Murine Blood Vessels Research Articles

DISTINCT PATTERNS OF ENDOTHELIAL CELL ACTIVATION PRODUCED BY EXTRACELLULAR HISTONES AND BACTERIAL LIPOPOLYSACCHARIDE.

Objective : Vascular endothelial cells (ECs) sense and respond to both trauma factors (histone proteins) and sepsis signals (bacterial lipopolysaccharide, LPS) with elevations in calcium (Ca 2+ ), but it is not clear if the patterns of activation are similar or different. We hypothesized that within seconds of exposure, histones but not LPS would produce a large EC Ca 2+ response. We also hypothesized that histones would produce different spatio-temporal patterns of Ca 2+ events in veins than in arteries. Methods : We studied cultured ECs (EA.hy926) and native endothelial cells from surgically opened murine blood vessels. High-speed live cell imaging of Ca 2+ events were acquired for 5 min before and after stimulation of cultured ECs with histones or LPS alone or in combination. Histone-induced EC Ca 2+ events were also compared in native endothelial cells from resistance-sized arteries and veins. Ca 2+ activity was quantified as "Ca 2+ prevalence" using custom spatiotemporal analysis. Additionally, cultured ECs were collected after 6 h of exposure to histones or LPS for RNA sequencing. Results : ECs-both in culture and in blood vessels-rapidly increased Ca 2+ activity within seconds of histone exposure. In contrast, LPS exposure produced only a slight increase in Ca 2+ activity in cultured ECs and no effect on blood vessels over 5-min recording periods. Histones evoked large aberrant Ca 2+ events (>30 s in duration) in both veins and arteries, but with different spatio-temporal patterns. Ca 2+ activity in arterial ECs often appeared as "rosettes", with Ca 2+ events that propagated from one cell to all adjacent surrounding cells. In veins, ECs responded individually without spreading. Surprisingly, exposure of cultured ECs to LPS for 5 min before histones potentiated EC Ca 2+ activity by an order of magnitude. Exposure of ECs to histones or LPS both increased gene expression, but different mRNAs were induced. Conclusions : LPS and histones activate ECs through mechanisms that are distinct and additive; only histones produce large aberrant Ca 2+ events. ECs in arteries and veins display different patterns of Ca 2+ responses to histones.

Endothelial-dependent relaxation of α-pinene and two metabolites, myrtenol and verbenol, in isolated murine blood vessels.

Epidemiological evidence shows that residential proximity to greenspaces is associated with lower risk of all-cause and cardiovascular mortality; however, the mechanism(s) underlying this link remains unclear. Plants emit biogenic volatile organic compounds such as α-pinene that could elicit beneficial cardiovascular effects. To explore the role of α-pinene more directly, we studied the metabolism and the vascular effects of α-pinene. We found that exposure of mice to α-pinene (1 ppm, 6 h) generated two phase I oxidation metabolites, cis- and trans-verbenol [(1R,2R,5R)-verbenol and (1 R,2S,5R)-verbenol)] and myrtenol [(1S,5R)-(+)-myrtenol] that were identified in urine by GC-MS. Precontracted naïve murine male and female aorta and superior mesenteric artery (SMA) were relaxed robustly (60% tension reduction) by increasing concentrations of α-pinene, myrtenol, and verbenol to 0.3 mM, whereas 1 mM α-pinene was vasotoxic. The SMA was six times more sensitive than the aorta to α-pinene. Both myrtenol and verbenol were equally potent and efficacious as parent α-pinene in male and female SMA. The sensitive portion of the α-pinene-, myrtenol-, and verbenol-induced relaxations in male SMA was mediated by 1) endothelium, 2) eNOS-derived NO, and 3) guanylyl cyclase (GC) activity. Moreover, α-pinene activated the transient receptor potential ankyrin-1 (TRPA1) channel whereas the metabolites did not. Endothelial-derived NO regulates blood flow, blood pressure, and thrombosis, and it is plausible that inhaled (and ingested) α-pinene (or its metabolites) augments NO release to mediate the cardiovascular benefits of exposure to greenness.NEW & NOTEWORTHY A common plant-derived biogenic volatile organic compound, α-pinene, and two of its metabolites, myrtenol and verbenol, stimulate vasorelaxation in murine superior mesenteric artery. Both α-pinene- and its metabolites induce vasorelaxation by activation of the endothelium, nitric oxide, and guanylyl cyclase. α-Pinene also activates the transient receptor potential ankyrin-1. Positive associations between greenness exposure and human cardiovascular health may be a result of the vascular action of α-pinene and its metabolites, a novel consideration.

