Lauri Eklund

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell–cell and cell–matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell–matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell–cell contacts and the formation of homotypic Tie2–Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell–matrix and cell–cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.

Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild‐type eyes, except Descemets membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss‐of‐function mutations in collagen XVIII.

Angiopoietin signaling in the vasculature.

The angiopoietin (Ang) growth factors and the endothelial Tie receptors regulate blood and lymphatic vessel development, and vascular permeability, inflammation, angiogenic remodeling and tumor vascularization in adult tissues. The angiopoietins activate the Tie receptors in unique in trans complexes at endothelial cell-cell and cell-matrix contacts. In addition, integrins have been implicated in the regulation of Ang-Tie signaling. Recent interest has focused on the function of angiopoietin-2 and its inhibition in the tumor vasculature and also in other pathological conditions associated with endothelial dysfunction. Here we review the current understanding of the signaling functions of the Ang-Tie pathway and its potential for future development of targeted vascular therapeutics.

Mouse models for studying angiogenesis and lymphangiogenesis in cancer

The formation of new blood vessels (angiogenesis) is required for the growth of most tumors. The tumor microenvironment also induces lymphangiogenic factors that promote metastatic spread. Anti‐angiogenic therapy targets the mechanisms behind the growth of the tumor vasculature. During the past two decades, several strategies targeting blood and lymphatic vessels in tumors have been developed. The blocking of vascular endothelial growth factor (VEGF)/VEGF receptor‐2 (VEGFR‐2) signaling has proven effective for inhibition of tumor angiogenesis and growth, and inhibitors of VEGF‐C/VEGFR‐3 involved in lymphangiogenesis have recently entered clinical trials. However, thus far anti‐angiogenic treatments have been less effective in humans than predicted on the basis of pre‐clinical tests in mice. Intrinsic and induced resistance against anti‐angiogenesis occurs in patients, and thus far the clinical benefit of the treatments has been limited to modest improvements in overall survival in selected tumor types. Our current knowledge of tumor angiogenesis is based mainly on experiments performed in tumor‐transplanted mice, and it has become evident that these models are not representative of human cancer. For an improved understanding, angiogenesis research needs models that better recapitulate the multistep tumorigenesis of human cancers, from the initial genetic insults in single cells to malignant progression in a proper tissue environment. To improve anti‐angiogenic therapies in cancer patients, it is necessary to identify additional molecular targets important for tumor angiogenesis, and to get mechanistic insight into their interactions for eventual combinatorial targeting. The recent development of techniques for manipulating the mammalian genome in a precise and predictable manner has opened up new possibilities for the generation of more reliable models of human cancer that are essential for the testing of new therapeutic strategies. In addition, new imaging modalities that permit visualization of the entire mouse tumor vasculature down to the resolution of single capillaries have been developed in pre‐clinical models and will likely benefit clinical imaging.

Abnormal maturation of the retinal vasculature in type XVIII collagen/endostatin deficient mice and changes in retinal glial cells due to lack of collagen types XV and XVIII

Type XVIII collagen is important in the early phase of retinal vascular development and for the regression of the primary vasculature in the vitreous body after birth. We show here that the retina in Col18a1−/− mice becomes densely vascularized by anomalous anastomoses from the persistent hyaloid vasculature by day 10 after birth. In situ hybridizations revealed normal VEGF mRNA expression, but the phenotype of collagen XVIII deficient mice closely resembled that of mice expressing VEGF120 and VEGF188 isoforms only, suggesting that type XVIII collagen may be involved in VEGF function. Type XVIII collagen was found to be indispensable for angiogenesis in the eye, as also oxygen‐induced neovascularization was less intense than normal in the Col18a1−/− mice. We observed a marked increase in the amount of retinal astrocytes in the Col18a1−/− mice. Whereas the retinal vessels of wild‐type mice are covered by astrocytes and the regressing, thin hyaloid vessels are devoid of astrocytes, the retinal vessels in the Col18a1−/− mice were similarly covered by astrocytes but not the persistent hyaloid vessels in the vitreous body. Interestingly, double null mice lacking type XVIII collagen and its homologue type XV collagen had the persistent hyaloid vessels covered by astrocytes, including the parts located in the vitreous body. We thus hypothesize that type XV collagen is a regulator of glial cell recruitment around vessels and that type XVIII collagen regulates their proliferation.

