Rat Mesentery Research Articles

Investigation into the diversity in the fractal dimensions of arterioles and venules in a microvascular network – A quantitative analysis

Fractal dimension is a robust fractal parameter for estimating the morphology of vascular networks. It reflects the property of vascular networks that may vary and thus, differentiate between individual networks and/or identify physiological and pathological conditions. As such, fractal dimension differs also between arteriolar and venular compartments, yet the underlying reason is so far unclear. In order to understand the mechanisms behind these differences, we quantitatively analyzed the impacts of vessel attributes on the fractal dimension. Fractal dimension and vessel attributes given by vessel density (VD), vessel length density (VL), and diameter index (DI=VD/VL) were analyzed in three microvascular networks of the rat mesentery, which were reconstructed from experimental data. The results show that differences in diameter between arterioles and venules are primarily responsible for arterio-venous differences in fractal dimension. Moreover, multiple linear regression analysis demonstrates that the sensitivity of the variation of fractal dimension to vessel length and diameter varies with the type of the vessels. While the change of vessel length contributes 57.8 ± 3.4% to the variation of arteriolar dimension, vessel diameter contributes 63.9 ± 4.8% to the variation of venular dimension. The present study provides an explanation for the different fractal dimension and dimension variation in arteriolar and venular compartments. It highlights the importance of estimating the fractal dimensions of arterioles and venules separately, which will enhance the ability of feature extraction by fractal analysis in physiological and clinical application.

Targeting Plasminogen Activator Inhibitor‐1 Alleviates Inflammatory Responses Invoked by Sepsis or Chlorinated Lipid

Plasminogen activator inhibitor‐1 (PAI‐1) promotes leukocyte‐endothelium interactions and transmigration secondary to ischemia/reperfusion (I/R). Our previous work demonstrated that 2‐chlorohexadecanal (2‐ClHDA), derived from myeloperoxidase (MPO) produced hypochlorous acid (HOCL) targeting of plasmalogen phospholipids, may serve as a mediator of the inflammatory responses to sepsis. Given the fact that the septic inflammatory responses invoked by cecal ligation and puncture (CLP) can be reversed by an MPO inhibitor, we have postulated that sepsis‐induced inflammation may be mediated through MPO/HOCL/2‐ClHDA axis. However, the utility of drug targeting of PAI‐1 to inhibit inflammatory responses induced by CLP or 2‐ClHDA superfusion remains to be evaluated. We have begun to explore this issue by pretreating rats with PAI‐1 inhibitor, PAI‐039 (tiplaxtinin, 44 ug/kg) one hour prior to CLP or Sham surgery and then again one hour before beginning to take measurements. Six hours after surgery, inflammatory responses in four groups of Sprague‐Dawley rats (n=6) were quantified and compared on leukocyte‐endothelial cell interactions and mast cell degranulation in rat mesenteries using intravital microscopy. In other groups of rats pretreated with PAI‐039 one hour beforehand, the exposed mesenteries were superfused with 2‐ClHDA at 10 mM (a dose within the range of plasma concentrations reported for septic patients or rats) for ninety minutes, with inflammatory responses recorded at baseline, thirty, sixty and ninety minutes. The data showed that CLP or mesenteric superfusion with 2‐ClHDA induced marked increases in the numbers of rolling and firmly adherent leukocytes, and significant mast cell activation that were similar in magnitude in both models, and were prevented by pretreatment with the PAI‐1 inhibitor PAI‐039. Our results indicate that the increases in leukocyte rolling, leukocyte adhesion, and mast cell degranulation invoked by 2‐ClHDA superfusion mimicked the responses to CLP. Moreover, these proinflammatory responses appear to be dependent on PAI‐1.Support or Funding InformationSupported by NIH grant GM‐115553.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Astrocyte Activation is Increased in a Rat Endometriosis Model

