Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease
2020; Elsevier BV; Volume: 190; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2020.06.013
ISSN1525-2191
AutoresLaura García-Posadas, Robin R. Hodges, Tor Paaske Utheim, Ole Kristoffer Olstad, Vanessa Delcroix, Helen P. Makarenkova, Darlene A. Dartt,
Tópico(s)Neuropeptides and Animal Physiology
ResumoThe purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1−/−, alias Thbs1−/−) mice and from mice expressing α-smooth muscle actin–green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α1-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca2+] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca2+]I with VIP and UTP was significantly smaller in MECs from TSP1−/− compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1−/− mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1−/− compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1−/− mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1−/− compared with WT mice. The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1−/−, alias Thbs1−/−) mice and from mice expressing α-smooth muscle actin–green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α1-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca2+] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca2+]I with VIP and UTP was significantly smaller in MECs from TSP1−/− compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1−/− mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1−/− compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1−/− mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1−/− compared with WT mice. The lacrimal gland is a tubuloacinar gland responsible for secreting the aqueous part of the tear film. This secretion is neurally regulated and can be altered in pathologic states, such as dry eye disease and Sjögren syndrome.1Dartt D.A. Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases.Prog Retin Eye Res. 2009; 28: 155-177Crossref PubMed Scopus (228) Google Scholar Afferent sensory nerves in the cornea and conjunctiva can be activated by different stimuli from the external environment; subsequently, these activated nerves stimulate efferent sympathetic (containing norepinephrine) and parasympathetic [containing acetylcholine and vasoactive intestinal peptide (VIP)] nerves that surround lacrimal gland cells. Release of these neurotransmitters stimulates lacrimal gland functions, most notably secretion of proteins, electrolytes, and water.1Dartt D.A. Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases.Prog Retin Eye Res. 2009; 28: 155-177Crossref PubMed Scopus (228) Google Scholar In addition, lacrimal gland secretion can also be mediated by a nonadrenergic, noncholinergic pathway that includes activation of purinergic receptors (P2X and P2Y).2Hodges R.R. Vrouvlianis J. Scott R. Dartt D.A. Identification of P2X(3) and P2X(7) purinergic receptors activated by ATP in rat lacrimal gland.Invest Ophthalmol Vis Sci. 2011; 52: 3254-3263Crossref PubMed Scopus (18) Google Scholar,3Ohtomo K. Shatos M.A. Vrouvlianis J. Li D. Hodges R.R. Dartt D.A. Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells.Invest Ophthalmol Vis Sci. 2011; 52: 9503-9515Crossref PubMed Scopus (21) Google Scholar The lacrimal gland is composed of three main cell types: acinar cells, ductal cells, and myoepithelial cells (MECs).4Hodges R.R. Dartt D.A. Signaling pathways of purinergic receptors and their interactions with cholinergic and adrenergic pathways in the lacrimal gland.J Ocul Pharmacol Ther. 2016; 32: 490-497Crossref PubMed Scopus (8) Google Scholar Lacrimal gland MECs surround acini but not ducts.5Makarenkova H.P. Dartt D.A. Myoepithelial cells: their origin and function in lacrimal gland morphogenesis, homeostasis, and repair.Curr Mol Biol Rep. 2015; 1: 115-123Crossref PubMed Google Scholar They have a stellate shape and multiple branching processes. The contractile function is possible based on the expression of α-smooth muscle actin (α-SMA), which also serves as the main