Homocysteine modulates the proteolytic potential of human vascular endothelial cells

Pathological levels of homocysteine induce a metalloproteinase-dependent degradation of the elastic structures in arterial wall. This elastolytic process is preferentially localized toward the internal elastic laminae and in the first layers of the media, suggesting endothelium could participate in extracellular matrix degradation induced by homocysteine. Therefore, we studied the effects of homocysteine on proteolytic potential of endothelial cells. Human umbilical vein endothelial cells were cultured with concentrations of homocysteine matching human physiological (10 microM) and pathological (50, 100, and 250 microM) plasma homocysteine levels. Pathological levels of homocysteine increased the secretion of elastolytic metalloproteinase-2 and -9, but not of metalloproteinase-3 and -7. Homocysteine also increased the expression of human tissue kallikrein, a potential activator of matrix metalloproteinase-2 and -9, while the expression of urokinase plasminogen activator was not altered. These results suggest vascular endothelial cells could participate in the subendothelial degradation of the arterial elastic structures occurring in hyperhomocysteinemia.     

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.     

An in vitro evaluation of the effects of homocysteine thiolactone on key steps of angiogenesis and tumor invasion

Homocysteine thiolactone is a highly reactive homocysteine derivative that can react easily with proteins. Protein homocysteinylation has been suggested as a possible mechanism underlying the pathological consequences of impaired homocysteine metabolism. Homocysteine inhibits key steps of angiogenesis and tumor invasion. It can be hypothesized that homocysteine thiolactone could mimic the described anti-angiogenic and anti-invasive effects of homocysteine. Therefore, we studied the effects of homocysteine thiolactone on different key steps of angiogenesis and tumor invasion, using model endothelial and tumor cell lines. This study demonstrates that homocysteine thiolactone, in high contrast to homocysteine, is not an anti-angiogenic compound. Furthermore, our results suggest that homocysteine thiolactone could behave as a pro-angiogenic compound.     

Effects of homocysteine on metabolic pathways in cultured astrocytes

Homocysteine is an amino acid that is an important risk factor for several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Increased homocysteine levels induce neuronal cell death in a variety of neuronal types. However, very few studies have probed the effects of homocysteine in astrocytes. The present study investigated the effects of homocysteine on primary cultures of astrocytes by exposing astrocytes to 400 microM homocysteine for 20 h. Metabolic extracts of cells were prepared following a 4-h incubation in minimum medium with 5.5 mM [U-(13)C]glucose in the presence or absence of homocysteine and analysed using (13)C NMR. The expression level of pyruvate dehydrogenase kinase isoform 2 (PDK-2), NAD(P)H levels and mitochondrial membrane potential responses were investigated following culture with homocysteine. Metabolomic analysis was performed using (1)H NMR spectroscopy and pattern recognition analysis. Following incubation with homocysteine there was a significant decrease (48%) in the ratio of flux through pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) which was due to an increased flux through PDH. In addition, homocysteine culture resulted in a significant reduction in PDK-2 protein expression. Following stimulation with glucose there was a significant increase in NAD(P)H levels and an impaired hyperpolarisation of the mitochondrial membrane in homocysteine-treated cells. Metabolomic analysis showed that the most discriminating metabolites following homocysteine treatment were choline and hypotaurine. In summary, the results demonstrated that sub-lethal concentrations of homocysteine caused significant metabolic changes and altered mitochondrial function in primary cultures of astrocytes.     

Inhibition of fibroblast-induced angiogenic phenotype of cultured endothelial cells by the overexpression of tissue inhibitor of metalloproteinase (TIMP)-3

In this study, we examined the effect of overexpression of tissue inhibitor of metalloproteinase (TIMP)-3 on the angiogenic phenotype expressed by vascular endothelial cells (ECs). ECs were infected with a recombinant adenovirus carrying the TIMP-3 gene at various multiplicities of infection, and TIMP-3 expression by transfected cells was confirmed by Western blotting and reverse zymography. At transfection doses of 6.25, 12.5, 25, 50 and 100 multiplicity of infection, EC migration was reduced to 66, 45, 25, 17 and 5%, respectively, of that of the control. At the multiplicity of infection of 20, capillary tube length was reduced by 80% compared to that of the control. Thus, expression of TIMP-3 by ECs effectively inhibited EC migration and tube formation. Overexpression of TIMP-3 by ECs may be considered a gene therapy strategy for the treatment of pathological angiogenesis such as cancer and diabetic retinopathy.     

