Vasoprotective effects of human CD34+ cells: towards clinical applications

<p>Abstract</p> <p>Background</p> <p>The development of cell-based therapeutics for humans requires preclinical testing in animal models. The use of autologous animal products fails to address the efficacy of similar products derived from humans. We used a novel immunodeficient rat carotid injury model in order to determine whether human cells could improve vascular remodelling following acute injury.</p> <p>Methods</p> <p>Human CD34+ cells were separated from peripheral buffy coats using automatic magnetic cell separation. Carotid arterial injury was performed in male Sprague-Dawley nude rats using a 2F Fogarty balloon catheter. Freshly harvested CD34+ cells or saline alone was administered locally for 20 minutes by endoluminal instillation. Structural and functional analysis of the arteries was performed 28 days later.</p> <p>Results</p> <p>Morphometric analysis demonstrated that human CD34+ cell delivery was associated with a significant reduction in intimal formation 4 weeks following balloon injury as compared with saline (I/M ratio 0.79 ± 0.18, and 1.71 ± 0.18 for CD34, and saline-treated vessels, respectively P < 0.05). Vasoreactivity studies showed that maximal relaxation of vessel rings from human CD34+ treated animals was significantly enhanced compared with saline-treated counterparts (74.1 ± 10.2, and 36.8 ± 12.1% relaxation for CD34+ cells and saline, respectively, P < 0.05)</p> <p>Conclusion</p> <p>Delivery of human CD34+ cells limits neointima formation and improves arterial reactivity after vascular injury. These studies advance the concept of cell delivery to effect vascular remodeling toward a potential human cellular product.</p

Magnetically Targeted Endothelial Cell Localization in Stented Vessels

ObjectivesA novel method to magnetically localize endothelial cells at the site of a stented vessel wall was developed. The application of this strategy in a large animal model is described.BackgroundLocal delivery of blood-derived endothelial cells has been shown to facilitate vascular healing in animal models. Therapeutic utilization has been limited by an inability to retain cells in the presence of blood flow. We hypothesized that a magnetized stent would facilitate local retention of superparamagnetically labeled cells.MethodsCultured porcine endothelial cells were labeled with endocytosed superparamagnetic iron oxide microspheres. A 500:1 microsphere-to-cell ratio was selected for in vivo experiments based on bromo-deoxyuridine incorporation and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assays. Stents were magnetized and implanted in porcine coronary and femoral arteries using standard interventional equipment. Labeled endothelial cells were delivered locally during transient occlusion of blood flow.ResultsThe delivered cells were found attached to the stent struts and were also distributed within the adjacent denuded vessel wall at 24 h.ConclusionsMagnetic forces can be used to rapidly place endothelial cells at the site of a magnetized intravascular stent. The delivered cells are retained in the presence of blood flow and also spread to the adjacent injured vessel wall. Potential applications include delivering a cell-based therapeutic effect to the local vessel wall as well as downstream tissue

Xenoantigenicity of porcine decellularized valves

Abstract Background The xenoantigenicity of porcine bioprosthetic valves is implicated as an etiology leading to calcification and subsequent valve failure. Decellularization of porcine valves theoretically could erase the antigenicity of the tissue leading to more durable prosthetic valves, but the effectiveness of decellularization protocols in regard to completely removing antigens has yet to be verified. Our hypothesis was that decellularization would remove the more abundant α-gal antigens but not remove all the non α-gal antigens, which could mount a response. Methods Porcine aortic valves were decellularized with 1% sodium dodecyl sulfate for 4 days. Decellularized cusps were evaluated for α-gal epitopes by ELISA. To test for non α-gal antigens, valves were implanted into sheep. Serum was obtained from the sheep preoperatively and 1 week, 1 month, and 2 months postoperatively. This serum was utilized for anti-porcine antibody staining and for quantification of anti-pig IgM and IgG antibodies and complement. Results Decellularized porcine cusps had 2.8 ± 2.0% relative α-gal epitope as compared to fresh porcine aortic valve cusps and was not statistically significantly different (p = 0.4) from the human aortic valve cusp which had a 2.0 ± 0.4% relative concentration. Anti-pig IgM and IgG increased postoperatively from baseline levels. Preoperatively anti-pig IgM was 27.7 ± 1.7 μg/mL and it increased to 71.9 ± 12.1 μg/mL average of all time points postoperatively (p = 0.04). Preoperatively anti-pig IgG in sheep serum was 44.9 ± 1.5 μg/mL and it increased to 72.6 ± 6.0 μg/mL average of all time points postoperatively (p = 0.01). There was a statistically significant difference (p = 0.00007) in the serum C1q concentration before valve implantation (2.5 ± 0.2 IU/mL) and at averaged time points after valve implantation (5.3 ± 0.3 IU/mL). Conclusions Decellularization with 1% sodium dodecyl sulfate does not fully eliminate non α-gal antigens; however, significant reduction in α-gal presence on decellularized cusps was observed. Clinical implications of the non α-gal antigenic response are yet to be determined. As such, evaluation of any novel decellularized xenografts must include rigorous antigen testing prior to human trials

Feasibility of selective cardiac ventricular electroporation.

IntroductionThe application of brief high voltage electrical pulses to tissue can lead to an irreversible or reversible electroporation effect in a cell-specific manner. In the management of ventricular arrhythmias, the ability to target different tissue types, specifically cardiac conduction tissue (His-Purkinje System) vs. cardiac myocardium would be advantageous. We hypothesize that pulsed electric fields (PEFs) can be applied safely to the beating heart through a catheter-based approach, and we tested whether the superficial Purkinje cells can be targeted with PEFs without injury to underlying myocardial tissue.MethodsIn an acute (n = 5) and chronic canine model (n = 6), detailed electroanatomical mapping of the left ventricle identified electrical signals from myocardial and overlying Purkinje tissue. Electroporation was effected via percutaneous catheter-based Intracardiac bipolar current delivery in the anesthetized animal. Repeat Intracardiac electrical mapping of the heart was performed at acute and chronic time points; followed by histological analysis to assess effects.ResultsPEF demonstrated an acute dose-dependent functional effect on Purkinje, with titration of pulse duration and/or voltage associated with successful acute Purkinje damage. Electrical conduction in the insulated bundle of His (n = 2) and anterior fascicle bundle (n = 2), was not affected. At 30 days repeat cardiac mapping demonstrated resilient, normal electrical conduction throughout the targeted area with no significant change in myocardial amplitude (pre 5.9 ± 1.8 mV, 30 days 5.4 ± 1.2 mV, p = 0.92). Histopathological analysis confirmed acute Purkinje fiber targeting, with chronic studies showing normal Purkinje fibers, with minimal subendocardial myocardial fibrosis.ConclusionPEF provides a novel, safe method for non-thermal acute modulation of the Purkinje fibers without significant injury to the underlying myocardium. Future optimization of this energy delivery is required to optimize conditions so that selective electroporation can be utilized in humans the treatment of cardiac disease

Additional file 1: of Xenoantigenicity of porcine decellularized valves

Methods Supplement (DOCX 19 kb

Bacitracin inhibited PDI attenuates NP-mediated generation of cGMP in vascular cells.

<p>Bacitracin (Ba) inhibits NPs activation in HMC (A), HVSMC (B) and HUVEC (C) * p ≤0.05 vs samples without bacitracin addition. (Error bars, +SD from 3 independent experiments, samples were triplicated in each experiment).</p