In this paper, we aimed to analyze the data collected in last 7 years from patients with AMD treated with rheohaemapheresis in order to elucidate the benefit of this procedure with respect to plasma lipids, selected biomarkers of inflammation, endothelial dysfunction, and a novel regulatory protein of lipid metabolism - PCSK9.
There are three main findings in this study. (i) Treatment with rheohaemapheresis was associated with the long-lasting, significant and desirable improvement of visual acuity concomitantly with the decrease in the original area of pathological changes (drusenoid pigment epithelium detachment, area of soft drusen, area of RPE-atrophy) in 10 from the total of 31 patients (32.2%), while therapeutic failure was present in 4 from 31 patients (12.9%). 17 patients (54.8%) did not have at least a 5-year follow-up, therefore they were not included for this evaluation. (ii) Treatment with rheohaemapheresis was associated with the significant reduction of lipids (TC, LDL-C, HDL-C, apoB, Lp(a)) and PCSK9; biomarkers of endothelial dysfunction (CD40L, MCP-1); and rheologically important parameters (fibrinogen, blood and plasma viscosity) after each procedure. (iii) The improvement of the best-corrected visual acuity and ophthalmoscopic findings in successfully treated AMD patients was associated with significantly lower lipid parameters (LDL-C, apoB and PCSK9).
We have shown in the present study, that the treatment of the in patients with advanced dry AMD by rheohaemapheresis resulted in the BCVA improvement during long-term follow up. These results confirmed our previous findings, where we demonstrated improved BCVA in the group of treated patients and statistically significant deterioration in the control group of patients . Similar results published the authors of the MAC-I study from the University of Cologne, MAC-II study from the University of Frankfurt, and MAC-III study from the University of Hamburg .
While the use of rheohaemapheresis was associated with successful treatment of AMD, the precise mechanisms have to be established. There are several plausible explanations resulting from this study with regard to lipid metabolism. The retina has multiple physiological demands for cholesterol utilization. There are physiological changes in cholesterol metabolism that occur with aging, and these affect the RPE. Drusen and basal linear deposit are quite specific for AMD, and cholesterol is present in drusen, which are pathognomonic disease markers both on clinical examination and in histopathological study . Eye pathology studies demonstrate a high cholesterol concentration in classical AMD lesions, such as drusen, aging BM, and newly discovered subretinal lesions . Numerous cholesterol and lipoprotein-related proteins and genes are expressed in human RPE and retina .
The beneficial effects of rheohaemapheresis treatment was associated with a significant decrease of total cholesterol, LDL-C, HDL-C, apoprotein B, and lipoprotein (a) levels in AMD patients after all rheohaemaphereses during the monitored interval of 7 years. Moreover, the baseline LDL-C and ApoB were significantly higher in AMD patients with therapeutic failure than in successfully treated patients in our study. One explanation could be that treatment with statins was more frequent in the group of successfully treated patients (n = 5; 50%) than in the patients with therapeutic failure (n = 1; 25%). There are a number of mechanisms by which statins may exert protective effects in AMD. These include, but are not limited to, serum lipid-lowering that may alter BM lipid deposition ; preservation of the outer retinal and choroidal vascular supply by an anti-atherogenic effect ; anti-inflammatory properties ; antioxidant effects that may counter increased plasma levels of oxidized LDL ; and inhibition of metalloproteinases that may contribute to fissuring and rupture of plaques that lead to neovascularization . HMG-CoA reductase inhibition may also have direct effects on cholesterol processing by outer retinal cells. The RPE is a native secretor of lipoproteins, and statins may affect lipidation of lipoproteins directly . However, a number of studies have examined the relationship between AMD and statin effectiveness compared to other treatment options, absence of treatment, or placebo, but the results remain mixed .
