Induced Pluripotent Stem Cell (iPSC)-Derived Extracellular Vesicles Are Safer and More Effective for Cardiac Repair Than iPSCs.

RATIONALE: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells (iPSCs) have not been directly compared with the source iPSCs. OBJECTIVE: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo. METHODS AND RESULTS: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV-treated mice exhibited improved left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement. iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone. Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe. CONCLUSIONS: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy. Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in patients with ischemic myocardial damage.

Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction.

RATIONALE: The role of interleukin (IL)-6 in the pathogenesis of cardiac myocyte hypertrophy remains controversial. OBJECTIVE: To conclusively determine whether IL-6 signaling is essential for the development of pressure overload-induced left ventricular (LV) hypertrophy and to elucidate the underlying molecular pathways. METHODS AND RESULTS: Wild-type and IL-6 knockout (IL-6(-/-)) mice underwent sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Serial echocardiograms and terminal hemodynamic studies revealed attenuated LV hypertrophy and superior preservation of LV function in IL-6(-/-) mice after TAC. The extents of LV remodeling, fibrosis, and apoptosis were reduced in IL-6(-/-) hearts after TAC. Transcriptional and protein assays of myocardial tissue identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 3 (STAT3) activation as important underlying mechanisms during cardiac hypertrophy induced by TAC. The involvement of these pathways in myocyte hypertrophy was verified in isolated cardiac myocytes from wild-type and IL-6(-/-) mice exposed to prohypertrophy agents. Furthermore, overexpression of CaMKII in H9c2 cells increased STAT3 phosphorylation, and exposure of H9c2 cells to IL-6 resulted in STAT3 activation that was attenuated by CaMKII inhibition. Together, these results identify the importance of CaMKII-dependent activation of STAT3 during cardiac myocyte hypertrophy via IL-6 signaling. CONCLUSIONS: Genetic deletion of IL-6 attenuates TAC-induced LV hypertrophy and dysfunction, indicating a critical role played by IL-6 in the pathogenesis of LV hypertrophy in response to pressure overload. CaMKII plays an important role in IL-6-induced STAT3 activation and consequent cardiac myocyte hypertrophy. These findings may have significant therapeutic implications for LV hypertrophy and failure in patients with hypertension.

Carbon monoxide induces a late preconditioning-mimetic cardioprotective and antiapoptotic milieu in the myocardium.

A growing body of evidence indicates that carbon monoxide (CO), once perceived merely as a poisonous gas, exerts antiapoptotic and cytoprotective effects. Using a water-soluble CO-releasing molecule (CORM) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), we previously reported that CO induces a delayed protection against myocardial infarction similar to that observed in the late phase of ischemic preconditioning (PC). In the current study, we investigated the molecular mechanisms underlying this cardioprotective effect. The impact on apoptotic signaling pathways was first examined in the setting of ischemia/reperfusion injury. Mice were pretreated with CORM-3 or iCORM-3 (which does not release CO) and subjected to coronary occlusion/reperfusion 24h later. In mice that received CORM-3, there was a significant reduction in markers of apoptosis (cleaved lamin A, cleaved caspase-3, and cleaved PARP-1) after ischemia/reperfusion injury. To elucidate the mechanism of CORM-3-induced cardioprotection we further examined the activation of transcription factors and induction of cardioprotective and apoptosis modulating proteins. Infusion of CORM-3 rapidly activated the stress-responsive transcription factors nuclear factor kappaB (NF-κB), signal transducers and activators of transcription (STAT)1, STAT3, and NF-E2-related factor-2 (Nrf2). This was followed 24h later by upregulation of cardioprotective proteins (heme oxygenase-1 [HO-1], cyclooxygenase-2 [COX-2], and extracellular superoxide dismutase [Ec-SOD]) and antiapoptotic proteins involving both the mitochondria-mediated (Mcl-1) and the death receptor-mediated (c-FLIP(S) and c-FLIP(L)) apoptosis pathways. We conclude that CO released by CORM-3 triggers a cardioprotective signaling cascade that recruits the transcription factors NF-κB, STAT1/3, and Nrf2 with a subsequent increase in cardioprotective and antiapoptotic molecules in the myocardium leading to the late PC-mimetic infarct-sparing effects. This article is part of a Special Issue entitled 'Possible Editorial'.

A murine model of inducible, cardiac-specific deletion of STAT3: its use to determine the role of STAT3 in the upregulation of cardioprotective proteins by ischemic preconditioning.

Pharmacological studies have shown that signal transducers and activators of transcription (STATs) are necessary for the delayed cardioprotection of ischemic preconditioning (PC). However, pharmacologic STAT inhibitors are not specific; furthermore, the individual role of STAT3 in late PC remains unknown. The objectives of the study were (i) to create an inducible, cardiac-specific STAT3 knockout mouse; (ii) to verify whether STAT3 deletion has any adverse effects in the short term (~1 month); and (iii) to use this novel tool to evaluate the role of STAT3 in the PC-induced upregulation of cardioprotective and anti-apoptotic proteins. We created an inducible, cardiomyocyte-restricted STAT3 deficient mouse (MCM TG:STAT3(flox/flox)) by interbreeding STAT3(flox/flox) mice and tamoxifen-inducible MCM TG mice. Treatment of MCM TG:STAT3(flox/flox) mice with tamoxifen resulted in deletion of STAT3 specifically in cardiac myocytes, concomitant with abrogation of ischemic PC-induced Tyr-705 and Ser-727 phosphorylation of STAT3 and increased STAT3 DNA-binding activity. In vehicle-treated MCM TG:STAT3(flox/flox) mice, ischemic PC increased the expression of cardioprotective (COX-2 and HO-1) and anti-apoptotic (e.g., Mcl-1, Bcl-x(L), c-FLIP(L), c-FLIP(S)) proteins 24h later; in contrast, in tamoxifen-treated MCM TG:STAT3(flox/flox) mice this increase was completely absent. Deletion of STAT3 had no apparent adverse effects on LV structure or function after 35 days. We have developed a novel inducible, cardiomyocyte-restricted STAT3 deficient mouse that can be used to specifically interrogate the role of this transcription factor in cardiovascular pathophysiology in vivo. Our data demonstrate, for the first time, that recruitment of STAT3 plays an obligatory role in the upregulation of cardioprotective and anti-apoptotic proteins and suggest that STAT3 activation is important in inhibiting both the death receptor pathway (which is modulated by c-FLIP(L) and c-FLIP(S)) and the mitochondrial pathway (which is mediated by Mcl-1 and Bcl-x(L)).

Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription1/3 pathway.

BACKGROUND: The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach. METHODS AND RESULTS: In the nonpreconditioned state, eNOS-/- mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC) epsilon and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS-/- mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC. CONCLUSIONS: In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC epsilon-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.

Role of the protein kinase C-epsilon-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning.

BACKGROUND: Although Janus kinase (JAK)-mediated Tyr phosphorylation of signal transducers and activators of transcription (STAT) 1 and 3 is essential for the upregulation of cyclooxygenase-2 (COX-2) and the cardioprotection of late preconditioning (PC), the role of Ser phosphorylation of STAT1 and STAT3 in late PC and the upstream signaling mechanisms responsible for mediating Ser phosphorylation of STAT1 and STAT3 remain unknown. METHODS AND RESULTS: In mice preconditioned with six 4-minute coronary occlusion/4-minute reperfusion cycles, we found that (1) ischemic PC activates the Raf1-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) 1/2-p44/42 MAPK signaling pathway, induces phosphorylation of STAT1 and STAT3 on the Ser-727 residue, and upregulates COX-2 expression; (2) pSer-STAT1 and pSer-STAT3 form complexes with pTyr-p44/42 MAPKs in preconditioned myocardium, supporting the concept that Ser phosphorylation of these 2 factors is mediated by activated p44/42 MAPKs; and (3) activation of the Raf-1-MEK-1/2-p44/42 MAPK-pSer-STAT1/3 pathway and induction of COX-2 during ischemic PC are dependent on protein kinase C (PKC)-epsilon activity, as determined by both pharmacological and genetic inhibition of PKCepsilon. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that ischemic PC causes Ser phosphorylation of STAT1 and STAT3 and that this event is governed by PKCepsilon via a PKCepsilon-Raf1-MEK1/2-p44/42 MAPK pathway. Furthermore, this is the first report that COX-2 expression in the heart is controlled by PKCepsilon. Together with our previous findings, the present study implies that STAT-dependent transcription of the genes responsible for ischemic PC is modulated by a dual signaling mechanism that involves both JAK1/2 (Tyr phosphorylation) and PKCepsilon (Ser phosphorylation).

Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria.

Although functional coupling between protein kinase Cepsilon (PKCepsilon) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKCepsilon remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKCepsilon and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKCepsilon with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKCepsilon complex at approximately 2%, approximately 0.2%, and approximately 1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKCepsilon can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKCepsilon resulted in a significant inhibition of Ca2+-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKCepsilon in mice, which is cardioprotective, greatly increased interaction of PKCepsilon with the pore components and inhibited Ca2+-induced pore opening. In contrast, cardiac expression of kinase-inactive PKCepsilon did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKCepsilon transgenesis. Collectively, these data demonstrate that PKCepsilon forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKCepsilon-induced cardioprotection.

The late phase of ischemic preconditioning induces a prosurvival genetic program that results in marked attenuation of apoptosis.

Although the cardioprotection afforded by the late phase of ischemic preconditioning (PC) in ischemia/reperfusion (I/R) injury has been well studied, it is unknown whether this beneficial effect can be attributed to inhibition of apoptosis. We hypothesized that ischemic PC affords protection by suppressing apoptosis and examined the underlying mechanisms. Myocardial infarction was produced in mice (30-min coronary occlusion). In animals preconditioned 24 h earlier with six 4-min coronary occlusion/4-min reperfusion (O/R) cycles, there was a marked decrease in apoptosis as assessed by three different parameters: hairpin-1 assay, caspase-3 activity, and immunohistochemical analysis of active caspase-3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1). This protective effect was accompanied by increased expression of multiple antiapoptotic proteins that regulate both the mitochondria-mediated (Bcl-x(L) and Mcl-1) and the death-receptor-mediated (c-FLIP(L) and c-FLIP(S)) pathway of apoptosis and by decreased expression of the proapoptotic protein Bad. This is the first demonstration that the late phase of ischemic PC attenuates cardiac apoptosis after ischemia/reperfusion injury and that this salubrious effect is associated with a complex genetic prosurvival program that results in modulation of several key proteins involved in both the mitochondrial and the death receptor pathways of apoptosis.

Tumor necrosis factor-alpha does not modulate ischemia/reperfusion injury in naïve myocardium but is essential for the development of late preconditioning.

The role of tumor necrosis factor (TNF)-alpha in myocardial ischemia/reperfusion injury remains controversial. We used homozygous TNF-alpha null mice (TNF-alpha(-/-)) to determine whether TNF-alpha modulates myocardial ischemia/reperfusion injury. Mice were subjected to a 30-min coronary occlusion followed by 24 h of reperfusion. When wild-type mice were preconditioned with six cycles of 4-min coronary occlusion/4-min reperfusion 24 h before the 30-min occlusion, infarct size was reduced from 58.6 +/- 1.9% of the risk region to 19.3 +/- 3.6%, indicating a late preconditioning (PC) effect. In non-preconditioned TNF-alpha(-/-) mice, infarct size was similar to that observed in wild-type mice (55.5 +/- 3.7%). However, in TNF-alpha(-/-) mice preconditioned with six occlusion/reperfusion cycles 24 h earlier, infarct size was not reduced (55.2 +/- 5.7%), indicating that the late PC protection against infarction was completely abolished. While minimal TNF-alpha immunoreactivity was detected in sham-operated hearts, extensive TNF-alpha expression was noted in the cytoplasm of cardiomyocytes in the ischemic/reperfused region 30 min after the PC ischemia. At 30 min after PC, wild-type mice exhibited increased DNA-binding activity of nuclear factor-kappa B (NF-kappa B) and activator protein-1 (AP-1) and nuclear translocation of p65, c-Jun and c-Fos; all of these changes were absent in TNF-alpha(-/-) mice. These data demonstrate that TNF-alpha does not modulate infarct size in the naïve (non-preconditioned) state but is essential for the development of the late phase of ischemic PC, possibly via the activation of NF-kappa B and AP-1 transcription factors.