Enhancement of gap junctional intercellular communication of normal human dermal fibroblasts cultured on polystyrene dishes grafted with poly-N-isopropylacrylamide

Technology developed to allow recovery of cells without enzyme treatment, involving a dish grafted with a thermoreactive polymer gel of poly-N-isopropylacrylamide (PIPAAm), was found to significantly enhance gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells). NHDF cells were cultured for 4 days on PIPAAm-grafted dishes irradiated with various doses of electron beams, and GJIC was assayed by the scrape-loading dye transfer method. The area of dye transfer was greater in the PIPAAm-grafted dishes than in the control culture dishes, indicating that the PIPAAm-grafted dishes enhanced the GJIC of NHDF cells. Connexin-43 (Cx43) expression was analyzed because Cx43 is considered to be a main component of the gap junctional channel. PIPAAm-grafted dishes irradiated with 100, 250, or 500 kGy of electron beams showed significantly enhanced expression of Cx43-NP, Cx43-P1, and especially Cx43-P2. Enhanced expression of Cx43-P2, a functional transmembrane protein, may be related to the promotion of GJIC. These results suggest that the PIPAAm-grafted dish not only enables the enzyme-free recovery of a cell monolayer for use in the construction of a three-dimensional artificial tissue, but also significantly contributes to the enhancement of GJIC, which may partly promote tissue strength on the surface of the PIPAAm-grafted dish.     

Increase in gap-junctional intercellular communications (GJIC) of normal human dermal fibroblasts (NHDF) on surfaces coated with high-molecular-weight hyaluronic acid (HMW HA)

Normal human dermal fibroblast (NHDF) cells were used to detect differences in gap-junctional intercellular communication (GJIC) by hyaluronic acid (HA), a linear polymer built from repeating disaccharide units that consist of N-acetyl-D-glucosamine (GlcNa) and D-glucuronic acid (GlcA) linked by a beta 1-4 glycosidic bond. The NHDF cells were cultured with different molecular weights (MW) of HA for 4 days. The rates of cell attachment in dishes coated with high-molecular-weight (HMW; 310 kDa or 800 kDa) HA at 2 mg/dish were significantly reduced at an early time point compared with low-molecular-weight (LMW; 4.8 kDa or 48 kDa) HA with the same coating amounts. HA-coated surfaces were observed by atomic force microscopy (AFM) under air and showed that HA molecules ran parallel in the dish coated with LMW HA and had an aggregated island structure in the dish coated with HMW HA surfaces. The cell functions of GJIC were assayed by a scrape-loading dye transfer (SLDT) method using a dye solution of Lucifer yellow. Promotion of the dye transfer was clearly obtained in the cell monolayer grown on the surface coated with HMW HA. These results suggest that HMW HA promotes the function of GJIC in NHDF cells. In contrast, when HMW HA was added to the monolayer of NHDF cells, the functions of GJIC clearly were lowered in comparison with the cells grown in the control dish or with those grown on the surface of HMW HA. Therefore it is concluded that the MW size of HA and its application method are important factors for generating biocompatible tissue-engineered products because of the manner in which the GJIC participates in cell differentiation and cell growth rate.     

Chemical, oncogene and growth factor inhibition gap junctional intercellular communication: an integrative hypothesis of carcinogenesis

Most, if not all, cancer cells have some dysfunction in gap-junction-mediated intercellular communication, either because of defects in cell adhesion or inability to have functional gap junctional communication. In addition, most, if not all, tumor-promoting chemicals and conditions down-regulate gap junction function, while some antitumor-promoting chemicals can up-regulate gap junctional communication. Several oncogenes are associated with down-regulation of gap junction function and several hormone and growth regulators, known to be tumor promoters, are also able to down-regulate gap junction function. On the other hand, some tumor suppressor genes have been linked to the up-regulation of gap junctions. Based on these observations, it is hypothesized that, if a progenitor cell is unable to perform gap junctional intercellular communication, normal growth control and cell differentiation would not be possible, thereby favoring the development of malignant neoplasia.     

Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E2

Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E₂ (PGE₂) and prostaglandin F_2α (PGF_2α) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE_2, we found enhanced GJIC with 1 nM PGE₂. This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE₂ was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE₂ secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE₂ to the cells. Our findings show that PGE₂ may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE₂ secretion by endometrial stromal cells may have the potential to suppress carcinogenesis in endometrial epithelial cells.     

Development of cultured dermal substitute composed of hyaluronic acid and collagen spongy sheet containing fibroblasts and epidermal growth factor

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5?days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6?days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3?days, but decreased slightly after 5?days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3–5?days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.     

Potential of wound dressing composed of hyaluronic acid containing epidermal growth factor to enhance cytokine production by fibroblasts

This study aimed to investigate the potential of a wound dressing composed of hyaluronic acid (HA) containing epidermal growth factor (EGF) to enhance cytokine production by fibroblasts. The present wound dressing has a two-layered spongy structure: an upper layer composed of crosslinked high-molecular-weight HA, and a lower layer composed of low-molecular-weight HA containing arginine (Arg) and vitamin C derivative (VC) with or without EGF. Human fibroblast-embedded collagen gel sheet (cultured dermal substitute: CDS) was elevated to the interface between the air and culture medium to create a wound surface model onto which each wound dressing was placed, which was followed by culture for 7 days. The EGF dressing (with EGF, Arg, VC) significantly enhanced the production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by CDS as compared to the EGF-free dressing (with Arg, VC). To evaluate if this enhanced production of VEGF and HGF achieved with the EGF dressing is sustained, a second experiment was conducted using a wound surface model. Each wound dressing was placed on the CDS in the wound surface model. Culture was then performed for 3 days (first period), after which each dressing was placed on another CDS for a further 3-day culture period (second period). The EGF dressing enhanced the production of VEGF and HGF by CDS during the first and second periods as compared to the corresponding production when using the EGF-free dressing. These results suggest that EGF can be maintained in the hydrated layer of a wound dressing composed of crosslinked high-molecular-weight HA.     

The inhibitory mechanism of gap junctional intercellular communication induced by polyethylene and the restorative effects by surface modification with various proteins

Gap junctional intercellular communication (GJIC) is a function that plays an important role in maintaining cell and tissue homeostasis and in regulating cell growth, development, and differentiation. Change in this function of V79 fibroblasts cultured on polyethylene films modified with albumin or collagen was estimated using fluorescence redistribution after photobleaching (FRAP) analysis. The GJIC function of V79 cells on nontreated polyethylene was strongly inhibited in comparison with those on a glass coverslip. When the cells were culture on collagen-immobilized polyethylene film, this function was recovered to about 70% of the cells cultured on the coverslip. However, albumin immobilization did not recover the function as much as collagen immobilization. Western blotting analysis and immunostaining of connexin 43, which is a major protein constituting gap junctional channel of these cells, revealed its abnormal expression and distribution in the cells on nontreated polyethylene, whereas its almost normal distribution was observed in the cells on collagen-immobilized polyethylene. This abnormal expression and distribution of connexin 43 induced by the surface of polyethylene may be ascribed to a strong inhibition of GJIC of V79 fibroblasts.     

Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type I collagen matrices

Healing of large open dermal wounds is associated with decreased values of the tensile strength even up to 6 months post-wounding. Results of previous studies have shown that healing is facilitated in the presence of a type I collagen sponge by promoting deposition of newly synthesized large-diameter collagen fibers parallel to the fibers of the sponge. In this study healing is evaluated in dermal wounds treated with a collagen sponge seeded with fibroblasts or coated with basic fibroblast growth factor (bFGF). Experimental results indicate that the presence of a collagen sponge results in increased wound tensile strength and increased collagen fiber diameters in the upper dermis 15 days post-wounding in an excisional guinea pig dermal wound model. In comparison, dermal wounds treated with collagen sponges seeded with fibroblasts or coated with bFGF showed increased tensile strengths 15 days postimplantation and increased degree of reepithelialization. These results indicate that fibroblast seeding and bFGF coating in conjunction with a type I collagen sponge matrix facilitate early dermal and epidermal wound healing.     

