Oral FXIIa inhibitor KV998086 suppresses FXIIa and single chain FXII mediated kallikrein kinin system activation.

Background: The kallikrein kinin system (KKS) is an established pharmacological target for the treatment and prevention of attacks in hereditary angioedema (HAE). Proteolytic activities of FXIIa and single-chain Factor XII (FXII) zymogen contribute to KKS activation and thereby may play roles in both initiating and propagating HAE attacks. In this report, we investigated the effects of potent small molecule FXIIa inhibitors on FXIIa and single chain FXII enzymatic activities, KKS activation, and angioedema in mice. Methods: We examined the effects of 29 structurally distinct FXIIa inhibitors on enzymatic activities of FXIIa and a mutant single chain FXII with R334A, R343A and R353A substitutions (rFXII-T), that does not undergo zymogen conversion to FXIIa, using kinetic fluorogenic substrate assays. We examined the effects of a representative FXIIa inhibitor, KV998086, on KKS activation and both carrageenan- and captopril-induced angioedema in mice. Results: FXIIa inhibitors designed to target its catalytic domain also potently inhibited the enzymatic activity of rFXII-T and the pIC50s of these compounds linearly correlated for rFXIIa and rFXII-T (R 2 = 0.93). KV998086, a potent oral FXIIa inhibitor (IC50 = 7.2 nM) inhibited dextran sulfate (DXS)-stimulated generation of plasma kallikrein and FXIIa, and the cleavage of high molecular weight kininogen (HK) in human plasma. KV998086 also inhibited rFXII-T mediated HK cleavage (p < 0.005) in plasma from FXII knockout mice supplemented with rFXII-T and stimulated with polyphosphate or DXS. Orally administered KV998086 protected mice from 1) captopril-induced Evans blue leakage in colon and laryngotracheal tissues and 2) blocked carrageenan-induced plasma HK consumption and paw edema. Conclusion: These findings show that small molecule FXIIa inhibitors, designed to target its active site, also inhibit the enzymatic activity of FXII zymogen. Combined inhibition of FXII zymogen and FXIIa may thereby suppress both the initiation and amplification of KKS activation that contribute to hereditary angioedema attacks and other FXII-mediated diseases.

Plasma Kallikrein Contributes to Intracerebral Hemorrhage and Hypertension in Stroke-Prone Spontaneously Hypertensive Rats.

Plasma kallikrein (PKa) has been implicated in contributing to hemorrhage following thrombolytic therapy; however, its role in spontaneous intracerebral hemorrhage is currently not available. This report investigates the role of PKa on hemorrhage and hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were fed with a high salt-containing stroke-prone diet to increase blood pressure and induce intracerebral hemorrhage. The roles of PKa on blood pressure, hemorrhage, and survival in SHRSP were examined in rats receiving a PKa inhibitor or plasma prekallikrein antisense oligonucleotide (PK ASO) compared with rats receiving control ASO. Effects on PKa on the proteolytic cleavage of atrial natriuretic peptide (ANP) were analyzed by tandem mass spectrometry. We show that SHRSP on high-salt diet displayed increased levels of PKa activity compared with control rats. Cleaved kininogen was increased in plasma during stroke compared to SHRSP without stroke. Systemic administration of a PKa inhibitor or PK ASO to SHRSP reduced hemorrhage and blood pressure, and improved neurological function and survival compared with SHRSP receiving control ASO. Since PKa inhibition was associated with reduced blood pressure in hypertensive rats, we investigated the effects of PKa on the cleavage of ANP. Incubation of PKa with ANP resulted in the generation fragment ANP5-28, which displayed reduced effects on blood pressure lowering compared with full length ANP. PKa contributes to increased blood pressure in SHRSP, which is associated with hemorrhage and reduced survival. PKa-mediated cleavage of ANP reduces its blood pressure lowering effects and thereby may contribute to hypertension-induced intracerebral hemorrhage.

PM2.5 induces vascular permeability increase through activating MAPK/ERK signaling pathway and ROS generation.

Although in-vivo exposure of PM2.5 has been suggested to initiate a disorder on vascular permeability, the effects and related mechanism has not been well defined. In this work, an obvious increase on vascular permeability has been confirmed in vivo by vein injection of PM2.5 into Balb/c mouse. Human umbilical vein vascular endothelial cells and the consisted ex-vivo vascular endothelium were used as model to investigate the effects of PM2.5 on the vascular permeability and the underlying molecular mechanism. Upon PM2.5 exposure, the vascular endothelial growth factor receptor 2 on cell membrane phosphorylates and activates the downstream mitogen-activated protein kinase (MAPK)/ERK signaling. The adherens junction protein VE-cadherin sheds and the intercellular junction opens, damaging the integrity of vascular endothelium via paracellular pathway. Besides, PM2.5 induces the intracellular reactive oxygen species (ROS) production and triggers the oxidative stress including activity decrease of superoxide dismutase, lactate dehydrogenase release and permeability increase of cell membrane. Taken together, the paracellular and transcellular permeability enhancement jointly contributes to the significant increase of endothelium permeability and thus vascular permeability upon PM2.5 exposure. This work provides an insight into molecular mechanism of PM2.5 associated cardiovascular disease and offered a real-time screening method for the health risk of PM2.5.

Retinal proteome associated with bradykinin-induced edema.

The plasma kallikrein-stimulated generation of bradykinin (BK) has been implicated in diabetic macular edema (DME). This study characterizes the effects of BK on the ultrastructure and proteome of the rat retina. The effects of intravitreal injection of BK on retinal thickness and vascular ultrastructure in Sprague Dawley rats were analyzed and compared with the effects of VEGF using spectral-domain optical coherence tomography. At 24 h post intravitreal injection of BK or saline vehicle retina were harvested and solubilized proteins were analyzed by mass spectrometry-based proteomics. Proteins were identified using X!Tandem and spectral counts were used as a semiquantitative measurement of protein abundance. Proteins identified from retinal extracts were annotated by Gene Ontology (GO) slim terms and compared with a human DME vitreous proteome. Intravitreal injection of BK and VEGF induced transient increases in retinal thickness of 46 µm (24.6%, p = 0.015) and 39 µm (20.3%, p = 0.004), respectively at 24 h, which were resolved to baseline thicknesses at 96 h post injection. BK and VEGF also increased retinal vessel diameters and tortuosity at 24 h post intravitreal injection. Proteomic analyses identified 1757 non-redundant proteins in the rat retina, including 1739 and 1725 proteins from BK- and saline control-injected eyes, respectively. Eighteen proteins, including two proteins associated with intercellular junctions, filamin A and actinin alpha 4, were decreased by at least 50% (p < 0.05) in retina from BK-injected eyes compare with retina from eyes injected with saline. In addition, 32 proteins were increased by > 2-fold (p < 0.05) in retina from BK-injected eyes. Eight proteins, including complement C3, were identified to be increased in both BK-stimulated rat retina and in human DME vitreous. Western blot analysis showed that Complement 3 levels in vitreous from BK-injected eyes in rats and clinical DME samples were increased by 6.6-fold (p = 0.039) and 4.3-fold (p = 0.02), compared with their respective controls. In summary, this study identifies protein changes in rat retina that are associated with BK-induced retinal thickening, including 8 proteins that were previously reported to be increased in the human DME vitreous proteome.

Retinol binding protein 3 is increased in the retina of patients with diabetes resistant to diabetic retinopathy.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

Iris Malformation and Anterior Segment Dysgenesis in Mice and Humans With a Mutation in PI 3-Kinase.

Purpose: To determine the ocular consequences of a dominant-negative mutation in the p85α subunit of phosphatidylinositol 3-kinase (PIK3R1) using a knock-in mouse model of SHORT syndrome, a syndrome associated with short stature, lipodystrophy, diabetes, and Rieger anomaly in humans. Methods: We investigated knock-in mice heterozygous for the SHORT syndrome mutation changing arginine 649 to tryptophan in p85α (PIK3R1) using physical examination, optical coherence tomography (OCT), tonometry, and histopathologic sections from paraffin-embedded eyes, and compared the findings to similar investigations in two human subjects with SHORT syndrome heterozygous for the same mutation. Results: While overall eye development was normal with clear cornea and lens, normal anterior chamber volume, normal intraocular pressure, and no changes in the retinal structure, OCT images of the knock-in mouse eyes revealed a significant decrease in thickness and width of the iris resulting in increased pupil area and irregularity of shape. Both human subjects had Rieger anomaly with similar defects including thin irides and irregular pupils, as well as a prominent ring of Schwalbe, goniosynechiae, early cataract formation, and glaucoma. Although the two subjects had had diabetes for more than 30 years, there were no signs of diabetic retinopathy. Conclusions: A dominant-negative mutation in the p85α regulatory subunit of PI3K affects development of the iris, and contributes to changes consistent with anterior segment dysgenesis in both humans and mice.

Plasma kallikrein mediates brain hemorrhage and edema caused by tissue plasminogen activator therapy in mice after stroke.

Thrombolytic therapy using tissue plasminogen activator (tPA) in acute stroke is associated with increased risks of cerebral hemorrhagic transformation and angioedema. Although plasma kallikrein (PKal) has been implicated in contributing to both hematoma expansion and thrombosis in stroke, its role in the complications associated with the therapeutic use of tPA in stroke is not yet available. We investigated the effects of tPA on plasma prekallikrein (PPK) activation and the role of PKal on cerebral outcomes in a murine thrombotic stroke model treated with tPA. We show that tPA increases PKal activity in vitro in both murine and human plasma, via a factor XII (FXII)-dependent mechanism. Intravenous administration of tPA increased circulating PKal activity in mice. In mice with thrombotic occlusion of the middle cerebral artery, tPA administration increased brain hemorrhage transformation, infarct volume, and edema. These adverse effects of tPA were ameliorated in PPK (Klkb1)-deficient and FXII-deficient mice and in wild-type (WT) mice pretreated with a PKal inhibitor prior to tPA. tPA-induced brain hemisphere reperfusion after photothrombolic middle cerebral artery occlusion was increased in Klkb1-/- mice compared with WT mice. In addition, PKal inhibition reduced matrix metalloproteinase-9 activity in brain following stroke and tPA therapy. These data demonstrate that tPA activates PPK in plasma and PKal inhibition reduces cerebral complications associated with tPA-mediated thrombolysis in stroke.

Evaluating anesthetic protocols for functional blood flow imaging in the rat eye.

The purpose of this study is to evaluate the suitability of five different anesthetic protocols (isoflurane, isoflurane­xylazine, pentobarbital, ketamine­xylazine, and ketamine­xylazine­vecuronium) for functional blood flow imaging in the rat eye. Total retinal blood flow was measured at a series of time points using an ultrahigh-speed Doppler OCT system. Additionally, each anesthetic protocol was qualitatively evaluated according to the following criteria: (1) time-stability of blood flow, (2) overall rate of blood flow, (3) ocular immobilization, and (4) simplicity. We observed that different anesthetic protocols produced markedly different blood flows. Different anesthetic protocols also varied with respect to the four evaluated criteria. These findings suggest that the choice of anesthetic protocol should be carefully considered when designing and interpreting functional blood flow studies in the rat eye.

Negatively charged silver nanoparticles cause retinal vascular permeability by activating plasma contact system and disrupting adherens junction.

