Folic Acid Mitigates Angiotensin-II-Induced Blood Pressure and Renal Remodeling

Clinical data suggests an association between systolic hypertension, renal function and hyperhomocysteinemia (HHcy). HHcy is a state of elevated plasma homocysteine (Hcy) levels and is known to cause vascular complications. In this study, we tested the hypothesis whether Ang II-induced hypertension increases plasma Hcy levels and contributes to renovascular remodeling. We also tested whether folic acid (FA) treatment reduces plasma Hcy levels by enhancing Hcy remethylation and thus mitigating renal remodeling. Hypertension was induced in WT mice by infusing Ang II using Alzet mini osmotic pumps. Blood pressure, Hcy level, renal vascular density, oxidative stress, inflammation and fibrosis markers, and angiogenic- and anti-angiogenic factors were measured. Ang II hypertension increased plasma Hcy levels and reduced renal cortical blood flow and microvascular density. Elevated Hcy in Ang II hypertension was associated with decreased 4, 5-Diaminofluorescein (DAF-2DA) staining suggesting impaired endothelial function. Increased expression of Nox-2, -4 and dihydroethidium stain revealed oxidative stress. Excess collagen IV deposition in the peri-glomerular area and increased MMP-2, and -9 expression and activity indicated renal remodeling. The mRNA and protein expression of asymmetric dimethylarginine (ADMA) was increased and eNOS protein was decreased suggesting the involvement of this pathway in Hcy mediated hypertension. Decreased expressions of VEGF and increased anti-angiogenic factors, angiostatin and endostatin indicated impaired vasculogenesis. FA treatment partially reduced hypertension by mitigating HHcy in Ang II-treated animals and alleviated pro-inflammatory, pro-fibrotic and anti-angiogenic factors. These results suggest that renovascular remodeling in Ang II-induced hypertension is, in part, due to HHcy.     

Secreted frizzled‐related protein 5 protects against oxidative stress‐induced apoptosis in human aortic endothelial cells via downregulation of Bax

This study was undertaken to determine the role of secreted frizzled‐related protein 5 (SFRP5) in endothelial oxidative injury. Human aortic endothelial cells (HAECs) were exposed to different oxidative stimuli and examined for SFRP5 expression. The effects of SFRP5 overexpression and knockdown on cell viability, apoptosis, and reactive oxygen species production were measured. HAECs treated with angiotensin (Ang) II (1 μM) or oxidized low‐density lipoprotein (oxLDL) (150 μg/mL) showed a significant increase in SFRP5 expression. Overexpression of SFRP5 significantly attenuated the viability suppression and apoptosis induction by Ang II and oxLDL, whereas the knockdown of SFRP5 exerted opposite effects. Overexpression of SFRP5 prevented ROS formation and β‐catenin activation and reduced Bax expression. Co‐expression of Bax significantly reversed the anti‐apoptotic effect of SFRP5 overexpression, whereas knockdown of Bax restrained Ang II‐ and oxLDL‐induced apoptosis in HAECs. Taken together, SFRP5 confers protection against oxidative stress‐induced apoptosis through inhibition of β‐catenin activation and downregulation of Bax.     

Cystathionine- -synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-β-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway. extracellular matrix; matrix metalloproteinase-9; intercellular cell adhesion molecule-1; vascular cell adhesion molecule-1; collagen type-1; hyperhomocysteinemia     

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

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, p16^INK4a 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.     

Cystathionine-beta-synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-beta-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway.     

SIRT1 reduction causes renal and retinal injury in diabetes through endothelin 1 and transforming growth factor β1

In diabetes, hyperglycaemia causes up‐regulation of endothelin 1 (ET‐1) and transforming growth factor beta 1 (TGF‐β1). Previously we showed glucose reduces sirtuin1 (SIRT1), a class III histone deacetylase. Here, we investigated the regulatory role of SIRT1 on ET‐1 and TGF‐β1 expression. Human microvascular endothelial cells were examined following incubation with 25 mmol/l glucose (HG) and 5 mmol/l glucose (NG) with or without SIRT1 or histone acetylase p300 overexpression or knockdown. mRNA expressions of ET‐1, TGF‐β1, SIRT1, p300 and collagen 1α(I) were examined. SIRT1 enzyme activity, ET‐1 and TGF‐β1 protein levels were measured. Histone acetylation and endothelial permeability were further investigated. Similar analyses were performed in the kidneys and retinas of SIRT1 overexpressing transgenic mice with or without streptozotocin induced diabetes. Renal functions were evaluated. In the endothelial cells (ECs), HG caused increased permeability and escalated production of ET‐1, TGF‐β1, collagen Iα(I). These cells also showed increased p300 expression, histone acetylation and reduced SIRT1 levels. These changes were rectified in the ECs following p300 silencing or by SIRT1 overexpression, whereas SIRT1 knockdown or p300 overexpression in NG mimicked the effects of HG. High ET‐1 and TGF‐β1 levels were seen in the kidneys and retinas of diabetic mice along with micro‐albuminuria and increased fibronectin protein (marker of glucose‐induced cell injury) levels. Interestingly, these detrimental changes were blunted in SIRT1 overexpressing transgenic mice with diabetes. This study showed a novel SIRT1 mediated protection against renal and retinal injury in diabetes, regulated through p300, ET‐1 and TGF‐β1.     

