罗格列酮对糖尿病大鼠胸主动脉钙化的影响

目的探讨罗格列酮对糖尿病大鼠胸主动脉钙化的影响。方法利用链脲佐菌素联合高能量饲料和维生素D3并尼古丁制备糖尿病血管钙化大鼠模型(DM+VDN组),并随机分为模型组、罗格列酮(RSG)治疗组和格列本脲(GLB)治疗组。检测大鼠一般代谢指标、血清糖基化终末产物(AGE)、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,胸主动脉钙含量及糖基化终末产物受体(RAGE)蛋白表达。结果 RSG治疗组和GLB治疗组血清AGE、MDA及SOD水平低于模型组,且RSG效果优于GLB(P0.05); RSG治疗组胸主动脉RAGE表达水平以及钙含量均低于模型组和GLB治疗组(P0.05)。结论罗格列酮可能通过降低AGE水平,下调RAGE表达,抑制氧化应激反应,延缓血管钙化进展。     

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。     

糖基化终末产物对活化肝星状细胞合成前胶原及致纤维化细胞因子的影响

目的探讨不同浓度的糖基化终末产物（AGEs）对肝星状细胞（HSC）活性的影响。方法体外合成糖基化牛血清白蛋白（AGE-BSA）,以不同浓度的AGE-BSA刺激HSC,观察AGE受体（RAGE）、转化生长因子（TGFβ1）,结缔组织生长因子（CTGF）、Ⅰ、Ⅲ型前胶原mRNA;ELISA法检测细胞上清液TGFβ1及Ⅰ、Ⅲ型前胶原蛋白的水平。结果高浓度的AGE-BSA（100μg/ml）培养24 h便能促进RAGE、TGFβ1、CTGF和Ⅰ、Ⅲ型前胶原mRNA的表达,并随着时间延长而升高,细胞上清液蛋白水平有同样改变。结论一定浓度的AGEs能促进HSC前胶原及致纤维化细胞因子的表达,并呈时效性改变。AGEs对肝纤维化可能起到促进作用。     

糖尿病皮肤病变及其机制的研究进展

糖尿病皮肤在未遭遇外源性损伤的情况下即已存在组织学和细胞功能学的异常改变.由于糖尿病皮肤存在含糖量增高以及晚期糖基化终末产物蓄积、基质金属蛋白酶及其抑制剂的比例失平衡、生长因子异常、炎性反应等异常改变,导致糖尿病皮肤厚度变薄,皮肤层次减少,结缔组织变性,细胞形态变异,黏附力及增殖能力F降,胶原合成紊乱.这种有别于皮肤组织完整性破坏的隐匿性损害可能是糖尿病皮肤易损或创面形成后难以愈合的重要原因之一.     

糖基化终末产物对大鼠肾皮质PAI-1表达和活性的影响

目的观察糖基化终末产物（AGEs）对大鼠肾皮质纤溶酶原激活物抑制物-1（PAI-1）表达和活性的影响。方法大鼠尾静脉注射AGE修饰大鼠血清蛋白（AGEs）6周，其中部分大鼠同时腹腔注射AGE形成抑制剂氨基胍（AG），以注射天然大鼠血清蛋白（RSP）和正常大鼠（Con）作为对照。免疫组织化学、Western blot、RT—PCR检测PAI-1表达水平，酶谱法分析纤溶酶原激活物（PA）活性，PAS染色评估细胞外基质（ECM）含量。结果与Con组及RSP组比较，AGEs组大鼠肾皮质ECM含量、PAI-1蛋白及mRNA表达水平均明显增高，PA活性明显降低（PAI-1 mRNAP〈0．01，其余P〈20．05），而同时注射AG的大鼠上述变化明显减轻。结论AGEs上调大鼠肾皮质PAI-1的表达，抑制PA活性，可能是糖尿病肾病ECM积聚的原因之一。     

