Oxygen sensitivity, potassium channels and regulation of placental vascular tone.
The human fetoplacental vasculature is a low resistance circulation with deoxygenated arterial relative to venous blood.
The placenta lacks neuronal innervation suggesting that local physical (e.g. oxygenation; flow rate), paracrine (e.g. endothelial cell nitric oxide) and circulating (e.g. angiotensin II) factors will contribute to blood flow regulation in small fetoplacental vessels.
Oxygenation (specifically hypoxia) has received particular attention. At the macro level, hypoxic challenge increases vascular resistance but the data's physiological relevance remains questionable.
Potassium (K+) channels are a diverse family of proteins known to play important roles in the normal physiological functions of endothelial and smooth muscle cells of a variety of vascular beds. K+ channels are categorized by their predicted transmembrane structure or gating properties.
A small number of perfused placental cotyledon and isolated blood vessels studies have assessed K+ channel activity. Specific activator/inhibitor application suggests functional voltage-gated (KV) channels whilst toxin inhibitor studies have documented calcium-activated (KCa) channel activity. Pharmacological ATP-sensitive (KATP) channel activation significantly dilates pre-constricted placental arteries and veins. There is a paucity of cell subtype specific expression studies of placental K+ channels. .
This review focuses on the roles of K+ channels and oxygenation in controlling reactivity of small fetoplacental blood vessels.
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Arterioles, capillaries and venules all actively change their cellular functions and phenotypes during inflammation in ways that are essential for maintenance of homeostasis and self-defense, and are also associated with many inflammatory disorders. Endothelial cells (ECs), together with pericytes and extracellular matrix proteins, can regulate blood flow, the coagulation cascade, fluid and solute exchange, and leukocyte trafficking. While capillary and venular functions in inflammation are well characterized, the arteriolar contribution to inflammation has only recently come into focus. Arterioles differ from venules in structure, EC morphology, shear environment, expression and distribution of surface ligands, hence regulation and function of arteriolar wall cells during inflammation may also be distinct from venules. Recent work indicates that in response to pro-inflammatory stimuli, arterioles alter barrier function, and support leukocyte and platelet interactions through upregulation of adhesion molecules. This suggests that in addition to their role in blood flow regulation, arterioles may also participate in inflammatory responses. In this review we will discuss mechanisms that characterize arteriolar responses to proinflammatory stimuli. We will detail how distinct arteriolar features contribute to regulation of barrier function and leukocyte-EC interactions in inflammation, and further highlight the potential priming effects of arteriolar responses on venular function and progression of inflammatory responses.
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To test whether long-term anti-hypertensive treatment with metoprolol succinate (a β1-adrenoceptor blocker) or olmesartan medoxomil (an angiotensin II AT1-receptor blocker) reverses microvascular dysfunction in hypertensive patients.
This study included 44 hypertensive outpatients and 20 age and sex-matched healthy controls. We used skin capillaroscopy to measure capillary density and recruitment at rest and during postocclusive reactive hyperemia (PORH). Endothelium-dependent vasodilation of skin microcirculation was evaluated with a laser Doppler perfusion monitoring system in combination with acetylcholine iontophoresis, PORH and local thermal hyperemia (LTH).
Pretreatment capillary density in hypertensive patients was significantly reduced compared to controls (71.3±1.5 vs 80.6±1.8 cap/mm2; p<0.001), as was PORH (71.7±1.5 vs 79.5±2.6 cap/mm2; p<0.05). After treatment for six months, capillary density increased to 75.4±1.1 cap/mm2 (p<0.01) at rest and 76.8±1.1 cap/mm2 during PORH. During LTH, cutaneous vascular conductance (CVC) in perfusion units (PU)/mm Hg was similar in patients (1.71 [1.31-2.12]) and controls (1.60 [1.12-1.91]) and increased significantly (1.82 [1.30-2.20]) after treatment. Maximal CVC during PORH was reduced in hypertensive patients (0.30 [0.22-0.39]) compared to controls (0.39 [0.31-0.49], p<0.001) and increased (0.41 [0.29-0.51], p<0.001) after treatment.
Capillary rarefaction and microvascular endothelial dysfunction in hypertensive patients responded favorably to long-term pharmacological treatment.
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Refractory angina is the occurrence of clinical symptoms despite maximal therapy. We investigated associations between microvascular function, atherosclerotic burden and clinical symptoms in subjects with coronary artery disease (CAD).
Skin microvascular response to heating and ischaemia was assessed in 167 male volunteers by laser Doppler fluximetry; 82 with CAD on maximal therapy and 85 with no known CAD (noCAD). Coronary artery calcification (CAC) scores, carotid intimal media thickness (IMT), and femoral IMT were measured and symptoms were scored using the Rose angina questionnaire.
