Abstract
Introduction
Recent studies suggested potential positive correlations between HLA-specific antibodies and development of cardiac allograft vasculopathy (CAV).
Methods
This prospective two-center study investigated early progression of CAV by coronary optical coherence tomography in 1 month and 12 months after heart transplantation (HTx) in 104 patients. Detection and characterization of donor specific (DSA) and MHC class-I polypeptide-related sequence A (MICA) antibodies were performed before, 1, 6 and 12 months after transplantation.
Results
During the first post-HTx year, we observed a significant reduction in the mean coronary luminal area (P < .001), and progression in mean intimal thickness (IT) (P < .001). DSA and anti-MICA occurred in 17% of all patients, but no significant relationship was observed between presence of DSA/anti-MICA and IT progression within 12 months after HTx. In contrast, we observed significant association between presence of DSA (p=0.031), de-novo DSA (p=0.031), HLA Class II DSA (p=0.017) and media thickness (MT) progression.
Conclusion
Results of our study did not identify a direct association between presence of DSA/anti-MICA and intimal thickness progression in an early period after HTx. However, we found significant relationships between DSA and media thickness progression that may identify a newly recognized immune-pathological aspect of CAV.
Keywords: cardiac allograft vasculopathy, donor specific antibodies, heart transplant, intimal thickness
Introduction
Human leukocyte antigen (HLA) matching is of significant importance in heart transplant (HTx) recipients as the presence of donor‐specific antibodies (DSA) directed against human leukocyte antigens (HLA) has been associated with allograft rejection, dysfunction, and loss [1,2]. Recent studies suggested positive correlation between anti-HLA antibodies and development of cardiac allograft vasculopathy (CAV), which is a main contributor of delayed mortality after HTx [3–4]. DSA mediate graft damage by binding to target HLA antigens expressed on the endothelium or smooth muscle cells of the allograft [5], activating complement via the classical pathway and contribute to endothelial cell injury and microvascular inflammation in arterial intima and media [6]. Furthermore, antibodies against non-classical MHC molecules, such as MHC class-I polypeptide-related sequence A (MICA) were shown to induce complement-dependent cytotoxicity and were implicated in acute and chronic solid organ allograft rejection, including CAV [7]. Nonetheless, a marked proportion of HTx patients do not have CAV even in the presence of antibodies against major histocompatibility antigens, indicating that the real genesis of this disease is still not completely understood.
Studies published on this subject are scarce, contradictory, and suffer from major limitations mainly due to small number of recruited patients, analysis of a paediatric cohorts, or use of low-sensitive coronary angiography as a CAV imaging modality [3–4,7–10]. Therefore, we aimed to evaluate the relationship between presence of donor specific anti-HLA and MICA antibodies and development of cardiac allograft vasculopathy in adult patients using highly automated 3-D optical coherence tomography analysis.
Methods
Patients
Between October 2014 and March 2017, 104 subjects, who survived first 12 months after HTx, from the Heart Centre at IKEM, Prague, and the Centre of Cardiovascular and Transplantation Surgery, Brno, Czech Republic were enrolled. The study (clinical trial NCT02503566) complies with the Declaration of Helsinki and was approved by the respective ethics committees. All HTx recipients ≥18 years of age were deemed eligible for inclusion into the study provided they were able and willing to give their informed consent. Exclusion criteria included kidney disease stage ≥IV (glomerular filtration <30 mL/min), unfavourable post‐HTx clinical conditions, such as severe nosocomial sepsis with prolonged antibiotic treatment during the first month, ongoing need for circulatory support using a ventricular assist device, and acute allograft failure.
Panel‐reactive antibody (PRA) testing and complement‐dependent cytotoxicity (CDC) crossmatch test
Prior to HTx, the presence of HLA‐specific antibodies was analysed in all recipients using the complement-dependent cytotoxicity (CDC) assay. Panel‐reactive antibodies (PRA) were expressed as a percentage of positive reactions within a panel of lymphocytes from 30 healthy donors. Patients’ serum samples were retested every three months or after a sensitizing event (blood transfusion, after VAD implantation, etc.). The last pre-transplant PRA were documented. CDC crossmatch test was performed before orthotopic HTx in all recipients. Individuals with pretransplant PRA ≥ 10% were transplanted only after confirmation of a prospective negative CDC crossmatch test.
Antibody detection by solid‐phase assays (SPA) and HLA typing
Patients were tested for presence of class I (HLA-A, -B, and -C), class II (HLA-DR, -DQ, and - DP) HLA and MICA antibodies using LABScreen Mixed and Single Antigen class I and class II techniques (One Lambda Inc., Canoga Park, CA, USA). Detection and characterization of antibodies were performed in all patients at 4 pre-defined time points - before HTx, 1, 6 and 12 months after transplantation (accepted time range of sample obtaining was ±1 week). DSA were considered positive if the mean fluorescent intensity (MFI) was > than 1000 for HLA Class I and > 2000 for HLA Class II antibodies, these cut off values were validated in the laboratory. Sera from patients on VAD were treated with AdsourbOut (One Lambda, Inc.) before analysis due to nonspecific binding on polystyrene beads. Antibody specificities defined in Luminex were compared with the mismatched HLA antigens of the donor allograft (donor‐specific antibodies, DSA).
Analysis of rejection in endomyocardial biopsies (EMB)
EMB specimens were stained with hematoxylin–eosin. Immunohistochemistry was performed on 3‐μm‐thick paraffin sections using immunoperoxidase‐based indirect method to detect C4d deposition and CD 68 expression on macrophages. Acute cellular rejection (ACR) and antibody-mediated rejection (AMR) were diagnosed according to ISHLT criteria [11,12].
ISHLT grade of 2R or higher was considered as significant cellular rejection. ISHLT grade <2R was considered as mild cellular rejection. AMR was diagnosed based on EMB findings, including histological and immune-pathological findings, and serial echocardiography examinations. The occurrence of pathological AMR (grade ≥ 1) was considered as positive [12].
OCT method
Coronary OCT imaging was performed at M1 and M12 after HTx as part of routine surveillance cardiac catheterization using a commercial intracoronary OCT system (ILUMIEN/DRAGONFLY OPTIS, St. Jude Medical). A 54 mm‐long segment of each HTx patient’s left anterior descending (LAD) artery, located within a proximal 100 mm segment, was imaged using automated pullback at 18 mm/sec and 10 frames/mm. Where the LAD artery exhibited unfavourable anatomical characteristics (small diameter, extreme tortuosity, muscle bridge), proximal segment of the left circumflex (LCx) or the right coronary artery (RCA) was imaged. The proximal fiduciary point was left main bifurcation in left coronary artery and first branch or well-defined calcification in RCA or LCx. After 12 months, patients underwent repeat cardiac catheterization and OCT of the same coronary artery with the same fiduciary points identified.
Image interpretation
For each frame of all OCT pullbacks, luminal, intimal-layer, and medial-layer surfaces were automatically segmented using a fully three‐dimensional LOGISMOS graph‐based approach developed at the University of Iowa [13,14], as previously documented [15,16]. Boundaries were identified as OCT brightness changes depicting tissue interfaces between adjacent wall layers. Automatically identified borders were efficiently edited using our Just‐Enough‐Interaction method adapted for the OCT segmentation environment [17,18] (Figure 1). This technique allows segmentation errors to be efficiently corrected in a 3D fashion on a regional basis if/as needed, an alternative to contour‐by‐contour/frame‐by‐frame manual retracing. Furthermore, portions of the OCT-imaged wall that were not analysable due to, e.g., guidewire shadow, excessive focal atherosclerosis, blood artefacts, etc. were excluded automatically using a deep learning network. This highly accurate, multilayer model ensures quantitative CAV analysis of every OCT frame of the imaged vessel for both the baseline and follow‐up image pullbacks. After identifying corresponding vascular landmarks, baseline and follow‐up pullback pairs were co‐registered, enabling location‐specific and fully three‐dimensional comparisons of layer‐based changes using quantitative indices.