褪黑素对糖尿病大鼠心肌MDA、CAT及GSH-Px含量和AGE、RAGE表达的影响

目的研究褪黑素(RAGE)对糖尿病大鼠心肌组织中丙二醛(MDA)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)含量的影响及糖基化终末产物(AGE)和糖基化终末产物受体(RAGE)表达水平的改变。方法清洁级雄性Wistar大鼠,高脂高糖饮食,配合腹腔注射链脲佐菌素诱导糖尿病模型,随后给予低剂量[10 mg/(kg体重·d)]和高剂量[20 mg/(kg体重·d)]MT灌胃16 w。比色法测定心肌组织中MDA、CAT和GSH-Px含量变化;HE染色观察心肌组织病理改变;超敏二步免疫组化染色测定心肌组织中AGE和RAGE蛋白表达。结果经过MT处理16 w后,与模型组比较,低剂量MT组和高剂量MT组血糖值无明显变化(P0.05);与模型组比较,低剂量MT组和高剂量MT组心肌组织中MDA含量明显降低,CAT和GSH-Px含量明显升高(P0.05);HE染色显示低剂量MT组和高剂量MT组心肌结构损伤程度均较模型组轻;与模型组比较,低剂量MT组和高剂量MT组心肌组织中AGE和RAGE蛋白表达明显降低(P0.05)。结论 MT可以降低糖尿病大鼠心肌组织MDA含量,提高CAT和GSH-Px含量;可改善心肌结构变化;降低心肌中AGE和RAGE蛋白的表达。     

糖基化终极产物对心血管系统的损伤作用及干预措施

目的糖基化终极产物(AGEs)是体内蛋白质与糖在无酶条件下发生反应后的产物.正常人体内AGE水平随年龄增长而缓慢增加,疾病状态下,如糖尿病时循环和组织中AGE修饰蛋白水平明显增加.AGEs主要通过与其受体RAGE相互作用介导一系列病理反应,研究表明AGEs可以通过损伤内皮功能、促进平滑肌细胞增殖、诱导心肌细胞凋亡、促进胶原蛋白交联、促进炎症因子及生长因子表达、干扰脂质代谢、影响凝血状态等机制对心血管系统产生损伤,阻滞AGEs/RAGE是心血管疾病治疗的新的靶点.     

晚期糖基化终末产物致动脉粥样硬化的机制

晚期糖基化终末产物(advanced glycation end products,AGE)是由还原糖的羰基与游离氨基反应形成的复合物。AGE通过与其受体(RAGE)结合,改变细胞内信号转导、诱导炎症、增强氧化应激;与胶原交联、修饰脂蛋白等损害血管的完整性;这些导致血管内皮细胞功能紊乱,刺激平滑肌细胞迁移增殖,启动及加速糖尿病动脉粥样硬化和血管并发症的发生发展。阻断AGE-RAGE系统对防治糖尿病并发症具有重要意义。     

晚期糖基化终末产物致动脉粥样硬化的机制

晚期糖基化终末产物(advanced glycation end products,AGE)是由还原糖的羰基与游离氨基反应形成的复合物.AGE通过与其受体(RAGE)结合,改变细胞内信号转导、诱导炎症、增强氧化应激;与胶原交联、修饰脂蛋白等损害血管的完整性;这些导致血管内皮细胞功能紊乱,刺激平滑肌细胞迁移增殖,启动及加速糖尿病动脉粥样硬化和血管并发症的发生发展.阻断AGE-RAGE系统对防治糖尿病并发症具有重要意义.     

恒定和波动高糖对大鼠肾系膜细胞损伤及RAGE表达的影响

目的 研究恒定和波动高糖对大鼠肾小球系膜细胞(GMC)损伤及细胞晚期糖基化终末产物受体(RAGE)表达的影响,探讨糖尿病肾病的病理损伤机制.方法 将体外培养的大鼠GMC在恒定和波动高糖培养基中培养,分别于24 h、48 h进行细胞学形态观察,四甲基偶氮唑蓝(MTT)法测定细胞存活率,生化法测定培养上清液中超氧化物歧化酶(SOD)活性和丙二醛的含量,RT-PCR检测细胞RAGE mRNA的表达情况.结果 波动高糖组在细胞形态、SOD活性、丙二醛含量、RAGE mRNA的表达与正常对照组和恒定高糖组相比均有明显差异(P＜0.05).结论 波动高糖造成大鼠GMC损伤比恒定高糖更为严重,是导致糖尿病肾病发生的重要原因之一.     

