More Related Content Similar to Book regeneration 2020 luisetto m et al extended version (20) More from M. Luisetto Pharm.D.Spec. Pharmacology (20) 1. BOOK
TITLE : Regeneration abilities of vertebrates and invertebrates
and relationship with pharmacological research: Hypothesis of
genetic evolution work and microenvironment inhibition role
Authors
1) Luisetto M applied pharmacologist, IMA academy Natural
science branch Italy 29121
2) Naseer Almukthar, Professor, Department of Physiology /College
of Medicine, University of Babylon, Iraq
3) Gamal Abdul Hamid Professor Hematology Oncology, University
of Aden, Aden, Yemen
4) Ibrahim G ,Department of Zoology, Alexandria University, Egypt
5) Behzad Nili Ahmadabadi , Innovative Pharmaceutical product
development specialist, USA
6) Ahmed Yesvi Rafa , Founder and President, Yugen Research
Organization; Undergraduate Student, Western Michigan
University, MI, USA 49008
7) Ghulam Rasool Mashori Department of Medical & Health
Sciences for Woman, Peoples University of Medical and Health
Sciences for Women,Pakistan
8) Tuweh Prince GADAMA the great, Professor,Cypress University
Malawi
9) Oleg Yurievich Latyshev IMA academy president
Keywords: Regeneration, cancer, stem cells, wound healing, reparative re-
generation, invertebrates, vertebrates, pathology, microenvironment , genotypic
-phenotypic expression ,heart regeneration, reexpression embryonic markers,
3. It is only one first aspect but that can produce relevant effect in human
health :
REGENERATION OF CELLS OF ANIMAL AND HUMAN TISSUES
THROUGH THE PROPER USE OF WATER
Prof. O. Latyshev,
President of IMA.
papa888@list.ru
We believe that there is an actual need to better study the
phenomenon of tissue regeneration in multicellular animals in order to
present a new strategy for the treatment of some disabling diseases such
as stroke, retinal degeneration, dementia, Parkinson's disease, as well as
for a better study of the pathology of cancer. We understand that the
regeneration of animal tissues is impossible without proper water
metabolism, from which energy-informational metabolism and
metabolism automatically follow.
As soon as the animal finds a proper source of water, the healing
moisture it has drunk is rapidly directed into the tissue cells, brings there
in sufficient quantities all the necessary nutrients and builds the cell in
many respects anew.
Waste materials from each cell are also transported outside by
water naturally through the pores of the skin and the excretory organs.
Thus, they practically do not have anything obsolete, old, and
unnecessary. The process of cell regeneration under conditions of
adequate water supply occurs continuously and regularly.
Similar positive changes should be expected in the cells of the tissues of
the human body if he consumes only pure water on an empty stomach.
(Whitout toxins or poisons or pollutants) .
Also the time of drunk can be relevant:
4. If a person wishes to take water or other liquid after a meal, as a dessert
or as a third course, by doing so he will render a disservice to various
tissues of his body.
Water drunk at the wrong time will interfere with the timely digestion
of food, and the hope for the complete extraction of nutrients from the
food consumed by a person will be almost completely lost.
As a result, not a single tissue of his body will receive the
appropriate nutrition in full.
Thus, tissue cells will not have enough nutrients not only to
organize regeneration processes, but even to maintain the intracellular
energy balance.
If the habit of drinking water improperly in a person takes root, then
the cells of his nervous tissue will not receive enough nutrition, and over
time,in example Parkinson's disease or other neurodegenerative disease
can develop. ( see the example of Well water and associated PD ).
If a person drank water, it would seem, on time - on an empty
stomach, but instead of pure water preferred artificial mineral water,
packed in a bottle or flask at the factory, he deceived the expectations of
his body, in this case - at the cellular and tissue level: it must be verified
the quality of this to avoid intake of dangerous substantie .
SC Bondy - 21 dic 2017 - Epidemiological evidence reveals that
aluminum levels in drinking water are related to the incidence
of Alzheimer's disease (AD)
Such water does not bring anything except original taste sensations
and a short-term uplift of mood.
In general, artificial- oxidized water(not of quality- or polluted ) will enter
the tissues, which will not allow the cells to restore their original structure,
to rejuvenate the tissues of the human body as a whole.
If the habit of drinking just this water takes root, at any time, it will
ultimately lead to defective cell regeneration, i.e. to the appearance of
5. immature cells capable of creating only cancer tissue.
The need to drink is saving life :
If a person does not drink water an hour and a half after eating, and after
eating meat dishes - after two hours, then water starvation occurs at the
cellular level.
Forgetfulness and unwillingness to drink water on time can lead a person
to many health problems and also stroke in example , since the vessels
of his circulatory system will pump mainly blood cells. And constantly be
in the risk zone of thrombosis of the vascular lumen.
According : article Nutr Rev. water, Hydration and Health
Barry M. Popkin et al :”Good hydration is associated with a reduction in
urinary tract infections, hypertension, fatal coronary heart disease,
venous thromboembolism, and cerebral infarct but all these effects need
to be confirmed by clinical trials.”
And in Int J Environ Res Public Health. 2018 The Effects of Hydration
Status on Cognitive Performances among Young Adults in Hebei, China:
A Randomized Controlled Trial (RCT)
Jianfen Zhang et al :“Dehydration may affect cognitive performances as
water accounts for 75% of brain mass”
In article published in J Cereb Blood Flow Metab. 2014 Water
deprivation induces neurovascular and cognitive dysfunction through
vasopressin-induced oxidative stress
Giuseppe Faraco et al :*
“Oxidative stress is responsible for cerebrovascular dysfunction in a
number of disease models, including ANGII-induced hypertension,
chronic intermittent hypoxia, or Alzheimer's disease”
In the case when a person uses water on time and regularly, but it is not
a dislike for water and a reluctance to drink a lot, then such other
widespread disease of our time (as dementia) can be related .
( see in example Brain wasting system activity ).
6. The cells of the nervous tissue of the human body, which are responsible
for the transmission of nerve impulses, also do not receive enough water
to recreate the proper acting volume.
Therefore, the quantity and quality of nerve impulses transmitted inside
tissue with poor trophies also becomes inappropriate.
As a result, it becomes increasingly difficult to use the arrays of human
memory day after day in work.
Some people prefer coffee, tea, energy drinks, cola, etc. to clean water.
At the same time, they should place a minimum of minimum hopes on a
healthy retina.
Its principle of action is akin to the work of nervous tissue in all parts of
the human body.
All processes taking place in the retina have a finer organization.
This delicate nervous tissue, which is responsible for supplying a person
with a significant amount of information essential for life, is polluted by
drinks, with which a person decided to replace clean drinking water.( also
sugar drinks ?)
Instead of clean water being able to promptly and fully remove waste
substances from the retinal cells and saturate the retinal tissue with full-
value nutrients, an increasing amount of toxins obtained from beverages
polluting the body are deposited on it, as in a kind of depot.
This gradually leads to progressive deterioration of vision. And then - and
to its complete loss due to atrophy of the retina, which has not received
proper nutrition. In addition - unexpectedly abundantly contaminated.
The above prompts us to draw the following conclusions:
1. Regeneration processes in the cells of animal and human tissues
are as successful as the quantity and quality of the water supplying them
corresponds to this.
2. Drinking pure water on an empty stomach help to frees the body
from significant risks - getting cancer, stroke, retinal atrophy, dementia or
7. Parkinson's disease.
In article Arctic Med Res. 1991.Hypothermia and cellular physiology
K E Zachariassen was written :
“Temperature has pronounced and complex effects on cellular
physiology. Rates of enzymatic processes display an exponential change
with temperature, as expressed by the Q10 relationship.
The basis of these effects may be temperature induced phase transitions
in membrane lipids and protein associated water, effects on bulk water
and effects on the relationship between water and inorganic solutes.
