David A. D'Alessandro

Human cardiac stem cells

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.

Vasopressin Deficiency Contributes to the Vasodilation of Septic Shock

BACKGROUND The hypotension of septic shock is due to systemic vasodilation. On the basis of a clinical observation, we investigated the possibility that a deficiency in vasopressin contributes to the vasodilation of septic shock. METHODS AND RESULTS In 19 patients with vasodilatory septic shock (systolic arterial pressure [SAP] of 92 +/- 2 mm Hg [mean +/- SE], cardiac output [CO] of 6.8 +/- 0.7 L/min) who were receiving catecholamines, plasma vasopressin averaged 3.1 +/- 1.0 pg/mL. In 12 patients with cardiogenic shock (SAP, 99 +/- 7 mm Hg; CO, 3.5 +/- 0.9 L/min) who were also receiving catecholamines, it averaged 22.7 +/- 2.2 pg/mL (P < .001). A constant infusion of exogenous vasopressin to 2 patients with septic shock resulted in the expected plasma concentration, indicating that catabolism of vasopressin is not increased in this condition. Although vasopressin is a weak pressor in normal subjects, its administration at 0.04 U/min to 10 patients with septic shock who were receiving catecholamines increased arterial pressure (systolic/diastolic) from 92/52 to 146/66 mm Hg (P < .001/P < .05) due to peripheral vasoconstriction (systemic vascular resistance increased from 644 to 1187 dyne.s/cm5; P < .001). Furthermore, in 6 patients with septic shock who were receiving vasopressin as the sole pressor, vasopressin withdrawal resulted in hypotension (SAP, 83 +/- 3 mm Hg), and vasopressin administration at 0.01 U/min, which resulted in a plasma concentration (approximately 30 pg/mL) expected for the level of hypotension, increased SAP from 83 to 115 mm Hg (P < .01). CONCLUSIONS Vasopressin plasma levels are inappropriately low in vasodilatory shock, most likely because of impaired baroreflex-mediated secretion. The deficiency in vasopressin contributes to the hypotension of vasodilatory septic shock.

Mitral-Valve Repair versus Replacement for Severe Ischemic Mitral Regurgitation

BACKGROUND Ischemic mitral regurgitation is associated with a substantial risk of death. Practice guidelines recommend surgery for patients with a severe form of this condition but acknowledge that the supporting evidence for repair or replacement is limited. METHODS We randomly assigned 251 patients with severe ischemic mitral regurgitation to undergo either mitral-valve repair or chordal-sparing replacement in order to evaluate efficacy and safety. The primary end point was the left ventricular end-systolic volume index (LVESVI) at 12 months, as assessed with the use of a Wilcoxon rank-sum test in which deaths were categorized below the lowest LVESVI rank. RESULTS At 12 months, the mean LVESVI among surviving patients was 54.6±25.0 ml per square meter of body-surface area in the repair group and 60.7±31.5 ml per square meter in the replacement group (mean change from baseline, -6.6 and -6.8 ml per square meter, respectively). The rate of death was 14.3% in the repair group and 17.6% in the replacement group (hazard ratio with repair, 0.79; 95% confidence interval, 0.42 to 1.47; P=0.45 by the log-rank test). There was no significant between-group difference in LVESVI after adjustment for death (z score, 1.33; P=0.18). The rate of moderate or severe recurrence of mitral regurgitation at 12 months was higher in the repair group than in the replacement group (32.6% vs. 2.3%, P<0.001). There were no significant between-group differences in the rate of a composite of major adverse cardiac or cerebrovascular events, in functional status, or in quality of life at 12 months. CONCLUSIONS We observed no significant difference in left ventricular reverse remodeling or survival at 12 months between patients who underwent mitral-valve repair and those who underwent mitral-valve replacement. Replacement provided a more durable correction of mitral regurgitation, but there was no significant between-group difference in clinical outcomes. (Funded by the National Institutes of Health and the Canadian Institutes of Health; ClinicalTrials.gov number, NCT00807040.).

