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1.  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.
PMCID: PMC3785654  PMID: 23771782
myocardial infarction; heart failure; ventricular function; stem cell; paracrine
2.  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.
PMCID: PMC3971949  PMID: 23921086
3.  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.
PMCID: PMC3939792  PMID: 22210280
4.  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.
PMCID: PMC3619429  PMID: 23000962
5.  Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload 
PLoS ONE  2013;8(7):e68528.
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.
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.
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.
PMCID: PMC3706399  PMID: 23874657
6.  Effect of the Use and Timing of Bone Marrow Mononuclear Cell Delivery on Left Ventricular Function After Acute Myocardial Infarction: The TIME Randomized Trial 
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.
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.
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.
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.
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 Number, NCT00684021
PMCID: PMC3652242  PMID: 23129008
7.  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 
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.
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.
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.
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).
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.
PMCID: PMC3600947  PMID: 22447880
Chronic CAD; Ischemic Heart Failure; Chronic Angina; bone marrow mononuclear cells; cardiac performance
8.  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 
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.
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).
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.
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.
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.
PMCID: PMC3600981  PMID: 22084195
Acute myocardial infarction; bone marrow mononuclear cells; LVEF; cardiac MRI
9.  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.
PMCID: PMC3154087  PMID: 21841940
U-statistic; clinical trials; score function; stem cells
10.  How Similar Are the Mice to Men? Between-Species Comparison of Left Ventricular Mechanics Using Strain Imaging 
PLoS ONE  2012;7(6):e40061.
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.
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.
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).
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.
PMCID: PMC3386935  PMID: 22768220
11.  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.
PMCID: PMC3152994  PMID: 21596004
12.  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.
PMCID: PMC3317210  PMID: 22481905
13.  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.
PMCID: PMC3177359  PMID: 21941558
14.  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.
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.
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.
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.
PMCID: PMC2921924  PMID: 20691824
15.  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.
PMCID: PMC3098280  PMID: 21625516
16.  Bone Marrow Support of the Heart in Pressure Overload Is Lost with Aging 
PLoS ONE  2010;5(12):e15187.
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.
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.
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.
PMCID: PMC3006343  PMID: 21203577
17.  Role of Cardiac Myocyte CXCR4 Expression in Development and Left Ventricular Remodeling After Acute Myocardial Infarction 
Circulation research  2010;107(5):667-676.
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.
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.
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.
PMCID: PMC2935208  PMID: 20634485
stem cells; myocardial infarction; cardiogenesis
18.  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.
PMCID: PMC2784639  PMID: 19699857
19.  Importance of the SDF-1:CXCR4 axis in myocardial repair 
Circulation research  2009;104(10):1133-1135.
PMCID: PMC2753196  PMID: 19461103
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.
PMCID: PMC2758651  PMID: 19184567
Stem cells; mesenchymal stem cells; in vitro lineage specification; myocardium infarct; cardiac differentiation; cardiac tissue recovery
21.  LateTIME 
Texas Heart Institute Journal  2010;37(4):412-420.
A realistic goal for cardiac cell therapy may be to attenuate left ventricular remodeling following acute myocardial infarction to prevent the development of congestive heart failure. Initial clinical trials of cell therapy have delivered cells 1 to 7 days after acute myocardial infarction. However, many patients at risk of developing congestive heart failure may not be ready for cell delivery at that time-point because of clinical instability or hospitalization at facilities without access to cell therapy. Experience with cell delivery 2 to 3 weeks after acute myocardial infarction has not to date been explored in a clinical trial. The objective of the LateTIME study is to evaluate by cardiac magnetic resonance the effect on global and regional left ventricular function, between baseline and 6 months, of a single intracoronary infusion of 150 × 106 autologous bone marrow mononuclear cells (compared with placebo) when that infusion is administered 2 to 3 weeks after moderate-to-large acute myocardial infarction. The 5 clinical sites of the Cardiovascular Cell Therapy Research Network (CCTRN) will enroll a total of 87 eligible patients in a 2:1 bone marrow mononuclear cells-to-placebo patient ratio; these 87 will have undergone successful percutaneous coronary intervention of a major coronary artery and have left ventricular ejection fractions ≤0.45 by echocardiography. When the results become available, this study should provide insight into the clinical feasibility and appropriate timing of autologous cell therapy in high-risk patients after acute myocardial infarction and percutaneous coronary intervention.
PMCID: PMC2929864  PMID: 20844613
Apoptosis; bone marrow cells; bone marrow transplantation; cell therapy; colony-stimulating factors; free radicals; heart failure; infusions, intra-arterial; inflammation/prevention & control; magnetic resonance imaging; myocardial infarction/therapy; myocardial ischemia/therapy; myocardial reperfusion injury; myocytes, cardiac; prospective studies; regeneration; research design; stem cells; stem cell transplantation; time factors; ventricular function, left; ventricular remodeling
The Journal of urology  2008;180(2):753-759.
To investigate the effect of duration of vaginal distention (VD) on the differential expression of stem cell homing and tissue repair cytokines and cytokine receptors in order to identify factors most important for recovery from injury.
Twenty 10-week-old virgin Sprague-Dawley rats were divided into 4 groups: 1-hour VD, 4-hour VD, 6-hour VD, and anesthetized shams. Vagina, bladder, urethra and rectum were harvested immediately after VD and Real-Time PCR was used to determine relative expression of cytokines and receptors of interest. Mixed models analysis was used to determine associations between expression level and VD duration.
