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1.  Rat Mesenchymal Stem Cell Secretome Promotes Elastogenesis and Facilitates Recovery From Simulated Childbirth Injury 
Cell transplantation  2013;23(11):1395-1406.
Vaginal delivery is a risk factor for stress urinary incontinence (SUI). Mesenchymal stem cells (MSCs) home to injured organs and can facilitate repair. The goal of this study was to determine if MSCs home to pelvic organs after simulated childbirth injury and facilitate recovery from SUI via paracrine factors. Three experiments were performed. Eighteen female rats received vaginal distension (VD) or sham VD and labeled intravenous (IV) MSCs to investigate if MSCs home to the pelvic organs. Whole-organ imaging and immunofluorescence were performed 1 week later. Thirty-four female rats received VD and IV MSCs, VD and IV saline, or sham VD and IV saline to investigate if MSCs accelerate recovery of continence. Twenty-nine female rats received VD and periurethral concentrated conditioned media (CCM), VD and periurethral control media, or sham VD and periurethral control media to investigate if factors secreted by MSCs accelerate recovery from VD. Urethral histology and function were assessed 1 week later. Significantly more MSCs were observed in the urethra, vagina, and spleen after VD compared to sham VD. Continence as measured by leak point pressure (LPP) was significantly reduced after VD in rats treated with saline or control media compared to sham VD but not in those given MSCs or CCM. External urethral sphincter (EUS) function as measured by electromyography (EMG) was not improved with MSCs or CCM. Rats treated with MSCs or CCM demonstrated an increase in elastin fibers near the EUS and urethral smooth muscle more similar to that of sham-injured animals than rats treated with saline or control media. MSCs homed to the urethra and vagina and facilitated recovery of continence most likely via secretion of paracrine factors. Both MSCs and CCM have promise as novel noninvasive therapies for SUI.
doi:10.3727/096368913X670921
PMCID: PMC4464671  PMID: 23866688
Paracrine factors; Urethra; External urethral sphincter (EUS); Electromyography (EMG); Female; Stress urinary incontinence (SUI); Elastin
2.  Functional Outcome After Anal Sphincter Injury and Treatment With Mesenchymal Stem Cells 
An animal model of mesenchymal stem cell (MSC) treatment in anal sphincter injury was studied. Results show that a single i.m. treatment with MSCs after injury resulted in increased anal pressures similar to those of serial i.v. injections, and these pressures were sustained 5 weeks after injection. Given that MSCs delivered i.m. or i.v. resulted in functional recovery, the i.m. route may be preferable as fewer cells seem to be needed.
This research demonstrates the regenerative effects of mesenchymal stem cells (MSCs) on the injured anal sphincter by comparing anal sphincter pressures following intramuscular and serial intravascular MSC infusion in a rat model of anal sphincter injury. Fifty rats were divided into injury (n = 35) and no injury (NI; n = 15) groups. Each group was further divided into i.m., serial i.v., or no-treatment (n = 5) groups and followed for 5 weeks. The injury consisted of an excision of 25% of the anal sphincter complex. Twenty-four hours after injury, 5 × 105 green fluorescent protein-labeled MSCs in 0.2 ml of phosphate-buffered saline (PBS) or PBS alone (sham) were injected into the anal sphincter for i.m. treatment; i.v. and sham i.v. treatments were delivered daily for 6 consecutive days via the tail vein. Anal pressures were recorded before injury and 10 days and 5 weeks after treatment. Ten days after i.m. MSC treatment, resting and peak pressures were significantly increased compared with those in sham i.m. treatment (p < .001). When compared with the NI group, the injury groups had anal pressures that were not significantly different 5 weeks after i.m./i.v. treatment. Both resting and peak pressures were also significantly increased after i.m./i.v. MSC treatment compared with treatment with PBS (p < .001), suggesting recovery. Statistical analysis was done using paired t test with Bonferroni correction. Marked decrease in fibrosis and scar tissue was seen in both MSC-treated groups. Both i.m. and i.v. MSC treatment after injury caused an increase in anal pressures sustained at 5 weeks, although fewer cells were injected i.m. The MSC-treated groups showed less scarring than the PBS-treated groups, with the i.v. infusion group showing the least scarring.
doi:10.5966/sctm.2013-0157
PMCID: PMC4039452  PMID: 24797828
Mesenchymal stem cells; Anal sphincter; Anal pressures; i.v. infusion; Fecal incontinence
3.  Impact of Parturition on Chemokine Homing Factor Expression in the Vaginal Distention Model of Stress Urinary Incontinence 
The Journal of urology  2012;189(4):1588-1594.
Purpose
Human childbirth simulated by vaginal distention is known to increase the expression of chemokines and receptors involved in stem cell homing and tissue repair. We hypothesized that pregnancy and parturition in rats contributes to the expression of chemokines and receptors after vaginal distention.
