Characterization and natural history of patients with LMNA-related dilated cardiomyopathy in the phase 3 REALM-DCM trial.

AIMS: LMNA-related dilated cardiomyopathy (DCM) is a rare disease with an incompletely defined phenotype. The phase 3 REALM-DCM trial evaluated a potential disease-modifying therapy for LMNA-related DCM but was terminated due to futility without safety concern. This study utilized pooled data from REALM-DCM to descriptively characterize the phenotype and progression of LMNA-related DCM in a contemporary cohort of patients using common heart failure (HF) measures. METHODS: REALM-DCM enrolled patients with stable LMNA-related DCM, an implanted cardioverter defibrillator or cardiac resynchronization therapy defibrillator, and New York Heart Association (NYHA) Class II/III HF symptoms. RESULTS: Between 2018 and 2022, 77 patients took part in REALM-DCM. The median patient age was 53 years (range: 23-72), and 57% were male. Overall, 88% of patients had a pathogenic or likely pathogenic LMNA variant, and 12% had a variant of uncertain significance with a concordant phenotype. Among patients with confirmed sequencing, 55% had a missense variant. Atrial fibrillation was present in 60% of patients; 79% of all patients had NYHA Class II and 21% had NYHA Class III HF symptoms at baseline. Median (range) left ventricular ejection fraction (LVEF), 6 min walk test (6MWT) distance, Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS) score and N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentration at baseline were 42% (23-62), 403 m (173-481), 67 (18-97) and 866 pg/mL (57-5248), respectively. LVEF, 6MWT distance and KCCQ-OS score were numerically lower in patients who had NYHA Class III versus II symptoms at baseline (LVEF: 38% vs. 43%; 6MWT distance: 326 vs. 413 m; and KCCQ-OS score: 43 vs. 70), whereas NT-proBNP concentration was higher (1216 vs. 799 pg/mL). Median follow-up was 73 weeks (range: 0.4-218; 73 in NYHA Class II and 75 in NYHA Class III). Patients displayed variable change from baseline in 6MWT, KCCQ-OS and NT-proBNP values during follow-up. Overall, 25% of patients experienced ventricular tachycardia, and 8% had ventricular fibrillation. Ten (13%) patients met the composite endpoint of worsening HF (adjudicated HF-related hospitalization or urgent care visit) or all-cause death; six had NYHA Class II and four had NYHA Class III at baseline. All-cause mortality occurred in 6 (8%) patients; three had NYHA Class II and three had NYHA Class III symptoms at baseline. CONCLUSIONS: Findings confirm the significant morbidity and mortality associated with LMNA-related DCM despite the standard of care management. Typical measures of HF, including 6MWT distance, KCCQ-OS score and NT-proBNP concentration, were variable but correlated with NYHA class. An unmet treatment need remains among patients with LMNA-related DCM. NCT03439514.

REALM-DCM: A Phase 3, Multinational, Randomized, Placebo-Controlled Trial of ARRY-371797 in Patients With Symptomatic LMNA-Related Dilated Cardiomyopathy.

BACKGROUND: LMNA (lamin A/C)-related dilated cardiomyopathy is a rare genetic cause of heart failure. In a phase 2 trial and long-term extension, the selective p38α MAPK (mitogen-activated protein kinase) inhibitor, ARRY-371797 (PF-07265803), was associated with an improved 6-minute walk test at 12 weeks, which was preserved over 144 weeks. METHODS: REALM-DCM (NCT03439514) was a phase 3, randomized, double-blind, placebo-controlled trial in patients with symptomatic LMNA-related dilated cardiomyopathy. Patients with confirmed LMNA variants, New York Heart Association class II/III symptoms, left ventricular ejection fraction ≤50%, implanted cardioverter-defibrillator, and reduced 6-minute walk test distance were randomized to ARRY-371797 400 mg twice daily or placebo. The primary outcome was a change from baseline at week 24 in the 6-minute walk test distance using stratified Hodges-Lehmann estimation and the van Elteren test. Secondary outcomes using similar methodology included change from baseline at week 24 in the Kansas City Cardiomyopathy Questionnaire-physical limitation and total symptom scores, and NT-proBNP (N-terminal pro-B-type natriuretic peptide) concentration. Time to a composite outcome of worsening heart failure or all-cause mortality and overall survival were evaluated using Kaplan-Meier and Cox proportional hazards analyses. RESULTS: REALM-DCM was terminated after a planned interim analysis suggested futility. Between April 2018 and October 2022, 77 patients (aged 23-72 years) received ARRY-371797 (n=40) or placebo (n=37). No significant differences (P>0.05) between groups were observed in the change from baseline at week 24 for all outcomes: 6-minute walk test distance (median difference, 4.9 m [95% CI, -24.2 to 34.1]; P=0.82); Kansas City Cardiomyopathy Questionnaire-physical limitation score (2.4 [95% CI, -6.4 to 11.2]; P=0.54); Kansas City Cardiomyopathy Questionnaire-total symptom score (5.3 [95% CI, -4.3 to 14.9]; P=0.48); and NT-proBNP concentration (-339.4 pg/mL [95% CI, -1131.6 to 452.7]; P=0.17). The composite outcome of worsening heart failure or all-cause mortality (hazard ratio, 0.43 [95% CI, 0.11-1.74]; P=0.23) and overall survival (hazard ratio, 1.19 [95% CI, 0.23-6.02]; P=0.84) were similar between groups. No new safety findings were observed. CONCLUSIONS: Findings from REALM-DCM demonstrated futility without safety concerns. An unmet treatment need remains among patients with LMNA-related dilated cardiomyopathy. REGISTRATION: URL: https://classic.clinicaltrials.gov; Unique Identifiers: NCT03439514, NCT02057341, and NCT02351856.

Long-term tafamidis efficacy in patients with transthyretin amyloid cardiomyopathy by baseline left ventricular ejection fraction.

AIMS: Patients with transthyretin amyloid cardiomyopathy (ATTR-CM) present with diverse left ventricular ejection fraction (LVEF). This study assessed tafamidis efficacy by baseline LVEF in the phase 3 Tafamidis in Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT) and its long-term extension (LTE) study. METHODS AND RESULTS: Patients were randomized to 30 months of tafamidis or placebo treatment in ATTR-ACT. On completion, patients could join an LTE study to receive tafamidis. All-cause mortality (death, heart transplant, or cardiac mechanical assist device implantation) from baseline to the end of follow-up was assessed in patients continuously treated with tafamidis (80 mg meglumine or 61 mg free acid) or delayed tafamidis treatment (placebo in ATTR-ACT; tafamidis in the LTE study) according to baseline LVEF (<50% or ≥50%). Supportive outcomes were evaluated over a shorter follow-up. Patients with baseline LVEF <50% (n = 177: 88 tafamidis- and 89 placebo-treated) had signs of more severe heart failure, a higher proportion were Black, and had variant ATTR-CM than those with LVEF ≥50% (n = 171: 85 tafamidis- and 86 placebo-treated). At the end of follow-up (median 60-64 months), all-cause mortality was numerically higher in patients with baseline LVEF <50%; however, consistent with supportive findings, continuous tafamidis treatment was associated with a 47% reduction in mortality risk compared with delayed tafamidis treatment in patients with LVEF <50% and ≥50% (hazard ratio 0.53 [95% confidence interval 0.367-0.758]; p < 0.001, and 0.53 [0.344-0.818]; p < 0.01, respectively). CONCLUSIONS: Early initiation of tafamidis is associated with reduced mortality in patients with ATTR-CM, irrespective of initial LVEF value. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT01994889, NCT02791230.

Long-term effectiveness of ARRY-371797 in people with dilated cardiomyopathy and a faulty LMNA gene: a plain language summary.

