Infliximab Limits Injury in Myocardial Infarction.

BACKGROUND: The purpose of this study was to investigate a therapeutic approach targeting the inflammatory response and consequent remodeling from ischemic myocardial injury. METHODS AND RESULTS: Coronary thrombus aspirates were collected from patients at the time of ST-segment-elevation myocardial infarction and subjected to array-based proteome analysis. Clinically indistinguishable at myocardial infarction (MI), patients were stratified into vulnerable and resilient on the basis of 1-year left ventricular ejection fraction and death. Network analysis from coronary aspirates revealed prioritization of tumor necrosis factor-α signaling in patients with worse clinical outcomes. Infliximab, a tumor necrosis factor-α inhibitor, was infused intravenously at reperfusion in a porcine MI model to assess whether infliximab-mediated immune modulation impacts post-MI injury. At 3 days after MI (n=7), infliximab infusion increased proregenerative M2 macrophages in the myocardial border zone as quantified by immunofluorescence (24.1%±23.3% in infliximab versus 9.29%±8.7% in sham; P<0.01). Concomitantly, immunoassays of coronary sinus samples quantified lower troponin I levels (41.72±7.34 pg/mL versus 58.11±10.75 pg/mL; P<0.05) and secreted protein analysis revealed upregulation of injury-modifying interleukin-2, -4, -10, -12, and -18 cytokines in the infliximab-treated cohort. At 4 weeks (n=12), infliximab treatment resulted in significant protective influence, improving left ventricular ejection fraction (53.9%±5.4% versus 36.2%±5.3%; P<0.001) and reducing scar size (8.31%±10.9% versus 17.41%±12.5%; P<0.05). CONCLUSIONS: Profiling of coronary thrombus aspirates in patients with ST-segment-elevation MI revealed highest association for tumor necrosis factor-α in injury risk. Infliximab-mediated immune modulation offers an actionable pathway to alter MI-induced inflammatory response, preserving contractility and limiting adverse structural remodeling.

Pulmonary Biodistribution of Platelet-Derived Regenerative Exosomes in a Porcine Model.

The purpose of this study was to evaluate the biodistribution of a platelet-derived exosome product (PEP), previously shown to promote regeneration in the setting of wound healing, in a porcine model delivered through various approaches. Exosomes were labeled with DiR far-red lipophilic dye to track and quantify exosomes in tissue, following delivery via intravenous, pulmonary artery balloon catheter, or nebulization in sus scrofa domestic pigs. Following euthanasia, far-red dye was detected by Xenogen IVUS imaging, while exosomal protein CD63 was detected by Western blot and immunohistochemistry. Nebulization and intravenous delivery both resulted in global uptake of exosomes within the lung parenchyma. However, nebulization resulted in the greatest degree of exosome uptake. Pulmonary artery balloon catheter-guided delivery provided the further ability to localize pulmonary delivery. No off-target absorption was noted in the heart, spleen, or kidney. However, the liver demonstrated uptake primarily in nebulization-treated animals. Nebulization also resulted in uptake in the trachea, without significant absorption in the esophagus. Overall, this study demonstrated the feasibility of pulmonary delivery of exosomes using nebulization or intravenous infusion to accomplish global delivery or pulmonary artery balloon catheter-guided delivery for localized delivery.

Nebulized platelet-derived extracellular vesicles attenuate chronic cigarette smoke-induced murine emphysema.

