Compositional differences between infant and adult human corneal basement membranes.

PURPOSE: Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. METHODS: Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. RESULTS: Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. CONCLUSIONS: The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.

Expression of protein kinase CK2 in astroglial cells of normal and neovascularized retina.

We previously documented protein kinase CK2 involvement in retinal neovascularization. Here we describe retinal CK2 expression and combined effects of CK2 inhibitors with the somatostatin analog octreotide in a mouse model of oxygen-induced retinopathy (OIR). CK2 expression in human and rodent retinas with and without retinopathy and in astrocytic and endothelial cultures was examined by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction. A combination of CK2 inhibitors, emodin or 4,5,6,7-tetrabromobenzotriazole, with octreotide was injected intraperitoneally from postnatal (P) day P11 to P17 to block mouse OIR. All CK2 subunits (alpha, alpha', beta) were expressed in retina, and a novel CK2alpha splice variant was detected by reverse transcriptase-polymerase chain reaction. CK2 antibodies primarily reacted with retinal astrocytes, and staining was increased around new intraretinal vessels in mouse OIR and rat retinopathy of prematurity, whereas preretinal vessels were negative. Cultured astrocytes showed increased perinuclear CK2 staining compared to endothelial cells. In the OIR model, CK2 mRNA expression increased modestly on P13 but not on P17. Octreotide combined with emodin or 4,5,6,7-tetrabromobenzotriazole blocked mouse retinal neovascularization more efficiently than either compound alone. Based on its retinal localization, CK2 may be considered a new immunohistochemical astrocytic marker, and combination of CK2 inhibitors and octreotide may be a promising future treatment for proliferative retinopathies.

Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture.

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.

Generation of neural progenitor cells from whole adult bone marrow.

The efficient and large-scale generation of neural progenitor cells for neural grafting in the treatment of neurological diseases has been a challenge. Here we describe the isolation and successful propagation of neural progenitor cells from adult rat bone marrow. Unfractionated bone marrow cultured in vitro with epidermal growth factor and basic fibroblast growth factor gave rise to cellular spheres which differentiated into neurons and glia. The cellular spheres expressed nestin, a neural stem cell marker as well as CD90, a marker of hematopoietic stem cells. This methodology addresses the ethical and tissue rejection problems associated with fetal neural stem cells and would circumvent the difficulty associated with generating neural progenitors from the adult brain. We demonstrate that bone marrow may offer a renewable autologous extracranial source of neural progenitor cells.

Neuronal injury affects expression of helix-loop-helix transcription factors.

Helix-loop-helix transcription (HLH) factors regulate several stages of neuronal development including differentiation of individual populations of neurons and neurite growth. Here we demonstrate that axonal injury of corticospinal and dorsal root ganglion neurons induces changes in the expression of several HLH transcription factors that function as negative regulators of neurogenesis and neurite outgrowth. However, the expression of HLH transcription factors that stimulate neurogenesis is not affected by the axonal injury. Expression of HES, SHARP and Id family members is suppressed shortly after axonal injury and expression returns to normal levels after 14 days. We hypothesize that down-regulation of these HLH transcription factors is required for initiation of regenerative response to axonal injury.

The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma.

Neural stem cells (NSCs) are capable of tracking migrating glioma cells. To exploit this tropism to generate an antitumor T-cell response, particularly against disseminating tumor pockets, we inoculated intracranial glioma-bearing mice with interleukin 12 (IL-12) producing NSCs. Intratumoral therapy with IL-12-secreting NSCs prolonged survival compared to treatment with nonsecretory NSCs or saline. NSCs demonstrated strong tropism for disseminating glioma, and IL-12-secreting NSC therapy was associated with enhanced T-cell infiltration in tumor microsatellites and long-term antitumor immunity. These results indicate that the use of tumor tracking NSCs represents a potent new therapeutic modality for glioma.

Blocking HES1 expression initiates GABAergic differentiation and induces the expression of p21(CIP1/WAF1) in human neural stem cells.

Mammalian neural stem cells can develop into a variety of neuronal and glial cell types. This involves a highly coordinated process of differentiation in which the Notch signaling pathway and the system of helix-loop-helix (HLH) transcriptional regulators play a key role. By exercising control over proliferation, initiation of differentiation, neurite outgrowth, and synaptogenesis, the network of HLH transcription factors regulates the fate of neural stem cells and progenitors. Here we show that the HLH transcription factor HES1 regulates the proliferation of human neural stem cells and that blocking its expression stimulates the expression of cyclin-dependent kinase inhibitor p21(CIP1/WAF1). Furthermore, we demonstrate that the suppression of HES1 expression initiates differentiation of neural stem cells into neurons, the majority of which develop the GABAergic phenotype. These findings underscore the importance of the HLH network, and HES1 in particular, in guiding the phenotypic development of neural stem cells.