marker for all smooth muscle cells, including lacrimal gland MECs.6Lemullois M. Rossignol B. Mauduit P. Immunolocalization of myoepithelial cells in isolated acini of rat exorbital lacrimal gland: cellular distribution of muscarinic receptors.Biol Cell. 1996; 86: 175-181Crossref PubMed Scopus (33) Google Scholar MECs also express other smooth muscle markers, such as calponin and α-actinin,7Chitturi R.T. Veeravarmal V. Nirmal R.M. Reddy B.V. Myoepithelial cells (MEC) of the salivary glands in health and tumours.J Clin Diagn Res. 2015; 9: ZE14-ZE18PubMed Google Scholar and epithelial cell markers, such as cytokeratin 14 (CK14). MECs are found in salivary, sweat, mammary, and lacrimal glands.5Makarenkova H.P. Dartt D.A. Myoepithelial cells: their origin and function in lacrimal gland morphogenesis, homeostasis, and repair.Curr Mol Biol Rep. 2015; 1: 115-123Crossref PubMed Google Scholar,7Chitturi R.T. Veeravarmal V. Nirmal R.M. Reddy B.V. Myoepithelial cells (MEC) of the salivary glands in health and tumours.J Clin Diagn Res. 2015; 9: ZE14-ZE18PubMed Google Scholar,8Gudjonsson T. Adriance M.C. Sternlicht M.D. Petersen O.W. Bissell M.J. Myoepithelial cells: their origin and function in breast morphogenesis and neoplasia.J Mammary Gland Biol Neoplasia. 2005; 10: 261-272Crossref PubMed Scopus (176) Google Scholar In the mammary glands, they play an important role in milk ejection, by contracting in response to oxytocin.9Haaksma C.J. Schwartz R.J. Tomasek J.J. Myoepithelial cell contraction and milk ejection are impaired in mammary glands of mice lacking smooth muscle alpha-actin.Biol Reprod. 2011; 85: 13-21Crossref PubMed Scopus (42) Google Scholar,10Weymouth N. Shi Z. Rockey D.C. Smooth muscle alpha actin is specifically required for the maintenance of lactation.Dev Biol. 2012; 363: 1-14Crossref PubMed Scopus (17) Google Scholar In the salivary gland, MECs play a role in contraction, basement membrane production, and tumor suppression.7Chitturi R.T. Veeravarmal V. Nirmal R.M. Reddy B.V. Myoepithelial cells (MEC) of the salivary glands in health and tumours.J Clin Diagn Res. 2015; 9: ZE14-ZE18PubMed Google Scholar Despite long-term interest, the specific functions of MECs in the lacrimal gland have only recently been investigated. First, studies have found that progenitor cell markers, such as nestin, Musashi 1, p63, and ABCG2, co-localize with α-SMA–positive cells in lacrimal glands, implying that MEC lineage contains progenitor cells and may play a role in regeneration and repair of the lacrimal gland.11Shatos M.A. Hodges R.R. Morinaga M. McNay D.E. Islam R. Bhattacharya S. Li D. Turpie B. Makarenkova H.P. Masli S. Utheim T.P. Dartt D.A. Alteration in cellular turnover and progenitor cell population in lacrimal glands from thrombospondin 1(-/-) mice, a model of dry eye.Exp Eye Res. 2016; 153: 27-41Crossref PubMed Scopus (9) Google Scholar,12Shatos M.A. Haugaard-Kedstrom L. Hodges R.R. Dartt D.A. Isolation and characterization of progenitor cells in uninjured, adult rat lacrimal gland.Invest Ophthalmol Vis Sci. 2012; 53: 2749-2759Crossref PubMed Scopus (38) Google Scholar Second, two studies found the contractile capacity of lacrimal gland MECs.13Hawley D. Tang X. Zyrianova T. Shah M. Janga S. Letourneau A. Schicht M. Paulsen F. Hamm-Alvarez S. Makarenkova H.P. Zoukhri D. Myoepithelial cell-driven acini contraction in response to oxytocin receptor stimulation is impaired in lacrimal glands of Sjogren's syndrome animal models.Sci Rep. 2018; 8: 9919Crossref PubMed Scopus (11) Google Scholar,14Satoh Y. Sano K. Habara Y. Kanno T. Effects of carbachol and catecholamines on ultrastructure and intracellular calcium-ion dynamics of acinar and myoepithelial cells of lacrimal glands.Cell Tissue Res. 1997; 289: 473-485Crossref PubMed Scopus (35) Google Scholar Using guinea pig lacrimal gland tissue pieces, Satoh et al14Satoh Y. Sano K. Habara Y. Kanno T. Effects of carbachol and catecholamines on ultrastructure and intracellular calcium-ion dynamics of acinar and myoepithelial cells of lacrimal