Peripheral inflammation produces an immune response in the brain by the activation of microglia and astrocytes. Astrocytes are also involved in the pain symptoms of chronic diseases such as endometriosis, which is characterized by the growth of uterine tissue outside the reproductive organs. Studies using a mouse model of endometriosis found an increase in astrocytes in the spinal cord but effects in the various brain regions such as the hippocampus are still unknown. However, decreased grey matter value in women with endometriosis experiencing chronic pelvic pain suggests the involvement of the central nervous system. Previous studies in our lab found an increase in hyperalgesia symptoms in a rat model of endometriosis, however, the activation of glia cells such astrocytes and microglia in the central nervous system is still unknown.AimStudy the effects of endometriosis on astrocyte and microglia activation in the hippocampus.MethodsEndometriosis was induced by implanting uterine tissue in the intestinal mesentery in female Sprague Dawley rats that weighed more than 200g (ENDO group). Control animals received only sutures in the same region (SHAM). Sixty days after the induction of endometriosis, both groups underwent behavioral testing using open field followed by sacrifice to collect brain and endometrial tissue. Brain sections were stained against the astrocyte marker, Glial Fibric Acid Protein (GFAP), and the microglia marker, ionized calcium‐binding adapter molecule 1 (IBA‐1), and both the percent area and number and length of projections was measured using ImageJ.ResultsThe ENDO group developed vesicles with a mean total area of 49.67 mm2, indicating development of endometriosis. The ENDO group showed no significant differences in entries to the center of the open field arena (p>0.05; n=4–6), however they had significantly less velocity and travelled less distance compared with the SHAM group (p<0.05; n=4–6). Brain immunofluorescence shows an increase in the percent of area of GFAP positive staining, and an increase in the number and length of astrocyte projections in the hippocampus in the ENDO group compared with the SHAM (p<0.05; n=4–6). No significant differences were found with microglia.ConclusionsOur results demonstrate the possible involvement of astrocytes in the brain of the rat endometriosis model. Astrocyte activation in the hippocampus may be associated with peripheral inflammation and contribute to the hyperalgesia and depressive behavior often associated with endometriosis.Support or Funding InformationSupported in part by P20 GM103475‐16 & R15AT009915.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stromal Vascular Fraction Vasculogenesis, Vessel Incorporation, and Integration with Intact Angiogenic Microvascular Networks in an Ex Vivo Cultured Tissue Model

Angiogenesis, the growth of new vessels from pre‐existing vessels, is vital to wound healing in the cases of myocardial infarction and peripheral arterial disease. Adipose‐derived stromal vascular fraction (SVF), consisting of endothelial cells, endothelial progenitor cells, pericytes, smooth muscle cells, fibroblasts and immune cells, can be transplanted to assist in wound healing as seen in various clinical trials. However, the spatiotemporal incorporation of the SVF within living tissues remains largely unknown. As such, understanding where and how SVF cells contribute to microvascular growth and remodeling will help guide their therapeutic use. The objective of this study was to evaluate the impact of SVF cells and their fate during microvascular growth utilizing a novel tissue culture model that enables time‐lapse observation of cell dynamics across intact networks. SVF was isolated from adipose of an adult male Wistar rat, labeled with DiI, and seeded onto mesentery tissues harvested from the same rat before culturing in MEM + 10% fetal bovine serum and 1% PenStrep for 3 days. Tissues were then labeled for lectin to identify vessels. Alternatively, SVF was isolated from adipose harvested from adult male or female SD‐EGFP rats, seeded onto lectin labeled mesentery from adult male Wistar rats, placed under the same culture conditions, and imaged every 24 hours. Vascularized area was increased in SVF treated versus control tissues (SVF = 84.5±1.3%; Control = 56.5%; p < 0.001; n = 4 tissues per group). The SVF related microvasculature was characterized by asterisk‐shaped clusters. GFP tracking of SVF revealed vessel segment formation via vasculogenesis containing endothelial cells and pericytes in previously avascular areas. In pre‐vascularized regions, SVF cells contributed to new vessel growth and integration with and along native vessels. SVF cells were observed both in apparent endothelial cell and wrapping pericyte locations. Interestingly, examples of new vessel segments derived from SVF cells were observed along LYVE‐1 positive lymphatic vessels. These results suggest that SVF transplantation increases vascular area through vasculogenesis and contributes to angiogenesis of pre‐existing vasculature. The results also support the use of the rat mesentery culture model as a novel tool for elucidating SVF cell transplant dynamics in an intact tissue environment.Support or Funding InformationFunded by NIH R01AG049821This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neovascularization and functional assessment of human pancreatic islets encapsulated with immuno‐modulatory stem cells

