Arteriolar Vasoconstriction Research Articles

The short-term effects of intravitreal aflibercept injections and dexamethasone implant on ocular hemodynamics in retinal vein occlusions.

PurposeTo determine the early effects of intravitreal anti-VEGF and dexamethasone application on blood flow velocities in patients with retinal vein occlusions.MethodsThe ophthalmic (OA) and the central retinal arteries (CRAs) of the affected and unaffected eyes of 21 patients with central retinal vein occlusion (CRVO), and 26 patients with branch retinal vein occlusion (BRVO) were investigated by Color Doppler imaging (CDI). Peak systolic volume (PSV), end diastolic volume (EDV), and average blood velocity (Vmean) of the CRA and OA were measured and resistive indexes (RI) were calculated at pre-injection and the 1st week and 1st month post-injection.ResultsThere was no statistically significant difference between the OA values for affected and unaffected eyes in the CRVO group before treatment. The CRA, EDV, and Vmean values were significantly lower in affected eyes. Blood flow velocities of both CRA and OA were not significantly different than in the fellow unaffected eyes in the BRVO group. The differences between PSV, EDV, and Vmean measurements of OA and CRA at different times pre- and post-injection in response to anti-VEGF and dexamethasone treatment in the CRVO group were significant; but there was no difference in RI value. OA blood flow velocity measurements were not statistically different post-injection in the BRVO group; however, the values of PSV and EDV of CRA decreased post-injection.ConclusionIntravitreal anti-VEGF and dexamethasone implant may induce retinal arteriolar vasoconstriction in patients with retinal vein occlusion.

Integration of purinergic and angiotensin II receptor function in renal vascular responses and renal injury in angiotensin II-dependent hypertension.

Glomerular arteriolar vasoconstriction and tubulointerstitial injury are observed before glomerular damage occurs in models of hypertension. High interstitial ATP concentrations, caused by the increase in arterial pressure, alter renal mechanisms involved in the long-term control of blood pressure, autoregulation of glomerular filtration rate and blood flow, tubuloglomerular feedback (TGF) responses, and sodium excretion. Elevated ATP concentrations and augmented expression of P2X receptors have been demonstrated under a genetic background or induction of hypertension with vasoconstrictor peptides. In addition to the alterations of the microcirculation in the hypertensive kidney, the vascular actions of elevated intrarenal angiotensin II levels may be mitigated by the administration of broad purinergic P2 antagonists or specific P2Y12, P2X1, and P2X7 receptor antagonists. Furthermore, the prevention of tubulointerstitial infiltration with immunosuppressor compounds reduces the development of salt-sensitive hypertension, indicating that tubulointerstitial inflammation is essential for the development and maintenance of hypertension. Inflammatory cells also express abundant purinergic receptors, and their activation by ATP induces cytokine and growth factor release that in turn contributes to augment tubulointerstitial inflammation. Collectively, the evidence suggests a pathophysiological activation of purinergic P2 receptors in angiotensin-dependent hypertension. Coexistent increases in intrarenal angiotensin II and activates Ang II AT1 receptors, which interacts with over-activated purinergic receptors in a complex manner, suggesting convergence of their post-receptor signaling processes.

Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage.

Delayed ischemic neurological deficit is the most common cause of neurological impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidation end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degradation, known as PDPs (propentdyopents), have been considered as potential additional effectors in the development of arterial vasoconstriction. To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. Via liquid chromatography/mass spectrometry, we measured increased PDP and BOX concentrations in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diameter in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concentration range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diameter changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we observed a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. For the first time, we have quantified significantly higher concentrations of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.

The histaminergic control of the iridal vascular tone in rats and its influencing by topical administration of olopatadine and ranitidine

Objective. We evaluated the histamine's role in regulating the iris vasomotricity in rats, using as a research tool topical olopatadine, a selective H1 blocker, which is indicated for the treatment of allergic conjunctivitis and ranitidine, a selective H2 blocker mainly used for the treatment of peptic ulcer disease. Methods. Two groups of six Wistar rats anesthetized with ketamine 200 mg/kg body weight were used. They received distilled water in conjunctival instillations, initially and after 5 minutes, olopatadine 2.5 mmol/ l for the first group, respectively ranitidine 2.5 mmol/ l for the second group. The changes of the iris arteriolar and venular diameters were recorded. Results. Both olopatadine and ranitidine produced statistically significant iridal arteriolar vasoconstriction and ranitidine determined statistically significant venuloconstriction, while distilled water did not produce any statistically significant effect. Conclusions. There is a vasodilator histaminergic tone exerted through the histaminergic H1 and H2 receptors in the iris arterioles and, respectively, through the H2 receptors in the iridal venules. Olopatadine, a topical H1 antagonist used in the treatment of ocular allergies, may interfere with the humoral regulation of the iris arteriolar tone. Ranitidine, an H2 antagonist, decreased the diameter of the iris arterioles and venules, when administered topically in rats.

Mastitis and Periareolar Necrosis by 1% Methylene Blue after Axilar Lymphadenectomy and Regional Mastectomy in a Bitch

Background: Mammary neoplasms are frequent in dogs, and surgical excision by radical unilateral mastectomy is the first-choice treatment for most cases. Typically, inguinal lymphadenectomy is performed during mammary inguinal excision, whereas axillar lymphadenectomy is performed using dyes to guide surgical dissection and avoid iatrogenic trauma. This study reports the case of a bitch that underwent axillar lymphadenectomy and regional mastectomy. In this way, this work has the objective of reporting the case of a bitch referred to mastectomy and lymphadenectomy, which presented complications due to the use of 1% methylene blue.Case: A 15-year-old female intact poodle weighing 6.2 kg presented with mammary nodules. Cytological examination confirmed malignant neoplasm in the mammary gland classified as Grade II (World Health Organization). Radical unilateral mastectomy and regional lymphadenectomy were performed. The surgical procedure involved intradermic injection of 0.5 mL of sterile 1% methylene blue solution around the right cranial thoracic mammary gland divided in four sites before skin incision. Following right axillar lymphadenectomy, instead of radical unilateral mastectomy, regional mastectomy was performed in two ulcerated nodules at the right cranial abdominal gland to shorten the surgery time after the bitch demonstrated severe trans-operative hypotension. The bitch was monitored during anesthetic recovery and was discharged after stabilization. At fourteen days after surgery, the patient was re-examined. Increased volume and pain in the mammary glands of both chains were observed. In addition, a whitish liquid secretion was noted primarily from the thoracic caudal left and thoracic cranial right mammary glands. The last mammary gland also presented a periareolar black area where the dye was applied. Progressive remission of the clinical signs was observed after therapy with amoxicillin and clavulanate (20 mg/kg/BID/15 days) and meloxicam (0.1 mg/kg/SID/3 days). At 60 days after surgery, the patient presented minor signs of mastitis at the left caudal thoracic mammary gland.Discussion: In humans, methylene blue is avoided because of rapid diffusion after injection, which could be associated with mastitis due to fat necrosis. The risk of this complication is reduced when tissue ipsilateral to the dyed lymph node is completely excised. In this case, the ipsilateral lymph node chain was not completed excised because of anesthetic complication, and periareolar necrosis was observed at the methylene blue injection site. This complication may be the result of methylene blue oxidation, which can produce formaldehyde and deamidated oxidation products. Methylene blue leads to vasoconstriction of arterioles and tissue ischemia when used in concentrated solutions. Furthermore, a 1% solution has a pH of 3-4.5, which increases its cytotoxic effect. Studies suggest that mammary neoplasms may promote local lymphatic alteration, as observed in this case, including inflammatory response at the contralateral chain that receives dye injection. This suggests that the dye is drained by lymphatic vessels and causes mastitis in the remaining mammary glands. Therefore, methylene blue can cause mastitis and tissue necrosis at the injection site, as well as in the contralateral chain in cases where radical unilateral mastectomy is not performed. This complication increases the morbidity associated with the procedure. However, in the present case, the treatment was sufficiently adequate to restore the patient’s condition.

Afferent arteriole responsiveness to endothelin receptor activation: does sex matter?

BackgroundThe pathogenesis of hypertension is distinct between men and women. Endothelin-1 (ET-1) is a potential contributor to sex differences in the pathophysiology of hypertension. ET-1 participates in blood pressure regulation through activation of endothelin A (ETA) and endothelin B (ETB) receptors including those in the vasculature. Previous studies demonstrated that sex and sex hormones evoke discrepancies in ET-1-mediated control of vascular tone in different vascular beds. However, little is known about sex- and sex hormone-related differences in ET-1-dependent renal microvascular reactivity. Accordingly, we hypothesized that loss of sex hormones impairs afferent arteriole reactivity to ET-1.MethodsMale and female Sprague Dawley rats were subjected to gonadectomy or sham surgery (n = 6/group). After 3 weeks, kidneys from those rats were prepared for assessment of renal microvascular responses to ET-1 (ETA and ETB agonist, 10−12 to 10−8 M) and sarafotoxin 6c (S6c, ETB agonist, 10−12 to 10−8 M) using the blood-perfused juxtamedullary nephron preparation.ResultsControl afferent arteriole diameters at 100 mmHg were similar between sham male and female rats averaging 14.6 ± 0.3 and 15.3 ± 0.3 μm, respectively. Gonadectomy had no significant effect on control arteriole diameter. In sham males, ET-1 produced significant concentration-dependent decreases in afferent arteriole diameter, with 10−8 M ET-1 decreasing diameter by 84 ± 1%. ET-1 induced similar concentration-dependent vasoconstrictor responses in sham female rats, with 10−8 M ET-1 decreasing the diameter by 82 ± 1%. The afferent arteriolar vasoconstrictor responses to ET-1 were unchanged by ovariectomy or orchiectomy. Selective ETB receptor activation by S6c induced a concentration-dependent decline in afferent arteriole diameter, with 10−8 M S6c decreasing diameter by 77 ± 3 and 76 ± 3% in sham male and female rats, respectively. Notably, ovariectomy augmented the vasoconstrictor response to S6c (10−12 to 10−9 M), whereas orchiectomy had no significant impact on the responsiveness to ETB receptor activation.ConclusionThese data demonstrate that sex does not significantly influence afferent arteriole reactivity to ET receptor activation. Gonadectomy potentiated the responsiveness of the afferent arteriole to ETB-induced vasoconstriction in females, but not males, suggesting that female sex hormones influence ETB-mediated vasoconstriction in the renal microcirculation.

A mechanism for injury through cerebral arteriole inflation.

An increase in arterial pressure within the cerebral vasculature appears to coincide with ischemia and dysfunction of the neurovascular unit in some cases of traumatic brain injury. In this study, we examine a new mechanism of brain tissue damage that results from excessive cerebral arteriole pressurization. We begin by considering the morphological and material properties of normotensive and hypertensive arterioles and present a computational model that captures the interaction of neighboring pressurized arterioles and the surrounding brain tissue. Assuming an axonal strain-induced injury criterion, we find that the injury depends on vessel spacing, proximity to an unconfined free surface, and the relative difference in stiffness between the arterioles and the surrounding tissue. We find that a steeper heterogeneity (stiffer vessels surrounded by softer brain tissue) causes larger axial strains to develop at some distance from the arteriole wall, within the brain parenchyma. For a more gradual heterogeneity (softer vessels), we observe more larger strain fields close to the arteriole walls. Both deformation patterns are comparable to damage seen in previous pathology studies on postmortem TBI patients. Finally, we use an analytical model to approximate the interplay between internal pressure, arteriole thickness, and the variation in mechanical properties of arterioles.

Brain correlates of stress-induced peripheral vasoconstriction in patients with cardiovascular disease.

The influence of acute psychological stress on cardiovascular disease is an emerging public health concern. Identification of brain mechanisms underlying this may aid in the discovery of possible treatments. Acute psychological stress may induce arteriolar vasoconstriction and reduce blood flow to vital organs. We hypothesized that functional changes in brain regions involved with memory and autonomic/emotional regulation are implicated in the vasoconstrictive stress response, including the medial prefrontal cortex (anterior cingulate), insula, and dorsolateral prefrontal cortex. Subjects with a history of coronary artery disease (N=59) underwent measurement of microvascular vasomotor tone with the EndoPAT device and O-15 positron emission tomography (PET) imaging of the brain during exposure to mental stress and control conditions. The peripheral arterial tonometry (PAT) ratio was calculated as the mean peripheral vasomotor tone during stress divided by the mean tone during rest. Whole brain contrasts were performed between groups above and below the median PAT ratio, and significant contrasts were defined with cutoff p<0.005. Stress-induced peripheral vasoconstriction (below median PAT ratio) was associated with increased stress activation in insula and parietal cortex, and decreased activation in the medial prefrontal cortex with stress tasks compared to control tasks. These findings demonstrate that stress-induced vasoreactivity is associated with changes in brain responses to stress in areas involved in emotion and autonomic regulation. These findings have important implications on possible treatments for mental stress-induced vascular toxicity.

