Intracellular Complement Component 3 Attenuated Ischemia-Reperfusion Injury in the Isolated Buffer-Perfused Mouse Heart and Is Associated With Improved Metabolic Homeostasis.

The innate immune system is rapidly activated during myocardial infarction and blockade of extracellular complement system reduces infarct size. Intracellular complement, however, appears to be closely linked to metabolic pathways and its role in ischemia-reperfusion injury is unknown and may be different from complement activation in the circulation. The purpose of the present study was to investigate the role of intracellular complement in isolated, retrogradely buffer-perfused hearts and cardiac cells from adult male wild type mice (WT) and from adult male mice with knockout of complement component 3 (C3KO). Main findings: (i) Intracellular C3 protein was expressed in isolated cardiomyocytes and in whole hearts, (ii) after ischemia-reperfusion injury, C3KO hearts had larger infarct size (32 ± 9% in C3KO vs. 22 ± 7% in WT; p=0.008) and impaired post-ischemic relaxation compared to WT hearts, (iii) C3KO cardiomyocytes had lower basal oxidative respiration compared to WT cardiomyocytes, (iv) blocking mTOR decreased Akt phosphorylation in WT, but not in C3KO cardiomyocytes, (v) after ischemia, WT hearts had higher levels of ATP, but lower levels of both reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) compared to C3KO hearts. Conclusion: intracellular C3 protected the heart against ischemia-reperfusion injury, possibly due to its role in metabolic pathways important for energy production and cell survival.

SNF472, a novel anti-crystallization agent, inhibits induced calcification in an in vitro model of human aortic valve calcification.

The purpose of the present study was to investigate whether SNF472, the hexasodium salt of myo-inositol hexaphosphate (IP6 or phytate): 1. Inhibits induced calcification in cultured aortic valve interstitial cells (VIC) as an in vitro model of aortic valve stenosis and 2. Whether inhibition is different in VIC obtained from healthy and calcified aortic valves. VIC from healthy (n = 5) and calcified (n = 7) human aortic valves were seeded in basic growth medium, osteogenic differentiation medium alone, or in osteogenic medium with SNF472 (3, 10, and 30 µM) and cultivated for 3 weeks. Calcification was quantified spectrophotometrically after Alizarin Red staining. In VIC from calcified valves, a complete inhibition of calcification was observed with SNF472 concentrations of 10 and 30 µM (p < .01), significantly stronger than in VIC from healthy valves. When SNF472 was added to VIC after 1 week in osteogenic medium, 30 and 100 µM SNF472 inhibited the progression of ongoing calcification by 81 and 100% (p < .01), respectively. The same concentrations of SNF472 given after 2 weeks reduced calcification by 35 and 40% respectively (not significant). SNF472 inhibited both the formation and the progression of calcification with the strongest effect in VIC from calcified valves.

Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves.

AIMS: Calcific aortic valve disease is the most common heart valve disease in the Western world. Bicuspid and tricuspid aortic valve calcifications are traditionally considered together although the dynamics of the disease progression is different between the two groups of patients. Notch signaling is critical for bicuspid valve development and NOTCH1 mutations are associated with bicuspid valve and calcification. We hypothesized that Notch-dependent mechanisms of valve mineralization might be different in the two groups. METHODS AND RESULTS: We used aortic valve interstitial cells and valve endothelial cells from patients with calcific aortic stenosis with bicuspid or tricuspid aortic valve. Expression of Notch-related genes in valve interstitial cells by qPCR was different between bicuspid and tricuspid groups. Discriminant analysis of gene expression pattern in the interstitial cells revealed that the cells from calcified bicuspid valves formed a separate group from calcified tricuspid and control cells. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notch-activated endothelial-to-mesenchymal transition and the corresponding expression of HEY1 and SLUG were also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves. CONCLUSION: Early signaling events including Notch-dependent mechanisms that are responsible for the initiation of aortic valve calcification are different between the patients with bicuspid and tricuspid aortic valves.

Mode of perfusion influences infarct size, coronary flow and stress kinases in the isolated mouse heart.

