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Mechanisms of Amelioration of Glucose-Induced Endothelial Dysfunction Following Inhibition of Protein Kinase C In Vivo
Abstract
Inhibition of protein kinase C (PKC) activity has been shown to improve the endothelial dysfunction associated with hyperglycemia and diabetes. The mechanisms by which inhibition of PKC activity ameliorates endothelial dysfunction in diabetes are not well understood. We investigated the relationship between PKC inhibition and leukocyte-endothelium interaction in the microcirculation of the rat mesentery exposed to 25 mmol/l D-glucose for 12 h. D-Glucose significantly increased leukocyte rolling and adherence in mesenteric postcapillary venules. This proinflammatory action of D-glucose was inhibited by superfusion of the mesentery with 30 nmol/l bisindolylmaleimide-I, a potent, selective PKC inhibitor (P < 0.01 vs. glucose alone after 90 min of superfusion). Immunohistochemical localization of the cell adhesion molecules P-selectin and intercellular adhesion molecule (ICAM)-1 on the endothelial cell surface was increased by 25 mmol/l D-glucose (P < 0.001 vs. control tissue from rats injected with saline), which was significantly reduced by bisindolylmaleimide-I (P < 0.001 vs. glucose alone). In addition, we studied adhesion of isolated neutrophils to rat superior mesenteric artery (SMA) vascular segments stimulated with 25 mmol/l D-glucose for 4 h in vitro. Pretreatment of the SMA vascular segments with either superoxide dismutase enzyme (100 units/ml) or bisindolylmaleimide-I (30 nmol/l) equally inhibited the increased neutrophil adherence to SMA endothelium in response to glucose. These data demonstrate that inhibition of PKC activity reduces leukocyte-endothelium interactions by suppressing surface expression of endothelial cell adhesion molecules in response to increased oxidative stress. These results provide a novel mechanism by which inhibition of PKC activity improves endothelial cell function in hyperglycemia and diabetes.
... We may speculate that this improvement in macular perfusion may be secondary to a favorable corticosteroidrelated effect on leukocyte stasis (leukostasis). Leukostasis has been shown to be involved in DR development and progress as it stimulates retinal non-perfusion and vascular leakage [38,39]. Specifically, diabetes has been shown to cause an increased activation of leukocytes with a following rise in the adhesion of these cells to vascular endothelium [38]. ...
... Leukostasis has been shown to be involved in DR development and progress as it stimulates retinal non-perfusion and vascular leakage [38,39]. Specifically, diabetes has been shown to cause an increased activation of leukocytes with a following rise in the adhesion of these cells to vascular endothelium [38]. Therefore, a decline in retinal perfusion and intensification in leukocyte adhesion have been suggested to be two concurrent processes that share common metabolic factors and are strictly inter-related [39]. ...
Introduction:
The aim of this study was to investigate changes in macular perfusion in patients affected by diabetic macular edema (DME) and treated with ILUVIEN® (fluocinolone acetonide intravitreal implant) 0.19 mg using optical coherence tomography angiography (OCTA).
Methods:
This was a retrospective cohort study that included patients aged > 18 years with type 2 non-proliferative diabetic retinopathy (DR) and DME at baseline. All patients were treated with the ILUVIEN® implant. A minimum of two 6 × 6-mm OCTA scans were required to ensure that all cases had a baseline OCTA and an OCTA performed at 4 months of follow-up. Qualitative and quantitative comparisons were performed.
Results:
Ten eyes from ten subjects were included in the analysis. Mean (± standard deviation) age of the study cohort was 57.1 ± 8.3 years. Mean parafoveal perfusion density (PD) at baseline was 64.1 ± 1.8% at baseline, increasing to 66.1 ± 2.9% (p = 0.013) at the 4-month follow-up visit. Mean parafoveal PD at baseline was 64.4 ± 2.1%, increasing to 65.2 ± 2.6% (p = 0.024) after 4 months. In the qualitative assessment, 60 regions (10 areas for each subject) were graded to assess changes in retinal perfusion between the baseline and follow-up visits. This assessment revealed that 24 regions (40.0%) were characterized by a qualitative increase in perfusion after treatment, while 22 (36.7%) and 14 (23.3%) regions were featured by a stability and reduction in retinal perfusion, respectively.
Conclusion:
OCTA analysis detects improvements in macular perfusion after treatment with ILUVIEN®. This improvement in macular perfusion may be associated with corticosteroid-related beneficial effects on leukostasis.
... We speculate that this corticosteroid-related increase in retinal perfusion might be related to positive effects on leukocyte stasis (leukostasis). Leukostasis has been implicated in the pathogenesis and progression of DR as it triggers retinal nonperfusion and vascular leakage [26,27]. In detail, diabetes was demonstrated to activate leukocytes with consequent increased adhesion to vascular endothelium of these cells [26]. ...
... Leukostasis has been implicated in the pathogenesis and progression of DR as it triggers retinal nonperfusion and vascular leakage [26,27]. In detail, diabetes was demonstrated to activate leukocytes with consequent increased adhesion to vascular endothelium of these cells [26]. Since decrease in retinal blood flow and increase in leukocyte adhesion are concomitant and are regulated by several metabolic factors in common, it was thus suggested that these two processes are strictly inter-related [27]. ...
Aim: To investigate late changes in peripheral ischemia in patients affected by diabetic macular edema (DME) and treated with repeated dexamethasone (DEX) intravitreal implants over a 1-year period.
Methods: In this retrospective cohort study, patients older than 18 years of age and with type 2 non-proliferative treatment-naïve diabetic retinopathy (DR) and DME at baseline were included. All patients were treated with two intravitreal DEX implants within 1 year of follow-up. A minimum of two annual ultra-widefield fluorescein angiography (UWF FA) were required to ensure that all cases had a baseline UWF FA (< 2 weeks before first treatment with dexamethasone) and a UWF FA performed at 12 months of follow-up. On baseline and 1-year UWFA images, peripheral retinal ischemia was quantified using the ischemic index (ISI).
Results: Six eyes of 5 patients (2 males, 3 females) met the inclusion criteria and were enrolled in this study. Best corrected visual acuity (BCVA) was 0.34±0.22 LogMAR at baseline and improved to 0.21±0.14 logMAR at the 1-year follow-up visit (p=.050). Mean±SD central macular thickness was 467.6±63.0 μm at baseline and 272.0±14.7 μm at the 1-year follow-up visit (p=.043). Mean±SD ISI was 26.7±14.1 % at baseline and reduced to 12.2±5.0 % at the 1-year follow-up visit (p = 0.012).
Conclusions: Improvement in retinal perfusion is still maintained 1 year after starting treatment with DEX implants. This improvement in retinal perfusion might be related to DEX implant-related positive effects on leukostasis.
... [55] Our findings also correlated with the in vivo studies. High glucose (25 mM) administration in rat mesenteric venules leads to increased immunoreactivity of P-selectin and ICAM-1, increasing inflammatory cell adhesion, [57] while CD44deficient mice showed less monocyte infiltration in type-1 diabetic condition. [58] The monocyte transmigration in our model might correlate with the endothelial dysfunction caused by the treatment introduced in our model. ...
Monocyte recruitment and transmigration are crucial in atherosclerotic plaque development. The multi‐disease complexities aggravate the situation and continue to be a constant concern for understanding atherosclerosis plaque development. Herein, a 3D hydrogel‐based model that integrates disease‐induced microenvironments is sought to be designed, allowing us to explore the early stages of atherosclerosis, specifically examining monocyte fate in multi‐disease complexities. As a proof‐of‐concept study, murine cells are employed to develop the model. The model is constructed with collagen embedded with murine aortic smooth muscle cells and a murine endothelial monolayer lining. The model achieves in vitro disease complexities using external stimuli such as glucose and lipopolysaccharide (LPS). Hyperglycemia exhibits a significant increase in monocyte adhesion but no enhancement in monocyte transmigration and foam cell conversion compared to euglycemia. Chronic infection achieved by LPS stimulation results in a remarkable augment in initial monocyte attachment and a significant increment in monocyte transmigration and foam cells in all concentrations. Moreover, the model exhibits synergistic sensitivity under multi‐disease conditions such as hyperglycemia and infection, enhancing initial monocyte attachment, cell transmigration, and foam cell formation. Additionally, western blot data prove the enhanced levels of inflammatory biomarkers, indicating the model's capability to mimic disease‐induced complexities during early atherosclerosis progression.
... They assessed ISI at baseline and 10 weeks after a single intravitreal injection of DEX, which seemed to significantly improve retinal perfusion, with stability of the clinical picture after 1 year. They hypothesized that their outcome resulted from the favorable effects of corticosteroids on leukostasis, that had been implied in the development of DR for its effects vascular leakage and retinal non-perfusion (81,82). However, these observations obtained from a small cohort of eyes have not found support by further studies. ...
Capillary non-perfusion (CNP) is one of the key hallmarks of diabetic retinopathy (DR), which may develop both in the periphery and at the posterior pole. Our perspectives on CNP have extended with the introduction of optical coherence tomography angiography (OCTA) and ultra-widefield imaging, and the clinical consequences of peripheral and macular CNP have been well characterized. Fluorescein angiography (FA) continues to be the gold standard for detecting and measuring CNP, particularly when ultra-widefield imaging is available. OCTA, on the other hand, is a quicker, non-invasive approach that allows for a three-dimensional examination of CNP and may soon be regarded as an useful alternative to FA. In this review, we provide an updated scenario regarding the characteristics, clinical impact, and management of central and peripheral CNP in DR.
... Während ein Größenrückgang der Plaques bei Cholesterinsenkung nicht regelhaft auftritt und das Vorliegen großer Plaques bei der Vorhersage kardiovaskulärer Ereignisse nur eine geringe Spezifität besitzt, bewirken Statine dennoch eine deutliche Reduktion kardiovaskulärer Ereignisse (29,35,36 (192,193). (194,195 (196). OxLDL entsteht durch vermehrte Oxidation von LDL, wobei diese Reaktion durch die Anwesenheit einer Hyperglykämie und die damit verbundene vermehrte Bildung von ROS begünstigt wird (111). ...
Das Vorliegen eines Diabetes mellitus erhöht die Wahrscheinlichkeit, an Atherosklerose zu erkranken und nachfolgend einen Gefäßverschluss zu entwickeln, erheblich. Während sich der Fokus der Forschung oft auf den Einfluss des Diabetes auf die Atheroskleroseprogression richtet, ist seine Rolle in der Atheroskleroseregression deutlich schlechter erforscht. Der Begriff der Atheroskleroseregression beschreibt Reparaturprozesse, die im Plaque nach Senkung des Plasmacholesterins auftreten. Das Ziel dieser Arbeit bestand darin, speziell den Aspekt der diabetischen Hyperglykämie in seinem Einfluss auf die Regression zu untersuchen.
Dazu wurde auf Grundlage des STZ-Diabetes-Modells sowie eines Antisense/Sense-basierten Regressionsmodells ein diabetisches Atheroskleroseregressionsmodell etabliert, in dem während der Regression in einer Gruppe die Plasmaglukose insulinunabhängig mit dem SGLT2-Hemmer Empagliflozin gesenkt wurde und in der anderen Regressionsgruppe die Hyperglykämie fortbestand.
Es zeigte sich, dass bei Glukosesenkung am Ende der Regression kleinere Plaques vorlagen und die Plaques einen geringeren Lipid- und Makrophagengehalt sowie einen höheren Kollagengehalt aufwiesen. Die geringere Plaquegröße und vor allem die histologischen Unterschiede sind Charakteristika einer verbesserten Atheroskleroseregression. Die drei genannten histologischen Merkmale werden zudem mit einer höheren Plaquestabilität und geringeren Rupturgefahr assoziiert.
Bei der mechanistischen Untersuchung der Ergebnisse zeigte sich, dass es bei Glukosesenkung sowohl zu einer verringerten Leukozytenadhäsion am Endothel als auch einer reduzierten Proliferation von Plaquemakrophagen kam. Beide Mechanismen trugen wahrscheinlich zur verbesserten Plaqueregression bzw. dem verstärkten Rückgang des Makrophagengehalts der Plaques bei Glukosesenkung bei. Das Leukozytenprofil im peripheren Blut blieb dagegen unverändert. Die Ergebnisse sind mit anderen in vitro- und in vivo-Arbeiten zum Einfluss hoher Glukosespiegel auf Leukozytenadhäsion und Makrophagenproliferation gut vereinbar.
Humane Studien zeigen, dass die Atheroskleroseregression bei Diabetikern verschlechtert ist. Diese Arbeit im diabetischen Mausmodell legt nahe, dass diese Beobachtung unter anderem auf eine hyperglykämieinduzierte, gesteigerte Proliferation von Plaquemakrophagen und eine gesteigerte Leukozytenadhäsion am Endothel zurückzuführen ist. Eine Bestätigung dieser Mechanismen im Menschen steht allerdings aus.
... Local vascular inflammation plays a central role in the progression of rupture-prone advanced plaques. Hyperglycemia and vascular inflammation are both known to increase leukocyte and monocyte adhesion and accumulation in atherosclerotic plaques [12,26,32]. In this study, we did not see differences in plaque macrophage content in aortic root, as plaque phenotype was already highly necrotic and acellular in both groups. ...
Background and aims
Diabetes is a major risk factor of atherosclerosis and its complications. The loss-of-function mutation E1506K in the sulfonylurea receptor 1 (SUR1-E1506K) induces hyperinsulinemia in infancy, leading to impaired glucose tolerance and increased risk of type 2 diabetes. In this study, we investigate the effect of SUR1-E1506K mutation on atherogenesis in hypercholesterolemic LDLR -/- mice.
Methods
SUR1-E1506K mutated mice were cross-bred with LDLR -/- mice (SUR1Δ/LDLR -/- ), 6 months old mice were fed a western-diet (WD) for 6 months to induce advanced atherosclerotic plaques. At the age of 12 months, atherosclerosis and plaque morphology were analyzed and mRNA gene expression were measured from aortic sections and macrophages. Glucose metabolism was characterized before and after WD. Results were compared to age-matched LDLR -/- mice.
Results
Advanced atherosclerotic plaques did not differ in size between the two strains. However, in SUR1Δ/LDLR -/- mice, plaque necrotic area was increased and smooth muscle cell number was reduced, resulting in higher plaque vulnerability index in SUR1Δ/LDLR -/- mice compared to LDLR -/- mice. SUR1Δ/LDLR -/- mice exhibited impaired glucose tolerance and elevated fasting glucose after WD. The positive staining area of IL-1β and NLRP3 inflammasome were increased in aortic sections in SUR1Δ/LDLR -/- mice compared to LDLR -/- mice, and IL-18 plasma level was elevated in SUR1Δ/LDLR -/- mice. Finally, the mRNA expression of IL-1β and IL-18 were increased in SUR1Δ/LDLR -/- bone marrow derived macrophages in comparison to LDLR -/- macrophages in response to LPS.
Conclusions
SUR1-E1506K mutation impairs glucose tolerance and increases arterial inflammation, which promotes a vulnerable atherosclerotic plaque phenotype in LDLR -/- mice.
