Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation

PURPOSE: To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation. METHODS: Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents. RESULTS: Immunohistochemical analysis indicated that type IV alpha1, alpha2, and alpha3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, alpha3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-beta1. alpha3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-beta1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels. CONCLUSIONS: The loss in the expression of alpha3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-beta1, and it accompanies the downregulation of keratocan expression.     

Preservation and expansion of the primate keratocyte phenotype by downregulating TGF-beta signaling in a low-calcium, serum-free medium

PURPOSE: To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-beta signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2+] medium. METHODS: Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco's modified Eagle's medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/10% FBS. TGF-beta signaling was monitored by measuring the promoter activity of TGF-beta1, -beta2, and -beta RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4. RESULTS: In KSFM, monkey keratocytes proliferated while maintaining the expression of keratocan, CD34, and ALDH proteins and keratocan promoter-driven ECFP for at least 15 passages. The nuclear accumulation of Smad2 and Smad4 and the promoter activities of TGF-beta1 and -beta RII were significantly downregulated in KSFM compared with DMEM/10% FBS. In KSFM, an increase of [Ca2+] to 1.8 mM and addition of 10% FBS synergistically downregulated the keratocan promoter activity, facilitated Smad2 and Smad4 nuclear translocation, and upregulated TGF-beta1 and -beta RII promoter activities. CONCLUSIONS: The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by downregulating Smad-mediated TGF-beta signaling.     

小鼠角膜基质细胞的KSFM培养基培养法

目的研究使用KSFM培养基能否获取具有增殖能力且保持生物学特性不变的小鼠角膜基质细胞（CSCs）。方法实验研究。将中央区角膜置于EDTA液（20mmol／L）内孵育45min后,用手术显微镊小心剥离角膜上皮层以及内皮层,并将获取的角膜基质置于含300U／mlⅠ型胶原酶的溶液中消化4h。离心后采用DMEM基础培养基、DMEM完全培养基（含10％FBS）以及KSFM培养基重悬细胞常规培养,并以含1U／m1分散酶的EDTA液消化传代细胞。同时,观察细胞并绘制细胞生长曲线;采用逆转录聚合酶链式反应（RT．PCR）检测细胞角膜蛋白多糖（keratocan）mRNA和乙醛脱氢酶（ALDH）mRNA表达情况;采用细胞免疫荧光染色以及蛋白质印迹方法检测细胞keratocan蛋白的表达情况。采用独立样本≠检验对数据进行统计学分析。结果通过胶原酶消化的方法可以从每只小鼠的角膜基质获取约l×l04个单个细胞。RT-PCR结果显示,原代细胞表达CSCs标记物keratocan和ALDH;免疫荧光染色和蛋白质印迹结果显示：原代细胞表达keratocan蛋白。培养于DMEM基础培养基内的原代CSCs无法增殖。培养于DMEM完全培养基内的CSCs可增殖,但第3代细胞不表达keratocan mRNA和ALDH mRNA以及keratocan蛋白。培养于KSFM培养基内的CSCs也可增殖,第3代细胞仍表达keratocan mRNA和ALDH mRNA以及keratocan蛋白,且与原代细胞相比,表达强度差异无统计学意义。结论KSFM培养基不仅能维持小鼠CSCs的生物学特性不变,还能有效促进细胞增殖。     

CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1

PURPOSE: To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation. METHODS: Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining. RESULTS: Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days. CONCLUSIONS: These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.     