Patients with CAD had poorer microvascular response to heating(114(95% CI 106-122)au CAD vs. 143(134-153)au noCAD; p<0.0001) and ischaemia (42(38-46)au CAD vs. 53(78-58)au. noCAD; p=0.001). 38% of the noCAD group had elevated CAC scores. There were no associations between markers of atherosclerosis and microvascular function.
42% of the CAD group had refractory angina. This was associated with impaired microvascular function compared to those with elevated CAC scores but no symptoms (109 (95-124)au vs. 131(122-140)au; p=0.008).
Men with symptomatic CAD have poorer microvascular function compared to individuals without CAD. Microvascular function does not correlate with atherosclerosis but is impaired in individuals with refractory angina. Microvascular dysfunction may play a role in the symptomatology of angina.
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Obese subjects exhibit decreased exercise capacity (VO2max). We have shown that vascular KATP channel mediates arteriolar dilation to muscle contraction. We hypothesize that exercise capacity is decreased in obesity due to impaired vascular KATP function.
VO2max was measured in LZR and OZR by treadmill running before and following treatment with the KATP blocker glibenclamide i.p. One week later the spinotrapezius muscle was prepared for in vivo microscopy. Arcade arteriolar diameters were measured following muscle contraction or application of the KATP opener cromakalim before and after glibenclamide application. In additional animals, LZR and OZR were treated with apocynin for 5 weeks. VO2max and arteriolar dilation experiments were repeated.
OZR exhibited decreased VO2max, functional and cromakalim-induced vasodilation as compared to LZR. Glibenclamide had no effect on VO2max and functional vasodilation in OZR but significantly inhibited responses in LZR. Vascular superoxide levels and NADPH oxidase activity were increased in OZR but reduced in apocynin-treated OZR. Apocynin increased the VO2max, functional and cromakalim-induced vasodilation in OZR with no effect in LZR.
Exercise capacity is dependent on vascular KATP channel function. The reduced exercise capacity in OZR appears to be due in part to superoxide-mediated impairment in vascular KATP function.
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Inflammation is involved in the pathogenesis of hypertension. Hypertensive animals have an increased number of perivascular macrophages in cerebral arteries. Macrophages might be involved in remodeling of the cerebral vasculature. We hypothesized that peripheral macrophage depletion would improve middle cerebral artery (MCA) structure and function in hypertensive rats.
for macrophage depletion, six-week-old stroke-prone spontaneously hypertensive rats (SHRSP) were treated with liposome-encapsulated clodronate (CLOD, 10ml/kg/every 3 or 4 days, I.P.), or vehicle (PBS lipo). MCA structure and function were analyzed by pressure and wire myography.
blood pressure was not affected by CLOD. The number of perivascular CD163 positive cells per microscopic field was reduced in the brain of SHRSP+CLOD. CLOD treatment caused an improvement in endothelium-dependent dilation after intralumenal perfusion of ADP and incubation with acetylcholine (Ach). Inhibition of nitric oxide production blunted the Ach response, and endothelium-independent dilation was not altered. At an intralumenal pressure of 80 mmHg, MCA from SHRSP+CLOD showed increased lumen diameter, decreased wall thickness and wall-to-lumen ratio. Cross-sectional area of pial arterioles from SHRSP+CLOD was higher than PBS lipo.
These results suggest that macrophage depletion attenuates MCA remodeling and improves MCA endothelial function in SHRSP.
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To determine the role of focal adhesion kinase (FAK) in the regulation of endothelial barrier function.
Stable FAK knockdown human pulmonary microvessel endothelial cells were generated by lentiviral infection of FAK shRNA. Measurements of isometric tension and transendothelial electrical resistance were performed.
A FAK knockdown human pulmonary endothelial cell line was generated by lentiviral infection with FAK shRNA and resulted in greater than 90% reduction in FAK protein with no change in Pyk2 protein. Loss of FAK alters cell morphology and actin distribution in both pre- and post-confluent endothelial cells. Large, polygonal shaped endothelial cells with randomly organized stress fibers were identified in pre-confluent cultures while in confluent monolayers, endothelial cells were irregularly shaped with actin bundles present at cell margins. An increase in the number and size of vinculin plaques was detected in FAK depleted cells. FAK knockdown monolayers generate a greater transendothelial electrical resistance than controls. Thrombin treatment induced similar changes in transendothelial resistance in both FAK knockdown and control cell lines. FAK depleted endothelial cells develop a higher stable basal isometric tension compared to control monolayers, but the increase in tension stimulated by thrombin does not differ between the cell lines. Basal myosin II regulatory light chain phosphorylation is unaltered in FAK depleted cells. In addition, loss of FAK enhances VE-cadherin localization to the cell membrane without altering VE-cadherin protein levels.
The loss of FAK in endothelial cells enhances cell attachment and strengthens cell-cell contacts resulting in greater basal tension leading to formation of a tighter endothelial monolayer.
