Konerirajapuram Natarajan Sulochana

Eales Disease—An Update

Eales disease, first described by Henry Eales in 1880, remains an enigma. The disease, observed more commonly in the Indian subcontinent than in the rest of the world, occurs in young healthy adult males, initially presenting as retinal periphlebitis and later as retinal ischemia that may lead to vascular alterations and neovascularization. Recurrent vitreous hemorrhage with or without retinal detachment is the common sequelae. In recent years, immunological, molecular biological, and biochemical studies have indicated the role of human leukocyte antigen, retinal autoimmunity, mycobacterium tuberculosis genome, and free radical mediated damage in the etiopathogenesis of this disease. However, its etiology appears to be multifactorial. The management depends on the stage of the disease and consists of medical treatment with oral corticosteroids in the active inflammatory stage and laser photocoagulation in the advanced retinal ischemia and neovascularization stages. The results of vitreoretinal surgery have been found to be satisfactory in case of vitreous hemorrhage with or without retinal detachment.

Histone deacetylase 3 (hdac3) is specifically required for liver development in zebrafish

Histone deacetylases (HDACs) are key transcription regulators that function by deacetylating histones/transcription factors and modifying chromatin structure. In this work, we showed that chemical inhibition of HDACs by valproic acid (VPA) led to impaired liver development in zebrafish mainly by inhibiting specification, budding, and differentiation. Formation of exocrine pancreas but not endocrine pancreas was also inhibited. The liver defects induced by VPA correlate with suppressed total HDAC enzymatic activity, but are independent of angiogenesis inhibition. Gene knockdown by morpholino demonstrated that hdac3 is specifically required for liver formation while hdac1 is more globally required for multiple development processes in zebrafish including liver/exocrine pancreas formation. Furthermore, overexpression of hdac3 but not hdac1 partially rescued VPA induced small liver. One mechanism by which hdac3 regulates zebrafish liver growth is through inhibiting growth differentiation factor 11 (gdf11), a unique target of hdac3 and a member of the transforming growth factor beta family. Simultaneous overexpression or morpholino knockdown showed that hdac3 and gdf11 function antagonistically in zebrafish liver development. These results revealed a novel and specific role of hdac3 in liver development and the distinct functions between hdac1 and hdac3 in zebrafish embryonic development.

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis.

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 310 helix and β-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.

Developing Antiangiogenic Peptide Drugs for Angiogenesis-Related Diseases

Angiogenesis is regulated by stimulators and inhibitors and involve multiple biological processes including endothelial cell proliferation, migration, cell-cell and cell-matrix adhesion, assembly into tube structures as well as apoptosis. Designing and developing peptides for therapeutic application to inhibit angiogenesis is an important area in antiangiogenic drug development. Small peptides have advantages over proteins for therapeutic application, due to their stability, solubility, increased bio-availability and lack of immune response in the host cell. Endogenous protein angiogenesis stimulators and inhibitors hold vital information for designing antiangiogenic peptides for drug development. These proteins function through their interaction with extracellular matrix molecules, cell surface receptors, proteases, as well as growth factors and cytokines. Conserved domains such as thrombospondin type 1 repeats (TSRs), kringle domains as well as critical amino acid residues present in these domains are involved in their functions. By exploiting these properties, several small peptides have been designed, synthetically made and being tested for therapeutic efficacy. Peptides derived from type 1 repeat of thrombospondin, alpha 4 and beta 1 chains of laminin, arginine rich N terminus of endostatin, leucine rich repeat 5 of decorin, pigment epithelium derived factor and N terminal of parathyroid hormone are examples of small antiangiogenic peptides derived from endogenous proteins. Such bioactive peptides are further modified physico-chemically to increase their potency and stability. In addition, phage-display library screening and combinatorial approach are also in use to identify novel antiangiogenic peptides targeting tumour and various proteins. This review will provide a comprehensive summary of the current status of the antiangiogenic peptides and their relevance for drug designing and development. Several critical issues that need to be resolved in translating this concept into clinical practice are also discussed.

