In vivo binding of topically applied human bFGF on rabbit corneal epithelial wound

We present the results of the first evaluation of human placenta extracted basic fibroblast growth factor (bFGF) in a rabbit corneal epithelium wound-healing model. Healing dose-response experiments after selective epithelial wounding with iodine vapors demonstrated that bFGF accelerated the repair process in a saturable manner. Corneal binding of topically applied 125I-labeled bFGF was investigated using radioassay and autoradiographic techniques. Basic FGF was shown to bind specifically to denuded epithelial basement membrane in a very stable fashion and not to the intact epithelium. No transfer of the topical bFGF to the aqueous humor or any intraocular structure could be observed. The stability of this interaction was further demonstrated by reextracting and characterizing the labeled factor from treated corneas. The specificity of the fixation was documented by in vivo topical competition with unlabeled bFGF or heparin. We propose that bFGF-basement membrane interactions play a role in corneal wound healing.     

Modulation of Transforming Growth Factor-β Actions in Rat Osteoblast-like Cells: The Effects of bFGF and EGF

Abstract

The present study examines how the mitogenic and differentiation functions of transforming growth factor-β (TGF-β) are modulated by basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in primary cultures of rat osteoblast-like (ROB) cells. TGF-β, bFGF, and EGF individually stimulated [³H]thymidine incorporation and cell proliferation in a dose range of 0.01-10 ng/ml. When studied in combination, high doses of bFGF and EGF were additive to low doses of TGF-β. The additive effects of bFGF and EGF on mitogenesis diminished with increasing doses of TGF-β. These three factors also decreased alkaline phosphatase activity individually within the same dose range. When cells were treated with the combined factors, only high doses of bFGF and EGF were additive to the TGF-β inhibition. We were unable to detect any change in collagen synthesis with each individual factor or in combined treatments. In addition, TGF-β or bFGF alone or in combination did not affect fibronectin synthesis. Our studies showed that the biological functions of TGF-β can be modulated by bFGF and EGF in ROB cells. The pattern of modulation is varied depending on the specific function examined.     

The Fate of Intravenously Administered bFGF and the Effect of Heparin

Abstract

The fate and effects of intravascular bFGF are unknown. We have investigated the fate of bFGF administered intravenously to rats in the presence and absence of heparin, and evaluated the effect of a 3-day IV infusion of bFGF on proliferation of endothelial and vascular smooth muscle cells in situ. [¹²⁵I]bFGF, administered as an IV bolus, was rapidly cleared from the circulation (t_1/2 = 1.5 min) by the liver. Nevertheless, it was maintained at a constant, predictable concentration in the blood (9.7 ± 4% of the amount infused) by continuous IV infusion. Heparin consistently altered the pattern: slowing the rate of clearance (t_1/2 = 4.5 min), increasing the plateau concentration in the blood during continuous infusion (32.5 ± 14.3% of the amount infused), and allowing intact (as determined by gel analysis) bFGF to cross from the circulation into the urine. A 3-day infusion of bFGF alone (2.5 ng/kg/min) and with adenosine (11.6 μM/kg/hr) did not increase [³H]thymidine incorporation in either endothelial cells or vascular smooth muscle cells, suggesting that they are refractory to this factor when it is administered intravascularly.     

Distribution of Acidic and Basic Fibroblast Growth Factors (FGF) in the Foetal Rat Eye: Implications for Lens Development