Chronic obstructive pulmonary disease (COPD) is a prevalent lung disease usually resulting from cigarette smoking (CS). Cigarette smoking induces oxidative stress, which causes inflammation and alveolar epithelial cell apoptosis and represents a compelling therapeutic target for COPD. Purified human platelet-derived exosome product (PEP) is endowed with antioxidant enzymes and immunomodulatory molecules that mediate tissue repair. In this study, a murine model of CS-induced emphysema was used to determine whether nebulized PEP can influence the development of CS-induced emphysema through the mitigation of oxidative stress and inflammation in the lung. Nebulization of PEP effectively delivered the PEP vesicles into the alveolar region, with evidence of their uptake by type I and type II alveolar epithelial cells and macrophages. Lung function testing and morphometric assessment showed a significant attenuation of CS-induced emphysema in mice treated with nebulized PEP thrice weekly for 4 weeks. Whole lung immuno-oncology RNA sequencing analysis revealed that PEP suppressed several CS-induced cell injuries and inflammatory pathways. Validation of inflammatory cytokines and apoptotic protein expression on the lung tissue revealed that mice treated with PEP had significantly lower levels of S100A8/A9 expressing macrophages, higher levels of CD4+/FOXP3+ Treg cells, and reduced NF-κB activation, inflammatory cytokine production, and apoptotic proteins expression. Further validation using in vitro cell culture showed that pretreatment of alveolar epithelial cells with PEP significantly attenuated CS extract-induced apoptotic cell death. These data show that nebulization of exosomes like PEP can effectively deliver exosome cargo into the lung, mitigate CS-induced emphysema in mice, and suppress oxidative lung injury, inflammation, and apoptotic alveolar epithelial cell death.

Reconstituted Extracellular Vesicles from Human Platelets Decrease Viral Myocarditis in Mice.

Patients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease-specific therapies exist to treat viral myocarditis. Here it is examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human-platelet-derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)-like module-containing mucin-like hormone receptor-like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll-like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL-1ß), transforming growth factor-beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7-9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T-cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.

Artificial Intelligence-Derived Electrocardiogram Assessment of Cardiac Age and Molecular Markers of Senescence in Heart Failure.

OBJECTIVE: To ascertain whether heart failure (HF) itself is a senescent phenomenon independent of age, and how this is reflected at a molecular level in the circulating progenitor cell niche, and at a substrate level using a novel electrocardiogram (ECG)-based artificial intelligence platform. PATIENTS AND METHODS: Between October 14, 2016, and October 29, 2020, CD34+ progenitor cells were analyzed by flow cytometry and isolated by magnetic-activated cell sorting from patients of similar age with New York Heart Association functional classes IV (n = 17) and I-II (n = 10) heart failure with reduced ejection fraction and healthy controls (n = 10). CD34+ cellular senescence was quantitated by human telomerase reverse transcriptase expression and telomerase expression by quantitative polymerase chain reaction, and senescence-associated secretory phenotype (SASP) protein expression assayed in plasma. An ECG-based artificial intelligence (AI) algorithm was used to determine cardiac age and difference from chronological age (AI ECG age gap). RESULTS: CD34+ counts and telomerase expression were significantly reduced and AI ECG age gap and SASP expression increased in all HF groups compared with healthy controls. Expression of SASP protein was closely associated with telomerase activity and severity of HF phenotype and inflammation. Telomerase activity was more closely associated with CD34+ cell counts and AI ECG age gap. CONCLUSION: We conclude from this pilot study that HF may promote a senescent phenotype independent of chronological age. We show for the first time that the AI ECG in HF shows a phenotype of cardiac aging beyond chronological age, and appears to be associated with cellular and molecular evidence of senescence.

Platelet-derived exosomes induce cell proliferation and wound healing in human endometrial cells.

Aim: To investigate the regenerative effects of a platelet-derived purified exosome product (PEP) on human endometrial cells. Materials & methods: Endometrial adenocarcinoma cells (HEC-1A), endometrial stromal cells (T HESC) and menstrual blood-derived stem cells (MenSC) were assessed for exosome absorption and subsequent changes in cell proliferation and wound healing properties over 48 h. Results: Cell proliferation increased in PEP treated T HESC (p < 0.0001) and MenSC (p < 0.001) after 6 h and in HEC-1A (p < 0.01) after 12 h. PEP improved wound healing after 6 h in HEC-1A (p < 0.01) and MenSC (p < 0.0001) and in T HESC between 24 and 36 h (p < 0.05). Conclusion: PEP was absorbed by three different endometrial cell types. PEP treatment increased cell proliferation and wound healing capacity.

