Spatial Distribution of Mast Cells Regulates Asymmetrical Angiogenesis at the Ocular Surface
2021; Elsevier BV; Volume: 191; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2021.02.016
ISSN1525-2191
AutoresWonkyung Cho, Sharad K. Mittal, Elsayed Elbasiony, Sunil K. Chauhan,
Tópico(s)melanin and skin pigmentation
ResumoMast cells, historically known for their function as effector cells in the induction of allergic diseases, reside in all vascularized tissues of the body, particularly, in proximity to blood and lymphatic vessels. Despite being neighboring sentinel cells to blood vessels, whether the spatial distribution of mast cells regulates the degree of angiogenesis remains to be investigated. Herein, an asymmetrical distribution of mast cells was shown at the murine ocular surface, with the higher number of mast cells distributed along the nasal limbus of the cornea compared with the temporal side. Using a well-characterized murine model of suture-induced corneal neovascularization, insult to the nasal side was shown to result in more extensive angiogenesis compared with that to the temporal side. To directly assess the impact of the spatial distribution of mast cell on angiogenesis, neovascularization was induced in mast cell–deficient mice (cKitw-sh). Unlike the wild-type (C57BL/6) mice, cKitw-sh mice did not show disproportionate growth of corneal blood vessels following the temporal and nasal insult. Moreover, cromolyn-mediated pharmacologic blockade of mast cells at the ocular surface attenuated the asymmetrical nasal and temporal neovascularization, suggesting that spatial distribution of mast cells significantly contributes to angiogenic response at the ocular surface. Mast cells, historically known for their function as effector cells in the induction of allergic diseases, reside in all vascularized tissues of the body, particularly, in proximity to blood and lymphatic vessels. Despite being neighboring sentinel cells to blood vessels, whether the spatial distribution of mast cells regulates the degree of angiogenesis remains to be investigated. Herein, an asymmetrical distribution of mast cells was shown at the murine ocular surface, with the higher number of mast cells distributed along the nasal limbus of the cornea compared with the temporal side. Using a well-characterized murine model of suture-induced corneal neovascularization, insult to the nasal side was shown to result in more extensive angiogenesis compared with that to the temporal side. To directly assess the impact of the spatial distribution of mast cell on angiogenesis, neovascularization was induced in mast cell–deficient mice (cKitw-sh). Unlike the wild-type (C57BL/6) mice, cKitw-sh mice did not show disproportionate growth of corneal blood vessels following the temporal and nasal insult. Moreover, cromolyn-mediated pharmacologic blockade of mast cells at the ocular surface attenuated the asymmetrical nasal and temporal neovascularization, suggesting that spatial distribution of mast cells significantly contributes to angiogenic response at the ocular surface. A transparent cornea, devoid of any blood vessels, is essential to maintaining visual acuity.1Foulsham W. Coco G. Amouzegar A. Chauhan S.K. Dana R. When clarity is crucial: regulating ocular surface immunity.Trends Immunol. 2018; 39: 288-301Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,2Cursiefen C. Chen L. Saint-Geniez M. Hamrah P. Jin Y. Rashid S. Pytowski B. Persaud K. Wu Y. Streilein J.W. Dana R. Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.Proc Natl Acad Sci U S A. 2006; 103: 11405-11410Crossref PubMed Scopus (219) Google Scholar Corneal neovascularization, characterized by abnormal new blood vessel growth from preexisting limbal vessel structures, occurs in various corneal pathologies, including inflammatory disorders, trauma, and corneal graft rejection.3Feizi S. Azari A.A. Safapour S. Therapeutic approaches for corneal neovascularization.Eye Vis (Lond). 2017; 4: 28Crossref PubMed Google Scholar,4Chen Y. Chauhan S.K. Lee H.S. Stevenson W. Schaumburg C.S. Sadrai Z. Saban D.R. Kodati S. Stern M.E. Dana R. Effect of desiccating environmental stress versus systemic muscarinic AChR blockade on dry eye immunopathogenesis.Invest Ophthalmol Vis Sci. 2013; 54: 2457-2464Crossref PubMed Scopus (40) Google Scholar Pathologic insults lead to a disruption of the equilibrium of pro-angiogenic and anti-angiogenic factors, resulting in proliferation and migration of vascular endothelial cells to form new blood vessels.5Azar D.T. Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis).Trans Am Ophthalmol Soc. 2006; 104: 264-302PubMed Google Scholar Interestingly, the pathologic growth of blood vessels is not always evenly distributed throughout the cornea. Ocular surface conditions, including peripheral hypertrophic subepithelial corneal opacification and pterygium, characterized by pathologic angiogenesis, have long been clinically observed to predominantly affect the nasal side of the cornea.6Dolezalova V. Is the occurrence of a temporal pterygium really so rare?.Ophthalmologica. 1977; 174: 88-91Crossref PubMed Scopus (21) Google Scholar, 7Kaufman S.C. Jacobs D.S. Lee W.B. Deng S.X. Rosenblatt M.I. Shtein R.M. Options and adjuvants in surgery for pterygium: a report by the American Academy of Ophthalmology.Ophthalmology. 2013; 120: 201-208Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 8Riedl J.C. Wasielica-Poslednik J. Weyer-Elberich V. Vossmerbaeumer U. Pfeiffer N. Lisch W. Gericke A. Visualization of corneal vascularization in peripheral hypertrophic subepithelial corneal opacification with OCT angiography.Acta Ophthalmol. 2018; 96: e974-e978Crossref PubMed Scopus (8) Google Scholar Despite such observations, the underlying mechanism that may contribute to the uneven distribution of neovascularization in tissues, such as the cornea, is yet to be uncovered. Mast cells, the tissue-resident cells, are present throughout vascularized tissues in the body, especially in abundance around the blood and lymph vessels.9Norrby K. Mast cells and angiogenesis.APMIS. 2002; 110: 355-371Crossref PubMed Scopus (301) Google Scholar,10Krystel-Whittemore M. Dileepan K.N. Wood J.G. Mast cell: a multi-functional master cell.Front Immunol. 2016; 6: 620Crossref PubMed Scopus (333) Google Scholar At the ocular surface, mast cells are distributed in the peripheral cornea, limbus, and conjunctiva.11Liu J. Fu T. Song F. Xue Y. Xia C. Liu P. Wang H. Zhong J. Li Q. Chen J. Li Y. Cai D. Li Z. Mast cells participate in corneal development in mice.Sci Rep. 2015; 5: 17569Crossref PubMed Scopus (30) Google Scholar,12Leonardi A. Motterle L. Bortolotti M. Allergy and the eye.Clin Exp Immunol. 2008; 1: 17-21Crossref Scopus (99) Google Scholar On activation, mast cells degranulate and release preformed and newly synthesized inflammatory mediators into the microenvironment.10Krystel-Whittemore M. Dileepan K.N. Wood J.G. Mast cell: a multi-functional master cell.Front Immunol. 2016; 6: 620Crossref PubMed Scopus (333) Google Scholar Granules are composed of various growth factors, cytokines, amines, and enzymes such as tryptase and β-hexosaminidase.9Norrby K. Mast cells and angiogenesis.APMIS. 2002; 110: 355-371Crossref PubMed Scopus (301) Google Scholar,13Wernersson S. Pejler G. Mast cell secretory granules: armed for battle.Nat Rev Immunol. 2014; 14: 478-494Crossref PubMed Scopus (546) Google Scholar The role of mast cells in ocular allergy is well established, and the use of a mast cell inhibitor, cromolyn sodium, to manage allergic symptoms, is a common practice in the clinic. Apart from their well-established role in allergy, mast cells also regulate innate and adaptive immune responses and angiogenesis.14Ribatti D. Crivellato E. Mast cells, angiogenesis, and tumour growth.Biochim Biophys Acta. 2012; 1822: 2-8Crossref PubMed Scopus (130) Google Scholar,15Matsuda K. Okamoto N. Kondo M. Arkwright P.D. Karasawa K. Ishizaka S. Yokota S. Matsuda A. Jung K. Oida K. Amagai Y. Jang H. Noda E. Kakinuma R. Yasui K. Kaku U. Mori Y. Onai N. Ohteki T. Tanaka A. Matsuda H. Mast cell hyperactivity underpins the development of oxygen-induced retinopathy.J Clin Invest. 2017; 127: 3987-4000Crossref PubMed Scopus (17) Google Scholar Ocular surface mast cells promote corneal neovascularization, in part, by secreting high levels of vascular endothelial growth factor-A.16Cho W. Mittal S.K. Elbasiony E. Chauhan S.K. Activation of ocular surface mast cells promotes corneal neovascularization.Ocular Surface. 