Effect of Metalloprotease Inhibition on Age-Associated Arterial Remodeling

金属蛋白酶抑制对年龄相关动脉重塑的影响

• 批准号: 10259324
• 负责人: Edward Lakatta
• 金额: $ 4.71万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10259324
• 关键词: Adenoviruses; Age; Age-Months; Aging; Amyloidosis; Angiotensins; Anti-Inflammatory Agents; Atherosclerosis; Attenuated; Biological Aging; Blood Pressure; CCL2 gene; Caloric Restriction; Cardiovascular Diseases; Cardiovascular Physiology; Cells; Cellularity; Chronic; Cleaved cell; Collagen; Collagen Type I; Complementary DNA; Cytoskeleton; Deposition; EDN1 gene; EGF gene; Elastic Fiber; Elastin; Elastin Fiber; Exhibits; Extracellular Matrix; Fibrosis; Gelatinase A; Gelatinases; Genetic Transcription; Genotype; Harvest; Health; Hypertension; Implant; In Situ; In Vitro; Incidence; Inflammatory; Infusion procedures; Inhibition of Matrix Metalloproteinases Pathway; Interstitial Collagenase; Knock-out; Length; Link; Longevity; Losartan; MMP2 gene; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Medial; Mediating; Metalloproteases; Molecular; Morbidity - disease rate; Mus; NF-kappa B; Phenotype; Phosphorylation; Platelet-Derived Growth Factor; Play; Production; Proteins; Rattus; Role; Saline; Signal Transduction; Signaling Molecule; Smooth Muscle Myocytes; Stains; Stroke; Structure; TGFB1 gene; TNF gene; Thick; Tissues; Translations; Vascular Smooth Muscle; arterial remodeling; arterial stiffness; calcification; density; healthspan; improved; in vivo; intimal medial thickening; mRNA Expression; medin; milk fat globule; mortality; novel strategies; osmotic minipump; overexpression; p65; platelet-derived growth factor BB; preservation; pressure; prevent; proendothelin 1; protein expression; receptor; response; senescence; transcription factor; young adult

Age-associated arterial structural and functional remodeling includes intimal vascular smooth muscle cell (VSMCS) cellularity ( invasion and proliferation), VSMCs senescence, collagen deposition, elastin fragmentation, amyloidosis, calcification as well as an increase in arterial pressure and stiffening. This adverse arterial cellular and matrix remodeling is linked to an increase in proinflammatory signaling molecules, including angiotensin II (Ang II), milk fat globule EGF-8 (MFG-E8) and its fragment medin, transforming growth factor-beta1 (TGF-1), monocyte chemoattractant protein 1 (MCP-1), and proendothelin 1 (pro-ET1), the activation of extracellular MMPs via the transcriptional factor, ETS-1, as well as a decreases in the anti-inflammatory molecule vasorin. We hypothesized that inhibiting MMP activation can decelerate adverse age-associated arterial remodeling leading to a decrease in arterial pressure. Indeed, chronic administration (8 months) of the broad-spectrum MMP inhibitor, PD166793, via a daily gavage, to 16-month-old FXBN rats markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by the following: (1) Age-associated decreases in aortic gelatinase and interstitial collagenase activity in situ; (2) The age-associated decrease aortic vasorin in situ was diminished; (3) the elastic fiber network integrity was preserved; (4) Collagen deposition was reduced; (5) MCP-1 and TGF- 1 activity levels were reduced; (6) Phosphorylation of the profibrogenic signaling molecule SMAD-2/3 was reduced; (7) Blockade of pro-ET1 activation; and (8) The expression of ETS-1 was downregulated. In addition, in vitro study shows that treating cultured VSMCs with pro-ET1 increased both the transcription and translation levels of ETS-1, and these effects were markedly reduced with the MMP inhibitor, PD166793. Furthermore, infecting VSMCs with an adenovirus harboring the full-length ETS-1 cDNA, increased the levels of activated forms of both TGF-1 and MCP-1 proteins. Collectively, our results indicate that MMP inhibition impedes age-associated arterial proinflammatory signaling and is accompanied by the preservation of the intact elastin fiber network, a reduction in collagen, and a blunting of age-associated increases in blood pressure. The glycosylated protein vasorin physically interacts with TGF-1 and functionally attenuates its fibrogenic signaling in VSMCs of the arterial wall. Ang II amplifies TGF-1 activation in aging VSMCs of the arterial wall via a decrease in vasorin signaling mediated by MMP2 cleavage. Vasorin mRNA and protein expression are significantly decreased both in the aortic wall and in VSMCs from old (30 mo) vs. young (8 mo) FXBN rats. Exposing young VSMCs to Ang II reduces vasorin protein expression to the levels of old untreated cells. Conversely treating