Similarities and differences between sexes in regional loss of cortical and trabecular bone in the mid‐femoral neck: The AGES‐Reykjavik longitudinal study

The risk of hip fracture rises rapidly with age, and is notably higher in women. After falls and prior fragility fractures, the main clinically recognized risk factor for hip fracture is reduced bone density. To better understand the extent to which femoral neck density and structure change with age in each sex, we carried out a longitudinal study in subjects not treated with agents known to influence bone mineral density (BMD), to investigate changes in regional cortical thickness, as well as cortical and trabecular BMD at the mid‐femoral neck. Segmental quantitative computed tomography (QCT) analysis was used to assess bone measurements in two anatomic subregions, the superolateral (superior) and inferomedial (inferior). A total of 400 older individuals (100 men and 300 women, aged 66–90 years) who were participants in the Age Gene/Environment Susceptibility‐Reykjavik Study (AGES‐Reykjavik), were studied. Participants had two QCT scans of the hip over a median follow‐up of 5.1 years (mean baseline age 74 years). Changes in bone values during follow‐up were estimated from mixed effects regression models. At baseline women had lower bone values in the superior region than men. At follow‐up all bone values were lower in women, except cortical volumetric bone mineral density (vBMD) inferiorly. The relative losses in all bone values estimated in the superior region were substantially (about threefold) and significantly greater compared to those estimated in the inferior region in both sexes. Women lost cortical thickness and cortical vBMD more rapidly than men in both regions; and this was only weakly reflected in total femoral neck dual‐energy X‐ray absorptiometry (DXA)‐like results. The higher rate of bone loss in women at critical locations may contribute materially to the greater femoral neck fracture incidence among women than men. © 2013 American Society for Bone and Mineral Research.     

Nucleolin enhances the proliferation and migration of heat‐denatured human dermal fibroblasts

Denatured dermis, a part of dermis in burned skin, has the ability to restore its normal morphology and functions after their surrounding microenvironment is improved. However, the cellular and molecular mechanisms by which the denatured dermis could improve wound healing are still unclear. This study aimed to investigate the role of nucleolin during the recovery of heat‐denatured human dermal fibroblasts. Nucleolin mRNA and protein expression were significantly increased time‐dependently during the recovery of heat‐denatured human dermal fibroblasts (52 °C, 30 seconds). Heat‐denaturation promoted a time‐dependent cell proliferation, migration, chemotaxis, and scratched wound healing during the recovery of human dermal fibroblasts. These effects were prevented by knockdown of nucleolin expression with small interference RNA (siRNA), whereas overexpression of nucleolin enhanced cell proliferation, migration, and chemotaxis of human dermal fibroblasts with heat‐denaturation. In addition, the expression of transforming growth factor‐beta 1(TGF‐β1) was significantly increased during the recovery of heat‐denatured dermis and human dermal fibroblasts. TGF‐β1 expression was up‐regulated by nucleolin in human dermal fibroblasts. The results suggest that nucleolin expression is up‐regulated, and play an important role in promoting cell proliferation, migration, and chemotaxis of human dermal fibroblasts during the recovery of heat‐denatured dermis with a mechanism probably related to TGF‐β1.     

Size distribution of leukocyte aggregates differentiate between stress‐ and infection/inflammation‐related leukocytosis

We evaluated the state of leukocyte adhesiveness/aggregation in the peripheral blood of 33 patients with stress and 33 patients with infection/inflammation. Both groups had a similar white blood cell count (9542±3067 and 10,512±2758 cells per cmm, respectively). It was found that the percentage of aggregated leukocytes in the peripheral blood of patients with infection/inflammation was significantly higher than in the patients with stress (24.7±11 per cent versus 15.8±6 per cent; p<0.0001). When we analyzed the size distribution of the aggregated cells we also found a significant difference between the number of couplets (2.7±1.9 versus 1.6±0.9; p=0.006), triplets (0.6±1.3 versus 0.2±0.2; p=0.05), quadruplets (0.14±0.2 versus 0.03±0.07; p=0.005) or quintets (0.04±0.1 versus 0.003±0.002; p=0.05) between these two groups. Thus, by using a simple slide test to reveal the state of leukocyte adhesiveness/aggregation in the peripheral blood one could favor the diagnosis of infection/inflammation induced leukocytosis as opposed to a stress‐related one. Copyright © 2000 John Wiley & Sons, Ltd.     

