Up-Regulation of Annexin-A1 and Lipoxin A4 in Individuals with Ulcerative Colitis May Promote Mucosal Homeostasis

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Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Detection of annexin-A1 NH₂-terminal-intact and -cleaved fragments in biopsies from healthy subjects without(Ctrl) or with a prior history (Hx) of UC, or UC patients with active disease (Active) or medically-induced remission (Rem).

<p>Annexin-1 was overexpressed in colonic mucosal biopsies of patients with UC compared to healthy patients with a prior history of UC (<b>A</b>). Alongside the native 37 kDa fragment, a NH₂-terminal cleaved isoform (33 kDa) could also be visualized, indicating specific degradation in response to externalization from activated cells. Analysis of relative intensity, where annexin-A1 immunoreactivity was normalized against β-actin (<b>B</b>). Data are expressed as mean ± SEM (**<i>P</i><.01, n = 5–11).</p

mRNA expression of FPR2/ALX in colonic mucosal biopsies from healthy subjects without(Ctrl) or with a prior history (Hx) of UC, or UC patients with active disease (Active) or medically-induced remission (Rem).

<p> Increased expression of FPR2/ALX was detected in biopsies from individuals with UC. Data are expressed as fold change relative to Ctrl (**<i>P</i><.01, n = 5–12).</p

Figure 3

<p>Expression of Annexin-A1 in colonic mucosal biopsies from healthy subjects without (Ctrl) or with a prior history (Hx) of UC, or UC patients with active disease (Active) or medically-induced remission (Rem) (<b>A</b>). Immunofluorescence detection of Annexin-A1 (red) demonstrates expression is increased in patients with UC, whether active or in medically-induced remission. Integrated pixel intensity revealed ∼3-fold increase in these groups, compared to healthy subjects (<b>B</b>). In biopsies from patients with active UC, Annexin-A1 (blue) staining could be localized to infiltrating PMNs (red; stained with anti-HNE) (<b>C</b>), however in biopsies from patients in medically-induced remission, Annexin-A1 staining (red) closely paralleled tissue infiltration by CD68⁺ macrophages (blue; stained with anti-CD68) (<b>D</b>). Annexin-A1 expression was also detected in crypt epithelial cells in subjects with UC, but not healthy subjects without/with a prior history of UC (<b>E</b>). Data are expressed as mean ± SEM (*<i>P</i><.05, ***<i>P</i><.001, n = 4). Magnification bar = 100 μm.</p

Figure 2

<p>Colonic mucosal lipoxin A₄ levels (<b>A</b>) in biopsies from healthy subjects without (Ctrl) or with a prior history (Hx) of UC, or UC patients with active disease (Active) or medically-induced remission (Rem). Lipoxin A₄ levels were significantly elevated in the samples from patients who were in medically-induced remission compared with healthy subjects and those with a prior history of UC (>4 years disease-free). Quantitative RT-PCR analysis of 5-LO (<b>B</b>), 12-LO (<b>C</b>) and 15-LO (<b>D</b>) expression revealed an increase of 5-LO expression in patients with active disease. This correlated with an increase in the number of 5-LO-positive cells as assessed by immunohistochemistry (<b>E</b>). Data are expressed as fold change relative to Ctrl (*<i>P</i><.05, **<i>P</i><.01 ***<i>P</i><.001, n = 5–20). Magnification bar = 100 μm.</p

Figure 1

<p>Expression of HNE and CD68 in colonic mucosal biopsies from healthy subjects without (Ctrl) or with a prior history (Hx) of UC, or UC patients with active disease (Active) or medically-induced remission (Rem) (<b>A</b>). HNE expression (Upper), used to detect PMNs was elevated in UC patients with active disease. CD68 expression (Lower), a pan macrophage marker, was elevated in active disease as well as in medically-induced remission. Cumulative analysis (mean counts/field) of HNE (<b>B</b>) and CD68 expression (<b>C</b>)<b>.</b> Dual-staining of macrophage populations with CD68 and CD206, markers for alternatively activated (M2) macrophages (<b>D</b>), Data are expressed as mean ± SEM (*<i>P</i><.05, ***<i>P</i><.001, n = 4). Magnification bar = 100μm.</p

Investigation of cardiac fibroblasts using myocardial slices

Aims: Cardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro , CFs undergo rapid phenotypic changes including increased expression of \u3b1-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices. Methods and Results: Unloading of myocardial slices induced CF proliferation without \u3b1-SMA expression up to 7 days in culture . CFs migrating onto the culture plastic support or cultured on glass expressed \u3b1SMA within 3 days. The cells on the slice remained \u3b1SMA(-) despite TGF-\u3b2 (20ng/mL) or angiotensin II (200\ub5M) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at day 3 and 7 (P\u2009&lt;\u20090.001). Conclusions: Myocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential target

Investigation of cardiac fibroblasts using myocardial slices

Aims: Cardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro , CFs undergo rapid phenotypic changes including increased expression of α-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices. Methods and Results: Unloading of myocardial slices induced CF proliferation without α-SMA expression up to 7 days in culture . CFs migrating onto the culture plastic support or cultured on glass expressed αSMA within 3 days. The cells on the slice remained αSMA(-) despite TGF-β (20ng/mL) or angiotensin II (200µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at day 3 and 7 (P &lt; 0.001). Conclusions: Myocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential target