Anti-IL-17A treatment reduces serum inflammatory, angiogenic and tissue remodeling biomarkers accompanied by less synovial high endothelial venules in peripheral spondyloarthritis.

Spondyloarthritis (SpA) is characterized by inflammation and new bone formation. The exact pathophysiology underlying these processes remains elusive. We propose that the extensive neoangiogenesis in SpA could play a role both in sustaining/enhancing inflammation and in new bone formation. While ample data is available on effects of anti-TNF on angiogenesis, effects of IL-17A blockade on serum markers are largely unknown. We aimed to assess the impact of secukinumab (anti-IL-17A) on synovial neoangiogenesis in peripheral SpA, and how this related to changes in inflammatory and tissue remodeling biomarkers. Serum samples from 20 active peripheral SpA patients included in a 12 week open-label trial with secukinumab were analyzed for several markers of angiogenesis and tissue remodeling. Synovial biopsies taken before and after treatment were stained for vascular markers. Serum levels of MMP-3, osteopontin, IL-6 (all P < 0.001), IL-31, S100A8, S100A9, Vascular Endothelial Growth Factor A (VEGF-A), IL-33, TNF-α (all P < 0.05) decreased significantly upon anti-IL17A treatment. Secukinumab treatment resulted in a decrease in the number of synovial high endothelial venules and lymphoid aggregate score. These results indicate that anti-IL-17A not only diminishes inflammation, but also impacts angiogenesis and tissue remodeling/new bone formation. This may have important implications for disease progression and/or structural damage.

Bam32/DAPP1-Dependent Neutrophil Reactive Oxygen Species in WKYMVm-Induced Microvascular Hyperpermeability.

B cell adaptor molecule of 32 kDa (Bam32), known as dual adapter for phosphotyrosine and 3-phosphoinositides 1 (DAPP1), has been implicated in regulating lymphocyte proliferation and recruitment during inflammation. However, its role in neutrophils during inflammation remains unknown. Using intravital microscopy, we examined the role of Bam32 in formyl peptide receptor agonist WKYMVm-induced permeability changes in post-capillary venules and assessed simultaneously neutrophil adhesion and emigration in cremaster muscles of Bam32-deficient (Bam32-/-) and wild-type (WT) control mice. We observed significantly reduced WKYMVm-induced microvascular hyperpermeability accompanied by markedly decreased neutrophil emigration in Bam32-/- mice. The Bam32-specific decrease in WKYMVm-induced hyperpermeability was neutrophil-dependent as this was verified in bone marrow transplanted chimeric mice. We discovered that Bam32 was critically required for WKYMVm-induced intracellular and extracellular production of reactive oxygen species (ROS) in neutrophils. Pharmacological scavenging of ROS eliminated the differences in WKYMVm-induced hyperpermeability between Bam32-/- and WT mice. Deficiency of Bam32 decreased WKYMVm-induced ERK1/2 but not p38 or JNK phosphorylation in neutrophils. Inhibition of ERK1/2 signaling cascade suppressed WKYMVm-induced ROS generation in WT neutrophils and microvascular hyperpermeability in WT mice. In conclusion, our study reveals that Bam32-dependent, ERK1/2-involving ROS generation in neutrophils is critical in WKYMVm-induced microvascular hyperpermeability during neutrophil recruitment.

The capability of detecting small vessels beyond the conventional MRI sensitivity using iron-based contrast agent enhanced susceptibility weighted imaging.

Imaging brain microvasculature is important in cerebrovascular diseases. However, there is still a lack of non-invasive, non-radiation, and whole-body imaging techniques to investigate them. The aim of this study is to develop an ultra-small superparamagnetic iron oxide (USPIO) enhanced susceptibility weighted imaging (SWI) method for imaging micro-vasculature in both animal (~10 µm in rat) and human brain. We hypothesized that the USPIO-SWI technique could improve the detection sensitivity of the diameter of small subpixel vessels 10-fold compared with conventional MRI methods. Computer simulations were first performed with a double-cylinder digital model to investigate the theoretical basis for this hypothesis. The theoretical results were verified using in vitro phantom studies and in vivo rat MRI studies (n = 6) with corresponding ex vivo histological examinations. Additionally, in vivo human studies (n = 3) were carried out to demonstrate the translational power of the USPIO-SWI method. By directly comparing the small vessel diameters of an in vivo rat using USPIO-SWI with the small vessel diameters of the corresponding histological slide using laser scanning confocal microscopy, 13.3-fold and 19.9-fold increases in SWI apparent diameter were obtained with 5.6 mg Fe/kg and 16.8 mg Fe/kg ferumoxytol, respectively. The USPIO-SWI method exhibited its excellent ability to detect small vessels down to about 10 µm diameter in rat brain. The in vivo human study unveiled hidden arterioles and venules and demonstrated its potential in clinical practice. Theoretical modeling simulations and in vitro phantom studies also confirmed a more than 10-fold increase in the USPIO-SWI apparent diameter compared with the actual small vessel diameter size. It is feasible to use SWI blooming effects induced by USPIO to detect small vessels (down to 10 µm in diameter for rat brain), well beyond the spatial resolution limit of conventional MRI methods. The USPIO-SWI method demonstrates higher potential in cerebrovascular disease investigations.

Activation of Notch signaling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway.

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.

The mediating role of the venules between smoking and ischemic stroke.

A potential mechanism by which smoking affects ischemic stroke is through wider venules, but this mediating role of wider venules has never been quantified. Here, we aimed to estimate to what extent the effect of smoking on ischemic stroke is possibly mediated by the venules via the recently developed four-way effect decomposition. This study was part of a population-based study including 9109 stroke-free persons participated in the study in 1990, 2004, or 2006 (mean age: 63.7 years; 58% women). Smoking behavior (smoking versus non-smoking) was identified by interview. Retinal venular calibers were measured semi-automatically on retinal photographs. Incident strokes were assessed until January 2016. A regression-based approach was used with venular calibers as mediator to decompose the total effect of smoking compared to non-smoking into four components: controlled direct effect (neither mediation nor interaction), pure indirect effect (mediation only), reference interaction effect (interaction only) and mediated interaction effect (both mediation and interaction). During a mean follow-up of 12.5 years, 665 persons suffered an ischemic stroke. Smoking increased the risk of developing ischemic stroke compared to non-smoking with an excess risk of 0.41 (95% confidence interval 0.10; 0.67). With retinal venules as a potential mediator, the excess relative risk could be decomposed into 77% controlled direct effect, 4% mediation only, 4% interaction only, and 15% mediated interaction. To conclude, in the pathophysiology of ischemic stroke, the effect of smoking on ischemic stroke may partly explained by changes in the venules, where there is both pure mediation and mediated interaction.

Vascular targeting of LIGHT normalizes blood vessels in primary brain cancer and induces intratumoural high endothelial venules.

High-grade brain cancer such as glioblastoma (GBM) remains an incurable disease. A common feature of GBM is the angiogenic vasculature, which can be targeted with selected peptides for payload delivery. We assessed the ability of micelle-tagged, vascular homing peptides RGR, CGKRK and NGR to specifically bind to blood vessels in syngeneic orthotopic GBM models. By using the peptide CGKRK to deliver the tumour necrosis factor (TNF) superfamily member LIGHT (also known as TNF superfamily member 14; TNFSF14) to angiogenic tumour vessels, we have generated a reagent that normalizes the brain cancer vasculature by inducing pericyte contractility and re-establishing endothelial barrier integrity. LIGHT-mediated vascular remodelling also activates endothelia and induces intratumoural high endothelial venules (HEVs), which are specialized blood vessels for lymphocyte infiltration. Combining CGKRK-LIGHT with anti-vascular endothelial growth factor and checkpoint blockade amplified HEV frequency and T-cell accumulation in GBM, which is often sparsely infiltrated by immune effector cells, and reduced tumour burden. Furthermore, CGKRK and RGR peptides strongly bound to blood vessels in freshly resected human GBM, demonstrating shared peptide-binding activities in mouse and human primary brain tumour vessels. Thus, peptide-mediated LIGHT targeting is a highly translatable approach in primary brain cancer to reduce vascular leakiness and enhance immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1ß, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (Selp(KI)). Selp(KI) (/) (KI) mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of Selp(KI) (/) (KI) mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in Selp(KI) (/-) mice. Higher basal P-selectin in Selp(KI) (/) (KI) mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.

