Perspectives On Medical Research
Volume 4, 1993
Increased Understanding of Sickle-Cell Anemia:
A Result of Clinical and In Vitro Research
Eric Dunayer
Sickle-cell anemia (SCA) is an inherited disorder
caused by abnormal hemoglobin. In humans, normal adult
hemoglobin consists of two pairs of globular proteins,
with each pair composed of an [alpha]-globin chain and
a [beta]-globin chain.(1) In most cases of SCA, a single
mutation causes the amino acid valine, located at a particular
point in hemoglobin's [beta]-globin chains, to be replaced
by another amino acid, glutamine.(2) As a result, the
body's hemoglobin becomes unstable under low-oxygen conditions:
individual hemoglobin molecules deform becoming insoluble
and gelatinous. When enough hemoglobin in a particular
red blood cell gels, the cell changes shape; instead
of resembling a disk centrally concave on both sides,
as is normal, the cell now resembles a sickle.(1)
Unlike normal red blood cells, sickled cells cannot bend in order to pass through narrow blood passageways. Because the spleen's blood passageways are particularly narrow, sickled red blood cells are especially likely to lodge in the spleen, which destroys them; when a sufficient number are destroyed, the result is anemia. In addition, sickled cells often adhere to blood vessels, causing ther to become blocked. Such vaso-occlusion leads to severe pain, cumulative organ dysfunction, and eventually death.(1)
The sickle-cell gene is inherited as an autosomal dominant. Those heterozygous for the gene carry the sickle-cell trait. Such "carriers" show no sign of the disease unless exposed to conditions of extremely low oxygen--for example at high altitudes. In the United States approximately 8% of blacks carry the sickle-cell trait. Individuals homozygous for the sickle-cell gene nearly always suffer from SCA. Approximately 0.2% (1 in 500) of American blacks ar homozygous for this gene.(3)
Currently no effective SCA therapy exists. Treatment is merely palliative aimed at relieving pain during vaso-occlusive crises and reducing organ damage.(4)
Information about sickle-cell trait and SCA has come almost exciusively from human clinical studies and in vitro studies of human-derived cells or molecules.
Until recently no animal "models" mimicked SCA's spontaneous
sickling of red blood cells or the long-term effects of SCA's vaso-occlusive
events.(4) In an effort to find an adequate animal model of SCA, researchers
have now inserted human genes that code for sickle-cell hemoglobin into
mouse embryos.(4-9) In some cases the resulting transgenic mice
have shown symptoms resembling those commonly seen in SCA; the mice's
symptoms, however, are milder.(4-9) To date research with transgenic
mice has failed to advance our understanding or treatment of SCA.
Fetal Hemoglobin's Capacity to Inhibit Sickling:
Insights from Clinical and In Vitro Studies
Most SCA research seeks to increase production of fetal hemoglobin, which has anti-sickling properties. These properties were uncovered by human clinical studies involving in vitro methods.
