Polydatin protects against lipopolysaccharide-induced endothelial barrier disruption via SIRT3 activation.

In a previous study, we demonstrated the role of polydatin (PD) in protecting against multiple organ dysfunction in sepsis. The aim of this study is to investigate whether PD protects against lipopolysaccharide (LPS)-induced endothelial barrier disruption through SIRT3 activation and to disclose the underlying mechanisms. Wild-type mice were injected with LPS and Evans Blue assay was performed to evaluate vascular permeability. Primary human umbilical vein endothelial cells (HUVECs) were stimulated with LPS. Endothelial permeability was evaluated by transendothelial electrical resistance (TER) and FITC-dextran leakage. SIRT3 activity was determined by a Deacetylase Fluorometric kit, and protein expression level of SIRT3 was detected by western blotting. Mitochondrial function was evaluated by determination of ROS level, mitochondrial membrane potential and mPTP opening. In endotoxemic mice, PD pretreatment attenuated vascular leakage in multiple organs while SIRT3 inhibition with 3-TYP reversed the effects of PD. PD treatment in late sepsis also exhibited barrier protective effects. In HUVECs, PD alleviated LPS-induced F-actin rearrangement, cadherin-catenin complex dissociation and endothelial hyperpermeability, whereas 3-TYP or SIRT3 siRNA attenuated the protective effects of PD. PD enhanced SIRT3 deacetylase activity, and attenuated LPS-induced decrease in SIRT3 expression as well. Furthermore, gain-of-function and loss-of-function strategies also confirmed the role of SIRT3 in enhancing endothelial barrier integrity. It was further ascertained that PD enhanced SIRT3-mediated deacetylation of SOD2 and cyclophilin D (CypD), thus suppressing mitochondrial dysfunction and subsequent endothelial barrier dysfunction. In addition, it was revealed that RAGE was involved in LPS-regulated SIRT3 signaling. Our results suggest that polydatin protects against LPS-induced endothelial barrier disruption dependent on SIRT3 and can be applied as a potential therapy for sepsis.

Altered MicroRNA Expression Is Responsible for the Pro-Osteogenic Phenotype of Interstitial Cells in Calcified Human Aortic Valves.

BACKGROUND: The transition of aortic valve interstitial cells (AVICs) to myofibroblastic and osteoblast-like phenotypes plays a critical role in calcific aortic valve disease progression. Several microRNAs (miRs) are implicated in stem cell differentiation into osteoblast. We hypothesized that an epigenetic mechanism regulates valvular pro-osteogenic activity. This study examined miR profile in AVICs of calcified valves and identified miRs responsible for AVIC phenotypic transition. METHODS AND RESULTS: AVICs were isolated from normal and diseased valves. The miR microarray analysis revealed 14 upregulated and 12 downregulated miRs in diseased AVICs. Increased miR-486 and decreased miR-204 levels were associated with higher levels of myofibroblastic biomarker α-smooth muscle actin and osteoblastic biomarkers runt-related transcription factor 2 (Runx2) and osterix (Osx). Cotransfection of miR-486 antagomir and miR-204 mimic in diseased AVICs reduced their ability to express Runx2 and Osx. The miR-486 mimic upregulated α-smooth muscle actin expression in normal AVICs through the protein kinase B pathway and moderately elevated Runx2 and Osx levels. Knockdown of α-smooth muscle actin attenuated Runx2 and Osx expression induced by miR-486. The miR-486 mimic and miR-204 antagomir synergistically promoted Runx2 and Osx expression and calcium deposition in normal AVICs and normal aortic valve tissue. CONCLUSIONS: In AVICs of calcified valves, increased levels of miR-486 induce myofibroblastic transition to upregulate Runx2 and Osx expression and synergize with miR-204 deficiency to elevate cellular and valvular pro-osteogenic activity. These novel findings indicate that modulation of the epigenetic mechanism underlying valvular pro-osteogenic activity has therapeutic potential for prevention of calcific aortic valve disease progression.

An epigenetic regulatory loop controls pro-osteogenic activation by TGF-ß1 or bone morphogenetic protein 2 in human aortic valve interstitial cells.

Calcific aortic valve disease (CAVD) is common in the elderly population, but pharmacological interventions for managing valvular calcification are unavailable. Transforming growth factor ß1 (TGF-ß1) and bone morphogenetic protein 2 (BMP-2) induce pro-osteogenic activation of human aortic valve interstitial cells (AVICs) that play an important role in valvular calcification. However, the molecular mechanism underlying pro-osteogenic activation in AVICs is incompletely understood. Here, we investigated an epigenetic regulatory mechanism in human AVIC pro-osteogenic activation induced by TGF-ß1 and BMP-2. Microarray and real-time PCR analyses revealed that microRNA (miR)-486 up-regulation and miR-204 down-regulation were characteristic changes in TGF-ß1- and BMP-2-stimulated normal AVICs and in AVICs from calcified valves. Both TGF-ß1 and BMP-2 down-regulated miR-204 through Smad pathways. Interestingly, an miR-486 antagomir diminished the effect of TGF-ß1 and BMP-2 on miR-204 levels and calcium deposit formation. Furthermore, the miR-486 antagomir increased the expression of Smurf2, a Smad inhibitor, in the presence or absence of TGF-ß1 or BMP-2 stimulation, whereas a miR-486 mimic reduced Smurf2 expression. Smurf2 knockdown augmented TGF-ß1- or BMP-2-induced miR-204 down-regulation and resulted in increased expression of the osteoblastic biomarkers Osx and Runx2. In summary, we found that TGF-ß1 and BMP-2 up-regulate miR-486 and down-regulate miR-204 in human AVICs to promote pro-osteogenic activity and that miR-486 inhibits Smurf2 expression to augment the miR-204 down-regulation. We conclude that the miR-486-Smurf2-Smad loop plays an important role in regulating AVIC pro-osteogenic activation in response to TGF-ß1 or BMP-2. Targeting this regulatory loop may have therapeutic potential for suppressing aortic valve calcification.

SIRT1 is required for mitochondrial biogenesis reprogramming in hypoxic human pulmonary arteriolar smooth muscle cells.

Although recent studies have reported that mitochondria are putative oxygen sensors underlying hypoxic pulmonary vasoconstriction, little is known concerning the sirtuin 1 (SIRT1)-mediated mitochondrial biogenesis regulatory program in pulmonary arteriolar smooth muscle cells (PASMCs) during hypoxia/reoxygenation (H/R). We investigated the epigenetic regulatory mechanism of mitochondrial biogenesis and function in human PASMCs during H/R. Human PASMCs were exposed to hypoxia of 24-48 h and reoxygenation of 24-48 h. The expression of SIRT1 was reduced in a time-dependent manner. Mitochondrial transcription factor A (TFAM) expression was increased during hypoxia and decreased during reoxygenation, while the release of TFAM was increased in a time-dependent manner. Lentiviral overexpression of SIRT1 preserved SIRT3 deacetylase activity in human PASMCs exposed to H/R. Knockdown of PGC-1α suppressed the effect of SIRT1 on SIRT3 activity. Knockdown of SIRT3 abrogated SIRT1-mediated deacetylation of cyclophilin D (CyPD). Notably, knockdown of SIRT3 or PGC-1α suppressed the incremental effect of SIRT1 on mitochondrial TFAM, mitochondrial DNA (mtDNA) content and cellular ATP levels. Importantly, polydatin restored SIRT1 levels in human PASMCs exposed to H/R. Knockdown of SIRT1 suppressed the effect of polydatin on mitochondrial TFAM, mtDNA content and cellular ATP levels. In conclusion, SIRT1 expression is decreased in human PASMCs during H/R. TFAM expression in mitochondria is reduced and the release of TFAM is increased by H/R. PGC-1α/SIRT3/CyPD mediates the protective effect of SIRT1 on expression and release of TFAM and mitochondrial biogenesis and function. Polydatin improves mitochondrial biogenesis and function by enhancing SIRT1 expression in hypoxic human PASMCs.

SIRT1/3 Activation by Resveratrol Attenuates Acute Kidney Injury in a Septic Rat Model.

Sepsis often results in damage to multiple organ systems, possibly due to severe mitochondrial dysfunction. Two members of the sirtuin family, SIRT1 and SIRT3, have been implicated in the reversal of mitochondrial damage. The aim of this study was to determine the role of SIRT1/3 in acute kidney injury (AKI) following sepsis in a septic rat model. After drug pretreatment and cecal ligation and puncture (CLP) model reproduction in the rats, we performed survival time evaluation and kidney tissue extraction and renal tubular epithelial cell (RTEC) isolation. We observed reduced SIRT1/3 activity, elevated acetylated SOD2 (ac-SOD2) levels and oxidative stress, and damaged mitochondria in RTECs following sepsis. Treatment with resveratrol (RSV), a chemical SIRT1 activator, effectively restored SIRT1/3 activity, reduced acetylated SOD2 levels, ameliorated oxidative stress and mitochondrial function of RTECs, and prolonged survival time. However, the beneficial effects of RSV were greatly abrogated by Ex527, a selective inhibitor of SIRT1. These results suggest a therapeutic role for SIRT1 in the reversal of AKI in septic rat, which may rely on SIRT3-mediated deacetylation of SOD2. SIRT1/3 activation could therefore be a promising therapeutic strategy to treat sepsis-associated AKI.

Effect of salvianolic acid B on TNF-α induced cerebral microcirculatory changes in a micro-invasive mouse model.

PURPOSE: To investigate the effects of salvianolic acid B (SAB) on tumor necrosis factor a (TNF-α) induced alterations of cerebral microcirculation with a bone-abrading model. METHODS: The influences of craniotomy model and bone-abrading model on cerebral microcirculation were compared. The bone-abrading method was used to detect the effects of intracerebroventricular application of 40 µg/kg·bw TNF-α on cerebral venular leakage of fluorescein isothiocyanate (FITC)- albulmin and the rolling and adhesion of leukocytes on venules with fluorescence tracer rhodamine 6G. The therapeutical effects of SAB on TNF-α induced microcirculatory alteration were observed, with continuous intravenous injection of 5 mg/kg·h SAB starting at 20 min before or 20 min after TNF-α administration, respectively. The expressions of CD11b/CD18 and CD62L in leukocytes were measured with flow cytometry. Immunohistochemical staining was also used to detect E-selectin and ICAM-1 expression in endothelial cells. RESULTS: Compared with craniotomy method, the bone-abrading method preserved a higher erythrocyte velocity in cerebral venules and more opening capillaries. TNF-α intervention only caused responses of vascular hyperpermeability and leukocyte rolling on venular walls, without leukocyte adhesion and other hemodynamic changes. Pre- or post-SAB treatment attenuated those responses and suppressed the enhanced expressions of CD11b/CD18 and CD62L in leukocytes and E-selectin and ICAM-1 in endothelial cells induced by TNF-α. CONCLUSIONS: The pre- and post-applications of SAB during TNF-α stimulation could suppress adhesive molecular expression and subsequently attenuate the increase of cerebral vascular permeability and leukocyte rolling.

Polydatin Protecting Kidneys against Hemorrhagic Shock-Induced Mitochondrial Dysfunction via SIRT1 Activation and p53 Deacetylation.