MMP-2及MMP-9与主动脉疾病相关性的研究进展

基质金属蛋白酶是一类可降解细胞外基质的蛋白酶,基质金属蛋白酶-2和-9为明胶酶,可降解细胞外基质中的胶原蛋白及弹性蛋白,其动态平衡对维持细胞外基质的稳定具有重要意义。主动脉的细胞外基质是主动脉中层重要的组成部分,细胞外基质成分的改变可导致主动脉中层结构的损伤,在主动脉疾病的发生、发展过程中起着重要作用。主动脉基质金属蛋白酶-2和-9的表达失衡可引起主动脉中层细胞外基质的降解,导致主动脉中层结构的损伤,从而促进主动脉疾病的发生。同时,主动脉疾病也可导致血浆中MMP-2、MMP-9浓度的升高。本文对近年来基质金属蛋白酶与主动脉疾病相关性的研究及进展作一综述,为心血管疾病发生机制的研究和治疗提供文献依据。     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Short- and long-term effects of silver nanoparticles on human microvascular endothelial cells

AIM: To study the response to silver nanoparticles (Ag NP) of human microvascular endothelial cells, protagonists of angiogenesis.METHODS: We cultured human microvascular endothelial cells and endothelial colony-forming cells in their corresponding growth medium. Stock solutions of Ag NP were prepared in culture medium and sonicated before use. They were added at different concentrations and for different times to culture media. The toxicity of Ag NP was investigated by measuring the reduction of yellow tetrazolium salt to dark purple formazan (MTT assay) at 575 nm. After staining with trypan blue, cell proliferation was assessed by counting viable cells. The lactate dehydrogenase leakage assay was performed on culture media by following the oxidation of NADH to NAD+ and monitoring the reaction kinetically at 340 nm. Reactive oxygen species production was quantified using 2’-7’-dichlorofluorescein diacetate. The alkaline comet assay was performed after mixing the cells with low melting-point agarose. Electrophoresis was then conducted and the samples were stained with ethidium bromide and analyzed with a fluorescence microscope.RESULTS: Ag NP are cytotoxic in a dose and time dependent fashion for HMEC. At high concentrations, Ag NP determine loss of membrane integrity as demonstrated by the increased activity of lactate dehydrogenase in the culture medium. Ag NP rapidly stimulate the formation of free radicals. However, pre-incubation with Trolox, apocynin, or N-acetyl-L-cysteine, antioxidants which have different structure and act through different mechanisms, is not sufficient to prevent cytotoxicity. Ag NP also induce DNA damage dose-dependently, as shown by comet assay. When exposed to sublethal concentrations of Ag NP for long times, the cells remain viable but are growth retarded. Interestingly, removal of Ag NP partially rescues cell growth. Also genotoxicity is reversible upon removal of Ag NP from culture medium, suggesting that no permanent modifications occur. It is noteworthy that Ag NP are cytotoxic and genotoxic also for endothelial progenitors, in particular for endothelial colony-forming cells, which participate to angiogenesis.CONCLUSION: Silver nanoparticles are cytotoxic and genotoxic for human microvascular endothelial cells and might become a useful tool to control excessive angiogenesis.     

