Lipofuscin: Wear Pigment or Alarm Signal for Cardiac AlloGraft Vasculopathy?

Abstract

Background: CAV (cardiac allograft vasculopathy) is considered the leading cause of late post-transplant mortality and affects approximately 50% of transplant patients at 10 years post-transplant. Its etiopathogenetic mechanism is considered to be immune-mediated, but the identification of other non-immunological risk factors could represent new therapeutic targets for this pathology. Lipofuscin is due to reactive oxygen species (ROS) and appears to have a determining role in CAV. The aim of this study is to investigate the association between the amount of lipofuscin identified on EMB (endomyocardial biopsy) from patients followed up after heart transplantation, and the presence of CAV detected by coronary angiography. Methods: This retrospective study includes 99 EMBs from 47 transplanted patients, who also had coronary angiography. The amount of lipofuscin, damage to intramyocardial small vessels, vasculitis, Quilty effect, and acute cellular and humoral rejection was evaluated microscopically. Results: 19 CAV cases (19.2%) were identified, of which 15 (68.18% of total CAV cases) were insignificant. Grade 3 lipofuscin affected equally the cases with insignificant and significant CAV, 3 cases (37.5%) from each group. Grade 2 lipofuscin was reported in 10 cases (58.8%) of insignificant CAV, respectively 1 case of significant CAV (5.9%), (p-value of 0.0001). Quantitative evaluation of lipofuscin on microscopic sections revealed 8 EMBs (8.1%) with grade 3 lipofuscin. Two cases (25.0%) with lipofuscin score 3 were associated with moderate ACR (acute cellular rejection), ISHLT 2R and 3 cases (37.5%) with lipofuscin score 3 were associated with mild ACR ISHLT 1R, the differences being statistically significant, (p = 0.0001). Lipofuscin grades 2 and 3 were associated with severe fibrosis in 6 cases (35.3%) and 2 cases (25.0%), respectively (p = 0.042). A statistically significant association between the degree of damage to the intramyocardial small vessels and the amount of intracytoplasmic lipofuscin was observed (p = 0.00014). Discussion: Our study revealed that lipofuscin was more frequently associated with CAV, fibrosis, and damaged small vessels. Oxidative stress influences lipofuscinogenesis and CAV, which leads to endothelial dysfunction and neointimal hyperplasia, which over time will produce progressive narrowing of the vascular lumen and dysfunction of the cardiac allograft. Of the total number of 19 cases with CAV, 17 (89.47%) presented a lipofuscin score of 2 or 3 concomitantly with CAV, (p = 0.0001). This could mean that lipofuscin is not a harmless degradation product. At the same time, the association of a large number of cases with lipofuscin score 2, 10 cases (58.8%) with insignificant CAV could lead to the idea of using lipofuscin as a potential biomarker in the early diagnosis of CAV. Conclusions: We evidenced a significant association between the amount of intracytoplasmic lipofuscin and CAV. Accordingly, lipofuscin might be involved in the pathogenesis of CAV. Further research is needed to clarify the exact mechanisms of this association.

1. Introduction

From Christian Barnard’s first transplant until today, cardiac allograft rejection remains a feared complication, and monitoring transplanted patients to limit rejection episodes and prevent cardiac allograft vasculopathy involves a multidisciplinary approach that harnesses the collective expertise of cardiologists, pathologists, and radiologists. Although standardized protocols have been introduced regarding immunosuppressive therapy in heart transplant patients, patient survival is still influenced by both complications of immunosuppressive medication and episodes of severe acute rejection or cardiac allograft vasculopathy (CAV) [1].

CAV is considered the leading cause of late post-transplant mortality and represents the progressive obliteration of both epicardial and intramyocardial arteries in a heart transplant patient. It affects 30% of heart transplant recipients at 5 years post-transplant and approximately 50% of transplant patients at 10 years post-transplant [2]. The etiopathogenetic mechanism is considered to be immune-mediated [3]. Therefore, identifying immunological and non-immunological risk factors involved in the occurrence of cardiac allograft vasculopathy could represent novel targets for post-transplant surveillance of patients.

Over time, factors such as vasculitis, fibrosis, Quilty effect (Q.E.), and myocyte damage have been identified. These previously mentioned factors seem to be more frequently associated with acute rejection episodes [4], but without proving a predictive value in CAV. However, a recent study has demonstrated that lipofuscin is frequently associated with cardiac allograft vasculopathy [5]. Lipofuscin is a non-degradable pigment from catabolism, located in the cytosol or lysosomes of postmitotic cells, with a role in inhibiting proteasome function, mitophagy, autophagy, lysosomal stability, and reactive oxygen species [6]. It is yellowish-brown in color and can be observed in the usual hematoxylin and eosin stain as small intracytoplasmic granules. The chemical composition of lipofuscin includes mostly oxidized proteins but also lipids; carbohydrates; metals such as iron, copper, or zinc; calcium; and fluorophores [7].

