Rene Schramm

RING+STRING Successful Repair Technique for Ischemic Mitral Regurgitation With Severe Leaflet Tethering

Background— Residual/recurrent mitral valve regurgitation is observed in 30% after undersized ring annuloplasty (RING) for ischemic mitral regurgitation (IMR). RING addresses primarily annular dilatation but does not correct severe leaflet tethering attributable to papillary muscle (PM) displacement. We proposed adjunctive PM repositioning under transesophageal echocardiography (TEE) guidance in the loaded beating heart using a transventricular suture (STRING). Methods and Results— Patients with tenting height ≥10 mm were identified as high-risk patients for repair failure. In these patients (n=30, age 68±11 years, ejection fraction 37±14%), RING (partial, median 29 mm) was combined with the adjunctive STRING-technique. A Teflon-pledgeted 3-0-polytetrafluoroethylene-suture was anchored in the posterior PM via horizontal aortotomy, exteriorized through the aorto-mitral continuity, and tied in the loaded beating heart under TEE guidance. Tenting height (14±2 mm versus 6±1 mm, P<0.001) and tenting area (3.9±0.9 cm2 versus 1.0±0.2 cm2, P<0.001) decreased. The distance between pPM and aorto-mitral continuity decreased (44±4 mm versus 37±3 mm, P<0.001). Survival at 2 years was similar compared with a historical matched control-group (89% versus 73%, P=0.13), whereas freedom from MR>II was higher in the RING+STRING-group (94% versus 71%, P=0.01). End-diastolic (61.7±7.2 mm versus 54.8±9.2 mm, P<0.001) and end-systolic (48.5±8.5 mm versus 42.7±7.8 mm, P=0.002) ventricular diameters decreased in the RING+STRING-group but persisted in the control-group (60.4±7.8 mm versus 58.9±7.5 mm, P=0.38; 47.8±9.6 mm versus 48.3±9.5 mm, P=0.52). During follow-up (median 26 months) only 1 patient of the study-group required reoperation for degenerative MR, while 2 control-group patients underwent reoperation for recurrent functional MR. Conclusions— Our novel approach for IMR attenuates high risk of repair failure in patients with severe leaflet tethering and results in reverse remodeling.

A statin‐based inhibitor of lymphocyte function antigen‐1 protects against ischemia/reperfusion‐induced leukocyte adhesion in the colon

Statins are mainly used to control hypercholesterolemia; however, recent studies have also ascribed anti‐inflammatory effects to the statins. LFA703 is a novel statin‐derived compound, which potently inhibits lymphocyte function antigen‐1 (LFA‐1, CD11a/CD18) but does not affect HMG‐CoA reductase activity. The objective of this study was to examine the anti‐inflammatory mechanisms of LFA703 in ischemia/reperfusion (I/R)‐induced leukocyte–endothelium interactions in the colon. For this purpose, the superior mesenteric artery was occluded for 30 min and leukocyte responses were analyzed in colonic venules after 120 min of reperfusion in mice using inverted intravital fluorescence microscopy. First, the inhibitory mechanisms of LFA703 on leukocyte adhesion were investigated in vitro using a mouse CD4+8+ thymocyte cell line. Immunoneutralization of LFA‐1 and ICAM‐1 abolished leukocyte adhesion, whereas inhibition of VLA‐4 had no effect in this in vitro assay. Indeed, it was found that LFA703 dose‐dependently reduced LFA‐1‐dependent leukocyte adhesion to mouse endothelial cells in vitro with an IC50 of 3.2 μM. I/R caused an increase in leukocyte rolling and adhesion in colonic venules. Immunoneutralization of LFA‐1 significantly reduced I/R‐induced leukocyte adhesion by 89% in colonic venules. In contrast, I/R‐provoked leukocyte rolling was insensitive to inhibition of LFA‐1 function. Administration of 30 mg kg−1 of LFA703 decreased reperfusion‐induced leukocyte adhesion by more than 91%, while the level of leukocyte rolling was unchanged, suggesting that LFA703 effectively blocked LFA‐1‐dependent firm adhesion of leukocyte in the colon. However, LFA703 did not decrease the expression of LFA‐1 on circulating leukocytes. This study demonstrates that LFA‐1 is indeed a critical adhesion molecule in mediating postischemic leukocyte adhesion in the colon. Moreover, this is the first study showing that a statin‐based synthetic compound has the capacity to abolish LFA‐1‐dependent leukocyte adhesion in I/R. These novel findings may have great implications in the clinical treatment of conditions associated with I/R‐induced tissue injury, such as organ transplantation, trauma and major surgery.

p38 Mitogen-Activated Protein Kinase-Dependent Chemokine Production, Leukocyte Recruitment, and Hepatocellular Apoptosis in Endotoxemic Liver Injury

