Cardiac endothelial ischemia/reperfusion injury-derived protein damage-associated molecular patterns disrupt the integrity of the endothelial barrier.

Human cardiac microvascular endothelial cells (HCMECs) are sensitive to ischemia and vulnerable to damage during reperfusion. The release of damage-associated molecular patterns (DAMPs) during reperfusion induces additional tissue damage. The current study aimed to identify early protein DAMPs in human cardiac microvascular endothelial cells subjected to ischemia-reperfusion injury (IRI) using a proteomic approach and their effect on endothelial cell injury. HCMECs were subjected to 60 min of simulated ischemia and 6 h of reperfusion, which can cause lethal damage. DAMPs in the culture media were subjected to liquid chromatography-tandem mass spectrometry proteomic analysis. The cells were treated with endothelial IRI-derived DAMP medium for 24 h. Endothelial injury was assessed by measuring lactate dehydrogenase activity, morphological features, and the expression of endothelial cadherin, nitric oxide synthase (eNOS), and caveolin-1. The top two upregulated proteins, DNAJ homolog subfamily B member 11 and pyrroline-5-carboxylate reductase 2, are promising and sensitive predictors of cardiac microvascular endothelial damage. HCMECs expose to endothelial IRI-derived DAMP, the lactate dehydrogenase activity was significantly increased compared with the control group (10.15 ± 1.03 vs 17.67 ± 1.19, respectively). Following treatment with endothelial IRI-derived DAMPs, actin-filament dysregulation, and downregulation of vascular endothelial cadherin, caveolin-1, and eNOS expressions were observed, along with cell death. In conclusion, the early protein DAMPs released during cardiac microvascular endothelial IRI could serve as novel candidate biomarkers for acute myocardial IRI. Distinct features of impaired plasma membrane integrity can help identify therapeutic targets to mitigate the detrimental consequences mediated of endothelial IRI-derived DAMPs.

The secretory leukocyte protease inhibitor (SLPI) in pathophysiology of non-communicable diseases: Evidence from experimental studies to clinical applications.

Non-communicable diseases (NCDs) are a worldwide health issue because of their prevalence, negative impacts on human welfare, and economic costs. Protease enzymes play important roles in viral and NCD diseases. Slowing disease progression by inhibiting proteases using small-molecule inhibitors or endogenous inhibitory peptides appears to be crucial. Secretory leukocyte protease inhibitor (SLPI), an inflammatory serine protease inhibitor, maintains protease/antiprotease balance. SLPI is produced by host defense effector cells during inflammation to prevent proteolytic enzyme-induced tissue damage. The etiology of noncommunicable illnesses is linked to SLPI's immunomodulatory and tissue regeneration roles. Disease phases are associated with SLPI levels and activity changes in regional tissue and circulation. SLPI has been extensively evaluated in inflammation, but rarely in NCDs. Unfortunately, the thorough evaluation of SLPI's pathophysiological functions in NCDs in multiple research models has not been published elsewhere. In this review, data from PubMed from 2014 to 2023 was collected, analysed, and categorized into in vitro, in vivo, and clinical studies. According to the review, serine protease inhibitor (SLPI) activity control is linked to non-communicable diseases (NCDs) and other illnesses. Overexpression of the SLPI gene and protein may be a viable diagnostic and therapeutic target for non-communicable diseases (NCDs). SLPI is also cytoprotective, making it a unique treatment. These findings suggest that future research should focus on these pathways using advanced methods, reliable biomarkers, and therapy approaches to assess susceptibility and illness progression. Implications from this review will help pave the way for a new therapeutic target and diagnosis marker for non-communicable diseases.

Proteomic Profiling of Early Secreted Proteins in Response to Lipopolysaccharide-Induced Vascular Endothelial Cell EA.hy926 Injury.