The Polyanionic Drug Suramin Neutralizes Histones and Prevents Endotheliopathy.

Drugs are needed to protect against the neutrophil-derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but challenges with dosing or side effects such as bleeding limit clinical application. In this study, we demonstrate that suramin, a widely available polyanionic drug, completely neutralizes the toxic effects of individual histones, but not citrullinated histones from neutrophil extracellular traps. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone-induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial-dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones invivo. Suramin also prevented histone-induced lung endothelial cell cytotoxicity invitro and lung edema, intra-alveolar hemorrhage, and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels.

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression.

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels.

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Acrolein but not its metabolite, 3-Hydroxypropylmercapturic acid (3HPMA), activates vascular transient receptor potential Ankyrin-1 (TRPA1): Physiological to toxicological implications

Acrolein, an electrophilic α,β-unsaturated aldehyde, is present in foods and beverages, and is a product of incomplete combustion, and thus, reaches high ppm levels in tobacco smoke and structural fires. Exposure to acrolein is linked with cardiopulmonary toxicity and cardiovascular disease risk. The hypothesis of this study is the direct effects of acrolein in isolated murine blood vessels (aorta and superior mesenteric artery, SMA) are transient receptor potential ankyrin-1 (TRPA1) dependent. Using isometric myography, isolated aorta and SMA were exposed to increasing levels of acrolein. Acrolein inhibited phenylephrine (PE)-induced contractions (approximately 90%) in aorta and SMA of male and female mice in a concentration-dependent (0.01–100 μM) manner. The major metabolite of acrolein, 3-hydroxypropylmercapturic acid (3HPMA), also relaxed PE-precontracted SMA. As the SMA was 20× more sensitive to acrolein than aorta (SMA EC50 0.8 ± 0.2 μM; aorta EC50 > 29.4 ± 4.4 μM), the mechanisms of acrolein-induced relaxation were studied in SMA. The potency of acrolein-induced relaxation was inhibited significantly by: 1) mechanically-impaired endothelium; 2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); 3) guanylyl cyclase (GC) inhibitor (ODQ); and, 4) a TRPA1 antagonist (A967079). TRPA1 positive immunofluorescence was present in the endothelium. Compared with other known TRPA1 agonists, including allyl isothiocyanate (AITC), cinnamaldehyde, crotonaldehyde, and formaldehyde, acrolein stimulated a more potent TRPA1-dependent relaxation. Acrolein, at high concentration [100 μM], induced tension oscillations (spasms) independent of TRPA1 in precontracted SMA but not in aorta. In conclusion, acrolein is vasorelaxant at low levels (physiological) yet vasotoxic at high levels (toxicological).

Stabilization of Nrf2 leading to HO-1 activation protects against zinc oxide nanoparticles-induced endothelial cell death

With the abundant production and wide application of zinc oxide nanoparticles (ZnONPs), the potential health risks of ZnONPs have raised serious concerns. Oxidative stress is recognized as the most important outcome of the toxicity induced by ZnONPs. The Nrf2-Keap1 system and its downstream antioxidative genes are the fundamental protective mechanisms for redox hemeostasis. However, the detailed mechanisms of Nrf2 activation in ZnONPs-treated endothelial cells and murine blood vessels have yet to be elucidated. Herein, we show that Nrf2 was activated and played a negative role in cell death induced by ZnONPs. Moreover, we demonstrate that HO-1 was the most extensively upregulated antioxidative gene-activated by Nrf2. Forced overexpression of HO-1, pharmacological activation of HO-1 with the agonists RTA-408 (omaveloxolone, an FDA-approved drug) and RTA-402 repressed cell death, and treatment with HO-1 antagonist SnPP exacerbated the cell death. Importantly, loss of HO-1 diminished the cytoprotective role induced by Nrf2 in ZnONPs-treated HUVEC cells, indicating that the Nrf2-HO-1 axis was the crucial regulatory mechanism for the antioxidative response in the context of ZnONPs-induced endothelial damage. Mechanistically, we demonstrate that the p62-Keap1 axis was not involved in the activation of Nrf2. Intriguingly, the degradation half-life of Nrf2 in HUVEC cells was increased from less than 1 h under quiescent conditions to approximately 6 h under ZnONPs treatment condition; moreover, ZnONPs treatment induced activation of Nrf2/HO-1 and accumulation of ubiquitin in the aorta ventralis of mouse, suggesting that the ubiquitin-proteasome system had been perturbed, which subsequently led to the stabilization of Nrf2 and activation of HO-1. This study might contribute to a better understanding of ZnONPs-associated toxicity.