Hypothermia may be lead to a collapse in ionic regulation, leading to an
uncontrollable and lethal calcium influx. Subfreezing temperatures may
cause injury due to cellular freezing with subsequent excessive osmotic
swelling, lyotropic effects or excessive osmotic shrinking due to
extracellular freezing. Cells may protect themselves by freeze avoidance
accomplished by removal of ice nucleators, production of proteinaceous
antifreeze agents and accumulation of polyols. Alternatively they may
secure extracellular freezing by production of extracellular ice nucleating
agents, and counteract lyotropic effects and osmotic shrinking by
accumulation of polyols which reduce ice content in a colligative manner.”
1. Hypotermia- glacier water helps the cells of the tissues of his
body to be in a state of constant self-renewal. This allows him
to regularly rejuvenate his body.
2. The presence of tea, coffee, cola and bottled mineral water in a
person's diet does not replace the need to drink clean water.
3. All of the above products for the most part pollute the human
body and destabilize its work.
4. Finally :The desire to drink mineral water, from which an
adequate effect is expected, should be satisfied directly at its
sources, in pump rooms and sanatoriums.
5. Only in this case, the accepted mineral water will really
contribute to the gradual rejuvenation of the human body.
8. This is an determinate approach to the best condition to permit to the
cells to mantein itself in a most possible balanced environment and any
Modify of this by iatrogenic substantia or uncorrect water intake
contribute to cellular toxicity or reduced renovation.
But more other factor are involved in homeostatic regenerative response
and in the best microenviromental condition to favour this ( inhibitor
substantie but also endogenous molecules as result of codified genetic
pattern as we can see next :
it is useful to observe metazoan evolution and genetic factor involved in
some phenomena like wound repair, regeneration of tissue and organs
and also cancer .
Finalistic or afinalistic process,gene evolution , inibithoty factors.
Conserved genes , not conserved genes , inhibition factors , phenotipic
expression, environmental pressure factors , immune response , body
complexity , movement necessity end many other :
In article Endogenous Archeological Sciences: Anatomy, Physiology,
Neuroscience, Biochemistry, Immunology, Pharmacology, Oncology,
Genetics as Instrument for A New Field of Investigation? Modern Global
Aspects for A New Discipline 2018 was written:
“”Today is interesting to submit to the scientific world a new field of
investigation more structured that We can denominate Endogenous
archeology. A new field in which collect different research works from
different discipline in order to correctly classify. Endogenous process and
structure of a sort of archeological provenience but today inside us..
Archeology science not to be consider only related human product and
manufacts but also an inside disciple to verify archeological process
related to mind- set kinetics and to other system or organs. Brain, mind,
immunologic system and other relevant physiological functions are
deeply influenced by a primitive structure and to deeply understand the
meaning of this complex system inside us make possible to better explain
today Human behavior and physiology and other process.”
But it it also interesting to observe some physiological- pathological
phenomena using an evolutionary approach : this make possible to verify
12. animals vary widely in their capacity to regenerate particular tissues.
IN-VERTEBRATES such as planarians and hydra, which can form
whole animals from small -segments, exhibit the greatest regenerative
aptitude .
Mammals, by contrast, fail to regenerate crucial structures, including
limbs, spinal- cord and cardiac muscle.
Certain vertebrates,including urodeles ( salamanders) and teleost fish (
zebrafish),retain the ability to regenerate these and other organs.
Fig. n 4 fish ancient and teleost
It is interesting to understand the degree to which fundamental aspects
of these organisms’ biology, rather than or to cardiac specific factors,
13. allow them to repair their hearts so effectively.
We briefly discuss certain non-cardiac influences on cardio-myocyte
proliferation in this Review, we refer interested readers to other, more
thorough reviews of the comparative biology of re-generation and its
mechanisms.”
Re-generation abilities seem related inverse to the more complexity of
vertebrates
The same inmmune -systems in evolutive pattern was different in more
primitive IN-VERTEBRATES ( innate immunity vs adaptative evolution ):
why ?
It seem that if evolute -organ there is a lost of regenerative abilities .
Relationship with repordutcitve patter?
Re-generation is the ability of tissues, organs or even organisms to
renew themselves or to recover after considerable physical damage. It is
due to the ability of un-affected cells to multiply and, as needed, to
differentiate in order to restore the injured part.
Tissue re-generation capacity depends on the type of cell, tissue or organ
affected by the injury and on the cell's ability to multiply and whether the
cells involved are able to regenerate, but at different levels of capacity.
Peripheral- nervous tissue has low re-generation power, but can recover
in the face of some aggressions, whereas in central nervous tissue
neurons cannot be regenerated.
Some animals are well known for the regenerative capacity of their
tissues, organs or even systems.
The planarians, the axolotes (salamander) and the starfish are examples.
The re-generation of the gecko tails is also an example of re-generation,
in this case of a complete organ.
The epithelium (skin) regenerates quickly and easily when destroyed.
Liver cells (liver) and bone tissue also have high- regenerative power.
Smooth muscle cells are able to regenerate in response to chemotactic
14. and mitogenic factors ( promote mitosis).
Already the muscle is often classified as permanent, being unable to
regenerate.
Connective tissue are also unable to undergo re-generation.
a failure in the mechanism that limits and controls the capacity and rate
of re-generation in specific tissues generally leads to tumor -formation.
The new cell types can come from different sources, such as: stem- cells
present in the body;
from de-differentiation, which is nothing more than the loss of
specification of a given cell type (it loses its "specificity" or its
differentiated state), which leads to the production of cells that will divide
and act as progenitors in the cell.
Repair of that damaged tissue;
the organism's already differentiated cells themselves can undergo
numerous divisions in order to repair the tissue formed by them; the new
cells that will act in the re-generation process may arise from trans-
differentiation, that is, a specific cell type is differentiated and then re-
differentiated into a new cell with a different function and type than
anterior, that is, it alters its state of differentiation (an epithelial cell, it can
be transdifferentiated into a muscle cell and can then act on the re-
generation of muscle -tissue that has been damaged and needs repair).
The trans-differentiation mechanism, specifically, can occur without
necessarily cell division, and can occur via a parent cell that was
obtained from a de-differentiation process.
In the case of porifers, in vitro tests showed that the placement of
aggregated cells from the animal allowed the formation of a new animal,
with complete and functional body.
The re-generation process in this case then generates a new, complete
animal :its cells have a high regenerative capacity. This is also shown
when cutting the animal into several pieces, as each of them gives rise to
a new animal after a while.
15. Hydra is a genus of living water polyp, belonging to the Cnidaria group,
which has a high capacity for re-generation, because it occurs throughout
the body, forming a new individual.
In vitro tests have shown that by allowing cells taken from the hydra to
form clusters, tentacles, other parts of the body and even the whole
animal can be formed again, showing that the cells of this animal have,
as in Porifera, an high regenerative capacity.
When you cut the animal into several pieces, one observes that, after a
certain period of time, each of the pieces gave rise to a whole new -
animal, which is why hydra is one of the greatest examples when thinking
about re-generation. Much of this is due to the ability of hydra cells to
organize themselves, which is due to a constant production of cells and
also signaling factors in adults.
The re-generation of the animal occurs either on the feet (“tentacles”) or
on the head part of the animal.
Tissues in the gastric region of the hydra contain polarity so that it is
possible for the cells to distinguish whether re-generation should occur at
the apical part of the head or at the bottom of the foot so that the new
animal contains all parts.
Head re-generation is more complex than that of the foot and requires
more elaborate gene- mechanisms. Hydra re-generation is defined as
“morphallaxis” because re-generation results from a rearrangement of the
animal's cellular and tissue content without cell proliferation.
Flatworms
Among the flatworms, the animal most popularly known for its great
regenerative capacity are the planarians. These animals are able to
completely regenerate their body after being cut into several pieces. After
cutting off its body, the neoblasts (pluripotent stem cells) begin to
proliferate, traversing the entire body (part of it) of the animal,
"completing it" and subsequently forming a new complete animal.
Due to their regenerative characteristics, planarians are widely studied.
This animal has mechanisms of cellular differentiation throughout its life -
cycle. Research contributes to works in the biomedical field related to
16. stem cell, tissue re-generation and degenerative disorders.
