Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : September 22, 2023
Accepted : December 14, 2023
Published online : March 31, 2024
doi:10.24659/gsr.11.1_7

Original article

The role of the low density lipoprotein receptor related protein 1 (LRP1) andthe receptor for advanced glycation end products (RAGE) in the ageing process
Nikola Barić
Private Family Medicine Practice in Labin, Labin, Croatia

Abstract

The process of ageing is currently seen as chronic progressive, unstoppable and irreversible changing of the living being's
structures and functions, which leads to the decline of its adaptive capacities and increased risk for injuries, diseases and death.
The ageing process can be broken down into two closely interwoven components: biological, i.e. normal or physiologicalageing, driven by a defined epigenetic/genetic program, and pathological or accelerated ageing, defined by the influence ofprimarily chronic uncontagious diseases. Presently, a number of theories are trying to answer the crucial questions, how andwhy this inevitable and complex biological process takes place in all living beings. On one side there are different variationsof the programmed ageing theory, and on the other side there are ageing theories based on chronic damage of organism
structures and functions that through time lead to irreparable damage, decline of adaptive capacities, weakening of a numberof vital functions and in the end inevitable death. It is important to mention the currently actual theories: theory of reactive
oxygen species effects (ROS), theory of long protein molecule cross-linking (cross linking theories), mutation theory,
autoimune theory, theory of free radical effects (Harman's free radical theory of ageing), non-enzymatic glycation theoryof ageing (advanced glycation end product [AGE] compounds), and the above mentioned programmed ageing theory. The
most recent experimental research emphasizes epigenetics as the crucial factor in this compound, unstoppable and essentiallypurposeful process. The close connection between the process of ageing and Alzheimer's disease (AD), and the role of lowdensity lipoprotein receptor-related protein1 (LRP1) receptor, and the receptor for AGEs (RAGE) in this degenerative disease
imply the question of the possible role of these two outstanding multifunctional receptors with numerous ligands in the ageing
process. The LRP1 receptor is particularly sensitive to epigenetic factors. Due to abundant methylation, its gene (location
12q13.3), is suppressed with the transcription drop, accompanied by the disorder of its many functions, especially those linked
to the drainage of harmful, toxic macromolecules from the brain. The continuing strong DNA methylation of the LRP1 genepromoter progressing during the life cycle, could be the crucial factor in the weakening of numerous processes, all of whichhave an impact in the accelerated ageing course of living beings. The exceptionally weak methylation of the RAGE promoter,also has a strong impact on the ageing acceleration. This study primarily deals with the essence of the normal biological, i. e.
physiological ageing, and tries to avoid as much as possible the impact of the other component, pathological ageing.

KEY WORDS: biological and pathological ageing, ageing theories, Alzheimer's disease, LRP1 receptor,
		

RAGE (Receptor for AGEs), epigenetics

Introduction

be the crucial factor in the brain ageing, perhaps even in thenormal, physiological ageing of the whole body 1- 4). LRP1, amultifunctional endocytic and cell signalling receptor, a large
endocytic receptor for more than 50 ligands, is dominantly
expressed on the abluminal side of endothelial cell membranesof the blood brain barrier (BBB). It is composed of 515 kDa
extracellular α-chain and 85 kDa intracellular β- chain
noncovalently connected. Its crucial function in the brain is
endocytosis, transcytosis and exocytosis of the mentioned toxic
metabolites, among them the amyloid beta peptide (Aβ) 5).
Of all its ligands, it is necessary to mention the following:
amyloid precursor protein (APP), bacterial by-products, tissue

Decreased clearance from the brain of a number ofpotentially toxic metabolites (for example: glutamate, Amyloidβ [Aβ], advanced glycation end products [AGEs]), could be apossible reason for pathophysiological events which can resultin the acceleration of fundamental processes included in theageing of the brain. The tight link between Alzheimer's
disease (AD), which is marked by an accelerated rate ofageing, and decreased low-density lipoprotein receptor-related
protein 1 (LRP1) receptor expression, lead to the logicalconclusion that the LRP1 receptor expression decline could
Corresponding to: Nikola Barić MD, PhD
Private Family Medicine Practice in Labin,
Presika 153, 52220 Labin, Croatia
TEL : + 385 - 052-852- 008 e-mail: nikolabaric100@gmail.com

Glycative Stress Research 2024; 11 (1): 7- 20(c) Society for Glycative Stress Research_ 7 _LRP1 and RAGE Receptors and Ageing

plasminogen activator (tPA), plasminogen activator inhibitor1(PAI-1), apoE, α2-macroglobulin (α2M), proteases, proteaseinhibitor complexes, extracellular matrix proteins, growth
factors, toxins, and viral proteins. LRP1 is also abundantly
expressed on the membranes of many cell types, such asneurons, astrocytes, macrophages, vascular cells, cerebral
glial cells, hepatocytes, fibroblasts, monocytes, adipocytes,
retinal Müller glial cells, and malignant cells. It is foundthat LRP1 has a crucial role in many processes included intumorigenesis and tumor progression 2, 6 -13).
The LRP1 gene promoter is enriched with CpG islands(regions where CG sequences are concentrated) and sites thatinduce the sensitivity of the LRP1 gene to DNA methylation(strong epigenetic event). Related to the DNA methylation levelrise, the LRP1 transcriptional activity decreases, and whenCpG sites are completely methylated, its transcriptional
activity completely disappears and LRP1 expression ceases( Fig. 1 ) 14). As opposed to the L RP1 gene, in the processof ageing and AD, there is a significant decline of DNA
methylation in the promoter of the gene for the receptor
for advanced glycation end products (RAGE). During theageing process and in AD, around this promoter there

is an intensified accumulation of AGEs, cytokines (i.e.,interleukin-1 [ IL-1], tumor necrosis factor-α [TNF-α]) andnuclear factor-кB (NF-кB), all of which can decrease thepromoter DNA methylation (blockade of the Dnmt [DNAmethyltransferase] approach to the RAGE promoter), andincrease RAGE gene expression (Fig. 2) 15).
RAGE, the multi-ligand and multifunctional receptor,
is a 50-55 kDa glycosylated protein with three extracellulardomains (res. 23-342 aa, V, C1, C 2 ), one hydrophobictransmembrane domain (343-363 aa) and the cytoplasmic
tail (364- 404 aa). Residues V- C1 contain numerous positive
charges of Arg and Lys residue, and they can easily bind withmany RAGE ligands that have high negative regions16). Why is
RAGE promoter protected from DNA methylation, and LRP1
promoter is not? The RAGE gene, by transcription, does notcode the inactive precursor protein, so that the completely
formed and active RAGE can promptly, even in ER (where it istranslated), contact and bind with the present AGEs, and forma close protective concentration (RAGE-AGE) around theclose promoter. The large Dnmt molecule can not approach thepromoter. Dnmt is the largest enzyme in humans, 1,620 AA.
LRP1 gene encodes the 600 kDa inactive precursor protein.

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LRP1 gene DNA methylation is abundant. RNA polymerase cannot access methylated Cm5. There exists actually a transcriptional
blockade. The LRP1 gene is silent. All biochemical and physiological processes caused by these genes are weakened or prevented.
Methylation adds a methyl group (-CH3) from SAM to the substrate. Dnmt (M.Hal) catalyzes the formation of mRNA molecules.
The DNA sequences that the proteins bind to, Adcat and L-met, initiate transcription of a single RNA transcript from the DNA
downstream of the promoter. The LRP-1 gene encodes a 600 kDa precursor protein (inactive protein) that is processed by furinin the trans-Golgi complex after passing through ER. As a result, a 515 kDa α chain and an 85 kDa β chain are formed noncovalently (active form). This means that LRP1, which reaches the Golgi apparatus through the endoplasmic reticulum, is stillinactive. No interaction with locally present AGE compounds is observed.
LRP1, low density lipoprotein receptor related protein 1; 5mC, 5-methylcytosine; CpG, regions where CG sequences areconcentrated, easily methylated; SAM, S-adenosylmethionine (also known as Admet); Dnmt, DNA methyltransferase (also known
as M.Hhal); Adcat, adenosyl cation; L-met, L-methionine; ER, endoplasmic reticulum; AGE, advanced glycation endproduct;
ROS, reactive oxygen species ; NF-κB, nuclear factor-κB; Sbs, SAM binding site; S, sulfur.

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Fig. 2. RAGE promoter-Dnmt and SAM can not approach the DNA.

Dnmt is the largest human enzyme (1,620 amino acids). It is a key factor in carrying out methylation of DNA cytosine C5 and theconsequent blockade of transcription. These events involve many biochemical compounds, which act as a barrier by staying aroundthe promoter region.The compounds include NF-κB, AGEs, TNFα, ICAM-1, VCAM-1, cytokines, and chemokines.
Dnmt, DNA methyltransferase (also known as M.Hhal); AGE, advanced glycation end product; RAGE, receptor for AGE; CML,
carboxymethyl lysine; CEL, carboxyethyl lysine; SAM, S-adenosylmethionine (also known as Admet); NF-κB, nuclear factorκB; TNFα, tumor necrosis factor α; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1;
MAP, mitogen-activated protein; CpG island, regions where CG sequences are concentrated, easily methylated; LRP1, low density
lipoprotein receptor related protein 1; 5mC, 5-methylcytosine; SAM, S-adenyl methionine (also known as Admet); Dnmt, DNA
methyltransferase (also known as M.Hhal); Adcat, adenosyl cation; L-met, L-methionine; ER, endoplasmic reticulum; ROS, reactive
oxygen species; Sbs, SAM binding site; S, sulfur.

This inactive form can not react with the locally present
AGEs, and after passing across the nucleus and ER, it
becomes processed by furin in the trans Golgi complex. The
result is the formation of the active LRP1 form. Now, faraway from the LRP1 promoter, a certain interaction of active
LRP1 with AGEs is probably possible. However, aroundthe LRP1 promoter there is no effective protective LRP1AGE accumulation against the Dnmt approach and DNA
methylation. Consequently, DNA methylation freely occurs(Fig. 1 ) 15).
Tohgi H. et al. 17), using the bisulfite method, the polymerase
chain reaction (PCR) and the direct sequencing of PCR
products have found a significant age-related decline ofmethylated cytosine in the RAGE promoter region during
the human cortex autopsy. This reduction in the number ofmethylcytosines (5mC) at transcription factor binding sites
increases the expression of RAGE, which may in turn play arole in the ageing of the brain.
The enhanced RAGE expression due to the significantly
enhanced AGE accumulation in the course of ageing resultsin the protein kinase C (PKC) activation, with the activationof nicotinamide adenine dinukleotide phosphate oxidase(NADPH oxidase), and oxidative stress (accumulation ofreactive oxygen species [ROS]; O 2* ¯, H 2 O 2, *OH). Oxidative
stress activates the NF-кB cascade, and induces the elevationof the following: platelet-derived growth factor (PDGF),_ 9 _LRP1 and RAGE Receptors and Ageing

vascular cell adhesion molecule-1 (VCAM-1), intercellularadhesion molecule-1 (ICAM-1), endothelial-leukocyte adhesion
molecule 1 ( E-selectin), monocyte chemoattractant protein-1(MCP-1), macrophage colony-stimulating factor (M-CSF),
cyclooxygenase -2 (COX-2), matrix metalloproteinase (MMP),
TNF-α, and interleukins ( ILs) 1, 15).
Kan S. et al. 18), analysed the effects of 5 - aza - 2' deoxycytidine (5'-aza-dC) demethylating agens on the
methylation status of the RAGE gene promoter in peripheral
blood mononuclear cells (PBMCs) of type-2 diabetes mellitus
retinopathy. The inhibition of the RAGE promoter DNA
methylation by 5-aza-dC resulted in a significant rise of IL-1β,
IL-6 and TNF-α in blood samples. The explanation lies in
the intensified RAGE receptor expression and its effects.
Oxidative stress induces astrocyte dysfunction and the NF-кB
cascade with signs of atherogenesis 1, 5, 15, 19). It is necesary toemphasize that LRP1 gene deletion (gene knockout) is lethal
for the embryo in mice 8, 12, 13, 20).
LRP1 is also included in a number of biological processes
as lipoprotein metabolism, degradation of proteases, activationof lysosomal enzymes, LRP1-mediated endocytosis, activationof cell signalling, phagocytosis of myelin debris, phagocytosis
of apoptotic cells, and cellular entry of bacterial toxins andviruses. Gonias SL et al. 8) and Nichols CE et al. 11), using
murine models, have investigated the effect of genetic disruptionof the LRP1 gene in smooth muscle cells on the pulmonary
function in naive animals and after exposure to bacterial
lipopolysacharides (LPS) or house dust mite extract. They
have found an increase in tissue resistance, elastance, and tissue
elastance at the baseline. The dysregulation of LRP1 in smoothmuscle cells affects the baseline pulmonary function andairway responsivenes.
LRP1 expression decline is typical for ageing. Analysing
the levels of LRP1 gene mRNA in isolated cerebral
microvessels, Osgood D. et al. 2) have found that these levels
were significantly reduced in ageing. This indicates that the
LRP1 gene transcriptional activity is also reduced. Considering
the crucial role of DNA methylation and demethylation in theregular embrional and later life cycle, the LRP1 gene functions
could indicate the LRP1 essential role in the complete ageing
process. Why would it not be possible that the LRP1 gene isthe crucial factor in accelerated ageing?
LRP1 receptor is very sensitive to oxidative stress(oxidative attack), which is otherwise increased in the brainsof the aged. The resulting LRP1 receptor oxidation results inthe alteration of a number of biological processes related tothis receptor 1, 7).
Actually, we do not know the crucial mechanisms
involved in the biology of ageing that is composed of complex
genetic and epigenetic events. A number of data indicate that
one universal program determined by evolution surely exists,
where epigenetic factors, interrelated with the genome, have avital role. Therefore it is possible to implicate the LRP1 effects
in this universal program, but, with the assumption that LRP1
effects are not the crucial ageing program per se.
L RP1 genes and their receptors are found in all living
beings, including mammals, reptiles, amphibia, fish, birds,
plants, fungi, mold, bacteria and viruses. All living beings
are susceptible to the ageing process and death. This study
deals exclusively with the ageing of mammals, and humans
in particular. The close functional link of AD and accelerated

ageing indicates that in the case of ageing without AD, as
well as without the genetic predisposition for this disease,
the LRP1 gene and its receptor can be included in themechanism of programmed ageing and inevitable death. Thisis especially credible if we consider epigenetic mechanisms,
particularly DNA methylation and demethylation. It has
already been emphasized that gene promoter LRP1 isinclined to DNA methylation 14). All this indicates that thepermanent programmed DNA methylation/demethylation
can be the component of programmed ageing and death (asemphasized before, without the presence of AD or its genetic
predisposition). However, the available literature does not give
direct facts about the possibility that the programmed ageing
is linked to the LRP1 gene and its receptor.

DNA methylation and demethylation related
to the life cycle

In the above text, it has already been stressed that
epigenetics does not deal with genetic mutations, but
exclusively with specific phenomena such as histone
acetylation and deacetylation, histone methylation anddemethylation, and especially DNA cytosine methylation anddemethylation. All these epigenetic phenomena are linked to
specific enzymes. The epigenetics of histones is not analysedin this study; however, epigenetic phenomena of DNA
methylation and demethylation should be explained. DNA
methylation is induced by DNA cytosine methyltransferase(Dnmt) enzyme, and DNA demethylation is induced by teneleven translocation (TET) family of dioxygenases and thymine
DNA glycosylase (TDG). By the epigenetic mechanism,
Dnmt enzyme transports the methyl group (-CH 3) from Sadenosylmethionine (SAM, also known as AdoMet, SAMe)on the C5 cytosine position with the 5-methylcytosine
formation. Methylation of LRP1 gene promoter CpG islands
leads to the disturbances of transcription factors binding
the accumulation of repressive methyl binding proteins
and the silencing gene expression and transcription. DNA
demethylation is achieved by the interplay of DNA oxidative
reactions and repair mechanisms 21).
Bernstein C. et al. 22), in their study show epigenetic eventsin the early embrional phase of mice on a graph where thepaternal level curve drastically falls in the fertilized female
gamete (oocyte, now transformed into zygote) within the firstsix hours after fertilisation The paternal chromosomes withinthese six hours are almost 100 % actively demethylated( TET3 and repair activity; there are no TET1 and TET2
effects). Maternal DNA methylation level in the zygote(fertilised female gamete) drops more slowly due to active
demethylation and achieves the exceptionally low paternal
level only after 4 - 5 days. The initial value of maternal DNA
methylation level in the moment of fertilisation was under
50 % of the value in paternal chromosomes. Only after 4 - 5days, both DNA methylation values grow slowly in parallel.
Slieker R.C. et al. 23) give a graphic presentation of therelation between the level of the DNA methylation promoter
ELOVL2 gene and the examinees' age. The graph shows
the relation of age and the total blood DNA methylation as
well as the linear statistically significant positive regression
line. There is a regular rise of the DNA methylation level
beginning approximately at age 10 up to almost 85 years.