In addition to its involvement in cardiomyocyte senescence, lipofuscin may constitute a critical factor in cardiac allograft vasculopathy, and studies such as that of Zakliczynski M. et al. [5] mention the predictive role of the development of vasculopathy. However, there are also studies such as that of Benvenuti L. A. et al. [8] that did not find an association between the amount of lipofuscin present on endomyocardial biopsies from heart transplant patients and the development of CAV. However, the aforementioned studies have proven the presence of a significantly higher amount of lipofuscin in patients in the late post-transplant period compared to patients of the same age group in the immediate post-transplant period. This raises the hypothesis of early aging of the transplanted heart, which could be a plausible explanation for the reduced period of functioning of transplanted hearts compared to native ones.

Given the conflicting evidence we found in the literature regarding the association of CAV with the presence of lipofuscin, the purpose of this study is to evaluate the presence of lipofuscin on endomyocardial biopsies (EMB) from heart transplant patients and to determine whether there is any relationship between the amount of lipofuscin identified on histological sections and the presence of cardiac allograft vasculopathy diagnosed by imaging. Associations between the amount of lipofuscin present on histopathological sections and other parameters such as age, sex, acute cellular or humoral rejection, the presence of fibrosis, vasculitis, the appearance of intramyocardial small vessels, and the Quilty effect were also assessed.

2. Materials and Methods

This retrospective study included 99 endomyocardial biopsies collected between January 2021 and March 2025, from 47 patients hospitalized for rejection monitoring, whose transplant was performed in ICvDT Târgu Mureș, Romania. The patients enrolled in the study are of both pediatric and adult age, as this heart transplant center performs transplants for both age groups. Only cases with concurrent coronary angiography were included. Patients whose transplantation was performed at the ICvDT Târgu Mureș center but whose monitoring was performed in other centers, as well as patients in whom no described angiographic protocols were found, were excluded from the study. Also, cases in which the paraffin blocks were damaged or contained insufficient material for the microscopic sections were excluded from the study. The study was approved by The Ethics and Research Committee of Clinical County Emergency Hospital, Targu Mures, Romania, under the Declaration of Helsinki.

After standard percutaneous EMB harvesting from the right ventricle, the myocardial fragments were fixed in formalin, embedded in paraffin blocks, and 3-micron-thick microscopic sections were made. These were stained with the usual hematoxylin-eosin stain as well as special stains such as Van Gieson and Trichrome Masson to highlight collagen fibers. The evaluation of microscopic slides was performed by two pathologists who quantified and staged both the changes suggestive of acute cellular rejection (ACR) and antibody mediated rejection (AMR), according to ISHLT protocols [9,10], as well as other parameters such as lipofuscin, fibrosis, Quilty effect, vasculitis, and damage of intramyocardial small vessels. Mapping of cell populations in the inflammatory infiltrate found on myocardial fragments was performed by immunohistochemical reactions, as follows: CD4, CD8, CD20, and CD 68.

2.1. Lipofuscin Assessment

Lipofuscin accumulation was evaluated on routine hematoxylin and eosin-stained myocardial sections by examining 10 non-overlapping high-power fields (HPFs) at 40× objective magnification for each case. A standardized graticule or counting frame was not used. The number of cardiomyocytes containing visible intracytoplasmic lipofuscin granules was recorded across the selected HPFs using the same evaluation protocol for all cases. All assessments were performed jointly by two experienced pathologists using a consensus-based approach, with each case reviewed simultaneously until agreement was reached. Consequently, no inter-observer variability was generated, and formal inter-observer agreement statistics (e.g., Cohen’s kappa or intraclass correlation coefficient) were not applicable.

Lipofuscin quantification was performed using four scores, from 0 to 3, by counting cardiomyocytes containing well-formed intracytoplasmic perinuclear lipofuscin granules in 10 high-power fields (HPFs). One HPF was defined as the microscopic field obtained using a 40× objective. A score of 0 was assigned when no lipofuscin was identified in the examined myocardial fragments. A score of 1 was assigned when lipofuscin granules were identified in up to 10 cardiomyocytes per 10 HPFs; a score of 2 when 11–20 cardiomyocytes per 10 HPFs contained lipofuscin granules; and a score of 3 when more than 20 cardiomyocytes per 10 HPFs exhibited well-defined perinuclear lipofuscin granules.

