Simulation of the initial stage of endochondral ossification: in vitro sequential culture of growth cartilage cells and bone marrow cells.

Growth cartilage cells were isolated from the ribs of young rats and cultured at high cell density in Ham's F-12 medium supplemented with 10% fetal calf serum. During 7 days, glycosaminoglycans and proteoglycans were actively synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured and biosynthetically prelabeled with 35SO4(2-) in their glycosaminoglycans, bone marrow cells caused release of 35S-labeled material into the culture medium. Glycosaminoglycan was also released by addition of conditioned medium obtained from cultures of bone marrow cells or peritoneal macrophages to the growth cartilage cell cultures. Electron microscopic studies of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of apatite mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells or macrophages removed glycosaminoglycan or proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was initiated. From these findings, sequential culture of growth cartilage cells and bone marrow cells is promising as an experimental system for investigating the mechanism of the initial stage of endochondral ossification.     

Reversible growth arrest in simian virus 40-transformed human fibroblasts.

A reversible growth arrest of simian virus 40-transformed human fibroblasts has been produced by replacement of methionine in the growth medium by its immediate metabolic precursor, homocysteine, Although these arrested cells exhibit a greatly reduced cloning efficiency when plated in methionine-supplemented medium, they resume rapid proliferation without a lag when subconfluent cells are refed with methionine-supplemented medium. This growth arrest is accompanied by a reduction in the percentage of mitotic cells in the cell population. Furthermore, data obtained using fluorescence-activated cell sorting techniques indicate that the cells are arrested i the S and G2 phases of the cell cycle. This is in contrast to a G1-phase accumulation of cells, which occurs only in methionine-supplemented medium at very high densities and which is similar to the G1 block seen in cultures of normal fibroblasts at high density. The apparent relationship between specific events in the DNA-synthetic and premitotic phase of the cell cycle and methionine dependence in these transformed cultures is discussed.     

Enhanced stimulation of human bone marrow macrophage colony formation in vitro by recombinant human macrophage colony-stimulating factor in agarose medium and at low oxygen tension.

Recombinant (r) and natural human (h) macrophage colony-stimulating factor (M-CSF, CSF-1) have been considered poor stimulators of macrophage progenitor cells present in human marrow, although they are potent stimulators of these cells in mouse marrow. We compared the growth characteristics of rhM-CSF-responsive human macrophage progenitor cells placed in semisolid agarose or agar culture medium and incubated for 14 days at ambient (approximately 20%) or lowered (5%) O2 tension. By itself, rhM-CSF was found to be a good stimulator of macrophage colony formation by human bone marrow cells cultured in agarose but not in agar; this growth was enhanced by incubation at 5% O2. Maximal numbers (up to 115/10(5) nonadherent low density cells plated) of macrophage colonies (50 to greater than 500 cells per colony) were stimulated by 500 to 1,000 units rhM-CSF/mL, with 1/2 maximal numbers stimulated by 250 to 500 units/mL. With agarose as the support medium, rhM-CSF was two- to fourfold more active on mouse than on human macrophage colony formation, in contrast to previous reports of 10- to 100-fold greater activity when agar was used as the support medium. Using nonadherent low density T lymphocyte-depleted human bone marrow cells growing in agarose at 5% O2, greater than additive effects on colony formation were observed when 31 to 500 units rhM-CSF were used in combination with either 10 ng rh interleukin-1 alpha (IL-1 alpha), 20, or 200 units rh granulocyte-macrophage (GM)-CSF or rhG-CSF. The agarose assay system should be useful for evaluating factors regulating the proliferation of human macrophage progenitor cells in vitro and during clinical trials with rhM-CSF.     

Isolation and culture of endothelial cells from human bone marrow

Summary : Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to re-establish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin-1 (UEA-1) to endothelial cells. Magnetic Dynabeads coupled with UEA-1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum-free medium together with 5% fetal calf serum for 24h. Cells were then cultured in endothelial serum-free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplemented with 5% fetal calf serum, endothlial cell growth supplement and heparin. After 2–4 weeks in culture, two morphologically different cell populations can be identified. One has a polygonal spindle-shaped morphology with a rapid growth rat, the other a rounded morphology and a slow growth rate. Both population have a vasiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM-1, VCAM-1, E-wlectin, CD31 and BMA120. Weibel-Palade bodies were observed by electron microscopy.
Culture of these cells will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.     