Inhibition of P53/miR‐34a improves diabetic endothelial dysfunction via activation of SIRT1

Endothelial dysfunction contributes to diabetic macrovascular complications, resulting in high mortality. Recent findings demonstrate a pathogenic role of P53 in endothelial dysfunction, encouraging the investigation of the effect of P53 inhibition on diabetic endothelial dysfunction. Thus, high glucose (HG)‐treated endothelial cells (ECs) were subjected to pifithrin‐α (PFT‐α)—a specific inhibitor of P53, or P53‐small interfering RNA (siRNA), both of which attenuated the HG‐induced endothelial inflammation and oxidative stress. Moreover, inhibition of P53 by PFT‐α or P53‐siRNA prohibited P53 acetylation, decreased microRNA‐34a (miR‐34a) level, leading to a dramatic increase in sirtuin 1 (SIRT1) protein level. Interestingly, the miR‐34a inhibitor (miR‐34a‐I) and PFT‐α increased SIRT1 protein level and alleviated the HG‐induced endothelial inflammation and oxidative stress to a similar extent; however, these effects of PFT‐α were completely abrogated by the miR‐34a mimic. In addition, SIRT1 inhibition by EX‐527 or Sirt1‐siRNA completely abolished miR‐34a‐I's protection against HG‐induced endothelial inflammation and oxidative stress. Furthermore, in the aortas of streptozotocin‐induced diabetic mice, both PFT‐α and miR‐34a‐I rescued the inflammation, oxidative stress and endothelial dysfunction caused by hyperglycaemia. Hence, the present study has uncovered a P53/miR‐34a/SIRT1 pathway that leads to endothelial dysfunction, suggesting that P53/miR‐34a inhibition could be a viable strategy in the management of diabetic macrovascular diseases.     

HIP/PAP protects against bleomycin‐induced lung injury and inflammation and subsequent fibrosis in mice

Hepatocarcinoma‐intestine‐pancreas/pancreatitis‐associated protein (HIP/PAP), a C‐type lectin, exerts anti‐oxidative, anti‐inflammatory, bactericidal, anti‐apoptotic, and mitogenic functions in several cell types and tissues. In this study, we explored the role of HIP/PAP in pulmonary fibrosis (PF). Expression of HIP/PAP and its murine counterpart, Reg3B, was markedly increased in fibrotic human and mouse lung tissues. Adenovirus‐mediated HIP/PAP expression markedly alleviated bleomycin (BLM)‐induced lung injury, inflammation, and fibrosis in mice. Adenovirus‐mediated HIP/PAP expression alleviated oxidative injury and lessened the decrease in pulmonary superoxide dismutase (SOD) activity in BLM‐treated mice, increased pulmonary SOD expression in normal mice, and HIP/PAP upregulated SOD expression in cultured human alveolar epithelial cells (A549) and human lung fibroblasts (HLF‐1). Moreover, in vitro experiments showed that HIP/PAP suppressed the growth of HLF‐1 and ameliorated the H₂O₂‐induced apoptosis of human alveolar epithelial cells (A549 and HPAEpiC) and human pulmonary microvascular endothelial cells (HPMVEC). In HLF‐1, A549, HPAEpiC, and HPMVEC cells, HIP/PAP did not affect the basal levels, but alleviated the TGF‐β1‐induced down‐regulation of the epithelial/endothelial markers E‐cadherin and vE‐cadherin and the over‐expression of mesenchymal markers, such as α‐SMA and vimentin. In conclusion, HIP/PAP was found to serve as a potent protective factor in lung injury, inflammation, and fibrosis by attenuating oxidative injury, promoting the regeneration of alveolar epithelial cells, and antagonizing the pro‐fibrotic actions of the TGF‐β1/Smad signaling pathway.     