Posttreatment with Ma-Xing-Shi-Gan-Tang, a Chinese medicine formula, ameliorates lipopolysaccharide-induced lung microvessel hyperpermeability and inflammatory reaction in rat.

OBJECTIVE: The aim of present study was to investigate the efficacy of MXSGT, a traditional Chinese medicine formula used for treatment of respiratory system diseases, in the LPS-induced rat ALI particularly with a focus on its effect on lung microvascular hyperpermeability and inflammatory reaction. METHODS: Male Sprague-Dawley rats were injected with LPS (7.5 mg/kg, 1.5 mg/mL) intraperitoneally. MXSGT (0.52 g or 2.61 g/kg) was given by gavage six hours after LPS injection. RESULTS: LPS stimulation resulted in a reduced survival rate, deteriorated vital signs, an increase in the number of leukocytes adhering to lung venules, the albumin leakage, the activity of MPO in lung tissues, the production of pro-inflammatory cytokines and lung perivascular edema. After LPS stimulation, western blot analysis revealed an increase in the expression of ICAM-1 and toll-like receptor 4, a decrease in tight junction proteins and an activation of cav-1, Src, and NF-κB. All the LPS-induced alterations were significantly attenuated by posttreatment with MXSGT. CONCLUSIONS: This study demonstrated MXSGT as a potential strategy for lung microvascular hyperpermeability and inflammatory reaction in ALI, and suggested that the beneficial role of MXSGT was correlated with toll-like receptor 4, Src, and NF-κB.

Quinic acid derivative KZ-41 exhibits radiomitigating activity in preclinical models of radiation injury.

Acute radiation syndrome is induced when a significant portion of the body receives high-dose, as well as high-dose rate, radiation. We have previously identified a quinic acid-based derivative, KZ-41, that protects from radiation injury. Further preclinical efficacy studies were conducted to determine the radiomitigating activity of KZ-41. C57BL/6 mice received total body irradiation (TBI-LD80/30, ¹³7Cs; ∼2 min) followed by either normal saline or KZ-41 (100 mg/kg sc ∼26 h post-TBI). KZ-41 increased 30-day survival by approximately 45% compared with vehicle controls (P < 0.05). To further investigate the potential radiomodulating mechanisms of KZ-41, we developed a combined radiation and vascular injury model. C57BL/6 mice surgically fixed with dorsal windows for dermal vasculature imaging received either sham or TBI (¹³7Cs; 6 Gray). Postcapillary venule injury was induced (24, 48, 72, and 96 h post-TBI) followed by imaging at 5 min and 24 h to assess clot formation and blood flow. Impairment in flow (P < 0.05) and clot formation (P < 0.05) were observed as early as 48 and 72 h, respectively. Thus, vascular injury 72 h post-TBI was used to evaluate intervention (KZ-41; 100 mg/kg i.p. at 12, 36, and 60 h post-TBI) on radiation-induced changes in both flow and clot formation. KZ-41, although not improving flow, increased clot formation (P < 0.05). Platelet counts were lower in both irradiated groups compared with sham controls (P < 0.05). In summary, KZ-41 exerts radiomitigating activity in lethally irradiated mice. Imaging results suggest KZ-41 exerts radiomitigating activity through mechanisms involving promotion of initial clot formation and vascular flow restoration. The imaging model described herein is useful for further examination of radiation-induced vascular injury repair mechanisms.

Ginsenoside Rb1 ameliorates lipopolysaccharide-induced albumin leakage from rat mesenteric venules by intervening in both trans- and paracellular pathway.

