Ahmad Y. Sheikh

Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts

Given their self-renewing and pluripotent capabilities, human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However, the host immune response against transplanted hESCs is not well characterized. In fact, controversy remains as to whether hESCs have immune-privileged properties. To address this issue, we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death, suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses, resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover, we have found CD4+ T cells to be an important modulator of hESC immune-mediated rejection. Finally, we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together, these data suggest that hESCs are immunogenic, trigger both cellular and humoral-mediated pathways, and, as a result, are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches.

Apelin signaling antagonizes Ang II effects in mouse models of atherosclerosis.

Apelin and its cognate G protein-coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II-mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II-mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.

Comparison of Different Adult Stem Cell Types for Treatment of Myocardial Ischemia

Background— A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study is designed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction. Methods and Results— Bone marrow mononuclear cells (MN), mesenchymal stem cells (MSC), skeletal myoblasts (SkMb), and fibroblasts (Fibro) expressing firefly luciferase (Fluc) and green fluorescence protein (GFP) were characterized by flow cytometry, bioluminescence imaging (BLI), and luminometry. Female FVB mice (n=70) underwent LAD ligation and intramyocardially received one cell type (5×105) or PBS. Cell survival was measured by BLI and by TaqMan PCR. Cardiac function was assessed by echocardiography and invasive hemodynamic measurements. Fluc expression correlated with cell number in all groups (r2>0.93). In vivo BLI revealed acute donor cell death of MSC, SkMb, and Fibro within 3 weeks after transplantation. By contrast, cardiac signals were still present after 6 weeks in the MN group, as confirmed by TaqMan PCR (P<0.01). Echocardiography showed significant preservation of fractional shortening in the MN group compared to controls (P<0.05). Measurements of left ventricular end-systolic/diastolic volumes revealed that the least amount of ventricular dilatation occurred in the MN group (P<0.05). Histology confirmed the presence of MN, although there was no evidence of transdifferentiation by donor MN into cardiomyocytes. Conclusions— This is the first study to show that compared to MSC, SkMB, and Fibro, MN exhibit a more favorable survival pattern, which translates into a more robust preservation of cardiac function.

Differentiation, Survival, and Function of Embryonic Stem Cell–Derived Endothelial Cells for Ischemic Heart Disease

Background— Embryonic stem (ES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of ES cell–derived endothelial cells (ESC-ECs), use molecular imaging techniques to examine their survival in vivo, and determine the therapeutic efficacy of ESC-ECs for restoration of cardiac function after ischemic injury. Methods and Results— Murine ES cells were transfected with a construct composed of a vascular endothelial cadherin promoter driving enhanced green fluorescence protein (pVE-cadherin-eGFP). Differentiation of ES cells to ECs was detected by FACS analysis using Flk-1 (early EC marker at day 4) and VE-cadherin (late EC marker at day 8). After isolation, these ESC-ECs express endothelial cell markers similar to adult mouse lung endothelial cells, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). For in vivo imaging, ES cells were transduced with an ubiquitin promoter driving firefly luciferase and monomeric red fluorescence protein (pUb-Fluc-mRFP). A robust correlation exists between Fluc signals and cell numbers by ex vivo imaging analysis (R2=0.98) and by in vitro enzyme assay (R2=0.94). Afterward, 5×105 ESC-ECs or PBS (as control) was injected into the hearts of mice undergoing LAD ligation (n=15 per group). Bioluminescence imaging showed longitudinal survival of transplanted ESC-ECs for ≈8 weeks. Echocardiogram demonstrated significant functional improvement in the ESC-EC group compared with control (P=0.04). Finally, postmortem analysis confirmed increased presence of small capillaries and venules in the infarcted zones by CD31 staining. Conclusions— This is the first study to track the fate and function of transplanted ESC-ECs in the heart. With further validation, these ESC-ECs could become a valuable source of cell therapy for induction of angiogenesis in the treatment of myocardial ischemia.

Collagen Matrices Enhance Survival of Transplanted Cardiomyoblasts and Contribute to Functional Improvement of Ischemic Rat Hearts

Background— Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices. Methods and Results— H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1×106 genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 &mgr;g/mL), or (5) GF/MG/FGF (10 &mgr;g/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function. Conclusions— Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.

