Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation.

Excessive retinal vascular permeability contributes to the pathogenesis of proliferative diabetic retinopathy and diabetic macular edema, leading causes of vision loss in working-age adults. Using mass spectroscopy-based proteomics, we detected 117 proteins in human vitreous and elevated levels of extracellular carbonic anhydrase-I (CA-I) in vitreous from individuals with diabetic retinopathy, suggesting that retinal hemorrhage and erythrocyte lysis contribute to the diabetic vitreous proteome. Intravitreous injection of CA-I in rats increased retinal vessel leakage and caused intraretinal edema. CA-I-induced alkalinization of vitreous increased kallikrein activity and its generation of factor XIIa, revealing a new pathway for contact system activation. CA-I-induced retinal edema was decreased by complement 1 inhibitor, neutralizing antibody to prekallikrein and bradykinin receptor antagonism. Subdural infusion of CA-I in rats induced cerebral vascular permeability, suggesting that extracellular CA-I could have broad relevance to neurovascular edema. Inhibition of extracellular CA-I and kallikrein-mediated innate inflammation could provide new therapeutic opportunities for the treatment of hemorrhage-induced retinal and cerebral edema.

Label-free quantitative analysis of one-dimensional PAGE LC/MS/MS proteome: application on angiotensin II-stimulated smooth muscle cells secretome.

A widely used method for protein identification couples prefractionation of protein samples by one-dimensional (1D) PAGE with LC/MS/MS. We developed a new label-free quantitative algorithm by combining measurements of spectral counting, ion intensity, and peak area on 1D PAGE-based proteomics. This algorithm has several improvements over other label-free quantitative algorithms: (i) Errors in peak detection are reduced because the retention time is based on each LC/MS/MS run and actual precursor m/z. (ii) Detection sensitivity is increased because protein quantification is based on the combination of peptide count, ion intensity, and peak area. (iii) Peak intensity and peak area are calculated in each LC/MS/MS run for all slices from 1D PAGE for every single identified protein and visualized as a Western blot image. The sensitivity and accuracy of this algorithm were demonstrated by using standard curves (17.4 fmol to 8.7 pmol), complex protein mixtures (30 fmol to 1.16 pmol) of known composition, and spiked protein (34.8 fmol to 17.4 pmol) in complex proteins. We studied the feasibility of this approach using the secretome of angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs). From the VSMC-conditioned medium, 629 proteins were identified including 212 putative secreted proteins. 26 proteins were differently expressed in control and Ang II-stimulated VSMCs, including 18 proteins not previously reported. Proteins related to cell growth (CYR61, protein NOV, and clusterin) were increased, whereas growth arrest-specific 6 (GAS6) and growth/differentiation factor 6 were decreased by Ang II stimulation. Ang II-stimulated changes of plasminogen activator inhibitor-1, GAS6, cathepsin B, and periostin were validated by Western blot. In conclusion, a novel label-free quantitative analysis of 1D PAGE-LC/MS/MS-based proteomics has been successfully applied to the identification of new potential mediators of Ang II action and may provide an alternative to traditional protein staining methods.

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.

Angiotensin II stimulates phosphorylation of an ectodomain-truncated platelet-derived growth factor receptor-beta and its binding to class IA PI3K in vascular smooth muscle cells.

PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85alpha mutant lacking the p110-binding domain (Deltap85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT1 (Ang II subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.

Effects of scorpion venom bioactive polypolypeptides on platelet aggregation and thrombosis and plasma 6-keto-PG F1alpha and TXB2 in rabbits and rats.

Effects of scorpion venom active polypeptide (SVAP) from scorpion venom of Buthus Martensii Karsch of Chinese on platelet aggregation in ex vivo and vitro in rabbits, thrombosis in carotid artery of rats and plasma 6-keto-PG F1alpha and TXB2 in rats were studied by the turbidimetry, the duplicated thrombosis model by electrostimulation and RIA, respectively. The results showed that SVAP 0.125, 0.25, 0.5 mg/ml inhibited significantly the rabbit platelet aggregation triggered by 0.3 U/ml thrombin, 10 microM ADP in vitro (P<0.05 or 0.01) and SVAP at the dose of 0.32, 0.64 mg/kg iv prolonged distinctively the occlusion time of thrombosis that were induced by electrical stimulation. Increased% of 0.16, 0.32 and 0.64 mg/kg were 30.16, 71.74, 98.27%, respectively, which showed a good dose-effect relationship. SVAP 0.22 mg/ml (in vitro) or 0.2, 0.4 mg/kg (in ex vivo) could obviously increase the plasma concentration of 6-keto-PG F1alpha, but slightly effect rats plasma concentration of TXB2 in vitro and in ex vivo and significantly increase of value of PG I2/TXA2, which suggested that the mechanism of the antithrombotic action of SVAP is related to the resistance against platelet aggregation, increase of the concentration of PG I2 in plasma.

Proteomic Analysis of Embryonic and Young Human Vitreous.

PURPOSE: The proteomic profile of vitreous from second-trimester human embryos and young adults was characterized using mass spectrometry and analyzed for changes in protein levels that may relate to structural changes occurring during this time. This vitreous proteome was compared to previous reports to confirm proteins already identified and reveal novel ones. METHODS: Vitreous from 17 human embryos aged 14 to 20 weeks gestation (WG) and from a 12-, a 14-, a 15-, and a 28-year-old was individually analyzed using tandem mass spectrometry-based proteomics. Peptide spectral count associations with embryonic age were assessed using a general linear model of fold changes and Spearman's rank correlation. Differences between embryonic and young adult vitreous proteomes were also compared. Immunohistochemistry was used to evaluate three proteins in five additional fetal (10-18 WG) human eyes. RESULTS: There were 1217 proteins identified in fetal and young adult human vitreous, 206 after quantile normalization and variance filtering. In embryos, the peptide counts of 37 proteins changed significantly from 14 to 20 WG: 75.7% increased, 24.3% decreased. Immunohistochemistry confirmed the absence of clusterin and cadherin in 10 and 14 WG eyes and their presence at 18 WG. Comparing embryonic to young adult vitreous, 47 proteins were significantly higher or lower. A total of 768 proteins not previously identified in the literature are presented. CONCLUSIONS: Proteins previously unreported in the human vitreous were identified. The human vitreous proteome undergoes significant changes during embryogenesis and young adulthood. A number of protein levels change considerably during the second trimester, with the majority decreasing.

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.

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.

Proteomic identification of novel plasma kallikrein substrates in the astrocyte secretome.

Plasma kallikrein (PK) is activated during hemorrhage and has been implicated in cerebral vascular permeability and edema. To further characterize the potential effects of PK on the brain that may follow cerebral vascular injury, we have utilized a proteomics approach to search for novel PK substrates in the astrocyte secretome. Extracellular proteins released by astrocytes are critical mediators of cerebral homeostasis, including roles in synapse function and vascular integrity. We identified 1,108 proteins in astrocyte condition medium and 295 of these were annotated as secreted proteins. The total abundance of nine proteins was changed after treatment with PK. Characterization of the secreted proteins revealed low molecular weight fragments for 59 proteins in conditioned media exposed to PK that were not observed in untreated controls. The most striking finding from this study was the appearance of fragmentation of 26 extracellular matrix-associated proteins including collagen isoforms 1-6 and11, nidogen-1 and -2, lysyl oxidase-like protein 1, and matrix metalloproteinase 19 in the presence of PK. We also demonstrated that PK induced the fragmentation of non-matrix proteins, including apolipoprotein E. This report further characterizes the astrocyte secretome and identifies novel potential targets of PK-induced proteolysis that may contribute to its effects on the brain following vascular injury.

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.

Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy.

An understanding of the diabetes-induced alterations in vitreous protein composition in the absence and in the presence of proliferative diabetic retinopathy (PDR) may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of vitreous samples from individuals with diabetes but without diabetic retinopathy (noDR) or with PDR and nondiabetic individuals (NDM). Using preparative one-dimensional SDS-PAGE and nano-LC/MS/MS of 17 independent vitreous samples, we identified 252 proteins from human vitreous. Fifty-six proteins were differentially abundant in noDR and PDR vitreous compared with NDM vitreous, including 32 proteins increased and 10 proteins decreased in PDR vitreous compared with NDM vitreous. Comparison of noDR and PDR groups revealed increased levels of angiotensinogen and decreased levels of calsyntenin-1, interphotoreceptor retinoid-binding protein, and neuroserpin in PDR vitreous. Biological pathway analysis revealed that vitreous contains 30 proteins associated with the kallikrein-kinin, coagulation, and complement systems. Five of them (complement C3, complement factor I, prothrombin, alpha-1-antitrypsin, and antithrombin III) were increased in PDR vitreous compared with NDM vitreous. Factor XII was detected in PDR vitreous but not observed in either NDM or noDR vitreous. PDR vitreous also had increased levels of peroxiredoxin-1 and decreased levels of extracellular superoxide dismutase, compared with noDR or NDM vitreous. These data provide an in depth analysis of the human vitreous proteome and reveal protein alterations that are associated with PDR.