Human Cardiac Stem Cell Differentiation Is Regulated by a Mircrine Mechanism

Background— Cardiac stem cells (CSCs) delivered to the infarcted heart generate a large number of small fetal-neonatal cardiomyocytes that fail to acquire the differentiated phenotype. However, the interaction of CSCs with postmitotic myocytes results in the formation of cells with adult characteristics. Methods and Results— On the basis of results of in vitro and in vivo assays, we report that the commitment of human CSCs (hCSCs) to the myocyte lineage and the generation of mature working cardiomyocytes are influenced by microRNA-499 (miR-499), which is barely detectable in hCSCs but is highly expressed in postmitotic human cardiomyocytes. miR-499 traverses gap junction channels and translocates to structurally coupled hCSCs favoring their differentiation into functionally competent cells. Expression of miR-499 in hCSCs represses the miR-499 target genes Sox6 and Rod1, enhancing cardiomyogenesis in vitro and after infarction in vivo. Although cardiac repair was detected in all cell-treated infarcted hearts, the aggregate volume of the regenerated myocyte mass and myocyte cell volume were greater in animals injected with hCSCs overexpressing miR-499. Treatment with hCSCs resulted in an improvement in ventricular function, consisting of a better preservation of developed pressure and positive and negative dP/dt after infarction. An additional positive effect on cardiac performance occurred with miR-499, pointing to enhanced myocyte differentiation/hypertrophy as the mechanism by which miR-499 potentiated the restoration of myocardial mass and function in the infarcted heart. Conclusions— The recognition that miR-499 promotes the differentiation of hCSCs into mechanically integrated cardiomyocytes has important clinical implications for the treatment of human heart failure.

Cardiomyogenesis in the Aging and Failing Human Heart

Background— Two opposite views of cardiac growth are currently held; one views the heart as a static organ characterized by a large number of cardiomyocytes that are present at birth and live as long as the organism, and the other views the heart a highly plastic organ in which the myocyte compartment is restored several times during the course of life. Methods and Results— The average age of cardiomyocytes, vascular endothelial cells (ECs), and fibroblasts and their turnover rates were measured by retrospective 14C birth dating of cells in 19 normal hearts 2 to 78 years of age and in 17 explanted failing hearts 22 to 70 years of age. We report that the human heart is characterized by a significant turnover of ventricular myocytes, ECs, and fibroblasts, physiologically and pathologically. Myocyte, EC, and fibroblast renewal is very high shortly after birth, decreases during postnatal maturation, remains relatively constant in the adult organ, and increases dramatically with age. From 20 to 78 years of age, the adult human heart entirely replaces its myocyte, EC, and fibroblast compartment ≈8, ≈6, and ≈8 times, respectively. Myocyte, EC, and fibroblast regeneration is further enhanced with chronic heart failure. Conclusions— The human heart is a highly dynamic organ that retains a remarkable degree of plasticity throughout life and in the presence of chronic heart failure. However, the ability to regenerate cardiomyocytes, vascular ECs, and fibroblasts cannot prevent the manifestations of myocardial aging or oppose the negative effects of ischemic and idiopathic dilated cardiomyopathy.

Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration.

Rationale: Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. Objective: Insulin-like growth factor (IGF)-1, IGF-2, and angiotensin II (Ang II), as well as their receptors, IGF-1R, IGF-2R, and AT1R, were characterized in c-kit+ hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. Methods and Results: C-kit+ hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation, and attenuated apoptosis. In addition to IGF-1, IGF-1R+ hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R+ hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit+ hCSCs. IGF-1R+ hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R+ hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. Conclusions: hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage, and myocyte differentiation, which points to this hCSC subset as the ideal candidate cell for the management of human heart failure.

Readmissions After Ventricular Assist Device: Etiologies, Patterns, and Days Out of Hospital

BACKGROUND Scarce literature exists describing the patterns of readmission after continuous flow left ventricular assist device (CF-LVAD) implantation. These carry significant cost and quality of life implications. We sought to describe the etiology and pattern of readmission among patients receiving CF-LVADs. METHODS Frequency, reason, urgency, and duration of readmission as well as freedom from readmission were examined in a retrospective review of our institutional experience. As an indirect means of quality of life, the ratio of days out of hospital (OOH)/days alive with device was calculated. RESULTS From 2006 to 2011, 71 adult patients implanted with a CF device were included. Indication for device implantation was bridge to transplant (n=19), potential bridge to transplant (n=25), or destination therapy (n=27). Length of support averaged 359 days. Total support time was 69.7 patient years. One hundred fifty-five readmissions accounted for a total of 1,659 hospital days. Fifty-six patients were readmitted during the study period. Median time to first readmission was 48 days (range 2 to 663 days). Median length of stay was 5 days. The single most common etiology for readmission was gastrointestinal bleeding accounting for 14% of readmissions. Readmissions were urgent (87%), elective (10%), or life-threatening (3%). Patients on the average enjoyed 92% of their time OOH. CONCLUSIONS Patients undergoing CF-LVAD support are often readmitted within 6 months of discharge. Readmissions tend to be of short duration and the most common reason is for gastrointestinal bleeding. Importantly, following discharge after implant procedure, 51 patients spent at least 90% of days OOH.