_ 10 _Glycative Stress Research

The DNA methylation level at the proximal age of 10 isabout 40 % and at about 85 years is 75 %. The analysis ofcertain brain regions, excluding the minimal methylation in
the region of cerebellum, practically shows the same results
as the analysis of the blood.
Hernandez D.G. et al. 24) analysed the DNA methylationlevels of certain CpG sites (10 CpG sites are shown) in the
cerebellum, frontal cortex, pons, and in temporal cortex, andfound a significantly marked increase of DNA methylation in
relation to age in all examined regions. The research includes
the age from 10 to 100 years. In fact, even the oldest groups
are affected by DNA methylation.
Osgood D. et al. 2) point out that in the course of ageing
there is a significant LRP1 reduction in expression. By
measuring the mRNA of isolated cerebral microvessels
during the age of 3, 6, 9, 12, 15, 20, 30 and 36 months inmice, the authors found a considerable drop of LRP1 genetranscription. Presently, very little is known about the causeof this phenomenon. However, the obtained data indicatethat the gene transcription is disturbed during the ageing
process by some upstream events rather than being posttranscriptional. The graphic presentation of LRP1 mRNA
expression indicates its constant growth from the 3rd to the15th month of life, then a constant drop to the 36th month.
This also relates to the receptor synthesis.
Again, the actual question is if it is possible that the
LRP1 gene is the crucial component of the program that
determines the process of programmed ageing. It has beenemphasized that this gene is evolutionarily present in theliving beings genome even from the primeval beginnings,
and that it is closely linked with the evolutionary present
epigenetics. The LRP1 gene is also closely linked with theaccelerated ageing in AD. It has been proven that LRP1 genedeletion in the mouse embrio is incompatible with further
developement and life 8, 12, 20).
It should be stressed that the LRP1 receptor is very
sensitive to glycative and oxidative stress 1).
The biological clock, i. e. the biological program,determines the velocity of the DNA methylation/
demethylation of particular genes, or their certain CpG
sites. The regression analysis curves shown in the graphsof this study indicate that the above mentioned velocity,i.e. the DNA methylation level, is determined by time 22, 23).
The higher velocity of DNA methylation has significantly
greater regression line angles, and lower velocities havesmaller ascending angles. This can be seen in the LRP1 andRAGE receptor curves that are significantly different fromeach other. RAGE has a much smaller ascending angle,
which has also been confirmed by the analysis of its mRNAlevel. The graphic presentation of 10 CpG sites in the study
by Hernandez DG et al. 24) indicates on all the curves (scatter
diagrams) a significantly higher methylation level at the ageof about 50-60 years, and an increased drop after that. At theage of about 100 years, there is practically no methylation.
The study by Slieker RC et al.23) shows similar results. The
biological program that determines the mentioned velocity
is most probably evolutionary determined so that the species
could have optimal conditions to survive. In this respect,
certain phases of human life are also determined by time.
This program also determines the human maximally possible
life span (about 120 years). The same applies to other living

being species.
The analysis of the DNA methylation process by human
DNA cytosine methyltransferase, M.Hhal, shows a remarkably
ageing-related increase in SAM and SAM synthetase levels(MATs). SAM (AdoMet, S-adenosyl-methionine, methyl
donor, cofactor) results from the synthesis of methionine andadenosine triphosphate (ATP) by the effect of 3 isoenzyme
of MATs, SAM synthetase. During the ageing process there
is an increase of SAM as well as MATs. Mammalian cells
express three genes: MAT1A (10q22.3), MAT2A (2p11.2)
and MAT2B (5q34). They encode a catalytic or regulatory
sub-unit used in the formation of three MATs isoenzymes 24).
The above mentioned authors have found a significant rise of
methyonine in aged tissues ( Fig. 3 ).
In the course of ageing, a weaker DNA methylation ofthese three gene promoters (as in the case of RAGE) should
most probably be expected, which would lead to a stronger
encoding of three MAT isozymes 25, 26).
Xing P. et al. 14) emphasize that the promoter region of
the LRP1 gene is enriched with CpG islands (methylation
targets) and sites that govern the sensitivity of the LRP1
gene to DNA methylation (the importance of this promoter
structure). They have found that, when CpG is completely
methylated, the transcriptional activity disappears and theexpression of LRP1 is silenced. CpG islands become moremethylated with increasing age. LRP1 expression declines
and the drainage from the brain of Aβ and other waste
products drops.
Silverberg G. D. et al. 15), in their experiments withmice have found a decreased methylation of the RAGE genepromoter region during the process of ageing. This leads
to a strong RAGE expression, PKC and NADPH oxidase
activation, as well as a significant increase of reactive oxygen
metabolites (ROS, O 2 *¯, H 2 O 2 , *OH ). The ROS elevation
leads to oxidative stress and NF-κB cascade. The increased
values of Aβ, AGEs, TNFα, and especially NF-κB prevent
promoter methylation, thus intensifying RAGE expression
and receptor transcription. Otherwise, RAGE receptor is astrong Aβ influx receptor. Oxidative stress leads to astrocyte
dysfunction and the onset of atherogenesis 27).
The mentioned events related to RAGE receptors and
RAGE genes have been presented in detail in papers by Barić
N. 27, 28).
The process of DNA gene promoter methylation anddemethylation continually takes place during the life cycleof the living being. The curves showing the relation of genepromoter DNA methylation and the age of the living being
have a continually ascending course. In humans, the scatter
diagram is abundant up to 60 years, and after that it obviously
thins out. The methylation of certain CpG sites occurs evenafter the age of 90. If the graphs of LRP1 regression curves
are compared with RAGE regression curves, it is evident thatthe ascending LRP1 angle is markedly steep, and for RAGE
the angle is has a shallow ascent. Obviously, in the LRP1
promoter, strong methylation and gene silencing occurs mucherlier. This silencing continuously takes place up to an
extremely old age in the living being, though gradually withless intensity. Considering the great number of LRP1 ligands
and LRP1 vital functions, it is clear that parallel to the growthof its promoter DNA methylation, these functons through time_ 11 _

LRP1 and RAGE Receptors and Ageing

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Fig. 3. Schematic presentation of S-adenosyl-methionine (SAM) synthesis.