Serum-free spheroid culture of mouse corneal keratocytes

PURPOSE: To develop a serum-free mass culture system for mouse keratocytes. METHODS: Corneas of C57BL6/J mice were enzyme digested after the epithelium and endothelium were removed. Stromal cells were cultured in serum-free DMEM/F12 (1:1) containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and B27 supplement. Primary spheres were dissociated by trypsin and subcultured as suspended secondary spheres. Cells from postnatal day (P)6 to P10 spheres were subcultured onto plastic dishes or type I collagen gels for phenotype analysis. The expression of the keratocyte markers keratocan, aldehyde dehydrogenase (Aldh), and CD34, were analyzed by RT-PCR, and vimentin and alpha-smooth muscle actin (alpha-SMA) were examined by immunocytochemistry. RESULTS: Primary keratocytes formed spheres, which were cultured for over 12 passages. Suspended sphere cells expressed vimentin, keratocan, CD34, and lumican, but were negative for cytokeratin K12 (K12) and Pax6. Sphere cells subcultured on plastic exhibited a dendritic morphology characteristic of keratocytes, and maintained keratocan, Aldh, and CD34 expression in serum-free medium. Sphere cells subcultured with 10% serum became fibroblastic, and expressed alpha-SMA when stimulated by transforming growth factor (TGF)-beta. alpha-SMA-positive cells demonstrated contractile properties on collagen gels, compatible with the myofibroblast phenotype. CONCLUSIONS: The phenotype of mouse keratocytes can be maintained in vitro for more than 12 passages by the serum-free sphere culturing technique.     

Mesenchymal stem cells derived from human limbal niche cells

Purpose. We investigated whether human limbal niche cells generate mesenchymal stem cells. Methods. Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. Results. Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. Conclusions. In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.     

Thy-1 distinguishes human corneal fibroblasts and myofibroblasts from keratocytes

After corneal injury, keratocytes become activated and transform into repair phenotypes-corneal fibroblasts or myofibroblasts, however, these important cells are difficult to identify histologically, compromising studies of stromal wound healing. Recent studies indicate that expression of the cell surface protein, Thy-1, is induced in fibroblast populations associated with wound healing and fibrosis in other tissues. We investigated whether keratocyte transformation to either repair-associated phenotype induced Thy-1 expression. Human corneal keratocytes were isolated by collagenase digestion. The cells were either processed immediately (i.e. freshly isolated keratocytes) or were cultured in the presence of 10% fetal bovine serum or transforming growth factor-beta to induce transformation to the corneal fibroblast and myofibroblast phenotypes, respectively. Thy-1 mRNA and protein expression by freshly isolated keratocytes and corneal fibroblasts were assessed by RT-PCR and Western blotting. mRNA also was extracted from the whole intact stroma and assessed by RT-PCR. Thy-1 was localised immunocytochemically in cultured human corneal fibroblasts, myofibroblasts, and in keratocytes in normal human corneal tissue sections. Thy-1 mRNA and protein were detectable in cultured human corneal fibroblasts, but not freshly isolated keratocytes. Whole uninjured stroma showed no detectable Thy-1 mRNA expression. Cultured human corneal fibroblasts and myofibroblasts both labelled for Thy-1, but keratocytes in the stroma of normal human cornea did not. We conclude that Thy-1 expression is induced by transformation of keratocytes to corneal fibroblasts and myofibroblasts, suggesting a potential functional role for Thy-1 in stromal wound healing and providing a surface marker to distinguish the normal keratocyte from its repair phenotypes.     

