Constitutive secretion of chemokines by cultured human trabecular meshwork cells

Trabecular meshwork endothelial (TME) cells secrete a number of factors, such as enzymes and cytokines, which modulate the functions of the cells and the extracellular matrix of the conventional aqueous outflow pathway. TME cells usually secrete these factors in response to stimuli such as mechanical stretching, laser irradiation and pro-inflammatory cytokines. Here, we report that cultured human TME cells isolated from two non-glaucomatous individuals secrete significant quantities of the chemotactic cytokines IL8, CXCL6 and MCP1 in the absence of any stimulation. The secretion of these chemokines was augmented by treatment with the pro-inflammatory cytokines TNFα and IL1β. By way of comparison, there was little or very low production of the three chemokines by human non-pigmented ciliary epithelial cells in the absence of stimulation. Our findings provide support to our recent observations that monocytes, presumably under the influence of chemotactic signals, circulate through the trabecular meshwork in the normal state and also that cytokines regulate the permeability of Schlemm’s canal endothelial cells. In addition, the fact that normal TME cells constitutively secrete chemotactic cytokines strengthens the notion that cytokines play a key role in the homeostasis of the outflow of the aqueous humor and, possibly, in the pathogenesis of glaucoma.     

Somatostatin receptor gene expression in human ocular tissues: RT-PCR and immunohistochemical study

PURPOSE: Somatostatin (SST) analogues have been used to treat proliferative diabetic retinopathy, pseudotumor cerebri, thyroid orbitopathy, and cystoid macular edema. There is a paucity of published data in regards to cell-specific distribution of SST receptors (SSTR) in normal human eye tissues. Gene expression for all five known SSTRs in normal human ciliary body/iris, retina, choroid, and cultured retinal pigment epithelial (RPE) cells were studied. METHODS: mRNA was isolated from human ocular tissues (iris/ciliary body, retina, and choroid) dissected from eight pairs of normal eyes (9-62 years) and from RPE cells grown in culture. RT-PCR was done for all five SSTRs in all analyzed tissues. Immunohistochemistry for SSTR1 and SSTR2 was performed on eight pairs of normal human eyes (28-74 years) imbedded in paraffin. RESULTS: SSTR1 to 5 genes are expressed in retina, SSTR1 and SSTR2 genes in cultured RPE cells, and SSTR1, 2, and 4 in ciliary body and choroid. SSTR1 and SSTR2 immunoreactivity (-ir) was observed on a variety of cells within all analyzed tissues including cornea, iris, trabecular meshwork, Schlemm's canal, ciliary processes, ciliary muscle, retina, choroid, cultured RPE cells, and optic nerve. CONCLUSIONS: SSTR genes are widely expressed in normal human eye tissues, with genes for SSTR1 and SSTR2 being the most widely expressed. Genes for all SSTRs are expressed in retina. SSTR1-ir and SSTR2-ir were observed in all analyzed ocular tissues. Detailed knowledge of SSTRs distribution and function in the human eye will result in a better understanding of their role in health and disease.     

The NEIBank project for ocular genomics: data-mining gene expression in human and rodent eye tissues

NEIBank is a project to gather and organize genomic resources for eye research. The first phase of this project covers the construction and sequence analysis of cDNA libraries from human and animal model eye tissues to develop an overview of the repertoire of genes expressed in the eye and a resource of cDNA clones for further studies. The sequence data are grouped and identified using the tools of bioinformatics and the results are displayed through a web site where they can be interrogated by keyword search, chromosome location, by Blast (sequence comparison) or by alignment on completed genomes. Many novel proteins and novel splice forms of known genes have already emerged from analysis of the accumulating data. This review provides an overview of the current state of the database for human eye tissues, with specific comparisons to some parallel data from mouse and rat, and with illustrative examples of the kinds of insights and discoveries these data can produce. One of the major themes that emerges is that at the molecular level human eye tissues have significant differences from those of rodents, encompassing species specific genes, alternative splice forms and great variation in levels of gene expression. These point to specific adaptations and mechanisms in the human eye and emphasize that care needs to be taken in the application of appropriate animal model systems.     

The expression and function of netrin-4 in murine ocular tissues

Netrin-4, a member of the netrin family, is a potent regulator of embryonic development. It promotes neurite extension and regulates pulmonary airway branching, vasculogenesis patterning, and endothelial proliferation in pathological angiogenesis. The initial characterization of netrin-4 expression was focused on epithelial-derived organs (kidney, lung and salivary gland) and the central nervous system. Ocular development is an ideal system to study netrin-4 expression and function, as it involves both ectodermal (cornea, lens and retina) and mesodermal (sclera and choroid) derivatives and has an extensive and well-characterized angiogenic process. Netrin-4 is expressed in all ocular tissues. It is a prominent component of the basement membranes of the lens and cornea, as well as all three basement membranes of the retina: the inner limiting membrane, vascular basement membranes, and Bruch's membrane. Netrin-4 is differentially deposited in vascular basement membranes, with more intense anti-netrin-4 reactivity on the arterial side. The retinal microcirculation also expresses netrin-4. In order to test the function of netrin-4 in?vivo, we generated a conventional mouse lacking Ntn4 expression. Basement membrane formation in the cornea, lens and retina is undisrupted by netrin-4 deletion, demonstrating that netrin-4 is not a major structural component of these basement membranes. In the Ntn4 homozygous null (Ntn4-/-) cornea, the overall morphology of the cornea, as well as the epithelial, stromal and endothelial stratification are normal; however, epithelial cell proliferation is increased. In the Ntn4-/- retina, neurogenesis appears to proceed normally, as does retinal lamination. In the Ntn4-/- retina, retinal ganglion cell targeting is intact, although there are minor defects in axon fasciculation. In the retinal vasculature of the Ntn4-/- retina, the distribution patterns of astrocytes and the vasculature are largely normal, with the possible exception of increased branching in the deep capillary plexus, suggesting that netrin-4 may act as a negative regulator of angiogenesis. These data, taken together, suggest that netrin-4 is a negative regulator of corneal epithelial cell proliferation and retinal vascular branching in?vivo, whereas netrin-4 may be redundant with other members of the netrin family in other ocular tissue development. Ntn4-/- mice may serve as a good model in which to study the role of netrins in?vivo of the pathobiologic vascular remodeling in the retina and cornea.     