We have shown for the first time that another key regulator of lipid metabolism which is serum PCSK9, has been significantly higher in AMD patients than in the healthy control group. This is unique finding with further implication into the possible pharmacological treatment of AMD by novel class of hypolipidemic drugs - inhibitors of PCSK9. So far, there are not any data in the literature studying the involvement of PCSK9 in the development of AMD. One experimental study used minipigs overexpressing a gain-of-function mutant (D374Y) of the human gene PCSK9 with a consequent experimental hypercholesterolemia and found advanced atheromatosis of retinal arterioles caused by lipid overload . The finding of increased plasma PCSK9 in the AMD patients predispose them to the long-lasting hypercholesterolemia and related atherogenic changes in the retinal arteries. On the other hand, efficient removal of LDL-C during the treatment by rheohaemapheresis (LDL-C is rapidly reduced on average by 40% per one session) will deplete cholesterol cell content and, via upregulation of sterol regulatory element binding protein (SREBP)  will down-regulate PCSK9 gene expression and thus lead to the decreased formation of PCSK9. In fact, rheohaemapheresis treatment in AMD patients was associated with a significant decrease of serum concentration of PCSK9 during the monitored interval of 7 years. We assume that the PCSK9 particles had been removed by the rheohaemapheresis filters, although we did not measure PCSK9 concentration directly in the adsorbers. However, we measured the difference of the PCSK9 concentration in the plasma at the inflow of the adsorbers and in the plasma outflow just after the adsorber in random 6 patients. We found that the immediate retention of PCSK9 in the adsorbers is in the range of 17%. Regardless the mechanism the resulting decrease of PCSK9 should lead to the decrease of LDL-C, similarly as does the therapy with PCSK9 inhibitors . Moreover, treatment by rheohaemapheresis also resulted in different results based on decrease of PCSK9 values post-rheohaemapheresis. Thus, the decrease of serum PCSK9 was significant post-rheohaemapheresis in AMD patients with successful treatment, but not in patients with therapeutic failure. These findings further imply that not only lower plasma LDL-C, but also lower plasma PCSK9 should be beneficial in order to successfully treat AMD. We assume that the significant decrease of PCSK9 after individual rheohaemapheresis is not long-lasting, similarly as the decrease of LDL-C. The rebound of LDL-C post LDL-apheresis occurs in 13 days post-treatment , and similar kinetics should be expected post-rheohaemapheresis. Body stores would be quickly depleted if not replaced by absorption of dietary cholesterol or newly synthesized cholesterol. The data has been shown indicating a threshold effect: reduction of LDL cholesterol to levels below 1.0 mmol/l induces an upregulation of the cholesterol biosynthesis in normocholesterolemic subjects . In our study LDL cholesterol was lowered to 0.92 mmol/l immediately after rheohaemapheresis. Despite prolonged, aggressive lipid lowering with rheohaemapheresis which should stimulate cholesterol biosynthesis and thus increase LDL-C, this was not noted in our present study. Most probable explanation is the frequency of aphereses (2 aphereses weekly at intervals of 2–4 days), which prevented the increase of LDL-C.
The beneficial impact of lowering LDL-C and PCSK9 by means of rheohaemapheresis in AMD patients was accompanied also with significant decrease of biomarkers of inflammation and endothelial dysfunction (CD40L, MCP-1) during the monitored interval of 7 years. We previously evaluated MCP-1 during our earlier research of rheohaemapheresis use due to the significant importance of macrophages in the microcirculation and we found a significant decrease in patients with AMD . This study demonstrated its decrease in patients with AMD. As stated in the literature, this may be a significant factor, indicating efficacy of the impact on activity of the inflammatory process or atherosclerosis . The mechanism of the inflammatory process modulation by rheohaemapheresis in the pathogenesis of AMD could also be documented by a significant decrease in inflammatory marker sCD40L in case of atherosclerotic mechanism, respectively. Evaluation of rheologically important parameters before and immediately after rheohaemapheresis showed significant decrease after rheohaemapheresis (fibrinogen decreased by 48%, viscosity of the blood by 9% and viscosity of plasma by 12%). The result is undoubtedly an improved microcirculation flow, which is also a basic prerequisite for increased flow in the choroid and improved retinal metabolism.
Our study had a small sample size, includes only the treatment group, and we did not incorporate a control treatment arm in this study. The patients were compared based on results of ophtalmological examination as the subjects who were clinically successfully treated and/or the individuals with therapeutic failure which shortened even more the number of evaluated patients. The small number of participants in the present study may affect the accuracy of our results. Furthermore, since the lipoprotein apheresis treatment technique is carried out only in our medical centers in the Czech Republic because of the technical and economic reasons, many AMD patients are unable to receive apheresis treatment, resulting in a particular bias in patient selection. Moreover, it is necessary to diagnose AMD disease in the early stage of the dry form, which is not frequently possible. Our study's strength is the collection of a large set of data comprising the long-term monitored interval of 7 years. We have experience with the rheohaemapheresis therapy in AMD patients in our center for more than 10 years, the total number of treated patients is 74, and the PCSK9 concentration was evaluated from the year 2012 on in 66 patients. To assess the outcome in AMD it is necessary to respect its slow progression rate, therefore we set up the interval of 5 years of follow-up and evaluate finally 31 patients who completed it. Finally, our study group is unique also because of the evaluation of the relationship of PCSK9 concentration to the AMD prognosis, markers of endothelial dysfunction, inflammation and rheologically important parameters. The results obtained in this study are valuable and will be evolved in our further research.