Development of Surgical and Visualization Procedures to Analyze Vasculatures by Mouse Tail Edema Model

Abstract Background Because of the high frequency of chronic edema formation in the current “aged” society, analyses and detailed observation of post-surgical edema are getting more required. Post-surgical examination of the dynamic vasculature including L.V. (Lymphatic Vasculature) to monitor edema formation has not been efficiently performed. Hence, procedures for investigating such vasculature are essential. By inserting transparent sheet into the cutaneous layer of mouse tails as a novel surgery model (theTailEdema bySilicone sheet mediatedTransparency protocol; TEST), the novel procedures are introduced and analyzed by series of histological analyses including video-based L.V. observation and 3D histological reconstruction of vasculatures in mouse tails. Results The dynamic generation of post-surgical main and fine (neo) L.V. connective structure during the edematous recovery process was visualized by series of studies with a novel surgery model. Snapshot images taken from live binocular image recording for TEST samples suggested the presence of main and elongating fine (neo) L.V. structure. After the ligation of L.V., the enlargement of main L.V. was confirmed. In the case of light sheet fluorescence microscopy (LSFM) observation, such L.V. connections were also suggested by using transparent 3D samples. Finally, the generation of neo blood vessels particularly in the region adjacent to the silicone sheet and the operated boundary region was suggested in 3D reconstruction images. However, direct detection of elongating fine (neo) L.V. was not suitable for analysis by such LSFM and 3D reconstruction procedures. Thus, such methods utilizing fixed tissues are appropriate for general observation for the operated region including of L.V. Conclusions The current surgical procedures and analysis on the post-surgical status are the first case to observe vasculatures in vivo with a transparent sheet. Systematic analyses including the FITC-dextran mediated snap shot images observation suggest the elongation of fine (neo) lymphatic vasculature. Post-surgical analyses including LSFM and 3D histological structural reconstruction, are suitable to reveal the fixed structures of blood and lymphatic vessels formation.

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Light sheet microscopy-based 3-dimensional histopathology of the lymphatic vasculature in Emberger syndrome

Phlebologie ◽

10.1055/a-1191-8380 ◽

2020 ◽

Vol 49 (04) ◽

pp. 242-248

Author(s):

René Hägerling

Keyword(s):

Lower Limb ◽

Lymphatic Vessels ◽

Light Sheet ◽

Lower Limbs ◽

Vascular Pathology ◽

Histopathological Analysis ◽

Upper Limbs ◽

3 Dimensional ◽

Light Sheet Microscopy ◽

Lymphatic Vasculature

Abstract Introduction Lymphovascular diseases represent a heterogenous group of inherited and sporadic disorders and refer to a range of possible underlying pathologies and pathogenesis.Emberger Syndrome, an inherited form of lymphedema, is characterized by bilateral lower limb lymphedema, however, upper limbs do not show any signs of swelling.To identify disease-associated histopathological alterations in patients with Emberger Syndrome and to elucidate potential histological differences between the lymphatic vasculature of upper and lower limbs, a detailed knowledge on the 3-dimensional tissue and vessel architecture is essential. However, the current gold standard in 2-dimensional histology provides only very limited spatial information. Material and methods To elucidate the underlying vascular pathology in Emberger Syndrome on the cellular level, we applied the 3-dimensional visualization and analysis approach VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction) to entire wholemount immunofluorescence-stained human tissue samples. VIPAR is a light sheet microscopy-based imaging technique, which allows 3-dimensional reconstruction of entire tissue biopsies followed by automated and semi-automated analysis of vascular parameters in 3-dimensional space. Results Using VIPAR we could show that in Emberger Syndrome the dermal lymphatic vasculature is intact and non-disrupted.However, lower limbs showed an hypoplastic lymphatic vasculature with absence of lymphatic valves in pre-collecting and collecting vessels. In contrast to the lower limbs, the lymphatic vasculature of the upper limbs showed no morphological alterations of lymphatic vessels and lymphatic valves compared to healthy controls. Discussion Based on the 3-dimensional histopathological analysis we were able to perform a detailed phenotyping of lymphatic vessels in the upper and lower limb in Emberger Syndrome and to identify the underlying vascular pathology. In addition, we could show vascular alteration between the upper and lower limbs indicating a vascular heterogeneity of dermal lymph vessels causing the lower limb lymphedema.