... Metformin inhibits PKC membrane translocation and activity induced by AngII [194]. AngII stimulates vascular NOX and is activated through the AngII receptor AT1R-PKC pathway [195], and is closely related to vascular oxidative stress in ED under diabetic conditions [196]. In addition, by inhibiting vasoconstrictor prostanoids, metformin also improves the endothelial function of mesenteric arteries in a T2D rat model [197]. ...
As a first-line treatment for diabetes, the insulin-sensitizing biguanide, metformin, regulates glucose levels and positively affects cardiovascular function in patients with diabetes and cardiovascular complications. Endothelial dysfunction (ED) represents the primary pathological change of multiple vascular diseases, because it causes decreased arterial plasticity, increased vascular resistance, reduced tissue perfusion and atherosclerosis. Caused by "biochemical injury", ED is also an independent predictor of cardiovascular events. Accumulating evidence shows that metformin improves ED through liver kinase B1 (LKB1)/5'-adenosine monophosphat-activated protein kinase (AMPK) and AMPK-independent targets, including nuclear factor-kappa B (NF-κB), phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), endothelial nitric oxide synthase (eNOS), sirtuin 1 (SIRT1), forkhead box O1 (FOXO1), krüppel-like factor 4 (KLF4) and krüppel-like factor 2 (KLF2). Evaluating the effects of metformin on endothelial cell functions would facilitate our understanding of the therapeutic potential of metformin in cardiovascular diabetology (including diabetes and its cardiovascular complications). This article reviews the physiological and pathological functions of endothelial cells and the intact endothelium, reviews the latest research of metformin in the treatment of diabetes and related cardiovascular complications, and focuses on the mechanism of action of metformin in regulating endothelial cell functions.
... This ROS increase leads to inflammation through the generation of IL-1, IL-6, IL-8, MCP-1, iNOS, IP-10, MMPs (especially MMP9), C5-9, and TNF-α [91]. In addition, endothelial adhesion molecules such as ICAM-1 (CD54) or E-Selectin (CD62E) [126][127][128] VCAM-1 (CD106) [129] and PECAM (CD31) are upregulated in ECs. ...
The management of diabetic retinopathy (DR) has evolved considerably over the past decade, with the availability of new technologies (diagnostic and therapeutic). As such, the existing Royal College of Ophthalmologists DR Guidelines (2013) are outdated, and to the best of our knowledge are not under revision at present. Furthermore, there are no other UK guidelines covering all available treatments, and there seems to be significant variation around the UK in the management of diabetic macular oedema (DMO). This manuscript provides a summary of reviews the pathogenesis of DR and DMO, including role of vascular endothelial growth factor (VEGF) and non-VEGF cytokines, clinical grading/classification of DMO vis a vis current terminology (of centre-involving [CI-DMO], or non-centre involving [nCI-DMO], systemic risks and their management). The excellent UK DR Screening (DRS) service has continued to evolve and remains world-leading. However, challenges remain, as there are significant variations in equipment used, and reproducible standards of DMO screening nationally. The interphase between DRS and the hospital eye service can only be strengthened with further improvements. The role of modern technology including optical coherence tomography (OCT) and wide-field imaging, and working practices including virtual clinics and their potential in increasing clinic capacity and improving patient experiences and outcomes are discussed. Similarly, potential roles of home monitoring in diabetic eyes in the future are explored. The role of pharmacological (intravitreal injections [IVT] of anti-VEGFs and steroids) and laser therapies are summarised. Generally, IVT anti-VEGF are offered as first line pharmacologic therapy. As requirements of diabetic patients in particular patient groups may vary, including pregnant women, children, and persons with learning difficulties, it is important that DR management is personalised in such particular patient groups. First choice therapy needs to be individualised in these cases and may be intravitreal steroids rather than the standard choice of anti-VEGF agents. Some of these, but not all, are discussed in this document.
... These findings suggest that AS2444697 improves the inflammatory microenvironment, which in turn leads to suppression of the aggravation of diabetic nephropathy. Studies have reported that hyperglycemia upregulates the expression of CAMs (Booth et al., 2002) and that there is a direct association between renal CAM expression and progressive renal injury in experimental animal models of renal disease including diabetic nephropathy (Matsui et al., 1996). CAM activation is an important marker of endothelial dysfunction and plays a pivotal role in the development of diabetic nephropathy. ...
Renal inflammation is a final common pathway of chronic kidney disease including diabetic nephropathy, which is the leading cause of end-stage renal disease and is associated with high cardiovascular risk and significant morbidity and mortality. Interleukin-1 (IL-1) receptor-associated kinase 4 (IRAK-4) is a pivotal molecule for IL-1 receptor- and Toll-like receptor-induced activation of proinflammatory mediators. In this study, we investigated the renoprotective properties of IRAK-4 inhibitor AS2444697 in KK/Ay type 2 diabetic mice. Four-week repeated administration of AS2444697 dose-dependently and significantly improved albuminuria; hyperfiltration, as measured by creatinine clearance; renal injury, including glomerulosclerosis; tubular injury markers, including urinary N-acetyl-β-D-glucosaminidase activity; and glomerular podocyte injury markers, including urinary nephrin excretion. In addition, AS2444697 attenuated plasma levels of proinflammatory cytokines, including IL-6; plasma levels of endothelial dysfunction markers, including intercellular adhesion molecule-1; and plasma levels and renal contents of oxidative stress markers. In contrast, AS2444697 did not significantly affect food intake or blood glucose levels. These results suggest that AS2444697 attenuates the progression of diabetic nephropathy mainly via anti-inflammatory mechanisms through inhibition of IRAK-4 activity under diabetic conditions and may represent a promising therapeutic option for the treatment of type 2 diabetic nephropathy.
... High glucose exposure induces monocyte activation, monocyte-endothelial cell adhesion and transmigration by increasing adhesion molecules such as VCAM-1 and cytokines such as MCP-1, Interleukin-1β [51][52][53] . In vivo, hyperglycemia increased expression of adhesion molecules on the endothelium, and increased accumulation of leukocytes on the endothelium has been observed in models of STZ-or alloxan-induced diabetes and hyperglycemia [54][55][56] . Our observation is in agreement with Nagareddy et al. who attributed a decrease of plaque resident macrophages to reduced monocyte entry into the vessel wall 5 . ...
Diabetes worsens atherosclerosis progression and leads to a defect in repair of arteries after cholesterol reduction, a process termed regression. Empagliflozin reduces blood glucose levels via inhibition of the sodium glucose cotransporter 2 (SGLT-2) in the kidney and has been shown to lead to a marked reduction in cardiovascular events in humans. To determine whether glucose lowering by empagliflozin accelerates atherosclerosis regression in a mouse model, male C57BL/6J mice were treated intraperitoneally with LDLR- and SRB1- antisense oligonucleotides and fed a high cholesterol diet for 16 weeks to induce severe hypercholesterolemia and atherosclerosis progression. At week 14 all mice were rendered diabetic by streptozotocin (STZ) injections. At week 16 a baseline group was sacrificed and displayed substantial atherosclerosis of the aortic root. In the remaining mice, plasma cholesterol was lowered by switching to chow diet and treatment with LDLR sense oligonucleotides to induce atherosclerosis regression. These mice then received either empagliflozin or vehicle for three weeks. Atherosclerotic plaques in the empagliflozin treated mice were significantly smaller, showed decreased lipid and CD68+ macrophage content, as well as greater collagen content. Proliferation of plaque resident macrophages and leukocyte adhesion to the vascular wall were significantly decreased in empagliflozin-treated mice. In summary, plasma glucose lowering by empagliflozin improves plaque regression in diabetic mice.
... Consistently, our data also found that hyperglycemia fluctuation led to exacerbation of the apoptotic effect on HUVECs. Oxidative stress generation [33], inflammation [34], and PKC activation [35] have been proposed as the factors for the damaging effect of hyperglycemia. Piconi et al. suggested that both stable and oscillating hyperglycemia increased HUVEC apoptosis through ROS overproduction at the mitochondrial electron transport chain [36]. ...
Hyperglycemia fluctuation is associated with diabetes mellitus (DM) complications when compared to persistent hyperglycemia. Previous studies have shown that paeoniflorin (PF), through its antiapoptosis, anti-inflammation, and antithrombotic properties, effectively protects against cardiovascular and cerebrovascular disease. However, the mechanism underlying the protection from PF against vascular injuries induced by hyperglycemia fluctuations remains poorly understood. Herein, we investigated the potential protective role of PF on human umbilical vein endothelial cells (HUVECs) subjected to intermittent glucose levels in vitro and in DM rats with fluctuating hyperglycemia in vivo . A remarkable increased apoptosis associated with elevated inflammation, increased oxidative stress, and high protein level of PKC β 1 was induced in HUVECs by intermittently changing glucose for 8 days, and PF recovered those detrimental changes. LY333531, a potent PKC β 1 inhibitor, and metformin manifested similar effects. Additionally, in DM rats with fluctuating hyperglycemia, PF protected against vascular damage as what has been observed in vitro . Taken together, PF attenuates the vascular injury induced by fluctuant hyperglycemia through oxidative stress inhibition, inflammatory reaction reduction, and PKC β 1 protein level repression, suggesting its perspective clinical usage.
... In clinical studies, VCAM-1 levels were positively associated with 24-hour urinary albumin excretion in hypertensive T2DM patients [33]. Hyperglycemia, oxidative stress and inflammatory cytokines induce VCAM-1 and ICAM-1 expression in a NF-κB dependent pathway [34]. Thus, NF-κB inhibitors or antioxidant strategies resulted effective to decrease synthesis of adhesion molecules in experimental DN [35]. ...
Introduction: Diabetic nephropathy (DN) is the leading cause of chronic kidney disease (CKD) and end stage renal disease (ESRD). Beyond the new anti-diabetic drugs that possess markedly cardiovascular and renal protective effects, no novel direct therapies for DN have become available on the market in the last twenty years. Recently well-designed clinical trials for the treatment of DN, with attractive pathogenetic rationale, e.g. bardoxolone and atrasentan, were cancelled or stopped because of safety concerns or lack of reaching the end-points, respectively.
Areas covered: In this review we focus on the involvement of inflammation in the pathogenesis of DN. We update information from recent experimental and clinical studies that reported beneficial effects of several agents targeting chemokines, cytokines, transcription factors and kinases as well as several compounds with anti-inflammatory properties on DN.
Expert opinion: Inflammation plays a key role on the DN progression. Preclinical studies have identified several anti-inflammatory molecules that effective decrease albuminuria and/or proteinuria. However, limited clinical trials in humans have been performed to confirm these results. Inhibitors of CCL2/CCR2, IL-1β and JAK/STAT pathways, and Nrf2 inducers are promising therapeutic options to improve the renal outcome of patients with DN, but appropriate clinical trials are necessary.
... The ROS leads to changes in the cell permeability and loss of membrane integrity [44], which can lead to tissue degeneration, cell death, and irreversible endothelial damage also diminishes NO production [45] and known to increase lipid peroxidation. Thus, the high levels of ROS and MDA creates a constant stressful environment thereby it intensifies the endothelial damage [46,47]. Thus, the supraphysiological levels of circulatory glucose induce high OS and their secondary cascades in multifaceted mechanism induce smooth muscle and endothelial degeneration and endothelial thickening in STZ rats. ...
INTRODUCTION:
Erectile dysfunction is one of the major secondary complications of diabetes. Mucuna pruriens (M. pruriens), a leguminous plant identified for its antidiabetic, aphrodisiac, and fertility enhancing properties, has been the choice of Indian traditional medicine.
AIM:
The objective of the present study was to analyze the efficacy of M. pruriens on free radicals-mediated penile tissue alterations in hyperglycemic male rats. Methods. Male albino rats were divided as group I (sham) control, group II (STZ) diabetes-induced (streptozotocin 60 mg/kg of body weight [bw] in 0.1 M citrate buffer), group III (STZ + MP) diabetic rats administered with 200 mg/kg bw of ethanolic extract of M. pruriens seed, group IV (STZ + SIL) diabetic rats administered with 5 mg/kg bw of sildenafil citrate, group V (sham + MP) administered with 200 mg/kg bw of extract alone, and group VI (sham + SIL) administered with 5 mg/kg bw of sildenafil citrate. The M. pruriens and sildenafil citrate were given (gavage) once daily for a period of 60 days. At the end of 60 days, the animals were sacrificed and subjected to analysis of reactive oxygen species levels, enzymic and nonenzymic antioxidant levels, levels of NOx, histological, and histomorphometrical study of penile tissue.
MAIN OUTCOME MEASURES:
Remedial use of M. pruriens seed extract on diabetes-induced erectile tissue damage.
RESULTS:
Significantly high levels of oxidative stress and low levels of antioxidants in the penile tissue seem to contribute to the increased collagen deposition and fibrosis of erectile tissue in STZ rats. Relatively, there was increased damage in STZ + SIL group. Supplementation of M. pruriens in STZ + MP group has revealed the potency to overcome oxidative stress, and good preservation of penile histoarchitecture.
CONCLUSION:
The ethanolic extract of M. pruriens seed significantly recovered or protected erectile tissue from the oxidative stress-induced degeneration by its antioxidant potentials. These findings propound to serve mankind by the treatment of diabetes-induced erectile dysfunction.
... Based on this evidence and our data showing partial and not complete inhibition of RA-stimulated glucose uptake with AMPK inhibition (Figure 4B), we hypothesized that PKCs may play a role in RA-stimulated glucose uptake. To address this hypothesis, we used bisindolylmaleimide I (BMD), a selective inhibitor of PKCα-, βI-, βII-, γ-, δ-and ε-isozymes, used extensively in other studies [38][39][40]. BMD did not have any effect on the basal glucose uptake (103 ± 3.4% of control) ( Figure 7A). Furthermore, RA-stimulated glucose uptake was not affected by BMD (RA: 190 ± 4.6% of control, BMD + RA: 188 ± 7.3% of control) ( Figure 7A). ...
Skeletal muscle is a major insulin-target tissue and plays an important role in glucose homeostasis. Impaired insulin action in muscles leads to insulin resistance and type 2 diabetes mellitus. 5′ AMP-activated kinase (AMPK) is an energy sensor, its activation increases glucose uptake in skeletal muscle and AMPK activators have been viewed as a targeted approach in combating insulin resistance. We previously reported AMPK activation and increased muscle glucose uptake by rosemary extract (RE). In the present study, we examined the effects and the mechanism of action of rosmarinic acid (RA), a major RE constituent, in L6 rat muscle cells. RA (5.0 µM) increased glucose uptake (186 ± 4.17% of control, p < 0.001) to levels comparable to maximum insulin (204 ± 10.73% of control, p < 0.001) and metformin (202 ± 14.37% of control, p < 0.001). Akt phosphorylation was not affected by RA, while AMPK phosphorylation was increased. The RA-stimulated glucose uptake was inhibited by the AMPK inhibitor compound C and was not affected by wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K). The current study shows an effect of RA to increase muscle glucose uptake and AMPK phosphorylation. RA deserves further study as it shows potential to be used as an agent to regulate glucose homeostasis.
... Up-regulation of cellular adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin, play an initial role in the formation of atheromatous plaques [3]. Moreover, for endothelial cells (ECs), PKC activation increases adhesion molecules expression, such as VCAM-1, ICAM-1, E-selectin, interleukin (IL)-6, IL-1, and the activation of NADPH oxidase [14,15]. There is convincing evidence demonstrating that PKC activation is involved in endothelial dysfunction and monocytes adhesion to ECs [16]. ...