Phenotypic differences between oral and skin fibroblasts in wound contraction and growth factor expression

Wounds of the oral mucosa heal in an accelerated fashion with reduced scarring compared with cutaneous wounds. The differences in healing outcome between oral mucosa and skin could be because of phenotypic differences between the respective fibroblast populations. This study compared paired mucosal and dermal fibroblasts in terms of collagen gel contraction, alpha-smooth muscle actin expression (alpha-SMA), and production of the epithelial growth factors: keratinocyte growth factor (KGF) and hepatocyte growth factor/scatter factor (HGF). The effects of transforming growth factor -beta1 and -beta3 on each parameter were also determined. Gel contraction in floating collagen lattices was determined over a 7-day period. alpha-SMA expression by fibroblasts was determined by Western blotting. KGF and HGF expression were determined by an enzyme-linked immunosorbent assay. Oral fibroblasts induced accelerated collagen gel contraction, yet surprisingly expressed lower levels of alpha-SMA. Oral cells also produced significantly greater levels of both KGF and HGF than their dermal counterparts. Transforming growth factor-beta1 and -beta3, over the concentration range of 0.1-10 ng/mL, had similar effects on cell function, stimulating both gel contraction and alpha-SMA production, but inhibiting KGF and HGF production by both cell types. These data indicate phenotypic differences between oral and dermal fibroblasts that may well contribute to the differences in healing outcome between these two tissues.     

自体及异体皮肤肌腱细胞中生长因子分子水平的差异研究

目的:对自体及异体皮肤成纤维细胞和肌腱细胞的生长因子在分子水平的表达差异进行研究.方法:体外培养自体及异体人肌腱细胞和人皮肤成纤维细胞,用基因芯片的方法对两种细胞的生长因子进行检测及比较,并用RT-PCR的方法进一步验证.结果:自体皮肤、肌腱细胞生长因子的分子水平表达无差异,而异体皮肤、肌腱细胞生长因子分子水平的表达略有差异.结论:自体皮肤成纤维细胞与肌腱细胞的生长因子表达相似,证实两种细胞具有同源性,自体皮肤成纤维细胞可以成为构建组织工程化肌腱的种子细胞.而异体皮肤成纤维细胞和肌腱细胞的生长因子表达略有差异,可能为个体差异所致.     

Secreted factors from equine mesenchymal stromal cells diminish the effects of TGF‐β1 on equine dermal fibroblasts and alter the phenotype of dermal fibroblasts isolated from cutaneous fibroproliferative wounds

The prevalence of cutaneous fibroproliferative disorders (CFPDs) is high and almost exclusively occurs in humans (keloids and hypertrophic scars) and horses (exuberant granulation tissue), making the horse a valuable translational model for studies on prevention and treatment of human CFPDs. CFPDs arise as a result of dysregulated wound healing characterized by persistently high levels of cytokines, such as transforming growth factor beta 1 (TGF‐β1), that contribute to excessive extracellular matrix deposition, and the physical disorganization of dermal fibroblasts (DF). The mesenchymal stromal cell (MSC) secretome, consisting of all factors secreted by MSC, has been shown to promote normal wound healing in both humans and horses, but its potential to treat CFPDs remains largely unexplored. Therefore, the objective of this study was to examine the effects of the equine MSC secretome on equine DF influenced by cytokines that contribute to the development of CFPDs. First, primary equine DF were treated with TGF‐β1 in vitro in the presence or absence of MSC secreted products. We found that MSC secreted products could block TGF‐β1‐induced changes in DF morphology, proliferation rate, gene expression, and contractile‐capacity. We then isolated primary DF from equine exuberant granulation tissue, to evaluate the potential of the MSC secretome to alter the phenotype of cells derived from a complex CFPD environment. These results showed that MSC secreted factors did not change proliferation or migration of these cells, but did lead to changes in expression of genes and proteins involved in extracellular matrix remodeling and did affect contractile capacity. These results warrant future studies designed to evaluate the potential of the MSC secretome to minimize the pathologies associated with CFPD in vivo.     

Keratinocyte conditioned medium abrogates the modulatory effects of IGF-1 and TGF-β1 on collagenase expression in dermal fibroblasts