Sepsis is a crucial public health problem with a high mortality rate caused by a dysregulated host immune response to infection. Vascular endothelial cell injury is an important hallmark of sepsis, which leads to multiple organ failure and death. Early biomarkers to diagnose sepsis may provide early intervention and reduce risk of death. Damage-associated molecular patterns (DAMPs) are host nuclear or cytoplasmic molecules released from cells following tissue damage. We postulated that DAMPs could potentially be a novel sepsis biomarker. We used an in vitro model to determine suitable protein-DAMPs biomarkers for early sepsis diagnosis. Low and high lipopolysaccharide (LPS) doses were used to stimulate the human umbilical vein endothelial cell line EA.hy926 for 24, 48, and 72 h. Results showed that cell viability was reduced in both dose-dependent and time-dependent manners. Cell injury was corroborated by a significant increase in lactate dehydrogenase (LDH) activity within 24 h in cell-conditioned medium. Secreted protein-DAMPs in the supernatant, collected at different time points within 24 h, were characterized using shotgun proteomics LC-MS/MS analysis. Results showed that there were 2233 proteins. Among these, 181 proteins from the LPS-stimulated EA.hy926 at 1, 12, and 24 h were significantly different from those of the control. Twelve proteins were up-regulated at all three time points. Furthermore, a potential interaction analysis of predominant DAMPs-related proteins using STITCH 5.0 revealed the following associations with pathways: response to stress; bacterium; and LPS (GO:0080134; 0009617; 0032496). Markedly, alpha-2-HS-glycoprotein (AHSG or fetuin-A) and lactotransferrin (LTF) potentially presented since the first hour of LPS stimulation, and were highly up-regulated at 24 h. Taken together, we reported proteomic profiling of vascular endothelial cell-specific DAMPs in response to early an in vitro LPS stimulation, suggesting that these early damage-response protein candidates could be novel early biomarkers associated with sepsis.

Wax apple (Syzygium samarangense) fruit extract ameliorates endothelial dysfunction and liver damage in high cholesterol diet-fed rats.

Background and aim: Wax apple fruit (Syzygium samarangense) is one of the most popular tropical fruit in Asia, and contains several essential nutrients. Therefore, this study explored the effects of the wax apple fruit extract on a high-cholesterol diet-induced vascular endothelial dysfunction and fatty liver in rats. Experimental procedure: Male Sprague Dawley rats were fed a diet with 1.5% cholesterol (HCD) for 8 weeks, and were given wax apple fruit extract (50 and 100 mg/kg/day) orally for the last 4 weeks. After 8 weeks, blood sample, thoracic aorta, and liver were collected and processed for biochemical and histological analysis. Additionally, vascular endothelial function and the protein expression of oxidative stress markers in aortae were evaluated. Results and conclusion: Wax apple reduced serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), but increased high-density lipoprotein cholesterol (HDL-C) levels. Furthermore, the liver levels of TG and TC were reduced in wax apple-treated hypercholesterolemic rats. Histological studies revealed that wax apple ameliorated HCD-induced morphologic changes of aortic and liver tissues of rats. In aortic tissues, the impaired endothelium-dependent responses to acetylcholine, the reduced nitric oxide (NO) contents, the elevated endothelin (ET)-1 contents, and the increased expression of NADPH oxidase subunit p47phox and 4-hydroxynonenal in HCD-fed rats were reversed by wax apple treatment. These results suggest that oral administration of wax apple improves vascular dysfunction and damage in hypercholesterolemic rats possibly through increasing NO bioavailability, decreasing ET-1 levels and reducing oxidative stress. Furthermore, wax apple ameliorates the HCD-induced fatty liver in rats.

Adeno-associated virus 9 vector-mediated cardiac-selective expression of human secretory leukocyte protease inhibitor attenuates myocardial ischemia/reperfusion injury.

