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Angiogenesis from a new perspective
Linköping University, Department of Clinical and Experimental Medicine, Ophthalmology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Ophthalmology in Linköping.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis is the emergence of new blood and lymph vessels from existing ones. In the pathologic form it contributes to the onset and progression of numerous different human disorders such as cancer, inflammation, atherosclerosis and blinding eye diseases. There exist a number of models to study angiogenesis, both in vitro and in vivo, but there is no single perfect model so far. Consequently there is a need to develop new angiogenesis assays for evaluating blood and lymph vessel behaviour in different physiologic settings.

The aim of this thesis was to gain insight into in vivo angiogenesis introducing a new technique in an inflammatory corneal model. The method involved in vivo examination of the cornea and subsequent comparison of in vivo findings with ex vivo immunohistochemical analysis of the same tissue samples. An existing suture model for inflammatory angiogenesis in the cornea was modified for in vivo observations with a clinically-approved corneal confocal microscope.

In this thesis, corneal lymph vessels were characterized for the first time in vivo and findings from the experimental bench could be applied in a clinical setting, where presumed lymphatics were observed in a corneal transplant patient with rejection. Furthermore, the technique was extended to investigate time-lapse processes in sprouting and regressing capillaries, and led to a number of new observations. CD11b+ myeloid cells constitute the first bulk of infiltrating inflammatory cells and contribute to inflammatory sprouting and regression in numerous ways including pre-patterning of the corneal stroma and guiding of capillary sprouts. Newly formed hemangiogenic sprouts are perfused with a slow-moving fluid and have a lumen. In blood vessel regression, capillary remodeling occurred by abandonment of sprout tips in close association with macrophages and vascular loops formed by presumed intussusceptive angiogenesis. In addition, a network of pericyte- and endothelium-free basement membrane tubes was formed after desertion or degradation of vascular endothelium in former corneal capillaries.

In conclusion, we introduce a new in vivo technique for investigating angiogenesis in a corneal model were in vivo findings can be interpreted with ex vivo definitions of specific cell types by immunohistochemistry. Findings from pre-clinical experiments have been possible to apply in a clinical setting when examining patients with corneal pathology.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2012. , 91 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1284
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-73137ISBN: 978-91-7519-999-3 (print)OAI: oai:DiVA.org:liu-73137DiVA: diva2:467111
Public defence
2012-01-20, Nils-Holger salen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2011-12-19 Created: 2011-12-19 Last updated: 2014-06-05Bibliographically approved
List of papers
1. Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy
Open this publication in new window or tab >>Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy
2010 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 51, no 2, 830-835 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE. To determine whether in vivo confocal microscopy (IVCM) of the cornea can be used for the label-free detection and monitoring of lymph vessels in live corneas.

METHODS. Parallel corneal hemangiogenesis and lymphangiogenesis was induced by the placement of a single suture in one cornea of male Wistar rats. Fourteen days after suture placement and under general anesthesia, laser-scanning IVCM was performed in the vascularized region. Corneas were subsequently excised for flat-mount double immunofluorescence with a pan-endothelial marker (PECAM-1/CD31) and a lymphatic endothelial specific marker (LYVE-1). Using the suture area and prominent blood vessels as points of reference, the identical microscopic region was located in both fluorescent and archived in vivo images. Additionally, vessel diameter, lumen contrast, and cell diameter and velocity within vessels were quantified from in vivo images.

RESULTS. Comparison of identical corneal regions in fluorescence and in vivo revealed prominent CD31(+)/LYVE-1(3+) lymph vessels that were visible in vivo. In vivo, corneal lymph vessels were located in the vascularized area in the same focal plane as blood vessels but had a darker lumen (P andlt; 0.001) sparsely populated by highly reflective cells with diameters similar to those of leukocytes in blood vessels (P = 0.61). Cell velocity in lymph vessels was significantly reduced compared with blood particle velocity (P andlt; 0.001). Morphologic characteristics enabled subsequent identification of corneal lymphatics in live, vascularized rat corneas before immunofluorescence labeling.

CONCLUSIONS. IVCM enabled the nondestructive, label-free, in vivo detection of corneal lymphatics. IVCM provides the possibility of observing lymphatic activity in the same live corneas longitudinally and, as a clinical instrument, of monitoring corneal lymphatics in live human subjects.

Place, publisher, year, edition, pages
Rockville, MD, United States: , 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-53820 (URN)10.1167/iovs.09-4407 (DOI)000273704700030 ()
Available from: 2010-02-05 Created: 2010-02-05 Last updated: 2017-12-12Bibliographically approved
2. Letter: In vivo confocal microscopy visualization of presumed lymph vessels in a case of corneal transplant rejection
Open this publication in new window or tab >>Letter: In vivo confocal microscopy visualization of presumed lymph vessels in a case of corneal transplant rejection
2011 (English)In: Clinical and Experimental Ophthalmology, ISSN 1442-6404, E-ISSN 1442-9071, Vol. 39, no 8, 832-834 p.Article in journal, Letter (Other academic) Published
Abstract [en]

n/a

Place, publisher, year, edition, pages
Wiley-Blackwell, 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-72259 (URN)10.1111/j.1442-9071.2011.02557.x (DOI)000296913900016 ()
Available from: 2011-11-24 Created: 2011-11-24 Last updated: 2017-12-08Bibliographically approved
3. Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting
Open this publication in new window or tab >>Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting
2011 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 52, no 6, 3060-3068 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE. To elucidate the temporal sequence of events leading to new capillary sprouting in inflammatory corneal angiogenesis.