Influence of high glucose and AGE environment on the proliferation,apoptosis, paracrine effects,and cytokine expression of human adipose stem cells in vitro

The incidence of delayed wound healing in patients with diabetes has increased in recent years. However, the reason of delayed diabetic wound healing and the changes of human adipose stem cells (hASCs) in the diabetic environment are still unclear. We simulated diabetic microenvironment with high glucose and glycation end products (AGEs) in vitro. CCK-8 and flow cytometry were used to study the proliferation and apoptosis of hASCs in the simulated diabetic microenvironment. The paracrine of hASCs was studied by transwell co-culture system. Protein chip was used to measure the expression of cytokines in hASCs. We found that high glucose and AGEs did not affect the proliferation of hASCs but arrested them in the S phase. More hASCs appeared early apoptosis in the simulated diabetic microenvironment. The promoting effect on the proliferation of fibroblasts and endothelial cells was weakened when hASCs were cultured in diabetic microenvironment for 6 days. The five cell factors, granulocyte colony-stimulating factor (G-CSF), transforming growth factor-α (TGF-α), hepatocyte growth factor (HGF), tissue inhibitor of metalloproteinases-1 (TIMP-1), and vascular endothelial growth factor (VEGF), were all downregulated in hASCs of AGEs and the high glucose group. In this study, we simulated diabetic microenvironment with high glucose and AGEs in vitro to evaluate the changes of proliferation, apoptosis, paracrine, and cytokine expression of hASCs in the diabetic environment and tried to find the possible reason of delayed diabetic wound healing.     

Topical secretoneurin gene therapy accelerates diabetic wound healing by interaction between heparan-sulfate proteoglycans and basic FGF

Diabetic foot ulcers represent a therapeutic problem of high clinical relevance. Reduced vascular supply, neuropathy and diminished expression of growth factors strongly contribute to wound healing impairment in diabetes. Secretoneurin, an angiogenic neuropeptide, has been shown to improve tissue perfusion in different animal models by increasing the amount of vessels in affected areas. Therefore, topical secretoneurin gene therapy was tested in a full thickness wound healing model in diabetic db/db mice. Secretoneurin significantly accelerated wound closure in these mice and immunohistochemistry revealed higher capillary and arteriole density in the wounded area compared to control mice. In-vitro, the mechanism of action of secretoneurin on human dermal microvascular endothelial cells was evaluated in normal and diabetic cells. Secretoneurin shows positive effects on in vitro angiogenesis, proliferation and apoptosis of these cells in a basic fibroblast growth factor dependent manner. A small molecular weight inhibitor revealed fibroblast growth factor receptor 3 as the main receptor for secretoneurin mediated effects. Additionally, we could identify heparan-sulfates as important co-factor of secretoneurin induced binding of basic fibroblast growth factor to human dermal endothelial cells. We suggest topical secretoneurin plasmid therapy as new tool for delayed wound healing in patients suffering from diabetes.     

Differential effects of PDGF-BB on matrix metalloproteases and cytokine release in fibroblasts of Type 2 diabetic patients and normal controls in vitro