Abstract

Previously we reported that, in vitro, lens cells proliferate, migrate or differentiate in response to low, medium and high concentrations of FGF respectively. To examine further the role of FGF in lens development we used immunohistochemistry to study the distribution of aFGF and bFGF in the eye of the 20 day rat foetus. Strong aFGF-like reactivity was localised in a band of cells near the lens equator which included the germinative zone where most cell proliferation occurs and the transitional zone where epithelial cells differentiate into fibres. The closely apposed inner epithelial layer of the ciliary and iridial retina also reacted strongly. Reactivity for aFGF was also found in the epidermis and in the corneal and conjunctival epithelia. In the neural retina, reactivity was found in the nerve fibre layer and in isolated cells of the inner plexiform layer. bFGF-like reactivity was found in the retinal ganglion cell layer, extra-ocular muscles and associated with endothelial cells of the hyaloid, lenticular and choroid vasculatures. Pre-digestion of sections with hyaluronidase caused loss of cell-associated reactivity but revealed strong bFGF-like reactivity in ocular basement membranes, in particular, the lens capsule. The sensitivity of this capsular bFGF localisation to heparinase indicates that bFGF in the extracellular matrix is complexed with heparan sulphate proteoglycans. The results of this study are consistent with the hypothesis that FGF plays an important role in lens development via both autocrine and paracrine mechanisms.     

Expression of epidermal growth factor receptors by odontogenic jaw cysts

The expression of epidermal growth factor receptor (EGFr) by odontogenic epithelium was studied in odontogenic cysts (n=35), ameloblastoma (n=6), and periapical granulomas containing proliferating epithelial rests of Malassez (n=7) using a panel of monoclonal antibodies to EGFr (clone E30, F4 and C11) known to react with formalin-fixed, paraffin-embedded sections. Odontogenic epithelium in all specimens demonstrated immunoreactivity with all three antibodies. Clone E30 consistently gave the most intense, membrane located staining pattern of the three antibodies tested. Generally, staining of epithelial cells progressively diminished with movement away from the basal cell layers toward the most superficial layers of cystic lining or centre of epithelial rests and tumour islands. Developmental odontogenic cysts (odontogenic keratocysts,n= 13; dentigerous cysts,n=11) and ameloblastoma (follicular type,n=5; unicystic type,n=1) expressed a higher level of EGFr staining than inflammatory cysts (radicular cysts,n=11) and the proliferating epithelial rests in periapical granulomas. However, foci of weak EGFr staining of odontogenic keratocyst lining, similar to that seen in radicular cysts, were found in areas associated with inflammation. In addition, epithelial rests not associated with inflammatory cell infiltrates exhibited stronger reactivity for EGFr than proliferating rests within periapical granulomas. These results indicate that the level of EGFr expression by odontogenic cysts and rests is related to the presence of inflammation within adjacent connective tissue and that there is no detectable difference in receptor expression between developmental cysts and ameloblastoma.     

Epidermal growth factor can replace thymic mesenchyme in induction of embryonic thymus morphogenesis in vitro

The thymus is surrounded by a thin layer of mesenchyme and the epithelial-mesenchymal interaction is known to be essential for the thymus development. To clarify the roles of mesenchyme in the thymus lobule formation that occurs around embryonic days 14–15 in vivo, we set up a three-dimensional organ culture system. The epithelium of embryonic day 13 thymic primordium was separated from the mesenchyme and cultured in Matrigel (reconstituted basement membrane). Addition of the mesenchyme to a chamber separated by a membrane filter induced the lobule formation of the thymic epithelium in vitro. We found that epidermal growth factor (EGF) can replace the mesenchyme for lobulation of the embryonic thymus in vitro. Among other growth factors tested, only transforming growth factor (TGF)-α was as effective as EGF, in agreement with the fact that EGF and TGF-α bind to the same receptor. These results suggest that EGF or its family members may be involved in morphogenesis and differentiation of the thymus gland epithelium, although we cannot exclude the possibility that other unknown factors are required in vivo.     

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.The cornea is typically avascular in its normal state. However, corneal neovascularization (NV) occurs in conjunction with several corneal diseases such as infection, injury, and autoimmune reactions and is one of the leading causes of blindness. Recent studies have identified several tyrosine kinases and their corresponding ligands that mediate NV, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF).^1,2,3bFGF was first identified as a pro-angiogenic factor and is studied extensively in corneal NV models because it is thought to be a major factor in the induction of corneal NV.^4,5,6 bFGF is secreted by corneal epithelial cells, stromal fibroblasts, and endothelial cells, and is localized to the corneal extracellular matrix.⁷ Low levels of bFGF are produced in unwounded corneas; however, enhanced bFGF production was detected in corneal epithelial cells after injury.⁸ VEGF was also shown to promote NV in corneal wounding models,⁹ and cross talk is thought to occur between bFGF and VEGF during corneal NV. For example, bFGF was shown to induce corneal NV by activating the VEGF/VEGFR system^10,11 and the systemic administration of anti-VEGF-A neutralizing antibodies dramatically reduces this effect.¹²Membrane type-1 matrix metalloproteinase (MT1-MMP) is the first transmembrane-containing matrix metalloproteinase to be identified.¹³ Based on previous reports using corneal wound-healing models, MT1-MMP mRNA is mainly localized to the corneal stroma.¹⁴ During NV, quiescent endothelial cells are activated and migration is facilitated by degrading the extracellular matrix through the action of specific proteases, including MT1-MMP.^15,16,17 The importance of the enzymatic function of MT1-MMP in corneal NV was shown using the corneal pocket assay in MT1-MMP-deficient mice.¹⁸ Interestingly, the expression of MT1-MMP is up-regulated by bFGF stimulation in prostate carcinoma cell lines,¹⁹ and it was also reported that MT1-MMP promotes VEGF secretion.^{20,21,22,23,24,25}In this study, we developed anti-MT1-MMP antibody to localize and characterize MT1-MMP protein in the mouse cornea. To assess the relationship between MT1-MMP and bFGF during corneal NV, we performed experiments that combined the corneal pocket assay using a bFGF pellet with the injection of naked MT1-MMP DNA. We observed an enhanced phosphorylation of MAP kinases in wild-type and MT1-MMP knockin (KI) cell lines over that of MT1-MMP knockout (KO) cell lines, suggesting a role of MT1-MMP in modulating bFGF-mediated signal transduction pathways.     

碱性成纤维细胞生长因子对大鼠实验性肾小管损伤和间质病变的作用

研究碱性成纤维细胞生长因子在大鼠肾小管损伤,再生以及肾间质病变过程中的作用。方法应用Ｎｏｒｔｈｅｒｎ印迹杂交,原位杂交及免疫组织化学ＳＰ方法,观察在大鼠的庆大霉素中毒肾小管损伤和再生过程,。ｂＦＧＦ及其受体表达的情况,＾３Ｈ－ＴｄＲ掺入法检测ｂＦＧＦ对培养的肾小管上皮细胞和肾间质成纤维细胞增殖的影响。结果肾小管上皮细胞损伤和再生过程中,ｂＦＧＦ ｍＲＮＡ有表达,随着修昨的增强,表达量逐渐增高;同时     

Upregulation of fibroblast growth factor-receptor messenger RNA expression in rat brain following transient forebrain ischemia

Recently, we demonstrated that transient forebrain ischemia in rats leads to an early and strong induction of basic fibroblast growth factor (bFGF) synthesis in astrocytes in the injured brain regions. In this study, in order to clarify the targets of such raised endogenous bFGF levels, the messenger RNA (mRNA) expression of its receptors (flg and bek) at in the hippocampus following transient forebrain ischemia induced by four-vessel occlusion for 20 min was investigated using an in situ hybridization technique. Transient forebrain ischemia induced an increase in the number of flg mRNA-positive cells from an early stage (24 h after ischemia) in the hippocampal CA1 subfield where delayed neuronal death occurred later (48–72 h after ischemia). This increase became more marked with the progression of neuronal death and was still evident in the same area 30 days later. The time course of the appearance and distribution pattern of flg mRNA-positive cells in the CA1 subfield were quite similar to those of bFGF mRNA-positive cells. On the other hand, in situ hybridization for bek mRNA showed only slight and transient (observed 72 h and 5 days after ischemia) increases in the number of mRNA-positive cells in the CA1 subfield following ischemia. The use of in situ hybridization and glial fibrillary acidic protein immunohistochemistry in combination demonstrated that the cells in the CA1 subfield that exhibited ischemia-induced flg or bek mRNA expression were astrocytes. These data indicate that transient forebrain ischemia induces upregulation of fibroblast growth factor-receptor expression, accompanied by increased bFGF expression in astrocytes, and suggest that the increased astrocytic bFGF levels in injured brain regions act on the astrocytes via autocrine systems and are involved in the development and maintenance of astrocytosis.     

