Nirmala SundarRaj

PAX6 expression identifies progenitor cells for corneal keratocytes

Keratocytes of the corneal stroma produce a transparent extracellular matrix required for vision. During wound‐healing and in vitro, keratocytes proliferate, becoming fibroblastic, and lose biosynthesis of unique corneal matrix components. This study sought identification of cells in the corneal stroma capable of assuming a keratocyte phenotype after extensive proliferation. About 3% of freshly isolated bovine stromal cells exhibited clonal growth. In low‐mitogen media, selected clonal cultures displayed dendritic morphology and expressed high levels of keratan sulfate, aldehyde dehydrogenase 3A1, and keratocan, molecular markers of keratocyte phenotype. In protein‐free media, both primary keratocytes and selected clonal cells aggregated to form attachment‐independent spheroids expressing elevated levels of those marker molecules. The selected clonal cells exhibited normal karyotype and underwent replicative senescence after 65–70 population doublings; however, they continued expression of keratocyte phenotypic markers throughout their replicative life span. The progenitor cells expressed elevated mRNA for several genes characteristic of stem cells and also for genes expressed during ocular development PAX6, Six2, and Six3. PAX6 protein was detected in the cultured progenitor cells and a small number of stromal cells in intact tissue but was absent in cultured keratocytes and fibroblasts. Cytometry demonstrated PAX6 protein in 4% of freshly isolated stromal cells. These results demonstrate the presence of a previously unrecognized population of PAX6‐positive cells in adult corneal stroma that maintain the potential to assume a keratocyte phenotype even after extensive replication. The presence of such progenitor cells has implications for corneal biology and for cell‐based therapies targeting corneal scarring.

Transforming growth factor-β stimulates collagen and fibronectin synthesis by human corneal stromal fibroblasts in vitro

The effects of transforming growth factor-beta (TGF beta) and epidermal growth factor (EGF) on the synthesis of collagen and fibronectin, and on the proliferation of human corneal stromal fibroblasts in vitro, were evaluated. Human corneal stromal fibroblasts in culture were incubated for 48 hours with TGF beta or EGF in the absence of serum. Collagen and fibronectin in the culture media were measured by a collagenase-digestion assay and a competitive ELISA, respectively. The effects of the growth factors on proliferation were assessed by 3H-thymidine incorporation. Collagen synthesis was dose-dependently stimulated by TGF beta; at a concentration of 1 ng/ml of TGF beta, a 120% increase in collagen synthesis was seen over that of controls (p < 0.01). EGF, at a concentration of 10 ng/ml, induced a 40% increase in collagen synthesis over that of controls (p < 0.01). The maximum stimulation by TGF beta was greater than that by EGF (p < 0.05). Fibronectin synthesis was stimulated by TGF beta and EGF in a dose-dependent manner; 230% (p < 0.001) and 210% (p < 0.01) increases in fibronectin synthesis were caused by 10 ng/ml TGF beta and EGF, respectively. TGF beta and EGF dose-dependently stimulated 3H-thymidine incorporation. The maximum increases in 3H-thymidine incorporation reached 180% (p < 0.001) and 190% (p < 0.001) over that in controls, at 10 ng/ml concentrations of TGF beta and EGF, respectively. In conclusion, both TGF beta and EGF are potent stimulants of collagen and fibronectin synthesis and proliferation. Therefore, these two growth factors may be effective alternatives or additional choices for the treatment of corneal ulcer.

The multipotential cells of the limbus.

Ample evidence exists that there is a centripetal movement of cells from the periphery of the cornea toward the centre. While conjunctival cells have the capacity to transdifferentiate into corneal epithelial cells, the limbal region appears to act as a barrier between the conjunctival and corneal epithelia, even after large epithelial defects are created.The existence of limbal stem cells is suggested by the apparent role of the limbus in acting as a source of peripheral corneal cells. While specific staining of limbal cells has been reported in the rabbit, there is no positive identification of such stem cells in the human. However, in the human there is negative staining for both a keratin cytoskeleton antigen and a cell surface antigen in the limbal epithelial zone. Efforts positively to identify human limbal stem cells continue, as do efforts to culture and transplant such cells.

Initial In Vitro Investigation of the Human Immune Response to Corneal Cells from Genetically Engineered Pigs

PURPOSE To compare the in vitro human humoral and cellular immune responses to wild-type (WT) pig corneal endothelial cells (pCECs) with those to pig aortic endothelial cells (pAECs). These responses were further compared with CECs from genetically engineered pigs (α1,3-galactosyltransferase gene-knockout [GTKO] pigs and pigs expressing a human complement-regulatory protein [CD46]) and human donors. METHODS The expression of Galα1,3Gal (Gal), swine leukocyte antigen (SLA) class I and class II on pCECs and pAECs, with or without activation by porcine IFN-γ, was tested by flow cytometry. Pooled human serum was used to measure IgM/IgG binding to and complement-dependent cytotoxicity (CDC) to cells from WT, GTKO, and GTKO/CD46 pigs. The human CD4(+) T-cell response to cells from WT, GTKO, GTKO/CD46 pigs and human was tested by mixed lymphocyte reaction (MLR). RESULTS There was a lower level of expression of the Gal antigen and of SLA class I and II on the WT pCECs than on the WT pAECs, resulting in less antibody binding and reduced human CD4(+) T-cell proliferation. However, lysis of the WT pCECs was equivalent to that of the pAECs, suggesting more susceptibility to injury. There were significantly weaker humoral and cellular responses to the pCECs from GTKO/CD46 pigs compared with the WT pCECs, although the cellular response to the GTKO/CD46 pCECs was greater than to the human CECs. CONCLUSIONS These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.

