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Hypertension as a proliferative disorder: contribution of apoptosisDr. Pavel Hamet CHUM Research Centre
Several arguments have been put forward suggesting that hypertension can be considered, at least in part, as a vascular proliferative disorder. Thus, first of all, hyperplasia is observed in vitro in vascular smooth muscle cells (VSMC) and fibroblasts from genetically hypertensive rats. It even persists in culture and is reversible in experimental models (1). Several groups have described neonatal heart hypertrophy in vivo but we were the first to report that the kidneys and aorta of genetically hypertensive rats have an increased amount of DNA which incorporates elevated quantities of thymidine (2-4). This persistence of hyperplasia in vitro and its presence in neonates indicate that hyperplasia is not simply due to heightened blood pressure, and can indeed constitute a primary element (5). (figure 1)
Hypertrophy is certainly apparent in VSMC in vitro in response to angiotensin and in the vascular wall in vivo with or without de novo DNA synthesis. The polyploidy demonstrated segregates even in F2 generations (6). Remodeling in vivo in the vessel wall and heart usually indicates a decreased lumen without hyperplasia and hypertrophy. It is frequently considered as a reshaping of the vascular wall without a change in cellular mass. Finally, rarefaction has been observed as a phenotype of hypertension, demonstrating cell loss in the brain and muscular capillaries with some potential of reversibility after treatment.
Our group has put forward the question whether or not apoptosis is involved in these processes. In our mind, initially, apoptotis would be in balance with increased cellular proliferation, which we demonstrated was mainly due to accelerated G1/S transition with a slight elevation in the G2 and M components in VSMC from the aorta of spontaneously hypertensive rats (SHR) compared to Wistar-Kyoto (WKY) or other normotensive controls (7). Thus, the first component of the imbalance in proliferation is the accelerated entry of smooth muscle cells into the S phase of the cell cycle (8). This led us to propose an imbalance between growth and programmed cell death (PCD) which would be in favor of growth in hyperplasia, in favor of PCD in rarefaction/atrophy, and both could be involved in remodeling (9). At that time, about 5 years ago, it was important to develop quantitative methodologies for apoptotic surveillance. (figure 2)
Initially, the cleavage to oligonucleotide fragments was considered a hallmark of apoptosis. Two types of fragments have been recognized (10): Type A represents large 20 to 50,000 kb fragments of DNA scaffold strength, while the more typical fragment, called Type B, is the result of internucleosomal cleavage into 180 bp and multiples of oligonucleotide strands. This is the typical ladder-forming picture used so much in the demonstration of apoptotic events. Such fragmentation can be employed for selective labeling of apoptosis where the apoptotic process increases fragmented DNA at internucleosomal sites which can be extracted and 3-OH radiolabeled by terminal deoxinucleotide (TDT) migration in agarose gel and autoradiography. (figure 3)
Of great help was the PhosphorImager System which permitted rapid and quantitative imaging of this DNA fragmentation (11,12). We have tested the feasibility and quantitative character of this labeling in an apoptotic model induced by gamma irradiation damage. The whole mouse was irradiated by 60 rads and injected with thymidine for monitoring of proliferation. The picture seen clearly demonstrated that [3H]-thymidine incorporation was nearly abolished while apoptosis was increased. This can be viewed in artificial colors where "Ø" radiation and "no TDT" serve as controls, and several animals are quantified in columns. A similar picture is demonstrated for the kidney, where the external cortex is particularly damaged by apoptosis since it is known that glomeruli are irradiation-sensitive. (figure 4)
Using this method, we have quantified and localized the apoptotic process as it occurs prior to the increase in left ventrical size induced by aortic coarctation. In collaborative work with Kathy Schwartz from Paris, we have demonstrated that the "apoptotic window" clearly appears on day 4 after aortic ligation. This is at the time of expression of early genes and prior to the increase in heart size. Whether or not the apoptotic process is directed to cardiomyocytes was the next question, and confocalmicroscopy allowed us to illustrate that indeed apoptosis occurs in patches throughout the myocardium without any clear anatomic concentration and predominantly in cardiomyocytes. Whether or not this apoptotic process is a requirement for hypertrophy and whether or not the modulation of existent apoptosis will be detrimental or helpful are the object of current studies (13).