Protease enzymes contribute to the initiation of cardiac remodeling and heart failure after myocardial ischemic/reperfusion (I/R) injury. Protease inhibitors attenuate protease activity and limit left ventricular dysfunction and remodeling. Previous studies showed the cardioprotective effect of secretory leukocyte protease inhibitor (SLPI) against I/R injury. However, overexpression of SLPI gene in cardiovascular diseases has only been investigated in an in vitro experiment. Here, cardiac-selective expression of the human secretory leukocyte protease inhibitor (hSLPI) gene and its effect on I/R injury were investigated. Adeno-associated virus (AAV) serotype 9 carrying hSLPI under the control of cardiac-selective expression promoter (cardiac troponin, cTn) was intravenously administered to Sprague-Dawley rats for 4 weeks prior to coronary artery ligation. The results showed that myocardial-selective expression of hSLPI significantly reduced infarct size, cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and myoglobin levels that all served to improve cardiac function. Moreover, overexpression of hSLPI showed a reduction in inflammatory cytokines, oxidatively modified protein carbonyl (PC) content, ischemia-modified albumin (IMA), and necrosis and cardiac tissue degeneration. In conclusion, this is the first study to demonstrate cardiac-selective gene delivery of hSLPI providing cardioprotection against myocardial I/R injury in an in vivo model.

Pineapple fruit improves vascular endothelial dysfunction, hepatic steatosis, and cholesterol metabolism in rats fed a high-cholesterol diet.

Hypercholesterolaemia is a significant risk factor for developing vascular disease and fatty liver. Pineapple (Ananas comosus), a tropical fruit widely cultivated in Asia, is reported to exhibit antioxidant and cholesterol-lowering activity; however, the potential hypolipidaemic mechanisms of pineapple fruit remain unknown. Therefore, we aimed to identify the anti-hypercholesterolaemic mechanism of pineapple fruit and to study the effect of pineapple fruit intake on hypercholesterolaemia-induced vascular dysfunction and liver steatosis in a high-cholesterol diet (HCD)-fed rats. Male Sprague Dawley rats were fed with standard diet or HCD, and the pineapple fruit was orally administered to HCD-fed rats for 8 weeks. At the end of treatment, vascular reactivity and morphology of aortas, as well as serum nitrate/nitrite (NOx), were determined. Liver tissues were also examined for histology, lipid content, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) activity, and protein expression of cholesterol metabolism-related enzymes. Results showed that pineapple fruit reduced the levels of hepatic cholesterol and triglycerides, and improved histological characteristics of a fatty liver in HCD-fed rats. Pineapple fruit also increased serum NOx, restored endothelium-dependent vasorelaxation, and reduced structural alterations in aortas of rats fed the HCD. In addition, a reduction of HMGCR activity and the downregulation of hepatic expression of HMGCR and sterol-regulatory element-binding protein 2 (SREBP2), as well as the upregulation of hepatic expression of cholesterol 7α-hydroxylase (CYP7A1) and LDL receptor (LDLR) were found in pineapple fruit-treated hypercholesterolaemic rats. These results indicate that pineapple fruit consumption can restore fatty liver and protect vascular endothelium in diet-induced hypercholesterolaemia through an improvement of hepatic cholesterol metabolism.

Tumor-Promoting Activity and Proteomic Profiling of Cisplatin/Oxaliplatin-Derived DAMPs in Cholangiocarcinoma Cells.

Damage-associated molecular patterns (DAMPs) are well recognized as the molecular signature of immunogenic cell death (ICD). The efficacy of drug-induced ICD function may be impacted by the precise ratio between immunostimulatory and immunoinhibitory DAMPs. Tumor-derived DAMPs can activate tumor-expressed TLRs for the promotion of tumor cell motility, invasion, metastatic spread and resistance to chemotherapeutic treatment. Herein, drug-induced DAMPs' expression and their role in tumor progression are utilized as one crucial point of evaluation regarding chemotherapeutic treatment efficacy in our study. Cisplatin and oxaliplatin, the conventional anticancer chemotherapy drugs, are emphasized as a cause of well-known DAMPs' release from cholangiocarcinoma (CCA) cells (e.g., HSP family, S100, CRT and HMGB1), whereby they trigger Akt, ERK and Cyclin-D1 to promote tumor activities. These findings strengthen the evidence that DAMPs are not only involved in immunomodulation but also in tumor promotion. Therefore, DAMP molecules should be considered as either targets of cancer treatment or biomarkers to evaluate treatment efficacy and tumor recurrence.