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to establish whether skin micro blood flow can be modified by exposure to the radiofrequency waves emitted by a mobile phone when the latter is held against the jaw and ear.
Variations in skin micro blood flow and skin temperature in adult volunteers were simultaneously recorded with a thermostatic laser Doppler system during a 20-minute “radiofrequency” exposure session and a 20-minute “sham” session. The skin microvessels’ vasodilatory reserve was assessed with a heat challenge at the end of the protocol.
During the radiofrequency exposure session, skin micro blood flow increased (vs. baseline) more than during the sham exposure session. The sessions did not differ significant in terms of the skin temperature time-course response. The skin microvessels’ vasodilatory ability was found to be greater during radiofrequency exposure than during sham exposure.
Our results reveal the existence of a specific vasodilatory effect of mobile phone radiofrequency emission on skin perfusion.
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Sepsis is a systemic inflammatory response syndrom. Emodin is a major ingredient of Rheum Palmatum, a Chinese herb that is widely used in China for treatment of endotoxemia-related diseases. This study intended to examine the effect of Emodin on lipopolysaccharide (LPS)-induced rat mesenteric microcirculatory disturbance and the underlying mechanisms. The male Wistar rats received LPS (5 mg/kg/hr) for 90 min, with or without administration of Emodin (10 mg/kg/hr) by enema 30 min before (pre-treatment) or after (post-treatment) LPS infusion, and the dynamics of mesenteric microcirculation were determined by inverted intravital microscopy. Expression of adhesion molecules and toll-like receptor 4 (TLR4), nuclear factor kappa B p65 (NF-κB p65), intercellular adhesion molecule-1 (ICAM-1), myeloperoxidase (MPO), and activator protein-1 (AP-1) in mesentery tissue was evaluated by flow cytometry and Western-blot, respectively. Pre or post-treatment with Emodin significantly ameliorated LPS-induced leukocyte emigration, reactive oxygen species production and albumin leakage, and the expression of TLR4, NF-κB p65, ICAM-1, MPO, and AP-1 in mesentery. These results demonstrate the beneficial role of Emodin in attenuating the LPS-induced microcirculatory disturbance, and support the use of Emodin for patients with endotoxemia.
© 2013 John Wiley & Sons Ltd
We hypothesised that trajectories of adiposity across childhood would be associated with retinal microcirculatory diameters at age 12 years, independent of blood pressure (BP).
The Avon Longitudinal Study of Parents and Children followed a cohort of children born in 1991-1992. The current study includes all children with retinal images acquired at the 12 year clinic and individual trajectories of ponderal index (PI) from 0-2 years and body mass index (BMI) from 2-10 years. Retinal microvascular measures included retinal arteriolar and venular diameters.
Children in this analysis had a birth weight of 3.5 ± 0.4 kg, a PI of 26.2 ± 2.4 kg/m3 and a gestational age of 39.7 ± 1.4 weeks (mean ± standard deviation (SD)). Analysis of growth trajectories showed that lower PI at birth was associated with narrower retinal arterioles. Higher PI at birth was associated with wider venular diameter, and a stronger positive association was evident between BMI change at 5-5.5 and 8.5-10 years with wider venular diameters. Current fat mass was also associated with wider venular diameters.
Retinal arterioles and venules are differentially associated with growth in early life and childhood adiposity. Early adiposity may adversely affect the microcirculation, with important implications for cardiovascular risk in adulthood.
© 2013 John Wiley & Sons Ltd
Endothelium-dependent vasodilation of coronary arterioles is impaired in obese rats and may be improved by a low-carbohydrate diet. The aim of this study is to elucidate the mechanism by which this improvement occurs.
We used four groups of male Zucker rats: lean and obese on either standard diet or low-carbohydrate diet. Coronary arterioles were cannulated and pressurized for diameter measurements during administration of acetylcholine or sodium nitroprusside or during flow. Real-time PCR was performed to quantify mRNA expression of super-oxide dismutase and catalase.
The low-carbohydrate diet significantly increased endothelium-dependent dilation in the obese rats. N-nitro-L-arginine methyl ester and indomethacin reduced responses to flow and acetylcholine in the lean rats without any effect on the obese on either diet. In contrast, tetraethylammonium and catalase blocked flow-dependent and acetylcholine-induced dilation in the obese on either diet while no effect was observed on the lean. The low-carbohydrate diet in the obese significantly up-regulated catalase mRNA expression and slightly increased super-oxide dismutase mRNA levels.
A low-carbohydrate diet improves endothelium-dependent vasodilation of coronary arterioles in obese rats through the production of hydrogen peroxide which acts as a hyperpolarizing factor, independent of nitric oxide and prostacyclin.
