Murat Ugurlucan

Is the intravascular administration of mesenchymal stem cells safe?: Mesenchymal stem cells and intravital microscopy

We investigated the kinetics of human mesenchymal stem cells (MSCs) after intravascular administration into SCID mouse cremaster vasculature by intravital microscopy. MSCs were injected into abdominal aorta through left femoral artery at two different concentrations (1 x 10(6) or 0.2 x 10(6) cell). Arterial blood velocity decrease by 60 and 18% 1 min after high/low dose MSCs injection respectively. The blood microcirculation was interrupted after 174+/-71 and 485+/-81 s. Intravital microscopy observation and histopathologic analysis of cremaster muscles indicated MSCs were entrapped in capillaries in both groups. 40 and 25% animals died of pulmonary embolism respectively in both high and low MSCs dose groups, which was detected by histopathologic analysis of the lungs. Intraarterial MSCs administration may lead to occlusion in the distal vasculature due to their relatively large cell size. Pulmonary sequestration may cause death in small laboratory animals. MSCs should be used cautiously for intravascular transplantation.

Intracardiac injection of erythropoietin induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Erythropoietin (EPO) protects the myocardium from ischaemic injury and promotes beneficial remodelling. We assessed the therapeutic efficacy of intracardiac EPO injection and EPO‐mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, EPO (3000 U/kg) or saline was delivered by intracardiac injection. Compared to myocardial infarction control group (MIC), EPO significantly improved left ventricular function (n= 11–14, P< 0.05) and decreased right ventricular wall stress (n= 8, P< 0.05) assessed by pressure‐volume loops after 6 weeks. MI‐EPO hearts exhibited smaller infarction size (20.1 ± 1.1%versus 27.8 ± 1.2%; n= 6–8, P< 0.001) and greater capillary density (338.5 ± 14.7 versus 259.8 ± 9.2 vessels per mm; n= 6–8, P< 0.001) than MIC hearts. Direct EPO injection reduced post‐MI myocardial apoptosis by approximately 41% (0.27 ± 0.03%versus 0.42 ± 0.03%; n= 6, P= 0.005). The chemoattractant SDF‐1 was up‐regulated significantly assessed by quantitative realtime PCR and immunohistology. c‐Kit+ and CD34+ stem cells were significantly more numerous in MI‐EPO than in MIC at 24 hrs in peripheral blood (n= 7, P< 0.05) and 48 hrs in the infarcted hearts (n= 6, P< 0.001). Further, the mRNAs of Akt, eNOS and EPO receptor were significantly enhanced in MI‐EPO hearts (n= 7, P< 0.05). Intracardiac EPO injection restores myocardial functions following MI, which may attribute to the improved early recruitment of c‐Kit+ and CD34+ stem cells via the enhanced expression of chemoattractant SDF‐1.

A transformed cell population derived from cultured mesenchymal stem cells has no functional effect after transplantation into the injured heart.

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent cells characterized by their self-renewal and differentiation potential. Accumulating clinical and preclinical evidence indicate MSCs are a promising cell source for regenerative medical therapies. However, undesirable immortalization, spontaneous transformation, and tumorigenic potential from long-term cultured MSCs have been reported in human and mouse. We report rat MSCs isolated from young donors could undergo transformation in early passage culture. We aimed to characterize the transformed population and determine their therapeutic effects after intracardiac transplantation in the infarcted myocardium. MSCs were isolated from bone marrow of Lewis rats according to standard protocols and cultured under standard conditions. Phenotype of growing cells was assessed by flow cytometry. Following acute myocardial infarction in rats, cells were delivered by intracardiac injection. Cardiac functions were assessed by pressure–volume loops. Infarction size and pathologic effects were evaluated after 6 weeks. The abnormal colonies were detected in culture as early at passage 3. They were noted to appear as distinctly different morphology from typical MSCs, which changed from a normal elongated spindle shape to a compact abnormal morphology. They exhibited rapid cell proliferation. Some subclones lost contact inhibition of cell division and formed multilayer aggregates. Chromosomal instability was detected. They were devoid of surface markers CD29, CD44, CD90, and CD117. Furthermore, there was no significant improvement on infarction size and cardiac function 6 weeks after cell transplantation. Our study highlights the need for establishment of biosafety criteria in regulating culture-expanded MSCs to achieve the full clinical therapeutic benefits.

Enhanced thoracic gene delivery by magnetic nanobead-mediated vector

Systemic gene delivery is limited by the adverse hydrodynamic conditions on the collection of gene carrier particles to the specific area. In the present study, a magnetic field was employed to guide magnetic nanobead (MNB)/polymer/DNA complexes after systemic administration to the left side of the mouse thorax in order to induce localized gene expression.

Autologous umbilical cord blood mononuclear cell transplantation preserves right ventricular function in a novel model of chronic right ventricular volume overload.

We aimed to evaluate the feasibility and efficacy of autologous umbilical cord blood mononuclear cell (UCMNC) transplantation on right ventricular (RV) function in a novel model of chronic RV volume overload. Four-month-old sheep (n = 20) were randomized into cell (n = 10) and control groups (n = 10). After assessment of baseline RV function by the conductance catheter method, a transannular patch (TAP) was sutured to the right ventricular outflow tract (RVOT). Following infundibulotomy the ring of the pulmonary valve was transected without cardiopulmonary bypass. UCMNC implantation (8.22 ± 6.28 × 107) in the cell group and medium injection in the control group were performed into the RV myocardium around the TAP. UCMNCs were cultured for 2 weeks after fluorescence-activated cell sorting (FACS) analysis for CD34 antigen. Transthoracic echocardiography (TTE) and computed tomography were performed after 6 weeks and 3 months, respectively. RV function was assessed 3 months postoperatively before the hearts were excised for immunohistological examinations. FACS analysis revealed 1.2 ± 0.22% CD34+ cells within the isolated UCMNCs from which AcLDL+ endothelial cells were cultured in vitro. All animals survived surgery. TTE revealed grade II–III pulmonary regurgitation in both groups. Pressure-volume loops under dobutamine stress showed significantly improved RV diastolic function in the cell group (dP/dtmin: p = 0.043; Eed: p = 0.009). CD31 staining indicated a significantly enhanced number of microvessels in the region of UCMNC implantation in the cell group (p < 0.001). No adverse tissue changes were observed. TAP augmentation and pulmonary annulus distortion without cardiopulmonary bypass constitutes a valid large animal model mimicking the surgical repair of tetralogy of Fallot. Our results indicate that the chronically volume-overloaded RV profits from autologous UCMNC implantation by enhanced diastolic properties with a probable underlying mechanism of increased angiogenesis.

Thrombocytopenia after aortic valve replacement with the Freedom Solo stentless bioprosthesis.

Stentless bioprostheses have been considered to achieve superior hemodynamics over stented bioprostheses for aortic valve replacement with improved long-term performance. We observed severe thrombocytopenia in patients who received the Sorin Freedom Solo aortic stentless pericardial bioprosthesis within the first days after implantation. Absolute and relative platelet counts within 2 weeks after implantation of either a stentless (Sorin Freedom Solo) or a stented (Sorin Mitroflow) bovine pericardial bioprosthesis were compared in a matched-pairs analysis in 40 patients. Except the preoperative values, absolute platelet count was higher at all time points in the Mitroflow group. In the Mitroflow group, the mean platelet count moderately dropped to a minimum of 60% of the initial value on POD 3 and fully recovered by POD 8. In the Freedom Solo group, platelet loss was significantly more severe (minimum relative value 25% on POD 4) with no recovery during follow-up (60% on POD 13). Eight patients of the Freedom Solo group experienced a critical platelet drop towards <20% of their initial values, in five of them absolute numbers decreased below 30,000/microl. No bleeding complications or other morbidity occurred. Attention should focus on the platelet count after implantation of the Freedom Solo bioprosthesis, especially in patients who are supposed to receive platelet inhibitors. However, the described phenomenon remains unexplained.