Annelids
Re-generation in annelids is quite diverse across the groups that form
this clade. Some animals are able to regenerate both anterior and
posterior, others regenerate only one of the 2 while others do not
regenerate at any time. The regenerative process in the animals that
present it occurs from the evolution of the blastema (similar to the
flatworms, the blastema is the name given to the animal's stem cell set)
that proliferates and evolves throughout the body, regenerating the lost
part.
In polychaetes, re-generation takes place from cellular de-differentiation
and re-differentiation of cells into those that will make up the lost tissue
being regenerated.
Arthropods
each group belonging to this clade has a different form of re-generation,
but this is much simpler and almost never involves the complete
formation of several limbs, not involving the formation of a new- animal,.
This re-generation mechanism is almost always regulated by some
hormones released by arthropods when they lose a limb, either due to
predation or autotomy. In this clade, re-generation also occurs from the
blastema, as in flatworms and annelids. arachnids, especially spiders
and scorpions, are able to regenerate their venom, whose content and
final volumes are different from the originals, because the proteins
contained therein are different.
Echinoderms
example of re-generation is the re-generation of the starfish's arms. Many
demonstrations are done where the arm of this animal is cut and in a
relatively short time, it regenerates itself. It is very common, when
collecting animals like the starfish, to find animals that have two or more
members in re-generation, each of which is at a stage of this process.
In addition to this re-generation of structures (limbs) the echinoderms,
especially sea cucumbers, have the ability to regenerate visceral
structures, which, in some types of defense mechanisms, are
17. regurgitated and "abandoned" and subsequently regenerated. The that
limb loss and re-generation is part of the asexual reproduction of this
group, because after specific fission mechanisms present in these
animals.
Ophiuroides and and oloturoides also present this reproductive
mechanism, with division of the body in some parts, which, individually,
from regenerative mechanisms, each form a new individual. This form of
reproduction is most commonly found in smaller animals and also
depends on the age of the animal. Usually larger animals tend to
reproduce sexually.
Amphibians
the salamander and newt are the most popular animals when thinking
about re-generation, as they are capable of regenerating a lost limb
altogether. Although limb re-generation is the most common example
when it comes to the regenerative process, retinal re-generation is a very
interesting aspect of amphibians, with the merman case being the most
interesting since it apparently retains this ability. retinal re-generation
throughout its adult life (an organ of the eye that often cannot regenerate
when lost or damaged when thinking of adult individuals).
Other amphibians, such as adult frogs, re-generation is quite limited, and
in adult Xenopus, after the loss of a limb, only a cartilaginous structure
with no digits is formed. It is believed that the full regenerative capacity of
the anuran limb is lost after the metamorphosis- process.
The re-generation of limbs in salamanders and newts happens in
different stages, being a very complex process.
First, the wound closes, that is, the place where the limb was previously
lost is closed. The cells then undergo a process of de-differentiation and
intense proliferation and migration to where the limb should be
regenerated. there is a constant growth of the regenerating limb and the
re-differentiation of these cells, to form again a complete limb, with
structure of bones and cartilage, as well as musculature and vessels for
blood circulation in the region. all of this has fine gene control.
Reptiles
18. re-generation is already quite limited, as it is in mammals. What is
observed is that in amniotes the re-generation of limbs is no longer
possible, which is quite curious, after all the limbs of amniotes and
amphibians have very similar embryonic formation and internal
structures. Little is known about these details yet.
In this group, the greatest example of regenerative process is the lizard-
tail. In the case of these animals, the regenerated structure is quite
similar to the original, both structurally and morphologically. Lizards are
the representatives of this group that have the highest regenerative
capacity, and this is due to the migration and proliferation of blastema,
mentioned in other previous animal groups. This stem cell proliferation
and subsequent differentiation allow the formation of a new functional tail
in these animals.
Mammals are a very diverse animal group, but compared to previous
animal groups they have a smaller and more limited regenerative
capacity.
They are not able, to regenerate complete and functional limbs, but
perform cellular re-generation almost constantly. Skin and blood cells
(especially red blood cells) are always renewed, so dead cells are
“discarded” and new cells are formed. In addition to these cells,
mammalian hair / hair also conforms
Animals that Regenerate
Lizards who lose all or part of their tails can grow new ones. ...
Planarians are flat worms. ...
Sea cucumbers have bodies that can grow to be three feet long. ...
Sharks continually replace lost teeth. ...
Spiders can regrow missing legs or parts of legs.
Sponges can be divided
For the aim of this work some questions are fundamental :
Most metazoans have at least some ability to regenerate damaged- cells
19. and tissues, although the regenerative capacity varies depending on the
species, organ, or developmental stage.
Why are some tissues,structures and species able to regenerate,
whereas others cannot?
Why in human can regenerate heart only in fetal life and not in adult?
Re-generation is a capacity of cells to re-synthesize in a sequential
manner to synthesize a tissue or an organ again. Many organisms have
the capacity to regenerate their lost parts but the extent of re-generation
capacity varies greatly among kingdoms and phyla.
IN-VERTEBRATES regenerate most of their parts as well as organs
whereas the vertebrates have limited -ability to regenerate.
Whereas in higher vertebrates only a few organs can regenerate and this
process also varies among different age groups of same organism.
A best example is vertebrate mammalians ( human beings), they possess
re-generation capacity in few organ cells such as hepatocytes o and
epithelial cells of skin e. few organs in humans can regenerate only in
fetal life but as the age progresses they lose the ability of re-generation
such as in heart cells myocardial cells.
The mechanism behind this loss of ability is not yet clear and also it gives
rise to a new horizon of research study that whether it is possible to
reactivate this lost ability in cells or not.
It could be a beneficial gate way to treat various diseases linked with
cellular destruction of such organs such as myocardial infarction.
A number of works have been conducted to check out the mechanism
and progression of re-generation.
In a study Zebra fish has been targeted to myocytic injury by four
different approaches : surgical resection of cardiac apex, cryoinjury-
induced myocardial ablation model, non-surgical, destruction of cardio
myocytes using a genetic ablation model and induction of hypoxia–
reoxygenation injury, where the re-generation output was revealed as
considerable extent of re-generation in 60 days, 180 days, 30 days and
21. indicated in green
(ability to regenerate), orange (incomplete capacity ) or red (incapacity ).
In each case, the approach used to induce cardiac damage and the
references associated are indicated. In warm-blooded species, cardiac
re-generation appears to be restricted
to a defined early-developmental period during embryonic and early-
neonatal life. In cold-blooded animals, 6 out of 9 species have the
ability to regenerate their heart during adult life, whereas three out of nine
species show an incomplete capacity or incapacity to undergo heart re-
generation.
From website http://archives.evergreen.edu/webpages/curricular/2011-
2012/m2o1112/web/amphibians.html:
“Amphibians have impressive regenerative- properties and amazing
plasticity in the neuronal connections throughout their body. Not only do
they have the ability to regenerate whole limbs (bone, skeletal muscle,
and other tissue) they also have the ability to regenerate neuronal-
connections. Many species of animals have been known to regenerate
nerves in damaged tissue of the body, but amphibians have been found
to regenerate neurons in the brain, spinal -cord, and large nerve fibers,
as the optic -nerve. the connections between the eye and optic tectum
have been greatly studied in many various amphibia. Orderly mapping by
the axons of ganglion cells in the retina, and the spatial arrangement of
the retina onto the tectum have been shown to depend upon
development and specificities that exist between retinal and tectal -
neurons. Both vision and topographic relations between retina and optic -
lobe is restored, and connections reform, when the optic nerve is cut and
allowed to regenerate. In contrast to certain amphibians, loss of neurons
or interruptions of connective- pathways in mammalian visual systems
lead to permanent damage and loss of vision.
Amphibians can also regenerate the neural- retina and lens of their eye
as they do their other tissues. Regeneration of these tissues are possible
by de-differentiation (loss of phenotype specific structure) of differentiated
stem- cells at the site where injury occured. These cells then re-
differentiate in response to signals from neighboring -cells (the damaged
cells) telling them to differentiate into the cells required for the
regeneration of lost tissue.
Another example of plasticity in amphibian vision is their ability to adapt
to permanent injuries, in order to correct their eye sight and visual field.
