    (American Association for the Advancement of Science, 2021-03-19) Lemmerman, Luke R.; Balch, Maria H.H.; Moore, Jordan T.; Alzate-Correa, Diego; Rincon-Benavides, Maria A.; Salazar-Puerta, Ana; Gnyawali, Surya; Harris, Hallie N.; Lawrence, William; Ortega-Pineda, Lilibeth; Wilch, Lauren; Risser, Ian B.; Maxwell, Aidan J.; Duarte-Sanmiguel, Silvia; Dodd, Daniel; Guio-Vega, Gina P.; McTigue, Dana M.; Arnold, W. David; Nimjee, Shahid M.; Sen, Chandan K.; Khanna, Savita; Rink, Cameron; Higuita-Castro, Natalia; Gallego-Perez, Daniel; Surgery, School of Medicine
    Ischemic stroke causes vascular and neuronal tissue deficiencies that could lead to substantial functional impairment and/or death. Although progenitor-based vasculogenic cell therapies have shown promise as a potential rescue strategy following ischemic stroke, current approaches face major hurdles. Here, we used fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis, intracranially, as a potential therapy for ischemic stroke. Perfusion analyses suggest that intracranial delivery of EFF-nanotransfected fibroblasts led to a dose-dependent increase in perfusion 14 days after injection. MRI and behavioral tests revealed ~70% infarct resolution and up to ~90% motor recovery for mice treated with EFF-nanotransfected fibroblasts. Immunohistological analysis confirmed increases in vascularity and neuronal cellularity, as well as reduced glial scar formation in response to treatment with EFF-nanotransfected fibroblasts. Together, our results suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming represent a promising strategy for the treatment of ischemic stroke.