Silver nanoparticles (AgNPs) have been extensively used as antibacterial component in numerous healthcare, biomedical and consumer products. Therefore, their adverse effects to biological systems have become a major concern. AgNPs have been shown to be absorbed into circulation and redistributed into various organs. It is thus of great importance to understand how these nanoparticles affect vascular permeability and uncover the underlying molecular mechanisms. A negatively charged mecaptoundeonic acid-capped silver nanoparticle (MUA@AgNP) was investigated in this work. Ex vivo experiments in mouse plasma revealed that MUA@AgNPs caused plasma prekallikrein cleavage, while positively charged or neutral AgNPs, as well as Ag ions had no effect. In vitro tests revealed that MUA@AgNPs activated the plasma kallikrein-kinin system (KKS) by triggering Hageman factor autoactivation. By using specific inhibitors aprotinin and HOE 140, we demonstrated that KKS activation caused the release of bradykinin, which activated B2 receptors and induced the shedding of adherens junction protein, VE-cadherin. These biological perturbations eventually resulted in endothelial paracellular permeability in mouse retina after intravitreal injection of MUA@AgNPs. The findings from this work provided key insights for toxicity modulation and biomedical applications of AgNPs.

Plasma Kallikrein-Kinin System as a VEGF-Independent Mediator of Diabetic Macular Edema.

This study characterizes the kallikrein-kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased twofold and 11.0-fold (both P < 0.0001), respectively, in vitreous from subjects with DME compared with those with a macular hole (MH). While the vascular endothelial growth factor (VEGF) level was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r = 0.266, P = 0.112). Using mass spectrometry-based proteomics, we identified 167 vitreous proteins, including 30 that were increased in DME (fourfold or more, P < 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin-induced retinal thickening in mice was not affected by blockade of VEGF receptor 2. Diabetes-induced RVP was decreased by up to 78% (P < 0.001) in Klkb1 (PPK)-deficient mice compared with wild-type controls. B2- and B1 receptor-induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (NOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF-independent mediator of DME.

Intraocular hemorrhage causes retinal vascular dysfunction via plasma kallikrein.

PURPOSE: Retinal hemorrhages occur in a variety of sight-threatening conditions including ocular trauma, high altitude retinopathy, and chronic diseases such as diabetic and hypertensive retinopathies. The goal of this study is to investigate the effects of blood in the vitreous on retinal vascular function in rats. METHODS: Intravitreal injections of autologous blood, plasma kallikrein (PK), bradykinin, and collagenase were performed in Sprague-Dawley and Long-Evans rats. Retinal vascular permeability was measured using vitreous fluorophotometry and Evans blue dye permeation. Leukostasis was measured by fluorescein isothiocyanate-coupled concanavalin A lectin and acridine orange labeling. Retinal hemorrhage was examined on retinal flatmounts. Primary cultures of bovine retinal pericytes were cultured in the presence of 25 nM PK for 24 hours. The pericyte-conditioned medium was collected and the collagen proteome was analyzed by tandem mass spectrometry. RESULTS: Intravitreal injection of autologous blood induced retinal vascular permeability and retinal leukostasis, and these responses were ameliorated by PK inhibition. Intravitreal injections of exogenous PK induced retinal vascular permeability, leukostasis, and retinal hemorrhage. Proteomic analyses showed that PK increased collagen degradation in pericyte-conditioned medium and purified type IV collagen. Intravitreal injection of collagenase mimicked PK's effect on retinal hemorrhage. CONCLUSIONS: Intraocular hemorrhage increases retinal vascular permeability and leukostasis, and these responses are mediated, in part, via PK. Intravitreal injections of either PK or collagenase, but not bradykinin, induce retinal hemorrhage in rats. PK exerts collagenase-like activity that may contribute to blood-retinal barrier dysfunction.

Measurement of pulsatile total blood flow in the human and rat retina with ultrahigh speed spectral/Fourier domain OCT.