SAM is synthesized intracellularly by MATs catalyzed by methionine, an essential amino acid, and ATP as substrates. As a methylgroup donor, it is an essential methionine derivative for the catalytic reaction of methyltransferases. With ageing, SAM concentrations
increase in the cerebrum, hippocampus, and other parts of the body (Hayashi Y et al. 25)). SAM is a coenzyme, and its presence isessential for the activity of the enzyme Dnmt. It is composed of an adenosyl cation bonded to the sulfur (S) of methionine, whichplays an important role as a sulfonium functional group. MAT1A, MAT2A, and MAT2B are three genes. The MAT1A gene isexpressed only in the adult liver. MAT2A and MAT2B are expressed in fetal liver and adult non-liver tissue. Three MATs isozymes,
Sams, isoenzymes AdoMet synthetases, and Methyonine adenosyltransferases; SAM, S-adenosylmethionine (also known as Admet);
MAT, methyonine adenosyltransferase; ATP, adenosine triphosohate; Dnmt, DNA methyltransferase (also known as M.Hhal); Adcat,
adenosyl cation; L-met, L-methionine; ROS, reactive oxygen species; AGE, advanced glycation end product; IL, interleukin; exp,
expression; Sbs, SAM binding site; S, sulfur.

_ 12 _Glycative Stress Research

are increasingly weakened and they are slowly exstinquished.
The weakening and slow exstinguishing of these functions
is actually equivalent to the process of ageing. The RAGE
gene has significantly slow methylation, and its receptor, in
relation to the LRP1 receptor, has a notably reversed function.
For example, as to Aβ peptides, LRP1 is a significant efflux
receptor, and RAGE is an influx receptor. The reduced
methylation of the RAGE gene promoter actually supports
its strong activity. The consequent oxidative stress, NF-κB
cascade and the astrocyte dysfunction contribute to thedevelopement of atherogenesis, and due to the positive
biofeedback mechanism with Aβ and TNF-α, for the further
strengthing of RAGE expression 1, 23, 24, 27, 29).
Yang J. et al. 30) point out that the main characteristicof DNA methylation is the transfer of the methyl group(-CH 3) from the methyl donor, SAM, onto the active siteof the DNA cytosine methyltransferase enzyme, and C5
everted cytosine from one chain of the corresponding DNA
molecule. It is evident that all phases of the human life cycle
take place under the intensive control of the genetic program
determined by evolution. However, besides this program, aparallel DNA methylation and demethylation program also
takes place. These two programs mutually interweave anddefine the velocity and course of the ageing process. It seems
that of all the genes, LRP1 and RAGE have a crucial role inthis process. When the optimal period for fertilization ends,
both of these genes increasingly change their expression,
thus changing a whole array of biological functions that
actually depend on these two genes. DNA methylation ispermanently in progress, but most probably, due to the lackof DNA demethylation, it gradually suppresses biological
functions linked to the L RP1 gene over time, and encourages
the negative, mostly atherogenic phenomena linked to the
RAGE gene (Fig. 4 ).
Three methionine adenosyltransferase genes, MAT1A(10q22.3), MAT 2A (2p11.2) and MAT 2B (5q34) encode acatalytic or regulatory sub-unit used in the formation of three
MATs isozymes. It has been found that the level of thesethree isozymes (Sams, isoenzymes AdoMet synthetases, andMethyonine adenosyltransferases; three MATs) is elevated inold age. They catalyze the interaction between methionine
and ATP with the formation of SAM. SAM transmits its
methyl group (-CH3) to the Dnmt enzyme and to C5 of the
DNA cytosine everted in the Dnmt enzyme pocket. SAM
level is also elevated in old age. What are the reasons forthese two mentioned elevations ( Fig. 3 ) 29) ?
As there is no other explanation, it can be justly assumed
that, in the case of DNA methylation of three MATs genepromoters, there is a similar situation as in the RAGE genes.
Due to the great accummulation around the promoters, AGEs,
ROS, TNF-α, cytokines, interleukines, NF-κB, PDGF,
VCAM-1, and ICAM-1, the access of DNMT and SAM tothe promoter is blocked as well as to CpG islands and sites.
This protective ring blocks the methylation of CpG islands
and sites on the promoter. However, the continually present
RNA polymerase can become active and provoke the MATs
isoenzymes coding. It should be emphasized that in thecourse of ageing there is a drop of the DNMT expression,and that the RNA polymerase is permanently attached to asliding clamp that prevents the polymerase from falling off

the DNA (Fig. 3 ) 29).
Cui D. et al. 31) have found that the expression levels of
DNMTs decrease with age. Comparing the levels of DNMT
enzymes: DNMT1 (gene location 19 p13. 2), DNMT3A (genelocation 2p23. 3) and DNMT3B (gene location 20q11.21),
in both human and mouse frontal cortex and hippocampal
tissues and in young and old examinees, they have found thatthe levels of DNMTs are evidently decreased with age. Cui D.
et al. 31) emphasize that the DNA methylation is an essential
dynamic biochemical process during the mammalian life
cycle. They also accentuate that DNMTs are abundant in theembryonic stage and significantly decrease after the terminal
differentiation stage. These three enzymes have a vital rolein mediating the DNA methylation process. The methylgroup (- CH 3) is transfered by the DNA methylation from themethyl donors (SAM) onto other genomic DNA sequences(Fig. 4 ).
Tsang S.Y. et al. 32), analysing variations of the global
DNA methylation levels in the ageing process, have found anincreasing trend up to the age of about 55-61 years, followedby the decreasing trend up to 75 years. In this way they haveobtained exact proof that the global hypomethylation in old
age is a strong risk marker for a whole spectrum of age-relateddisorders such as cancer, cardiovascular and neurodegenerative
disorders, and type 2 diabetes. The frequent AD onset at theage of about 65 years and more (LOAD) is in concordancewith the steep decline of the global DNA methylation between
55 and 70 years.
Earlier in the study it has been stressed that there is agreat difference between the global DNA methylation andDNA methylation connected to gene promoters and CpG
islands and sites. While global DNA methylation declines
with age, the latter methylation increases with age. Bothmethylation types are in progress during the whole life
cycle of the living being. Global DNA methylation refers
to the average methylation status that occurs across thewhole genome. Gene-specific DNA methylation refers to
the analysis of the methylation status of specific genes. This
methylation increases with age resulting in the decreaseof the methylated genes expression, and the decrease of anumber of biological functions related to these genes 33).
It is obvious that the DNMTs genes are exposed to a
type of age-related hypermethylation in promoter regions ofspecific genes, with a consequent decrease of correspondent
mRNA levels and decrease of DNMT encoding (as in LRP1
genes) ( Fig. 4 ) 33, 34).
Based on in vitro models and studies carried out in tissues
and blood DNA samples, Bellizzi D. et al. 35) demonstrated adecrease in global cytosine methylation during ageing. On
the other hand, additional studies have been reported with anage-related hypermethylation in promoter regions of specificgenes, with a consequent decrease of correspondent mRNAlevels. In humans, this hypermethylation primarily has been
observed for genes involved in cell cycle signalling, tumorcell invasion, DNA repair, apoptosis, metabolism and cellsignalling.
Lin L. et al. 36) point out that RAGE is a patternrecognition receptor (PRR) that interacts with diverse
endogenous ligands. This receptor is encoded by the RAGE
gene. Ligation of RAGE receptors triggers a series of cellular_ 13 _

