Publications by authors named "Ian A Darby"

30 Publications

  • Page 1 of 1

Protection against reperfusion injury by 3',4'-dihydroxyflavonol in rat isolated hearts involves inhibition of phospholamban and JNK2.

Int J Cardiol 2018 03 28;254:265-271. Epub 2018 Jan 28.

School of Health and Biomedical Sciences, RMIT University, Australia. Electronic address:

Background: Flavonols, including 3',4'-dihydroxyflavonol (DiOHF), reduce myocardial ischemia and reperfusion (I/R) injury but their mechanism remains uncertain. To better understand the mechanism of the cardioprotective actions of flavonols we investigated the effect of DiOHF on cardiac function and the activation of protective and injurious signalling kinases after I/R in rat isolated hearts.

Methods: We assessed the effect of global ischemia (20min) and reperfusion (5-30min) on cardiac function and injury in rat isolated, perfused hearts in the absence or presence of DiOHF (10μM) during reperfusion. Western blotting was used to assess changes in the phosphorylation state of kinases known to be involved in injury or protection.

Results: DiOHF improved cardiac contractility and reduced perfusion pressure and cell death in the isolated hearts. Phosphorylation of p38MAPK and CaMKII increased during ischemia with no further increase during reperfusion. Phosphorylation of other kinases increased during reperfusion. Phosphorylation of phospholamban (PLN) peaked at 5min of reperfusion whereas phosphorylation of Akt, Erk, STAT3 and JNK2 was highest after 30min. The presence of DiOHF during reperfusion significantly inhibited the activation of PLN and JNK without affecting phosphorylation of the protective kinases Erk1/2 and STAT3. Experiments in vitro demonstrated that DiOHF inhibited CaMKII by competing with ATP but not Ca/calmodulin.

Conclusions: It is proposed that DiOHF confers protection against myocardial reperfusion injury by inhibiting CaMKII and subsequent PLN-induced leak of Ca from the sarcoplasmic reticulum as well as by inhibiting JNK2 activation to reduce apoptosis.
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http://dx.doi.org/10.1016/j.ijcard.2017.11.101DOI Listing
March 2018

Hypoxia in tissue repair and fibrosis.

Cell Tissue Res 2016 09 16;365(3):553-62. Epub 2016 Jul 16.

Department of Nephrology, Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia.

Hypoxia and hypoxia signalling through the transcription factor hypoxia inducible factor-1 (HIF-1), play an important role in normal tissue repair processes. Tissue injury generally produces at least the transient loss of normal vascular perfusion and the resulting hypoxia induces the expression of many genes that allow the tissue to adapt to hypoxia, to start the repair process and, in time, to re-establish oxygen delivery to the tissue. In most cases, transient hypoxia and the activation of the HIF-1 pathway are beneficial and promote the repair process, producing tissue that might not perfectly reform its original architecture but that has its function substantially restored. However, in some cases of chronic injury, chronic hypoxia and pathological repair, the hypoxia pathway might be responsible for driving the process of fibrosis and can lead to excessive scarring and compromised organ function.
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http://dx.doi.org/10.1007/s00441-016-2461-3DOI Listing
September 2016

The myofibroblast, a key cell in normal and pathological tissue repair.

Cell Mol Life Sci 2016 Mar 17;73(6):1145-57. Epub 2015 Dec 17.

Department of Physiology, Faculty of Pharmacy, University of Limoges, 2 rue du Dr. Marcland, 87025, Limoges Cedex, France.

Myofibroblasts are characterized by their expression of α-smooth muscle actin, their enhanced contractility when compared to normal fibroblasts and their increased synthetic activity of extracellular matrix proteins. Myofibroblasts play an important role in normal tissue repair processes, particularly in the skin where they were first described. During normal tissue repair, they appear transiently and are then lost via apoptosis. However, the chronic presence and continued activity of myofibroblasts characterize many fibrotic pathologies, in the skin and internal organs including the liver, kidney and lung. More recently, it has become clear that myofibroblasts also play a role in many types of cancer as stromal or cancer-associated myofibroblast. The fact that myofibroblasts are now known to be key players in many pathologies makes understanding their functions, origin and the regulation of their differentiation important to enable them to be regulated in normal physiology and targeted in fibrosis, scarring and cancer.
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http://dx.doi.org/10.1007/s00018-015-2110-0DOI Listing
March 2016

Cytoskeletal protein Flightless I inhibits apoptosis, enhances tumor cell invasion and promotes cutaneous squamous cell carcinoma progression.

Oncotarget 2015 Nov;6(34):36426-40

Regenerative Medicine, Future Industries Institute, University of South Australia, Adelaide, South Australia, Australia.

Flightless I (Flii) is an actin remodeling protein that affects cellular processes including adhesion, proliferation and migration. In order to determine the role of Flii during carcinogenesis, squamous cell carcinomas (SCCs) were induced in Flii heterozygous (Flii+/-), wild-type and Flii overexpressing (FliiTg/Tg) mice by intradermal injection of 3-methylcholanthrene (MCA). Flii levels were further assessed in biopsies from human SCCs and the human SCC cell line (MET-1) was used to determine the effect of Flii on cellular invasion. Flii was highly expressed in human SCC biopsies particularly by the invading cells at the tumor edge. FliiTg/Tg mice developed large, aggressive SCCs in response to MCA. In contrast Flii+/- mice had significantly smaller tumors that were less invasive. Intradermal injection of Flii neutralizing antibodies during SCC initiation and progression significantly reduced the size of the tumors and, in vitro, decreased cellular sphere formation and invasion. Analysis of the tumors from the Flii overexpressing mice showed reduced caspase I and annexin V expression suggesting Flii may negatively regulate apoptosis within these tumors. These studies therefore suggest that Flii enhances SCC tumor progression by decreasing apoptosis and enhancing tumor cell invasion. Targeting Flii may be a potential strategy for reducing the severity of SCCs.
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http://dx.doi.org/10.18632/oncotarget.5536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742187PMC
November 2015

Quantitative proteomics of bronchoalveolar lavage fluid in lung adenocarcinoma.