Figure 1.
Three-dimensional segmentation of luminal (red), intimal (green), and medial (orange) surfaces performed volumetrically in the entire OCT pullback with intimal and medial layer thickness, brightness, and roughness indices calculated in 360 radial directions in each OCT frame. Changes of layer-specific indices were evaluated in a location-specific manner after M1—M12 OCT registration of the paired pullbacks.
Quantitative indices of vessel wall morphology and tissue appearance
Intimal thickness (IT) and medial thickness (MT) were determined as averaged values over all local pointwise measurements for each image frame of the OCT pullback obtained 1M and 12M after HTx. Lumen area (LA) was also obtained for each image frame. CAV risk of each patient was quantified as averaged OCT-frame-specific progression (ΔIT, ΔMT, ΔLA) of all paired frames from 1M to 12M after registration.
OCT analysis was blinded to the HLA data analysis.
Statistical analysis
Numerical variables were described as mean values ± standard deviations or median and interquartile ranges (IQR), where appropriate. The coronary morphological changes or patient characteristics changes from M1 to M12 were evaluated using Paired Student’s t test or Wilcoxon signed‐rank test, where appropriate. Categorical variables, presented as counts and percentages, were compared using Fisher’s exact test. Spearman’s rank correlation coefficient was used to evaluate the relationship between 12‐month IT progression and HR‐related variables. A Mann-Whitney test was used to compare coronary morphology and patient characteristics between groups with different antibodies. Bonferroni correction was used to avoid false positives where several tests were performed simultaneously. Renvironment was employed for statistical computing, with a P‐value of .05 used to denote statistical significance.
Definitions
DSA positive/anti-MICA positive – presence of class I/II anti HLA/anti-MICA in any of assessed periods (before HTx, 1, 6, 12 months after HTx). De-novo DSA – presence of class I/II HLA antibodies that did not pre-exist but developed after HTx in any of assessed periods (1, 6, 12 months after HTx). Persistent DSA – presence of class I/II anti HLA in more than one period (before HTx, 1, 6, 12 months after HTx). HLA class II DSA positive - presence of class II anti HLA antibodies in any of assessed periods (before HTx, 1, 6, 12 months after HTx).
Results
In total, 104 out of 108 originally recruited patients were enrolled into the study (four patients died prior to M12 follow‐up visit). Patient characteristics are summarized in Table 1. In this cohort, 90 left anterior descending, 5 left circumflex, and 9 right coronary arteries were analysed. In the 104 1M/12M pairs of registered OCT pullbacks, the median analysable angular range was 277° [255°−294°] per frame (corresponding to 277 locally specific pointwise measurements per frame). No full frames were excluded after image registration and the average overlapping pullback length was 40.0 ± 5.6 mm. A total of 41,649 registered frame pairs and 9,226,399 local pointwise measurements were analysed resulting in an average co-registered intimal surface area of 309.2 ± 69.8 mm2 per vessel pullback.
Table 1.