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2* 2*, --$)(  ( ()$("  ()*&-'$, .$/&/'  Glycative Stress Research

signalling events including the activation of NF-κB, leading
to the production of pro-inflammatory cytokines, and causing
inflammation. RAGE signalling has been implicated in
multiple detrimental human illnesses including diabetes,
atherosclerosis, arthritis, and Alzheimer's disease. RAGE is
expressed in tissues and cell types that are critical for immune
control, including lung, liver, vascular endothelium, monocytes,
dendritic cells, and innate neurons. The ligand activated by
RAGE interacts with integrins and facilitates the recruitmentof pro-inflammatory leukocytes to the sites of inflammation
and worsens, the inflammation. RAGE plays an importantrole in innate immunity. AGEs are the major in vivo ligandsthat activate RAGE to elicit inflammation.
Isermann B. et al. 37), analysing the interaction between
AGEs and RAGE, have found that the interaction results inan activation of NF-κB cascade increased the expression ofcytokines, chemokines, and adhesion molecules, and theinduction of oxidative stress ( ROS: O 2*¯, superoxide radical;
H 2 O 2 , hydrogen peroxide; *OH, hydroxyl radical). Theyemphasize that except for RAGE, AGEs interact with a numberof other receptors, while RAGE interacts with a diverse groupof ligands. A detailed description of the mentioned interactions
is shown in the study by Barić N 27).
Semba R.D. et al. 38) emphasize that AGEs in the bodyare permanently generated through the non-enzymatic
glycation process resulting from the covalent bonding ofsugar molecules, such as glucose or fructose, to protein or
lipid molecules. The whole process develops slowly andcontinually, and without the influence of enzymes. This
process is accelerated in diabetes. It has been found that thebonding of AGEs with their RAGE does not lead to the AGE
clearance and degradation. On the contrary, it provokes thepermanent postreceptor signallization including the NFκB and MAP kinase activation, with prolonged cellular
dysfunction and localized tissue destruction. The mentioned
bonding causes the enhancement of oxidative stress andinflammatory effects. AGEs form covalent cross-links withproteins. This cross-linking of long protein molecules causes
the increase of tissue density in the aorta, carotides, and othermajor arteries. The pulse wave velocity increases as well as
the systolic and pulse pressure. In all tissues and organs, thetissue density increases and elasticity decreases. Clearly,
these conditions are the ageing phenotype.
Senatus L.M. et al. 39) emphasize that AGEs are formed
throughout life via the process of non-enzymatic glycation ofproteins and lipids. Humans and animals are also exposed toexogenous sources of AGEs ingested through food-derived
AGEs and tobacco products. It is evident that AGE production
and accumulation strictly accompany the normal ageing
process. This results in the high AGE crosslinking of collagenand elastin molecules. The consequent increased stiffness of
the aorta and other conduit arteries is associated with a greaterrisk of ageing-associated cardio- and cerebro-vascular diseases
and mortality. AGE accumulation causes upregulation of
inflammation and destruction of collagen and elastin. RAGE
is expressed in a number of important cell types implicatedin arterial ageing. AGE interaction with RAGE provides amechanism to link AGE-RAGE to arterial ageing and its
consequences, such as stroke, hypertension, atherosclerosis,
myocardial infarction, and heart failure.
Kim H. J. et al. 16) point out that RAGE binds and mediates