姜黄素对角膜基质细胞纤维化的抑制作用

背景角膜的创伤或手术可导致角膜基质细胞纤维化,进而形成瘢痕。研究表明姜黄素可明显减轻组织的纤维化程度,但姜黄素是否会影响角膜基质细胞纤维化的研究尚少。目的观察不同质量浓度的姜黄素对小鼠角膜基质细胞向成纤维细胞转化过程的影响,探讨姜黄素抗角膜基质纤维化的作用。方法150只6～8周龄BALB／c小鼠,分离角膜基质细胞并在含质量分数10％胎牛血清（FBS）的DMEM中进行培养,以原代角膜基质细胞重悬于DMEM中并分为5组：（1）空白对照组（DMEM＋10％FBS,含质量分数1％。DMSO,CG组）。（2）低剂量组（CG＋7．5mg／L姜黄素组）。（3）中剂量组（CG＋10mg／L姜黄素组）。（4）高剂量组（CG＋12．5mg／L姜黄素组）。（5）无诱导剂组（DMEM,含1％oDMSO）。上述因素干预7d后,用逆转录聚合酶链反应（RT—PCR）法检测各组中细胞表型keratocan、醛脱氢酶（ALDH）、CD90、decorin、fibronectin一1的表达。用MTS法检测姜黄素对角膜基质细胞增生的影响。制备小鼠角膜冰冻切片,采用免疫荧光技术检测角膜基质细胞内fibronectin-1的表达。结果原代培养的角膜基质细胞呈梭形,为细胞质丰富且核大的角膜基质成纤维细胞。随着姜黄素质量浓度的增加,角膜基质细胞中keratocanmRNA、ALDHmRNA的表达量增加,CD90mRNA和decorinmRNA的表达量减少,差异均有统计学意义（P〈O．05）,fibronectin-1mRNA的表达变化差异无统计学意义（P〉0．05）。MTS法检测发现,随着姜黄素质量浓度的增加,对角膜基质细胞增生的抑制率逐渐增加（F=956．00,P〈0．05）。免疫荧光技术检测发现角膜基质细胞中fibronectin-1的表达呈红色荧光。结论姜黄素对离体小鼠角膜基质细胞纤维化有明显的抑制作用,可减轻角膜基质创伤修复过程中的过度纤维化。     

Epigenetic silencing of maspin expression occurs early in the conversion of keratocytes to fibroblasts

Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.     

低剂量TGF-β1维持三维培养模型中牛角膜基质细胞生长和缓解细胞外基质纤维化的作用

背景 转化生长因子(TGF)-β1在角膜损伤修复过程中发挥重要作用,不同剂量的TGF-β1对角膜细胞外基质(ECM)的合成具有不同影响,从而影响瘢痕形成的程度.既往研究多集中于高质量浓度TGF-β1对二维培养的角膜基质细胞的影响,而低质量浓度TGF-β1对角膜基质细胞ECM合成的影响鲜见研究.目的研究低质量浓度TGF-β1对体外三维培养的角膜基质细胞生长状况及ECM合成的影响. 方法 采用两步胶原酶消化法从新鲜牛眼中分离牛角膜基质细胞并置于含体积分数10％胎牛血清(FBS)的DMEM/F12培养基,利用Pellet体外三维培养模型对牛角膜基质细胞进行培养.将细胞分为0.25 ng/ml TGF-β1+5％ FBS组和0.50 ng/ml TGF-β1+5％ FBS组,分别于培养后48 h、1周、2周、3周观察Pellet培养模型的形态变化.于培养后3周采用苏木精-伊红染色法对Pellet培养模型进行常规组织形态学检查,采用钙黄绿素-AM/碘化丙啶法(Calcein-AM/PI法)于激光扫描共焦显微镜下检查角膜基质细胞的死亡率;采用实时荧光定量PCR及免疫荧光细胞化学法分别检测细胞中α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、Ⅰ型胶原(Col Ⅰ)和ColⅢmRNA及其蛋白的相对表达量;采用实时荧光定量PCR法检测细胞中角膜蛋白(KERA)mRNA和基膜聚糖(LUM)mRNA的表达.结果 培养后48 h、1周、2周和3周Pellet细胞均成团生长.苏木精-伊红染色可见,Pellet球内均有大量红色淡染的胶原纤维以及大部分正常的成纤维细胞和少量坏死的成纤维细胞,坏死细胞可见细胞形态破坏,呈均质红染,且0.25 ng/ml TGF-β1+5％ FBS组和0.50 ng/ml TGF-β1+5％ FBS组培养细胞形态无明显差别.0.25 ng/ml TGF-β1+5％ FBS组和0.50 ng/ml TGF-β1+5％ FBS组细胞死亡率分别为(33.60±1.65)％和(30.90±0.78)％,差异无统计学意义(t=0.144,P=0.887).0.25 ng/ml TGF-β1+5％FBS组Pellet培养模型中α-SMA、FN、ColⅢ蛋白表达量均低于0.50 ng/ml TGF-β1+5％ FBS组,差异均有统计学意义(tα-SMA=4.622,P=0.010;tFN=2.973,P=0.040;tCol Ⅲ=7.845,P＜0.001),0.25 ng/ml TGF-β1 +5％ FBS组Col Ⅰ的表达量明显高于0.50 ng/ml TGF-β1+5％ FBS组,差异有统计学意义(tColⅠ=4.022,P=O.016).在转录水平和蛋白表达水平,0.25 ng/ml TGF-β1 +5％ FBS组ColⅢ/Col Ⅰ比值均低于0.50 ng/ml TGF-β1+5％FBS组,差异均有统计学意义(tmRNA=-3.039,P=0.038;t蛋白=3.215,P=0.032).培养后48 h、1周、2周Pellet培养模型中KERA mRNA和LUM mRNA均呈阳性表达,0.25 ng/ml TGF-β1+5％ FBS组LUM mRNA相对表达量随培养时间延长而逐渐增加;0.50 ng/ml TGF-β1 +5％ FBS组LUM mRNA相对表达量于培养后1周时达峰值.2个组Pellet培养模型中KERA mRNA相对表达量均在培养后1周达峰值. 结论 低剂量的TGF-β1既能够维持Pellet体外三维培养模型中角膜基质细胞的生长并合成ECM,也使ECM组成成分的合成倾向于正常状态,以减少瘢痕化修复的趋势.     