Several studies have reported a strong association between high plasma level of Trimethylamine N-oxide (TMAO) and atherosclerosis development. However, the exact mechanism underlying this correlation is unknown. In this study, we try to explore the impact of TMAO on endothelial dysfunction. After TMAO treatment, Human umbilical vein endothelial cells (HUVECs) showed significant impairment in cellular proliferation and HUVECs-ECM adhesion compared to control. Likewise, TMAO markedly suppressed HUVECs migration in transwell migration assay and wound healing assay. In addition, we found TMAO up-regulated VCAM-1 expression, promoted monocyte adherence, activated PKC and phosphorylated NF-κB. Interestingly, TMAO-stimulated VCAM-1 expression and monocyte adherence were diminished by PKC inhibitor. These results demonstrate that TMAO promotes early pathological process of atherosclerosis by accelerating endothelial dysfunction, including decreasing endothelial self-repair and increasing monocyte adhesion. Furthermore, TMAO-induced monocyte adhesion is partly attributable to activation of PKC/NF-κB/VCAM-1.
... It has been shown that oxidative stress may be both causes of apoptosis of pancreatic beta cells and insulin resistance [3]. ROS may cause lipids, proteins and nucleic acids oxidative damage Furthermore, ROS can activate cellular proliferation, increase expression of adhesive molecules and proinflammatory cytokines, as well as accelerate fibrosis [4,5]. Oxidative stress may also led to the activation of protein kinase C, nuclear factor kB, and alter the intracellular calcium signalling pathway. ...
Chronic hyperglycemia is the main cause of increased oxidative stress in type 2 diabetes mellitus patients. The over-production of oxygen free radicals and nitric oxide radicals leads to the damage of many cellular coumpounds including lipids, proteins and nucleic acids. DNA damage in various cells may cause mutations and genomic instability, thus contributing to cancer. Therefore, hypoglycemic drugs with antioxidant activity might have an impact on the level of oxidative stress and probably, the risk of cancer. It has been suggested that metformin administration to diabetic patients may decrease the incidence of a malignant neoplasms. Some hypoglycemic drugs, including gliclazide, inhibiting the adhesion of plateletes and cancer cells to endothelium may decrease the risk of cancer embolism in the pulmonary circulation of rats. Therefore, it is tempting to speculate that this phenomenon may also occur in patients treated with gliclazide. The rationality of this presumption is partially supported by the data indicating free radical scavenging activity and beneficial effect of this agent on platelet reactivity.
... The inhibition of calpain activity decreases the expression of pro-inflammatory adhesion molecules, ICAM and VCAM, on the endothelial surface during hyperglycemia (Stalker et al., 2003). In addition, the inhibition of protein kinase reduces the interaction between leukocytes and the vascular endothelium through the suppression of endothelial cell surface expression in response to oxidative stress (Booth et al., 2002). Perhaps those key reactions, which result in inhibition, can lead to reduction of the inflammatory process in the kidney glomerulus. ...
Uncontrolled diabetes has become a major cause of mortality and morbidity by reason of vascular angiopathy. The aim of this study was to evaluate the concentrations of soluble forms of vascular adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), E-selectin, and thrombomodulin in patients with well-controlled and uncontrolled diabetes type 2.
The study was conducted on 62 patients with diabetes. Group I consisted of 35 patients with well-controlled diabetes. The second group included 27 patients with uncontrolled diabetes with micro-albuminuria. A control group was made up of 25 healthy volunteers. The concentrations of sVCAM-1, sICAM-1, sE-selectin, and soluble thrombomodulin were assayed in plasma. Serum concentration of creatinine was measured and the plasma concentrations of fasting glucose and glycated hemoglobin (HbA1c) determined.
Lower concentrations of ICAM-1 were found in the group of uncontrolled diabetes patients compared with those with well-controlled disease. In patients with uncontrolled diabetes, VCAM-1 levels were significantly higher compared with the group with well-controlled diabetes. In patients with uncontrolled diabetes a positive correlation was obtained between glomerular filtration rate and sE-selectin and a negative correlation between the levels of creatinine and ICAM-1, although there was a positive correlation between (HbA1c) and ICAM-1.
The study confirmed the participation of the inflammatory process associated with impaired vascular endothelial function in the pathogenesis of type 2 diabetes. The opposite effect of uncontrolled hyperglycemia on adhesion molecules suggests different functions of VCAM-1 and ICAM-1 in complications of diabetes.
... It is reported that mechanisms of islet β cell failure are different in the progression of T1DM and T2DM [28]. In individuals without diabetic, insulin secretion of beta cells is linked to peripheral insulin sensitivity through a postulated negative feedback loop that allows the beta cells to compensate for any change in whole body insulin sensitivity by a proportionate and reciprocal change in insulin secretion [30]. It is generally accepted that insulin resistance is the primary defect and that pancreatic beta cell dysfunction occurs later that contributes to the progression of diabetes. ...
Diabetes mellitus (DM) is considered an inflammatory process with systemic involvement of the vascular tree. Asymptomic coronary artery disease is common in diabetic patients and is a strong predictor for future adverse coronary vascular events as well as early death. The objective of this study was to determine serum levels Intercellular adhesion molecule-1 (ICAM-1), Vascular adhesion molecule-1 (VCAM-1), interleukin 1-β (IL1-β), and C-Reactive protein (CRP) in relation to some markers indicative of type II diabeticT2DM.
Methods:
50 patients in early stage of T2DM with no cardiovascular history and 30 healthy subjects were enrolled in this study. All anthropometrical indexes were measured in two groups. Laboratory investigations including: Fasting blood sugar (FBS) and lipid profile were measured by enzymatic colorimetric methods. Serum insulin, ICAM-1, VCAM-1, IL1-β, and CRP were measured by enzyme linked immunosorbent assay ELISA.
Results:
The levels of FBS (195.94±12.178 mg/dL), Insulin (18.627±1.224 µIU/ml). VCAM-1 (2017.3±108.908 ng/dl), IL1-β (29.559±1.225 pg/ml), CRP (23.989±2.526 mg/l), and lipid abnormality, were highest in diabetic patients with significant differences (P< 0.05) when compared with those of control group, while ICAM show no significant difference (57.620±0.960 ng/dl). Positive significant correlation was found between VCAM and CRP (r= 0.415, P= 0.01). Atherogenic Index of Plasma (AIP) show no significant differences with ICAM, VCAM, CRP, and IL1-β.
... 7,8 For EC, PKC activation increased the expression of adhesion molecules, such as selectins, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), IL-6, IL-1, and the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and superoxide production. 9,10 Schmidt and Yan's group previously published supportive data on the potential role of PKC-b in atherosclerotic plaque formation. Depletion of PKC-b gene or treatment with ruboxistaurin (RBX; LY333531) in apolipoprotein E (apoE)-deficient mice decreased atherosclerosis by inhibiting the early growth response (Egr)-1 protein, which regulated VCAM expression and MMP-2 activity preferentially in EC. 11 More recently, the same group reported that inhibition of PKC-b reduced CD11c, chemokine (C-C motif) ligand 2, and IL-1b in macrophages induced by high glucose (HG) levels and diabetes. ...
Aims:
Clinical observations showed a correlation between accelerated atherosclerosis in diabetes and high plasmatic level of IL-18, a pro-inflammatory cytokine. IL-18 enhances the production of inflammatory cytokines and cellular adhesion molecules contributing to atherosclerotic plaque formation and instability. Previous studies indicated that protein kinase C (PKC)-β inhibition prevented macrophage-induced cytokine expression involved in diabetic (DM) atherosclerotic plaque development. However, the role of PKC-β activation on IL-18/IL-18-binding protein (IL-18BP) pathway causing endothelial dysfunction and monocyte adhesion in diabetes has never been explored.
Methods and results:
Apoe(-/-) mice were rendered DM and fed with western diet containing ruboxistaurin (RBX), a PKC-β inhibitor. After 20 weeks, atherosclerotic plaque composition was quantified. Compared with non-diabetic, DM mice exhibited elevated atherosclerotic plaque formation, cholestoryl ester content and macrophage infiltration, as well as reduced IL-18BP expression in the aorta which was prevented with RBX treatment. Endothelial cells (ECs) and macrophages were exposed to normal or high glucose (HG) levels with or without palmitate and recombinant IL-18 for 24 h. The combined HG and palmitate condition was required to increase IL-18 expression and secretion in macrophages, while it reduced IL-18BP expression in EC causing up-regulation of the vascular cell adhesion molecule (VCAM)-1 and monocyte adhesion. Elevated VCAM-1 expression and monocyte adherence were prevented by siRNA, RBX, and IL-18 neutralizing antibody.
Conclusion:
Our study unrevealed a new mechanism by which PKC-β activation promotes EC dysfunction caused by the de-regulation of the IL-18/IL-18BP pathway, leading to increased VCAM-1 expression, monocyte/macrophage adhesion, and accelerated atherosclerotic plaque formation in diabetes.
... Diabetes mellitus through hyperglycemia is widely known to be a major factor that leads to microvascular and neural complication [1]. Hyperglycemia-induced end organ damage in diabetes mellitus is associated with 1) accumulation of advanced glycation end products [2,3]; 2) increased oxidative stress [4,5]; 3) polyol metabolic pathway [6,7]; and 4) activation of protein kinase C pathway [8,9]. Insulin deficiency in diabetes mellitus stimulates lipolysis in the adipose tissue, and gives rise to hyperlipidemia and fatty liver [10]. ...
Streptozotocin (STZ)-induced diabetic mice increased levels of serum glucose, triglyceride and cholesterol, and decreased level of serum insulin. Effects of Bofutsushosan (BOF: Pulvis ledebouriellae compositae: 防風通聖散) and its composed crude drug, gardeniae fructus (GF: 山梔子) were investigated on levels of these diabetic parameters (serum glucose, insulin, triglyceride and cholesterol) in STZ-diabetic mice. BOF and GF were extracted in 10 volumes of distilled water with an automatic extractor “Torobi”. STZ-induced diabetic mice with serum glucose level of over 600 mg/dl at 3 - 4 weeks after intravenous injection of 150 mg/kg STZ were used for experiments. BOF extract, GF extract, geniposide (a main constituent of GF), and glibenclamide were administered intraperitoneally into 3-hour-fasted STZ-diabetic mice. At 6 hours after administration, BOF extract (100 - 300 mg/kg) decreased high levels of serum glucose, triglyceride and cholesterol, and also increased low level of serum insulin in STZ-diabetic mice in a dose-dependent manner, respectively. Anti-diabetic drug glibenclamide (0.3 - 1 mg/kg) as positive control significantly decreased serum glucose and cholesterol levels, and increased serum insulin level in the diabetic mice. GF extract (30 - 300 mg/kg) decreased serum glucose, triglyceride and cholesterol levels but did not affect serum insulin level in the diabetic mice. Geniposide (10 - 100 mg/kg), decreased serum glucose level but did not affect serum insulin and triglyceride levels in the diabetic mice. These results demonstrated that intraperitoneally administrated BOF extract improved abnormal levels of serum glucose, insulin, triglyceride and cholesterol in the STZ-diabetic mice as being similar to glibenclamide. GF extract has an important role in a part of improving actions of BOF in the diabetic mice. The action of GF extract on serum glucose was parallel with the action of geniposide in the diabetic mice, supporting roles of geniposide in anti-hyperglycemic action of GF.
... leukostasis) to the retinal vascular endothelial cells occurs 28 early in diabetic patients and animals. [139][140][141][142] Diabetes induces upregulation of adhesion molecules [142][143][144] and promotes vascular leakage [145][146][147] by upregulation of intra-cellular adhesion molecule (ICAM), vascular endothelial growth factor (VEGF) or advanced glycation end-products. 148,149 Platelet-fibrin thrombi in retinal capillaries are increased in DR secondary to chronic hyperglycemia and platelet activating factor which contributes to thrombus formation. ...
... Leukocyte rolling and adhesion are characteristics of vascular inflammation, and vascular reactivity to acetylcholine (ACh) is an indicator of normal vascular function. Because increased leukocyte rolling (34) and decreased ACh-induced vasodilation (35) have been reported in diabetes mellitus (DM) (22), we used this model to study vasoprotective effects of pitavastatin. In an STZtreated mouse diabetic model, leukocyte rolling was increased and ACh-induced vasodilation was dampened compared with those of the vehicle-treated mice (Fig. 5A-D, Supplemental Videos 1-3). ...
Because ERK5 inhibits endothelial inflammation and dysfunction, activating ERK5 might be a novel approach to protecting vascular endothelial cells (ECs) against various pathological conditions of the blood vessel. We have identified small molecules that protect ECs via ERK5 activation and determined their contribution to preventing cardiac allograft rejection. Using high-throughput screening, we identified certain statins and antimalarial agents including chloroquine, hydroxychloroquine, and quinacrine as strong ERK5 activators. Pitavastatin enhanced ERK5 transcriptional activity and Kruppel-like factor-2 expression in cultured human and bovine ECs, but these effects were abolished by the depletion of ERK5. Chloroquine and hydroxychloroquine upregulated ERK5 kinase activity and inhibited VCAM-1 expression in an ERK5-dependent but MAPK/ERK kinase 5- and Kruppel-like factor 2/4-independent manner. Leukocyte rolling and vascular reactivity were used to evaluate endothelial function in vivo, and we found that EC-specific ERK5 knockout (ERK5-EKO) mice exhibited increased leukocyte rolling and impaired vascular reactivity, which could not be corrected by pitavastatin. The role of endothelial ERK5 in acute cardiac allograft rejection was also examined by heterotopic grafting of the heart obtained from either wild-type or ERK5-EKO mice into allomismatched recipient mice. A robust increase in both inflammatory gene expression and CD45-positive cell infiltration into the graft was observed. These tissue rejection responses were inhibited by pitavastatin in wild-type but not ERK5-EKO hearts. Our study has identified statins and antimalarial drugs as strong ERK5 activators and shown that ERK5 activation is preventive of endothelial inflammation and dysfunction and acute allograft rejection.
... Diabetes mellitus through hyperglycemia is widely known to be a major factor that leads to microvascular and neural complication [1]. Hyperglycemia-induced end organ damage in diabetes mellitus is associated with 1) accumulation of advanced glycation end products [2,3]; 2) increased oxidative stress [4,5]; 3) polyol metabolic pathway [6,7]; and 4) activation of protein kinase C pathway [8,9]. Insulin deficiency in diabetes mellitus stimulates lipolysis in the adipose tissue, and gives rise to hyperlipidemia and fatty liver [10]. ...
... Hyperglycaemia is undoubtedly of significance in increased monocyte-endothelial cell adhesion, at least some of which effect is due to stimulation of endothelial expression of adhesion molecules. Consistently, hyperglycemia has been shown to induce monocyte adhesion molecule expression on the endothelium, and increased accumulation of white blood cells on the endothelium has been observed in models of streptozotocin-or alloxan-induced diabetes and hyperglycemia (Booth, 2002). Our research group has recently shown that exposing decidual endothelial cells to high glucose levels in vitro caused an increase in adhesion molecule expression and monocyte adhesion, indicating that hyperglycemia is sufficient for initiation of macrophage-rich lesions (Renard, 2004, Xie et al submitted for publication). ...
... Moreover, similar effects are mimicked in vitro in endothelial cell culture exposed to sera of diabetic patients or advanced glycation endproduct-albumin and are significantly reduced in vivo by protein C inhibition or the administration of insulin or SOD. In addition, a possible role for the receptor for advanced glycosylated endproducts (RAGE) as endothelial adhesion molecule binding to beta 2 integrin on leukocyte surface, has been proposed [65,[79][80][81][82]. ...
Atherogenesis is the pathogenetic process leading to formation of the atheroma lesion. It is associated to a chronic inflammatory state initially stimulated by an aberrant accumulation of lipid molecules beyond the endothelial barrier. This event triggers a cascade of deleterious events mainly through immune cell stimulation with the consequent liberation of potent pro-inflammatory and tissue damaging mediators. The atherogenetic process implies marked modifications of endothelial cell functions and a radical change in the endothelial–leukocyte interaction pattern. Moreover, accumulating evidence shows an important link between microvascular and inflammatory responses and major cardiovascular risk factors. This review illustrates the current knowledge on the effects of obesity, hypercholesterolemia and diabetes on microcirculation; their pathophysiological implications will be discussed.
... The endothelial dysfunction is caused by an imbalance between vasoconstrictor and vasodilator molecules, and between pro-atherogenic and pro-coagulant states. Importantly, chronic exposure to harmful physical and chemical stimuli can lead to endothelial dysfunction (Caballero, 2003), among which, oxidised low-density lipoprotein (oxLDL) and hyperglycemia are the most relevant factors (Booth, Stalker, Lefer, & Scalia, 2002). The hallmark of endothelial dysfunction is the inhibition of nitric oxide (NO)-mediated vasodilatation, the increase of ROS production and the enhanced expression of endothelial cell adhesion molecules (Cominacini et al., 2001). ...
... Vascular insulin signaling can be improved using inhibitors of protein kinase C (PKC), such as ruboxistaurin [51]. Especially PKCβ has emerged as a promising target, and PKC inhibition has been shown in multiple studies in mice and humans to improve endothelial insulin signaling, nitric oxide synthesis and barrier function in diabetes [51,138,139]. In addition, PKCθ has been shown to impair endothelial insulin signaling and may be an additionally relevant PKC isoform for improving endothelial function [44,46], but evidence in humans has yet to be obtained. ...
Endothelial dysfunction associated with diabetes and cardiovascular disease is characterized by changes in vasoregulation, enhanced generation of reactive oxygen intermediates, inflammatory activation, and altered barrier function. These endothelial alterations contribute to excess cardiovascular disease in diabetes, but may also play a role in the pathogenesis of diabetes, especially type 2. The mechanisms underlying endothelial dysfunction in diabetes differ between type 1 (T1D) and type 2 diabetes (T2D): hyperglycemia contributes to endothelial dysfunction in all individuals with diabetes, whereas the causative mechanisms in T2D also include impaired insulin signaling in endothelial cells, dyslipidemia and altered secretion of bioactive substances (adipokines) by adipose tissue. The close association of so-called perivascular adipose tissue with arteries and arterioles facilitates the exposure of vascular endothelium to adipokines, particularly if inflammation activates the adipose tissue. Glucose and adipokines activate specific intracellular signaling pathways in endothelium, which in concert result in endothelial dysfunction in diabetes. Here, we review the characteristics of endothelial dysfunction in diabetes, the causative mechanisms involved and the role of endothelial dysfunction(s) in the pathogenesis of T2D. Finally, we will discuss the therapeutic potential of endothelial dysfunction in T2D.
... First, all nutrients absorbed post-prandial must traverse the microcirculation to be stored in adipocytes (see also paragraph above). Accordingly, studies have extensively shown that overload of nutrients such as glucose and FFA acutely induces microvascular dysfunction in non-obese laboratory rodents [39,40]. Similarly, release of FFA from adipose storage during fasting is acutely associated with accumulation of macrophages in visceral fat depots [41]. ...
In most humans, obesity is associated with a chronic low-grade inflammatory reaction occurring in several organ tissues, including the adipose tissue. Infiltration of bone marrow derived leukocytes (granulocytes, monocytes, lymphocytes) into expanding adipose depots appears to be an integral component of inflammation in obesity. Circulating leukocytes invade organ tissues mainly through post-capillary venules in the microcirculation. The endothelium of the post-capillary venules acts as a gatekeeper to leukocyte adhesion and extravasation by displacing on its luminal surface adhesion molecules that bind the adhesive receptors expressed on circulating leukocytes. Several studies investigating the impact of obesity on the microcirculation have demonstrated the occurrence of microvascular dysfunction in experimental animal model of obesity, as well as in obese humans. To date though, working hypotheses and study designs have favored the view that microvascular alterations are secondary to adipose tissue dysfunction. Indeed, a significant amount of data exists in the scientific literature to support the concept that microvascular dysfunction may precede and cause adipose tissue inflammation in obesity. Through review of key published data, this article prospectively presents the concept that in response to nutrients overload the vascular endothelium of the microcirculation acutely activates inflammatory pathways that initiate infiltration of leukocytes in visceral adipose tissue, well before weight gain and overt obesity. The anatomical and physiological heterogeneity of different microcirculations is also discussed toward the understanding of how obesity induces different inflammatory phenotypes in visceral and subcutaneous fat depots.
... Clinical evidence suggests that endothelial adhesion molecule expression is enhanced in the aorta and the internal mammary artery of diabetic patients [15] . Additionally, hyperglycemiamediated adhesion of neutrophils to rat endothelial cells could be attenuated by superoxide dismutase in vivo [16]. These findings support the hypothesis that intracellular oxidative stress might be critical to high-glucose-mediated, diabetes-associated atherosclerosis. ...
Chronic elevation of glucose level activates vascular inflammation and increases endothelial adhesiveness to monocytes, an early sign of atherogenesis. This study aimed to elucidate the detailed mechanisms of high-glucose-induced endothelial inflammation, and to investigate the potential effects of Ginkgo biloba extract (GBE), an antioxidant herbal medicine, on such inflammation.
Human aortic endothelial cells were cultured in high glucose or mannitol as osmotic control for 4 days. The expression of cytokines and adhesion molecules and the adhesiveness of endothelial cells to monocytes were examined. The effects of pretreatment of GBE or N-acetylcysteine, an antioxidant, were also investigated.
Either high glucose or mannitol significantly increased reactive oxygen species (ROS) production, interleukin-6 secretion, intercellular adhesion molecule-1 (ICAM-1) expression, as well as endothelial adhesiveness to monocytes. The high-glucose-induced endothelial adhesiveness was significantly reduced either by an anti-ICAM-1 antibody or by an interleukin-6 neutralizing antibody. Interleukin-6 (5 ng/ml) significantly increased endothelial ICAM-1 expression. Piceatannol, a signal transducer and activator of transcription (STAT) 1/3 inhibitor, but not fludarabine, a STAT1 inhibitor, suppressed high-glucose-induced ICAM-1 expression. Pretreatment with GBE or N-acetylcysteine inhibited high-glucose-induced ROS, interleukin-6 production, STAT1/3 activation, ICAM-1 expression, and endothelial adhesiveness to monocytes.
Long-term presence of high glucose induced STAT3 mediated ICAM-1 dependent endothelial adhesiveness to monocytes via the osmotic-related redox-dependent interleukin-6 pathways. GBE reduced high-glucose-induced endothelial inflammation mainly by inhibiting interleukin-6 activation. Future study is indicated to validate the antioxidant/anti-inflammatory strategy targeting on interleukin-6 for endothelial protection in in vivo and clinical hyperglycemia.
... However, there is circumstantial evidence from in vivo studies both in animals and humans that the endothelium is particularly sensitive to changes in glucose concentrations. Investigators have evaluated the effects of acute (≤ 12 h) glucose administration on leukocyte adhesion to microvascular ECs in rodents using intravital microscopy and found evidence of increased leukocyte rolling and adhesion to microvessels in the absence (Booth et al., 2002) or presence of co-administered IL-1β or TNF-α (Azcutia et al., 2010a). The effects of elevated glucose in these studies were most likely mediated by increased expression of the adhesion molecules P-selectin, VCAM-1, and ICAM-1, through pathways mediated by PKC and increased oxidative stress and/or activation of the NF-κB pathway (Booth et al., 2002; Azcutia et al., 2010b). ...
Progress in preventing atherosclerotic coronary artery disease (CAD) has been stalled by the epidemic of type 2 diabetes. Further advances in this area demand a thorough understanding of how two major features of type 2 diabetes, insulin resistance and hyperglycemia, impact atherosclerosis. Insulin resistance is associated with systemic CAD risk factors, but increasing evidence suggests that defective insulin signaling in atherosclerotic lesional cells also plays an important role. The role of hyperglycemia in CAD associated with type 2 diabetes is less clear. Understanding the mechanisms whereby type 2 diabetes exacerbates CAD offers hope for new therapeutic strategies to prevent and treat atherosclerotic vascular disease.
Altered blood glucose metabolism is ultimately responsible for all the clinical complications of type-2 diabetes. In this overview, we explain some of the mechanisms that are influenced or modulated by increased blood glucose levels.
Deep learning (DL) is a subset of artificial intelligence (AI), which uses multilayer neural networks modelled after the mammalian visual cortex capable of synthesizing images in ways that will transform the field of glaucoma. Autonomous DL algorithms are capable of maximizing information embedded in digital fundus photographs and ocular coherence tomographs to outperform ophthalmologists in disease detection. Other unsupervised algorithms such as principal component analysis (axis learning) and archetypal analysis (corner learning) facilitate visual field interpretation and show great promise to detect functional glaucoma progression and differentiate it from non-glaucomatous changes when compared with conventional software packages. Forecasting tools such as the Kalman filter may revolutionize glaucoma management by accounting for a host of factors to set target intraocular pressure goals that preserve vision. Activation maps generated from DL algorithms that process glaucoma data have the potential to efficiently direct our attention to critical data elements embedded in high throughput data and enhance our understanding of the glaucomatous process. It is hoped that AI will realize more accurate assessment of the copious data encountered in glaucoma management, improving our understanding of the disease, preserving vision, and serving to enhance the deep bonds that patients develop with their treating physicians. 深度学习 (Deep learning,DL) 是人工智能的一个分支, 通过模仿哺乳动物视皮层合成影像的能力, 建立多层神经网络模型。 这种技术会在青光眼领域起到变革的作用。自主DL算法能够最大化地收集眼底图像和OCT中包含的信息, 在疾病探查方面甚至能够超越眼科医生。其他的无监管算法例如主成分分析 (纵学习) 和原型分析 (角点学习) 有助于对视野结果进行解释, 并且与传统软件包相比能够更好地检测青光眼功能性进展, 并与非青光眼相鉴别。此外, 如卡尔曼滤波器等预测性工具可收录一系列影响因素后确定维持视力的目标眼压值, 从而彻底改变了青光眼的管理。DL算法通过处理青光眼数据可生成激活图, 引导我们关注高通量数据中嵌入的关键数据元素, 并加强我们对青光眼发展过程的理解。最后, 希望AI能够更加精准地评估青光眼治疗管理过程中的大量数据, 提高我们对青光眼、保护视力的认识, 成为患者与医生之间的深厚纽带。
Metabolic syndrome is a critically important precursor to the onset of many diseases, such as cardiovascular disease, and cardiovascular disease is the leading cause of death worldwide. The primary risk factors of metabolic syndrome include hyperglycaemia, abdominal obesity, dyslipidaemia, and high blood pressure. It has been well documented that metabolic syndrome alters vascular endothelial and smooth muscle cell functions in the heart, brain, kidney and peripheral vessels. However, there is less information available regarding how metabolic syndrome can affect pulmonary vascular function and ultimately increase an individual's risk of developing various pulmonary vascular diseases, such as pulmonary hypertension. Here, we review in detail how metabolic syndrome affects pulmonary vascular function. image
About 30% of stroke patients are diabetic and more than 90% of them comprise type 2 diabetes (T2D). Diabetic stroke patients have higher mortality and worse neurological outcomes. Emerging clinical and experimental data suggest that blood-brain barrier (BBB) disruption, neuroinflammation, and stroke recovery impairment are exacerbated in diabetic patients. Hence, finding therapeutic approaches that can target these specific diabetic mechanisms in stroke is the thrust of the present translational study. Here, we summarize the ischemia-reperfusion injury in stroke, presenting the clinical evidence for involvement of hyperglycemia in severe damage of cerebral ischemia-reperfusion. We go on to consider the mechanisms that underlie such pathology, and highlight areas for future basic research and clinical studies into diabetic ischemia and reperfusion.
Neurons in the ischemic penumbra or peri-infarct zone may undergo delayed cell death which called programmed cell death (PCD) and thus they are potentially recoverable for some time after the onset of stroke. There were three major morphologies of PCD in the cerebral ischemic injury, including apoptosis, autophagy and programmed necrosis (also known as necroptosis). In this review we will discuss the characteristics, molecular mechanism of each PCD mode and their role in cerebral ischemia and reperfusion injury (CIRI), furthermore crosstalk between various modes of PCD is also dicussed.
The eye serves as a mirror to the body vasculature. Changes in the retinal vasculature translate into the prediction of risk from cardiovascular disease. The mammalian retinal vasculature is the most widely used tissue to study physiological angiogenesis. Thus, the retinal vasculature plays a particular role in research linking various fields of expertise. Diabetic retinopathy is often cited as the paradigm for an angiogenic disease. However, there is much more to the diabetic eye, as the disease characteristics need to be considered to understand commonalities and differences f.e. to tumor angiogenesis. Primarily. diabetic retinopathy is vasoregressive in nature. Progressive capillary dropout drives sight-threatening proliferative diabetic retinopathy and macular edema by inflaming a hypoxic response. Although there is no animal model that qualitatively and quantitatively mimicks advanced human eye disease, they reflect the complex hyperglycemia-driven interaction of the neurovascular unit better than any in vitro systems. Downstream of high ambient glucose, multiple biochemical abnormalities exist which affect not only vascular cells of the capillary network but the entire neurovascular unit. Novel concepts that arise from tumor research can be useful to limit the neovascular of permeability-enhancing response to hypoxia in the diabetic retina.
Diabetic macular oedema (DMO), a leading cause of preventable visual loss in the working population, is caused by an increase in microvascular endothelial cell permeability, and its prevalence is on the increase in parallel with the rising worldwide prevalence of diabetes. It is known that retinal vascular leakage in DMO is contributed to by VEGF upregulation as well as non-VEGF dependent inflammatory pathways, and the potential use of anti-inflammatory agents such as the glucocorticoids, including dexamethasone are being extensively studied. However, the mechanisms of action of dexamethasone in DMO reduction are not fully understood. Using human primary retinal endothelial cells (REC) the in vitro effect of dexamethasone in modulating the proliferation, permeability and gene expression of key tight and adheren junction components, and the expression of angiopoietins (Ang) 1 and 2 in high (25 mM) glucose conditions were investigated. High glucose decreased REC proliferation, an effect that was reversed by dexamethasone. High glucose conditions significantly increased REC permeability and decreased claudin-5, occludin and JAM-A gene expression; dexamethasone was effective in partially reversing these changes, restoring EC permeability to the normal or near normal state. High glucose levels resulted in reduction of Ang1 secretion, although Ang2 levels were consistently high. DEX increased Ang1 and decreased Ang2, indicating that the balance of Ang1/Ang2 may be important in determining functional changes in REC under high glucose conditions.