Collagen XV Is Necessary for Modeling of the Extracellular Matrix and Its Deficiency Predisposes to Cardiomyopathy

Rationale: The extracellular matrix (ECM) is a major determinant of the structural integrity and functional properties of the myocardium in common pathological conditions, and changes in vasculature contribute to cardiac dysfunction. Collagen (Col) XV is preferentially expressed in the ECM of cardiac muscle and microvessels. Objective: We aimed to characterize the ECM, cardiovascular function and responses to elevated cardiovascular load in mice lacking Col XV (Col15a1−/−) to define its functional role in the vasculature and in age- and hypertension-associated myocardial remodeling. Methods and Results: Cardiac structure and vasculature were analyzed by light and electron microscopy. Cardiac function, intraarterial blood pressure, microhemodynamics, and gene expression profiles were studied using echocardiography, telemetry, intravital microscopy, and PCR, respectively. Experimental hypertension was induced with angiotensin II or with a nitric oxide synthesis inhibitor. Under basal conditions, lack of Col XV resulted in increased permeability and impaired microvascular hemodynamics, distinct early-onset and age-dependent defects in heart structure and function, a poorly organized fibrillar collagen matrix with marked interstitial deposition of nonfibrillar protein aggregates, increased tissue stiffness, and irregularly organized cardiomyocytes. In response to experimental hypertension, Col15a1 gene expression was increased in the left ventricle of wild-type mice, and mRNA expression of natriuretic peptides (ANP and BNP) and ECM modeling were abnormal in Col15a1−/− mice. Conclusions: Col XV is necessary for ECM organization in the heart, and for the structure and functions of microvessels. Col XV deficiency leads to a complex cardiac phenotype and predisposes the subject to pathological responses under cardiac stress.

Automatic detection and analysis of cell motility in phase‐contrast time‐lapse images using a combination of maximally stable extremal regions and Kalman filter approaches

Phase‐contrast illumination is simple and most commonly used microscopic method to observe nonstained living cells. Automatic cell segmentation and motion analysis provide tools to analyze single cell motility in large cell populations. However, the challenge is to find a sophisticated method that is sufficiently accurate to generate reliable results, robust to function under the wide range of illumination conditions encountered in phase‐contrast microscopy, and also computationally light for efficient analysis of large number of cells and image frames. To develop better automatic tools for analysis of low magnification phase‐contrast images in time‐lapse cell migration movies, we investigated the performance of cell segmentation method that is based on the intrinsic properties of maximally stable extremal regions (MSER). MSER was found to be reliable and effective in a wide range of experimental conditions. When compared to the commonly used segmentation approaches, MSER required negligible preoptimization steps thus dramatically reducing the computation time. To analyze cell migration characteristics in time‐lapse movies, the MSER‐based automatic cell detection was accompanied by a Kalman filter multiobject tracker that efficiently tracked individual cells even in confluent cell populations. This allowed quantitative cell motion analysis resulting in accurate measurements of the migration magnitude and direction of individual cells, as well as characteristics of collective migration of cell groups. Our results demonstrate that MSER accompanied by temporal data association is a powerful tool for accurate and reliable analysis of the dynamic behaviour of cells in phase‐contrast image sequences. These techniques tolerate varying and nonoptimal imaging conditions and due to their relatively light computational requirements they should help to resolve problems in computationally demanding and often time‐consuming large‐scale dynamical analysis of cultured cells.

Joint cell segmentation and tracking using cell proposals

Time-lapse microscopy imaging has advanced rapidly in last few decades and is producing large volume of data in cell and developmental biology. This has increased the importance of automated analyses, which depend heavily on cell segmentation and tracking as these are the initial stages when computing most biologically important cell properties. In this paper, we propose a novel joint cell segmentation and tracking method for fluorescence microscopy sequences, which generates a large set of cell proposals, creates a graph representing different cell events and then iteratively finds the most probable path within this graph providing cell segmentations and tracks. We evaluate our method on three datasets from ISBI Cell Tracking Challenge and show that our greedy nonoptimal joint solution results in improved performance compared with state of the art methods.

Angiopoietins and Tie Receptors

The Tie1 and Tie2 receptor tyrosine kinases and the angiopoietin growth factor ligands, Ang1-4, are essential for vascular maturation. Targeted deletion of any of the Tie1, Tie2 or Ang1 genes in mice results in embryonic lethality during embryonic days 9.5–13.5. The receptors are expressed mainly in endothelial cells, while Ang1 is produced by perivascular cells and thought to stabilize quiescent endothelium. In contrast, Ang2 is secreted by endothelial cells in angiogenic vasculature, such as in tumors, leading to destabilization of the endothelium. Ang1 multimers stimulate the phosphorylation of Tie1 and Tie2, while Ang2 functions as a context-dependent agonist/antagonist for Tie2. Ang1 has promising vascular protective effects as an anti-permeability, anti-inflammatory and cell survival factor, but it can also induce vessel remodelling. The angiopoietin-Tie signalling pathway may be a therapeutically useful target in the treatment of a number of diseases, including oedema, endotoxaemia, transplant arteriosclerosis and cancer.