Homocysteine altered ROS generation and NO accumulation in endothelial cells

Mild hyperhomocysteinemia (HHcy) is a risk factor for vascular disease and is closely associated with endothelial dysfunction. Oxidative stress and decreased nitric oxide (NO) bioavailability were reported in HHcy-induced vascular injury; however, the exact relationship is not understood. We thus directly determine the production of reactive oxygen species (ROS) and NO in cultured endothelial cells (HUVECs) to demonstrate the correlated variation between ROS and NO induced by Hcy (homocysteine), Cys (cysteine), another thiol compound, and Met (methionine), precursor of HHcy in animal study. HUVECs were treated with Hcy, Cys, or Met for 0.5 or 22-24 h; ROS generation was detected by DCF fluorescence with flow cytometry and NO by chemiluminescence. In non-cytotoxic (<1.0 mM) concentration ranges, Met exerted no effects on either ROS production or NO concentration, Cys decreased ROS production and increased NO in both short-term (0.5 h) and long-term (22-24 h) treatments; Hcy, however, induced a biphasic effect on ROS production, i.e., inhibitory at 0.5 h but stimulatory at 24 h. The maximal stimulation by Hcy (0.25 mM) was significantly reduced by co-incubation (12 h) with estrogen (1 microM). Hcy caused an early (0.5 h) increase of medium NO which was absent in long-term Hcy treatment. The oxidative stress caused by long-term Hcy incubation could be ameliorated by estrogen, consistent with earlier in vivo observations that estrogen prevents HHcy-induced injury.     

Celastrol exerts anti‐inflammatory effect in liver fibrosis via activation of AMPK‐SIRT3 signalling

Celastrol, a pentacyclic tritepene extracted from Tripterygium Wilfordi plant, showing potent liver protection effects on several liver‐related diseases. However, the anti‐inflammatory potential of celastrol in liver fibrosis and the detailed mechanisms remain uncovered. This study was to investigate the anti‐inflammatory effect of celastrol in liver fibrosis and to further reveal mechanisms of celastrol‐induced anti‐inflammatory effects with a focus on AMPK‐SIRT3 signalling. Celastrol showed potent ameliorative effects on liver fibrosis both in activated hepatic stellate cells (HSCs) and in fibrotic liver. Celastrol remarkably suppressed inflammation in vivo and inhibited the secretion of inflammatory factors in vitro. Interestingly, celastrol increased SIRT3 promoter activity and SIRT3 expression both in fibrotic liver and in activated HSCs. Furthermore, SIRT3 silencing evidently ameliorated the anti‐inflammatory potential of celastrol. Besides, we found that celastrol could increase the AMPK phosphorylation. Further investigation showed that SIRT3 siRNA decreased SIRT3 expression but had no obvious effect on phosphorylation of AMPK. In addition, inhibition of AMPK by employing compound C (an AMPK inhibitor) or AMPK1α siRNA significantly suppressed SIRT3 expression, suggesting that AMPK was an up‐stream protein of SIRT3 in liver fibrosis. We further found that depletion of AMPK significantly attenuated the inhibitory effect of celastrol on inflammation. Collectively, celastrol attenuated liver fibrosis mainly through inhibition of inflammation by activating AMPK‐SIRT3 signalling, which makes celastrol be a potential candidate compound in treating or protecting against liver fibrosis.     

SIRT1: A novel target to prevent atherosclerosis

Atherosclerosis is a chronic immuno‐inflammatory disease associated with blood lipids disorder. Many studies have demonstrated that caloric restriction (CR) can prevent atherosclerosis and extend lifespan. Sir2 protein, mammal's SIRT1, has been reported to at least partly contribute to the protective effect of CR. Hence, we hypothesize that SIRT1 is a key regulator in the pathogenesis of atherosclerosis and that upregulation of SIRT1 in endothelial cells may mimic CR's beneficial effect on vascular health. The recent studies have demonstrated that endothelial SIRT1 is an anti‐atherosclerosis factor and the possible mechanism may be related to inhibit oxidized low‐density lipoprotein (oxLDL)‐induced apoptosis, upregulate endothelial nitric oxide synthase (eNOS) expression, and improve endothelium relaxation function. We infer that SIRT1 may be a novel target for atherosclerosis prevention and treatment. J. Cell. Biochem. 108: 10–13, 2009. © 2009 Wiley‐Liss, Inc.     

Increased endogenous H2S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia

Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.     

Matrix metalloproteinase-9 in homocysteine-induced intestinal microvascular endothelial paracellular and transcellular permeability

Although elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with inflammatory bowel disease (IBD), the mechanism of Hcy action is unclear. In the present study, we tested the hypothesis that HHcy activates matrix metalloproteinase-9 (MMP-9), which in turn enhances permeability of human intestinal microvascular endothelial cell (HIMEC) layer by decreasing expression of endothelial junction proteins and increasing caveolae formation. HIMECs were grown in Transwells and treated with 500 μM Hcy in the presence or absence of MMP-9 activity inhibitor. Hcy-induced permeability to FITC-conjugated bovine serum albumin (FITC-BSA) was assessed by measuring fluorescence intensity of solutes in the Transwells' lower chambers. The cell-cell interaction and cell barrier function was estimated by measuring trans-endothelial electrical impedance. Confocal microscopy and flow cytometry were used to study cell junction protein expressions. Hcy-induced changes in transcellular transport of HIMECs were estimated by observing formation of functional caveolae defined as caveolae labeled by cholera toxin and antibody against caveolin-1 and one that have taken up FITC-BSA. Hcy instigated HIMEC monolayer permeability through activation of MMP-9. The increased paracellular permeability was associated with degradation of vascular endothelial cadherin and zona occludin-1 and transcellular permeability through increased caveolae formation in HIMECs. Elevation of Hcy content increases permeability of HIMEC layer affecting both paracellular and transcellular transport pathways, and this increased permeability was alleviated by inhibition of MMP-9 activity. These findings contribute to clarification of mechanisms of IBD development.     