Cancer Genomics Proteomics 2015 Jan-Feb;12(1):39-48

Cancer and Tissue Repair Laboratory, School of Medical Sciences, RMIT University, Bundoora, VIC, Australia.

Background: The most commonly reported primary lung cancer subtype is adenocarcinoma, which is associated with a poor prognosis and short survival. Proteomic studies on human body fluids such as bronchoalveolar lavage fluid (BALF) have become essential methods for biomarker discovery, examination of tumor pathways and investigation of potential treatments.

Aim: This study used quantitative proteomics to investigate the up-regulation of novel proteins in BALF from patients with primary lung adenocarcinoma in order to identify potential biomarkers.

Materials And Methods: BALF samples from individuals with and without primary lung adenocarcinoma were analyzed using liquid chromatography-mass spectrometry.

Results: One thousand and one hundred proteins were identified, 33 of which were found to be consistently overexpressed in all lung adenocarcinoma samples compared to non-cancer controls. A number of overexpressed proteins have been previously shown to be related to lung cancer progression including S100-A8, annexin A1, annexin A2, thymidine phosphorylase and transglutaminase 2.

Conclusion: The overexpression of a number of specific proteins in BALF from patients with primary lung adenocarcinoma may be used as a potential biomarker for lung adenocarcinoma.
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September 2015

Blood classical monocytes phenotype is not altered in primary non-small cell lung cancer.

World J Clin Oncol 2014 Dec;5(5):1078-87

Saleh A Almatroodi, Applied Medical Sciences College, Qassim University, Buraidah, Saudi Arabia.

Aim: To evaluate the M1 and M2 monocyte phenotype in patients with non-small cell lung cancer (NSCLC) compared to controls. Also, to examine the expression of Th1 and Th2 cytokines in plasma of NSCLC vs controls.

Methods: Freshly prepared peripheral blood mononuclear cells samples were obtained from patients with NSCLC (lung adenocarcinoma and squamous cell lung carcinoma) and from non-cancer controls. Flow cytometry was performed to investigate M1 and M2 phenotypes in peripheral monocytes (classical monocytes CD14+, CD45+ and CD16-) using conventional surface markers. Th1 and Th2 cytokine production was also analysed in the plasma using cytometric bead array technique.

Results: There were no significant difference in expression of M1 (HLA-DR) and/or M2 markers (CD163 and CD36) markers on classical monocytes in patients with NSCLC compared to non-cancer controls. Expression of CD11b, CD11c, CD71 and CD44 was also shown to be similar in patients with NSCLC compared to non-cancer controls. Th1 and Th2 cytokines [interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12 (p70), tumor necrosis factor (TNF)-α, TNF-β, and interferon-γ] analysis revealed no significant difference between patients with NSCLC and non-cancer controls.

Conclusion: This study shows no alteration in peripheral monocyte phenotype in circulating classical monocytes in patients with NSCLC compared to non-cancer controls. No difference in Th1 and Th2 cytokine levels were noted in the plasma of these patients.
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http://dx.doi.org/10.5306/wjco.v5.i5.1078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259935PMC
December 2014

Fibroblasts and myofibroblasts in wound healing.

Clin Cosmet Investig Dermatol 2014 6;7:301-11. Epub 2014 Nov 6.

Department of Physiology and EA 6309, FR 3503, Faculties of Medicine and Pharmacy, University of Limoges, Limoges, France.

(Myo)fibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myo)fibroblasts are embedded in a sophisticated extracellular matrix (ECM) that they secrete, and a complex and interactive dialogue exists between (myo)fibroblasts and their microenvironment. In addition to the secretion of the ECM, (myo)fibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myo)fibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myo)fibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis), or during aging, this dialogue between the (myo)fibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myo)fibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment.
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http://dx.doi.org/10.2147/CCID.S50046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226391PMC
November 2014

Temporal variation in the deposition of different types of collagen within a porous biomaterial implant.

J Biomed Mater Res A 2014 Oct 16;102(10):3550-5. Epub 2013 Nov 16.

CSIRO Materials Science and Engineering, Bayview Avenue, Clayton, Victoria, 3169, Australia.

The deposition of new collagen in association with a medical implant has been studied using expanded polytetrafluoroethylene vascular replacement samples implanted subcutaneously in sheep, for up to 28 days. New type I collagen mRNA synthesis was followed by in situ hybridization, while the accumulation of new collagen types III, V, VI, XII, and XIV was followed by immunohistochemistry. All the collagen detected in the pores of the implant were newly deposited at various times after implantation and were not due to any pre-existing dermal collagen that may have been present around the implant. Collagen deposition was seen initially surrounding the implant and, with time, was seen to infiltrate within its pores. In situ hybridization showed that the majority of infiltrating cells had switched on mRNA that coded for type I collagen production. Histology showed that cellular infiltration increased with time, accompanied by increasing collagen deposition. The deposition of different collagen types happened at different rates. The type V and VI collagens preceded the major interstitial collagens in the newly deposited tissue, although at longer time points, detection of type V collagen appeared to decrease. After disruption of the interstitial collagens with enzyme, the "masked" type V collagen was clearly still visible by immunohistochemistry. Little type XII collagen could be seen within the porous mesh, although it was seen in the surrounding tissues. By contrast, type XIV was seen throughout the porous structure of the implanted mesh, with less being visible outside the material where type XII was more abundant.
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http://dx.doi.org/10.1002/jbm.a.35027DOI Listing
October 2014

Siah2-deficient mice show impaired skin wound repair.