25. The assumption of a terrestrial- lifestyle required many adaptations not
only for the eye but also in the management of reproduction. Extant
amphibians can not move far from water, because they are required to
lay their eggs in water. The cleidoic egg changed that. A cleidoic -egg (or
amniotic egg) is one with a leathery or hard shell that limits evaporation
and does not require constant contact with water. For some animals such
as placental- mammals, the amniotic egg is internal and thus does not
need the outer and impervious shell. This permitted newly terrestrial
animals to move away from water- sources.
Sauropsids diverged into 2 distinct lineages: Lepidosaurs, including
lizards, the tuatara, and eventually snakes and Archosaurs, including
turtles, crocodiles, and eventually birds.
This was a major step for the terrestrial eye because the crystalline lens
slimmed to the shape of a lentil and accommodation was accomplished
by lens deformation instead of lens- movement. This key step using
striated musculature in most sauropsids and was faster, more accurate
and produced a brighter image than lens movementll lineages of
sauropsids and synapsids are believed to have developed
accommodation by lens- deformation. Snakes are an exception. Snakes
radiated from the lizard lineage and lost this ability during their fossorial
sojourn, only to revert to lens movement for accommodation when they
emerged from underground. they had lost the ability for accommodation
of any kind. evolution found a different manner of accommodation using
lens- movement analogous to that of fish. This rather clumsy method of
accommodation by lens movement consists of squeezing the vitreous to
push the lens forward and backward is analogous to, but not homologous
to accommodation in fish.”
So it is possible to say that more superios vertebrates need less
regenerative abilities then amphibian? And this due by the eye
characteristic? And what role in evolution played the different need of
accommodation in eyes to focalize light in retina?
An retina regeneration in zone out of light incidence is not usefull.? And
so evolutive Suppressed?
28. Cell lineage tracing techniques have shown that cardio-myocyte
migration is essential in heart- re-generation.
The chemical signaling pathway Cxcl12a/Cxcr4b has also been shown to
be essential in this kind of process.
the transcription factor hand2 has been shown to expressed in heart- re-
generation in zebrafish, and its over-expression has been shown to
promote cardio-myocyte proliferation.
The hearts of neonatal -mice can regenerate after ventricular resection,
Neuregulin 1 (Nrg1) has been shown to play an important role in the
cardiac- re-generation of zebrafish and neonatal mice.
After cardiac injury, the perivascular cells of zebrafish have been shown
to highly express Nrg1,21 and blocking the Nrg1 receptor Erbb2 with
AG1478 inhibited cardiac- re-generation in zebrafish.
Adult zebrafish cardio-myocytes are mainly mononuclear cells, while
adult mammalian cardio-myocytes are mainly bi-nuclear and multi-
nuclear cells.
Similar to zebrafish, neonatal mouse cardio-myocytes are mainly
mononuclear. Nrg1 can stimulate the proliferation of neonatal- mouse
cardio-myocytes.” (3)
Thomas P. Lozito et al :
“ humans, like most mammals, suffer from very minimal natural-
regenerative capabilities.
As the closest relatives of mammals that exhibit enhanced regenerative-
abilities as adults, lizards potentially represent the most relevant model
for direct- comparison and subsequent improvement of mammalian
healing.
Lizards are able to regenerate amputated tails, and exhibit adaptations
31. Fig. n 12 Toad
Neotenic salamanders, which never fully develop and retain non-ossified,
cartilaginous skeletons into adulthood, are able to regenerate fully formed
limbs , with all the original cartilaginous- skeletal elements of the originals
.
Regenerated salamander limbs also recreate the musculature of the
amputated arms/legs.
Frogs, which do fully develop and exhibit ossified skeleton as adults,
regenerate cartilage spikes rather than limbs following amputation .
Cartilage spikes are continuous with the radio-ulna bone of the original
limb, and no other skeletal elements are formed, and very little muscle is
regenerated . (This inverse relationship between skeletal
development/maturity and re-generation fidelity, as well as the preference
for producing cartilage, are also observed in tail re-generation.)
Lizards are the only group of amniotes capable of tail re-generation as
adults, and, unlike the anamniotic salamanders, adult lizard axial-
skeletons are fully ossified.
34. generation over bone.
This is particularly interesting given that cartilage is a tissue that most
mammals, and humans, are completely unable to heal, let alone
regenerate.
Among the regenerative vertebrates, only lizards are grouped with
mammals as amniotes, and that many of the regenerative properties and
processes exemplified in lizards is shared with amphibians, the bulk of
this review will focus on the lizard in its discussion of enhanced wound
healing capabilities.
Lizard-tail re-generation follows waves of process of de-generation,
proliferation, and differentiation.
Regardless of whether the lizard-tail is amputated or autotomized, the
first stage of re-generation is actually characterized by tail stump tissue
de-generation and breakdown.
Within days of tail loss, macrophages and osteoclasts home to stump
tissues, where they proliferate and secrete proteases such as matrix
metallo-peptidase 9 (MMP-9)
These proteases breakdown stump tissues, including the terminal tail-
vertebra, which is effectively cut in half by osteoclasts .
The exception to this tissue de-generation is the epidermis, which
proliferates around the wound surface and migrates through the break in
the terminal tail vertebra created by the osteoclasts .
Several days later, when the most distal portion of the tail stump is shed,
a process known as ablation, the stump is completely covered by new
epidermal- tissues, which is referred to as wound -epidermis .
Macrophages, osteoclasts, and wound epidermis secrete proteases that
degenerate stump -bone, muscle, and connective tissue, releasing a
variety of cell types directly under the wound- epidermis .
Cells- derived from degenerated stump tissues secrete signals, such as
IGF-2 , which have been shown to potentiate wound epidermis
development in other models of re-generation .
35. Wound epidermis thickens and stratifies as it forms the structure known
as the apical cap.
In turn, the apical cap produces another set of signals, including Wnt5a
and FGF-2.
FGF-2 signals induce proliferation and chemo-taxis in tail stump
ependymal cells, which line the central canal of the spinal- cord .
As they proliferate and migrate towards the apical cap, ependymal cells
self-organize into a structure known as the ependymal tube, which forms
the bulk of the regenerated spinal- cord.
Implantation of FGF-soaked beads attracts ependymal cells toward
implantation sites, resulting in ependymal- tube branching .
implantation of beads soaked in the drug SU5402, a specific inhibitor of
FGF receptors, blocks ependymal- tube extension .
As the ependymal tube infiltrates the mass of cells released from
degenerated stump tissues, stump cell populations proliferate and swell
beneath the apical cap, forming the lizard- tail blastema .
This ends the de-generation phase of tail re-generation, also known as
the latent period due to the lack of tail elongation.
Blastema cell proliferation now drives rapid tail growth , which can reach
4–5 mm per day in some animal species .
blastema -cells begins differentiating into regenerated tissues, including
muscle and skeletal tissues .
Lizard-tail re-generation is dependent upon the spinal- cord.
One questions that arises when studying re-generation in reptiles and in
amphibians is “Why can these organisms regenerate, while mammals
can not?”.
Comparisons between non-regenerative and regenerative organisms
have identified 2 tissues/structures both unique to regenerative- species
and required for re-generation: the apical cap and the tail spinal cord ,
Wound epidermis that forms over the stumps of amputate mouse tails
38. events .
While many of the key mechanisms involved in wound
healing including re-epithelialization , cell proliferation, angiogenesis, and
extra-cellular matrix deposition and remodeling] are widely conserved,
the fidelity of repair often varies .
In humans and most other mammals, non-lethal injuries
typically result in the replacement of damaged tissues with a fibrous
substitute known as scar .
Although scars participate in re-establishing homeostasis and barrier
functions, they lack the organization, tensile strength and specialized
functions of theoriginal tissues.
In contrast, other kind of vertebrates – including various
species of bony- fish (teleosts), salamanders and lizards – are capable of
wound- healing without scar- formation.
Instead of replacing damaged tissue with a fibrous infill, these species
undergo a tissue-specific program to restore tissue- architecture and
function.
Although vertebrates lack the capacity for whole-body
re-generation, a broad range of organs can be partially replaced,
including portions of the skin (epidermis , dermis), heart (ventricle),
forebrain (tel-encephalon), spinal cord and even multi-tissue
appendages, such as limbs and the tail.
