Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells was entirely abrogated, while EL adhesion was significantly increased. The chemokine SDF-1alpha mediates firm adhesion c-kit+ cells only in the presence of TNF-alpha stimulation via an ICAM-1- and CXCR4-dependent mechanism. The presence of eNOS appears to be a crucial and specific factor for firm c-kit+-cell adhesion to the vascular endothelium.

Minimizing cardiopulmonary bypass attenuates myocardial damage after cardiac surgery.

The standard heart-lung machine is deemed a major trigger of systemic inflammatory reactions, potentially inducing organ failure. The strict reduction of blood-artificial surface and blood-air contact might represent meaningful improvements of the extracorporeal technology with respect to organ preservation. In this study, we assessed perioperative myocardial damage by using a novel minimal extracorporeal circuit (MECC) and a conventional cardiopulmonary bypass (CPB) system. Sixty patients scheduled for coronary artery bypass surgery were randomly assigned to either the MECC or the standard CPB system. Myocardial markers were determined by specific immunoassays 6, 12, and 24 hours after CPB initiation. Results were corrected for hemodilution.Demographics, hemodynamics, the number of anastomoses, CPB, and cross-clamp time were comparable between the groups. MECC patients demonstrated significantly lower levels of Troponin T (ng/ml) at 6, 12, and 24 hours (0.07 +/- 0.01 vs. 0.16 +/- 0.04, p < 0.005; 0.12 +/- 0.03 vs. 0.28 +/- 0.08, p < 0.008; 0.21 +/- 0.05 vs. 0.35 +/- 0.09, p < 0.03, respectively) and creatine kinase-MB (U/l) at 6 and 12 hours (22.5 +/- 1.5 vs. 40.6 +/- 3.3, p < 0.0001; 23.3 +/- 3.4 vs. 40.8 +/- 8.0, p < 0.001, respectively). Creatine kinase-MB at 24 hours tended to lower values in the MECC group but did not quite reach statistical significance. The MECC system may not only provide a less invasive solution to meet the requirements during cardiac surgery but also a more organ-preserving alternative to standard CPB.

Pericardial suction blood separation attenuates inflammatory response and hemolysis after cardiopulmonary bypass.

OBJECTIVES: Retransfusion of pericardial suction blood (PSB) is critically considered under the aspect of the biocompatibility of the cardiopulmonary bypass (CPB). We investigated various indicators of inflammation and blood cell activation associated with CPB and re-transfusion of PSB during cardiac surgery. DESIGN: Thirty-five patients undergoing elective coronary artery bypass grafting were prospectively randomized into two groups. In group A (n = 15, retransfusion group) the pericardial suction blood was continuously retransfused during CPB, in group B (n = 20, no-retransfusion group) the suction blood was separated. Parameters indicating the status of the inflammation and blood cell activation were analyzed before and at the end of CPB, latest after 90 minutes on CPB. RESULTS: Patients' perioperative data did not differ between groups. The inflammatory markers C-reactive protein, PMN-Elastase and Interleukin-6 increased in both groups after CPB (p < 0.04) with significantly lower values in the no-retransfusion group (p < 0.02). Leukocytes and platelet activation markers beta-Thromboglobulin and soluble P-Selectin also experienced a significant elevation during observation time (p < 0.02) without any difference between the groups. Free hemoglobin and LDH tremendously increased during CPB with lower values in the no-retransfusion group. CONCLUSIONS: Cardiotomy suction is a major cause of hemolysis and contributes significantly to the systemic inflammatory response.

Effects of poly-2-methoxyethylacrylate (PMEA)-coating on CPB circuits.