PPARß/δ priming enhances the anti-apoptotic and therapeutic properties of mesenchymal stromal cells in myocardial ischemia-reperfusion injury.

BACKGROUND: Mesenchymal Stromal Cells (MSC) have been widely used for their therapeutic properties in many clinical applications including myocardial infarction. Despite promising preclinical results and evidences of safety and efficacy in phases I/ II, inconsistencies in phase III trials have been reported. In a previous study, we have shown using MSC derived from the bone marrow of PPARß/δ (Peroxisome proliferator-activated receptors ß/δ) knockout mice that the acute cardioprotective properties of MSC during the first hour of reperfusion are PPARß/δ-dependent but not related to the anti-inflammatory effect of MSC. However, the role of the modulation of PPARß/δ expression on MSC cardioprotective and anti-apoptotic properties has never been investigated. OBJECTIVES: The aim of this study was to investigate the role of PPARß/δ modulation (inhibition or activation) in MSC therapeutic properties in vitro and ex vivo in an experimental model of myocardial infarction. METHODS AND RESULTS: Naïve MSC and MSC pharmacologically activated or inhibited for PPARß/δ were challenged with H2O2. Through specific DNA fragmentation quantification and qRT-PCR experiments, we evidenced in vitro an increased resistance to oxidative stress in MSC pre-treated by the PPARß/δ agonist GW0742 versus naïve MSC. In addition, PPARß/δ-priming allowed to reveal the anti-apoptotic effect of MSC on cardiomyocytes and endothelial cells in vitro. When injected during reperfusion, in an ex vivo heart model of myocardial infarction, 3.75 × 105 PPARß/δ-primed MSC/heart provided the same cardioprotective efficiency than 7.5 × 105 naïve MSC, identified as the optimal dose in our experimental model. This enhanced short-term cardioprotective effect was associated with an increase in both anti-apoptotic effects and the number of MSC detected in the left ventricular wall at 1 h of reperfusion. By contrast, PPARß/δ inhibition in MSC before their administration in post-ischemic hearts during reperfusion decreased their cardioprotective effects. CONCLUSION: Altogether these results revealed that PPARß/δ-primed MSC exhibit an increased resistance to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARß/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the therapeutic injected doses. These results could be of major interest to improve MSC efficacy for the cardioprotection of injured myocardium in AMI patients.

Novel Coconut Vinegar Attenuates Hepatic and Vascular Oxidative Stress in Rats Fed a High-Cholesterol Diet.

Background: Hypercholesterolemia is an independent modifiable risk factor that accelerates the development of both non-alcoholic fatty liver and atherosclerosis. Coconut water contains a variety of phytochemicals that make it appealing for producing vinegar. Coconut vinegar is rapidly gaining popularity for health benefits in Southeast Asia. The purpose of this study is to evaluate the effect of daily supplementation of coconut vinegar on hepatic and vascular oxidative stress in rats fed a high-cholesterol diet (HCD). Methods: Mature coconut water was fermented with coconut sap sugar using Saccharomyces cerevisiae and Acetobacter aceti vat Europeans, respectively. Bioactive compounds and antioxidant capacity of coconut vinegar were examined in vitro. Adult male Sprague-Dawley rats were randomly divided into four groups; the control group fed a standard diet (S), a group that received HCD (SC), a group that received HCD supplemented with coconut vinegar at a dose of 1 mL/kg/day (SCV), and a group that received HCD with atorvastatin at a dose of 30 mg/kg/day (SCA). After 8 weeks, serum metabolic profiles, fatty liver, hepatic, and vascular oxidative stress were determined. Results: In in vitro studies, coconut vinegar was rich in phenolic compounds and organic acids. The antioxidant capacity of 30 µL of coconut vinegar was 181.55 ± 8.15 µM Trolox equivalent antioxidant capacity (TEAC). In the HCD fed rats, daily supplementation of coconut vinegar reduced weight gain, serum triglycerides, and fasting blood sugar levels without renal or liver toxicity. In the liver, coconut vinegar reduced the accumulation of both hepatic cholesterol and hepatic triglyceride, and it also reduced hepatic 4-hydroxynonenal (4-HNE) lipid peroxidation. In the aortic tissues, coconut vinegar increased nitric oxide bioavailability and reduced aortic 4-HNE lipid peroxidation. Conclusion: Novel coconut vinegar is the source of antioxidants, and daily supplementation of coconut vinegar was found to attenuate dyslipidemia-induced hepatic and vascular oxidative stress by protective against cellular lipid peroxidation.