Isthmin is a novel secreted angiogenesis inhibitor that inhibits tumour growth in mice

Anti‐angiogenesis represents a promising therapeutic strategy for the treatment of various malignancies. Isthmin (ISM) is a gene highly expressed in the isthmus of the midbrain–hindbrain organizer in Xenopus with no known functions. It encodes a secreted 60 kD protein containing a thrombospondin type 1 repeat domain in the central region and an adhesion‐associated domain in MUC4 and other proteins (AMOP) domain at the C‐terminal. In this work, we demonstrate that ISM is a novel angiogenesis inhibitor. Recombinant mouse ISM inhibited endothelial cell (EC) capillary network formation on Matrigel through its C‐terminal AMOP domain. It also suppressed vascular endothelial growth factor (VEGF)‐basic fibroblast growth factor (bFGF) induced in vivo angiogenesis in mouse. It mitigated VEGF‐stimulated EC proliferation without affecting EC migration. Furthermore, ISM induced EC apoptosis in the presence of VEGF through a caspase‐dependent pathway. ISM binds to αvβ5 integrin on EC surface and supports EC adhesion. Overexpression of ISM significantly suppressed mouse B16 melanoma tumour growth through inhibition of tumour angiogenesis without affecting tumour cell proliferation. Knockdown of isthmin in zebrafish embryos using morpholino antisense oligonucleotides led to disorganized intersegmen‐tal vessels in the trunk. Our results demonstrate that ISM is a novel endogenous angiogenesis inhibitor with functions likely in physiological as well as pathological angiogenesis.

The first but not the second thrombospondin type 1 repeat of ADAMTS5 functions as an angiogenesis inhibitor

Angiogenesis is critical for tumour growth and metastasis where factors that regulate this process are potential targets for development of anti-cancer drugs. In this study, we show that the first TSR domain of the extracellular matrix protease ADAMTS5, unlike the second TSR, has anti-angiogenic activities where it inhibits endothelial cell tube formation on Matrigel, reduces cell proliferation and attachment, while promoting cell apoptosis and migration, all in a dose-dependent manner. Furthermore, it influences the architecture of endothelial cells by disrupting actin stress fibres and reducing focal adhesions, likely via suppressing RhoA activation. TSR1 of ADAMTS5 is therefore a novel anti-angiogenic peptide and could serve as a prototype for future development into anti-cancer drugs.

Involvement of oxidative and nitrosative stress in promoting retinal vasculitis in patients with Eales' disease.

OBJECTIVESnEales disease (ED) is an idiopathic retinal vasculitis condition, which affects retina of young adult males. The histopathological hallmark in ED is the adhesion of leukocytes to the endothelium and the infiltration of these cells into the retinal parenchyma. Phagocyte generated free radicals have been implicated in mediating tissue damage associated with various inflammatory vasculopathies. In the present study, we have investigated the possible role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in causing retinal tissue damage in ED.nnnDESIGN AND METHODSn35 patients with ED and 20 healthy control subjects were included in the study. Monocytes (MC) were separated from peripheral blood of the respective study participants. Inducible nitric oxide synthase (iNOS) protein expression was assessed using Western blot and 3 nitrotyrosine (3NTYR) by reversed phase high performance liquid chromatography (RP HPLC). Thiobarbituric acid reactive substances (TBARS) were determined by measuring malondialdehyde (MDA) formed. Superoxide dismutase (SOD) activity was assayed based on the ability of SOD to inhibit auto-oxidation of epinephrine. Iron, copper and zinc content were determined using Atomic Absorption Spectrophotometer. Immunolocalization of iNOS and 3NTYR was performed on the surgically excised epiretinal membranes (ERM) from patients with ED.nnnRESULTSnThere was a significant increase in the expression of iNOS, as well as 3NTYR accumulation, diminished SOD activity, elevated lipid peroxides, iron, copper and decreased zinc content in the MC of patients with ED when compared with healthy control subjects. The elevated levels of ROS and RNS products correlated with diminished antioxidant status in patients with ED. Strong immunoreactivity for iNOS and 3NTYR was observed in inflammatory cells and endothelial cells in ERM obtained from patients with ED.nnnCONCLUSIONSnOur findings from this study clearly reveal the involvement of RNS and ROS in the development of retinal vasculitis in ED. Based on our present study and earlier studies we confirm the role of free radicals in mediating retinal tissue damage in ED. Hence we believe selective inhibition of iNOS or supplementation with antioxidants vitamin E and C might be beneficial in controlling retinal vasculitis in patients with ED.