The primary cause for mortality and morbidity in patients with diabetes is atherosclerosis, an inflammatory vascular disease. Over the past decade, it has become increasingly clear that in a diabetic milieu, oxidative stress plays a vital role in the pathogenesis of diabetes-associated atherosclerosis. In this chapter, we focus on mechanisms whereby oxidative stress contributes to the accelerated progression of atherosclerosis in diabetes. In particular, our emphasis is on experimental evidence from animal models, in which specific components leading to oxidative stress are targeted via genetic manipulation. We also explore the use of targeted pharmacological compounds to lower oxidative stress levels. Furthermore, we discuss novel antioxidant compounds that are proving to be beneficial in preclinical trials. Thus, by gaining an understanding of the molecular mechanisms by which diabetes-induced oxidative stress accelerates the progression of atherosclerosis, it is hoped that this will lead to the generation of new therapies that are more efficacious in reducing the burden of diabetic complications. © Springer-Verlag Berlin Heidelberg 2014. All rights are reserved.
Objective:
Recent studies have demonstrated that Ang1-7 has anti-inflammatory effects. Since the formation of Ang1-7 is significantly altered in the setting of diabetes, here we aimed to evaluate whether Ang1-7 infusion could ameliorate diabetes-induced leukocyte recruitment.
Methods:
Wild-type male Wistar rats were randomly allocated to the following groups: control + saline, control + Ang1-7, diabetes + saline, diabetes + Ang1-7. Diabetes was induced by streptozotocin. Saline and Ang1-7 (576 μg/kg/day) were injected intraperitoneally daily. After 4 weeks leukocyte trafficking was studied in vivo by intravital microscopy in the mesenteric bed, where the expression of pro-oxidative, proinflammatory, and profibrotic molecules was also assessed. In parallel in vitro studies, HUVEC were grown in 5 mM, 22 mM, 30 mM, 40 mM, 50 mM, and 75 mM glucose media for 48 h, 72 h and 6 days and were treated either with placebo, or with Ang1-7, or with Ang1-7 and its inhibitor A779 in order to evaluate the expression of ICAM-1 and VCAM-1. We further studied leukocytes recruitment in vitro by evaluating PMN-HUVEC adhesion.
Results:
Ang1-7 prevented in vivo diabetes-induced leukocyte adhesion and extravasation, and it significantly reduced vascular hypertrophy and the other molecular changes due to diabetes. Ang 1-7 prevented also in vitro the hyperglycemia-induced increase of ICAM-1 and VCAM-1 as well as the hyperglycemia-induced PMN adhesion. A779 inhibited Ang 1-7 effects.
Conclusions:
Ang1-7 significantly reduced diabetes-induced leukocyte recruitment both in vivo and in vitro. These findings emphasize the potential utility of ACE2/Ang1-7/Mas repletion as a strategy to reduce diabetes-induced atherosclerosis.
An increase in intracellular calcium represents one of the early events during an ischaemic stroke. It triggers many downstream processes which promote the formation of brain oedema, the leading cause of death after an ischaemic stroke. As impairment of blood-brain barrier (BBB) accounts for much of oedema formation, the current study explored the impact of intracellular calcium on barrier integrity in relation to protein kinase C, caspase-3/7, plasminogen activators and the pro-oxidant enzyme NADPH oxidase. Human brain microvascular endothelial cells alone or in co-culture with human astrocytes were subjected to 4h of oxygen-glucose deprivation alone or followed by 20h of reperfusion (OGD±R) in the absence or presence of inhibitors for urokinase plasminogen activator (amiloride), NADPH oxidase (apocynin), intracellular calcium (BAPTA-AM) and protein kinase C-α (RO-32-0432). Endothelial cells with protein kinase C-α knockdown, achieved by siRNA, were also exposed to the above conditions. BBB permeability was measured by transendothelial electrical resistance and Evan's blue-albumin and sodium fluorescein flux. Intracellular calcium and total superoxide anion levels, caspase-3/7, NADPH oxidase, plasminogen activator and protein kinase C activities, stress fibre formation, the rate of apoptosis and BBB permeability were increased by OGD±R. Treatment with the specific inhibitors or knockdown of protein kinase C-α attenuated them. This study reveals successive increases in intracellular calcium levels and protein kinase C-α activity are key mechanisms in OGD±R-mediated impairment of BBB. Furthermore inhibition of protein kinase C-α may be therapeutic in restoring BBB function by reducing the rate of cytoskeletal reorganisation, oxidative stress and apoptosis.
Diabetic macular oedema (DMO) is responsible for significant visual impairment in diabetic patients. The primary cause of DMO is fluid leakage resulting from increased vascular permeability through contributory anatomical and biochemical changes. These include endothelial cell (EC) death or dysfunction, pericyte loss or dysfunction, thickened basement membrane, loss or dysfunction of glial cells, and loss/change of EC Glycocalyx. The molecular changes include increased reactive oxygen species, pro-inflammatory changes: advanced glycation end products, intracellular adhesion molecule-1, Complement 5-9 deposition and cytokines, which result in increased paracellular permeability, tight junction disruption, and increased transcellular permeability. Laser photocoagulation has been the mainstay of treatment until recently when pharmacological treatments were introduced. The current treatments for DMO target reducing vascular leak in the macula once it has occurred, they do not attempt to treat the underlying pathology. These pharmacological treatments are aimed at antagonising vascular endothelial growth factor (VEGF) or non-VEGF inflammatory pathways, and include intravitreal injections of anti-VEGFs (ranibizumab, aflibercept or bevacizumab) or steroids (fluocinolone, dexamethasone or triamcinolone) as single therapies. The available evidence suggests that each individual treatment modality in DMO does not result in a completely dry macula in most cases. The ideal treatment for DMO should improve vision and improve morphological changes in the macular (eg, reduce macular oedema) for a significant duration, reduced adverse events, reduced treatment burden and costs, and be well tolerated by patients. This review evaluates the individual treatments available as monotherapies, and discusses the rationale and potential for combination therapy in DMO. A comprehensive review of clinical trials related to DMO and their outcomes was completed. Where phase III randomised control trials were available, these were referenced, if not available, phase II trials have been included.Eye advance online publication, 26 June 2015; doi:10.1038/eye.2015.110.
Background:
Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis.
Methods and results:
Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR(-/-) mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and upregulated vascular cell adhesion molecule 1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced endothelial nitric oxide synthase expression. p90RSK activity was increased in diabetic mouse vessels, and fluoromethyl ketone-methoxyethylamine, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in diabetic mice. Interestingly, in ERK5-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of fluoromethyl ketone-methoxyethylamine, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of fluoromethyl ketone-methoxyethylamine on endothelial dysfunction. Fluoromethyl ketone-methoxyethylamine also inhibited atherosclerosis formation in ApoE(-/-) mice.
Conclusions:
Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in diabetes mellitus and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.
Diabetic retinopathy (DR) is the leading cause of blindness amongst the working-age population, and diabetes acceleratedcardiovascular disease (CVD) the commonest cause of death in diabetic patients. Although, there is evidence suggesting a close association between DR and CVD, particularly in patients with Type 2 diabetes, the pathophysiology underlying the link is unclear. Here we review common risk factors and pathogenic mechanisms linking DR and CVD, and aim to highlight the need for a more holistic view of the management of diabetes and its complications. The understanding of the link between the two complications could eventually lead to refined management strategies and improved patient outcomes in the expanding diabetes epidemic.
Purpose of the review: Mortality from cardiovascular complications is largely responsible for the guarded prognosis of patients with diabetes mellitus. One important event that contributes to these cardiovascular complications is endothelial dysfunction, an early, yet reversible feature of diabetes. Endothelial function is closely associated with nitric oxide biology and signaling and is particularly sensitive to oxidative stress that is associated with diabetes mellitus. The purpose of this brief review, therefore, is to highlight recent findings concerning the mechanisms of diabetic endothelial dysfunction with a particular focus on oxidative stress. Recent findings: Several sources of superoxide have been identified in diabetes including NADPH oxidases, mitochondria, and endothelial nitric oxide synthase that cause endothelial dysfunction through peroxynitrite formation. In addition, tyrosine nitration of the prostacyclin synthase by peroxynitrite leads to decreased prostacyclin formation and is a novel mechanism of oxidative stress impairing endothelial function independent of nitric oxide bioavailability. Although unrelated to oxidative stress, exciting evidence points to a role for endothelial progenitor cells with regard to their decreased availability and impaired function in diabetes that may play a role in shaping future investigative strategies. Summary: Thus, increasing evidence over the past few years has implicated oxidative stress as a central feature of impaired endothelial function in diabetes. These recent findings should help to identify strategies to improve endothelial function in diabetes and, perhaps, reduce the vascular the complications of this disease.
The effects of intermittent and constant high glucose in the formation of nitrotyrosine and 8-hydroxydeoxyguanosine (markers of oxidative stress), as well as the possible linkage between oxidative stress and apoptosis in endothelial cells, have been evaluated. Stable high glucose increased nitrotyrosine, 8-hydroxydeoxyguanosine (8-OHdG), and apoptosis levels. However, these effects were more pronounced in intermittent high glucose. Protein kinase C (PKC) was elevated in both such conditions, particularly in intermittent glucose. The adding of the PKC inhibitors bisindolylmaleimide-I and LY379196, a specific inhibitor of PKC-beta isoforms, normalized nitrotyrosine and reduced 8-OHdG concentration and cell apoptosis in both stable and intermittent high glucose. Similar results were obtained with the MnSOD mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride that normalized nitrotyrosine, 8-OHdG, and apoptosis and inhibited PKC activation. NAD(P)H oxidase was also measured. NAD(P)H oxidase components p47phox, p67phox, and p22phox was overexpressed during both stable and intermittent high glucose. PKC inhibition and MnSOD mimetic normalized this phenomenon. In conclusion, our study shows that the exposure of endothelial cells to both stable and intermittent high glucose stimulates reactive oxygen species overproduction also through PKC-dependent activation of NAD(P)H oxidase, leading to increased cellular apoptosis. Our data suggest that glucose fluctuations may also be involved in the development of vascular injury in diabetes.
This chapter examines the unique problems and alterations of the diverse forms of vascular regulation caused by obesity, insufficient insulin, and insulin resistance. Much of the chapter focuses on changes in microvascular regulation in obesity as a prelude to the more serious consequences of insulin-independent diabetes in the obese animal and human. The issues of insulin resistance of obesity and eventual development of hyperglycemia despite very high circulating concentrations of insulin appear to synergistically interact to compromise both microvascular and macrovascular functions. Medical intervention with pharmacology, diet, and exercise allows the vast majority of persons with Types I and II diabetes with even modestly successful treatment to have very few bouts of severe hyperglycemia. Although there are similarities in the final macro- and microvascular outcomes of both Type I (insulin-deficient) and Type II (insulin-resistant) diabetes mellitus, the vast majority of Type I diabetes occurs in juvenile life while the body is growing. This raises an interesting possibility that disturbances of microvascular growth could impact both the juvenile vasculature and type of microvascular morphology present once the juvenile reaches adult life. Despite intervention, microvascular disease leading to impaired renal function, blindness, and skin damage and accelerated atherosclerosis of the macrovessels associated with stroke and myocardial infarction continue to be major issues for the Type I diabetic population. Probable mechanisms of impaired endothelial and vascular smooth muscle function due to inadequate insulin action and hyperglycemia are discussed.
A clear relationship between diabetes and cardiovascular disease has been established for decades. Despite this, the mechanisms by which diabetes contributes to plaque formation remain in question. Some of this confusion derives from studies in type 2 diabetics where multiple components of metabolic syndrome show proatherosclerotic effects independent of underlying diabetes. However, the hyperglycemia that defines the diabetic condition independently affects atherogenesis in cell culture systems, animal models, and human patients. Endothelial cell biology plays a central role in atherosclerotic plaque formation regulating vessel permeability, inflammation, and thrombosis. The current paper highlights the mechanisms by which hyperglycemia affects endothelial cell biology to promote plaque formation.
It is becoming increasingly clear that suboptimal blood glucose control results in adverse effects on large blood vessels, thereby accelerating atherosclerosis and cardiovascular disease, manifested as myocardial infarction, stroke, and peripheral vascular disease. Cardiovascular disease is accelerated by both type 1 and type 2 diabetes. In type 1 diabetes, hyperglycemia generally occurs in the absence of elevated blood lipid levels, whereas type 2 diabetes is frequently associated with dyslipidemia. In this review article, we discuss hyperglycemia versus hyperlipidemia as culprits in diabetes-accelerated atherosclerosis and cardiovascular disease, with emphasis on studies in mouse models and isolated vascular cells. Recent studies on LDL receptor-deficient mice that are hyperglycemic, but exhibit no marked dyslipidemia compared with nondiabetic controls, show that diabetes in the absence of diabetes-induced hyperlipidemia is associated with an accelerated formation of atherosclerotic lesions, similar to what is seen in fat-fed nondiabetic mice. These effects of diabetes are masked in severely dyslipidemic mice, suggesting that the effects of glucose and lipids on lesion initiation might be mediated by similar mechanisms. Recent evidence from isolated endothelial cells demonstrates that glucose and lipids can induce endothelial dysfunction through similar intracellular mechanisms. Analogous effects of glucose and lipids are also seen in macrophages. Furthermore, glucose exerts many of its cellular effects through lipid mediators. We propose that diabetes without associated dyslipidemia accelerates atherosclerosis by mechanisms that can also be activated by hyperlipidemia.
Micro- and macro-vascular complications are the leading causes of morbidity and mortality in type 1 and type 2 diabetic patients. Despite the vast clinical experience linking diabetic metabolic abnormalities to cardiovascular lesions, the molecular basis of individual susceptibility to diabetic cardiovascular injury is still largely unknown. Significant advances in this area may come from studies on suitable animal models. Although no animal model can accurately reproduce the human disease, experimental studies in animals have the great advantage to eliminate factors such as ethnicity, economic and geographic variables, drug interactions, diet, gender and age differences that importantly limit clinical studies. Indeed, appropriate animal models have provided important information on genetic and environmental risks of diabetes, and helped to dissect molecular mechanisms underlying the development, progression and therapeutic control of this disease. Unfortunately, none of the diabetic models presently available fully mimics the human syndrome. Therefore, the current knowledge on the pathogenesis of cardiovascular complications relies on the evaluation of distinct phenotypes from various diabetic models. In addition to strains prone to diabetes, this disease can be induced by surgical, pharmacological or genetic manipulation in several animal species. Rodents are the most used, although some studies are still performed in larger animals as rabbits, cats, pigs or monkeys. Far from being exhaustive, this work should serve as a general overview of the most relevant clues provided by major species and models for the overall comprehension of cardiovascular complications in type 1 and type 2 diabetes.
