Impact of a durable left ventricular assist device on inflammation and cardiac remodelling in subjects with advanced heart failure

Abstract

Background

This study aimed to explore the association of long-term left ventricular assist device (LVAD) support with changes in inflammation and cardiac remodelling in subjects with advanced heart failure (HF).

Methods

A single-centre prospective observational trial was conducted in 16 consecutive subjects with advanced HF who were indicated for implantation of HeartMate 3 LVAD. Blood samples were collected both during the procedure (V1) and 12 months postoperatively (V2) for comprehensive analysis. Additionally, in a subgroup of seven LVAD subjects listed for heart transplantation (HTx), a histological examination was performed on myocardial samples obtained during LVAD implantation and from the explanted heart following HTx.

Results

LVAD implantation was associated with reductions in brain natriuretic peptide (324.7±47.0 vs 124.6±50.3 pg/mL) and C reactive protein (15.2±3.8 vs 6.0±1.3 mg/L) levels. Decreased inflammation markers coincided with reduced levels of lymphocyte- and macrophage-derived cytokines and their stimulating factors. LVAD implantation was associated with modulation of circulating biomarkers related to negative cardiac remodelling processes, including reductions in fibrosis- and remodelling-associated mediators (Extracellular Matrix Metalloproteinase Inducer, fibroblast growth factor (FGF)-2, FGF-19, receptor for advanced glycation endproducts, suppression of tumourigenicity 2, interleukin 27) and angiogenesis-related factors (stromal cell-derived factor-1α, growth differentiation factor (GDF)-15, somatotropin, vascular endothelial growth factor, angiopoietin 1, hepatocyte growth factor). In parallel, markers of metabolism and systemic catabolic state were significantly modulated, with decreases in GDF-15, somatotropin, trefoil factor 3 and resistin and increases in leptin levels. Histological analysis of myocardial samples available from a subgroup of subjects showed reduced macrophage infiltration following LVAD implantation.

Conclusions

Long-term LVAD support was associated with improved biochemical and haematological profiles and with coordinated changes in circulating markers of inflammation, metabolism and cardiac remodelling. These exploratory findings provide further insight into the biological changes associated with durable mechanical unloading in subjects with advanced HF.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Advanced heart failure is associated with chronic subclinical inflammation, immune activation and progressive adverse cardiac remodelling, while durable left ventricular assist device (LVAD) support improves haemodynamics, survival and end-organ perfusion.

• However, the biological changes associated with mechanical unloading across molecular and remodelling pathways remain incompletely understood.

WHAT THIS STUDY ADDS

• This study combines longitudinal biochemical and proteomic profiling with paired myocardial histology to characterise biological changes associated with long-term LVAD support.

• We demonstrate that LVAD support was associated with coordinated changes in circulating inflammatory, fibrotic, angiogenic and extracellular matrix remodelling-related markers, accompanied by reduced myocardial macrophage infiltration and fibrosis.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Our findings provide a basis for future studies investigating prognostic biomarkers, personalised monitoring strategies and therapeutic approaches targeting residual inflammation and other biological pathways during long-term LVAD support.

Introduction

Heart failure (HF) is a complex clinical syndrome associated with substantial morbidity, mortality and healthcare burden worldwide.¹ Its development and progression are influenced by multiple demographic and cardiometabolic factors, including sex, age, obesity, diabetes and hypertension.² Many of these factors are associated with chronic subclinical inflammation and accelerated cellular senescence, both of which contribute to HF pathophysiology and disease progression.³

Subclinical inflammation is characterised by sustained immune cell activation and increased production of inflammatory cytokines, chemokines and growth factors. These cells infiltrate insulin-sensitive tissues, particularly adipose tissue, where they promote the secretion of pro-inflammatory cytokines, chemokines and growth factors. This inflammatory milieu activates resident immune cells and adipocytes, leading to dysregulated adipokine secretion, typically increased leptin and resistin levels and reduced adiponectin production. Impaired adipose tissue function can subsequently affect neighbouring tissues and organs,^{4 5} including the myocardium via epicardial adipose tissue.⁶ Together, chronic inflammation and adipose tissue dysfunction also contribute to the development of cachexia, a frequent and adverse complication in subjects with advanced HF.⁷

As HF progresses, the myocardium undergoes profound structural and cellular alterations, including cardiomyocyte necrosis, immune cell infiltration, angiogenesis, fibrosis and extracellular matrix remodelling.⁸ In parallel, these pathological changes are accompanied by metabolic disturbances, such as neurohumoral activation and shifts in myocardial substrate utilisation, which collectively drive adverse ventricular remodelling and further impair cardiac function. Declining cardiac performance triggers compensatory mechanisms, notably activation of the sympathetic nervous system and the renin–angiotensin–aldosterone system. These responses are initially adaptive but become maladaptive, promoting volume overload, higher peripheral vascular resistance, tachycardia and progressive cardiac decompensation, ultimately worsening clinical outcomes.⁹

Therapeutic options for subjects with advanced HF have expanded considerably over the past two decades. Heart transplantation (HTx) remains the gold standard for eligible subjects,¹⁰ but its availability is limited by the persistent shortage of donor organs. A durable left ventricular assist device (LVAD) has therefore become an established treatment option for patients with advanced HF, either as a bridge to transplantation or as a destination therapy. Durable LVADs provide adequate cardiac output and restore end-organ perfusion. Technological advances have markedly improved device durability and reduced complication rates, enabling prolonged support and resulting in improved survival and quality of life for subjects receiving LVAD therapy.^11–13 Despite these clinical benefits, the biological changes associated with long-term LVAD remain incompletely understood. In particular, how mechanical unloading and improved end-organ perfusion are reflected in circulating inflammatory, metabolic and cardiac remodelling-related factors, and how these alterations contribute to HF progression, recovery or long-term outcomes, remain to be fully elucidated.

The present study aimed to explore the biological changes associated with long-term LVAD support, focusing on subclinical inflammation and cardiac remodelling-related pathways. We hypothesised that durable LVAD support would be associated with coordinated changes in inflammatory mediators and biomarkers related to myocardial remodelling and maladaptive compensatory pathways. We further explored the relationship between systemic biomarker changes and myocardial histological findings in subjects with paired tissue samples. This integrated approach may improve understanding of the biological responses associated with durable mechanical unloading and provide a foundation for future studies investigating biomarkers and molecular pathways in patients with advanced HF.

Methods

Study subjects

This single-centre prospective observational trial included 16 consecutive subjects with advanced HF (stage D) undergoing HeartMate 3 (HM3) LVAD implantation. During the 5-year post-implantation follow-up (January 2020–January 2025), seven subjects (43.7%) underwent HTx from LVAD, according to standard institutional and national allocation criteria, primarily based on donor organ availability, overall clinical status, comorbidities and transplant eligibility. Four subjects (25%) died within the follow-up period. Detailed subject-level information regarding the histological subgroup, duration of LVAD support, and major clinical events is provided in online supplemental tables.