The uterus has a remarkable ability to heal itself. Every month the inside lining of the uterus grows in preparation for pregnancy and sheds if no pregnancy occurs. Unfortunately, this cycle of growth, shedding and repair can be injured and lead to menstrual changes, pain or even infertility. In this study, we looked how special cell messengers ­ called exosomes ­ could help uterine cells. Exosomes are special messengers that contain substances to help the body heal and regenerate injured cells and tissues. We obtained exosomes created from human transfusion-grade platelets. We studied the exosomes' effects in three different cell types that all are important inside the uterine lining. Specifically, we studied the ability of the exosomes to help cells proliferate and migrate into a wound. In this study, exosomes were recognized by the human endometrial cells and were absorbed. Once they were inside the cells, they increased cell proliferation as well as the ability of the cells to heal a scratch wound. Furthermore, the more exosomes we presented to the cells, the more the cells were able to proliferate and move into a wound for healing. These findings lay the groundwork for future studies in animal models of uterine injury.

Exosome biopotentiated hydrogel restores damaged skeletal muscle in a porcine model of stress urinary incontinence.

Urinary incontinence afflicts up to 40% of adult women in the United States. Stress urinary incontinence (SUI) accounts for approximately one-third of these cases, precipitating ~200,000 surgical procedures annually. Continence is maintained through the interplay of sub-urethral support and urethral sphincter coaptation, particularly during activities that increase intra-abdominal pressure. Currently, surgical correction of SUI focuses on the re-establishment of sub-urethral support. However, mesh-based repairs are associated with foreign body reactions and poor localized tissue healing, which leads to mesh exposure, prompting the pursuit of technologies that restore external urethral sphincter function and limit surgical risk. The present work utilizes a human platelet-derived CD41a and CD9 expressing extracellular vesicle product (PEP) enriched for NF-κB and PD-L1 and derived to ensure the preservation of lipid bilayer for enhanced stability and compatibility with hydrogel-based sustained delivery approaches. In vitro, the application of PEP to skeletal muscle satellite cells in vitro drove proliferation and differentiation in an NF-κB-dependent fashion, with full inhibition of impact on exposure to resveratrol. PEP biopotentiation of collagen-1 and fibrin glue hydrogel achieved sustained exosome release at 37 °C, creating an ultrastructural "bead on a string" pattern on scanning electron microscopy. Initial testing in a rodent model of latissimus dorsi injury documented activation of skeletal muscle proliferation of healing. In a porcine model of stress urinary incontinence, delivery of PEP-biopotentiated collagen-1 induced functional restoration of the external urethral sphincter. The histological evaluation found that sustained PEP release was associated with new skeletal muscle formation and polarization of local macrophages towards the regenerative M2 phenotype. The results provided herein serve as the first description of PEP-based biopotentiation of hydrogels implemented to restore skeletal muscle function and may serve as a promising approach for the nonsurgical management of SUI.

Exosome-Induced Vaginal Tissue Regeneration in a Porcine Mesh Exposure Model.

OBJECTIVES: The purpose of this study was to explore the utility of an injectable purified exosome product derived from human apheresis blood to (1) augment surgical closure of vaginal mesh exposures, and (2) serve as a stand-alone therapy for vaginal mesh exposure. METHODS: Sixteen polypropylene meshes (1×1-3×3 cm) were implanted in the vaginas of 7 Yorkshire-crossed pigs by urogynecologic surgeons (day 0). On day 7, group 1 underwent surgical intervention via vaginal tissue suture reclosure with (n=2 pigs, n=4 meshes) or without (n=2 pigs, n=4 meshes) exosome injection; group 2 underwent medical intervention with an exosome injection (n=3, n=8 meshes). One animal in group 2 was given oral 2'-deoxy-5-ethynyluridine to track cellular regeneration. Euthansia occurred at 5 weeks. RESULTS: Mesh exposures treated with surgical closure alone experienced reexposure of the mesh. Exosome treatment with or without surgical closure resulted in partial to full mesh exposure resolution up to 3×3 cm. Exosome-treated tissues had significantly thicker regenerated epithelial tissue (208 µm exosomes-only and 217 µm surgery+exosomes, versus 80 µm for surgery-only; P < 0.05); evaluation of 2'-deoxy-5-ethynyluridine confirmed de novo regeneration throughout the epithelium and underlying tissues. Capillary density was significantly higher in the surgery+exosomes group (P = 0.03). Surgery-only tissues had a higher inflammatory and fibrosis response as compared with exosome-treated tissues. CONCLUSIONS: In this pilot study, exosome treatment augmented healing in the setting of vaginal mesh exposure, reducing the incidence of mesh reexposure after suture closure and decreasing the area of mesh exposure through de novo tissue regeneration after exosome injection only. Further study of varied local tissue conditions and mesh configurations is warranted.