OBJECTIVES: To ascertain if mitochondrial dysfunction (MD) of kidney cells is present in severe hemorrhagic shock and to investigate whether polydatin (PD) can attenuate MD and its protective mechanisms. RESEARCH DESIGN AND METHODS: Renal tubular epithelial cells (RTECs) from rat kidneys experiencing HS and a cell line (HK-2) under hypoxia/reoxygenation (H/R) treatment were used. Morphology and function of mitochondria in isolated RTECs or cultured HK-2 cells were evaluated, accompanied by mitochondrial apoptosis pathway-related proteins. RESULT: Severe MD was found in rat kidneys, especially in RTECs, as evidenced by swollen mitochondria and poorly defined cristae, decreased mitochondrial membrane potential (ΔΨm), and reduced ATP content. PD treatment attenuated MD partially and inhibited expression of proapoptotic proteins. PD treatment increased SIRT1 activity and decreased acetylated-p53 levels. Beneficial effect of PD was abolished partially when the SIRT1 inhibitor Ex527 was added. Similar phenomena were shown in the H/R cell model; when pifithrin-α (p53 inhibitor) was added to the PD/Ex527 group, considerable therapeutic effects were regained compared with the PD group apart from increased SIRT1 activity. CONCLUSIONS: MD is present in severe HS, and PD can attenuate MD of RTECs via the SIRT1-p53 pathway. PD might be a promising therapeutic drug for acute renal injury.

Polydatin ameliorates injury to the small intestine induced by hemorrhagic shock via SIRT3 activation-mediated mitochondrial protection.

BACKGROUND: Previously, we demonstrated that sirtuin (SIRT)1 plays vital roles in the small intestine (SI), protecting against severe hemorrhagic shock (HS), and that polydatin (PD) can attenuate SI injury via SIRT1 activation. OBJECTIVE: To explore the role of SIRT3 and mitochondria in SI injury after HS, and explore SIRT3 as a therapeutic target of PD in HS. METHODS: An H2O2-induced model of oxidative stress and an HS model were created in IEC-6 cells and Sprague-Dawley rats, respectively. Protein content and activity of SIRT1/3 and SOD2, acetylated-SOD2 level, and mitochondrial morphology/function were determined. RESULTS: Expression and activity of SIRT1/3 were reduced in SI tissue and IEC-6 cells after HS or oxidative stress, accompanied by an increased acetylated-SOD2 level and damaged mitochondria. Treatment with PD or resveratrol restored SIRT1/3 activity considerably, restored SIRT1/3 expression slightly, and reduced acetylated-SOD2 levels, which lead to elevated SOD2 activity and ameliorated mitochondrial function. The addition of 3-TYP (SIRT3 inhibitor) partially blocked the mitochondrial-protective effects of PD, but did not affect increased SIRT1 activity. CONCLUSIONS: The SIRT3-SOD2 signaling pathway is involved in mitochondrial dysfunction induced by HS. PD attenuates mitochondrial dysfunction via activation of the SIRT3-SOD2 pathway, and may be a new approach for HS treatment.

Activation of sirtuin 1/3 improves vascular hyporeactivity in severe hemorrhagic shock by alleviation of mitochondrial damage.

Vascular hyporeactivity is one of the major causes responsible for refractory hypotension and associated mortality in severe hemorrhagic shock. Mitochondrial permeability transition (mPT) pore opening in arteriolar smooth muscle cells (ASMCs) is involved in the pathogenesis of vascular hyporeactivity. However, the molecular mechanism underlying mitochondrial injury in ASMCs during hemorrhagic shock is not well understood. Here we produced an in vivo model of severe hemorrhagic shock in adult Wistar rats. We found that sirtuin (SIRT)1/3 protein levels and deacetylase activities were decreased in ASMCs following severe shock. Immunofluorescence staining confirmed reduced levels of SIRT1 in the nucleus and SIRT3 in the mitochondria, respectively. Acetylation of cyclophilin D (CyPD), a component of mPT pore, was increased. SIRT1 activators suppressed mPT pore opening and ameliorated mitochondrial injury in ASMCs after severe shock. Furthermore, administration of SIRT1 activators improved vasoreactivity in rats under severe shock. Our data suggest that epigenetic mechanisms, namely histone post-translational modifications, are involved in regulation of mPT by SIRT1/SIRT3- mediated deacetylation of CyPD. SIRT1/3 is a promising therapeutic target for the treatment of severe hemorrhagic shock.

Polydatin Alleviates Small Intestine Injury during Hemorrhagic Shock as a SIRT1 Activator.