Air Pollution Exposure and Pulmonary Vascular Morphology in the Framingham Heart Study

Background: Controlled exposure of rodents to particle pollution has been found to cause pulmonary arteriolar vasoconstriction and increased right ventricular pressure, but few studies have assessed if outdoor pollution exposure affects the pulmonary vasculature in generally healthy adults.Aims: To evaluate associations of long-term exposure to fine particulate matter (PM2.5), elemental carbon (EC, an indicator of combustion-related pollution), and distance to a major road with radiographic measures of pulmonary blood volume and distal vascular pruning among community-dwelling adults. Methods: We determined the total blood volume (TBV) of the pulmonary arterial and venous vasculature, and the percent of pulmonary blood volume in vessels with a cross-sectional area less than 5 mm2 (BV5%, a measure of distal vascular pruning), using an automated CT algorithm among 2,470 Framingham Heart Study chest CT sub-study participants from 2008-2011. We assigned distance to major roadway, average PM2.5 from 2004-2008 using a spatio-temporal model based on satellite data, and 2004-2008 average EC using a spatio-temporal prediction model using chemical transport data. Linear regression models of each exposure and TBV and BV5% were adjusted for age, sex, height, weight, cigarette smoking, second-hand tobacco, census tract median housing value, cohort (Offspring or 3rd Generation), and date of CT scan.Results: Mean (standard deviation, SD) TBV was 143.1 (31.0) mL and mean (SD) BV5% was 39.3 (4.1) %. Median (interquartile range, IQR) 5-year average exposure was 9.9 (1.3) µg/m3 for PM2.5 and 0.44 (0.14) µg/m3 for EC. In fully adjusted models, living close to a major road, and the 5-year average of PM2.5 and EC were not associated with TBV or BV5%. Conclusions: We did not observe any associations between exposure to traffic- and combustion-related pollution during adulthood and pulmonary vascular morphology on CT at contemporary pollution levels in the Northeastern United States.

Impaired microcirculation after subarachnoid hemorrhage in an in vivo animal model

The influence of aneurysmal subarachnoid hemorrhage (SAH) on brain microcirculation has not yet been systematically investigated. We established an animal model to examine (1) the brain surface microcirculation (2) the influences of cerebrospinal fluid (CSF) from aneurysmal SAH on the brain surface microcirculation. A rat SAH model was induced by injection of autologous arterial blood into the cisterna magnum, and the brain surface microcirculation was evaluated by a capillary videoscope with craniotomy at the fronto-parietal region. CSF from SAH rats and SAH patients was applied on the brain surface of naïve rats to assess the resulting microcirculatory changes. In the SAH rats, diffuse constriction of cortical arterioles within 24 hours of SAH was observed. Similar patterns of microcirculation impairment were induced on normal rat brain surfaces via application of CSF from SAH rats and SAH patients. Furthermore, the proportion of subjects with arteriolar vasoconstriction was significantly higher in the group of SAH patients with delayed ischemic neurological deficits (DIND) than in those without DIND (p < 0.001). This study demonstrated impaired microcirculation on brain surface arterioles in a rat model of SAH. CSF from SAH rats and patients was responsible for impairment of brain surface microcirculation.

Abstract 034: Resilience of Isolated, Perfused Cerebral Penetrating Microarterioles to Angiotensin II (Ang II) Contractions Depends on Local Generation of Prostaglandin D 2 (PGD 2 )

The brain depends on a continuous supply of blood for its oxygenation, whereas the kidney is over-perfused for its metabolic needs in order to provide sufficient plasma to form a glomerular filtrate. Thus, the brain requires resilience to vasoconstriction to prevent ischemia and vascular cognitive impairment, but the mechanisms are unclear. Methods and Results: Single penetrating cerebral microarterioles (CMAs, 12-18μM) were dissected from the frontal cortex and single renal afferent arterioles (RAAs, 8-12μM) from the kidney cortex to investigate the hypothesis that CMAs deploy unique mechanisms to provide resilience to Ang II vasoconstriction. Individual arteriolar genes were assessed by RNAseq or RT-PCR of endothelial cells (ECs). The mRNA for lipocalin type PGD 2 synthase (LPGDS) and the PGD 1 receptor (PD1R) were &gt; 3,000-fold higher in CMAs than RAAs whereas RAAs expressed 3-fold more mRNA for thromboxane A 2 synthase. Both microarterioles had similar expression of AT1Rs. Endothelial cells cultured from these vessels had similar patterns of gene expression. Single isolated perfused RAAs contracted strongly with Ang II (at 10 -6 mol·l -1 ; -47 ± 2%; P&lt;0.005) whereas CMAs were totally resistant to Ang II (0.1 ± 0.1%; NS), yet both contracted similarly to endothelin I or perfusion pressure (n = 6 per group). However, CMAs from COX 1 -/- (vs +/+) mice did contract with Ang II (-15 ± 2 vs 0.1 ± 0.1%; P&lt;0.01) and contracted with Ang II after incubation with parecoxib (vs vehicle) to block COX2 (-7 ± 3 vs 0.1 ± 0.1%; P&lt;0.01) or after dual blockade of COX1 + 2 (-20 ± 2%; P&lt;0.01) or after incubation with AT-56 (vs vehicle) to block LPGDS (-20 ± 3 vs 0.1 ± 0.1%; P&lt;0.01). During LPGDS blockade, incubation of CMAs with BW245c (stable PD1R agonist) reduced Ang II contraction &gt; 65% (-8 ± 2%; P&lt;0.01). In contrast, COX blockade reduced Ang II contractions of RAAs, indicating opposing effects of PGs on cerebral and renal vessels. Measurements of cerebral and renal blood flow and MAP in anesthetized mice confirmed selective renal vasoconstriction with Ang II, yet selective cerebral vasodilation with BW245c. Perspective: Resilience against Ang II vasoconstriction in cerebral arterioles depends on the generation of PGD 2 and could be a therapeutic target for vascular dementia and stroke.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Neuropeptide Y1 and alpha-1 adrenergic receptor-mediated decreases in functional vasodilation in gluteus maximus microvascular networks of prediabetic mice.