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Gene-expression profiling of different arms of lymphatic vasculature identifies candidates for manipulation of cell traffic

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602357113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10643-10648 ◽

Cited By ~ 23

Author(s):

Imtiaz Iftakhar-E-Khuda ◽

Ruth Fair-Mäkelä ◽

Anu Kukkonen-Macchi ◽

Kati Elima ◽

Marika Karikoski ◽

...

Keyword(s):

Scavenger Receptor ◽

Lymphatic Vessels ◽

In Vivo Studies ◽

Molecular Characteristics ◽

Lymphatic Endothelium ◽

Macrophage Scavenger Receptor ◽

Subcapsular Sinus ◽

Cancer Spread ◽

Lymphatic Vasculature

Afferent lymphatic vessels bring antigens and diverse populations of leukocytes to draining lymph nodes, whereas efferent lymphatics allow only lymphocytes and antigens to leave the nodes. Despite the fundamental importance of afferent vs. efferent lymphatics in immune response and cancer spread, the molecular characteristics of these different arms of the lymphatic vasculature are largely unknown. The objective of this work was to explore molecular differences behind the distinct functions of afferent and efferent lymphatic vessels, and find possible molecules mediating lymphocyte traffic. We used laser-capture microdissection and cell sorting to isolate lymphatic endothelial cells (LECs) from the subcapsular sinus (SS, afferent) and lymphatic sinus (LS, efferent) for transcriptional analyses. The results reveal marked differences between afferent and efferent LECs and identify molecules on lymphatic vessels. Further characterizations of Siglec-1 (CD169) and macrophage scavenger receptor 1 (MSR1/CD204), show that they are discriminatively expressed on lymphatic endothelium of the SS but not on lymphatic vasculature of the LS. In contrast, endomucin (EMCN) is present on the LS endothelium and not on lymphatic endothelium of the SS. Moreover, both murine and human MSR1 on lymphatic endothelium of the SS bind lymphocytes and in in vivo studies MSR1 regulates entrance of lymphocytes from the SS to the lymph node parenchyma. In conclusion, this paper reports surprisingly distinct molecular profiles for afferent and efferent lymphatics and a function for MSR1. These results may open avenues to explore some of the now-identified molecules as targets to manipulate the function of lymphatic vessels.

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EMILIN1/α9β1 Integrin Interaction Is Crucial in Lymphatic Valve Formation and Maintenance

Molecular and Cellular Biology ◽

10.1128/mcb.00872-13 ◽

2013 ◽

Vol 33 (22) ◽

pp. 4381-4394 ◽

Cited By ~ 38

Author(s):

Carla Danussi ◽

Lisa Del Bel Belluz ◽

Eliana Pivetta ◽

Teresa Maria Elisa Modica ◽

Andres Muro ◽

...

Keyword(s):

Lymphatic Vessels ◽

Lymphatic Endothelial Cell ◽

Cell Receptor ◽

Regulatory Function ◽

Lymphatic Valve ◽

Lymphatic Vasculature ◽

And Migration ◽

Valve Formation

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometricin vitroandin vivofunctional assays, we conclude that this impaired phenotype depends on the lack of α9β1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a “guiding” molecule in migration of lymphatic endothelial cells.

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The adaptor protein Grb2b is an essential modulator for lympho-venous sprout formation in the zebrafish trunk

Angiogenesis ◽

10.1007/s10456-021-09774-w ◽

2021 ◽

Author(s):

Cristina Mauri ◽

Andreas van Impel ◽

Eirinn William Mackay ◽

Stefan Schulte-Merker

Keyword(s):

Genetic Interaction ◽

Lymphatic Vessels ◽

Adaptor Protein ◽

Cardinal Vein ◽

Cell Behaviors ◽

Downstream Signaling ◽

Vein Formation ◽

Lymphatic Vasculature ◽

Sprout Formation

AbstractVegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts.