Materials and Methods
We used 72 age matched female Lewis rats, including virgin rats with and without vaginal distention, and delivered rats with and without vaginal distention. Each rat was sacrificed immediately, or 3 or 7 days after vaginal distention and/or parturition, and the urethra was harvested. Relative expression of chemokines and receptors was determined by real-time polymerase chain reaction. Mixed models were used with the Bonferroni correction for multiple comparisons.
Results
Vaginal distention up-regulated urethral expression of CCL7 immediately after injury in virgin and postpartum rats. Hypoxia inducible factor-1α and vascular endothelial growth factor were up-regulated only in virgin rats immediately after vaginal distention. CD191 expression was immediately up-regulated in postpartum rats without vaginal distention compared to virgin rats without vaginal distention. CD195 was up-regulated in virgin rats 3 days after vaginal distention compared to virgin rats without vaginal distention. CD193 and CXCR4 showed delayed up-regulation in virgin rats 7 days after vaginal distention. CXCL12 was up-regulated in virgin rats 3 days after vaginal distention compared to immediately after vaginal distention. Interleukin-8 and CD192 showed no differential expression.
Conclusions
Vaginal distention results in up-regulation of the chemokines and receptors expressed during tissue injury, which may facilitate the spontaneous functional recovery previously noted. Pregnancy and delivery up-regulated CD191 and attenuated the expression of hypoxia inducible factor-1α and vascular endothelial growth factor in the setting of vaginal distention, likely by decreasing hypoxia.
doi:10.1016/j.juro.2012.09.096
PMCID: PMC4383296  PMID: 23022009
urethra; delivery; obstetric; vagina; wounds; injuries; cytokines
4.  Reduced Physical Fitness in Patients With Heart Failure as a Possible Risk Factor for Impaired Driving Performance 
The American Journal of Occupational Therapy  2015;69(2):6902260010p1-6902260010p8.
Examination of the associations among physical fitness, cognition, and driving performance in 18 people with heart failure found that reduced physical fitness was associated with worse simulated driving.
OBJECTIVE. Reduced physical fitness secondary to heart failure (HF) may contribute to poor driving; reduced physical fitness is a known correlate of cognitive impairment and has been associated with decreased independence in driving. No study has examined the associations among physical fitness, cognition, and driving performance in people with HF.
METHOD. Eighteen people with HF completed a physical fitness assessment, a cognitive test battery, and a validated driving simulator scenario.
RESULTS. Partial correlations showed that poorer physical fitness was correlated with more collisions and stop signs missed and lower scores on a composite score of attention, executive function, and psychomotor speed. Cognitive dysfunction predicted reduced driving simulation performance.
CONCLUSION. Reduced physical fitness in participants with HF was associated with worse simulated driving, possibly because of cognitive dysfunction. Larger studies using on-road testing are needed to confirm our findings and identify clinical interventions to maximize safe driving.
doi:10.5014/ajot.2015.013573
PMCID: PMC4480054  PMID: 26122681
automobile driving; cognition disorders; computer simulation; heart failure; physical fitness; risk factors
5.  Stem Cell Therapy for Heart Disease 
Journal of General Internal Medicine  2013;28(10):1353-1363.
Coronary artery disease is the leading cause of death in Americans. After myocardial infarction, significant ventricular damage persists despite timely reperfusion and pharmacological management. Treatment is limited, as current modalities do not cure this damage. In the past decade, stem cell therapy has emerged as a promising therapeutic solution to restore myocardial function. Clinical trials have demonstrated safety and beneficial effects in patients suffering from acute myocardial infarction, heart failure, and dilated cardiomyopathy. These benefits include improved ventricular function, increased ejection fraction, and decreased infarct size. Mechanisms of therapy are still not clearly understood. However, it is believed that paracrine factors, including stromal cell-derived factor-1, contribute significantly to stem cell benefits. The purpose of this article is to provide medical professionals with an overview on stem cell therapy for the heart and to discuss potential future directions.
doi:10.1007/s11606-013-2508-z
PMCID: PMC3785654  PMID: 23771782
myocardial infarction; heart failure; ventricular function; stem cell; paracrine
6.  Interference with Akt signaling protects against myocardial infarction and death by limiting the consequences of oxidative stress 
Science signaling  2013;6(287):ra67.