Patient characteristics and their association with presence of DSA and anti-MICA
| N=104 | p | DSA positive N=18 | DSA negative N=86 | p | Anti-MICA positive N=18 | Anti-MICA negative N=86 | p | |
|---|---|---|---|---|---|---|---|---|
| Age (years) | 51.7±12.4 | - | 52.7±12.6 | 51.5±12.4 | ns | 52.1±11.4 | 51.6±12.6 | ns |
| Female | 26 (25%) | - | 10 (55.6%) | 16 (18.6%) | 0.004 | 3 (16.7%) | 23 (26.7%) | ns |
| Ischaemic cardiomyopathy | 25 (24%) | - | 5 (27.8%) | 20 (23.3%) | ns | 4 (22.2%) | 21 (24.4%) | ns |
| VAD before HTx | 27 (26.0%) | - | 6 (33.3%) | 21 (24.4%) | ns | 4 (22.2%) | 23 (26.7) | ns |
| Cold ischaemia time (min) | 124[90–161] | - | 122 [100–152] | 125 [87–166] | ns | 124[98–151] | 124[88–164] | ns |
| Donor age (years) | 41.2±11.9 | - | 37.7±11.3 | 41.9±12.0 | ns | 39.6±15.2 | 41.5±11.2 | ns |
| Donor gender (female) | 78 (75%) | - | 8 (44.4%) | 18 (19%) | ns | 14(77.8%) | 64(76.2%) | ns |
| BMI (kg/m2) 1M/12 M | 26.4±4.1/28.5±4.6 | 0.006 | 25.8±3.7/27.4±3.8 | 26.5±4.1/28.8±4.8 | ns/ns | 27.0±3.2/29.2±4.7 | 26.3±4.2/28.4±4.6 | ns/ns |
| CMV infection within 12 months | 12 (11.5%) | - | 4 (22.2%) | 8 (9.3%) | ns | 4(22.2%) | 8 (9.3%) | ns |
| Mild cellular rejection within 12 months | 82(78.8%) | - | 17 (94.4%) | 65 (75.6%) | ns | 13(72.2%) | 69(80.2%) | ns |
| Significant cellular rejection within 12 months | 15 (14.4%) | - | 5 (27.8%) | 10 (11.6%) | ns | 3(16.7%) | 12(14.0%) | ns |
| Humoral rejection within 12 months | 5 (4.8%) | - | 3 (16.7%) | 2 (2.3%) | ns | 0(0%) | 5(5.8%) | ns/ns |
| LV ejection fraction (%) 1M/12M | 60.5±3.0/60.3±4.1 | ns | 61.4±4.1/61.1±3.2 | 60.4±2.7/60.1±4.3 | ns/ns | 60.3±2.3/59.9±2.6 | 60.6±3.1/60.4±4.4 | ns/ns |
| HbA1C (mmol/mol) 1M/12M | 40.4±9.2/46.5±16. 7 | 0.015 | 43.3±11.6/46.9±16.4 | 39.7±8.3/46.3±16.7 | ns/ns | 40.5±5.3/43.1±9.5 | 40.3±9.6/47.1±17.7 | ns/ns |
| Creatinine (μmol/l) 1M/12M | 87.0±25.3/108.9±39.4 | <0.00 1 | 81.5±21.9/108.0±45.7 | 88.1±25.9/109.1±38.3 | ns/ns | 89.1±21.0/114.3±44.3 | 86.5±26.2/107.8±38.5 | ns/ns |
| hsTnT (ng/l) 1M/12M | 111.4[78.0–152.0]/15.7[10.0–26.0] | <0.001 | 107 [83.7–134.0]/12.0[10.0–15.9] | 113.2[77.8–161.0]/17.0[10.8–26.2] | ns/ns | 109[86.9–152.0]/16.5[10.8–25.2] | 112.4[77.8–156.5]/15.3[10.0–26.5] | ns/ns |
| BNP (ng/l) 1M/12M | 338.5[207.0–598.0]/87.4[55.0–134.3] | <0.001 | 377.0[224.0–820.0]/93.3[46.0–136.0] | 330.4[207.0–564.9]/85.8[55.8–134.0] | ns/ns | 336.5[208.7–581.1]/85.0[49.0–102.0] | 338.5[205.0–619.1]/90.5[56.2–149.2] | ns/ns |
| Aspirin 1M/12M | 83(79.8%)/85(81.7%) | ns | 13 (72.2%)/12(66.7%) | 70 (81.4%)/73(84.9%) | ns/ns | 17(94.4%)/17(94.4%) | 66 (76.7%)/68(79.1%) | ns/ns |
| Statin 1M/12M | 89(85.6%)/91(87.5%) | ns | 15 (83.3%)/14(77.8%) | 74 (86.0%)/77(89.5%) | ns/ns | 16(88.9%)/17(94.4%) | 73(84.9%)/74(86.0%) | ns/ns |
| Steroids 1M/12M | 104(100%)/95(91.3) | ns | 18 (100%)/18(100.0%) | 86 (100%)/77(89.5%) | ns/ns | 18 (100%)/15(83.3%) | 86 (100%)/80(93.0%) | ns/ns |