Objective:To determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in endotoxin-induced liver injury. Background:MAPKs have been reported to play a potential role in regulating inflammatory responses, but the role of p38 MAPK signaling in chemokine production, leukocyte recruitment, and hepatocellular apoptosis in the liver of endotoxemic mice is not known. Methods:Endotoxin-induced leukocyte-endothelium interactions were studied by use of intravital fluorescence microscopy in the mouse liver. Tumor necrosis factor-α (TNF-α) and CXC chemokines, liver enzymes, and apoptosis were determined 6 hours after endotoxin challenge. The specific p38 MAPK inhibitor SB 239063 was given immediately prior to endotoxin exposure. Phosphorylation and activity of p38 MAPK were determined by immunoprecipitation and Western blot. Results:Endotoxin increased phosphorylation and activity of p38 MAPK in the liver, which was markedly inhibited by SB 239063. Inhibition of p38 MAPK signaling dose-dependently decreased endotoxin-induced leukocyte rolling, adhesion, and sinusoidal sequestration of leukocytes. SB 239063 markedly reduced endotoxin-induced formation of TNF-α and CXC chemokines in the liver. Indeed, the endotoxin-provoked increase of liver enzymes and hepatocellular apoptosis were abolished and sinusoidal perfusion was restored in endotoxemic mice treated with SB 239063. Conclusions:This study demonstrates that p38 MAPK signaling plays an important role in regulating TNF-α and CXC chemokine production in endotoxemic liver injury and that inhibition of p38 MAPK activity abolishes endotoxin-induced leukocyte infiltration as well as hepatocellular apoptosis. These novel findings suggest that interference with the p38 MAPK pathway may constitute a therapeutic strategy against septic liver damage.

Staphylococcal Enterotoxin B-Induced Acute Inflammation Is Inhibited by Dexamethasone: Important Role of CXC Chemokines KC and Macrophage Inflammatory Protein 2

ABSTRACT This study was conducted to examine the anti-inflammatory mechanisms of dexamethasone during leukocyte recruitment and expression of the CXC chemokines macrophage inflammatory protein 2 (MIP-2) (CXCL2) and cytokine-induced neutrophil chemoattractant (KC) (CXCL1) in staphylococcal enterotoxin B (SEB)-induced acute inflammation. To do this, SEB was injected into murine air pouches with or without dexamethasone pretreatment for 2 h. SEB induced infiltration of leukocytes in a dose- and time-dependent manner, with the maximal response observed after 4 h of treatment with 10 μg of SEB. The recruited leukocytes comprised more than 77% neutrophils. Moreover, SEB challenge (10 μg) provoked time-dependent secretion of CXC chemokines, which peaked after 1 h. Local administration of antibodies against MIP-2 and KC significantly reduced SEB-triggered neutrophil accumulation by 38 and 59%, respectively. Dexamethasone (10 mg kg−1) significantly decreased neutrophil recruitment by 82% and reduced secretion of MIP-2 and KC by 89 and 85%, respectively, in response to SEB challenge. Our data demonstrate that dexamethasone potently inhibits neutrophil recruitment in SEB-induced inflammation. Moreover, we provide evidence that MIP-2 and KC are key mediators in the neutrophil response to SEB. Furthermore, our findings demonstrate that dexamethasone attenuates SEB-induced expression of MIP-2 and KC. Thus, this study elucidates important signaling pathways of SEB-induced neutrophil recruitment and anti-inflammatory mechanisms of action of dexamethasone.

Fundamental and distinct roles of P-selectin and LFA-1 in ischemia/reperfusion-induced leukocyte-endothelium interactions in the mouse colon

ObjectiveTo study the adhesive mechanisms underlying ischemia/reperfusion (I/R)-induced leukocyte–endothelium interactions in the colon. Summary Background DataLeukocyte recruitment is a key feature in I/R-induced tissue injury, but the mechanisms regulating leukocyte rolling and adhesion in the colon are not known. The authors recently developed a new model to study the molecular mechanisms of I/R-provoked leukocyte–endothelium interactions in the colon microcirculation using inverted intravital fluorescence microscopy. MethodsThe superior mesenteric artery was occluded for 30 minutes and leukocyte responses were analyzed after 120 minutes of reperfusion in colonic venules in mice. The adhesive mechanisms underlying I/R-induced leukocyte rolling and adhesion were investigated using monoclonal antibodies against L-, E- and P-selectin, and CD11a gene-targeted mice were used to examine the role of lymphocyte function antigen-1 (LFA-1, CD11a/CD18). ResultsReperfusion provoked a clear-cut increase in leukocyte rolling and adhesion in colonic venules compared to negative controls. Both P- and E-selectin mRNA were expressed in the colon after this I/R insult. Pretreatment with an anti-P-selectin antibody reduced leukocyte rolling and adhesion by 88% and 85%, respectively, whereas antibodies against L- and E-selectin had no effect. Moreover, I/R-induced leukocyte adhesion in LFA-1-deficient mice was reduced by more than 95%. ConclusionsThis study provides evidence that leukocyte rolling is exclusively and nonredundantly mediated by P-selectin and that firm adhesion is supported by LFA-1 in I/R-induced leukocyte recruitment in the colon. Taken together, both P-selectin and LFA-1 may be important targets to control pathologic inflammation in I/R-induced tissue injury in the colon.

