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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.