WHAT IS THIS PLAIN LANGUAGE SUMMARY ABOUT?: This summary explains the results of a long-term extension study on the effects of a specific medicine. A long-term extension study allows people who have already completed a research study to continue taking treatment. Researchers can then look at how a treatment works over a long period of time. This extension study looked at the effects of a medicine called ARRY-371797 (also known as PF-07265803) in people with dilated cardiomyopathy (DCM for short) caused by a faulty lamin A/C gene (also known as the LMNA gene). This condition is called LMNA-related DCM. In people with LMNA-related DCM, the heart muscle becomes thinner and weaker than normal. This can lead to heart failure, where the heart is unable to pump enough blood around the body. The extension study allowed people who had completed an earlier 48-week study to continue taking ARRY-371797 for another 96 weeks (around 22 months). WHAT WERE THE RESULTS OF THE EXTENSION STUDY?: 8 people joined the extension study and continued with the dose of ARRY-371797 that they had taken in the first study. This means that people could have taken ARRY-371797 continuously for up to 144 weeks (around 2 years and 9 months). Using the 6-minute walk test (6MWT for short), researchers regularly checked people taking ARRY-371797 to see how far they could walk. Throughout the extension study, people were able to walk further than they could before they started taking ARRY-371797. This suggests that people could maintain the improvements in their ability to do daily activities with long-term ARRY-371797 treatment. Researchers also looked at how severe people's heart failure was by using a test that measures levels of a biomarker called NT-proBNP. A biomarker is something found in the body that can be measured to indicate the extent of a disease. Throughout this study, the levels of NT-proBNP in people's blood was lower than before they started taking ARRY-371797. This suggests that they maintained stable heart function. Using the Kansas City Cardiomyopathy Questionnaire (KCCQ for short), researchers asked people about their quality of life, and if they experienced any side effects. A side effect is something that people feel while taking a treatment. Researchers evaluate if a side effect is related to the treatment or not. Some improvement in KCCQ response during the study was seen, although results were varied. There were no serious side effects that were considered related to treatment with ARRY-371797. WHAT DO THE RESULTS OF THE EXTENSION STUDY MEAN?: Researchers found that the improvements in functional capacity and heart function seen with ARRY-371797 treatment in the original study were maintained with long-term treatment. Larger studies are needed to determine if ARRY-371797 could be an effective treatment for people with LMNA-related DCM. One such study (called REALM-DCM) was started in 2018 but ended early, as it was unlikely to show a clear treatment benefit of ARRY-371797. Phase 2 long-term extension study (NCT02351856) Phase 2 study (NCT02057341) Phase 3 REALM-DCM study (NCT03439514).

Prevalence and Clinical Burden of Idiopathic Dilated Cardiomyopathy in the United States.

Background: Dilated cardiomyopathy (DCM) contributes significantly to heart failure prevalence, yet supporting epidemiologic data is sparse. This study sought to estimate the period prevalence of DCM and the proportion of idiopathic DCM in the United States using a large, diverse electronic health records (EHR) database. Methods: This retrospective, observational study included 56,812,806 deidentified patients in Optum EHR with visits between 2017 and 2019. Suspected DCM cases were identified using ICD-10 coding. Deidentified clinical notes from 1000 randomly selected cases were manually reviewed to determine the diagnosis of DCM and estimate the proportion of idiopathic DCM. The period prevalence and clinical burden of DCM and idiopathic DCM were estimated. Results: Manual clinical review demonstrated that our definition had a positive predictive value of 92.5% for DCM, with 46.3% estimated as the idiopathic DCM proportion. The estimated period prevalence of DCM between 2017 and 2019 was 118.33 per 100,000. Prevalence increased for adults ≥65 years of age, males, and African Americans. Extrapolation to the 2019 US population led to an overall estimated burden of roughly 388,350 patients. Adjusting for the proportion of cases with idiopathic DCM yielded an idiopathic DCM prevalence of 59.23 per 100,000 and a burden of 194,385 patients. Evidence of clinical genetic testing in this population was scarce, with less than 0.43% of DCM cases reporting a testing code. Conclusions: This study establishes a conservative period prevalence for DCM and idiopathic DCM and demonstrates very low molecular genetic testing for DCM. These findings suggest that the clinical burden of genetic DCM may be underestimated.

Mechanisms of type 2 diabetes resolution after Roux-en-Y gastric bypass.