© 2013 John Wiley & Sons Ltd
Recent findings attested erythropoietin (EPO) tissue-protective effects in ischemically challenged tissues. Therefore, the study aimed at elaborating the effect of systemic pre- and postconditioning using EPO in a mouse model of persistent ischemia of the skin.
Three groups of nine C57Bl/6-mice each were analyzed, including untreated controls, EPO-preconditioning and EPO-postconditioning (500IU EPO/kg body weight/day for 10 days). Critically perfused skin flaps undergoing necrosis, if kept untreated, were mounted into dorsal skinfold chambers. Intravital epi-fluorescence microscopy was performed over 10 days, assessing tissue necrosis, microcirculation, inflammation and angiogenesis. Protein expression analysis of endothelial nitric oxide synthase (eNOS) was performed. Hematocrit analyses were carried out in separate animals (n=8).
Only EPO-preconditioning was able to significantly reduce necrosis, when compared to controls. This correlated with a significantly increased capillary density in the critically perfused tissue. Administration of EPO only slightly increased eNOS-expression at day 10, when compared to controls. EPO induced angiogenesis and increased hematocrit. Finally, EPO significantly reduced leukocytic inflammation in arterioles in all EPO-receiving mice.
EPO-preconditioning effectively reduces skin necrosis predominantly by capillary maintenance and reperfusion as well as improved tissue regeneration. Thus, EPO-preconditioning might represent a promising, non-invasive approach to reduce complications in ischemically challenged skin.
© 2013 John Wiley & Sons Ltd
The pathophysiology underlying hyperthyroidism-induced left ventricle (LV) dysfunction and hypertrophy directly involves the heart and indirectly involves the neuroendocrine systems. The effects of hyperthyroidism on the microcirculation are still controversial in experimental models. We investigated the effects of hyperthyroidism on the cardiac function and microcirculation of an experimental rat model
Male Wistar rats (170-250 g) were divided into two groups: the euthyroid group (n = 10), which was treated with 0.9% saline solution, and the hyperthyroid group (n = 10), which was treated with L-thyroxine (600 μg/kg/day, i.p.) during 14 days. An echocardiographic study was performed to evaluate the alterations in cardiac function, structure and geometry. The structural capillary density and the expression of angiotensin II AT1 receptor in the LV were analyzed using histochemistry and immunohistochemistry, respectively
Hyperthyroidism was found to induce profound cardiovascular alterations, such as systolic hypertension, tachycardia, LV dysfunction, cardiac hypertrophy and myocardial fibrosis. The present study demonstrates the existence of structural capillary rarefaction and the down-regulation of the cardiac angiotensin II AT1 receptor in the myocardium of hyperthyroid rats in comparison with euthyroid rats
Microvascular rarefaction may be involved in the pathophysiology of hyperthyroidism-induced cardiovascular alterations.
© 2013 John Wiley & Sons Ltd
To use the obese Zucker rat (OZR) model of the metabolic syndrome to determine the impact of dilator stimuli on myogenic activation (MA) of gracilis arterioles (GA) and middle cerebral arteries (MCA). We tested the hypothesis that increased oxidant stress and thromboxane A2 (TxA2) exacerbate MA, and prevent its blunting with dilator stimuli, in OZR
GA/MCA from OZR and lean Zucker rats (LZR) were pressurized ex vivo. MA was determined under control conditions and following challenge with acetylcholine, hypoxia and adenosine. Responses were also evaluated after pre-treatment with TEMPOL (antioxidant) and SQ-29548 (PGH2/TxA2 receptor antagonist)
MA was increased (and dilator responses decreased) in GA/MCA from OZR, dependent on the endothelium and ROS. In GA, the impact of ROS on MA and dilator effects was largely via TxA2, while in MCA, this appeared was more dependent on NO bioavailability. Intrinsic responses of GA/MCA to carbacyclin, U46619, and NO donors were similar between strains.
A developing ROS-based endothelial dysfunction in MCA and GA of OZR contributes to an enhanced MA of these vessels. While treatment of GA/MCA with TEMPOL attenuates MA in OZR, the mechanistic contributors to altered MA, distal to ROS, differ between the two resistance vessels.
© 2013 John Wiley & Sons Ltd
We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion.
Male Sprague Dawley rats subjected to middle cerebral artery occlusion and reperfusion (MCAO/R) were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0-4 hours); cerebral infarct size, edema and neurological function were assessed at 24 h. Curcuminoid effects on TNFα-stimulated human brain microvascular endothelial cell (HBMVEC) were assessed.
Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC.
Turmeric derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium.
© 2013 John Wiley & Sons Ltd
IL-27 belongs to the IL-12 family of cytokines and is recognized for its role in Th cell differentiation and as an inhibitor of tumor-angiogenesis. The purpose of this study was to investigate the effect of IL-27 on proliferation of lymphatic endothelial cells to gain insight into the interplay between the immune system and development of the lymphatic system.