Matrix metalloproteinase-2 contributes to tumor necrosis factor alpha induced apoptosis in cultured rat cardiac myocytes

Tumor necrosis factor alpha (TNFalpha) is associated with a higher risk of cardiovascular disease. Matrix metalloproteinase-2 (MMP-2) has been implicated in the pathophysiology of ischemic heart disease. However, the role of interactions between MMP-2 and TNFalpha, associated with cardiac apoptosis, is unknown. We hypothesized that MMP-2 will contribute to TNFalpha-induced myocardial apoptosis. After treatment with TNFalpha (1-20 ng/ml) for 24 h, or with TNFalpha (10 ng/ml) for 0, 6, 12, 24, or 48 h, MMP-2 activity, percent of TUNEL-positive myocytes, and DNA fragmentation dose, and time-dependently increased compared to control. However, TNFalpha blockade (neutralizing antibodies against human TNFalpha, 25 microg/ml) significantly reduced the activity of MMP-2 and markers of apoptosis induced by TNFalpha. Interestingly, MMP-2 antibody (30 microg/ml), or the MMP-2 inhibitors Doxycycline (Dox, 1-50 micromol/l) or GM6001 (GM, 10 micromol/l), prior to TNFalpha insult, decreased myocardial MMP-2 activity and reduced the percent of TUNEL-positive myocytes and DNA fragmentation. Moreover, MMP-2 inhibition reduced Bax expression and caspase3 activity, as well as increasing Bcl2 expression. MMP-2 inhibition was associated with decreased cardiac MMP-2 activity and decreased myocardial apoptosis induced by TNFalpha. These results suggest that MMP-2 contributes to TNFalpha-induced apoptosis in cultured rat cardiac myocytes.     

Anti-angiogenic Property of Edible Berries

Recent studies show that edible berries may have potent chemopreventive properties. Anti-angiogenic approaches to prevent and treat cancer represent a priority area in investigative tumor biology. Vascular endothelial growth factor (VEGF) plays a crucial role for the vascularization of tumors. The vasculature in adult skin remains normally quiescent. However, skin retains the capacity for brisk initiation of angiogenesis during inflammatory skin diseases such as psoriasis and skin cancers. We sought to test the effects of multiple berry extracts on inducible VEGF expression by human HaCaT keratinocytes. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seed, and strawberry) and a grape seed proanthocyanidin extract (GSPE) were studied. The extracts and uptake of their constituents by HaCaT were studied using a multi-channel HPLC-CoulArray approach. Antioxidant activity of the extracts was determined by ORAC. Cranberry, elderberry and raspberry seed samples were observed to possess comparable ORAC values. The antioxidant capacity of these samples was significantly lower than that of the other samples studied. The ORAC values of strawberry powder and GSPE were higher than cranberry, elderberry or raspberry seed but significantly lower than the other samples studied. Wild bilberry and blueberry extracts possessed the highest ORAC values. Each of the berry samples studied significantly inhibited both H 2 O 2 as well as TNF &#102 induced VEGF expression by the human keratinocytes. This effect was not shared by other antioxidants such as &#102 -tocopherol or GSPE but was commonly shared by pure flavonoids. Matrigel assay using human dermal microvascular endothelial cells showed that edible berries impair angiogenesis.     

Antioxidative effects of lovastatin in cultured human endothelial cells

The effects of lovastatin on glutathione peroxidase activity, hydrogen peroxide consumption, [3H]cholesterol uptake and [14C]acetate incorporation were investigated in cultured human endothelial cells. Treatment of endothelial cells with lovastatin in a medium without serum for 4 hr significantly increased both glutathione peroxidase activity and hydrogen peroxide consumption. This treatment also significantly inhibited cholesterol synthesis and cholesterol esterification. However, lovastatin stimulated cholesterol uptake by the cells. These alterations produced by lovastatin continued up to 24 hr. When serum was present in the culture medium, only decreased cholesterol synthesis and esterification were detected. We suggest that the in vitro antioxidative ability of lovastatin resulted, in part at least, from its activating effect on glutathione peroxidase, its stimulative effect on the ability of endothelial cell to scavenge H(2)O(2), and its hypolipidemic effect.     

