Ptf1a/Rbpj complex inhibits ganglion cell fate and drives the specification of all horizontal cell subtypes in the chick retina.

During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix-loop-helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.

Inducible nitric oxide synthase mediates the change from retinal to vitreal neovascularization in ischemic retinopathy.

Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in the avascular retina, mediated at least in part by a downregulation of VEGF receptor 2 (VEGFR2) in cells adjacent to iNOS-expressing cells. At the same time, pathological intravitreal neovascularization is considerably stronger in iNOS-expressing animals. These findings demonstrate that iNOS plays a crucial role in retinal neovascular disease and show that it offers an ideal target for the control of vitreal neovascularization through improvement of the vascularization of the hypoxic retina.

The protein tyrosine phosphatase interacting protein 51 (PTPIP51) is required for the differentiation of photoreceptors.

Proliferation and differentiation of retinal progenitor cells (RPCs) are tightly controlled by extrinsic cues and distinct combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we investigated the role of the protein tyrosine phosphatase interacting protein 51 (PTPIP51) in the differentiation of RPCs. The expression pattern of PTPIP51 was analyzed by immunostaining during post-natal retinal development in the rat. Ex vivo electroporation has been used to silence or misexpress PTPIP51 in post-natal retinal explants, and the retinal phenotype was investigated after 3-7days in vitro (div). PTPIP51 expression in the retina started postnatally and was maintained throughout adulthood, especially in retinal ganglion cells and in the inner segment of photoreceptor cells. Silencing of Ptpip51 expression in postnatal retina failed to modify the commitment of late RPCs in the different lineages but severely impaired the final differentiation of photoreceptors, observed by a decrease in the fraction of Rhodopsin-positive cells after 7div. By contrast, misexpression of PTPIP51 in early or late RPCs failed to modify the differentiation of the RPCs. Our data demonstrate that PTPIP51 is implicated in the differentiation process of immature photoreceptors. Because PTPIP51 is specifically localized in the inner segment, PTPIP51 may contribute to the complex stage of maturation of the apical segment of these cells.

Characterization of G proteins in rat myometrium. A differential modulation of Gi2 alpha and Gi3 alpha during gestation.

Myometrial membranes, obtained from estrogen-dominated (day 0) rat uteri, were immunoblotted with antiserum (SG1), which recognizes the alpha subunits of both Gi1 and Gi2, with antiserum (LE2) specific for Gi2 alpha, and with I3B antiserum, specific for Gi3 alpha. The data revealed the absence of detectable levels of Gi1 alpha and the simultaneous presence of Gi2 alpha and Gi3 alpha as Gi subunits in rat myometrium. The expression of Gi proteins during gestation (days 0, 12, 21) was studied with the above antibodies. No qualitative change in the nature of Gi alpha species was observed during gestation: Gi1 alpha remained undetectable, Gi2 alpha and Gi3 alpha were both present on days 12 and 21. Of significance was the increase (160%) in the amount of Gi2 alpha at midgestation (day 12) compared to days 0 and 21. A different pattern was observed with Gi3 alpha, which decreased with advancing gestation (day 0 greater than 12 greater than 21). Immunodetection of beta subunits of G proteins indicated the presence of a 35/36 kDa doublet on days 0, 12 and 21, with an increase at midgestation. The simultaneous increase in Gi2 alpha and beta subunits may provide an explanation for the previously demonstrated alteration in adenylate cyclase stimulability detected at midgestation.

Identification of both Gi2 and a novel, immunologically distinct, form of Go in rat myometrial membranes.

Immunoblotting of rat myometrial membranes with an antiserum (SG1) which recognizes the alpha-subunits of both Gi1 and Gi2 indicated the presence of detectable levels of an apparently single form of some 40 kDa. A second antiserum (LE2) specific for Gi2 also recognized this protein, confirming its identity. Immunoblotting of the myometrial membranes with a series of antipeptide (OC1, IM1, ON1) and polyclonal (RV3) antisera, all of which recognize rat brain Go, produced a more complex pattern. Antisera OC1 and ON1 recognized a single polypeptide which essentially comigrated with rat brain Go. In contrast, antisera RV3 and IM1 did not recognize the myometrial protein. These data provide evidence for the presence of Gi2 and of a novel G-protein, related immunologically to Go, in rat myometrial membranes.

Delayed onset and decreased severity of experimental autoimmune uveoretinitis in mice lacking nitric oxide synthase type 2.

To investigate the role of nitric oxide (NO), produced by the inducible form of NO synthase (NOS-2) in the development of experimental autoimmune uveoretinitis (EAU), we immunized C57BL/6x129Sv (H-2(b)) mice carrying a targeted disruption of the gene encoding NOS-2 (NOS-2[-/-]), and wild-type (WT) C57BL/6x129Sv controls with interphotoreceptor retinoid binding protein (IRBP). NOS-2[-/-] mice developed a clinical EAU with delayed onset and decreased severity compared to WT controls. The ocular tissues from WT mice contained activated F4/80 macrophages with NOS-2 expression and retinal destruction whereas less intense EAU was detected in NOS-2[-/-] mice. The expression of NOS-2 mRNA was detected in the retina at the peak of EAU in WT. Analysis of cytokine production in the spleen from NOS-2[-/-] mice by RT-PCR showed high levels of IL-10 mRNA. Our results demonstrate that NO is clearly involved in EAU and may be important for the regulation of immune responses through the regulation of IL-10.

Nitric oxide synthase-II is expressed in severe corneal alkali burns and inhibits neovascularization.

PURPOSE: Inducible nitric oxide synthase (NOS-II) is expressed in many inflammatory conditions. The implication of nitric oxide (NO) in angiogenesis remains controversial. The role of NOS-II and its influence on angiogenesis in corneal neovascularization is unknown and was investigated in this study. METHODS: A mouse model of corneal neovascularization induced by chemical cauterization was used. NOS-II mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction, and NOS-II protein was studied in situ by immunohistochemical analysis of the cornea. The influence of NOS-II on neovascularization was determined by comparison of vessel development in "normal" wild-type mice and mice with a targeted disruption of the NOS-II gene. RESULTS: NOS-II mRNA was induced to very high levels after corneal cauterization and remained upregulated throughout the disease. Migratory cells in the center of the cauterization area expressed NOS-II protein. The neovascular response in mice lacking the NOS-II gene was significantly stronger than in wild-type mice, and the difference increased over time. CONCLUSIONS: These data are the first evidence that NOS-II is expressed in this model of sterile corneal inflammation. NOS-II expression inhibited angiogenesis in severe corneal alkali burns.

Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.

PURPOSE: To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors. METHODS: Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells. CONCLUSIONS: The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.

Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitis rats.

PURPOSE: Inducible nitric oxide (NO) synthase (iNOS) has been implicated in the pathogenesis of endotoxin-induced uveitis (EIU). This study was undertaken to localize the cells, in the eye, which express iNOS during EIU in the rat. METHODS: EIU was induced in Lewis rats by a single foot pad injection of 150 micrograms lipopolysaccharide (LPS) from Salmonella typhimurium. At different time intervals after LPS injection, the authors evaluated ocular inflammation (slit lamp observation), iNOS localization by in situ hybridization, and comparison of OX-42- and ED1-positive cell appearance and of glial response by specific immunohistochemistry. RESULTS: iNOS mRNA was not detected in the iris-ciliary body nor in the retina of control rats. It was detected strongly in the epithelial cells of the iris-ciliary body at 6 hours and also in stromal cells of the ciliary processes at 16 hours after LPS injection. In the neuroretina, iNOS mRNA was observed in the inner layers 16 hours after LPS injection. iNOS-positive cells were also present on the vitreous at this time. At 6 and approximately 16 hours after LPS injection, immunohistochemistry experiments revealed a large number of OX-42- and ED1-positive cells (microglia, macrophages, or polymorphonuclear leukocytes) colocalized in part with some iNOS-positive cells in the ciliary body and in the retina. Furthermore, expression of iNOS in Müller cells cannot be excluded. CONCLUSIONS: These observations confirm that subcutaneous injection of endotoxin dramatically induces NOS mRNA expression in the eye, and they demonstrate that epithelial cells of the iris-ciliary body and cells infiltrating the anterior segment of the eye and the retina are the major source of NO. These results support the hypothesis that both inflammatory and resident ocular cells are involved in iNOS expression during EIU.

Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye.

PURPOSE: To evaluate the effect of intraocular administration of nitric oxide (NO) donors in the rabbit eye on intraocular pressure (IOP), inflammation, and toxicity. METHODS: Intravitreal and intracameral injections of two NO donors, SIN-1 and SNAP, and SIN-1C and BSS were performed. Clinical examination, IOP measurements, protein evaluation in aqueous humor, and histologic analysis of the ocular globes were realized. Nitric oxide release was demonstrated by nitrite production in the aqueous humor and in the vitreous using the Griess reaction. RESULTS: The drastic decrease of IOP, observed after a single NO donor injection, was correlated directly with nitrite production and, thus, to NO release. Injection of inactive metabolite of SIN-1, SIN-1C, which is not able to release NO, did not modulate IOP. When administered in the aqueous humor or in the vitreous, NO did not diffuse from one segment of the eye to another. No inflammation or histologic damage was observed as a result of a single NO donor administration. CONCLUSIONS: Nitric oxide is implicated directly in the regulation of IOP and its acute, and massive release into the rabbit eye did not induce inflammation or other growth toxic effects on the ocular tissues.

Effect of cytokines and nitric oxide on tight junctions in cultured rat retinal pigment epithelium.

PURPOSE: These experiments were designed to study the effect of cytokines and nitric oxide (NO) on rat retinal pigment epithelial (RPE) cell tight junctions in vitro. METHODS: Cultures of confluent RPE cells from retinas of PVG rats (a strain susceptible to development of experimental uveitis) were prepared on filters and incubated with various stimulants. The function of the tight junction was evaluated by measuring the transepithelial electrical resistance (TER) of the cell monolayer and the passive permeation of [3H]inulin across confluent RPE cells. The morphology of the intercellular junctions was visualized by immunolocalization of the tight junction-associated protein zonula occludens-1 (ZO-1) and F-actin. RESULTS: Seventy-two hours after plating, the RPE cell monolayer showed a mean TER level of 67.6+/-18.8 omega/cm2. A decrease in TER was observed after treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). The addition of tumor necrosis factor-alpha (TNF-alpha) accelerated the decrease of TER, whereas NG-monomethyl-L-arginine (L-NMMA) (an NO synthase [NOS] inhibitor) did not further modify the resistance decrease. In contrast, 3-morpholino-sydnonimine (SIN-1), a sydnonimine analog and NO donor, increased the TER. The variations of TER were correlated with the transepithelial fluxes of [3H]inulin and with tight junction morphologic changes of ZO-1 and F-actin immunostaining. CONCLUSIONS: Incubation with LPS associated with IFN-gamma and TNF-alpha induces alterations of RPE tight junctions, whereas NO is involved in the maintenance of their integrity. Cytokines and NO production could play a role in regulation of the blood-ocular barrier function and of the development of ocular inflammation.

Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis.

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.

Nitric oxide in the eye: multifaceted roles and diverse outcomes.

Recent works have highlighted the role of nitric oxide in a wide array of disease entities, including septic shock, hypertension, cerebral ischemia, and chronic degenerative diseases of the nervous system. The functions of nitric oxide appear very diverse, having actions on vascular tone, neurotransmission, immune cytotoxicity, and many others. Nitric oxide is an important mediator of homeostatic processes in the eye, such as regulation of aqueous humor dynamics, retinal neurotransmission and phototransduction. Changes in its generation or actions could contribute to pathological states such as inflammatory diseases (uveitis, retinitis) or degenerative diseases (glaucoma, retinal degeneration). Localization in the eye and biochemical characteristics of nitric oxide will be reviewed. A better understanding of the nitric oxide pathway will be the key to the development of new approaches to the management and treatment of various ocular diseases.