Although it may be tempting to summarize scar-forming versus
scar-free wound healing responses simply along phylogenetic lines
( mammals scar, salamanders and lizards do not), the reality is far
more complex.
Fetal- mammals can heal cutaneous wounds scar-free prior to the early-
to mid-gestation period , while postnatal mice, rats, rhesus -monkeys and
39. human children can also spontaneously regenerate amputated digit- tips.
Several species of African spiny mice are able to perfectly heal holes
created in their ears, and even lose and then regenerate large portions of
skin (∼60% of the total dorsal body surface area;)
the mechanisms involved in scar-free wound healing and re-generation
are taxonomically wide-spread, which leads to the riddle: why are some
tissues,structures and species able to regenerate, whereas others can
not do this ?
We begin by considering the benefits of the lizard
model, followed by a discussion of select examples of the wound
healing and regenerative responses of lizards to injury.
Another region of the CNS demonstrating variable responses to injury is
the optic- nerve.
In mammals and birds, damage to these axons can result in vision- loss,
as retinal ganglion cells degenerate and undergo cell- death
Cellular de-generation and the inability to restore the visual pathway in
these species appears to be the result of a complex inhibitory micro-
environment, related to the formation of a glial- scar (rich in
proteoglycans and glial cells) and various axon impeding proteins such
as Nogo-A.
As might be expected, species capable of restoring vision
after injury to the optic- nerve ( zebrafish) are characterized by retinal -
ganglion cell survival , and the absence of axon inhibitory proteins such
as Nogo and a glial scar .
The optic nerve of some lizard species can regenerate, even though they
express Nogo-A and form a glial scar .
Optic nerve re-generation is particularly efficient in Ctenophorus
ornatus ( ornate dragon lizard), with the crushed optic nerve outgrowing
to re-contact the optic tectum within 1 month.
40. Although excitatory and inhibitory neurotransmission is dysfunctional
following re-generation, and vision is not spontaneously returned, lizards
can regain sight with training .
in vitro experiments show that retinal ganglion cells of lizards are
insensitive to the inhibitory signals that otherwise obstruct mammalian
axon- outgrowth.
Using an explant strategy, mammalian (rat) dorsal root ganglia and lizard
retina were cultured on each of mammalian and lizard glial- cells.
Whereas both these environments inhibited regrowth of mammalian
axons, neither inhibited the regrowth of lizard -axons .
These data reveal a surprising diversity across vertebrates in how the
optic- nerve responds to injury, with lizards uniquely interposed between
full functional restoration and regenerative- failure.
Many mechanisms and cellular participants involved in wound healing
and re-generation in lizards are conserved with those of salamanders
and teleosts ( even some mammals).
how do lizards prevent (or limit) microbial invasion following tail or skin-
loss? Early evidence showed the production of anti-microbial peptides
(such as beta-defensins) as an important adaptation with obvious
biomedical -implications.
Although genomic - transcriptomic data are now available for several tail-
regenerating species, it is instructive to compare these findings with
those of closely related but re-generation-incompetent lizards. .“(5)
Elizabeth D. Hutchins ET al :
“Lizards, amniote vertebrates like humans, are able to lose and
regenerate a functional tail.
Understanding the molecular -basis of this process would advance
regenerative approaches in amniotes, including humans. We have
carried out transcriptomic analysis of tail re-generation in a lizard, the
green anole Anolis carolinensis, which revealed 326 differentially
expressed genes activating multiple developmental and repair -
41. mechanisms, genes involved in wound response, hormonal- regulation,
Musculo-skeletal development, and the Wnt and MAPK/FGF pathways
were differentially expressed along the re-generating tail axis.
We identified 2 micro-RNA precursor families, 22 unclassified non-coding
RNAs, and 3 novel protein-coding genes significantly enriched in the
regenerating tail.
high levels of progenitor/stem cell markers were not observed in any
region of the regenerating -tail.
We observed multiple tissue-type specific clusters of proliferating cells
along the regenerating -tail, not localized to the tail tip.
These findings predict a different- mechanism of re-generation in the
lizard than the blastema model described in the salamander and the
zebrafish, which are anamniote- vertebrates”.
“ lizard-tail regrowth involves the activation of conserved developmental
and wound- response pathways, which are potential targets for
regenerative medical therapies. Re-generation of appendages in the
adult is observed in various vertebrates, including in the lizard-tail, the
salamander limb , tail, and the zebrafish caudal fin .
Molecular- cellular analyses in these model organisms are beginning to
reveal conserved versus divergent mechanisms for tissue re-generation..
Re-generation in newts is associated with proteins specific to urodele
amphibians, casting doubt on the conservation of these re-generative
pathways with other vertebrates .
muscle formation during limb re-generation differs between newts and
the axolotl .
Mammals possess some neonatal regenerative- capabilities, including
mouse and human digit tip re-generation and heart re-generation in the
mouse, but these kind of processes are limited in the adult -organism .
Lizards are capable of re-growing appendages, and as amniote
vertebrates, are evolutionarily more closely related to humans than
42. other models of re-generation, ( salamander , zebrafish).
A deep examination of the genetic regulation of re-generation in an
amniote model will advance our understanding of the conserved
processes of re-generation in vertebrates.
In response to threats, lizards have evolved their ability to autotomize, or
self-amputate, their tails and regenerate a replacement .
The patterning and final structure of the lizard- tail is quite distinct
between embryonic -development and the process of re-generation .
Whereas the original tail skeleton and the muscular groups are
segmentally- organized, reflecting embryonic -patterning, the re-
generated tail consists of a single un-segmented cartilaginous tube
surrounded by un-segmented muscular bundles .
the segmental organization of the spinal cord and dorsal root ganglia in
the original tail are absent in the replacement, with regenerated axons
extending along the length of the endo-skeleton .
De-differentiation has been proposed to be a major source of proliferating
cells in the an-amniote salamander blastema model .
No clear evidence of de-differentiation has been identified in tail re-
generation in the lizard, an amniote vertebrate .
A temporal-spatial gradient of tissue patterning and differentiation
along the re-generating tail axis has been showed .
While transcriptomic analysis has been carried out in anamniote
regenerative models, including the zebrafish tail, the newt limb,
and the axolotl limb , the genetic profile of pathways
activated in re-generation of amniote appendages has not been
well described.
Through transcriptomic- analysis of lizard- tail re-generation,
43. It was identified that genes in path-ways involved in
developmental processes, myogenesis, chondrogenesis,
and neurogenesis, adult processes, as wound and
immune -responses, and are differentially expressed along the
regenerating tail- axis.
The Wnt pathway was significantly enriched along the regenerating
lizard- tail axis, and the activation of this path-way has also been verified
in the salamander tail- tip and mouse digit tip re-generation .
The activation of Wnt signaling in 2 amniote lineages, mammals and
squamate reptilesm and urodele- amphibians supports a role for this
path-way in re-generation that is conserved among tetrapod vertebrates.
Transcriptomic analysis also showed that genes involved in thyroid
hormone generation were differentially expressed, suggesting a
regulatory connection between re-generation of the lizard -tail and
Musculo-skeletal transformations during amphibian- metamorphosis.
The lizard dio2 gene is the ortholog of deiodinase, iodothyronine, type
I,which in mammals converts thyroxine pro-hormone (T4) to
bioactive 3,3’,5-triiodo-thyronine (T3) .
In Xenopus -laevis, T3 is the key signal for the process of metamorphosis
from tadpole to adult frog .
Many of the changes associated with meta-morphosis are also observed
in remodeling of the tail- stump and outgrowth of the lizard- tail.
The lizard cga -gene is the ortholog of chorionic gonadotropin, alpha
chain, which encodes the alpha chain of TSH and other crucial
hormones.
During tadpole meta-morphosis, thyroid hormone (TH) and TSH rise,
despite the normal expectation that TH would down-regulate TSH .
Changes in TH regulation of TSH may also be altered in re-generation.
45. Other tissues are formed from A distributed growth of distinct cell types,
as development of the eye from neural crest, mesenchymal, and
placodal ecto-dermal tissue .