The intricacy of multiple feedback loops in the pathways downstream of Akt allows Akt to control multiple cellular processes in the cardiovascular system and precludes inferring consequences of its activation in specific pathological conditions. Akt1, the major Akt isoform in heart and vasculature, has a protective role in the endothelium during atherosclerosis. However, Akt1 activation has been proposed to have detrimental consequences in the cardiovascular system. Mice lacking the apolipoprotein E (ApoE), which promotes clearance of remnant lipoproteins, and the high-density lipoprotein receptor SR-BI are a model of spontaneous myocardial infarction and severe dyslipidemia. Akt1 was activated in these mice, and this activation correlated with cardiac dysfunction, hypertrophy, and fibrosis; increased infarct area; macrophage cholesterol accumulation and atherosclerosis; and reduced lifespan. Akt1 activation was associated with inflammation, oxidative stress, accumulation of oxidized lipids and increased abundance of CD36, a major sensor of oxidative stress, which created a positive feedback loop that exacerbated the consequences of oxidative stress. Thus, interference with Akt1 signaling in vivo could be protective and improve survival in dyslipidemia in the absence of SR-BI by reducing oxidative stress and responses to oxidized lipids.
doi:10.1126/scisignal.2003948
PMCID: PMC3971949  PMID: 23921086
7.  Coronary Collateral Growth—Back to the Future 
The coronary collateral circulation is critically important as an adaptation of the heart to prevent the damage from ischemic insults. In their native state, collaterals in the heart would be classified as part of the microcirculation, existing as arterial-arterial anastomotic connections in the range of 30 to 100 μM in diameter. However, these vessels also show a propensity to remodel into components of the macrocirculation and can become arteries larger than a 1000 μM in diameter. This process of outward remodelling is critically important in the adaptation of the heart to ischemia because the resistance to blood flow is inversely related to the fourth power of the diameter of the vessel. Thus, an expansion of a vessel from 100 to 1000 μM would reduce resistance (in this part of the circuit) to a negligible amount and enable delivery of flow to the region at risk. Our goal in this review is to highlight the voids in understanding this adaptation to ischemia—the growth of the coronary collateral circulation. In doing so we discuss the controversies and unknown aspects of the causal factors that stimulate growth of the collateral circulation, the role of genetics, and the role of endogenous stem and progenitor cells in the context of the normal, physiological situation and under more pathological conditions of ischemic heart disease or with some of the underlying risk factors, e.g., diabetes. The major conclusion of this review is that there are many gaps in our knowledge of coronary collateral growth and this knowledge is critical before the potential of stimulating collateralization in the hearts of patients can be realized.
doi:10.1016/j.yjmcc.2011.12.006
PMCID: PMC3939792  PMID: 22210280
8.  Stimulus-dependent phosphorylation of profilin-1 in angiogenesis 
Nature cell biology  2012;14(10):1046-1056.
Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell (EC) migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show VEGF-A-inducible phosphorylation of Pfn-1 at Tyr129 is critical for EC migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induce Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Furthermore, Pfn-1 phosphorylation is robustly and preferentially elevated in blood vessels during tissue repair after myocardial infarction in humans. Conditional endothelial knock-in of phosphorylation-deficient Pfn-1Y129F in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signaling events to initiate EC migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy.
doi:10.1038/ncb2580
PMCID: PMC3619429  PMID: 23000962
9.  Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload 
PLoS ONE  2013;8(7):e68528.
Rationale
Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.
Objective
We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.
Methods and Results
Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.
Conclusions
These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.
doi:10.1371/journal.pone.0068528
PMCID: PMC3706399  PMID: 23874657
10.  Effect of the Use and Timing of Bone Marrow Mononuclear Cell Delivery on Left Ventricular Function After Acute Myocardial Infarction: The TIME Randomized Trial 
Context
While the delivery of cell therapy following ST segment myocardial infarction (STEMI) has been evaluated in previous clinical trials, the influence of the timing of cell delivery on the effect on left ventricular (LV) function has not been analyzed in a trial that randomly designated the time of delivery.
Objective
To determine 1) the effect of intracoronary autologous bone marrow mononuclear cell (BMC) delivery following STEMI on recovery of global and regional LV function and 2) if timing of BMC delivery (3 versus 7 days following reperfusion) influences this effect.
Design, Setting, and Patients
Between July 17, 2008 and November 15, 2011, 120 patients were enrolled in a randomized, 2×2 factorial, double-blind, placebo-controlled trial of the National Heart, Lung, and Blood Institute (NHLBI)-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) of patients with LV dysfunction (LV Ejection Fraction (LVEF) ≤45%) following successful primary percutaneous coronary intervention (PCI) of anterior STEMI.
Interventions
Intracoronary infusion of 150 × 106 BMCs or placebo (randomized 2:1 BMC:placebo) within 12 hours of aspiration and processing administered at Day 3 or Day 7 (randomized 1:1) post-PCI.
Main Outcome Measures
Co-primary endpoints were: 1) Change in global (LVEF) and regional (wall motion) LV function in infarct and border zones at 6 months measured by cardiac magnetic resonance imaging and 2) Change in LV function as affected by timing of treatment on Day 3 versus Day 7. Secondary endpoints included major adverse cardiovascular events as well as changes in LV volumes and infarct size.
