Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway

Abstract Kallistatin, a plasma protein, protects against vascular and organ injury. This study is aimed to investigate the role and mechanism of kallistatin in endothelial senescence. Kallistatin inhibited H2O2‐induced senescence in human endothelial cells, as indicated by reduced senescence‐associated‐β‐galactosidase activity, p16INK 4a and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked H2O2‐induced superoxide formation, NADPH oxidase levels and VCAM‐1, ICAM‐1, IL‐6 and miR‐34a synthesis. Kallistatin reversed H2O2‐mediated inhibition of endothelial nitric oxide synthase (eNOS), SIRT1, catalase and superoxide dismutase (SOD)‐2 expression, and kallistatin alone stimulated the synthesis of these antioxidant enzymes. Moreover, kallistatin's anti‐senescence and anti‐oxidant effects were attributed to SIRT1‐mediated eNOS pathway. Kallistatin, via interaction with tyrosine kinase, up‐regulated Let‐7g, whereas Let‐7g inhibitor abolished kallistatin's effects on miR‐34a and SIRT1/eNOS synthesis, leading to inhibition of senescence, oxidative stress and inflammation. Furthermore, lung endothelial cells isolated from endothelium‐specific kallistatin knockout mice displayed marked reduction in mouse kallistatin levels. Kallistatin deficiency in mouse endothelial cells exacerbated senescence, oxidative stress and inflammation compared to wild‐type mouse endothelial cells, and H2O2 treatment further magnified these effects. Kallistatin deficiency caused marked reduction in Let‐7g, SIRT1, eNOS, catalase and SOD‐1 mRNA levels, and elevated miR‐34a synthesis in mouse endothelial cells. These findings indicate that endogenous kallistatin through novel mechanisms protects against endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐SIRT1‐eNOS pathway.

dysfunction marker plasminogen activator inhibitor-1 (PAI-1) expression. [6][7][8] Moreover, cellular senescence is characterized by shortened telomere length or reduced telomerase activity. 9 Oxidative stress and inflammation have been implicated in the ageing process and adversely affect endothelial availability and function. 10,11 Oxidative stress compromises telomere integrity and nitric oxide (NO) bioavailability to accelerate cellular senescence in endothelial cells. 12 Vascular senescence is also associated with enhanced expression of the inflammatory molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), which leads to augmented adhesion of monocytes into vascular wall, facilitating the ignition and progression of ageing-associated vascular diseases. 13,14 Therefore, inhibition of oxidative stress and inflammation is of great significance for controlling endothelial senescence.
MicroRNAs (miRNAs) are essential regulators for a plethora of cellular processes. 15 A highly evolutionarily conserved miRNA  has been reported to inhibit oxLDL-induced apoptosis of endothelial cells and alleviate atherosclerosis in mice. 16,17 Let-7g also has the ability to improve multiple endothelial functions by stimulating sirtuin 1 (SIRT1) signalling. 18 Furthermore, miR-34a, a well-recognized tumour suppressor and a senescent inducer, triggers endothelial senescence by directly suppressing the target gene SIRT1. 19 SIRT1, an NAD + -dependent deacetylase and anti-ageing molecule, is decreased in vascular tissue undergoing senescence. 20 Evidence has indicated a positive feedback regulation between SIRT1 and endothelial nitric oxide synthase (eNOS). SIRT1 promotes the expression of eNOS and activates eNOS by its deacetylase activity, while eNOS through NO production stimulates SIRT1 expression. [21][22][23] NO produced from eNOS has a wide range of biological properties to maintain vascular homeostasis, including vascular dilation, and inhibition of oxidative stress and inflammation. [24][25][26] Therefore, Let-7g, miR-34a and antioxidant genes SIRT1 and eNOS are key signalling molecules for regulating endothelial senescence.
Kallistatin was discovered in human plasma as a tissue kallikreinbinding protein and identified as a serine proteinase inhibitor. [27][28][29][30][31][32] Kallistatin plays a protective role in vascular and organ injury. 33 Kallistatin gene or protein delivery attenuates vascular and multi-organ injuries by its pleiotropic activities in vasodilation, and inhibition of oxidative stress, inflammation and apoptosis in animal models and cultured cells. [33][34][35][36][37][38][39][40] Kallistatin inhibits vascular inflammation and oxidative stress by stimulating eNOS expression and activation, and antagonizing tumour necrosis factor (TNF)-a-mediated inflammatory gene expression in endothelial cells. 38,40,41 Recently, we showed that kallistatin treatment alleviates endothelial progenitor cell (EPC) senescence, mouse aortic aging and prolonged Caenorhabditis elegans (C. elegans) lifespan by inhibiting miR-34a and increasing SIRT1 levels. 42 Moreover, plasma kallistatin levels were shown to be positively associated with leucocyte telomere length in young African Americans, implicating the involvement of kallistatin in anti-ageing process. 43 H 2 O 2 is widely used to achieve oxidative stress-induced premature senescence in endothelial cells and fibroblasts. 44,45 According to previous findings, the dose of H 2 O 2 (100 lmol/L) represents subcytotoxic conditions to induce senescence. 46,47 In this study, we investigated the role and mechanism of kallistatin in oxidative stress-induced endothelial senescence by 2 approaches: (1) exogenous kallistatin treatment in human umbilical vein endothelial cells (HUVECs), and (2) endogenous kallistatin deficiency in mouse lung endothelial cells isolated from endothelium-specific kallistatin-deficient (KS endoÀ/À ) mice.