Transforming growth factor-β regulates growth as well as collagen and fibronectin synthesis of human marrow fibroblasts

Three growth factors present in platelets, namely platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF), have been implicated in the pathogenesis of bone marrow fibrosis frequently associated with myeloproliferative disorders. In this study, regulation of the proliferation, as well as collagen and fibronectin synthesis from marrow fibroblasts by TGF-beta was investigated. TGF-beta alone at high plating density stimulated the proliferation of cells at low concentrations, but rather showed inhibition at high concentrations in both MPD patients and control subjects. In the presence of PDGF, which has been confirmed to be a main growth factor for marrow fibroblasts, low concentration of TGF-beta inhibited the proliferation at low cell density, but there was no inhibition at high cell density. The synthesis of both type I and type III procollagen was enhanced by high concentrations of TGF-beta in both MPD patients and control subjects, while PDGF or EGF showed no effect. The fibronectin synthesis was also enhanced by TGF-beta, but not by PDGF or EGF. These results suggest that growth and stromal protein synthesis of fibroblasts causing marrow fibrosis are regulated by TGF-beta as well as PDGF and EGF, when these factors are released or leaked from platelets or megakaryocytes into marrow environment in MPD patients.     

The effect of plating density on granulosa cell growth and differentiation in vitro

The extent of FSH-mediated LH/hCG receptor induction and of basal and FSH-stimulated progesterone production by porcine granulosa cells in vitro, in serum-containing medium, is directly related to the plating density. Relative to pre-culture levels, low- and high-density cultures of cells routinely exhibited 1-2- and 10-11-fold increases in [125I]iodo-hCG binding, respectively. Monolayer growth, i.e. cell division, as measured by increases in cell protein or DNA content, was inversely related to plating density. This density-directed inverse relationship between growth and differentiation did not appear to be coupled under the conditions utilized. Whereas monolayer growth was dependent upon the cell surface density, i.e. the number of cells per unit surface area, differentiation was dependent upon cell concentration, i.e. cells per unit volume of medium. Cells plated at low density in medium containing 10% serum exhibited 50% less [125I]iodo-hCG binding than cells in 5% serum (P less than 0.025). Conversely, cells plated at high density exhibited a 14% increase (P less than 0.025) in binding at the higher serum level. Thus, it appears that the extent of differentiation depends upon the capacity of cells to neutralize serum inhibition which in turn is dependent upon the cell concentration. Serum neutralization by granulosa cells is an FSH-dependent process. Conditioned medium derived from insulin-treated, high-density cultures did not facilitate optimum LH/hCG receptor induction in low-density cultures. Conditioned medium from cultures treated with insulin plus FSH, however, facilitated LH/hCG receptor induction in low-density cultures to the same extent as obtained in high-density cultures. The enhancement by FSH-conditioned medium cannot be attributed to residual FSH or to dilution of serum components during the preparation of the conditioned medium. The phenomena of serum-attenuated granulosa cell differentiation in vitro, and of a density-dependent reversal of this process, may have regulatory implications in vivo since follicular fluid contains many serum components and since the granulosa cell complement is an important determinant of follicle maturation.     

Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro.

Purified fibrinogen at concentrations of 3-30 nM has been found to stimulate continuous growth of human lymphoid and myeloid cell lines under serum-free conditions. A strong proliferative response resulted from the synergism elicited by the addition of fibrinogen to transferrin-supplemented medium. This effect was observed with the pre-B-cell line Raji, the T lymphoma-derived JM, and the monocytic cell line U 937, either at high or low cell densities. With the promyelocytic cell line HL 60, fibrinogen did not shorten the doubling time of cultures seeded at high cell densities (2 x 10(5) cells per ml). However, at cell densities lower by 2 orders of magnitude and in the same medium, it promoted growth with a doubling time similar to that obtained at high cell concentrations. Fibrinogen also was found to increase the plating efficiency and colony size when human bone marrow cells were cultured in semisolid medium containing serum. In long-term bone marrow liquid cultures without fibrinogen, colony-forming cells were no longer detected after 6 weeks. In those cultured with fibrinogen, approximately equal to 50 granulocyte-macrophage colonies per 10(5) cells were obtained after 6 weeks, and 10, after 12 weeks. Purified fibrinogen fragment D possessed a stimulating activity similar to that of the intact fibrinogen molecule. This fragment cannot form fibrin, thus eliminating fibrin as a source of the mitogenic effect.     