Sialyl Lewis X modification of the epidermal growth factor receptor regulates receptor function during airway epithelial wound repair

Background Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N‐glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLe^x) in airway epithelial repair. Objective To examine whether an sLe^x decoration of EGFR can modulate receptor function during AEC repair. Methods Primary normal human bronchial epithelial (NHBE) cells were cultured in vitro. Co‐localization of sLe^x and EGFR was examined using confocal microscopy. Expressions of RNA and protein were analysed using RT‐PCR and Western blotting. The final step in the synthesis of sLe^x was catalysed by a specific α‐1,3‐fucosyltransferase (FucT‐IV). To evaluate the role of sLe^x in EGFR activation, a knockdown of the FucT‐IV gene with small interfering RNA (siRNA) and an inhibitory anti‐sLe^x antibody (KM‐93) was used. Results We demonstrated a co‐localization of sLe^x with EGFR on NHBE cells using confocal microscopy. Using a blocking antibody for sLe^x after a mechanical injury, we observed a reduction in EGFR phosphorylation and epithelial repair following injury. FucT‐IV demonstrates a temporal expression coordinate with epithelial repair. Down‐regulation of FucT‐IV expression in NHBE by specific siRNA suppressed sLe^x expression. The use of FucT‐IV siRNA significantly reduced phosphorylation of EGFR and prevented epithelial repair. An immunohistochemical analysis of human normal and asthmatic airways showed a significant reduction in sLe^x and tyrosine‐phosphorylated EGFR (pY⁸⁴⁵‐EGFR) in the epithelium of asthmatic subjects compared with that of normal subjects. Conclusion The present data demonstrate that sLe^x, in association with EGFR, in NHBE is coordinate with repair. This glycosylation is important in modulating EGFR activity to affect the repair of normal primary AEC. Cite this as: S. Allahverdian, A. Wang, G. K. Singhera, B. W. Wong and D. R. Dorscheid, Clinical & Experimental Allergy, 2010 (40) 607–618.     

Dramatic promotion of wound healing using a recombinant human-like collagen and bFGF cross-linked hydrogel by transglutaminase

Abstract

The basic fibroblast growth factor (bFGF) plays an important role in the wound repair process. However, lacking of better biomaterials to carry bFGF still is a challenge in skin repair and regeneration. In this study, the human-like collagen (HLC) cross-linked with transglutaminase (TG) to fabricate a HLC/TG hydrogel to load bFGF. The physical properties of hydrogel, such as interior structure, mechanical property, were characterized in vitro using scanning electron microscopy (SEM), rheometer. Then, the effects of the HLC/TG hydrogel on the bFGF and cell attachmentwere evaluated, and the results showed that the HLC/TG hydrogel has good biocompatibility towards bFGF and cells. Finally, skin wound healing test was performed for the evaluation of HLC/TG hydrogels with bFGF in a mouse model. All results of macroscopic and microscopic analysis indicated that not only our HLC/TG hydrogel provide a delivery of growth factors, but also the HLC/TG hydrogel with bFGF achieving better skin regeneration in the structure and function.     

Sustained release and activation of the growth factor basic fibroblast growth factor from loaded scaffolds in heart valve tissue engineering

Objectives. Loading of biological matrices offers an opportunity to induce specific cell behaviour. We previously reported the use of growth factors to promote cell invasion and proliferation in tissue valve engineering.

We investigated biological matrices preloaded with heparin as an ionically attractive template for the binding, activation and sustained release of basic fibroblast growth factor (bFGF).