Open Heart Surgery in Patients With Sickle Cell Hemoglobinopathy

Background— In patients with sickle cell trait or disease, reduced life expectancy and a tendency for complications are believed to negatively affect likelihood of survival after open heart surgery. The aim of this study was to review retrospectively the perioperative results of patients undergoing cardiac surgery at our institution. Methods and Results— Between January 1995 and December 2006, 47 patients with either sickle cell disease or sickle cell trait underwent open heart surgery at our institution. The average age of the 29 male and 18 female patients was 20 years. Patient outcomes were analyzed through the use of the institutional database. Clinical and echocardiographic follow-up was complete in all patients except 3, with a mean follow-up period of 46 months. Current status could be confirmed in 32 patients. The most common operations included the treatment of congenital and valvular heart diseases. There were no coronary artery bypass grafting procedures. Average weight of the patients was 45 kg. Exchange transfusion was performed both preoperatively and during surgery. Mean preoperative hemoglobin S concentration was 30.4±3.2% and decreased to 8.1±2.6% while on pump. Average on-pump hematocrit value was 25.4±3.7%; in the postoperative period, it increased to 32.7±4.9%. Mean cardiopulmonary bypass and cross-clamp times were 95 and 69 minutes, respectively. None of the patients had sickling crisis or acidosis. Postoperative complications included exploration for hemorrhage in 3 patients (6.4%), stroke in 2 patients (4.3%), renal failure in 2 patients (4.3%), and prolonged ventilation in 1 patient (2.1%). Average hospital stay was 8.3 days (range, 4 to 27 days). Early in-hospital death occurred in 1 patient (2.1%); currently, 31 patients (66%) remain alive and free of cardiac symptoms. Conclusion— Heart valve surgery and surgery for congenital heart diseases can be performed safely in patients with sickle cell disease or sickle cell trait with acceptable outcome and survival rates.

Intracardiac Erythropoietin Injection Reveals Antiinflammatory Potential and Improved Cardiac Functions Detected by Forced Swim Test

Systemic administration of erythropoietin (Epo) protects the myocardium from an ischemic insult and promotes beneficial remodeling. We hypothesized that intracardiac injection of Epo may exhibit cardioprotective potential with reduced systemic toxicity. Following myocardial infarction (MI), Epo was injected directly into the border of the infarction. Six weeks after an MI, we evaluated infarction size, angiogenesis, and pathologic effects of the treatment. Myocardial performance was assessed with a Forced Swim Test adapted to the study. Anti-inflammatory and cellular proliferative effects of Epo were analyzed by measuring expression of integrin-beta and CdK4 by reverse transcriptase-polymerase chain reaction (RT-PCR). The findings indicated improved cardiac status with direct Epo administration. Exercise capacity detected by the Forced Swim Test was significantly increased. There was radical reduction of absolute infarction size, ventricular dilatation, and hypertrophy in the Epo group. Integrin-beta was down-regulated and CdK4 expression was increased significantly with Epo. In conclusion, the study demonstrated that intramyocardial Epo injection, following MI, reduced inflammation, enhanced angiogenesis and proliferation, improved myocardial functions, and did not lead to intramural thrombus formation.

HMGB-1 induces c-kit+ cell microvascular rolling and adhesion via both toll-like receptor-2 and toll-like receptor-4 of endothelial cells.

