Arterial wall remodeling under sustained axial twisting in rats

Blood vessels often experience torsion along their axes and it is essential to understand their biological responses and wall remodeling under torsion. To this end, a rat model was developed to investigate the arterial wall remodeling under sustained axial twisting in vivo. Rat carotid arteries were twisted at 180° along the longitudinal axis through a surgical procedure and maintained for different durations up to 4 weeks. The wall remodeling in these twisted arteries was examined using histology, immunohistochemistry and fluorescent microscopy. Our data showed that arteries remodeled under twisting in a time-dependent manner during the 4 weeks post-surgery. Cell proliferation, MMP-2 and MMP-9 expressions, medial wall thickness and lumen diameter increased while collagen to elastin ratio decreased. The size and number of internal elastic lamina fenestrae increased with elongated shapes, while the endothelial cells elongated and aligned towards the blood flow direction gradually. These results demonstrated that sustained axial twisting results in artery remodeling in vivo. The rat carotid artery twisting model is an effective in vivo model for studying arterial wall remodeling under long-term torsion. These results enrich our understanding of vascular biology and arterial wall remodeling under mechanical stresses.     

Modelling pulse wave propagation in the rabbit systemic circulation to assess the effects of altered nitric oxide synthesis

Pulse wave propagation in the mature rabbit systemic circulation was simulated using the one-dimensional equations of blood flow in compliant vessels. A corrosion cast of the rabbit circulation was manufactured to obtain arterial lengths and diameters. Pulse wave speeds and inflow and outflow boundary conditions were derived from in vivo data. Numerical results captured the main features of in vivo pressure and velocity pulse waveforms in the aorta, brachiocephalic artery and central ear artery. This model was used to elucidate haemodynamic mechanisms underlying changes in peripheral pulse waveforms observed in vivo after administering drugs that alter nitric oxide synthesis in the endothelial cells lining blood vessels. According to our model, these changes can be explained by single or combined alterations of blood viscosity, peripheral resistance and compliance, and the elasticity of conduit arteries.     

Engineering of fibrin-based functional and implantable small-diameter blood vessels

We engineered implantable small-diameter blood vessels based on ovine smooth muscle and endothelial cells embedded in fibrin gels. Cylindrical tissue constructs remodeled the fibrin matrix and exhibited considerable reactivity in response to receptor- and nonreceptor-mediated vasoconstrictors and dilators. Aprotinin, a protease inhibitor of fibrinolysis, was added at varying concentrations and affected the development and functionality of tissue-engineered blood vessels (TEVs) in a concentration-dependent manner. Interestingly, at moderate concentrations, aprotinin increased mechanical strength but decreased vascular reactivity, indicating a possible relationship between matrix degradation/remodeling, vasoreactivity, and mechanical properties. TEVs developed considerable mechanical strength to withstand interpositional implantation in jugular veins of lambs. Implanted TEVs integrated well with the native vessel and demonstrated patency and similar blood flow rates as the native vessels. At 15 wk postimplantation, TEVs exhibited remarkable matrix remodeling with production of collagen and elastin fibers and orientation of smooth muscle cells perpendicular to the direction of blood flow. Implanted vessels gained significant mechanical strength and reactivity that were comparable to those of native veins. Our work demonstrates that fibrin-based TEVs hold significant promise for treatment of vascular disease and as a biological model for studying vascular development and pathophysiology.     

FeCo/graphite nanocrystals for multi-modality imaging of experimental vascular inflammation

Background

FeCo/graphitic-carbon nanocrystals (FeCo/GC) are biocompatible, high-relaxivity, multi-functional nanoparticles. Macrophages represent important cellular imaging targets for assessing vascular inflammation. We evaluated FeCo/GC for vascular macrophage uptake and imaging in vivo using fluorescence and MRI.

