Laura C. Tumiati

Beneficial effect of autologous cell transplantation on infarcted heart function: comparison between bone marrow stromal cells and heart cells

BACKGROUND Cell transplantation may restore function after myocardial infarction, but the optimal cell type remains controversial. We compared autologous bone marrow stromal cells (BMCs) with autologous heart cells (HCs) in a porcine myocardial infarction model. METHODS Yorkshire pigs underwent coil occlusion of the left anterior descending artery. Bone marrow stromal cells were obtained from sternal marrow and HCs were obtained by left ventricular biopsy, then cultured for 4 weeks. Four weeks after infarction, a 99mTc-sestamibi single-photon emission tomography (99mTc-MIBI SPECT) scan was performed and the pigs were then transplanted with BMCs (n = 7), HCs (n = 7), or culture medium (n = 14). Four weeks after transplantation, 99mTc-MIBI SPECT scanning was repeated to evaluate regional perfusion. Pressure-volume loops were constructed from micromanometer and conductance catheter data to evaluate left ventricular function. Hearts were evaluated histologically. RESULTS Bone marrow stromal cells and HCs engrafted within the infarct and assumed a myocyte morphology. SPECT MIBI scans showed increased perfusion in the infarct in cell-transplanted pigs, while perfusion decreased in the control pigs. Heart cell transplantation improved preload-recruitable stroke work and HC and BMC transplantation both shifted the end-systolic pressure-volume relation to the left. Both BMCs and HCs prevented thinning and expansion of the infarct region, and some BMCs differentiated into endothelial cells in newly formed blood vessels perfusing the infarct. CONCLUSIONS Both BMCs and HCs engrafted in the infarct region and improved let ventricular function by preventing infarct thinning. Bone marrow stromal cells demonstrated greater plasticity in vivo, and may offer a practical alternative to HC transplantation to restore function and perfusion after a myocardial infarction.

Markers of inflammation in recipients of continuous-flow left ventricular assist devices.

Although the newer continuous-flow left ventricular assist devices (CF-LVADs) provide clinical advantages over the pulsatile pumps, the effects of low pulsatility on inflammation are incompletely understood. The objective of our study was to examine the levels of inflammatory mediators in CF-LVAD recipients compared with both healthy control subjects and heart failure patients who were candidates for CF-LVAD support. Plasma levels of chemokines, cytokines, and inflammatory markers were measured in 18 CF-LVAD recipients and compared with those of 14 healthy control subjects and 14 heart failure patients who were candidates for CF-LVADs. The levels of granulocyte macrophage-colony stimulating factor, macrophage inflammatory proteins-1&bgr;, and macrophage-derived chemokine were significantly higher in the CF-LVAD group compared with both the heart failure and the healthy control groups, whereas no significant differences were observed between the healthy control subjects and the heart failure groups. Compared with the healthy controls, C-reactive protein, interferon gamma-induced protein-10, monocyte chemotactic protein-1, and interleukin-8 levels were significantly higher in both the CF-LVAD and heart failure groups, but no significant differences were observed between the CF-LVAD recipients and the heart failure patients. Inflammatory markers were elevated in CF-LVAD recipients compared with healthy control subjects and the heart failure patients. Further studies should investigate the clinical implications of elevated levels of inflammation in CF-LVAD recipients.

Correlation between circulating endothelial progenitor cell function and allograft rejection in heart transplant patients

Endothelial progenitor cells (EPCs) may contribute to rejection and cardiac allograft vasculopathy (CAV) by being intrinsically involved in the rejection process and causing neointimal hyperplasia. The mammalian target of rapamycin inhibitors (mTORi), sirolimus and everolimus, have been demonstrated to attenuate the progression of CAV and are cytotoxic to EPC. Thus, one mechanism by which mTORi may protect against CAV is by altering EPC function. Our study measured circulating EPC function and correlated this assessment with rejection episodes in heart transplant (HT) recipients. In addition, we examined the effect of mTORi on EPCs. Patients who received HT at our institution between 1995 and 2007 were included and stratified by International Society for Heart and Lung Transplantation (ISHLT) rejection grade. Group A (n = 13) consisted of patients with at least one moderate/severe rejection episode (grade ≥ 2). Group B (n = 28) patients had no moderate/severe episodes (grade < 2). Patients were also independently stratified based on exposure as mTORi (n = 21) vs. non mTORi (n = 20). To assess EPC functional capacity, we counted the number of colony‐forming units (CFU) of EPCs in peripheral blood samples from HT recipients. There were no significant differences in baseline characteristics between groups. The mean EPC‐CFU counts/plate for group A (rejecting) were 30 ± 6 vs.16 ± 3 for group B (nonrejecting) (P = 0.03). The EPC‐CFU counts/plate in the mTORi group (15 ± 3) were lower compared to the non mTORi (27 ± 5) group (P = 0.04). We found that EPC colony‐forming capacity was higher in HT patients who experienced moderate/severe rejection episodes. Patients on mTORi showed a reduced EPC colony count consistent with our previous findings of EPC cytotoxicity. Detection of circulating EPC function post‐transplant may reliably identify patient risk level for subsequent allograft rejection and allow for appropriate adjustments to immunosuppression. Converting to mTORi therapy may reduce EPC function and provide a novel mechanism to prevent rejection and possibly attenuate the development of CAV.

Increased cyclic guanosine monophosphate levels and continuous-flow left-ventricular assist devices: Implications for gastrointestinal bleeding

OBJECTIVES We examine the hypothesis that cyclic guanosine monophosphate (cGMP) levels are elevated in recipients of continuous-flow left ventricular assist devices (CF-LVADs) and that elevated cGMP levels are associated with a risk of gastrointestinal (GI) bleeding events. METHODS The levels of cGMP, nitric oxide, platelet activation markers, platelet-derived growth factors (PDGF) AB/BB and AA, and the inflammatory mediator C-reactive protein (CRP) were examined in 19 CF-LVAD recipients, 21 patients who had heart failure, and 19 healthy control-group participants. RESULTS The median level of cGMP was significantly higher in CF-LVAD recipients, compared with healthy participants (6.6 vs 2.1 pmol/mL, u = 62.5; P = .001; r = -0.55). Median cGMP levels in the heart failure group (12.5 pmol/L) were higher, compared with both CF-LVAD recipients (u = 75.0; P = .001; r = -0.53) and healthy participants (u = 4.0; P < .001; r = -0.83). Compared with the healthy group, median CRP levels were significantly higher in CF-LVAD recipients (2.9 vs 8.0 mg/L; u = 58.0; P < .001; r = -0.63) and heart failure patients (2.9 vs 7.0 mg/L; u = 59.0; P < .001; r = -0.65). In the subgroup of patients supported with the HeartMate II (Thoratec Corporation, Pleasanton, Calif), pulsatility index was significantly negatively correlated with cGMP levels (r = -0.73; P < .05), indicating that low pulsatility index is associated with higher cGMP levels. High cGMP levels were significantly associated with GI bleeding events, but not with bleeding events in general. CONCLUSIONS The primary finding of this study is that GI bleeding in CF-LVAD recipients is associated with significantly elevated cGMP levels, despite high levels of CRP, which interfere with cGMP production. Further studies are required to determine whether elevated cGMP levels can be used as a clinical marker for increased risk of GI bleeding in CF-LVAD recipients.

Longitudinal Assessment of Inflammation in Recipients of Continuous-Flow Left Ventricular Assist Devices

BACKGROUND The long-term effects of continuous-flow left ventricular assist device (CF-LVAD) support on trends of inflammatory markers over time are unknown. We examined the hypothesis that the levels of inflammatory markers in CF-LVAD recipients are higher than in healthy controls and that these levels increase over time with long-term CF-LVAD support. METHODS We examined the levels of inflammatory markers longitudinally at baseline before CF-LVAD implantation and at 3, 6, and 9 months after implantation. We then compared the levels of inflammatory markers to those in a healthy control group. RESULTS Compared with baseline values before CF-LVAD implantation, left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVESd) decreased significantly at 3, 6, and 9 months after CF-LVAD implantation. Brain natriuretic peptide (BNP) levels dropped significantly after CF-LVAD implantation but did not normalize. Improvements in ejection fraction at 3, 6, and 9 months after CF-LVAD implantation did not reach significance. Monocyte chemoattractant protein-1, interferon γ-induced protein, and C-reactive protein levels were higher in the CF-LVAD recipients at each of the time points (baseline before CF-LVAD implantation and 3, 6, and 9 months after implantation) compared with levels in healthy controls. In CF-LVAD recipients, serum interleukin-8, tumour necrosis factor-α, and macrophage inflammatory protein-β increased significantly at 9 months, and macrophage-derived chemokine increased at 6 months after CF-LVAD implantation compared with baseline. CONCLUSIONS Despite improvements in LV dimensions and BNP levels, markers of inflammation remained higher in CF-LVAD recipients. High levels of inflammation in CF-LVAD recipients may result from heart failure preconditioning or the long-term device support, or both. Because inflammation may be detrimental to CF-LVAD recipients, future studies should determine whether inflammatory pathways are reversible.

Endothelin-1 antagonism and nitric oxide augmentation prevents cyclosporine-induced vasomotor impairment.

BACKGROUND We previously demonstrated that cyclosporine (CyA) impairs endothelial function as a result of alterations in nitric oxide (NO) and endothelin-1 (ET-1) regulation. Bosentan (BOS), an ET-1 antagonist, and tetrahydrobiopterin (BH₄), an eNOS cofactor, may reduce endothelial dysfunction by improving ET-1/NO homeostasis. METHODS Lewis rats received intraperitoneal injections of CyA with BOS or with BOS+BH₄ daily for 2 weeks. Control (Con) animals received saline injections. Thoracic aortic segments were assessed for endothelial-dependent (E(dep)) and -independent (E(ind)) relaxation (E(max%)) after exposure to acetylcholine and sodium nitroprusside. Vessel sensitivity to ET-1-induced vasospasm was evaluated. RESULTS CyA use resulted in impaired E(dep) vasorelaxation when compared with Con, whereas BOS and BH₄ treatment preserved E(dep) vasorelaxation. CyA significantly altered E(ind) vasorelaxation, whereas BOS and BH₄ therapy attenuated CyA-induced effects. Compared with Con, CyA and BH₄ exposure demonstrated increased sensitivity to ET-1 vasospasm. BOS therapy abrogated the CyA and BH₄-induced sensitivity to vasospasm. CyA treatment resulted in higher 8-isoprostane levels compared with Con. CyA-mediated vascular dysfunction is characterized by impaired NO and ET-1 homeostasis. CONCLUSIONS Our study suggests potential therapeutic strategies to prevent endothelial dysfunction as combined therapy with ET-1 antagonism and NO augmentation completely abrogated CyA-induced vascular injury.