Presence of ornithine ketoacid aminotransferase in human ocular tissues

Ornithine ketoacid aminotransferase activity in the human ocular tissues was biochemically studied. The choroid plus retina, ciliary body, and iris showed high enzyme activity. The enzyme had a pH optimum at near 8.0–8.1, and the K_m value for pyridoxal phosphate was 8 M. The possible role of ornithine ketoacid aminotransferase in the human ocular tissues was discussed.     

Studies of gamma-glutamyl transpeptidase in human ocular tissues.

γ-Glutamyl transpeptidase was found to be in relatively much higher concentrations in human retina and iris than in the lens. In the lens, the enzyme was localized to a major extent in the epithelium plus capsule fraction. The lens cortex and nucleus had very little enzyme activity.γ-Glutamyl transpeptidase was purified about 200-fold to a specific activity of 897 mU per mg protein from cultured human lens epithelium. The purification steps included heating at 37° for 2 hr, deoxycholate extraction, DE-52 column chromatography, Sephadex G-200 gel filtration and affinity chromatography. The enzyme preparation was found to have a K_m of about 0·7 mm for γ-glutamyl p-nitroanilide and a pH optimum of 8·2. Magnesium had no significant effect on the enzyme activity, whereas, sodium and potassium inhibited the enzyme slightly. The competitive inhibition of γ-glutamyl transpeptidase by GSH and GSSG when γ-glutamyl p-nitroanilide was used as substrate indicates that the artificial substrate binds to the protein at the same site as the natural substrate, i.e. GSH. The enzyme was not inhibited by sulfhydryl blocking reagents up to a concentration of 1 mm indicating that the enzyme does not require the presence of sulfhydryl groups for its activity.Antibodies raised against an apparently homogeneous γ-glutamyl transpeptidase from human kidney precipitated the enzyme from ocular tissues and the enzyme purified from the cultured lens epithelial cells. This indicates that the enzyme of the ocular tissues and the kidney are of similar genetic origin.     

Prevalence of SARS-CoV-2 in human post-mortem ocular tissues

BackgroundSARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues.MethodsThe expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in additional 10 research-intent COVID-19 positive donors.FindingsOf 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP–I) disinfection.InterpretationsOur study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.     

Angiotensin-converting enzyme in bovine, feline, and human ocular tissues

Angiotensin-converting enzyme was shown to be present in retinal vessels and neural retina of feline, bovine, and human eyes. It was also demonstrated in the other ocular tissues of feline eyes, in especially high concentration in the highly vascular uveal layer. Its role in the physiology of ocular blood flow and neurophysiology is uncertain, especially in the retina where circulating angiotensin and bradykinin are confined to the intravascular space by the blood-retina barrier, and sufficient data are not available to describe these peptides as transmitters or modulator molecules in the retina.     

Histologic localization of sodium fluorescein in human ocular tissues.

We studied the distribution pattern of sodium fluorescein in human eyes microscopically. The ciliary body showed early and diffuse leakage, with staining of the basement membrane of the nonpigmented ciliary epithelium, indicating movement of fluorescein from the ciliary body into the aqueous humor. After five minutes the iris stroma stained with fluorescein, probably from the aqueous humor. The retinal vessels and retinal pigment epithelium were impermeable to fluorescein. Corresponding to the background fluorescence seen in fluorescein angiography, fluorescence was present in Bruch's membrane and in the stroma of the inner one third of the choroid. Drusen stained most intensely in areas of greatest PAS positively. Early fluorescence of the optic disk was the result of intravascular perfusion of the dye. Minimal diffusion of fluorescein from the fenestrated choroidal vessels across the border tissue of Elschnig into the peripheral optic nerve bundles was observed. Late fluorescene of the optic disk was due mainly to fluorescein staining of the lamina scleralis and glial columns.     

The human leukocyte antigen complex and chronic ocular inflammatory disorders

PURPOSE: To review the role of gene products from the human leukocyte antigen (HLA) complex in the normal functioning of the immune system, ocular inflammation, and models of autoimmunity. METHOD: A review of recently published reports. RESULTS: Many chronic ocular inflammatory diseases are associated with specific alleles of the HLA complex. Understanding how HLA gene products function normally provides clues to the mechanism of disease associations. In the thymus, these molecules control the shape of the developing T-cell repertoire, leading to self-tolerance. In the periphery, HLA molecules bind and present peptide fragments to T cells, leading to a variety of effector functions. Although effector functions are for the most part beneficial, models are reviewed in which peptide-HLA interactions lead to T-cell responses with pathologic consequences. Herpes stromal keratitis is an informative animal model highlighting the role of self-tolerance, infection, and molecular mimicry in the development of autoimmunity. CONCLUSIONS: Human leukocyte antigen gene products may be associated with chronic inflammatory disorders through the unique presentation of "disease-inducing" peptides or the development of a T-cell repertoire prone to autoreactivity and molecular mimicry.