All participants signed written informed consent before enrolment into the study. The subjects were not compensated since the samples were taken during a planned surgery.¹⁴

Surgical methods

Myocardial samples were taken perioperatively after 6–12 hours of fasting for further analysis. All procedures were performed via median sternotomy, providing optimal access to the heart. The myocardial samples were retrieved from the left ventricular apex during HM3 LVAD implantation and subsequently from the explanted heart at the time of HTx. Freshly collected specimens in phosphate-buffered saline (PBS; 0.01 mol/L, pH 7.4; Sigma, St. Louis, MO, USA) were stored at −80 °C or fixed immediately after collection in 2% formaldehyde at room temperature for at least 24 hours and then used for histological analysis.

Concomitant procedures

The most frequent concomitant procedure was the left atrial appendage exclusion (in six subjects, 37.5%). The tricuspid valve repair was performed in four subjects (25%) with severe preoperative insufficiency and/or annulus size greater than or equal to 40 mm. In cases of aortic insufficiency (more than mild), the valve was coaptated in four subjects (25%) using Park’s stitch technique.¹⁵ A temporary right ventricular assist device (RVAD) was implanted in one subject (6.3%). In one subject (6.3%), a previously implanted mechanical aortic valve had to be replaced with a biological prosthesis.

The device used in this study, the HM3 LVAD (Abbott, Chicago, USA), is a centrifugal, fully magnetically levitated, continuous-flow blood pump designed to improve haemocompatibility and reduce shear stress on blood components.¹⁶

Biochemical analysis

Blood samples were taken before surgery, after 6–12 hours of fasting. Samples were centrifuged for 10 min at 1000×g within 30 min after withdrawal at room temperature. Plasma or serum aliquots were subsequently stored at −80 °C. Biochemical parameters (blood glucose, glycated haemoglobin, sodium, potassium, chloride, urea, creatinine, bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, C reactive protein (CRP), total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein cholesterol, non-HDL cholesterol, triglycerides) and blood count analyses were done at the Laboratory Methods Division, IKEM, Prague, Czech Republic.

Plasma protein detection

All protein detection was performed using a semiquantitative method with the Proteome Profiler Human XL Cytokine Array (Bio-Techne R&D Systems s.r.o., Minneapolis, USA) according to the manufacturer’s instructions. All samples were analysed in technical duplicates. Paired samples obtained from the same subject at V1 and V2 were exposed and imaged simultaneously in a single chemiluminescent detection run using a multi-membrane imaging system, Alliance Q9 Advanced (UVItec Ltd., Cambridge, UK), thereby minimising variability associated with signal acquisition and exposure conditions. Spot intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For each analyte, the local background signal was subtracted, and the resulting signal intensity was normalised to the internal positive control spots present on the same membrane to account for inter-membrane variability. Data are presented as normalised arbitrary units (sample peak area). To minimise technical variability, all samples were processed using the same assay lot and under identical experimental conditions. Signals that were indistinguishable from background after background correction were considered below the detection limit and were excluded from subsequent statistical analyses. Image acquisition and quantification were performed by investigators blinded to the sampling time point.

Histological analysis

Fixed tissue specimens were processed manually for paraffin embedding. Briefly, tissues were washed in running tap water to remove residual fixative and subsequently dehydrated through a graded series of ethanol solutions (70%, 80%, 96% and 100%), with each step performed for 60–90 min depending on tissue size. Complete dehydration was ensured by two changes of absolute ethanol.

After dehydration, tissues were cleared in xylene (two changes, 45–60 min each) until the specimens became translucent, indicating effective replacement of ethanol. The cleared tissues were then infiltrated with molten paraffin wax (melting point 56 °C–58 °C) in a paraffin oven. Paraffin infiltration was performed in three consecutive paraffin baths, each lasting 60 min, to ensure complete penetration of the tissue.

Following infiltration, specimens were embedded in fresh paraffin using metal embedding moulds and allowed to solidify at room temperature. Paraffin blocks were subsequently cooled to enhance hardness and stored at room temperature until sectioning. Serial sections of 3–5 µm thickness were cut using a rotary microtome, mounted onto glass slides and dried overnight at 37 °C.

For CD68+ cells assessment, paraffin sections (3–5 µm) were deparaffinised in xylene (two changes, 10 min each) and rehydrated through a graded ethanol series (100%, 96%, 80% and 70%) to distilled water. Antigen retrieval was performed by heat-induced epitope retrieval in citrate buffer (10 mM, pH 6.0) using a water bath or microwave oven for 20 min, followed by gradual cooling to room temperature. To reduce nonspecific antibody binding, sections were incubated in a blocking solution containing 5% bovine serum albumin in PBS for 30 min at room temperature. Sections were then incubated with a primary monoclonal anti-CD68 antibody (clone KP1, cat M0814, Dako Agilent, Glostrup, Denmark) overnight at 4 °C in a humidified chamber. After washing in PBS, sections were incubated with an appropriate biotinylated secondary antibody for 30 min at room temperature, followed by incubation with streptavidin–horseradish peroxidase complex according to the manufacturer’s instructions. Sections were counterstained with Mayer’s haematoxylin, dehydrated through graded ethanol, cleared in xylene and mounted with a permanent mounting medium.

For Picrosirius Red staining, paraffin sections were deparaffinised, covered with resorcin-fuchsin for 30 min, then nuclei were stained with Wiegert’s haematoxylin for 10 min, slides were coloured with Picro-Sirius Red solution and incubated for 15 min, dehydrated through graded ethanol, cleared in xylene and mounted with a permanent mounting medium.

For the Gomori’s blue trichrome, paraffin sections were deparaffinised, placed into Blouin’s fluid for 25 min at 56 °C, then cooled in running tap water until yellow colour was completely removed, nuclei were stained by Wiegert’s haematoxylin for 10 min, and slides were placed in the Gomori’s Trichrome Stain (blue) for 12 min, quickly rinsed in distilled water, dehydrated through graded ethanol, cleared in xylene and mounted with a permanent mounting medium.

Accumulation of collagen and fibrotic tissue was assessed by a blinded observer in randomly selected fields (n=3, magnification 100×) per section, and the measurements were averaged for each sample.

Statistical analysis

Statistical analysis was performed using SigmaPlot V.13.0 (SPSS), and graphs were created using GraphPad Prism V.10.2.3 (GraphPad Software, Boston, MA, USA). Given the small sample size, the results are expressed as median (IQR). The normality of all data was assessed by the Shapiro-Wilk test. A paired t-test or a Wilcoxon signed-rank test was used to assess differences between visits, as appropriate. The Spearman correlation analysis (due to non-parametric data) was used to assess the association between proteomic data and quantification of fibrosis, collagen and macrophage percentage in myocardium. The statistical significance of all tests was assigned to p≤0.05. The statistical significance of Proteome Profiler Human XL Cytokine Array results was adjusted using the Benjamini-Hochberg procedure, with statistical significance defined as a corrected p≤0.05 and a false discovery rate 25%. Both p values are present in table 3, while figures 1 and 2 contain graphs with significance at p≤0.05, along with the calculated corrected p value.