glands.Cell Tissue Res. 1997; 289: 473-485Crossref PubMed Scopus (35) Google Scholar found that MECs contracted in response to cholinergic, but not to adrenergic, agonists. Meanwhile, Hawley et al13Hawley D. Tang X. Zyrianova T. Shah M. Janga S. Letourneau A. Schicht M. Paulsen F. Hamm-Alvarez S. Makarenkova H.P. Zoukhri D. Myoepithelial cell-driven acini contraction in response to oxytocin receptor stimulation is impaired in lacrimal glands of Sjogren's syndrome animal models.Sci Rep. 2018; 8: 9919Crossref PubMed Scopus (11) Google Scholar found that MEC contraction in response to oxytocin was impaired in lacrimal gland lobules from two mouse models of aqueous-deficient dry eye, nonobese diabetic mice, and MRL/lymphoproliferation mice compared with wild-type (WT) mice. Supporting an MEC functional response to neural agonists, lacrimal gland MECs express muscarinic,6Lemullois M. Rossignol B. Mauduit P. Immunolocalization of myoepithelial cells in isolated acini of rat exorbital lacrimal gland: cellular distribution of muscarinic receptors.Biol Cell. 1996; 86: 175-181Crossref PubMed Scopus (33) Google Scholar VIP,15Hodges R.R. Zoukhri D. Sergheraert C. Zieske J.D. Dartt D.A. Identification of vasoactive intestinal peptide receptor subtypes in the lacrimal gland and their signal-transducing components.Invest Ophthalmol Vis Sci. 1997; 38: 610-619PubMed Google Scholar and purinergic receptors.3Ohtomo K. Shatos M.A. Vrouvlianis J. Li D. Hodges R.R. Dartt D.A. Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells.Invest Ophthalmol Vis Sci. 2011; 52: 9503-9515Crossref PubMed Scopus (21) Google Scholar Involvement of MEC purinergic receptors in intracellular Ca2+ responses of cultured MECs was reported,3Ohtomo K. Shatos M.A. Vrouvlianis J. Li D. Hodges R.R. Dartt D.A. Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells.Invest Ophthalmol Vis Sci. 2011; 52: 9503-9515Crossref PubMed Scopus (21) Google Scholar suggesting a potential role of MECs in tear secretion regulation. Thus, lacrimal gland MECs could play a role in both secretion and lacrimal gland repair. Thrombospondin (TSP) 1 is a large matricellular protein that is found intracellularly and extracellularly. It has multiple domains, allowing it to support a large number of cellular functions, including cell migration, proliferation, and cell death, and to prevent inflammation. Although dry eye disease in humans has not been associated with TSP1 loss of function mutation, there is a polymorphism in TSP1 that predisposes individuals to develop dry eye after refractive surgery.16Contreras-Ruiz L. Ryan D.S. Sia R.K. Bower K.S. Dartt D.A. Masli S. Polymorphism in THBS1 gene is associated with post-refractive surgery chronic ocular surface inflammation.Ophthalmology. 2014; 121: 1389-1397Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Because corneal neural dysfunction is a critical component of dry eye in humans, corneal nerves are damaged in refractive surgery, and as corneal nerves are damaged in the TSP1−/− (Thbs1−/−) mice, we feel that the TSP1−/− mouse model is a relevant model for a portion, although not all types of dry eye disease.17Tatematsu Y. Khan Q. Blanco T. Bair J.A. Hodges R.R. Masli S. Dartt D.A. Thrombospondin-1 Is necessary for the development and repair of corneal nerves.Int J Mol Sci. 2018; 19: 3191Crossref Scopus (7) Google Scholar,18Belmonte C. Nichols J.J. Cox S.M. Brock J.A. Begley C.G. Bereiter D.A. Dartt D.A. Galor A. Hamrah P. Ivanusic J.J. Jacobs D.S. McNamara N.A. Rosenblatt M.I. Stapleton F. Wolffsohn J.S. TFOS DEWS II pain and sensation report.Ocul Surf. 2017; 15: 404-437Crossref PubMed Scopus (227) Google Scholar In addition, in TSP1 null (TSP1−/−) mice the appearance of inflammatory infiltrates corresponds to development of functional defects in the lacrimal gland, indicating its use as a model of dry eye disease.11Shatos M.A. Hodges R.R. Morinaga M. McNay D.E. Islam R. Bhattacharya S. Li D. Turpie B. Makarenkova H.P. Masli S. Utheim T.P. Dartt D.A. Alteration in cellular turnover and progenitor cell population in lacrimal glands from thrombospondin 1(-/-) mice, a model of dry eye.Exp Eye Res. 2016; 153: 27-41Crossref PubMed Scopus (9) Google Scholar,19Bhattacharya S. Garcia-Posadas L. Hodges R.R. Makarenkova H.P. Masli S. Dartt D.A. Alteration in nerves and neurotransmitter stimulation of lacrimal gland secretion in the TSP-1(-/-) mouse model of aqueous deficiency dry eye.Mucosal Immunol. 