cellular responses to a range of damage-associated molecularpattern molecules (DAMPs), such as AGEs, High MobilityGroup Box1 (HMGB1-nuclear protein that binds to DNA andacts as an architecture chromatin-binding factor), and S100/
calgranulins (a group of protein sensors of intracellular
calcium levels). RAGE and its ligands stimulate the activationof diverse pathways, such as p38MAPK, ERK1/2, Cd42/Rac,and JNK (c-Jun amino terminal kinase). Upregulation of
RAGE expression has been reported in atherosclerosis, AD,
cardiovascular diseases, and immune/inflammatory diseases.
As distinguished from LRP1, RAGE knockout mice arehealthy and developmentally normal. This suggests partial
RAGE knockdown might be a safe therapeutic strategy. RAGE
receptor elevation is recognized as the hallmark of ageing.
RAGE -/- mice present a pro-longevity phenotype. Once again,
the authors emphasize that ageing leads to the accumulationof AGEs in tissues and plasma.
Noroozi R. et al. 40) emphasize that ageing, as anirretrievable phenomenon during the whole life, is marked
by a progressive decline in physiological funcionality anddisease variability. Epigenetic modifications, Especially DNA
methylation, correlate with ageing and age-related diseases.
The analysis of the functions of both genes (LRP1 andRAGE) and their receptors clearly shows that during theageing process and old age their functions change through the
DNA methylation process. The LRP1functions increasingly
weaken (e.g., Aβ drainage), and RAGE functions areintensified. RAGE promoter has free access to transcription
factors, thus enabling the transcription. In LRP1 genes, most
CpG sites in the promoter are methylated and transcription
factors (TFs) do not have direct access to CpG sites in the
promoter. It should be noted that TF regulator proteinsapproach the beginning part of the gene. They provoke or
prevent gene transcription, enabling RNA polymerasis torecognise the gene and to bind with the promoter. It should
be noted that RAGE is expressed at low levels in normal
tissues. The analysis of these changes indicates that they
actually correspond with the damages and changes related
to ageing and old age, and that they essentially correspondto the process of ageing. LRP1, RAGE, and their receptors
likely play the crucial role in the primeval programme of thisinevitable and purposeful process.
It is clear that the DNA methylation /demethylation
processes continuously go on, faster or more slowly throughout
the life cycle of the living being. In the early embrional
phase, DNA methylation drastically declines with the DNA
demethylation rise. In this phase, active are Ten-eleventranslocation 3 (TET3) -dependent active demethylation and
DNA replication-dependent passive demethylation. The volumeof DNA methylation falls to the value 43 % of the earlier
value. After the forming of blastocyste (fourth day after
fertilisation) starts the epigenetic reprogramming, and bymeans of DNMTs, DNA methylation is again restored. In
a very small space there are everted DNA cytosines, RNApolymerase, DNMT enzymes, ATP, methionine, SAM, andMATs isoenzymes. Following is the DNA methylation withthe subsequent transcription blockade (Fig. 5 ).
Epigenetic events continuously go on, and among them,
DNA methylation is especially important. It correlates withageing and age-related diseases. Using the age-related
alterations in the DNA of certain CpG sites, several_ 15 _

LRP1 and RAGE Receptors and Ageing$#&&/+#&!,#*"#&!' &,%)
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**&+#$("0*#'$'!#$()'***#&,
0+"*!&*$$("**' +"",%&$# 0$)*+)'&!$0'&+)'$$0+"-'$,+#'&+)%#&!&+#()'!)%% *#%,$+&',*$0.#+"+"#*()'!)%+" %+"0$+#'& %+"0$+#'&()'!)%#*$*'+,$ +"*+.'()'!)%*)
$#&&*%'+")#&!' $'*$0#&+))$+&,%)' -#+$#'$'!#$ ,%,$+#'& ,&+#'&*
Fig. 5. Schematic presentation of permanent DNA methylation/demethylation during the eukariotic life cycle.

The black broad line presents the father's life cycle, and the pink broad line presents the mother's life cycle. DNA methylation
and DNA demethylation are two tightly connected epigenetic events. The global DNA methylation decreases with age. Agerelated hypermethylation is found in promoter regions of specific genes. The active demethylation is induced by TET family of
dioxygenases. LRP1 deletion (knock-down) in the embrionic phase is incompatible with life. Events during the life cycle have an
essential epigenetic biological program (biological clock).
TET3, methylcytosine dioxygenase – band 2p13.1; TET2, methylcytosine dioxygenase – band 4q24; TET1, methylcytosine
dioxygenase – band 10q21.3; Thimine DNA glycosylase (TDG) – band 12q23.3; TF, transcription factor; LRP1, low density
lipoprotein receptor related protein 1; AGE, advanced glycation end product; RAGE, receptor for AGE; Dnmt, DNA
methyltransferase (also known as M.Hhal); TET, ten-eleven translocation; TF, transcription factor; DNA rep, DNA replication;
RP, repair process; LR, LRP1 and RAGE; Meth, DNA methylation; Demeth, DNA demethylation; B, birth; F, fertilization; M,morula, 16 cells; Z, zygote; D, death.

_ 16 _Glycative Stress Research

investigations have attemped to predict chronological age 41-43).
Sanada F. et al. 43) emphasize that ageing is a complex
process that results from a combination of environmental,
genetic, and epigenetic factors. It is marked with a chronic
low-grade inflammation in the absence of overt infection.
In contrast to young individuals, aged individuals haveconstantly elevated levels of inflammatory cytokines,
especially IL-6 and TNF-α. They have also found in visceralfat of obese individuals the accumulation of macrophages,
which are a major source of low-grade persistent systemic
inflammation and insulin resistance. Cigarette smoke is fullof ROS with the production of IL-6, TNF-α, and IL-1β.
Many tissues in the elderly are chronically inflammed, andIL -6, IL-1β, and TNF-α are known to weaken the anabolic
signalling cascade, including insulin and erythropoietin
signalling with sarcopenia development. Presented
inflammatory cytokines are the same as cytokines connectedwith RAGE and its expression. No comment is necessary.
The analysis of scientific papers that comprehensively
deal with the human ageing process shows remarkableconcordance of detected biomarker characteristics typical ofageing and old age and the effects of LRP1 and RAGE 44-47).

Attempts to slow down the ageing process in humans

If we accept the hypothesis related to the elevatedlevel of RAGE expression as the crucial factor in the ageing
process, the question arises whether the use of RAGE
blockers to slow down the course of ageing is justified.
Kong Y. et al. 47) exclusively deal with animal models of
AD therapy. They investigate therapeutic possibilities ofthe strong RAGE blockator, FPS-ZM1, with its toxic side
effects and earlier detected biological effects. They pointout the damaging effects on the human body of high RAGE
expression levels. RAGE activates the NF-κB cascade withintensive secretion of proinflammatory cytokines, TNF-α,
IL-6, and macrophage colony-stimulating factor 1 (CSF-1).
RAGE activation leads to the production and aggregationof Aβ and the forming of neurofibrillary tangles (NFTs),
as well as to the destruction of synaptic transmission andneurons. All this promotes the onset and development of
AD. The above mentioned authors have found that thecompound, FPS-ZM1, effectively passes across the BBB,
affects the V-type region of RAGE, and after binding to
the receptor, it blocks its intracranial functions. FPS-ZM1,
after binding, completely restores the brain blood flow,
inhibits neurotoxicity, decreases the microglial activity andthe neuroinflammatory response, and improves the cognitive
behavior. There are no toxic effects, even when the applied
dose was 500 times higher than the therapeutic dose. The
above mentioned facts are related to the experiments withmice. The FPS-ZM1 molecular formula is C 20 H 22 CINO or
N-benzyl- 4 -chloro-N- ciclohexylbenzamide.
Huang J. et al. 48) present the results of exceptional
therapeutic effect of the FPS-ZM1 in the treatment of
periodontal disease (gingivitis and periodontitis) in a groupof patients. There were no side effects. These results give
great hope for future therapeutic attempts in humans.
Deane R. et al. 49) synthesized the FPS-ZM1 in the year
2012. This is an extracellular polymeric substance, organic