2020; 18: 857-864Crossref PubMed Scopus (9) Google Scholar In the current study, a series of experiments were conducted to investigate whether mast cells contribute to the observed asymmetry in vessel growth between the nasal and temporal side of the cornea. Specifically, the effect of the spatial distribution of mast cells were investigated on pathologic vessel formation using a well-characterized murine model of inflammatory corneal neovascularization and genetically modified mast cell–deficient cKitw-sh mice. Herein, suture placement on the nasal side resulted in more extensive corneal neovascularization compared with that on the temporal side. Moreover, a higher number of mast cells were observed on the nasal half of the cornea compared with the temporal half. However, mast cell deficiency and pharmacologic blockade of mast cell activation abrogated the difference in the degree of angiogenesis following nasal and temporal insult, suggesting a critical contribution of mast cells in promoting disproportionate angiogenic response at the ocular surface. BALB/c mice, aged 6 to 8 weeks, were used for the described experiments. A fully congenic cKitw-sh mouse strain on a C57BL/6J genetic background (stock number 012861) and sex- and age-matched C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME).17Grimbaldeston M.A. Chen C.C. Piliponsky A.M. Tsai M. Tam S.Y. Galli S.J. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar Littermates were used for each set of experiments. cKitw-sh mice were confirmed for their deficiency in mast cells at the ocular surface and in the peritoneum and for their comparable generation of total CD45+ cells and myeloid cells in the bone marrow compared with wild-type mice.17Grimbaldeston M.A. Chen C.C. Piliponsky A.M. Tsai M. Tam S.Y. Galli S.J. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar,18Elbasiony E. Mittal S.K. Foulsham W. Cho W. Chauhan S.K. Epithelium-derived IL-33 activates mast cells to initiate neutrophil recruitment following corneal injury.Ocular Surface. 2020; 18: 633-640Crossref PubMed Scopus (15) Google Scholar There is an increased accumulation of neutrophils and platelets in different lymph organs of cKitw-sh.19Nigrovic P.A. Gray D.H.D. Jones T. Hallgren J. Kuo F.C. Chaletzky B. Gurish M. Mathis D. Benoist C. Lee D.M. Genetic inversion in mast cell-deficient (Wsh) mice interrupts corin and manifests as hematopoietic and cardiac aberrancy.Am J Pathol. 2008; 173: 1693-1701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar All protocols were approved by the Animal Care and Use Committee of Schepens Eye Research Institute. The mice were housed in the Schepens Eye Research Institute animal vivarium and treated according to the Association for Research in Vision and Ophthalmology Guidelines for Use of Animals in Ophthalmic and Vision Research. Corneal neovascularization was induced by placing a single intrastromal suture on anesthetized mice, as previously described.20Jin Y. Chauhan S.K. El Annan J. Annan J.E. Sage P.T. Sharpe A.H. Dana R. A novel function for programmed death ligand-1 regulation of angiogenesis.Am J Pathol. 2011; 178: 1922-1929Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar,21Saban D.R. Bock F. Chauhan S.K. Masli S. Dana R. Thrombospondin-1 derived from APCs regulates their capacity for allosensitization.J Immunol. 2010; 185: 4691-4697Crossref PubMed Scopus (37) Google Scholar Briefly, a single figure-of-eight suture was intrastromally placed on either the nasal or the temporal side of the cornea, 1.0 mm from the limbal area using 11.0 nylon sutures (MANI, Tochigi, Japan). The sutures remained in the cornea and induced inflammation and neovascularization (Figure 1). Following suture placement, a triple antibiotic ointment was applied topically. To subside suture-induced pain, buprenorphine was administered via s.c. injection. Ocular surface tear wash (5 μL/wash) was collected at 0, 1, 3, and 6 hours after suture placement to measure mast cell activation. Mice were clinically assessed using a slit lamp, and subsequently, on day 4, were euthanized, and their corneas (including the corneal limbus and conjunctiva) were harvested for further analysis. Three microliters of phosphate-buffered saline (PBS) or 2% sodium cromolyn in PBS (Sigma-Aldrich Corp., St. Louis, MO) was administered topically to sutured corneas at five time points on the day of suture placement (–3, –1, 1, 3, and 6 hours postoperatively). Thereafter, PBS or cromolyn was administered topically five times per day (every 3 hours) for 4 days. Corneas were harvested and lysed using the freeze-thaw method. Briefly, corneas were digested by placing the corneas alternatively in dry ice and 37°C water bath. Corneal tissue was mechanically lysed using a pellet pestle (Life Sciences, Waltham, MA) between every cycle. A total of seven complete freeze-thaw cycles were completed before centrifuging the cells. The supernatant was