Wound Repair Regen 2013 May-Jun;21(3):437-47. Epub 2013 Apr 29.

Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora.

Hypoxia is associated with the dermal wound healing process and hypoxia signaling is presumed to be crucial for normal wound repair. The Siah2 ubiquitin ligase controls the abundance of hypoxia-inducible factor-1 alpha, and loss of Siah2 results in destabilization of hypoxia-inducible factor-1 alpha under hypoxia. Utilizing Siah2(-/-) mice we demonstrate that cutaneous wound healing is impaired in these mice. Wounds in Siah2(-/-) mice heal slower and are associated with delayed induction of myofibroblast infiltration and reduced collagen deposition. This coincides with delayed angiogenesis and reduced macrophage infiltration into the wounds of Siah2(-/-) mice. We furthermore demonstrate that primary Siah2(-/-) dermal fibroblasts have reduced migratory capacities and produce less collagen than wild-type fibroblasts. Additionally, Siah2(-/-) fibroblasts showed conserved responses to transforming growth factor-β at the receptor level (pSmad 2C activation) but reduced responses downstream. Together, our data show, for the first time, that Siah2 is involved as a positive regulator in the wound healing response. Understanding the role of hypoxia signaling in tissue repair and fibrosis and interference with the hypoxia signaling pathway via regulation of Siah2 may provide new targets for clinical regulation of fibrosis and scarring.
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http://dx.doi.org/10.1111/wrr.12045DOI Listing
December 2013

The myofibroblast, multiple origins for major roles in normal and pathological tissue repair.

Fibrogenesis Tissue Repair 2012 6;5(Suppl 1):S5. Epub 2012 Jun 6.

Facultés de Médecine et de Pharmacie, Université de Limoges, EA 6309 "Maintenance Myélinique et Neuropathies Périphériques", FR 3503, Limoges F-87025, France.

Myofibroblasts differentiate, invade and repair injured tissues by secreting and organizing the extracellular matrix and by developing contractile forces. When tissues are damaged, tissue homeostasis must be re-established, and repair mechanisms have to rapidly provide harmonious mechanical tissue organization, a process essentially supported by (myo)fibroblasts. Under physiological conditions, the secretory and contractile activities of myofibroblasts are terminated when the repair is complete (scar formation) but the functionality of the tissue is only rarely perfectly restored. At the end of the normal repair process, myofibroblasts disappear by apoptosis but in pathological situations, myofibroblasts likely remain leading to excessive scarring. Myofibroblasts originate from different precursor cells, the major contribution being from local recruitment of connective tissue fibroblasts. However, local mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and cells derived from an epithelial-mesenchymal transition process, may represent alternative sources of myofibroblasts when local fibroblasts are not able to satisfy the requirement for these cells during repair. These diverse cell types probably contribute to the appearance of myofibroblast subpopulations which show specific biological properties and which are important to understand in order to develop new therapeutic strategies for treatment of fibrotic and scarring diseases.
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http://dx.doi.org/10.1186/1755-1536-5-S1-S5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368789PMC
May 2015

Hypoxia and hypoxia signaling in tissue repair and fibrosis.

Int Rev Cell Mol Biol 2012 ;296:139-85

Department of Surgery, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria, Australia.

Following injury, vascular damage results in the loss of perfusion and consequent low oxygen tension (hypoxia) which may be exacerbated by a rapid influx of inflammatory and mesenchymal cells with high metabolic demands for oxygen. Changes in systemic and cellular oxygen concentrations induce tightly regulated response pathways that attempt to restore oxygen supply to cells and modulate cell function in hypoxic conditions. Most of these responses occur through the induction of the transcription factor hypoxia-inducible factor-1 (HIF-1) which regulates many processes needed for tissue repair during ischemia in the damaged tissue. HIF-1 transcriptionally upregulates expression of metabolic proteins (GLUT-1), adhesion proteins (integrins), soluble growth factors (TGF-β and VEGF), and extracellular matrix components (type I collagen and fibronectin), which enhance the repair process. For these reasons, HIF-1 is viewed as a positive regulator of wound healing and a potential regulator of organ repair and tissue fibrosis. Understanding the complex role of hypoxia in the loss of function in scarring tissues and biology of chronic wound, and organ repair will aid in the development of pharmaceutical agents that can redress the detrimental outcomes often seen in repair and scarring.
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http://dx.doi.org/10.1016/B978-0-12-394307-1.00003-5DOI Listing
October 2012

Explanting is an ex vivo model of renal epithelial-mesenchymal transition.

J Biomed Biotechnol 2011 24;2011:212819. Epub 2011 Nov 24.

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, VIC 3050, Australia.

Recognised by their de novo expression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts is key to the pathogenesis of fibrosis in chronic kidney disease. Increasingly, we realise that epithelial-mesenchymal transition (EMT) may be an important source of these cells. In this study we describe a novel model of renal EMT. Rat kidney explants were finely diced on gelatin-coated Petri dishes and cultured in serum-supplemented media. Morphology and immunocytochemistry were used to identify mesenchymal (vimentin+, α-smooth muscle actin (SMA)+, desmin+), epithelial (cytokeratin+), and endothelial (RECA+) cells at various time points. Cell outgrowths were all epithelial in origin (cytokeratin+) at day 3. By day 10, 50 ± 12% (mean ± SE) of cytokeratin+ cells double-labelled for SMA, indicating EMT. Lectin staining established a proximal tubule origin. By day 17, cultures consisted only of myofibroblasts (SMA+/cytokeratin-). Explanting is a reproducible ex vivo model of EMT. The ability to modify this change in phenotype provides a useful tool to study the regulation and mechanisms of renal tubulointerstitial fibrosis.
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http://dx.doi.org/10.1155/2011/212819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227440PMC
April 2012

Cardioprotection from ischaemia-reperfusion injury by a novel flavonol that reduces activation of p38 MAPK.