In vivo microscopy was used to assess the relationships among shear rate (and shear stress), leukocyte rolling velocity, and leukocyte adherence in a cat mesentery preparation. Shear rate in individual venules and arterioles of 25-35 microns diameter were varied over a wide range by graded occlusion of an arterial loop. There was a linear decline in leukocyte rolling velocity (Vwbc) as red cell velocity (Vrbc) was reduced. The ratio Vwbc/Vrbc remained constant despite variations in shear stress from 5-25 dyn/cm2. A reduction in shear stress was associated with an increased leukocyte adherence, particularly when Vwbc was reduced below 50 microns/s. Reduction in wall shear rate below 500 s-1 in arterioles allowed 1-3 leukocytes to adhere per 100 microns length of vessel, while venules exposed to the same shear rates had 5-16 adherent leukocytes. In arterioles, leukocyte rolling was only observed at low shear rates. At shear rates less than 250 s-1 leukocyte rolling velocity was faster in arterioles than venules, and the ratio Vwbc/Vrbc for arterioles was 0.08 +/- 0.02, which was fourfold higher than the ratio obtained in venules at similar shear rates. Pretreatment with the CD18-specific antibody (mAb) IB4 increased leukocyte rolling velocity in venules by approximately 20 microns/s at red cell velocities below 2,000 microns/s. mAb IB4 largely prevented the leukocyte adherence to arterioles and venules, and increased the ratio Vwbc/Vrbc observed in venules at low shear elicit a CD18-dependent adhesive interaction between leukocytes and microvascular endothelium, and that differences in shear rates cannot explain the greater propensity for leukocyte rolling and adhesion in venules than arterioles.
The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.
The present experiments were undertaken to examine the hypothesis that glucose-induced increased de novo synthesis of 1,2-diacyl-sn-glycerol (which has been observed in a number of different tissues, including retinal capillary endothelial cells exposed to elevated glucose levels in vitro) and associated activation of protein kinase C may play a role in mediating glucose-induced vascular functional changes. We report here that twice daily instillation of 30 mM glucose over 10 d in a rat skin chamber granulation tissue model induces approximately a 2.7-fold increase in diacylglycerol (DAG) levels (versus tissues exposed to 5 mM glucose) in association with marked increases in vascular clearance of albumin and blood flow. The glucose-induced increase in DAG levels as well as the vascular functional changes are prevented by addition of 3 mM pyruvate. Pharmacological activation of protein kinase C with the phorbol ester TPA in the presence of 5 mM glucose increases microvascular albumin clearance and blood flow, and similar effects are observed with 1-monoolein (MOG), a pharmacological inhibitor of the catabolism of endogenous DAG. A pharmacological inhibitor of protein kinase C (staurosporine) greatly attenuates the rise in microvascular albumin clearance (but not the rise in blood flow) induced by glucose or by MOG. These findings are compatible with the hypothesis that elevated concentrations of glucose increase tissue DAG content via de novo synthesis, resulting in protein kinase C activation, and that these biochemical events are among the factors that generate the increased microvascular albumin clearance.
125I-labeled albumin permeation (IAP) has been assessed in various tissues in spontaneously diabetic insulin-dependent female BB/W rats and in male Sprague-Dawley rats with severe or mild forms of streptozocin-induced diabetes (SS-D and MS-D, respectively). In BB/W diabetic rats and in rats with SS-D, indices of IAP were significantly increased in tissues and vessels predisposed to diabetic vascular disease in humans, including the eyes (anterior uvea, posterior uvea, and retina), sciatic nerve, aorta, kidney, and new vessels formed after induction of diabetes. No evidence of increased IAP was observed in heart, brain, testes, or skeletal muscle in BB/W or SS-D rats. In MS-D rats, indices of IAP were increased only in the kidney and in new vessels formed after the onset of diabetes.
Marked tissue differences were observed in the effects of two structurally different aldose reductase inhibitors (sorbinil and tolrestat) and of castration on diabetes-induced increases in IAP and in tissue levels of polyols in SS-D rats. Both aldose reductase inhibitors and castration completely prevented diabetes-induced increases in IAP in new vessels and in sciatic nerve in BB/W and SS-D rats. Both aldose reductase inhibitors also markedly decreased IAP in the anterior uvea (∼85%), posterior uvea (∼65-75%), retina (∼65-70%), and kidney (∼70-100%); castration reduced IAP in the anterior uvea (∼55%), kidney (∼50%), and retina (∼30%) but had no effect on the posterior uvea. The diabetes-induced increases in IAP in the aorta were reduced only slightly (∼20%) by aldose reductase inhibitors and castration.
In SS-D rats, tissue polyol levels were markedly increased in sciatic nerve, retina, anterior uvea, and posterior uvea but not in the aorta. Castration significantly reduced polyol levels only in the sciatic nerve and retina, whereas both aldose reductase inhibitors markedly decreased polyol levels in anterior and posterior uvea and in sciatic nerve and retina.
These observations demonstrate that 1) the most characteristic functional alteration in vessels in human diabetics, i.e., increased vascular permeability, is evident in rats with diabetes of only 3 wk duration; 2) the most marked increases in IAP in the diabetic rat occur in vessels associated with clinically significant vascular disease in human diabetics; 3) even very mild diabetes is associated with increased IAP in the kidney and in new vessels induced by angiogenesis in the diabetic milieu; 4) diabetes-induced increases in IAP in all tissues examined are aldose reductase-linked phenomena and, with the exception of the posterior uvea, are modulated by sex steroids; and 5) the extent to which diabetes-induced increases in IAP are modulated by sex steroids and reduced or completely prevented by aldose reductase inhibitors varies markedly in different vascular beds.
The time course of endothelial P-selectin, ICAM-1, and E-selectin expression was studied in a feline model of myocardial ischemia and reperfusion. Cats were subjected to 90 min of myocardial ischemia followed by 0, 10, 20, 60, 150, or 270 min of reperfusion. At the end of reperfusion, the coronary vasculature was examined immunohistochemically to localize monoclonal antibodies (mAbs) PB1.3, RR1/1, and Cy1787 directed against P-selectin, ICAM-1, and E-selectin, respectively. Immunohistochemical localization for P-selectin, recognized by mAb PB1.3, was maximally expressed 20 min after reperfusion in 60 +/- 6% of coronary venules (P < 0.05 compared to non-reperfused controls), and covered 59 +/- 3% of the endothelial cell perimeter of immunostained coronary venules. Immunolocalization of mAb PB1.3 gradually declined at 60, 150, and 270 min of reperfusion. Immunohistochemical localization of mAb RR1/1 (anti-ICAM-1) in endothelial cells of coronary venules was observed to a modest extent in non-ischemic myocardium and at 10, 20, and 60 min of reperfusion, but was significantly increased following 150 and 270 min of reperfusion (P < 0.05 compared non-reperfused controls). At 270 min post-reperfusion, mAb RR1/1 was seen in 50 +/- 4% of coronary venules. Endothelial immunolocalization of mAb Cy1787 (anti-E-selectin) was only observed in 13 +/- 1 and 14 +/- 3% of coronary venules after 150 and 270 min of reperfusion, respectively, suggesting that pronounced expression of E-selectin does not occur within 270 min after reperfusion. These results demonstrate sequential expression of three major endothelial cell adherence molecules in situ following myocardial ischemia and reperfusion. The timing of endothelial cell expressed P-selectin and ICAM-1 could coordinate neutrophil trafficking during the early stages of reperfusion.
Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase receptors. We have characterized the VEGF-activated intracellular signal transduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects. VEGF induced concentration- and time-dependent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10-10 M within 10 min as measured both by in situ and translocation assays. Immunoblotting analysis of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca2+-sensitive PKC isoforms (α and βII) was increased in the membrane fractions, whereas no changes were observed for PKC isoforms δ and ε. The stimulation of PKC activity by VEGF was preceded by the activation of phospholipase Cγ (PLCγ). This was demonstrated by parallel increases in PLCγ tyrosine phosphorylation, [3H]inositol phosphate production, and [3H]arachidonic acid-labeled diacylglycerol formation in bovine aortic endothelial cells. In addition, VEGF increased phosphatidylinositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF-induced increase in PKC activity or endothelial cell growth. Interestingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inhibitors, abolished both VEGF- induced PKC activation and endothelial cell proliferation. VEGF's mitogenic effect was inhibited by a PKC isoform β-selective inhibitor, LY33-3531, in a concentration-dependent manner. In contrast, antisense PKC-α oligonucleotides enhanced VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC-α protein content. Thus, VEGF appears to mediate its mitogenic effects partly through the activation of the PLCγ and PKC pathway, involving predominately PKC-β isoform activation in endothelial cells.
We addressed the role of hyperglycemia in leukocyte-endothelium interaction under flow conditions by exposing human umbilical vein endothelial cells for 24 h to normal (5 mM), high concentration of glucose (30 mM), advanced glycosylation end product-albumin (100 microg/ml), or hyperglycemic (174-316 mg/dl) sera from patients with diabetes and abnormal hemoglobin A1c (8.1+/-1.4%). At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dyn/cm2). Rolling and adherent cells were evaluated by digital image processing. Results showed that 30 mM glucose significantly (P < 0. 01) increased the number of adherent leukocytes to endothelial cells in respect to control (5 mM glucose; 151+/-19 versus 33+/-8 cells/mm2). A similar response was induced by endothelial stimulation with IL-1beta, here used as positive control (195+/-20 cells/mm2). The number of rolling cells on endothelial surface was not affected by high glucose level. Stable adhesion of leukocytes to glucose-treated as well as to IL-1beta-stimulated endothelial cells was preceded by short interaction of leukocytes with the endothelial surface. The distance travelled by leukocytes before arrest on 30 mM glucose, or on IL-1beta-treated endothelial cells, was significantly (P < 0.01) higher than that observed for leukocytes adhering on control endothelium (30 mM glucose: 76.7+/-3.5; IL1beta: 69.7+/-4 versus 5 mM glucose: 21.5+/-5 microm). Functional blocking of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells with the corresponding mouse mAb significantly inhibited glucose-induced increase in leukocyte adhesion (67+/-16, 83+/-12, 62+/-8 versus 144+/-21 cells/ mm2). Confocal fluorescence microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Electrophoretic mobility shift assay of nuclear extracts of human umbilical vein endothelial cells (HUVEC) exposed for 1 h to 30 mM glucose revealed an intense NF-kB activation. Treatment of HUVEC exposed to high glucose with the NF-kB inhibitors pyrrolidinedithiocarbamate (100 microM) and tosyl-phe-chloromethylketone (25 microM) significantly reduced (P < 0.05) leukocyte adhesion in respect to HUVEC treated with glucose alone. A significant (P < 0.01) inhibitory effect on glucose-induced leukocyte adhesion was observed after blocking protein kinase C activity with staurosporine (5 nM). When HUVEC were treated with specific antisense oligodesoxynucleotides against PKCalpha and PKCepsilon isoforms before the addition of 30 mM glucose, a significant (P < 0.05) reduction in the adhesion was also seen. Advanced glycosylation end product-albumin significantly increased the number of adhering leukocytes in respect to native albumin used as control (110+/-16 versus 66+/-7, P < 0.01). Sera from diabetic patients significantly (P < 0.01) enhanced leukocyte adhesion as compared with controls, despite normal levels of IL-1beta and TNFalpha in these sera. These data indicate that high glucose concentration and hyperglycemia promote leukocyte adhesion to the endothelium through upregulation of cell surface expression of adhesive proteins, possibly depending on NF-kB activation.
Recent studies have identified that the activation of protein kinase C (PKC) and increased diacylglycerol (DAG) levels initiated by hyperglycemia are associated with many vascular abnormalities in retinal, renal, and cardiovascular tissues. Among the various PKC isoforms, the beta- and delta-isoforms appear to be activated preferentially in the vasculatures of diabetic animals, although other PKC isoforms are also increased in the renal glomeruli and retina. The glucose-induced activation of PKC has been shown to increase the production of extracellular matrix and cytokines; to enhance contractility, permeability, and vascular cell proliferation; to induce the activation of cytosolic phospholipase A2; and to inhibit Na+-K+-ATPase. The synthesis and characterization of a specific inhibitor for PKC-beta isoforms have confirmed the role of PKC activation in mediating hyperglycemic effects on vascular cells, as described above, and provide in vivo evidence that PKC activation could be responsible for abnormal retinal and renal hemodynamics in diabetic animals. Transgenic mice overexpressing PKC-beta isoform in the myocardium developed cardiac hypertrophy and failure, further supporting the hypothesis that PKC-beta isoform activation can cause vascular dysfunctions. Interestingly, hyperglycemia-induced oxidative stress may also mediate the adverse effects of PKC-beta isoforms by the activation of the DAG-PKC pathway, since treatment with D-alpha-tocopherol was able to prevent many glucose-induced vascular dysfunctions and inhibit DAG-PKC activation. Clinical studies are now in progress to determine whether PKC-beta inhibition can prevent diabetic complications.
Diabetic hyperglycaemia causes a variety of pathological changes in small vessels, arteries and peripheral nerves. Vascular endothelial cells are an important target of hyperglycaemic damage, but the mechanisms underlying this damage are not fully understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C isoforms; increased formation of glucose-derived advanced glycation end-products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycaemia-induced abnormalities. Hyperglycaemia increases the production of reactive oxygen species inside cultured bovine aortic endothelial cells. Here we show that this increase in reactive oxygen species is prevented by an inhibitor of electron transport chain complex II, by an uncoupler of oxidative phosphorylation, by uncoupling protein-1 and by manganese superoxide dismutase. Normalizing levels of mitochondrial reactive oxygen species with each of these agents prevents glucose-induced activation of protein kinase C, formation of advanced glycation end-products, sorbitol accumulation and NFkappaB activation.
Objective: Previous studies suggest a role of superoxide anion radicals (·O2−) in impaired endothelium-dependent relaxation of diabetic blood vessels; however, the role of secondary reactive oxygen species remains unclear. In the present study, we investigated a role of various potential reactive oxygen species in diabetic endothelial dysfunction. Methods: Thoracic aortic rings from 8-week streptozotocin-induced diabetic and age-matched control rats were mounted in isolated tissue baths. Endothelium-dependent relaxation to acetylcholine (ACH) and endothelium-independent relaxation to nitroglycerin (NTG) were assessed in precontracted rings. Results: ACH-induced relaxation was impaired in diabetic compared to control rings and was not improved with either indomethacin or daltroban. ACH-induced relaxation in both control and diabetic rings was completely blocked with the nitric oxide synthase inhibitors, l-nitroarginine methyl ester or l-nitroarginine (l-NA). NTG-induced relaxation was insensitive to l-NA and was unaltered by diabetes. Pretreatment with superoxide dismutase (SOD) at activities which did not alter contractile tone failed to alter responses to ACH in diabetic rings. Similar results were obtained using either catalase or mannitol. In contrast, the combination of SOD plus catalase or DETAPAC, an inhibitor of metal-facilitated hydroxyl radical (·OH) formation, markedly enhanced relaxation to ACH in diabetic but not in control rings. Neither the combination of SOD plus catalase nor DETAPAC altered the sensitivity or relaxation to NTG in control rings with or without endothelium. In diabetic rings with endothelium, both DETAPAC or SOD plus catalase increased sensitivity but not maximum relaxation to NTG. In diabetic rings without endothelium, relaxation and sensitivity to NTG were unaltered by either treatment. In l-NA-treated diabetic rings with endothelium, sensitivity and relaxation to NTG was unaltered by either DETAPAC or SOD plus catalase. Conclusion: Diabetic endothelium produces increases in both ·O2− and H2O2 leading to enhanced intracellular production of ·OH. Thus, ·OH are implicated in diabetes-induced endothelial dysfunction.