Collagen fibers were highlighted using Masson trichrome. Their thickness was subsequently measured using an Olympus BX 46 microscope, Olympus Corporation, Tokyo, Japan with a built-in capture system, which allowed the measurement of the thickness of collagen fibers both in the usual hematoxylin eosin staining, and in Masson trichrome stain. The CellSens program [Ver. 3.2] was used to perform the measurements. Fibrosis was also stratified using 4 scores, as follows: score zero for the absence of fibrosis; score 1 (mild fibrosis) when fine connective tissue trames with thicknesses up to 15 microns were observed; score 2 (moderate fibrosis) for the presence of collagen fibers with thicknesses between 16 and 30 microns; and score 3 (severe fibrosis) for collagen bands exceeding 30 microns.

Another microscopic aspect that was evaluated was the changes in the intramyocardial small vessels. Any pathological changes such as ballooning of endothelial cells, intimal thickening, intra or perivascular edema, endothelial dystrophic changes, and the presence of intraluminal or perivascular inflammatory infiltrate were recorded. The stratification of vascular changes was done as follows: if 3 pathological aspects were present simultaneously, a score of 3 was given; if 2 pathological changes were present, a score of 2 was given; if only one pathological change was present, a score of 1 was given; and for the normal appearance of the intramyocardial small vessels, a score of 0 was given. Vasculitis was also evaluated on EMB, being mild or moderate.

For myocyte damage, parameters such as premyocytolysis, microvacuolization, hypereosinophilia, cytoplasmic granularity, hydropic dystrophy, coagulation necrosis, cardiomyocyte hypertrophy, and nuclear integrity were monitored. Quantification of pathological changes was performed using a principle similar to that used for “intramyocardial small vessel damage”. Thus, for normal cardiomyocytes the score was 0, and the presence of one pathological change was given a score of 1; for two or three concomitant pathological changes, a score of 2 was given; and for the presence of more than 3 concomitant changes, a score of 3 was given.

2.2. Statistical Analysis

Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 23, Chicago, IL, USA). Data were considered as nominal or quantitative variables. Nominal variables were characterized using frequencies. Quantitative variables were tested for normality of distribution using the Kolmogorov–Smirnov test and were characterized by median and percentiles (25–75%) or by mean and standard deviation (SD), when appropriate. A chi-square test was used to compare the frequencies of nominal variables. The statistical analysis of the data also included generalized estimating equation (GEE) and mixed-effects models. Multivariate analysis was carried out using linear regressions. We used as dependent variable the lipofuscin grade 2 or 3 (coded with 1). we included as potential independent variables the data that achieved the criterion of significance at p < 0.2 in univariate analysis. We used the Bonferroni correction in order to account for multiple comparisons. The level of statistical significance was set at p < 0.05.

3. Results

Ninety-nine EMB samples were included in this study, collected from 47 transplanted patients, 7 patients (14.89%) of pediatric age and 40 patients (85.11%) of adult age. Of the total, 68 endomyocardial biopsies (EMBs) (68.7%) were obtained from male patients. The maximum number of biopsies obtained from a single patient was five, while the minimum was one. Between two and six myocardial tissue fragments were collected per biopsy, with four fragments being the most common, observed in 65 (65.7%) EMBs. The patients ranged in age from 14 to 66 years. The largest proportion of biopsies, 27 (27.3%), was obtained from patients aged 51–60 years. For analysis, patients were divided into six age groups, as follows: 11–20, 21–30, 31–40, 41–50, 51–60, and 61–70 years.

To identify a possible association between the amount of lipofuscin and the age of the patients, lipofuscin scores from different age ranges were compared. Of the total of 8 EMBs with score 3 lipofuscin, 3 EMBs (37.5%) were recorded in the age range 31–40 years and 2 EMBs (25%) were recorded in the age range 11–20 years. At the opposite end, of the total of 34 EMBs quantified with grade 0 lipofuscin, 14 EMBs (41.2%) were recorded in the age group 51–60 years and 11 EMBs (32.4%) in the age range 41–50 years. Grade 2 lipofuscin was observed most frequently in the age group 31–40 years, 5 EMBs (29.4%) and grade 1 lipofuscin was most frequently observed in the age group 41–50 years, 11 EMBs (27.5%). These differences are statistically significant, p-value being 0.008 (p = 0.008)

There were no statistically significant differences in the amount of lipofuscin present on EMBs from women and men; of the total cases with grade 0 lipofuscin, 24 EMBs (70.6%) were recorded in men and of the total of 8 cases with grade 3 lipofuscin, 5 EMBs (62.5%) were men (p = 0.95).

In Figure 1, microscopic images illustrating lipofuscin score 1, score 2, and score 3, respectively, can be observed. However, readers should keep in mind that the quantification of the scores was done by counting cells containing intracytoplasmic lipofuscin from 10 HPF.

Figure 1.

Intracytoplasmic lipofuscin marked with arrows. (A): Lipofuscin score 1 (HE, ob 20×); (B): Lipofuscin score 2 (HE, ob 20×); (C): Lipofuscin score 3 (HE, ob 40×).

