Folin Marcella, Baiguera Silvia, Tommasini Mara, Guidolin Diego, Conconi Maria Teresa, De Carlo Eugenio, Nussdorfer Gastone G, Parnigotto Pier Paolo
Department of Biology, University of Padua, Padua I-35121, Italy.
Int J Mol Med. 2005 Jun;15(6):929-35.
Several studies have shown that beta-amyloid (beta A) deposits are associated with damage of cerebral vessels and that in Alzheimer's disease (AD) beta A peptides are cytotoxic for cerebral endothelial cells (ECs). However, little is known about the mechanisms underlying these effects of beta A peptides. Hence, we have investigated the effects of beta A(1-40) and beta A(1-42) on rat neuromicrovascular ECs (NECs) cultured in vitro. NECs were isolated, plated (1.5x10(4) cells/cm2) on collagen/fibronectin-coated Petri dishes and cultured in EC growth medium MV2. After 24 h of culture, NECs were incubated with beta A(1-40) and beta A(1-42) (10(-9) or 10(-7) M) and cultured for another 3, 24 or 48 h. Cell viability was assayed by either trypan blue exclusion or by measuring redox activity (MTS assay). Cell proliferation was assessed by measuring the incorporation of 5'-bromo-2'-deoxyuridine into DNA, cell apoptosis by TUNEL assay and cell necrosis by evaluating the release of lactate dehydrogenase. The morphology of cultured NECs was examined by transmission electron microscopy. Other NECs were plated (2.5x10(4) cells/cm2) on Matrigel coated-wells and incubated for 18 h in the presence or absence of beta A peptides for in vitro angiogenesis assay. Beta A peptides significantly decreased NEC viability and enhanced cell apoptosis and necrosis rates. NEC proliferation was not significantly affected. The effects were seen after an incubation period of 3 h (and also 24 h in the case of the redox activity) but not 48 h and beta A(1-42) was much more effective in its toxic effects than beta A(1-40). NECs incubated for 24 h with beta A peptides displayed ultrastructural signs of cell degeneration. beta A peptides prevented NECs cultured on Matrigel to form a capillary-like network and image analysis showed that the downloading of dimensional and topological parameters of the meshwork was significant only in the case of the incubation with beta A(1-42). Collectively our findings allow us to conclude that i) beta A peptides exert a marked toxic effect on cultured NECs, which conceivably reduces their in vitro angiogenic activity; ii) beta A(1-42) is the more toxic form, which could suggest its main role in the pathogenesis of cerebral vessel lesions in AD and iii) the maximum toxic action occurs after a short incubation period, which could be explained by assuming that beta A peptides are unable to accumulate in NECs due to their rapid degradation, thereby allowing NECs to fully recover within 48 h.
多项研究表明，β-淀粉样蛋白（βA）沉积物与脑血管损伤相关，且在阿尔茨海默病（AD）中，βA肽对脑内皮细胞（ECs）具有细胞毒性。然而，关于βA肽这些作用的潜在机制知之甚少。因此，我们研究了βA（1-40）和βA（1-42）对体外培养的大鼠神经微血管内皮细胞（NECs）的影响。分离NECs，接种（1.5×10⁴个细胞/cm²）于胶原/纤连蛋白包被的培养皿中，并在EC生长培养基MV2中培养。培养24小时后，将NECs与βA（1-40）和βA（1-42）（10⁻⁹或10⁻⁷M）孵育，并再培养3、24或48小时。通过台盼蓝排斥法或测量氧化还原活性（MTS法）检测细胞活力。通过测量5'-溴-2'-脱氧尿苷掺入DNA来评估细胞增殖，通过TUNEL法检测细胞凋亡，通过评估乳酸脱氢酶的释放检测细胞坏死。通过透射电子显微镜检查培养的NECs的形态。将其他NECs接种（2.5×10⁴个细胞/cm²）于基质胶包被的孔中，在有或无βA肽的情况下孵育18小时以进行体外血管生成测定。βA肽显著降低NEC活力，提高细胞凋亡和坏死率。NEC增殖未受到显著影响。在孵育3小时后观察到这些效应（对于氧化还原活性，24小时时也观察到），但48小时时未观察到，且βA（1-42）在其毒性作用方面比βA（1-40）更有效。用βA肽孵育24小时的NECs显示出细胞变性的超微结构迹象。βA肽阻止在基质胶上培养的NECs形成毛细血管样网络，图像分析表明，仅在与βA（1-42）孵育的情况下，网络的尺寸和拓扑参数的下载才有显著变化。总体而言，我们的研究结果使我们能够得出以下结论：i）βA肽对培养的NECs具有显著的毒性作用，这可能会降低其体外血管生成活性；ii）βA（1-42）是毒性更强的形式，这可能表明其在AD脑血管理病变发病机制中的主要作用；iii）最大毒性作用在短时间孵育后出现，这可以通过假设βA肽由于快速降解而无法在NECs中积累来解释，从而使NECs能够在48小时内完全恢复。