Prediabetes is associated with impaired contraction‐evoked dilation of skeletal muscle arterioles, which may be due to increased sympathetic activity accompanying this early stage of diabetes disease. Herein, we sought to determine whether blunted contraction‐evoked vasodilation resulted from enhanced sympathetic neuropeptide Y1 receptor (Y1R) and alpha‐1 adrenergic receptor (α1R) activation. Using intravital video microscopy, second‐, third‐, and fourth‐order (2A, 3A, and 4A) arteriolar diameters were measured before and following electrical field stimulation of the gluteus maximus muscle (GM) in prediabetic (PD, Pound Mouse) and control (CTRL, c57bl6, CTRL) mice. Baseline diameter was similar between groups; however, single tetanic contraction (100 Hz; 400 and 800 msec) and sustained rhythmic contraction (2 and 8 Hz, 30 sec) evoked rapid onset vasodilation and steady‐state vasodilatory responses that were blunted by 50% or greater in PD versus CTRL. Following Y1R and α1R blockade with sympathetic antagonists BIBP3226 and prazosin, contraction‐evoked arteriolar dilation in PD was restored to levels observed in CTRL. Furthermore, arteriolar vasoconstrictor responses to NPY (10−13–10−8 mol/L) and PE (10−9–10−5 mol/L) were greater in PD versus CTRL at higher concentrations, especially at 3A and 4A. These findings suggest that contraction‐evoked vasodilation in PD is blunted by Y1R and α1R receptor activation throughout skeletal muscle arteriolar networks.

Renal Hemodynamic Function at the Extremes of T1D Duration-Adolescents vs. Adults with T1D for =50 Years