Rho-Mediated Regulation of TGF-β1– and FGF-2–Induced Activation of Corneal Stromal Keratocytes

PURPOSE To investigate the role of Rho GTPase signaling in FGF-2- and TGF-beta1-induced activation of corneal keratocytes. METHODS Keratocytes isolated from rabbit corneal stroma and plated in a serum-free medium were treated with FGF-2/heparin or TGF-beta1 in the presence or absence of Rho inhibitor (C3 exoenzyme) or ROCK (Rho kinase) inhibitor (Y27632). Specific phenotypic changes were analyzed by immunocytochemistry and Western blot analysis, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR. RESULTS TGF-beta1-induced expression of alpha-SMA and transcription of alpha-SMA mRNA in activated keratocytes were reduced by Rho or ROCK inhibition during the activation. In nonactivated keratocytes, the expression of alpha3(IV) collagen was downregulated by Rho-inhibition. TGF-beta1- or FGF-2-induced downregulation of the expression of alpha3(IV) collagen and its mRNA was not significantly altered by Rho or ROCK inhibition. TGF-beta1- and FGF-2-induced decreases in cell-associated and secreted KS, and lumican mRNA levels were prevented by Rho or ROCK inhibition. However, FGF-2-induced decreases in keratocan mRNA levels were prevented by Rho inhibition but not by ROCK inhibition. Whereas Rho inhibition downregulated both TGF-beta1- and FGF-2-induced tenascin-C expression, ROCK inhibition was found to downregulate only TGF-beta1-induced expression. CONCLUSIONS Rho signaling has a significant role in the activation of keratocytes. Rho, via ROCK-independent and/or -dependent pathways differentially regulates the TGF-beta1-induced expression of alpha-SMA and TGF-beta1- and FGF-2-induced de novo expression of tenascin-C and the downregulation of alpha3(IV) collagen and KSPGs, lumican and keratocan.

Development and characterization of monoclonal antibodies to human type III procollagen

Abstract Hybridomas which secrete monoclonal antibodies against human type III procollagen have been developed. By an enzyme-linked immunosorbent assay, three of the monoclonal antibodies have been determined to be against non-helical extensions of the molecules while two of the antibodies are against helical portion of the molecules which is sensitive to bacterial collagenase action. These findings have been further confirmed by carrying out immuno-reaction of the pro α-chains transferred on nitrocellulose paper from sodium dodecyl sulfate polyacrylamide gels. These monoclonal antibodies have been found to be suitable reagents for immunohistochemical studies as well as for immunoassays of type III procollagen and collagen.

Alterations of post-translational modifications of procollagen by SV40-transformed human fibroblasts

I t is n o w well known that collagen is initially synthesized as a precursor called proeoliagen, which then undergoes a var iety o f postt ranslat ional modif i cat ions to yield collagen. These modif icat ions include hyd roxy la t i on o f prol ine and lysine residues, glycosylat ion o f certain hydroxy lys ine residues, and cleavage o f the ca rboxyand aminoterminal pept ide extensions f rom the procollagen molecule . Al though the procoiiagen intermediates are shortqived in vivo, their convers ion into collagen is relatively slow in cells growing in cul ture [ 1 ] . Normal h u m a n dermal f ibroblasts growhng in cul ture are known to secrete procol lagen into the m ed i um [ 1 6 ] . These cells also secrete procoHagen peptidase(s) which cleave the terminal pept ide extensions o f procollagen to fo rm collagen [7] . Viral t ransformat ion o f ce~ls in cul ture is k n o w n to result in changes in g rowth pat terns , specific act ivi ty o f enzymes , cell surface, and nuclear and m e m b r a n e associated antigens [8] . This c o m m u nicat ion gives evidence that viral t r ans format ion also affects the pos tsynthe t ic modif ica t ion o f procollagen. Those activities tha t appear to be al tered by transfo rmat ion include procol lagen peptidase activity as well as the ex ten t o f lysine hydroxy la t ion and glycosylat ion in the procollagen m o l e c u l e .