Atorvastatin and ezetimibe protect against hypercholesterolemia-induced lung oxidative stress, inflammation, and fibrosis in rats.

Background: Hypercholesterolemia is a major risk factor for interstitial lung disease (ILD). Atorvastatin and ezetimibe are antilipemic drugs that have pleiotropic effects. However, their effects on pulmonary fibrosis prevention and the mechanisms underlying hypercholesterolemia have not been fully investigated. This study aimed to evaluate the individual effects of atorvastatin and ezetimibe on lung inflammation and fibrosis in high-cholesterol diet (HCD)-fed rats. Materials and methods: Male Sprague-Dawley rats were divided into four groups - standard diet (S), standard diet + 1% cholesterol (SC), standard diet + 1% cholesterol with 30 mg/kg/day atorvastatin (SCA), and standard diet + 1% cholesterol with 10 mg/kg/day ezetimibe (SCE). At the end of an 8-week dietary schedule, serum lipid parameters and the levels of lung oxidative stress, inflammatory cytokines, and fibrotic mediators were determined. Results: Atorvastatin and ezetimibe treatment remarkably reduced serum lipid profiles with reversed pulmonary histological alterations, in addition to reducing the levels of lung oxidative stress, inflammation, and fibrosis in hypercholesterolemic rats. Conclusion: Atorvastatin and ezetimibe treatment showed a protective effect against hypercholesterolemia-induced pulmonary fibrosis in rats. This information appears potentially useful in the prevention of PF in a hypercholesterolemia model; however, further rigorous investigations are needed to prove their clinical utility on antifibrosis.

Cold Pressor Test Influences the Cardio-Ankle Vascular Index in Healthy Overweight Young Adults.

OBJECTIVE: The cold pressor test (CPT) has been shown a potential sympathoexcitatory stimulus which increases aortic pulse wave velocity and the aortic augmentation index, suggesting that noninvasively, arterial stiffness parameters are altered by the CPT. The cardio-ankle vascular index (CAVI) is widely used for reflecting arterial stiffness, and the ankle-brachial index (ABI) for evaluating peripheral artery disease in obesity. We aimed to assess CAVI and ABI in overweight young adults in the context of sympathetic activation by using the CPT. METHODS: 160 participants were divided into 2 groups: 86 normal-weight (body mass index [BMI] 18.50-22.99 kg/m2) and 74 overweight (BMI ≥23 kg/m2). The CPT was performed by immersing a participant's left hand into cold water (3-5°C) for 3 min, and CAVI and ABI assessment. RESULTS: At baseline, the CAVI in the overweight group was significantly less than that in the normal-weight group (5.79 ± 0.85 vs. 6.10 ± 0.85; p < 0.05). The mean arterial pressure (MAP) for overweight was significantly greater than that for normal-weight subjects (93.89 ± 7.31 vs. 91.10 ± 6.72; p < 0.05). During the CPT, the CAVI increased in both normal-weight and overweight subjects, the CAVI value was greater during the CPT in overweight subjects by 14.36% (6.62 ± 0.95 vs. 5.79 ± 0.85, p < 0.05) and in normal-weight subjects by 8.03% (6.59 ± 1.20 vs. 6.10 ± 0.85, p < 0.05) than those baseline values. The CPT evoked an increase in systolic blood pressure (SBP), diastolic BP (DBP), heart rate (HR,) and pulse pressure (PP) in both groups. After a 4-min CPT period, the CAVI returned values similar to the baseline values in both groups, and the SBP, DBP, MAP, and PP in overweight participants were significantly higher than those in normal-weight participants. However, there was no significant difference in the ABI at baseline, during CPT, and post-CPT in either group. CONCLUSIONS: Our results indicated that the CAVI was influenced by sympathetic activation response to the CPT in both normal-weight and overweight young adults. Specifically, during the CPT, the percentage change of the CAVI in overweight response was greater in normal-weight participants than baseline values in each group. The ABI was not found significantly associated with CPT. These findings suggesting that sympathoexcitatory stimulus by CPT influence CAVI results.