Our aim was to better understand kidney function changes and RAAS activation over the natural history of T1D in patients with a wide range of T1D duration. GFRinulin and ERPFPAH were measured during a euglycemic clamp before and after an angiotensin II infusion (ANGII, 3 ng·kg-1·min-1-a measure of RAAS activation) in patients with T1D: 28 adolescents (age 16.2±2.0 years), 54 young adults (25.4±5.6 years) and 66 adults with ≥50 years of T1D (65.7±7.5 years) from the Canadian Study of Longevity. Gomez’s equations were used to estimate afferent (RA) and efferent (RE) arteriolar resistances and glomerular hydrostatic pressure (PGLO). In a step-wise fashion, GFRinulin, ERPFPAH, PGLO decreased, renal vascular resistance (RVR) and RA increased in adolescents vs. young adults vs. adults with ≥50 years of T1D (Figure 1). RE was similar in adolescents vs. young adults, but was higher in patients with ≥50 years of T1D. ANGII resulted in blunted renal hemodynamic responses in patients with ≥50 years of T1D (RVR increase of 0.033±0.016 vs. 0.049±0.019mmHg/L/min in adolescents, p=0.0006) suggesting a state of enhanced RAAS activation. With longer duration of disease, patients with T1D have evidence of renal vasoconstriction, leading to lower GFRinulin, ERPFPAH, elevated RVR and RAAS activation. Further work is required to understand the potential role of non-RAAS, pharmacologic agents that target RA in patients with early T1D. Disclosure Y. Lytvyn: None. P. Bjornstad: Consultant; Self; Boehringer Ingelheim GmbH. J.A. Lovshin: Other Relationship; Self; AstraZeneca. Consultant; Self; Novo Nordisk Inc.. Research Support; Self; Sanofi, Merck Sharp &amp; Dohme Corp.. Other Relationship; Self; Novo Nordisk Inc. G. Boulet: Advisory Panel; Self; Medtronic, Sanofi, Novo Nordisk Inc.. Other Relationship; Self; Janssen Global Services, LLC., Abbott. M. Farooqi: None. V.S. Lai: None. J.M. Tse: None. L. Cham: None. L. Lovblom: None. A. Weisman: None. H.A. Keenan: Research Support; Self; Sanofi. Employee; Self; Sanofi Genzyme. M.H. Brent: Research Support; Self; Novartis Canada. Advisory Panel; Self; Novartis Canada. Research Support; Self; Bayer Canada. Advisory Panel; Self; Bayer Canada, Allergan Canada. Research Support; Self; Roche Canada. N. Paul: None. V. Bril: Consultant; Self; Alexion Pharmaceuticals, Inc.. Research Support; Self; CSL Behring, Grifols. Advisory Panel; Self; CSL Behring. Consultant; Self; Grifols. Research Support; Self; Shire. Advisory Panel; Self; Pfizer Inc. A. Advani: Research Support; Self; AstraZeneca, Boehringer Ingelheim GmbH. Other Relationship; Self; Boehringer Ingelheim GmbH. E.B. Sochett: None. B.A. Perkins: Advisory Panel; Self; Boehringer Ingelheim GmbH. Research Support; Self; Boehringer Ingelheim GmbH, Novo Nordisk Inc.. Advisory Panel; Self; Novo Nordisk Inc., Abbott. Speaker's Bureau; Self; Abbott, Janssen Pharmaceuticals, Inc.. Advisory Panel; Self; Insulet Corporation. Speaker's Bureau; Self; Insulet Corporation, Dexcom, Inc. D. Cherney: Consultant; Self; AbbVie Inc.. Other Relationship; Self; AstraZeneca, Boehringer Ingelheim GmbH, Eli Lilly and Company. Consultant; Self; Sanofi. Other Relationship; Self; Merck &amp; Co., Inc.. Consultant; Self; Mitsubishi Tanabe Pharma Corporation. Other Relationship; Self; Janssen Pharmaceuticals, Inc..

The mechanism of botulinum A on Raynaud syndrome.

BackgroundBotulinum neurotoxin type A (BoNT/A) is emerging as a treatment modality for Raynaud’s phenomenon (RP). However, the mechanism of the role of BoNT/A in antagonizing the constriction of arteriola in RP remains unclear.Materials and methodsWe tested the constriction of arteriole diameter and the distribution of adrenergic receptors on the rat cremaster modle. Moreover, we measured the secretion of norepinephrine (NE), protein level changes and related receptors on cultured rat superior cervical ganglia neurons(SCGNs), a model of sympathetic neuron.ResultsBased on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/A, prazosin, and BQ123 treatment can result in significant inhibition of arteriole vasoconstriction with the same electrical stimulation. The inhibition effect of prazosin was equivalent to BoNT/A, while BQ123 has a synergistic effect with BoNT/A. After treating SCGNs using BoNT/A for 30 min, the decrease in fluorescence intensity of FM1-43 slowed down which was correlated with the doses of BoNT/A. Furthermore, release of NE in the supernatant was significantly decreased as measured by enzyme-linked immunosorbent assay, 24 h after a high dose of BoNT/A (25 µ/mL). Cleaved-SNAP-25 was detected by Western blotting 24 h following BoNT/A (50 µ/mL) treatment. Moreover, receptor SV2C, GM1, and FGFR3 were detected on sympathetic neurons, similarly to cholinergic neurons.ConclusionOur study showed that BoNT/A could significantly inhibit electrical stimulation-induced arteriole vasoconstriction through the sympathetic pathway. The mechanism was similar to the cholinergic one, in which the vesicle release of sympathetic neurons could be inhibited by cleavage of SNAP-25. The end result was blocked vesicle fusion with the presynaptic membrane after BoNT/A treatment, inhibiting the release of the NE.