AIM: The isolated, retrogradely perfused heart (modified Langendorff model) is a widely used method in experimental heart research. The presence of an intraventricular balloon is necessary to get functional measurements. We have previously shown that the balloon induces phosphorylation of some suggested cardioprotective mitogen-activated protein kinases (MAPK): P38-MAPK, ERK 1/2 and JNK. We hypothesized that the balloon could influence cardioprotection, protect against ischaemia reperfusion injury and interfere with coronary flow. METHODS AND RESULTS: Isolated mouse hearts were perfused for 5, 10, 20, 40 and 60 min with a balloon in the left ventricle. We found a wavelike phosphorylation of all MAPK while AKT displayed a gradual dephosphorylation when compared to non-perfused hearts. Hearts were subjected to 20 min of stabilization with or without the balloon, followed by 35 min of ischaemia and 120 min of reperfusion. Although the MAPK were phosphorylated, the infarcts were larger in the balloon group. When the balloon was present during the entire protocol, compared to removal at the end of ischaemia, the infarct size was also larger, especially in the endocardial layer. The balloon reduced post-ischaemic endocardial coronary flow, despite a higher average flow, indicating a hyperperfused epicard. Blocking the balloon-induced ERK 1/2 phosphorylation during stabilization did not affect infarct size. The effect of post-conditioning was influenced by the balloon, showing reduced infarct size when the balloon was present. CONCLUSION: The balloon used for pressure measurements may contributes to cell death possibly by reducing endocardial coronary flow.

Mitochondrial DNA damage and repair during ischemia-reperfusion injury of the heart.

Ischemia-reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1(-/-) mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1(-/-)/myh(-/-), inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."

Dedicated immunosensing of the mouse intestinal epithelium facilitated by a pair of genetically coupled lectin-like receptors.

The integrity of the intestinal epithelium is constantly surveyed by a peculiar subset of innate-like T lymphocytes embedded in the epithelial cell layer, hence called intestinal intraepithelial lymphocytes (IELs). IELs are thought to act as "first-line" sentinels sensing the state of adjacent epithelial cells via both T-cell receptors and auxiliary receptors. Auxiliary receptors modulating IEL activity include C-type lectin-like receptors encoded in the natural killer gene complex such as NKG2D. Here, we report that the CTLR Nkrp1g is expressed by a subpopulation of mouse CD103(+) IELs allowing immunosensing of the intestinal epithelium through ligation of the genetically coupled CTLR Clr-f that is almost exclusively expressed on differentiated intestinal epithelial cells (IECs). Most of these Nkrp1g-expressing IELs exhibit a γδTCR(bright)Nkg2a(-) phenotype and are intimately associated with the intestinal epithelium. As Clr-f expression strongly inhibits effector functions of Nkrp1g-expressing cells and is upregulated upon poly(I:C) challenge, Clr-f molecules may quench reactivity of these IELs towards the epithelial barrier that is constantly provoked by microbial and antigenic stimuli. Altogether, we here newly characterize a genetically linked C-type lectin-like receptor/ligand pair with a highly restricted tissue expression that apparently evolved to allow for a dedicated immunosurveillance of the mouse intestinal epithelium.

Mesenchymal stromal cells fail to alleviate experimental graft-versus-host disease in rats transplanted with major histocompatibility complex-mismatched bone marrow.

Mesenchymal stromal cells (MSC) can be used to treat graft-versus-host disease (GVHD) caused by allogeneic stem cell transplantation (allo-SCT). The effectiveness of this therapy has been variable in clinical trials and in experimental animal models. In this study, we investigated the ability of bone marrow (BM)-derived MSC to alleviate GVHD in an experimental rat model of allo-SCT using two different combinations of major histocompatibility complex (MHC) mismatch with survival as the primary endpoint. Recipient rats received total body irradiation and a transplant of T cell-depleted donor BM cells with either a full [PVG.7B → BN] or a partial MHC mismatch [PVG.1U → PVG.R23] restricted to the class II and non-classical class I sub-regions (RT1-B/D-CE/N/M). GVHD was invoked by infusion of graded doses of donor leukocytes 2 weeks after allo-SCT. Weekly doses of MSC were injected starting on the day of donor leukocyte infusion. No significant overall improvement of mortality and morbidity was observed in the two transplantation settings. Stimulation of MSC with exogenous tumor necrosis factor α and interferon (IFN)γ prior to infusion could not rescue BM-transplanted rats from lethal acute GVHD. In conclusion, repeated administrations of MSC failed to alleviate GVHD after fully or partially MHC-mismatched allo-SCT in the rat.

Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts.

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability (R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, ß-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1-50.1%). When ß-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

Natural killer cell subsets in man and rodents.