IL-27-stimulated signal transduction in human dermal lymphatic endothelial cells was measured by western blotting and synthesis of CXCL10 and CXCL11 by use of RT-PCR and ELISA. Proliferation was measured using MTT and BrdU kits and the role of STAT1 and chemokines was determined by use of siRNA and recombinant proteins.
Stimulation of lymphatic endothelial cell cultures with IL-27 induced JAK dependent phosphorylation of STAT1 and STAT3 and inhibited lymphatic endothelial cell proliferation and migration. Expression of CXCL10 and CXCL11, both STAT1 target genes, were profoundly up-regulated upon IL-27 stimulation, and recombinant CXCL10 and CXCL11 inhibited FGF-2-induced proliferation in vitro. siRNA targeting of STAT1 almost completely abrogated CXCL10 and CXCL11 expression as well as the proliferative effect of IL-27.
IL-27 function as an anti-lymphangiogenic regulator in vitro by up-regulating chemokines and interfering with the mitogenic effect of growth factors through STAT1 activation.
© 2013 John Wiley & Sons Ltd
To determine if the diabetic ketoacidosis (DKA)-induced inflammation in juvenile mice provokes activation and dysfunction of cerebrovascular endothelial cells (CVECs).
DKA in juvenile mice was induced with administration of streptozocin and alloxan. Blood from DKA mice was assessed for cytokines and soluble cell adhesion proteins, and either DKA plasma or exogenous compounds were applied to an immortalized mouse brain endothelial cell line (bEND3).
DKA increased circulating levels of IL-6, IL-8(KC), MCP-1, IL-10, sE-selectin, sICAM-1 and sVCAM-1. Stimulation of bEND3 with DKA plasma caused cellular activation (increased reactive oxygen species and activation of NF-κΒ), up-regulation of a pro-adhesive phenotype (E-selectin, ICAM-1 and VCAM-1) and increased leukocyte-bEND3 interaction (leukocyte rolling/adhesion). Transendothelial electric resistance, a measure of bEND3 monolayer integrity, was decreased by DKA plasma. Activation and dysfunction of bEND3 with DKA plasma was supressed by plasma heat-treatment (56°C, 1hr) and replicated with the application of DKA recombinant cytomix (IL-6, IL-8(KC), MCP-1 and IL-10), implicating circulating inflammatory protein(s) as mediators. Treatment of bEND3 with β-OH-butyrate, the main ketone elevated in DKA, failed to mimic the DKA-plasma induced activation and dysfunction of bEND3.
DKA elicits systemic inflammation associated with CVEC activation and dysfunction, possibly contributing to DKA-associated intracranial microvascular complications.
© 2013 John Wiley & Sons Ltd
The control of arteriolar diameters in microvasculature has been in the focus of studies on mechanisms matching oxygen demand and supply at the tissue level. Functionally, important vascular elements include endothelial cells (EC), vascular smooth muscle cells (VSMC) and red blood cells (RBC). Integration of these different cell types into functional units aimed at matching tissue oxygen supply with tissue oxygen demand is only achieved when all these cells can respond to the signals of tissue oxygen demand. Many vasoactive agents that serve as signals of tissue oxygen demand have their receptors on all these types of cells (VSMC, EC, and RBC) implying that there can be a coordinated regulation of their behavior by the tissue oxygen demand. Such functions of RBC as oxygen carrying by hemoglobin (Hb), rheology, and release of vasoactive agents are considered. Several common extra- and intracellular signaling pathways that link tissue oxygen demand with control of VSMC contractility, EC permeability, and RBC functioning are discussed.
© 2013 John Wiley & Sons Ltd
The classical model of metabolic regulation of blood flow in muscle tissue implies the maintenance of basal tone in arterioles of resting muscle and their dilation in response to exercise and/or tissue hypoxia via the evoked production of vasodilator metabolites by myocytes. A century-long effort to identify specific metabolites responsible for explaining active and reactive hyperemia has not been successful. Furthermore, the metabolic theory is not compatible with new knowledge on the role of physiological radicals (e.g., nitric oxide, NO, and superoxide anion, O2-) in the regulation of microvascular tone. We propose a model of regulation in which muscle contraction and active hyperemia are considered the physiologically normal state. We employ the “bang-bang” or “on/off” regulatory model which makes use of a threshold and hysteresis; a float valve to control the water level in a tank is a common example of this type of regulation. Active bang-bang regulation comes into effect when the supply of oxygen and glucose exceeds the demand, leading to activation of membrane NADPH oxidase, release of O2- into the interstitial space and subsequent neutralization of the interstitial NO. Switching arterioles on/off when local blood flow crosses the threshold is realized by a local cell circuit with the properties of a bang-bang controller, determined by its threshold, hysteresis and dead-band. This model provides a clear and unambiguous interpretation of the mechanism to balance tissue demand with a sufficient supply of nutrients and oxygen.