Rho Kinase Inhibitors Are Involved in L‐Type Voltage‐dependent Calcium Channel Pathway in Vascular Smooth Muscle Cells

Voltage‐dependent L‐type calcium channels (L‐VDCC) and the rho/rho kinase pathway are two predominate intracellular pathways that regulate renal microvascular tone and reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests these pathways are convergent. We hypothesized that the rho kinase inhibitors influenced L‐VDCC signaling. The effects of rho kinase inhibitors Y‐27632 or RKI‐1447 on KCl‐depolarization were assessed in afferent arterioles using the in vitro blood‐perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30, 60, and 90mM) led to concentration‐dependent vasoconstriction of afferent arterioles reducing the diameter to 95±6, 56±4 and 42±3% of the baseline, respectively (p&lt;0.05). Addition of Y‐27632 (1, 5 and 10 μM) or RKI‐1447 (0.1, 1 and 10 μM) significantly increased starting diameter by 16–65% (n=6/each, p&lt;0.05). The KCl‐induced vasoconstriction with 1 μM Y‐27632 or 0.1 and 1 μM RKI‐1447 was similar to control, but markedly attenuated with 5 and 10 μM Y‐27632, and 10 μM RKI‐1447 (p&lt;0.05 vs KCl alone). The afferent arteriole diameter averaged 99±2, 83±2, 71±4%, and 98±0.8, 89±5, 81±6% of the baseline for 5 and 10 μM Y‐27632, and 97±2, 89±3 and 88±4% of the baseline for 10 μM RKI‐1447, respectively (n=6/each, p&lt;0.05 vs KCl alone). Changes in intracellular calcium concentration [Ca2+]i were estimated by fura‐2‐AM fluorescence (ratio F340/380) during KCl‐depolarization in cultured A7r5 cells (n=46–118/each group) and in freshly isolated renal microvascular smooth muscle cells (RMVSMCs, n=32–118/each group). Administration of 90 mM KCl increased fura‐2 fluorescence markedly in both cell types. The KCl‐induced increase of the F340/F380 ratio was unaltered by 0.1 or 0.5 μM Y‐27632 but was significantly attenuated by Y‐27632 between 1–10 μM by 50–56% (p&lt;0.05 vs KCl alone) in A7r5 cells but only at 10 μM in RMVSMCs by 42% (p&lt;0.05 vs KCl alone). RKI‐1447 however, significantly attenuated KCl‐mediated elevation of [Ca2+]i in both cell types regardless of the concentrations applied (0.1–10 μM, p&lt;0.05), indicating that Y‐27632 and RKI‐1447 decrease L‐VDCC function. These studies indicate that the rho kinase inhibitors, Y‐27632 and RKI‐1447 partially inhibit L‐VDCC function and participate in L‐VDCC signaling.Support or Funding InformationThis study was supported by a Grant‐in‐Aid (15GRNT25240015) from the American Heart Association Great Southeast Affiliate and a grant from NIH (DK106500) to ZG, and by grants from NIH (DK044628, HL074167, HL098135 and HL095499) to EWI.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Phenylephrine and Norepinephrine on the Diameter of the Epineurial Vessels Supplying the Sciatic Nerve

BackgroundPhenylephrine (PE) and norepinephrine (NE) are known vasoconstrictors; however, little is known about their role in blood flow regulation at the sciatic nerve. The purpose of this study was to determine the effects of PE and NE on the epineurial vessels supplying the sciatic nerve.MethodsAdult male Sprague‐Dawley rats (323.1 ± 5.2 g) were anaesthetized using urethane, α‐chloralose, then were tracheotomized, catheterized, and had the sciatic nerve exposed. Alexa Fluor 488 Isolectin GS‐IB4 conjugate [178.6 μm/ml] was used to label the endothelial cells of the vessel wall allowing for visualization of the epineurial vessels using intravital video microscopy. PE (10−5 M) or NE (10−5 M) was topically applied to the sciatic region for 4 minutes, separately. Images were obtained at baseline, immediately prior to drug application, and every minute for 4 minutes during application. Data are expressed at the time point that maximum change in diameter occurred. Data are expressed as mean ± standard error.ResultsApplication of PE (n=11) resulted in a vasoconstriction (baseline: 22.0 ± 2.1 μm, PE: 19.3 ± 2.0 μm; P&lt;0.05). Similarly, topical application of NE (n=8) induced a vasoconstriction (baseline: 31.2 ± 4.5 μm, NE: 26.5 ± 4.6 μm; P&lt;0.05). Mean arteriolar pressure was unaffected during either PE (baseline: 99.1 ± 5.6 mmHg, PE: 97.4 ± 5.4 mmHg) or NE (baseline: 104.0 ± 4.5 mmHg, NE: 107.3 ± 7.7 mmHg) application.ConclusionBoth PE and NE caused vasoconstriction at the sciatic nerve, which is consistent with previous studies in other tissues observing vasoconstriction through activation of α‐1 adrenergic receptors. As PE and NE are both α‐1 receptor agonists, these findings suggest that α‐1 receptors may be involved in blood flow regulation at the peripheral nerves.Support or Funding InformationThis study was supported by the SEED Research Grant from The University of Western Ontario.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscarinic Receptor‐Mediated Regulation of Epineurial Blood Vessels of the Sciatic Nerve