The Ephrin A1–EphA2 System Promotes Cardiac Stem Cell Migration After Infarction

Rationale: Understanding the mechanisms that regulate trafficking of human cardiac stem cells (hCSCs) may lead to development of new therapeutic approaches for the failing heart. Objective: We tested whether the motility of hCSCs in immunosuppressed infarcted animals is controlled by the guidance system that involves the interaction of Eph receptors with ephrin ligands. Methods and Results: Within the cardiac niches, cardiomyocytes expressed preferentially the ephrin A1 ligand, whereas hCSCs possessed the EphA2 receptor. Treatment of hCSCs with ephrin A1 resulted in the rapid internalization of the ephrin A1–EphA2 complex, posttranslational modifications of Src kinases, and morphological changes consistent with the acquisition of a motile cell phenotype. Ephrin A1 enhanced the motility of hCSCs in vitro, and their migration in vivo following acute myocardial infarction. At 2 weeks after infarction, the volume of the regenerated myocardium was 2-fold larger in animals injected with ephrin A1–activated hCSCs than in animals receiving control hCSCs; this difference was dictated by a greater number of newly formed cardiomyocytes and coronary vessels. The increased recovery in myocardial mass with ephrin A1–treated hCSCs was characterized by further restoration of cardiac function and by a reduction in arrhythmic events. Conclusions: Ephrin A1 promotes the motility of EphA2-positive hCSCs, facilitates their migration to the area of damage, and enhances cardiac repair. Thus, in situ stimulation of resident hCSCs with ephrin A1 or their ex vivo activation before myocardial delivery improves cell targeting to sites of injury, possibly providing a novel strategy for the management of the diseased heart.

Progenitor Cells From the Explanted Heart Generate Immunocompatible Myocardium Within the Transplanted Donor Heart

Rationale: Chronic rejection, accelerated coronary atherosclerosis, myocardial infarction, and ischemic heart failure determine the unfavorable evolution of the transplanted heart in humans. Objective: Here we tested whether the pathological manifestations of the transplanted heart can be corrected partly by a strategy that implements the use of cardiac progenitor cells from the recipient to repopulate the donor heart with immunocompatible cardiomyocytes and coronary vessels. Methods and Results: A large number of cardiomyocytes and coronary vessels were created in a rather short period of time from the delivery, engraftment, and differentiation of cardiac progenitor cells from the recipient. A proportion of newly formed cardiomyocytes acquired adult characteristics and was integrated structurally and functionally within the transplant. Similarly, the regenerated arteries, arterioles, and capillaries were operative and contributed to the oxygenation of the chimeric myocardium. Attenuation in the extent of acute damage by repopulating cardiomyocytes and vessels decreased significantly the magnitude of myocardial scarring preserving partly the integrity of the donor heart. Conclusions: Our data suggest that tissue regeneration by differentiation of recipient cardiac progenitor cells restored a significant portion of the rejected donor myocardium. Ultimately, immunosuppressive therapy may be only partially required improving quality of life and lifespan of patients with cardiac transplantation.

Outcomes of cardiac transplantation in septuagenarians

BACKGROUND Cardiac transplantation in many centers is programmatically limited to patients aged younger than 70 years. We investigated the trends and outcomes for cardiac transplantation in recipients aged 70 years and older in the United States. METHODS De-identified data were provided by United Network of Organ Sharing. Transplant recipients were grouped by age 60-69 years and 70 years and older. Univariate comparisons were performed using Students t-test or the Pearson chi-square test. Survival was estimated using the Kaplan-Meier technique and compared with the log-rank test. Cox regression was used to determine predictors of death after transplant. Statistical significance was assigned to p < 0.05. RESULTS Between January 1, 1998, and June 15, 2010, 5,807 sexagenarians and 332 septuagenarians received allografts. The septuagenarian cohort had more men, less diabetes, was less likely to have a ventricular assist device, and more likely to be status II. Donors for septuagenarians were older and died more frequently from intracranial hemorrhage. Median unadjusted survival was 9.8 years for sexagenarians vs 8.5 years for septuagenarians (p = 0.003). There was no difference in the incidence of cerebrovascular accident, length of stay, or pacemaker need between groups. Septuagenarians were less likely to be treated for rejection the first year (p = 0.001). Age was a multivariate predictor of death (hazard ratio, 1.289; 95% confidence interval, 1.039-1.6; p = 0.021). CONCLUSIONS Selected septuagenarians with advanced heart failure can derive great benefit from cardiac transplantation, although survival is inferior to that of an immediately younger sexagenarian cohort. Most of the mortality risk is seen in the first year after transplantation. A reduced incidence of rejection was observed and warrants further study.