collected for tryptase and β-hexosaminidase analysis. Mast Cell Degranulation Assay Kits (Sigma-Aldrich) were used to quantify levels of tryptase enzyme. The kit detects the chromophore p-nitroaniline cleaved from the labeled substrate tosyl-gly-pro-lys-p-nitroaniline.22Summers S.A. Gan P.Y. Dewage L. Ma F.T. Ooi J.D. O'Sullivan K.M. Nikolic-Paterson D.J. Kitching A.R. Holdsworth S.R. Mast cell activation and degranulation promotes renal fibrosis in experimental unilateral ureteric obstruction.Kidney Int. 2012; 82: 676-685Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar In brief, ocular surface tear wash or corneal lysates were incubated with 0.1 mg/mL tosyl-gly-pro-lys-p-nitroaniline (substrate) for 3 hours at 37°C. A SpectraMax Plus 384 Microplate Reader (Molecular Devices, San Jose, CA) was used to quantify free p-nitroaniline at 405 nm. Levels of β-hexosaminidase enzyme were quantified using β-n-acetylglucosaminidase assay kits (Sigma-Aldrich). The kit measures the level of 4-nitrophenyl N-acetyl-β-d-glucosaminide hydrolysis.23Wolf A.J. Reyes C.N. Liang W. Becker C. Shimada K. Wheeler M.L. Cho H.C. Popescu N.I. Coggeshall K.M. Arditi M. Underhill D.M. Hexokinase is an innate immune receptor for the detection of bacterial peptidoglycan.Cell. 2016; 166: 624-636Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar In brief, corneal lysates or ocular surface tear wash was incubated with 0.1 mg/mL 4-nitrophenyl N-acetyl-β-d-glucosaminide (substrate) for 1 hour at 37°C. The enzyme-substrate reaction was stopped with 5 mg/mL sodium carbonate, and absorbance at 405 nm was measured using SpectraMax Plus 384 Microplate Reader (Molecular Devices). β-Hexosaminidase levels were estimated using the formula: U/mL = (A405sample – A405blank) × 0.05 × 0.3 × Dilution Factor/A405 standard × time × volume of sample in milliliters. Corneas with limbus were harvested and immunostained, as previously described.24Chauhan S.K. Jin Y. Goyal S. Lee H.S. Fuchsluger T.A. Lee H.K. Dana R. A novel pro-lymphangiogenic function for Th17/IL-17.Blood. 2011; 118: 4630-4634Crossref PubMed Scopus (78) Google Scholar,25Ferrari G. Hajrasouliha A.R. Sadrai Z. Ueno H. Chauhan S.K. Dana R. Nerves and neovessels inhibit each other in the cornea.Invest Ophthalmol Vis Sci. 2013; 54: 813-820Crossref PubMed Scopus (51) Google Scholar Briefly, corneal stroma and epithelium were separated after EDTA treatment for 30 minutes at 37°C, and the stroma was fixed using 4% paraformaldehyde. Tissues were incubated at 4°C with fluorescein isothiocyanate–conjugated CD31 (Biolegend, San Diego, CA) overnight. To stain for avidin, the harvested cornea was fixed using 4% paraformaldehyde and was subsequently incubated at 4°C with Texas red–conjugated avidin (ThermoFisher, Waltham, MA) for 6 hours. Stained corneas were whole mounted on slides using VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and visualized using a confocal microscope (Leica TCS-SP5; Buffalo Grove, IL). The area covered by blood vessel (CD31+) and the number of mast cells (avidin+) was calculated using ImageJ version 1.52v software (NIH, Bethesda, MD; https://imagej.nih.gov/ij, last accessed November 20, 2020).26Sahu S.K. Mittal S.K. Foulsham W. Li M. Sangwan V.S. Chauhan S.K. Mast cells initiate the recruitment of neutrophils following ocular surface injury.Invest Ophthalmol Vis Sci. 2018; 59: 1732-1740Crossref PubMed Scopus (21) Google Scholar Slit-lamp biomicroscopy was used to clinically evaluate corneal neovascularization following suture placement and to acquire images.27Chung E.S. Saban D.R. Chauhan S.K. Dana R. Regulation of blood vessel versus lymphatic vessel growth in the cornea.Invest Ophthalmol Vis Sci. 2009; 50: 1613-1618Crossref PubMed Scopus (32) Google Scholar The slit-lamp analysis was performed on days 2 and 4 after suture placement. Slit-lamp images were converted into binary images, and vascular density as percentage area of the vessels in the total cornea was calculated using the vessel analysis plugin in ImageJ version 1.52v. Unpaired two-tailed t-tests were used to compare means between two groups. The significance level was set at P < 0.05. Data are presented as the means ± SEM. The results shown are representative of at least three independent experiments. Sample sizes were estimated on the basis of previous studies on corneal angiogenesis.16Cho W. Mittal S.K. Elbasiony E. Chauhan S.K. Activation of ocular surface mast cells promotes corneal neovascularization.Ocular Surface. 