Eur J Pharmacol 2011 May 1;658(2-3):160-7. Epub 2011 Mar 1.

Howard Florey Institute, University of Melbourne, Victoria, Australia.

Oxidative stress, activation of intracellular protein kinases and cardiomyocyte apoptosis are known mediators of cardiac ischaemia/reperfusion injury. The sites at which NP202, a novel water soluble pro-drug of 3',4'-dihydroxyflavonol (DiOHF), acts in this cascade to cause cardioprotection are unknown. In this study we examined the ability of NP202 to reduce infarct size after a prolonged period of ischaemia and reperfusion. In addition, we tested whether NP202 inhibits pro-apoptotic signalling, apoptosis and inflammation following myocardial ischaemia and reperfusion. Sheep were anaesthetised, the heart exposed and the 2nd branch of the left anterior descending coronary artery isolated. The artery was occluded for 3h and, five minutes before 3h of reperfusion was commenced, sheep were treated with intravenous vehicle or NP202. At the end of reperfusion infarct size was measured and normal left ventricle, non-infarcted area-at-risk and infarcted myocardium were collected to identify polymorphonuclear leukocytes (PMN) or apoptotic cells (TUNEL-positive), or assessed for activation of mitogen-activated protein kinase (MAPK) pathways by Western blot analysis. Compared with vehicle treatment, NP202 reduced infarct size (-20 ± 4%, P<0.05) and decreased the number of PMNs and TUNEL-positive cells in the area-at-risk (-35 ± 16% and -52 ± 19%, respectively) and infarcted tissue (-57 ± 9 and -81 ± 5%, respectively, P<0.05). Furthermore, NP202 significantly reduced I/R-induced elevated p38 MAPK phosphorylation (by 67 ± 4%, P<0.05) in the area-at-risk zone. In conclusion, the novel aqueous flavonol NP202 provided significant cardioprotection from clinically relevant prolonged myocardial ischaemia when administered just before reperfusion. Efficacy of NP202 was also associated with reduced p38 MAPK activation, inflammation and apoptotic cell death.
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http://dx.doi.org/10.1016/j.ejphar.2011.02.041DOI Listing
May 2011

Proteomic analysis of differentially expressed proteins in peripheral cholangiocarcinoma.

Cancer Microenviron 2010 Jun 26;4(1):73-91. Epub 2010 Jun 26.

Cholangiocarcinoma is an adenocarcinoma of the liver which has increased in incidence over the last thirty years to reach similar levels to other liver cancers. Diagnosis of this disease is usually late and prognosis is poor, therefore it is of great importance to identify novel candidate markers and potential early indicators of this disease as well as molecules that may be potential therapeutic targets. We have used a proteomic approach to identify differentially expressed proteins in peripheral cholangiocarcinoma cases and compared expression with paired non-tumoral liver tissue from the same patients. Two-dimensional fluorescence difference gel electrophoresis after labeling of the proteins with cyanines 3 and 5 was used to identify differentially expressed proteins. Overall, of the approximately 2,400 protein spots visualised in each gel, 172 protein spots showed significant differences in expression level between tumoral and non-tumoral tissue with p < 0.01. Of these, 100 spots corresponding to 138 different proteins were identified by mass spectrometry: 70 proteins were over-expressed whereas 68 proteins were under-expressed in tumoral samples compared to non-tumoral samples. Among the over-expressed proteins, immunohistochemistry studies confirmed an increased expression of 14-3-3 protein in tumoral cells while α-smooth muscle actin and periostin were shown to be overexpressed in the stromal myofibroblasts surrounding tumoral cells. α-Smooth muscle actin is a marker of myofibroblast differentiation and has been found to be a prognostic indicator in colon cancer while periostin may also have a role in cell adhesion, proliferation and migration and has been identified in other cancers. This underlines the role of stromal components in cancer progression and their interest for developing new diagnostic or therapeutic tools.
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http://dx.doi.org/10.1007/s12307-010-0047-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3047624PMC
June 2010

In situ localization of apoptosis using TUNEL.

Methods Mol Biol 2010 ;611:161-70

Department of Nephrology, The Royal Melbourne Hospital, Melbourne, VIC, Australia.

Apoptosis is an important process both in normal biology and in various pathologies and disease states. Apoptosis in tissue or cells can be detected in a number of ways. In tissue sections, electron microscopy can identify apoptosis by cellular and nuclear morphology, and in live cells, changes in the membrane and membrane permeability allow apoptosis and necrosis to be observed. Histologically, apoptosis is best detected using the partial DNA degradation that is present in apoptotic cell nuclei. Terminal transferase-mediated UTP nick end-labeling (TUNEL) has been used successfully for detection of DNA degradation in paraffin-embedded tissue sections and can be combined with immunohistochemistry if desired to allow more precise identification of apoptotic cells.
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http://dx.doi.org/10.1007/978-1-60327-345-9_13DOI Listing
February 2010

Using in situ hybridisation to localize renal gene expression in tissue sections.

Methods Mol Biol 2009 ;466:119-132

Department of Nephrology, School of Medical Sciences, RMIT University, Bundoora, VIC, Australia.