© 2013 John Wiley & Sons Ltd
Proinflammatory cytokine tumor necrosis factor-alpha(TNF-α) during myocardial ischemia/reperfusion(MI/R) injury has been studied extensively. However, how TNF-α induce microvascular dysfunction in MI/R is still unclear. This study investigates whether TNF-α regulates fibrinogen-like protein 2(fgl2) expression, a procoagulant resulting in the formation of fibrin-rich microthrombus in MI/R injury.
Microthrombosis, TNF-α and fgl2 expression were assessed in rats with MI/R injury. The effect of TNF-α on fgl2 expression and fgl2 prothrombinase activity were investigated in cardiac microvascular endothelial cells(CMECs), then CMECs were pretreated with selective inhibitors of two major signaling pathways by activated by TNF-α. TNF-α and fgl2 expression were both upregulated in MI/R group. When neutralization of TNF-α, fgl2 expression was decreased in vivo. Fgl2 expression was upregulated in CMECs exposed to TNF-α. Accordingly, the ability of thrombin generation was increased in CMECs. Besides, TNF-α-induced fgl2 expression in the cells was suppressed by nuclear factor kappa B (NF-κB) inhibitor pyrrolidinethiocarbamate(PDTC) and/or p38 mitogen-activated protein kinase(MAPK) inhibitor SB203580.
TNF-α upregulates fgl2 expression via activation of NF-kB and p38 MAPK in CMECs. TNF-α-induced flg2 in CMECs mediates the formation of fibrin-rich microthrombus, which may be one of the mechanisms of microvascular dysfunction or obstruction due to MI/R injury.
© 2013 John Wiley & Sons Ltd
Oxygen delivery, underpinned by vascular tone, is the principle limiting factor in the study of skeletal muscle physiology, particularly during muscle contraction. The aim of this study was to develop an autologous perfused rat hindlimb preparation for the study of skeletal muscle contractile function.
Adult Wistar rats were surgically prepared using a by-pass system for pump-controlled arterial blood flow to, and venous return from the hindlimb during periods of quiescence and twitch contraction of the gastrocnemius-plantaris-soleus muscle bundle.
During rest hindlimb perfusion pressure (102±5 mmHg) was not different to systemic arterial pressure (99±4 mmHg). Hindlimb pressure was responsive to vasoconstrictors and vasodilators (±50 mmHg). The arterial PO2 (100±3 mmHg), O2 saturation, and acid-base balance (pH: 7.42±0.01) contributed to resting hindlimb (a-v)O2 difference (4.8±0.5 mL.100 mL−1) and VO2 (0.31±0.03 μmol.min−1.gww−1). Repetitive isometric twitch tension (1 Hz, 0.05 ms, 10 min) was best maintained at a flow rate of 2 mL.min−1 (VO2 increased 5-fold during muscle contraction) and efficiency of oxygen-use increased from 0.27±0.08 to 0.52±0.07 N.μmol−1.min−1.
The autologous rat hindlimb provided resting vascular tone allowing maintenance of perfusion pressure at flows within the physiological range. Oxygen delivery supported repetitive twitch contractions and facilitated measurement of active metabolism.
© 2013 John Wiley & Sons Ltd
Compromised perfusion of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous injection of ascorbate inhibits platelet adhesion and plugging in septic capillaries. In the present study we hypothesized that ascorbate reduces aggregation of platelets and their surface expression of P-selectin (a key adhesion molecule) in mice.
Platelets were isolated from control mice and subjected to agents known to be released into the bloodstream during sepsis (thrombin, ADP or U46619, thromboxane A2 analog). Platelet aggregation was analysed by aggregometry and P-selectin expression by flow cytometry.
Platelet-activating agents increased aggregation and P-selectin expression. Ascorbate inhibited these increases. This inhibitory effect was NOS-independent (LNAME had no effect). In contrast to the platelet-activating agents, direct stimuli lipopolysaccharide, TNFα or plasma from septic mice did not increase aggregation/expression, a finding consistent with the literature. The results suggest a complex mechanism of platelet aggregation and P-selectin expression in sepsis, where generation of platelet-activating stimuli is required first, before platelet aggregation and adhesion in capillaries occur.
The ability of ascorbate to reduce platelet aggregation and P-selectin expression could be an important mechanism by which ascorbate inhibits capillary plugging in sepsis.
© 2013 John Wiley & Sons Ltd
To investigate how red blood cell aggregation could modulate the spatial variations of cell-free layer formation in the vicinity of an arteriolar bifurcation.
Visualization of blood flow was performed in upstream and downstream vessels of arteriolar bifurcations in the rat cremaster muscles under reduced flow conditions before and after induction of red blood cell aggregation to both physiological normal and pathological hyper levels seen in humans.