polymer, of bacterial origin that for bacteria serves as
prevention from unfavourable environmental effects.
Singh H. et al. 50) emphasize that the accumulation ofAGE compounds in the body has a significant role in theonset and course of many inflammatory health disorders,
including cardiovascular diseases, diabetes mellitus, immune
inflammation, cancer, and neurodegenerative disorders. The
detection and binding of AGE compounds is dominantly
performed by the already mentioned RAGE. The binding ofAGEs, as well as other ligands, to RAGE activates multiple
signalling pathways, such as STAT3 (signal transducer andactivator of transcription 3), MAPK/ERK (mitogen-activatedprotein inase/extracellular signal-regulated kinase), and JNK,
which results in the increase of transcription factors, including
NF-κB. The mentioned binding is included in the pathogenesisof numerous inflammatory diseases, such as atherosclerosis,
diabetes mellitus, cancer, neurodegenerative disorders,rheumatoid arthritis, and chronic renal failure. The RAGE
inhibitor FPS-ZM1 has shown significant effectivenes inblocking an array of RAGE ligands, resulting in a significant
drop in the oxidative stress level and in inflammatory events.
Hong Y. et al. 51) have found an increased level of AGEs
in observed brains of AD patients. AGEs and RAGE haveimportant roles in the pathogenesis of AD. Experimenting
with rats, they have found that after intrahippocampal
injection of AGEs, the intraperitoneal administration of FPSZM1 has significantly reduced Aβ elevated production
induced by AGEs, inflammation, and oxidative stress. They
conclude that FPS-ZM1 might be a novel therapeutic
agent to treat AD patients. Barić N. 1), in his paper, gives adetailed review of key anti-AGE and anti-RAGE therapeutic
approaches. These methods are also comprehensively presented
in the study by Senatus L.M. et al. 39).
Kang D. E. et al. 52), Shibata M. et al. 53), and Silverberg
G.D. et al. 15) point out that the LRP1 expression at the BBB
is reduced during both normal ageing and AD. To correct
the decreased LRP1 expression in the brain, Sagare A. P. et
al. 54) suggest three sets of actions: a) lifestyle changes (e.g.,diet, exercise, and enriched environment), b) pharmacologicalagents (e. g., statins and plant-based active principles), andc) gene therapy. Sagare A. P. et al. 54) emphasize the great
importance of antioxidant nutrients (polyphenol-rich foods,
vitamins A, C, and E, green tea, and extra virgin olive oil )which could protect LRP1 from oxidative damage. Physicalactivity can improve cerebral angiogenesis in rodents andhumans. Statins are the target cells of cholesterol loweringdrugs. Simvastatin and atorvastatin are especially effectivein the brain. The authors stress that effective gene transfer
can be achieved by viral based systems which are highly
effective in mediating cell entry and transfer of genes.
Adeno-associated virus (AAV) is frequently used as a vector
for these therapies. The use of AAV2, which carries LRP1
DNA or its smaller fragments, leads to the restoration of
LRP1 expression in the brain vascular endothelial cells in
AD. The detailed explanation of the LRP1 gene deficiency
gene therapy is presented in the paper by Barić N 1). The
elevated LRP1 expression in BBB is obtained by rifampicin 55).
The portal infusion of insulin leads to the evident rise of
LRP1 expression on the hepatocyte plasma membrane.
According to Shinohara M. et al. 13), the environmental
enrichment improves the vascular dysfunction by increasing_ 17 _

LRP1 and RAGE Receptors and Ageing

angiogenesis accompanied by the upregulation of LRP1.
Exercise ameliorates brain Aβ deposition and cognitive
decline in APP transgenic mice with the upregulation of
LRP1. Oleocanthol (a type of natural phenolic compound)from extra virgin olive oil upregulates LRP1 at the BBB andenhances Aβ clearance 1, 56, 57). Cannabis sativa plant treatment
also enhances Aβ transit at the BBB with increased LRP1
levels at the BBB 1, 58). Statin treatment can also upregulate
LRP1 in the liver as well as in the brain 1, 59). Special attention
in the anti-ageing treatments has recently been directed toGinkgo biloba, especially to Ginkgo biloba leaves extract(standardized special extract, Egb761) 60-63). Reddy V. P. et al. 64),in their comprehensive study, present detailed facts related to
Alagebrium (ALT-711), aminoguanidine, and pyridoxamine.

hypothesis probably sounds unrealistic, but many times it has
happened that something so obvious has not been recognised.
During the entire life cycle of eukariotes, starting from theirprimeval beginnings, the DNA methylation and demethylation
processes, simultaneously and with a certain balance,
permanently take place in their cell nuclei, thus determining
the program of the biological time clock. These processes
separated by fertilization into events related to parents, andevents related to descendants, represent the base, primarily inparents, for the ageing process with the crucial role of LRP1
and RAGE.

Conflict of interest declaration
None.

Conclusion

Based on the analysis of numerous ligands linked to LRP1
receptor, RAGE, and a number of important biochemical
processes that they control, these two genes, associated with
DNA methylation/demethylation, could possibly be crucial
factors in the program that drives the ageing process. This

Research grant

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