The presence or absence of CAV was assessed by invasive coronary angiography. The patients were categorized into insignificant and significant CAV according to the following definition: CAV is a complication of heart transplantation that consists of remodeling of the epicardial arterial vasculature and micro-vascularization, due to chronic rejection [3]. According to the ISHLT reporting system, CAV is considered insignificant when the arterial lumen is narrowed less than 50% and CAV is significant when the lumen shows a narrowing of more than 50% of the lumen [11]. Nineteen CAV cases (19.2%) were identified, of which 15 (68.18% of total CAV cases) were insignificant. Grade 3 lipofuscin affected equally both groups of cases with insignificant and significant CAV, 3 cases (37.5%) from each group. Grade 2 lipofuscin was reported in 10 cases of insignificant CAV (58.8%), respectively 1 case of significant CAV (5.9%), the differences recorded being statistically significant, with a p-value of 0.0001.

Regarding acute cellular rejection (ACR), 8 cases (8.1%) presented mild rejection ISHLT 1R, 2 cases (2.0%) presented moderate rejection ISHLT 2R, and 1 case (1.01%) presented severe acute cellular rejection 3R. Also, 11 cases (11.11%) presented AMR. Quantitative evaluation of lipofuscin on microscopic sections revealed 34 EMBs (34.3%) without the presence of lipofuscin, 40 EMBs (40.4%) with grade 1 lipofuscin (less than 10 cells with intracytoplasmic lipofuscin granules/10HPF), 17 EMBs (17.2%) with grade 2 lipofuscin (between 10 and 20 cells with lipofuscin granules/10 HPF), and 8 EMBs (8.1%) showed grade 3 lipofuscin (>21 cells with intracytoplasmic lipofuscin granules/10 HPF). Several microscopic images showing the association between ACR and lipofuscin are shown in Figure 2.

Figure 2.

Association between lipofuscin and ACR (A): Moderate ACR ISHLT 2R—foci of inflammatory infiltrate associated with myocyte damage can be observed. (HE, ob 40×); (B): Another picture from the same slide showing numerous cardiomyocytes with intracytoplasmic lipofuscin—arrows; The lipofuscin score, assigned after evaluating 10 high-power fields (HPF) at 40× objective magnification, was 3. (HE, ob 40×); (C): Severe ACR ISHLT 3R—diffuse inflammatory infiltrate with multiple foci of myocyte damage; The black arrow highlights intracytoplasmic lipofuscin. The black arrow highlights intracytoplasmic lipofuscin. (HE, OB 40x); (D): Lipofuscin (black arrows) and inflammatory infiltrate; The image shows a case of moderate acute cellular rejection (ISHLT grade 2R) associated with a lipofuscin score of 3. (HE, ob 40×).

Statistically significant differences were also found in the case of the association between lipofuscin and ACR. Thus, 33 EMBs (97.1%) with lipofuscin grade 0 presented ISHLT 0; 3 EMBs (37.5%) of the biopsies with lipofuscin grade 3 were associated with mild ACR ISHLT 1R; and 2 EMBs (25.0%) with lipofuscin grade 3 were associated with moderate ACR, ISHLT 2R. Also, in 3 cases (17.6%) with grade 2 lipofuscin microscopic changes of ACR ISHLT 1R were observed, (p = 0.0001). For AMR, a statistically significant association with the amount of intracytoplasmic lipofuscin was also found, as follows: in the case of 4 EMB (50.0%) with grade 3 lipofuscin, they were associated with AMR, respectively 4 EMB (23.5%) with grade 2 lipofuscin were associated with AMR (p = 0.0001), compared to 71 EMB without AMR (87.5% of all EMB without AMR) who presented lipofuscin score 0 or 1. For all immunohistochemical markers listed above, a positive association of the number of positive inflammatory cells with the amount of lipofuscin visualized on HE slides was found.

The Quilty effect (Q.E.) represents a localized collection of inflammatory cells—primarily lymphocytes, macrophages, and plasma cells—and small capillaries, that forms within the endocardium of a transplanted heart, being often considered a tertiary lymphoid organ [12]. In our study, the Q.E. was observed on 15 EMBs, of which 6 EMBs (40.0%) did not present intracytoplasmic lipofuscin and 4 EMBs (26.66%) had associated grade 2 lipofuscin, without registering a statistically significant association between the presence of Q.E. and the amount of intracytoplasmic lipofuscin. (p = 0.34). In Figure 3, the microscopic appearance of QE can be observed, as well as the positive CD 20 immunostaining.

Figure 3.

Quilty effect The micrograph shows a tissue section with a lipofuscin score of 1 and the presence of the Quilty effect. (A): Q.E.—The endocardium marked with blue arrow and inflammatory cells with black arrow) (HE, ob 10×); (B): Q.E.—CD20-positive B lymphocytes are stained brown. (CD 20 immunostain; ob 10×).