Results
Patient mean age was 56.9±10.9 years with 87.5% male. At 6 months, LVEF increased similarly in both BMC (45.2±10.6 to 48.3±13.3 %) and placebo groups (44.5±10.8 to 47.8±13.6 %). No detectable treatment effect on regional LV function was observed in either infarct or border zones. Differences between therapy groups in the change in global LV function over time when treated at Day 3 (−0.9±2.9%, 95% CI 6.6 to 4.9%, p=0.763) or Day 7 (1.1±2.9%, 95% CI −4.7 to 6.9, p=0.702) were not significant, nor were they different from each other. Also, timing of treatment had no detectable effect on recovery of regional LV function. Major adverse events were rare with no difference between groups.
Conclusions
Patients with STEMI, who underwent successful primary PCI and administration of intra-coronary BMCs at either 3 or 7 days following the event, had recovery of global and regional LV function similar to placebo
Trial Registration
ClinicalTrials.gov Number, NCT00684021
doi:10.1001/jama.2012.28726
PMCID: PMC3652242  PMID: 23129008
11.  Effect of Transendocardial Delivery of Autologous Bone Marrow Mononuclear Cells on Functional Capacity, Left Ventricular Function, and Perfusion in Chronic Ischemic Heart Failure: The FOCUS-CCTRN Trial 
Context
Previous studies utilizing autologous bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy have demonstrated safety and suggested efficacy. The FOCUS protocol was designed to assess efficacy of a larger cell dose in an adequately well-powered phase II study.
Objective
To determine if administration of BMCs through transendocardial injections improves myocardial perfusion, reduces left ventricular (LV) end systolic volume, or enhances maximal oxygen consumption in patients with coronary artery disease (CAD), LV dysfunction, and limiting heart failure and/or angina.
Design, Setting, and Patients
This is a 100 million cell, first-in-man randomized, double-blind, placebo-controlled trial was performed by the National Heart, Lung, and Blood Institute-sponsored Cardiovascular Cell Therapy Research Network (CCTRN) in symptomatic patients (NYHA II-III and/or CCS II-IV) receiving maximal medical therapy, with a left ventricular ejection fraction (LVEF)≤45%, perfusion defect by single-photon emission tomography (SPECT), and CAD not amenable to revascularization.
Intervention
All patients underwent bone marrow aspiration, isolation of BMCs using a standardized automated system performed locally, and transendocardial injection of 100 million BMCs or placebo (2:1 BMC: placebo).
Main Outcome Measures
Three co-primary endpoints assessed at 6 months were changes in (a) LV end systolic volume (LVESV) by echocardiography, (b) maximal oxygen consumption (MVO2), and (c) reversibility on SPECT. Secondary measures included other SPECT measures, magnetic resonance imaging (MRI), echocardiography, clinical improvement, and major adverse cardiac events (MACE). Phenotypic and functional analyses of the cell product were performed by the CCTRN Biorepository lab.
Results
Of 153 consented patients, a total of 92 (82 men; average age, 63 years) were randomized (n= 61 BMC, 31 placebo) at 5 sites between April 29, 2009 and April 18, 2011. Changes in LVESV index, (−0.9 ± 11.3 mL/m2; P = 0.733; 95% CI, −6.1 to 4.3), MVO2 (1.0 ± 2.9; P = 0.169; 95% CI, −0.42 to 2.34), percent reversible defect change, (−1.2 ± 23.3; P = 0.835; 95% CI, −12.50 to 10.12), and incidence of MACEwere not statistically significant. However, in an exploratory analysis the change in LVEF across the entire cohort by therapy group was significant (2.7 ± 5.2%; P = 0.030; 95% CI, 0.27 to 5.07).
Conclusions
This is the largest cell therapy trial of autologous BMCs in patients with ischemic LV dysfunction. In patients with chronic ischemic heart disease, transendocardial injection of BMCs compared to placebo did not improve LVESV, MVO2, or reversibility on SPECT.
doi:10.1001/jama.2012.418
PMCID: PMC3600947  PMID: 22447880
Chronic CAD; Ischemic Heart Failure; Chronic Angina; bone marrow mononuclear cells; cardiac performance
12.  Effect of Intracoronary Delivery of Autolologous Bone Marrow Mononuclear Cells Two to Three Weeks Following Acute Myocardial Infarction on Left-Ventricular Function: The LateTIME Randomized Trial 
Context
Clinical trial results suggest that intracoronary delivery of autologous bone marrow mononuclear cells (BMCs) may improve left ventricular (LV) function when administered within the first week following myocardial infarction (MI). However, since a substantial number of patients may not present for early cell delivery, we investigated the efficacy of autologous BMC delivery 2–3 weeks post-MI.