Background & ObjectiveDeveloping successful therapies based on re‐introduction of healthy pancreatic islets in diabetic patients requires new strategies to combat the immune response caused by implantation, as well as improving neovascularization and engraftment of the implant. Human amniotic epithelial cells (AECs) are immuno‐modulatory stem cells that can confer a local immune‐privileged environment in vivo, and block grafted tissue rejection [1]. The objective of this study was to develop a protocol for encapsulating islets with AECs, and examine the effects of encapsulation on islet function, and neovascularization.MethodsHuman pancreatic islets were individually placed in ultra‐low attachment round bottom wells with human islet medium, and were divided into three experimental groups: 1) islet‐only, 2) adipose‐derived stem cells (ASCs)+islet, and 3) AECs+islet. For groups 2 and 3, each islet was assumed to have ~2000 beta cells, and the stem cells were added to each well to encapsulate individual islets at 2:1 cell ratio. The temporal c‐peptide secretion level was measured after 3 and 6 days by perfusing the islets with basal and high glucose solutions using dynamic readout assay, and confirmed the functionality of the encapsulated islets. To assess the angiogenic properties of encapsulation, cell culture supernates were collected after 6 days of culture, and used for an angiogenesis array kit, to identify and compare the level of 52 angiogenic factors present in each group. The same test was repeated for ASC‐ and AEC‐only cultures to identify any effects caused by the cell encapsulation.Results & ConclusionsThe level of pro‐angiogenic factors increased in encapsulated groups compared to the islet‐only group (Fig. 1). A larger increase in angiogenic factors in encapsulated islets compared to stem cell‐only groups demonstrated a synergetic angiogenic effect caused by encapsulation of islets with ASCs and AECs. To test the angiogenic response in a physiological scenario, the islets were seeded on rat mesentery tissues, and cultured for 6 days, and the level of angiogenic response was compared between different groups. Our results highlight the effect of encapsulating human islets with ASCs and AECs on engraftment and neovascularization, and showcase the potential of the islet cell encapsulation method for a successful long‐term implantation in patients with diabetes.Support or Funding InformationThe Virginia Catalyst and UVA Center for Advanced BiomanufacturingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A hybrid discrete-continuum approach for modelling microcirculatory blood flow.

In recent years, biological imaging techniques have advanced significantly and it is now possible to digitally reconstruct microvascular network structures in detail, identifying the smallest capillaries at sub-micron resolution and generating large 3D structural data sets of size >106 vessel segments. However, this relies on ex vivo imaging; corresponding in vivo measures of microvascular structure and flow are limited to larger branching vessels and are not achievable in three dimensions for the smallest vessels. This suggests the use of computational modelling to combine in vivo measures of branching vessel architecture and flows with ex vivo data on complete microvascular structures to predict effective flow and pressures distributions. In this paper, a hybrid discrete-continuum model to predict microcirculatory blood flow based on structural information is developed and compared with existing models for flow and pressure in individual vessels. A continuum-based Darcy model for transport in the capillary bed is coupled via point sources of flux to flows in individual arteriolar vessels, which are described explicitly using Poiseuille's law. The venular drainage is represented as a spatially uniform flow sink. The resulting discrete-continuum framework is parameterized using structural data from the capillary network and compared with a fully discrete flow and pressure solution in three networks derived from observations of the rat mesentery. The discrete-continuum approach is feasible and effective, providing a promising tool for extracting functional transport properties in situations where vascular branching structures are well defined.

Hypertension exhibits 5-HT4 receptor as a modulator of sympathetic neurotransmission in the rat mesenteric vasculature.