ObjectiveThe objective of this study was to determine the role of muscarinic receptors on the diameter of the epineurial vessels of the sciatic nerve in situ.MethodsAn anesthetized rat model (318.4±7.1 g) was used to expose the sciatic nerve and directly visualize its microvasculature using intravital video microscopy. Rats were tracheotomized and catheters were inserted into the jugular vein for supplemental anesthesia, and the carotid artery for measures of mean arteriolar pressure. Alexa Fluor 488 Isolectin GS‐IB4 conjugate (178.6 μm/ml) was topically applied for 10 minutes to the exposed sciatic nerve to stain the endothelial cells of the epineurial vessels of the nerve. Following a 30‐min equilibration period, acetylcholine (10−4 M, n=8) or atropine (10−5 M, n=6) was topically applied for 4 and 5 minutes, respectively. Images were captured at baseline (immediately prior to drug application) and every minute during the application period. The images were analyzed offline using ImageJ to measure the diameter of the vessels at each time point. All data are expressed as mean ± standard error.ResultsAcetylcholine caused a vasodilation of the epineurial vessels (baseline: 22.9±2.4 μm, acetylcholine: 29.2±2.9 μm, p&lt;0.01). There was no difference in mean arteriolar pressure from baseline (103.8±1.5 mmHg) to maximum vasodilation (102.3±2.7 mmHg). Atropine caused a vasoconstriction of the epineurial vessels (baseline: 26.7±3.2 μm, atropine: 15.5±3.6 μm, p&lt;0.05). There was no difference in mean arteriolar pressure from baseline (104.0±1.9 mmHg) to maximum vasoconstriction (105.2±2.2 mmHg).ConclusionApplication of acetylcholine, a muscarinic receptor agonist, resulted in a vasodilation while application of atropine, a muscarinic receptor antagonist, caused vasoconstriction. This finding suggests that muscarinic receptors are involved in blood flow regulation of peripheral nerves. Further, since atropine constricted the vessels at baseline conditions, muscarinic receptors may be involved in maintaining baseline diameter.Support or Funding InformationThis study was supported by the SEED Research Grant from The University of Western Ontario.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lack of contribution of nitric oxide synthase to cholinergic vasodilation in murine renal afferent arterioles.

We have previously reported significant increases in neuronal nitric oxide synthase (NOS) immunostaining in renal arterioles of angiotensin type 1A receptor (AT1A) knockout mice, and in arterioles and macula densa cells of AT1A/AT1B knockout mice. The contribution of nitric oxide derived from endothelial and macula densa cells in the maintenance of afferent arteriolar tone and acetylcholine-induced vasodilation was functionally determined in kidneys of wild-type, AT1A, and AT1A/AT1B knockout mice. Acetylcholine-induced changes in arteriolar diameters of in vitro blood-perfused juxtamedullary nephrons were measured during control conditions, in the presence of the nonspecific NOS inhibitor, Nω-nitro-l-arginine methyl ester (NLA), or the highly selective neuronal NOS inhibitor, N5-(1-imino-3-butenyl)-l-ornithine (VNIO). Acetylcholine (0.1 mM) produced a significant vasoconstriction in afferent arterioles of AT1A/AT1B mice (-10.9 ± 5.1%) and no changes in afferent arteriolar diameters of AT1A knockout mice. NLA (0.01-1 mM) or VNIO (0.01-1 μM) induced significant dose-dependent vasoconstrictions (-19.8 ± 4.0% 1 mM NLA; -7.8 ± 3.5% 1 μM VNIO) in afferent arterioles of kidneys of wild-type mice. VNIO had no effect on afferent arteriole diameters of AT1A knockout or AT1A/AT1B knockout mice, suggesting nonfunctional neuronal nitric oxide synthase. These data indicate that acetylcholine produces a significant renal afferent arteriole vasodilation independently of nitric oxide synthases in wild-type mice. AT1A receptors are essential for the manifestation of renal afferent arteriole responses to neuronal nitric oxide synthase-mediated nitric oxide release.

Targeting microthrombosis and neuroinflammation with vepoloxamer for therapeutic neuroprotection after traumatic brain injury.

Targeting microthrombosis and neuroinflammation with vepoloxamer for therapeutic neuroprotection after traumatic brain injury.