High‐mobility group box 1 (HMGB‐1) is a strong chemo‐attractive signal for both inflammatory and stem cells. The aim of this study is to evaluate the mechanisms regulating HMGB‐1–mediated adhesion and rolling of c‐kit+ cells and assess whether toll‐like receptor‐2 (TLR‐2) and toll‐like receptor‐4 (TLR‐4) of endothelial cells or c‐kit+ cells are implicated in the activation of downstream migration signals to peripheral c‐kit+ cells. Effects of HMGB‐1 on the c‐kit+ cells/endothelial interaction were evaluated by a cremaster muscle model in wild‐type (WT), TLR‐2 (−/−) and Tlr4 (LPS‐del) mice. The mRNA and protein expression levels of endothelial nitric oxide synthase were determined by quantitative real‐time PCR and immunofluorescence staining. Induction of crucial adhesion molecules for rolling and adhesion of stem cells and leukocytes were monitored in vivo and in vitro. Following local HMGB‐1 administration, a significant increase in cell rolling was detected (32.4 ± 7.1% in ‘WT’ versus 9.9 ± 3.2% in ‘control’, P < 0.05). The number of firmly adherent c‐kit+ cells was more than 13‐fold higher than that of the control group (14.6 ± 5.1 cells/mm2 in ‘WT’ versus 1.1 ± 1.0 cells/mm2 in ‘control’, P < 0.05). In knockout animals, the fraction of rolling cells did not differ significantly from control levels. Firm endothelial adhesion was significantly reduced in TLR‐2 (−/−) and Tlr4 (LPS‐del) mice compared to WT mice (1.5 ± 1.4 cells/mm2 in ‘TLR‐2 (−/−)’ and 2.4 ± 1.4 cells/mm2 in ‘Tlr4 (LPS‐del)’ versus 14.6 ± 5.1 cells/mm2 in ‘WT’, P < 0.05). TLR‐2 (−/−) and Tlr4 (LPS‐del) stem cells in WT mice did not show significant reduction in rolling and adhesion compared to WT cells. HMGB‐1 mediates c‐kit+ cell recruitment via endothelial TLR‐2 and TLR‐4.

Statins in the prevention of postoperative atrial fibrillation: is there really no effect?

We read with interest the recent article by Virani et al regarding the influence of preoperative statin therapy on occurrence of postoperative atrial fibrillation (POAF). In the retrospective study, the authors included 4044 patients subjected to cardiac surgery and showed that preoperative statin therapy was not associated with decreased incidence of POAF. We believe there are certain points to be addressed concerning the data presented. The relationship between statin therapy and POAF has been widely studied by different groups. A recent study by Lertsburapa et al and an extended review by Howard and Barnes present interestingly diverse outcomes. In the ARMYDA-3 trial, statin treatment resulted in a 61% reduction in risk of atrial fibrillation and by 42% in the Mariscalco et al study. We believe the results obtained by Virani et al are not connected with a lack of statin activity but rather with the study limitations. First of all, the manuscript has a retrospective background, and the findings of such studies are not necessarily consistent. The authors also included patients receiving different groups of statin medications. Interestingly, some of their patients, who were subjected to cardiac surgery from January 2003, were administered cerivastatin, which was withdrawn from the US market in August 2001. Another issue to be discussed is the duration of the statin therapy and dosages that had been used. The effects of statins in cardiosurgical patients depend on their pleiotropic action. This is strictly dependent on the type of statin and is dosedependent. In addition, unfortunately, the groups of patients in the manuscript are very heterogeneous, which we believe could have resulted in a significant influence on POAF occurrence. Although the authors make their remarks also on patients with heart failure, we believe this should be regarded as novel because available data lack convincing results for effects of statins on heart failure. It may also be associated with small doses or selected statins which may result from their pleiotropic activity. Cardiac surgery, depending on the procedure, for example, coronary artery bypass graft and ischemic or rheumatic valve pathologies, has different aspects and different cardiovascular disorders induce atrial fibrillation with different pathophysiologic mechanisms. We would like to congratulate the authors for their tremendous effort in their manuscript; however, the presented study does not add anything new to the current knowledge as to the role of statins in POAF occurrence. There are still many aspects of this therapy that need to be discussed: specifically, the type of statins with the highest efficacy in this group of patients; the size of dose; the role of statins in patients undergoing coronary artery bypass graft, concomitant valve surgery, or valve surgery alone; and finally, their role in patients with heart failure subjected to cardiac surgery.