Sympathetic overdrive is a key player in hypertension, where the mesenteric vasculature plays a relevant role in modulating blood pressure. Although 5-HT inhibits noradrenergic mesenteric neurotransmission in normotensive rats, its effect on the mesenteric sympathetic drive in hypertensive rats has not been studied. We investigated the influence of in vivo 5-HT by characterizing the implicated serotonergic receptors on the mesenteric sympathetic outflow in rats with N-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. Hypertension was induced in male Wistar rats by L-NAME administration (30 mg/kg per day; 21 days) in drinking water. The rats were anesthetized (sodium pentobarbital; 60 mg/kg, i.p.), prepared for the in situ autoperfused rat mesentery, and subjected for monitoring their systemic blood pressure (SBP), heart rate (HR), and mesenteric perfusion pressure (MPP). Electrical stimulation of mesenteric sympathetic nerves resulted in frequency-dependent increases in MPP without altering SBP or HR. The 5-HT and cisapride (5-HT4 agonist) i.a. bolus (1-25 µg/kg) inhibited vasopressor responses by electrical stimulation of the mesenteric nerves, unlike an i.a. bolus (25 µg/kg each) of the agonist 5-carboxamidotryptamine (5-HT1/7 agonist), α-methyl-5-HT (5-HT2), or 1-PBG (5-HT3). However, i.a. cisapride (25 µg/kg) did not affect the noradrenaline-induced vasoconstriction in the mesenteric vasculature. Administration of the selective 5-HT4 receptor antagonist GR 125487 (1 mg/kg, i.v.) completely abolished cisapride- and 5-HT-evoked mesenteric sympatholytic effects. Additionally, ELISA analysis demonstrated higher 5-HT4 receptor expression in mesenteric arteries from L-NAME-hypertensive compared with normotensive rats. Our findings suggest that L-NAME-induced hypertension modifies the 5-HT modulation of the rat mesenteric sympathetic drive: prejunctional 5-HT4 receptors are involved in the serotonergic sympathoinhibitory effect.

Oral ingestion of a novel oxygenating compound, Ox66™, is non-toxic and has the potential to increase oxygenation

Ox66™ is a novel solid state oxygenating compound. In order to support the use of Ox66™ as a potential oxygenating supplement to injured cells, this study evaluated the safety of Ox66™, its ability to withstand the conditions in the digestive tract, and its potential to increase oxygenation in the mesentery in rats. The toxicity of Ox66™ was evaluated by performing acute (10-day) and chronic (90-day) feeding studies on rats, the stability of the compound in the digestive tract was evaluated via ex vivo simulated digestion and subsequent CFDA viability assay on gut epithelial cells, and its capacity for oxygenation in the mesenteric microcirculation was determined by interstitial fluid pressure (PISF) O2 measurements upon injection into the small intestine of rats. No toxicity was found associated with acute or chronic oral administration of the compound in rats, and the compound was able to withstand the environment of the digestive tract in vitro. Based on the acute animal feeding study, the NOAEL was considered to be 1000 mg/kg/day. This proof-of-concept study further demonstrates the potential of Ox66™ to function as an oxygenating supplement that might be useful for treating either pathological hypoxic-related conditions or to improve oxygenation levels during or after exercise under healthy conditions.

A one-dimensional mathematical model of collecting lymphatics coupled with an electro-fluid-mechanical contraction model and valve dynamics.

