Prestress Due to Dimensional Changes Caused by Demineralization: A Potential Mechanism for Microcracking in Bone

Microcracking in bone due to internal strains caused by mineralization is a possible mechanism of damage. Similar damage can be seen in other biological composites such as trees experiencing growth-related prestresses. Dimensional changes in cortical bone due to demineralization and experimental glycation were studied to test whether mineralization-related prestrains are consistent with observed microcracking patterns in bone. A microscopy technique that enables wet measurements of length and angle of milled bone specimens was used. Demineralization of bovine and human bones caused significant anisotropic changes in tissue size. Dimensional changes due to demineralization in bovine bone were prevented or reduced when collagen cross linking was increased by glycation. The dimensional changes of bone caused by demineralization are consistent with the hypothesis that mineralization-caused stresses in remodeling tissue can cause microcracks. © 2002 Biomedical Engineering Society. PAC2002: 8719Rr     

Good glycaemic control reduces oxidation and glycation end-products in collagen of diabetic rats

Summary Blood glucose control plays a prominent role in the aetiology of diabetic complications. Recent data support the hypothesis that non-enzymatic pathways (glycation and oxidation) are involved in the pathogenesis of tissue damage in diabetes mellitus. In this study the level of pentosidine, a marker of glycation, and the intensity of collagen-linked fluorescence glycation (370/440 and 335/385 nm) and oxidation-related (356/460 and 390/460 nm), have been examined in spontaneously diabetic rats with good and poor glycaemic control. Pentosidine increased dramatically in rats with poor control, and slightly in those with good control. At the end of the study, after 6 months of diabetes, pentosidine levels were 13 ± 5 and 2.1 ± 0.5 pmol/mg collagen, respectively (control rats: 1.1 ± 0.1 pmol/mg collagen). A similar pattern was observed for both glycation or oxidation-related fluorescence. The group of rats with poor control always showed elevated average values when compared to rats with good control, with a relative increase of over 200 %. The results emphasize the role of good glycaemic control in preventing the growth of glycation or oxidation end-products in collagen. On comparison between the general mean level of all glycated haemoglobin and the mean pentosidine level of the three groups, a very good exponential correlation was found (r = 0.993, p < 0.001). The fluorescence values presented a less strong relationship, but a correlation with glycaemic control was still present. If the post-translational modifications of proteins play a leading role in the pathogenesis of complications it is possible to conclude that strict glycaemic control, obtained by accurate insulin therapy can prevent them by inhibiting the non-enzymatic modification of proteins and delaying their accumulation in collagen. The therapeutic implications are obvious. [Diabetologia (1996) 39: 1440–1447] Received: 23 March 1996 and in final revised form: 29 August 1996     

Possible Involvement of Altered RGD Sequence in Reduced Adhesive and Spreading Activities of Advanced Glycation End Product-Modified Fibronectin to Vascular Smooth Muscle Cells

Although fibronectin (FN) modified by advanced glycation end products (AGEs) has been shown to contribute to the development of diabetic vascular complications through its reduced adhesive activity to vascular cells, little is known about changes in the cell binding domain of AGE-modified FN. Here we examined the mechanism of reduced adhesive and spreading activities of AGE-modified FN to vascular smooth muscle cells (SMCs), particularly the contribution of modification of Arg-Gly-Asp (RGD) sequence. Incubation with glucose caused not only the formation of N-carboxymethyllysine and pentosidine, but also polymerization of FN in a dose- and time-dependent manner. AGE-modified FN had significantly low adhesive and spreading activities to cultured SMCs. On the other hand, multimeric FN formed by disulfide bonds did not show any effect on either cell adhesion or spreading. The adhesive activity of type I collagen, one of the RGD sequence-containing proteins, to SMCs also decreased by AGE-modification. The inhibitory effect of AGE-modification on cell adhesion was significantly greater in type I collagen than in FN. Although the extent of AGE-modification of type I collagen was indistinguishable from that of FN, AGE-modification decreased the arginine content of type I collagen by 69.5% and of FN by 30.6%, compared with their non-glycated forms. The addition of RGD peptides caused a decrease in adhesion of SMCs to non-glycated FN, but not to AGE-modified FN. Modification of RGD sequence with glyoxal eliminated its inhibitory effect on cell adhesion. Our results suggest that a marked decrease in adhesive and spreading activities of AGE-modified FN to SMCs might largely be due to a modification of its RGD sequence by AGE, thus suggesting a potential link between AGE modification of FN and the pathogenesis of diabetic angiopathy.     