Figure 1. Dynamic regulation of inflammatory cytokines and macrophage infiltration in advanced heart failure. Soluble levels of (A) adipokines (leptin, resistin) and growth differentiation factor-15 (GDF-15) and (B) inflammatory proteins, including B-cell activating factor (BAFF), cluster of differentiation 30 (CD30), granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNγ), interleukin (IL)-2, IL-3, IL-4, IL-10, IL-17A and tumour necrosis factor alpha (TNFα) were measured during (V1) and 12 m (V2) after LVAD implantation by Proteome Profiler Human XL Cytokine Array. (C) Representative histological images and statistical analysis of myocardial tissue obtained at the time of LVAD implantation and from explanted hearts, immunohistochemically stained for CD68 to visualise macrophage infiltration. Data are expressed as mean±SEM. ^xp<0.05 V1 vs V2; paired t-test or Wilcoxon signed-rank test. Calculated corrected p value are present.

Figure 2. Longitudinal changes in angiogenic and remodelling proteins. Soluble levels of (A) angiogenic and growth-related factors, including angiopoietin-1, hepatocyte growth factor (HGF), stromal cell-derived factor 1α (SDF-1α), somatotropin, vascular endothelial growth factor (VEGF) and trefoil factor 3 (TFF3) and (B) remodelling-associated mediators, including emmprin, fibroblast growth factors (FGF-2 and FGF-19), interleukin 27 (IL-27), pentraxin-3, receptor for advanced glycation end products (RAGE), suppression of tumourigenicity 2 (ST2) and urokinase plasminogen activator receptor (uPAR) were measured during (V1) and 12 m (V2) after LVAD implantation by Proteome Profiler Human XL Cytokine Array. Data are expressed as mean±SEM. ^xp<0.05 V1 vs V2; paired t-test or Wilcoxon signed-rank test. Calculated corrected p value are present. AU, arbitrary units.

Results

Baseline characteristics of study subjects

16 male subjects with advanced HF stage D (ejection fraction (EF) ≤30%, New York Heart Association (NYHA) III–IV) undergoing LVAD implantation were examined at the time of procedure and 12 months post-implantation. Among the subjects, 12.5% were categorised as Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profile 2, 50% as INTERMACS profile 3 and 37.5% as INTERMACS profile 4. The mean baseline left ventricular ejection fraction (LVEF) was approximately 20%. Common comorbidities included type 2 diabetes mellitus in 7 subjects, atrial fibrillation in 11 subjects, chronic kidney disease and pulmonary hypertension in 10 subjects each. The average age at implantation was 59 years (table 1).

Table 1. Baseline characteristics of study subjects.

Demographics
Age (years)	59.31±3.48
Gender male/female	16/0
Type 2 diabetes, n (%)	7 (43.7)
Atrial fibrillation, n (%)	11 (68.8)
Chronic kidney disease, n (%)	10 (62.50)
Pulmonary hypertension, n (%)	10 (62.50)
Temporary RVAD implantation, n (%)	1
Ischaemic cardiomyopathy, n (%)	3 (18.75)
Other cardiomyopathy, n (%)	3 (18.75)
Transplanted, n (%)	7 (43.8)
NYHA class, n (%)
III	10 (62.50)
IV	6 (37.50)
INTERMACS profiles, n (%)
2	2 (12.50)
3	8 (50.00)
4	6 (37.50)
Haemodynamics
Left ventricular ejection fraction (%)	20.39±1.32
Left ventricular end-diastolic diameter (mm)	75.06±2.23
Mean pulmonary artery pressure (mm Hg)	37.07±2.01
Pulmonary vascular resistance (Woods Units)	3.33±0.36
Mean capillary wedge pressure (mm Hg)	25.33±2.17
Cardiac index (L/m2/min)	1.74±0.11

INTERMACS, Interagency Registry for Mechanically Assisted Circulatory Support; NYHA, New York Heart Association; RVAD, right ventricular assist device .

HTx was performed according to standard institutional and national allocation criteria, considering transplant eligibility, donor availability and clinical status. Subjects receiving LVAD as destination therapy were not considered transplant candidates. One subject underwent LVAD decommissioning following myocardial recovery (online supplemental table 1). Major clinical events and interventions during LVAD support and after HTx are summarised in online supplemental table 2.

Baseline and 12-month follow-up medication data are summarised in online supplemental table 3. As the cohort was enrolled before the publication of the ARIES-HM3 trial results and widespread adoption of aspirin withdrawal strategies in LVAD HM3 recipients, most subjects received standard antithrombotic therapy consisting of warfarin and aspirin. Medication data are presented descriptively, as the observational design and limited sample size precluded evaluation of the independent effects of individual medications on the measured biomarkers, and concomitant treatment may have contributed to the observed changes.

Effects of LVAD implantation on metabolic profile and inflammation

LVAD implantation was associated with significant improvements in both biochemical and haematological parameters (table 2). After 12 months of LVAD support, circulating levels of brain natriuretic peptide (BNP), a well-established marker of HF severity, were markedly reduced, indicating improved cardiac status and the effectiveness of mechanical unloading.¹⁷ In parallel, increases in body mass index (BMI), prealbumin and albumin levels were observed, suggesting improved nutritional status and a reduced risk of cachexia in the studied cohort.¹⁸ An increase in serum calcium levels was also noted following LVAD implantation, and haematological analysis revealed a significant reduction in total leucocyte counts accompanied by increases in erythrocyte count, haemoglobin concentration and haematocrit values (table 2). The observed decrease in CRP levels (table 2) may reflect reduced inflammatory response.

Table 2. The effect of LVAD implantation on biochemical parameters.