We propose a one-dimensional model for collecting lymphatics coupled with a novel Electro-Fluid-Mechanical Contraction (EFMC) model for dynamical contractions, based on a modified FitzHugh-Nagumo model for action potentials. The one-dimensional model for a deformable lymphatic vessel is a nonlinear system of hyperbolic Partial Differential Equations (PDEs). The EFMC model combines the electrical activity of lymphangions (action potentials) with fluid-mechanical feedback (circumferential stretch of the lymphatic wall and wall shear stress) and lymphatic vessel wall contractions. The EFMC model is governed by four Ordinary Differential Equations (ODEs) and phenomenologically relies on: (1) environmental calcium influx, (2) stretch-activated calcium influx, and (3) contraction inhibitions induced by wall shear stresses. We carried out a stability analysis of the stationary state of the EFMC model. Contractions turn out to be triggered by the instability of the stationary state. Overall, the EFMC model allows emulating the influence of pressure and wall shear stress on the frequency of contractions observed experimentally. Lymphatic valves are modelled by extending an existing lumped-parameter model for blood vessels. Modern numerical methods are employed for the one-dimensional model (PDEs), for the EFMC model and valve dynamics (ODEs). Adopting the geometrical structure of collecting lymphatics from rat mesentery, we apply the full mathematical model to a carefully selected suite of test problems inspired by experiments. We analysed several indices of a single lymphangion for a wide range of upstream and downstream pressure combinations which included both favourable and adverse pressure gradients. The most influential model parameters were identified by performing two sensitivity analyses for favourable and adverse pressure gradients.

Occlusion and rupture of ex vivo capillary bifurcation due to acoustic droplet vaporization

Gas embolotherapy (GE) consists in the occlusion of tumor blood vessels using gas emboli induced by acoustic droplet vaporization (ADV), to create tumor starvation and localized drug delivery. Therefore, the occlusion and rupture of capillary bifurcation due to ADV was investigated in an ex vivo rat mesentery model using a confocal acousto-optical high-speed microscope system. Following ADV bubble formation, coalescence, and translational movement, the growing bubbles lodged in and then occluded two different capillary bifurcations. Capillary rupture was induced at the bubble lodging area, immediately followed by gas extravasation and bubble dislodging. Before and after bubble lodgment/occlusion, a local microvessel invagination was observed due to the interactions between ADV bubbles and the microvessel itself, indicating a contribution to the capillary rupture. Understanding the transient dynamics of ADV bubble, the bubble–microvessel interaction and the consequent mechanical bio-effects in GE is of the paramount importance for developing and applying this approach in clinical practice.

Changes in the Microvasculature of Small Bowel Mesentery in Rats with Prehepatic Portal Hypertension: the Preliminary Study in vivo

According to the literature, neoangiogenesis in the mesentery of the small intestine is the trigger of portosystemic collateral circulation in portal hypertension. However, the types of blood vessels involved in this process have not been established. The aim of this study was to determine which type of the mesenteric microvessels participates in angiogenesis in portal hypertension. Studies were performed in 12 adult outbred female rats weighing 250 ± 50 g which were divided into two groups: sham-operated (n = 5) and experimental (n = 7). The mesenteric microcirculation of rats in both groups was examined by intravital microscopy during the first laparotomy and relaparotomy on the 15th day of the experiment. In contrast to the sham-operated animals, prehepatic portal hypertension was induced in rats of the experimental group by partial portal vein ligation during the first operation. Portal pressure was measured in both groups at the time of the second operation. The portal pressure in rats of the experimental group was significantly higher than in sham-operated rats: 12,53 ± 1,26 mm Hg and 9,34 ± 0,14 mm Hg, respectively (p<0,01). There was a significant increase in the number of capillaries (p <0,05) and total vascular density (p <0,05) in rats of both groups by the 15th day of the experiment. At the same time, a greater increase in the capillary network was observed in rats of the experimental group (p <0,05), whereas no significant differences were found in the values of vascular density of the other types of blood vessels. Changes in the mesenteric microvasculature in portal hypertension lay in the increase in the values of vascular density, which occurred mostly at the expense of capillaries.

A Novel ex vivo Mouse Mesometrium Culture Model for Investigating Angiogenesis in Microvascular Networks

Background: The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study’s objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Methods: Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. Results: These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. Conclusion: These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks.

Differential Effects of Brain Death on Rat Microcirculation and Intestinal Inflammation: Female Versus Male.

