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1.  Reduced RhoA activity mediates acute alcohol intoxication-induced inhibition of lymphatic myogenic constriction despite increased cytosolic [Ca2+] 
We previously showed that acute alcohol intoxication (AAI) reduces lymphatic myogenic constriction in response to step increases in luminal pressure. Because of the known role of Ca2+ in smooth muscle contractile responses, we investigated how alcohol impacts cyclic Ca2+ and whether changes in RhoA/ROCK mediated Ca2+ sensitivity underlie the alcohol-induced reduction of myogenic responsiveness.
AAI was produced by intragastric administration of 30% alcohol in rats. Mesenteric lymphatics were cannulated and loaded with Fura-2 AM to [Ca2+]i for 30 min after AAI. Active GTP-bound RhoA levels were determined by ELISA. To determine ROCK's ability to restore myogenic responsiveness following AAI, isolated lymphatics were transfected with constitutively active ca-ROCK protein.
Lymphatics from alcohol-treated rats displayed significantly larger Ca2+ transients. Also, step increases in luminal pressure caused a gradual rise in the basal [Ca2+]i between transients that was greater in lymphatics submitted to AAI, compared to vehicle control. RhoA-GTP was significantly reduced in lymphatics from the AAI group, compared to vehicle control. Transfection with ca-ROCK protein restored the myogenic response of lymphatic vessels isolated from AAI animals.
The data strongly suggest that the alcohol-induced inhibition of mesenteric lymphatic myogenic constriction is mediated by reduced RhoA/ROCK-mediated Ca2+ sensitivity.
PMCID: PMC3610832  PMID: 23237297
calcium; lymphatic smooth muscle; myogenic tone; ROCK; ethanol
2.  Lymphatic filariasis: Perspectives on lymphatic remodeling and contractile dysfunction in filarial disease pathogenesis 
Lymphatic filariasis, one of the most debilitating diseases associated with the lymphatic system, affects over a hundred million people worldwide and manifests itself in a variety of severe clinical pathologies. The filarial parasites specifically target the lymphatics and impair lymph flow, which is critical for the normal functions of the lymphatic system in maintenance of body fluid balance and physiological interstitial fluid transport. The resultant contractile dysfunction of the lymphatics causes fluid accumulation and lymphedema, one of the major pathologies associated with filarial infection. In this review, we take a closer look at the contractile mechanisms of the lymphatics, its altered functions and remodeling during an inflammatory state and how it relates to the severe pathogenesis underlying a filarial infection. We further elaborate on the complex host parasite interactions, and molecular mechanisms contributing to the disease pathogenesis. The overall emphasis is on elucidating some of the emerging concepts and new directions that aim to harness the process of lymphangiogenesis or enhance contractility in a dysfunctional lymphatics, thereby restoring the fluid imbalance and mitigating the pathological conditions of lymphatic filariasis.
PMCID: PMC3613430  PMID: 23237232
3.  Divergent effects of aging and sex on vasoconstriction to endothelin in coronary arterioles 
The risk for cardiovascular disease increases with advancing age; however, the chronological development of heart disease differs in males and females. The purpose of this study was to determine whether age-induced alterations in responses of coronary arterioles to the endogenous vasoconstrictor, endothelin, are sex-specific. Coronary arterioles were isolated from young and old male and female rats to assess vasoconstrictor responses to endothelin (ET), and ETa and ETb receptor inhibitors were used to assess receptor-specific signaling. In intact arterioles from males, ET-induced vasoconstriction was reduced with age, whereas age increased vasoconstrictor responses to ET in intact arterioles from female rats. In intact arterioles from both sexes, blockade of either ETa or ETb eliminated age-related differences in responses to ET; however, denudation of arterioles from both sexes revealed age-related differences in ETa-mediated vasoconstriction. In arterioles from male rats, ETa receptor protein decreased, whereas ETb receptor protein increased with age. In coronary arterioles from females, neither ETa nor ETb receptor protein changed with age, suggesting age-related changes in ET signaling occur downstream of ET receptors. Thus, aging-induced alterations in responsiveness of the coronary resistance vasculature to endothelin are sex-specific, possibly contributing to sexual dimorphism in the risk of cardiovascular disease with advancing age.