Characterization of growth factor(s) produced by chemically transformed hamster dermal fibroblasts

Summmary Cell lysates from transformed hamster dermal fibroblasts (HDF cells) induced colony formation of normal HDF cells in soft agar medium and also stimulated proliferation of normal HDF cells in medium supplemented with plasma-derived serum (PDS). The colony forming activites of cell lysates correlated well with their growth stimulating activities in PDS-supplemented medium. The growth factor(s) were shown to be sensitive to trypsin and dithiothreitol (DTT), but heat stable. However, these transformed HDF cell lines did not all produce tumor nodules when injected into hamsters. This was not due to immunological rejection because similar results were obtained in hamsters treated with anti-hamster thymocyte serum (ATS). Thus it was concluded that production of these growth factor(s) by transformed HDF cell lines is not itself sufficient for acquisition of complete malignancy in vivo.     

The effect of low affinity platelet factor 4 (LAPF4) secreted by human megakaryoblastic cell line (MEG-01) upon human bone marrow fibroblasts.

BACKGROUND. The role of low affinity platelet factor 4 (LAPF4) in the hemopoietic microenvironment has not yet been clarified. METHODS. Low affinity platelet factor 4 (LAPF4) was purified from normal human platelets and the culture medium of the human megakaryoblastic cell line (MEG-01), and their effects upon the growth of human bone marrow fibroblasts were assessed in order to investigate the biological role of LAPF4. The purified LAPF4 was added to the culture media of human bone marrow fibroblasts up to the concentration of 200 ng/m1, and the growth rate of fibroblasts and the uptake of 3H-thymidine into fibroblasts were measured. RESULTS. The molecular weight of LAPF4 from MEG-01 was approximately 8,800, which corresponded to the monomer type of LAPF4 from normal platelets. The density of fibroblasts after 10 days of culture was 4.6 +/- 0.9 x 10(5)/ml, 7.8 +/- 0.8 x 10(5)/ml and 11.3 +/- 0.6 x 10(5) in control medium, in the medium with LAPF4 from platelets and in the medium with LAPF4 from MEG-01, respectively, which indicates that LAPF4 from MEG-01 enhanced the growth rate of bone marrow fibroblast almost 2.5 times. The uptake of 3H-thymidine into fibroblasts was significantly increased by 1,000 ng/ml of LAPF4 from MEG-01. CONCLUSIONS. These results suggest that LAPF4 may play a role in the proliferation of fibroblasts.     

Release of somatomedin-like activity by cultured WI-38 human fibroblasts

Confluent cultures of normal diploid WI-38 human embryonic lung fibroblasts released somatomedin (SM)-like activity into their incubation medium during culture in serum-free medium. This postculture medium (conditioned medium) stimulated cell division in these same cultured WI-38 fibroblasts and 35SO4 uptake by hypophysectomized rat cartilage in vitro. The conditioned medium also contained immunoreactive SM (IRSM) activity which yielded parallel dose-response curves to human serum in a RIA for SM. The IRSM activity measured in conditioned medium was not the artifactual result of effects of possible SM-binding proteins or proteolytic enzymes in conditioned medium. These studies suggest that cultured WI-38 fibroblasts produce and release SM-like activity which has SM-like biological activity and is immunoreactive with a basic SM purified from human plasma Cohn fraction and having similarity with SM-C and insulin-like growth factor-I. Human GH appears to stimulate production and release of IRSM activity by these cells.     