OBJECTIVE: To evaluate the role of SIRT1 in small intestine damage following severe hemorrhagic shock and to investigate whether polydatin (PD) can activate SIRT1 in shock treatment. RESEARCH DESIGN AND METHODS: The severe hemorrhagic shock model was reproduced in Sprague Dawley rats. MAIN OUTCOME MEASURES: Two hours after drug administration, half of the rats were assessed for survival time evaluation and the remainder were used for small intestinal tissue sample collection. RESULTS: Bleeding and swelling appeared in the small intestine with epithelial apoptosis and gut barrier disturbance during hemorrhagic shock. SIRT1 activity and PGC-1α protein expression of the small intestine were decreased, which led to an increase in acetylated SOD2 and decreases in the expression and activity of SOD2, resulting in severe oxidative stress. The decreased SIRT1 activity and expression were partially restored in the PD administration group, which showed reduced intestine injury and longer survival time. Notably, the effect of PD was abolished after the addition of Ex527, a selective inhibitor of SIRT1. CONCLUSIONS: The results collectively suggest a role for the SIRT1-PGC-1α-SOD2 axis in small intestine injury following severe hemorrhagic shock and that PD is an effective SIRT1 activator for the shock treatment.

Polydatin protects hepatocytes against mitochondrial injury in acute severe hemorrhagic shock via SIRT1-SOD2 pathway.

OBJECTIVE: The aim of the study was to determine whether hepatocyte mitochondrial injury instigates severe shock and to explore effective therapy. METHODS: Wistar rats were randomly divided into five groups: Control (sham) group, shock + normal saline, shock + cyclosporine A, shock + resveratrol (Res) and shock + polydatin (PD) group. Mitochondrial morphology and function in hepatocytes following treatment were determined. RESULTS: Hepatocytes following severe shock exhibited mitochondrial dysfunction characterized with opening of mitochondrial permeability transition pores, mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm) and reduced ATP levels. Moreover, severe shock induced oxidative stress with increased lipid peroxidation and reactive oxygen species, decreased SOD2 (Superoxide Dismutase 2) and GSH/GSSG, which resulted in increased lysosomal membrane permeabilization and hepatocyte mitochondrial injury. Additionally, Res and PD restored decreased deacetylase sirtuin1 activity and protein expression in liver tissue following severe shock, suppressed oxidative stress-induced lysosomal unstability and mitochondrial injury by increasing the protein expression of SOD2, and thereby contributed to the prevention of hepatocyte mitochondria dysfunction and liver injury. CONCLUSIONS: PD effectively preserved hepatocytes from mitochondrial injury via SIRT1-SOD2 pathway and may be a new approach to treatment of irreversible shock.

Polydatin: a new therapeutic agent against multiorgan dysfunction.

BACKGROUND: Polydatin (PD), a monocrystalline and polyphenolic drug isolated from a traditional Chinese herb (Polygonum cuspidatum), is protective against mitochondrial dysfunction and has been approved for clinical trials in the treatment of shock. However, whether the administration of PD has a therapeutic effect on multiple-organ dysfunction syndrome (MODS) requires investigation. MATERIAL AND METHODS: MODS was induced in Sprague-Dawley rats via hemorrhage and ligation and puncture of cecum-induced sepsis. The rats were divided into three groups as follows: MODS + PD, MODS + normal saline, and a control group (no treatment). Survival time, blood biochemical indexes, and histopathologic changes in various organs were evaluated; serum oxidative stress (advanced oxidative protein products [AOPPs]) and proinflammatory cytokines (tumor necrosis factor-α, interleukin 1ß, and interleukin 6) were assayed using enzyme-linked immunosorbent assay. Apoptosis-related protein expression (B-cell lymphoma-2 [Bcl-2] and Bax) was assayed by immunohistochemical and Western blotting methods, whereas caspase-3 activity was assayed by spectrophotometry. RESULTS: PD improved organ function, prolonged survival time, and reduced MODS incidence and serum levels of AOPPs and proinflammatory cytokines. It also decreased Bax levels and caspase-3 activity and increased Bcl-2 levels in the kidney and liver. CONCLUSIONS: PD may serve as a potential therapeutic for MODS, as it suppresses oxidative stress, inhibits inflammatory response, attenuates apoptosis, and protects against mitochondrial dysfunction.

