An in vivo neovascularization assay for screening regulators of angiogenesis and assessing their effects on pre-existing vessels

Therapeutic regulation of tissue vascularization has appeared as an attractive approach to treat a number of human diseases. In vivo neovascularization assays that reflect physiological and pathological formation of neovessels are important in this effort. In this report we present an assay where the effects of activators and inhibitors of angiogenesis can be quantitatively and qualitatively measured. A provisional matrix composed of collagen I and fibrin was formed in a plastic cylinder and implanted onto the chick chorioallantoic membrane. A nylon mesh separated the implanted matrix from the underlying tissue to distinguish new from pre-existing vessels. Vascularization of the matrix in response to fibroblast growth factor-2 or platelet-derived growth factor-BB was scored in a double-blinded manner, or vessel density was measured using a semi-automated image analysis procedure. Thalidomide, fumagillin, U0126 and TGFβ inhibited neovessel growth while hydrocortisone exerted a negative and wortmannin a toxic effect on the pre-existing vasculature. This quantitative, inexpensive and rapid in vivo angiogenesis assay might be a valuable tool in screening and characterizing factors that influence wound or tumor induced vascularization and in assessing their effects on the normal vasculatur

Discovery of microvascular miRNAs using public gene expression data: miR-145 is expressed in pericytes and is a regulator of Fli1

International audienceBACKGROUND: A function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets. METHODS: Bioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber. RESULTS: miR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro. CONCLUSIONS: miR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature

Myofibroblasts and the Vascular Endothelium : Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing. We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A. MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts. miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA. Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors. These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.

Myofibroblasts and the Vascular Endothelium : Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing. We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A. MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts. miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA. Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors. These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.

Myofibroblasts and the Vascular Endothelium : Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing. We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A. MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts. miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA. Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors. These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.

Fibrin fragment E potentiates TGF-beta-induced myofibroblast activation and recruitment

Fibrin is an essential constituent of the coagulation cascade, and the formation of hemostatic fibrin clots is central to wound healing. Fibrin clots are over time degraded into fibrin degradation products as the injured tissue is replaced by granulation tissue. Our goal was to study the role of the fibrin degradation product fragment E (FnE) in fibroblast activation and migration. We present evidence that FnE is a chemoattractant for fibroblasts and that FnE can potentiate TGF-beta-induced myofibroblast formation. FnE forms a stable complex with alpha(v)beta(3) integrin, and the integrin beta(3) subunit is required both for FnE-induced fibroblast migration and for potentiation of TGF-beta-induced myofibroblast formation. Finally, subcutaneous infusion of FnE in mice results in a fibrotic response in the hypodermis. These results support a model where FnE released from clots in wounded tissue promote wound healing and fibrosis by both recruitment and activation of fibroblasts. Fibrin fragment E could thus represent a therapeutic target for treatment of pathological fibrosis

Von Frames und Slots bis Krambambuli : Beiträge zur zweisprachigen Lexikographie : Referate der zweiten internationalen Lexikographiekonferenz Jyväskylä, Finnland 24. - 26. 3. 1994

The present volume contains a general overview of current theoretical and practical issues in lexicography with special emphasis on bilingual applications. The contributions are divided into four sections. The first three record the plenary sessions of the Second International Conference on Lexicography in Jyvaskyla in March 1994. The fourth section contains papers from workshops thematically linked more or less closely to the plenary sessions. The first section, "General theory of lexicography and lexicology" is opened by R. R. K. Hartmann (Exeter) who reviews the four traditional aspects of research on dictionaries, the history, typology, critical assessment and use of dictionaries, discussing their particular relevance to bilingual lexicography. W. Martin (Amsterdam) then discusses dictionary definitions and models for the representation of knowledge. Martin favours what he calls deliberate inconsistency whereby the lexicographer, while basing his work on an explicit system such as the frames and slots of artificial intelligence, in fact restricts his definitions to the most relevant information. The definition of meaning is but one aspect of the lemma. A further essential aspect is the question of how words can be combined. M. Järventausta (Savonlinna) shows how recent considerations in valency theory can be used to describe and compare the respective combinatory characteristics of (partial) semantic equivalents in German and Finnish. U. Heid (Stuttgart) presents a similar topic from a different theoretical perspective in his workshop paper on the ordering of information on syntactic structures in a contrastive dictionary for machine translation. The problem of combination is also discussed in the second (plenary) section on "Aspects of contrastive lexicograpy". In H.-P. Kromann's (Kopenhagen) contribution, "How much grammar does a bilingual dictionary need?" the matter is firstly reviewed from a macro-, medio- and microstructural viewpoint and then critically demonstrated using entries for the preposition über in mono- and bilingual dictionaries as an example. J. Korhonen (Helsinki) then suggests a model for describing verb-idioms in German-Finnish and Finnish-German dictionaries. The same theme is taken up by J. Toomar (Jyväskylä) in her workshop paper, an analysis of the lemmatization of standard verbal idioms in a German-Finnish dictionary. R. Frisch (Jyväskylä/Wurzburg) and I. Hyvärinen (Jyväskylä) concern themselves with word formation and dictionaries. Frisch critically assesses the treatment of lemmatized word-formation elements in German-Finnish dictionaries whereas Hyvärinen looks at the formation of antonyms to see whether lemmatized antonyms can be used to reveal systematic contrasts between languages. L. Tossavainen (Jyväskylä) also deals with word formation and lexicography in her workshop paper, concentrating especially on the lemmatization of compound nouns. The two contributions in the section on "Particular questions of lexicography" lend the volume a historical dimension. A. F. Kelletat (Germersheim) takes a look at Comenius's "Orbis sensualium pictus" from 1658, pointing out how its didactic innovations anticipate bi- or multilingual illustrated learners' dictionaries. K Keinästö (Oulu) presents a wide range of more recent German loan words in Finnish and then traces the fate of the word Krambambuli through German, Scandinavian and Finnish dictionaries and encyclopedias. The workshop papers in the fourth section draw attention to various specific aspects and problems. Apart from the papers already mentioned above they include work by B. Fuchs (Jyväskylä) and D. Neuendorff (Oulu) connecting the critical evaluation of dictionaries with dictionary user research, further lexicosemantic papers by S. Tschirpke (Helsinki/ Greifswald) and A. Uurasjärvi (Tampere) on lexical families and lexical fields and a paper by J. Boger (Jyväskylä) illustrating the special problems of dialect lexicography with experience from the Low Saxon Dictionary