In humans, red blood cells normally produce fetal hemoglobin (composed of [alpha]-globin and [gamma]-globin chains) in utero until about 30 weeks of gestation. Hemoglobin production then almost entirely switches to production of adult hemoglobin ([alpha]-globin and [beta]-globin chains).(10) However, red blood cells containing fetal hemoglobin continue to circulate for some time after an infant's birth. Normally a newborn's blood is about 80% fetal hemoglobin; by the time the infant is 4 1/2 months old, adult hemoglobin has largely replaced fetal hemoglobin, which few cells then contain.(11)
In 1948 pediatrician Janet Watson tested 226 black mothers and their newborns for sickled red blood cells. Eight percent of the mothers and 8.4% of the infants tested positive, showing red blood cells that sickled in vitro under low-oxygen conditions. The percentage of red cells that sickled in vitro, however, differed sharply between the two "positive" groups: in the mothers it was nearly 100%, but in the infants it averaged only 11 % (ranging from 0.5% to 30%).(11) Watson then hypothesized that fetal hemoglobin might inhibit sickling--in which case the rate of sickling should increase as adult hemoglobin replaces fetal hemoglobin. To continue study on infants who showed sickling, Watson followed 11 of them to four months of age, by which time over 90% of each infant's red blood cells sickled in vitro.(11) In further support of Watson's theory, other researchers soon found that, among adults with SCA, those few with at least 20% fetal hemoglobin have milder clinical symptoms.(12,13)
After researchers had demonstrated that fetal hemoglobin inhibits sickling
and lessens SCA's severity, hematologist Thalia Papayannopoulou and colleagues
examined whether or not it was possible to increase the number of red
blood cells producing fetal hemoglobin in adult humans. In 1976 Papayannopoulou's
research group cultured bone-marrow cells from adult humans and found
that as the number of red blood cells increased so did the number of
cells producing fetal hemoglobin. Consequently they hypothesized that
it might be possible to reactivate fetal hemoglobin production in adults.(14)
Screening for Drugs to Increase Fetal Hemoglobin:
The Futile Use of Baboons
However, research on fetal hemoglobin soon became increasingly animal-oriented--after Joseph DeSimone and colleagues discovered in 1978 that baboons, like humans, normally show a progression from fetal to adult hemoglobin production.(15,16)
Because Papayannopoulou et al.'s work with human bone-marrow culture had suggested that an increase in red blood cell production can be accompanied by an increase in fetal-hemoglobin production, DeSimone and colleagues next hypothesized that it might be possible to increase fetal-hemoglobin production in baboons by increasing their red blood cell production.(16) Since anemia induced by particular drugs or by blood loss ordinarily leads to a compensatory increase in red blood cell production, DeSimone's group artificially induced anemia in baboons with drugs and/or bloodletting.(15,16) One group of baboons responded by producing 10% of their replacement hemoglobin as fetal hemoglobin; these baboons were designated "high responders." Those baboons who produced less than 2% fetal hemoglobin were designated "low responders."(15)
DeSimone et al. then explored using baboons to screen for drugs capable of increasing fetal-hemoglobin production.(16) The first drug to be tested, 5-Azacytidine (5-AZA), was known to counter normal biochemical inhibition of the expression of some genes, including the gene for fetal hemoglobin.(1) In 1982 DeSimone and colleagues bled two groups of high-responder baboons to induce anemia; they then administered 5-AZA to one of the groups. On average about 60% of the replacement hemoglobin produced by the baboons who had received 5-AZA was fetal hemoglobin; in contrast, the control-group baboons produced only about 10% fetal hemoglobin.(15)
When, in 1983, 5-AZA was tried in SCA patients, preliminary results
were not nearly as promising as in high-responder baboons: in the patients
5-AZA generally increased fetal-hemoglobin levels to only 6-18%.(1,17)
Epidemiological studies of SCA patients with naturally occurring fetal
hemoglobin had previously shown that the percentage of fetal hemoglobin
must be 20-40% to be therapeutic.(17) In any case, human trials of 5-AZA
were soon abandoned, after researchers found that the drug causes carcinomas
in rats.(16)
Hematologist Adil Al-Khatti and colleagues soon
tested another potential anti-sickling drug on anemic
high-responder baboons--recombinant erythropoietin, a
genetically engineered form of a naturally occurring
hormone that regulates production of red blood cells.
In 1987 they reported that erythropoietin increased the
percentage of fetal hemoglobin from approximately 10%
to as high as 50%.(18) However, a decade earlier Papayannopoulou
et al. had already shown, in human bone-marrow culture,
that erythropoietin increases the percentage of cells
containing fetal hemoglobin.(1,19) In addition, when
Al-Khatti et al. conducted their baboon research on erythropoietin,
the hormone was already being used to treat the anemia
that often accompanies kidney failure in humans. The
hypothesis that erythropoietin increases fetal-hemoglobin
production could easily have been tested clinically,
in patients with anemia related to kidney failure or
with SCA.(18) When in 1991 erythropoietin was finally
clinically tested in humans with SCA, it did not dramatically
increase fetal-hemoglobin production, as it had in the
baboon subjects.(1) The baboons used in fetal-hemoglobin
research cannot serve to predict the magnitude of humans'
fetal-hemoglobin response to drugs.