Accumulation of NO synthase (type-I) at the neuromuscular junctions in adult mice.

Recent data have shown that NOS-I is localized almost exclusively to the sarcolemma of fast-twitch fibers, where it probably interacts with the dystrophin-glycoprotein complex. The concentration of dystrophin-related protein at the neuromuscular junctions and the possible involvement of NO in synaptic suppression led us to investigate the presence of NOS-I at the adult motor endplates. Our data clearly show that NOS-I protein, detected by immunohistochemistry accumulates at the adult endplates. Furthermore, the absence of NOS-I protein at the denervated neuromuscular junctions suggest a neural origin of this enzyme. The putative roles of NO at the endplate are discussed.

Protection against light-induced retinal degeneration by an inhibitor of NO synthase.

The existence of nitric oxide synthase (NOS) in retinal rod outer segments and pigmented epithelial cells suggests that NO in excess could impair the interaction between these cells, resulting in photoreceptor degeneration. To test this hypothesis, NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, was intraperitoneally injected daily into rats subjected to constant illumination for 7 days in order to destroy their photoreceptors. By measuring photoreceptor nuclear layer thicknesses, we found that L-NAME partially protects (by up to 35%) against the degeneration of photoreceptors and acts to maintain their organization. Thus NO may be involved in the process by which photoreceptor degeneration results from constant illumination of the retina.

Expression of inducible nitric oxide synthase in cytomegalovirus-infected glial cells of retinas from AIDS patients.

The inducible isoform of nitric oxide synthase has been detected in cytomegalovirus (CMV)-infected retinas from acquired immunodeficiency syndrome (AIDS) patients by immunohistochemistry and NADPH-diaphorase staining. Subsequent immunohistochemistry using antibodies against CMV antigens and glial fibrillary acidic protein (GFAP) demonstrated that inducible NOS was localized in CMV-infected glial cells, particularly Müller cells. These findings indicate that inducible NOS is expressed in vivo in the human retina as a result of viral infection, and suggest that high levels of NO production might be involved in CMV-induced retinitis.

Fibroblast growth factors alter light responses and dark voltage in retinal rods of the frog (Rana temporaria).

Fibroblast growth factors (FGF-1 and FGF-2) were applied intracellularly via whole-cell patch-clamp electrodes while the membrane voltage was recorded simultaneously. During recording the exchange of substances by diffusion between cytosol and pipette medium affects the cell's function. Under control conditions, the loss of nucleotides is reflected by a slow hyperpolarization of the dark voltage and prolongated light responses. Addition of FGF-1 and FGF-2 to the pipette medium accelerated the time course of the hyperpolarization and intensified the prolongation of the light responses. The depolarization of photoreceptor cells after intracellular application of the nitric oxide (NO)-synthase cofactor nicotinamide adenine dinucleotide phosphate (NADPH) and the stabilization of light response recovery by L-arginine is abolished by FGF-2. FGF-2 was ineffective when it was applied together with the calcium chelator ethylene glycol-bis(2-aminoethylether)tetraacetate (EGTA). The results indicate a possible role of FGF in the regulation of NO and calcium in photoreceptor cells and may explain protective effects of FGF in degenerative processes of photoreceptor cells.

Differential regulation of nitric oxide synthase-II mRNA by growth factors in rat, bovine, and human retinal pigmented epithelial cells.