| Tacrolimus 1M/12M | 104(100%)/103(99%) | ns | 18 (100%)/18(100.0%) | 86 (100%)/84(97.7%) | ns/ns | 18 (100%)/18(100.0%) | 86 (100%)/84(97.7%) | ns/ns |
| m-TOR inhibitor 1M/12M | 0 (0%)/6 (5.8%) | ns | 0 (0%)/1 (5.6%) | 0 (0%)/5 (5.8%) | ns/ns | 0 (0%)/1 (5.6%) | 0 (0%)/5 (5.8%) | ns/ns |
| MMF 1M/12M | 103(99%)/92 (88.5%) | 0.021 | 18 (100%)/17 (94.4%) | 86 (100%)/75 (87.2%) | ns/ns | 18 (100%)/16 (88.9%) | 86 (100%)/76 (88.4%) | ns/ns |
Anti-MICA – Anti -MHC class I chain-related antibodies; BMI – body mass index; BNP – brain natriuretic peptide; CMV – cytomegalovirus; DSA – donor specific antibodies; HDL – high-density lipoprotein; hsTnT – high sensitive troponin T; LDL – low-density lipoprotein; LV – left ventricle; M – month; MMF - mycophenolate mofetil; TAG – triacylglycerol; VAD – ventricular assist device
During the first year after HTx, DSA and MICA were equally detected in 17% (18/104) of patients. DSA to HLA class II antigens occurred in 10% (10/104), de-novo DSA in 13% (13/104) and persistent DSA were detected in 12% (12/104) of all patients. Positive retrospective complement-dependent cytotoxicity/CDC crossmatch was observed in 3% of patients (3/104). Female gender of HTx recipient was significantly associated with presence of DSA (p<0.01, Table 1). Table 2 illustrates the types and prevalence of DSA in the cohort.
Table 2.
Specificities of donor specific anti-HLA antibodies
| Patient number | HLA class I/II | Specificities | Mean fluorescence intensity | Number of mismatches |
|---|---|---|---|---|
| 1 | I | B18 | 6590 | 5 |
| 2 | II | DQ2 | 2892 | 3 |
| 3 | I/II | A1, A3/DQ2 | 6173, 1918/5850 | 6 |
| 4 | II | DR4, DQ6 | 2524, 2830 | 6 |
| 5 | I | A2 | 2173 | 3 |
| 6 | I | A2 | 3258 | 3 |
| 7 | I | B44 | 1511 | 6 |
| 8 | I/II | A1, B57/DQ6, DQ9 | 1021, 1397/8744, 2344 | 6 |
| 9 | I/II | B38/DR15, DQ6 | 4131/2532, 2215 | 4 |
| 10 | I | A2 | 2057 | 6 |
| 11 | I | A32, B35 | 3029, 3082 | 5 |
| 12 | I/II | A25/DQ6, DR15, DR51 | 1129/5167, 3281, 11263 | 4 |
| 13 | II | DR 52 | 3821 | 5 |
| 14 | I/II | B18/DQ6, DQ7 | 3872/3053, 3083 | 5 |
| 15 | I | A11 | 7157 | 5 |
| 16 | I | B56 | 14065 | 5 |
| 17 | II | DR4 | 5259 | 6 |
| 18 | II | DR8 | 5000 | 4 |
HLA – human leukocyte antigen
We observed significant luminal area (LA) reduction (p<0.001) and intimal thickness progression (p<0.001) within the first post-transplant year (Table 3). None of the studied parameters related to the presence of DSA and anti-MICA (DSA/anti-MICA positive, de-novo DSA, persistent DSA and DSA to HLA Class II antigens) were associated with changes in luminal area and/or IT within 12 months after HTx. According to analysis of patient-based OCT findings (Δ mean LA, Δ mean IT, Δ mean MT) among selected donor-related characteristics and non-immunologic risk factors (blood pressure, lipid profile, glycemia, CMV infection and immunosuppressive regimen), significant negative correlation was observed between the difference in HDL at M1 and M12 and Δ mean LA (p=0.042) while all other studied associations lacked statistical significance.