	Before LVAD	12 months after LVAD	P value
Number (n; males/females)	16 (16/0)	16 (16/0)
BNP (pg/mL)	299.5 (161.0; 498.0)	65.8 (32.2; 125.3)*	0.022
Hs troponin T (ng/mL)	58.33 (34.39; 143.8)	–
BMI (kg/m2)	26.55 (24.20; 29.33)	28.60 (25.90; 32.40)*	0.003
Glycaemia (mmol/L)	5.25 (4.45; 6.96)	5.27 (4.96; 6.36)	0.900
HbA1c (mmol/mol)	44.50 (41.25; 51.75)	41.00 (39.50; 59.50)	0.588
Sodium (mmol/L)	136.1 (131.4; 137.6)	137.9 (137.0; 141.8)	0.019
Potassium (mmol/L)	4.365 (4.015; 4.478)	4.345 (3.957; 4.698)	0.520
Chloride (mmol/L)	99.95 (96.00; 104.8)	104.4 (101.5; 106.9)*	0.022
Calcium (mmol/L)	2.12 (1.962; 2.217)	2.43 (2.23; 2.49)*	0.002
Iron (µmol/L)	12.6 (6.5; 22.4)	11.4 (8.9; 13.9)	0.352
Phosphorus (mmol/L)	1.230 (1.043; 1.402)	–
Copper (µmol/L)	20.54 (16.10; 21.6)	–
Creatinine (µmol/L)	118.3 (103.0; 130.6)	105.9 (90.7; 132.5)	0.202
Urea (mmol/L)	12.0 (6.6; 14.6)	8.2 (6.3; 11.3)*	0.025
Uric acid (µmol/L)	397.5 (281.8; 582.8)	394.5 (348.8; 419.8)	0.122
Prealbumin (g/L)	0.17 (0.12; 0.21)	0.23 (0.19; 0.30)*	0.010
Albumin (g/L)	32.7 (29.8; 36.6)	43.6 (40.1; 45.4)*	<0.001
Total protein (g/L)	60.8 (54.5; 63.4)	–
Total bilirubin (µmol/L)	20.1 (16.5; 34.8)	11.9 (10.7; 27.8)	0.194
Ceruloplasmin (g/L)	0.35 (0.31; 0.44)	–
Transferrin (g/L)	2.41 (1.86; 2.84)	–
Transferrin saturated (g/L)	17.4 (11.4; 47.7)	–
Ferritin (µg/L)	157.5 (103.3; 486.8)	–
Total iron-binding capacity (µmol/L)	60.5 (47.0; 71.3)	–
ALT (µkat/L)	0.740 (0.323; 1.275)	0.525 (0.415; 0.645)	0.561
AST (µkat/L)	0.525 (0.352; 1.320)	0.450 (0.340; 0.548)	0.632
ALP (µkat/L)	1.610 (1.385; 1.930)	1.635 (1.265; 1.910)	0.687
GGT (µkat/L)	1.830 (1.077; 2.760)	0.815 (0.542; 1.218)*	0.044
C reactive protein (mg/L)	9.9 (3.5; 24.8)	5.0 (3.1; 6.2)*	0.018
Total cholesterol (mmol/L)	3.2 (2.6; 3.8)	4.0 (2.9; 4.8)	0.164
HDL cholesterol (mmol/L)	0.73 (0.59; 0.91)	0.95 (0.84; 1.20)	0.204
LDL cholesterol (mmol/L)	1.80 (1.15; 2.55)	2.40 (1.28; 3.05)	0.216
Triglycerides (mmol/L)	1.12 (0.78; 1.24)	1.52 (0.59; 2.19)	0.213
Apolipoprotein B (g/L)	0.76 (0.48; 1.00)	–
Apolipoprotein A1 (g/L)	0.84 (0.69; 0.97)	–
TSH (mIU/L)	2.105 (1.583; 4.670)	–
T3 free (pmol/L)	2.650 (2.313; 3.510)	–
T4 free (pmol/l)	16.140 (12.973; 17.887)	–
Cortisol (nmol/L)	372.0 (328.8; 457.3)	–
Leucocytes (109/L)	6.30 (12.85; 8.28)	4.30 (7.60; 5.13)*	0.001
Neutrophils (109/L)	3.39 (5.89; 4.47)	2.91 (5.12; 3.34)	0.117
Lymphocytes (109/L)	0.8 (1.385; 0.963)	0.3 (1.38; 0.787)	0.724
Monocytes (109/L)	0.42 (0.76; 0.535)	0.21 (0.575; 0.455)	0.138
Eosinophils (109/L)	0 (0.15; 0.0825)	0.08 (0.16; 0.103)	0.825
Basophils (109/L)	0.02 (0.055; 0.03)	0.01 (0.05; 0.04)	0.677
Erythrocytes (1012/L)	2.39 (3.89; 3.422)	2.92 (4.625; 4.155)*	0.002
Granulocytes immature (109/L)	0.01 (0.04; 0.02)	0.01 (0.035; 0.02)	0.064
Neutrophils (%)	56.6 (73.3; 63.7)	50.0 (67.2; 63.2)	0.190
Lymphocytes (%)	9.6 (13.85; 12.725)	2.4 (18.5; 16.4)	0.127
Monocytes (%)	4.4 (8.55; 7.025)	1.7 (8.8; 7.075)	0.751
Eosinophils (%)	0 (1.7; 0.9)	0.7 (2.3; 1.55)	0.431
Basophils (%)	0.2 (0.65; 0.3)	0.2 (0.75; 0.525)*	0.044
Granulocytes immature (%)	0.2 (0.55; 0.325)	0.2 (0.4; 0.225)	0.093
Haemoglobin (g/L)	79 (116; 98)	84 (135; 118.5)*	<0.001
Haematocrit	0.217 (0.353; 0.295)	0.268 (0.402; 0.358)*	0.002
Mean corpuscular volume (fL)	74 (90.85; 84.725)	75.3 (89.7; 84.05)	0.822
Mean cell haemoglobin (pg)	23.4 (30; 27.975)	23.6 (30.05; 27.625)	0.836
Mean corpuscular haemoglobin concentration (g/L)	307 (329.5; 323.5)	313 (333; 326.25)	0.991
Red cell distribution width (%)	13.2 (16.25; 15.4)	13.4 (14.55; 13.7)*	0.010
Thrombocytes (109/L)	71 (195; 142.5)	73 (208.5; 176.5)	0.599
Thrombocyte volume (fL)	8.9 (10.45; 9.9)	9.4 (10.95; 10.05)	0.105
Thrombocrit (mL/L)	0.9 (2.05; 1.5)	0.7 (2.35; 1.8)	0.323
Thrombocytes >12 fL (%)	16.5 (29; 23.7)	19.8 (32.9; 25.925)*	0.041

Data are expressed as median (IQR).

^*p≤0.05 V1 vs V2; paired t-test or Wilcoxon signed-rank test.

ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BNP, brain natriuretic peptide; GGT, γ-glutamyl transferase; HbA1c, glycated haemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

At the molecular level, LVAD implantation influenced adipokine and cytokine profiles. Circulating leptin levels significantly increased following LVAD support, likely reflecting the observed rise in BMI and improvement in nutritional status, whereas resistin levels were reduced (figure 1A). LVAD implantation was associated with a reduction in circulating growth differentiation factor (GDF)-15 levels, an emerging prognostic marker of HF morbidity and mortality that reflects cellular stress, inflammation and adverse myocardial remodelling, but also an anorexigenic marker implicated in cardiac cachexia (figure 1A). LVAD support was further associated with decreased levels of several lymphocyte-derived (interferon gamma (IFN-γ), interleukin (IL) 2, IL-3 and IL-17A) pro-inflammatory and macrophage-derived (IL-4 and IL-10) anti-inflammatory cytokines. A strong trend towards reduced tumour necrosis factor alpha (TNF-α) levels was also observed (p=0.057, p cor = 0.068), together with a significant decrease in CD30 (figure 1B). In contrast, the levels of several other inflammatory mediators, including IL-1, IL-6, IL-8, selected chemokines (eg, intercellular adhesion molecule (ICAM)-1, VCAM-1) and adiponectin, remained unchanged following LVAD implantation (table 3), which may mirror the absence of visible histological signs of severe inflammation in the myocardial samples taken before LVAD implantation as well as during HTx (online supplemental table 3). In addition, LVAD support was associated with reduced circulating levels of B-cell activating factor (BAFF), a key regulator of B lymphocyte survival and proliferation, together with granulocyte colony-stimulating factor (G-CSF) and granulocyte–macrophage colony-stimulating factor (GM-CSF), both of which promote neutrophil and macrophage differentiation and expansion (figure 1B). Except of G-CSF, IL-3, IL-4, and TNF-α, all factors were significant even after correction for multiple testing.