The basics of in situ hybridization have been widely applied to a diverse range of situations where we need to localize the distribution of nucleic acids. Advances in other molecular techniques such as the advent of gene microarrays has not diminished the significance of in situ hybridization, but rather highlight the importance of being able to identify the topology of gene expression. In situ hybridization offers a degree of precision that is unavailable with other molecular techniques. This chapter outlines techniques used to examine the spatial distribution of gene expression in the kidney using complementary RNA (cRNA) probes with both radioactive and non-radioactive labels.
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http://dx.doi.org/10.1007/978-1-59745-352-3_9DOI Listing
February 2009

Fibroblast differentiation in wound healing and fibrosis.

Int Rev Cytol 2007 ;257:143-79

School of Medical Sciences, RMIT University, Melbourne, Australia.

The contraction of granulation tissue from skin wounds was first described in the 1960s. Later it was discovered that during tissue repair, fibroblasts undergo a change in phenotype from their normal relatively quiescent state in which they are involved in slow turnover of the extracellular matrix, to a proliferative and contractile phenotype termed myofibroblasts. These cells show some of the phenotypic characteristics of smooth muscle cells and have been shown to contract in vitro. In the 1990s, a number of researchers in different fields showed that myofibroblasts are present during tissue repair or response to injury in a variety of other tissues, including the liver, kidney, and lung. During normal repair processes, the myofibroblastic cells are lost as repair resolves to form a scar. This cell loss is via apoptosis. In pathological fibroses, myofibroblasts persist in the tissue and are responsible for fibrosis via increased matrix synthesis and for contraction of the tissue. In many cases this expansion of the extracellular matrix impedes normal function of the organ. For this reason much interest has centered on the derivation of myofibroblasts and the factors that influence their differentiation, proliferation, extracellular matrix synthesis, and survival. Further understanding of how fibroblast differentiation and myofibroblast phenotype is controlled may provide valuable insights into future therapies that can control fibrosis and scarring.
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http://dx.doi.org/10.1016/S0074-7696(07)57004-XDOI Listing
June 2007

Role of the phosphatidylinositol 3-kinase and mTOR pathways in the regulation of renal fibroblast function and differentiation.

Int J Biochem Cell Biol 2007 18;39(1):206-19. Epub 2006 Aug 18.

Department of Nephrology, The Royal Melbourne Hospital, Parkville, Vic. 3050, Australia.

Tubulointerstitial fibrosis is largely mediated by (myo)fibroblasts present in the interstitium. In this study, we investigated the role of mTOR and phosphatidylinositol 3-kinase in the regulation of fibroblast kinetics, fibroblast differentiation, and collagen synthesis. Rat renal fibroblasts were propagated from kidneys 3 days post-ureteric obstruction and specific inhibitors of mTOR (RAD) and phosphatidylinositol 3-kinase (LY294002) were used to examine the regulation of fibrogenesis. LY294002 but not RAD completely inhibited phosphorylation of Akt, while both inhibitors decreased phosphorylation of the S6 ribosomal protein. RAD and LY decreased foetal calf serum stimulated proliferation and DNA synthesis. In addition to their individual effects, treatment with both RAD and LY294002 decreased serum-induced fibroblast proliferation and DNA synthesis significantly more than either drug alone. TUNEL positive cells (apoptosis) in RAD and LY294002 treated groups were not different from control groups. In addition to their effect on proliferation, both inhibitors also reduced total collagen synthesis. Differentiation studies indicated an increase in alpha-smooth muscle actin expression relative to beta-actin (western blotting), with cytochemistry confirming that all doses of RAD and LY294002 increased the proportion of alpha-smooth muscle actin positive cells, and hence myofibroblasts. Effects were independent of cell toxicity. These results highlight the potential significance of PI3K and mTOR, in the regulation of renal (myo)fibroblast activity. The synergistic effects of LY and RAD on proliferation suggest that mTOR signalling involves pathways other than phosphatidylinositol 3-kinase. These results provide a novel insight into the mechanisms of fibroblast regulation during fibrogenesis.
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http://dx.doi.org/10.1016/j.biocel.2006.08.004DOI Listing
February 2007

Time course analysis of hypoxia, granulation tissue and blood vessel growth, and remodeling in healing rat cutaneous incisional primary intention wounds.

Wound Repair Regen 2006 May-Jun;14(3):277-88

Bernard O'Brien Institute of Microsurgery and University of Melbourne Department of Surgery, St. Vincent's Hospital, Melbourne, Australia.

Hypoxia and the development and remodeling of blood vessels and connective tissue in granulation tissue that forms in a wound gap following full-thickness skin incision in the rat were examined as a function of time. A 1.5 cm-long incisional wound was created in rat groin skin and the opposed edges sutured together. Wounds were harvested between 3 days and 16 weeks and hypoxia, percent vascular volume, cell proliferation and apoptosis, alpha-smooth muscle actin, vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-beta1 expression in granulation tissue were then assessed. Hypoxia was evident between 3 and 7 days while maximal cell proliferation at 3 days (123.6+/-22.2 cells/mm2, p<0.001 when compared with normal skin) preceded the peak percent vascular volume that occurred at 7 days (15.83+/-1.10%, p<0.001 when compared with normal skin). The peak in cell apoptosis occurred at 3 weeks (12.1+/-1.3 cells/mm2, p<0.001 when compared with normal skin). Intense alpha-smooth muscle actin labeling in myofibroblasts was evident at 7 and 10 days. Vascular endothelial growth factor receptor-2 and vascular endothelial growth factor-A were detectable until 2 and 3 weeks, respectively, while transforming growth factor-beta1 protein was detectable in endothelial cells and myofibroblasts until 3-4 weeks and in the extracellular matrix for 16 weeks. Incisional wound granulation tissue largely developed within 3-7 days in the presence of hypoxia. Remodeling, marked by a decline in the percent vascular volume and increased cellular apoptosis, occurred largely in the absence of detectable hypoxia. The expression of vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-beta1 is evident prior, during, and after the peak of vascular volume reflecting multiple roles for these factors during wound healing.
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http://dx.doi.org/10.1111/j.1743-6109.2006.00122.xDOI Listing
December 2006

Histochemical localization of apoptosis with in situ labeling of fragmented DNA.