Table 3.
Optical coherence tomography findings of CAV
| M1 | M12 | Change (M12-M1) | P | |
|---|---|---|---|---|
| Δ mean LA (mm2) | 8.5 ± 2.1 | 7.6 ± 2.2 | −0.9 ± 1.6 | <0.001 |
| Δ mean IT (μm) | 102.9 ± 38.7 | 133.2 ± 69.0 | 30.4 ± 45.9 | <0.001 |
| Δ mean MT (μm) | 81.9 ± 24.5 | 84.1 ± 24.0 | 2.2 ± 12.5 | 0.074 |
In contrast, we observed significant association between presence of DSA (p=0.031), denovo DSA (p=0.031), HLA Class II DSA (p=0.027) and MT progression (Table 4). Figure 2 visualizes the changes in intimal and medial thickness in relation to presence of DSA/anti-MICA in the cohort.
Table 4.
Optical coherence tomography findings and comparison between DSA/anti-MICA
| DSA | De-novo DSA | Persistent DSA | Class II HLA DSA | DSA>5000 MFI | Anti-MICA | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Positive (17%, 18/104) | Negative (83%, 86/104) | Positive (13%, 13/104) | Negative (87%, 91/104) | Positive (12%, 12/104) | Negative (88%, 92/104) | Positive (10%, 10/104) | Negative (90%, 94/104) | Positive (8%, 8/104) | Negative (92%, 94/104) | Positive (17%, 18/104) | Negative (83%, 86/104 | ||
| Δ mean LA (mm2) | −1.3 ± 1.9 | −0.8 ± 1.6 | −1.3 ± 2.1 | −0.8 ± 1.6 | −1.3 ± 2.1 | −0.8 ± 1.6 | −1.6 ± 2.5 | −0.8 ± 1.5 | −0.9 ± 0.9 | −0.9 ± 1.7 | −1.4 ± 1.5 | −0.8 ± 1.6 | |
| p=ns | p=ns | p=ns | p=ns | p=ns | p=ns | ||||||||
| Δ mean IT (μm) | 32.5 ± 34.5 | 30.0 ± 48.1 | 35.8 ± 32.0 | 29.6 ± 47.6 | 29.7 ± 32.2 | 30.5 ± 47.5 | 34.9 ± 36.7 | 30.0 ± 46.8 | 16.4 ± 22.9 | 31.6 ± 47.2 | 42.2 ± 66.9 | 27.9 ± 40.3 | |
| p=ns | p=ns | p=ns | p=ns | p=ns | p=ns | ||||||||
| Δ mean MT (μm) | 9.7 ± 11.8 | 0.7 ± 12.2 | 11.2 ± 11.2 | 0.9 ± 12.2 | 7.1 ± 10.8 | 1.6 ± 12.6 | 14.1 ± 11.7 | 1.1 ± 12.0 | 3.2 ± 6.2 | 2.1 ± 12.9 | 4.8 ± 12.0 | 1.7 ± 12.6 | |
| p=0.031 | p=0.031 | p=ns | p=0.017 | p=ns | p=ns | ||||||||
Figure 2. Changes in intimal and medial thickness in relation to presence of DSA/anti-MICA in the cohort.
DSA – donor specific antibodies; IT - intimal thickness; anti-MICA – anti-MHC class-I polypeptide-related sequence A; MT – media thickness; μm – micrometres.
By comparing the presence of DSA and anti-MICA with humoral rejections, we observed significant association between de-novo DSA and humoral rejection (p=0.013). Positive retrospective CDC crossmatch was not related to IT or MT progression (p=NS).
Discussion
The main findings of our study can be summarized as follows: 1) We observed significant luminal area reduction, and intimal thickness progression within the first post-transplant year. 2) DSA, de-novo DSA and HLA Class II DSA presence were significantly associated with media thickness progression. 3) DSA and anti-MICA both occurred in 17% of all patients, and their presence was not related to intimal thickness progression within the first post HTx year.