BMP-2 and TGF-ß1 mediate biglycan-induced pro-osteogenic reprogramming in aortic valve interstitial cells.

UNLABELLED: Biglycan accumulates in aortic valves affected by calcific aortic valve disease (CAVD), and soluble biglycan upregulates BMP-2 expression in human aortic valve interstitial cells (AVICs) via Toll-like receptor (TLR) 2 and induces AVIC pro-osteogenic reprogramming, characterized by elevated pro-osteogenic activities. We sought to identify the factors responsible for biglycan-induced pro-osteogenic reprogramming in human AVICs. Treatment of AVICs with recombinant biglycan induced the secretion of BMP-2 and TGF-ß1, but not BMP-4 or BMP-7. Biglycan upregulated TGF-ß1 expression in a TLR4-dependent fashion. Neutralization of BMP-2 or TGF-ß1 attenuated the expression of alkaline phosphatase (ALP), osteopontin, and runt-related transcription factor 2 (Runx2) in cells exposed to biglycan. However, neutralization of both BMP-2 and TGF-ß1 abolished the expression of these osteogenic biomarkers and calcium deposition. Phosphorylated Smad1 and Smad3 were detected in cells exposed to biglycan, and knockdown of Smad1 or Smad3 attenuated the effect of biglycan on the expression of osteogenic biomarkers. While BMP-2 and TGF-ß1 each upregulated the expression of osteogenic biomarkers, an exposure to BMP-2 plus TGF-ß1 induced a greater upregulation and results in calcium deposition. We conclude that concurrent upregulation of BMP-2 and TGF-ß1 is responsible for biglycan-induced pro-osteogenic reprogramming in human AVICs. The Smad 1/3 pathways are involved in the mechanism of AVIC pro-osteogenic reprogramming. KEY MESSAGE: Biglycan upregulates BMP-2 and TGF-ß1 in human aortic valve cells through TLRs. Both BMP-2 and TGF-ß1 are required for aortic valve cell pro-osteogenic reprogramming. Smad signaling pathways are involved in mediating the pro-osteogenic effects of biglycan.

Soluble biglycan induces the production of ICAM-1 and MCP-1 in human aortic valve interstitial cells through TLR2/4 and the ERK1/2 pathway.

OBJECTIVE: Mononuclear cell infiltration in valvular tissue is one of the characteristics in calcific aortic valve disease. The inflammatory responses of aortic valve interstitial cells (AVICs) play an important role in valvular inflammation. However, it remains unclear what may evoke AVIC inflammatory responses. Accumulation of biglycan has been found in diseased aortic valve leaflets. Soluble biglycan can function as a danger-associated molecular pattern to induce the production of proinflammatory mediators in cultured macrophages. We tested the hypothesis that soluble biglycan induces AVIC production of proinflammatory mediators involved in mononuclear cell infiltration through Toll-like receptor (TLR)-dependent signaling pathways. METHODS: Human AVICs isolated from normal aortic valve leaflets were treated with specific siRNA and neutralizing antibody against TLR2 or TLR4 before biglycan stimulation. The production of ICAM-1 and MCP-1 was assessed. To determine the signaling pathway involved, phosphorylation of ERK1/2 and p38 MAPK was analyzed, and specific inhibitors of ERK1/2 and p38 MAPK were applied. RESULTS: Soluble biglycan induced ICAM-1 expression and MCP-1 release in human AVICs, but had no effect on IL-6 release. TLR4 blockade and knockdown reduced ICAM-1 and MCP-1 production induced by biglycan, while knockdown and neutralization of TLR2 resulted in greater suppression of the inflammatory responses. Biglycan induced the phosphorylation of ERK1/2 and p38 MAPK, but ICAM-1 and MCP-1 production was reduced only by inhibition of the ERK1/2 pathway. Further, inhibition of ERK1/2 attenuated NF-κB activation following biglycan treatment. CONCLUSIONS: Soluble biglycan induces the expression of ICAM-1 and MCP-1 in human AVICs through TLR2 and TLR4 and requires activation of the ERK1/2 pathway. AVIC inflammatory responses induced by soluble biglycan may contribute to the mechanism of chronic inflammation associated with calcific aortic valve disease.