BACKGROUND: Bariatric surgery is the most effective treatment for the reduction of weight and resolution of type 2 diabetes mellitus (T2 DM). The objective of this study was to longitudinally assess hormonal and tissue responses after RYGB. METHODS: Eight patients (5 with T2 DM) were studied before and after RYGB. A standardized test meal (STM) was administered before and at 1, 3, 6, 9, 12, and 15 months. Separately, a 2-hour hyperinsulinemic-euglycemic clamp (E-clamp) and a 2-hour hyperglycemic clamp (H-clamp) were performed before and at 1, 3, 6, and 12 months. Glucagon-like peptide-1 (GLP-1) was infused during the last hour of the H-clamp. Body composition was assessed with DXA methodology. RESULTS: Enrollment body mass index was 49±3 kg/m(2) (X±SE). STM glucose and insulin responses were normalized by 3 and 6 months. GLP-1 level increased dramatically at 1, 3, and 6 months, normalizing by 12 and 15 months. Insulin sensitivity (M of E-clamp) increased progressively at 3-12 months as fat mass decreased. The insulin response to glucose alone fell progressively over 12 months but the glucose clearance/metabolism (M of H-clamp) did not change significantly until 12 months. In response to GLP-1 infusion, insulin levels fell progressively throughout the 12 months. CONCLUSION: The early hypersecretion of GLP-1 leads to hyperinsulinemia and early normalization of glucose levels. The GLP-1 response normalizes within 1 year after surgery. Enhanced peripheral tissue sensitivity to insulin starts at 3 months and is associated with fat mass loss. ß-cell sensitivity improves at 12 months and after the loss of ≈33% of excess weight. There is a tightly controlled feedback loop between peripheral tissue sensitivity and ß-cell and L-cell (GLP-1) responses.

GLP-1 (7-36) amide restores myocardial insulin sensitivity and prevents the progression of heart failure in senescent beagles.

BACKGROUND: We previously demonstrated that older beagles have impaired whole body and myocardial insulin responsiveness (MIR), and that glucagon-like peptide-1 (GLP-1 [7-36] amide) improves MIR in young beagles with dilated cardiomyopathy (DCM). Here, we sought to determine if aging alone predisposes to an accelerated course of DCM, and if GLP-1 [7-36] amide would restore MIR and impact the course of DCM in older beagles. METHODS: Eight young beagles (Young-Control) and sixteen old beagles underwent chronic left ventricle (LV) instrumentation. Seven old beagles were treated with GLP-1 (7-36) amide (2.5 pmol/kg/min) for 2 weeks prior to instrumentation and for 35 days thereafter (Old + GLP-1), while other 9 served as control (Old-Control). All dogs underwent baseline metabolic determinations and LV biopsy for mitochondria isolation prior to the development of DCM induced by rapid pacing (240 min-1). Hemodynamic measurements were performed routinely as heart failure progressed. RESULTS: At baseline, all old beagles had elevated non-esterifed fatty acids (NEFA), and impaired MIR. GLP-1 reduced plasma NEFA (Old-Control: 853 ± 34; Old + GLP-1: 531 ± 33 µmol/L, p < 0.02), improved MIR (Old-Control: 289 ± 54; Old + GLP-1: 512 ± 44 mg/min/100 mg, p < 0.05), and increased uncoupling protein-3 (UCP-3) expression in isolated mitochondria. Compared to the Young-Control, the Old-Controls experienced an accelerated course of DCM (7 days versus 29 days, p < 0.005) and excess mortality, while the Old + GLP-1 experienced increased latency to the onset of DCM (7 days versus 23 days, p < 0.005) and reduced mortality. CONCLUSION: Aging is associated with myocardial insulin resistance, which predispose to an accelerated course of DCM. GLP-1 treatment is associated with increased MIR and protection against an accelerated course of DCM in older beagles.

GLP-1(32-36)amide, a novel pentapeptide cleavage product of GLP-1, modulates whole body glucose metabolism in dogs.

We have previously demonstrated in human subjects who under euglycemic clamp conditions GLP-1(9-36)amide infusions inhibit endogenous glucose production without substantial insulinotropic effects. An earlier report indicates that GLP-1(9-36)amide is cleaved to a nonapeptide, GLP-1(28-36)amide and a pentapeptide GLP-1(32-36)amide (LVKGR amide). Here we study the effects of the pentapeptide on whole body glucose disposal during hyperglycemic clamp studies. Five dogs underwent indwelling catheterizations. Following recovery, the dogs underwent a 180 min hyperglycemic clamp (basal glucose +98 mg/dl) in a cross-over design. Saline or pentapeptide (30 pmol kg(-1) min(-1)) was infused during the last 120 min after commencement of the hyperglycemic clamp in a primed continuous manner. During the last 30 min of the pentapeptide infusion, glucose utilization (M) significantly increased to 21.4±2.9 mg kg(-1) min(-1)compared to M of 14.3±1.1 mg kg(-1)min(-1) during the saline infusion (P=0.026, paired t-test; P=0.062, Mann-Whitney U test). During this interval, no significant differences in insulin (26.6±3.2 vs. 23.7±2.5 µU/ml, P=NS) or glucagon secretion (34.0±2.1 vs. 31.7±1.8 pg/ml, P=NS) were observed. These findings demonstrate that under hyperglycemic clamp studies the pentapeptide modulates glucose metabolism by a stimulation of whole-body glucose disposal. Further, the findings suggest that the metabolic benefits previously observed during GLP-1(9-36)amide infusions in humans might be due, at least in part, to the metabolic effects of the pentapeptide that is cleaved from the pro-peptide, GLP-1(9-36)amide in the circulation.