肝癌细胞中connexin32、connexin43的表达及其与间隙连接通讯功能的相关性

目的：研究肝细胞肝癌和正常肝细胞间隙连接蛋白connexin32（Cx32)、connexin43(Cx43）的表达，及其对间隙连接通讯功能（GJIC）的影响。方法：应用应用培养及流式细胞分析技术（FCM），研究肝癌细胞系HHCC、SMMC－7721和正常肝系QZG细胞中Cx32和Cx43的表达。结合Ｌucifer Yellow划痕标记荧光传输技术（SLDT），检测上述细胞的间隙连接通讯功能。结果：流式细胞仪分析证实，Cx32蛋白在肝癌细胞系HHCC、SMMC－7721和正常肝细胞系QZG细胞中表达的阳性率分别为1．9％、0．7％和99．0％；Cx43蛋白在HHCC、SMMC－7721和QZG细胞中表达的阳性率分别为7．3％、26．5％和99．1％。SLDT检测发现肝癌细胞HHCC，SMMC－7721的间隙连接通讯功能较正常肝细胞QZG明显减弱。结论；Cx3、Cx43蛋白在正常肝细胞中具有较高水平的表达，在肝癌细胞中表达水平显著降低，肝癌细胞的间隙连接通讯功能较正常肝细胞亦明显减弱。Cx32、Cx43表达调控异常引起的间隙连接通讯障碍可能与肝癌的发生密切相关。     

Myofibroblast keratinocyte growth factor reduces tight junctional integrity and increases claudin-2 levels in polarized Caco-2 cells

The colonic epithelium is composed of a polarized monolayer sheathed by a layer of pericryptal myofibroblasts (PCMFs). We mimicked these cellular compartments in vitro to assess the effects of paracrine-acting PCMF-derived factors on tight junction (TJ) integrity, as measured by transepithelial electrical resistance (TER). Coculture with 18Co PCMFs, or basolateral administration of 18Co conditioned medium, significantly reduced TER of polarized Caco-2 cells. Among candidate paracrine factors, only keratinocyte growth factor (KGF) reduced Caco-2 TER; basolateral KGF treatment led to time- and concentration-dependent increases in claudin-2 levels. We also demonstrate that amphiregulin (AREG), produced largely by Caco-2 cells, increased claudin-2 levels, leading to epidermal growth factor receptor-mediated TER reduction. We propose that colonic epithelial TJ integrity can be modulated by paracrine KGF and autocrine AREG through increased claudin-2 levels. KGF-regulated claudin-2 induction may have implications for inflammatory bowel disease, where both KGF and claudin-2 are upregulated.     

Induction of gap junctional intercellular communication,connexin43 expression,and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway

Expression of gap junction proteins and cell–cell communication was studied in the human neural-glial cell line, SVG, as a first step in defining whether the SVG cells could be used as a model system to study the role of gap junctions in neuronal precursor cells. SVG cells were found to express connexin43 protein that co-migrated with WB-F344 rat liver connexin43 and that reacted with connexin43-specific antibodies on Western blots. However, fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein-loaded cells failed to show significant dye coupling. Agents that stimulate the adenylyl cyclase/cAMP pathway were used to induce gap junctional intercellular communication in the SVG cultures. A 24–48 h treatment of SVG cells with 5 μM forskolin or 5 μM forskolin+200 μM 3-isobutyl-1-methylxanthine increased the percentage of dye-coupled cells from 5–65%, using the fluorescent recovery after photobleaching method. The increase in dye coupling induced by forskolin or forskolin+3-isobutyl-1-methylxanthine was inhibited by octanol, which is known to block gap junction-mediated cell communication. Western blot analysis of total protein extracts revealed the appearance of a higher molecular weight connexin43 protein band after treatment of SVG cells with forskolin or forskolin+3-isobutyl-1-methylxanthine, that was not observed in vehicle-treated controls. Alkaline phosphatase treatment of total protein extracts from forskolin or forskolin+3-isobutyl-1-methylxanthine-treated cells reduced the higher molecular weight band to ≈41,000 the same as observed in the control extracts. The alkaline phosphatase treatment demonstrates that the higher molecular weight band was due to a phosphorylation event stimulated by forskolin or the forskolin+3-isobutyl-1-methylxanthine combination. In addition, treatment of the SVG cells with the forskolin or forskolin+3-isobutyl-1-methylxanthine stimulated outgrowth of neurite-like processes from the cell body which immunostained positive for the connexin43 protein as well as protein markers for neurons and oligodendrocytes.We hypothesize that the SVG cells may represent a neuronal progenitor cell population that has the ability to differentiate when exposed to the appropriate signals.     

Induction of gap junctional intercellular communication, connexin43 expression, and subsequent differentiation in human fetal neuronal cells by stimulation of the cyclic AMP pathway

Expression of gap junction proteins and cell-cell communication was studied in the human neural-glial cell line, SVG, as a first step in defining whether the SVG cells could be used as a model system to study the role of gap junctions in neuronal precursor cells. SVG cells were found to express connexin43 protein that co-migrated with WB-F344 rat liver connexin43 and that reacted with connexin43-specific antibodies on Western blots. However, fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein-loaded cells failed to show significant dye coupling. Agents that stimulate the adenylyl cyclase/cAMP pathway were used to induce gap junctional intercellular communication in the SVG cultures. A 24-48 h treatment of SVG cells with 5 microM forskolin or 5 microM forskolin + 200 microM 3-isobutyl-1-methylxanthine increased the percentage of dye-coupled cells from 5-65%, using the fluorescent recovery after photobleaching method. The increase in dye coupling induced by forskolin or forskolin + 3-isobutyl-1-methylxanthine was inhibited by octanol, which is known to block gap junction-mediated cell communication. Western blot analysis of total protein extracts revealed the appearance of a higher molecular weight connexin43 protein band after treatment of SVG cells with forskolin or forskolin + 3-isobutyl-1-methylxanthine, that was not observed in vehicle-treated controls. Alkaline phosphatase treatment of total protein extracts from forskolin or forskolin + 3-isobutyl-1-methylxanthine-treated cells reduced the higher molecular weight band to approximately 41,000 the same as observed in the control extracts. The alkaline phosphatase treatment demonstrates that the higher molecular weight band was due to a phosphorylation event stimulated by forskolin or the forskolin + 3-isobutyl-1-methylxanthine combination. In addition, treatment of the SVG cells with the forskolin or forskolin + 3-isobutyl-l-methylxanthine stimulated outgrowth of neurite-like processes from the cell body which immunostained positive for the connexin43 protein as well as protein markers for neurons and oligodendrocytes. We hypothesize that the SVG cells may represent a neuronal progenitor cell population that has the ability to differentiate when exposed to the appropriate signals.     

Effects of epidermal growth factor and keratinocyte growth factor on the growth of oropharyngeal keratinocytes in coculture with autologous fibroblasts in a three-dimensional matrix

Tissue engineering of oropharyngeal mucosa is rendered complex by the fact that oropharyngeal keratinocytes are difficult to culture in the long term and do not grow well after several subcultivations. Three populations of oropharyngeal keratinocytes were isolated by a method based on different levels of beta(1)-integrin expression. In particular, keratinocytes were isolated between cell fractions that adhere rapidly on collagen-IV-coated culture dishes (RAC-IV) and populations that are less adherent (RAC-IV-D). The total fraction of both subpopulations served as a control (RAC-IV-T). The epidermal growth factor (EGF) and the keratinocyte growth factor (KGF) were examined with regard to their effects on the growth of the three populations. Growth curves of all three cell fractions grown with or without EGF were generated, and different concentrations of EGF and KGF were tested. EGF did not change any growth characteristics of the cells, with the exception of the speed of growth. Best growth was achieved with a physiologic EGF concentration of 0.15-1.5 ng/ml and a KGF concentration of 15 ng/ml. Finally, we cocultured oropharyngeal keratinocytes and their autologous fibroblasts in a three-dimensional matrix using Matrigeltrade mark. Oropharyngeal keratinocytes grown in coculture formed larger colonies than keratinocytes grown without fibroblasts. In conclusion, we were able to optimize the supplement of EGF and KGF in standard medium for the long-term culture of primary oropharyngeal keratinocytes. The use of Matrigel as a scaffold for three-dimensional cocultures of oropharyngeal keratinocytes and fibroblasts might signify a step forward in the development of a transplantable mucosa construct.     