The rate of allosensitized patients on waiting lists is increasing as a consequence of growing numbers of retransplants and assist-device implantations [2,19]. There is now considerable evidence demonstrating the detrimental effects of anti–human leukocyte antigen (anti-HLA) donor-specific antibodies (DSA) on outcomes such as rejection, graft failure, and survival [2]. According to ISHLT consensus statement, it is estimated that 15% of heart transplant recipients develop DSA in the first-year post-transplant [2], whereas Moayedi et al. reported their development in up to 23% of all patients [20]. As per our results, female gender was significantly associated with presence of DSA (p=0.004), which is in agreement with other studies on the topic [21], and support the existing data on the significant association between higher level of allosensitization in female patients before HTx. Also, we observed significant association between de-novo DSA and humoral rejection. These patients may require enhanced immunosuppression, increased HLA antibody monitoring, or additional physiological assessment.
Scarce and conflicting data have been published on the relationship between DSA and cardiac allograft vasculopathy. Some of them demonstrated positive association of DSA presence and CAV [3–4]. Of note is that the vast majority of performed studies used coronary angiography as an imaging modality for CAV detection [3,9]. This is an important limitation considering its lack of sensitivity when compared with IVUS and OCT. It is well accepted that lumen loss in CAV may be caused not only by intimal thickening and plaque progression but also by vascular remodelling via changes in external elastic membrane area [22,23]. Topilsky et al. showed that patients with pre transplant Class II DSA have an accelerated rate of coronary plaque progression compared to DSA negative recipients when assessed by 3-D IVUS [4].
Smith et al. performed coronary angiography at 1, 3, 5, 7 and 10 years after transplantation. When considering HLA antibodies, both DSA and non‐DSA, de novo, persistent, and transient, respectively - no association was found between post‐transplant HLA‐specific antibodies and subsequent development of CAV [9]. Likewise, Tran et al. reported in a pediatric cohort that DSA were not an independent predictor of CAV on multivariate analysis [10]. Clemmensen et al. provided comprehensive characterization of prognostic significance of DSA presence and CAV using optical coherence tomography on top of coronary angiography [8]. While they found a strong relation between DSA presence and that of ISHLT CAV grade ≥II, no difference was reported in terms of OCT-derived mean intimal area increase between DSA positive and negative patients.
CAV is characterized by accelerated concentric fibrous intimal hyperplasia along the length of coronary vessels, and its early diagnosis is of crucial importance, as identification of such patients may favourably affect CAV-related allograft failure rates [24]. Optical coherence tomography (OCT) enables the thin intimal layer to be clearly differentiated from the tunica media, a crucial factor in determining early progression of CAV, as early as during the first year after HTx [16,25]. Using our highly automated 3D-OCT analysis, after registering the overlapping baseline and follow-up pullbacks, 400 ± 56 frames were analysed per pullback. It is this surface-based fully three-dimensional analysis of the layers in over 9 million wall locations over the full length of each pair of registered pullbacks that yields the ability to accurately and reproducibly determine small sub-voxel size changes of local layer thicknesses between baseline and follow-up indices (Fig. 1). Such measurements are unavailable if a small number of selected frames is analysed, contributing to the value of our highly automated fully three-dimensional analysis. This approach is the most detailed quantitative OCT method available for assessing early changes in cardiac vasculature [16].
In our cohort, we have not observed significant changes of intimal thickness within one year after HTx that would be attributable to DSA. None of the monitored variables related to DSA occurrence showed any close association with intimal thickening, namely no statistically significant association with Delta IT was found when considering DSA, de-novo DSA, persistent DSA and HLA Class II DSA. But, as Kaczmarek et al. showed, CAV was significantly more frequent in patients with DSA-positive findings over long-term follow-up, difference versus DSA-negative patients [3]. However, such differences only emerged after about 6 years post-transplant. Similarly, in a recent robust study by Loupy et al., most of the 1301 patients from the cohort started at <1 ISHLT CAV grade in 1 year after HTx, further following 4 distinct CAV trajectories up to 10 years post-transplant [26]. Hence, we cannot exclude the possibility of DSA-related intimal thickness progression later in the post-transplant period.