The re-generation of the amphibian -limb involves a region
of highly proliferative cells adjacent to the wound epithelium, the
blastema, with tissues differentiating as they grow more distant from
the blastema.
re-generation of the lizard- tail seem to follow a more distributed model.
Stem cell markers and PCNA and MCM2 positive cells are not highly
elevated in any particular region of the regenerating tail, suggesting
multiple foci of regenerative growth.
This contrasts with PNCA and MCM2 immunostaining of
developmental and regenerative growth zone models such as skin
appendage formation , liver development , neuronal
re-generation in the newt , and the regenerative blastema ,
which all contain localized regions of proliferative growth.
Skeletal muscle and cartilage differentiation occurs along the length of
the regenerating tail during outgrowth; it is not limited to the most
proximal regions.
the distal tip region of the regenerating tail is highly vascular, unlike a
blastema, which is avascular .
This suggest that the blastema model of anamniote limb re-generation
does not reflect the regenerative process in tail re-generation of the
lizard, an amniote vertebrate.
Re-generation requires a cellular source for tissue growth.
Satellite- cells, which reside along mature myo-fibers in adult
skeletal muscle, have been studied extensively for their involvement
47. burning, freezing, mechanical injury, and chemical injury .
Most researcher agree that this work and sub-sequent experiments to
date involving modern capabilities to detect bona fide re-generation,
generated little evidence to conclude that there is significant myocardial
re-generation after cardiac injury.
Most also agree that the key limitation to cardiac muscle re-generation is
likely to be the poor ability of adult mammalian cardio-myocytes to enter
the cell cycle and undergo division .
Cardio-myocytes in the fetal mammalian heart are mononucleated and
proliferative; but shortly after birth the vast majority of cardio-myocyte
DNA replication occurs without cytokinesis or karyokinesis.
most cardio-myocytes are binucleated with diploid nuclei in the adult
mouse heart, and mononucleated with polyploid nuclei in the adult
human heart .
After this postnatal- switch, it is rare for cardio-myocytes to enter the cell
cycle .
observations suggest that injury may influence the propensity for adult
mammalian cardio-myocyte proliferation.
In injured rodent- ventricles, histological examination of 3Hthymidine
incorporation identified detectable DNA replication in nuclei of myofibers
bordering necrotic tissue .
better resolution using transgenic mice in which cardio-myocytes were
labeled by a nuclear-localized lacZ reporter protein , although no
distinction between karyokinesis and cytokinesis was provided.
These labeled cardio-myocytes were detected near the border zone of
myocardial damage at exceptionally low levels (~0.0083%).
Frisen et al overcame this hurdle by taking advantage of the high levels
of the radiocarbon 14C released from nuclear bomb tests during the
Cold- War .
In the atmosphere, 14C reacts with oxygen to form 14CO2, which is then
48. captured in plants and eventually incorporated in humans through the
food chain.
The genomic 14C concentration was quantified in cardio-myocyte nuclei
purified by flow- cytometry, facilitating retrospective dating of cardio-
myocytes from recently deceased people of various ages.
Mathematical -modeling of the radiocarbon data suggested that human
cardio-myocytes renew throughout life with a capacity that gradually
decreases from ~1% annual turnover at the age of 25 to 0.45% at the
age of 75.
inferred from these data is that nearly 50% of cardio-myocytes are
replenished during a normal life span .
Anversa et al used human tissue- samples collected from cancer-
patients who had received infusion of iodo-deoxyuridine, a thymidine
analog used as radio-sensitizer for therapy: 22% of cardio- myocytes in
the human heart are renewed every year .
And that about 13% of endothelial -cells and 20% of fibroblasts under-go
turnover each year in the heart, suggesting that cardio-myocytes have
the highest renewal capacity among cardiac- cell types examined in the
study.
these results suggest that the mammalian heart possesses a measurable
capacity for renewal.
It is not yet clear if cardio-myocytes are renewed through differentiation
from a stem population or through cell division
( existing cardio-myocytes.)
The possibility of natural cardiac re-generation in amphibians has been
examined first by Soviet- scientists in the 1960’s .
Salamanders present great re-generation among vertebrates, renew
removed or injured body parts like lens, retina, spinal- cord, jaws,
portions of intestine, brain tissue, and major -appendages.
Amphibians survive massive mechanical injury to the ventricle, including
removal of as much as ¼ of the chamber.
49. This resilience is a feat in itself, and is likely to reflect a lesser reliance
than mammalian species on vigorous circulation.
Resection injury penetrates the ventricular lumen, releasing a large
amount of blood.
In the newt heart, the formation of connective scar -tissue seems to be a
dominant response after resection of the ventricular apex, and there is
only minor replacement of cardiac- muscle .
when the resected myocardium was minced and grafted back to the
wound area, the tissue graft could assemble into a contiguous, contractile
mass .
resection injuries at the base of the newt heart were reported to
regenerate with much less scarring .
the outcome of re-generation could be influenced by the level and type of
tissue damage, a warning of sorts that heart re-generation should be
assessed in multiple injury contexts.
there is definitive evidence for proliferative activity in newt and axolotl
cardio-myocytes.
These include the presence of mitotic figures in cardio-myocyte nuclei as
visualized by standard histology and transmission electron microscopy,
and multiple indicators of DNA synthesis .
Teleost hearts The zebrafish is highly amenable to genetic approaches,
and has become a popular model system for understanding vertebrate
embryonic development.
adult zebrafish effectively regenerate multiple structures that mammals
fail to regenerate, including retinae, brain tissues, spinal cord, and the
major appendages .
An study examined the effects of removing ~20% of the ventricle by
surgical resection .
As with the mechanically injured amphibian- heart, the injury seals by a
quick clotting mechanism and the organ sustains sufficient contractile
force to continue to drive circulation.
50. Over the next month, a series of events occurs in response to ventricular
resection.
In the infarcted mammalian -ventricle, fibrin deposition attracts the
fibroblasts and inflammatory- cells, and is a precursor to scarring.
The fibrin clot is typically not replaced by scar tissue during cardiac repair
in zebrafish; little or no collagen is retained by 1–2 months after resection
injury.
the clot is supplanted by cardiac -muscle, restoring a contiguous wall of
vascularized cardiac -muscle.
Elevated indices of cardio-myocyte proliferation were detectable at the
end of the first -week after injury, and observable for weeks after this .
As for amphibian, it is possible that different injury types introduce
distinct outcomes of myocardial re-generation.
Wang et al produced a transgenic system to facilitate cell type-specific
ablation in zebrafish .
This system employs 2 transgenes:
1) a 4-hydroxytamoxifen (4-HT)-inducible Cre recombinase (CreER)
restricted to cardio-myocytes by the regulatory sequences of cardiac
myosin light chain 2 (cmlc2);
2) a cytotoxic DTA (diphtheria toxin A chain) gene that can be inducibly
targeted to CreER-expressing cells. In these transgenic fish, referred to
as Z-CAT (zebrafish cardio-myocyte ablation transgenes),
a single injection of 4-HT could eliminate more than 60% of cardio
myocytes throughout the heart.
While such massive loss of myo-cardium did not normally affect survival,
it caused lethargy, a gasping phenotype, and reduced exercise capacity,
classic indicators of heart -failure that are not seen after resection -injury.
these signs of heart- failure reversed within several days, a
recovery that correlated with massive cardio-myocyte proliferation
51. detected throughout the ventricle.
By 30 days after the injury, the ventricle was filled with new muscle and
displayed little or no scar- tissue.
It is important to test if re-generated cardio-myocytes incorporate
functionally with existing cardiac muscle and do not generate
arrhythmias.
This was studied using optical- voltage mapping of surface myocardium
at various stages of re-generation.
At 7 days after injury, when cardio-myocytes begin to proliferate, muscle
at the re-generating apex was uncoupled.
A week later, coupling was evident, and by 30 dpa, electrical conduction
through the apex occurred at normal velocities .
Loss and recovery of conduction velocities was also evident after genetic
ablation, and then re-generation, of cardio-myocytes
in the Z-CAT model .
