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Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Ophthalmology in Linköping.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Ophthalmology in Linköping.ORCID iD: 0000-0001-8722-9155
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 7616Article in journal (Refereed) Published
Abstract [en]

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P amp;lt; 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP , 2017. Vol. 7, article id 7616
National Category
Immunology in the medical area
Identifiers
URN: urn:nbn:se:liu:diva-140047DOI: 10.1038/s41598-017-07129-4ISI: 000407569300001PubMedID: 28811496OAI: oai:DiVA.org:liu-140047DiVA, id: diva2:1136615
Note

Funding Agencies|Swedish Research Council [2012-2472]; Swedish Foundation Stiftelsen Synframjandets Forskningsfond/Ogonfonden

Available from: 2017-08-28 Created: 2017-08-28 Last updated: 2019-04-18
In thesis
1. Regulation of inflammation and angiogenesis in the cornea
Open this publication in new window or tab >>Regulation of inflammation and angiogenesis in the cornea
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Inflammation and angiogenesis, the growth of new blood vessels from pre-existing ones, are involved in tumor growth, ocular diseases and wound healing. In ocular angiogenesis, new pathological vessels grow into a specific eye tissue, leak fluid, and disrupt vision. The development of safe and effective therapies for ocular angiogenesis is of great importance for preventing blindness, given that current treatments have limited efficacy or are associated with undesirable side effects. The search for alternative treatment targets requires a deeper understanding of inflammation and how it can lead to angiogenesis in the eye in pathologic situations. This thesis provides new insights into the regulation of inflammation and angiogenesis, particularly at the gene expression and phenotypic levels, in different situations characterized by angiogenesis of the cornea, often called corneal neovascularization. For instance, specific genes and pathways are either endogenously activated or suppressed during active inflammation, wound healing, and during resolution of inflammation and angiogenesis, serving as potential targets to modulate the inflammatory and angiogenic response. In addition, as part of the healing response to restore corneal transparency, inflammation and angiogenesis subside with time in the cornea. In this context, LXR/RXR signaling was found to be activated in a time-dependent manner, to potentially regulate resolution of inflammation and angiogenesis. During regression of new angiogenic capillaries, ghost vessels and empty basement membrane sleeves are formed, which can persist in the cornea for a long time. Here, ghost vessels were found to facilitate subsequent revascularization of the cornea, while empty basement membrane sleeves did not revascularize. The revascularization response observed here was characterised by vasodilation, increased inflammatory cell infiltration and by sprouting at the front of the reperfused vessels. Importantly, reactive oxygen species and nitrous oxide signaling among other pro-inflammatory pathways were activated, and at the same time anti-inflammatory LXR/RXR signaling was inhibited. The interplay between activation and inhibition of these pathways highlights potential mechanisms that regulate corneal revascularization. When treating corneal neovascularization clinically, corticosteroids are in widespread use due to their effectiveness. To minimize the many undesirable side effects associated with corticosteroid use, however, identifying new and more selective agents is of great importance. Here, it was observed that corticosteroids not only suppressed pro-inflammatory chemokines and cytokines, but also activated the classical complement pathway. Classical complement may represent a candidate for further selective therapeutic manipulation to investigate its effect on treatment of corneal neovascularization.

In summary, this thesis identifies genes, pathways, and phenotypic responses involved in sprouting and remodeling of corneal capillaries, highlights novel pathways and factors that may regulate inflammation and angiogenesis in the cornea, and provides insights into regulation of capillary regression and reactivation. Further investigation of these regulatory mechanisms may offer alternative and effective treatment targets for the treatment of corneal inflammation and angiogenesis.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2018. p. 55
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1625
National Category
Ophthalmology Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:liu:diva-147979 (URN)10.3384/diss.diva-147979 (DOI)9789176852842 (ISBN)
Public defence
2018-06-01, Nils-Holger salen, Campus US, Linköping, 13:06 (English)
Opponent
Supervisors
Available from: 2018-05-21 Created: 2018-05-21 Last updated: 2019-09-30Bibliographically approved
2. Inhibitors of corneal inflammation and angiogenesis: Prospectives and challenges
Open this publication in new window or tab >>Inhibitors of corneal inflammation and angiogenesis: Prospectives and challenges
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pathologic angiogenesis is involved in cancer and several blinding conditions such as wet age-related macular degeneration, proliferative retinopathies and corneal neovascularization.