2020; 18: 857-864Crossref PubMed Scopus (9) Google Scholar,27Chung E.S. Saban D.R. Chauhan S.K. Dana R. Regulation of blood vessel versus lymphatic vessel growth in the cornea.Invest Ophthalmol Vis Sci. 2009; 50: 1613-1618Crossref PubMed Scopus (32) Google Scholar To investigate the degree of neovascularization depending on the site of insult, a model of inflammatory corneal neovascularization was used.28Giacomini C. Ferrari G. Bignami F. Rama P. Alkali burn versus suture-induced corneal neovascularization in C57BL/6 mice: an overview of two common animal models of corneal neovascularization.Exp Eye Res. 2014; 121: 1-4Crossref PubMed Scopus (31) Google Scholar A single intrastromal figure-of-eight suture 1 mm from the limbus was placed on either the nasal or the temporal side of the cornea of BALB/c mice (Figure 1A). Nonsutured corneas served as controls. Corneal neovascularization was clinically assessed every 2 days using a slit-lamp biomicroscope (Figure 1B). Binary images from slit-lamp images were generated to quantify vascular density using the ImageJ version 1.52v software. Nasal suture resulted in significantly more neovascularization compared with temporal suture on day 4 after suture placement (P = 0.01) (Figure 1C). To quantify the growth of the microvessels not visible to the naked eye, corneas were harvested on day 4 for immunohistochemistry analysis. Whole-mounted corneas were immunostained with fluorochrome-conjugated CD31, and blood vessel growth was measured by calculating the vessel area covered by the new branching blood vessels using ImageJ version 1.52v software. Consistent with previous clinical observations,6Dolezalova V. Is the occurrence of a temporal pterygium really so rare?.Ophthalmologica. 1977; 174: 88-91Crossref PubMed Scopus (21) Google Scholar,7Kaufman S.C. Jacobs D.S. Lee W.B. Deng S.X. Rosenblatt M.I. Shtein R.M. Options and adjuvants in surgery for pterygium: a report by the American Academy of Ophthalmology.Ophthalmology. 2013; 120: 201-208Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar significantly higher nasal neovascularization was observed compared with temporal neovascularization following suture placement (P = 0.04) (Figure 1D). These results suggest that the location of the insult mediates the degree of neovascularization, specifically that suture placed on the nasal side results in increased neovascularization compared with that placed on the temporal side of the cornea. Having observed greater corneal neovascularization following nasal suture, the mechanism underlying the difference in blood vessel growth was investigated. Mast cells, distributed along the limbal area of the cornea,18Elbasiony E. Mittal S.K. Foulsham W. Cho W. Chauhan S.K. Epithelium-derived IL-33 activates mast cells to initiate neutrophil recruitment following corneal injury.Ocular Surface. 2020; 18: 633-640Crossref PubMed Scopus (15) Google Scholar are associated with angiogenesis in various pathologies15Matsuda K. Okamoto N. Kondo M. Arkwright P.D. Karasawa K. Ishizaka S. Yokota S. Matsuda A. Jung K. Oida K. Amagai Y. Jang H. Noda E. Kakinuma R. Yasui K. Kaku U. Mori Y. Onai N. Ohteki T. Tanaka A. Matsuda H. Mast cell hyperactivity underpins the development of oxygen-induced retinopathy.J Clin Invest. 2017; 127: 3987-4000Crossref PubMed Scopus (17) Google Scholar,29Coussens L.M. Raymond W.W. Bergers G. Laig-Webster M. Behrendtsen O. Werb Z. Caughey G.H. Hanahan D. Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.Genes Dev. 1999; 13: 1382-1397Crossref PubMed Scopus (786) Google Scholar; therefore, the hypothesis that mast cell distribution and activation account for the difference in neovascularization was tested. To investigate mast cell distribution, naïve corneas were harvested with limbus and cut into nasal and temporal halves. Each half of the cornea was immunostained with fluorochrome-conjugated avidin, which specifically binds to mast cells (Figure 2A). The numbers of avidin+ mast cells were quantified using ImageJ version 1.52v software. A significantly higher number of mast cells were distributed along the limbus of the nasal half of the cornea compared with the temporal half (P = 0.02) (Figure 2B). To further confirm the spatial distribution of mast cells, nasal and temporal halves of the naïve cornea were lysed, and levels of mast cell–specific markers, tryptase, and β-hexosaminidase, were quantified.30Moon T.C. Befus A.D. Kulka M. Mast cell mediators: their differential release and the secretory pathways involved.Front Immunol. 