The impact of patient sex on the response to intramyocardial mesenchymal stem cell administration in patients with non-ischaemic dilated cardiomyopathy

Abstract

Aims

Sex differences impact the occurrence, presentation, prognosis, and response to therapy in heart disease. Particularly, the phenotypic presentation of patients with non-ischaemic dilated cardiomyopathy (NIDCM) differs between men and women. However, whether the response to mesenchymal stem cell (MSC) therapy is influenced by sex remains unknown. We hypothesize that males and females with NIDCM respond similarly to MSC therapy.

Methods and results

Male (n = 24) and female (n = 10) patients from the POSEIDON-DCM trial who received MSCs via transendocardial injections were evaluated over 12 months. Endothelial function was measured at baseline and 3 months post-transendocardial stem cell injection (TESI). At baseline, ejection fraction (EF) was lower (P = 0.004) and end-diastolic volume (EDV; P = 0.0002) and end-systolic volume (ESV; P = 0.0002) were higher in males vs. females. In contrast, baseline demographic characteristics, Minnesota Living with Heart Failure Questionnaire (MLHFQ), and 6-min walk test (6MWT) were similar between groups. EF improved in males by 6.2 units (P = 0.04) and in females by 8.6 units (P = 0.04; males vs. females, P = 0.57). EDV and ESV were unchanged over time. The MLHFQ score, New York Heart Association (NYHA) class, endothelial progenitor cell-colony forming units, and serum tumour necrosis factor alpha improved similarly in both groups.

Conclusion

Despite major differences in phenotypic presentation of NIDCM in males and females, this study is the first of its kind to demonstrate that MSC therapy improves a variety of parameters in NIDCM irrespective of patient sex. These findings have important clinical and pathophysiologic implications regarding the impact of sex on responses to cell-based therapy for NIDCM.

Graphical Abstract

Graphical Abstract.

Time for primary review: 34 days

1. Introduction

Patient sex differences impact the occurrence, presentation, prognosis, and response to therapy of heart disease, frequently with women having a worse outcome.¹ With respect to non-ischaemic dilated cardiomyopathy (NIDCM), there are major differences in clinical phenotype and gene expression profiles between men and women; women present with milder disease, higher ejection fraction (EF), lower left ventricular (LV) internal diameter in diastole,² and better prognosis than men.³ Sex-specific patterns of cardiovascular ageing play an important role in these discrepancies, yet the mechanisms underlying these differences remain elusive.⁴ The physiologic basis for sex-related differences involves multiple systems, including activation of the renin–angiotensin and sympathetic systems,⁵ calcium handling by the sarcoplasmic reticulum,^6,7 mitochondrial metabolism,⁷ and cell apoptosis in the failing hearts.⁸ Withdrawal of oestrogen at the time of menopause is associated with vascular and cardiac ageing in females and induces endothelial dysfunction, arterial stiffness, LV hypertrophy, and prevalence of heart failure (HF) with preserved ejection fraction (HFpEF).^7,9

There is growing evidence that endogenous stem cell function contributes to this sex disparity. The anti-atherosclerotic effects of oestrogen in fertile females have been attributed to oestrogen-mediated regulation of endothelial progenitor cells (EPCs), increasing circulating CD34⁺KDR⁺ EPCs and improving vascular homeostasis.¹⁰ Mesenchymal stem cells (MSCs) also display sex-specific differences. Female MSCs exhibit decreased apoptosis, IL-6 production, and tumour necrosis factor alpha (TNF-α), and increased vascular endothelial growth factor (VEGF) expression compared with male MSCs.¹¹ Furthermore, in a mouse myocardial infarction model, treatment with female MSCs produced greater recovery of LV pressures and maximum and minimum dP/dT compared with treatment with male MSCs.⁷ Owing to their immunoprivileged profile and abundant capacity for paracrine signalling, MSCs are an important candidate cell therapy for cardiac regeneration.¹² MSCs reduce scar size, augment vascularity, and promote regeneration of cardiomyocytes in the failing heart.^12,13 While previous studies have investigated the effect of sex and oestrogen exposure on the efficacy of donor MSCs, patient sex differences may also impact the recipient’s response to cell therapy.¹⁴ However, there are no studies in humans assessing the difference between men and women in response to cell-based therapy for cardiac repair, and whether intracardiac delivery is impaired or enhanced in females remains unknown.