Overexpression of wound healing-promoting factors such as transforming growth factor-1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1) during the healing process has been implicated in the development of dermal fibrosis in patients following thermal injury, surgical incision, and deep trauma. However, the mechanism through which the expression of these two fibrogenic factors is slowed down and/or abrogated in the late stages of the healing process is not known. Here, we hypothesize that keratinocyte-releasable factors counteract the fibrogenic role of both IGF-1 and TGF-beta1 in fibroblasts. To test this hypothesis, the levels of collagenase (MMP-1), as an index for extracellular matrix degradation, in dermal fibroblasts in response to either keratinocyte-conditioned medium (KCM) or our recently identified keratinocyte-releasable stratifin in the presence and absence of either IGF-1, TGF-beta1, or both were evaluated. The results of Northern analysis showed a significant increase in collagenase mRNA expression in cells treated with KCM in the presence of both IGF-1 and TGF-beta1. The effect was, at least in part, due to keratinocyte-derived stratifin that was present in KCM. This was ascertained as the levels of MMP-1 mRNA were markedly reduced when cells were treated with stratifin-immuno-depleted KCM. The results of Western blot analysis showed an increase in the level of MMP-1 protein in stratifin-treated fibroblasts and this was consistent with the level of MMP-1 mRNA expression detected by Northern analysis. However, in contrast to KCM, whose efficacy on MMP-1 expression was modestly reduced by either IGF-1 and TGF-beta1, or a combination of both, these factors abrogated the MMP-1 stimulatory effect of stratifin in fibroblasts. In summary, the results of this study revealed that both stratifin and KCM stimulate the expression of MMP-1-in fibroblasts and this effect can be abrogated by either IGF-1, TGF-beta1, or a combination of both.     

Interspecies comparison of prostate cancer gene‐expression profiles reveals genes associated with aggressive tumors

Prostate cancer (PC) is a heterogeneous disease whose aggressive phenotype is the second leading cause of cancer‐related death in men. The identification of key molecules and pathways that play a pivotal role in PC progression towards an aggressive form is crucial. A major effort towards this end has been taken by global analyses of gene expression profiles. However, the large body of data did not provide a definitive idea about the genes which are associated with the aggressive growth of PC. In order to identify such genes, we performed an interspecies comparison between several human data sets and high quality microarray data that we generated from the transgenic adenocarcinoma of mouse prostate (TRAMP) strain. The TRAMP PC mimics the histological and pathological appearance as well as the aggressive phenotype of human PC (huPC). Analysis of the microarray data, derived from microdissected TRAMP specimens removed at different stages of the disease yielded genetic signatures delineating the TRAMP PC development and progression. Comparison of the TRAMP data with a set of genes representing the core expression signature of huPC yielded a limited set genes. Some of these genes are known predictors of poor prognosis in huPC. Interestingly, the modulation of genes responsible for the invasive phenotype of huPC occurs in TRAMP already during the transition to prostate intraepithelial neoplasia (PIN) and onwards to localized tumors. We therefore suggest that critical oncogenic events leading to an aggressive phenotype of huPC can be studied in the PIN stage of TRAMP. Prostate 69:1034–1044, 2009. © 2009 Wiley‐Liss, Inc.     

Alternatively activated macrophages derived from THP‐1 cells promote the fibrogenic activities of human dermal fibroblasts

Macrophages play a key role in the wound healing process and can be divided into classically activated macrophages (M1) and alternatively activated macrophages (M2). Fibroblasts maintain the physical integrity of connective tissue, participate in wound closure as well as produce and remodel extracellular matrix. Macrophages have a close relationship with fibroblasts by increasing the production of matrix metalloproteinase‐1 (MMP‐1) for faster wound closure and remodeling and myofibroblast differentiation from fibroblasts. In this study, resting state (M0), M1 and M2 macrophages differentiated from the human monocytic THP‐1 cell line were used to co‐culture with human dermal fibroblasts (HDF) for 48, 96 and 144 hours to investigate the effect of macrophages subsets on the fibrogenic activity of fibroblasts. The differentiation and polarization from THP‐1 cells to M0, M1 and M2 macrophages were characterized by flow cytometry and cell cycle analysis. Cell sorting was performed to purify M0 and M2 macrophages. Cell proliferation, collagen synthesis, myofibroblast formation, gene expression of anti‐fibrotic and pro‐fibrotic factors, MMP‐1 activity, and cytokine concentration were investigated. Results showed differentiation of M0 and polarization of M1 and M2 macrophages. M2 macrophages promoted the fibrogenic activities of co‐cultured HDF by facilitating cell proliferation, increasing the collagen content, alpha‐smooth muscle actin expressed cells, expression of the pro‐fibrotic genes and concentration of M2 macrophage related factors, as well as decreasing the expression of the anti‐fibrotic genes and MMP‐1 activity. These findings reinforce the pro‐fibrotic role of M2 macrophages, suggesting therapeutic strategies in fibrotic diseases should target M2 macrophages in the future.     

