Activation of diverse signaling pathways by ex-vivo delivery of multiple cytokines for myocardial repair.

We tested the hypothesis that simultaneous transgenic overexpression of a select quartet of growth factors activates diverse signaling pathways for mobilization and participation of various stem/progenitor cells for cardiogenesis in the infarcted heart. Human insulin growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1a), and hepatocyte growth factor (HGF) plasmids were synthesized and transfected into skeletal myoblasts (SM) from young male wild-type or transgenic rats expressing green fluorescent protein (GFP). Overexpression of growth factors in transfected SM ((Trans)SM) was confirmed by reverse transcription polymerase chain reaction, western blotting, and fluorescence immunostaining. Using our custom-made growth factor array and western blotting, multiple angiogenic and prosurvival factors were detected in (Trans)SM, including secreted frizzled related protein-1,2,4,5, matrix metalloproteinases-3 and 9, connexin-43, netrin-1, Nos-2, Wnt-3, Akt, MAPK42/44, Stat3, nuclear factor kappa B (NFκB), hypoxia-inducible factor 1 (HIF-1α), and protein kinase C (PKC). The conditioned medium (CM) from (Trans)SM was cytoprotective for cardiomyocytes following H(2)O(2) treatment [P<0.01 vs. CM from native SM ((Nat)SM)], promoted a higher transwell migration of human umbilical cord vein endothelial cells (223.3±1.8, P<0.01) and in vitro tube formation (47.8±1.9, P<0.01). Intramyocardial transplantation of 1.5×10(6) (Trans)SM (group-3) in a rat model of acute myocardial infarction induced extensive mobilization of cMet(+), ckit(+), ckit(+)/GATA(4+), CXCR4(+), CD44(+), CD31(+), and CD59(+) cells into the infarcted heart on day 7 and improved integration of (Trans)SM in the heart compared to (Nat)SM (group 2) (P<0.05). Extensive neomyogenesis and angiogenesis in group-3 (P<0.01 vs. group-2), with resultant attenuation of infarct size (P<0.01 vs. group-2) and improvement in global heart function (P<0.01 vs. group-2) was observed at 8 weeks. In conclusion, simultaneous activation of diverse signaling pathways by overexpression of multiple growth factors caused massive mobilization and homing of stem/progenitor cells from peripheral circulation, the bone marrow, and the heart for accelerated repair of the infarcted myocardium.

MicroRNA-143 is a critical regulator of cell cycle activity in stem cells with co-overexpression of Akt and angiopoietin-1 via transcriptional regulation of Erk5/cyclin D1 signaling.

We report that simultaneous expression of Akt and angiopoietin-1 (Ang-1) transgenes supported mitogenesis in stem cells with a critical role for microRNA-143 (miR-143) downstream of FoxO1 transcription factor. Mesenchymal stem cells (MSC) from young male rats were transduced with Ad-vectors encoding for Akt ((Akt)MSC) and Ang-1 ((Ang-1)MSC) transgenes for their individual or simultaneous overexpression ((AA)MSC; > 5-fold gene level and > 4-fold Akt and Ang-1 protein expression in (AA)MSC vs. Ad-Empty transduced MSC; (Emp)MSC). (AA)MSC had higher phosphorylation of FoxO1, which activated Erk5, a distinct mitogen-induced MAPK that drove transcriptional activation of cyclin D1 and Cdk4. Flow cytometry showed > 10% higher S-phase cell population that was confirmed by BrdU assay (15%) and immunohistology for Ki67 (11%) in (AA)MSC using (Emp)MSC as controls. miR array supported by real-time PCR showed induction of miR-143 in (AA)MSC (4.73-fold vs.. (Emp)MSC). Luciferase assay indicated a dependent relationship between miR-143 and Erk5 in (AA)MSC. FoxO1-specific siRNA upregulated miR-143, whereas inhibition of miR-143 did not change FoxO1 activation. However, miR-143 inhibition repressed phosphorylation of Erk5 and abrogated cyclin D1 with concomitant reduction in cells entering cell cycle. During in vivo studies, male GFP+ (AA)MSC transplanted into wild-type female infarcted rat hearts showed significantly higher number of Ki67 expressing cells (p < 0.05 vs. (Emp)MSC) 7 days after engraftment (n = 4 animals/group). In conclusion, co-overexpression of Akt and Ang-1 in MSC activated cell cycle progression by upregulation of miR-143 and stimulation of FoxO1 and Erk5 signaling.

Non-hypoxic stabilization of HIF-Iα during coordinated interaction between Akt and angiopoietin-1 enhances endothelial commitment of bone marrow stem cells.

We previously reported that mesenchymal stem cells (MSC) co-expressing Akt and angiopoietin-1 (Ang-1) preserved infarcted heart function via angiomyogenesis. The present study determined the mechanism of co-overexpression of Akt and Ang-1 in promoting endothelial commitment of MSC. The cells were transduced with vectors encoding for Akt ((Akt)MSC), Ang-1 ((Ang-1)MSC), and both Akt and Ang-1 ((AA)MSC) using Empty vector transduced MSC ((Emp)MSC) as control. Molecular studies indicated a coordinated interaction between Akt and Ang-1 in (AA)MSC and led to non-hypoxic stabilization of hypoxia inducible factor-1α (HIF-Iα) which accentuated under 4-h anoxia. We also observed HIF-Iα dependent induction of hemeoxygenase-1, endothelial specific markers and VEGF in (AA)MSC. Vascular commitment of (AA)MSC was confirmed by immunostaining, Western blotting and flow cytometry for endothelial specific early and late markers including Flt1, Flk1, Tie2, VCAM-1, and von Willebrand Factor-VIII (vWF-VIII) in HIF-Iα dependent fashion besides exhibiting higher emigrational activity and angiogenesis in vitro. (AA)MSC transplanted into rat model of myocardial infarction showed higher Flk1 and Flt1 positivity and also promoted intrinsic Flk1(+) and Flt1(+) cell mobilization into the infarcted heart. Given the ease of availability of MSC and simplicity of approach to co-overexpress Ang-1 and Akt to enhance their endothelial commitment, the strategy will be significant for cellular angiogenesis to treat ischemic heart.

The effect of diabetes and poor left ventricular function on bone marrow cell-induced myocardial protection.

OBJECTIVES: The myocardium of patients with diabetes and poor left ventricular (LV) function cannot be protected by interventions such as ischemic preconditioning (IP). We investigated whether these clinical conditions influence the protection elicited by the paracrine effect of bone marrow cells (BMCs) and whether the cause for loss in protection resides in the BMCs, the myocardium, or both. METHODS: BMCs and right atrial appendage were obtained from patients with and without diabetes and from poor (EF < 30%) and preserved LV function undergoing elective cardiac surgery. Muscles (n = 6/group) were co-cultured with BMCs and subjected to 90 min ischemia/120 min reoxygenation at 37°C. The degree of protection was assessed by measuring creatine kinase (CK) released, and myocardial cell necrosis and apoptosis. RESULTS: Ischemia-induced CK release, cell necrosis, and apoptosis in the diabetic myocardium were not significantly affected by IP or by co-incubation with autologous or non-diabetic allogenic BMCs. Conversely, significant reduction in CK release, cell necrosis, and apoptosis were observed when non-diabetic myocardium was co-incubated with allogenic diabetic BMCs. Interestingly, while allogenic BMCs from subjects with preserved LV function exerted a modest but significant reduction in CK leakage and cell necrosis, but not apoptosis, on failing myocardium, the BMCs from patients with poor LV function failed to protect their own and the allogenic myocardium from subjects with normal LV function. CONCLUSIONS: The failure to protect the myocardium of patients with poor LV function against ischemia/reoxygenation-induced injury is mainly due to a deficit in their BMCs and the myocardium itself, whereas in patients with diabetes the deficit remains within the myocardium and not in the BMCs.

Protection of human myocardium by bone marrow cells: role of long-term administration of the mitochondrial K(ATP) channel opener nicorandil.

BACKGROUND: We have previously demonstrated that bone marrow cells (BMCs) afford myocardial protection as potent as ischemic preconditioning (IP) and also that the myocardium of patients treated with the mitoK(ATP) channel opener nicorandil cannot be protected by IP. Here, we investigated whether nicorandil influences the cardioprotection elicited by BMCs and whether any loss in protection can be rescued by naïve allogenic BMCs. MATERIALS AND METHODS: BMCs and right atrial appendage were obtained from patients on long-term treatment and nontreated with nicorandil. The atrial myocardium was subjected to 90 min ischemia/120 min reoxygenation at 37°C in the presence and absence of autologous and allogenic BMCs. Some muscles were subjected to IP prior to ischemia and served as positive controls. Tissue injury was assessed by creatine kinase released during reoxygenation, and cell necrosis and apoptosis were determined by propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). RESULTS: Creatine kinase (CK) release and cell necrosis and apoptosis induced by ischemia were not significantly reduced by IP in the myocardium from nicorandil subjects and values were also unaffected by the co-incubation with autologous or allogenic BMCs from subjects not treated with nicorandil (naïve BMCs). However, when the myocardium from subjects not treated with nicorandil was co-incubated with autologous BMCs or with allogenic BMCs from subjects treated with nicorandil, there was a similar significant reduction in CK release, cell necrosis and apoptosis. CONCLUSIONS: The cardioprotective properties of BMCs from subjects treated with the mitoK(ATP) channel opener nicorandil are preserved; however, the myocardium of these patients cannot benefit from the cardioprotective effect of BMCs due to an unresponsive myocardium.

Bone marrow cell-induced protection of the human myocardium: characterization and mechanism of action.

OBJECTIVES: The mechanism of the putative beneficial effect of myocardial transplantation of bone marrow cells remains unclear. We studied the protective properties of bone marrow cells on the human myocardium and investigated the underlying mechanism. METHODS: Bone marrow cells and the right atrial appendage were obtained from patients undergoing elective cardiac surgery. Myocardial slices were subjected to 90 minutes of simulated ischemia/120 minutes of reoxygenation at 37 degrees C following various protocols. Tissue injury was assessed by creatine kinase released into the media during the reoxygenation period, and myocardial necrosis and apoptosis were determined by propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (percent of aerobic control). RESULTS: Autologous unfractionated bone marrow cells significantly reduced myocardial injury. Maximal protection was obtained with 5 x 10(6) autologous cells (approximately 1.5 x 10(5) cells/mg wet myocardium) that caused a reduction in creatine kinase release and cell death by necrosis and apoptosis of 70% to 80%. Allogenic bone marrow cells were as protective as the autologous cells and their effect was unaffected by prior frozen storage or culturing. Similar myocardial protection was also attained when bone marrow cells were present only before or during ischemia, or during reoxygenation, a benefit that was comparable with that of ischemic preconditioning. Conditioned media by the bone marrow cells was sufficient to induce protection, which was abolished by the selective insulin-like growth factor-1 receptor blocker PQ401. CONCLUSIONS: Bone marrow cells possess potent myocardial protective properties that are triggered by a secreted factor or factors and mediated by insulin-like growth factor-1 receptor. These results have important clinical implications for the therapeutic use of bone marrow cells in ischemic heart disease and for the design of future clinical studies.

Randomized controlled trial on the cardioprotective effect of bone marrow cells in patients undergoing coronary bypass graft surgery.

AIMS: This randomized study investigates whether bone marrow cells (BMCs) can reduce ischaemic injury during cardiac surgery. METHODS AND RESULTS: Forty-four elective coronary artery bypass grafting patients were randomized to control group or BMCs group (whereby autologous BMCs were administered with each dose of cardioplegia antegradely into the coronaries). Troponin I and CK-MB were measured during the first 48 h after surgery and were not significantly different between the control and BMCs groups. The role of cardiopulmonary bypass (CPB) on the cardioprotective effects of BMCs was also studied using an in vitro model of stimulated ischaemia and reoxygenation on right atrial appendages obtained from controls either before or 10 min after the initiation of CPB. Bone marrow cells significantly reduced myocardial injury in muscles obtained prior to CPB. This effect was comparable with ischaemic preconditioning (IP), although their combination did not afford additional benefit. However, when muscles were harvested after CPB, myocardial injury in the ischaemic group alone was less, and BMCs or IP did not exert further protection. CONCLUSION: Bone marrow cells did not afford additional benefit when used as an additive to cardioplegia during CPB. However, BMCs offer cardioprotection as potent as IP, when the heart is not subjected to stress, such as CPB, that per se can precondition the myocardium.