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Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation

Blood ◽

10.1182/blood-2004-08-3382 ◽

2005 ◽

Vol 105 (12) ◽

pp. 4649-4656 ◽

Cited By ~ 168

Author(s):

Tohru Morisada ◽

Yuichi Oike ◽

Yoshihiro Yamada ◽

Takashi Urano ◽

Masaki Akao ◽

...

Keyword(s):

Endothelial Cells ◽

Lymphatic Vessel ◽

Matrix Protein ◽

Lymphatic Vessels ◽

Vascular Endothelial ◽

Vessel Formation ◽

Lymphatic Vasculature ◽

In Vitro Effects

Abstract Angiopoietin (Ang) signaling plays a role in angiogenesis and remodeling of blood vessels through the receptor tyrosine kinase Tie2, which is expressed on blood vessel endothelial cells (BECs). Recently it has been shown that Ang-2 is crucial for the formation of lymphatic vasculature and that defects in lymphangiogenesis seen in Ang-2 mutant mice are rescued by Ang-1. These findings suggest important roles for Ang signaling in the lymphatic vessel system; however, Ang function in lymphangiogenesis has not been characterized. In this study, we reveal that lymphatic vascular endothelial hyaluronan receptor 1-positive (LYVE-1+) lymphatic endothelial cells (LECs) express Tie2 in both embryonic and adult settings, indicating that Ang signaling occurs in lymphatic vessels. Therefore, we examined whether Ang-1 acts on in vivo lymphatic angiogenesis and in vitro growth of LECs. A chimeric form of Ang-1, cartilage oligomeric matrix protein (COMP)-Ang-1, promotes in vivo lymphatic angiogenesis in mouse cornea. Moreover, we found that COMP-Ang-1 stimulates in vitro colony formation of LECs. These Ang-1-induced in vivo and in vitro effects on LECs were suppressed by soluble Tie2-Fc fusion protein, which acts as an inhibitor by sequestering Ang-1. On the basis of these observations, we propose that Ang signaling regulates lymphatic vessel formation through Tie2. (Blood. 2005;105:4649-4656)

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ДИНИТРОЗИЛЬНЫЕ КОМПЛЕКСЫ ЖЕЛЕЗА C ТИОЛСОДЕРЖАЩИМИ ЛИГАНДАМИ ПРЕДСТАВЛЕНЫ В ЖИВЫХ ОРГАНИЗМАХ В ОСНОВНОМ ИХ БИЯДЕРНОЙ ФОРМОЙ

Биофизика ◽

10.31857/s0006302920060149 ◽

2020 ◽

Vol 65 (6) ◽

pp. 1142-1153

Author(s):

В.Д. Микоян ◽

◽

Е.Н. Бургова ◽

Р.Р. Бородулин ◽

А.Ф. Ванин ◽

...

Keyword(s):

Electron Paramagnetic Resonance ◽

Sodium Nitrite ◽

Iron Complexes ◽

Paramagnetic Resonance ◽

First Case ◽

Dinitrosyl Iron ◽

Electron Paramagnetic ◽

Dinitrosyl Complexes

The number of mononitrosyl iron complexes with diethyldithiocarbamate, formed in the liver of mice in vivo and in vitro after intraperitoneal injection of binuclear dinitrosyl iron complexes with N-acetyl-L-cysteine or glutathione, S-nitrosoglutathione, sodium nitrite or the vasodilating drug Isoket® was assessed by electron paramagnetic resonance (EPR). The number of the said complexes, in contrast to the complexes, formed after nitrite or Isoket administration, the level of which sharply increased after treatment of liver preparations with a strong reducing agent - dithionite, did not change in the presence of dithionite. It was concluded that, in the first case, EPR-detectable mononitrosyl iron complexes with diethyldithiocarbamate in the absence and presence of dithionite appeared as a result of the reaction of NO formed from nitrite with Fe2+-dieth- yldithiocarbamate and Fe3+-diethyldithiocarbamate complexes, respectively. In the second case, mononitrosyl iron complexes with diethyldithiocarbamate appeared as a result of the transition of iron-mononitosyl fragments from ready-made iron-dinitrosyl groups of binuclear dinitrosyl complexes, which is three to four times higher than the content of the mononuclear form of these complexes in the tissue...