AIMS/HYPOTHESIS: The complex process of wound healing is regulated by various growth factors. The systemic character of diabetes mellitus favors the chronification of diabetic wounds. In this study, the in vitro effects of platelet-derived growth factor (PDGF)-BB on the expression of cytokines and matrix metalloproteases (MMPs) in fibroblasts of Type 2 diabetic patients and healthy controls were investigated. METHODS: We studied six Type 2 diabetic patients (mean Hba1(c)=7.5%) and six healthy controls. For proliferation studies, cultivated fibroblasts, prepared from biopsies taken from the thigh, were stimulated with different concentrations of PDGF. After 48 h, the expression of MMPs and cytokines was measured. We analysed the mRNA expression by RT-PCR (TaqMan), tissue protein levels by zymography, and cell supernatant levels by ELISA. RESULTS: Levels of MMP-mRNA were elevated in diabetic fibroblasts compared with healthy controls. At baseline, MMP-2 protein levels were significantly increased in the fibroblast of diabetic patients (P=.019). For MMP-9, a trend towards higher levels (P=.3) was found. After incubation with PDGF, a significant reduction of MMP-9 (P=.01) and MMP-13 (P=.04) was found. Analysis of cytokine release in cell culture supernatant showed elevated levels of interleukin (IL)-8 at baseline conditions. MMP-1 and MMP-2 levels in the supernatant were concentration-dependently reduced. CONCLUSIONS: This study, for the first time, demonstrates elevated MMPs in cultivated fibroblasts (derived from intact skin and not from an open wound) of diabetic patients compared with healthy controls under in vitro conditions. Therefore, our data support the hypothesis of alterations of wound healing in diabetic patients on the cellular level, reflecting the systemic character of the disease.     

Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor-binding protein-related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus

Expansion of extracellular matrix with fibrosis occurs in many tissues as part of the end-organ complications in diabetes, and advanced glycosylation end products (AGE) are implicated as one causative factor in diabetic tissue fibrosis. Connective tissue growth factor (CTGF), also known as insulin-like growth factor-binding protein-related protein-2 (IGFBP-rP2), is a potent inducer of extracellular matrix synthesis and angiogenesis and is increased in tissues from rodent models of diabetes. The aim of this study was to determine whether CTGF is up-regulated by AGE in vitro and to explore the cellular mechanisms involved. AGE treatment of primary cultures of nonfetal human dermal fibroblasts in confluent monolayer increased CTGF steady state messenger RNA (mRNA) levels in a time- and dose-dependent manner. In contrast, mRNAs for other IGFBP superfamily members, IGFBP-rP1 (mac 25) and IGFBP-3, were not up-regulated by AGE. The effect of the AGE BSA reagent on CTGF mRNA was due to nonenzymatic glycosylation of BSA and, using neutralizing antisera to AGE and to the receptor for AGE, termed RAGE, was seen to be due to late products of nonenzymatic glycosylation and was partly mediated by RAGE. Reactive oxygen species as well as endogenous transforming growth factor-beta1 could not explain the AGE effect on CTGF mRNA. AGE also increased CTGF protein in the conditioned medium and cell-associated CTGF. Thus, AGE up-regulates the profibrotic and proangiogenic protein CTGF (IGFBP-rP2), a finding that may have significance in the development of diabetic complications.     

Localization and regulation of pregnancy-associated plasma protein a expression in healing human skin

Pregnancy-associated plasma protein A (PAPP-A) is an IGF-binding protein-4 (IGFBP-4) metalloproteinase that cleaves inhibitory IGFBP-4 to amplify local IGF-I bioavailability in vitro. Thus it has functional implications in injury/repair responses. In this study we determined PAPP-A expression in healing human skin. Wounds were induced with a scalpel on the forearms of three normal subjects and were allowed to heal by first intention. Biopsies obtained on d 0, 2, 8, and 14 were processed for immunohistochemical detection of PAPP-A, IGF-I, and IGFBP-4. In uninjured skin (d 0), strong staining for PAPP-A was present in the epidermis, sweat and sebaceous gland epithelial cells, hair follicles, and blood vessels; no PAPP-A was detected in dermal fibroblasts or with mature collagen bundles. IGF-I localized strongly to epithelial cells of skin glands was weak to moderate in epidermis and blood vessels, and was absent in dermal cells. Weak focal staining for IGFBP-4 was found within uninjured epidermis. During wound healing, PAPP-A expression was induced in dermal granulation tissue within and adjacent to the injury. PAPP-A was present in dermis on d 2 and was increased in intensity and extent on d 8 and 14. PAPP-A expression also increased in the epidermis. PAPP-A expression in cells of granulation tissue colocalized with alpha-smooth actin staining of myofibroblasts and new blood vessels as well as with CD68 staining of macrophages and was associated with the compact, newly synthesized collagen of the healing wound. IGF-I staining was enhanced in the epidermis localized to the area of the incision and in granulation tissue associated with lymphoid cells. IGFBP-4 staining of the epidermis remained unchanged during wound healing, but was induced in the fibroblastic cells of granulation tissue over time. These data demonstrate localized and regulated expression of PAPP-A in human skin and suggest that PAPP-A may play an important role in an integrated IGF system in wound healing and tissue remodeling in vivo.     