Crotonaldehyde-induced vascular relaxation and toxicity: Role of endothelium and transient receptor potential ankyrin-1 (TRPA1)

IntroductionCrotonaldehyde (CR) is an electrophilic α,β-unsaturated aldehyde present in foods and beverages and is a minor metabolite of 1,3-butadiene. CR is a product of incomplete combustion, and is at high levels in smoke of cigarettes and structural fires. Exposure to CR has been linked to cardiopulmonary toxicity and cardiovascular disease. ObjectiveThe purpose of this study was to examine the direct effects of CR in murine blood vessels (aorta and superior mesenteric artery, SMA) using an in vitro system. Methods and resultsCR induced concentration-dependent (1–300 μM) relaxations (75–80%) in phenylephrine (PE) precontracted aorta and SMA. Because the SMA was 20× more sensitive to CR than aorta (SMA EC50 3.8 ± 0.5 μM; aorta EC50 76.0 ± 2.0 μM), mechanisms of CR relaxation were studied in SMA. The CR-induced relaxation at low concentrations (1–30 μM) was inhibited by: 1) mechanically-impaired endothelium; 2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); 3) guanylyl cyclase (GC) inhibitor (ODQ); 4) transient receptor potential ankyrin-1 (TRPA1) antagonist (A967079); and, 5) by non-vasoactive level of nicotine (1 μM). Similarly, a TRPA1 agonist, allyl isothiocyanate (AITC; mustard oil), stimulated SMA relaxation dependent on TRPA1, endothelium, NO, and GC. Consistent with these mechanisms, TRPA1 was present in the SMA endothelium. CR, at higher concentrations (100–300 μM), induced tension oscillations (spasms) and irreversibly impaired contractility (a vasotoxic effect enhanced by impaired endothelium). ConclusionsCR relaxation depends on a functional endothelium and TRPA1, whereas vasotoxicity is enhanced by endothelium dysfunction. Thus, CR is both vasoactive and vasotoxic along a concentration continuum.

Role of Transient Receptor Potential Ankyrin‐1 (TRPA1) in Endothelial‐Specific Effects of Diverse Aldehydes

IntroductionStructurally diverse aldehydes are found in abundance in the environment including in foods, beverages and tobacco‐derived aerosols. Some aldehydes are associated with cardiovascular disease risk especially in cigarettes smokers, however, the detailed mechanisms by which aldehydes influence cardiovascular health and disease are incompletely known.ObjectiveBecause reactive aldehydes, in general, are considered instigators of endothelium dysfunction, we explored the direct effects of a diverse set of aldehydes in isolated murine blood vessels, i.e., aorta and superior mesenteric artery (SMA).MethodsAldehydes: acetaldehyde (AA); formaldehyde (FA); cinnamaldehyde (CA); crotonaldehyde (CR); and a TRPA1 agonist, allyl isothiocyanate (AITC, mustard oil), were tested for vascular effects in isolated murine aorta and SMA using isometric myography.ResultsAll aldehydes induced endothelium‐dependent relaxation in SMA. AA (1–100 mM), FA (30–1,500 μM), CA (30–300 μM) and CR (1–100 μM) strongly and reversibly relaxed (>75% relaxation) agonist‐induced contractions. High levels of FA (1.2–1.6 mM) and CR (>300 μM) irreversibly altered vascular function in aorta and SMA. To identify the mechanism of aldehyde‐induced relaxations, isolated blood vessels were pre‐contracted with phenylephrine (PE), U46,619 (thromboxane A2 analog), or 60 mM K+ (High K) and then exposed to AA, FA, CA, CR or AITC with intact or mechanically‐impaired endothelium (air‐perfused) as well as in the absence or presence of: 1) Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME); 2) an inhibitor of guanylyl cyclase (ODQ); and, 3) a TRPA1 antagonist (A967079; 1 μM). The sensitivity (EC50) but not the efficacy (% decreased tension) of FA‐ and AA‐induced relaxations was dependent on the contractile agonist (i.e., PE < U46,619 ≤ High K) indicating multiple mechanisms. In PE pre‐contracted SMA, vasorelaxations at the lowest levels of AA, FA, CA, CR and AITC were inhibited significantly by mechanically‐disrupted endothelium, L‐NAME, ODQ, and by TRPA1 antagonist except for AA, which was TRPA1 independent. TRPA1 was co‐localized with isolectin immunofluorescent staining in SMA endothelium but not in aorta.ConclusionBecause low levels (physiological) of several aldehydes (FA, CA, CR) induce reversible and efficacious vasorelaxations via TRPA1 channels, we propose that endogenous and exogenous sources of these aldehydes may significantly impact gut (mesenteric) blood flow to augment postprandial hyperemia in aid of digestion and nutrient uptake.Support or Funding InformationResearch funding from the National Institutes of Health: GM127607, HL122676, U54HL120163.