In 12 EMBs (35.3%) that did not show any lipofuscin deposits, the absence of fibrosis was also noted, in two other biopsies (5.9%) with lipofuscin grade 0, severe fibrosis was present. Lipofuscin grades 2 and 3 were associated with severe fibrosis in 6 cases (35.3%) and 2 cases (25.0%), respectively. Moderate fibrosis was more frequently found in biopsies on which a reduced amount of lipofuscin was observed, in 7 EMBs (17.5%) with lipofuscin grade 1 and 6 EMBs (17.6%) without lipofuscin, respectively (p = 0.042).

Data analysis revealed a statistically significant association between the degree of damage to the intramyocardial small vessels and the amount of intracytoplasmic lipofuscin. Thus, 5 cases (62.5%) were recorded with grade 3 lipofuscin and vascular damage score 3, 11 EMB (64.7%) with grade 2 lipofuscin were also associated with changes in the intramyocardial small vessels score 2. At the opposite pole, 13 EMB (38.2%) were noted with normal-appearing intramyocardial vessels, without the presence of lipofuscin in cardiomyocytes (p = 0.00014).

In Table 1, the detailed results of the association of the amount of lipofuscin and various parameters such as the presence/absence of CAV, ACR, AMR, fibrosis, Q.E., and the appearance of intramyocardial small vessels can be observed.

Table 1.

Association between lipofuscin and different parameters.

Parameter		Lipofuscin				p Value
		Score 0	Score 1	Score 2	Score 3
Angiocoronaro graphy	CAV absent	34 (100%)	38 (95.0%)	6 (35.3%)	2 (25.0%)	p = 0.0001
	insignificant CAV	0 (0.0%)	2 (5.0%)	10 (58.8%)	3 (37.5%)
	significant CAV	0 (0.0%)	0 (0.0%)	1 (5.9%)	3 (37.5%)
ACR	ISHLT 0	33 (97.1%)	38 (95.0%)	14 (82.4%)	3 (37.5%)	p = 0.0001
	ISHLT 1R	1 (2.9%)	1 (2.5%)	3 (17.6%)	3 (37.5%)
	ISHLT 2R	0 (0.0%)	0 (0.0%)	0 (0.0%)	2 (25.0%)
	ISHLT 3R	0 (0.0%)	1 (2.5%)	0 (0.0%)	0 (0.0%)
AMR	AMR absent	33 (97.1%)	38 (95.0%)	13 (76.5%)	4 (50.0%)	p = 0.0001
	AMR present	1 (2.9%)	2 (5.0%)	4 (23.5%)	4 (50.0%)
Fibrosis	Score 0	12 (35.3%)	5 (12.5%)	4 (23.5%)	0 (0.0%)	p = 0.042
	Score 1	14 (41.2%)	17 (42.5%)	6 (35.3%)	6 (75.0%)
	Score 2	6 (17.6%)	7 (17.5%)	1 (5.9%)	0 (0.0%)
	Score 3	2 (5.9%)	11 (27.5%)	6 (35.3%)	2 (25.0%)
Q.E.	Absent	28 (82.4%)	37 (92.5%)	13 (76.5%)	6 (75.0%)	p = 0.34
	Present	6 (17.6%)	3 (7.5%)	4 (23.5%)	2 (25.0%)
Appearance of intramyocardi al small vessels	Score 0	13 (38.2%)	4 (10.0%)	1 (5.9%)	1 (12.5%)	p = 0.00014
	Score 1	9 (26.5%)	13 (32.5%)	2 (11.8%)	0 (0.0%)
	Score 2	8 (23.5%)	15 (37.5%)	11 (64.7%)	2 (25.0%)
	Score 3	4 (11.8%)	8 (20.0%)	3 (17.6%)	5 (65.5%)

3.1. Figures, Tables and S

The association of vasculitis with the amount of intracytoplasmic lipofuscin proved to be statistically significant. In this study, 24 cases (70.6%) without vasculitis were recorded who also did not present intracytoplasmic lipofuscin, and 9 cases (26.5%) with mild vasculitis without intracytoplasmic lipofuscin. Also, 5 EMB (62.5%) with mild vasculitis were recorded in which an abundant amount of intracytoplasmic lipofuscin was observed (score 3) and 5 EMB (29.4%) with moderate vasculitis who associated lipofuscin score 2 (p = 0.0001).

Six EMBs (17.6%) of the total cases with lipofuscin score 0 were associated with myocyte changes score 3 and 16 EMB (47.1%) were associated with myocyte changes score 2. There were also 3 cases (37.5%) of the total with lipofuscin score 3, which had myocyte changes score 3 and, respectively, 7 cases (41.2%) of the total cases with lipofuscin score 2, with myocyte changes score 2. The differences recorded are not statistically significant, the p value being 0.58.