Inhibition of TIMP1 enhances angiogenesis in vivo and cell migration in vitro

Neovascular invasion into a 3-dimensional matrix is controlled, in part, by matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). We tested the hypothesis that increasing MMP activity, via a specific blocking antibody to TIMP1, would enhance fibrovascular invasion into a PVA sponge. In vivo, inhibition of TIMP1 doubled the amount of angiogenic invasion (percentage area of invasion 33.5 +/- 3.5 vs 16.9 +/- 9.5, P = 0.003). The blocking antibody to TIMP1 did not increase the proportion of cells that were proliferating in the sponge implants, underscoring the importance of migration. In vitro, human microvascular endothelial cells (hmEC) and dermal fibroblasts treated with the antibody did not secrete greater amounts of collagenase but migrated significantly farther on collagen I (increase in distance migrated 26.6 +/- 9.4%, P = 0.003). Human dermal microvascular endothelial cells exposed to the TIMP1 blocking antibody exhibited a significant change in cell shape to a more elongated morphology. In conclusion, inhibition of TIMP1 increased angiogenesis into a PVA sponge in vivo and enhanced the migration of dermal hmEC and fibroblasts on collagen I in vitro. We propose that blocking TIMP1 improves angiogenesis by increasing cell motility during fibrovascular invasion.     

Cellular senescence controls fibrosis in wound healing

Mammalian wound healing involves the rapid synthesis and deposition of extracellular matrix (ECM) to maintain tissue integrity during repair. This process must be tightly controlled, as its deregulation may result in fibrosis, scarring, and loss of tissue function. Recent studies have uncovered an efficient and parsimonious mechanism for rendering fibrogenesis self-limiting in wound healing: in such diverse organs as the liver and skin, the myofibroblasts that initially proliferate and produce ECM are themselves eventually driven into senescence, blocking their further proliferation and converting them into matrix-degrading cells. Myofibroblast senescence in skin wounds is triggered by a dynamically expressed matricellular protein, CCN1/CYR61, which acts through integrin-mediated induction of oxidative stress. We propose that the onset of myofibroblast senescence is a programmed wound healing response that functions as a self-limiting mechanism for fibrogenesis, and this process may be regulated by the ECM microenvironment through the expression of CCN1/CYR61.     

Receptor for advanced glycation end products mediates inflammation and enhanced expression of tissue factor in vasculature of diabetic apolipoprotein E-null mice

Advanced glycation end products (AGEs) and their cell surface receptor, RAGE, have been implicated in the pathogenesis of diabetic complications. Here, we studied the role of RAGE and expression of its proinflammatory ligands, EN-RAGEs (S100/calgranulins), in inflammatory events mediating cellular activation in diabetic tissue. Apolipoprotein E-null mice were rendered diabetic with streptozotocin at 6 weeks of age. Compared with nondiabetic aortas and kidneys, diabetic aortas and kidneys displayed increased expression of RAGE, EN-RAGEs, and 2 key markers of vascular inflammation, vascular cell adhesion molecule (VCAM)-1 and tissue factor. Administration of soluble RAGE, the extracellular domain of the receptor, or vehicle to diabetic mice for 6 weeks suppressed levels of VCAM-1 and tissue factor in the aorta, in parallel with decreased expression of RAGE and EN-RAGEs. Diabetic kidney demonstrated increased numbers of EN-RAGE-expressing inflammatory cells infiltrating the glomerulus and enhanced mRNA for transforming growth factor-beta, fibronectin, and alpha(1) (IV) collagen. In mice treated with soluble RAGE, the numbers of infiltrating inflammatory cells and mRNA levels for these glomerular cytokines and components of extracellular matrix were decreased. These data suggest that activation of RAGE primes cells targeted for perturbation in diabetic tissues by the induction of proinflammatory mediators.     