Methods and Results

Hyperlipidemic and diabetic mice underwent carotid ligation to produce a macrophage-rich vascular lesion. In situ and ex vivo fluorescence imaging were performed at 48 hours after intravenous injection of FeCo/GC conjugated to Cy5.5 (n = 8, 8 nmol of Cy5.5/mouse). Significant fluorescence signal from FeCo/GC-Cy5.5 was present in the ligated left carotid arteries, but not in the control (non-ligated) right carotid arteries or sham-operated carotid arteries (p = 0.03 for ligated vs. non-ligated). Serial in vivo 3T MRI was performed at 48 and 72 hours after intravenous FeCo/GC (n = 6, 270 µg Fe/mouse). Significant T2* signal loss from FeCo/GC was seen in ligated left carotid arteries, not in non-ligated controls (p = 0.03). Immunofluorescence staining showed colocalization of FeCo/GC and macrophages in ligated carotid arteries.

Conclusions

FeCo/GC accumulates in vascular macrophages in vivo, allowing fluorescence and MR imaging. This multi-functional high-relaxivity nanoparticle platform provides a promising approach for cellular imaging of vascular inflammation.     

Single Cell FRET Analysis for the Identification of Optimal FRET-Pairs in Bacillus subtilis Using a Prototype MEM-FLIM System

Protein-protein interactions can be studied in vitro, e.g. with bacterial or yeast two-hybrid systems or surface plasmon resonance. In contrast to in vitro techniques, in vivo studies of protein-protein interactions allow examination of spatial and temporal behavior of such interactions in their native environment. One approach to study protein-protein interactions in vivo is via Förster Resonance Energy Transfer (FRET). Here, FRET efficiency of selected FRET-pairs was studied at the single cell level using sensitized emission and Frequency Domain-Fluorescence Lifetime Imaging Microscopy (FD-FLIM). For FRET-FLIM, a prototype Modulated Electron-Multiplied FLIM system was used, which is, to the best of our knowledge, the first account of Frequency Domain FLIM to analyze FRET in single bacterial cells. To perform FRET-FLIM, we first determined and benchmarked the best fluorescent protein-pair for FRET in Bacillus subtilis using a novel BglBrick-compatible integration vector. We show that GFP-tagRFP is an excellent donor-acceptor pair for B. subtilis in vivo FRET studies. As a proof of concept, selected donor and acceptor fluorescent proteins were fused using a linker that contained a tobacco etch virus (TEV)-protease recognition sequence. Induction of TEV-protease results in loss of FRET efficiency and increase in fluorescence lifetime. The loss of FRET efficiency after TEV induction can be followed in time in single cells via time-lapse microscopy. This work will facilitate future studies of in vivo dynamics of protein complexes in single B. subtilis cells.     

Evaluation of Compliance of Arterial Vessel Using Coupled Fluid Structure Interaction Analysis

The in vivo and ex vivo compliance of arteries are expected to be closely related and estimated. Fluid-structure interaction analysis can assess the agreement between the two compliances. To evaluate this hypothesis, a pulsatile fluid-structure interaction analysis of blood flow in femoral artery of a dog was conducted using: (1) measured in vivo mean pressure (72.5 mmHg), mean pressure drop (0.59 mmHg), mean velocity (15.1 cm/sec); and (2) ex vivo measurements of non -- linear elastic properties of femoral artery. Additional analyses were conducted for physiological pressures (104.1 and 140.7 mmHg) and blood flow using a characteristic linear pressure -- flow relationship. The computed compliance decreased from 0.198% diameter change/mmHg at 72.5 mmHg to 0.145% diameter change/mmHg at 140.7 mmHg. The computed compliance tends to match well with in vivo compliance of femoral artery at lower pressure but is overestimated at higher pressure. This suggests an alteration in the compliance of the artery during ex vivo elasticity measurements.     

Coupled fluid–structure interaction hemodynamics in a zero-pressure state corrected arterial geometry

Hemodynamic conditions in large arteries are significantly affected by the interaction of the pulsatile blood flow with the distensible arterial wall. A numerical procedure for solving the fluid–structure interaction problem encountered in cardiovascular flows is presented. We consider a patient-specific carotid bifurcation geometry, obtained from 3D reconstruction of in vivo acquired tomography images, which yields a geometrical representation of the artery corresponding to its pressurized state. To recover the geometry of the artery in its zero-pressure state which is required for a fluid–structure interaction simulation we utilize inverse finite elastostatics. Time-dependent flow simulations with in vivo measured inflow volume flow rate in the 3D undeformed artery are performed through the finite element method. The coupled-momentum method for fluid–structure interaction is adopted to incorporate the influence of wall compliance in the numerical computation of the time varying flow domain. To demonstrate the importance in recovering the zero-pressure state of the artery in hemodynamic simulations we compute the time varying flow field with compliant walls for the original and the zero-pressure state corrected geometric configurations of the carotid bifurcation. The most important resulting effects in the hemodynamic environment are evaluated. Our results show a significant change in the wall shear stress distribution and the spatiotemporal extent of the recirculation regions.     

小口径血液导流管的实验研究

目的:探讨小口径血液导流管在动物离断肢体模型中快速恢复通血的实验基础应用,研究小口径血液导流管实验幼猪离断肢体维持通血效果的评价。方法:20只实验幼猪随机分为A、B两组,制成后肢完全离断模型模型,采用内径为2.0 mm、外径2.5mm的血液导流管,A组长度10 cm;B组长度20 cm,进行血管桥接后定期观察血液导流管通畅性,观察终点为血液导流管完全堵塞,血管超声探测仪无血流信号,远端血管搏动消失,离断肢体以远皮下毛细血管网无渗血。比较两组到达观察终点的时间有无差异。结果:建立临时血管通路后,离断肢体远端股动脉的远端有搏动,血管超声探测仪可检测到血液导流管内有血流信号,随着时间的延长,血液导流管动脉段逐渐由鲜红色变为暗红色,导流管段逐渐形成附壁血栓,远端血管搏动及皮下毛细管网渗血逐渐减弱直至消失,血流信号消失,两组到达观测终点的时间分别为A组365±47.4 min;B组359±31.5 min,两者比较其差异没有统计学意义(P0.05)。说明长度在10 cm-20 cm的小口径血液导流管在实验动物离断肢体血管通血方面无明显差异。结论:小口径血液导流管能够用于动物离断肢体的血管临时桥接,维持通血时间可达6-8小时,有效通血时间长。实验数据说明小口径血液导流管适合于动物离断肢体模型中的血管桥接,在下一步临床应用中在四肢复杂血管损伤中有着较为广阔的临床应用前景。     

An Accurate,Flexible and Small Optical Fiber Sensor: A Novel Technological Breakthrough for Real-Time Analysis of Dynamic Blood Flow Data In Vivo

Because of the limitations of existing methods and techniques for directly obtaining real-time blood data, no accurate microflow in vivo real-time analysis method exists. To establish a novel technical platform for real-time in vivo detection and to analyze average blood pressure and other blood flow parameters, a small, accurate, flexible, and nontoxic Fabry-Perot fiber sensor was designed. The carotid sheath was implanted through intubation of the rabbit carotid artery (n = 8), and the blood pressure and other detection data were determined directly through the veins. The fiber detection results were compared with test results obtained using color Doppler ultrasound and a physiological pressure sensor recorder. Pairwise comparisons among the blood pressure results obtained using the three methods indicated that real-time blood pressure information obtained through the fiber sensor technique exhibited better correlation than the data obtained with the other techniques. The highest correlation (correlation coefficient of 0.86) was obtained between the fiber sensor and pressure sensor. The blood pressure values were positively related to the total cholesterol level, low-density lipoprotein level, number of red blood cells, and hemoglobin level, with correlation coefficients of 0.033, 0.129, 0.358, and 0.373, respectively. The blood pressure values had no obvious relationship with the number of white blood cells and high-density lipoprotein and had a negative relationship with triglyceride levels, with a correlation coefficient of –0.031. The average ambulatory blood pressure measured by the fiber sensor exhibited a negative correlation with the quantity of blood platelets (correlation coefficient of −0.839, P<0.05). The novel fiber sensor can thus obtain in vivo blood pressure data accurately, stably, and in real time; the sensor can also determine the content and status of the blood flow to some extent. Therefore, the fiber sensor can obtain partially real-time vascular rheology information and may thus enable the early diagnosis of blood rheology disorders and diseases.     