部分肝切除后的肝损伤血清和肝细胞生长因子促进大鼠骨髓细胞表达甲胎蛋白和白蛋白

目的 检测大鼠骨髓中是否存在肝脏干细胞，并用肝损伤血清和肝细胞生长因子(HGF)刺激骨髓细胞向肝细胞转化。方法 SD大鼠骨髓单个核细胞分3组进行培养：(1)单纯培养基对照组；(2)肝损伤血清组(15％，血清来自于2-AAF 75％肝切除大鼠)；(3)HGF(20ng／ml)组。用甲胎蛋白(AFP)和白蛋白作为细胞标志，用免疫组织化学、逆转录聚合酶链反应(PCR)、巢式PCR和western blot方法，观察大鼠肝损伤血清和HGF对骨髓细胞转化的促进作用。结果 肝损伤血清组和HGF组培养后10d和20d AFP免疫组织化学和western blot染色阳性；RT-PCR AFP mRNA阳性，新鲜骨髓细胞和单纯IMDM／F12培养基组AFP蛋白和mRNA均阴性。新鲜骨髓细胞存在白蛋白mRNA表达，在肝损伤血清组和HGF组培养后10d和20d，白蛋白mRNA表达增强。结论 大鼠肝损伤血清和HGF可促使体外培养骨髓细胞表达AFP mRNA和AFP。骨髓中可能存在骨髓源性肝干细胞，并微弱表达白蛋白mRNA；肝损伤血清和HGF可以促进骨髓细胞表达白蛋白mRNA。     

小鼠骨髓基质细胞的生物学特性及血管新生能力

目的 探讨体外培养的骨髓基质细胞的一些生物学特性及体内移植后在缺血区新血管生成中的作用。方法分离5—6周龄的小鼠胫骨、股骨，用预冷的DMEM／F12培养基冲洗出骨髓，经密度梯度离心分离出骨髓单个核细胞，接种后12～16d形成单层贴壁的成纤维样细胞。体外诱导分化鉴定分离的细胞，用传代的细胞进行生长曲线测定，观察其接种贴壁率、分裂指数，检测细胞周期和超微结构，并建立下肢缺血模型。荧光标记的体外扩增的骨髓单个核细胞被移植入缺血组织。移植后2周，荧光显微镜及内皮细胞碱性磷酸酶染色，检查荧光阳性细胞与染色阳性细胞的时空关系。结果体外传代培养的单个核细胞倍增时间约为42h。传代10h贴壁率达90％以上。分裂指数曲线与生长曲线相似。细胞周期显示约83％的细胞处于G1期。结论 体外培养的骨髓基质细胞生长稳定，传代后的细胞适应性强，增殖较快，表现出较早期细胞特点，在体外及移植入体内缺血区能分化为血管内皮细胞，有望用于改善组织缺血。     

Interleukin-1 induces human bone marrow-derived fibroblasts to produce multilineage hematopoietic growth factors

Interleukin-1 (IL-1) has been shown to induce stromal cells, including endothelial cells and fibroblasts, to produce multilineage hematopoietic growth factors. Although both of these cell types are well-described elements of the hematopoietic microenvironment, previous studies of IL-1-inducible colony-stimulating factor responses have utilized fibroblasts and endothelial cells from nonhematopoietic sites. Since we hypothesize that this intercellular growth network is active in the hematopoietic microenvironment, we sought to determine the responsiveness of bone marrow fibroblasts to IL-1. We demonstrate here that recombinant human IL-1 alpha and beta stimulate the dose-dependent release of granulocyte-macrophage colony-stimulating activity (GM-CSA) and burst-promoting activity (BPA) by cultured human bone marrow fibroblasts. We conclude that bone marrow fibroblasts produce hematopoietic growth factors in response to interleukin-1, and that this may be a mechanism by which stromal cells regulate cellular growth and differentiation within the hematopoietic microenvironment.     

Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration- dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage- independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL- 6 may have indirect as well as direct influences on hematopoietic cell proliferation.     