2018; 11: 1138-1148Crossref PubMed Scopus (8) Google Scholar,20Turpie B. Yoshimura T. Gulati A. Rios J.D. Dartt D.A. Masli S. Sjogren's syndrome-like ocular surface disease in thrombospondin-1 deficient mice.Am J Pathol. 2009; 175: 1136-1147Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Moreover, in the lacrimal gland, TSP1 is highly expressed in MECs20Turpie B. Yoshimura T. Gulati A. Rios J.D. Dartt D.A. Masli S. Sjogren's syndrome-like ocular surface disease in thrombospondin-1 deficient mice.Am J Pathol. 2009; 175: 1136-1147Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar and that MEC function is strongly affected by the inflammation that occurs in these mice,13Hawley D. Tang X. Zyrianova T. Shah M. Janga S. Letourneau A. Schicht M. Paulsen F. Hamm-Alvarez S. Makarenkova H.P. Zoukhri D. Myoepithelial cell-driven acini contraction in response to oxytocin receptor stimulation is impaired in lacrimal glands of Sjogren's syndrome animal models.Sci Rep. 2018; 8: 9919Crossref PubMed Scopus (11) Google Scholar suggesting a critical role of TSP1 in MEC function. We hypothesize that inflammation that mimics dry eye spontaneously developed with age in TSP1−/− mice and alters MEC function regulated by neural agonists.20Turpie B. Yoshimura T. Gulati A. Rios J.D. Dartt D.A. Masli S. Sjogren's syndrome-like ocular surface disease in thrombospondin-1 deficient mice.Am J Pathol. 2009; 175: 1136-1147Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar The calcium dynamics and contractile capacity of lacrimal gland MECs isolated from the WT and TSP1−/− mice in response to parasympathetic and sympathetic neurotransmitters and purinergic agonists that can mediate lacrimal gland protein secretion were analyzed. RPMI 1640 cell culture medium, penicillin/streptomycin, and l-glutamine were purchased from Lonza (Walkerville, IL). Fetal bovine serum was purchased from Atlanta Biologicals (Norcross, GA). Antibodies used are listed in Table 1. VIP was from Anaspec (Fremont, CA), whereas fura-2/AM was from Invitrogen (Carlsbad, CA). The 35-mm glass-bottom culture dishes were from MatTek Corporation (Ashland, MA). Alamar blue was purchased from Thermo Fisher Scientific (Waltham, MA). TRIZOL, thapsigargin, carbachol (Cch), phenylephrine (Ph), ATP, and UTP were from Sigma-Aldrich (St. Louis, MO).Table 1Sources of AntibodiesAntibodySupplierCatalog no.DilutionCloneα-SMADiagnostic Biosystems, Pleasanton, CAMOB0011:10001A4α-SMASigma-Aldrich, St. Louis, MOA25471:4001A4CK14Abcam, Cambridge, United KingdomAb78001:500LL002p63Santa Cruz Biotechnology, Dallas, TXsc-252681:200D9P63AbcamAb2147901:300EPR5701P2X3Alomone Labs, Jerusalem, IsraelAPR-0261:200PolyclonalP2X7Alomone LabsAPR-0081:200PolyclonalP2Y1Alomone LabsAPR-0211:200PolyclonalP2Y11Alomone LabsAPR-0151:200PolyclonalP2Y13Alomone LabsAPR-0171:200Polyclonalβ-actinSigma-AldrichA22281:1000AC-74α-SMA, α-smooth muscle actin; CK14, cytokeratin 14; p63, tumor protein p63; P2X3, P2X7, P2Y1, P2Y11, and P2Y13, purinergic receptors. Open table in a new tab α-SMA, α-smooth muscle actin; CK14, cytokeratin 14; p63, tumor protein p63; P2X3, P2X7, P2Y1, P2Y11, and P2Y13, purinergic receptors. All experiments were conducted following the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Schepens Eye Research Institute Animal Care and Use Committee. Male and female C57BL/6J mice with ages ranging from 4 to 12 weeks were purchased from Jackson Laboratories (Bar Harbor, ME) and used to optimize all protocols and to study normal lacrimal gland MEC function. C57BL/6J mice were then used as controls when compared with TSP1−/− mice. TSP1−/− mice were originally