Impact of Repeat Dosing and Mesh Exposure Chronicity on Exosome-Induced Vaginal Tissue Regeneration in a Porcine Mesh Exposure Model.

OBJECTIVE: The aim of the study was to compare vaginal wound healing after exosome injection in a porcine mesh exposure model with (1) single versus multiple dose regimens and (2) acute versus subacute exposure. METHODS: Six 80-kg Yorkshire-crossed swine each had 2 polypropylene meshes implanted to create the vaginal mesh exposure model. Animals were divided into 3 groups based on number and timing of exosome injection: (1) single purified exosome product (PEP) injection (acute-single), (2) weekly PEP injections (acute-weekly, 4 total injections), and (3) delayed single injection (subacute-single). Acute and subacute injections occurred 1 and 8 weeks after mesh implantation, respectively. EdU, a thymidine analog, was given twice weekly after the first injection to track tissue regeneration. Euthanasia and tissue analysis occurred 4 weeks after the first injection. ImageJ was used to quantify epithelial thickness, cellular proliferation, and capillary density. Statistical analysis was performed using analysis of variance and post hoc Tukey test. RESULTS: Acute-single PEP injection tissues mirrored pilot study results, validating replication of protocol. Within the acute groups, weekly dosing resulted in 1.5× higher epithelial thickness (nonsignificant), 1.8× higher epithelial proliferation (P < 0.05), and 1.5× higher regenerated capillary density (P < 0.05) compared with single injection. Regarding chronicity of the exposure, the subacute group showed 1.7× higher epithelial proliferation (nonsignificant) and similar capillary density and epithelial thickness as compared with the acute group. CONCLUSIONS: Exosome redosing resulted in significantly greater epithelial proliferation with significantly higher regenerated capillary density, leading to a trend toward thicker epithelium. Subacute exposure exhibited similar regeneration to acute exposure despite a delayed injection timeline. These results contribute to a growing body of preclinical research demonstrating utility of exosomes in pelvic floor disorders.

Feasibility of selective cardiac ventricular electroporation.

INTRODUCTION: The 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. METHODS: In 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. RESULTS: PEF 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. CONCLUSION: PEF 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.

M3RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction.

IMPACT STATEMENT: The M3RNA (microencapsulated modified messenger RNA) platform is an approach to deliver messenger RNA (mRNA) in vivo, achieving a nonintegrating and viral-free approach to gene therapy. This technology was, in this study, tested for its utility in the myocardium, providing a unique avenue for targeted gene delivery into the freshly infarcted myocardial tissue. This study provides the evidentiary basis for the use of M3RNA in the heart through depiction of its performance in cultured cells, healthy rodent myocardium, and acutely injured porcine hearts. By testing the technology in large animal models of infarction, compatibility of M3RNA with current coronary intervention procedures was verified.

Collagen and Fractionated Platelet-Rich Plasma Scaffold for Dermal Regeneration.

BACKGROUND: Current options for in vivo regeneration of dermal tissue remain limited. The purpose of this study was to engineer a unique scaffold capable of recruiting dermal stem cells from adjacent tissue, thus circumventing the need to seed the scaffolds with stem cells before implantation, leading to skin regeneration. METHODS: A hydrogel scaffold was created through combination of type I collagen along with fractionated platelet-rich plasma. This was compared to a control hydrogel consisting of type I collagen and fetal bovine serum. Hydrogels were cultured with fresh human skin tissue and incubated with supplemental media. Gels were digested weekly for cellular content as examined by flow cytometry at the 4- and 8-week time points. The fractionated platelet-rich plasma and collagen gels were then implanted onto full-thickness skin defects on the backs of rats and compared to wounds healing by secondary intention. Wound area was evaluated for epithelialization and neovascularization. RESULTS: Platelet-rich plasma fractionation increased platelet-derived growth factors. In contrast to collagen scaffolds, fractionated platelet-rich plasma-supplemented scaffolds recruited more dermal-derived stem cells from fresh skin tissue compared with collagen hydrogels at the 4- and 8-week time points. Furthermore, fractionated platelet-rich plasma-supplemented hydrogels accelerated wound healing, angiogenesis, and hair and sweat gland formation, ultimately regenerating a dermis-like tissue. CONCLUSIONS: Generation of hydrogels with fractionated platelet-rich plasma was able to improve cellular recruitment and growth and differentiation of dermal-derived stem cells, leading to hair growth and sweat gland formation. This provides a novel approach to regenerate skin for treating large defects.