PPARß/δ Is Required for Mesenchymal Stem Cell Cardioprotective Effects Independently of Their Anti-inflammatory Properties in Myocardial Ischemia-Reperfusion Injury.

Myocardial infarction ranks first for the mortality worldwide. Because the adult heart is unable to regenerate, fibrosis develops to compensate for the loss of contractile tissue after infarction, leading to cardiac remodeling and heart failure. Adult mesenchymal stem cells (MSC) regenerative properties, as well as their safety and efficacy, have been demonstrated in preclinical models. However, in clinical trials, their beneficial effects are controversial. In an experimental model of arthritis, we have previously shown that PPARß/δ deficiency enhanced the therapeutic effect of MSC. The aim of the present study was to compare the therapeutic effects of wild-type MSC (MSC) and MSC deficient for PPARß/δ (KO MSC) perfused in an ex vivo mouse model of ischemia-reperfusion (IR) injury. For this purpose, hearts from C57BL/6J mice were subjected ex vivo to 30 min ischemia followed by 1-h reperfusion. MSC and KO MSC were injected into the Langendorff system during reperfusion. After 1 h of reperfusion, the TTC method was used to assess infarct size. Coronary effluents collected in basal condition (before ischemia) and after ischemia at 1 h of reperfusion were analyzed for their cytokine profiles. The dose-response curve for the cardioprotection was established ex vivo using different doses of MSC (3.105, 6.105, and 24.105 cells/heart) and the dose of 6.105 MSC was found to be the optimal concentration. We showed that the cardioprotective effect of MSC was PPARß/δ-dependent since it was lost using KO MSC. Moreover, cytokine profiling of the coronary effluents collected in the eluates after 60 min of reperfusion revealed that MSC treatment decreases CXCL1 chemokine and interleukin-6 release compared with untreated hearts. This anti-inflammatory effect of MSC was also observed when hearts were treated with PPARß/δ-deficient MSC. In conclusion, our study revealed that the acute cardioprotective properties of MSC in an ex vivo model of IR injury, assessed by a decreased infarct size at 1 h of reperfusion, are PPARß/δ-dependent but not related to their anti-inflammatory effects.

p38 MAPK Inhibitor (SB203580) and Metformin Reduces Aortic Protein Carbonyl and Inflammation in Non-obese Type 2 Diabetic Rats.

Microvascular and macrovascular diseases are the main causes of morbidity in type 2 diabetes patients through chronic hyperglycaemic condition via oxidative stress and inflammation. Reactive oxygen species (ROS) activate p38 MAPK phosphorylation and inflammation which enhances protein modification by carbonylation. The use of metformin and a p38 MAPK inhibitor is hypothesised to reduce ROS production and inflammation but effects of metformin and p38 MAPK inhibitor (SB203580) on ROS production and inflammation in vascular type 2 diabetes mellitus non-obese (T2DM) have not been investigated. The Goto-Kakizaki rat T2DM model was divided into three groups as T2DM, T2DM treated with 15 mg/kg bw metformin and T2DM treated with 2 mg/kg bw SB203580 for 4 weeks. Rat aortas were isolated and protein carbonyl (PC) contents were measured by spectrophotometric DNPH assay. Aortic IL-1ß level was determined by ELISA. Results showed that aortic PC contents in the T2DM group were significantly higher than in non-diabetic rats. Treatment with metformin or SB203580 significantly reduced PC contents while only metformin significantly reduced IL-1ß levels. Findings indicated that metformin reduced ROS production and inflammation in diabetic vessels and possibly reduce vascular complications in non-obese T2DM.