obtained through Dr. J. Lawler (Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA) and bred by Dr. Sharmila Masli (Boston University, Boston, MA). These mice were generated on the C57BL/6J background. Only female, 4- to 12-week–old TSP1−/− mice were used because of the higher prevalence of dry eye disease in females. α-SMA–green fluorescent protein (GFP) mice bred on a C57BL/6J background (3 male and 3 female) were a kind gift from Dr. Ivo Kalajzic (University of Connecticut, Storrs, CT). In these mice, α-SMA present in MECs is labeled with GFP, allowing for easy detection of MECs in culture. All mice were euthanized with carbon dioxide for 5 minutes, followed by cervical dislocation as a secondary physical means to ensure animal death. Then, both exorbital lacrimal glands were excised. MECs were cultured from mouse lacrimal glands using the following protocol: exorbital lacrimal glands from C57BL/6J, SMA-GFP, and TSP1−/− mice were isolated, minced into small pieces, and incubated at 37°C in collagenase III (5 mg/mL) dissolved in Krebs-Ringer Bicarbonate buffer (KRB, 120 mmol/L NaCl, 25 mmol/L NaHCO3, 10 mmol/L HEPES, 4.8 mmol/L KCl, 1.2 mmol/L MgCl2, 1.2 mmol/L NaH2PO4, and 1 mmol/L CaCl2) containing 0.5% bovine serum albumin (BSA) for three 10-minute cycles. The fragments were triturated between each cycle. After incubation, the fragments and cell suspension were filtered through a 50-μm nylon mesh into a plastic centrifuge tube and centrifuged for 2 minutes at 500 × g. The pellet was washed twice with KRB buffer containing 4% BSA. The pellet was resuspended in KRB buffer plus 0.5% BSA, and cells were placed in culture dishes. Cells were fed with RPMI 1640 culture medium supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 μg/mL of penicillin/streptomycin, 1% 1 mol/L HEPES, nonessential amino acids mixture, and 1% sodium pyruvate. Cells were maintained for approximately 2 to 3 weeks in standard culture condition (37°C, 5% CO2), and medium was changed every other day. All experiments were performed with first passage cells that were grown for 1 day after trypsinization. To confirm that the cultured cells were MECs, the expression of MEC markers α-SMA and CK14, which in the lacrimal gland is only expressed in MECs21Kuony A. Michon F. Epithelial markers aSMA, Krt14, and Krt19 unveil elements of murine lacrimal gland morphogenesis and maturation.Front Physiol. 2017; 8: 739Crossref PubMed Scopus (8) Google Scholar (data not shown), were evaluated. The cell markers α-SMA and CK14 in our cultured cells were regularly checked. During the optimization of the protocol, the expression of these markers at different time points between 2 and 28 days of culture was checked. For the remaining experiments, the cellular identity at the end of the primary culture period of 2 to 3 weeks was checked. Cells maintained their MEC markers throughout the entire period. The expression of p63, a progenitor cell marker, was analyzed. First-passage cultured cells were grown on 8-well multichamber slides and fixed in methanol. Slides with methanol-fixed lacrimal gland tissue sections were used as controls. Cells were incubated for 1 hour with a blocking solution [1% BSA and 2% Triton X-100 in phosphate-buffered saline (PBS)]. Thereafter, primary antibodies against α-SMA (1:200 dilution in PBS), CK14 (1:500 in PBS), and p63 (1:100 in PBS) were added. After 1-hour incubation, slides were washed with PBS, and then secondary antibodies conjugated with Cy2 or Cy3 (at 1:200 dilution in PBS) were added for 1 hour. To detect cell nuclei, DAPI was added to the mounting medium. Negative controls included the omission of primary antibodies. Cells were viewed by fluorescence microscopy (Eclipse E80i, Nikon, Tokyo, Japan), and micrographs were taken using a digital camera (Spot, Diagnostic Instruments Inc., Sterling Heights, MI) or using a Zeiss LSM 710 confocal laser scanning microscope (Zeiss, Oberkochen, Germany). After isolation by collagenase digestion, cells were suspended in KRB and 0.5% BSA, and