PMCID: PMC3594502  PMID: 23198990
rat; vasodilation; BQ123; BQ788
4.  A chronic scheme of cranial window preparation to study pial vascular reactivity in murine cerebral malaria 
The acute implantation of a cranial window for studying cerebroarteriolar reactivity in living animals involves a highly surgically-invasive craniotomy procedure at the time of experimentation, which limits its application in severely ill animals such as in the experimental murine model of cerebral malaria (ECM). To overcome this problem, a chronic window implantation scheme was designed and implemented.
A partial craniotomy is first performed by creating a skull bone flap in the healthy mice, which are then left to recover for 1–2 weeks, followed by infection to induce ECM. Uninfected animals are utilized as control. When cranial superfusion is needed, the bone flap is retracted and window implantation completed by assembling a perfusion chamber for compound delivery to the exposed brain surface. The presurgical step is intended to minimize surgical trauma on the day of experimentation.
Chronic preparations in uninfected mice exhibited remarkably improved stability over acute ones by significantly reducing periarteriolar tissue damage and enhancing cerebroarteriolar dilator responses. The chronic scheme was successfully implemented in ECM mice which unveiled novel preliminary insights on impaired cerebroarteriolar reactivity and eNOS dysfunction.
The chronic scheme presents an innovative approach for advancing our mechanistic understanding on cerebrovascular dysfunction in ECM.
PMCID: PMC3632649  PMID: 23279271
cerebroarteriolar responses; cranial window superfusion; endothelial nitric oxide synthase; experimental cerebral malaria; Plasmodium berghei ANKA
5.  Endothelial cells actively concentrate insulin during its transendothelial transport 
We examined insulin's uptake and transendothelial transport (TET) by cultured bovine aortic endothelial cells (BAECs) in order to: a) ascertain whether insulin accumulates within the cells to concentrations greater than in the media; b) compare the TET of insulin to that of inulin (using the latter as a tracer for passive transport or leak); and c) determine whether insulin's TET depended on insulin action. Using 125I-insulin at physiologic concentrations, we found that BAECs accumulate insulin >5-fold above media concentrations and that the TET of insulin, but not inulin, is saturable and requires intact PI-3-kinase and MEK-kinase signaling. We conclude that the insulin receptor and downstream signaling from the receptor regulate endothelial insulin transport. Based on comparison of the kinetics of BAEC insulin uptake with insulin TET, we suggest that insulin uptake is rate limiting for insulin TET.
PMCID: PMC3654094  PMID: 23350546
6.  Glycocalyx volume measurements: a critical review of tracer dilution methods for its measurement 
A clinical measure of endothelial glycocalyx structure would have great potential importance because lesions of the glycocalyx may be the first changes to occur in diabetes and in a wide range of vascular diseases. A method recently described by Nieuwdorp et al [15, 16] for estimating the volume of the luminal glycocalyx of the entire human vascular system would seem to be the first attempt to develop a measure of this kind. It is based on the tracer dilution principle and this review considers the principles and conditions that underlie this method and the extent to which the conditions appear to have been fulfilled in this case. Our analysis raises two questions about (a) the estimation of the concentration of the tracer (dextran 40) at zero time and (b) the estimation of plasma volume, both of which can be answered by changes in experimental protocol. A third question, concerning the partition coefficient of the tracer between plasma and the fluid within the glycocalyx, cannot be answered at the present time and until it has been resolved, glycocalyx volume cannot be estimated from the dilution of a macromolecular tracer.
PMCID: PMC4041983  PMID: 19184776
7.  Vascular Tone and Ca2+ Signaling in Murine Cremaster Muscle Arterioles In Vivo 
We sought to determine some of the molecular requirements for basal state “tone” of skeletal muscle arterioles in vivo, and whether asynchronous Ca2+ waves are involved or not.
Cremaster muscles of anesthetized exMLCK and smGCaMP2 biosensor mice were exteriorized, and the fluorescent arterioles were visualized with wide-field, confocal or multiphoton microscopy to observe Ca2+ signaling and arteriolar diameter.