OBJECTIVES: In this study, the immuno- and neuroprotective effect of a novel cardiopulmonary bypass coating was investigated. DESIGN: Thirty nine patients scheduled for elective coronary artery bypass grafting were randomly assigned to either PMEA-coated (n = 19) or non-coated CPB circuits (n = 20). Pericardial suction blood was separated and retransfused only if needed at the end of operation. Neurocognitive functions were examined preoperatively and 7-10 days postoperatively using a standard neuropsychological test battery. Assuming an inflammatory etiology, the most cogent inflammatory markers were perioperatively analyzed. RESULTS: Postoperatively, patients of the PMEA-coated group performed better in Go/NoGo and Mini-Mental-test than patients of the non-coated group (p < 0.04). Other neurocognitive testing did not reveal significant differences between the groups. Although most inflammatory parameters showed a significant intraindividual increase during or shortly after CPB, there was no difference in inflammatory alteration between the groups. CONCLUSIONS: PMEA-coating of cardiopulmonary bypass surfaces revealed some minor benefits in preservation of neurocognitive functions after surgery. The immediate inflammatory response remained mostly unaffected. Suction blood separation may additionally contribute to proper postoperative outcome.

Circulating endothelial cells demonstrate an attenuation of endothelial damage by minimizing the extracorporeal circulation.

OBJECTIVE: Detachment of endothelial cells may represent serious injury of the endothelium after cardiopulmonary bypass. We investigated whether the extent of endothelial injury is related to the type of cardiopulmonary bypass system used (conventional or minimized) and determined circulating endothelial cells as well as von Willebrand factor and soluble thrombomodulin. METHODS: Twenty patients scheduled for elective coronary bypass grafting were randomly assigned to either the minimal extracorporeal circulation system or the standard cardiopulmonary bypass. Ten healthy volunteers served as controls. Circulating endothelial cells per milliliter of full blood were perioperatively determined by immunomagnetic cell separation technique. Endothelial plasma markers were measured by enzyme-linked immunosorbent assay. RESULTS: Preoperative circulating endothelial cell numbers did not differ between the experimental groups, but were significantly higher than in the healthy controls (18.6 +/- 5.6 vs 7.2 +/- 3.8, P < .001). At 6 hours, circulating endothelial cell numbers increased significantly compared with baseline in both experimental groups and peaked at 12 hours after cardiopulmonary bypass initiation, each time with significantly lower values in the minimal extracorporeal circulation group (6 hours: 44.0 +/- 9.9 vs 29.6 +/- 9.8, P = .007; 12 hours: 48.1 +/- 6.8 vs 31.8 +/- 7.1, P < .001). Likewise, von Willebrand factor and soluble thrombomodulin postoperatively increased in both groups with a tendency toward lower levels in the minimal extracorporeal circulation group. Although circulating endothelial cells gradually declined, continually with lower numbers in the minimal extracorporeal circulation group, the endothelial plasma markers remained elevated during observation time. CONCLUSIONS: Circulating endothelial cells represent a novel marker of the intrinsic endothelial damage caused by cardiopulmonary bypass. Its analysis facilitates the evaluation of cardiopulmonary bypass modifications as the minimal extracorporeal circulation system could be proven to be less injurious to endothelium and myocardium.

Evaluation of coated oxygenators in cardiopulmonary-bypass systems and their impact on neurocognitive function.

INTRODUCTION: Coronary artery bypass graft surgery (CABG) using cardiopulmonary bypass (CPB) is assumed to be associated with a decline of neurocognitive functions. This study was designed to analyse the neurocognitive function of patients with coronary heart disease before and after CABG and to determine possible protective effects of oxygenator surface coating on neurological outcome. METHODS: Forty patients scheduled for selective CABG were prospectively randomized into two groups of 20 patients each according to the type of hollow-fibre membrane oxygenator used. Non-coated oxygenators (Group A) were compared to phosphorylcholine (PC)coated oxygenators (Group B). A battery of six neurological tests was administered preoperatively, 7-10 days and 4-6 months after surgery. RESULTS: One patient of Group A suffered from a perioperative stroke and died on postoperative day 3, presumably because of sudden heart failure. Two patients of Group A (10%) developed a symptomatic transitory delirious psychotic syndrome (STPT) on postoperative days 3 and 5. None of the patients of Group B had perioperative complications. The test analysis revealed a trend of declined neurocognitive function early after CABG, but did not show any difference in neurocognitive outcome between the two groups. DISCUSSION: PC coating of the oxygenators did not show any significant benefit on neurocognitive function after CABG using CPB.

Placement of a left ventricular assist device in a patient with dextrocardia.