| Purification and characterization of recombinant human kallistatin
Recombinant human kallistatin was expressed and secreted into serum-free medium of cultured HEK293T cells, and purified by ammonium sulphate precipitation followed by nickel-affinity chromatography as described previously. 30,48 The purity and identity of human kallistatin were verified by SDS-polyacrylamide gel electrophoresis and Western blot with a specific monoclonal antibody. 48 Similarly, cultured primary mouse lung endothelial cells at 80% confluency were treated with or without H 2 O 2 (100 lmol/L) for 3 days to evaluate senescent markers and oxidative stress indicators, and for 24 hours to measure gene expression.

| Telomerase activity assay
Human umbilical vein endothelial cells were preincubated with or without 1 lmol/L kallistatin for 30 minutes prior to the addition of H 2 O 2 (100 lmol/L) for 24 hours. Cells were lysed with non-denaturing lysis buffer. Telomerase activity was measured using TRAPEZE RT telomerase detection kit (EMD Millipore, Billerica, MA, USA).

| NADPH oxidase activity assay
The enzymatic activity of NADPH oxidase was assessed by a luminescence assay in the presence of lucigenin (250 lmol/L; Sigma) and NADPH (100 lmol/L; Sigma) as described. 50 Fluorescence intensity was continuously monitored for 15 minutes with a TD20/20 luminometer. Protein concentrations were measured by Pierce BCA protein assay kit (Thermo Fisher Scientific, Bremen, Germany). The chemiluminescent signal was normalized by protein concentration.

| Detection of superoxide formation
Cellular superoxide generation was detected using the fluorescent probe 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma) as described. 38 Treated HUVECs in six-well plates were incubated for

| Isolation and Identification of mouse lung endothelial cells
Mouse lung endothelial cells were isolated from 8-week-old KS endoÀ/À and WT mice as described. 51 Mice were euthanized by

| Kallistatin inhibits H 2 O 2 -induced oxidative stress and inflammation
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34a-mediated inhibition of SIRT1-eNOS pathway, and reduces endothelial senescence, oxidative stress and inflammation
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| Identification of kallistatin deficiency in mouse lung endothelial cells
We generated endothelium-specific kallistatin knockout (KS endoÀ/À ) mice by crossing KS fl/fl mice with Tie2Cre + KS fl/+ mice. Lung endothelial cells were isolated from WT mice and KS endoÀ/À mice.
Cultured primary mouse lung endothelial cells were identified by CD31 expression and immunostaining ( Figure 5A,B). In lysed mouse endothelial cell DNA samples, kallistatin null allele (del) was detected only in KS endoÀ/À mice, but not in WT mice ( Figure 5C), indicating that endogenous kallistatin is ablated in mouse endothelial cells.
Moreover, the protein and mRNA levels of mouse kallistatin were markedly diminished in endothelial cells from KS endoÀ/À mice, compared with endothelial cells from WT mice ( Figure 5D,E). These results confirm that mouse endogenous kallistatin is deficient in KS endoÀ/À mouse endothelial cells.