Pineapple consumption reduced cardiac oxidative stress and inflammation in high cholesterol diet-fed rats.

BACKGROUND: Hypercholesterolemia is a major risk factor for cardiovascular disease. It has been reported that pineapple contains healthy nutrients and phytochemicals associated with antioxidant and anti-inflammatory capacities. No investigation exists concerning the effect of pineapple consumption modulating hypercholesterolemia-induced cardiac damage in high-cholesterol diet (HCD)-fed rats. This study evaluated the effect of pineapple consumption on lipid-lowering, cardiac oxidative stress and inflammation in HCD-fed rats. METHODS: Male Sprague-Dawley rats were fed with HCD, in the presence and absence of Pineapple (Ananas comosus L.) cv. Pattavia powder for 8 weeks. Then, serum lipid profiles, liver and renal function tests, cardiac oxidative stress and pro-inflammatory cytokines were determined. RESULTS: Daily pineapple consumption reduced weight gain, serum lipid profiles, atherogenic coefficient (AC), cardiac risk ratio (CRR), and liver enzyme activity, without causing renal dysfunction. Pineapple consumption also restores cardiac protein carbonyl (cPC) content, reduces cardiac malondialdehyde (MDA), cardiac pro-inflammation cytokine IL-6 and IL-1ß levels. CONCLUSION: Pineapple possesses antioxidant and lipid-lowering properties and daily consumption alleviates hypercholesterolemia-induced cardiac lipid peroxidation and pro-inflammation elevation in an in vivo model. This study demonstrates that pineapple is a potential candidate for cardioprotection against hypercholesterolemia.

Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury.

Vascular endothelial cell (EC)-derived factors play an important role in endothelial-cardiomyocyte crosstalk and could save cardiomyocytes (CMs) from injury. The manipulation of endothelial cells to secrete protective factors could enhance cardioprotection. Secretory leukocyte protease inhibitor (SLPI) has been known to protect the heart. The goal of this study was to evaluate the in vitro paracrine protective effect and mechanisms of EC-derived human SLPI on cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury. Stable endothelial cells overexpressing human SLPI were generated from an endothelial cell line (EA.hy926). The cytoprotective effect was determined by cell survival assay. The results showed that endothelial-derived recombinant human SLPI (rhSLPI) reduced simulated ischemia/reperfusion (I/R)-(81.75% ± 1.42% vs. 60.27% ± 2.52%, p < 0.05) and hypoxia/reoxygenation (H/R)-induced EC injury (83.57% ± 1.78% vs. 63.07% ± 1.93%, p < 0.05). Moreover, co-culture of ECs overexpressing rhSLPI with CMs at ratios 1:1 and 1:3 or treatment with conditioned medium enhanced cell viability by 10.51-16.7% (co-culture) and 15.25-20.45% (conditioned medium) by reducing intracellular reactive oxygen species (ROS) production, the Bax/Bcl-2 expression ratio, caspase-3, and caspase-8, and in preconditioned CMs by activation of p38 MAPK and Akt survival kinase. In conclusion, this study showed for the first time that EC-derived rhSLPI provided cardio-vasculoprotective effects against I/R injury as a possible alternative therapeutic strategy for cardioprotection.

Overexpression and pre-treatment of recombinant human Secretory Leukocyte Protease Inhibitor (rhSLPI) reduces an in vitro ischemia/reperfusion injury in rat cardiac myoblast (H9c2) cell.