Retinal pigmented epithelial cells (RPE cells) express the inducible isoform of NO synthase (NOS-II) after stimulation by cytokines and endotoxin. We have attempted to describe and compare the effect of fibroblast growth factor (FGF) and transforming growth factor beta (TGFbeta) on NOS-II mRNA level in three different cell species: rat, bovine, and human. Northern blot analysis demonstrated that, in the rat RPE cells, FGF upregulates and TGFbeta inhibits NOS-II mRNA accumulation, whereas in the bovine cells, the opposite effect appears. For the human RPE cells, RT-PCR analysis demonstrated that FGF upregulates and TGFbeta inhibits NOS-II mRNA accumulation, as in rat cells, even though the effect of TGFbeta was weaker. Thus, this study demonstrates that marked differences in growth factors regulating NOS-II occur between species, suggesting fundamental signal transduction differences at some level between rat, human, and cow.

Tumor necrosis factor and nitric oxide production by resident retinal glial cells from rats presenting hereditary retinal degeneration.

The inherited retinal dystrophy observed in Royal College of Surgeons (RCS) rats is a widely used model for the study of the photoreceptor degeneration that occurs in retinitis pigmentosa and macular degeneration. The visual cell degeneration is accompanied by an abnormal accumulation of microglial cells in the retina of RCS rats presenting the dystrophy. In the present study, we show that combined stimulation of RCS dystrophic retinal Müller glial (RMG) cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) induced the release in culture supernatants of significantly higher amounts of tumor necrosis factor (TNF) and nitric oxide (NO) compared to nondystrophic congenic controls. In contrast, the levels of TNF and NO found in the supernatants from microglial cells were not significantly different in both strains. Interestingly, as shown by thymidine incorporation, microglial cells from RCS dystrophic rats have a prominent capacity of proliferation in culture medium compared to microglia isolated from RCS non dystrophic controls. Incubation of RMG cells and microglia with the stereoselective inhibitor of NOS, NG-monomethyl-L-arginine (L-NMMA), inhibited nitrite release in LPS + IFN-gamma-stimulated RMG cells and microglia. The addition of TGF-beta with LPS + IFN-gamma clearly inhibited TNF release in supernatants from both dystrophic and control rat RMG cells and microglia. While TGF-beta significantly inhibited nitrite synthesis in RMG cells, the effect on nitrite synthesis by microglia was very low. The retinal dystrophy observed in RCS dystrophic rats could result from an abnormal reactivity of RMG and microglial cells to release TNF and NO in response to stimulants. The immunomodulatory cytokine TGF-beta and inhibitors of NOS could be negative regulators in the cytokine network and nitrite synthesis thus interfering with the development of photoreceptor cell death.

Differential tumor necrosis factor and nitric oxide production in retinal Müller glial cells from C3H/HeN and C3H/HeJ mice.

Tumor necrosis factor (TNF) and nitric oxide (NO) have been shown to play a role in the pathogenesis of endotoxin-induced uveitis (EIU) in rats. Susceptibility to develop EIU in vivo is correlated with the extent of TNF production by retinal Müller glial cells (RMG). Moreover, RMG cells from the susceptible Lewis rat strain synthesize high amounts of nitrite under in vitro stimulation. Variations in susceptibility to endotoxin are observed among mice strains: C3H/HeN mice are known to be susceptible to develop EIU while C3H/HeJ are refractory. We show here that treatment of RMG cells from both strains with LPS + IFN-gamma does not induce TNF-synthesis in culture supernatants but produces high amounts of NO only in the supernatants from activated C3H/HeN RMG cells. The addition of TNF in the culture medium containing LPS/IFN-gamma further increases nitrite production in C3H/HeN RMG cells and allows the synthesis of low levels of nitrite in C3H/HeJ RMG cells. Addition of a specific NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), blocks NO release. We have previously shown that intraperitoneal injections of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) which inhibited nitrite and TNF release in the ocular media reduced EIU in rat. We conclude here that the in vivo susceptibility to develop EIU in mice is correlated with the extent of in vitro nitrite production by RMG cells confirming the implication of NO in the induction of ocular inflammation. The low level of retinal inflammation observed during EIU in C3H/HeN mice compared to rats could be related to the absence of TNF production by RMG cells.