Methods Mol Biol 2006 ;326:227-34

Department of Nephrology, The Royal Melbourne Hospital and Department of Medicine, University of Melbourne, Melbourne, Australia.

Cell death by apoptosis is now recognized widely as an important constituent of cell turnover and disease pathology. Characterized by the cleavage of DNA into oligonucleosome-sized DNA fragments, end-labeling of fragmented DNA often is used as an in situ histological marker of apoptosis. The judicious and appropriate use of this technique therefore provides us with an important tool for assessing cell kinetics. Protocols for both terminal transferase-mediated UTP nick end-labeling, so-called TUNEL, and the combination of TUNEL with immunohistochemical staining are presented here, along with a discussion of its significance and interpretation.
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http://dx.doi.org/10.1385/1-59745-007-3:227DOI Listing
July 2006

Histochemical localization of cell proliferation using in situ hybridization for histone mRNA.

Methods Mol Biol 2006 ;326:219-26

Department of Nephrology, The Royal Melbourne Hospital and Department of Medicine, University of Melbourne, Melbourne, Australia.

Monoclonal antibodies to proliferation associated antigens have long been used to histologically localize mitogenesis. However, techniques that distinguish cells in the synthetic or S phase have tended to rely on the in vivo incorporation of tritiated thymidine or thymidine analogs such as bromodeoxyuridine. The necessity to pulse with these labels before retrieving tissue means that they cannot be used in humans and are not available retrospectively. Measuring expression of histones serves as a useful adjunct to these techniques. As expression of histone proteins (H2A, H2B, H3, H4) are restricted to the synthetic phase of the cell cycle, hybridization for histone mRNA precisely distinguishes those cells in the S phase. Measuring their expression can easily be applied to the histological localization of proliferation, and can be used both prospectively and with archived tissue specimens. Several histone in situ hybridization probes and nonradioactive detection systems are now available commercially. A generalized protocol for their use in measuring in situ proliferation is provided in this chapter.
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http://dx.doi.org/10.1385/1-59745-007-3:219DOI Listing
July 2006

In situ hybridization using cRNA probes: isotopic and nonisotopic detection methods.

Methods Mol Biol 2006 ;326:17-31

School of Medical Sciences, RMIT University, Melbourne, Australia.

In this chapter we describe the use of cRNA (riboprobes) in the detection of gene expression in tissue sections. Riboprobes offer good sensitivity and allow the detection of low-level mRNA expression. In some cases, the use of radiolabeling is justified because this method is still sensitive. However, recent advances in nonisotopic detection methods mean that in some cases digoxigenin (DIG) or biotin labeling also may be sufficiently sensitive to detect mRNA expression in tissues of interest. The use of alkaline phosphatase conjugated anti-DIG antibodies improves the sensitivity of DIG detection over peroxidase systems, and the use of amplification systems based on biotinyl tyramide has improved the sensitivity of biotin labelled probe detection. Finally, it can be shown that low-level mRNA expression is easier to detect in frozen sections than in paraffin-embedded material, with a consequent loss in quality of morphology.
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http://dx.doi.org/10.1385/1-59745-007-3:17DOI Listing
July 2006

Osteopontin expression in normal and fibrotic liver. altered liver healing in osteopontin-deficient mice.

J Hepatol 2006 Feb 15;44(2):383-90. Epub 2005 Aug 15.

Groupe de Recherches pour l'Etude du Foie, Inserm E0362, Institut Fédératif de Recherche 66, Pathologies Infectieuses et Cancers, Université Victor Segalen Bordeaux 2, Bordeaux, France.

Background/aims: Osteopontin has been implicated in numerous physiopathological events. Osteopontin expression in normal and fibrotic liver and liver fibrogenesis in osteopontin-deficient mice were studied.

Methods: Fibrosis was induced in mice and rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. The liver was used for conventional histology, osteopontin immunohistochemistry and in situ hybridization, or protein and RNA extraction. In mice, necrotic areas and fibrosis were evaluated by quantitative image analysis.

Results: In normal liver, osteopontin mRNA expression was very low. After CCl4 treatment or bile duct ligation, osteopontin mRNA expression was increased. Osteopontin was expressed by biliary epithelial cells in normal and fibrotic liver. Soon after the beginning of the CCl4 treatment, osteopontin was also present in inflammatory cells of the necrotic areas. In osteopontin-deficient mice, necrotic areas after a single dose of CCl4, and fibrosis after chronic CCl4 treatment were significantly increased as compared with wild-type treated mice.

Conclusions: Our results show that osteopontin expression increases during liver fibrogenesis. Furthermore, osteopontin-deficient mice were more susceptible to CCl4 treatment, displaying more necrosis during the initial steps (probably due to a deficiency in nitric oxide production) and more fibrosis thereafter. The increase in osteopontin expression observed during liver fibrogenesis may play a protective role.
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http://dx.doi.org/10.1016/j.jhep.2005.07.024DOI Listing
February 2006

Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: evidence of fiber type specificity.

FASEB J 2004 Jun 1;18(9):992-4. Epub 2004 Apr 1.

Skeletal Muscle Research Laboratory, Centre for Nutrition, Metabolism & Endocrinology, School of Medical Sciences, RMIT University, Melbourne, Australia.