Our ability to detect small changes of coronary wall layer thickness over time yielded the observed highly statistically significant association between DSA variables and medial thickness progression in our HTx cohort. Progression of intima/media thickness is predominantly based on vascular smooth muscle cells (SMCs) proliferation [27]. SMCs most probably arise from the medial layer of a donor or from progenitor cells resident at the medial/adventitial border [28]. In experimental models, alloantibody activation of medial SMCs was demonstrated to be a first step of CAV, followed by their subsequent proliferation and migration to intimal layer [29]. Hiemann et al. reported that luminal size reduction in the small arteries (detectable by histology from biopsies) is present as early as in the first year after HTx, which is in 90% caused by thickening of the medial layer [30]. Thus, it is possible that medial thickening is an important early indicator of CAV development, reflecting the increased mitotic activity of SMCs that precedes intimal infiltration and the progression of intimal changes. Moreover, as previously reported in IVUS studies [31,32], early CAV changes were associated with long-term prognostic significance, therefore diagnosis of such changes as early as 1 year after HTx is of paramount importance.
The clinical significance of antibodies directed against major histocompatibility complex class-I chain‐related antigen A (MICA) is not well understood [7,33,34], but few studies reported that anti-MICA was strongly associated with acute rejection and CAV [7,34]. The incidence of anti-MICA varies considerably, between 3% in healthy individuals to more than 30% after kidney and heart transplantation [35]. In our population, we observed its occurrence in 17% of patients, but no relation to intimal or medial thickness progression was found.
Limitations
Baseline OCT imaging results were only available at M1 after HTx. Thus, we were unable to differentiate between donor‐transmitted coronary artery disease and de novo progression of CAV during the first post‐HTx month. Due to the additional need to administer angiographic contrast and the associated risk of renal function deterioration, only one vessel per patient was examined by OCT. Due to the limited depth of OCT tissue penetration (1.5‐3 mm), layered coronary structures, especially the external elastic lamina, were not always visible in some patients with extensive donor‐transmitted coronary disease, thus impacting our ability to precisely detect changes in the coronary vasculature.
Anti-HLA DSA/anti-MICA were collected 1, 6 and 12 months after HTx, nevertheless we cannot exclude scenarios, in which patients might have developed antibodies in other periods within 1 year after HTx, during which antibody presence was not tested.
Immunology testing did not include testing to distinguish complement‐binding donor‐specific HLA antibodies from non‐complement‐binding antibodies.
Our relatively short follow-up (1 year after HTx) could have impacted results of our study, therefore, we cannot exclude the possibility of DSA-related intimal and medial thickness progression later in the post-transplant period.
Conclusions
Our study did not identify a direct association between presence of DSA and progression of intimal thickening within 12 months after HTx. Nonetheless, DSA, de-novo DSA and HLA Class-II DSA presence were significantly associated with media thickness progression, which may describe a newly recognized early-stage immune-pathological process of CAV. As reported above, medial thickening over time can be reliably quantified using 3D OCT imaging and may potentially serve as the earliest quantitative indicator of the CAV onset.
Highlights.
Using our highly automated 3D-OCT analysis, we observed significant luminal area reduction, and intimal thickness progression within the first post-transplant year.
DSA, de-novo DSA and HLA Class II DSA presence were significantly associated with media thickness progression, which may describe a newly recognized early-stage immune-pathological process of CAV.
DSA and anti-MICA both occurred in 17% of all patients, and their presence was not related to intimal thickness progression within the first post HTx year.
Acknowledgements
We would like to thank Associate Professor Andreas Wahle for his contribution to data management.
Funding:
This project was supported by research grants from the Czech Ministry of Health (16-27465A), MH-CZDRO (IKEM-IN 00023001), and NIH (R01-EB004640).
Footnotes
Conflict of interest: Authors have nothing to disclose.
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