In these dieseases, the angiogenic triggers are hypoxia and inflammation, and both involve the main angiogenic mediator, which is Vascular Endothelial Growth Factor (VEGF). Among available treatments, anti-VEGF often shows limited or temporary efficacy, while steroids are potentially responsible for many side-effects. This thesis presents a series of linked studies aimed at elucidating the early pathologic changes leading to inflammation and corneal neovascularization, and how various treatments affect this process. In this thesis, anti-inflammatory and anti-angiogenic treatments are applied in corneal neovascularization models, to identify VEGF-independent pathways and other novel factors as future therapy targets, as well as to investigate the endogenous modulation of angiogenesis.

A model of experimental neovascularization in the rat cornea was used as main model, where the neovascular response is triggered by a surgical suture placed into the cornea. Investigational treatments (anti-Vegf, dexamethasone, IMD0354, Gap27, or control substances) were then given topically, with the exception of IMD0354, which was given systemically. The effects in the cornea were studied in vivo with slit lamp photography to assess and quantify macroscopic vessel growth and using in vivo confocal microscopy (IVCM) to study cell infiltration and limbal vessel dilation and detect microscopic vessel sprouts; these examinations were performed longitudinally. Genomic analysis with RNA microarray, selected gene expression with q-RT-PCR, and selected protein expression in tissue (immunohistochemistry, immunofluorescence, Western blot) were performed at different time-points. Moreover, other experiments on cell cultures (HUVEC and HCEC), organ cultures (human corneas), ex vivo models (aortic rings) and in vivo studies (zebrafish vasculogenesis) were performed.

Dexamethasone suppressed limbal vasodilation and corneal neovascularization more than anti-Vegf, despite no difference in inflammatory cell infiltration into the cornea. Five-hundred eleven fewer genes were differentially expressed in dexamethasone-treated corneas relative to naïve corneas, compared to anti-Vegf. Among them, several major pro-angiogenic and pro-inflammatory factors and chemokines were suppressed only by dexamethasone and represent novel candidate factors to target in order to improve anti-VEGF treatment. On the other hand, selective inhibition of a single inflammatory pathway (NF-κB), despite showing similar early effects as dexamethasone in suppressing tissue inflammation, was not effective enough to suppress new vessel growth. The same factors suppressed by dexamethasone are also inhibited in endogenous modulation of angiogenesis. Surprisingly, dexamethasone activated several complement factors, which could possibly be beneficial in the anti-angiogenic response.

In a different therapeutic approach, promoting cell migration to accelerate epithelial wound closure similarly was not sufficient to avoid inflammation and angiogenesis in the cornea.

In conclusion, new and more effective treatments are needed for corneal inflammation and neovascularization with fewer side-effects. In this thesis, several novel factors and mechanisms related to inflammation are identified, factors that are not addressed by anti-Vegf therapy, and therefore represent interesting objects for further study, as they have the potential to be targets for adjuvant therapy. Specific anti-inflammatory treatment as well as therapeutic activation of endogenous regulatory pathways, and potentially complement modulation, might represent new strategies to improve anti-angiogenic therapy, but when used alone they do not seem to avoid corneal neovascularization.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2019. p. 91
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1685
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-156415 (URN)10.3384/diss.diva-156415 (DOI)9789176850640 (ISBN)
Public defence
2019-05-17, Nils Holgersalen, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2019-04-30 Created: 2019-04-18 Last updated: 2019-04-30Bibliographically approved

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Department of Clinical and Experimental MedicineFaculty of Medicine and Health SciencesDepartment of Ophthalmology in LinköpingDivision of Neuro and Inflammation Science
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