Objective
To determine if intracoronary delivery of autologous BMCs improves global and regional LV function when delivered 2–3 weeks following first MI.
Design, Setting, and Patients
LateTIME is a randomized, double-blind, placebo-controlled trial of the National Heart, Lung, and Blood Institute - sponsored Cardiovascular Cell Therapy Research Network (CCTRN) of 87 patients with significant LV dysfunction (LVEF ≤ 45%) following successful primary percutaneous coronary intervention (PCI).
Interventions
Intracoronary infusion of 150 × 106 autologous BMCs (total nucleated cells) or placebo (2:1 BMC:placebo) was performed within 12 hours of bone marrow aspiration after local automated cell processing.
Main Outcome Measures
The primary endpoints were changes in global (LVEF) and regional (wall motion) LV function in the infarct and border zone from baseline to 6 months as measured by cardiac MRI at a core lab blinded to treatment assignment Secondary endpoints included changes in LV volumes and infarct size.
Results
87 patients were randomized between July 2008 and February 2011: mean age = 57 ± 11 yrs, 83% male. Harvesting, processing, and intracoronary delivery of BMCs in this setting was feasible and safe. The change from baseline to six months in the BMC group, when compared to the placebo group, for LVEF (48.7 to 49.2% vs. 45.3 to 48.8%; Difference = −3.0, 95% CI −7.0 to 0.9), wall motion in the infarct zone (6.2 to 6.5 vs. 4.9 to 5.9 mm; Difference = −0.7, 95% CI −2.8 to 1.3), and wall motion in the border zone (16.0 to 16.6 mm vs. 16.1 to 19.3 mm; Difference = −2.6; 95% CI −6.0 to 0.8) were not statistically significant. There was no significant change in LV volumes and infarct volumes decreased by a similar amount in both groups at 6 months compared to baseline.
Conclusions
Among patients with MI and LV dysfunction following reperfusion with PCI, intracoronary infusion of autologous BMCs compared to intracoronary placebo infusion, 2–3 weeks after PCI did not improve global or regional function at 6 months.
doi:10.1001/jama.2011.1670
PMCID: PMC3600981  PMID: 22084195
Acute myocardial infarction; bone marrow mononuclear cells; LVEF; cardiac MRI
13.  Combining Censored and Uncensored Data in a U-Statistic: Design and Sample Size Implications for Cell Therapy Research 
The assumptions that anchor large clinical trials are rooted in smaller, Phase II studies. In addition to specifying the target population, intervention delivery, and patient follow-up duration, physician-scientists who design these Phase II studies must select the appropriate response variables (endpoints). However, endpoint measures can be problematic. If the endpoint assesses the change in a continuous measure over time, then the occurrence of an intervening significant clinical event (SCE), such as death, can preclude the follow-up measurement. Finally, the ideal continuous endpoint measurement may be contraindicated in a fraction of the study patients, a change that requires a less precise substitution in this subset of participants.
A score function that is based on the U-statistic can address these issues of 1) intercurrent SCE's and 2) response variable ascertainments that use different measurements of different precision. The scoring statistic is easy to apply, clinically relevant, and provides flexibility for the investigators' prospective design decisions. Sample size and power formulations for this statistic are provided as functions of clinical event rates and effect size estimates that are easy for investigators to identify and discuss. Examples are provided from current cardiovascular cell therapy research.
doi:10.2202/1557-4679.1286
PMCID: PMC3154087  PMID: 21841940
U-statistic; clinical trials; score function; stem cells
14.  How Similar Are the Mice to Men? Between-Species Comparison of Left Ventricular Mechanics Using Strain Imaging 
PLoS ONE  2012;7(6):e40061.
Background
While mammalian heart size maintains constant proportion to whole body size, scaling of left ventricular (LV) function parameters shows a more complex scaling pattern. We used 2-D speckle tracking strain imaging to determine whether LV myocardial strains and strain rates scale to heart size.
Methods
We studied 18 mice, 15 rats, 6 rabbits, 12 dogs and 20 human volunteers by 2-D echocardiography. Relationship between longitudinal or circumferential strains/strain rates (SLong/SRLong, SCirc/SRCirc), and LV end-diastolic volume (EDV) or mass were assessed by the allometric (power-law) equation Y = kMβ.
Results
Mean LV mass in individual species varied from 0.038 to 134 g, LV EDV varied from 0.015 to 102 ml, while RR interval varied from 81 to 1090 ms. While SLong increased with increasing LV EDV or mass (β values 0.047±0.006 and 0.051±0.005, p<0.0001 vs. 0 for both) SCirc was unchanged (p = NS for both LV EDV or mass). Systolic and diastolic SRLong and SRCirc showed inverse correlations to LV EDV or mass (p<0.0001 vs. 0 for all comparisons). The ratio between SLong and SCirc increased with increasing values of LV EDV or mass (β values 0.039±0.010 and 0.040±0.011, p>0.0003 for both).