These indicated that the newly created Cardio-myocytes show evidence
of functional integration in the regenerated zebrafish heart.
MI is caused by ischemic injury, and coronary artery occlusion is routinely
used as an injury model in small and large mammalian model systems.
the zebrafish- ventricle is diminutive (~1 mm3), and the coronary vascular
network perfuses a relatively small proportion of ventricular- muscle,
making this type of injury a difficult task.
Cryo-cauterization has been used as an alternative model to coronary
artery ligation in mouse .
this injury model was applied to zebrafish .
In the initial report of this injury model, collagen deposits formed during
the 3 weeks following injury, yet were subsequently replaced with new
cardiac muscle by 130 days post-injury .
Although cardiac muscle and coronary vasculature were recovered at the
cryo-injured area, perfect ventricular shape was not restored, as also
52. observed in the resection model. Whereas the amount of cauterized
ventricular tissue is similar to that removed in the resection
model,dynamics of cardiac re-generation differed with injury type.
This delay likely reflects the need to remove necrotic tissue after
cryoinjury for re-generation in the damaged area to take place.
A recent study has shown the capacity of heart re-generation after an
analogous necrotic injury in the giant danio, a teleost fish closely related
to zebrafish .
These 3 quite different injury models each stimulate robust myocardial re-
generation in zebrafish, although at different rat What limits the
regenerative capacity of the mammalian heart?
Explaining these clear differences in cardiac regenerative capacity
among vertebrate species is a central pursuit of the field. One possible
reason for this may be intrinsic differences in cardio-myocytes.
Lower vertebrate cardio-myocytes tend to be mononucleated, smaller in
size, and containing fewer myofibrils, as compared to those of adult
mammals. In fact, these characters are typical of cardio-myocytes in
young mammals, and might facilitate cell cycle reentry after injury .
Sadek and colleagues have applied a resection injury model to the
neonatal mouse heart .
approximately 15% of the muscle was removed from the left ventricular
apex of one day-old mice.
At this age, mice are in the process of major growth, as are their cardiac
chambers.
Similar to the zebrafish and salamander models, a large blood clot
quickly sealed the wound after injury.
After surgery and sutures, pups were cared for by mothers until weaning.
Strikingly, during this 3-week period, the ventricles fully healed without
major scarring. Cardio-myocyte proliferation
indices were boosted both near to and away from the resection plane to
53. levels even higher than normally seen in growing hearts.
By contrast, resection injuries performed at 7 days after birth led to the
formation of a fibrotic scar.
the capacity of myocardial re-generation is transiently present in the
neonatal mouse heart, but is quickly lost by 7 days after birth.
Postnatal switches in cardio-myocyte proliferation and regenerative
capacity coincide with changes of the expression of cell cycle regulator
genes ,and a recent study suggested the role of microRNAs (miRNAs) in
this regulation .
In this work, microarray analysis was carried out to identify subsets of
miRNAs of which expression is changed in murine cardiac ventricles
between 1 and 10 days after birth. The analysis identified miR-195, a
member of the miR-15 family that is reported to regulate B
cell proliferation and contribute to leukemogenesis, as being highly
upregulated during the postnatal period.
Checkpoint kinase 1 (Check1) was shown to be directly regulated
through a binding site in its 3′UTR.
While cardio-myocyte characteristics would appear to have a primary role
in regenerative capacity, another basis for the poor regenerative potential
of the mammalian heart may be the activity of non-myocardial cardiac
cells in response to injury.
For instance, fibroblasts make up a high percentage of adult mammalian
cardiac cells, and a much lower percentage of fetal mammalian or adult
non-mammalian vertebrate hearts.
These fibroblasts not only have the capacity to form scar tissue, but also
appear to impact the proliferative capacity of
Cardiom-yocytes.
To this point, a recent study found that adult cardiac fibroblasts co-
cultured with neonatal cardio-myocytes inhibited their proliferation, while
embryonic cardiac fibroblasts had no such effect .
54. age-related changes in fibroblast characters might modify cardiac
regenerative capacity.
the hearts of lower vertebrates such as zebrafish have long,
exaggerated trabeculae that protrude into the ventricular lumen.
These trabeculae are lined by a large total surface area of endocardial
cells.
The mammalian cardiac chambers briefly display similar anatomy in their
fetal form.
During maturation, mammalian ventricles then acquire a thick,
vascularized wall with limited trabeculation and low relative endocardial
surface area.
the zebrafish endo-cardium quickly responds to injury and induces a
signal(s) that is required for myocardial proliferation, while the endo-
cardium of the adult mouse heart does not appear to mount an
analogous response .
CELLULAR SOURCES OF CARDIAC MUSCLE RE-GENERATION
Identification of endogenous cardiac stem and progenitor cells in the
postnatal mammalian heart
adult mammalian cardio-myocytes have low proliferative character, and it
makes good sense for biologists to search for undifferentiated progenitor
cells that have the potential to mature into contractile cells.
Several cell types with the potential to create
Cardio-myocytes postnatally have been described, expressing either the
pan-stem cell marker c-Kit , the transcription factor Islet1 , or the cell
surface marker stem cell antigen 1 (Sca-1).
Other candidate cardiac progenitor cells include “side population” cells
that possess physiological properties to efflux fluorescent dye , or to form
multicellular clusters, referred to as cardiospheres, in culture .
Harvey and colleagues reported colony-forming cells (cardiac resident
colony forming units - fibroblasts, cCFU-Fs) in the adult mouse heart that
55. have long-term growth potential in culture .
Clonally derived cCFUFs were shown to give rise to multiple mesodermal
lineages in vitro including cardio-myocytes, endothelial cells, smooth
muscles, adipocytes, cartilage and bone.
Injection of GFP-tagged cCFU-Fs into the infarcted heart demonstrated
that those cells have the capacity to create cardio-myocytes, endothelial
cells, and smooth muscle cells in vivo.
differentiation of endodermal and ectodermal cell types, such as
hepatocytes, neurons, and oligo-dendrocytes, was also detected in vitro,
suggesting trans-germ layer plasticity of this population.
Gene expression profiles and marker expression, as well as
perivascular localization of cCFU-Fs, were analogous to those of
mesenchymal stem cells derived from bone marrow. transplantation
assays and Cre-based genetic fate mapping
indicated that cCFU-Fs are likely to derive from the epicardium, a finding
of interest given other findings that suggest epicardial trans-differentiation
capacity .
Marbán and colleagues reported findings from a randomized phase 1 trial
with cardio-sphere-derived stem cells .
cardiospheres were grown from explant culture biopsies of MI patients
suffering from left ventricular dysfunction.
Autologous cardiosphere-derived cells (CDCs) were then infused into the
artery associated with the infarct.
Functional examinations of CDC-treated
hearts showed that although there were some beneficial effects, overall
ejection fraction of left ventricle was not significantly recovered.
MRI examinations revealed that mean scar mass was significantly
reduced in CDC groups, suggesting recovery of myocardium.
This change was likely induced by indirect mechanisms, as human CDCs
exert beneficial effects through paracrine mechanisms when injected into
59. unidentified signal(s) released from the transplanted cells, a signal that
can enable de novo cardio-myocyte creation from a still unidentified
source.
Fate-mapping experiments described above do not address the
possibility that cardio-myocytes are a heterogeneous population with
respect to their regenerative capacity, containing certain muscle cells that
may be better suited for division after injury.
That is,myocardial re-generation might depend on such “elite cardio-
myocytes”, perhaps even identifiable by a specific gene expression
signature. This seems logical, given that the heart is initially contributed
by 2 recognized cardiac fields , and that various
cardio-myocytes have different physiologic and/or functional properties .
re-generation of the zebrafish ventricular apex involves activation of
gata4 regulatory sequences in a sub-population of cardio-myocytes
within the wall near the injury.
newt cardio-myocytes isolated from the adult ventricle showed
heterogenous proliferation in cell culture.
Only one-third of these cells progressed through mitosis and underwent
successive cell divisions .
In adult mammals, a recent study by Bersell and colleagues reported that
Neuregulin1(NRG1) promotes proliferation of differentiated adult mouse
cardio-myocytes in cell cultureand when introduced in vivo .