Recipient Hypertonic Saline Infusion Prevents Cardiac Allograft Dysfunction

Objective Hypertonic saline (HTS) has potent immune and vascular effects. We assessed recipient pretreatment with HTS on allograft function in a porcine model of heart transplantation and hypothesized that HTS infusion would limit endothelial and left ventricular (LV) dysfunction following transplantation. Methods Heart transplants were performed after 6 hours of cold ischemic storage. Recipient pigs were randomized to treatment with or without HTS (7.5% NaCl) before cardiopulmonary bypass (CPB). Using a myograft apparatus, coronary artery endothelial‐dependent (Edep) and ‐independent (Eind) relaxation was assessed. LV performance was determined using pressure‐volume loop analysis. Pulmonary interleukin (IL)‐2, IL‐6, and tumor necrosis factor (TNF)‐&agr; expression was measured. Results Weaning from CPB and LV performance after transplantation were improved in HTS‐treated animals. Successful weaning from CPB was greater in the HTS‐treated hearts (8 of 8 vs 2 of 8; P < .05). Mean LV functional recovery was improved in the HTS‐treated animals, as assessed by preload recruitable stroke work (65 ± 10% vs 27 ± 10%; P < .001) and end‐systolic elastance (55 ± 7% vs 37 ± 4%; P < .001). Treatment with HTS resulted in improved Edep (mean maximum elastance [Emax], 56 ± 5% vs 37 ± 7%; P < .001) and Eind (mean Emax%, 77 ± 6% vs 52 ± 4%; P < .001) vasorelaxation compared with control. Pulmonary expression of IL‐2, IL‐6, and TNF‐&agr; increased following transplantation, whereas HTS therapy attenuated IL production (P < .001). Transplantation increased plasma TNF‐&agr; levels and LV TNF‐&agr; expression, whereas HTS prevented this up‐regulation (P < .001). Conclusions Recipient HTS pretreatment preserves allograft vasomotor and LV function, and HTS therapy limits CPB‐induced injury. HTS may be a novel recipient intervention to prevent graft dysfunction.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

Tacrolimus preserves vasomotor function and maintains vascular homeostasis

BACKGROUND Post-transplant immunosuppression is associated with endothelial dysfunction that may lead to vasculopathy. We have previously demonstrated that cyclosporine causes vascular dysfunction. In this we study examined the effect of tacrolimus (Tac) in an identical model. METHODS Lewis rats (n = 8 per group) were injected with Tac (low, medium or high dose) or saline (Con) daily for 2 weeks. Segments of thoracic aorta (TAo) were assessed for endothelium-dependent (Edep) and -independent (Eind) vasorelaxation (E(max)) and sensitivity to endothelin (ET)-induced vasoconstriction (C(max)). ET(A) and ET(B) receptor (Rc) expression levels were determined in TAo. Tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) expression were determined in myocardial (LV) tissue. Plasma ET levels and tissue oxidative injury were quantified by enzyme-linked immunoassay. RESULTS Tac did not impair Edep relaxation when compared with Con (p = 0.69). Impairment of sodium nitroprusside-mediated Eind vasorelaxation was noted with Tac (E(max): Con 69 ± 2%, Tac high 54 ± 2%; p = 0.0001), whereas no such impairment was seen with diltiazem-mediated Eind vasorelaxation (p = 0.06). Tac decreased sensitivity to ET (C(max): Con 222 ± 19%, Tac high 162 ± 11%; p = 0.0002) and ET levels (Con 0.8 ± 0.1 fmol/ml, Tac 0.4 ± 0.1 fmol/ml; p = 0.02). Tac did not alter ET(A) Rc expression (p = 0.28), but increased ET(B) Rc levels (p = 0.02). Oxidative injury was similar in both LV (p = 0.43) and Ao (p = 0.73) tissue. Similarly, TNF-α expression (p = 0.16) was not different between groups, whereas expression of TGF-β demonstrated a significant decrease with Tac treatment (p = 0.02). CONCLUSION Our findings suggests that tacrolimus has beneficial effects with respect to endothelial function.

10-Year Experience with HLA-G in Heart Transplantation

The Human Leukocyte Antigen-G (HLA-G) is a MHC-class Ib molecule with robust immunomodulatory properties; in transplant, it inhibits cytotoxic activity of immune cells and thus has a pivotal role in protecting the allograft from immune attack. The present review details a 10-year experience investigating the influence of HLA-G on heart transplantation, allograft rejection and cardiac allograft vasculopathy development. Exploration of HLA-G in transplantation began with the initial findings of its increased expression in allograft hearts. Since then, HLA-G has been recognized as an important factor in transplant immunology. We discuss inducers of HLA-G expression, and the importance of HLA-G as a potential biomarker in allograft rejection and heart failure. We also highlight the importance of polymorphisms and how they may influence both HLA-G expression and clinical outcomes. There remains much to be done in this field, however we hope that findings from our group and other groups will ignite interest and facilitate further expansion of HLA-G research in transplantation.