Heat Shock Preconditioning Reduces Ischemic Tissue Necrosis by Heat Shock Protein (hsp)-32-mediated Improvement of the Microcirculation Rather Than Induction of Ischemic Tolerance

Introduction:Supraphysiologic stress induces a heat shock response, which may exert protection against ischemic necrosis. Herein we analyzed in vivo whether the induction of heat shock protein (HSP) 32 improves survival of chronically ischemic myocutaneous tissue, and whether this is based on amelioration of microvascular perfusion or induction of ischemic tolerance. Methods:The dorsal skin of mice was subjected to local heat preconditioning (n = 8) 24 hours before surgery. In additional heat-preconditioned animals (n = 8), HSP-32 was inhibited by tin-protoporphyrin-IX. Unconditioned animals served as controls (n = 8). A random-pattern myocutaneous flap was elevated in the back of the animals and fixed into a dorsal skinfold chamber. The microcirculation, edema formation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day period using intravital fluorescence microscopy. Results:HSP-32 protein expression was observed only in heat-preconditioned but not in unconditioned flaps. Heat preconditioning induced arteriolar dilation, which was associated with a significant improvement of both arteriolar blood flow and capillary perfusion in the distal part of the flap. Further, heat shock reduced interstitial edema formation, attenuated apoptotic cell death, and almost completely abrogated the development of flap necrosis (4% ± 1% versus controls: 53% ± 5%; P[r] < 0.001). Most strikingly, inhibition of HSP-32 by tin-protoporphyrin-IX completely blunted the preconditioning-induced improvement of microcirculation and resulted in manifestation of 72% ± 4% necrosis. Conclusion:Local heat preconditioning of myocutaneous tissue markedly increases flap survival by maintaining adequate nutritive perfusion rather than inducing ischemic tolerance. The protection is caused by the increased arteriolar blood flow due to significant arteriolar dilation, which is mediated through the carbon monoxide-associated vasoactive properties of HSP-32.

Erythropoietin protects critically perfused flap tissue

Objective:The objective of this study was to analyze whether erythropoietin (EPO) protects from necrosis of critically perfused musculocutaneous tissue and the mechanisms by which this protection is achieved. Background:EPO is the regulator of erythropoiesis and is used to treat patients with anemia of different causes. Recent studies suggest that EPO has also other tissue-protective effects, irrespective of its erythropoietic properties. Material and Methods:C57BL/6-mice were treated with 3 doses of EPO at 500 IU/kg intraperitoneally. EPO was given either before (preconditioning, n = 7), before and after (overlapping treatment, n = 7), or after (treatment, n = 7) surgery. Animals receiving only saline served as controls (CON). Acute persistent ischemia was induced by elevating a randomly perfused flap in the back of the animals. This critically perfused tissue demonstrates an initial microvascular failure of ∼40%, resulting in ∼50% tissue necrosis if kept untreated. Repetitive fluorescence microscopy was performed over 10 days, assessing angiogenesis, functional capillary density, inflammatory leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis. Additional molecular tissue analyses included the determination of inducible nitric oxide synthase, erythropoietin receptor (EPO-R), and vascular endothelial growth factor (VEGF). Results:EPO preconditioning did not affect hematocrit and EPO-R expression, but increased inducible nitric oxide synthase in the critically perfused tissue. This correlated with a significant arteriolar dilation, which resulted in a maintained functional capillary density (CON: 0 ± 0 cm/cm2; preconditioning: 37 ± 21 cm/cm2; overlapping treatment: 72 ± 26 cm/cm2; P < 0.05). EPO pretreatment further significantly reduced microvascular leukocyte adhesion and apoptotic cell death. Moreover, EPO pretreatment induced an early VEGF upregulation, which resulted in new capillary network formation (CON: 0 ± 0 cm/cm2; preconditioning: 40 ± 3 cm/cm2; overlapping treatment: 33 ± 3 cm/cm2; P < 0.05). Accordingly, EPO pretreatment significantly reduced tissue necrosis (CON: 48% ± 2%; preconditioning: 26% ± 3%; overlapping treatment: 20% ± 3%; P < 0.05). Of interest, EPO treatment was only able to alleviate ischemia-induced inflammation but could not improve microvascular perfusion and tissue survival. Conclusions:EPO pretreatment improves survival of critically perfused tissue by nitric oxide -mediated arteriolar dilation, protection of capillary perfusion, and VEGF-initiated new blood vessel formation.