More generally, baboons and humans show species differences that preclude the validity of SCA work in baboons. For example, the two species differ in the way fetal hemoglobin is distributed among their red blood cells. In baboons fetal hemoglobin tends to be evenly distributed among all red blood cells; in humans it tends to appear only in a small percentage of cells.(19) Therefore, drugs intended to increase fetal-hemoglobin production in humans can increase it only in proportionately few cells. Since only these cells might then resist sickling, the problems associated with sickling would continue.(17)
Clearly the use of baboons is an ineffective way of seeking information on fetal-hemoglobin production in humans.Butyrates' Capacity to Increase Fetal Hemoglobin:
Another Discovery from Clinical and In Vitro Research
Insights into another potential way of increasing fetal-hemoglobin production have also derived exclusively from clinical and in vitro research.
Diabetics experience bouts of abnormally high blood-glucose levels. During the pregnancy of a diabetic woman, excess glucose periodically crosses the placenta; the fetus responds by producing more insulin, which reduces glucose levels.(10) Because insulin has been shown to affect gene expression, in the 1980s Susan Perrine and colleagues examined red blood cells from newborns of diabetic mothers to see whether insulin affected expression of the gene for fetal hemoglobin. The group found that the newborns did show a delayed switch from fetal to adult hemoglobin production.(10) Using cultured blood cells from the umbilical cords of newborns whose mothers were not diabetic, Perrine et al. then tested various substances known to be elevated in diabetes. They found that some butyrates (fatty acids) increase fetal-hemoglobin production.(20)
Next the researchers infused sheep fetuses--in utero--with butyrates. The fetuses showed a delayed switch from fetal to adult hemoglobin.(21) Providing no new insights, this finding simply paralleled the prior clinical and in vitro findings.
The therapeutic implications of butyrates' effect on hemoglobin production have yet to be demonstrated.
Unsuccessful Efforts to Contrive an Adequate Animal Model of SCA
In various nonhuman animals--including deer, goats, hamsters, mongooses, raccoons, sheep, and squirrels--red blood cells sometimes naturally sickle.(22) However, as pathologists Robert Nalbandian and colleagues note in a letter to The New England Journal of Medicine, the sickling that occurs in these animals' red blood cells is generally independent of oxygen concentration, may be reversed by changes in pH, and occurs (in vitro) in high-concentration salt solutions or after prolonged storage of cells. Sickling of human cells shows none of these characteristics. Nalbandian et al. conclude, "The mechanisms involved and very probably the molecular pathology in these animal models differ considerably from that postulated [for SCA]."(22)
Because the naturally occurring mutation that causes SCA is apparently unique to humans, in 1988 Edward Rubin et al, inserted the human gene that codes for sickle-cell hemoglobin into mouse embryos, in the hope that the red blood cells of the resulting transgenic mice would then sickle.(4) Unlike humans, mice normally have two types of [beta]-globin in their red blood cells--major and minor.(4) Since in humans normal [beta]-globin inhibits sickling, the researchers used a strain of mice who suffer from [beta]-thalassemia, a condition in which major [beta]-globin is absent. Some of the resulting transgenic mice produced sickle-cell hemoglobin and passed the gene for such hemoglobin on to their offspring. In no mouse, however, did any red blood cells sickle.(4) (The mice's minor [beta]-globin may have prevented sickling.)(4)
