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Stroke. 1996;27:2102-2109

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(Stroke. 1996;27:2102-2109.)
© 1996 American Heart Association, Inc.

Articles

Role of Extracellular Matrix in Experimental Vasospasm

Inhibitory Effect of Antisense Oligonucleotide on Collagen Induction

Keisuke Onoda, MD; Shigeki Ono, MD; Kotaro Ogihara, MD; Tomomi Shiota, MD; Shoji Asari, MD; Takashi Ohmoto, MD Yoshifumi Ninomiya, MD, PhD

the Departments of Neurological Surgery (K. Onoda, S.O., K. Ogihara, T.S., S.A., T.O.) and Molecular Biology and Biochemistry (K.Onoda, S.O., T.S., Y.N.), Okayama University Medical School (Japan).


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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 down arrowIntroduction 
 down arrowReferences 
 
Background and Purpose Although it has been suggested that collagen plays a role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage, there has been no constructive research to prove it directly. In this study we stopped the transcription of the procollagen type I gene by introducing antisense oligonucleotides for its mRNA in a rat femoral artery model of vasospasm induced by blood and assayed the changes in the vasoconstrictive activity of the vessel and expression of the procollagen mRNA.

Methods We applied antisense, sense, or missense oligonucleotides, located at the carboxyl propeptide region for {alpha}1(I) procollagen mRNA, onto the femoral artery in a rat femoral artery model of vasospasm. The diameter of the artery was measured by angiography. The transcription level of the procollagen gene in the arterial tissue was assayed by use of reverse transcription–polymerase chain reaction. Morphological change in the artery was observed with aldehyde-fuchsin-Masson-Goldner staining.

Results In the model, when the artery was exposed to antisense oligonucleotides in pluronic gel for 5 days to prevent arterial contraction, the contraction was inhibited at a significant level (76.0%±5.6) when compared with that in control experiments using sense oligonucleotides (64.0%±2.4), missense oligonucleotides (63.5%±3.5), or gel alone (62.1%±5.8). The application of antisense oligonucleotide resulted in a marked decrease in {alpha}1(I) procollagen mRNA expression as determined by polymerase chain reaction, indicating that the collagen reduction by antisense oligonucleotides occurred at the transcription level. Histological staining suggested that collagen accumulation at the site in the artery where antisense oligonucleotide had been administered was indeed less than that in the control artery.

Conclusions The results indicate that the induction of procollagen type I could cause pathogenesis of the arterial contraction induced by blood in a rat femoral vasospasm model.


Key Words: collagen • gene expression • vasospasm • rats


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Although intensive efforts have been made to investigate the pathogenesis of cerebral vasospasm after SAH, the issue remains unresolved. Therefore, the essential therapeutic approach for preventing vasospasm has not been fully established. Phenomena that occur in cerebral vasospasm are similar to those seen in the wound-healing stage after vascular injury due to SAH. In the process of wound healing,1 2 induction of collagen synthesis was observed in the injured vascular wall. The deposition of collagen has been found in experimental SAH models, as well as in vasospasm in humans. Intimal expansion consists of an increase in collagen fiber content and accumulation of smooth muscle cell–like cells that migrate into the intima.3 4 Also in these models, degenerating smooth muscle cells with vacuole formation are associated with an increased collagen content in the tunica media,3 4 5 and the adventitia of vessels is thickened and demonstrates an increase in collagen fibers along with inflammatory cell infiltration.5 6 The increase in collagen content of the arterial wall after SAH may be responsible for the increased stiffness and thickening of the arterial wall,7 8 9 10 11 12 as well as the acute smooth muscle contraction, resulting in delayed cerebral vasospasm. However, several investigators13 14 indicated no evidence for an increase in collagen in the arterial wall during vessel narrowing and thus questioned the importance of increased collagen in the pathogenesis of vasospasm. Few studies15 16 have carried out a quantitative analysis of collagen content in the arterial wall after SAH, and the results of those studies are rather conflicting. Consequently, to clarify the role of collagen in the pathogenesis of vasospasm, we introduced a new approach, the "antisense method," that uses regulation of gene expression17 to inhibit any increase in collagen in a vascular model using the rat femoral arterial wall.