Impaired endothelium-dependent relaxation occurs in diabetic rabbit aorta and normal aorta exposed to elevated concentrations of glucose and is prevented by cyclooxygenase inhibitors. The role of free radicals in the endothelial cell impairment was examined with free radical scavengers and in aortas from rabbits fed with probucol (1% wt/wt, a lipid-soluble antioxidant). Rings of aorta suspended for measurement of isometric tension were incubated for 6 h in control (5.5 mM) or elevated (44 mM) glucose. Impairment of endothelium-dependent relaxation to acetylcholine caused by exposure to elevated glucose was prevented by superoxide dismutase, catalase, deferoxamine, or allopurinol and did not occur in aortas from probucol-fed rabbits. Similarly, impairment of acetylcholine relaxations in aortas from alloxan-induced diabetic rabbits was restored to normal by superoxide dismutase. Oxygen-derived free radicals generated by xanthine oxidase also caused impaired acetylcholine relaxations. Exposure of aortic segments to elevated glucose or to xanthine oxidase caused a significant increase in release of immunoreactive prostanoids. These data indicate that the endothelial cell dysfunction caused by elevated glucose is mediated by free radicals that are likely generated through the increased cyclooxygenase catalysis occurring in the endothelium. Treatment with antioxidants protects against impaired endothelium-dependent relaxations caused by elevated glucose.
Nodular expansion of glomerular mesangium with increased amounts of extracellular matrix (ECM) material is pathognomic of diabetic nephropathy. The precise mechanisms involved in this accumulation are unknown. Recently, we reported using a solid-phase enzyme-linked immunosorbent assay (ELISA) technique that glomerular mesangial cells, the principal cell type residing in glomerular mesangium, accumulate 50-60% more fibronectin (FN), laminin (LM), and type IV collagen (T-IV) when cultured in medium containing high glucose (30 mM) (S. H. Ayo, R. A. Rodnik, J. Garoni, W. F. Glass II, and J. I. Kreiberg. Am. J. Pathol. 136: 1339-1348, 1990). ECM assembly is controlled by its rate of synthesis and degradation, as well as its binding and rate of incorporation into the ECM. To elucidate the mechanisms involved, pulse-chase experiments were designed to estimate ECM protein synthesis from the incorporation of Trans-35S [( 35S]methionine, [35S]cysteine) into immunoprecipitated FN, LM, and T-IV. mRNA levels were examined, and degradation rates were estimated from the disappearance of radioactivity from matrix proteins in mesangial cells previously incubated with Trans-35S. One week of growth in 30 mM glucose resulted in approximately 40-50% increase in the synthesis of all three matrix proteins compared with 10 mM glucose-grown cells. This was accompanied by a significant increase in the transcripts for all three matrix proteins (approximately twofold). The specific activity of the radiolabel in trichloroacetic acid-precipitable cell protein showed no difference between cells grown in 10 or 30 mM glucose, indicating that total protein synthesis was unchanged. After 1 wk, the rate of FN, LM, and T-IV collagen degradation was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
A possible relationship between protein kinase C activation and impaired receptor-mediated endothelium-dependent relaxation in diabetes mellitus was examined in isolated aorta from normal rabbit exposed to elevated glucose. Aorta treated for 10 min with 4-phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, showed decreased relaxations to the endothelium-dependent vasodilator, acetylcholine, similar to normal aorta exposed to elevated glucose (22 and 44 mM) for 6 h. Relaxations to the receptor-independent endothelium-dependent vasodilator, A23187, and those caused by the direct smooth muscle vasodilator, sodium nitroprusside, were unaffected by treatment with PMA or exposure to elevated glucose. Indomethacin increased relaxations to acetylcholine of aorta treated with PMA indicating a role for vasoconstrictor prostanoids. PMA caused a significant increase in basal and acetylcholine-stimulated release of vasoconstrictor prostanoids including thromboxane A2 from aortic segments with, but not without endothelium. Protein kinase C inhibitors, H-7 or sphingosine, restored the abnormal acetylcholine-induced relaxations as well as suppressed the abnormal release of prostanoids in aorta exposed to elevated glucose. These findings suggest that the dysfunction of receptor-mediated endothelium-dependent relaxation associated with exposure to elevated glucose is due to increased production of vasoconstrictor prostanoids by the endothelium as a consequence of protein kinase C activation.
Exposure of skin chamber granulation tissue vessels in nondiabetic rats to 11 or 15 mM D-glucose (but not L-glucose or 3-O-methylglucose) twice daily for 10 d induces vascular functional changes (increased albumin permeation and blood flow) identical to those in animals with mild or severe streptozotocin diabetes, respectively. These vascular changes are strongly linked to increased metabolism of glucose via the sorbitol pathway and are independent of nonenzymatic glycosylation as well as systemic metabolic and hormonal imbalances associated with the diabetic milieu. (J. Clin. Invest. 1990. 85:1167-1172.)
Isolation of polymorphonuclear leukocytes (PMN) provides an opportunity to study PMN activity in vitro and to label PMN for study of in vivo kinetics. However, simple phlebotomy (SP) of a small animal frequently yields too few PMN for in vitro handling, while PMN harvested from an induced-peritonitis may not accurately reflect PMN in a less stimulated state. We report a novel method of harvesting PMN from the circulation of rats, using hetastarch exchange transfusion (HET), which is both time and animal sparing. HET harvested 8-fold more PMN than SP. In vitro cell function was examined with assays of adherence, chemotaxis, bacterial killing, and superoxide generation. No significant (p less than 0.05) difference was found between PMN obtained by HET and pooled-PMN obtained by SP. In vivo function was examined following labeling with indium111-oxine. The kinetics pattern described suggested normal migratory activity when compared to previous reports. The data demonstrate that rats possess a relatively large, noncirculating pool of PMN which is readily accessible by HET.
GMP-140 is an integral membrane glycoprotein found in secretory granules of platelets and endothelial cells. After cellular activation, it is rapidly redistributed to the plasma membrane. The cDNA-derived primary structure of GMP-140 predicts a cysteine-rich protein with multiple domains, including a "lectin" region, an "EGF" domain, nine tandem consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Some cDNAs also predict a soluble protein with a deleted transmembrane segment. The domain organization of GMP-140 is similar to that of ELAM-1, a cytokine-inducible endothelial cell receptor that binds neutrophils. This similarity suggests that GMP-140 belongs to a new family of inducible receptors with related structure and function on vascular cells.
Glomerular inositol content and the turnover of polyphosphoinositides was reduced by 58% in 1-2 wk streptozotocin diabetic rats. Addition of inositol to the incubation medium increased polyphosphoinositide turnover in glomeruli from diabetic rats to control values. Despite the reduction in inositol content and polyphosphoinositide turnover, protein kinase C was activated in glomeruli from diabetic rats, as assessed by an increase in the percentage of enzyme activity associated with the particulate cell fraction. Total protein kinase C activity was not different between glomeruli from control and diabetic rats. Treatment of diabetic rats with insulin to achieve near euglycemia prevented the increase in particulate protein kinase C. Moreover, incubation of glomeruli from control rats with glucose (100-1,000 mg/dl) resulted in a progressive increase in labeled diacylglycerol production and in the percentage of protein kinase C activity which was associated with the particulate fraction. These results support a role for hyperglycemia per se in the enhanced state of activation of protein kinase C seen in glomeruli from diabetic rats. Glucose did not appear to increase diacylglycerol by stimulating inositol phospholipid hydrolysis in glomeruli. Other pathways for diacylglycerol production, including de novo synthesis and phospholipase C mediated hydrolysis of phosphatidylcholine or phosphatidyl-inositol-glycan are not excluded.
A system has been implemented for measurement of red blood cell velocity in microvessels by using an optical Doppler technique. Ronchi rulings are used to stimulate a differential grating to translate red blood cell movement to light intensity variations. These variations are sensed by two photodiodes coupled in a resistive subtraction mode. The nonelectronic subtraction allows high transimpedance gains (2 X 10(9) V/I) while noise is held to a minimum (4.5 mv RMS in a 5-kHz bandwidth). To derive average velocity the average frequency determination of the amplified signal is performed with a thresholding frequency-to-voltage functional block. The velocimeter provides the typical performance features of an optical Doppler system, including high-frequency response, without the need for the complications of the laser Doppler technique or the requirement of custom micro-prism gratings. The device represents a cost-effective approach to intravital work, and offers significant improvements in performance over standard techniques.
Nitric oxide (NO) is an important molecular messenger accounting for endothelium-derived relaxing factor. Recently, NO synthase (NOS) from cultured endothelial cells has been purified and molecularly cloned. To evaluate the effect of phosphorylation by protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) on endothelial constitutive NOS catalytic activity, we incubated purified endothelial NOS with PKC or PKA. Endothelial NOS was stoichiometrically phosphorylated by PKC and PKA. In intact bovine aortic endothelial cells (BAECs), NOS was phosphorylated by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). NOS activity measured by the conversion of [3H]arginine to [3H]citrulline in homogenates of BAECs treated with TPA or phorbol 12,13-dibutyrate was reduced by 30%, whereas dibutylyl cyclic AMP did not affect NOS activity. Moreover, we measured NO release from cultured BAECs by a chemiluminescence method to examine the effect of PKC and PKA on endothelial NOS activity. In cultured BAECs, ATP gamma S and A23187 induced NO release in time- and dose-dependent manners. Phorbol esters such as TPA and phorbol 12,13-dibutyrate dose dependently inhibited NO release stimulated by A23187 as well as ATP gamma S. Reduction of NO release by TPA was almost completely prevented by pretreatment with staurosporine, an inhibitor of PKC. NO release by A23187 was increased in PKC-downregulated BAECs. In contrast, dibutylyl cyclic AMP or 8-bromo cyclic GMP had no effect on NO release from BAECs induced by A23187 or ATP gamma S. These results indicate that phosphorylation of NOS by PKC is associated with a reduction of its catalytic activity in vascular endothelial cells.
Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because No has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or L-NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or L-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7-hydroxystaurosporine). Furthermore, L-NAME-induced P-selectin expression was significantly attenuated by either L-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly, L-NAME further potentiated P-selectin upregulation by thrombin. L-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti-P-selectin monoclonal antibody PB1.3 or by UCN-01, L-arginine, 8-bromo-cGMP or SNP but not by D-arginine or he nonblocking anti-P-selectin monoclonal antibody NBP1.6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of L-NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression.
The increases in diacylglycerol (DAG) level and protein kinase C (PKC) activity have been characterized biochemically and functionally in the retina and the brain of diabetic rats as well as in cultured vascular cells. PKC specific activities were increased in the membraneous fraction of retina from streptozotocin (STZ)-induced diabetic rats and the genetically determined diabetic BB rats, respectively, after 1 or 2 wk of diabetes, compared with control. The ratio of total PKC activities from membraneous and cytosol fractions was also increased in the retina of diabetic rats. With diabetes, all the isoenzymes and the total DAG level were increased in the rat retina, whereas no changes were found in the rat brain. Insulin treatment normalized plasma glucose levels and partially prevented the increases in the membraneous PKC activity and all the isoenzymes in the retina. In the retinal endothelial cells, the total DAG level and PKC specific activities are increased by 36 and 22%, respectively, in the membraneous pool when the glucose levels are changed from 5.5 to 22 mM. Activation of PKC activity and isoform beta II by the vitreal injection of phorbol dibutyrate mimicked the abnormal retinal blood circulation observed in diabetic rats (2.22 +/- 0.24 vs. 1.83 +/- 0.40 s). Thus diabetes and elevated glucose levels will increase DAG level and PKC activities and its isoenzyme specifically in vascular cells and may affect retinal hemodynamics.
We measured changes in basal release of nitric oxide and its effect on polymorphonuclear leukocyte (PMN) adherence to endothelial cells (ECs) in a feline model of myocardial ischemia (90 minutes) and reperfusion. Basal release of nitric oxide from the left anterior descending coronary artery (LAD) after myocardial ischemia/reperfusion and from the control left circumflex coronary artery (LCX) was assessed by NG-nitro L-arginine methyl ester (L-NAME)-induced vasocontraction. L-NAME induced a significant EC-dependent vasocontraction in control LCX rings (0.28 +/- 0.04 g), which was fully reversed by L-arginine but not D-arginine. L-NAME-induced vasocontraction of LAD rings was not significantly changed after 90 minutes of myocardial ischemia without reperfusion. However, 10 minutes of reperfusion reduced the L-NAME-induced vasocontraction to 0.13 +/- 0.04 g (p < 0.05), and this was restored by addition of 3 mM L-arginine but not D-arginine. Longer periods of reperfusion progressively decreased L-NAME-induced vasocontraction. After 270 minutes of reperfusion, L-NAME-induced vasocontraction was virtually abolished. Myocardial ischemia without reperfusion did not increase PMN adherence to ECs. However, PMN adherence to LAD ECs was significantly increased after 20 minutes of reperfusion (39 +/- 6 to 105 +/- 9 PMNs/mm2, p < 0.01), and incubation of LAD segments with L-arginine significantly attenuated this increase in PMN adherence. After 270 minutes of reperfusion, PMN adherence to LAD ECs was further increased to 224 +/- 10 PMNs/mm2 (p < 0.001). This increase in PMN adherence was almost completely blocked by MAb R15.7, a monoclonal antibody against CD18 of PMNs, and was significantly attenuated by MAb RR1/1, a monoclonal antibody against intercellular adhesion molecule-1 of ECs (p < 0.01). These results indicate that decreased basal release of endothelium-derived relaxing factor after myocardial ischemia/reperfusion precedes enhanced PMN adherence to the coronary endothelium, which may lead to PMN-induced myocardial injury.
The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531,
a specific inhibitor of the β isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC
β1 and β2, with a half-maximal inhibitory constant of ∼5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth
of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion
rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.
Diabetes is associated with an increased incidence of ischemic organ damage. The objectives of present study were to compare the leukocyte-endothelial cell adhesive interactions and albumin leakage response of mesenteric venules to ischemia-reperfusion between control rats, rats with streptozotocin-induced diabetes, and rats with hyperglycemia induced by glucose infusion and to define the molecular determinants of the leukocyte accumulation elicited by ischemia-reperfusion in diabetic rats.
Under baseline conditions, lower venular shear rates and an increased number of rolling leukocytes were noted in diabetic rats, whereas the number of adherent and emigrated leukocytes did not differ from that in control rats. Spontaneous albumin leakage from mesenteric venules was markedly increased in diabetic rats but not in hyperglycemic nondiabetic rats. Ischemia-reperfusion elicited significantly larger increases in leukocyte adhesion and emigration and albumin leakage in diabetic rats. Acute elevation of glucose levels did not modify the microvascular responses to ischemia-reperfusion compared with control rats. Antibodies directed against CD11/CD18, intercellular adhesion molecule-1 (ICAM-1), or P-selectin but not L-selectin significantly decreased the number of adherent and emigrated leukocytes after ischemia-reperfusion in diabetic rats. However, none of the antibodies significantly attenuated the increased albumin leakage response to ischemia-reperfusion in diabetic rats.