We present an approach to measure pulsatile total retinal arterial blood flow in humans and rats using ultrahigh speed Doppler OCT. The axial blood velocity is measured in an en face plane by raster scanning and the flow is calculated by integrating over the vessel area, without the need to measure the Doppler angle. By measuring flow at the central retinal artery, the scan area can be very small. Combined with ultrahigh speed, this approach enables high volume acquisition rates necessary for pulsatile total flow measurement without modification in the OCT system optics. A spectral domain OCT system at 840nm with an axial scan rate of 244kHz was used for this study. At 244kHz the nominal axial velocity range that could be measured without phase wrapping was ±37.7mm/s. By repeatedly scanning a small area centered at the central retinal artery with high volume acquisition rates, pulsatile flow characteristics, such as systolic, diastolic, and mean total flow values, were measured. Real-time Doppler C-scan preview is proposed as a guidance tool to enable quick and easy alignment necessary for large scale studies. Data processing for flow calculation can be entirely automatic using this approach because of the simple and robust algorithm. Due to the rapid volume acquisition rate and the fact that the measurement is independent of Doppler angle, this approach is inherently less sensitive to involuntary eye motion. This method should be useful for investigation of small animal models of ocular diseases as well as total blood flow measurements in human patients in the clinic.

Hyperinsulinemia does not change atherosclerosis development in apolipoprotein E null mice.

OBJECTIVE: To determine the contribution of hyperinsulinemia to atherosclerosis development. METHODS AND RESULTS: Apolipoprotein E (Apoe) null mice that had knockout of a single allele of the insulin receptor (Insr) gene were compared with littermate Apoe null mice with intact insulin receptors. Plasma insulin levels in Insr haploinsufficient/Apoe null mice were 50% higher in the fasting state and up to 69% higher during a glucose tolerance test, but glucose tolerance was not different in the 2 groups. C-peptide levels, insulin sensitivity, and postreceptor insulin signaling in muscle, liver, fat, and aorta were not different between groups, whereas disappearance in plasma of an injected insulin analog was delayed in Insr haploinsufficient/Apoe null mice, indicating that impaired insulin clearance was the primary cause of hyperinsulinemia. No differences were observed in plasma lipids or blood pressure. Despite the hyperinsulinemia, atherosclerotic lesion size was not different between the 2 groups at time points up to 52 weeks of age when measured as en face lesion area in the aorta, cross-sectional plaque area in the aortic sinus, and cholesterol abundance in the brachiocephalic artery. CONCLUSIONS: Hyperinsulinemia, without substantial vascular or whole-body insulin resistance and without changes in plasma lipids or blood pressure, does not change susceptibility to atherosclerosis.

Hyperglycemia-induced cerebral hematoma expansion is mediated by plasma kallikrein.

Hyperglycemia is associated with greater hematoma expansion and poor clinical outcomes after intracerebral hemorrhage. We show that cerebral hematoma expansion triggered by intracerebral infusion of autologous blood is greater in diabetic rats and mice compared to nondiabetic controls and that this augmented expansion is ameliorated by plasma kallikrein (PK) inhibition or deficiency. Intracerebral injection of purified PK augmented hematoma expansion in both diabetic and acutely hyperglycemic rats, whereas injection of bradykinin, plasmin or tissue plasminogen activator did not elicit such a response. This response, which occurs rapidly, was prevented by co-injection of the glycoprotein VI agonist convulxin and was mimicked by glycoprotein VI inhibition or deficiency, implicating an effect of PK on inhibiting platelet aggregation. We show that PK inhibits collagen-induced platelet aggregation by binding collagen, a response enhanced by elevated glucose concentrations. The effect of hyperglycemia on hematoma expansion and PK-mediated inhibition of platelet aggregation could be mimicked by infusing mannitol. These findings suggest that hyperglycemia augments cerebral hematoma expansion by PK-mediated osmotic-sensitive inhibition of hemostasis.

Vitreous proteomics and diabetic retinopathy.

Diabetic retinopathy is the major cause of acquired blindness in working-age adults. Studies of the vitreous proteome have provided insights into the etiology of diabetic retinopathy and suggested potential molecular targets for treatments. Further characterization of the protein changes associated with the progression of this disease may suggest additional therapeutic approaches as well as reveal novel factors that may be useful in predicting risk and functional outcomes of interventional therapies. This article provides an overview of the various techniques used for proteomic analysis of the vitreous and details results from various studies evaluating vitreous of diabetic patients using the proteomic approach.

Angiotensin AT1 receptor antagonism ameliorates murine retinal proteome changes induced by diabetes.

Diabetic retinopathy is the most common microvascular complication caused by diabetes mellitus and is a leading cause of vision loss among working-age adults in developed countries. Understanding the effects of diabetes on the retinal proteome may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of retina from C57BL/6 mice with 2 months of streptozotocin-induced diabetes and age-matched nondiabetic control mice. To explore the role of the angiotensin AT1 receptor in the retinal proteome in diabetes, a subgroup of mice were treated with the AT1 antagonist candesartan. We identified 1792 proteins from retinal lysates, of which 65 proteins were differentially changed more than 2-fold in diabetic mice compared with nondiabetic mice. A majority (72%) of these protein changes were normalized by candesartan treatment. Most of the significantly changed proteins were associated with metabolism, oxidative phosphorylation, and apoptotic pathways. An analysis of the proteomics data revealed metabolic and apoptotic abnormalities in the retina from diabetic mice that were ameliorated with candesartan treatment. These results provide insight into the effects of diabetes on the retina and the role of the AT1 receptor in modulating this response.

Plasma kallikrein mediates angiotensin II type 1 receptor-stimulated retinal vascular permeability.

Hypertension is a leading risk factor for the development and progression of diabetic retinopathy and contributes to a variety of other retinal diseases in the absence of diabetes mellitus. Inhibition of the renin-angiotensin system has been shown to provide beneficial effects against diabetic retinopathy, both in the absence and presence of hypertension, suggesting that angiotensin II (Ang II) and the Ang II type 1 receptor may contribute to retinal vascular dysfunction. We investigated the effects of the Ang II type 1 receptor antagonist candesartan on retinal vascular permeability (RVP) in normotensive rats with streptozotocin-induced diabetes mellitus and in rats with Ang II-induced hypertension. We showed that candesartan treatment decreased diabetes mellitus- and Ang II-stimulated RVP by 58% (P<0.05) and 79% (P<0.05), respectively, compared with untreated controls, suggesting that activation of the Ang II type 1 receptor contributes to blood-retinal barrier dysfunction. We found that plasma kallikrein levels are increased in the retina of rats with Ang II-stimulated hypertension and that intravitreal injection of either plasma kallikrein or bradykinin is sufficient to increase RVP. We showed that a novel small molecule inhibitor of plasma kallikrein, 1-benzyl-1H-pyrazole-4-carboxylic acid 4-carbamimidoyl-benzylamide, delivered systemically via a subcutaneous pump, decreased Ang II-stimulated RVP by 70% (P<0.05) and ameliorates Ang II-induced hypertension, measured from the carotid artery by telemetry, but did not reduce Ang II-induced retinal leukostasis. These findings demonstrate that activation of the Ang II type 1 receptor increases RVP and suggest that systemic plasma kallikrein inhibition may provide a new therapeutic approach for ameliorating blood-retinal barrier dysfunction induced by hypertension.

Retinal blood flow in diabetes.

OBJECTIVE: This is a review of work focused on characterizing retinal blood flow in diabetes. The review describes results on validation of the methodology for retinal blood flow measurements, the mechanisms of action of various factors that contribute to abnormalities in retinal blood flow in diabetic rodent models, and the translation of these results to clinical studies demonstrating the effectiveness of different therapeutic agents in normalizing retinal blood flow abnormalities in patients with diabetes. METHODS: Retinal blood flow measurements were performed using video fluorescein angiography, a methodology that is based on the measurement of fluorescein dye circulation times through the retinal circulation. RESULTS: The results of a number of experiments are summarized, detailing the effects of hyperglycemia and the roles of factors such as protein kinase C activation, endothelin-1 and endothelin-3, angiotensin-II, and nitric oxide in the development of retinal blood flow abnormalities in diabetes. CONCLUSION: The measurement of retinal blood flow both in animals and in clinical trials using the same retinal blood flow measurement methodology can provide a valuable method of quantitation allowing characterization of physiological effects and their association with metabolic alterations in diabetes and their effects on the development and incidence of microvascular complications.