These treatments appeared topredominantly affect a sub-population of
mono-nucleated (versus bi-nucleated) cardio-myocytes, a finding
consistent with the idea that some cardio-myocytes are more receptive to
re-generation signals.
Trans-differentiation is a regenerative phenomenon in which one cell type
converts to another, sometimes using an undifferentiated intermediate.
A classic example of trans-differentiation occurs after removal of the lens
from an adult next.
a new lens emerges from the dorsal, but not the ventral, pigmented iris
60. tissue .
Using elegant tissue transplantation experiments, Tanaka and colleagues
demonstrated that while dermal cells might show multi-potency, most
cells in the limb stump are restricted to contributing their own tissue type
during re-generation .
Bone marrow-derived cells like hematopoietic stem cells and
mesenchymal stem cells were thought to differentiate to cardiac muscle
and contribute to functional recovery after MI.
results from subsequent works indicate that these cell types may
contribute to cardiac muscle survival/repair by indirect paracrine
mechanisms, as opposed todirect differentiation into myocardium.
it seems a rare and difficult task for differentiated cells to switch a
determined lineage under natural conditions.
cumulative evidence demonstrates that experimental manipulations can
overcome this hurdle.
Recent findings indicate that forced expression of fate-determining
transcription factors can eventually wrest control of
the developmental program of a cell type that has previously been
committed to a specific lineage.
Notable examples are the derivation of induced pluripotent stem cells
from adult somatic cells, the direct reprogramming of pancreatic β-cells
from exocrine cells , and the conversion of fibroblasts into neurons.
Relevant to cardiac cells, direct differentiation of non-cardiogenic
mesoderm into beating cardio-myocytes and direct reprogramming of
cardiac or dermal fibroblasts to cardiac muscle cells have been
demonstrated.
In the infarcted adult mouse heart, lineage tracing experiments indicated
that the epicardium does not differentiate into cardiac muscle;
instead, epicardial cells contribute to the canonical epicardial lineage
(epicardium,fibroblasts, smooth muscle, perivascular cells) .
61. While data indicate that epicardial cells lack natural myogenic potential
under most contexts , a recent study suggests that this restriction can be
modulated.
Thymosin β4 (Tβ4) is a peptide that has been shown to enhance
vascular potential to adult epicardial- derived cells (EPDCs) and improve
responses to MI
When Tβ4 was injected into mice prior to infarction, epicardial cells
induced the expression of the embryonic epicardial gene
Wt1 and cardiac progenitor markers.
Genetic fate-mapping analysis combined with transplantation assays with
purified epicardial cells provided evidence that EPDCs near the infarcted
area turn into functional cardio-myocytes at low frequency .
Although the low re-programming efficiency and the preconditioning with
Tβ4 injections may not be realistic for therapy, this study provided
rationale for considering the adult epicardium as a source for creating
new myocardium.
A hallmark of zebrafish heart re-generation is the presence of injury
responses that occur not only near trauma, but also in an organ-wide
manner. Works thus far have found that all
major cardiac tissues - epicardium, endo-cardium and myocardium -
employ this strategy in response to injury .
The endo-cardium stands out among these tissues, as it shows the
earliest responses yet seen after cardiac injury.
Within an hour of local injury, endocardial cells throughout the heart take
on a rounded morphology and show detachment from underlying
myofibers.
Concomitant with these morphological changes, endocardial cells induce
the expression of developmental marker genes, raldh2 and heg, in an
organ-wide manner by 3 hours postinjury .
- this activation does not occur in the vascular endothelium, suggesting a
distinct role of the endocardial endothelium in this response.
62. Similarly, embryonic epicardial markers tbx18 and raldh2 are induced in
adult epicardial cells as early as 1 day after injury, and become
detectable around the periphery of the entire heart by 3 days
post trauma .
In the myocardium gata4 regulatory sequences are activated in
ventricular cardio myocytes located in the subepicardial compact layer of
the entire ventricle by 7 days post-injury, before this signature localizes to
regenerating cardio-myocytes .
At different time courses depending on the cell type, these injury-
activated expression signatures disappear globally and localize to the
injury site, where they aid or indicate cardiac muscle re-generation, as
described later.
The organ-wide response is not unique to the adult zebrafish heart.
When neonatal mouse ventricles are injured by resection, cardio-myocyte
mitoses and sarcomere disassembly are
increased not only near the injury but also in areas distant from the injury
.
Like the zebrafish, the neonatal mouse activates this organ-wide
response quickly;
indices are boosted a day after injury and peak at 7 days after apical
resection of the ventricle.
local injury can induce global cardio-myocyte morphology changes and
proliferation in the neonatal mouse heart.
Results from various injury models suggest that the activation process in
zebrafish does not require tissue removal or direct injury to the endo-
cardium and epicardial tissue, and is not maintained by circulating
systemic factors.
when zebrafish are intraperitoneally injected with Lipopolysaccharide
(LPS), an agent that can induce systemic inflammation, the expression of
the retinoic acid (RA) synthesizing enzyme raldh2 is induced in the entire
endo-cardium and epicardium of the uninjured heart.
63. During mammalian liver re-generation, partial hepatectomy is known to
affect tissue distant from trauma and activates compensatory hepatocyte
proliferation in spare lobes, partly through inflammatory factors such as
interleukin-6 and TNFα .
factors released during cardiac inflammation may help to trigger organ-
wide injury responses during heart re-generation.
It seems natural to imagine that in tissues that are competent for re-
generation, local signals provoked by injury target regenerative events.
cumulative examples of natural heart re-generation indicate that, instead,
injury responses are initially activated throughout the entire chamber or
organ, a property that might be key to regenerative success.
Regulation by non-myocardial cells
The epicardium and the endo-cardium appear to play important signaling
and structural roles during heart re-generation in zebrafish.
morphological changes in endocardial cells start organ-wide but become
localized to the wound area by around 1 dpa.
By 7–14 dpa, epicardial cells that have amplified in response to injury
accumulate in the wound site .
endocardial cells near the injury site and epicardial cells integrated into
the wound maintain high expression of raldh2 while
re-generation continues .
Recent transgenic experiments involving overexpression of a dominant-
negative form of RA receptor alpha, or an RA-degrading enzyme,
Cyp26a1, indicated that RA produced by activated endocardial and
epicardial cells is essential to maintain myocardial proliferation at the
injury site.
Establishing new vasculature is critical for tissue re-generation.
as during embryonic heart development in higher vertebrates
the creation of new vascular components appears to be facilitated by
epicardial cells.
65. As mentioned earlier, cardio-myocyte de-differentiation is typically
characterized by reduction of sarcomere structures and expression of
fetal gene markers, and appears to be a shared mechanism associated
with cardiac muscle re-generation.
Braun and colleagues recently investigated heart- tissue samples from
chronic dilated cardio-myopathy (DCM)patients, in an effort to discover
factors that cause de-differentiated phenotypes in human
cardio-myocytes.
By using proteomics and biochemical approaches, Oncostatin M (OSM)
was found to be highly expressed in DCM hearts but not healthy hearts.
OSM is a cytokine that has pleiotropic- functions and transduces signals
through a heterodimeric receptor composed of gp130, a co-receptor
shared with many other cytokines, and OSM receptor (Oβ) or LIF -
receptor.
The authors found that OSM induced loss of sarcomeric structures and
re-expression of embryonic markers in rat adult cardio-myocytes in vitro
and in vivo, through signals mediated by Oβ.
It could also enhance cell-cycle entry in neonatal cardio-myocytes in
vitro, and Oβ was required for dedifferentiation phenotypes in cardio-
myocytes at the border zone in mouse MI- models.” (7)
Jensen B et al :
“Birds and mammals both developed high performance- hearts from a
heart that must have been reptile-like and the hearts
of extant reptiles have an unmatched variability in design.
We studied the growth of cardiac- compartments and changes in
morphology principally in the model organism corn snake ,but also in the
genotyped anole and the Philippine sailfin lizard .
In the corn snake, we found that the ventricle and atria grow
exponentially, whereas the myocardial- volumes of the
atrio-ventricular canal and the muscular outflow tract are stable.