Here, we analysed sex-specific differences in the response to cell-based therapy in patients enrolled in the POSEIDON-DCM trial, a Phase I/II randomized comparison of safety and efficacy of bone marrow-derived human MSCs in a NIDCM population. In this trial, transendocardial cell injection with autologous or allogeneic MSCs was safe, and improved global EF, quality of life, and functional capacity. Patients with NIDCM who received allogeneic MSCs exhibited improved endothelial function. We generated the hypothesis that males and females with NIDCM respond similarly to MSC treatment, regardless of baseline differences, resulting in a greater chance of EF recovery in females.

2. Methods

Patients from the POSEIDON-DCM trial were included in analysis and divided into male and female groups. The associations between patient sex and cardiac function, quality of life, functional status, and endothelial function were assessed. Although POSEIDON-DCM was an open-label study, all data analysis was masked to those assessing all study endpoints and statistical analysis was performed by a third party for unmasking.

The POSEIDON-DCM trial¹⁵ was conducted at the University of Miami under an Investigational New Drug Application from the US Food and Drug Administration. All patients provided written informed consent and the investigation conformed to the principles outlined in the Declaration of Helsinki. The study design of the POSEIDON-DCM trial has been previous published.¹⁶ In brief, patients were eligible to enroll after their diagnosis of NIDCM was confirmed, as defined by an EF less than 40% and either a LV end-diastolic diameter greater than 5.9 cm in male subjects, and >5.6 cm in female subjects or a LV end-diastolic volume index >125 mL/m², verified by gated blood pool scan, two-dimensional echocardiogram, cardiac computed tomography (CT), cardiac magnetic resonance imaging (MRI), or left ventriculogram at least 6 months prior to study inclusion.^15,16 Patients had to be treated with appropriate maximal medical therapy for HF. For beta blockers, the patient must have been on a stable dose of clinically appropriate beta blockers for 3 months. For angiotensin-converting enzyme (ACE) inhibitors, the patient must have been on a stable dose of clinically appropriate agent for 1 month. Additional eligibility criteria included being 18–95 years old and a candidate for cardiac catheterization within 5–10 weeks of screening as determined by the study team. Patients were excluded from the study if they had a non-cardiac condition that limited their lifespan to <1-year, a glomerular filtration rate <45 mL/min/1.73 m², serious radiographic contrast allergy, a need for coronary artery revascularization, diagnosis of myocarditis,¹⁷ evidence of a life-threatening arrhythmia in the absence of a defibrillator, or clinical history of a malignancy within 5 years of screening.

MSCs were isolated from bone marrow aspirates of either healthy male donors (allogeneic) (mean age 25.4 ± 3.3 years old) or the NIDCM patients (autologous), and manufactured at the University of Miami Interdisciplinary Stem Cell Institute, as previously described.^18,19 In brief, bone marrow mononuclear cells were prepared by centrifugation of whole bone marrow against a low-density gradient using Ficoll-Paque Premium (d = 1.077) according to the manufacturer’s protocol. Cells were collected at the interface. According to the International Stem Cell Therapy guidelines and as described in the published POSEIDON-DCM trial, the cells used in the POSEIDON-DCM study had the following characteristics: cell viability >70%, positive for CD105, and negative for CD45 by flow cytometry analysis, no growth of bacteria via Gram stain, negative for mycoplasma via polymerase chain reaction, and endotoxin level ≤5 EU/mL. MSCs were delivered by transendocardial cell injection at passage 2 using the Biosense Webster MyoStar NOGA Catheter System (Johnson & Johnson) into 10 LV sites to target a uniform distribution throughout the LV area and at least 5 mm distant from adjacent injection sites.^15,16 We compared data at baseline, and 3, 6, and 12 months post-transendocardial stem cell injection (TESI) to assess patient sex differences.

Cardiac function was assessed at baseline and 12 months post-TESI by cardiac CT or MRI. Cardiac imaging parameters included absolute global EF, end-diastolic volume (EDV), end-systolic volume (ESV), and sphericity index. The measurements and analysis were performed using iNtuition software ver.4.4.7.47; TeraRecon, Inc., Foster City, CA, USA for CT and Qmass MR 7.2 (Medis Inc., The Netherlands) for MRI.¹⁶ MRI was conducted only on those patients without implanted devices or with scannable permanent pacemakers and implantable cardioverter-defibrillators.¹⁶ Patient functional capacity was assessed by the 6-min walk test (6MWT). Quality of life parameters included Minnesota Living with Heart Failure Questionnaire (MLHFQ) total score and New York Heart Association (NYHA) functional class.¹⁵

Endothelial function was assessed at baseline and 3 months post-TESI. EPC-colony forming units (EPC-CFUs) from peripheral blood samples and flow-mediated diameter (FMD) measurements were performed as previously described.^15,20 Shortly, peripheral blood samples were obtained from patients before and there months after MSC administration. EPCs were isolated from samples using Ficoll-Paque and 5 million cells were seeded on 6-well fibronectin-coated dishes (BD biosciences) in CFU-Hill medium (stem cell technologies, cat#05900).^20,21 The non-adherent cells were collected 48 h later and 1 million cells were seeded on 24-well fibronectin-coated dishes. On Day 5, EPC-CFUs were counted in five wells and the average was obtained. Furthermore, cells were stained with anti-CD31 and anti-VEGFR to characterize EPCs using IHC.

Serum TNF-α was measured at baseline and at 6 months using a human TNF-α enzyme-linked immunoadsorbent assay high-sensitivity kit (eBiosciences).

Continuous variables are presented as mean ± standard deviation [or median and interquartile range (IQR) as appropriate]. Data distribution were determined by the Pearson normality test. Categorical variables were assessed using the Pearson χ² and Fisher’s exact tests. For parameters with a significant departure from normality, a non-parametric Wilcoxon-Mann–Whitney test was used. Normally distributed parameters were evaluated using two-way ANOVA and multiple comparison was performed using the Bonferroni and Tukey corrections. Endothelial function parameters were compared using the two-sided t-tests. All statistical tests were performed at alpha = 0.05. Analyses were conducted using GraphPad Prism system (Version 6.0, La Jolla, CA, USA).

3. Results

3.1 Patient population

Thirty-four patients with NIDCM who received human MSCs from the POSEIDON-DCM trial were included in the analysis (Table 1). Twenty-four patients (71%) were male and 10 (29%) were female. The mean age at TESI was 54.3 ± 10.4 years for males and 55.1 ± 11.6 years for females (P = 0.84). Ninety percent of the patients in both groups were White. The mean time from NIDCM diagnosis to cell therapy was 6.5 ± 5.7 years for males and 6.5 ± 9.3 years for females (P = 0.99). Ten (41.7%) male patients received autologous MSCs, and 14 (58.3%) received allogeneic MSCs. Six (60.0%) females received autologous, and 4 (40.0%) received allogeneic MSCs (P = 0.46).

Table 1.

Baseline characteristics

	Males (n = 24)	Females (n = 10)	P-value
Age at transplantation (years)	54.3 ± 10.4	55.1 ± 11.6	0.8
Weight (kg)	93.2 (77.3–101.7)	73.9 (64.7–81.7)	0.003
BSA (m2)	2.1 ± 0.3	1.8 ± 0.1	0.0009
Systolic blood pressure (mmHg)	115.7 ± 13.1	121.2 ± 19.3	0.3
Diastolic blood pressure (mmHg)	72.5 ± 12.2	69.0 ± 11.2	0.4
Ethnicity			0.7
Hispanic or Latino	8 (33.3%)	4 (40.0%)
Not Hispanic or Latino	16 (66.6%)	6 (60.0%)
Race			0.4
White	21 (87.5%)	9 (90.0%)
African American	2 (8.3%)	1 (10.0%)
Unknown	1 (4.2%)	0 (0.0%)
NIDCM diagnosis before TESI (years)	6.5 ± 5.7	6.5 ± 9.3	0.99
AICD or BiV/CRT	20 (83.3%)	9 (90.0%)	0.99
History of hypertension	9 (37.5%)	1 (10.0%)	0.2
History of smoking	13 (54.2%)	4 (40.0%)	0.7
History of diabetes	0 (0.0%)	1 (10.0%)	0.3
New York Heart Association functional class			0.053
I	8 (33.3%)	2 (20.0%)
II	9 (37.5%)	8 (80.0%)
III	7 (29.2%)	0 (0.0%)
6-min walk test (m)	430.9 ± 92.0	401.5 ± 73.9	0.4
MLHFQ total score	37.9 ± 25.0	42.6 ± 21.0	0.6
Serum TNF-α	12.0 ± 4.7	14.3 ± 6.2	0.2
Ejection fraction (%)	23.5 ± 9.0	33.6 ± 7.3	0.004
End-diastolic volume (mL)	360.4 (291.2–470.0)	251.4 (192.5–267.1)	0.0002
End-systolic volume (mL)	288.9 (231.9–358.5)	162.5 (119.1–190.5)	0.0002
LV end-diastolic volume index (mm/m2)	183.0 (139.7–208.7)	139.9 (107.0–151.2)	0.0028
LV end-systolic volume index (mm/m2)	143.4 (108.3–159.0)	91.58 (67.34–105.1)	0.0013
End-diastolic internal diameter (LVIDD) (mm)	79.2 (68.9–85.9)	63.8 (57.7–67.1)	0.0004
End-systolic internal diameter (LVIDS) (mm)	69.4 (60.5–77.4)	53.4 (46.07–60.95)	0.0005
Long-axis diameter (mm)	113.1 ± 16.9	93.8 ± 10.7	0.002

Values are expressed as mean ± SD, median (interquartile range), or n (%).

The POSEIDON-DCM trial enrolled 37 patients (n = 18 for autologous MSCs and n = 19 for allogeneic MSCs), of whom 34 received TESI (n = 16 autologous and n = 18 allogeneic). In this study, we included 34 patients, of whom 24 were males and 10 were females. Ten patients were excluded from the 34 that were treated, 2 due to death (both unrelated to treatment), 2 received an LV assist device (LVAD), 3 underwent heart transplant, and 3 withdrew from the study.

Table 1 shows the baseline characteristics and comparisons between groups. Compared with females, males had lower EF (P = 0.004), higher EDV corrected for body surface area (BSA) (EDV/BSA; P = 0.0028), and ESV corrected for BSA (ESV/BSA; P = 0.0013), similar to previous studies² of NIDCM. In contrast, demographic characteristics, MLHFQ, and 6MWT were similar between the groups at baseline (Table 1).

3.2 Left ventricular function and structure

We examined the impact of patient sex on improvement of cardiac function. Cardiac CT or MRI was used to assess cardiac volumes and functionality. At baseline, the average EF was 26.5 ± 9.6%, EDV 296.7 mL (IQR 246.4–429.3), and ESV 233.7 mL (IQR 168.5–325.7). At 12 months post-TESI, both groups had substantial increases in EF from baseline: males by 6.2 EF units [95% confidence interval (CI) 0.3–12.0; P = 0.04], and females by 8.6 EF units (95% CI 0.3–16.9; P = 0.04), however, the absolute change from baseline to 12 months post-TESI was similar for males and females (P = 0.6, Figure 1A). Stroke volume (Figure 1B), EDV (Figure 1C), and ESV (Figure 1D) did not change from baseline in either group. The LV EDV index (EDV/BSA) and LV ESV index (ESV/BSA) also did not change in either group (Supplementary material online, Figure S1).

Figure 1.

Changes in left ventricular function and structure over 12 months. (A) Male and female patients demonstrate an increased ejection fraction from baseline to 12 months post-TESI (P = 0.04, n = 16 and P = 0.04, n = 8, respectively; t-test and Mann–Whitney test). (B–D) Neither group improved stroke volume, end-diastolic volume, or end-systolic volume.

Although the absolute change from baseline EF and cardiac volumes were not different between groups, LV function and structure parameters at 12 months were better in females compared with males. EF was 29.4% (95% CI 22.4–36.5) in males and 43.2% (95% CI 37.8–48.6) in females, P = 0.01 (Figure 2A). Overall, EF raised above 40% in 25.0% of males and 62.5% of females at 12 months post-TESI (P = 0.07). EDV index was 162.0 mL (IQR 130.8–209.5) in males and 123.7 mL (IQR 99.02–126.5) in females, P = 0.01 (Figure 2B); and ESV index was 118.9 mL (IQR 82.84–154.8) in males and 67.18 mL (IQR 52.69–79.20) in females, P = 0.006 (Figure 2C). Post-TESI, three male patients underwent heart transplant. Similarly, LVADs were implanted in two male patients, but no females underwent heart transplantation or LVAD.

Figure 2.

Left ventricular function and structure at 12 months. (A) EF was significantly higher in females (n = 8) compared with males (n = 16) at 12 months post-TESI (P = 0.01, t-test). (B) End-diastolic volume index and (C) end-systolic volume index were lower in females than males (P = 0.01, P = 0.006, respectively; Mann–Whitney test).

In order to increase the sample size, we combined two studies that recruited NIDCM patients, this POSEIDON DCM study¹⁴ and that of Heidecker et al.,² to characterize baseline cardiac function and structure and NYHA functional class. Interestingly, the combined data showed similar findings as our POSEIDON DCM study alone. LV ejection fraction (LVEF) was significantly higher in female (n = 44) compared with male (n = 23) NIDCM patients at presentation (females: 30.7 ± 14.3%, males: 20.0 ± 11.5%, P = 0.0098; Supplementary material online, Figure S2A). Moreover, end-diastolic internal diameter (LVIDD) and end-systolic internal diameter (LVIDS) were significantly smaller in females vs. males at baseline (P = 0.0001, P < 0.0001, respectively; Supplementary material online, Figure S2B and C). However, NYHA functional class was similar between the groups (P = 0.29, Supplementary material online, Figure S3).

3.3 Functional status and quality of life

Functional capacity was measured by the 6MWT and quality of life was assessed using the MLHFQ and NYHA functional class. The 6MWT at baseline was similar in both groups: males: 430 m (95% CI 391.1–470.7) and females: 401.5 m (95% CI 348.7–454.3, P = 0.4). The 6MWT increased in males by 29.9 m (95% CI −10.9 to 70.8; P = 0.2) and females by 33.1 m (95% CI −20.9 to 87.2; P = 0.3, Figure 3A) with no significant change at 12 months post-TESI relative to baseline. Moreover, there was no between-group difference (P = 0.9). The mean MLHFQ score at baseline was 37.9 (95% CI 27.3–48.4) in males and 42.6 (95% CI 27.6–57.6) in females. A repeated-measures model was used to evaluate MLHFQ at 6- and 12-month follow-up after MSC treatment. The MLHFQ total score improved over time in both groups: −14.3 (95% CI −28.7 to 0.2) in males, P = 0.05 and −29.9 (95% CI −50.3 to −9.5) in females, P = 0.003, with no difference between groups (P = 0.1, Figure 3B). At 12 months, 50% of male and female patients improved in NYHA functional class (between-group change in NYHA functional class P = 0.3, Figure 3C).

Figure 3.

Impact of human MSCs on functional status and quality of life over 12 months. (A) Changes in 6MWT from baseline to 12 months in males (P = 0.2, n = 14) and females (P = 0.3, n = 8), two-way ANOVA multiple comparison. (B) Minnesota Living with Heart Failure Questionnaire (MLHFQ) total score values in males and females at each time point are depicted, using two-way ANOVA multiple comparisons model. The total score improved at 12 months post-TESI in both groups (P = 0.05, n = 16 and P = 0.003, n = 8, respectively). (C) NYHA functional classification improved by 50% in both male (n = 18) and female (n = 8) patients after 1-year post-TESI (between group P = 0.25, χ² test).

3.4 Endothelial function

Endothelial function also improved from baseline in both sexes. Males and females had similar mean EPC-CFUs and FMD at baseline (P = 0.2 and P = 0.4, respectively). EPC-CFUs increased in males by 5.3 CFU (95% CI 1.9–8.8, P = 0.005) and females by 4.1 CFU (95% CI 0.1–8.1, P = 0.045) at 3 months (Figure 4A). While FMD improved by 1.9% (95% CI 0.2–3.6) in males (P = 0.03), there was no change in females at 3 months relative to baseline [1.2% (95% CI −1.0 to 3.4, P = 0.3, Figure 4B]. There was no sex difference associated with EPC-CFUs or FMD at 3 months post-TESI (P = 0.6 and P = 0.6, respectively).

Figure 4.

Endothelial function changes over 3 months. (A) The production of EPC-CFU substantially improved over 3 months post-TESI in both males and females (P = 0.005, n = 15 and P = 0.045, n = 8, respectively, t-test). (B) Flow-mediated vasodilation (FMD) improved in males only at 3 months post-TESI (P = 0.03, n = 15, t-test).

3.5 Inflammatory biomarkers

Serum TNF-α levels decreased at 6 months in both males: 7.8 pg/mL (95% CI 4.9–10.8 P < 0.0001) and females: 11.3 pg/mL (95% CI 6.6–16.0, P < 0.0001, Figure 5), with no difference between groups.

Figure 5.

Effects of human MSCs on serum TNF-α. Serum TNF-α decreased in pg/mL from baseline to 6 months in both groups (male: P < 0.0001, n = 20, female: P < 0.0001, n = 9; two-way ANOVA), with no difference between groups at 6 months post-TESI (P = 0.15, two-way ANOVA).

4. Discussion

The major new findings of this study are that women and men with NIDCM respond similarly to transendocardial injections of MSCs. Importantly, since women present with less LV remodelling than do men, women likely have a more substantial clinical recovery from NIDCM than do men (Figure 6). This finding has important clinical implications for the ongoing development of cell therapy and must be taken into consideration for ongoing clinical trial design.

Figure 6.

MSC therapy for male and female NIDCM patients. Representative examples of cardiac CT images from male and female patients with NIDCM before and after MSC therapy. As shown, female NIDCM patient has higher global ejection fraction and smaller left ventricular structure at baseline, thereby giving female patient a higher recovery rate at 12 months after TESI.

Patient sex plays a significant role in the pathophysiology and clinical manifestations of cardiovascular disease (CVD). Although women represent one-third of the HF population,²² their treatment outcomes have historically been subpar when compared with men with the same disease.⁹ The underrepresentation of women in clinical trials with regard to sex-specific treatments of HF may be an important factor contributing to this discrepancy.²³ Given the sex-based differences in CVD, studies need to evaluate the response of novel therapeutics in both men and women and compare the impact of patient sex on efficacy. While previous studies have examined whether donor sex influences the efficacy of cell-based therapies,^24,25 this study is the first to examine the relationship between recipient sex and MSC efficacy. We found that despite profound differences in the phenotype of LV remodelling at presentation, MSC treatment improves the NIDCM phenotype irrespective of patient sex. There were no differences between men and women in the improvement of cardiac functional parameters, quality of life, NYHA functional class, endothelial function, and serum TNF-α in response to MSC treatment.

The differences in clinical presentation in male and female NIDCM patients plays a major role in the present findings. Differences in sex chromosomes and hormones can lead to differences in gene expression and CV system function (myocardial remodelling under stress, vascular function, NO signalling, and metabolism of drugs by sex-specific cytochrome expression).²⁶ In our study and in agreement with previous investigations, female patients at baseline had higher EF and lower end-diastolic and ESVs, and shorter long-axis diameters.² Importantly, when the data from two separate studies were combined, it showed similar findings: EF was higher in female vs. male NIDCM patients at baseline. Moreover, LVIDD and LVIDS were smaller in female patients compared with male. Not only are there differences related to cardiac function, but Heidecker et al.² identified myocardial transcriptomic differences between the sexes in subjects with new-onset NIDCM. Similarly, Luchner et al.²⁷ demonstrated sex-specific differences in LV function with higher EF noted in women when compared with men with HF, supporting the hypothesis that women are better protected against systolic dysfunction and ventricular dilatation than men.²⁸ Importantly, MSC administration led to an increased rate of heart failure with recovered ejection fraction (HFrecEF) in females than males. This subset of patients with HFrecEF have a notably improved clinical course compared with patients with HF with reduced EF (HFrEF), with less frequent hospitalizations, LVAD, heart transplants, and lower all-cause mortality.^29,30 Improvement in LVEF has been associated with improved quality of life.³¹ The need for cardiac transplantation or mechanical circulatory support is lower in patients with HFrecEF when compared with HFrEF and HFpEF. In our study, only male patients underwent heart transplantation and LVAD post-TESI.²⁹

Given the discrepancy between males and females with regard to intrinsic MSC function,¹¹ this study provides evidence that recipient sex does not influence the therapeutic efficacy of MSCs for NIDCM. This novel finding is of particular importance because of the dissimilarities in CVD between men and women.^1–3 Men typically develop coronary artery disease and experience myocardial infarction 10 years earlier than females.³² NIDCM compared with ischaemic cardiomyopathy has a greater prevalence of females.³³ Interestingly, the severity of NYHA class has a stronger association with death in men, whereas older age is more strongly correlated with mortality in women with HF.³⁴ Prior to menopause, females have a much lower risk of developing CVD presumably secondary to the protective effects of endogenous oestrogen production.^7,9 The withdrawal of oestrogen predisposes women to develop HF because of the increased activity of the renin–angiotensin system along with the inhibition of nitric oxide and natriuretic peptides.²⁵ Furthermore, post-menopausal women have a higher risk of HF and mortality rate following acute ischaemic event than men.⁷ This discrepancy may be a result of sex-based differences in cardiac fibrous tissue composition, relaxation kinetics, calcium handling at the level of the sarcoplasmic reticulum and the preservation of ventricular contour.^6,7 Taken together, these factors may contribute to the differences seen in baseline characteristics between men and women with HF. In addition, studies have shown that stem cells, including MSCs, are affected by sex steroids, with oestrogen conferring a positive influence on their survival via inhibition of apoptosis, improvement of growth kinetics, and induction of telomerase activity.^25,35 Oestrogen also exerts positive effects on MSCs in particular by reducing production of pro-inflammatory TNF-α, while enhancing proliferation,³⁶ differentiation, and production of vascular endothelial growth factor and other paracrine signalling molecules.²⁵

There is little information on differences in the use of pharmacologic therapy between men and women with NIDCM. Most large multicentre trials of chronic HF have not included sufficient numbers of women to draw conclusions about safety and efficacy of pharmacological treatment, and sex-specific results were not reported in the original trials. It’s known that sex hormones play an important role in the regulation of β-receptors, thus different responses would be expected. However, pooled meta-analysis data from three clinical studies (MERIT-HF, COPERNICUS, and CIBIS II) suggest that β-blockers lead to a similar decrease in mortality in men and women.³⁷ Similarly, the available data suggest that there are no significant sex-specific differences in the therapeutic effect of angiotensin receptor blockers^38,39 and aldosterone antagonists^40–42 in patients with systolic HF. Interestingly, meta-analysis of ACE inhibitor therapy showed a greater decrease in mortality among men, possibly due to the relatively small number of women included in these trials. The patients included in the POSEIDON DMC study had to be treated with appropriate maximal medical therapy for HF. Taking together all of these factors, effects of MSC treatment do not appear to be biased by a medical therapy factor.

MSC therapy for cardiac diseases continues to expand with more clinical trials and it is vital to understand the factors that influence its efficacy.^43,44 Golpanian et al.⁴⁵ examined the effects of recipient age on MSC efficacy and found that MSC therapy in patients with ischaemic cardiomyopathy improves functional capacity, quality of life, and reduces myocardial infarction size similarly, regardless of age. In our study, cardiac functionality as measured by EF also improved significantly in NIDCM patients regardless of patient sex. MSC injection also produced consistent improvements in total MLHFQ score, an effective measure of quality of life in HF patients.⁴⁶ MLHFQ total score improved in both males and females at 12 months from baseline, independently of patient sex. 6MWT is widely applied to clinical and research settings for assessing functional status, prognosis, and the response to therapeutic interventions²⁷ in patients with HF⁴⁷ and is a prognostic marker of subsequent cardiac death and a predictor of hospitalization for cardiovascular reasons in patients with HF.⁴⁸ Our findings show that improvements in 6MWT in females and males did not reach significance (only ∼30 m increase in each group), likely due to the small sample size. Importantly, there was no difference between men and women in 6MWT changes. Similar improvements in EF, quality of life, and NYHA functional class strongly suggest that despite major differences in the phenotypic presentation of NIDCM in males and females, MSC therapy provides functional recovery irrespective of patient sex.

Endothelial dysfunction is an essential component of the pathophysiology of the failing heart in NIDCM patients.⁴⁹ In a landmark paper in 2003, Hill et al.⁵⁰ found that EPC colony number significantly correlated to both Framingham risk score and FMD in subjects with cardiovascular risk. Moreover, FMD also revealed a significant relation between endothelial function and the number of circulation EPCs.⁵⁰ Since then, multiple studies have not only confirmed these findings, but furthermore, have intimately linked diminished EPC-CFUs to impaired endothelial function in patients with CVD.^20,51–53 Moreover, a low level of circulating progenitor cells (CPCs) is an independent predictor of higher mortality.⁵¹ CPCs play important role in recovery and heart remodelling, participating in angiogenesis and vasculogenesis.⁵⁴ An underlying mechanism of MSC therapy is the stimulation of endogenous progenitor cells,^55,56 including EPCs.²⁰ Specifically, in our previous study, we found that both EPC-CFUs and FMD were dramatically reduced in patients with NIDCM compared with young healthy adults and that after allogeneic MSC treatment, these levels significantly improved.²⁰ In addition, we measured VEGF in patient serum and found that patients with NIDCM had severely elevated VEGF, purporting an important role of endothelial dysfunction in patients with heart disease. These levels were significantly reduced following allogeneic MSC administration. Together, these findings combined with the EPC-CFU and FMD findings further support the significant role endothelial function plays in heart disease, and, moreover, the role MSC therapy plays in ameliorating endothelial dysfunction.²⁰ This study demonstrated that MSC administration to male and female NIDCM patients stimulates EPC function but improves FMD towards normal in males only. These findings offer important clinical insights into the therapeutic bioactivity and effects of MSC for NIDCM patients and support the need for further studies in larger cohorts of patients.

4.1 Study limitations

The primary goal of this hypothesis-generating study was to compare the effects of MSCs in male and female NIDCM patients; however, the number of patients was relatively small, which limits the generalizability of these results. In addition, this study combined the donor source of the cell (autologous and allogeneic MSCs) in order to increase the power of the study by increasing the number of treated patients/group. However, despite this limitation, the number of patients treated with autologous or allogeneic MSCs in each group was similar. A lack of comparison with a placebo control in both groups makes it difficult to draw firm conclusions. However, despite these limitations, we believe that addressing sex differences in response to MSC therapy in NIDCM patients is crucial and enhances the understanding of how MSCs may modulate cardiac function and structure regardless of recipient’s sex. Finally, a more detailed analysis of NIDCM aetiology, genetic profile, and molecular assays, including hormonal levels is needed. We did not incorporate these assessments, as they were not part of the POSEIDON-DCM study design. Further placebo-controlled and double-blinded studies with larger sample sizes are needed to examine the effect of recipient sex on response to MSC therapy in a more diverse patient population.

5. Conclusions

Previous studies have demonstrated the safety and potential efficacy of transendocardial MSC administration for HF. Given the paucity of data generated by clinical trials with regard to patient sex differences in HF therapy, this study is the first to address sex-specific differences in the response to cell-based therapy in patients with NIDCM. In this study, women and men with NIDCM benefited from transendocardial injections of MSCs. Further analysis of the impact of patient sex and of sex hormones on the efficacy of cell-based therapeutics may provide insights into the mechanism(s) of action and the role(s) of the hormonal milieu in stem cell function. Future trials need to be designed with an adequate statistical power to obtain meaningful results on sex differences in response to cell-based therapy. Overall, our findings have important implications and support the enrolment of more women for examining patient sex as a modifier in larger randomized clinical trials of MSC efficacy in CVD.

Authors’ contributions

J.M.H. served as PI of POSEIDON-DCM study and oversaw its conduct. V.F. and J.M.H. conceived and designed the study. V.F., A.R., M.N., B.A.T., M.N.B., and C.P. acquired, analysed, and interpreted the data. R.D.M. did the intramyocardial injection of the cells. A.K. and K.V. manufactured and characterized the cells at the GMP facility. V.F. did primary statistical analyses. V.F. and J.M.H. wrote the initial draft. A.R., I.H.S., and W.B. reviewed and edited all versions of the manuscript. B.H. provided clinical data for additional analysis. All authors read and approved the final manuscript.

Conflict of interest: J.M.H. reported having a patent for cardiac cell-based therapy. He holds equity in Vestion Inc. and maintains a professional relationship with Vestion Inc. as a consultant and member of the Board of Directors and Scientific Advisory Board. J.M.H. is the Chief Scientific Officer, a compensated consultant and advisory board member for Longeveron and holds equity in Longeveron. J.M.H. is also the co-inventor of intellectual property licensed to Longeveron. A.K. discloses a relationship with Longeveron LLC that includes consulting. K.V. discloses a relationship with Vestion Inc. that includes equity. Longeveron LLC and Vestion Inc. did not participate in funding this work. All other authors have no conflict of interest to declare.

Funding

The POSEIDON-DCM study was funded by the National institutes of Health [R01 HL R0110737].

 

Translational perspective

Important patient sex differences exist in non-ischaemic dilated cardiomyopathy (NIDCM) clinical phenotype and gene expression profiles. The effectiveness of pharmacological treatment relative to patient sex requires evaluation due to the paucity of sex-specific data in clinical trials. Herein, we report that despite baseline phenotypic differences in left ventricular remodelling, mesenchymal stem cell (MSC) treatment improves the NIDCM phenotype irrespective of patient sex. Importantly, MSC administration to female patients may lead to an increased rate of heart failure with recovered ejection fraction, which carries an improved mortality, transplant rate, and hospitalization rate. This hypothesis-generating study encourages the design of future trials that evaluate patient sex differences in response to cell-based therapy.