Table 3. The effect of LVAD implantation on selected parameters.

	Before LVAD	12 months after LVAD	P value	Corrected p value
Metabolism
Adiponectin	3923.3 (3132.9; 4426.7)	3549.9 (3219.1; 4178.7)	0.828	0.233
ApoA1	2545.5 (2363.5; 4126.0)	2769.3 (2062.6; 3603.4)	0.199	0.124
DPP4	2290.6 (1771.5; 3786.1)	2575.4 (1554.1; 3440.8)	0.573	0.192
NGAL	4246.2 (3264.4; 5796.2)	3914.5 (3094.1; 4822.7)	0.404	0.167
RBP-4	2614.0 (2286.6; 3211.3)	2726.5 (2236.8; 2937.3)	0.366	0.158
SHBG	2700.6 (2037.6; 3549.4)	2585.2 (2002.5; 3142.4)	0.948	0.250
Cystatin C	2553.6 (1882.6; 2802.6)	2049.0 (1468.2; 2813.2)	0.167	0.112
Vitamin D BP	3165.1 (2397.6; 3828.5)	3432.6 (2569.1; 3893.5)	0.644	0.204
TfR	1944.0 (1691.4; 2413.3)	1702.3 (1297.1; 2062.2)	0.120	0.092
Angiogenesis
Angiogenin	2732.5 (2266.6; 3853.1)	3121.5 (2205.4; 3884.4)	0.709	0.218
Angiopoetin 2	1606.5 (1114.5; 2108.5)	1390.1 (577.2; 2344.1)	0.279	0.148
ENA-78	1371.2 (832.2; 1628.2)	1540.5 (964.4; 2509.1)	0.426	0.175
Endoglin	4804.7 (3919.5; 5443.7)	4417.0 (3877.9; 4836.5)	0.087	0.083
KLK3	263.2 (202.8; 403.5)	170.7 (120.1; 302.3)	0.252	0.143
ECM remodelling and regeneration
FGF7	732.7 (391.1; 1525.9)	684.9 (266.4; 1102.3)	0.096	0.087
GROa	1002.7 (474.8; 1448.3)	593.4 (402.6; 1076.2)	0.129	0.095
IGFBP2	3408.0 (2607.1; 5574.4)	3514.8 (2314.6; 5260.6)	0.420	0.172
IGFBP3	3194.3 (2314.8; 4395.4)	3630.9 (2093.2; 5460.7)	0.665	0.211
IL-11	809.7 (198.3; 1640.0)	614.7 (241.3; 1240.2)	0.117	0.090
IL-22	1468.0 (1144.9; 1784.4)	1130.3 (711.3; 1777.0)	0.190	0.119
MMP9	3424.8 (2608.6; 4561.9)	3053.3 (2691.5; 3748.8)	0.245	0.141
Serpin E1	2788.1 (2326.0; 3527.8)	2783.8 (2090.0; 3108.9)	0.691	0.216
TGFa	685.9 (445.7; 1058.3)	451.3 (392.9; 710.9)	0.159	0.109
Osteopontin	3245.3 (2630.7; 4519.3)	3087.2 (2769.1; 4072.7)	0.802	0.231
Thrombospondin-1	3696.2 (2871.3; 4631.4)	3059.2 (2662.5; 3959.4)	0.277	0.146
Cardiovascular homeostasis
BDNF	2262.9 (1957.0; 2958.5)	1974.1 (1339.3; 3265.3)	0.930	0.245
DKK-1	2498.6 (1758.4; 3301.0)	2077.9 (1650.1; 2681.0)	0.138	0.100
LIF	505.0 (341.4; 669.0)	417.4 (301.9; 647.6)	0.462	0.182
PF4	3326.3 (2795.9; 4201.9)	3199.5 (2927.8; 3717.6)	0.471	0.184
Relaxin 2	1283.2 (1079.8; 2287.6)	1082.9 (868.9; 1263.9)	0.138	0.100
Growth factors
EGF	1187.2 (622.4; 2453.1)	1347.4 (671.0; 2155.2)	0.788	0.228
M-CSF	646.2 (528.5; 833.7)	543.0 (363.3; 856.4)	0.562	0.189
PDGF-AA	3885.4 (2320.2; 4416.7)	2975.4 (1972.4; 4012.1)	0.240	0.138
PDGF-AB/BB	4163.5 (2515.4; 4985.2)	3103.5 (2375.4; 4623.0)	0.072	0.078
Immune reaction
Chemotaxis
CD31	3634.0 (3000.3; 4038.0)	3662.1 (2789.7; 4492.4)	0.765	0.226
ICAM-1	2293.2 (1554.6; 3294.1)	2135.8 (1493.7; 2561.3)	0.227	0.131
VCAM-1	3643.8 (3001.9; 4332.8)	3523.3 (3284.3; 3991.9)	0.754	0.223
IL-8	1765.2 (1317.2; 2003.6)	119.7 (1002.0; 1322.7)	0.151	0.104
MCP-1	1463.7 (1091.3; 2367.5)	1454.8 (943.7; 1973.4)	0.584	0.194
MCP-3	900.0 (621.8; 1102.1)	635.7 (487.3; 1094.3)	0.186	0.117
MIF	1108.0 (653.4; 1489.1)	1080.1 (765.5; 1342.2)	0.661	0.209
MIG	529.5 (367.4; 950.7)	616.0 (383.9; 872.5)	0.940	0.248
MIP1a/b	400.1 (274.8; 767.7)	417.3 (259.7; 606.6)	0.433	0.177
MIP13a	551.1 (402.2; 1128.3)	610.0 (355.4; 904.9)	0.611	0.199
MIP13b	1498.5 (870.1; 1999.3)	1347.8 (955.1; 2106.5)	0.917	0.240
RANTES	3093.4 (2378.8; 3598.1)	2612.6 (2354.6; 2944.0)	0.218	0.126
TARC	271.8 (151.9; 1125.4)	265.0 (104.2; 532.3)	0.323	0.150
Pro-inflammatory factors
C5/C5a	2765.6 (2282.6; 4450.8)	2974.7 (2239.7; 3673.9)	0.374	0.160
Complement factor D	2588.6 (1699.0; 2748.7)	2120.5 (1659.5; 2489.0)	0.072	0.078
CD14	1956.6 (1728.2; 2625.7)	1804.5 (1317.4; 2151.9)	0.090	0.085
CD40 ligand	1421.6 (706.7; 2746.0)	1437.6 (448.2; 2518.9)	0.237	0.136
FTL-3 ligand	1603.2 (860.8; 2446.3)	1454.3 (941.5; 1776.5)	0.228	0.133
IL-1a	2291.7 (1817.3; 2917.0)	2143.3 (1604.2; 2991.2)	0.477	0.187
IL-1b	1086.2 (713.3; 1592.3)	1046.2 (573.5; 1404.8)	0.389	0.165
IL-6	1021.2 (670.4; 1270.0)	744.4 (614.6; 1125.6)	0.065	0.075
IL12p70	1319.3 (935.9; 2042.7)	1470.7 (975.3; 1967.1)	0.656	0.206
IL-15	1418.1 (608.6; 1736.0)	1003.3 (711.8; 1474.4)	0.455	0.180
IL-16	779.2 (537.0; 1336.1)	724.5 (481.3; 974.4)	0.067	0.073
IL-23	189.2 (120.3; 331.7)	194.1 (104.5; 291.3)	0.349	0.153
IL-31	1393.2 (862.4; 1662.8)	1184.3 (890.0; 1554.5)	0.377	0.163
IL-32	850.1 (545.1; 1072.9)	680.7 (582.4; 1205.2)	0.405	0.170
IL-34	1026.6 (638.6; 1687.2)	1134.0 (671.6; 1735.5)	0.862	0.238
IP-10	1365.8 (723.0; 2443.3)	1369.8 (1014.0; 1560.0)	0.744	0.221
I-TAC	536.2 (404.1; 692.2)	408.4 (218.2; 509.1)	0.140	0.102
Myeloperoxidase	1332.9 (695.8; 2985.3)	1125.5 (640.5; 2727.0)	0.619	0.201
TIM-3	3019.7 (2487.8; 3523.4)	2337.1 (1939.0; 3908.1)	0.193	0.121
Anti-inflammatory factors
IL-1ra	1161.1 (573.0; 2112.0)	770.9 (483.8; 1318.4)	0.223	0.129
IL-13	1178.5 (767.0; 1815.6)	1229.6 (373.5; 1497.3)	0.073	0.080
IL-18	3641.8 (2265.8; 5166.0)	3304.4 (2037.1; 4882.3)	0.358	0.155
IL-19	292.9 (203.5; 504.3)	244.0 (145.1; 462.5)	0.588	0.197
IL-24	517.7 (414.3; 798.2)	517.1 (309.1; 952.6)	0.670	0.214
Pro- and anti-inflammatory factors
Chitinase 3-like 1	2376.6 (1893.2; 3144.5)	2420.8 (1825.3; 3167.7)	0.922	0.243
IL-5	274.8 (153.2; 458.4)	237.6 (157.6; 331.1)	0.159	0.109
IL-33	744.7 (294.8; 1166.4)	748.5 (376.0; 920.5)	0.168	0.114
Apoptotic signal
FAS ligand	1072.1 (600.9; 1463.6)	1016.8 (603.7; 1371.5)	0.854	0.235