Modification of prostacyclin-stimulatory activity in sera by glucose, insulin, low density lipoprotein, linoleic acid and linoleic acid hydroperoxide

Reduced prostacyclin (PGI2) production by the vascular wall has been proposed as one of the possible causes of diabetic vascular complications. We found an activity which stimulated PGI2 production by cultured endothelial cells (PGI2-stimulatory activity, PSA) in human plasma-derived serum (PDS). The PSA was less in patients with diabetes mellitus. The present study was undertaken to evaluate how metabolic factors relevant to diabetic angiopathy modify the PSA. Pooled PDS was prepared from 10 healthy volunteers. The 6-keto-PGF1 alpha (6KF, a stable metabolite of PGI2) production by cultured bovine aortic endothelial cells was maximally stimulated by Dulbecco's modified Eagle's medium (DMEM) containing 10% pooled PDS after incubation for 60 min. The production of 6KF was reduced in a dose-dependent manner by the addition of 10% pooled PDS with glucose and linoleic acid hydroperoxide (lipid peroxide). In contrast, human low density lipoprotein (LDL) and linoleic acid (unsaturated fatty acid) enhanced the production of 6KF by 10% pooled PDS in a dose-dependent manner. Insulin, however, showed no effect on the production of 6KF by 10% pooled PDS. These results suggest that the reduced PSA in diabetics may be the result, in part, of a modification of the PSA by diabetic metabolic factors such as glucose and lipid peroxide.     

Human alpha fetoprotein enhances epidermal growth factor proliferative activity upon porcine granulosa cells in monolayer culture.

Alpha fetoprotein (AFP) is present at high concentrations in fetal fluids, certain neoplasias, and regenerating liver. Its physiological function remains largely unknown. Using a primary monolayer culture system, we investigated the proliferative activity of human (h) cord blood (CB) and highly purified AFP. hAFP, purified from hCB by Cibacron blue and immunoaffinity chromatography was homogeneous on SDS-PAGE and silver stain. Porcine granulosa cells from ovarian small follicles were cultured (25,000/cm2) for 2 days in medium (Ham's F-12:DMEM, 1:1) + 5% fetal calf serum (FCS) to facilitate attachment, followed by 6 days in medium containing: FCS, hCB or h amniotic fluid (1-20%)+/- EGF (10 ng/ml); or 0.25% plasma-derived serum (PDS) containing human low density lipoprotein (LDL, 25 ug/ml), +/- AFP (0.05-5 ug), and +/- EGF and IGF-I (10 ng/ml). In this system, single growth factors do not stimulate proliferation, a characteristic also exhibited by AFP. When combined with EGF, however, AFP dose-dependently increased proliferation to levels equal to that obtained with 10% FCS (2.3-fold increase vs PDS/LDL controls). When combined with EGF+IGF-I, AFP again dose-dependently increased proliferation to levels equal to that obtained with 10% FCS+EGF (6.7-fold increase vs controls). Purified human albumin used in place of AFP was not effective. TGF-a but not PDGF could replace the proliferative activity of EGF. These results suggest that AFP at physiological levels, although not itself mitogenic, can enhance the mitogenic activity of EGF and TGF-a and may function to modulate growth factor-mediated proliferation during development and neoplasia.     

Elevation of membrane tyrosine phosphatase activity in density-dependent growth-arrested fibroblasts.

Swiss 3T3 cells harvested at high density contain a membrane protein-tyrosine-phosphatase (EC 3.1.3.48) whose specific activity is on average 8-fold higher than that of cells harvested at low or medium densities. Investigation of the conditions affecting this elevation of specific activity suggests that it is associated with density-dependent growth arrest. Fibroblasts in the exponentially doubling phase have a relatively low level of membrane phosphatase specific activity, which rises only as the rate of cell proliferation decreases and is maximal when cell growth is contact inhibited. These observations have been extended to BALB/c 3T3 fibroblasts and normal human diploid fibroblasts. The increase in membrane tyrosine phosphatase activity is coupled to density arrest and not to cellular quiescence in general, as no increase in phosphatase specific activity is detected when non-contact-inhibited cells are induced to arrest their growth through serum deprivation. The observed alterations in specific activity are attributable to a tyrosine phosphatase of Mr 37,000 that was purified and characterized from solubilized membrane fractions of Swiss 3T3 cells. A regulatory mechanism controlling tyrosine phosphatase activity may play a role in cell proliferation and growth arrest caused by cell contact.     