双侧颈总动脉结扎对大鼠学习记忆相关脑区血流量的影响

目的为建立双侧颈总动脉结扎致大鼠血管性痴呆模型,观察了大鼠双侧颈总动脉结扎后不同脑区脑血流量的影响。方法采用激光多普勒血流仪,测定麻醉大鼠双侧颈总动脉结扎后１０ｍｉｎ内不同脑区脑血流量变化。结果大鼠双侧颈总动脉结扎后,平均脑血流量减少额区６７８３％、顶区５６８２％、枕区１６１６％、Ｍｙｎｅｒｔ基底核５１２１％、尾壳核４１１８％,海马ＣＡ１５１８３和海马ＣＡ２４１２１。结论大鼠双侧颈总动脉结扎后与学习记忆有关的脑区脑血流量均显著下降     

Resveratrol Improved Flow-Mediated Outward Arterial Remodeling in Ovariectomized Rats with Hypertrophic Effect at High Dose

Objectives

Chronic increases in blood flow in resistance arteries induce outward remodeling associated with increased wall thickness and endothelium-mediated dilatation. This remodeling is essential for collateral arteries growth following occlusion of a large artery. As estrogens have a major role in this remodeling, we hypothesized that resveratrol, described as possessing phytoestrogen properties, could improve remodeling in ovariectomized rats.

Methods

Blood flow was increased in vivo in mesenteric arteries after ligation of adjacent arteries in 3-month old ovariectomized rats treated with resveratrol (5 or 37.5 mg/kg per day: RESV5 or RESV37.5) or vehicle. After 2 weeks arterial structure and function were measured in vitro in high flow (HF) and normal flow (NF) arteries isolated from each rat.

Results

Arterial diameter was greater in HF than in NF arteries in ovariectomized rats treated with RESV5 or RESV37.5, not in vehicle-treated rats. In mice lacking estrogen receptor alpha diameter was equivalent in HF and NF arteries whereas in mice treated with RESV5 diameter was greater in HF than in NF vessels. A compensatory increase in wall thickness and a greater phenylephrine-mediated contraction were observed in HF arteries. This was more pronounced in HF arteries from RESV37.5-treated rats. ERK1/2 phosphorylation, involved in hypertrophy and contraction, were higher in RESV37.5-treated rats than in RESV5- and vehicle-treated rats. Endothelium-dependent relaxation was greater in HF than in NF arteries in RESV5-treated rats only. In HF arteries from RESV37.5-treated rats relaxation was increased by superoxide reduction and markers of oxidative stress (p67phox, GP91phox) were higher than in the 2 other groups.

Conclusion

Resveratrol improved flow-mediated outward remodeling in ovariectomized rats thus providing a potential therapeutic tool in menopause-associated ischemic disorders. This effect seems independent of the estrogen receptor alpha. Nevertheless, caution should be taken with high doses inducing excessive contractility and hypertrophy in association with oxidative stress in HF arteries.     

Arterial adaptations to altered blood flow

Arterial remodeling in response to altered blood flow is believed to be critical to vascular adaptations to developmental, physiological, pathological, and therapeutically induced changes in blood flow. To assess this remodeling, we used left-to-right carotid anastomosis to increase blood flow in the right common carotid arteries of adult rabbits by 60%. After 2 months, these vessels exhibited no compensatory enlargement. In contrast, the same procedure performed in 5- to 6-week-old weanling rabbits resulted in accelerated growth of the vessels: diameters exceeded those of control arteries by 19% after 2 months. Common carotid arteries in adult rabbits remodeled to produce a diameter reduced by 23% when blood flow was reduced by 63% by external carotid ligation. This adaptation restored shear stress exerted on the vessel wall to control levels. The reduced diameter was not reversed when the vessels were maximally dilated with nitroprusside, adenosine, and forskolin; however, normal diameters were restored within 1 week when normal blood flows were reestablished. Thus, the adult arteries did not respond to increased blood flow produced by the anastomosis, but this procedure did reverse adaptations to decreased flow. In contrast, immature arteries were responsive to this increase in blood flow, even in the absence of prior flow modulation.     

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.     