Improvement of microvascular graft equilibration and preservation in non-heart-beating donors by warm preflush with streptokinase.

Using in situ fluorescence microscopy with Sprague Dawley rats, we studied the hypothesis of compromised microvascular kidney perfusion on organ harvest in non-heart-beating donors (NHBDs), and we evaluated the potential benefit of an additional preflush with saline solution containing streptokinase. Aortal flush of NHBD kidneys solely with University of Wisconsin solution resulted in a significantly (P <0.05) reduced functional capillary density (FCD) with increased perfusion heterogeneity compared with kidneys of heart-beating controls. This was associated with an increased lactate dehydrogenase (LDH) release on 24 hr postpreservation rinse of the grafts (76.7±18.9 U/L). Warm preflush with low-viscosity Ringer’s lactate (RL) solution alone did not influence the decreased renal FCD and the postpreservation LDH release (76.2±29.1 U/L). In contrast, the addition of streptokinase to the RL preflush solution resulted in a significant (P <0.05) improvement of FCD with values not statistically different from those of heart-beating controls. This was associated with an attenuation of perfusion heterogeneity and a significantly lowered postpreservation LDH release (17.0±2.5 U/L). Furthermore, in transplanted and reperfused NHBD kidney grafts, the use of streptokinase-supplemented RL for preflush during organ harvest significantly (P <0.05) reduced early manifestation of tubular necrosis (29%±8%) when compared with kidneys preflushed exclusively with University of Wisconsin solution (56%±4%). Thus, we conclude that kidney harvest from NHBDs is prone to severe microvascular perfusion deficits, which are likely to preclude successful preservation of organ integrity during cold storage. Temporary fibrinolytic preflush with streptokinase may represent a feasible tool to improve microvascular graft equilibration, which effectively protects the renal integrity during both cold storage and posttransplant reperfusion.

Statins inhibit lymphocyte homing to peripheral lymph nodes.

Lymphocyte homing to peripheral lymph nodes is governed by adhesion molecules, including lymphocyte function‐associated antigen 1 (LFA‐1). Statins are 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors and exert anti‐inflammatory effects, e.g. inhibition of LFA‐1. It is still not known whether statin compounds are capable of inhibiting lymphocyte homing in vivo. We used a cervical lymph node preparation to study the effects of simvastatin on lymphocyte adhesion to high endothelial venules (HEVs) by means of intravital fluorescence microscopy (IVM). IVM revealed that firm adhesion of lymphocytes to HEV endothelium critically depends on the adhesive function of LFA‐1. The number of firmly adherent lymphocytes was reduced by 58% in LFA‐1‐deficient mice (P < 0·05 versus wild‐type controls). As in mutant mice, acute treatment with simvastatin (i.p. injection at 2 hr prior to IVM) inhibited the firm adhesion of lymphocytes to HEV endothelium of wild‐type animals by 63% (P < 0·05 versus vehicle‐treated wild‐type controls). In addition, acute treatment with the synthetic statin‐derivate LFA878 also reduced firm lymphocyte adhesion in HEVs by 63% (P > 0·05 versus placebo‐treated controls). Histological analysis after a 10‐day treatment with simvastatin showed reduced cellularity of cervical lymph nodes, as indicated by a reduction of the relative area of haematoxylin‐stained cell nuclei in cervical lymph node cross‐sections from 94 ± 0% in vehicle‐treated controls to 77 ± 3% in simvastatin‐treated mice (P < 0·05). We conclude that statin compounds are capable of inhibiting lymphocyte homing to murine peripheral lymph nodes in vivo. This may have novel implications for the treatment of adaptive immune responses, e.g. transplant rejection.

Intraoperative inhalation of the long-acting prostacyclin analog iloprost for pulmonary hypertension

Pulmonary hypertension (PHT) contributes significantly to morbidity and mortality in cardiac surgery—especially in patients with long-standing mitral valve disease. Vasodilators administered intravenously can reduce pulmonary vascular resistance but also have an effect on the systemic circulation. Arterial hypotension must be expected, which results in reduction of right coronary blood flow and may lead to further deterioration of right ventricular performance. Inhaled nitric oxide (NO) is a more selective pulmonary vasodilator with virtually no systemic side effects. Recently, the inhalational use of the long-acting prostacyclin analog iloprost has been reported to offer superior selective pulmonary vasodilating properties relative to NO inhalation in primary PHT. We report our initial experience with the intraoperative use of iloprost inhalation in a case series of cardiac surgical patients.