3.2. Generalized Estimating Equation (GEE) Analysis Results

A total of 99 endomyocardial biopsy obtained from 47 patients were included in the analysis. Because some patients contributed more than one biopsy, generalized estimating equation (GEE) logistic regression models with patient ID as the clustering variable were used to account for within-patient correlation.

Higher lipofuscin scores were strongly associated with the presence of imaging-detected CAV. For each one-point increase in the lipofuscin score, the odds of CAV (CAV insignificant and significant versus absence of CAV) increased more than sevenfold (β = 1.963, robust SE = 0.506; OR = 7.12, 95% CI 2.64–19.19; p < 0.001).

A similar and stronger association was observed when the analysis focused on angiographic significant CAV. For each one-point increase in the lipofuscin score, the odds of angiographic significant CAV (versus absence of CAV—insignificant CAV) increased more than elevenfold (β = 2.430, robust SE = 0.629; OR = 11.36, 95% CI 3.31–38.99; p < 0.001).

The descriptive distribution was consistent with a dose–response pattern. CAV was absent in all biopsies with a lipofuscin score of 0, whereas the proportion of biopsies with CAV increased progressively at higher lipofuscin scores. Among biopsies with lipofuscin scores of 2 or 3, CAV was substantially more frequent, and angiographic significant CAV was observed only in this higher-score range. Overall, these findings support a positive dose–response association between increasing lipofuscin accumulation and the presence and severity of imaging-detected CAV.

3.3. Mixed-Effects Model

As a sensitivity analysis, a mixed-effects logistic regression model with a patient-specific random intercept was fitted to account for repeated biopsies from the same patient. The year of biopsy was not included in the model.

Higher lipofuscin scores were strongly associated with the presence of imaging-detected CAV. For each one-point increase in the lipofuscin score, the odds of CAV (insignificant and significant versus absent CAV) increased approximately ninefold (β = 2.191, SE = 0.200; OR = 8.94, 95% CI 6.05–13.22; p < 0.001).

When significant CAV was analyzed as the outcome, each one-point increase in the lipofuscin score was associated with a significantly increased odds of significant CAV (versus absent–insignificant CAV) (β = 1.181, SE = 0.236; OR = 3.26, 95% CI 2.05–5.17; p < 0.001).

The direction and statistical significance of the association between lipofuscin score and CAV were consistent with the findings from the GEE analysis, supporting the robustness of the primary association.

For the multivariate logistic regression model, in the relationship between lipofuscin (the dependent variable) and the independent variables we identified significant relationships for CAV (OR = 104.8; C.I. 95% 17.7 to 619.6, p < 0.001) and humoral rejection (OR = 18.7; C.I. 95% 3.0 to 117.7; p = 0.002). In Table 2, the results of the multivariate logistic regression model are presented in detail.

Table 2.

The results of the multivariate logistic regression model.

Variable	Odds Ratio (OR)	95% C.I.	p Value	GEE * or (95% C.I.)
Angiocoronarography	104.8	17.7–619.6	<0.0001	132.1 (16.0–1091)
Humoral rejection (AMR)	18.7	3.0–117.7	0.002	20.2 (3.3–122)
Vasculitis	3.1	0.63–15.5	0.16	3.1 (0.57–16.7)

* GEE = generalized estimating equations.

4. Discussion

In our study we found a more frequent association of CAV with a higher amount of lipofuscin. Also, ACR and AMR were more frequently associated with higher scores for lipofuscin. Another finding of this study is that pathological changes in the small intramyocardial vessels were more marked on EMB, which showed more abundant amounts of lipofuscin.

Lipofuscin is considered by many authors to be an insoluble waste localized to the lysosomes and cytosol of postmitotic cells associated with cellular aging [13,14], but recent research has proven the involvement of lipofuscin in certain neurological degenerative diseases [15] as well as its potential role as a biomarker of retinal oxidative damage [16,17]. At the same time, recent research mentions the role of lipofuscin accumulation in the following processes: decreasing cardiomyocyte contractility [18], the association of serum concentration of this pigment with the presence of hypertrophic cardiomyopathy [19], and the association of intracytoplasmic concentration of this pigment with cardiac allograft vasculopathy [5]. However, the exact mechanism by which this association occurs requires detailed studies.

One of the hypotheses of lipofuscinogenesis supports the idea that oxidized proteins resulting from reactions of reactive oxygen species—ROS become non-functional, are no longer degraded due to impaired proteasome function, and accumulate intracellularly in the form of lipofuscin granules. In turn, lipofuscin has the potential to generate reactive oxygen species through its ability to bind iron, its accumulation being closely linked to the occurrence of diseases such as Parkinson’s or Alzheimer’s [20]. But also in cardiac pathology, lipofuscin seems to play a crucial role in the senescence and degenerative processes of cardiomyocytes, by decreasing Sirt 1 proteins with a cardioprotective role [21,22].

