Diquafosol tetrasodium elicits total cholesterol release from rabbit meibomian gland cells via P2Y2 purinergic receptor signalling.

Diquafosol tetrasodium (DQS), a purinergic P2Y2 receptor agonist, stimulates secretion of both water and mucins from the conjunctiva into tears. Hence, DQS-containing eye drops have been approved as a therapeutic option for dry eye disease in some Asian countries, including Japan. Recent clinical reports state that instilling DQS-containing eye drops significantly increases the lipid layer thickness in tears. Therefore, we examined this compound's direct actions on holocrine lipid-secreting meibomian gland cells and their function. Isolated meibomian gland cells (meibocytes) were procured from rabbits and cultivated in serum-free culture medium. Differentiated meibocytes with pioglitazone were used for the subsequent experiments. Intracellular Ca2+ signalling of the cells was dramatically elevated with DQS addition in a dose-dependent manner. This DQS-induced elevation was almost completely cancelled by the coexistence of the selective P2Y2 receptor antagonist AR-C118925XX. DQS treatment also facilitated total cholesterol (TC) release from cells into the medium. This effect of DQS on TC was suppressed significantly by the intracellular Ca2+ chelator BAPTA-AM as well as by AR-C118925XX. DNA fragmentation analysis revealed that DQS may have enhanced the apoptotic DNA fragmentation caused spontaneously by cells. Thus, DQS could stimulate meibocytes to release lipids through the P2Y2 receptor and possibly facilitate holocrine cell maturation.

Novel use of a chemically modified siRNA for robust and sustainable in vivo gene silencing in the retina.

Despite efficient and specific in vitro knockdown, more reliable and convenient methods for in vivo knockdown of target genes remain to be developed particularly for retinal research. Using commercially available and chemically modified siRNA so-called Accell siRNA, we established a novel in vivo gene silencing approach in the rat retina. siRNA designed for knockdown of the house keeping gene Gapdh or four retinal cell type-specific genes (Nefl, Pvalb, Rho and Opn1sw) was injected into the vitreous body, and their retinal mRNA levels were quantified using real-time PCR. Intravitreal injection of siRNA for Gapdh resulted in approximately 40-70% reduction in its retinal mRNA levels, which lasted throughout a 9-day study period. Furthermore, all the selected retinal specific genes were efficiently down-regulated by 60-90% following intravitreal injection, suggesting injected siRNA penetrated into major retinal cell types. These findings were consistent with uniform distribution of a fluorescence-labeled siRNA injected into the vitreous body. Interestingly, gene silencing of Grin1, a core subunit of NMDA receptor, was accompanied by significant prevention from NMDA-induced retinal ganglion cell death. Thus, we provide single intravitreal injection of Accell siRNA as a versatile technique for robust and sustainable in vivo retinal gene silencing to characterize their biological functions under physiological and pathophysiological conditions.

Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.

Identification of human oral keratinocyte stem/progenitor cells by neurotrophin receptor p75 and the role of neurotrophin/p75 signaling.

This study was undertaken to determine whether human oral keratinocyte stem cells characteristically express higher levels of the low-affinity neurotrophin receptor p75 and to elucidate the function of p75 in oral keratinocytes. Examination of their expression patterns and cell-cycling status in vivo showed that p75 was exclusively expressed in the basal cell layer of both the tips of the papillae and the deep rete ridges. These immunostaining patterns suggest a cluster organization; most p75(+) cells did not actively cycle in vivo. Cell sorting showed that cells in the p75(+) subset were smaller and possessed higher in vitro proliferative capacity and clonal growth potential than the p75(-) subset. Clonal analysis revealed that holoclone-type (stem cell compartment), meroclone-type (intermediate compartment), and paraclone-type (transient amplifying cell compartment) cells, previously identified in skin and the ocular surface, were present in human oral mucosal epithelium. Holoclone-type cells showed stronger p75 expression at both the mRNA and protein level than did meroclone- and paraclone-type cells. Among the several neurotrophins, nerve growth factor (NGF) and neurotrophin-3 stimulated p75(+) oral keratinocyte cell proliferation, and only NGF protected them from apoptosis. Our in vivo and in vitro findings indicate that p75 is a potential marker of oral keratinocyte stem/progenitor cells and that some neurotrophin/p75 signaling affects cell growth and survival.

Porcine corneal epithelial cells consist of high- and low-integrin beta1-expressing populations.

PURPOSE: Immunohistochemically, all cells in the porcine corneal epithelium, from the superficial to the basal layer, express integrin beta1. Flow cytometric study has shown that they comprise integrin beta1 high-expressing (beta1 2+) and low-expressing (beta1+) populations. This study was undertaken to determine their proliferation characteristics. METHODS: Epithelial cells from porcine corneas were sorted and labeled with anti-integrin beta1 antibody and a fluorescent-dye-conjugated secondary antibody. The fluorescent intensity of labeled cells was analyzed and beta1 2+ and beta1+ cells were cultured in an adhesive-coated culture plate. RESULTS: Flow cytometry demonstrated that the epithelial cells comprised two distinct populations with a similar ratio throughout the cornea. Whereas beta1 2+ cells attached and grew to confluence in the plate, beta1+ attached only transiently to the plate and exhibited minimal growth. CONCLUSIONS: The data indicate that the porcine cornea contains two distinct populations of epithelial cells, one exhibiting high and the other low integrin beta1 expression. The observation that beta1 2+ cells had greater growth potential suggests that they may represent an enriched population of transit-amplifying cells.

Effects of dexamethasone and cyclosporin A on human beta-defensin in corneal epithelial cells.