Basal state tone of the arterioles was~50%. Local block of Ang-II receptors (AT1) or α1-adrenoceptors (α1-AR) had no effect on diameter, nor did complete block of sympathetic nerve activity (SNA). Inhibition of phospholipase C caused dilation nearly to the Ca2+-free (passive) diameter, as did exposure to nifedipine or 2-APB. Arterioles were also dilated when treated with SKF96365. High-resolution imaging of exMLCK fluorescence (ratio) or GCaMP2 fluorescence in smooth muscle cells failed to reveal Ca2+ waves (although Ca2+ waves/transients were readily detected by both biosensors in small arteries, ex vivo).
Arterioles of cremaster muscle have vascular tone of ~50%, which is not due to α1-AR, AT1R, or SNA. PLC activity, L-type Ca2+ channels, 2-APB- and SKF96365-sensitive channels are required. Propagating Ca2+ waves are not present. A key role for PLC and InsP3R in vascular tone in vivo, other than producing Ca2+ waves, is suggested.
PMCID: PMC4019383  PMID: 23140521
calcium; vascular tone; smooth muscle; arterioles; sympathetic nerve activity
8.  Regulation of Cerebral Artery Smooth Muscle Membrane Potential by Ca2+-activated Cation Channels 
Arterial tone is dependent on the depolarizing and hyperpolarizing currents regulating membrane potential and governing the influx of Ca2+ needed for smooth muscle contraction. Several ion channels have been proposed to contribute to membrane depolarization, but the underlying molecular mechanisms are not fully understood. In this review, we will discuss the historical and physiological significance of the Ca2+-activated cation channel, TRPM4, in regulating membrane potential of cerebral artery smooth muscle cells. As a member of the recently described transient receptor potential super family of ion channels, TRPM4 possesses the biophysical properties and upstream cellular signaling and regulatory pathways that establish it as a major physiological player in smooth muscle membrane depolarization.
PMCID: PMC3573261  PMID: 23116477
Cerebral Artery Smooth Muscle Cells; TRPM4; Membrane Potential
9.  Ryanodine receptors, calcium signaling and regulation of vascular tone in the cerebral parenchymal microcirculation 
The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of cerebral blood flow, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of parenchymal arterioles. There are, moreover, notable functional differences between pial arteries and parenchymal arterioles. For example, in pial vascular smooth muscle cells (VSMCs), local calcium release events (“calcium sparks”) through ryanodine receptor (RyR) channels in sarcoplasmic reticulum membrane activate large conductance, calcium-sensitive potassium (BK) channels to modulate vascular diameter. In contrast, VSMCs in parenchymal arterioles express functional RyR and BK channels, but under physiological conditions these channels do not oppose pressure-induced vasoconstriction. Here we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of cerebral blood flow.
PMCID: PMC3612564  PMID: 23216877
brain parenchymal arteriole; cerebral blood flow; acidosis; ryanodine receptor; potassium channel
10.  Calcium dynamics in vascular smooth muscle 
Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells. These signals vary with respect to their mechanisms of generation, temporal properties, and spatial distributions. The calcium signals discussed include calcium waves, junctional calcium transients, calcium sparks, calcium puffs, and L-type calcium channel sparklets. For each calcium signal we address underlying mechanisms, general properties, physiological importance, and regulation.
PMCID: PMC3651748  PMID: 23384444
11.  Local regulation of L-type Ca2+ channel sparklets in arterial smooth muscle 
This review addresses the latest advances in our understanding of the regulation of a novel Ca2+ signal called L-type Ca2+ channel sparklets in arterial smooth muscle. L-type Ca2+ channel sparklets are elementary Ca2+ influx events produced by the opening of a single or a small cluster of L-type Ca2+ channels. These Ca2+ signals were first visualized in the vasculature in arterial smooth muscle cells. In these cells, L-type Ca2+ channel sparklets display two functionally distinct gating modalities that regulate local and global intracellular Ca2+ concentration ([Ca2+]i). The activity of L-type Ca2+ channel sparklets varies regionally within a cell depending on the dynamic activity of a cohort of protein kinases and phosphatases recruited to L-type Ca2+ channels in the arterial smooth muscle sarcolemma in a complex coordinated by the scaffolding molecule A kinase anchoring protein 150 (AKAP150). We also described a mechanism whereby clusters of L-type Ca2+ channels gate cooperatively to amplify intracellular Ca2+ signals with likely pathological consequences.