NK cells are important contributors to the early immune defence against infected or transformed cells. They are rapidly activated in response to cytokines, whereby they exert their effector functions. NK cell responses are controlled by a multitude of receptors, which are expressed by subpopulations of NK cells with distinct phenotypes and functionalities. Direct comparisons between species are often difficult because of differences in the expression of NK cell receptors and other markers. In addition, NK cells change their phenotype and effector functions during differentiation, by tissue-specific factors, or upon activation, complicating interpretations. We will here review the similarities and differences between the major NK cell subsets in man and two well-characterized rodent models.

Proteasome inhibitors eliminate protective effect of postconditioning in cultured neonatal cardiomyocytes.

A role of proteasomal proteolysis in the pathogenesis of ischemia-reperfusion is being actively studied. To evaluate the participation of the proteasome in postconditioning phenomenon, we used primary culture of neonatal cardiomyocytes. 30 minutes of anoxia followed by 60 minutes of reoxygenation was undergone. Postconditioning was modeled by 3 cycles of 1-minute reoxygenation followed by 1-minute anoxia, respectively. Clasto-lactacystin b-lactone, a specific proteasome inhibitor, in the dose that does not cause cell death (2.5 mM) was added to the culture medium just before the cycles of postconditioning. Percentages of living, necrotic, and apoptotic cells were determined by staining with bisBenzimide and propidium iodide. Autophagy was demonstrated by staining vacuolar structures with monodansyl cadaverine. Proteasomal activity was determined by cleavage intensity of specific fluorogenic substrates. Trypsin-like, chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were decreased after anoxia. Reoxygenation led to an increase in trypsin-like and chymotrypsin-like activities comparing to anoxia, but these parameters never reached the control levels. PGPH activity was restored up to the initial level. Postconditioning increased numbers of living cells and decreased that of necrotic, apoptotic and autophagic cells. Paradoxically, it was established, that proteasome inhibitors prevented the necrotic and apoptotic cell death of cardiomyocytes in anoxia-reoxygenation, but in the same concentration abolished the effects of postconditioning. The data obtained permit to suppose that proteasome inhibitors can be used for pharmacological postconditioning.

Postconditioning prevents apoptotic necrotic and autophagic cardiomyocyte cell death in culture.

In the paper the data concerning the possibility of the reproduction of the postconditioning phenomena in the cardyomicyte culture are presented. Primary cultures of cardiomyocytes from neonatal rats underwent 30 minutes of anoxia followed by 60 minutes of reoxygenation. Three different models of postconditioning were used: 3 cycles of 1, 3, or 5 minutes of reoxygenation followed by 1, 3, or 5 minutes of anoxia, respectively. The percentage of living, necrotic, and apoptotic cells were determined by staining with Hoechst 33342 and propidium iodide. Autophagy was demonstrated by the staining of vacuolar structures in vivo by monodansyl cadaverine. After anoxia and reoxygenation the amount of living, necrotic and apoptotic cells were 79 +/- 1.5, 7.8 +/- 0.9 and 13 +/- 1.5 %, respectively (in unstimulated cell culture 90 +/- 0.8, 3.3 +/- 0.3, and 5.5 +/- 0.7, P < 0.0001 for all). Postconditioning with 1 min anoxia 3-fold increased the amount of living cells and decreased the number of necrotic and apoptotic cells (P = 0.002, P = 0.02 and P = 0.043 respectively). Postconditioning with cycles of 3 and 5 minutes had a gradually reduced effect compared to cycles of 1 minute. The percentage of autophagic cells in control cell culture was 4.3 +/- 0.3%. This number increased after anoxia-reoxygenation to 14 +/- 0.8%, and was reduced by postconditioning (P < 0.001). The data obtained indicate that postconditioning is one of the effective methods of cardioprotection and could effectively decrease the amount of cardiomyocytes with traits of programmed or non-programmed cell death.

Effects of sex, gonadectomy, and oestrogen substitution on ischaemic preconditioning and ischaemia-reperfusion injury in mice.

