PHARMACOGENOMICS OF CEREBRAL VASOSPASM
Pharmacogenomics is defined to identify the genes or loci that are involved in determining the responsiveness and to distinguish responders and nonresponders to a given drug (1). Genome sequencing, and transcriptome and proteome analysis are of particular significance in pharmacogenomics. Sequencing is used to locate polymorphisms, and monitoring of gene expression can provide clues about the genomic response to disease and treatment. The transcriptome analysis can be carried out by methods of random cDNA sequencing (expressed sequence tag project, body map project, serial analysis of gene expression, etc.), mRNA display (differential display, fluorescent differential display, RNA arbitrarily primed polymerase chain reaction, molecular indexing, gene expression fingerprinting, etc.) and differential hybridization (cDNA high-density filter, cDNA microarray, oligomicrochip, etc.). We used transcriptome analysis to identify therapeutic target genes by studying change of gene expression in animal models of cerebral vasospasm (2) and found novel drug target candidates through this pharmacogenomic strategy (3).
Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) causes cerebral ischemia and infarction. The present study uses transcriptome analysis to identify therapeutic target genes in the rat model of SAH.
Despite considerable advances in diagnostic, surgical, and anesthetic techniques and perioperative management, the outcome for patients with SAH remains poor, with overall mortality rates of 25% and significant morbidity among approximately 50% of survivors (2). Cerebral vasospasm is the delayed narrowing of large capacitant arteries at the base of the brain, and is a major cause of morbidity and mortality after SAH (2). In about one-half of cases, a delayed neurological ischemic deficit occurs, and 15-20% of such patients suffer stroke or die from vasospasm despite maximal therapy (2). Accumulating evidence suggests that hemoglobin (Hb)-induced oxidative stress plays a central role in the pathogenesis of vasospasm after SAH (4). However, the mechanism by which delayed cerebral vasospasm resolves spontaneously has not been sufficiently investigated. If the intrinsic mechanism of spasmolysis was clarified, then it might lead to a novel therapeutic strategy. In the present study, we employed an improved differential display technique (5), fluorescent differential display, to identify differentially expressed genes and evaluate the functional significance of such genes in the basilar artery with vasospasm.
TARGET VALIDATION FOR CEREBRAL VASOSPASM
Rats were allocated into one of four groups; one-hemorrhage, two-hemorrhage, one-saline and two-saline injected groups. The one- and two-hemorrhage rats were given one or two (48 h apart) injections of autologous blood into the cisterna magna. The saline groups were given saline injections by the same protocol. No rats developed neurological deficits. Comparisons of mean values for body weight, mean arterial blood pressure, heart rate and end-tidal CO2 revealed no significant differences among the groups. The one- and two-hemorrhage rats showed biphasic vasospasms that occurred at 10 min (early vasospasm) and on days 2 and 7 (delayed vasospasm) after blood injections (3). Delayed vasospasm in the two-hemorrhage rats resembled human vasospasm. No significant vasospasm was observed in the saline groups.
From fluorescent differential display fingerprints, we identified changes in intensity in approximately 3% (280 bands) of the 9642 bands specific for delayed vasospasm in the two-hemorrhage rats (Fig. 1). Once upregulated, the cDNA fragment that coincided with the development of vasospasm proved the rat heme oxygenase-1 (HO-1) gene. Thus, we focused on this gene to clarify the target validation for vasospasm.
HO-1 mRNA levels in the basilar artery and brain obtained at identical time points were determined by two quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis methods. HO-1 mRNA levels were expressed as a ratio to HO-2 mRNA levels for normalization, because the mRNA levels of HO-2 and β-actin in the basilar artery and brain after blood or saline injections were unchanged. Real-time RT-PCR analysis demonstrated that HO-1 mRNA was exclusively induced in the basilar artery and modestly induced in the whole brain in SAH groups, but not in the saline groups. The expression levels of HO-1 mRNA were higher in the two-hemorrhage rats, where the amount of blood was larger than the one-hemorrhage rats. Interestingly, the expression levels of HO-1 mRNA in the basilar artery were the most prominent among the tissues evaluated (3). In spite of the identical distribution of SAH, the levels of HO-1 mRNA in the basilar artery with delayed vasospasm were about seven times higher than in the ventral brain stem on day 7 in the two-hemorrhage group. A significant linear correlation was observed between the degree of delayed vasospasm and HO-1 mRNA levels in the basilar artery, ventral brain stem, and cerebellum. HO-1 mRNA was not induced in the basilar artery with early vasospasm, and in the brain 10 min after the blood injection on day 0. Competitive RT-PCR analysis of HO-1 and HO-2 mRNA yielded similar results.
To determine the functional significance of HO-1 gene induction in the basilar artery and brain, we examined the effects of selective HO-1 inhibition using antisense HO-1 ODN on angiographic vasospasm (6). Antisense HO-1 ODN, sense HO-1 ODN, or scrambled ODN treatment was performed on one-hemorrhage rats. In the basilar artery, a successful blockage of HO-1 mRNA upregulation was observed after two injections of antisense HO-1 ODN (3). The HO-1 antisense ODN also prevented the upregulation of HO-1 protein in the basilar artery. The levels of HO-2 and β-actin mRNA were not affected by antisense HO-1 ODN, sense HO-1 ODN, or scrambled ODN treatment. Sense HO-1 ODN or scrambled ODN treatment did not affect HO-1 gene expression levels and delayed vasospasm in the basilar artery. On the other hand, the antisense HO-1 ODN significantly inhibited HO-1 induction in the basilar artery until day 4 after SAH. Moreover, antisense HO-1 ODN significantly aggravated vasospasm (3). The peak time of vasospasm was delayed from days 2 to 4. Vasospasm was present up until day 7, when it had already resolved in the control rats. It is noteworthy that HO-1 mRNA in the basilar artery was significantly induced during days 5 to 7 after the antisense ODN treatment, when vasospasm was significantly attenuated. The antisense HO-1 ODN treatment did not affect the diameter of the basilar artery in the saline groups, suggesting that antisense HO-1 ODN does not itself exacerbate vasospasm.