Positive associations between VD duration and level of urethral expression were found for one of the receptors of MCP-3 [CCR1 (p=0.0001)], as well as, for MCP-3 (p=0.025), CCR5 (p=0.032), and HIF1-alpha, (p=0.023). A positive relationship between VD duration and MCP-3 expression was also observed in rectal tissue (p=0.035). Urethral expression of CCR2, another receptor for MCP-3, approached significance (p=0.066). An inverse relationship between VD duration and expression of IL8 was found in the bladder (p=0.0008). No association was demonstrated between VD duration and expression of SDF-1, CXCR4, CCR2, CCR3, and VEGF in any pelvic organs.
These data support a relationship between VD duration and subsequent expression of MCP-3 and one of its associated receptors (CCR1) in the urethra immediately following VD. The increase in HIF1-alpha expression in the urethra with prolonged VD suggests a limited role of tissue ischemia in the immediate response of pelvic organs to VD.
PMCID: PMC2728595  PMID: 18554634
stress urinary incontinence; vaginal childbirth; MCP-3; SDF-1; IL8; HIF; Real-time PCR; female
23.  Serum Myeloperoxidase Levels Independently Predict Endothelial Dysfunction in Humans 
Circulation  2004;110(9):1134-1139.
In vitro and animal studies demonstrate that myeloperoxidase catalytically consumes nitric oxide as a substrate, limiting its bioavailability and function. We therefore hypothesized that circulating levels of myeloperoxidase would predict risk of endothelial dysfunction in human subjects.
Methods and Results
Serum myeloperoxidase was measured by enzyme-linked immunoassay, and brachial artery flow–mediated dilation and nitroglycerin-mediated dilation were determined by ultrasound in a hospital-based population of 298 subjects participating in an ongoing study of the clinical correlates of endothelial dysfunction (age, 51±16; 61% men, 51% with cardiovascular disease). A strong inverse relation between brachial artery flow–mediated dilation and increasing quartile of serum myeloperoxidase level was observed (11.0±6.0%, 9.4±5.3%, 8.6±5.8%, and 6.4±4.5% for quartiles 1 through 4, respectively; P<0.001 for trend). Using the median as a cut point to define endothelial dysfunction, increasing quartile of myeloperoxidase predicted endothelial dysfunction after adjustment for classic cardiovascular disease risk factors, C-reactive protein levels, prevalence of cardiovascular disease, and ongoing treatment with cardiovascular medications (OR, 6.4; 95% CI, 2.6 to 16; P=0.001 for highest versus lowest quartile).
Serum myeloperoxidase levels serve as a strong and independent predictor of endothelial dysfunction in human subjects. Myeloperoxidase-mediated endothelial dysfunction may be an important mechanistic link between oxidation, inflammation, and cardiovascular disease.
PMCID: PMC2718053  PMID: 15326065
atherosclerosis; free radicals; inflammation; peroxidase; nitric oxide
24.  Genetic Enhancement of Stem Cell Engraftment, Survival and Efficacy 
Circulation research  2008;102(12):1471-1482.
Cell based therapies for the prevention and treatment of cardiac dysfunction offer the potential to significantly modulate cardiac function and improve outcomes in patients with cardiovascular disease. To date several clinical studies have suggested the potential efficacy of several different stem cell types; however, the benefits seen in clinical trials have been inconsistent and modest. In parallel, pre-clinical studies have identified key events in the process of cell based myocardial repair, including stem cell homing, engraftment, survival, paracrine factor release and differentiation that need to be optimized in order to maximize cardiac repair and function. The inconsistent and modest benefits seen in clinical trials combined with the preclinical identification of mediators responsible for key events in cell based cardiac repair, offers the potential for cell based therapy to advance to cell based gene therapy in an attempt to optimize these key events in the hope of maximizing clinical benefit. Below we discuss potential key events in cardiac repair, and the mediators of these events that could be of potential interest for genetic enhancement of stem cell based cardiac repair.
PMCID: PMC2668244  PMID: 18566313
Gene Transfer; Myocardial Repair; Stem Cells; Heart Failure; Acute Myocardial Infarction
25.  Affinity Manipulation of Surface-conjugated RGD-peptide to Modulate Binding of Liposomes to Activated Platelets 
Biomaterials  2008;29(11):1676-1685.
Platelet adhesion, activation and fibrinogen-mediated aggregation are primary events in vascular thrombosis and occlusion. An injectable delivery system that can carry thrombolytics selectively to the sites of active platelet aggregation has immense potential in minimally invasive targeted therapy of vascular occlusion. To this end we are studying liposomes surface-modified by fibrinogen-mimetic RGD-motifs that can selectively target and bind integrin GPIIb-IIIa on activated platelets. Here we report liposome surface-modification with a conformationally constrained high-affinity cyclic RGD-motif to modulate the GPIIb-IIIa-binding capability of the liposomes. Such affinity enhancement is important for practical in vivo applications to compete with native fibrinogen towards binding GPIIb-IIIa. The platelet-binding of RGD-modified liposomes were studied by fluorescence and scanning electron microscopy, and flow cytometry, in vitro. Binding of RGD-modified liposomes was also tested in vivo in a rat carotid injury model and analyzed ex vivo by fluorescence microscopy. The results from all experiments show that cyclic RGD-liposomes bind activated platelets significantly higher compared to linear RGD-liposomes. Hence, the results establish the feasibility of modulating the platelet-targeting and binding ability of vascularly targeted liposomes by manipulating the affinity of surface-modifying ligands.
PMCID: PMC2278119  PMID: 18192005

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