old VSMCs with the Ang II type AT1 receptor antagonist, Losartan, upregulates vasorin protein expression up to the levels of young. The physical interactions between vasorin and TGF-1 are significantly decreased in old vs. young VSMCs. Further, treating young VSMCs with Ang II increases the levels of MMP-2 activation and TGF-1 downstream molecules p-SMAD-2/3 and collagen type I production up to the levels of old untreated VSMCs, and these effects are substantially inhibited by overexpressing vasorin. Treating young rats (8 mo) with Ang II for 28 days via an osmotic minipump markedly reduced the expression of vasorin and improved arterial health. Importantly, the vasorin protein is effectively cleaved by activated MMP-2 both in vitro and in vivo. Administration of the MMP inhibitor, PD 166793, for 6 months, to young adult rats (18 mo) via a daily gavage markedly increased the levels of vasorin in the aortic wall. Thus, reduced vasorin amplifies Ang II profibrotic signaling via the activation of MMP-2 in VSMCs within the aging arterial wall. Aging exponentially increases the incidence of morbidity and mortality of quintessential inflammatory cardiovascular disease such as hypertension, atherosclerosis, and stroke mainly due to arterial proinflammatory shifts at the molecular, cellular, and tissue levels within the arterial wall. Calorie restriction (CR) in rats improves cardiovascular function and extends both healthspan and lifespan. How CR effects the proinflammatory landscape of molecular, cellular, and tissue proinflammatory phenotypic shifts within the arterial wall in rats are examined in the current study. Aortae were harvested from young (6-month-old) and old (24-month-old) Fisher 344 rats, fed ad libitum (AL) and a second group maintained on a 40% CR beginning at one month of age. Histopathologic and morphometric analysis of the arterial wall demonstrated that CR markedly reduced age-associated intimal medial thickening, collagen deposition, and increased elastin fraction within the arterial walls. Aortic wall immunostaining/blotting showed that CR effectively prevented an age-associated increase in the density of platelet derived growth factor (PDGF-BB), MMP2 activity, TGF-1 and its downstream signaling molecule, p-SMAD-2/3. In early passage cultured VSMCs isolated from both AL and CR rat aortae, CR alleviated the age-associated VSMC phenotypic shifts, pro-fibrogenic signaling, and proliferation in response to PDGF-BB. Collectively, CR reduces matrix and cellular proinflammation associated with aging that occurs within the aortic wall and is attributable to PDGF signaling. Thus, CR reduces PDGF-associated MMP activation signaling cascade and contributes to the postponement of biological aging, preserving a more youthful aortic wall phenotype. Recently, we found that both proinflammatory molecules Ang II and MFG-E8 increase with age or hypertension associated arterial remodeling. MFG-E8 is a downstream molecule of Ang II signaling and effectively activates MMP2 in cells VSMC in vitro and in vivo. MFG-E8 plays a crucial role in Ang II-induced blood pressure, arterial proinflammation and structural remodeling. We identified that MFG-E8 knock out (MFG-E8-/-) and wide-type (WT) mice chronically infused with Ang II or a saline control via an osmotic mini-pump for four weeks showed an significant increase in systolic blood pressure in both WT and MFG-E8-/- mice infused with Ang II vs saline after day 7 post-implant onward. Histochemical staining and immunostaining demonstrated that the intimal-medial thickness, VSMC proliferation, elastin lamina breaks, and collagen type I deposit were significantly increased within the aortae harvested from WT vs. age-matched MFG-E8-/- mice despite the change in systolic blood pressure seen similarly in both genotypes . Furthermore, chronic Ang II infusion significantly enhanced the expression of inflammatory molecule MFG-E8, MMP2 protein and up-regulated the activation of TGF-1 and its downstream signaling molecule p-Smad2 in the aortic walls of WT vs MFG-E8-/- mice. Notably, Ang II treated WT mice exhibited increased expression of proinflammatory leading molecules such as nuclear factor-kappa beta p65 (Nf-kB p65), MCP-1 and tumor necrosis factor-alpha 1 (TNF-a1) in aortic walls. However, these effects were substantially diminished in MFG-E8-/- mice. Taken together, the current findings demonstrate that MFG-E8 modulates Ang II-induced arterial cellular and matrix remodeling via the reduction of proinflammation. Thus, targeting the MFG-E8 molecule is a novel approach to retard arterial aging.