Polydatin--a new mitochondria protector for acute severe hemorrhagic shock treatment.

OBJECTIVE: The aim of the study was find out whether neuronal mitochondrial injury does take place in severe shock and to explore effective therapy for severe shock. RESEARCH DESIGN AND METHODS: Rats were divided in the following group: sham, shock + normal saline (NS), shock + cyclosporine A (CsA), shock + resveratrol (Res) and shock + polydatin (PD). Rats were subjected to shock for 2 h, followed by administration of NS, CsA, Res and PD, and infusion of shed blood. Morphology, metabolism and function of mitochondria were measured. RESULTS: Increased lipid peroxides (LPO) levels, lysosomal injury and mitochondrial permeability transition pore opening took place in neurons, resulting in swollen mitochondria with poorly defined cristae, decreased mitochondrial membrane potential (ΔΨ) and reduced ATP content in shock + NS group, indicating mitochondrial dysfunction. Mitochondrial protectors, such as CsA, Res and PD, partially inhibited these alterations, especially following PD protection, ATP level increased from 44.14 ± 13.81% in shock + NS group to 89.57 ± 9.21% and the survival time was prolonged from 6.3 ± 5.9 h in the shock + NS group to 31.6 ± 13.7 h in shock + PD group. CONCLUSIONS: The study shows that neuronal mitochondrial injury is involved in the genesis of severe shock and PD may be the best choice for protection of neuron against mitochondrial injury in severe shock.

Biglycan induces the expression of osteogenic factors in human aortic valve interstitial cells via Toll-like receptor-2.

OBJECTIVE: Although biglycan (BGN) and oxidized low-density lipoprotein (oxLDL) accumulation has been observed in calcific, stenotic aortic valves, their role in the pathogenesis of calcific aortic valve disease is poorly understood. We hypothesized that soluble BGN induces the osteogenic response in human aortic valve interstitial cells via Toll-like receptor (TLR) 2 and TLR4 and mediates the proosteogenic effect of oxLDL. METHODS AND RESULTS: Aortic valve interstitial cells of stenotic valves express higher levels of BGN. Stimulation of cells from normal valves with BGN increased the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) among the chondrogenic/osteogenic markers examined and caused accumulation of calcium deposits. TLR2 silencing, but not TLR4 silencing, reduced BMP-2 and ALP levels after BGN stimulation although coimmunoprecipitation revealed that BGN interacts with both TLR2 and TLR4. BGN induced the phosphorylation of extracellular signal-regulated protein kinase-1/2, p38 mitogen-activated protein kinase and nuclear factor-κB. Inhibition of extracellular-regulated kinase-1/2 markedly reduced the upregulation of BMP-2 and ALP expression by BGN whereas inhibition of p38 mitogen-activated protein kinase or nuclear factor-κB had a moderate effect. Stimulation of aortic valve interstitial cells with oxLDL upregulated BGN expression and release. Knockdown and neutralization of BGN reduced the effect of oxLDL on BMP-2 and ALP expression. CONCLUSIONS: Extracellular soluble BGN induces the expression of BMP-2 and ALP in human aortic valve interstitial cells primarily via TLR2 and contributes to the proosteogenic effect of oxLDL. These findings highlight the potential role of soluble BGN and oxLDL in the development of calcific aortic valve disease.

Acute lung inflammatory response and injury after hemorrhagic shock are more severe in postpartum rabbits.