the number of cells was counted using a hemocytometer before plating for culture. First-passage cultured lacrimal gland MECs were grown on 35-mm glass-bottom culture dishes for 24 hours. Then cells were incubated in KRB buffer with 0.5% BSA, 8 μmol/L pluronic acid F127, 250 μmol/L sulfinpyrazone, and 0.5 μmol/L of fura-2/AM for 1 hour at 37°C. Fura-2 is a fluorescent molecule that indicates the intracellular [Ca2+]. After incubation, cells were washed with KRB buffer that contained 250 μmol/L sulfinpyrazone, and the dishes were observed using a Ca2+ imagining system (InCyt Im2, Intracellular Imaging, Cincinnati, OH). This system allows measurement of the ratio of fura-2 using excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. A mean of 30 cells per dish was selected, and intracellular [Ca2+] was measured in each cell. A basal reading was performed for at least 15 seconds. The effect of the cholinergic agonist Cch (10−5 to 10−2 mol/L), VIP (10−9 mol/L to 10−7 mol/L), the α1-adrenergic agonist Ph (10−6 to 10−3 mol/L), the P2X agonist ATP (10−7 to 10−4 mol/L), the P2Y agonist UTP (10−7 to 10−4 mol/L), and high potassium chloride (40 mmol/L) in cultured MECs was evaluated by adding 100 μL of each agonist to 900 μL of buffer. For the potassium chloride experiments, the sodium chloride concentration was decreased by 40 mmol/L. Data are presented as the change in peak intracellular [Ca2+], which was calculated by subtracting the basal value from the intracellular [Ca2+] peak. At least three independent experiments were performed for each condition. To calculate intracellular Ca2+ stores, extracellular Ca2+ was removed. Then cells were treated for 15 minutes with 10−5 mol/L thapsigargin. Peak intracellular [Ca2+] and total area under the curve were measured. After that, Ca2+ was reintroduced in the bath, and the rate of refilling was analyzed. To analyze the contraction of MECs in response to the different agonists (Cch, VIP, Ph, ATP, UTP, and high potassium chloride), the Leica DMI 6000 live-cell imaging system from Leica Microsystems (Wetzlar, Germany) was used. Live MECs cultured from α-SMA–GFP WT and TSP1−/− mice were trypsinized and plated on 35-mm glass-bottomed culture dishes as described for measurement of intracellular [Ca2+]. After 24 hours, cells were observed under the microscope under fluorescence or brightfield. After 1 minute of basal observation, the different neurotransmitters were added. Images were taken automatically every 15 seconds for 30 minutes. Time-lapse movies were created using the individual images, and the response of MECs was evaluated. The expression of purinergic receptors P2X3, P2X7, P2Y1, P2Y11, and P2Y13 and the housekeeping protein β-actin was measured by Western blot analysis. Cultured MECs were homogenized in radioimmunoprecipitation assay buffer [(10 mmol/L Tris-HCl pH 7.4), 150 mmol/L NaCl, 1% deoxycholic acid, 1% Triton X-100, 0.1% SDS, and 1 mmol/L EDTA] that contained a protease inhibitor cocktail (Sigma-Aldrich), and cells were scraped. The homogenates were collected, sonicated, and centrifuged at 14,500 × g for 10 minutes at 4°C. Proteins were separated by SDS-PAGE using a 10% gel and processed for Western blotting. Antibodies against purinergic receptors were used at 1:500 dilution. The β-actin antibody was used at a dilution of 1:1000. Secondary antibody conjugated to horseradish peroxidase was used at a dilution of 1:2000, and immunoreactive bands were visualized by the enhanced chemiluminescence method. The films were analyzed with ImageJ software version 1.48 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Data are expressed as percentage of WT values, which were set to 100%. The expression of genes in cultured MECs derived from female C57BL/6J mice (n = 3) and TSP1−/− mice (n = 3), aged 6 to 8 weeks, were analyzed using RNA microarrays. Total RNA was isolated with TRIZol. A NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) was used for RNA quantification. A total of 150 ng of total RNA was processed with a GeneChip