Lipopolysaccharide (LPS) is one of the common pathogens that causes mesentery hyperpermeability- and intestinal edema-related diseases. This study evaluated whether ginsenoside Rb1 (Rb1), an ingredient of a Chinese medicine Panax ginseng, has beneficial effects on mesentery microvascular hyperpermeability induced by LPS and the underlying mechanisms. Male Wistar rats were continuously infused with LPS (5 mg · kg(-1) · h(-1)) via the left jugular vein for 90 min. In some rats, Rb1 (5 mg · kg(-1) · h(-1)) was administrated through the left jugular vein 30 min after LPS infusion. The dynamics of fluorescein isothiocynate-labeled albumin leakage from mesentery venules was assessed by intravital microscopy. Intestinal tissue edema was evaluated by hematoxylin and eosin staining. The number of caveolae in endothelial cells of microvessels was examined by electron microscopy. Confocal microscopy and Western blotting were applied to detect caveolin-1 (Cav-1) expression and phosphorylation, junction-related proteins, and concerning signaling proteins in intestinal tissues and human umbilical vein endothelial cells. LPS infusion evoked an increased albumin leakage from mesentery venules that was significantly ameliorated by Rb1 posttreatment. Mortality and intestinal edema around microvessels were also reduced by Rb1. Rb1 decreased caveolae number in endothelial cells of microvessels. Cav-1 expression and phosphorylation, VE-Cadherin phosphorylation, ZO-1 degradation, nuclear factor-κB (NF-κB) activation, and Src kinase phosphorylation were inhibited by Rb1. Rb1 ameliorated microvascular hyperpermeability after the onset of endotoxemia and improved intestinal edema through inhibiting caveolae formation and junction disruption, which was correlated to suppression of NF-κB and Src activation.

[Investigation of the venodilatory effect of vascular endothelial growth factor (VEGF) in rat gingiva]. / A vaszkularis endoteliális növekedési faktor (VEGF) venulákra kifejtett hatásának vizsgálata patkány fogínyben.

VEGF induces proliferation of endothelial cells, stimulates angiogenesis, and increases vascular permeability in many organs. Nevertheless, we have only limited information about its role on gingival hemodynamics, especially in venules. Therefor the aim of this study was to assess the acute circulatory effects of VEGF on rat gingival venules by means of the following protocol. Wister rats (n=63) were devided into five study groups after anesthesia; each animal received 10 microl of experimental solution dripped onto the lower interincisal gingiva. The groups included: 1) saline control (after the experiment, gingiva was excised for VEGF receptor 2 [VEGFR2] immunohistochemistry); 2) VEGF (0.1, 1, 10, or 50 microg/ml); 3) VEGF2 receptor antagonist 5-((7-benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methyl-phenol-hydrochloride (ZM323881; 20 microg/ml); 4) ZM323881 (20 microg/ml) followed by VEGF application (50 microg/ml after 15 minutes); and 5) VEGF (10 microg/ml), these rats were premedicated with nitric oxide (NO) synthase blocker (NG-nitro-L-arginine-methyl-ester [L-NAME]; 1 mg/ml in drinking water) for 1 week before the experiment. Changes in gingival superficial venule diameter were measured by vital microscopy prior to and 1, 5, 15, 30, and 60 minutes after the administration of the experimental solutions. According to our findings, VEGF dose-dependently increased the venular diameter compared to saline. ZM323881 alone did not cause any alteration. Premedication with ZM323881 or L-NAME decreased the dilatory effects of VEGF. Occassionally moderate VEGFR2 immunohistochemical labeling was observed in the wall components of the venules. Concluding our results we can say, that there is no remarkable VEGF production under physiologic circumstances in rat gingiva, but VEGF is able to increase gingival blood flow through the activation of VEGF2 receptors and consequent NO release.

Evaluation of resuscitation fluids on endothelial glycocalyx, venular blood flow, and coagulation function after hemorrhagic shock in rats.