PMCID: PMC3577995  PMID: 23116449
12.  What Role for Store-Operated Ca2+ Entry in Muscle? 
Store-operated Ca2+ entry (SOCE) is a receptor-regulated Ca2+ entry pathway that is both ubiquitous and evolutionarily conserved. SOCE is activated by depletion of intracellular Ca2+ stores through receptor-mediated production of inositol 1,4,5-trisphosphate (IP3). The depletion of endoplasmic reticulum (ER) Ca2+ is sensed by stromal interaction molecule 1 (STIM1). On store depletion, STIM1 aggregates and moves to areas where the ER comes close to the plasma membrane (PM; within 25nm) to interact with Orai1 channels and activate Ca2+ entry. Ca2+ entry through store-operated Ca2+ (SOC) channels, originally thought to mediate the replenishment of Ca2+ stores, participate in active downstream signaling by coupling to the activation of enzymes and transcription factors that control a wide variety of long-term cell functions such as proliferation, growth and migration. SOCE has also been proposed to contribute to short-term cellular responses such as muscle contractility. While there are significant STIM1/Orai1 protein levels and SOCE activity in adult skeletal muscle, the precise role of SOCE in skeletal muscle contractility is not clear. The dependence on SOCE during cardiac and smooth muscle contractility is even less certain. Here, we will hypothesize on the contribution of SOCE in muscle and its potential role in contractility and signaling.
PMCID: PMC3646967  PMID: 23312019
13.  Diversity in Mechanisms of Endothelium-Dependent Vasodilation in Health and Disease 
Small arterioles (40–150 μm) contribute to the majority of vascular resistance within organs and tissues. Under resting conditions, the basal tone of these vessels is determined by a delicate balance between vasodilator and vasoconstrictor influences. Cardiovascular homeostasis and regional tissue perfusion is largely a function of the ability of these small blood vessels to constrict or dilate in response to the changing metabolic demands of specific tissues. The endothelial cell layer of these microvessels is a key modulator of vasodilation through the synthesis and release of vasoactive substances. Beyond their vasomotor properties, these compounds importantly modulate vascular cell proliferation, inflammation, and thrombosis. Thus the balance between local regulation of vascular tone and vascular pathophysiology can vary depending upon which factors are released from the endothelium. This review will focus on the dynamic nature of the endothelial released dilator factors depending on species, anatomic site, and presence of disease, with a focus on the human coronary microcirculation. Knowledge how endothelial signaling changes with disease may provide insights into the early stages of developing vascular inflammation and atherosclerosis, or related vascular pathologies.
PMCID: PMC3625248  PMID: 23311975
14.  Perivascular innervation: A multiplicity of roles in vasomotor control and myoendothelial signaling 
The control of vascular resistance and tissue perfusion reflect coordinated changes in the diameter of feed arteries and the arteriolar networks they supply. Against a background of myogenic tone and metabolic demand, vasoactive signals originating from perivascular sympathetic and sensory nerves are integrated with endothelium-derived signals to produce vasodilation or vasoconstriction. PVNs release adrenergic, cholinergic, peptidergic, purinergic, and nitrergic neurotransmitters that lead to SMC contraction or relaxation via their actions on SMCs, ECs, or other PVNs. ECs release autacoids that can have opposing actions on SMCs. Respective cell layers are connected directly to each other through GJs at discrete sites via MEJs projecting through holes in the IEL. Whereas studies of intercellular communication in the vascular wall have centered on endothelium-derived signals that govern SMC relaxation, attention has increasingly focused on signaling from SMCs to ECs. Thus, via MEJs, neurotransmission from PVNs can evoke distinct responses from ECs subsequent to acting on SMCs. To integrate this emerging area of investigation in light of vasomotor control, the present review synthesizes current understanding of signaling events that originate within SMCs in response to perivascular neurotransmission in light of EC feedback. Though often ignored in studies of the resistance vasculature, PVNs are integral to blood flow control and can provide a physiological stimulus for myoendothelial communication. Greater understanding of these underlying signaling events and how they may be affected by aging and disease will provide new approaches for selective therapeutic interventions.