CTRP3 protects against uric acid-induced endothelial injury by inhibiting inflammation and oxidase stress in rats

Hyperuricemia, which contributes to vascular endothelial damage, plays a key role in multiple cardiovascular diseases. This study was designed to investigate whether C1q/tumor necrosis factor (TNF)-related protein 3 (CTRP3) has a protective effect on endothelial damage induced by uric acid and its underlying mechanisms. Animal models of hyperuricemia were established in Sprague-Dawley (SD) rats through the consumption of 10% fructose water for 12 weeks. Then, the rats were given a single injection of Ad-CTRP3 or Ad-GFP. The animal experiments were ended two weeks later. In vitro, human umbilical vein endothelial cells (HUVECs) were first infected with Ad-CTRP3 or Ad-GFP. Then, the cells were stimulated with 10 mg/dL uric acid for 48 h after pretreatment with or without a Toll-like receptor 4 (TLR4)-specific inhibitor. Hyperuricemic rats showed disorganized intimal structures, increased endothelial apoptosis rates, increased inflammatory responses and oxidative stress, which were accompanied by reduced CTRP3 and elevated TLR4 protein levels in the thoracic aorta. In contrast, CTRP3 overexpression decreased TLR4 protein levels and ameliorated inflammatory responses and oxidative stress, thereby improving the morphology and apoptosis of the aortic endothelium in rats with hyperuricemia. Similarly, CTRP3 overexpression decreased TLR4-mediated inflammation, reduced oxidative stress, and rescued endothelial damage induced by uric acid in HUVECs. In conclusion, CTRP3 ameliorates uric acid-induced inflammation and oxidative stress, which in turn protects against endothelial injury, possibly by inhibiting TLR4-mediated inflammation and downregulating oxidative stress.     

Kallistatin reduces vascular senescence and aging by regulating microRNA‐34a‐SIRT1 pathway

Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)‐induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF‐α‐induced cellular senescence in EPCs, as indicated by reduced senescence‐associated β‐galactosidase activity and plasminogen activator inhibitor‐1 expression, and elevated telomerase activity. Kallistatin blocked TNF‐α‐induced superoxide levels, NADPH oxidase activity, and microRNA‐21 (miR‐21) and p16^INK4a synthesis. Kallistatin prevented TNF‐α‐mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR‐34a synthesis, whereas miR‐34a overexpression abolished kallistatin‐induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR‐34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ‐induced aortic senescence, oxidative stress, and miR‐34a and miR‐21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild‐type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR‐34 or sir‐2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR‐34, but stimulated sir‐2.1 and sod‐3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR‐34a‐SIRT1 pathway.     

Activation of ITLN-1 attenuates oxidative stress injury via activating SIRT1/PGC1-α signaling in neuroblastoma cells

Cerebral injury is closely associated with enhanced oxidative stress. A newly discovered secretory adipocytokine, intelectin-1 (ITLN-1), has been shown to have beneficial effects in neuroprotection in epidemiological studies. However, the specific molecular mechanism of ITLN-1 in protecting against cerebral oxidative stress needs further investigation. In this study, we hypothesize that ITLN-1 plays a protective role against oxidative stress injury through the SIRT1/PGC1-α signaling pathway in neuromatocytes. We used hydrogen peroxide (H₂O₂) as a oxidative stress model to simulate oxidative stress injury. Then, small interfering RNAs (siRNAs) was used to knock down SIRT1 in N2a cells with or without ITLN overexpression, followed by H₂O₂-induced injury. We observed that H₂O₂ injury significantly decreased the levels of ITLN-1, SIRT1, and PGC-1α. However, ITLN overexpression reversed H₂O₂-induced decline in cell viability and rise in apoptosis and intracellular ROS levels in N2a cells, while ITLN siRNA worsened the neurocyte injury. Furthermore, SIRT1 knockdown reversed the positive effect of ITLN overexpression on oxidative stress injury in N2a cells. Taken together, these findings suggest that ITLN-1 exerts neuroprotective effects against oxidative stress injury primarily through the SIRT1/PGC-1α axis.