In this study, we aimed to determine whether skeletal muscle cells per se are a source of interleukin (IL)-6 during contraction and whether IL-6 production is fiber type specific. Muscle biopsy samples were collected from seven males before (PRE) and after (POST) completing 120 min of continuous bicycle ergometry. Biopsies were sectioned and analyzed for the following: IL-6 protein detected by immunohistochemistry (IHC), IL-6 mRNA content detected by in situ hybridization, fiber type measured by either IHC or myofibrillar ATPase activity stain, and glycogen content measured by periodic acid schiff (PAS) assay. Fibers were qualitatively categorized according to glycogen content to one of five groups (1-5), with 1 being very low (LOW) and 5 being very high (HIGH) glycogen. Total fluorescence (PRE vs. POST) and glycogen-dependent fluorescence (LOW vs. HIGH) of IL-6 protein were quantitated using Metamorph software. Total IL-6 protein was elevated from PRE to POST exercise (P<0.05). At PRE, IL-6 protein was evenly distributed across all fibers at low levels, consistent with glycogen distribution. At POST, IL-6 protein was greater (P<0.05) in HIGH compared with LOW glycogen fibers, which coincided with type 2 fibers. IL-6 mRNA was distributed peripherally in all fibers at PRE. At POST, however, IL-6 mRNA appeared predominantly in type 2 fibers, which also had higher glycogen content (P<0.05). These data demonstrate that myocytes per se are a source of IL-6 produced during contraction. Our data also suggest that type 2 fibers predominantly produce IL-6 during muscle contractile activity.
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http://dx.doi.org/10.1096/fj.03-1259fjeDOI Listing
June 2004

Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis.

Lab Invest 2003 Dec;83(12):1689-707

Groupe de Recherches pour l'Etude du Foie (AD), INSERM E0362, Université Victor Segalen Bordeaux 2, Bordeaux, France.

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http://dx.doi.org/10.1097/01.lab.0000101911.53973.90DOI Listing
December 2003

Fibrillin-1 expression in normal and fibrotic rat liver and in cultured hepatic fibroblastic cells: modulation by mechanical stress and role in cell adhesion.

Lab Invest 2004 Feb;84(2):203-12

Groupe de Recherches pour l'Etude du Foie, INSERM E0362, and Institut Fédératif de Recherche 66, Pathologies Infectieuses et Cancers, Université Victor Segalen Bordeaux 2, Bordeaux, France.

Fibrillin-1, together with elastin, is the main component of elastic fibers found throughout the extracellular space and responsible for the biomechanical properties of most tissues and organs. In this work, fibrillin-1 expression and modulation were explored in experimental rat liver fibrosis and in vitro; furthermore, the role of fibrillin-1 fragments on cell adhesion was analyzed. Fibrosis was induced by subjecting rats to common bile duct ligation for 72 h and 7 days or carbon tetrachloride (CCl(4)) treatment for 2 and 6 weeks. Immunohistochemistry showed that, after bile duct ligation, fibrillin-1, elastin, and alpha-smooth muscle actin colocalized in the developing portal connective tissue. In CCl(4)-treated animals, a similar colocalization was observed in septa; however, elastin deposition was not observed around activated alpha-smooth muscle actin-positive stellate cells of the parenchyma. Treatment with the profibrogenic mediator transforming growth factor-beta1 (TGF-beta1) greatly increased the fibrillin-1 expression of cultured liver fibroblasts. The level of fibrillin-1 expression was significantly higher in cells grown in restrained (stressed) collagen lattices compared with those grown in unrestrained collagen lattices. Cell adhesion on the C-terminal fragment of fibrillin-1 containing the RGD sequence (rF6H) slightly increased (between 0.3 and 2.5 microg/ml) and decreased at higher concentrations, while adhesion on the N-terminal fragment of fibrillin-1 (rF16) was dose-dependently decreased. In addition, the rF16 fragment decreased cell adhesion to fibronectin. In conclusion, our study illustrates the important deposition of fibrillin-1 that occurs in two mechanistically distinct settings of liver fibrogenesis. Furthermore, the induction of fibrillin-1 expression by TGF-beta1 and mechanical stress, and the antiadhesive properties of fibrillin-1 fragments suggest important implications for physiological and pathological fibrillin-1 catabolism during tissue remodeling.
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http://dx.doi.org/10.1038/labinvest.3700023DOI Listing
February 2004

Approaches for introducing peptides into intact and functional arteriolar smooth muscle: manipulation of protein kinase-based signalling.

Clin Exp Pharmacol Physiol 2003 Sep;30(9):653-8

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia.

1. An exact understanding of signal transduction pathways within intact and functional arteriolar smooth muscle is made difficult by limited access to the intracellular environment due to the cell membrane. The aim of the present studies was to determine the feasibility of using polycationic lipids and reverse permeabilization for the introduction of peptide inhibitors into smooth muscle cells of the intact arteriolar wall. 2. Isolated cannulated arterioles were exposed to polycationic lipid preparations together with varying concentrations of the protein beta-galactosidase (30-90 microg/mL). Similar experiments were also performed using cultured smooth muscle cells. Staining for the chromogenic substrate of beta-galactosidase (5-bromo-4-chloro-3-indolyl-beta-d-galactosidase; X-gal) demonstrated incorporation of the protein into cultured cells but not intact arteriolar smooth muscle. Similarly, polycationic lipid treatment did not enable loading of arteriolar smooth muscle (as assessed by cAMP-mediated vasodilation) with the protein kinase (PK) A inhibitory peptide PKI. 3. In contrast, reverse permeabilization, using high ATP concentrations in the presence of EGTA enabled introduction of PKI and inhibition of forskolin-mediated vasodilatation. Furthermore, arterioles maintained full viability following reverse permeabilization, as demonstrated by an ability to develop spontaneous myogenic tone. 4. Reverse permeabilization provides a method for introducing peptide inhibitors into functional arteriolar smooth muscle and manipulating signal transduction. Protein transfection using polycationic lipids appears to be limited by the barrier provided by the adventitia or inherent differences between cells under cultured conditions compared within the intact arteriole.
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http://dx.doi.org/10.1046/j.1440-1681.2003.03892.xDOI Listing
September 2003

Matrix protein glycation impairs agonist-induced intracellular Ca2+ signaling in endothelial cells.