Brain death (BD) affects organs by multiple mechanisms related to hemodynamic effects, hormonal changes, and the systemic inflammatory response, which reduce organ function and viability. BD reduces microcirculatory perfusion in rat mesentery; this disturbance is also observed in the pancreas and lungs. Sex hormones can affect microcirculatory function, altering tissue perfusion and influencing the inflammatory process. Here, we present differences between sexes in the microcirculatory alterations generated by BD and in inflammatory infiltrate. Male, female, and ovariectomized-female Wistar rats were submitted to BD by intracranial balloon catheter sudden inflation. BD was confirmed by maximally dilated and fixed pupils, apnea, absence of reflexes, and a drop in mean arterial pressure. Perfusion and flow of the mesenteric microcirculation were analyzed. Intestinal myeloperoxidase activity and leukocyte infiltration were quantified. ELISA quantified serum estradiol, corticosterone, and inflammatory mediators, whereas expression of eNOS, endothelin, and endothelial adhesion molecule was measured by immunohistochemistry. Male rats presented lower percentages of mesenteric perfused microvessels and reduced blood flow compared to females. The female group presented higher eNOS and endothelin expression. Leukocyte infiltration into intestinal walls was higher in females in comparison to that in males. Moreover, the female group showed higher mesenteric vessel ICAM-1 expression than males, whereas serum TNF-α, IL-1β, and IL-10 levels did not differ between sexes. The high estradiol concentration before BD and high eNOS expression apparently favored the maintenance of microvascular perfusion/flow; however, BD caused an acute reduction of female sex hormone concentration and higher ICAM-1 level; thus, the proinflammatory organ status after BD is favored.

A computational modeling of blood flow in asymmetrically bifurcating microvessels and its experimental validation.

Microvascular transport is complex due to its heterogeneity. Many researchers have been developing mathematical and computational models in predicting microvascular geometries and blood transport. However, previous works were focused on developing simulation models, not on validating suggested models with microvascular geometry and blood flow in the real microvasculature. In this paper, we suggest a computational model for microvascular transport with experimental validation in its geometry and blood flow. The geometry is generated by controlling asymmetric conditions of microvascular network. Also, the blood flow in microvascular networks is predicted by considering in vivo viscosity, Poiseuille flow model, and hematocrit redistribution by plasma skimming. The suggested model is validated by the measured data in rat mesentery. Also, the microvascular transport in a case of mouse cortex is predicted and compared against experimental data to check applicability of the suggested model.

Endothelial Cell Phenotypes are Maintained During Angiogenesis in Cultured Microvascular Networks

A challenge in tissue engineering biomimetic models for studying angiogenesis is building the physiological complexity of real microvascular networks. Our laboratory recently introduced the rat mesentery culture model as an ex vivo experimental platform for investigating multicellular dynamics involved in angiogenesis within intact microvascular networks. The objective of this study was to compare endothelial cell phenotypes along capillary sprouts in cultured ex vivo rat mesentery microvascular networks to in vivo endothelial cell phenotypes. For Day 3 (Ex Vivo) tissues, adult rat mesentery tissues were cultured for three days in media supplemented with 10% serum. For Day 3 (In Vivo) tissues, adult rats were anesthetized and the mesentery was exteriorized for twenty minutes to induce angiogenesis. Microvascular networks from Day 3 (Ex Vivo) and Day 3 (In Vivo) groups were angiogenic, characterized by an increase in vessel density, capillary sprouting, and identification of similar BrdU-positive endothelial cell distributions along sprouts. Endothelial cells in both groups extended pseudopodia at the distal edge of capillary sprouts and displayed similar endothelial cell UNC5b, VEGFR-2, and CD36 labeling patterns. The results from this study support the physiological relevance of the rat mesentery culture model and highlight its novelty as a biomimetic tool for angiogenesis research.