In the last few years, apoptosis has become much better
defined as a sequence of biochemical events where a membrane-triggering mechanism, either
a dependant or independant death domain containing proteins specific for different types
of apoptotic stimuli, engages an intracellular process which usually leads to a fall in
the proton gradient at the marker of mitochondrial cristae. A series of pro-apoptotic and
anti-apoptotic proteins controls these mechanisms by their agregation, including bax,
Bcl-x, bad and their derivatives. Mitochondria release cytochrome c, which is one of the
initial steps in the final outcome controlled by caspases. These proteolytic enzymes,
working in cascade, are auto-activated and finally elicit DNA damage and cell resorption
by a process not involving inflammation but rather the participation of neighboring cells.
To demonstrate apoptosis, monitoring of DNA destruction is still required as the amount of oligonucleosomal laddering increases, but at least 1 biological step, usually involving caspase activation, occurs in parallel as a confirmation of the apoptotic event.
It is important to distinguish between different modes of cell death. In the past, we spoke only of necrosis where sheets of tissues are destroyed by external intervention. Now, our attention is directed predominantly to apoptosis. In several situations, however, such as myocardial infarction, both apoptosis and necrosis are manifest. The work by Anthony Anversa from New York has clearly demonstrated the presence of apoptotic cells, usually surrounding the necrotic component of myocardial infarction (14). Whether or not the apoptotic component can be better controlled than the necrotic component is currently under investigation. The studies of Currie et al. have shown that heat shock protein (HSP) expression may be protective in cardiac ischemia. We have, therefore, undertaken experiments in collaboration with Dr. Johanne Tremblay on prophylaxis against necrosis and apoptosis by HSP in VSMC (15). These studies have divulged that the expression of HSP, including HSP70 and HSP27, indeed protects cells in acute heat shock after mild heat stress, but only against necrosis and not at all against apoptosis. The phenomenon has been investigated by DNA fragmentation in serum deprivation-induced apoptosis, contrasting with severe heat shock and leading to DNA smear, evoked by necrosis. We proposed that apoptosis may be a continuum of necrosis after serum deprivation, where HSP expression is not protective, while in acute heat shock, HSP expression induced by mild heat shock can prevent necrosis but not apoptosis, at least in VSMC. (figure 5)
Ion transport abnormalities have been widely described in hypertension. Several of these abnormalities produce changes in ionic content as well as proton concentration. Proton gradient alterations may result in proliferative abnormalities encountered with hypertension. We have, therefore, investigated whether intracellular ion concentrations can modify the extent of apoptosis in VSMC. These studies performed in collaboration with Dr. Sergei N. Orlov, have clearly demonstrated that inversion of intracellular Na+/K+ ratio blocks apoptosis in VSMC at a site upstream of caspase-3 (16). The tool we have used is ouabain, which inhibited DNA fragmentation in VSMC. To ensure that significant apoptosis is seen, we have transfected VSMC with E1A gene which evokes up to 25% apoptosis by serum withdrawal. The addition of ouabain abolished this induction of apoptosis by serum withdrawal, and control experiments showed that reversion of the Na+/K+ ratio itself does not affect apoptosis but abolishes its modulation by ouabain, suggesting that indeed intracellular Na+/K+ content and not the ouabain molecule itself modulates apoptosis. Our studies have revealed that ouabain inhibits caspase-3 activation. Additional new results demonstrate that other ouabain-related compounds, such as digitalis, have similar activity. Protection is secondary to new protein synthesis in the presence of ouabain, which is responsible for the anti-apoptotic event. This novel step in apoptotic modulation is currently under study.