AIM: Ischaemic preconditioning (IPC) has been demonstrated to protect heart function and viability, but has been predominantly studied in male animals. METHODS: We studied a possible influence of sex and oestrogen for protection in IPC. Infarct size and heart function after 40 min global ischaemia and 60 min reperfusion with or without preceding classic IPC was investigated in Langendorff-perfused hearts. Hearts were harvested from 10-week-old male and female C57BL6 mice with or without gonadectomy 6 weeks earlier, or gonadectomy and substitution with 17 beta-oestradiol for 4 weeks (n = 104). RESULTS: Classic IPC reduced depression of left ventricular developed pressure (P < 0.01), attenuated the increase of end-diastolic pressure (P < 0.01), and reduced infarct size (P < 0.01) in hearts of untreated male mice, but failed to protect untreated females which had improved functional recovery and smaller infarctions than untreated males. After gonadectomy of female mice, developed pressure was reduced (P < 0.01) and infarct size increased (P < 0.01) compared with normal females, with no protection of preconditioning. The changes were not reversed by 17 beta-oestradiol substitution. In hearts of gonadectomized males, the post-ischaemic increase of end-diastolic pressure was attenuated (P < 0.01), and enhanced after substitution with 17 beta-oestradiol (P < 0.01). The preconditioning effect disappeared after gonadectomy and gonadectomy with substitution in male mice. CONCLUSION: There is a sex difference in evoking preconditioning in male and female mice which is only partially dependent on sex hormones.

Importance of preanalytical handling of samples for measurement of cardiac troponin T in coronary effluent from isolated rat hearts.

The isolated, buffer-perfused heart is probably the most widely used model in experimental heart research, and the coronary effluent is often analysed for markers of myocardial injury. Adsorption to surrounding materials may be a serious problem of protein measurements in solutions with low protein concentrations. The aims of the present study were to investigate the importance of the preanalytical phase when measuring cardiac troponin T (cTnT) in a buffer perfusate and to investigate whether addition of albumin to the effluent might increase recovery of cTnT and improve the assay. Coronary effluent was collected in tubes of different materials and in tubes with 40 g/L bovine albumin, and then frozen. cTnT was analysed at different time points after withdrawal from the freezer. cTnT was 2.3-119 times higher in effluent with albumin. In effluent without albumin, cTnT concentration declined to 2% of the initial concentration after two episodes of freezing and thawing. The cTnT loss could not be prevented by using polystyrene or siliconized glass, but was partially inhibited in effluent with albumin. Furthermore, creatine kinase and lactate dehydrogenase levels were higher in effluent with albumin. The within-series coefficient of variation for cTnT was markedly improved when using effluent with albumin.

Exposure of rats to hyperoxia enhances relaxation of isolated aortic rings and reduces infarct size of isolated hearts.

Exposure of rats to hyperoxia before organ harvesting protected their isolated hearts against global ischaemia-reperfusion injury in a previous study. The present study investigates whether hyperoxia influences vasomotor function and regional ischaemia of the heart. Isolated rings of the thoracic aorta were obtained from rats immediately or 24 h after in vivo exposure to 60 min of hyperoxia (>95% O2), and the in vitro dose-response to phenylephrine (PHE), prostaglandin F2alpha (PGF2alpha) and endothelin-1 (ET-1), acetylcholine (Ach) and sodium nitroprusside (SNP) was assessed. Hyperoxia in vivo increased the relaxation of aortic rings to Ach and SNP, while it delayed contraction to PHE. The effect was more evident when the vessels were harvested immediately rather than 24 h after hyperoxic exposure. In separate experiments rat hearts were isolated immediately after hyperoxia, buffer-perfused, and subjected to 30 min of regional ischaemia and reperfused for 120 min. Infarct size was determined by triphenyl tetrazolium chloride staining. Hyperoxia significantly reduced infarct size. In normoxic controls 23.0 +/- 8.3% of the area at risk was infarcted, while in hyperoxic animals infarct size was 14.8 +/- 5.6% of the area at risk (P = 0.012). Exposure of rats to hyperoxia modifies the vasomotor response of isolated aortic rings, and reduces the infarct size of isolated rat heart. These novel aspects of hyperoxic treatment require further studies to explore the potential of its clinical application.

Cardioprotection by breathing hyperoxic gas-relation to oxygen concentration and exposure time in rats and mice.