Reduction of diabetes-induced oxidative stress, fibrotic cytokine expression, and renal dysfunction in protein kinase Cbeta-null mice.

Diabetes induces the activation of several protein kinase C (PKC) isoforms in the renal glomeruli. We used PKC-beta(-/-) mice to examine the action of PKC-beta isoforms in diabetes-induced oxidative stress and renal injury at 8 and 24 weeks of disease. Diabetes increased PKC activity in renal cortex of wild-type mice and was significantly reduced (<50% of wild-type) in diabetic PKC-beta(-/-) mice. In wild-type mice, diabetes increased the translocation of PKC-alpha and -beta1 to the membrane, whereas only PKC-alpha was elevated in PKC-beta(-/-) mice. Increases in urinary isoprostane and 8-hydroxydeoxyguanosine, parameters of oxidative stress, in diabetic PKC-beta(-/-) mice were significantly reduced compared with diabetic wild-type mice. Diabetes increased NADPH oxidase activity and the expressions of p47(phox), Nox2, and Nox4 mRNA levels in the renal cortex and were unchanged in diabetic PKC-beta(-/-) mice. Increased expression of endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-beta, connective tissue growth factor (CTGF), and collagens IV and VI found in diabetic wild-type mice was attenuated in diabetic PKC-beta(-/-) mice. Diabetic PKC-beta(-/-) mice were protected from renal hypertrophy, glomerular enlargement, and hyperfiltration observed in diabetic wild-type mice and had less proteinuria. Lack of PKC-beta can protect against diabetes-induced renal dysfunction, fibrosis, and increased expressions of Nox2 and -4, ET-1, VEGF, TGF-beta, CTGF, and oxidant production.

Hepatocyte growth factor induces retinal vascular permeability via MAP-kinase and PI-3 kinase without altering retinal hemodynamics.

PURPOSE: Although vascular endothelial growth factor (VEGF) is a key mediator of retinal vascular permeability (RVP), there may be additional humoral contributors. Hepatocyte growth factor (HGF) induces endothelial cell separation, regulates expression of cell adhesion molecules and is increased in the vitreous fluid of patients with proliferative diabetic retinopathy. The purpose of this study was to evaluate the in vivo effects of HGF on RVP and retinal hemodynamics and delineate the signaling pathways. METHODS: RVP was assessed by vitreous fluorescein fluorophotometry in rats. Time course and dose-response were determined after intravitreal HGF injection. MAP kinase (MAPK), phosphatidylinositol 3-kinase (PI-3 kinase), and protein kinase C (PKC) involvement were examined by using selective inhibitors. Retinal blood flow (RBF) and mean circulation time (MCT) were evaluated by video fluorescein angiography. RESULTS: HGF increased RVP in a time- and dose-dependent manner. HGF-induced RVP was evident 5 minutes after injection, and reached maximal levels after 25 minutes (+107% versus vehicle, P=0.002). This effect was comparable to that of maximum VEGF stimulation (134%+/-128% at 25 ng/mL). Selective inhibitors of MAPK (PD98059) and PI-3 kinase (LY294002) suppressed HGF-induced RVP by 86%+/-44% (P=0.015) and 97%+/-59% (P=0.021), respectively. Non-isoform-selective inhibition of PKC did not significantly decrease HGF-induced RVP. Although VEGF increases RBF and reduces MCT, HGF did not affect either. CONCLUSIONS: HGF increases RVP in a time- and dose-dependent manner at physiologically relevant concentrations with a magnitude and profile similar to that of VEGF, without affecting retinal hemodynamics. Thus, HGF may represent another clinically significant contributor to retinal edema distinct from the actions of VEGF.