Data are expressed as median (IQR). All data are expressed as arbitrary unit (sample peak area).

^*p≤0.05 V1 vs V2; paired t-test or Wilcoxon signed-rank test. No parameter reaches critical value p≤0.05 or B-H corrected p≤0.05 value.

BDNF, brain-derived neutrophic factor; FGF7, fibroblast growth factor 7; ICAM, intercellular adhesion molecule; IL, interleukin; LVAD, left ventricular assist device; MCP-1, monocyte chemoattractant protein 1; MIP1a/b, macrophage inflammatory protein 1 alpha/beta; MMP9, matrix metalloproteinase 9; RANTES, regulated on activation, normal T cell expressed and secreted; VCAM, vascular cell adhesion molecule.

Importantly, analysis of myocardial tissue samples from a subgroup of seven subjects at the time of LVAD implantation and during HTx demonstrated a reduction in macrophage infiltration following LVAD support in most subjects (figure 1C). In contrast to some earlier reports,¹⁹ our histological findings suggest attenuation of chronic myocardial inflammation. This tissue-level observation aligns with the observed reductions in circulating inflammatory cytokines and further supports the concept of a dampened immune response following LVAD implantation.

LVAD implantation is associated with reduced circulating markers of angiogenesis and tissue remodelling

LVAD implantation was associated with a reduction in circulating factors involved in tissue regeneration, angiogenesis and vascular signalling. Specifically, levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) significantly decreased and their reduction was accompanied by statistically weaker decrease of stromal cell-derived factor 1α (SDF-1α), somatotropin, trefoil factor 3 (TFF-3) and angiopoietin 1 (figure 2A). These markers are commonly elevated in advanced HF and are linked to ongoing myocardial stress, remodelling and vascular adaptation.^{20 21} In addition, we observed significantly reduced circulating levels of established cardiovascular risk and remodelling markers, such as pentraxin 3, urokinase plasminogen activator receptor (uPAR) and soluble ST2 (figure 2B). Other several proteins related to extracellular matrix remodelling, inflammation and fibrosis, including receptor for advanced glycation end products (RAGE), basic fibroblast growth factor (FGF-2), extracellular matrix metalloproteinase inducer (EMMPRIN), IL-27 and fibroblast growth factor 19 (FGF-19) were decreased. Angiopoietin 1, TFF-3, FGF-19, and IL-27 did not reach statistical significance after correction for multiple testing (figure 2B).

Despite these systemic changes, histological analyses suggested that myocardial fibrosis was already established at the time of LVAD implantation and continued to progress until HTx in most patients. The majority of subjects exhibited mild interstitial fibrosis at implantation, which evolved to moderate or severe fibrosis at the time of transplantation, accompanied by myomalacia and moderate cardiomyocyte hypertrophy without evidence of active inflammation. Perivascular lipomatosis was observed in myocardial samples obtained at the time of transplantation (online supplemental table 4).

Quantitative assessment of collagen and fibrosis was consistent with the histological findings and demonstrated an increase in the collagen (2.042 (1.557; 4.339) vs 6.020 (4.927; 14.103), p=0.016) and a tendency to increase in the fibrotic area (1.438 (1.070; 1.680) vs 12.761 (5.523; 16.546), p=0.075) over time (online supplemental figure 1). However, interpretation of these results is complicated by a timing mismatch: myocardial tissue at transplantation was often collected significantly later than the blood, which was sampled at the 12-month time point; in some subjects, tissue samples were collected 29 or 34 months after LVAD implantation (online supplemental table 1), limiting direct comparability between tissue and circulating biomarkers.

Correlation analysis demonstrated positive associations between myocardial CD68-positive macrophage infiltration and circulating levels of ST2, uPAR, HGF, GDF-15, RAGE, resistin, IL-17A and TFF3 (online supplemental table 5), whereas the extent of myocardial collagen deposition and fibrosis was not associated with any of the analysed parameters.

Discussion

Durable LVADs are designed to restore cardiac output and ensure adequate end-organ perfusion in subjects with advanced HF. Accordingly, LVAD implantation is used across a broad clinical spectrum, including as a bridge to candidacy, bridge to decision, bridge to recovery, bridge to HTx or as destination therapy in selected subjects with advanced HF,¹² as was also the case in our study. While the primary purpose of LVADs is haemodynamic support, accumulating evidence suggests that mechanical unloading of the failing left ventricle is associated with pleiotropic biological effects. These include modulation of inflammatory pathways, attenuation of cellular senescence and partial reversal of adverse cardiac remodelling.²² The purpose of this study was to evaluate changes in inflammatory mediators and markers associated with myocardial remodelling and maladaptive compensatory pathways following LVAD implantation to better understand its associated molecular effects. Our results confirmed that changes in leptin, GDF-15, CD30, HGF, VEGF, pentraxin 3, ST2 and uPAR remained significant after correction for multiple testing, supporting the robustness of these observations. These biomarkers may therefore represent promising candidates for further investigation as markers of biological responses to long-term LVAD support in subjects with advanced HF.

LVAD implantation was associated with improvements in several biochemical and haematological parameters. Specifically, it was accompanied by an increase in calcium levels²³ and an improvement in haematological status.²⁴ Furthermore, the reduction in BNP levels was accompanied by decreases in pentraxin 3, uPAR and ST2. Pentraxin 3 is a marker of immune activation, vascular endothelial dysfunction and angiogenesis, and has been associated with left ventricular diastolic dysfunction and adverse outcomes in HF.^{25 26} Similarly, uPAR is linked to immune activation, fibrosis and apoptosis.²⁷ Importantly, reductions in Pentraxin-3, uPAR and ST2 remained significant after correction for multiple testing, supporting the robustness of these findings. Their observed reduction is consistent with modulation of pathways associated with inflammation, fibrosis and adverse remodelling in HF during long-term LVAD support. In addition, LVAD implantation was associated with reduced levels of GDF-15, a member of the transforming growth factor-β family, which is abundantly secreted in response to cellular stress, hypoxia, inflammation and oxidative stress, and has been associated with fibrosis, hypertrophy, endothelial dysfunction and adverse outcomes in HF.^{28 29} Besides, it plays a role in regulating appetite and energy balance and has been linked to the development of cardiac cachexia. Thus, elevated GDF-15 links myocardial stress to systemic wasting. Consistent with this, Monzo et al³⁰ showed that higher GDF-15 levels were associated with more severe anorexia, greater weight loss, higher cachexia prevalence, worse congestion and RV dysfunction and independently predicted adverse outcomes. The observed reduction in circulating GDF-15, therefore, likely reflects changes in biological pathways associated with cellular stress and metabolic dysregulation during long-term LVAD support, although the present study was not designed to assess prognostic implications.

Despite several adverse events during the 5-year follow-up, CRP levels were lower at 12 months after LVAD implantation than at baseline, which may reflect a combination of factors, including mechanical unloading, improved end-organ perfusion, clinical stabilisation and reduced neurohumoral activation, all of which may lessen the stimuli sustaining chronic immune activation.³¹ However, in the absence of a comparator group, the relative contribution of these mechanisms cannot be determined. The reduction in inflammatory burden may be related to changes in immune cell availability and activity rather than altered chemokine signalling. This interpretation is supported by the observed decreases in leucocyte counts and growth factor levels involved in immune cell proliferation and differentiation. Although changes in molecules associated with blood and immune cell growth factors did not reach significance after correction for multiple testing in all cases, the observed decline in IL-2, GM-CSF and G-CSF, factors involved in T-cell proliferation and regulation of granulocyte and macrophage differentiation and expansion, may suggest reduced immune cell turnover and be associated with mechanical unloading. These findings are further supported by the reduction in myocardial CD68-positive macrophage infiltration observed in the subgroup of subjects. These cytokines not only promote leucocyte proliferation but also facilitate neovascularisation and limit myocardial injury after ischaemia. The observed decline may therefore reflect reduced activation of inflammatory pathways accompanying advanced HF rather than direct suppression of specific immune responses.³² Additionally, GM-CSF and IL-3 are key mediators of emergency myelopoiesis. By driving myeloid cell proliferation and infiltration in cardiac tissue, their decline may indicate reduced inflammatory activation. Cardiac fibroblasts produce GM-CSF, activating resident macrophages through a process regulated by IL-17A, which itself also decreases after LVAD implantation. This interpretation is supported by the observed positive correlation between myocardial macrophage infiltration and circulating IL-17A levels. Consequently, GM-CSF may further amplify this inflammatory circuit indirectly via dendritic cells.³³ Similarly, reductions in the pro-inflammatory cytokines IFN-γ, IL-3, IL-2 and IL-17A are consistent with reduced activation of Th1 and Th17 cells, T cytotoxic lymphocytes and NK cells, all of which may contribute to inflammatory signalling in HF.⁴ Nevertheless, the reduction in adaptive immune activation is also consistent with decreased CD30 levels, a lymphocyte surface receptor associated with immunologic risk in HTx.³⁴ The significant decrease in CD30 following LVAD implantation may reflect changes in adaptive immune activation. However, its biological significance remains uncertain.³⁵ Humoral immunity was also affected, as reflected by reduced levels of BAFF, a key regulator of B cells. Elevated BAFF has been linked to adverse cardiovascular outcomes and increased mortality following myocardial infarction.³⁶ Macrophage-related pathways were similarly modulated. IFN-γ and TNF-α levels were decreased, along with the anti-inflammatory cytokines IL-4 and IL-10. This pattern suggests an overall reduction in macrophage activation rather than a shift toward a specific phenotype.⁴ Histological and proteomic analyses showed concordant reductions in myocardial macrophage infiltration, supporting an integrated systemic and tissue-level response. Furthermore, circulating uPAR, ST2 and GDF-15 levels showed positive correlations with myocardial macrophage infiltration, suggesting that these biomarkers may reflect myocardial inflammatory activity during long-term LVAD support. Notably, IL-4 may contribute to myocardial fibrosis, given its role in promoting collagen synthesis in HF.³⁷

Cardiac remodelling is a central pathological feature of HF. It results from sustained injury caused by inflammation, oxidative stress, cellular senescence and chronic volume and pressure overload.²⁹ Cardiac fibrosis is a key component of this process and involves excessive deposition of extracellular matrix. This process impedes myocardial compliance, disrupts electrical conduction and promotes cardiomyocyte hypertrophy and apoptosis.³⁸ Progressive fibrosis increases myocardial stiffness and leads to both systolic and diastolic dysfunction.³⁹ In our study, transplant samples showed progression of interstitial fibrosis. In contrast, circulating levels of several mediators associated with profibrotic signalling, including EMMPRIN, FGF-2, FGF-19, RAGE, IL-27 and ST2, were reduced following LVAD support. Because myocardial fibrosis represents a late structural endpoint, whereas these circulating proteins reflect active fibrogenic signalling, these findings should not be interpreted as evidence that LVAD prevents or reverses myocardial fibrosis. Rather, they suggest modulation of molecular pathways involved in extracellular matrix remodelling that may precede measurable histological regression of collagen. Thus, our findings support the concept that long-term LVAD support is associated with attenuation of profibrotic signalling despite persistence of established myocardial fibrosis. EMMPRIN, a cell surface glycoprotein, induces MMP-2 and MMP-9 synthesis in response to hypoxia or ischaemia and promotes atherosclerotic plaque instability.⁴⁰ In experimental studies, EMMPRIN has been linked to myocardial fibrosis and adverse ventricular remodelling.⁴¹ FGF-2 has been associated with HF severity, reduced LVEF and elevated NT-proBNP levels⁴² and may promote hypertrophy and fibrosis. FGF-19 exhibits context-dependent effects. It can exert protective effects under oxidative stress⁴³ and prevent ventricular dysfunction.⁴⁴ RAGE, expressed in cardiomyocytes, vascular cells, fibroblasts and immune cells, is upregulated in response to ischaemia, diabetes, inflammation and HF. Activation of RAGE by advanced glycation end products promotes inflammation, hypertrophy and fibrosis.⁴⁵ Soluble RAGE levels correlate with HF severity,⁴⁶ and their reduction has been shown to confer cardioprotection in experimental models.⁴⁷ In our study, RAGE was positively correlated with macrophage content in the myocardium, likely reflecting its association with macrophage biology.⁴⁸ IL-27, produced by activated immune antigen-presenting cells, promotes a pro-inflammatory Th1 phenotype⁴⁹ and activates cardiac fibroblasts, driving their transformation into myofibroblasts and enhancing collagen production.⁵⁰ The observed reduction in these circulating mediators is therefore consistent with decreased activation of profibrotic molecular pathways, although it does not necessarily indicate structural regression of myocardial fibrosis. A similar pattern was observed for ST2, a well-established marker of myocardial stress, fibrosis and ventricular remodelling,⁵¹ which inhibits the cardioprotective effects of IL-33. The significantly reduced ST2 levels associated with LVAD implantation are consistent with previously published results.⁵² Taken together, these observations suggest modulation of profibrotic signalling during long-term LVAD support rather than direct regression of established myocardial fibrosis.

Neovascularisation and angiogenesis are other essential components of cardiac remodelling, ensuring adequate oxygen and nutrient delivery to myocardial tissue. HF with reduced EF is associated with impaired coronary flow reserve and microvascular dysfunction.²¹ Following LVAD implantation, levels of angiogenesis-related mediators, including SDF-1α, GDF-15, VEGF, HGF and angiopoietin-1, were reduced. These findings may reflect altered molecular responses associated with long-term mechanical unloading and reduced hypoxic or inflammatory signalling. However, because the present study assessed circulating biomarkers rather than myocardial angiogenesis directly, these observations should not be interpreted as evidence of reduced angiogenesis or vascular remodelling.

The coordinated expression of VEGF and angiopoietin-1 improves myocardial perfusion and function by promoting angiogenesis and cardiomyocyte survival, and by reducing apoptosis.⁵³ Moreover, angiopoietin-1 promotes endothelial survival, vascular stabilisation and anti-inflammatory effects through Tie2 signalling.^{54 55} Likewise, HGF regulates cell migration, morphogenesis, apoptosis, and proliferation, with cardioprotective and proangiogenic effects.⁵⁶ In addition, HGF has been associated with the modulation of macrophage function and may promote polarisation towards an anti-inflammatory phenotype.⁵⁷ It supports endothelial regeneration and neovascularisation after infarction and stimulates the release of mitogens such as VEGF. Vascular maturation and arteriogenesis involve angiopoietin/Tie2 signalling, which induces HGF secretion and promotes smooth muscle cell recruitment, thereby contributing to the stabilisation of newly formed vessels.⁵⁸ The biological significance of both VEGF and HGF is highlighted by their reduction after correction analysis. Reduced levels of VEGF and HGF factors associated with LVAD implantation may therefore indicate a diminished requirement for compensatory angiogenesis.

The above-mentioned reductions in GDF-15 and somatotropin levels point to altered heart–liver communication. GDF-15 suppresses hepatic growth hormone signalling in heart disease,⁵⁹ while somatotropin is essential for maintaining cardiac structure and function.⁶⁰ The decline of both markers may reflect normalisation of endocrine signalling following improved cardiac output. Similarly, reduced TFF-3 levels, previously linked to myocardial ischaemia and cardioprotection,^61–63 may reflect reduced ischaemic stress and altered metabolic adaptation during long-term LVAD support.

LVAD implantation also influenced adipose tissue biology. The significant rise in leptin may reflect increased BMI and improved nutritional status with attenuation of cardiac cachexia, which is beneficial given the association between leptin deficiency or resistance and impaired cardiac metabolism and function.⁶⁴ Additionally, leptin has been implicated in the progression of cellular senescence and ageing.^{65 66} Conversely, reduced resistin levels may indicate reduced macrophage-associated inflammatory activity, which is consistent with the observed reduction in myocardial CD68-positive cell infiltration.⁶⁷ This interpretation is further supported by the positive correlation between resistin levels and myocardial macrophage infiltration.

Taken together, our findings suggest that long-term LVAD support is associated with coordinated changes in inflammatory, metabolic, fibrosis-related and angiogenesis-related biomarkers. The accompanying reduction in myocardial macrophage infiltration is consistent with these systemic molecular changes, although the histological observations should be interpreted cautiously, given the limited subgroup size. Persistent low-grade inflammation may nevertheless remain due to ongoing blood–device interactions and mechanical stress.⁶⁸ Importantly, reductions in circulating profibrotic mediators should not be interpreted as evidence of regression of myocardial fibrosis but may reflect attenuation of active profibrotic signalling preceding measurable structural changes. Overall, these findings provide exploratory insight into the biological responses associated with long-term LVAD support and warrant confirmation in larger studies integrating circulating biomarkers with comprehensive myocardial tissue characterisation.

Conclusion

In this study, long-term LVAD support in subjects with advanced HF was associated with favourable changes in a broad range of biomarkers related to inflammation, cardiac remodelling, angiogenesis, profibrotic signalling molecules and systemic metabolism, together with histological evidence of decreased myocardial macrophage infiltration. Implantation of LVAD support is associated with biological changes beyond haemodynamic unloading, influencing key biological processes associated with HF progression. Although long-term mechanical circulatory support is inherently associated with persistent inflammatory activation, the observed findings suggest modulation of molecular pathways associated with HF progression and provide further insight into the biological effects accompanying long-term LVAD support in subjects with advanced HF.

Further studies in larger multicentre cohorts are warranted to confirm these findings, clarify their long-term clinical significance and determine whether targeting these biological pathways may further enhance the management of subjects with advanced HF supported with LVADs.

Study limitations

This was a single-centre observational study with a relatively small cohort size, which limits the generalisability of the findings and precludes definitive conclusions regarding causality. The absence of a control group reflects the difficulty of identifying a clinically comparable cohort managed exclusively with medical therapy among patients meeting established indications for durable LVAD support or HTx. All enrolled subjects were male, reflecting the characteristics of consecutive patients treated during the enrolment period, which further limits the generalisability of the findings. Changes in pharmacological therapy and interval clinical events may have influenced biomarker profiles, but their independent effects could not be evaluated given the limited sample size. Circulating biomarkers were assessed uniformly at 12 months, whereas paired myocardial tissue was obtained at the time of HTx or LVAD decommissioning, limiting direct comparisons between molecular and histological findings. Finally, myocardial tissue was available only in a subgroup of subjects, and the histological assessment was restricted to fibrosis, collagen and macrophage infiltration without a comprehensive evaluation of other remodelling processes.

Data availability statement

Data are available upon reasonable request.