OBJECTIVE: The acute respiratory distress syndrome may complicate postpartum hemorrhagic shock and resuscitation, but its mechanisms are not yet well defined. We studied the lung inflammatory response to postpartum hemorrhagic shock and resuscitation in a rabbit model and the role of the nuclear factor-κB pathway. DESIGN: Randomized, controlled, prospective study. SETTING: University hospital laboratory. SUBJECTS: Nonobstetric (not pregnant nor postpartum) and obstetrical (within 2 hrs postpartum) rabbits. INTERVENTIONS: Nonobstetric and obstetric female New Zealand white rabbits underwent fixed-pressure or fixed-volume hemorrhagic shock for 30 mins and then were rapidly resuscitated with the shed blood and Ringer's solution. Finally, they were either monitored for survival time or euthanized by exsanguination for lung tissue examination 24 hrs after hemorrhage. MEASUREMENTS AND MAIN RESULTS: After hemorrhagic shock and resuscitation, median survival time in obstetric rabbits (3 days) was significantly shorter (p<.05) than that in nonobstetric rabbits (5 days). Compared with nonobstetric rabbits, obstetric rabbits had more severe lung injury as indicated by alveolar and interstitial fluid accumulation and marked neutrophil sequestration and greater lung injury score, myeloperoxidase activity, expression of intercellular adhesion molecule-1, serum tumor necrosis factor-α levels, and nuclear factor-κB activation, and lower serum interleukin-10 levels (p<.05 for all). CONCLUSIONS: After hemorrhage and resuscitation, obstetric rabbits had significantly shorter survival time and more severe lung injury than nonobstetric rabbits. The mechanism may be through upregulation of the signal transductions of the nuclear factor-κB pathways.

Polydatin, a natural polyphenol, protects arterial smooth muscle cells against mitochondrial dysfunction and lysosomal destabilization following hemorrhagic shock.

The main objective of this study was to investigate the activity of polydatin on mitochondrial dysfunction and lysosomal stability of arteriolar smooth muscle cells (ASMCs) in severe shock. The experimental animals (rats) were divided into five groups: control, hemorrhagic shock, shock + CsA, shock + Res, and shock + PD (exposed to cyclosporin A, resveratrol, or polydatin following induction of hemorrhagic shock, respectively). The calcein-Co(2+) technique revealed opening of ASMC mitochondrial permeability transition pores (mPTP) after shock with resulting mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm), and reduced intracellular ATP levels. These alterations were all inhibited by exposure to PD, which was significantly more effective than CsA and Res. PD also preserved lysosomal stability, suppressed activation of K(ATP) channels, ASMC hyperpolarization, and reduced vasoresponsiveness to norepinephrine that normally follows severe shock. The results demonstrate that exposure to PD after initiation of severe shock effectively preserves ASMC mitochondrial integrity and has a significant therapeutic effect in severe shock. The effects may partially result from lysosomal stabilization against shock-induced oxidative stress and depressed relocation of hydrolytic enzymes and redox-active lysosomal iron that, in turn, may induce mPTP opening.

Atractyloside induces low contractile reaction of arteriolar smooth muscle through mitochondrial damage.

Atractyloside is the principal naturally occurring active ingredient in ethnomedicines and animal grazing forage. Evidence that atractyloside can induce opening of the mitochondrial permeability transition pore (mPTP) indicates that mitochondrial mechanisms may play an important role in pathophysiological lesions of the heart, liver and kidney after atractyloside poisoning. Therefore, in this study we investigated the association of atractyloside-induced mitochondrial damage in arteriolar smooth muscle cells (ASMCs) with contractile reaction. Atractyloside led to depolarized and swollen or damaged ASMC mitochondria, which might be related to the concentration-dependent induction of mPTP opening. Relative ATP content in ASMCs was significantly reduced by 48%, 63% and 66% of control when cells were treated with 7.5, 10, and 15 µm atractyloside for 10 min, respectively, and ASMCs were hyperpolarized. In addition, the contractile responsiveness of ASMCs was eventually weakened. These results suggest that atractyloside has a toxic effect on vasoreactivity, which is possibly related to mitochondrial damage.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.