Large asymmetries of layer widths on opposite sides of the downstream vessel were attenuated along the vessel and this effect could be prominently enhanced by the hyper-aggregation due to a higher formation rate of the layer which was greater on one side than the other of the vessel. The proportion of downstream layer formation constituted by the smaller downstream vessel generally increased with a thicker layer width at the wall of the upstream vessel adjacent to it. A greater tendency of the layer formation in the smaller downstream vessel was found under the hyper-aggregating condition than normal-aggregating and non-aggregating conditions.
Red blood cell aggregation could attenuate the asymmetry in cell-free layer formation on opposite sides of the downstream vessel but enhances the heterogeneity of the layer formation between downstream vessels.
© 2013 John Wiley & Sons Ltd
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.
© 2013 John Wiley & Sons Ltd
The purpose of this study was to visualize glomerular hyperfiltration in rats at the early stage of diabetes under in vivo conditions and to quantitatively examine the effect of C-peptide on filtration.
Type 1 diabetes was induced by streptozotocin (50 mg/kg) in Wistar rats. The rats were divided into 4 groups: control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide. C-peptide was continuously infused (50 pmol·kg-1min-1). Filtration was visualized by a bolus shot of various sizes of dextrans (3k to 70k Da) conjugated with Texas Red and observed with a multiphoton microscope under inhaled anesthesia. Relative sieving coefficients were used to quantify filtration.
Almost all smaller particles (3–10k Da) were filtered both in control and diabetic rats. Filtration of larger particles (40–70k Da) was seen in normal rats but was more apparent in diabetic rats, where it was progressive according to the duration of diabetes. C-peptide administration restored the leakage of larger particles to the level seen for the control.
We visualized and analyzed hyperfiltration and confirmed that C-peptide has a nephroprotective effect. Furthermore, we found that the leakage of larger particles increased as the duration of diabetes increased.
© 2013 John Wiley & Sons Ltd
Granulocyte colony stimulating factor (G-CSF) and Erythropoietin (EPO) have shown a notable capability in neovascularization. However, their use is limited because of untoward leucocytosis, erythrogenesis, and short half-life in the plasma. Herein we examined whether G-CSF and EPO released from fibrin gel injected into ischemic tissues would synergistically promote neovascularization with limited systematic effects in a rat hindlimb ischemic model.
In vivo study, group Gel received an intramuscular injection of fibrin gel; group Gel+G-CSF received fibrin gel containing human G-CSF; group Gel+EPO received fibrin gel containing human EPO; group Gel+G-CSF&EPO received fibrin gel containing G-CSF and EPO; group G-CSF&EPO received G-CSF and EPO. Through promoting the expression of SDF-1, local high concentration of EPO could traffic CXCR4+ cells mobilized by G-CSF to enhance neovascularizaton in ischemic muscle. The treatment with Gel+G-CSF&EPO was superior to the other treatments on blood flow reperfusion, capillary density and α smooth muscle actin positive vessel density. And this treatment induced modest WBCs count increase in peripheral blood.
G-CSF and EPO released from fibrin gel had a combined effect on postischemia neovasculariztion. This treatment may be a novel therapeutic modality for ischemic peripheral artery disease.
© 2013 John Wiley & Sons Ltd
Hypertension is characterized by microvascular remodeling resulting in increased wall/lumen ratio and elevated microvascular stiffness. Aiming to transform the measurement of macrovascular stiffness into a microvascular environment we introduce a non-invasive method to assess retinal pulse wave velocity (rPWV). rPWV alterations in early hypertension are investigated in detail. The developed methodology is compared with its possible computational alternatives.
Time dependent alterations of retinal arterial diameter were assessed non-invasively by the Dynamic Vessel Analyzer in 65 male normoalbuminuric normotensive to mildly hypertensive subjects (age: 28.7±6.0 years). rPWV was computed using three different methods. ‘Method1’ used filtration at heart rate (HR), ‘Method2’ filtered at higher HR multiples, ‘Method3’ used additionally linear fit for data averaging.
‘Method2’ and ‘Method3’ applying filtration at high HR multiples showed strong associations with systolic BP throughout the cohort (r=0.49, r=0.63, P<0.001). Based on the highest association ‘Method3’ was proposed to characterize rPWV. Hypertensive patients showed higher rPWV (1243±694 units/second) than subjects with high-normal BP (786±486 units/second, P<0.01) or normotensive subjects (442±148 units/second, P<0.001).
rPWV demonstrated a strong association with BP and can discriminate between optimal, high-normal and mildly hypertensive BP values. rPWV may add detailed insights to early microvascular pathophysiology, potentially beyond microalbuminuria.
© 2013 John Wiley & Sons Ltd
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.