In the whole brain tissue, including the neuronal tissue and vessels, HO-1 mRNA was significantly induced on day 1. The magnitude of HO-1 induction in the whole brain tissue was much smaller than in the basilar artery. The HO-1 responses in the whole brain tissue after the antisense HO-1 ODN treatment were not significantly different from those of the control. These results suggest that the HO-1 levels in the whole brain tissue may reflect the HO-1 induction in the neuronal tissue that was not affected by antisense HO-1 ODN. In the neuronal tissue, HO-1 induction has been observed in glial cells after SAH (7). The inhibitory effects of antisense HO-1 ODN treatment on HO-1 induction in the basilar artery could be masked by the greater amounts of HO-1 induction in glial cells. Thus, intrathecal injections of antisense HO-1 ODN affected the HO-1 levels in the basilar artery, but not in the neuronal tissue.
We estimated the sum of OxyHb and DeoxyHb, and MetHb levels because our method of measuring Hb levels did not allow us to continue anaerobic conditions. The cisternal levels of OxyHb and DeoxyHb in the control rats reached a peak on day 2, when delayed vasospasm was most prominent, and rapidly fell on day 3. On the other hand, the levels of OxyHb and DeoxyHb after the antisense HO-1 ODN treatment were higher on day 3 than on day 2, and started to fall on day 4. The levels of OxyHb and DeoxyHb after antisense HO-1 ODN treatment were significantly higher than in the control on days 4 and 5. In contrast to OxyHb and DeoxyHb, the levels of MetHb did not change appreciably in the control rats. The levels of MetHb were significantly higher on day 5 after antisense HO-1 ODN treatment than the baseline, but not significantly different from the control. Thus, aggravation of vasospasm after antisense HO-1 treatment may be partly due to the delayed clearance of OxyHb and DeoxyHb in the subarachnoid space.
HO-1 GENE INDUCTION AS A NOVEL DRUG TARGET
Recent studies support the hypothesis that HO-1 induction plays an important role in cellular protection against both heme- and nonheme-mediated oxidative injury (8). The magnitude of HO-1 induction after oxidative stress, the wide distribution of this enzyme in systemic tissues, and the intriguing biological activities of its catalytic by-products make HO-1 a highly attractive candidate for maintaining cellular homeostasis in response to oxidative stress (8). This study demonstrated the upregulation of HO-1 mRNA in the cerebral arteries and, more importantly, the augmentation and prolongation of delayed vasospasm by antisense HO-1 ODN treatment after SAH.
Moreover, we found the protective effects of HO-1 gene induction by endogenous or clinical compounds in cerebral vasospasm (manuscript in preparation). HO is the rate-limiting enzyme in the heme degradative pathway (9). All isozymes of HO, HO-1 (an inducible isozyme), HO-2 (a noninducible isozyme) and HO-3 (a recently identified isozyme), metabolize heme in Hb and generate carbon monoxide (CO), free iron (ferric iron) and biliverdin (subsequently reduced to bilirubin) (9). Hb is a potentially toxic molecule. The available evidence suggests that the initial reaction leading to the formation of all potentially toxic compounds is the autoxidation of OxyHb and DeoxyHb to MetHb. A major factor that mediates delayed vasospasm also has been reported to be OxyHb and DeoxyHb, but not MetHb (4). The induction of HO can have dual beneficial effects against the toxicities of Hb: it removes Hb itself and synthesizes an antioxidant bilirubin.
In the vascular cells, the expressions of HO-1 have been observed in the endothelial and smooth muscle cells in some pathophysiological conditions (8). CO, a by-product of HO, is a gas molecule that shares some of the properties of nitric oxide, in as much as CO binds to the heme moiety of cytosolic guanylyl cyclase to produce cGMP (10). In the vascular smooth muscle cells, HO-1 is induced by hypoxia and its by-product, CO, promotes the accululation of cGMP. Increased cGMP causes smooth muscle relaxation. Furthermore, smooth muscle cell-derived CO inhibits the production of the endothelium-derived vasoactive agents, endothelin-1 and platelet-derived growth factor-B.
In conclusion, we found remarkable upregulation of HO-1 mRNA in the basilar artery, which might be closely related to the occurrence of delayed vasospasm after SAH. In this report, we clearly demonstrate, for the first time, that intrathecal administration of antisense HO-1 ODN aggravates vasospasm, suggesting HO-1 gene induction has spasmolytic effects. Furthermore, we found the protective effects of HO-1 gene induction by endogenous or clinical compounds in cerebral vasospasm (Fig. 2). Therapeutic gene induction of HO-1 could be a novel strategy for the prevention and treatment of Hb-induced pathologic conditions including delayed cerebral vasospasm. Our results suggest that the pharmacogenomics, especially transcriptome analysis, have the potential for strategy to define novel drug targets in various diseases.
Acknowledgement: This work was supported in part by Grants-in-Aid for Scientific Research and for International Scientific Research from the Ministry of Education, Science, Sports and Culture, the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, and the Grant for Pediatric Diseases from the Ministry of Health and Welfare, Japan. It was also supported in part by Grants-in-Aid for the Research Projects from the Mie Medical Research Foundation.
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The symposium and the publication of this supplement were supported by an educational grant from Novartis Pharma K.K. Tokyo, Japan.