PMCID: PMC3771679  PMID: 23289720
sympathetic nerves; sensory nerves; cell-cell communication; Ca2+ signaling
15.  Recruitment of Dynamic Endothelial Ca2+ Signals by the TRPA1 Channel Activator AITC in Rat Cerebral Arteries 
Stimulation of endothelial TRP channels, specifically TRPA1, promotes vasodilation of cerebral arteries through activation of Ca2+-dependent effectors along the myoendothelial interface. However, presumed TRPA1-triggered endothelial Ca2+ signals have not been described. We investigated whether TRPA1 activation induces specific spatial and temporal changes in Ca2+ signals along the intima that correlate with incremental vasodilation.
Confocal imaging, immunofluorescence staining and custom image analysis were employed.
We found that endothelial cells of rat cerebral arteries exhibit widespread basal Ca2+ dynamics (44 ± 6 events/minute from 26 ± 3 distinct sites in a 3.6x104 μm2 field). The TRPA1 activator AITC increased Ca2+ signals in a concentration-dependent manner, soliciting new events at distinct sites. Origination of these new events corresponded spatially with TRPA1 densities in IEL holes, and the events were prevented by the TRPA1 inhibitor HC-030031. Concentration-dependent expansion of Ca2+ events in response to AITC correlated precisely with dilation of pressurized cerebral arteries (p = 0.93 by F-test). Correspondingly, AITC caused rapid endothelium-dependent suppression of asynchronous Ca2+ waves in subintimal smooth muscle.
Our findings indicate that factors that stimulate TRPA1 channels expand Ca2+ signal-effector coupling at discrete sites along the endothelium to evoke graded cerebral artery vasodilation.
PMCID: PMC3524345  PMID: 22928941
cerebral artery; endothelium; calcium; TRPA1; AITC
16.  Shear Dependent Adhesion of Leukocytes and Lectins to the Endothelium and Concurrent Changes in Thickness of the Glycocalyx of Post-capillary Venules in the Low Flow State 
To elucidate shear dependent effects of deformation of the endothelial glycocalyx on adhesion of circulating ligands in post-capillary venules, and delineate effect of matrix metalloproteases (MMPs).
Adhesion of leukocytes (WBCs) and lectin-coated fluorescently labeled microspheres (FLMs, 0.1 μm diameter), to endothelium (EC) of post-capillary venules in mesentery was examined during acute reductions in shear rates (γ̇, hemorrhagic hypotension). Adhesion was examined with or without superfusion with 0.5 μM doxycycline to inhibit MMPs. Thickness of the glycocalyx was measured by exclusion of fluorescent 70 kDa dextran from the EC surface.
During superfusion with Ringers, rapid reductions in γ̇ resulted in a significant rise in WBC adhesion and a two-fold rise in microsphere adhesion. With addition of doxycycline WBC and FLM adhesion increased two-fold under high and low flow conditions. FLM adhesion was invariant with γ̇ throughout the network in the normal (high) flow state. With reductions in γ̇, thickness of the glycocalyx increased significantly, with or without doxycycline.
The concurrent increase in WBC and FLM adhesion with increased thickness of the glycocalyx during reductions in shear suggests that glycocalyx core proteins recoil from their deformed steady state configuration, which increases exposure of binding sites for circulating ligands.
PMCID: PMC3541453  PMID: 22963321
Low flow state; hemorrhagic hypotension; leukocyte adhesion; glycocalyx; matrix metalloproteases; doxycycline
17.  Exercise training-enhanced, endothelium-dependent dilation mediated by altered regulation of BKCa channels in collateral-dependent porcine coronary arterioles 
Test the hypothesis that exercise training increases the contribution of large-conductance, Ca2+-dependent K+ (BKCa) channels to endothelium-mediated dilation in coronary arterioles from collateral-dependent myocardial regions of chronically occluded pig hearts and may function downstream of H2O2.
An ameroid constrictor was placed around the proximal left circumflex coronary artery to induce gradual occlusion in Yucatan miniature swine. Eight weeks postoperatively, pigs were randomly assigned to sedentary or exercise training (treadmill; 14 wk) regimens.