Human beta-defensins (hBDs), which are mainly expressed in epithelial tissues, may contribute to infection-protective mechanisms in the ocular surface. We examined hBD1 and hBD2 expression in the ocular surface by RT-PCR and immunohistochemistry and studied the effects of immunosuppressive agents on their expression in human corneal epithelial cells in vitro. mRNA expression of hBD1 and hBD2 was confirmed in corneal epithelial cells. While the hBD1 peptide was immunohistochemically detected in corneal, limbal, and conjunctival epithelium, the level of expression was stronger in limbal- and conjunctival- than in corneal epithelium. Very weak expression of the hBD2 peptide was detected only in corneal epithelium. After 48-h culture of human corneal epithelial cells in the presence of 10(-5) M dexamethasone or 1 mg l(-1) cyclosporin A, total RNA was extracted and hBD1 and hBD2 mRNA expressions compared using semi-quantitative RT-PCR and introduced amplified fragment length polymorphism (iAFLP) assay. The two methods yielded almost identical results. hBD1 mRNA expression was not changed by dexamethasone but was down-regulated by cyclosporin A. hBD2 mRNA expression was up-regulated by dexamethasone and down-regulated by cyclosporin A. Our findings suggest that hBD1 and hBD2 are regulated by different mechanisms and that hBD1 may contribute to infection-protective mechanisms in the relatively immunosuppressed status induced by dexamethasone.

Human amniotic membrane, like corneal epithelial basement membrane, manifests the alpha5 chain of type IV collagen.

PURPOSE: To reexamine whether the alpha5 chain of type IV (alpha5[IV]) collagen, thought to be absent, is in fact present in human amniotic membrane. METHODS: Cryosections of human amniotic membrane obtained at Cesarean section were immunohistochemically examined for the presence of alpha5(IV), with or without inclusion of the denaturing step. Amniotic membrane was digested with collagenase to release the noncollagenous NC1 domain from the alpha-chain. The NC1 domain of alpha5(IV) was then assayed on Western blot analysis. Identical experiments were performed with human corneas and conjunctivae obtained from an American eye bank. RESULTS: The basement membrane of denatured samples of amniotic membrane and cornea stained positive for alpha5(IV). Without the denaturing step, only corneal samples were positive. With or without denaturing, conjunctival epithelium did not stain. Western blot analysis detected NC1 domains of alpha5(IV) in amniotic membrane and corneal samples. CONCLUSIONS: The basement membrane of amniotic membrane resembles that of corneal epithelium but not conjunctiva. Amniotic membrane may be an excellent substrate for corneal epithelial cells.

The presence of keratin 5 as an IgG Fc binding protein in human corneal epithelium.

Rabbit immunoglobulin G (IgG) binds specifically to the human corneal epithelium. We investigated this phenomenon to identify the binding material in the tissue. Sections of human cornea were immunostained with negative-control grade rabbit IgG, F(ab')(2) (the antigen recognition fragment of IgG yielded by pepsin digestion), and Fc fragments. Human corneal epithelium was homogenized in a buffer containing Triton X-100 and the water-insoluble residue was subjected to two-dimensional gel electrophoresis. Western blotting was performed on the gels to detect materials that bound with rabbit IgG Fc fragments. Spots in the gel that bound with the Fc fragments were collected and analysed by peptide mass fingerprinting (PMF). Using an antibody against a binding candidate, keratin 5 (suggested by PMF analysis), we checked the occurrence of this phenomenon. Immunohistochemistry showed that rabbit IgG bound to corneal epithelium via the Fc region but not F(ab')(2). Western blots using rabbit Fc fragments showed that some protein spots in the gel bound to the fragments. All of the binding materials were basic and had molecular weights of approximately 55 kDa. PMF analysis showed that the binding material was keratin 5; all binding materials on the transferred membrane were recognized by an anti-keratin 5 antibody. On corneal sections, anti-keratin 5 produced staining similar to that of rabbit IgG. These findings suggest that rabbit IgG binds to keratin 5 in the human corneal epithelium via the Fc region of the molecule.

The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane.

PURPOSE: To determine the feasibility of using human amniotic membrane (AM) as a substrate for culturing oral epithelial cells and to investigate the possibility of using autologous cultivated oral epithelial cells in ocular surface reconstruction. METHODS: An ocular surface injury was created in one eye of each of eight adult albino rabbits by a lamellar keratectomy, and a conjunctival excision was performed, including and extending 5 mm outside the limbus. Oral mucosal biopsy specimens were obtained from these eight adult albino rabbits and cultivated for 3 weeks on a denuded AM carrier. The cultivated epithelium was examined by electron microscopy (EM) and immunohistochemically labeled for several keratins. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces of the eight rabbits were surgically reconstructed by transplanting the autologous cultivated oral epithelial cells on the AM carrier. RESULTS: The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. EM revealed that the epithelial cells were very similar in appearance to those of normal corneal epithelium, had numerous desmosomal junctions, and were attached to a basement membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the keratin pair 4 and 13 and keratin-3 in the cultivated oral epithelial cells. Corneas that were grafted with the cultivated oral epithelial cells on an AM carrier were clear and were all epithelialized 10 days after surgery. CONCLUSIONS: Cultures of oral epithelial cells can be generated to confluence on AM expanded ex vivo from biopsy-derived oral mucosal tissue. Autologous transplantation was performed with these cultivated oral epithelial cells onto the ocular surfaces of keratectomized rabbit eyes. Autologous transplantation of cultivated oral epithelium is a feasible method for ocular surface reconstruction. The long-term outcome of such transplantation is not yet clear, and its feasibility in clinical use should be evaluated further.