OBJECTIVES: Breathing a hyperoxic gas (> or =95% O(2)) protects against ischaemia-reperfusion injury in rat and mouse hearts. The present study investigated how oxygen concentration and duration of hyperoxic exposure influenced cardioprotection, and whether hyperoxia might induce delayed cardioprotection (after 24 h). METHODS: Animals were kept in normal air or in a hyperoxic environment, and their hearts were isolated and Langendorff-perfused immediately or 24 h thereafter. Global ischaemia was induced for 25 min in rats and 40 min in mice, followed by 60 min of reperfusion. Infarct size was determined by triphenyl tetrazolium chloride staining. RESULTS: In rats exposure to > or =95, 80, and 60%, but not to 40% of oxygen immediately before heart isolation and perfusion improved postischaemic functional recovery. Eighty or more percent of oxygen also reduced infarct size. A preconditioning-like effect could be evoked by 60 or 180 min of hyperoxia, giving both immediate and delayed protection. In the mouse heart protection could be induced by pretreatment for 15 or 30, but not by 60 min with > or =95% oxygen. The protective effect of hyperoxia in mice could be evoked in the immediate model only. CONCLUSIONS: Hyperoxia protects the isolated rat and mouse heart against ischaemia-reperfusion injury, but some species-different responses exist. The protection depends on both oxygen concentration in inspired air, and duration of hyperoxic exposure.

Postoperative mediastinitis in cardiac surgery - microbiology and pathogenesis.

OBJECTIVE: During 1992-2000, postoperative mediastinitis developed after 126 (1.32%) of 9557 consecutive cardiac surgery procedures. The study was done to describe the variation in clinical characteristics and microbiological etiology in mediastinitis. METHODS: The records of 126 cases of postoperative mediastinitis were reviewed. RESULTS: The median time from operation to the development of mediastinitis was 7 days. Sternal dehiscence was seen in 86 patients (68%). Coagulase negative staphylococci (CNS) were isolated in 46% of the cases with a verified microbiological etiology, Staphylococcus aureus in 26% and gram-negative bacteria in 18%. CNS were more frequently isolated in patients with sternal dehiscence (44/80, 55%) than in patients with stable sternum (10/38, 26%) (P=0.003). However, S. aureus was more frequent in patients with stable sternum (18/38, 47%) than in patients with sternal dehiscence (13/80, 16%) (P<0.001). High body mass index was associated with coagulase negative staphylococci (P<0.001) and with sternal dehiscence (P=0.008). Chronic obstructive pulmonary disease was also associated with sternal dehiscence (P<0.001) and with coagulase negative staphylococci (P=0.04). Patients who had been reoperated before onset of mediastinitis tended to have an increased risk for a gram-negative etiology (32 vs. 15% in patients not reoperated, P=0.06). The overall 90-day all cause mortality in patients with mediastinitis was 19%. High age, need for reoperation before mediastinitis, and a long primary operation time was associated with increased mortality (P=0.02, P=0.007 and P=0.001, respectively). No specific bacterial etiology was associated with increased mortality nor was the presence of bacteriemia. CONCLUSIONS: Three different types of postoperative mediastinitis can be distinguished: (1) mediastinitis associated with obesity, chronic obstructive pulmonary disease, and sternal dehiscence, typically caused by coagulase negative staphylococci; (2) mediastinitis following peroperative contamination of the mediastinal space, often caused by S. aureus, and (3) mediastinitis mainly caused by spread from concomitant infections in other sites during the postoperative period, often caused by gram negative rods. The proposed classification of mediastinitis into three groups with different pathogenic mechanisms may be useful in understanding which prophylactic counter measures have the potentials to be effective in a given situation.

The effect of in vivo depletion of NKR-P1+ or CD8+ lymphocytes on the acute rejection of allogeneic lymphocytes (ALC) in the rat.

We have depleted lymphocyte subsets in PVG and AO rats with MoAbs 3.2.3 (against NKR-P1 on NK and NK/T cells) and OX-8 (against CD8 on CTL and NK cells), and examined the effect on the killing of YAC-1 target cells in vitro and the effect on the acute rejection of small allogeneic lymphocytes in vivo (allogeneic lymphocyte cytotoxicity, ALC). While 3.2.3 treatment led to only a partial depletion of 3.2.3-positive cells in PVG rats, this treatment drastically reduced the number of NKR-P1+ cells in AO rats, abolished splenic NK activity against the NK-sensitive tumour target YAC-1, and markedly diminished the ALC response. Rats treated with OX-8 for 1 day showed a similar loss of NK cell function in vivo and in vitro. However, in rats treated with OX-8 for 3 days a 3.2.3+ and OX-8- population consisting of NK cells appeared, restoring ALC. The results demonstrate that NK cell responses can be greatly diminished after in vivo treatment with these MoAbs. Furthermore, they demonstrate that ALC is not necessarily linked to expression of the CD8 molecule.