Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Inhibitors of corneal inflammation and angiogenesis: Prospectives and challenges
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.
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: urn:nbn:se:liu:diva-156415DOI: 10.3384/diss.diva-156415ISBN: 9789176850640 (print)OAI: oai:DiVA.org:liu-156415DiVA, id: diva2:1305836
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
List of papers
1. Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model
Open this publication in new window or tab >>Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model
Show others...
2014 (English)In: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 125, p. 118-127Article in journal (Refereed) Published
Abstract [en]

Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-alpha, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-alpha, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2. Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-alpha at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-alpha, CCL2, CCL3, CXCL2, or DLL4.

Place, publisher, year, edition, pages
Elsevier, 2014
Keywords
angiogenesis; neovascularization; cornea; inflammation; dexamethasone; anti-VEGF; confocal microscopy; rat
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-110279 (URN)10.1016/j.exer.2014.06.006 (DOI)000340079300014 ()24933712 (PubMedID)
Note

Funding Agencies|Crown Princess Margaretas Foundation; County Council of Ostergotland; Swedish Research Council [2012-2472]

Available from: 2014-09-05 Created: 2014-09-05 Last updated: 2019-04-18Bibliographically approved
2. Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea
Open this publication in new window or tab >>Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea
Show others...
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
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:liu:diva-140047 (URN)10.1038/s41598-017-07129-4 (DOI)000407569300001 ()28811496 (PubMedID)
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
3. Selective IKK2 inhibitor IMD0354 disrupts NF-kappa B signaling to suppress corneal inflammation and angiogenesis
Open this publication in new window or tab >>Selective IKK2 inhibitor IMD0354 disrupts NF-kappa B signaling to suppress corneal inflammation and angiogenesis
Show others...
2018 (English)In: Angiogenesis, ISSN 0969-6970, E-ISSN 1573-7209, Vol. 21, no 2, p. 267-285Article in journal (Refereed) Published
Abstract [en]

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-kappa B signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-kappa B activation through selective blockade of the IKK complex I kappa B kinase beta (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNF alpha-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-alpha expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-kappa B by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-kappa B signaling in the development of pathologic corneal neovascularization.

Place, publisher, year, edition, pages
Springer Netherlands, 2018
Keywords
Cornea; Neovascularization; NF-kappa B; IMD0354; IKK2; VEGF
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-147373 (URN)10.1007/s10456-018-9594-9 (DOI)000428924500007 ()29332242 (PubMedID)2-s2.0-85041334437 (Scopus ID)
Note

Funding Agencies|Swedish Research Council [2012-2472]; Swedish Foundation Stiftelsen Synframjandets Forskningsfond/Ogonfonden; Svenska Sallskapet for Medicinsk Forskning; Linkoping Universitet; Jeanssons Stiftelser

Available from: 2018-05-18 Created: 2018-05-18 Last updated: 2019-05-01Bibliographically approved
4. Effect of connexin 43 inhibition by the mimetic peptide Gap27 on corneal wound healing, inflammation and neovascularization
Open this publication in new window or tab >>Effect of connexin 43 inhibition by the mimetic peptide Gap27 on corneal wound healing, inflammation and neovascularization
Show others...
2016 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 173, no 19, p. 2880-2893Article in journal (Refereed) Published
Abstract [en]

Background and PurposeThe connexin 43 (Cx43) mimetic peptide Gap27 was designed to transiently block the function of this gap junction. This study was undertaken to investigate the effect of Gap27 on corneal healing, inflammation and neovascularization. Experimental ApproachThe effect of Gap27 on wound healing, inflammation and vascularization was assessed in primary human corneal epithelial cells (HCEC) in vitro and whole human corneas ex vivo, and in an in vivo rat wound healing model. Key ResultsGap27 enhanced the wound closure of HCEC in vitro and accelerated wound closure and stratification of epithelium in human corneas ex vivo, but did not suppress the corneal release of inflammatory mediators IL-6 or TNF- in vivo. In human corneas ex vivo, F4/80 positive macrophages were observed around the wound site. In vivo, topical Gap27 treatment enhanced the speed and density of early granulocyte infiltration into rat corneas. After 7days, the expressions of TNF- and TGF1 were elevated and correlated with inflammatory cell accumulation in the tissue. Additionally, Gap27 did not suppress VEGF release in organotypic culture, nor did it suppress early or late VEGFA expression or neovascularization in vivo. Conclusions and ImplicationsGap27 can be effective in promoting the healing of superficial epithelial wounds, but in deep stromal wounds it has the potential to promote inflammatory cell migration and accumulation in the tissue and does not suppress the subsequent neovascularization response. These results support the proposal that Gap27 acts as a healing agent in the transient, early stages of corneal epithelial wounding.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2016
National Category
Ophthalmology
Identifiers
urn:nbn:se:liu:diva-132050 (URN)10.1111/bph.13568 (DOI)000383668900006 ()27472295 (PubMedID)
Note

Funding Agencies|European Union - Regional, National and International Programs Initiative for the Mobility and Development of researchers careers (I-MOVE) [267232]; European Cooperation in Science and Technology (EU-COST) program [BM1302, COST-STSM-BM1302-24766]

Available from: 2016-10-19 Created: 2016-10-17 Last updated: 2019-04-18

Open Access in DiVA

Inhibitors of corneal inflammation and angiogenesis: Prospectives and challenges(6154 kB)108 downloads
File information
File name FULLTEXT01.pdfFile size 6154 kBChecksum SHA-512
4fd70366e3a4a148c33432490c8d5e10bbceae298524106820d3c31d4cb898e4587b0c704e2353ab4c579b19c26d1a5b002f2b9437fed33d7b4c4578d9448d3e
Type fulltextMimetype application/pdf

Other links

Publisher's full text

Search in DiVA

By author/editor
Mirabelli, Pierfrancesco
By organisation
Department of Clinical and Experimental MedicineFaculty of Medicine and Health SciencesDepartment of Ophthalmology in Linköping
Cell and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 108 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
isbn
urn-nbn

Altmetric score

doi
isbn
urn-nbn
Total: 154 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf