Cardiac Regeneration and Stem Cells.

After decades of believing the heart loses the ability to regenerate soon after birth, numerous studies are now reporting that the adult heart may indeed be capable of regeneration, although the magnitude of new cardiac myocyte formation varies greatly. While this debate has energized the field of cardiac regeneration and led to a dramatic increase in our understanding of cardiac growth and repair, it has left much confusion in the field as to the prospects of regenerating the heart. Studies applying modern techniques of genetic lineage tracing and carbon-14 dating have begun to establish limits on the amount of endogenous regeneration after cardiac injury, but the underlying cellular mechanisms of this regeneration remained unclear. These same studies have also revealed an astonishing capacity for cardiac repair early in life that is largely lost with adult differentiation and maturation. Regardless, this renewed focus on cardiac regeneration as a therapeutic goal holds great promise as a novel strategy to address the leading cause of death in the developed world.

Repressive histone methylation regulates cardiac myocyte cell cycle exit.

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo.

Outcomes of biventricular mechanical support patients discharged to home to await heart transplantation.

BACKGROUND: The use of left ventricular assist devices has grown rapidly in recent years for patients with end-stage heart failure. A significant proportion of patients require both left- and right-sided support with biventricular assist devices (BiVADs) as a bridge to transplantation. Traditionally, these patients have waited in the hospital until they receive a transplant. PURPOSE: The aim of this study was to characterize the clinical course of BiVAD patients discharged to home to await heart transplantation. METHODS: Between November 2009 and July 2011, 24 adult patients underwent Thoratec paracorporeal BiVAD placement at the University of California Los Angeles, all with an Interagency Registry for Mechanically Assisted Circulatory Support score 1 or 2. The disposition, complications, and rehospitalizations of these subjects were retrospectively reviewed. RESULTS: Fourteen of the 24 patients were successfully discharged to home, with a mean time of 60 ± 27 days from BiVAD implantation to discharge. Ninety-three percent (13/14) of the patients sent home went on to be transplanted. Eleven of the 14 (79%) came in from home to receive their transplant. The mean time from BiVAD implantation to transplantation was 100 ± 65 days. Of the 14 patients discharged to home, there were 18 readmissions in 8 patients. CONCLUSION: In this small single-center review, we found that complex medical patients with BiVADs can be discharged to home and can await a heart transplant from home under the close management of multidisciplinary acute care and outpatient teams.

Divergent mitochondrial biogenesis responses in human cardiomyopathy.

BACKGROUND: Mitochondria are key players in the development and progression of heart failure (HF). Mitochondrial (mt) dysfunction leads to diminished energy production and increased cell death contributing to the progression of left ventricular failure. The fundamental mechanisms that underlie mt dysfunction in HF have not been fully elucidated. METHODS AND RESULTS: To characterize mt morphology, biogenesis, and genomic integrity in human HF, we investigated left ventricular tissue from nonfailing hearts and end-stage ischemic (ICM) or dilated (DCM) cardiomyopathic hearts. Although mt dysfunction was present in both types of cardiomyopathy, mt were smaller and increased in number in DCM compared with ICM or nonfailing hearts. mt volume density and mtDNA copy number was increased by ≈2-fold (P<0.001) in DCM hearts in comparison with ICM hearts. These changes were accompanied by an increase in the expression of mtDNA-encoded genes in DCM versus no change in ICM. mtDNA repair and antioxidant genes were reduced in failing hearts, suggestive of a defective repair and protection system, which may account for the 4.1-fold increase in mtDNA deletion mutations in DCM (P<0.05 versus nonfailing hearts, P<0.05 versus ICM). CONCLUSIONS: In DCM, mt dysfunction is associated with mtDNA damage and deletions, which could be a consequence of mutating stress coupled with a peroxisome proliferator-activated receptor γ coactivator 1α-dependent stimulus for mt biogenesis. However, this maladaptive compensatory response contributes to additional oxidative damage. Thus, our findings support further investigations into novel mechanisms and therapeutic strategies for mt dysfunction in DCM.

"Good enough solutions" and the genetics of complex diseases.

In this Emerging Science Review, we discuss a systems genetics strategy, which we call gene module association study (GMAS), as a novel approach complementing genome-wide association studies (GWAS), to understand complex diseases by focusing on how genes work together in groups rather than singly. The first step is to characterize phenotypic differences among a genetically diverse population. The second step is to use gene expression microarray (or other high-throughput) data from the population to construct gene coexpression networks. Coexpression analysis typically groups 20 000 genes into 20 to 30 modules containing tens to hundreds of genes, whose aggregate behavior can be represented by the module's "eigengene." The third step is to correlate expression patterns with phenotype, as in GWAS, only applied to eigengenes instead of single nucleotide polymorphisms. The goal of the GMAS approach is to identify groups of coregulated genes that explain complex traits from a systems perspective. From an evolutionary standpoint, we hypothesize that variability in eigengene patterns reflects the "good enough solution" concept, that biological systems are sufficiently complex so that many possible combinations of the same elements (in this case eigengenes) can produce an equivalent output, that is, a "good enough solution" to accomplish normal biological functions. However, when faced with environmental stresses, some "good enough solutions" adapt better than others, explaining individual variability to disease and drug susceptibility. If validated, GMAS may imply that common polygenic diseases are related as much to group interactions between normal genes, as to multiple gene mutations.

Dynamic Visualization of DNA Methylation in Cell Cycle Genes during iPSC Cardiac Differentiation.

Background: Epigenetic DNA methylation is an essential mechanism controlling gene expression and cellular function. Existing analyses with conventional assays have generated significant insights into static states of DNA methylation, but were unable to visualize the dynamics of epigenetic regulation. Aim: We utilized a genomic DNA methylation reporter (GMR) system to track changes in DNA methylation during cardiac differentiation. Methods and Results: The promoter region of Cdk1 (Cyclin-dependent kinase 1) or Sox2 (SRY-Box Transcription Factor 2) gene was cloned upstream of the small nuclear ribonucleoprotein polypeptide N (Snrpn) minimal promoter followed by a fluorescent reporter gene. Mouse induced pluripotent stem cells (iPSCs) carrying Sox2 GMR rapidly lost fluorescent reporter signal upon the induction of differentiation. Cdk1 GMR reporter signal was strong in undifferentiated iPSCs, and gradually decreased during directed cardiomyocyte (CM) differentiation. RT-qPCR and pyrosequencing demonstrated that the reduction of Sox2 and Cdk1 was regulated by hypermethylation of their CpG regions during cardiac differentiation. The present study demonstrated the dynamic DNA methylation along the course of cell cycle withdrawal during CM differentiation. Conclusion: The GMR reporter system can be a useful tool to monitor real-time epigenetic DNA modification at single-cell resolution.

Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells.

Our understanding of how mesodermal tissue is formed has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESCs) is initiated by epithelial-to-mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that the earliest CD326-CD56+ cells, generated from hESCs in the presence of activin A, BMP4, VEGF, and FGF2, represent a multipotent mesoderm-committed progenitor population. CD326-CD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle, and cardiomyocytes, while lacking the pluripotency of hESCs. CD326-CD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a unique approach to study how germ layer specification is regulated and offer a promising target for tissue engineering.

Hippo signaling and histone methylation control cardiomyocyte cell cycle re-entry through distinct transcriptional pathways.

AIMS: Accumulating data demonstrates that new adult cardiomyocytes (CMs) are generated throughout life from pre-existing CMs, although the absolute magnitude of CM self-renewal is very low. Modifying epigenetic histone modifications or activating the Hippo-Yap pathway have been shown to promote adult CM cycling and proliferation. Whether these interventions work through common pathways or act independently is unknown. For the first time we have determined whether lysine demethylase 4D (KDM4D)-mediated CM-specific H3K9 demethylation and Hippo pathways inhibition have additive or redundant roles in promoting CM cell cycle re-entry. METHODS AND RESULTS: We found that activating Yap1 in cultured neonatal rat ventricular myocytes (NRVM) through overexpressing Hippo pathway inhibitor, miR-199, preferentially increased S-phase CMs, while H3K9me3 demethylase KDM4D preferentially increased G2/M markers in CMs. Together KDM4D and miR-199 further increased total cell number of NRVMs in culture. Inhibition of Hippo signaling via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1) also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducible activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle, while miR-199 and si-Sav1 preferentially up-regulated genes involved in G1/S phase. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Using transgenic mice over-expressing KDM4D together with Myc, we demonstrated that KDM4D/Myc significantly increased CM cell cycling but did not affect cardiac function. CONCLUSIONS: KDM4D effects on CM cell cycle activity are additive with the Hippo-Yap1 pathway and appear to preferentially regulate different cell cycle regulators. This may have important implications for strategies that target cardiac regeneration in treating heart disease.

Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.

Mithramycin is a gene-selective Sp1 inhibitor that identifies a biological intersection between cancer and neurodegeneration.

Oncogenic transformation of postmitotic neurons triggers cell death, but the identity of genes critical for degeneration remain unclear. The antitumor antibiotic mithramycin prolongs survival of mouse models of Huntington's disease in vivo and inhibits oxidative stress-induced death in cortical neurons in vitro. We had correlated protection by mithramycin with its ability to bind to GC-rich DNA and globally displace Sp1 family transcription factors. To understand how antitumor drugs prevent neurodegeneration, here we use structure-activity relationships of mithramycin analogs to discover that selective DNA-binding inhibition of the drug is necessary for its neuroprotective effect. We identify several genes (Myc, c-Src, Hif1α, and p21(waf1/cip1)) involved in neoplastic transformation, whose altered expression correlates with protective doses of mithramycin or its analogs. Most interestingly, inhibition of one these genes, Myc, is neuroprotective, whereas forced expression of Myc induces Rattus norvegicus neuronal cell death. These results support a model in which cancer cell transformation shares key genetic components with neurodegeneration.

Abcc6 deficiency causes increased infarct size and apoptosis in a mouse cardiac ischemia-reperfusion model.

OBJECTIVE: ABCC6 genetic deficiency underlies pseudoxanthoma elasticum (PXE) in humans, characterized by ectopic calcification, and early cardiac disease. The spectrum of PXE has been noted in Abcc6-deficient mice, including dystrophic cardiac calcification. We tested the role of Abcc6 in response to cardiac ischemia-reperfusion (I/R) injury. METHODS AND RESULTS: To determine the role of Abcc6 in cardioprotection, we induced ischemic injury in mice in vivo by occluding the left anterior descending artery (30 minutes) followed by reperfusion (48 hours). Infarct size was increased in Abcc6-deficient mice compared with wild-type controls. Additionally, an Abcc6 transgene significantly reduced infarct size on the background of a naturally occurring Abcc6 deficiency. There were no differences in cardiac calcification following I/R, but increased cardiac apoptosis was noted in Abcc6-deficient mice. Previous studies have implicated the bone morphogenetic protein (BMP) signaling pathway in directing calcification, and here we showed that the BMP responsive transcription factors pSmad1/5/8 were increased in hearts of Abcc6 mice. Consistent with this finding, BMP4 and BMP9 were increased and activin receptor-like kinase-2 and endoglin were downregulated in cardiac extracts from Abcc6-deficient mice versus controls. CONCLUSIONS: These data identify Abcc6 as a novel modulator of cardiac myocyte survival after I/R. This cardioprotective mechanism may involve inhibition of the BMP signaling pathway, which modulates apoptosis.

The role of E2F-1 and downstream target genes in mediating ischemia/reperfusion injury in vivo.

E2Fs are a family of transcription factors that regulate proliferation, differentiation and apoptosis in many cell types. E2F-1 is the prototypical E2F and the family member that has most often been implicated in also mediating apoptosis. To better understand the role of E2F-1 in mediating cardiomyocyte injury we initially analyzed E2F family member expression after ischemia/reperfusion (I/R) in vivo or simulated ischemia in vitro. I/R injury in vivo caused a 3.4-fold increase specifically in E2F-1 protein levels. Expression of other E2F family members did not change. To establish the role of E2F-1 in I/R we examined the response of germline deleted E2F-1 mice to I/R injury. Infarct size as a percentage of the area at risk was decreased 39.8% in E2F-1(-/-) mice compared to E2F-1(+/+) controls. Interestingly, expression of classic, E2F-1 apoptotic target genes was not altered in E2F-1 null cardiomyocytes after I/R. However, upregulation of the primary member of the Forkhead family of transcription factors, FoxO-1a, was attenuated. Consistent, with a role for FoxO-1a as an important target of E2F-1 in I/R, a number of proapoptotic FoxO-1a target genes were also altered. These results suggest that E2F-1 and FoxO-1a belong to a complex transcriptional network that may modulate myocardial cell death during I/R injury.

Statins do not significantly affect muscle sympathetic nerve activity in humans with nonischemic heart failure: a double-blind placebo-controlled trial.

BACKGROUND: Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) have been shown to reduce sympathetic nervous system (SNS) activation in experimental heart failure (HF). However, this potential mechanism of action of statins in HF has not been well studied in humans. METHODS AND RESULTS: Twenty-six patients with nonischemic systolic HF (left ventricular ejection fraction [LVEF] ≤35%) were randomized to atorvastatin (10 mg) or placebo for 3 months. Pre- and posttreatment testing included echocardiography, laboratory assays, quality of life (QOL) questionnaires, and peroneal nerve muscle sympathetic nerve activity (MSNA) via microneurography. Eighteen subjects had technically adequate MSNA tracings before and after treatment. The cohort was 65% male, 81% New York Heart Association functional class II, LVEF 26 ± 6%, and low-density lipoprotein cholesterol (LDL-C) 108 ± 26 mg/dL. Baseline MSNA was 41 ± 2 bursts/min. LDL-C significantly decreased in the atorvastatin (-36.8%) versus the placebo (-0.1%) group (P < .0001). However, there was no significant change in MSNA (-16.2% vs -2.5%), LVEF, B-type natriuretic peptide, or QOL score in the atorvastatin compared with the placebo group. CONCLUSIONS: Short-term statin therapy in patients with nonischemic HF does not result in a significant decrease in SNS activation as measured by MSNA. These findings are consistent with the neutral outcomes of large clinical trials of statins in HF.

High-resolution 3D fluorescent imaging of intact tissues.

Histological analysis of fluorescently labeled tissues has been a critical tool to understand molecular organization in situ. However, assessing molecular structures within large cells and in the context of human organ anatomy has been challenging because it requires penetration of staining reagents and light deep into opaque tissues, while also conforming to the spatial constraints of high-resolution objective lenses. This methodology article describes optimized sample preparation for sub-micron resolution 3D imaging in human and rodent tissues, yielding imaging depth (>100 µm) and resolution (<0.012 µm3 voxel size) that has previously been limited to whole-mount in vitro organoid systems, embryos, and small model organisms. Confocal images of adult human and rodent organs, including heart, kidney, and liver, were generated for several chemical and antibody stains in cleared tissue sections >100 µm thick. This method can be readily adopted by any lab performing routine histology and takes 3 days from the start of tissue preparation to 3D images.

Pharmacologic therapy for engraftment arrhythmia induced by transplantation of human cardiomyocytes.

Heart failure remains a significant cause of morbidity and mortality following myocardial infarction. Cardiac remuscularization with transplantation of human pluripotent stem cell-derived cardiomyocytes is a promising preclinical therapy to restore function. Recent large animal data, however, have revealed a significant risk of engraftment arrhythmia (EA). Although transient, the risk posed by EA presents a barrier to clinical translation. We hypothesized that clinically approved antiarrhythmic drugs can prevent EA-related mortality as well as suppress tachycardia and arrhythmia burden. This study uses a porcine model to provide proof-of-concept evidence that a combination of amiodarone and ivabradine can effectively suppress EA. None of the nine treated subjects experienced the primary endpoint of cardiac death, unstable EA, or heart failure compared with five out of eight (62.5%) in the control cohort (hazard ratio = 0.00; 95% confidence interval: 0-0.297; p = 0.002). Pharmacologic treatment of EA may be a viable strategy to improve safety and allow further clinical development of cardiac remuscularization therapy.

Lymphocytic infiltration leads to degradation of lacrimal gland extracellular matrix structures in NOD mice exhibiting a Sjögren's syndrome-like exocrinopathy.

We previously reported that lacrimal glands (LGs) of male non-obese diabetic (NOD) mice, an established mouse model of autoimmune inflammatory LG disease that displays many features of human LGs in patients afflicted with Sjögren's syndrome (SjS), exhibit significant degradation of extracellular matrix (ECM) structures as well as increased expression of matrix metalloproteinases (MMPs). The purpose of the current study was to expand the spectrum of proteases identified, to clarify their probable origin as well as to identify the contribution of these changes to disease pathogenesis. We explored in depth the changes in ECM structures and ECM protease expression at the onset of disease (6 weeks) versus late stage disease (18 weeks) in male NOD mouse LGs, relative to LGs of age-matched male NODscid, a severely immunocompromised congenic strain, and healthy BALB/c mice. LG tissues were examined using routine histological, immunohistochemical, Western Blot and gene expression analyses novel multiphoton imaging technologies. We further characterized the profile of infiltrating immune cells under each condition using flow cytometry. Our results show that the initial infiltrating cells at 6 weeks of age are responsible for increased MMP and cathepsin H expression and therefore initiate the LG ECM degradation in NOD mice. More importantly, NODscid mice exhibited normal LG ECM structures, indicating the lymphocytes seen in the LGs of NOD mice are responsible for the degradation of the LG ECM. The disease-related remodeling of LG ECM structures may play a crucial role in altering the acinar signaling environment, disrupting the signaling scaffolds within the cells, which are required to mobilize the exocytotic trafficking machinery, ultimately leading to a loss of LG function in patients afflicted with SjS.

A cyclin D2-Rb pathway regulates cardiac myocyte size and RNA polymerase III after biomechanical stress in adult myocardium.

Normally, cell cycle progression is tightly coupled to the accumulation of cell mass; however, the mechanisms whereby proliferation and cell growth are linked are poorly understood. We have identified cyclin (Cyc)D2, a G(1) cyclin implicated in mediating S phase entry, as a potential regulator of hypertrophic growth in adult post mitotic myocardium. To examine the role of CycD2 and its downstream targets, we subjected CycD2-null mice to mechanical stress. Hypertrophic growth in response to transverse aortic constriction was attenuated in CycD2-null compared with wild-type mice. Blocking the increase in CycD2 in response to hypertrophic agonists prevented phosphorylation of CycD2-target Rb (retinoblastoma gene product) in vitro, and mice deficient for Rb had potentiated hypertrophic growth. Hypertrophic growth requires new protein synthesis and transcription of tRNA genes by RNA polymerase (pol) III, which increases with hypertrophic signals. This load-induced increase in RNA pol III activity is augmented in Rb-deficient hearts. Rb binds and represses Brf-1 and TATA box binding protein (TBP), subunits of RNA pol III-specific transcription factor B, in adult myocardium under basal conditions. However, this association is disrupted in response to transverse aortic constriction. RNA pol III activity is unchanged in CycD2(-/-) myocardium after transverse aortic constriction, and there is no dissociation of TBP from Rb. These investigations identify an essential role for the CycD2-Rb pathway as a governor of cardiac myocyte enlargement in response to biomechanical stress and, more fundamentally, as a regulator of the load-induced activation of RNA pol III.

Reprogrammed mouse fibroblasts differentiate into cells of the cardiovascular and hematopoietic lineages.

Forced expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the murine fibroblast genome, generating induced pluripotent stem (iPS) cells. Although the differentiation potential of these cells is thought to be equivalent to that of embryonic stem (ES) cells, it has not been rigorously determined. In this study, we sought to identify the capacity of iPS cells to differentiate into Flk1-positive progenitors and their mesodermal progeny, including cells of the cardiovascular and hematopoietic lineages. Immunostaining of tissues from iPS cell-derived chimeric mice demonstrated that iPS cells could contribute in vivo to cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. To compare the in vitro differentiation potential of murine ES and iPS cells, we either induced embryoid body (EB) formation of each cell type or cultured the cells on collagen type IV (ColIV), an extracellular matrix protein that had been reported to direct murine ES cell differentiation to mesodermal lineages. EB formation and exposure to ColIV both induced iPS cell differentiation into cells that expressed cardiovascular and hematopoietic markers. To determine whether ColIV-differentiated iPS cells contained a progenitor cell with cardiovascular and hematopoietic differentiation potential, Flk1-positive cells were isolated by magnetic cell sorting and exposed to specific differentiation conditions, which induced differentiation into functional cardiomyocytes, smooth muscle cells, endothelial cells, and hematopoietic cells. Our data demonstrate that murine iPS cells, like ES cells, can differentiate into cells of the cardiovascular and hematopoietic lineages and therefore may represent a valuable cell source for applications in regenerative medicine. Disclosure of potential conflicts of interest is found at the end of this article.

Adipose tissue-derived cells improve cardiac function following myocardial infarction.

BACKGROUND: Adipose tissue consists of mature adipocytes and a mononuclear cell fraction termed adipose tissue-derived cells (ADCs). Within these heterogeneous ADCs exists a mesenchymal stem cell-like cell population, termed adipose tissue-derived stem cells. An important clinical advantage of adipose tissue-derived stem cells over other mesenchymal stem cell populations is the fact that they can be isolated in real time in sufficient quantity, such that ex vivo expansion is not necessary to obtain clinically relevant numbers for various therapeutic applications. MATERIALS AND METHODS: The aim of this investigation was to evaluate the therapeutic potential of freshly isolated ADCs in treating rats acutely following myocardial infarction. Rats underwent 45 min of left anterior descending artery occlusion followed by reperfusion. Fifteen minutes post-myocardial infarction, saline or 5 x 10(6) ADCs from green fluorescent protein-expressing transgenic rats were injected into the chamber of the left ventricle. Left ventricular function and morphometry was followed with 2-D echocardiography for 12 wk, at which point hearts were harvested for histological analysis. RESULTS: Twelve weeks following cell therapy, left ventricular end-diastolic dimension was less dilated while the ejection fraction and cardiac output of ADC-treated rats were significantly improved compared to control rats (P < 0.01). Despite this benefit, absolute engraftment rates were low. This paradox may be partially explained by ADC-induced increases in both capillary and arteriole densities. CONCLUSIONS: These data confirm the therapeutic benefit of freshly isolated ADCs delivered post-MI and suggest a novel beneficial mechanism for ADCs through a potent proangiogenic effect.

Cyclin-dependent kinase 2 signaling regulates myocardial ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury to the heart is accompanied by the upregulation and posttranslational modification of a number of proteins normally involved in regulating cell cycle progression. Two such proteins, cyclin-dependent kinase-2 (Cdk2) and its downstream target, the retinoblastoma gene product (Rb), also play a critical role in the control of apoptosis. Myocardial ischemia activates Cdk2, resulting in the phosphorylation and inactivation of Rb. Blocking Cdk2 activity reduces apoptosis in cultured cardiac myocytes. Genetic or pharmacological inhibition of Cdk2 activity in vivo during I/R injury led to a 36% reduction in infarct size (IFS), when compared to control mice, associated with a reduction in apoptotic myocytes. To confirm that Rb was the critical target in Cdk2-mediated I/R injury, we determined the consequences of I/R injury in cardiac-specific Rb-deficient mice (CRb(L/L)). IFS was increased 140% in CRb(L/L) mice compared to CRb+/+ controls. TUNEL positive nuclei and caspase-3 activity were augmented by 92% and 36%, respectively, following injury in the CRb(L/L) mice demonstrating that loss of Rb in the heart significantly exacerbates I/R injury. These data suggest that Cdk2 signaling pathways are critical regulators of cardiac I/R injury in vivo and support a cardioprotective role for Rb.