Magnesium supplementation and deoxycorticosterone acetate--salt hypertension: effect on arterial mechanical properties and on activity of endothelin-1

The aim of this study was to show whether the decrease in blood pressure induced by Mg supplementation in deoxycorticosterone acetate - salt (DOCA-salt) hypertensive rats is associated with mechanical modifications of blood vessels and (or) changes in tissular production and (or) vasoconstrictor activity to endothelin-1. DOCA-salt treatment increased blood pressure, media thickness, cross-sectional area, and lumen diameter of carotid arteries. Distensibility and incremental elastic modulus versus stress were not altered in carotid arteries, suggesting that the DOCA-salt vessel wall adapts structurally to preserve its blood pressure buffering capacity. Magnesium supplementation attenuated DOCA-salt hypertension. In comparison with normotensive rats, systolic, mean, and pulse pressures were higher whereas diastolic pressure was not different in Mg-supplemented DOCA-salt rats. Magnesium supplementation did not significantly modify the elastic parameters of carotid arteries. In resistance mesenteric arteries, DOCA-salt hypertension induces an inward hypertrophic remodeling. Magnesium supplementation attenuates wall hypertrophy and increases lumen diameter to the normotensive diameter, suggesting a decrease in peripheral resistance. Magnesium supplementation normalizes the altered vasoconstrictor activity of endothelin-1 in mesenteric arteries and attenuates endothelin-1 overproduction in kidney, left ventricle, and aorta of DOCA-salt rats. These findings suggest that Mg supplementation prevents blood pressure elevation by attenuating peripheral resistance and by decreasing hypertrophic effect of endothelin-1 via inhibition of endothelin-1 production.     

Engineering an Endothelialized Vascular Graft: A Rational Approach to Study Design in a Non-Human Primate Model

After many years of research, small diameter, synthetic vascular grafts still lack the necessary biologic integration to perform ideally in clinical settings. Endothelialization of vascular grafts has the potential to improve synthetic graft function, and endothelial outgrowth cells (EOCs) are a promising autologous cell source. Yet no work has established the link between endothelial cell functions and outcomes of implanted endothelialized grafts. This work utilized steady flow, oscillatory flow, and tumor necrosis factor stimulation to alter EOC phenotype and enable the formulation of a model to predict endothelialized graft performance. To accomplish this, EOC in vitro expression of coagulation and inflammatory markers was quantified. In parallel, in non-human primate (baboon) models, the platelet and fibrinogen accumulation on endothelialized grafts were quantified in an ex vivo shunt, or the tissue ingrowth on implanted grafts were characterized after 1mth. Oscillatory flow stimulation of EOCs increased in vitro coagulation markers and ex vivo platelet accumulation. Steady flow preconditioning did not affect platelet accumulation or intimal hyperplasia relative to static samples. To determine whether in vitro markers predict implant performance, a linear regression model of the in vitro data was fit to platelet accumulation data—correlating the markers with the thromboprotective performance of the EOCs. The model was tested against implant intimal hyperplasia data and found to correlate strongly with the parallel in vitro analyses. This research defines the effects of flow preconditioning on EOC regulation of coagulation in clinical vascular grafts through parallel in vitro, ex vivo, and in vivo analyses, and contributes to the translatability of in vitro tests to in vivo clinical graft performance.     

Age and sex dependence of hemodynamic parameters of human internal carotid arteries

The study of the ultrasound diameter, linear velocity, and resistance of the internal carotid arteries in 647 subjects of both sexes aged from 1 to 74 years has been performed. Additionally, shear stress and the Reynolds number have been calculated. During the period from early childhood to adolescence and from the first mature to younger elderly ages, there is an increase in the diameter of the internal carotid arteries. Phases of an increase in the vascular resistance by the first period of childhood, adolescence, and younger elderly age are observed. The space flow velocity has relatively stable parameters till youth, and then it declines by younger elderly age. The average linear velocity, shear stress, and the Reynolds number progressively diminish twice by younger elderly age. Laminar blood flow with local twists in the early stages of postnatal ontogenesis is characteristic of the internal carotid arteries. The diameter of the internal carotid arteries, vascular resistance index, and blood flow velocity are higher in men than in women during most age periods. Shear stress in both internal carotid arteries during all the age periods studied is symmetrical and has no sex differences.     