BACKGROUND: Endothelial glycocalyx (EG) plays an essential role in endothelium integrity and may be compromised by hemorrhagic shock. The effects of currently available resuscitation fluids such as Hextend (HEX) or lactated Ringer's solution (LR) on vascular function and coagulation are not well understood. The aim of the present study was to compare the effects of fresh frozen plasma (FFP) with HEX or LR in their ability to repair EG structure, promote volume expansion, increase blood flow, and prevent coagulopathy. METHODS: A total of 121 microvessels from cremaster muscle were studied in 32 anesthetized instrumented rats. After baseline systemic and microvascular measurements, 40% hemorrhage followed by resuscitation was performed, and measurements were repeated. Coagulation was evaluated using ROTEM to assay clot formation time, clotting time, firmness, strength, and lysis. Velocity and "platelet component" of strength were calculated. Fluorescein isothiocyanate or Texas Red bound to Dextrans was injected to estimate EG thickness in vivo. RESULTS: Respiratory rate, blood pH, base excess, and lactate returned to near-baseline levels in all treatments. Hemodilution caused by LR and HEX decreased firmness, prolonged clotting time, and lowered platelet counts. EG thickness in HEX- and LR-treated rats was 50% lower, and plasma syndecan 1 was 50% higher than sham and FFP groups. Blood flow and shear rate were restored in the HEX group. Resuscitation with FFP improved coagulation and blood flow. CONCLUSION: Our findings support the concept of cardiovascular and microvascular stabilization by infused FFP, in which the increase in microvascular perfusion associated with restored EG is essential for an optimal resuscitation strategy.

Preconditioning with soluble guanylate cyclase activation prevents postischemic inflammation and reduces nitrate tolerance in heme oxygenase-1 knockout mice.

Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²âº. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function.

High-dose chlorogenic acid induces inflammation reactions and oxidative stress injury in rats without implication of mast cell degranulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Chlorogenic acid (CA) exits widely in those Chinese herbal injections that have antibacterial and antiphlogistic effects and belongs to the ethnopharmacological family of medicines. Chinese herbal injections containing high levels of CA have been reported to increase the adverse drug reactions, but the mechanism for which is still unclear. In this study, we investigated the mechanism of the CA derived adverse drug reactions. AIM OF THE STUDY: The present study was to explore the potential role of CA in initiating inflammatory reaction and the underlying mechanism. MATERIALS AND METHODS: Male Wistar rats were treated with different dosages of CA for different time period. The variables examined included microcirculation by intravital microscopy, histology of ileum tissue, expression of adhesion molecules CD11b and CD18 on leukocytes by flow cytometry, myeloperoxidase activity and maleic dialdehyde content in ileum tissue by spectrophotometry, activity of superoxide dismutase and catalase in serum by ELISA, and expression of NADPH oxidase subunits by PCR and Western blot. RESULTS: High-dose CA increased the number of adherent leukocytes, generation of peroxides in the venular walls and induced albumin leakage from mesentery venules. High-dose CA induced changes also included an increase in maleic dialdehyde, myeloperoxidase, inflammatory cytokines and NADPH oxidase activities, and a decline in activity of superoxide dismutase and catalase. CONCLUSION: High-dose, but not Low-dose CA induced inflammation reaction, and in this process an imbalance between oxidant and antioxidant mechanism may be involved, providing more information for better understanding the rationale behind the adverse effects of CA.

Microvascular regulatory role and increased expression of vascular endothelial growth factor receptor type 2 in experimental gingivitis.

OBJECTIVE: The aim of the present study was to investigate the possible microvascular regulatory role of vascular endothelial growth factor receptor type 2 (VEGFR2) in experimental gingivitis in rats. BACKGROUND: Our previous results demonstrated that functionally active VEGFR2s are located in the venules of rat gingiva. While there is no remarkable endogenous gingival VEGF production under normal circumstances, exogenous VEGF, via VEGFR2, shows venodilatory effects. We assumed that VEGF plays an important role in vasoregulatory processes (vasodilation, increased permeability, angiogenesis) of gingival inflammation. METHODS: Gingivitis was induced by placing ligatures and composite material around and between the lower incisors of anesthetized Wistar rats next to the gingival margin. Seven days later, VEGFR2 antagonist (ZM323881), was dripped upon the labial gingiva next to the lower incisors. Diameter changes of the selected gingival venules were measured by vital microscopy. Animals with healthy gingiva served as controls. Venule diameter changes were compared to the baseline and to control groups (no ligature). Immunohistochemical and Western blot analysis for VEGFR2 were utilized. RESULTS: After 15, 30 and 60 min of local application of ZM323881, there was a significant venoconstriction in the inflamed gingiva compared to the baseline, while no change was recorded in controls. Endothelium, smooth muscle cells and pericytes of the gingivitis group showed increased VEGFR2 expression. CONCLUSION: Our findings suggest that there is an increased VEGF production in gingivitis, which may play an important role in vasodilation of rat gingival venules.

Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II.

OBJECTIVE: Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX(3)CL1) was explored. METHODS AND RESULTS: Enhanced CX(3)CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 µmol/L Ang-II increased CX(3)CL1 expression, yet neutralization of CX(3)CL1 activity only significantly inhibited Ang-II-induced mononuclear cell-human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-α in Ang-II-induced CX(3)CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-α, and interferon-γ, CX(3)CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. CONCLUSIONS: These results suggest that Ang-II induces functional CX(3)CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX(3)CL1-mononuclear leukocyte CX(3)CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II-associated cardiovascular diseases.

Leukocyte-dependent responses of the microvasculature to chronic angiotensin II exposure.

Angiotensin II (Ang II) contributes to the pathogenesis of hypertension and other cardiovascular diseases. Ang II induces a pro-oxidative, proinflammatory, and prothrombogenic phenotype in vascular endothelial cells. Although the peptide promotes the recruitment of leukocytes and platelets and induces oxidative stress in the microvasculature, it remains unclear whether and how the blood cell recruitment is linked to the production of reactive oxygen species. In this study, we addressed the contributions of Ang II type 1 receptors (AT(1)r) and gp91(phox) to the recruitment of leukocytes and platelets and reactive oxygen species production in venules during chronic (2-week) infusion of Ang II in wild-type (WT) and mutant mice. Intravital video microscopy was used to measure the adhesion and emigration of leukocytes, the adhesion of fluorescently labeled platelets, and dihydrorhodamine oxidation (a measure of oxidative stress) in cremaster muscle postcapillary venules. In WT mice, Ang II infusion induced a time-dependent increase in the adhesion of leukocytes and platelets and enhanced reactive oxygen species production in venules. These changes in blood cell adhesion and reactive oxygen species production were not observed in AT(1)r(-/-) mice, AT(1)r(-/-) bone marrow chimeras (blood cells deficient in AT(1)r), gp91(phox-/-) mice, gp91(phox-/-) chimeras (blood cells or endothelial cells deficient in gp91(phox)), and in WT mice rendered granulocytopenic via intraperitoneal injection of antimouse granulocyte receptor 1 antibody. Thrombocytopenic WT mice (platelets depleted by intraperitoneal injection of rabbit antimouse thrombocyte antiserum) responded similar to WT mice. These findings implicate leukocyte-associated AT(1)r and gp91(phox) in the induction of the pro-oxidative, proinflammatory, and prothrombogenic phenotype assumed by microvessels that is chronically exposed to elevated Ang II.

Retinal vascular changes following intravitreal ranibizumab injections for neovascular AMD over a 1-year period.

PURPOSE: To assess retinal vascular calibre changes in eyes with neovascular age-related macular degeneration (AMD), treated with intravitreal anti-vascular endothelial growth factor agents, over a 1-year period and compare any such changes to untreated fellow eyes. METHODS: Treatment naïve patients with neovascular AMD received three consecutive intravitreal injections of ranibizumab, followed by a pro re nata dosing regimen up to 1 year, with the aim of maintaining a 'fluid-free' macula. Retinal arteriolar and venular calibre was measured from digital fundus photographs at baseline and at three monthly intervals to 1 year, and summarised as central retinal artery equivalent (CRAE) and central retinal venular equivalent (CRVE), respectively. RESULTS: A total of 53 injected eyes and 41 fellow, non-injected eyes were analysed. At baseline, there were no differences in retinal vascular calibre between injected and non-injected eyes (mean CRAE (SD) 144.93 (14.07) vs 145.74 (13.10) µm, P=0.80 and mean CRVE (SD) 216.23 (25.93) vs 219.91 (22.82) µm, P=0.53). Over a 12-month period, retinal venular calibre dilatation occurred in injected eyes (mean CRVE change +5.71 (14.71) µm, P=0.007), with no change in retinal arterioles, +0.69 (14.71) µm, P=0.68. In non-injected eyes, arteriolar narrowing occurred as a whole, mean CRAE change -4.20 (7.00) µm, P=0.001, over 12 months, with a trend for narrowing in venules, -2.16 (11.56) µm, P=0.28. In injected eyes, after controlling for covariates, the changes in CRVE over 12 months mirrored improvements in macular thickness, -0.06 (-0.005, -0.11) µm, P=0.04, and visual acuity, +9.66 (-0.30, +19.32) µm, P=0.06. CONCLUSION: Intravitreal ranibizumab significantly dilated retinal venules after a 1-year period.