METHODS. Angiogenesis was induced by corneal suture placement in Wistar rats. The inflamed region was examined by time-lapse in vivo confocal microscopy for up to 7 days. At 6 and 12 hours and 1, 2, 4, and 7 days, corneas were excised for flat mount immunofluorescence with primary antibodies for CD31, CD34, CD45, CD11b, CD11c, Ki-M2R, NG2, and alpha-SMA. From days 0 to 4, the in vivo extravasation and expansion characteristics of single limbal vessels were quantified.

RESULTS. Starting hours after induction and peaking at day 1, CD45(+)CD11b(+) myeloid cells extravasated from limbal vessels and formed endothelium-free tunnels within the stroma en route to the inflammatory stimulus. Limbal vessel diameter tripled on days 2 to 3 as vascular buds emerged and transformed into perfused capillary sprouts less than 1 day later. A subset of spindle-shaped CD11b(+) myeloid-lineage cells, but not dendritic cells or mature macrophages, appeared to directly facilitate further capillary sprout growth. These cells incorporated into vascular endothelium near the sprout tip, co-expressing endothelial marker CD31. Sprouts had perfusion characteristics distinct from feeder vessels and many sprout tips were open-ended.

CONCLUSIONS. Time-lapse in vivo corneal confocal microscopy can be used to track a temporal sequence of events in corneal angiogenesis. The technique has revealed potential roles for myeloid cells in promoting vessel sprouting in an inflammatory corneal setting.

Place, publisher, year, edition, pages
Research in Vision and Opthalmology, 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-69178 (URN)10.1167/iovs.10-6101 (DOI)000291100800026 ()
Note
Original Publication: Beatrice Bourghardt Peebo, Per Fagerholm, Catharina Traneus-Rockert and Neil Lagali, Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting, 2011, INVESTIGATIVE OPHTHALMOLOGY and VISUAL SCIENCE, (52), 6, 3060-3068. http://dx.doi.org/10.1167/iovs.10-6101 Copyright: Research in Vision and Opthalmology http://www.arvo.org/Available from: 2011-06-17 Created: 2011-06-17 Last updated: 2017-12-11Bibliographically approved
4. Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model
Open this publication in new window or tab >>Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model
2011 (English)In: Angiogenesis, ISSN 0969-6970, E-ISSN 1573-7209, Vol. 14, no 3, 393-405 p.Article in journal (Refereed) Published
Abstract [en]

In this study, we introduce a technique for repeated, microscopic observation of single regressing capillaries in vivo in inflamed murine corneas. Natural capillary regression was initiated by removal of inflammatory stimulus during an active pro-angiogenic phase, while the additional impact of anti-angiogenic treatment with triamcinolone or bevazicumab was investigated. Capillaries regressed naturally within 1 week and treatments did not further enhance the natural regression. Morphologically, early-phase regression was characterized by significant lumen narrowing and a significant reduction in CD11b+ myeloid cell infiltration of the extracellular matrix. By 1 week, vascular remodeling occurred concomitant with CD11b+CD68+KiM2R+ mature macrophage localization on capillary walls. Empty conduits without blood flow, positive for collagen IV and devoid of vascular endothelium and pericytes, were apparent in vivo and by 3 weeks were more numerous than perfused capillaries. By 3 weeks, macrophages aggregated around remaining perfused capillaries and were observed in apposition with degrading capillary segments. Abrupt termination of capillary sprouting in our regression model further revealed vascular endothelial abandonment of sprout tips and perfused capillary loop formation within a single angiogenic sprout, possibly as an intussusceptive response to cessation of the stimulus. Finally, we observed lumen constriction and macrophage localization on capillary walls in vivo in a clinical case of corneal capillary regression that paralleled findings in our murine model.

Place, publisher, year, edition, pages
Springer Verlag (Germany), 2011
Keyword
Inflammation, Capillary regression, In vivo confocal microscopy, Cornea
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-70325 (URN)10.1007/s10456-011-9223-3 (DOI)000293922300015 ()
Note
The original publication is available at www.springerlink.com: Beatrice Bourghardt Peebo, Per Fagerholm, Catharina Traneus-Rockert and Neil Lagali, Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model, 2011, Angiogenesis, (14), 3, 393-405. http://dx.doi.org/10.1007/s10456-011-9223-3 Copyright: Springer Verlag (Germany) http://www.springerlink.com/Available from: 2011-09-02 Created: 2011-09-02 Last updated: 2017-12-08

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