The mechanism by which lipofuscin is formed is thought to be the decrease in the activity of lysosomal enzymes such as cysteine proteases, the most involved of which are cathepsins B, H and L [23,24]. The mechanism of lipofuscinogenesis affects the activity of cathepsins in two possible ways, as follows: The first is represented by Primary Enzymatic Deficiency, in which lipofuscin-like material accumulates massively due to the genetic damage of cathepsins B and L [25]. The second way is represented by the vicious circle of oxidative stress through which increased levels of ROS produce the oxidation of lipids and proteins that become resistant to the degradation of cathepsins B, H, and L, which leads to the accumulation of lipofuscin. Another factor that completes this oxidative stress path is the disruption of lysosomal acidity, resulting in a decrease in the activity of acid-dependent proteases [16].

Ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, is closely interconnected with lipofuscinogenesis through a self-amplifying cycle of oxidative stress. Lipofuscin-associated iron promotes ROS generation and ferroptosis, while ferroptosis-related lipid peroxidation further drives lipofuscin accumulation [26,27,28]. In CAV, ferroptosis contributes to endothelial dysfunction and vascular smooth muscle cell alterations, promoting severe intimal thickening and vascular lumen obliteration [29,30].

At the same time, oxidative stress also plays a crucial role in CAV, which through the imbalance between free radicals and antioxidants, leads to endothelial dysfunction and neointimal hyperplasia, which over time will produce progressive narrowing of the vascular lumen and dysfunction of the cardiac allograft [31]. In this way, oxidative stress could be the “common pathway” that could explain the close association between cardiomyocyte lipofuscin loading and the presence of CAV.

Of the total number of 19 cases with CAV, 17 (89.47% of the total CAV cases) presented a lipofuscin score of 2 or 3 concomitantly with CAV (p = 0.0001). This could be a signal leading to the abandonment of the idea that lipofuscin is a harmless degradation product. At the same time, the association of many cases with lipofuscin score 2, 10 cases (58.8%) with insignificant CAV could lead to the idea of using lipofuscin as a potential biomarker in the early diagnosis of CAV. Also, lipofuscin observed on EMB could lead to the establishment of effective treatment protocols in slowing down the progression of CAV by decreasing oxidative stress and activating proteasome function, autophagy, and mitophagy, thus interrupting the vicious circle through which lipofuscin maintains the elevated level of oxidative stress which in turn drives further accumulation of lipofuscin.

Our study also found a significant association of increased lipofuscin levels with acute cellular and humoral rejection, respectively. Three EMBs (37.5%) with lipofuscin grade 3 were associated with mild ACR, and 2 EMBs (25.0%) with lipofuscin grade 3 were associated with moderate ACR, p = 0.0001. The link between lipofuscin and acute cellular rejection can be explained through interconnected mechanisms involved in pathology and transplant biology. ACR involves a robust inflammatory response with dense lymphocyte infiltrate, generating severe oxidative stress. This environment rapidly degrades and oxidizes cellular macromolecules, increasing the production of wear-and-tear pigment. An electron microscopy study [32] has also found a specific phenomenon where mitochondria cluster close to the nuclear envelope due to rejection-induced stress, the phenomenon being encountered only in ACR. This stress and increased autophagy result in dense bodies (degraded mitochondria) and higher levels of lipofuscin accumulation in the tissue. Another mechanism that could justify the association of lipofuscin with acute rejection is the fact that ACR involves an inflammatory process that metabolically exhausts cardiomyocytes and subsequently impairs lysosomal degradation with lipofuscin accumulation.

Another statistically significant association found in this study was between lipofuscin and fibrosis. Both parameters are essential in the structural and metabolic damage of the transplanted heart, as it is known that the prevalence of fibrosis is positively associated with the severity of CAV [33]. As oxidative stress increases, damaged cells trigger inflammation, and inflammatory cells secrete fibrogenic mediators, all of which accelerate the accumulation of connective tissue [34]. Figure 4 illustrates the association between lipofuscin, fibrosis, and changes in intramyocardial small vessels.

Figure 4.

Fibrosis and intramyocardial small vessels changes (A): Interstitial fibrosis—blue circle and lipofuscin—blue arrows. The lipofuscin score assigned to this case was 2, and the fibrosis score was 3. (HE, ob 20×) (B): Fibrosis score 3; Masson’s trichrome stain was used to highlight collagen fibers, which are 496 visible in the image stained green. (Masson trichrome, ob 5×) (C): Vasculitis and cardiomyocites with lipofuscin—blue arrows; The case shown in the image is associated with mild vasculitis and a lipofuscin 498 score of 3. (HE, ob 20×) (D): Small vessels with ballooning of endothelial cells (black arrows), intimal thickening or perivascular edema (green arrows) along with cardiomyocites with lipofuscin—blue arrows (HE, ob 10×).