An Ex Vivo Platform for Studying Angiogenesis in Perfused Microvascular Networks

A challenge in tissue engineering biomimetic models for studying angiogenesis is building the physiological complexity of real microvascular networks. Utilizing an alternative top‐down approach, our laboratory recently introduced the rat mesentery culture model as an ex vivo experimental platform for investigating the multi‐cellular dynamics involved in angiogenesis within an intact microvascular network. However, a current limitation of the model is that microvessels are not perfused. The objective of this study was to demonstrate the feasibility of perfusing microvascular networks during the culture of rat mesentery tissue. Adult Wistar rat mesentery tissues were harvested along with a feeding second order arteriole and venule, and then transferred to a customized tissue chamber for perfusion. The arteriole was cannulated with a glass pipette (40 – 60 μm diameter) and secured in place with 7–0 suture. Injection with blue food dye through the cannulated arteriole identified the lumens of vessels across the hierarchy of intact microvascular networks and confirmed successful perfusion. Tissues with cannulated arterioles were cultured in the perfusion chamber up to three days with either minimum essential media (MEM) or MEM supplemented with 10% serum. Live/dead labeling confirmed that cannulated mesentery tissues remain viable after three days in culture. Additional labeling with BSI‐lectin identified endothelial cells along microvascular networks and confirmed the tissues were undergoing angiogenesis, characterized by increases in vascular density and capillary sprouting. These results demonstrate the ability to perfuse intact microvascular networks ex vivo in cultured mesentery tissues and supports the novelty of the rat mesentery culture model as a biomimetic tool for investigating angiogenesis.Support or Funding InformationNIH R01AG049821This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of Microvascular Network Growth in the Mouse Mesentery

Ex Vivo biomimetic models have emerged as powerful experimental tools for basic science discovery and pre‐clinical drug testing. A challenge for microvascular research is recapitulating the complexity associated with intact networks in real tissue scenarios. To meet this challenge, our laboratory recently introduced the rat mesentery culture model, which enables the real‐time investigation of multicellular interactions during angiogenesis and lymphangiogenesis at the same time. The impact of this approach could be increased with the use of analogous transgenic mouse tissue. However, mouse mesentery is normally avascular raising the question: Can we induce microvascular growth into the mouse mesentery? Preliminary observations of mesenteric tissues harvested from mice treated with tamoxifen for conditional knockout studies suggests that vascularization in the mouse mesentery might be possible. The objective of this study was to test the hypothesis that tamoxifen treatment causes microvascular growth into avascular mouse mesenteric tissue. Female C57BL/6 mice were given a daily IP injection of 0.1 mL for 5 consecutive days containing the following experimental groups: Saline, Sunflower Seed Oil (Oil), and Tamoxifen (Tmx) dissolved in sunflower seed oil (10 mg/mL). Following 21 days post the last injection, mesenteric tissues were harvested and labeled with anti‐mouse platelet endothelial cell adhesion molecule (PECAM), neuron‐glial antigen 2 (NG2), and alpha‐smooth muscle actin (a‐SMA) antibodies to identify endothelial cells, pericytes, and smooth muscle cells, respectively. Zero tissues (0/8) from the Saline group contained a branching microvascular network. In contrast, all tissues from the Oil (8/8) and Tmx (8/8) groups contained networks with arterioles, venules, and capillaries. Smooth muscle cells and pericytes were present in their expected wrapping morphologies and locations along the hierarchy of the networks. In contrast to Saline tissues, Oil and Tmx treated tissues also contained branching LYVE‐positive lymphatic networks. The dramatic increase of vascularized tissue area and the vascularized tissue density were not significantly different between the Oil and Tmx groups (vascular area: Saline = 0.05 ± 0.05 %, Oil = 28.34 ± 8.61 %, Tmx = 35.56 ± 9.47 %; vascular density: Saline = 4.19 ± 4.19 #/mm2, Oil = 290.26 ± 32.00 #/mm2, Tmx = 275.61 ± 31.82 #/mm2; n = 8 tissues from 2 mice per group). The results of this study provide the first evidence that microvascular network growth in mouse mesenteric tissues can be induced and that sunflower seed oil is sufficient for the stimulation. More importantly, the results enable the use of mesentery tissue culture for the novel investigation of microvascular cell dynamics with transgenic mouse strains.Support or Funding InformationNIH R01AG049821This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacological dissection of the cellular mechanisms associated to the spontaneous and the mechanically stimulated ATP release by mesentery endothelial cells: roles of thrombin and TRPV.