© 2012 John Wiley & Sons Ltd
Hypoxia-inducible factor (HIF) is a hypoxia-responsive transcriptional factor that controls the expression of proteins contributing to homeostatic responses to hypoxia. Spatial heterogeneity of tissue oxygenation has been postulated as a determinant of structure and function of hepatic lobules, while its molecular mechanisms remain unknown. This study aimed to examine the role of HIF-1 expressed in hepatocytes in regulation of hepatic microcirculation.
We have generated mice harboring a floxed HIF-1α allele, and employed the albumin-Cre transgenic line to inactivate the gene site-specifically in hepatocytes.
Intravital observation of the hepatic microcirculation revealed extension of hepatic lobules in HIF-1α-deficient mice. Measurement of microvascular diameter, velocity, and local oxygen tension by laser-assisted phosphorimetry showed that the oxygen consumption in the lobules of HIF-1α-deficient mice was greater than that in those of control mice. Isolated hepatocytes from HIF-1α-deficient mice also stimulated oxygen consumptions with increased contents of mitochondrial DNA. Overexpression of HIF-1α decreased the expression of PGC-1α mRNA, whereas the knockdown of the HIF-1α gene increased it, suggesting that HIF-1 regulates cellular respiration through mitochondrial biogenesis.
Our results suggest that constitutive expression of HIF-1α in hepatocytes acts as a determinant of hepatic lobular structure and oxygen consumption by changing mitochondrial contents.
© 2012 John Wiley & Sons Ltd
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.
© 2012 John Wiley & Sons Ltd
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.
© 2012 John Wiley & Sons Ltd
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.
© 2012 John Wiley & Sons Ltd
Dynamic changes in intracellular Ca2+ levels in vascular smooth muscle cells are critically important for cardiovascular regulation. This Special Topic Issue highlights a series of expert opinion articles focused on this important subject. After a brief overview, novel discoveries surrounding smooth muscle cell Ca2+ influx via L-type and T-type channels are reviewed. Current work revealing the functional importance of dynamic Ca2+ signaling in the control of the parenchymal microvasculature and the emerging role of mitochondrial Ca2+ signaling and store-operated Ca2+ entry in smooth muscle cells is discussed. Finally, recent data describing a new target of localized Ca2+ signaling in arterial myocytes that is responsible for membrane depolarization is reviewed. Authors were encouraged to write in an opinionated and provocative manner with the hope of stimulating discussion in this area of research.
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.
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 [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 AKAP150. We also described a mechanism whereby clusters of L-type Ca2+ channels gate cooperatively to amplify intracellular Ca2+ signals with likely pathological consequences.
Cerebral blood flow is controlled by a network of resistance arteries that dilate and constrict to mechanical and chemical stimuli. Vasoactive stimuli influence arterial diameter through alterations in resting membrane potential and the influx of Ca2+ through voltage-gated Ca2+ channels. Historically, L-type Ca2+ channels were thought to be solely expressed in cerebral arterial smooth muscle. Recent studies have, however, challenged this perspective, by providing evidence of T-type Ca2+ channels in vascular tissues. This perspective piece will introduce T-type Ca2+ channels, their electrophysiological properties, and potential roles in arterial tone development. We begin with a brief overview of Ca2+ channels and a discussion of the approaches used to isolate this elusive conductance. We will then speculate on how the two T-type Ca2+ channels expressed in cerebral arterial smooth muscle might differentially influence arterial tone. This discovery of T-type Ca2+ channels alters our traditional understanding of Ca2+ dynamics in vascular tissue and fosters new avenues of research and insight into the basis of arterial tone development.
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 CBF, 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 PAs. There are, moreover, notable functional differences between pial arteries and PAs. For example, in pial VSMCs, local calcium release events (“calcium sparks”) through ryanodine receptor (RyR) channels in SR membrane activate large conductance, calcium-sensitive potassium channels to modulate vascular diameter. In contrast, VSMCs in PAs 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 CBF.
Mitochondrial Ca2+ uptake contributes important feedback controls to limit the time course of Ca2+signals. Mitochondria regulate cytosolic [Ca2+] over an exceptional breath of concentrations (~200 nM to >10 μM) to provide a wide dynamic range in the control of Ca2+ signals. Ca2+ uptake is achieved by passing the ion down the electrochemical gradient, across the inner mitochondria membrane, which itself arises from the export of protons. The proton export process is efficient and on average there are less than three protons free within the mitochondrial matrix. To study mitochondrial function, the most common approaches are to alter the proton gradient and to measure the electrochemical gradient. However, drugs which alter the mitochondrial proton gradient may have substantial off target effects that necessitate careful consideration when interpreting their effect on Ca2+ signals. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the electrochemical gradient and are altered by changes in plasma membrane potential. Care is again needed in interpreting results. This review provides a brief description of some of the methods commonly used to alter and measure mitochondrial contribution to Ca2+ signaling in native smooth 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 25 nm) 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.
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.