In vivo gene delivery and visualization of corneal stromal cells using an adenoviral vector and keratocyte-specific promoter

PURPOSE: This study was conducted to determine whether intrastromal injection of adenoviral construct could be used to transfect corneal stroma cells effectively in vivo and to determine whether a tissue-specific promoter could be used to express exogenous genes in keratocytes. METHODS: An adenoviral construct with a cytomegalovirus (pCMV)-driven enhanced green fluorescent protein (EGFP) reporter gene was injected into the stroma of murine corneas. In vivo expression was quantitated and samples were analyzed by in vivo stereomicroscopy, and ex vivo expression was determined by confocal three dimensional (3-D) reconstruction. The 3.2-kb keratocan promoter was used to drive tissue-specific reporter gene expression in vivo. RESULTS: EGFP expression was first detected in vivo 11 hours after injection of adeno-EGFP in the corneal stroma, with a duration of approximately 3 weeks. Ex vivo wholemount cornea confocal analysis with 3-D reconstruction allowed visualization of EGFP expression in corneal stroma cells, to accurately assess cellular architecture and distribution in the corneal stroma. Naked pCMV-EGFP plasmid DNA did not express the reporter gene to the levels of the adeno-EGFP. The 3.2-kb keratocan promoter was capable of driving EGFP tissue-specific expression in the cornea. CONCLUSIONS: Intrastromal injection of adenovirus packaged DNA constructs is a rapid and efficient way to deliver and express genes in the corneal stroma. Intrastromal injection is also capable of delivering tissue-specific promoter constructs to the corneal stroma for gene expression. Furthermore, 3-D reconstruction provides a powerful tool for enhanced visualization of the corneal stroma environment and cellular biology.     

Isolation of a putative keratocyte activating factor from the corneal stroma

Fetal bovine serum has commonly been used to expand the population of keratocytes in culture. Tissue extracts, however, have also been used to grow other cell types. We prepared a DMEM/F12 extract of corneal stroma and compared the growth and morphology of collagenase-isolated keratocytes cultured in DMEM/F12, or DMEM/F12 containing either stromal extract or fetal bovine serum. Cell proliferation was measured by 3H-thymidine and BrdU incorporation as well as by DNA quantitation. The extract was fractionated by gel filtration. Cell morphology was assessed by phase-contrast microscopy. Culture in both extract and serum stimulated keratocytes to proliferate, but keratocytes cultured in the extract grew more slowly due to a longer cell cycle and to a lower final density because of greater sensitivity to contact inhibition. Keratocytes cultured in serum became fibroblastic while those cultured in extract retained the dendritic morphology of quiescent keratocytes. The stimulating factors in the corneal extract were more sensitive to heat inactivation and of higher molecular weight than the stimulating factors in serum. These results indicate that the mitogenic activity in extract and serum are different and that the phenotypes resulting from growth in serum and extract are also different. Keratocytes cultured at low cell densities in the corneal extract may mimic keratocyte activation, an initial and crucial event for keratocytes during the corneal wound healing process.     

IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro

Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-beta/ml over a 7-day culture period. We found that IGF-II and TGF-beta, alone or combined, increased both (3)H-thymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to alpha-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no alpha-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-beta expressed alpha-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-beta, however, expressed alpha-SM actin, fibronectin, SPARC, lumican, and keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-beta mediated down regulation of keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-beta.     

Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing

PURPOSE: To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS: The expression of VSX1 was evaluated through semiquantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS: Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS: The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.     

Corneal epithelial cells and stromal keratocytes efficently produce CC chemokine-ligand 20 (CCL20) and attract cells expressing its receptor CCR6 in mouse herpetic stromal keratitis

PURPOSE: CC chemokine-ligand 20 (CCL20) is known to be selectively expressed by surface-lining mucosal epithelial cells and skin epidermal keratinocytes and to attract cells such as immature dendritic cells and effector T cells via CCR6. This study evaluated the ability of corneal epithelial cells and stromal keratocytes to produce CCL20 in vitro and in vivo. METHODS: Human corneal epithelial cells (HCE) and corneal keratocytes (HCK) were treated without or with various cytokines and expression of CCL20 mRNA and secreion of its protein were evaluated by RT-PCR and ELISA. Induction of CCL20 mRNA in HCE and HCK was also examined upon in vitro infection with HSV-1. Using a mouse model of herpetic stromal keratitis (HSK), induction of CCL20 expression and accumulation of cells expressing CCR6 were evaluated by RT-PCR and immunohistochemistry. RESULTS: Not only corneal epithelial cells but also stromal keratocytes efficiently expressed CCL20 mRNA and protein upon stimulation with IL-1beta and TNF-alpha. In vitro infection with HSV-1 also induced CCL20 mRNA in both types of cells. In a mouse herpetic stromal keratitis model, prominent accumulation of CCL20 and CCR6 mRNA was revealed in HSV-1-infected corneas. Furthermore, immunohistochemistry demonstrated production of CCL20 by corneal epithelial cells as well as stromal keratocytes and stromal infiltration of DEC205+ dendritic cells, CD4+ T cells and CD8+ T cells. Double staining revealed that CCR6-expressing cells were mostly MHC class II+ dendritic cells. CONCLUSIONS: Not only epithelial cells but also stromal keratocytes are efficient producers of CCL20 in the cornea and recruit CCR6-expressing cells such as dendritic cells into inflamed cornea.     

Expression of haematopoietic stem cell markers, CD133 and CD34 on human corneal keratocytes

AIM: To study the expression of CD133 and CD34 antigens on cultured human keratocytes over time. METHODS: Primary cultures of human corneal stromal cells were established from explants derived from cadaver eye donors. The cultures were sorted for CD133+ and CD34+ cells using magnetic beads. Both the primary cultures and secondary passages of sorted cells were further analysed by flow cytometry and western blot analysis for expression of the same antigens over time. RESULTS: Four different cell populations-namely, CD133+, CD133-, CD34+ and CD34-, were identified in the culture samples. Two further specific subgroups were identified by flow cytometry: CD133+/CD34- cells and CD133+/CD34+ cells. Expression of CD133 declines more than CD34 with time in cell cultures. Although most cells lost expression of these markers, small populations retained staining up to 5 weeks in culture. CONCLUSION: Human keratocytes express the haematopoietic stem cell markers CD133 and CD34. This expression decreases with time in culture, with most but not all cells losing expression. On the basis of these markers, the corneal stroma shows a heterogeneous population of cells. Expression or down regulation of expression of these molecules could represent different stages of activation of these cells.     

The heterogeneous murine corneal stromal cell populations in vitro

PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.