Methods. Heparin loading concentrations were evaluated and different incubation times were tested. Heparin and heparin-bound bFGF uptake and release were evaluated by ¹²³I radio-labelling. Biological activity of bFGF was evaluated in vitro.

Results. Maximum heparin uptake was observed for 2000 μg/ml at 2 h and stabilized thereafter. bFGF-loaded matrices showed an initial burst release of 15% within 4 h and thereafter sustained release reaching 21% at 24 h. Released bFGF was bioactive.

Conclusions. This model would be useful in tissue engineering using porcine aortic matrices and could be applied using other growth factors or combinations.     

Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in treatment of corneal alkali burns in mice

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.     

Effect of EGF and bFGF on Fibroblast Proliferation and Angiogenic Cytokine Production from Cultured Dermal Substitutes

Abstract

Growth factors accelerate wound healing but the underlying mechanisms remain poorly understood. The aim of this study was to investigate the effect of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on fibroblast proliferation and production of angiogenic factors from cultured dermal substitutes (CDS). In the first experiment, fibroblasts were seeded into a flask at a density of 1 × 10⁴ cells/cm².Cell proliferation was assessed after culturing in media containing EGF or bFGF at concentrations ranging from 2 to 50 μg. The number of fibroblasts increased significantly in the presence of EGF or bFGF, but fibroblasts detached from the flasks in the presence of 50 μg bFGF. In the second experiment, CDS were prepared by incorporating fibroblasts into collagen gels. To make a wound surface model, the CDS was elevated to the air–liquid interface, on which a spongy sheet of hyaluronic acid (HA) containing EGF or bFGF was placed. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS after 1 week of cultivation was measured by ELISA. When the CDS was covered with a HA sponge containing EGF (Group 1), fibroblasts released 3.5-times more VEGF compared with a HA-alone sponge (control group). When covered with a HA sponge containing bFGF (Group 2), 8.7-times more VEGF was released compared with the control group. Fibroblasts in Groups 1 and 2 released 9.6- and 9.3-times more HGF, respectively, compared with the control group. Thus, EGF stimulates fibroblasts to produce VEGF and HGF, in addition to its ability to enhance epidermal cell proliferation.     

Immunohistochemical localization of c-erbB-2 protein and epidermal growth factor receptor in normal surface epithelium,surface inclusion cysts,and common epithelial tumours of the ovary

Summary The c-erbB-2 (HER-2/neu) protein is a membrane glycoprotein growth factor receptor showing molecular homology with the epidermal growth factor receptor (EGFR). We examined the immunohistochemical reactivity of monoclonal antibodies against both of these proteins in normal surface epithelium, surface inclusion cysts, and common epithelial tumours of the ovary. The ovarian tumours were classified as benign (16), borderline malignant (2), and malignant (19). Normal surface ovarian epithelium was weakly positve for both c-erbB-2 protein and EGFR. In surface inclusion cysts, however, the epithelial cells lining the lumen exhibited stronger staining for c-erbB-2 protein, but no staining for EGFR. All 16 benign ovarian tumours and the 2 borderline malignant ovarian tumours were positive for c-erbB-2 protein and negative for EGFR. Of the ovarian carcinomas, 13 of the 19 (68.4%) were positive for c-erbB-2 protein and negative for EGFR, while 4 showed positivity for both c-erbB-2 protein and EGFR. Two cases were negative for both proteins. Expression of both c-erbB-2 protein and EGFR was found in endometrioid carcinoma with squamous differentiation and in clinically advanced poorly differentiated serous carcinomas. Expression of c-erbB-2 protein appears to be increased and that of EGFR is reduced in the early stage of epithelial ovarian oncogenesis. The expression of EGFR with c-erbB-2 protein in ovarian carcinoma is related both to histological differentiation and/or advanced clinical stage.     

Triptolide Suppresses Alkali Burn‐Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression

Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti‐inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical‐induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real‐time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn‐induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose‐dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn‐induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348–1355, 2017. © 2017 Wiley Periodicals, Inc.     