Blood flow augmentation by intrinsic venular contraction in vivo.

Venomotion, spontaneous cyclic contractions of venules, was first observed in the bat wing 160 years ago. Of all the functional roles proposed since then, propulsion of blood by venomotion remains the most controversial. Common animal models that require anesthesia and surgery have failed to provide evidence for venular pumping of blood. To determine whether venomotion actively pumps blood in a minimally invasive, unanesthetized animal model, we reintroduced the batwing model. We evaluated the temporal and functional relationship between the venous contraction cycle and blood flow and luminal pressure. Furthermore, we determined the effect of inhibiting venomotion on blood flow. We found that the active venous contractions produced an increase in the blood flow and exhibited temporal vessel diameter-blood velocity and pressure relationships characteristic of a peristaltic pump. The presence of valves, a characteristic of reciprocating pumps, enhances the efficiency of the venular peristaltic pump by preventing retrograde flow. Instead of increasing blood flow by decreasing passive resistance, venular dilation with locally applied sodium nitroprusside decreased blood flow. Taken together, these observations provide evidence for active venular pumping of blood. Although strong venomotion may be unique to bats, venomotion has also been inferred from venous pressure oscillations in other animal models. The conventional paradigm of microvascular pressure and flow regulation assumes venules only act as passive resistors, a proposition that must be reevaluated in the presence of significant venomotion.

Intravenous immunoglobulins modulate neutrophil activation and vascular injury through FcγRIII and SHP-1.

RATIONALE: Intravascular neutrophil recruitment and activation are key pathogenic factors that contribute to vascular injury. Intravenous immunoglobulin (IVIG) has been shown to have a beneficial effect in systemic inflammatory disorders; however, the mechanisms underlying IVIG's inhibitory effect on neutrophil recruitment and activation are not understood. OBJECTIVE: We studied the mechanisms by which IVIG exerts protection from neutrophil-mediated acute vascular injury. METHODS AND RESULTS: We examined neutrophil behavior in response to IVIG in vivo, using real-time intravital microscopy. We found that an antibody that blocks both FcγRIII and its inhibitory receptor counterpart, FcγRIIB, abrogated the inhibitory effect of IVIG on leukocyte recruitment and heterotypic red blood cell (RBC) interactions with adherent leukocytes in wild-type mice. In the context of sickle cell disease, the blockade of both FcγRIIB and III abrogated the protective effect of IVIG on acute vaso-occlusive crisis caused by neutrophil recruitment and activation. Analysis of FcγRIIB- and FcγRIII-deficient mice revealed the predominant expression of FcγRIII on circulating neutrophils. FcγRIII mediated IVIG-triggered inhibition of leukocyte recruitment, circulating RBC capture, and enhanced Mac-1 activity, whereas FcγRIIB was dispensable. In addition, FcγRIII-induced IVIG anti-inflammatory activity in neutrophils was mediated by recruitment of Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1). Indeed, the protective effect of IVIG on leukocyte recruitment and activation was abrogated in SHP-1-mutant mice. CONCLUSIONS: FcγRIII, a classic activating receptor, has an unexpected inhibitory role on neutrophil adhesion and activation via recruitment of SHP-1 in response to IVIG. Our results identify SHP-1 as a therapeutic target in neutrophil-mediated vascular injury.