2014; 5: 569Crossref PubMed Scopus (216) Google Scholar The nasal half showed significantly higher levels of tryptase and β-hexosaminidase, compared with the temporal half (P = 0.02 and P = 0.007, respectively) (Figure 2C). To assess whether this dichotomy of mast cell distribution perpetuates during neovascularization, tear wash was collected following suture placement to analyze secretion of tryptase and β-hexosaminidase (Figure 2D). The suture placed on the nasal side resulted in a higher fold increase in tryptase [4.3-fold versus 1.4-fold (temporal); P = 0.0002] and β-hexosaminidase levels [2.1-fold versus 1.1-fold (temporal); P = 0.006], compared with that placed on the temporal side. Our data indicate a higher distribution of mast cell on the nasal half of the cornea, suggesting that observed differences in suture-induced mast cell activation result from the underlying difference in spatial distribution of mast cells. Having observed increased neovascularization and enhanced mast cell activation following insult to the nasal side, whether the spatial distribution of mast cells was critical to the asymmetrical angiogenic response was assessed. To determine this, corneal neovascularization was indiced in mast cell–deficient mice (cKitw-sh) and wild-type C57BL/6 mice (control). cKitw-sh mice are deficient in mast cells at the ocular surface and in the peritoneum with comparable generation of total CD45+ cells and myeloid cells in the bone marrow compared with wild-type mice.17Grimbaldeston M.A. Chen C.C. Piliponsky A.M. Tsai M. Tam S.Y. Galli S.J. Mast cell-deficient W-sash c-kit mutant Kit W-sh/W-sh mice as a model for investigating mast cell biology in vivo.Am J Pathol. 2005; 167: 835-848Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar,18Elbasiony E. Mittal S.K. Foulsham W. Cho W. Chauhan S.K. Epithelium-derived IL-33 activates mast cells to initiate neutrophil recruitment following corneal injury.Ocular Surface. 2020; 18: 633-640Crossref PubMed Scopus (15) Google Scholar However, there is an increased accumulation of neutrophils and platelets in different lymph organs of cKitw-sh.19Nigrovic P.A. Gray D.H.D. Jones T. Hallgren J. Kuo F.C. Chaletzky B. Gurish M. Mathis D. Benoist C. Lee D.M. Genetic inversion in mast cell-deficient (Wsh) mice interrupts corin and manifests as hematopoietic and cardiac aberrancy.Am J Pathol. 2008; 173: 1693-1701Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar Immunohistochemistry analysis using avidin staining confirmed the deficiency of mast cells at the ocular surface in cKitw-sh mice (Figure 3A). Neovascularization was clinically assessed in naïve cornea following suture placement on days 2 and 4 using a slit-lamp biomicroscope (Figure 3B). Slit-lamp images were converted into binaries using ImageJ version 1.52v software to quantify vascular density. As in BALB/c mice (Figure 1B), C57BL/6 mice showed a higher degree of neovascularization following suture placement on the nasal side compared with that on the temporal side (Figure 3B). However, no significant difference in corneal neovascularization was observed following nasal and temporal suture in cKitw-sh mice on day 4 (Figure 3, B and C). Collectively, current data suggest that asymmetrical neovascularization is dependent on the spatial distribution of mast cells at the ocular surface. Finally, to assess whether inhibiting mast cell activation nullifies the difference in the degree of neovascularization, corneas were topically treated with 2% cromolyn, a clinically used mast cell stabilizer.31Altounyan R.E. Review of clinical activity and mode of action of sodium cromoglycate.Clin Allergy. 1980; : 481-489Crossref PubMed Scopus (89) Google Scholar Corneas were treated, as outlined in the experimental design, with cromolyn or PBS control (Figure 4A). Topical treatment of cromolyn significantly suppressed ocular surface mast cell activation, as confirmed by lower levels of tryptase in cromolyn-treated corneas (Figure 4B). Corneal neovascularization was clinically assessed every 2 days using a slit-lamp biomicroscope (Figure 4C). Slit-lamp images were converted into binary images to measure vascular density using ImageJ version 1.52sv software. Cromolyn treatment abrogated the difference in suture-induced nasal and temporal neovascularization, with a dramatic reduction in total vessel growth at the ocular surface (Figure 4D). Furthermore, cromolyn-mediated attenuation of asymmetrical