Proliferative Role of Kv11 Channels in Murine Arteries.

K+ channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K+ currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries.Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries. Immunofluorescence was performed on single vascular smooth muscle cells using antibodies against the ERG1 expression product (Kv11.1). Single cell electrophysiology was performed on myocytes from portal vein and several different arteries, complimented by isometric tension recordings. Proliferation assays were undertaken on smooth muscle cells isolated from femoral arteries.Results: ERG1 transcripts were detected in all murine blood vessels, and Kv11.1 immunofluorescence was observed in all smooth muscle cells. However, K+ currents with properties consistent with ERG channels were only recorded in portal vein myocytes. Moreover, ERG channel blockers (E4031 or dofetilide, 1 μM) failed to depolarize carotid arteries or produce contraction. Proliferation of arterial smooth muscle cells was associated with a marked increase in ERG1 expression and ERG blockers suppressed proliferation significantly.Conclusions: These data reveal that arterial blood vessels express ERG channels that appear to be functional silent in contractile smooth muscle but contribute to proliferative response.

Relief from vascular occlusion using photothermal ablation of thrombus with a multimodal perspective

Fibrinolytic therapy for arterial or venous thrombotic disorders involves the systemic administration of thrombolytics such as streptokinase, which is associated with serious bleeding complications. With this study, we provide a proof-of-concept of photothermal thrombus ablation with gold nanorods exposed to near-infrared irradiation, both in vitro using materials generated from purified fibrinogen or plasma and in vivo in murine blood vessels. This is the first report of the application of photothermal therapy as an anti-thrombotic measure. Remarkably, the addition of streptokinase had a multimodal additive effect with regard to acceleration of photothermal lysis of thrombi even at a dose significantly below the therapeutic concentration, thus minimizing the life-threatening side effects and adverse complications. This combinatorial approach exhibits great promise for lysing pathological clots while effectively overcoming the drawbacks of existing therapies.

Ischemia and reperfusion injury of the spinal cord: experimental strategies to examine postischemic paraplegia

Thoracoabdominal aortic replacement, necessary in case of injuries, aneurysms and dissections, shows a high complication rate as a consequence of the perioperative ischemia / reperfusion-sequence (I/R). Clamping above and below the lesion leads to the spinal cord suffering from ischemia. Clamping times of less than 30 minutes show only a small risk of neurological deficit, while longer periods increase paraplegia rates disproportionately. The subsequent reperfusion as a second hit causes additional damage to the spinal cord. Up to 30 % of all patients who require being treated in the thoracoabdominal part of the aorta suffer from paraplegia within the first 24 postoperative hours (Kahn et al., 2012).

Isoform 165 of vascular endothelial growth factor in collagen matrix improves ovine cryopreserved ovarian tissue revascularisation after xenotransplantation in mice.

BackgroundAggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still requires improvement.MethodsFrozen/thawed sheep ovarian cortical biopsies were embedded in collagen matrix with or without isoform 165 of vascular endothelial growth factor (VEGF165) and transplanted into ovaries of immunodeficient mice. Ovaries were chosen as transplantation sites to more closely resemble clinical conditions in which orthotopic transplantation has previously allowed several spontaneous pregnancies.ResultsWe found that VEGF165 significantly increased the number of Dextran-FITC positive functional vessels 3 days after grafting. Dextran- fluorescein isothiocyanate (FITC) positive vessels were detectable in 53% and 29% of the mice in the VEGF-treated and control groups, respectively. Among these positive fragments, 50% in the treated group displayed mature smooth-muscle-actin-alpha (alpha-SMA) positive functional vessels compared with 0% in the control group. CD31 positive murine blood vessels were observed in 40% of the VEGF165 transplants compared with 21% of the controls. After 3 weeks, the density of murine vessels was significantly higher in the VEGF165 group.ConclusionThe encapsulation of ovarian tissue in collagen matrix in the presence of VEGF165 before grafting has a positive effect on functional blood vessel recruitment. It can be considered as a useful technique to be improved and further developed before human clinical applications in female cancer patients in the context of fertility preservation.

Xenograft Tumors Vascularized with Murine Blood Vessels May Overestimate the Effect of Anti-Tumor Drugs: A Pilot Study

Recent evidence demonstrated that endothelial cells initiate signaling events that enhance tumor cell survival, proliferation, invasion, and tumor recurrence. Under this new paradigm for cellular crosstalk within the tumor microenvironment, the origin of endothelial cells and tumor cells may have a direct impact on the pathobiology of cancer. The purpose of this pilot study was to evaluate the effect of endothelial cell species (i.e. murine or human) on xenograft tumor growth and response to therapy. Tumor xenografts vascularized either with human or with murine microvascular endothelial cells were engineered, side-by-side, subcutaneously in the dorsum of immunodefficient mice. When tumors reached 200 mm3, mice were treated for 30 days with either 4 mg/kg cisplatin (i.p.) every 5 days or with 40 mg/kg sunitinib (p.o.) daily. Xenograft human tumors vascularized with human endothelial cells grow faster than xenograft tumors vascularized with mouse endothelial cells (P<0.05). Notably, human tumors vascularized with human endothelial cells exhibited nuclear translocation of p65 (indicative of high NF-kB activity), and were more resistant to treatment with cisplatin or sunitinib than the contralateral tumors vascularized with murine endothelial cells (P<0.05). Collectively, these studies suggest that the species of endothelial cells has a direct impact on xenograft tumor growth and response to treatment with the chemotherapeutic drug cisplatin or with the anti-angiogenic drug sunitinib.

The nucleus of endothelial cell as a sensor of blood flow direction

SummaryHemodynamic shear stresses cause endothelial cells (ECs) to polarize in the plane of the flow. Paradoxically, under strong shear flows, ECs disassemble their primary cilia, common sensors of shear, and thus must use an alternative mechanism of sensing the strength and direction of flow. In our experiments in microfluidic perfusion chambers, confluent ECs developed planar cell polarity at a rate proportional to the shear stress. The location of Golgi apparatus and microtubule organizing center was biased to the upstream side of the nucleus, i.e. the ECs polarized against the flow. These in vitro results agreed with observations in murine blood vessels, where EC polarization against the flow was stronger in high flow arteries than in veins. Once established, flow-induced polarization persisted over long time intervals without external shear. Transient destabilization of acto-myosin cytoskeleton by inhibition of myosin II or depolymerization of actin promoted polarization of EC against the flow, indicating that an intact acto-myosin cytoskeleton resists flow-induced polarization. These results suggested that polarization was induced by mechanical displacement of EC nuclei downstream under the hydrodynamic drag. This hypothesis was confirmed by the observation that acute application of a large hydrodynamic force to ECs resulted in an immediate downstream displacement of nuclei and was sufficient to induce persistent polarization. Taken together, our data indicate that ECs can sense the direction and strength of blood flow through the hydrodynamic drag applied to their nuclei.

Sphingosine-1-phosphate accelerates neoangiogenesis, reduces hypoxia, and improves primordial follicle survival in human ovarian xenografts: a prelude to improving ovarian transplantation outcomes

Sphingosine-1-phosphate accelerates neoangiogenesis, reduces hypoxia, and improves primordial follicle survival in human ovarian xenografts: a prelude to improving ovarian transplantation outcomes