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Faculty Opinions recommendation of Direct detection of abortive RNA transcripts in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1159625.620939 ◽

2009 ◽

Author(s):

Katsuhiko Murakami

Keyword(s):

Direct Detection ◽

Rna Transcripts

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Efficient aortic lymphatic drainage is necessary for atherosclerosis regression induced by ezetimibe

Science Advances ◽

10.1126/sciadv.abc2697 ◽

2020 ◽

Vol 6 (50) ◽

pp. eabc2697

Author(s):

Kim Pin Yeo ◽

Hwee Ying Lim ◽

Chung Hwee Thiam ◽

Syaza Hazwany Azhar ◽

Caris Tan ◽

...

Keyword(s):

Lymphatic Vessels ◽

Lymphatic Drainage ◽

Lymphatic Transport ◽

Atherosclerotic Plaques ◽

Tissue Fluid ◽

Atherosclerosis Progression ◽

Lymphatic Vasculature ◽

Lesion Regression ◽

Transport Of Macromolecules

A functional lymphatic vasculature is essential for tissue fluid homeostasis, immunity, and lipid clearance. Although atherosclerosis has been linked to adventitial lymphangiogenesis, the functionality of aortic lymphatic vessels draining the diseased aorta has never been assessed and the role of lymphatic drainage in atherogenesis is not well understood. We develop a method to measure aortic lymphatic transport of macromolecules and show that it is impaired during atherosclerosis progression, whereas it is ameliorated during lesion regression induced by ezetimibe. Disruption of aortic lymph flow by lymphatic ligation promotes adventitial inflammation and development of atherosclerotic plaque in hypercholesterolemic mice and inhibits ezetimibe-induced atherosclerosis regression. Thus, progression of atherosclerotic plaques may result not only from increased entry of atherogenic factors into the arterial wall but also from reduced lymphatic clearance of these factors as a result of aortic lymph stasis. Our findings suggest that promoting lymphatic drainage might be effective for treating atherosclerosis.

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Development of UHPLC/Q-TOF Analysis Method to Screen Glycerin for Direct Detection of Process Contaminants 3-Monochloropropane-1,2-diol Esters (3-MCPDEs) and Glycidyl Esters (GEs)

Molecules ◽

10.3390/molecules26092449 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2449

Author(s):

Lauren Girard ◽

Kithsiri Herath ◽

Hernando Escobar ◽

Renate Reimschuessel ◽

Olgica Ceric ◽

...

Keyword(s):

Renal Failure ◽

Solid Phase ◽

Direct Detection ◽

Screening Method ◽

Extraction Procedure ◽

Biodiesel Production ◽

Food Contaminants ◽

Exact Mass ◽

Glycidyl Esters

The U.S. Food and Drug Administration’s (FDA′s) Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats since 2007. Renal failure accounted for 30% of reported cases. Jerky pet treats contain glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Glycidyl esters (GEs) and 3-monochloropropanediol esters (3-MCPDEs) are food contaminants that can form in glycerin during the refining process. 3-MCPDEs and GEs pose food safety concerns, as they can release free 3-MCPD and glycidol in vivo. Evidence from studies in animals shows that 3-MCPDEs are potential toxins with kidneys as their main target. As renal failure accounted for 30% of reported pet illnesses after the consumption of jerky pet treats containing glycerin, there is a need to develop a screening method to detect 3-MCPDEs and GEs in glycerin. We describe the development of an ultra-high-pressure liquid chromatography/quadrupole time-of-flight (UHPLC/Q-TOF) method for screening glycerin for MCPDEs and GEs. Glycerin was extracted and directly analyzed without a solid-phase extraction procedure. An exact mass database, developed in-house, of MCPDEs and GEs formed with common fatty acids was used in the screening.

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