Conclusions
While SCirc is unchanged, SLong increases with increasing heart size, indicating that large mammals rely more on long axis contribution to systolic function. SRLong and SRCirc, both diastolic and systolic, show an expected decrease with increasing heart size.
doi:10.1371/journal.pone.0040061
PMCID: PMC3386935  PMID: 22768220
15.  Youth is wasted on the young 
Amphibians and zebrafish are able to regenerate lost myocardial tissue without loss of cardiac function; whereas mammals, in response to myocardial injury, develop scar and lose cardiac function. This dichotomy of response has been thought to be due to the fact that adult mammalian cardiac myocytes are multinucleated and have limited proliferative capacity. Neonatal mammalian cardiac myocytes do have a limited capacity to proliferate. What has been unknown is whether this limited proliferative capacity is associated with the ability to regenerate myocardial tissue soon after birth. Recently, it has been demonstrated that 1-day-old neonatal mice do have the ability to regenerate resected cardiac tissue, and that the capacity to regenerate cardiac tissue is lost by 7 days after birth. The present commentary reviews these results and attempts to offer perspective as to how these important findings relate to current and future strategies to prevent and treat cardiac dysfunction in clinical populations.
doi:10.1186/scrt65
PMCID: PMC3152994  PMID: 21596004
16.  miR-145 is differentially regulated by TGF-β1 and ischaemia and targets Disabled-2 expression and wnt/β-catenin activity 
Abstract
The effect of wnt/β-catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled-2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down-regulation of Dab2 regulates cardiac protein expression and wnt/β-catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor-β1 (TGF-β1). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR-145 in the 3′-UTR of Dab2. In MSC in culture, we observed that TGF-β1 treatment led to rapid and sustained up-regulation of pri–miR-145. Through gain and loss of function studies we demonstrate that miR-145 up-regulation was required for the down-regulation of Dab2 and increased β-catenin activity in response to TGF-β1. To begin to define how Dab2 might regulate wnt/β-catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham-operated myocardium. Following AMI, Dab2 levels were rapidly up-regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down-regulation of myocardial pri–miR-145 expression following AMI. Our data demonstrate a novel and critical role for miR-145 expression as a regulator of Dab2 expression and β-catenin activity in response to TGF-β1 and hypoxia.
doi:10.1111/j.1582-4934.2011.01385.x
PMCID: PMC4365889  PMID: 21762377
myocardial infarction; growth factors/cytokines; gene expression
17.  Detection and Quantification of Fluorescent Cell Clusters in Cryo-Imaging 
We developed and evaluated an algorithm for enumerating fluorescently labeled cells (e.g., stem and cancer cells) in mouse-sized, microscopic-resolution, cryo-image volumes. Fluorescent cell clusters were detected, segmented, and then fit with a model which incorporated a priori information about cell size, shape, and intensity. The robust algorithm performed well in phantom and tissue imaging tests, including accurate (<2% error) counting of cells in mouse. Preliminary experiments demonstrate that cryo-imaging and software can uniquely analyze delivery, homing to an organ and tissue distribution of stem cell therapeutics.
doi:10.1155/2012/698413
PMCID: PMC3317210  PMID: 22481905
18.  Pelvic Organ Distribution of Mesenchymal Stem Cells Injected Intravenously after Simulated Childbirth Injury in Female Rats 
The local route of stem cell administration utilized presently in clinical trials for stress incontinence may not take full advantage of the capabilities of these cells. The goal of this study was to evaluate if intravenously injected mesenchymal stem cells (MSCs) home to pelvic organs after simulated childbirth injury in a rat model. Female rats underwent either vaginal distension (VD) or sham VD. All rats received 2 million GFP-labeled MSCs intravenously 1 hour after injury. Four or 10 days later pelvic organs and muscles were imaged for visualization of GFP-positive cells. Significantly more MSCs home to the urethra, vagina, rectum, and levator ani muscle 4 days after VD than after sham VD. MSCs were present 10 days after injection but GFP intensity had decreased. This study provides basic science evidence that intravenous administration of MSCs could provide an effective route for cell-based therapy to facilitate repair after injury and treat stress incontinence.
doi:10.1155/2012/612946
PMCID: PMC3177359  PMID: 21941558
19.  Rationale and Design for the Intramyocardial Injection of Autologous Bone Marrow Mononuclear Cells for Patients with Chronic Ischemic Heart Disease and Left Ventricular Dysfunction Trial (FOCUS) 
American heart journal  2010;160(2):215-223.