Intravital Fluorescence Microscopy Improves Thrombosis Phenotype Scoring in Mice

Over the past 15 years, the laboratory mouse has yielded a wealth of information on the pathogenesis of thrombosis. To a large extent, the driving force behind the use of mice to study thrombosis has been the remarkable advances in molecular approaches to selectively modifying gene expression, thereby allowing analyses of the function of specific blood and vascular wall proteins in vivo. Although all murine blood vessels might be considered small, studies of thrombosis in mice have been categorized as macrovascular and microvascular. The former pertains to conductance arteries and large veins, such as the carotid artery, aorta, inferior vena cava, and jugular vein. Studies of these blood vessels are relevant to human diseases such as myocardial infarction, stroke, and deep venous thrombosis. Microvascular thrombosis models have involved analyses of arterioles (diameters approximately 30 to 100 μm) within vascular beds that can be exteriorized en bloc and imaged microscopically as thin sections, such as those of the cremaster muscle and mesentery.1,2 Microvascular studies are relevant to …

The Use of Immunofluorescent Array Tomography to Study the Three-Dimensional Microstructure of Murine Blood Vessels

The purpose of this study was to develop the methods needed to enable the application of Immunofluorescent Array Tomography (IAT), a novel three-dimensional (3D) microscopy technique, to murine blood vessels. The anterior and posterior regions of the infrarenal aorta of 8–10 week old C57BL6 mice were evaluated. Staining and image analysis methods were developed. Antibody selection, primary antibody concentration, co-staining with multiple primary antibodies, and the multi-cycle staining design were optimized to produce positive and specific staining of elastin (1:50 dilution), smooth muscle cell actin (SMCA, 20 μg mL−1), and collagen type I (10 μg mL−1). Non-specific interactions were limited by optimizing secondary antibody staining conditions (1:200 dilution, 30 min). SMCA and collagen type I were stained in the first cycle, elastin was stained in the second cycle, and nuclei were stained in both cycles. Algorithms were developed to quantify volume fractions of medial elastin, SMCA, and nuclei, as well as adventitial collagen type I. Elastin thickness, spacing between elastin lamellae, elastin fragmentation, media wall thickness, nuclei aspect ratio, and nuclei amount were also quantified. We have qualitatively and quantitatively characterized the 3D microstructure and cellular morphology of the anterior and posterior infrarenal murine aorta using IAT.

Abstract 1581: Determination of the cellular origin of stroma in osteosarcoma

Abstract Introduction- Solid tumors like osteosarcoma have a distinct structure that is comprised of two interdependent compartments, the parenchyma (neoplastic cells) and the stroma that the neoplastic cells induce or produce and in which they are dispersed. The stroma may be induced as a result of tumor cell-host interactions or alternatively tumors may undergo different patterns of programmed differentiation to perform the necessary stromal support functions. Since stroma development is critical to the growth of tumor, identification of origin and mechanism of development of stroma can help target potential therapies against the tumor. Therefore we performed this study to elucidate the origin of stroma in a osteoarcoma xenograft, with a hypothesis that, the stroma of osteosarcoma is host (mouse) derived. Methods- (1) Frozen sections from xenograft osteosarcomas, were stained with vWF and CD31 (endothelial marker antigens) to identify the blood vessels. (2) The endothelial cells were laser capture dissected from frozen sections by pattern recognition. The Human and mice vWF and CD31 gene expression was quantified by real time PCR. (3) Detection of human and mice vWF gene by F.I.S.H. (4) Double immunofluorescent staining used to further delineate the mouse or human origin of the blood vessels. Anti Human Vimentin used to stain all human tissues, and anti vWF(Human and mice) to stain blood vessels of human and mice, fluorophore labeled secondary antibodies used to identify the bound primary antibodies. Results-The Immuno staining for vWF (reactive for both human and mice) and CD 31 (reactive only for human) revealed deficient staining for CD31, implying that the blood vessels were mostly murine. The gene expression assays on the microdissected samples for human and mouse VWF and CD 31, showed that there is expression of both human and murine vWF and CD31 in the xenografts, with more mouse genes than human in the microdissected blood vessels. The findings were confirmed by F.I.S.H for vWF probes for human and mice, whose initial results show that 95% of the tumors is human but at least 5% of the xenograft section stained for the mouse vWF probes. Double immunofluorescent studies are still in progress but preliminary results show, predominant human blood vessels with interspread murine blood vessels. Conclusions – Although confirmatory experiments are still in progress, initial suggestions in the context of human heterotopic osteosarcoma xenografts, show that most of blood vessels are murine derived. Further studies will be undertaken to determine whether the blood vessels in human osteosarcomas are tumor derived or a result of vascular in-growth as is the case in the murine system. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1581. doi:10.1158/1538-7445.AM2011-1581