An Athymic Rat Model for Mandibular Osteoradionecrosis Allowing for Direct Translation of Regenerative Treatments.

OBJECTIVE: We aim to create a model of mandibular osteoradionecrosis in athymic rats. Athymic rats provide an immunosuppressed environment whereby human stem cells and biomaterials can be used to investigate regenerative solutions for osteoradionecrosis, bridging the gap between in vivo testing and clinical application. STUDY DESIGN: Prospective animal study. SETTING: Academic otolaryngology department laboratory. SUBJECTS AND METHODS: After Institutional Animal Care and Use Committee approval, 10 athymic nude rats were divided into 2 groups. The experimental group (n = 6) underwent irradiation (20 Gy), while the control group (n = 4) underwent sham irradiation catheter placement only. All 10 rats underwent extraction of the second mandibular molar 7 days later. The rats were sacrificed 28 days after dental extraction, and their mandibles were harvested. The mandibles were examined with histologic analysis and bone volume analysis based on 3-dimensional micro-computed tomography. RESULTS: All 10 rats survived the experiment period. Radiographic and histologic analysis revealed decreased bone formation in the experimental group compared with the control group. Jaw region volume ratio was 0.83 for the experimental group versus 0.97 in the control group (P = .003). The region-of-interest volume ratio was 0.75 in the experimental group and 0.97 in the control group (P = .005). Histologically, there were increased osteoclasts (P = .02) and decreased osteoblasts (P = .001) as well as increased fibrosis in the experimental group versus the control group. CONCLUSION: Mandibular osteoradionecrosis can be effectively and reproducibly produced in an athymic rat model. This will allow further research to study regenerative medicine in an athymic rat model.

Adipose-derived Mesenchymal Stem Cells Are Phenotypically Superior for Regeneration in the Setting of Osteonecrosis of the Femoral Head.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (bmMSCs) have been used as a cellular therapeutic option for treatment of osteonecrosis of the femoral head. However, use of bmMSCs as a treatment adjuvant for orthopaedic disorders in general has achieved limited success. Adipose-derived MSCs (aMSCs) may be a more-efficient regenerative cell source given their greater quantity and protection from physiologic stress. QUESTIONS/PURPOSES: We asked the following questions in a paired analysis of MSCs from patients with osteonecrosis: (1) Is there a difference in proliferation potential between aMSCs and bmMSCs? (2) Is there a difference in osteogenic differentiation potential between aMSCs and bmMSCs? (3) Are genetic pathways differentially expressed between aMSCs and bmMSCs that may govern functional phenotypic discrepancies? METHODS: Periarticular samples of adipose tissue and bone marrow from the femoral canal were obtained from 15 patients undergoing hip replacement for late-stage (Steinberg Stages III-VI) osteonecrosis. MSCs were isolated from both tissue sources and taken through a standardized 20-day cell division protocol to establish cumulative cell count. They also were grown in osteogenic differentiation media for 14 days with subsequent measurement of alkaline phosphatase in units of optical density. RNA was isolated from aMSCs and bmMSCs in five patients to assess differentially expressed genetic pathways using the Affymetrix GeneChip Human Transcriptome Array 2.0 platform. RESULTS: Proliferation capacity was increased by fourfold in aMSCs compared with bmMSCs after 20 days in culture. The mean difference in cumulative cell count was 3.99 × 10(8) cells (SD = 1.67 × 10(8) cells; 95% CI, 3.07 × 10(8)-4.92 × 10(8) cells; p < 0.001). Bone differentiation efficiency as measured by optical density was increased by 2.25-fold in aMSCs compared with bmMSCs. The mean difference in optical density was 1.27 (SD = 0.34; 95% CI, 1.08-1.46; p < 0.001). RNA transcriptome analysis showed 284 genes that met statistical (p < 0.05) and biological (fold change > 1.5) significance cutoffs for differential expression between cell populations. Subsequent network topology of differentially expressed genes showed alterations in pathways critical for musculoskeletal tissue development in addition to many nonspecific findings. CONCLUSIONS: aMSCs outperform bmMSCs in growth rate and bone differentiation potential in the setting of osteonecrosis, suggesting they may provide a more-potent regenerative therapeutic strategy in this population. Differential expression of genes and cellular pathways highlighted in this study may provide therapeutic targets for cellular optimization or acellular treatment strategies. CLINICAL RELEVANCE: aMSCs may provide a more robust cellular therapeutic than bmMSCs for treatment of osteonecrosis. Ideally, a well-designed prospective study will be able to evaluate the efficacy of these cellular therapies side-by-side in patients with bilateral early stage disease.