晚期糖基化终末产物对小鼠骨髓干细胞向血管内皮细胞分化的影响

观察晚期糖基化终末产物(AGEs)对骨髓干细胞(BMSC)向内皮细胞分化的影响.结果 发现,20μg/ml AGEs对BMSC分化的影响不明显,100μg/ml AGEs使BMSC分化能力明显下降,血管内皮生长因子受体2阳性细胞计数和百分数均明显降低.     

Simvastatin attenuates the endothelial pro-thrombotic shift in saphenous vein grafts induced by Advanced glycation endproducts

Background

Advanced glycation endproducts (AGEs) and its receptors (RAGEs) are heterogeneous signaling proteins associated to diabetes and responsible of endothelial alterations leading to atherosclerosis progression and graft failure. The aim of this study was to investigate the role of statin in reducing AGEs related endothelial damage.

Methods

Endothelial cell(EC) obtained from leftovers of saphenous vein grafts of non-diabetic patients were incubated with AGEs (2 and 20 μM) and subsequently treated with Simvastatin. Neutrophils (PNM) adherence, ROS production and RAGE and peroxisome proliferator-activated receptors-gamma (PPAR-γ) expression were analyzed. As clinical validation of the in vitro findings, ECs of diabetic patients in optimized glycaemic control administered with a 3 weeks Simvastatin regimen were similarly processed.

Results

Simvastatin blunted the rise in PMN adhesion and ROS generation following stimulation of saphenous vein EC culture with AGEs in vitro. This effect was time dependent and was associated to an increase in PPAR-γ induction paralleled by a decrease in RAGEs expression. Parallely, data from diabetic patients administered with Simvastatin showed a similar significant reduction in PNM adhesion and ROS generation. Simvastatin treatment significantly decreased RAGEs expression in ECs from diabetic patients and determined a slight increase in PPAR-γ expression but the latter failed to reach statistical significance. Interference in the function of these two crucial pathways might be at the root of the statin antinflammatory and antithrombotic effect in the context of AGEs-associated damage.

Conclusions

Despite the recently raised warning on the use of statins in the diabetic population, this study elucidates their cornerstone position in endothelial homeostasis of saphenous grafts in patients with controlled diabetes.     

Cytotoxicity of Acetaldehyde‐Derived Advanced Glycation End‐Products (AA‐AGE) in Alcoholic‐Induced Neuronal Degeneration

Abstract : Background: The Maillard reaction that leads to the formation of advanced glycation end‐products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients, in aging and in neurodegenerative processes. We hypothesize that acetaldehyde (AA), one of the main metabolites of alcohol, may be involved in alcohol‐induced neurotoxicity in vivo by formation of AA‐derived AGEs (AA‐AGE) with brain proteins. Methods: AA‐AGE‐bovine serum albumin (BSA) and AA‐AGE‐rabbit serum albumin (RSA) were prepared as described previously. Antibody specific for AA‐AGE was isolated from rabbit antiserum by affinity chromatography. Primary cortical neuronal cell cultures were prepared as described previously. Results: Incubation of cortical neurons with AA‐AGE produced a dose‐dependent increase in neuronal cell‐death, and the neurotoxicity of AA‐AGE was neutralized by the addition of an anti‐AA‐AGE specific antibody, but not by anti‐N‐ethyllysine (NEL) antibody. The AA‐AGE epitope was detected in human brain of alcoholism. Conclusions: We propose that the structural epitope AA‐AGE is an important toxic moiety for neuronal cells in alcoholism.     

高糖环境中真皮成纤维细胞生物学行为的变化

目的 探讨糖尿病（DM）状态对真皮成纤维细胞（FB）生物学行为的影响。方法 通过DM大鼠模型，对皮肤的组织生物学特征及真皮FB的生物学行为进行检测。结果 （1）在DM状态下，皮肤出现一系列组织学改变（厚度明显变薄和炎性细胞浸润等）、皮肤糖含量与炎症介质水平及糖基化终末产物（AGEs）的表达异常升高、真皮FB的真皮增殖细胞核内抗原表达上升，但增殖周期显示处于S期细胞比例升高而G2／M期细胞却未相应增加。（2）在高糖和AGE-人血清白蛋白（HSA）干预下，真皮FB的生长抑制和凋亡细胞百分率显著高于正常对照（NC）组（P〈0．05或0．01），且生长抑制和凋亡率呈AGE-HSA的剂量依赖性。结论 在DM状态下，高糖环境和AGEs蓄积是导致FB功能异常的重要原因，可能也是造成DM患者皮肤“隐性损害”以及创面难愈的重要原因之一。     