FGF-2– and TGF-β1–Induced Downregulation of Lumican and Keratocan in Activated Corneal Keratocytes by JNK Signaling Pathway

PURPOSE Downregulation of lumican and keratocan expression is an undesirable phenotypic change that occurs during corneal wound healing. The present study was intended to determine whether the activation of Jun N-terminal kinase (JNK)-signaling pathway is involved in their downregulation in TGF-β1- and FGF-2-activated keratocytes. METHODS Keratocytes, isolated from rabbit corneal stroma, and cultured in a serum-free medium, pretreated or not treated with JNK inhibitor (SP600125), were activated with FGF-2/heparin sulfate (HS) or TGF-β1 in the presence or absence of SP600125. In another set of experiments, keratocytes were transfected with JNK1/2 Dicer-substrate RNA (DsiRNA) and then activated with TGF-β1 or FGF-2/HS. Specific phenotypic changes were analyzed immunocytochemically and correlated with Western blot analyses. The relative levels of specific mRNAs were estimated by quantitative RT-PCR using specific reagents. RESULTS The FGF-2/HS- or TGF-β-induced activation of corneal stromal keratocytes to fibroblast- or myofibroblast-phenotype, respectively, resulted in marked decreases in cell surface-associated and secreted keratan sulfate proteoglycans (KSPGs). Both keratocan and lumican proteins and their mRNAs were downregulated in the activated keratocytes. However, JNK inhibition during the activation of keratocytes, pretreated with the JNK inhibitor, suppressed the reduction in the cell-surface associated and secreted KSPGs (lumican and keratocan), and their mRNA transcripts. Downregulation of total KSPGs and their mRNAs was also inhibited by decreasing JNK1 and JNK2 levels via JNK1/2 DsiRNA transfection of keratocytes before their activation. CONCLUSIONS Extrapolating from the present study, FGF-2- and TGF-β1-activation of JNK signaling pathway may be partly responsible for the downregulation of keratocan and lumican expression in activated corneal keratocytes observed during corneal stromal wound healing.

Unique Parameters in the Healing of Linear Partial Thickness Penetrating Corneal Incisions in Rabbit: Immunohistochemical Evaluation

The healing of penetrating nonperforating linear corneal incisions in rabbit was analysed immunohistochemically. Regeneration of the basement membrane (BM) zone was evaluated by analysing the following components using monoclonal antibodies (MAbs): 1) an antigen designated AgHEBM1 (a 66k Mr polypeptide), 2) type VII collagen which is associated with anchoring fibrils and 3) an antigen designated Ag63/D7 which is associated with the lamina densa and anchoring plaques. Concomitant activity in the regenerating stroma was evaluated by analysing the distribution of 1) fibronectin, 2) keratan sulfate and 3) a fetal antigen, designated AgM12, (an intermediate filament associated protein with a Mr of 130k). Synthesis of the BM zone components was not evident at day 2 postwounding but was seen at day 7, only at the deepest aspects of the wounds. Re-establishment of a continuous BM zone was evident by day 60. The stromal regeneration had started by day 7, at the deepest aspect of the wounds, as judged from the distribution of stromal antigens including the fetal antigen AgM12. The number of activated cells (cells expressing AgM12) had decreased significantly by day 30 and diminished by day 60. Between day 30 and day 180 the thickness of the regenerated tissues had not increased significantly although the stroma had not yet reached its normal thickness. Even after six months, the epithelial plug persisted and the concentration of the sulfated epitopes of keratan sulfates in the regenerated stromal matrix were less than in the surrounding nonwounded regions. The cessation of expression of AgM12, by the cells in regenerating stroma, coincided with the relative decrease in extracellular matrix syntheses. The return of the stromal cells to the quiescent state also coincided with re-establishment of a continuous BM zone between the epithelium and the stroma in the regenerated tissue. These observations suggested that the synthesis, assembly and remodeling of stromal and epithelial extracellular matrices during the healing of penetrating corneal wounds may be influenced by the interactions between activated stromal cells and epithelial cells.

Responses of Cultured Human Keratocytes and Myofibroblasts to Ethyl Pyruvate: A Microarray Analysis of Gene Expression

PURPOSE Ethyl pyruvate (EP) has pharmacologic effects that remediate cellular stress. In the organ-cultured murine lens, EP ameliorates oxidative stress, and in a rat cataract model, it attenuates cataract formation. However, corneal responses to EP have not been elucidated. In this study, the potential of EP as a therapeutic agent in corneal wound healing was determined by examining its effects on the transition of quiescent corneal stromal keratocytes into contractile myofibroblasts. METHODS Three independent preparations of cultured human keratocytes were treated with TGF-beta1, to elicit a phenotypic transition to myofibroblasts in the presence or absence of 10 or 15 mM EP. Gene expression profiles of the 12 samples (keratocytes +/- EP +/- TGF-beta1 for three preparations) were produced by using gene microarrays. RESULTS TGF-beta1-driven twofold changes in at least two of three experiments defined a group of 1961 genes. Genes showing twofold modulation by EP in at least two experiments appeared exclusively in myofibroblasts (857 genes), exclusively in keratocytes (409 genes), or in both phenotypes (252 genes). Analysis of these three EP-modulated groups showed that EP (1) inhibited myofibroblast proliferation with concomitant modulation of some cell cycle genes, (2) augmented the NRF2-mediated antioxidant response in both keratocytes and myofibroblasts, and (3) modified the TGF-beta1-driven transition of keratocytes to myofibroblasts by inhibiting the upregulation of a subset of profibrotic genes. CONCLUSIONS These EP-induced phenotypic changes in myofibroblasts indicate the potential of EP as a therapeutic agent in corneal wound healing.