The role of nitric oxide synthase isoforms and arginase in the pathogenesis of diabetic foot ulcers: possible modulatory effects by transforming growth factor beta 1

Aims/hypothesis. l-arginine, an amino acid involved in wound healing, is metabolised by one of two pathways; nitric oxide synthase and arginase. If metabolised by nitric oxide synthase, this can result in tissue destruction, or matrix deposition if metabolised by arginase. The aim therefore was to investigate the role of these enzymes in the pathogenesis of diabetic foot ulcers. Methods. The activity, proteins by Western blot analysis and cellular distribution (using immunocytochemistry) of these enzymes were measured in diabetic foot ulcers, diabetic skin and normal skin. Results. Total and inducible nitric oxide synthase (p < 0.001) and endothelial nitric oxide synthase were increased in diabetic ulcers compared with diabetic and normal skin and were associated with increased plasma nitrite concentrations in diabetic ulcers (p < 0.05). Inducible nitric oxide synthase was the major isoform, with the macrophage being the predominant cellular source. Similarly arginase activity was increased (p < 0.01) in diabetic ulcers. The protein levels corroborated with the activity data, with the fibroblast being the major cellular source. The spatial and cellular distribution of the two enzyme systems was distinct. Transforming growth factor-beta1 was decreased in diabetic ulcers in comparison with diabetic skin and normal skin. Conclusion/interpretation. Increased nitric oxide synthase activity in diabetic foot ulcers may be responsible for the impaired healing in this disease. Furthermore, the increased activity of arginase could account for the characteristic callus formation around these ulcers. In addition, the lower concentrations of transforming growth factor-beta1 in diabetic ulcers may explain the raised concentrations of nitric oxide in this condition. [Diabetologia (1999) 42: 748–757] Received: 23 September 1998 and in revised form: 10 December 1998     

Angiogenic properties of myofibroblasts isolated from normal human skin wounds

During wound healing, angiogenesis plays a crucial role in inducing adequate perfusion of the new tissue, thereby allowing its survival. This angiogenic process contributes to the formation of granulation tissue, alongside myofibroblasts. Myofibroblasts are cells specialized in wound contraction and synthesis of new extracellular matrix. Fibroblasts, considered by some to be at the origin of myofibroblasts, have already been shown to promote neovascularization. Thus, we hypothesized that myofibroblasts play a key role during angiogenic development in wound healing. We isolated myofibroblasts from normal human skin wounds and dermal microvascular endothelial cells (HDMVEC) and fibroblasts from skin. Using an in vitro fibrin-based model, we compared the proangiogenic activity of wound myofibroblasts to that of fibroblasts in the presence of HDMVEC. By immunostaining with collagen IV antibodies, we observed the formation of a capillary network significantly more developed when HDMVEC were cultured with myofibroblasts compared to the network formed in the presence of fibroblasts. The differences between these cell types did not result from a differential secretion of Vascular Endothelial Growth Factor or basic Fibroblast Growth Factor. However, in the presence of myofibroblasts, a significant decrease in matrix metalloproteinase activity was observed. This finding was correlated with a significant increase in Tissue Inhibitor of MetalloProteinase (TIMP)-1 and TIMP-3. Furthermore, inhibition of TIMP-1 secretion using shRNA significantly decreased myofibroblasts induced angiogenesis. These results led to the hypothesis that normal wound myofibroblasts contribute to the vascular network development during wound healing. Our data emphasize the critical role of wound myofibroblasts during healing.