Background
The increasing worldwide prevalence of coronary artery disease (CAD) continues to challenge the medical community. Management options include medical and revascularization therapy. Despite advances in these methods, CAD is a leading cause of recurrent ischemia and heart failure, posing significant morbidity and mortality risks along with increasing health costs in a large patient population worldwide.
Trial Design
The Cardiovascular Cell Therapy Research Network (CCTRN) was established by the National Institutes of Health to investigate the role of cell therapy in the treatment of chronic cardiovascular disease. FOCUS is a CCTRN-designed randomized Phase II, placebo-controlled clinical trial that will assess the effect of autologous bone marrow mononuclear cells delivered transendocardially to patients with left ventricular (LV) dysfunction and symptomatic heart failure or angina. All patients need to have limiting ischemia by reversible ischemia on SPECT assessment.
Results
After thoughtful consideration of both statistical and clinical principles, we will recruit 87 patients (58 cell treated and 29 placebo) to receive either bone marrow–derived stem cells or placebo. Myocardial perfusion, LV contractile performance, and maximal oxygen consumption are the primary outcome measures.
Conclusions
The designed clinical trial will provide a sound assessment of the effect of autologous bone marrow mononuclear cells in improving blood flow and contractile function of the heart. The target population is patients with CAD and LV dysfunction with limiting angina or symptomatic heat failure. Patient safety is a central concern of the CCTRN, and patients will be followed for at least 5 years.
doi:10.1016/j.ahj.2010.03.029
PMCID: PMC2921924  PMID: 20691824
20.  Significance of Thymosin β4 and Implication of PINCH-1-ILK-α-Parvin (PIP) Complex in Human Dilated Cardiomyopathy 
PLoS ONE  2011;6(5):e20184.
Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression—the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI.
doi:10.1371/journal.pone.0020184
PMCID: PMC3098280  PMID: 21625516
21.  Bone Marrow Support of the Heart in Pressure Overload Is Lost with Aging 
PLoS ONE  2010;5(12):e15187.
Rationale
Exogenous stem cell delivery is under investigation to prevent and treat cardiac dysfunction. It is less studied as to the extent endogenous bone marrow derived stem cells contribute to cardiac homeostais in response to stress and the affects of aging on this stress response.
Objective
To determine the role of bone marrow (BM) derived stem cells on cardiac homeostasis in response to pressure overload (PO) and how this response is altered by aging.
Methods and Results
Young (8 weeks) and old (>40 weeks) C57/b6 mice underwent homo- and heterochronic BM transplantation prior to transverse aortic constriction (TAC). We found that older BM is associated with decreased cardiac function following TAC. This decreased function is associated with decrease in BM cell engraftment, increased myocyte apoptosis, decreased myocyte hypertrophy, increased myocardial fibrosis and decreased cardiac function. Additionally, there is a decrease in activation of resident cells within the heart in response to PO in old mice. Interestingly, these effects are not due to alterations in vascular density or inflammation in response to PO or differences in ex vivo stem cell migration between young and old mice.
Conclusions
BM derived stem cells are activated in response to cardiac PO, and the recruitment of BM derived cells are involved in cardiac myocyte hypertrophy and maintenance of function in response to PO which is lost with aging.
doi:10.1371/journal.pone.0015187
PMCID: PMC3006343  PMID: 21203577
22.  Role of Cardiac Myocyte CXCR4 Expression in Development and Left Ventricular Remodeling After Acute Myocardial Infarction 
Circulation research  2010;107(5):667-676.
Rationale
Stromal cell–derived factor (SDF)-1/CXCR4 axis has an instrumental role during cardiac development and has been shown to be a potential therapeutic target for optimizing ventricular remodeling after acute myocardial infarction (AMI) and in ischemic cardiomyopathy. Although a therapeutic target, the specific role of cardiac myocyte CXCR4 (CM-CXCR4) expression following cardiogenesis and survival of cardiac myocyte and left ventricular remodeling after AMI is unknown.
Objective
We hypothesized that cardiac myocyte derived CXCR4 is critical for cardiac development, but it may have no role in adulthood secondary to the short transient expression of SDF-1 and the delayed expression of CM-CXCR4 following AMI. To address this issue, we developed congenital and conditional CM-CXCR4−/− mouse models.