These results indicate that diabetes mellitus is associated with exaggerated leukocyte-endothelial cell adhesion and albumin leakage responses to ischemia-reperfusion. The enhanced leukocyte accumulation in response to ischemia-reperfusion is mediated by CD11/CD18-ICAM-1 interactions (firm adhesion) and P-selectin (rolling). The exaggerated albumin leakage response to ischemia-reperfusion in diabetics is not mediated by the recruited inflammatory cells.
Previous studies suggest a role of superoxide anion radicals (.O2-) in impaired endothelium-dependent relaxation of diabetic blood vessels; however, the role of secondary reactive oxygen species remains unclear. In the present study, we investigated a role of various potential reactive oxygen species in diabetic endothelial dysfunction.
Thoracic aortic rings from 8-week streptozotocin-induced diabetic and age-matched control rats were mounted in isolated tissue baths. Endothelium-dependent relaxation to acetylcholine (ACH) and endothelium-independent relaxation to nitroglycerin (NTG) were assessed in precontracted rings.
ACH-induced relaxation was impaired in diabetic compared to control rings and was not improved with either indomethacin or daltroban. ACH-induced relaxation in both control and diabetic rings was completely blocked with the nitric oxide synthase inhibitors, L-nitroarginine methyl ester or L-nitroarginine (L-NA). NTG-induced relaxation was insensitive to L-NA and was unaltered by diabetes. Pretreatment with superoxide dismutase (SOD) at activities which did not alter contractile tone failed to alter response to ACH in diabetic rings. Similar results were obtained using either catalase or mannitol. In contrast, the combination of SOD plus catalase or DETAPAC, an inhibitor of metal-facilitated hydroxyl radical (.OH) formation, markedly enhanced relaxation to ACH in diabetic but not in control rings. Neither the combination of SOD plus catalase nor DETAPAC altered the sensitivity or relaxation to NTG in control rings with or without endothelium. In diabetic rings with endothelium, both DETAPAC or SOD plus catalase increased sensitivity but not maximum relaxation to NTG. In diabetic rings without endothelium, relaxation and sensitivity to NTG were unaltered by either treatment. In L-NA-treated diabetic rings with endothelium, sensitivity and relaxation to NTG was unaltered by either DETAPAC or SOD plus catalase.
Diabetic endothelium produces increases in both .O2- and H2O2 leading to enhanced intracellular production of .OH. Thus, .OH are implicated in diabetes-induced endothelial dysfunction.
Telomerase is a specialized ribonucleoprotein polymerase that adds hexanucleotides (TTAGGG) onto human chromosomal ends. The expression of telomerase activity has been associated with cell immortalization and the malignant phenotype in most cancers. How the telomerase activity is regulated in cancer cells is presently not known. In this work, the effects of cell cycle blockers, DNA damaging agents, TopII inhibitors and proteins kinase inhibitors on the telomerase activity were examined in cultured nasopharyngeal carcinoma cells NPC-076. Agents which interfere with tubulin assembly (Taxol and vinblastine) and agents which arrest cells at S phase (methotrexate and 5-fluorouracil) did not inhibit telomerase activity of treated cells. Agents which damage DNA (cisplatin, methyl methanesulfonate, and UV radiation) and TopII inhibitors (etoposide and daunorubicin) also did not inhibit telomerase activity of treated cells. Among the protein kinase inhibitors examined, no significant inhibition of telomerase activity was observed with cells treated with quercetin, H-89, or herbimycin A. On the other hand, two protein kinase C (PKC) inhibitors (bisindolylmaleimide I and H-7) were found to produce a big inhibition of telomerase activity in treated cells. Staurosporine produced a moderate inhibition, and sphingosine had a small inhibitory effect. The inhibition of telomerase activity by PKC inhibitors appears to be specific since the treated cells were mostly viable (i.e., greater than 75%) and still retained significant levels of protein synthesis capability. These results implicate that protein kinase C is involved in the regulation of telomerase activity in vivo.
The early effects of hypercholesterolemia on leukocyte-endothelium interaction were studied in vivo in the rabbit mesenteric microcirculation. Rabbits fed a 0.5% high-cholesterol (HC) diet showed elevated plasma cholesterol levels during the 1 to 2 weeks of HC feeding (P<0.001 versus control diet-fed rabbits). Intravital microscopy of mesenteric venules revealed that leukocyte rolling had increased 10-fold (P<0.001 versus control-fed group) at the end of the first week of the HC diet, which was sustained after 2 weeks of HC feeding (P<0.001 versus control-fed rabbits). Firm adherence of leukocytes to the endothelium was moderately increased after a 1-week period of hypercholesterolemia (P<0.05) but increased by 12-fold at 2 weeks (P<0.001 versus control diet-fed and P<0.01 versus 1-week HC-fed rabbits). Upregulation of the endothelial cell adhesion molecules P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was observed immunohistochemically on the intestinal microvascular endothelium of HC-fed rabbits. P-selectin was maximally expressed within the first week of the HC diet and remained elevated during the second week of cholesterol feeding (P<0.01 versus control). In contrast, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were moderately upregulated at 1 week but were highly expressed after 2 weeks of the HC diet (P<0.05 and P<0.001 versus control, respectively). Basal release of NO from both mesenteric microvascular and aortic endothelium in cholesterol-fed rabbits was progressively reduced after 1 (P<0.05) and 2 (P<0.01) weeks. Our data suggest that enhanced leukocyte-endothelium interaction occurs in vivo in the rabbit microcirculation during the first 2 weeks of hypercholesterolemia. This phenomenon is associated with impaired basal NO release and progressive endothelial surface expression of endothelial cell adhesion molecules (ie, P-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1) in the microvasculature.
This article examines the evidence for nitric oxide (NO) as a protective agent in splanchnic ischemia-reperfusion and other forms of acute intestinal inflammation. Four major points emerge from this body of data. First, acute intestinal inflammation results in an early (i.e., <5 min) and severe decrease in endothelium-derived NO. Thus the early trigger event in this condition is a functional loss of NO. Second, administration of exogenous NO, NO donors, or NO precursors ameliorate splanchnic ischemia-reperfusion and other forms of acute intestinal inflammation (i.e., splanchnic trauma). These beneficial effects occur at physiological levels of NO when given early in the course of the inflammatory state. Third, blockade of nitric oxide synthase (NOS) or gene deletion of NOS exacerbates intestinal inflammation. Fourth, there are a variety of signaling mechanisms that may mediate the protective effect of NO.
Recently, we demonstrated in vivo effects of acutely induced hyperglycemia, diabetes and mannitol infusions on rat mesenteric microcirculation concerning leukocyte-endothelial-cell interaction (Schäffler et al. EJCI 28: 886-893, 1998).
In this study we have investigated the possible involvement of the protein kinase C (PKC) and p38 MAP kinase cascade as signal transducing elements during hyperglycemic and osmotic stress in an in vivo rat model.
Acutely induced hyperglycemia resulted in a significant increase in leukocyte adhesion. This effect could be mimicked by mannitol. Both PKC and p38 MAP kinase were involved in the effects mediated by glucose and mannitol. Acutely induced hyperglycemia resulted in a significant increase in leukocyte emigration. This effect could be imitated by mannitol. However, PKC and p38 MAP kinase were only involved under osmotic stimulation. The hyperglycemia-induced reduction in leukocyte rolling velocity seemed to be a glucose-specific effect, since mannitol did not influence leukocyte rolling velocity. This glucose effect on leukocyte rolling velocity was mediated by an activation of the PKC/p38 MAP kinase cascade. Both hyperglycemia and osmotic stimuli alone were able to reduce venular shear rate without recruitment of the p38 MAP kinase cascade. The observed reduction of shear rate seems to be mediated only by the osmotic effects of glucose via activation of the PKC system.
The observed effects of glucose on adhesion, emigration and shear rate are due to osmotic effects. The PKC/MAP kinase cascade is involved as a signal transducing component. The reduction of leukocyte rolling velocity is a glucose-specific effect, mediated by the activation of both the PKC and the p38 MAP kinase cascade. Venular shear rate and leukocyte emigration can be influenced by glucose and mannitol due to different regulation mechanisms. It is concluded, that isoform-specific inhibitors of PKC and specific MAP kinase inhibitors represent a potential drug target for preventing microvascular dysfunction in diabetes.
The activity of protein kinase C (PKC), preferentially beta isoform of PKC, has been shown to be elevated in the diabetic retina. Recently, LY333531, a specific inhibitor of PKC-beta, has been reported to improve the decrease of retinal blood flow in early diabetes. Increased leukocyte entrapment has been suggested to be involved in blood flow disturbances in the early diabetic retina. This study was designed quantitatively to evaluate leukocyte entrapment in the retinal microcirculation of diabetic rats and the effect of LY333531 on leukocyte entrapment.
Diabetes was induced in male Long-Evans rats by intraperitoneal injection of streptozotocin (60 mg/kg). LY333531 (0.1, 1.0, or 10.0 mg/kg/d) was administered orally during a 4-week diabetic period. Leukocyte entrapment in the retinal microcirculation was quantitatively evaluated in vivo with acridine orange digital fluorography.
The number of leukocytes trapped in the retinal microcirculation of diabetic rats (mean +/- SEM; 14.3 +/- 1.3 cells/mm2) was significantly increased, compared with nondiabetic control rats (7.5 +/- 0.3 cells/mm2; P < 0.0001). Oral administration of LY333531 significantly decreased the number of leukocytes trapped in the retinal microcirculation of diabetic rats (10.9 +/- 0.6, 11.3 +/- 0.7, and 10.4 +/- 0.4 cells/mm2 with LY333531 0.1, 1.0, and 10.0 mg/kg/d, respectively; P < 0.05).
Treatment with LY333531 attenuated the increase of leukocyte entrapment in the retinal microcirculation during the period of early diabetes. This effect may contribute to the improvement of abnormal retinal blood flow in early diabetes with LY333531. LY333531 might have a therapeutic efficacy in preventing microcirculatory flow disturbances by trapped leukocytes in the early diabetic retina.
While a damaged endothelium is recognised to be a key accessory to diabetic macroangiopathy, awareness is developing that impairments concerning endothelium- and nitric oxide (NO)-dependent microvascular function, may contribute to several other corollaries of diabetes, such as hypertension, dyslipidaemia and in vivo insulin resistance. There are now several reports describing elevations in specific oxidant stress markers in both insulin resistance syndrome (IRS) and diabetes, together with determinations of reduced total antioxidant defence and depletions in individual antioxidants. Such a pro-oxidant environment in diabetes may disrupt endothelial function through the inactivation of NO, resulting in the attenuation of a fundamental anti-atherogenic and euglycaemic vascular influence. Indeed, experimental and clinical data suggest that the supplementation of insulin resistant or diabetic states with antioxidants such as vitamin E, normalises oxidant stress and improves both endothelium-dependent vasodilation and insulin sensitivity. However, the promising potential efficacy of antioxidant therapy in cardiovascular disease and diabetes, in either a primary or secondary preventative role, awaits definitive clinical demonstration.
Incubation of endothelial cells in vitro with high concentrations of glucose activates protein kinase C (PKC) and increases nitric oxide synthase (NOS III) gene expression as well as superoxide production. The underlying mechanisms remain unknown. To address this issue in an in vivo model, diabetes was induced with streptozotocin in rats. Streptozotocin treatment led to endothelial dysfunction and increased vascular superoxide production, as assessed by lucigenin- and coelenterazine-derived chemiluminescence. The bioavailability of vascular nitric oxide (as measured by electron spin resonance) was reduced in diabetic aortas, although expression of endothelial NOS III (mRNA and protein) was markedly increased. NOS inhibition with N:(G)-nitro-L-arginine increased superoxide levels in control vessels but reduced them in diabetic vessels, identifying NOS as a superoxide source. Similarly, we found an activation of the NADPH oxidase and a 7-fold increase in gp91(phox) mRNA in diabetic vessels. In vitro PKC inhibition with chelerythrine reduced vascular superoxide in diabetic vessels, whereas it had no effect on superoxide levels in normal vessels. In vivo PKC inhibition with N:-benzoyl-staurosporine did not affect glucose levels in diabetic rats but prevented NOS III gene upregulation and NOS-mediated superoxide production, thereby restoring vascular nitric oxide bioavailability and endothelial function. The reduction of superoxide in vitro by chelerythrine and the normalization of NOS III gene expression and reduction of superoxide in vivo by N:-benzoyl-staurosporine point to a decisive role of PKC in mediating these phenomena and suggest a therapeutic potential of PKC inhibitors in the prevention or treatment of vascular complications of diabetes mellitus. The full text of this article is available at http://www.circresaha.org.
We employed intravital microscopy of the rat mesenteric microvasculature to study the effects of local hyperglycemia on leukocyte-endothelial cell interactions. Intraperitoneal injection of 6, 12.5, and 25 mmol/l D-glucose to the rat significantly and time-dependently increased leukocyte rolling and leukocyte adherence in, and leukocyte transmigration through mesenteric venules compared with control rats injected with Krebs-Henseleit (K-H) solution alone or given 25 mmol/l L-glucose intraperitoneally. The response elicited by D-glucose was associated with significant attenuation of endothelial nitric oxide (NO) release, as demonstrated by direct measurement of NO release in inferior vena caval segments isolated from rats exposed to 25 mmol/l D-glucose for 4 h (P < 0.01 vs. vena caval segments from control rats). Local application of 0.05 U/min insulin for 90 min significantly attenuated glucose-induced leukocyte rolling, adherence, and migration (P < 0.01 from 25 mmol/l D-glucose alone). Immunohistochemical localization of P-selectin expressed on endothelial surface was significantly increased 4 h after exposure of the mesenteric tissue to high ambient glucose (P < 0.01 vs. ileal venules from rats injected with K-H solution alone or 25 mmol/l L-glucose). Insulin markedly inhibited endothelial cell surface expression of P-selectin in ileal venules exposed to elevated ambient glucose in vivo (P < 0.01 vs. control rats injected with 25 mmol/l L-glucose). These data demonstrate that acute increases in ambient glucose comparable to those seen in diabetic patients are able to initiate an inflammatory response within the microcirculation. This inflammatory response to glucose is associated with upregulation of the endothelial cell adhesion molecule P-selectin and can be blocked by local application of insulin.
the ileum was first washed free of blood by perfusion with K-H buffer (warmed to 37°C and bubbled with 95% O 2 and 5% CO 2 ). Once the venous retina to retinal circulation
- Briefly
Briefly, the ileum was first washed free of blood by perfusion with K-H buffer (warmed to 37°C and bubbled with 95% O 2 and 5% CO 2 ). Once the venous retina to retinal circulation. Am J Physiol 265:E783–E793, 1993
Nitric oxide: an endogenous modulator of leukocyte adhesion
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- M Suzuki
- D N Granger
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Borders JL, Granger HJ: An optical doppler intravital velocimeter. Microvasc Res 27:117-127, 1984