The core planar cell polarity gene,Vangl2, maintains apical‐basal organisation of the corneal epithelium

The role of the core planar cell polarity (PCP) pathway protein, Vangl2, was investigated in the corneal epithelium of the mammalian eye, a paradigm anatomical model of planar cell migration. The gene was conditionally knocked out in vivo and knocked down by siRNA, followed by immunohistochemical, behavioural and morphological analysis of corneal epithelial cells. The primary defects observed in vivo were of apical‐basal organisation of the corneal epithelium, with abnormal stratification throughout life, mislocalisation of the cell membrane protein, Scribble, to the basal side of cells, and partial loss of the epithelial basement membrane. Planar defects in migration after wounding and in the presence of an applied electric field were noted. However, knockdown of Vangl2 also retarded cell migration in individual cells that had no contact with their neighbours, which precluded a classic PCP mechanism. It is concluded that some of the planar polarity phenotypes in PCP mutants may arise from disruption of apical‐basal polarity.     

The Impact of Type 1 Diabetes Mellitus on Corneal Epithelial Nerve Morphology and the Corneal Epithelium

Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. Although the exact etiology is unknown, it is believed that a reduction in corneal sensitivity and loss of neurotrophic support contributes to corneal disease. Information regarding the relationship between nerve loss and effects on the corneal epithelium is limited. We investigated changes in the corneal epithelium and nerve morphology using three-dimensional imaging in vivo and in situ in a streptozotocin-induced diabetic mouse model. Streptozotocin-treated mice showed increased levels of serum glucose and growth retardation consistent with a severe diabetic state. A reduction in the length of the subbasal nerve plexus was evident after 6 weeks of disease. Loss of the subbasal nerve plexus was associated with corneal epithelial thinning and a reduction in basal epithelial cell density. In contrast, loss of the terminal epithelial nerves was associated with animal age. Importantly, this is the first rodent model of type 1 diabetes that shows characteristics of corneal epithelial thinning and a reduction in basal epithelial cell density, both previously have been documented in humans with diabetic corneal neuropathy. These findings indicate that in type 1 diabetes, nerve fiber damage is evident in the subbasal nerve plexus before terminal epithelial nerve loss and that neurotrophic support from both the subbasal nerve plexus and terminal epithelial nerves is essential for the maintenance of corneal epithelial homeostasis.Diabetes mellitus (DM) is a severe metabolic disease with increasing prevalence worldwide. Diabetic neuropathy is one of the most common complications in DM and increases in incidence with both age and duration of disease.¹ At the ocular surface, loss of corneal innervation in DM is associated with reduced corneal sensitivity and altered tear secretion.^2–4 Reduced aqueous tear production from decreased lacrimal gland innervation and an abnormal blink reflex contribute to the high incidence of dry eye that frequently is encountered clinically. In addition, loss of trophic support, which is essential for homeostasis of the epithelium, is believed to contribute to epithelial fragility, corneal erosions, and persistent epithelial defects.⁵ Changes in corneal thickness also have been reported. In the earlier stages of disease, central corneal thickening occurs as a result of endothelial dysfunction and increased corneal edema.^6,7 Later stages of disease, however, result in thinning of the corneal epithelium, which is associated with a more severe neurotrophic state.⁸ Restoration of corneal epithelial homeostasis and the associated corneal nerve plexus presents an important clinical challenge because many of these neurotrophic conditions are highly refractory to conventional therapies.In vivo confocal microscopy (IVCM) is used increasingly in clinical studies to evaluate disease-related changes in the subbasal nerve plexus (SBNP). In diabetes, morphologic alterations in the SBNP have been associated with both retinopathy and peripheral neuropathy.^9–12 Although rapid and noninvasive, the use of confocal microscopy in clinical studies prohibits examination of the terminal epithelial nerves (TENs) and raises many questions regarding whether the loss documented by the confocal examination represents one or more fibers, and the type of fibers affected.¹³ To address this gap, immunohistochemical studies using rodent models of diabetes have investigated the effects of hyperglycemia on the SBNP and TENs. By using two-dimensional imaging techniques, these studies have reported reductions in both the SBNP and TENs, with early loss of TENs preceding SBNP damage.¹⁴ However, because of the tortuosity and complex branching patterns of the TENs as they run throughout the corneal epithelium, analysis of TENs from two-dimensional data sets limit the data that can be extracted and therefore requires more advanced three-dimensional (3D) modeling. Moreover, none of the rodent studies to date have shown a detectable effect on central corneal epithelial thickness or basal epithelial cell density (BECD), as seen in humans, as a consequence of disease state or nerve loss.^14,15Here, we investigated the effects of type 1 DM on the mouse corneal epithelium. Our primary objective was to evaluate the effects of type 1 DM on total corneal and sublayer thickness and BECD.^16,17 Our secondary objective was to assess the corresponding effects on the neural architecture using 3D volumetric reconstruction of the SBNP and TENs in situ. This allowed for a systematic evaluation of corneal nerve thickness and cellular changes in response to disease duration and animal age.     