One of the major causes of cardiac cell death during myocardial ischemia is the oversecretion of protease enzymes surrounding the ischemic tissue. Therefore, inhibition of the protease activity could be an alternative strategy for preventing the expansion of the injured area. In the present study, we investigated the effects of Secretory Leukocyte Protease Inhibitor (SLPI), by means of overexpression and treatment of recombinant human SLPI (rhSLPI) in an in vitro model. Rat cardiac myoblast (H9c2) cells overexpressing rhSLPI were generated by gene delivery using pCMV2-SLPI-HA plasmid. The rhSLPI-H9c2 cells, mock transfected cells, and wild-type (WT) control were subjected to simulated ischemia/reperfusion (sI/R). Moreover, the treatment of rhSLPI in H9c2 cells was also performed under sI/R conditions. The results showed that overexpression of rhSLPI in H9c2 cells significantly reduced sI/R-induced cell death and injury, intracellular ROS level, and increased Akt phosphorylation, when compared to WT and mock transfection (p <0.05). Treatment of rhSLPI prior to sI/R reduced cardiac cell death and injury, and intra-cellular ROS level. In addition, 400 ng/ml rhSLPI treatment, prior to sI, significantly inhibited p38 MAPK phosphorylation and rhSLPI at 400-1000 ng/ml could increase Akt phosphorylation.

An in vitro endothelial cell protective effect of secretory leukocyte protease inhibitor against simulated ischaemia/reperfusion injury.

Endothelial dysfunction is an essential deleterious modulator of ischaemia/reperfusion (I/R) injury. Secretory leukocyte protease inhibitor (SLPI) has demonstrated myocardial protection in cardiac transplantation; however, the effect of SLPI in endothelial I/R injury remains unexplored. In the present study, the effect of recombinant human SLPI (rhSLPI) treatment against endothelial cells (ECs) subjected to simulated I/R injury and the effect of treatment at different time points were determined. Human umbilical vein ECs (HUVECs) were subjected to normoxic or simulated I/R (sI/R) conditions, and rhSLPI at concentrations of 1, 10, 100 and 1,000 ng/ml was added to the cells prior to ischaemia, during ischaemia or at the onset of reperfusion. Endothelial injury and cytoskeleton disruption were assessed, and western blot analysis was conducted. The results revealed that rhSLPI treatment at 1,000 ng/ml significantly increased the HUVEC viability under sI/R injury (P<0.05). In addition, treatment with rhSLPI prior to or during ischaemia markedly attenuated the activity of lactase dehydrogenase compared with that in the sI/R group. In addition, the H2O2-induced reactive oxygen species production was reduced by ~17% upon rhSLPI pretreatment. Endothelial cytoskeleton disruption was also preserved by rhSLPI added prior to the reperfusion period. Furthermore, pretreatment with rhSLPI promoted protein kinase B activation, as well as reduced p38 mitogen-activated protein kinase phosphorylation and B-cell lymphoma 2-associated X protein expression in response to I/R injury. These findings indicated that rhSLPI possesses antioxidant and antiapoptotic properties against endothelial responses to I/R injury. Therefore, the cytoprotective effect of rhSLPI may provide a potential pharmaceutical target to limit endothelial-mediated I/R injury.

Obesity alters the peripheral circadian clock in the aorta and microcirculation.

OBJECTIVE: Perturbation of daily rhythm increases cardiovascular risk. The aim of this study was to determine whether obesity alters circadian gene expression and microvascular function in lean mice and obese (db/db) mice. METHODS: Mice were subjected to normal LD or DD to alter circadian rhythm. Metabolic parameters and microvascular vasoreactivity were evaluated. Array studies were conducted in the am and pm cycles to assess the rhythmicity of the entire genomics. Rhythmic expression of specific clock genes (Bmal1, Clock, Npas2, Per1, Per2, and Cry1), clock output genes (dbp), and vascular relaxation-related genes (eNOS, GTPCH1) were assessed. RESULTS: Obesity was associated with metabolic dysfunction and impaired endothelial dilation in the microvasculature. Circadian rhythm of gene expression was suppressed 80% in both macro- and microcirculations of obese mice. Circadian disruption with DD increased fasting serum glucose and HbA1c in obese but not lean mice. Endothelium-dependent dilation was attenuated in obese mice and in lean mice subjected to DD. Rhythmic expression of per1 and dbp was depressed in obesity. Expression of eNOS expression was suppressed and GTPCH1 lost rhythmic expression both in obesity and by constant darkness. CONCLUSION: These results suggest that obesity reduces circadian gene expression in concert with impaired endothelial function. The causal relationship remains to be determined.