Exercise training significantly enhanced bradykinin-mediated dilation in collateral-dependent arterioles (~125 μm diameter) compared with sedentary pigs. The BKCa-channel blocker, iberiotoxin alone or in combination with the H2O2 scavenger, polyethylene glycol catalase, reversed exercise training-enhanced dilation in collateral-dependent arterioles. Iberiotoxin-sensitive whole-cell K+ currents (i.e., BKCa-channel currents) were not different between smooth muscle cells of nonoccluded and collateral-dependent arterioles of sedentary and exercise trained groups.
These data provide evidence that BKCa-channel activity contributes to exercise training-enhanced endothelium-dependent dilation in collateral-dependent coronary arterioles despite no change in smooth muscle BKCa-channel current. Taken together, our findings suggest that a component of the bradykinin signaling pathway, which stimulates BKCa channels, is enhanced by exercise training in collateral-dependent arterioles and suggest a potential role for H2O2 as the mediator.
PMCID: PMC3574200  PMID: 23002811
ischemia; reactive oxygen species; ischemic heart disease; free radicals
Cells require energy to carry out their functions and they typically use oxidative phosphorylation to generate the needed ATP. Thus, cells have a continuous need for oxygen which they receive by diffusion from the blood through the interstitial fluid. The circulatory system pumps oxygen-rich blood through a network of increasingly minute vessels, the microcirculation. The structure of the microcirculation is such that all cells have at least one nearby capillary for diffusive exchange of oxygen and red blood cells release the oxygen bound to hemoglobin as they traverse capillaries. This review focuses first on the historical development of techniques to measure oxygen at various sites in the microcirculation, including the blood, interstitium and cells. Next, approaches are described as to how these techniques have been employed to make discoveries about different aspects of oxygen transport. Finally, ways in which oxygen might participate in the regulation of blood flow toward matching oxygen supply to oxygen demand is discussed. Overall, the transport of oxygen to the cells of the body is one of the most critical functions of the cardiovascular system and it is in the microcirculation where the final local determinants of oxygen supply, oxygen demand and their regulation are decided.
PMCID: PMC3574207  PMID: 23025284
Oxygen transport; red blood cell; hemoglobin; oxygen tension; oxygen saturation; oxygen consumption; phosphorescence quenching; microspectrophotometry; arteriole; capillary; intravital video microscopy
19.  Polycystic kidneys have decreased vascular density: a microCT study 
Polycystic kidney disease (PKD) is a common cause of end stage renal failure and many of these patients suffer vascular dysfunction and hypertension. It remains unclear whether PKD is associated with abnormal microvascular structure. Thus, this study examined the renovascular structure in PKD.
PKD rats (PCK model) and controls were studied at 10 weeks of age, and mean arterial pressure (MAP), renal blood flow and creatinine clearance were measured. Microvascular architecture and cyst number and volume were assessed using micro-computed tomography, and angiogenic pathways evaluated.
Compared to controls, PKD animals had an increase in MAP (126.4±4.0 vs. 126.2±2.7mmHg) and decreased clearance of creatinine (0.39±0.09 vs. 0.30±0.05ml/min), associated with a decrease in microvascular density, both in the cortex (256±22 vs. 136±20 vessels per cm2) and medullar (114±14 vs. 50±9 vessels/cm2) and an increase in the average diameter of glomeruli (104.14±2.94 vs. 125.76±9.06 mm). PKD animals had increased fibrosis (2.2±0.2 fold vs. control) and a decrease in the cortical expression in hypoxia inducible factor 1-α and vascular endothelial growth factor.
PKD animals have impaired renal vascular architecture, which can have significant functional consequences. The PKD microvasculature could represent a therapeutic target to decrease the impact of this disease.
PMCID: PMC3698948  PMID: 23167921
polycystic kidney disease; vasculature; cysts; kidney; micro-computed tomography; angiogenesis; renal function
20.  Is The Real In Vivo Nitric Oxide Concentration Pico or Nano Molar? Influence of Electrode Size on Unstirred Layers and NO Consumption 
There is a debate if the nitric oxide concentration ([NO]) required to influence vascular smooth muscle is below 50 nM or much higher. 30 μm and larger diameter electrodes report [NO] below 50 nM, whereas diameters of < 10–12 μm report hundreds of nM. This study examined how size of electrodes influenced [NO] measurement due to NO consumption and unstirred layer issues.