Characterization of two Preparations of Antibodies to Basic Fibroblast Growth Factor which Exhibit Distinct Patterns of Immunolocalization

Abstract

Immunoglobulins reactive against basic fibroblast growth factor (bFGF) were obtained from the serum of a single rabbit immunized against residues [1-24] of bFGF conjugated to keyhole limpet hemocyanin (KLH). Pure immunoglobulin preparations no. 1 and no. 2 were prepared using different affinity chromatography columns and preabsorption to KLH-coupled Sepharose for preparation no. 1. Both preparations no. 1 and no. 2 were specific for bFGF in in vitro assays. Competition with synthetic peptides suggests that preparations no. 1 and no. 2 recognize predominantly epitope(s) within residues [16-24]bFGF or residues [1-10]bFGF, respectively, in situ Furthermore, no. 2 (but not no. 1) antibodies can react with tissue-(heparin-)-bound antigen. When used in indirect immunofluorescence for bFGF in frozen heart sections, preparation no. 1 stained predominantly muscle intercalated discs (IcDs); muscle nuclei were also stained, in an overall punctate fashion. Preparation no. 2 stained muscle nuclei strongly, in association with the nuclear envelope; it also stained basement-membrane associated bFGF. Differences in immunostaining were also observed in uterine smooth muscle and kidney sections but not in skeletal muscle. It is plausible that accessibility of various epitopes within the amino-terminal region depends strongly on the local interactions of bFGF. Our data illustrate the importance of using several different antibodies to localize bFGF in a tissue     

Sodium channels in ocular epithelia

Voltage-gated, tetrodotoxin(TTX)-blockable sodium channels are found in most excitable cells and are the primary contributors to action potentials generated by many of these cells. To date, there has only been one report of a non-cultured vertebrate epithelial cell type containing TTX-blockable Na⁺ channels: rabbit non-pigmented ciliary body epithelial cells [Cilluffo MC et al. (1991) Invest Opthalmol Vis Sci 32:1619–1629], and three reports of cultured epithelial cells containing TTX-blockable Na⁺ channels: rabbit non-pigmented and pigmented ciliary body epithelium [Ciluffo MC et al. (1991) Invest Opthalmol Vis Sci 32:1619–1629; Fain GL, Farahbakhsh (1989) J Physiol (Lond) 417:83–103] and human lens epithelium [Cooper K et al. (1990) J Membr Biol 117:285–298]. We report here the presence of sodium currents in two different non-cultured, freshly dissociated transporting epithelial cell types: the rabbit corneal endothelium and the frog lens epithelium. We also report the occurrence of sodium currents in six additional cultured ocular epithelial cell types from three different species. These currents have a current/voltage (I/V) relationship consistent with traditional voltage-gated Na⁺ currents, are quinidine- and TTX-blockable (of the low-affinity TTX-sensitive type), and disappear following bath substitution of Na⁺ with Cs⁺ or K⁺.