Lipoteichoic acid and interleukin 1 stimulate synergistically production of hepatocyte growth factor (scatter factor) in human gingival fibroblasts in culture.

Lipoteichoic acids (LTA) from various gram-positive bacteria, including oral streptococci such as Streptococcus sanguis, enhanced the production of hepatocyte growth factor (HGF) (scatter factor) by human gingival fibroblasts in culture, whereas lipopolysaccharides (LPS) from various gram-negative bacteria did not. In contrast, LPS induced interleukin 1 activity in human gingival epithelial cells in culture, while LTA had little effect. LTA and recombinant human interleukin 1 alpha enhanced synergistically the production of HGF/SF in human gingival fibroblast cultures. Recombinant human HGF, in turn, enhanced the proliferation of human gingival epithelial cells in culture.     

Different human B cell subsets respond to interleukin 2 and to a high molecular weight B cell growth factor (BCGF)

After activation by anti-μ antibody human B cells acquire the ability to proliferate in the presence of recombinant interleukin 2 (IL2), a 20-kDa mol. mass B cell growth factor (BCGF) and a high mol. mass BCGF (50-kDa BCGF). An anti-IL2 receptor (IL 2R) monoclonal antibody inhibits the IL2-dependent proliferation without affecting that induced by BCGF. B cells expressing the IL2R after anti-μ antibody activation (IL2R⁺ cells) were separated from those not expressing IL2R (IL2R^? cells). IL2 stimulated the proliferation of only IL2R⁺ cells whereas the 20-kDa BCGF acted on both IL2R⁺ and IL2R^? cells. Importantly, the 50-kDa BCGF supported the proliferation of IL2R^? cells whereas it was inactive on IL2R⁺ cells. Thus, the B cell subset responding to the 50-kDa BCGF after anti-μ antibody activation is distinct from that responding to IL2.     

成纤维细胞生长因子2、成纤维细胞生长因子受体4蛋白在人甲状腺乳头状癌中的表达及意义

目的探讨甲状腺乳头状癌(PTC)组织中成纤维细胞生长因子2(FGF-2)和成纤维细胞生长因子受体4(FGFR-4)的表达及其是否存在相关性。方法收集89例甲状腺乳头状癌及30例癌旁正常甲状腺组织标本,采用免疫组织化学和免疫印迹法(Western blotting)检测FGF-2和FGFR-4蛋白在标本中的表达,并进行统计学分析。结果免疫组织化学方法结果显示,与癌旁正常组织相比,FGF-2及FGFR-4在人类甲状腺乳头状癌组织中均高表达(P0.01,P0.01),两者差异有统计学意义;FGF-2和FGFR-4在甲状腺乳头状癌中的表达与淋巴结转移(χ2=14.798,P0.01;χ2=7.27,P0.01)和分化程度(χ2=13.824,P0.01;χ2=16.921,P0.01)相关,而与性别、年龄、肿瘤大小无关(P0.05);通过Western blotting技术分析,FGF-2和FGFR-4癌组织中的表达明显高于正常组织,随着癌组织分化程度的降低,表达明显上调(P0.05),其结果和免疫组织化学染色的检测结果一致;并且两者在甲状腺乳头状癌中的表达呈正相关(rs=0.434,P0.01)。结论 FGF-2和FGFR-4与甲状腺乳头状癌的发生、侵袭和转移有关,两者具有正协同作用,联合检测对判断甲状腺乳头状癌的恶性程度及生物学行为是一项有意义的综合性指标。