As a next step, we examined the impact of several antihypertensive classes of drugs on cardiovascular remodeling. It is known that cardiac as well as vascular hypertrophy/hyperplasia can be prevented or reverted by antihypertensive agents. Apoptosis in these studies was examined in collaboration with Dr. Denis deBlois. First, we demonstrated that several classes of medications, including angiotensin II receptor antagonists, converting enzyme inhibitors and calcium channel blockers can decrease aortic DNA content in contrast to hydralazine in spite of the fact that it has at least an equal effect in lowering blood pressure. This suggests that some of these antiproliferation events are blood pressure-independent (12,17). (figure 6)
Many studies have reported organ-specific and time-specific actions of these antihypertensive classes of medications. Initially, we observed that soon after administration of an antihypertensive agent, there was an apoptotic event, usually within 1 to 3 weeks, followed by suppression of DNA synthesis. We have been able to pinpoint such an "apoptotic window" in the aorta, carotid and heart. Our further questions were addressed to the anatomical localization of these apoptotic events. Thus, we have demonstrated recently that agents, such as nifedipine, concentrate apoptotic effectiveness in the sub-epicardium region without affecting myocardial mass. Current data illustrate that apoptosis is actually confined to fibroblasts, leaving cardiomyocytes intact after intervention with such agents as angiotensin receptor blockers (17). With the latter class, we have also addressed the question whether or not AT1 or AT2 receptors are involved. Our studies have demonstrated convincingly that the apoptotic peak induced by Valsartan can be clearly abolished by the AT2 receptor antagonist PD123319, preceded by an increase of Bax and a decrease of Bcl-2, allowing apoptosis to occur. (figure 7)
One final question, which we would like to discuss here, is whether or not this apoptotic abnormality in hypertension is genetically determined. For this purpose, we have elected to investigate the early stage in hypertension development, the neonatal state, where cardiac hyperplasia is already evident with augmented DNA. We interpret this hyperplasia as evidence for a primary event not yet modified by hemodynamic changes. In collaboration with Dr. Pierre Moreau, we evaluated the degree of apoptosis in the neonatal heart and discovered that it is highly suppressed in neonates, perhaps contributing to increased heart size as the two are actually negatively correlated with each other (18).
Recently, we examined the genetic determinants of decreased apoptosis in neonates by applying the segregating model. (figure 8)
In collaboration with Drs. V. Kren, M. Pravenec and J. Kunes from Charles University in Prague and the Academy of Science in the Czech Republic, we used recombinant inbred strains. These strains are replicas of hypertensive and normotensive ancestors crossed with the F1 generation from which F2 hybrids are derived. At this level, each animal is heterozygous for most of the loci and is thereafter inbred for 25 generations. These strains are replicas of the initial F2 generation except that all the animals are homozygous at all loci and each strain can be studied in repetition, having the same defined genotype. As these animals have been densely genotyped on all chromosomes, we thus have a set of models in which each phenotype can be localized not only in individual animals, but also in response to medication and during evolution of the disease. We have recently established that this model can serve in the evaluation of kidney hyperplasia (19). (figure 9)
By applying the model in neonates for the detection of apoptosis, we have indeed demonstrated that both Type A and Type B apoptoses are decreased in SHR, and there is up to a 5-fold gradient between different strains of the recombinant inbred panel. This allows the localization and identification of at least 2 loci, the first on chromosome 1, syntenic to human chromosomes 12 and 16 with several interesting candidate genes, and the second on chromosome 18, syntenic to human chromosome 5. Such an approach now permits us to search for positional candidates and identify, eventually, the genes responsible for the modulation of apoptosis favoring hypertrophy in neonates. Furthermore, we have identified loci responsible for cardiac hypertrophy in newborns vs adults, and indeed different loci appear to be involved. Thus, genes of susceptibility, development and maintenance of cardiac hypertrophy seems to display their functions at different times.
Genes of susceptibility, development and maintenance of
Heart weight/Body weight was associated with distinct markers
When we selected the lowest and highest quartiles of heart weight/body weight mass from birth to adulthood. Two genetic loci, one on chromosome 4 and the other on chromosome 14, appear with the highest correlation of heart weight/body weight mass (r = 0.72), suggesting shared genetic determinants from birth to adulthood with different genes apparently adding to heart rate as blood pressure develops. We believe that genetic methods will permit us to identify putative genes involved in the cardiac and vascular remodeling of hypertension. As the pathways of both apoptosis and proliferation are being uncovered, a novel target for therapeutic intervention should be unveiled.
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