Dextrocardia most commonly presents in the setting of situs inversus, but it may occur as an isolated anomaly with normal position of the abdominal organs. Herein we present a 54-year-old man with ischemic cardiomyopathy and dextrocardia with normal position of the abdominal organs who presented with an exacerbation of congestive heart failure requiring inotropic support as well as mechanical ventilation. An implantable, wearable left ventricular assist device was placed in this patient to allow for ambulation and eventual discharge home. The patient survived 4 months before he developed pneumonia and expired.

Plasma neurohormone levels correlate with left ventricular functional and morphological improvement in LVAD patients.

OBJECTIVE: Brain natriuretic peptide (BNP) and endothelin-1 (ET-1) have been shown to be markers of left ventricular (LV) function. To determine the feasibility of using serial assays of these neurohormones in the assessment of cardiac status in the left ventricular assist device (LVAD) setting, we examined the relationship between LV function, myocardial morphology, and plasma levels of these hormones in LVAD recipients. METHODS: Plasma BNP and ET-1 levels were serially assayed in 19 end-stage congestive heart failure (CHF) patients before and after LVAD implantation with various devices (i.e., MicroMed DeBakeyVAD/DVAD, Novacor/NVAD, TCI Heartmate/TCI, Thoratec/TVAD). Echocardiography performed correspondingly at the time points of the hormonal assays and immunohistochemical collagen staining of left ventricular tissue samples, derived from six non-failing hearts as well as from LVAD patients at the time of device insertion and removal, were then contrasted. Patients were grouped according to device used and etiology of heart disease (ischemic or dilated cardiomyopathy, ICM/DCM). RESULTS: LVAD therapy significantly improved LV ejection fraction (EF%: 21 +/- 3.8% to 28.11 +/- 3.57%), cardiac output (CO: 3.49 +/- 1.3 to 7.3 +/- 0.2 l/m), and left ventricular end-diastolic diameter (LVEDD: 6.68 +/- 0.92 versus 4.79 +/- 1.54 cm, P < 0.0001) in all patients. Absolute BNP and ET-1 plasma levels remained significantly lower in all patients after LVAD implantation (both P < 0.001). The NVAD group exhibited the most BNP reduction and EF% increase (P < 0.0004 and P < 0.038, respectively). Average collagen levels were reduced in all patients (P < 0.0005). Among the devices, the NVAD group demonstrated the most evident change (P < 0.0036), while there was comparable reduction in the DCM and ICM groups (both P < 0.03). In general, postoperative BNP and ET-1 trends exhibited a notable parallelism with both manifesting bi-phasic tendencies and an inverse proportionality to corresponding EF% measurements. CONCLUSIONS: Device selection appears to influence the cardiac morphological and neurohormonal expressive tendencies exhibited by recipients. Plasma BNP and ET-1 levels correlate with both LV function and myocardial morphological improvement. Alterations in the levels of these hormones during LVAD support may be real-time indicators of prevailing myocardial autocrine/paracrine activity and as such may be of potential use in future algorithms of cardiac assessment and therapeutic decision-making with regard to transplant urgency and/or possible device explantation.

Organ-specific regulation of pro-inflammatory molecules in heart, lung, and kidney following brain death.

BACKGROUND: Nonspecific inflammatory events following brain death may increase the intensity of the immunological host response. The present study investigated the course of pro-inflammatory molecules in heart, lung, kidney, and plasma after brain death induction. MATERIALS AND METHODS: Brain death was induced in five pigs by inflation of an intracranial Foley catheter and five pigs were sham-operated as controls. Each experiment was terminated 6 h after brain death/sham operation and the organs were harvested. We measured the mRNA and protein levels for TNF-alpha, IL-1beta, and IL-6 in heart, lung, kidney, and plasma. Additionally, the mRNA expression for IL-6R, ICAM-1, MCP-1, and TGF-beta was determined in each organ. RESULTS: After 6 h, the plasma cytokine levels were higher in the brain-dead animals than in the sham-operated. In heart, lung, and kidney there was an increase in IL-6 and IL-1beta following brain death, while TNF-alpha was up-regulated in lung only (P < 0.05). MCP-1 and TGF-beta were significantly higher in heart and lung and IL-6R increased in heart after brain death (P < 0.05). CONCLUSIONS: Brain death was associated with non-uniform cytokine expression patterns in the investigated organs. These expression patterns may cause variable pro-inflammatory priming resulting in different degrees of damage and explain the organ-specific variation in outcomes after transplantations.