Electrodes were 2 mm disk, 30μm X 2 mm carbon fiber, and single 7μm diameter carbon fiber within open tip microelectrode, and exposed 7 μm carbon fiber of ~15 μm to 2 mm length.
All electrodes demonstrated linear calibrations with sufficient stirring. As stirring slowed, 30 μm and 2 mm electrodes reported much lower [NO] due to unstirred layers and high NO consumption. The three 7 μm microelectrodes had minor stirring issues. With limited stirring with NO present, 7 μm open tip microelectrodes advanced toward 30 μm and 2 mm electrodes experienced dramatically decreased current within 10–50μm of the larger electrodes due to high NO consumption. None of the 7 μm microelectrodes interacted.
The data indicate large electrodes underestimate [NO] due to excessive NO consumption under conditions where unstirred layers are unavoidable and true microelectrodes are required for valid measurements.
PMCID: PMC3542416  PMID: 22925222
Nitric Oxide; Microelectrodes; Arteriole
21.  Microvascular Repair: Post-Angiogenesis Vascular Dynamics 
Microcirculation (New York, N.Y. : 1994)  2012;19(8):10.1111/j.1549-8719.2012.00207.x.
Vascular compromise and the accompanying perfusion deficits cause or complicate a large array of disease conditions and treatment failures. This has prompted the exploration of therapeutic strategies to repair or regenerate vasculatures thereby establishing more competent microcirculatory beds. Growing evidence indicates that an increase in vessel numbers within a tissue does not necessarily promote an increase in tissue perfusion. Effective regeneration of a microcirculation entails the integration of new stable microvessel segments into the network via neovascularization. Beginning with angiogenesis, neovascularization entails an integrated series of vascular activities leading to the formation of a new mature microcirculation and includes vascular guidance and inosculation, vessel maturation, pruning, arterio-venous specification, network patterning, structural adaptation, intussusception, and microvascular stabilization. While the generation of new vessel segments is necessary to expand a network, without the concomitant neovessel remodeling and adaptation processes intrinsic to microvascular network formation, these additional vessel segments give rise to a dysfunctional microcirculation. While many of the mechanisms regulating angiogenesis have been detailed, a thorough understanding of the mechanisms driving post-angiogenesis activities specific to neovascularization has yet to be fully realized, but is necessary in order to develop effective therapeutic strategies for repairing compromised microcirculations as a means to treat disease.
PMCID: PMC3842172  PMID: 22734666
22.  Voluntary wheel running selectively augments insulin-stimulated vasodilation in arterioles from white skeletal muscle of insulin resistant rats 
Voluntary wheel running (RUN) prevents declines in insulin-mediated vasodilation, an important component of insulin-mediated glucose disposal, in rats prone to obesity and insulin resistance.
Determine whether RUN: 1) improves insulin-stimulated vasodilation after insulin resistance has been established, and 2) differentially affects arterioles from red and white muscle.
Insulin signaling and vasoreactivity to insulin (1–1000 μIU/mL), were assessed in second order arterioles (2A) from the white (Gw) and red (Gr) gastrocnemius of sedentary OLETF rats at 12 and 20 weeks of age (SED12; SED20) and those undergoing RUN (RUN20) or caloric restriction (CR20; to match body weight of RUN) from 12–20 weeks.
Glucose and insulin responses to i.p. glucose were reduced in RUN20, elevated in SED20 (P<0.05 vs. SED12), and maintained in CR20. Insulin-stimulated vasodilation was greater in Gw, but not Gr, 2As of RUN20 (P<0.01 vs. all groups) and was improved by ET-1 receptor inhibition in Gw 2As from SED20 and CR20 (P<0.05). There were no differences in microvascular insulin signaling among groups or muscle beds.
RUN selectively improved insulin-mediated vasodilation in Gw 2As, in part through attenuated ET-1 sensitivity/production, an adaptation that was independent of changes in adiposity and may contribute to enhanced insulin-stimulated glucose disposal.