In 1990 David Greaves and colleagues also inserted the SCA gene into mouse embryos. Because in humans sickle-cell hemoglobin is composed of [alpha]-globin and abnormal [beta]-globin chains, the researchers inserted genes, joined together, for both of these globin chains. Three of the five transgenic mice born showed sickle-cell hemoglobin, comprising 10%, 35%, and 83% of their total hemoglobin. These three mice also produced red blood cells that sickled in vitro. However, as revealed by blood smears, two of the three mice with blood cells that sickled in vitro showed no in vivo sickling.(5) Further, whereas in humans with SCA the percentage of sickled cells in vivo is 6-32%,(23) in the transgenic mouse who showed in vivo sickling the percentage was only about 0.1 %. None of the mice had any SCA-like symptoms, including anemia.(5) The mice were then crossbred with [beta]-thalassemic mice. The resulting offspring had over 90% sickled cells in vitro. Although these mice showed anemia and enlarged spleens, the symptoms were milder than in most SCA and the mice were otherwise healthy.(6)
Since implanting the human gene for the most common form of sickle-cell hemoglobin did not cause SCA in mice, researchers turned to two less common forms (S-Antilles and D Punjab) that contain two abnormal amino acids (rather than one) and so cause disease even in humans heterozygous for the sickle-cell gene. In 1991, Marie Trudel et al. reported joining S-Antilles and D Punjab hemoglobin genes and inserting them into mouse embryos. As the researchers had desired, the resulting transgenic mice produced sickle-cell hemoglobin, which the researchers dubbed "hemoglobin SAD" (for S-Antilles/D Punjab).(7) When these mice were bred with normal mice, the offspring produced approximately 20% of their hemoglobin as hemoglobin SAD. The offspring also had slightly enlarged spleens and some sickled red blood cells but were not anemic.(7) When the first-generation transgenic mice were bred with [beta]-thalassemic mice, the offspring had approximately 26% hemoglobin SAD as well as more sickled cells in vivo and more destruction of red blood cells than the mice with 20% hemoglobin SAD. Exposed to conditions of very low oxygen (atmospheric concentration of only 8% as opposed to the normal 21 %), these mice died, possibly from vaso-occiusion caused by severe sickling.(7)
Although hemoglobin-SAD mice possess red blood cells that sickle in vivo, particularly under conditions of very low oxygen, there are serious scientific problems with using these mice to investigate SCA.
First, unlike most sickle-cell hemoglobin, hemoglobin SAD has two abnormal amino acids rather than one; arising from a different mutation, it has a different molecular basis for its instability.(7) Researchers have claimed that hemoglobin-SAD mice can be useful in the search for drugs to reduce or prevent sickling,(4) but agents that stabilize hemoglobin SAD may not stabilize the more common sickle-cell hemoglobin, and vice versa.
More importantly, species differences render the use of mice for SCA research fundamentally unsound. Unlike humans, mice have no distinct fetal hemoglobin: until 11 days of gestation, fetal mice produce three [beta]-like globins that closely resemble adult mouse hemoglobin; for the remainder of gestation, they produce adult hemoglobin.(8) Further, in humans the switch from fetal to adult hemoglobin is reversible, whereas in mice the switch from the three initial [beta]-like globins to two adult [beta]-globins is apparently irreversible.(8) Therefore, possible methods of reactivating fetal hemoglobin--the focus of most SCA research--cannot be tested in mice. Also, human SCA patients often suffer kidney damage, even kidney failure, from a succession of vaso-occlusive episodes; in the mouse kidney, blood vessels are proportionately wider and therefore at less risk of becoming obstructed.(9) As hematologist Mary Fabray and colleagues conclude, "The transgenic mouse (as any other animal model) will necessarily differ substantially from the sickle cell disease patient because mouse anatomy and physiology differ from human."(9)
Conclusion
Advances in understanding SCA have come almost exclusively from human clinical research and in vitro studies of human-derived cells or molecules. Although animal “models” have been used in efforts to discover ways of preventing or countering the sickling of red blood cells, fundamental species differences have undermined such efforts. These differences will, almost certainly, continue to prevent animal experimentation from making any significant contribution to the prevention or treatment of SCA.
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