Expression of the VEGF gene family during retinal vaso-obliteration and hypoxia.

Vascular insufficiency and retinal ischaemia precede many proliferative retinopathies and stimulate secretion of vasoactive growth factors. Vascular endothelial growth factor (VEGF) plays a major role and we therefore investigated the other members of the VEGF family: Placental growth factor (PlGF), VEGF-B, -C, and -D, and platelet derived growth factors (PDGF) A and B. Neonatal mice were exposed to hyperoxia for 5 days and then returned to room air (resulting in acute retinal ischaemia). RT-PCR demonstrated that all the members of the VEGF family are expressed in the retina and in situ hybridization (ISH) located their mRNAs primarily in ganglion cells. Similarly to VEGF itself, VEGF-C, PDGF-A, and PDGF-B were upregulated during retinal ischaemia (P < 0.05). Only PlGF gene expression increased during hyperoxia (P < 0.01). The expression pattern of these growth factors suggests a role in the normal retina and during vaso-obliterative and ischaemic phases.

eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage

Purpose: In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina.

Methods: Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA.

Results: In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels.

Conclusions: In hyperoxia, reduced BH bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia. © 2012 The Association for Research in Vision and Ophthalmology, Inc.

Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis?

Retinopathy is a major complication of diabetes mellitus and this condition remains a leading cause of blindness in the working population of developed countries. As diabetic retinopathy progresses a range of neuroglial and microvascular abnormalities develop although it remains unclear how these pathologies relate to each other and their net contribution to retinal damage. From a haemodynamic perspective, evidence suggests that there is an early reduction in retinal perfusion before the onset of diabetic retinopathy followed by a gradual increase in blood flow as the complication progresses. The functional reduction in retinal blood flow observed during early diabetic retinopathy may be additive or synergistic to pro-inflammatory changes, leucostasis and vaso-occlusion and thus be intimately linked to the progressive ischaemic hypoxia and increased blood flow associated with later stages of the disease. In the current review a unifying framework is presented that explains how arteriolar dysfunction and haemodynamic changes may contribute to late stage microvascular pathology and vision loss in human diabetic retinopathy.

HPLC Determination of Angiotensin-Converting Enzyme Activity on Toyopearl HW-40S Column

Angiotensin-converting enzyme (EC3.4.15. I; ACE), isa membrane-bounddipeptidyl carboxypeptidase that mediates the cleavage of the C-terminal dipeptide His-Leu of the decapeptide angiotensin, generating the most powerful endogenous vaso-constricting angiotensin.
Some ACE inhibitors, such as Captopril, have been used as anti-hypertensive drugs. Moreover in recent years, large quantities of ACE inhibitors have been identijied and isolated from peptides derivedfrom food material such as casein, soy protein, jish protein and so on. Functional food with hypotensive effect has been developed on the basis of these works.
Typicalprocedures for screening hypotensive peptides offood origins are separationof products of peptic and tryptic digestion of proteins followed by inhibitory activitydetermination of each fraction. A method developed by Cushman has been the mostwidely used, in which ACE activity is determined by the amount of hippuric acid
generated as a product of enzymatic reaction of ACE with tripeptide of hippuryl-Lhistidyl-L-leucine. Hippuric acid is determined spectrophotometrically at 228 nm after its isolation from the reaction system by ethylacetate extraction, which not only requires alarge quantity of reagent but also results in large error.
An improved method based on Cushman ’s method is proposed in this paper. In this method, an enzymatic reaction system is based on Cushman’s method, while isolation and determination of hippuric acid is performed by medium perjormance gel chromatography on a Toyopearl HW-40s column. Due to the size exclusion nature of the column with somewhat hydrophobic properties, complete separation of four existing fractions in the reaction system is obtained within a smallfraction of the time necessary in Cushman’s method, with ideal reproducibility.