Post-implant evaluation of the anastomotic mechanical and geometrical coupling between human native arteries and arterial cryografts implanted in lower-limb: mechanical, histological and ultraestructural studies of implanted cryografts

BackgroundThere is an urgent need of vascular substitutes (VS) to be used in lower limb revascularization procedures when autologous veins are not available and synthetic prosthesis are contraindicated. Since the mechanical differences with respect to native vessels are determinants of the VS failure, the substitutes should have mechanical properties similar to those of the recipient vessels. The use of cryopreserved arteries (cryografts) could overcome limitations of available VS. These work aims were to characterize (a) native vessels/implanted cryografts mechanical and geometrical coupling, (b) cryografts capability to ensure mismatch levels lesser than those expected for expanded polytetrafluoroethylene (ePTFE), (c) cryografts functional properties considering their histological and ultra-structural characteristics.MethodsInstantaneous pressure (mechano-transducers) and diameter (B-mode echography) were obtained in implanted femoro-popliteal, ileo-femoro-popliteal and axilo-humeral cryografts (n = 8), in femoral arteries from recipients (n = 8), recipient-like (n = 15) and multiorgan donors-like (n = 15) subjects, and in ePTFE segments (n = 10). Calculus: (a) Mechanical parameters: elastic modulus, arterial compliance, distensibility and characteristic impedance; (b) Arterial remodeling: diameter, wall thickness, cross-sectional area and wall-to-lumen ratio; (c) Native vessels/VS coupling. Histological and structural analysis were done in explanted femoro-popliteal and axilo-humeral cryografts (n = 7).ResultsPost-implant the cryografts remodeled. Their stiffness increased and the conduit function diminished. Remodeling resulted in an improvement in native vessels/cryograft coupling, which was always better than native vessels/ePTFE coupling.ConclusionsPost-implant cryograft remodeling improved native vessels/cryografts coupling. Cryografts would have mechanical and geometrical advantages over ePTFE. Anastomotic cryograft remodeling differed from that expected only due to haemodynamic factors. The structural properties of the remodeled cryografts contribute to explain their functional characteristics.     

The Vascular Effects of Sodium Tanshinone IIA Sulphonate in Rodent and Human Pregnancy

Danshen, in particular its derivative tanshinone IIA (TS), is a promising compound in the treatment of cardiovascular diseases and has been used for many years in traditional Chinese medicine. Although many actions of TS have been researched, its vasodilator effects in pregnancy remain unknown. There have been a few studies that have shown the ability of TS to reduce blood pressure in women with hypertensive pregnancies; however, there are no studies which have examined the vascular effects of TS in the pregnant state in either normal or complicated pregnancies. Our aim was to determine the vasoactive role of TS in multiple arteries during pregnancy including: rat resistance (mesenteric and uterine) and conduit (carotid) arteries. Further, we aimed to assess the ability of TS to improve uterine blood flow in a rodent model of intrauterine growth restriction. Wire myography was used to assess vascular responses to the water-soluble derivative, sodium tanshinone IIA sulphonate (STS) or to the endothelium-dependent vasodilator, methylcholine. At mid-pregnancy, STS caused direct vasodilation of rat resistance (pEC₅₀ mesenteric: 4.47±0.05 and uterine: 3.65±0.10) but not conduit (carotid) arteries. In late pregnancy, human myometrial arteries responded with a similar sensitivity to STS (pEC₅₀ myometrial: 3.26±0.13). STS treatment for the last third of pregnancy in eNOS^-/- mice increased uterine artery responses to methylcholine (E_max eNOS^-/-: 55.2±9.2% vs. eNOS^-/- treated: 75.7±8.9%, p<0.0001). The promising vascular effects, however, did not lead to improved uterine or umbilical blood flow in vivo, nor to improved fetal biometrics; body weight and crown-rump length. Further, STS treatment increased the uterine artery resistance index and decreased offspring body weight in control mice. Further research would be required to determine the safety and efficacy of use of STS in pregnancy.     

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca²⁺]_cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.     

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease², stroke³ and heart failure⁴. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species⁵. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers⁶ however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration^7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry¹⁰. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model⁹ which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter¹¹ as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.