Transition of asthmatic bronchial fibroblasts to myofibroblasts is inhibited by cell-cell contacts

The role of airway wall remodelling in bronchial asthma is well established. Myofibroblasts, the cells displaying features intermediate between fibroblasts and smooth muscle cells, are involved in this process but the mechanism of myofibroblasts activation in the onset of the disease remains obscure. Myofibroblasts can differentiate from various cell types, including resident fibroblasts, and the fibroblasts to myofibroblasts transition (FMT) can be reproduced in?vitro. We aimed to investigate the process of FMT in human bronchial fibroblasts (HBF) derived from non-asthmatic (n?=?7) and asthmatic (n?=?7) subjects. We also tested whether cell-cell contacts affect FMT by using N-cadherin blocking antibody. HBF plated in low or high cell density were treated with TGF-β(1) up to one week to induce FMT. The percentage of myofibroblsts was counted and expression of α-smooth muscle actin was evaluated by cytoimmunofluorescence, flow cytometry and immunobloting. We demonstrated that the intensity of FMT induced by TGF-β(1)in?vitro was strongly enhanced in asthmatic as compared to non-asthmatic HBF populations. This process was facilitated by low cell plating density in both groups of cultures. Furthermore, we proved that neither HBF-conditioned medium nor growth arrest in G(0)/G(1) phase of cell cycle could stop the TGF-β(1)-induced FMT in asthmatic cell populations. However, even in sparse asthmatic HBF, the blocking of N-cadherin resulted in the inhibition of FMT. Our findings show for the first time that the initial absence or an induced loss of cell-cell adhesions in asthmatic HBF populations is important for the completion of FMT.     

Effects of basic fibroblast growth factor (FGF-2) on proliferation of human skin fibroblasts in type II diabetes mellitus.

Skin fibroblasts from patients with diabetes mellitus display abnormalities in cell proliferation. The use of exogenous growth factors on diabetic wounds has been found to stimulate fibroblast proliferation and facilitate wound healing. However, the results of application of FGF-2 alone to diabetic wounds in clinical trials have been disappointing. The objective of this experiment was to study the effects of FGF-2 and media supplements on in vitro proliferation of skin fibroblasts from patients with type II diabetes and nondiabetic controls, and to evaluate the association between fibroblast proliferation and cAMP production. Fibroblast cell lines (n = 5 from diabetic and n = 5 from control individuals) were cultured in DMEM + 20% FBS for 7 days. Cells were then counted, plated into 24-well plates at a concentration of 2 x 10(4) cells/well and incubated for 24 h in DMEM with serum. The next day, medium was changed to serum-free DMEM alone or DMEM with supplements (albumin, transferrin, insulin and hydrocortisone). Cells were cultured in the presence or absence of varying doses of FGF-2 (0, 0.3, 1, 3, 10 and 30 ng/ml) for 72 hrs then counted and medium was collected for cAMP radioimmunoassay. The doubling time for cell number tended to be greater (p < 0.2) for diabetic fibroblasts than for control fibroblasts. The addition of supplements to the medium reduced (p < 0.05) the doubling time for both fibroblast types. FGF-2 stimulated (p < 0.05) proliferation of diabetic fibroblasts only in medium containing supplements. In contrast, FGF-2 stimulated proliferation of control fibroblasts in medium with or without supplements. The maximal effects of FGF-2 on fibroblast proliferation were greater (p < 0.02) in medium with supplements than in medium without supplements. The K(D) of FGF-2 for fibroblast proliferation was greater (p < 0.06) for diabetic than for control fibroblasts, and lower (p < 0.02) for medium with supplements than for medium without supplements. Fibroblasts from patients with diabetes mellitus produced more (p < 0.05) cAMP than control fibroblasts. These results demonstrate that FGF-2 requires the presence of supplements to enhance proliferation of fibroblasts from patients with type II diabetes mellitus. In addition, fibroblasts from diabetic patients showed a greater K(D) for FGF-2 in terms of cell proliferation. These data suggest a defective FGF receptor or down-regulation of the FGF receptor-mediated cascade that leads to cell proliferation. Identifying methods of reducing the K(D) of FGF-2 in stimulating the proliferation of diabetic fibroblasts may improve the clinical response of diabetic wounds to FGF-2.