Methods and Results
Two strains of CM-CXCR4flox/flox mice were generated by crossing CXCR4flox/flox mice with MCM-Cre+/− mouse and MLC2v-Cre+/− mouse on the C57BL/6J background, yielding CXCR4flox/flox MCM-Cre+/− and CXCR4flox/floxMLC2v-Cre+/− mice. Studies demonstrated recombination in both models congenitally in the MLC2v-Cre+/− mice and following tamoxifen administration in the MCM-Cre+/− mice. Surprisingly the CXCR4flox/floxMLC2v-Cre+/− are viable, had normal cardiac function, and had no evidence of ventricular septal defect. CXCR4flox/floxMCM+/− treated with tamoxifen 2 weeks before AMI demonstrated 90% decrease in cardiac CXCR4 expression 48 hours after AMI. Twenty-one days post AMI, echocardiography revealed no statistically significant difference in the wall thickness, left ventricular dimensions or ejection fraction (40.9±7.5 versus 34.4±2.6%) in CXCR4flox/flox mice versus CM-CXCR4−/− mice regardless of strategy of Cre expression. No differences in vascular density (2369±131 versus 2471±126 vessels/mm2; CXCR4flox/flox versus CM-CXCR4−/− mouse), infarct size, collagen content, or noninfarct zone cardiac myocyte size were observed 21 days after AMI.
Conclusions
We conclude that cardiac myocyte–derived CXCR4 is not essential for cardiac development and, potentially because of the mismatch in timings of peaks of SDF-1 and CXCR4, has no major role in ventricular remodeling after AMI.
doi:10.1161/CIRCRESAHA.110.223289
PMCID: PMC2935208  PMID: 20634485
stem cells; myocardial infarction; cardiogenesis
23.  Rationale and Design for TIME: A Phase-II, Randomized, Double-Blind, Placebo-Controlled Pilot Trial Evaluating the Safety and Effect of Timing of Administration of Bone Marrow Mononuclear Cells Following Acute Myocardial Infarction 
American heart journal  2009;158(3):356-363.
Several previous studies have demonstrated that administration of autologous bone marrow-derived mononuclear cells (BMMNCs) improve cardiac function in patients following acute myocardial infarction (AMI). However, optimum timing of administration has not been investigated in a clinical trial. The Cardiovascular Cell Therapy Research Network (CCTRN) was developed and funded by the NHLBI to address important questions such as timing of cell delivery and to accelerate research in the use of cell-based therapies. The TIME trial is a randomized, Phase II, double-blind, placebo-controlled clinical trial. The five member clinical sites of the CCTRN will enroll a total of 120 eligible patients with moderate-to-large anterior AMIs who have undergone successful PCI of the LAD coronary artery, and have an LVEF ≤45% by echocardiography. Participants will have bone marrow aspirations and intra-coronary infusions of 150 × 106 BMMNCs or placebo on day 3 or day 7 post-AMI. Objectives of this study are 1) To evaluate effects of BMMNCs on regional and global left-ventricular (LV) function compared to placebo therapy in patients with acute AMI as assessed by cardiac magnetic resonance imaging (cMRI) at 6 months, and 2) To assess whether effects of BMMNC infusion on global and regional LV function and safety are influenced by the time of administration. This study will provide further insight into the clinical feasibility and appropriate timing of autologous BMNNC therapy in high-risk patients following AMI and PCI.
doi:10.1016/j.ahj.2009.06.009
PMCID: PMC2784639  PMID: 19699857
24.  Importance of the SDF-1:CXCR4 axis in myocardial repair 
Circulation research  2009;104(10):1133-1135.
doi:10.1161/CIRCRESAHA.109.198929
PMCID: PMC2753196  PMID: 19461103
25.  PRE-TRANSPLANTATION SPECIFICATION OF STEM CELLS TO CARDIAC LINEAGE FOR REGENERATION OF CARDIAC TISSUE 
Stem Cell Reviews  2009;5(1):51-60.
Myocardial infarction (MI) is a lead cause of mortality in the Western world. Treatment of acute MI is focused on restoration of antegrade flow which inhibits further tissue loss, but does not restore function to damaged tissue. Chronic therapy for injured myocardial tissue involves medical therapy that attempts to minimize pathologic remodeling of the heart. End stage therapy for chronic heart failure (CHF) involves inotropic therapy to increase surviving cardiac myocyte function or mechanical augmentation of cardiac performance. Not until the point of heart transplantation, a limited resource at best, does therapy focus on the fundamental problem of needing to replace injured tissue with new contractile tissue. In this setting, the potential for stem cell therapy has garnered significant interest for its potential to regenerate or create new contractile cardiac tissue. While to date adult stem cell therapy in clinical trials has suggested potential benefit, there is waning belief that the approaches used to date lead to regeneration of cardiac tissue. As the literature has better defined the pathways involved in cardiac differentiation, preclinical studies have suggested that stem cell pretreatment to direct stem cell differentiation prior to stem cell transplantation may be a more efficacious strategy for inducing cardiac regeneration. Here we review the available literature on pre-transplantation conditioning of stem cells in an attempt to better understand stem cell behavior and their readiness in cell-based therapy for myocardial regeneration.
doi:10.1007/s12015-009-9050-8
PMCID: PMC2758651  PMID: 19184567
Stem cells; mesenchymal stem cells; in vitro lineage specification; myocardium infarct; cardiac differentiation; cardiac tissue recovery

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