| DISCUSSION
This study demonstrates that endogenous kallistatin plays a protective role in endothelial senescence, and kallistatin through a novel mechanism inhibits cellular senescence, oxidative stress and inflammation.
Kallistatin antagonized oxidative stress-induced senescence, as was recently recognized as an anti-aging miRNA to improve endothelial functions. 18 Let-7g negatively regulated apoptosis, senescence and inflammation in endothelial cells. [16][17][18] Up-regulation of SIRT1 levels was involved in Let-7g -mediated inhibition of endothelial senescence. 18 In this study, we showed that kallistatin increased Let-7g synthesis, and kallistatin, via Let-7g induction, stimulated SIRT1 and eNOS, leading to inhibition of endothelial senescence, oxidative stress and inflammation in endothelial cells. Importantly, kallistatin harbours 2 structural elements, an active site and a heparin-binding domain. [30][31][32] Kallistatin's active site is essential for eNOS and SIRT1 expression through interacting with a tyrosine kinase in EPCs and endothelial cells. 42,58 Kallistatin via its active site stimulates tyrosine kinase-PKC-ERK signalling, leading to the synthesis of suppressor of cytokine signaling-3 (SOCS3), an anti-inflammatory gene in cultured macrophages. 61 It is likely that kallistatin via the active site interacts with tyrosine kinase to stimulate Let-7g synthesis. Moreover, miR-34a-SIRT1 axis has been regarded to be one of critical pathways to regulate endothelial senescence. 19 Our previous study showed that kallistatin reduced EPC senescence and aorta ageing by inhibiting the synthesis of miR-34a, a pro-senescent miRNA. 42 Consistently, the current study indicates that kallistatin treatment inhibited miR-34a and prevented H 2 O 2 -induced miR-34a synthesis in endothelial cells. Importantly, we found that Let-7g is the upstream regulator of miR-34a in response to kallistatin treatment.
These results indicate that kallistatin protects against oxidative stress-induced endothelial senescence by up-regulating Let-7g to inhibit miR-34a-SIRT1-eNOS pathway.
To further investigate the role of endogenous kallistatin in vascular senescence, we generated KS endoÀ/À mice by loxp/Cre technology, and isolated lung endothelial cells from KS endoÀ/À mice and WT Genistein, tyrosine kinase inhibitor; NAM, SIRT1 inhibitor; L-NAME, NOS inhibitor reduction in Let-7g, SIRT1, eNOS, catalase and SOD-1. Thus, Let-7gmediated SIRT1-eNOS pathway is suppressed when kallistatin is deficient. We previously showed that human kallistatin protein treatment reduced vascular senescence and ROS formation, but increased SIRT1 and eNOS levels in aortas of diabetic mice. 42 Moreover, kallistatin gene delivery decreased aortic superoxide levels and glomerular endothelial loss in hypertensive deoxycorticosterone acetate-salt rats. 38 Conversely, kallistatin depletion by neutralizing kallistatin antibody injection exacerbated renal and cardiovascular oxidative stress, inflammation and organ injury in hypertensive rats. 50 These combined findings support the notion that endogenous kallistatin acts as a protective molecule in vascular injury and senescence by stimulating Let-7g, SIRT1 and eNOS, and suppressing miR-34a synthesis.
Overall, this study indicates that kallistatin inhibits cellular senescence, oxidative stress and inflammation by promoting Let-7gmediated inhibition of miR-34a-SIRT1-eNOS pathway in human endothelial cells. Moreover, endogenous kallistatin plays a protective role in endothelial senescence, oxidative stress and inflammation, as confirmed by kallistatin-deficient mouse endothelial cells. Therefore, kallistatin could potentially be used as a promising new therapeutic strategy for vascular ageing and dysfunction in humans.