Regarding microscopic pathological changes in intramyocardial small vessels, they were positively associated with the amount of intracytoplasmic lipofuscin. Similar data were also found in the study conducted by Zakliczynski M. et al. [5], suggesting the idea that damage to intramyocardial small vessels and lipofuscin could be associated with CAV.

Generalized Estimating Equation (GEE) Analysis

This association between lipofuscin and imaging detected CAV remained statistically significant after accounting for the non-independence of repeated biopsies obtained from the same patient using a GEE framework. Importantly, the magnitude of the association was substantial, as follows: each one-point increase in the lipofuscin score was associated with approximately sevenfold higher odds of CAV.

The analysis also demonstrated a dose–response pattern. The association became even stronger when the outcome was restricted to significant CAV, with an approximately elevenfold increase in the odds of significant CAV for each one-point increase in the lipofuscin score. This graded relationship could have a biological and clinical significance because it suggests that increasing lipofuscin accumulation may reflect a progressive process associated not only with the presence of CAV but also with more advanced disease.

CAV was not observed in biopsies with a lipofuscin score of 0, while the frequency of CAV increased markedly among biopsies with scores of 2 and 3. Although the observational nature of the study does not permit causal inference, the consistency between the strong statistical association and the observed dose–response gradient can lead to the hypothesis that lipofuscin accumulation may serve as a histopathological marker associated with the presence and severity of CAV.

However, to verify the hypothesis that lipofuscin scoring may have potential value as a histological marker for identifying patients at increased risk of CAV, further studies with larger cohorts and independent validation are warranted to determine the diagnostic and prognostic utility of lipofuscin quantification and to establish clinically meaningful score thresholds.

The data presented in this study are preliminary, and studies on larger cohorts of patients are required to better investigate the results of this study and lead to the standardization of lipofuscin quantification visualized on myocardial fragments as well as the exact mechanism by which lipofuscin is associated with cardiac allograft vasculopathy and other parameters such as acute rejection, fibrosis, or damage to intramyocardial small vessels.

The study has several limitations, primarily due to its observational, single-center design and the relatively small number of endomyocardial biopsies included. Another limitation of this study is that the assessment of CAV via coronary angiography is a highly operator-dependent procedure and can yield false-negative results, particularly for non-significant CAV. For the findings of this study to be incorporated into standardized protocols for the prevention of cardiac allograft vasculopathy (CAV) and for guiding adjustments in immunosuppressive therapy, they must be validated in multicenter studies. Nevertheless, we believe that the results presented here represent an important first step toward elucidating the relationship between lipofuscin and cardiac allograft vasculopathy.

5. Conclusions

Endomyocardial biopsy remains one of the most valuable diagnostic methods for acute cellular and humoral rejection, but it can provide additional information such as the appearance of intramyocardial small vessels, vasculitis, fibrosis, or lipofuscin that seem to be related to chronic rejection (cardiac allograft vasculopathy). Thus, our study proves a significant association between the amount of intracytoplasmic lipofuscin and CAV detected by angiography. Lipofuscin was also observed more frequently in association with fibrosis or on myocardial fragments that presented intramyocardial small vessels with more severe damage. Lipofuscin might not be just a trivial wear pigment but a factor involved in CAV pathogenesis. The exact role and mechanism of this association require further research.

Abbreviations

The following abbreviations are used in this manuscript:

CAV	Cardiac allograft vasculopathy
EMB	endomyocardial biopsies
ROS	Reactive oxygen species
Q.E.	Quilty effect
HPF	High power field
HE	Hematoxilin-eosin
ICvDT	Emergency Institute for Cardiovascular Diseases and Transplantation
ISHLT	International Society for Heart and Lung Transplantation
ACR	Acute cellular rejection
AMR	Acute mediated rejection

Author Contributions

Conceptualization, A.O.F. and H.S.; investigation, writing—original draft, A.O.F., L.H. and M.C.S.; methodology, resources, software, M.C.S., A.O.F., S.V., L.G., H.S. and C.C.R.; statistical analysis of the data, S.V.; writing—review and editing, A.O.F., M.C.S., L.G. and C.C.R.; project administration and supervision, A.I.S. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted in accordance with the 431 Declaration of Helsinki, and approved by the Institutional Ethics Committee of Emergency 432 County Clinical Hospital Targu Mures (protocol code Ad. 5220/04.03.2022).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data presented in this study are available on request from the corresponding author due to privacy, legal reasons, and ethical reasons.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.