Endogenous bone morphogenetic proteins mediate 1α, 25‐dihydroxyvitamin D3‐induced expression of osteoblast differentiation markers in human dermal fibroblasts

Human dermal fibroblasts are generally considered to be restricted to a fibroblastic lineage. Although dermal fibroblasts do not typically express markers of osteoblastic differentiation, they have previously been shown to undergo osteoinduction when stimulated with bone morphogenetic proteins (BMPs) or vitamin D₃. However, involvement of BMP signaling in vitamin D₃‐mediated osteoinduction has not been reported. In this study, human dermal fibroblasts were cultured in chemically defined medium containing vitamin D₃, in the presence of the BMP antagonist noggin or neutralizing antibodies specific for BMP‐4 or BMP‐6, and characterized for markers of osteoblastic differentiation. Treatment of dermal fibroblasts with vitamin D₃ induced expression of BMP‐4 (1.2 ± 0.2, 1.7 ± 0.2, and 1.8 ± 0.2 relative fold increase) and BMP‐6 (9.1 ± 0.3, 23.3 ± 2.1, and 30.4 ± 3.0 relative fold increase) at 3, 14, and 21 days, respectively. Vitamin D₃ was also shown to induce the expression of the osteoblast‐specific markers, alkaline phosphatase and osteocalcin, in a dose‐dependent manner in human dermal fibroblasts. Addition of noggin, BMP‐4 antibodies, and BMP‐6 antibodies resulted in a downregulation of alkaline phosphatase activity (by 42%, 22%, and 20%, respectively) and secreted osteocalcin (by 20%, 31%, and 49%, respectively) after 21 days in culture. However, blocking BMP signaling did not result in complete recovery of a fibroblastic phenotype. Taken together, these results suggest that BMP signaling plays a role in the induction of an osteoblastic phenotype in human dermal fibroblasts in response to vitamin D₃ stimulation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:162–168, 2009     

Avoidance of potentially traumatic stimuli mediates the relationship between accumulated lifetime trauma and late‐life depression and anxiety

This study examined the influence of lifetime accumulated trauma on late‐life mental health in a sample of 1,216 older adults, 65–94 years old, residing in New Zealand. Multiple regression analyses indicated that accumulated trauma predicted both depression and anxiety in this sample. The hypothesis that avoidance of memories and situations surrounding prior trauma mediates relationships between cumulative trauma and depression and anxiety was supported. Avoidance of prior traumatic memories and situations explained 49% of the variance between accumulated trauma and depression and 46% of the variance between accumulated trauma and anxiety. Results also suggest that traumatic experiences during young adulthood and middle age are stronger predictors of anxiety and depression among older adults than trauma experienced in childhood and adolescence.     

人毛乳头细胞生长相关蛋白作用下细胞VEGF的表达

目的研究人毛乳头生长相关蛋白作用下不同代人毛乳头细胞VEGF的表达。方法通过体外培养低传代的人毛乳头细胞,收集其上清配制成条件培养基,用此条件培养基培养高传代人毛乳头细胞,通过RT-PCR观察各代毛乳头细胞的VEGF变化。结果用低传代人毛乳头细胞的上清液培养过的9代人毛乳头细胞的VEGF表达不仅明显好于对照组(P<0.01),而且明显好于基础培养基作用下的7代人毛乳头细胞的VEGF表达(P<0.01)。结论低传代人毛乳头细胞的培养液在体外能明显地促进VEGF的表达。     

血竭素高氯酸盐对成纤维细胞增殖与成纤维细胞生长因子mRNA表达的影响

目的 观察血竭素高氯酸盐(Dp)对人皮肤成纤维细胞(Fb)生物学特性的影响,探讨其促进创而愈合的机制.方法 分离培养人正常Fb,将Dp以不同浓度(0.063、0.125、0.250、0.625、1.250、2.500 mg/L)分别加入培养液,采用噻唑蓝(MTT)比色法检测Dp在不同浓度以及不同时间点对体外培养的Fb增殖作用的影响.通过流式细胞仪,实时荧光定量聚合酶链式反应( real-time PCR)分别检测最适浓度培养条件下Fb的细胞周期变化以及成纤维细胞生长因子(FGF) mRNA的合成表达.结果 Dp在0.625～1.250 mg/L浓度范围内,其吸光度值(0.237±0.012、0.243±0.017)均高于对照组(0.208±0.011)差异有统计学意义(t值分别为2.11、2.23,P＜0.05),此浓度范围可促进Fb增殖,且呈剂量依赖性,在浓度为1.250 mg/L时(A值0.243±0.017),促增殖作用最为显著(t =2.23,P＜0.01),流式细胞仪结果显示,在浓度为1.250 mg/L时,Dp可明显促进Fb通过G1/S及S/G2期限制点,S期及G2/M期细胞与对照组比较明显增多,G0/G1期细胞与对照组比较明显减少(t值分别为4.32、7.53、3.27,P＜0.05).细胞因子mRNA表达测定中,1.250 mg/L Dp组与对照组比较表达明显上调,差异亦有统计学意义(=1.48,p＜0.05).结论 Dp能显著促进Fb增殖,加快Fb周期进程,同时可促进FGF的mRNA表达,可能与血竭促进创面愈合的机制有关.