neovascularization was confirmed using immunohistochemistry analysis of corneas harvested on day 4 after suture placement. Consistent with the slit-lamp observation, no significant difference was observed in the suture-induced growth of CD31+ vessels between the nasal and temporal side following cromolyn treatment (Figure 4E). In addition, the effect of cromolyn on vascular endothelial cells was tested using an in vitro culture system. No difference in the proliferation of vascular endothelial cells between PBS- and cromolyn-treated cultures (Supplemental Figure S1) was observed. Taken together, the data demonstrate that pharmacologic blockade of mast cells abrogates the difference in the degree of suture-induced neovascularization. Corneal neovascularization, a prevalent adverse effect of various ocular surface conditions, disrupts the corneal clarity by inducing persistent inflammation.32Clements J.L. Dana R. Inflammatory corneal neovascularization: etiopathogenesis.Semin Ophthalmol. 2011; 26: 235-245Crossref PubMed Scopus (49) Google Scholar,33Chauhan S.K. Dohlman T.H. Dana R. Corneal lymphatics: role in ocular inflammation as inducer and responder of adaptive immunity.J Clin Cell Immunol. 2014; 5: 1000256Crossref PubMed Google Scholar This study advances our understanding of corneal neovascularization and the contribution of the spatial distribution of mast cells to the disproportionate angiogenic response. Specifically, the current data show that i) suture placement on the nasal side results in greater corneal neovascularization than that on the temporal side, ii) higher number of mast cells are distributed on the nasal side of the cornea, iii) mast cell deficiency results in comparable levels of corneal neovascularization following nasal and temporal insult, and iv) pharmacologic blockade of mast cells abrogates the difference in degree of suture-induced nasal and temporal neovascularization. Despite disproportionate neovascularization affecting various corneal pathologies, only a few studies have investigated the spatial distribution of pathologic neovascularization across the cornea. Pterygium, a thickened triangular tissue growth of the cornea, is marked by a high degree of vascularization, particularly on the nasal side.7Kaufman S.C. Jacobs D.S. Lee W.B. Deng S.X. Rosenblatt M.I. Shtein R.M. Options and adjuvants in surgery for pterygium: a report by the American Academy of Ophthalmology.Ophthalmology. 2013; 120: 201-208Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar,34Chan C.M.L. Chew P.T.K. Alsagoff Z. Wong J.S. Tan D.T.H. Vascular patterns in pterygium and conjunctival autografting: a pilot study using indocyanine green anterior segment angiography.Br J Ophthalmol. 2001; 85: 350-353Crossref PubMed Scopus (27) Google Scholar Similarly, significantly more neovascularization was observed on the nasal side of the cornea following suture placement compared with the temporal side. This study shows, for the first time, that spatial distribution of mast cells contributes to the nasal preferences of corneal neovascularization. Mast cells, the tissue-resident secretory cells, have been implicated in various pathologic conditions, including angiogenesis.35Woolley D.E. The mast cell in inflammatory arthritis.N Engl J Med. 2003; 348: 1709-1711Crossref PubMed Scopus (115) Google Scholar,36Chen Y. Li C. Xie H. Fan Y. Yang Z. Ma J. He D. Li L. Infiltrating mast cells promote renal cell carcinoma angiogenesis by modulating PI3K→AKT→GSK3beta→AM signaling.Oncogene. 2017; 36: 2879-2888Crossref PubMed Scopus (34) Google Scholar The current data, consistent with previous studies, show that mast cells are primarily located around the limbus in the cornea.11Liu J. Fu T. Song F. Xue Y. Xia C. Liu P. Wang H. Zhong J. Li Q. Chen J. Li Y. Cai D. Li Z. Mast cells participate in corneal development in mice.Sci Rep. 2015; 5: 17569Crossref PubMed Scopus (30) Google Scholar,12Leonardi A. Motterle L. Bortolotti M. Allergy and the eye.Clin Exp Immunol. 2008; 1: 17-21Crossref Scopus (99) Google Scholar Interestingly, a significantly higher number of mast cells were seen to be distributed along the nasal limbus of the cornea. 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Targeted mast cell silencing protects against joint destruction and angiogenesis in experimental arthritis in mice.Arthritis Rheum. 