J Cell Physiol 2002 Oct;193(1):80-92

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.

Studies have shown diabetes to be associated with alterations in composition of extracellular matrix and that such proteins modulate signal transduction. The present studies examined if non-enzymatic glycation of fibronectin or a mixed matrix preparation (EHS) alters endothelial cell Ca(2+) signaling following agonist stimulation. Endothelial cells were cultured from bovine aorta and rat heart. To glycate proteins, fibronectin (10 microg/ml), or EHS (2.5 mg/ml) were incubated (37 degrees C, 30 days) with 0.5 M glucose-6-phosphate. Matrix proteins were coated onto cover slips after which cells (10(5) cells/ml) were plated and allowed to adhere for 16 h. For measurement of intracellular Ca(2+), cells were loaded with fura 2 (2 microM) and fluorescence intensity monitored. Bovine cells on glycated EHS showed decreased ability for either ATP (10(-6) M) or bradykinin (10(-7) M) to increase Ca(2+) (i). In contrast, glycated fibronectin did not impair agonist-induced increases in Ca(2+) (i). In the absence of extracellular Ca(2+), ATP elicited a transient increase in Ca(2+) (i) consistent with intracellular release. Re-addition of Ca(2+) resulted in a secondary rise in Ca(2+) (i) indicative of store depletion-mediated Ca(2+) entry. Both phases of Ca(2+) mobilization were reduced in cells on glycated mixed matrix; however, as the ratio of the two components was similar in all cells, glycation appeared to selectively impair Ca(2+) release from intracellular stores. Thapsigargin treatment demonstrated an impaired ability of cells on glycated EHS to increase cytoplasmic Ca(2+) consistent with decreased endoplasmic reticulum Ca(2+) stores. Further support for Ca(2+) mobilization was provided by increased baseline IP(3) levels in cells plated on glycated EHS. Impaired ATP-induced Ca(2+) release could be induced by treating native EHS with laminin antibody or exposing cells to H(2)O(2) (20-200 microM). Glycated EHS impaired Ca(2+) signaling was attenuated by treatment with aminoguanidine or the antioxidant alpha-lipoic acid. The results demonstrate that matrix glycation impairs agonist-induced Ca(2+) (i) increases which may impact on regulatory functions of the endothelium and implicate possible involvement of oxidative stress.
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http://dx.doi.org/10.1002/jcp.10153DOI Listing
October 2002

Skin flap-induced regression of granulation tissue correlates with reduced growth factor and increased metalloproteinase expression.

J Pathol 2002 May;197(1):117-27

Wound Healing and Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.

Previous studies have shown that covering granulation tissue of a full-thickness skin wound by a vascularized skin flap induces tissue remodeling, with a rapid loss of granulation tissue cells by apoptosis. In the present study, in situ hybridization has been used to examine mRNA expression for several factors that may be implicated in the apoptosis seen in this tissue. Skin wounds were made on the dorsal skin of 8-week-old rats. Ten days after wounding, skin flaps were created surgically and sutured over the granulation tissue. Tissue sections of granulation tissue from various times after addition of the skin flap were hybridized with 33P-labelled cRNA probes for transforming growth factor-beta1 (TGF-beta1), beta-inducible gene H3 (beta-ig-h3), alpha1 (1) procollagen, alpha-smooth muscle actin, matrix metalloproteinase-13 (MMP-13) and -2 (MMP-2), tissue inhibitor of metalloproteinase-1 (TIMP-1), and inducible nitric oxide synthase (iNOS). Control granulation tissue prior to addition of the skin flap showed high levels of TGF-beta1, beta-ig-h3, alpha1 (1) procollagen, alpha-smooth muscle actin, and TIMP-1 expression. MMP-13, MMP-2, and iNOS mRNA were low in 10-day granulation tissue. Addition of a skin flap resulted in a decrease in the expression of TGF-beta1, beta-ig-h3, alpha1 (I) procollagen, alpha-smooth muscle actin, and TIMP-1, but increased expression of MMP-13 and MMP-2. Similarly, an increase in iNOS mRNA expression was observed in the granulation tissue after addition of the skin flap. Addition of a vascularized skin flap may result in rapid remodelling of granulation tissue due to a decrease in expression of the trophic growth factor TGF-beta1 and increased degradation of extracellular matrix due to an alteration in the balance between MMPs and their inhibitor, TIMP-1. Additionally, increased iNOS expression may also favour apoptosis through the generation of free radicals. The additive effect of reduced growth factor expression, increased extracellular matrix turnover, and nitric oxide generation may result in the fibroblast and vascular cell apoptosis seen during the rapid remodelling of this tissue.
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http://dx.doi.org/10.1002/path.1074DOI Listing
May 2002

Collagen fibril formation in a wound healing model.

J Struct Biol 2002 Jan-Feb;137(1-2):23-30

CSIRO Molecular Science, 343 Royal Parade, Parkville, Australia.

Control of tissue composition and organization will be a key feature in the development of successful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial implant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days. Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that approximately 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal domains; the remaining fibrils had no polarity reversal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collagens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be readily followed.
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http://dx.doi.org/10.1006/jsbi.2002.4460DOI Listing
December 2002