Beyond glycemic control: cardiovascular effects of incretin-based therapies.

As cardiovascular (CV) disease remains the major cause of mortality and morbidity in type 2 diabetes mellitus, reducing macrovascular complications has been a major target of antiglycemic therapies. Emerging evidence suggests that incretin-based therapies are safe and may provide CV and cerebrovascular (CBV) benefits beyond those attributable to glycemic control, making the class an attractive therapeutic option. However, the mechanisms whereby the various classes of incretin-based therapies exert CV and CBV benefits may be distinct and may not necessarily lead to similar outcomes. In this chapter, we will discuss the potential mechanisms and current understanding of CV and CBV benefits of native glucagon-like peptide (GLP)-1, GLP-1 receptor agonists and analogues, and of dipeptidyl peptidase-4 inhibitor therapies as a means to better understand differences in safety and efficacy.

Incretin-based therapies: can we achieve glycemic control and cardioprotection?

Glucagon-like (GLP-1) is a peptide hormone secreted from the small intestine in response to nutrient ingestion. GLP-1 stimulates insulin secretion in a glucose-dependent manner, inhibits glucagon secretion and gastric emptying, and reduces appetite. Because of the short circulating half-life of the native GLP-1, novel GLP-1 receptor (GLP-1R) agonists and analogs and dipeptidyl peptidase 4 (DPP-4) inhibitors have been developed to facilitate clinical use. Emerging evidence indicates that GLP-1-based therapies are safe and may provide cardiovascular (CV) benefits beyond glycemic control. Preclinical and clinical studies are providing increasing evidence that GLP-1 therapies may positively affect CV function and metabolism by salutary effects on CV risk factors as well as via direct cardioprotective actions. However, the mechanisms whereby the various classes of incretin-based therapies exert CV effects may be mechanistically distinct and may not necessarily lead to similar CV outcomes. In this review, we will discuss the potential mechanisms and current understanding of CV benefits of native GLP-1, GLP-1R agonists and analogs, and of DPP-4 inhibitor therapies as a means to compare their putative CV benefits.

Dietary flavanol intervention lowers the levels of endothelial microparticles in coronary artery disease patients.

Current evidence suggests that regenerative v. degenerative endothelial responses can be integrated in a clinical endothelial phenotype, reflecting the net result between damage from risk factors and endogenous repair capacity. We have previously shown that a cocoa flavanol (CF) intervention can improve endothelial function and increase the regenerative capacity of the endothelium by mobilising circulating angiogenic cells in patients with coronary artery disease (CAD). The aim of the present study was to investigate whether CF can lower the levels of circulating endothelial microparticles (EMP), markers of endothelial integrity, along with improvements in endothelial function. The levels of EMP in the frozen plasma samples of CAD patients were measured along with endothelial function (flow-mediated vasodilation, FMD); n 16, FMD data published previously), and these data were compared with those of young (n 12) and age-matched (n 12) healthy control subjects. The CAD patients exhibited significantly increased levels of EMP along with impaired FMD when compared with the healthy control subjects. The levels of CD144⁺ and CD31⁺/41⁻ EMP were inversely correlated with FMD (r -0.67, P=0.01 and r -0.59, P=0.01, respectively). In these CAD patients, the levels of EMP were measured after they had consumed a drink containing 375 mg of CF (high-CF intervention, HiFI) or 9 mg of CF (macro- and micronutrient-matched low-CF control, LoFl) twice daily over a 30-d period in a randomised, double-blind, cross-over study. After 1 month of HiFI, the levels of CD31⁺/41⁻ and CD144⁺ EMP decreased (-25 and -23%, respectively), but not after LoFl. Our data show that flavanols lower the levels of EMP along with higher endothelial function, lending evidence to the novel concept that flavanols may improve endothelial integrity.

Injection of human bone marrow and mononuclear cell extract into infarcted mouse hearts results in functional improvement.

BACKGROUND: We have previously shown that mouse whole bone marrow cell (BMC) extract results in improvement of cardiac function and decreases scar size in a mouse model of myocardial infarction (MI), in the absence of intact cells. It is not clear if these results are translatable to extracts from human BMC (hBMC) or mononuclear cells (hMNC), which would have significant clinical implications. METHODS: Male C57BL/6J (10-12 weeks old) mice were included in this study. MI was created by permanent ligation of the left anterior descending artery. Animals were randomized into three groups to receive ultrasound-guided myocardial injections with either hBMCs extract (n=6), hMNCs extract (n=8) or control with 0.5% bovine serum albumin (BSA) (n=7). Cardiac function was assessed by echocardiography at baseline, 2 and 28 days post-MI. Infarct size and vascularity was assessed at 28 days post-MI. RESULTS: hBMC and hMNC extract preserve cardiac function and result in smaller scar size post-MI when compared with the control group. CONCLUSIONS: The current study for the first time reports that hBMC and hMNC extracts improve cardiac function post-MI in a mouse MI model. Further studies are necessary to fully address the potential clinical benefits of these therapies.

Fractionation of mouse bone-marrow cells limits functional efficacy in non-reperfused mouse model of acute myocardial infarction.

BACKGROUND AND OBJECTIVES: Clinical trials of bone-marrow (BM)-derived cells for therapy after acute myocardial infarct (MI) have been controversial. The most commonly used cells for these trials have been mononuclear cells (MNC), obtained by fractionation of BM cells (BMCs) via different protocols. In this study, we performed a head-to-head comparison of: 1) whole BMC; 2) fractionated BM (fBM) using the commonly used Ficoll protocol; 3) the extract derived from the fBM (fBM extract) versus 4) saline (HBSS) control for treatment of acute MI. METHODS: In total, 155 male C57BL/6J (10-12-week old) mice were included. Echocardiography was performed at baseline and 2 days after permanent ligation of the left anterior descending artery to induce MI. Echocardiography and histology were employed to measure outcome at 28 days post-MI. RESULTS: Whole BMC therapy improved left ventricular ejection fraction (LVEF) post-MI, but fBM or fBM extract was not beneficial compared to control (change of LVEF of 4.9% ±4.6% (P = 0.02), -0.4% ±5.8% (P = 0.86), -2.0% ±6.2% (P = 0.97) versus -1.4% ±5.3%, respectively). The histological infarct size or numbers of arterioles or capillaries at infarct or border zone did not differ between the groups. CONCLUSIONS: Clinical studies should be performed to test whether whole BMC therapy translates into better outcome also after human MI.

Evidence of diminished coronary flow in pulmonary hypertension: explaining angina pectoris in this patient group?

BACKGROUND: Many patients with pulmonary hypertension (PH) have symptoms of angina without evidence of occlusive coronary artery disease. For the first time, this study addresses the influence of progressively increasing pulmonary artery pressure (PAP) on left anterior descending artery flow in a rat model of PH. The role of pulmonary artery dilatation, septal wall motion abnormality, cardiac output or diastolic blood pressure in determining coronary blood flow (CBF) during PH was determined. METHODS: Pulmonary hypertension was induced in 6-week-old female nude rats (n = 44) using monocrotaline. Animals underwent right heart catheterization and echocardiography, and blood pressure measurement was taken at baseline, 21 and 35 days. RESULTS: A total of 103 echocardiographic studies were carried out at three fixed time points in rats with variable PAP. CBF decreased from 46·6 ± 14·3 to 24·7 ± 12·3 cm s(-1) (P<0·001) over time. Pulmonary artery diameter increased from 2·30 ± 0·19 to 2·83 ± 0·30 mm (P<0·001), and left ventricular (LV) cardiac output decreased from 143 ± 23 to 78 ± 30 ml min(-1) (P<0·001). Using observed solution estimates of 0·00170 (P = 0·0005) and -1·75 (P = 0·006) for these variables, we calculated that CBF increased by 5·90 cm s(-1) (15·6%, CI: 14·5-17·1%) or decreased by -4·86 cm s(-1) (-12·9%, CI: -14·1-11·9%) for every standard deviation increase in LV cardiac output or pulmonary artery diameter, respectively. CBF decreased significantly with increasing PAP. Pulmonary artery diameter and LV cardiac output appear to be independent determinants of coronary flow in PH. CONCLUSIONS: Coronary flow reduction in murine PH has potential to be clinically meaningful and should therefore further studied in a clinical trial.

A dosing study of bone marrow mononuclear cells for transendocardial injection in a pig model of chronic ischemic heart disease.

We studied the effect of the dose of bone marrow mononuclear cells, delivered via transendocardial injection, upon capillary density and fibrosis in pigs with chronic ischemic heart disease.Pigs (n = 16) that had undergone ameroid constrictor placement (left circumflex coronary artery) to induce chronic ischemia were divided equally into 4 groups on the basis of bone marrow mononuclear cell dose: control (saline injection) and 50, 100, or 200 × 10(6) bone marrow mononuclear cells. Thirty days after ameroid placement, each pig received 13 transendocardial NOGA-guided injections. An implantable loop recorder monitored possible arrhythmias caused by cell transplantation. Thirty days later, the pigs were killed, and their hearts were evaluated histopathologically for fibrosis and capillary density; the number of cells per segment was correlated with fibrosis and capillary density. No adverse events, arrhythmias, or cardiac inflammatory reactions were associated with cell therapy. Less fibrosis was seen in pigs that received 100 × 10(6) cells than in control pigs. A trend toward higher capillary density was seen with higher cell concentrations. Segments injected with more than 20 × 10(6) million cells had the highest capillary density and the least amount of fibrosis (P < 0.05 vs controls).In conclusion, transendocardial injections (up to 200 × 10(6) bone marrow mononuclear cells) were safe. Analyses of individual injected segments suggest potential benefit from higher cell concentrations per segment.

Treatment of pulmonary arterial hypertension with circulating angiogenic cells.

Despite advances in the treatment of pulmonary arterial hypertension, a truly restorative therapy has not been achieved. Attention has been given to circulating angiogenic cells (CACs, also termed early endothelial progenitor cells) because of their ability to home to sites of vascular injury and regenerate blood vessels. We studied the efficacy of human CAC therapy in the treatment of pulmonary arterial hypertension at two different stages of disease severity. Cells were isolated from peripheral blood and administered to nude rats on day 14 ("early") or day 21 ("late") after monocrotaline injection. The control group received monocrotaline but no cell treatment. Disease progression was assessed using right heart catheterization and echocardiography at multiple time points. Survival differences, right ventricular hypertrophy (RVH), and vascular hypertrophy were analyzed at the study endpoint. Quantitative PCR was performed to evaluate cell engraftment. Treatment with human CACs either at the early or late time points did not result in increased survival, and therapy did not prevent or reduce the severity of disease compared with control. Histological analysis of RVH and vascular muscularization showed no benefit with therapy compared with control. No detectable signal was seen of human transcript in transplanted lungs at 14 or 21 days after cell transplant. In conclusion, CAC therapy was not associated with increased survival and did not result in either clinical or histological benefits. Future studies should be geared toward either earlier therapeutic time points with varying doses of unmodified CACs or genetically modified cells as a means of delivery of factors to the pulmonary arterial circulation.

Timing of bone marrow cell therapy is more important than repeated injections after myocardial infarction.