Endothelial cells participate in extracellular ATP release elicited by mechanosensors. To characterize the dynamic interactions between mechanical and chemical factors that modulate ATP secretion by the endothelium, we assessed and compared the mechanisms participating in the spontaneous (basal) and mechanically stimulated secretion using primary cultures of rat mesentery endothelial cells. ATP/metabolites were determined in the cell media prior to (basal) and after cell media displacement or a picospritzer buffer puff used as mechanical stimuli. Mechanical stimulation increased extracellular ATP that peaked within 1min, and decayed to basal values in 10min. Interruption of the vesicular transport route consistently blocked the spontaneous ATP secretion. Cells maintained in media lacking external Ca2+ elicited a spontaneous rise of extracellular ATP and adenosine, but failed to elicit a further extracellular ATP secretion following mechanical stimulation. 2-APB, a TRPV agonist, increased the spontaneous ATP secretion, but reduced the mechanical stimulation-induced nucleotide release. Pannexin1 or connexin blockers and gadolinium, a Piezo1 blocker, reduced the mechanically induced ATP release without altering spontaneous nucleotide levels. Moreover, thrombin or related agonists increased extracellular ATP secretion elicited by mechanical stimulation, without modifying spontaneous release. In sum, present results allow inferring that the spontaneous, extracellular nucleotide secretion is essentially mediated by ATP containing vesicles, while the mechanically induced secretion occurs essentially by connexin or pannexin1 hemichannel ATP transport, a finding fully supported by results from Panx1-/- rodents. Only the latter component is modulated by thrombin and related receptor agonists, highlighting a novel endothelium-smooth muscle signaling role of this anticoagulant.

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

Possible mechanisms of diverse spaceflight effects on endothelial function in different vascular beds

Nitrite-Mediated Hypoxic Vasodilation Predicted from Mathematical Modeling and Quantified from in Vivo Studies in Rat Mesentery.

Nitric oxide (NO) generated from nitrite through nitrite reductase activity in red blood cells has been proposed to play a major role in hypoxic vasodilation. However, we have previously predicted from mathematical modeling that much more NO can be derived from tissue nitrite reductase activity than from red blood cell nitrite reductase activity. Evidence in the literature suggests that tissue nitrite reductase activity is associated with xanthine oxidoreductase (XOR) and/or aldehyde oxidoreductase (AOR). We investigated the role of XOR and AOR in nitrite-mediated vasodilation from computer simulations and from in vivo exteriorized rat mesentery experiments. Vasodilation responses to nitrite in the superfusion medium bathing the mesentery equilibrated with 5% O2 (normoxia) or zero O2 (hypoxia) at either normal or acidic pH were quantified. Experiments were also conducted following intraperitoneal (IP) injection of nitrite before and after inhibiting XOR with allopurinol or inhibiting AOR with raloxifene. Computer simulations for NO and O2 transport using reaction parameters reported in the literature were also conducted to predict nitrite-dependent NO production from XOR and AOR activity as a function of nitrite concentration, PO2 and pH. Experimentally, the largest arteriolar responses were found with nitrite >10 mM in the superfusate, but no statistically significant differences were found with hypoxic and acidic conditions in the superfusate. Nitrite-mediated vasodilation with IP nitrite injections was reduced or abolished after inhibiting XOR with allopurinol (p < 0.001). Responses to IP nitrite before and after inhibiting AOR with raloxifene were not as consistent. Our mathematical model predicts that under certain conditions, XOR and AOR nitrite reductase activity in tissue can significantly elevate smooth muscle cell NO and can serve as a compensatory pathway when endothelial NO production is limited by hypoxic conditions. Our theoretical and experimental results provide further evidence for a role of tissue nitrite reductases to contribute additional NO to compensate for reduced NO production by endothelial nitric oxide synthase during hypoxia. Our mathematical model demonstrates that under extreme hypoxic conditions with acidic pH, endogenous nitrite levels alone can be sufficient for a functionally significant increase in NO bioavailability. However, these conditions are difficult to achieve experimentally.