与年龄相关的动脉结构和功能重塑包括内膜血管平滑肌细胞（VSMCS）细胞结构（侵袭和增殖）、VSMC衰老、胶原沉积、弹性蛋白破碎、淀粉样变性、钙化以及动脉压力增加和硬化。这种不利的动脉细胞和基质重塑与促炎信号分子的增加有关，包括血管紧张素 II (Ang II)、乳脂球 EGF-8 (MFG-E8) 及其片段 medin、转化生长因子-β1 (TGF-1)、单核细胞趋化蛋白 1 (MCP-1) 和内皮素原 1 (pro-ET1)， 通过转录因子 ETS-1 激活细胞外 MMP，以及抗炎分子 vasorin 的减少。 我们假设抑制 MMP 激活可以减缓与年龄相关的不良动脉重塑，从而导致动脉压下降。事实上，通过每日强饲法对 16 个月大的 FXBN 大鼠长期（8 个月）施用广谱 MMP 抑制剂 PD166793 显着减弱了预期的与年龄相关的动脉压升高。伴随着以下结果：（1）与年龄相关的主动脉明胶酶和间质胶原酶原位活性降低； (2)与年龄相关的主动脉血管素原位减少减弱； (3)保留了弹性纤维网络的完整性； (4)胶原沉积减少； (5) MCP-1和TGF-1活性水平降低； (6)促纤维化信号分子SMAD-2/3的磷酸化降低； (7) 阻断pro-ET1激活； (8)ETS-1表达下调。此外，体外研究表明，用 pro-ET1 处理培养的 VSMC 会增加 ETS-1 的转录和翻译水平，而使用 MMP 抑制剂 PD166793 会显着降低这些影响。此外，用含有全长 ETS-1 cDNA 的腺病毒感染 VSMC，可增加 TGF-1 和 MCP-1 蛋白激活形式的水平。总的来说，我们的结果表明，MMP 抑制会阻碍与年龄相关的动脉促炎信号传导，并伴随着完整弹性蛋白纤维网络的保存、胶原蛋白的减少以及与年龄相关的血压升高的减弱。 糖基化蛋白血管素与 TGF-1 发生物理相互作用，并在功能上减弱其在动脉壁 VSMC 中的纤维化信号传导。 Ang II 通过减少 MMP2 裂解介导的血管素信号传导，增强动脉壁老化 VSMC 中 TGF-1 的激活。与年轻（8 个月）FXBN 大鼠相比，老年（30 个月）大鼠的主动脉壁和 VSMC 中 Vasorin mRNA 和蛋白表达均显着降低。将年轻的 VSMC 暴露于 Ang II 可以将血管素蛋白表达降低至未处理的老细胞的水平。相反，用 Ang II 型 AT1 受体拮抗剂氯沙坦治疗老年 VSMC，可将血管素蛋白表达上调至年轻水平。与年轻 VSMC 相比，老年 VSMC 中血管素和 TGF-1 之间的物理相互作用显着减少。此外，用Ang II处理年轻的VSMC会增加MMP-2激活和TGF-1下游分子p-SMAD-2/3和I型胶原蛋白产生的水平，直至达到未处理的年老VSMC的水平，并且这些作用基本上被过度表达血管素所抑制。通过微型渗透泵用 Ang II 治疗幼年大鼠（8 个月）28 天，显着降低了血管素的表达并改善了动脉健康。重要的是，血管素蛋白在体外和体内均可被激活的 MMP-2 有效裂解。通过每日强饲法对年轻成年大鼠（18 个月）施用 MMP 抑制剂 PD 166793 6 个月，可显着增加主动脉壁中血管素的水平。