Immunolocalization and quantification of advanced glycation end products in retinal neovascular membranes and serum: A possible role in ocular neovascularization

Purpose. Earlier studies have revealed the association of advanced glycation end products (AGE) with the pathogenesis of various micro and macro vascular complications. The purpose of the present study is to localize AGEs, namely carboxy methyl lysine (CML-AGE) and methyl glyoxal-derived AGEs (MG-AGE), in retinal neovascular membranes and to quantify them in serum samples. Methods. Surgically excised retinal neovascular membranes and serum samples obtained from patients with diabetic retinopathy, Eales disease and nondiabetics were studied. Immunolocalization of AGEs namely CML-AGE and MG-derived AGEs was done using avidin biotin complex method and quantification was done by enzyme linked immunosorbent assay (ELISA). Results. CML-AGE immunoreactivity was detected in all cases of Eales disease and 61% cases of diabetic retinopathy and none in idiopathic epiretinal membrane (ERM). MG-AGE immunoreactivity was observed in ~15% of diabetic retinopathy and none in Eales disease and and idiopathic ERM. Quantification of AGEs in serum samples revealed statistically significant increased levels of MG-AGE in diabetes, in relation to nondiabetics with idiopathic ERM and CML-AGE in Eales disease, in relation to diabetics and nondiabetics with idiopathic ERM. Conclusion. Results from this study suggest that AGEs formed through glycation and glycoxidation may play an important role in the development of retinal neovascularization. The immunoreactivity of CML-AGEs in neovascular membrane and its increased levels in serum suggest that inspite of the normoglycemic status, glycoxidation and lipid peroxidation due to oxidative stress may trigger retinal neovascularization in Eales disease, while MG-AGEs in diabetic membrane and serum suggest the role of glycation. Thus the mechanism of neovascularization in different pathological conditions could be different.

Primary vitrectomy for combined rhegmatogenous retinal detachment and choroidal detachment with or without oral corticosteroids: a pilot study.

Purpose: The occurrence of choroidal detachment (CD) in eyes with primary rhegmatogenous retinal detachment (RRD) is relatively uncommon (2%–4.5%). Recent reports suggest that primary vitrectomy yields better anatomic success than scleral buckling. However, for these inflamed eyes with low intraocular pressure, the influence of preoperative oral steroids on reattachment rates has not been elucidated yet. Methods: Twenty eyes with combined RRD and CD that underwent primary vitrectomy were randomized to receive oral steroids (for 1 week) or no oral steroids before surgery. Results: Preoperative clinical data such as mean age, lens status, Snellen visual acuity, duration of macular detachment, CD (size and extent), and retinal detachment characteristics (e.g., extent, number of retinal breaks, atrophic or tractional retinal break, size of retinal break, and location of retinal break) were similarly distributed in both groups. Single-operation anatomic success was 81.8% (9/11) among those patients who received preoperative oral steroids and was 66.7% (6/9) among those who did not receive preoperative oral steroids. After reoperation, anatomic success was 100% in both groups. The mean follow-up was 20.1 months. Conclusion: The results suggest that administration of oral steroids before primary vitrectomy in eyes with combined RRD and CD improves reattachment rates.

Accumulation of 8-hydroxydeoxyguanosine and its relationship with antioxidant parameters in patients with Eales' disease: Implications for antioxidant therapy

Purpose. Eales’ disease (ED) is an idiopathic retinal inflammatory disease, which affects the retina of young adult males. Oxidative stress has been associated with the development of various inflammatory diseases. Accumulation of DNA oxidation products has been considered as an early biomarker for assessing oxidative tissue damage. To our knowledge there are no human clinical reports available on the accumulation of DNA adducts in ocular inflammatory diseases. Hence the aim of the present study was to determine the levels of 8-hydroxy-2deoxyguanosine (8-OHdG) and to correlate it with the antioxidant and oxidant parameters in patients with ED and healthy control subjects. Methods. Twenty-two patients with ED [12 in active perivasculitis (AV) and 10 in healed vasculitis stages (HV)] were recruited for the study. 14 healthy volunteers were included as control. 8-OHdG was determined in their leukocytes by Gas chromatography-Mass Spectrometry (GC/MS) technique. Glutathione levels were determined in plasma, while Superoxide dismutase (SOD) activity and thiobarbituricacid reactive substances (TBARS) content were determined in their erythrocytes. Results. Our results indicated that 8-OHdG levels were elevated by 3 and 1.9 folds respectively in AV and HV stages of patients with ED, when compared with healthy control subjects. Increased 8-OHdG levels correlated with diminished SOD activity and decreased GSH content in patients with ED. Conclusions. To our knowledge, this is the first report of DNA adduct accumulation in patients with ED. Increased DNA adduct accumulation correlated with the severity of the disease, and they lie in parallel with diminished antioxidant capacity observed in patients with ED. Based on the observations from our present work and our earlier studies, we reiterate that oxidative stress is involved in the disease process. Hence we believe antioxidant vitamin E and C supplementation might be beneficial to patients with ED.