Effect of positive end-expiratory pressure on porcine right ventricle function assessed by speckle tracking echocardiography.

BACKGROUND: Right ventricle (RV) dysfunction and hypotension can be induced by high levels of positive end-expiratory pressure (PEEP). We sought to determine in an animal model if a novel ultrasound analysis technique: speckle tracking echocardiography (STE), could determine deterioration in RV function induced by PEEP and to compare this to a conventional method of RV analysis: fractional area change (FAC). STE is a sensitive, angle-independent method for describing cardiac deformation ('strain') and is particularly useful in analyzing RV function as has been shown in pulmonary hypertension cohorts. METHODS: Ten pigs, 40-90 kg, anaesthetized, fully mechanically ventilated at 6 ml/kg were subject to step-wise escalating levels of PEEP at two-minute intervals (0, 5, 10, 15, 20, 25 and 30 cmH20). Intracardiac echocardiography was used to image the RV as transthoracic and transesophageal echocardiography did not give sufficient image quality or flexibility. Off-line STE analysis was performed using Syngo Velocity Vector Imaging (Seimens Medical Solutions Inc., USA). STE systolic parameters are RV free wall strain (RVfwS) and strain rate (RVfwSR) and the diastolic parameter RV free wall strain rate early relaxation (RVfwSRe). RESULTS: With escalating levels of PEEP there was a clear trend of reduction in STE parameters (RVfwS, RVfwSR, RVfwSRe) and FAC. Significant hypotension (fall in mean arterial blood pressure of 20 mmHg) occurred at approximately PEEP 15 cmH2O. Comparing RVfwS, RVfwSR and RVfwSRe values at different PEEP levels showed a significant difference at PEEP 0 cmH2O vs PEEP 10 cmH2O and above. FAC only showed a significant difference at PEEP 0 cmH2O vs PEEP 20 cmH2O and above. 30% of pigs displayed dyssychronous RV free wall contraction at the highest PEEP level reached. CONCLUSIONS: STE is a sensitive method for determining RV dysfunction induced by PEEP and deteriorated ahead of a conventional assessment method: FAC. RVfwS decreased to greater extent compared to baseline than FAC, earlier in the PEEP escalation process and showed a significant decrease before there was a clinical relevant decrease in mean arterial blood pressure. Studies in ICU patients using transthoracic echocardiography are warranted to further investigate the most sensitive echocardiography method for detecting RV dysfunction induced by mechanical ventilation.

Cloning and expression of porcine ß1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen.

BACKGROUND: Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine ß1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. METHODS: The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. RESULTS: The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti-Sd(a) and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. CONCLUSION: The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response detected after pig-to-baboon cardiac xenotransplantation.

Optimized delivery system achieves enhanced endomyocardial stem cell retention.

BACKGROUND: Regenerative cell-based therapies are associated with limited myocardial retention of delivered stem cells. The objective of this study is to develop an endocardial delivery system for enhanced cell retention. METHODS AND RESULTS: Stem cell retention was simulated in silico using 1- and 3-dimensional models of tissue distortion and compliance associated with delivery. Needle designs, predicted to be optimal, were accordingly engineered using nitinol, a nickel and titanium alloy displaying shape memory and superelasticity. Biocompatibility was tested with human mesenchymal stem cells. Experimental validation was performed with species-matched cells directly delivered into Langendorff-perfused porcine hearts or administered percutaneously into the endocardium of infarcted pigs. Cell retention was quantified by flow cytometry and real-time quantitative polymerase chain reaction methodology. Models, computing optimal distribution of distortion calibrated to favor tissue compliance, predicted that a 75°-curved needle featuring small-to-large graded side holes would ensure the highest cell retention profile. In isolated hearts, the nitinol curved needle catheter (C-Cath) design ensured 3-fold superior stem cell retention compared with a standard needle. In the setting of chronic infarction, percutaneous delivery of stem cells with C-Cath yielded a 37.7±7.1% versus 10.0±2.8% retention achieved with a traditional needle without effect on biocompatibility or safety. CONCLUSIONS: Modeling-guided development of a nitinol-based curved needle delivery system with incremental side holes achieved enhanced myocardial stem cell retention.

Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts.

BACKGROUND: Transgenic expression of human complement regulatory proteins reduces the frequency of hyperacute rejection (HAR) in Gal-positive cardiac xenotransplantation. In this study, we examined the impact of human CD55 (hCD55) expression on a Gal knockout (GTKO) background using pig-to-primate heterotopic cardiac xenotransplantation. METHODS: Cardiac xenotransplantation was performed with GTKO (group 1; n=6) and GTKO.hCD55 (group 2; n=5) donor pigs using similar immunosuppression. Cardiac biopsies were obtained 30 min after organ reperfusion. Rejection was characterized by histology and immunohistology. Intragraft gene expression, serum non-Gal antibody, and antibody recovered from rejected hearts were analyzed. RESULTS: HAR of a GTKO heart was observed. Remaining grafts developed delayed xenograft rejection. Median survival was 21 and 28 days for groups 1 and 2, respectively. Vascular antibody deposition was uniformly detected 30 min after organ reperfusion and at explant. A higher frequency of vascular C5b deposition was seen in GTKO organs at explant. Serum non-Gal antibody, antibody recovered from the graft, and intragraft gene expression were similar between the groups. CONCLUSION: HAR of GTKO hearts without hCD55 may occur. Expression of hCD55 seemed to restrict local complement activation but did not improve graft survival. Chronic vascular antibody deposition with evidence of protracted endothelial cell activation was seen. These observations suggest that non-Gal antibody-induced chronic endothelial cell activation coupled to possible hemostatic incompatibilities may be the primary stimulus for delayed xenograft rejection of GTKO hearts. To avoid possible HAR, future clinical studies should use donors expressing human complement regulatory proteins in the GTKO background.

Identification of new carbohydrate and membrane protein antigens in cardiac xenotransplantation.

BACKGROUND: α1,3-Galactosyltransferase gene knockout (GTKO) pigs reduced the significance of antibody to galactose alpha 1,3-galactose (Gal) antigens but did not eliminate delayed xenograft rejection (DXR). We hypothesize that DXR of GTKO organs results from an antibody response to a limited number of non-Gal endothelial cell (EC) membrane antigens. In this study, we screened a retrovirus expression library to identify EC membrane antigens detected after cardiac xenotransplantation. METHODS: Expression libraries were made from GT:CD46 and GTKO porcine aortic ECs. Viral stocks were used to infect human embryonic kidney cells (HEK) that were selected by flow cytometry for IgG binding from sensitized cardiac heterotopic xenograft recipients. After three to seven rounds of selection, individual clones were assessed for non-Gal IgG binding. The porcine complementary DNA was recovered by polymerase chain reaction amplification, sequenced, and identified by homology comparisons. RESULTS: A total of 199 and 317 clones were analyzed from GT:CD46 and GTKO porcine aortic EC complementary DNA libraries, respectively. Sequence analysis identified porcine CD9, CD46, CD59, and the EC protein C receptor. We also identified porcine annexin A2 and a glycosyltransferase with homology to the human ß1,4 N-acetylgalactosaminyl transferase 2 gene. CONCLUSION: The identified proteins include key EC functions and suggest that non-Gal antibody responses may compromise EC functions and thereby contribute to DXR. Recovery of the porcine ß1,4 N-acetylgalactosaminyl transferase 2 suggests that an antibody response to a SD-like carbohydrate may represent a new carbohydrate moiety involved in xenotransplantation. The identification of these porcine gene products may lead to further donor modification to enhance resistance to DXR and further reduce the level of xenograft antigenicity.