Circulating Matrix Metalloproteinase-2 and -9 Enzyme Activities in the Children with Ventricular Septal Defect

Ventricular septal defect (VSD) is the most common form of congenital heart diseases. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in causal cardiac tissue remodeling. We studied the changes of circulating MMP-2 and MMP-9 activities in the patients with VSD severity and closure. There were 96 children with perimembranous VSD enrolled in this study. We assigned the patients into three groups according to the ratio of VSD diameter/diameter of aortic root (Ao). They were classified as below: Trivial (VSD/Ao ratio ≤ 0.2), Small (0.2 < VSD/Ao ≤ 0.3) and Median (0.3 < VSD/Ao) group. Plasma MMP-2 and MMP-9 activities were assayed by gelatin zymography.There was a significant higher MMP-2 activity in the VSD (Trivial, Small and Median) groups compared with that in Control group. The plasma MMP-9 activity showed a similar trend as the findings in MMP-2 activity. After one year follow-up, a significant difference in the MMP-9 activity was found between VSD spontaneous closure and non-closure groups. In conclusion, a positive trend between the severity of VSD and activities of MMP-2 and MMP-9 was found. Our data imply that MMP-2 and MMP-9 activities may play a role in the pathogenesis of VSD.     

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.     

阿托伐他汀钙片治疗脑梗死的疗效及其对MMP-2表达的影响

摘要 目的：探讨阿托伐他汀钙片治疗脑梗死的疗效及其对基质金属蛋白酶（MMP-2）表达的影响。方法：选择2019年5月-2021年2月在本院诊治的脑梗死患者64例,根据随机信封抽签原则将患者分为阿托伐他汀组32例与对照组32例。对照组给予常规双抗治疗,阿托伐他汀组以对照组为基础给予阿托伐他汀治疗,两组都治疗观察3个月,记录MMP-2表达的变化。结果：治疗后阿托伐他汀组的总有效率为93.8 %,高于对照组的68.8 %（P<0.05）。两组治疗后的低密度脂蛋白胆固醇（LDL-C）、甘油三酯（TG）、总胆固醇（TC）水平均低于治疗前（P<0.05）,高密度脂蛋白胆固醇（HDL-C）水平高于治疗前（P<0.05）,组间对比无差异（P>0.05）。两组治疗后的大脑中动脉平均血流速度（Vm）明显高于治疗前,搏动指数（PI）明显低于治疗前,阿托伐他汀组与对照组对比差异明显（P<0.05）。两组治疗后的血清MMP-2含量低于治疗前,阿托伐他汀组低于对照组（P<0.05）。两组治疗期间的不良反应主要为恶心呕吐、低血压、静脉血栓、头晕脑胀,组内对比无差异（P>0.05）。结论：阿托伐他汀治疗脑梗死能降低MMP-2水平,可在平衡血脂水平的基础上提高患者的治疗效果,还可提高大脑中动脉的血流速度,且为增加不良反应。     

The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.     

Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.     

Characterization of porcine arterial endothelial cells cultured on amniotic membrane, a potential matrix for vascular tissue engineering

The existing of basement membrane improves the development of endothelium while constructing blood vessel equivalent. The amniotic membrane (AM) provides a natural basement membrane and has been used in ocular surface reconstruction. This study evaluated the molecular and cellular characteristics of porcine vascular endothelial cells (ECs) cultured on AM. ECs cultured on AM expressed the endothelial marker vWF and exhibited normal endothelial morphology. Here, we demonstrated that AM enhanced the expression of intercellular molecules, platelet-endothelial cell adhesion molecule-1 (PECAM-1), and adhesion molecule VE-cadherin at the intercellular junctions. The expression level of integrin was markedly higher in ECs cultured on AM than on plastic dish. Furthermore, the AM downregulated the expression of E-selectin and P-selectin in both LPS-activated and non-activated ECs. Consistently, adhesion of leukocytes to both activated and non-activated cells was decreased in ECs cultured on AM. Our results suggest that AM is an ideal matrix to develop a functional endothelium in blood vessel equivalent construction.