罗格列酮对动脉粥样硬化大鼠血清晚期糖基化终产物的影响及可能机制

目的:观察晚期糖基化终产物在动脉粥样硬化大鼠血清中的水平,以及应用胰岛素增敏剂罗格列酮对其的影响,并探讨其可能机制。方法:通过一次性维生素D3注射+高脂喂养法建立动脉粥样硬化模型。动物分组:A组:动脉粥样硬化模型组,B组:罗格列酮治疗组,C组:正常对照组,24周后处死全部大鼠,测定大鼠血清晚期糖基化终产物(AGE)、丙二醛(MDA)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、总胆固醇(TC)、甘油三脂(TG)水平。计算血浆致动脉硬化指数(AIP)。取主动脉全段,HE染色分析大鼠主动脉病变,PHOTOSHOP系统测定粥样斑块面积占内膜面积的百分比。免疫组化法观察主动脉丙二醛修饰低密度脂蛋白(MDA-LDL)的沉积。结果:大鼠血清AGE水平A组>B组>C组(P均<0.01)。MDA血清水平亦有A组>B组>C组(P值分别<0.05和0.01)。AIP数值A组>B组>C组(P均<0.01)。TC、LDL血清水平A组和B组均高于C组(P均<0.01),A组和B组之间无统计学差异。除C组外,其他两组大鼠主动脉均见粥样斑块形成,A组动脉粥样斑块面积/内膜面积百分比(38%±6%)高于B组(26%±8%)(P<0.05),A组粥样斑块病变部位MDA-PLDL的沉积面积较B组大(P<0.01,AGE与AIP值呈正相关(r=0.698,P<0.01),AGE与血清MDA呈正相关(r=0.514,P<0.05)。结论:血清AGE增高是动脉粥样硬化的危险因素之一。应用罗格列酮可以降低AGE水平,改善动脉损伤。AGE导致动脉粥样硬化的机制可能与增加氧化应激和增高血浆致动脉硬化指数有关。     

糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响

目的 探讨糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响.方法在培养的小鼠巨噬细胞株(J774A.1)中分别加入不同浓度(50、100、200及400 mg/L)的糖基化终产物干预24 h和同一浓度(200 mg/L)的糖基化终产物干预12、24及48 h,以无血清培养基和相应浓度的牛血清白蛋白为对照.用逆转录聚合酶链方法检测基质金属蛋白酶诱导物mRNA的表达,用酶联免疫吸附法检测上清中基质金属蛋白酶诱导物蛋白水平,用酶谱法检测上清中基质金属蛋白酶9的活性.结果 糖基化终产物干预组的基质金属蛋白酶诱导物mRNA表达水平和上清中蛋白水平与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05).糖基化终产物干预组的上清中基质金属蛋白酶9的活性与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05).结论 糖基化终产物促进小鼠巨噬细胞基质金属蛋白酶诱导物mRNA表达、蛋白分泌及基质金属蛋白酶9的活性;提示糖基化终产物可能通过调节巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9的活性而影响糖尿病动脉粥样硬化斑块的稳定性.     

晚期糖基化终产物诱导血管内皮细胞骨架形态改变的机制

目的研究晚期糖基化终产物修饰蛋白对内皮细胞细胞骨架肌动蛋白的形态学影响及特异的糖基化终产物受体和氧化应激在此病理过程中的作用。方法用不同浓度的糖基化终产物修饰人血清白蛋白与人脐静脉内皮细胞株ECV304在体外共同培养不同时间，并设立对照组进行比较，采用免疫荧光染色法显示细胞骨架的形态学改变。结果与对照组相比，糖基化终产物修饰人血清白蛋白以时间和剂量依赖的方式影响内皮细胞骨架肌动蛋白聚合丝状和可溶性单体（或球状）形态的改变。随着糖基化终产物修饰人血清白蛋白作用浓度和时间的增加，丝状肌动蛋白所形成的外周致密带边缘出现锯齿样断裂，趋于变细崩解消散，最终形成由非极性单行排列的肌动蛋白丝组成的应力纤维。可溶性单体表现为向细胞浆和胞膜移位，胞浆区出现点状和丝状染色，细胞间距离明显增大。可溶性糖基化终产物受体的抗体和烟酰胺腺嘌呤二核苷酸磷酸盐氧化酶抑制剂均可阻断糖基化终产物对细胞骨架的影响。结论糖基化终产物修饰蛋白对细胞骨架的影响是通过与内皮细胞上的糖基化终产物受体结合并引起细胞内的氧化应激所介导的，这一作用可能与糖基化终产物所致血管通透性升高有关。