Role of mast cells and their mediators in failing myocardium under mechanical ventricular support.

BACKGROUND: Mast cells have been implicated in tissue remodeling and fibroblast stimulation. We explored the effect of mechanical support by left ventricular assist device (LVAD) in failing myocardium and looked into grade and distribution of interstitial fibrosis, mast cell density, mast cell phenotypes and basic fibroblast growth factor (bFGF) expression pre- and post-LVAD. METHODS: Myocardial tissue was obtained from 20 patients with end-stage cardiomyopathy at the time of LVAD implantation and LVAD removal and from 7 donor hearts not used for transplantation. Tissue sections were stained for mast cells using tryptase as a marker and the myocardial fibrosis was measured. Double staining for tryptase and chymase was performed for detection of chymase-positive mast cells. Fluorescent microscopy showed the relationship of mast cells to bFGF, and bFGF expression was quantified by Western blot. RESULTS: There was a significant increase in mast cells in heart failure vs normal myocardium. A secondary increase in mast cells occurred after long-term (>40 days) support compared with matched pre-LVAD samples (mean +/- SEM; 57.4 +/- 8.6 cells/10 fields vs 45.1 +/- 7.6 SEM cells/10 fields, p < 0.01). The secondary increase in mast cells was associated specifically with an increase in chymase-negative mast cells (p < 0.01). These findings are statistically significant with concurrent decreased expression of bFGF and decreased fibrosis in the same patient tissues (p < 0.01). CONCLUSIONS: We suggest that, under long-term support, there is a change in phenotypic expression in mast cells, which can alter fibroblast functions. The decreased myocardial bFGF levels might be the result of these phenotypically altered mast cells.

Interaction between isolated human myocardial mast cells and cultured fibroblasts.

INTRODUCTION: Previously, we reported an increase in interstitial collagen and total mast-cell numbers in heart failure versus normal myocardium. A secondary increase, primarily in chymase-negative mast cells, occurred following LVAD support compared to matched pre-LVAD tissue samples and was associated with a decrease in interstitial collagen and bFGF. To further elucidate the changes in interstitial collagen, we investigated the direct interaction between mast cells, isolated from failing myocardium with or without previous LVAD support, and human fibroblasts in a coculture model. Additionally, the expression of HSP-47, the pro-collagen-specific chaperone protein, was determined in the particular myocardium. MATERIALS AND METHODS: Myocardial tissue was obtained from 10 patients with end-stage dilated cardiomyopathy (DCM) at the time of transplantation. Five patients were transplanted following LVAD support, five patients without previous LVAD support. Mast cells were isolated according to a standard protocol, including collagenase digestion and cell separation. The isolated mast cells were co-cultured with human fibroblasts for 12 h, with or without stimulation of degranulation, and protein synthesis was measured by [(3)H]-proline incorporation. HSP-47 immunostaining was performed in the different myocardial samples and the positive cells were quantified. RESULTS: Stimulated mast cells isolated from DCM tissue (without previous LVAD support) caused a 92% increase in [(3)H]-proline incorporation and consequently in protein production in fibroblasts compared to mast-cell free culture (P < 0.01), while conversely stimulated mast cells isolated from LVAD supported myocardium decreased the [(3)H]-proline incorporation by 63% (P < 0.01) below baseline. Nonstimulated mast cells did not significantly alter the protein production over baseline. There was also a significant increase in the number of HSP-47-positive cells in DCM myocardium compared to normal (P < 0.01) and there was a shift toward normal after LVAD support (P < 0.01). CONCLUSION: We demonstrate that fibroblast protein production in vitro is significantly altered by mast cells and that the direction of change is dependent on whether myocardium was supported by LVAD. We suggest that under long-term LVAD support there is a phenotypic alteration in myocardial mast cells, which leads to a change in concentration and/or composition of mediators, capable of re-remodeling the myocardial matrix.