Comparison of Transplantation of Adipose Tissue- and Bone Marrow- Derived Mesenchymal Stem Cells in the Infarcted Heart

Background. Mesenchymal stem cells hold promise for cardiovascular regenerative therapy. Derivation of these cells from the adipose tissue might be easier compared with bone marrow. However, the in vivo fate and function of adipose stromal cells (ASC) in the infarcted heart has never been compared directly to bone marrow-derived mesenchymal cells (MSC). Methods. ASC and MSC were isolated from transgenic FVB mice with a &bgr;-actin promoter driving firefly luciferase and green fluorescent protein double fusion reporter gene, and they were characterized using flow cytometry, microscopy, bioluminescence imaging and luminometry. FVB mice (n=8 per group) underwent myocardial infarction followed by intramyocardial injection of 5×105 ASC, MSC, fibroblasts (Fibro, positive control), or saline (negative control). Cell survival was measured using bioluminescence imaging for 6 weeks and cardiac function was monitored by echocardiography and pressure-volume analysis. Ventricular morphology was assessed using histology. Results. ASC and MSC were CD34−, CD45−, c-Kit−, CD90+, Sca-1+, shared similar morphology and had a population doubling time of ∼2 days. Cells expressed Fluc reporter genes in a number-dependent fashion as confirmed by luminometry. After cardiac transplantation, both cell types showed drastic donor cell death within 4 to 5 weeks. Furthermore, transplantation of either cell type was not capable of preserving ventricular function and dimensions, as confirmed by pressure-volume-loops and histology. Conclusion. This is the first study comparing the in vivo behavior of both cell types in the infarcted heart. ASC and MSC do not tolerate well in the cardiac environment, resulting in acute donor cell death and a subsequent loss of cardiac function similar to control groups.

Molecular Imaging of Bone Marrow Mononuclear Cell Homing and Engraftment in Ischemic Myocardium

Bone marrow mononuclear cell (BMMC) therapy shows promise as a treatment for ischemic heart disease. However, the ability to monitor long‐term cell fate remains limited. We hypothesized that molecular imaging could be used to track stem cell homing and survival after myocardial ischemia‐reperfusion (I/R) injury. We first harvested donor BMMCs from adult male L2G85 transgenic mice constitutively expressing both firefly luciferase (Fluc) and enhanced green fluorescence protein reporter gene. Fluorescence‐activated cell sorting analysis revealed ∼0.07% of the population to consist of classic hematopoietic stem cells (lin‐, thy‐int, c‐kit+, Sca‐1+). Afterward, adult female FVB recipients (n = 38) were randomized to sham surgery or acute I/R injury. Animals in the sham (n = 16) and I/R (n = 22) groups received 5 × 106 of the L2G85‐derived BMMCs via tail vein injection. Bioluminescence imaging (BLI) was used to track cell migration and survival in vivo for 4 weeks. BLI showed preferential homing of BMMCs to hearts with I/R injury compared with sham hearts within the first week following cell injection. Ex vivo analysis of explanted hearts by histology confirmed BLI imaging results, and quantitative real‐time polymerase chain reaction (for the male Sry gene) further demonstrated a greater number of BMMCs in hearts with I/R injury compared with the sham group. Functional evaluation by echocardiography demonstrated a trend toward improved left ventricular fractional shortening in animals receiving BMMCs. Taken together, these data demonstrate that molecular imaging can be used to successfully track BMMC therapy in murine models of heart disease. Specifically, we have demonstrated that systemically delivered BMMCs preferentially home to and are retained by injured myocardium.

Endogenous regulation of cardiovascular function by apelin-APJ

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.

Adenoviral Human BCL-2 Transgene Expression Attenuates Early Donor Cell Death After Cardiomyoblast Transplantation Into Ischemic Rat Hearts

Background— Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts. Methods and Results— H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1×106 mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a “working heart” model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21±0.03; mH9c2: 0.21±0.04; control: 0.15±0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0±6.2; mH9c2: 48.7±6.1; control: 34.3±6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology. Conclusion— Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.

In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells After Transplantation Into Ischemic Myocardium

Objective— Clinical trials of bone marrow–derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model. Methods and Results— We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis. Conclusion— Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.