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.

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.

In Vitro Activity of Cefiderocol, Cefepime-Zidebactam, and ß-Lactam Combinations Versus Other Antibiotic Classes Against Various Sequence Types of Clinically Isolated Carbapenemase-Producing Klebsiella pneumoniae.

Aim: This study aimed to establish the in vitro efficacy and susceptibility profiles of new ß-lactam antibiotics against clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) strains. Materials and Methods: A total of 117 nonduplicated CPKP isolates were tested against cefiderocol, cefepime-zidebactam, ceftazidime-avibactam, tigecycline, and other 20 antibiotics by broth microdilution. The carbapenemase genes were identified using PCR and sequencing, while multilocus sequence typing established the bacterial strains. Results: Three significant sequence types (STs), including ST147, ST16, and ST11, were shown to be the dominant STs, which occupied ∼90% of the tested population. Three carbapenemase genes, blaNDM-1, blaOXA-181, and blaOXA-232, were detected. The blaNDM-1 was found in ST147 and ST16 but not in ST11, while the blaOXA-232 was not detected in ST147. The majority of ST16 isolates contained both blaNDM-1 and blaOXA-232, which was not seen in other strains. Cefiderocol, cefepime-zidebactam, and tigecycline were the most active agents against CPKP. Both MIC50 and MIC90 of these three antibiotics remained within the susceptible categories, while nearly all other antibiotics were in the resistant levels. However, in ST11, which carried only blaOXA genes without blaNDM-1, ceftazidime-avibactam was effective with the MIC90 at 2 µg/mL. In addition, amikacin was shown to have good activity in ST11. In contrast, gentamicin was active in only ST16 and ST147. Conclusions: This study is the first report that demonstrates the prevalence of CPKP, distribution of strains, resistant genes, and antimicrobial susceptibility profiles in northern Thailand. These data would contribute to appropriate individual treatment and the selection of infection control strategies.

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.

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.