BACKGROUND: Bone marrow cell treatment has been proposed as a therapy for myocardial infarction, but the optimal timing and number of injections remain unknown. METHODS: Myocardial infarction was induced in mice followed by ultrasound-guided injection of mouse bone marrow cells at different time points post myocardial infarction (Days 3, 7, and 14) as monotherapy and at Days 3+7 as "double" therapy and at Days 3+7+14 as "triple" therapy. Controls received saline injections at Day 3 and Days 3+7+14. Left ventricular ejection fraction was evaluated post myocardial infarction prior to any therapy and at Day 28. Hearts were analyzed at Day 28 for infarct size and survival of donor cells. RESULTS: Left ventricular ejection fraction decreased from 55.3±0.9% to 37.6±0.6% (P<.001) 2 days post myocardial infarction in all groups. Injection of bone marrow cells at Day 3 post myocardial infarction resulted in smaller infarct size (17.8±3.6% vs. 36.6±7.1%; P=.05) and improved LV function (left ventricular ejection fraction 40.3±2.0% vs. 31.1±8.3%; P<.05) compared to control. However, delayed therapy at Day 7 or 14 did not. Multiple injections of bone marrow cells, either double therapy or triple therapy, did not result in reduction in infarct size, but led to improvements in left ventricular ejection fraction at Day 28 compared to control (39.9±3.6% and 38.8±5.5% vs. 34.8±5.3%; all P<.05). The number of donor cells surviving at Day 28 did not correlate with improvement in left ventricular ejection fraction. CONCLUSIONS: Injection of bone marrow cells at Day 3 reduced infarct size and improved left ventricular function. Multiple injections of bone marrow cells had no additive effect. Delaying cell therapy post myocardial infarction resulted in no functional benefit at all. These results will help inform future clinical trials.

Cytokine combination therapy with erythropoietin and granulocyte colony stimulating factor in a porcine model of acute myocardial infarction.

PURPOSE: Erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF) have generated interest as novel therapies after myocardial infarction (MI), but the effect of combination therapy has not been studied in the large animal model. We investigated the impact of prolonged combination therapy with EPO and GCSF on cardiac function, infarct size, and vascular density after MI in a porcine model. METHODS: MI was induced in pigs by a 90 min balloon occlusion of the left anterior descending coronary artery. 16 animals were treated with EPO+GCSF, or saline (control group). Cardiac function was assessed by echocardiography and pressure-volume measurements at baseline, 1 and 6 weeks post-MI. Histopathology was performed 6 weeks post-MI. RESULTS: At week 6, EPO+GCSF therapy stabilized left ventricular ejection fraction, (41 ± 1% vs. 33 ± 1%, p < 0.01) and improved diastolic function compared to the control group. Histopathology revealed increased areas of viable myocardium and vascular density in the EPO+GCSF therapy, compared to the control. Despite these encouraging results, in a historical analysis comparing combination therapy with monotherapy with EPO or GCSF, there were no significant additive benefits in the LVEF and volumes overtime using the combination therapy. CONCLUSION: Our findings indicate that EPO+GCSF combination therapy promotes stabilization of cardiac function after acute MI. However, combination therapy does not seem to be superior to monotherapy with either EPO or GCSF.

Cytokine combination therapy with long-acting erythropoietin and granulocyte colony stimulating factor improves cardiac function but is not superior than monotherapy in a mouse model of acute myocardial infarction.

BACKGROUND: Erythropoietin (EPO) and granulocyte colony stimulating factor (GCSF) are potential novel therapies after myocardial infarction (MI). We first established the optimal and clinically applicable dosages of these drugs in mobilizing hematopoietic stem cells (HSC), and then tested the efficacy of monotherapy and combination therapy post-MI. METHODS AND RESULTS: Optimal doses were established in enhanced green fluorescent protein (eGFP) + chimeric mice (n = 30). Next, mice underwent MI and randomized into 4 groups (n = 18/group): 1) GCSF; 2) EPO; 3) EPO+GCSF; and 4) control. Left ventricular (LV) function was analyzed pre-MI, at 4 hours and at 28 days post-MI. Histological assessment of infarct size, blood vessels, apoptotic cardiomyocytes, and engraftment of eGFP+ mobilized cells were analyzed at day 28. LV function in the control group continued to deteriorate, whereas all treatments showed stabilization. The treatment groups resulted in less scarring, increased numbers of mobilized cells to the infarct border zone (BZ), and a reduction in the number of apoptotic cardiomyocytes. Both EPO groups had significantly more capillaries and arterioles at the BZ. CONCLUSION: We have established the optimal doses for EPO and GCSF in mobilizing HSC from the bone marrow and demonstrated that therapy with these agents, either as monotherapy or combination therapy, led to improvement of cardiac function post-MI. Combination therapy does not seem to have additive benefit over monotherapy in this model.