2007; 56: 1806-1816Crossref PubMed Scopus (75) Google Scholar Topical administration of cromolyn effectively inhibits nonallergic ocular inflammation.26Sahu S.K. Mittal S.K. Foulsham W. Li M. Sangwan V.S. Chauhan S.K. Mast cells initiate the recruitment of neutrophils following ocular surface injury.Invest Ophthalmol Vis Sci. 2018; 59: 1732-1740Crossref PubMed Scopus (21) Google Scholar,45Li M. Mittal S.K. Foulsham W. Amouzegar A. Sahu S.K. Chauhan S.K. Mast cells contribute to the induction of ocular mucosal alloimmunity.Am J Transplant. 2019; 19: 662-673Crossref PubMed Scopus (9) Google Scholar The current data show cromolyn treatment results in no significant difference in suture-induced neovascularization between the nasal and temporal side of the cornea, corroborating the observation in cKitw-sh mice. Additionally, cromolyn treatment resulted in significantly reduced corneal neovascularization. These findings suggest that mast cell blockade could be a potential therapeutic strategy to suppress neovascularization at the ocular surface and other vascularized organs, regardless of the site of the insult. Moreover, the current data showing a strong correlation between nasal mast cell distribution and neovascularization along with previous reports on increased mast cell density in promoting tumor angiogenesis suggest that tissue-specific immunologic niche may determine the severity of pathologic angiogenesis in different tissues.46Takanami I. Takeuchi K. Naruke M. Mast cell density is associated with angiogenesis and poor prognosis in pulmonary adenocarcinoma.Cancer. 2000; 88: 2686-2692Crossref PubMed Scopus (197) Google Scholar,47Dundar E. Oner U. Peker B.C. Metintas M. Isiksoy S. Ak G. The significance and relationship between mast cells and tumour angiogenesis in non-small cell lung carcinoma.J Int Med Res. 2008; 36: 88-95Crossref PubMed Scopus (41) Google Scholar Taken together, our data demonstrate that the mast cells orchestrate site-specific neovascularization at the ocular surface and that inhibition of mast cell function abrogates the asymmetrical angiogenic response between the nasal and temporal portion of the cornea. These findings provide novel insights into the importance of the spatial distribution of mast cells in regulating the disproportionate pathologic growth of blood vessels. Download .jpg (.1 MB) Help with files Supplemental Figure S1Cromolyn does not regulate the proliferation of microvascular endothelial cells. A total of 5 × 103 vascular endothelial cells (VECs) were cultured in a 96-well flat-bottom plate in EBM-2 basal medium (Lonza) or supplemented media (5% fetal bovine serum, vascular endothelial growth factor, FGF, EGF, or IGF) and treated with phosphate-buffered saline (PBS) or 2% cromolyn for 36 hours. A: Proliferation was assessed using bromodeoxyuridine (BrdU) assay, as per manufacturer's protocol (Millipore). VEC proliferation, as indicated by BrdU assay. Proliferation was calculated as percentage proliferation of VECs in supplemented media from basal media. B: Total RNA was purified and reverse transcribed into cDNA using Superscript III (Invitrogen, Carlsbad, CA). Real-time PCR was conducted using TaqMan Universal PCR Mastermix and preformulated primers for murine VEGFR2 (Mm00440099_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Mm99999915_gl). Fold change in mRNA expression of vascular endothelial growth factor receptor 2 (VEGFR2) in VECs in supplement media, from VECs in basal media. Representative data from three independent experiments are shown. Data are represented as means ± SD (A and B). Download .jpg (.11 MB) Help with files Supplemental Figure S2Down-regulation of angiogenic factor expression in cromolyn-treated corneas One figure-of-eight intrastromal suture was placed in BALB/c mice. Mice were treated topically every 3 hours using 2% cromolyn eye drops of phosphate-buffered saline (PBS; 3 μL) for five times a day for 4 days. Corneas were harvested on day 4 after suture placement and were lysed to evaluate the expression of VEGF-A (Mm00437304_m1) and FGF2 (Mm01285715_m1) using real-time PCR. mRNA was normalized to GAPDH (internal control). Representative data from two independent experiments are shown, and each experiment consisted of four animals. Data are represented as means ± SD. ∗P < 0.05, ∗∗P < 0.01 (t-test). W.C., S.K.M., and S.K.C. assisted in designing the study, W.C. and E.E. performed experiments, W.C., S.K.M., and S.K.C. analyzed the data, and W.C., S.K.M., and S.K.C. wrote the manuscript. S.K.C. contributed the underlying hypothesis.
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