PMCID: PMC3498513  PMID: 22804760
type 2 diabetes; exercise; insulin; microvascular; endothelin-1
23.  Cerebral microcirculatory responses of insulin resistant rats are preserved to physiological and pharmacological stimuli 
Previously we have shown that insulin resistance impairs the vascular reactivity of the major cerebral arteries of Zucker obese (ZO) rats prior to the occurrence of type-2 diabetes mellitus. However, the functional state of the microcirculation in the cerebral cortex is still being explored.
We tested the local cortical blood flow (CoBF) responses of 11–13 week-old ZO (n=31) and control Zucker lean (ZL, n=32) rats to several stimuli measured by laser Doppler flowmetry using a closed cranial window setup.
The topical application of 1–100μM bradykinin elicited the same degree of CoBF elevation in both ZL and ZO groups. There was no significant difference in the incidence, latency, and amplitude of the N-methyl-D-Aspartate-induced cortical spreading depression-related hyperemia between the ZO and ZL groups. Hypercapnic CoBF response to 5% carbon-dioxide ventilation did not significantly change in the ZO compared to the ZL. Topical bicuculline-induced cortical seizure was accompanied by the same increase of CoBF in both the ZO and ZL at all bicuculline doses.
CoBF responses of the microcirculation are preserved in the early period of the metabolic syndrome, which creates an opportunity for intervention to prevent and restore the function of the major cerebral vascular beds.
PMCID: PMC3502673  PMID: 22845548
Zucker obese; cortical spreading depression; N-methyl-D-aspartate; bicuculline; hypercapnia
24.  Coordination of Intercellular Ca2+ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries 
To test the hypothesis that Ca2+ responses to G-protein coupled receptor (GPCR) activation are coordinated between neighboring endothelial cells of resistance arteries.
Endothelial cell tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca2+ responses to ACh were imaged with confocal microscopy.
Cell-to-cell transfer of propidium iodide confirmed functional gap junction channels. 1 μM ACh evoked Ca2+ responses [9.8±0.8/min, (F/F0)=3.11±0.2] which pseudo-linescan analysis revealed to be composed of Ca2+ waves and spatially-restricted Ca2+ release events. 100 nM ACh induced Ca2+ responses of lower frequency (4.5±0.7/min) and amplitude (F/F0=1.95±0.11) composed primarily of spatially-restricted events. The interval between Ca2+ waves in Adjacent cells (0.79±0.12 s) was shorter (P<0.05) than between Nonadjacent cells (1.56±0.25 s). Spatially-restricted Ca2+ release events had similar frequencies and latencies between Adjacent and Nonadjacent cells. Inhibiting intracellular Ca2+ release with 2-APB, Xestospongin C or thapsigargin eliminated Ca2+ responses.
With moderate GPCR stimulation, localized Ca2+ release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca2+ waves via the endoplasmic reticulum. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells.
PMCID: PMC3502682  PMID: 22860994
endothelium; gap junctions; G protein coupled receptors; acetylcholine
Although the causal relationship between acute myocardial edema and cardiac dysfunction has been established, resolution of myocardial edema and subsequent recovery of cardiac function have not. The time to resolve myocardial edema and the degree that cardiac function is depressed after edema resolves are not known. We therefore characterized temporal changes in cardiac function as acute myocardial edema formed and resolved.
Acute myocardial edema was induced in the canine model by elevating coronary sinus pressure for three hours. Myocardial water content and cardiac function were determined before and during coronary sinus pressure elevation, and after coronary sinus pressure restoration.
Although no change in systolic properties was detected, accumulation of water in myocardial interstitium was associated with increased diastolic stiffness. When coronary sinus pressure was relieved, myocardial edema resolved within 180 min. Diastolic stiffness, however, remained significantly elevated compared to baseline values, and cardiac function remained compromised.
The present work suggests that the cardiac dysfunction caused by the formation of myocardial edema may persist after myocardial edema resolves. With the advent of new imaging techniques to quantify myocardial edema, this insight provides a new avenue for research to detect and treat a significant cause of cardiac dysfunction.
PMCID: PMC3502725  PMID: 22708850
Edema-induced dysfunction; edema resolution

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