HT One-Cycle cDNA Synthesis Kit and a GeneChip HT IVT Labeling Kit (Affymetrix, Santa Clara, CA). Labeled and fragmented single-stranded cDNA was hybridized to the GeneChip Mouse Gene 2.0 ST Array (41,345 transcripts) (Affymetrix). Thereafter, the arrays were rinsed and stained using a FS-450 fluidics station (Affymetrix). Signal intensities were measured with a Hewlett Packard Gene Array Scanner 3000 7 G (Hewlett Packard, Palo Alto, CA), and the scanned images were processed by the Affymetrix GeneChip Command Console. The CEL files were imported into Partek Genomics Suite software version 6.6 (Partek Inc., St. Louis, MO). Robust microarray analysis was applied for normalization. Gene transcripts with a maximal signal value <5 (log2) across all arrays were removed to filter for low and nonexpressed genes, reducing the number of gene transcripts to 20,900. Differentially expressed genes between groups were identified using one-way analysis of variance analysis in the Partek Genomics Suite Software. Clustering analysis was performed using the same name module in a Partek Genomics Suite Software. For expression comparisons of different groups, profiles were compared using a one-way analysis of variance model. Data are presented as fold changes and P values. Data are presented as means ± SD. The t-test was performed when comparing two groups. For comparing more than two groups, a one-way analysis of variance was performed after ensuring equality of variance. A Tukey test was then performed ad hoc. In the absence of variance equality, a Kruskal-Wallis test was performed. Differences were considered to be significant at P ≤ 0.05. No difference was found in the number of cells obtained from male (474,667 ± 199,613 cells) compared with female WT mice (307,333 ± 45,553 cells) (P = 0.2298) (Figure 1A). The number of cells obtained from α-SMA–GFP mice was similar to the number obtained from C57BL/6J WT mice. In contrast, fewer cells were obtained from female TSP1−/− mice compared with WT mice (199,833 ± 62,317 versus 291,800 ± 49,357, P = 0.0257) (Figure 1B). MECs obtained from the three different types of mice used in this study: C57BL/6J (WT), α-SMA–GFP, and TSP1−/− mice, were isolated and expanded in vitro. MEC growth over time is shown (Figure 2) for α-SMA–GFP mice because of their visibility. There were no evident morphologic or behavioral differences between the cells grown from the three strains of mice. To characterize the cultured cells and confirm their identity at the end of the cell culture period, immunofluorescence staining was performed using antibodies against different MEC markers: α-SMA, CK14, and the proliferation marker p63 in WT lacrimal gland as well as MECs cultured from WT and TSP1−/− mouse lacrimal gland (Figure 3). Most cells in culture expressed α-SMA and CK14, as do MECs in the lacrimal gland. In addition, a large number of cultured cells expressed p63. Therefore, the overwhelming majority of cells in culture were MECs. An increase in intracellular [Ca2+] is a well-established second messenger system for cell contraction as well as a myriad of other cellular functions, and for that reason, intracellular [Ca2+] is tightly regulated in cells. Intracellular [Ca2+] was measured in cultured MECs before and after addition of the different neural agonists (Figure 4). Cch, a cholinergic agonist, significantly increased intracellular [Ca2+] at 10−4 mol/L and 10−3 mol/L to maximum values of 376.98 ± 226.29 and 403.60 ± 298.54 nmol/L, respectively (Figure 4, A and B). VIP at 10−8 mol/L increased intracellular [Ca2+] to a maximum value of 635.04 ± 328.00 nmol/L (Figure 4, C and D). Ph, an α-adrenergic agonist, produced a significant increase in peak intracellular [Ca2+] with maximum values of 474.40 ± 313.60 and 388.56 ± 341.36 nmol/L at 10−6 mol/L and 10−5 mol/L, respectively (Figure 4, E and F). ATP, an activator of P2X receptors, increased peak intracellular [Ca2+] to 350.68 ± 220.39 nmol/L at 10−6 mol/L (Figur
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