因此，减少的血管素通过激活老化动脉壁内 VSMC 中的 MMP-2 来放大 Ang II 促纤维化信号传导。 衰老使高血压、动脉粥样硬化和中风等典型炎症性心血管疾病的发病率和死亡率成倍增加，这主要是由于动脉壁内分子、细胞和组织水平上的动脉促炎性变化所致。大鼠的热量限制（CR）可改善心血管功能并延长健康寿命和寿命。 本研究探讨了 CR 如何影响大鼠动脉壁内分子、细胞和组织促炎表型变化的促炎景观。从年轻（6 个月大）和年老（24 个月大）Fisher 344 大鼠身上采集主动脉，随意喂养（AL），第二组从 1 个月龄开始维持 40% CR。动脉壁的组织病理学和形态测量分析表明，CR 显着减少了与年龄相关的内膜内侧增厚、胶原沉积和动脉壁内弹性蛋白分数的增加。主动脉壁免疫染色/印迹表明，CR 有效阻止了与年龄相关的血小板衍生生长因子 (PDGF-BB)、MMP2 活性、TGF-1 及其下游信号分子 p-SMAD-2/3 密度的增加。 在从 AL 和 CR 大鼠主动脉分离的早期传代培养的 VSMC 中，CR 减轻了与年龄相关的 VSMC 表型变化、促纤维化信号传导和 PDGF-BB 反应的增殖。总的来说，CR 减少了与主动脉壁内发生的衰老相关的基质和细胞促炎症，该炎症可归因于 PDGF 信号传导。因此，CR 减少 PDGF 相关的 MMP 激活信号级联，有助于推迟生物衰老，保持更年轻的主动脉壁表型。 最近，我们发现促炎分子 Ang II 和 MFG-E8 随着年龄或高血压相关的动脉重塑而增加。 MFG-E8 是 Ang II 信号传导的下游分子，可在体外和体内有效激活细胞 VSMC 中的 MMP2。 MFG-E8 在 Ang II 诱导的血压、动脉促炎症和结构重塑中发挥着至关重要的作用。我们发现，通过微型渗透泵长期输注 Ang II 或生理盐水对照 4 周的 MFG-E8 敲除 (MFG-E8-/-) 和宽型 (WT) 小鼠显示，与输注 Ang II 的 WT 和 MFG-E8-/- 小鼠相比，植入后第 7 天之后，与生理盐水相比，WT 和 MFG-E8-/- 小鼠的收缩压显着升高。组织化学染色和免疫染色表明，与年龄匹配的 MFG-E8-/- 小鼠相比，尽管两种基因型的收缩压变化相似，但从 WT 收获的主动脉内内膜中层厚度、VSMC 增殖、弹性蛋白层断裂和 I 型胶原沉积显着增加。此外，慢性Ang II输注显着增强WT与MFG-E8-/-小鼠主动脉壁中炎症分子MFG-E8、MMP2蛋白的表达，并上调TGF-1及其下游信号分子p-Smad2的激活。值得注意的是，Ang II 治疗的 WT 小鼠主动脉壁中促炎先导分子的表达增加，例如核因子-kappa beta p65 (Nf-kB p65)、MCP-1 和肿瘤坏死因子-α 1 (TNF-a1)。然而，这些作用在 MFG-E8-/- 小鼠中显着减弱。综上所述，目前的研究结果表明，MFG-E8 通过减少促炎症来调节 Ang II 诱导的动脉细胞和基质重塑。因此，靶向 MFG-E8 分子是延缓动脉衰老的一种新方法。