The rat femoral artery model is one of the vasospasm animal models and was first developed by applying blood or blood components to the adventitia surface of the femoral artery.18 Characteristic features of the model include chronic narrowing of the femoral artery and marked ultrastructural and immunohistological changes. These morphological changes throughout the vessel wall exposed to periadventitial blood are similar to those seen in human cerebral arterial vasospasm after SAH.18 Furthermore, the stiffening and thickening of the femoral artery observed in the model may reflect increased extracellular components such as collagen. In fact, Kasuya et al19 observed that the expression of TGF-ß and procollagen types I and III mRNA started increasing at day 3 and day 7, respectively, after the application of blood, and both reached their peak at day 7 in the same model, suggesting that the chronic narrowing of the artery observed in the model is attributable to induction of procollagen types I and III. The purpose of this study was to determine, by selectively inhibiting expression of collagen type I mRNA using AD, whether the increased stiffness and thickness of the artery exposed to periadventitial blood was directly due to collagen type I biosynthesis.

The antisense method is often efficient for analyzing gene function in vitro. It is considered that AD specifically binds to the targeted complementary sequences at the level of transcription, RNA processing, translocation, or translation and thereby reduces the specific gene expression.17 Recently AD was used to regulate gene expression in the vascular system in vivo20 21 ; AD for the c-myb gene introduced into the rat carotid artery with balloon injury suppressed the proliferation and migration of smooth muscle cells.22 In the same model, data indicated that AD for cdc2 kinase and proliferation cell nuclear antigen mRNAs suppressed neointimal hyperplasia on intraluminal application.23 24

In the present investigation, we introduced AD complementary to mRNA for {alpha}1(I) procollagen into a rat femoral artery model of vasospasm to clarify the role of collagen type I in the pathogenesis of vasospasm.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
OligoDNA Production
On the basis of the report of Laptev et al,25 we synthesized the most effective AD to inhibit the rat procollagen type I gene expression in the rat femoral artery vasospasm model.18 The target sequence of AD is located in exon 51 of the gene, which encodes a part of the carboxyl propeptide of the procollagen {alpha}1(I) chain. To obtain the nucleotide sequence of the target region, we used PCR, cloning, and nucleotide sequencing based on the reported nucleotide sequences.26 The sequences of the oligoDNAs used were 5'-TGTTGCCTTCGCCCCTGAG-3' (+3658 to +3676) for AD, 5'-CTCAGGGGCGAAGGCAACA-3' for SD, and 5'-TGTTGCCTTAGCACTTGAT-3' for MD, which were modified with phosphorothioate to protect against nonspecific DNases27 and dissolved in 25% pluronic gel. Phosphorothioated ADs labeled with fluorescein were also synthesized by adding fluor-12-deoxynucleotides at the 5' ends of the oligonucleotides. The sequence similarities were checked against the reported DNA sequences present in the Genebank/EMBL database, and we found no sequences significantly related to them.

Fluorescence Microscopy
To confirm whether oligoDNAs applied onto the adventitia of the femoral artery were incorporated into the vascular wall, we labeled ADs or MDs with fluorescin and chased the fluorescence-labeled oligoDNAs. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium (30 mg/kg IP). The right femoral artery was exposed under a surgical microscope, and 0.1 mL of autologous blood was applied onto the artery. The wound was covered. Two days later, the artery was exposed again, and 100 µL (2 nmol/100 µL) of fluorescein-labeled oligoDNA dissolved in pluronic gel or gel alone (as a negative control) was applied from the adventitia side of the artery. Twenty-four hours or 5 days later, the femoral artery was rapidly removed after perfusion-fixation with 4% paraformaldehyde. Cryostat sections were prepared with a standard technique and observed under a fluorescence microscope. We repeated the same experiments three more times to confirm the results.

Rat Femoral Artery Vasospasm Model
The femoral artery model for vasospasm developed by Okada et al18 was modified in this present investigation. Namely, male Sprague-Dawley rats weighing from 450 to 650 g were anesthetized with pentobarbital sodium (30 mg/kg IP) and allowed to breathe spontaneously. Using a sterile microsurgical technique, we exposed 1-cm proximal segments of both femoral arteries in the inguinal region and placed 1x1-cm nylon sheets under them. Fresh autologous blood (0.1 mL) or saline for the control was applied directly to the adventitia of the arteries. After the application, the sheets were sutured with 6-0 nylon to cover the arteries bearing an arterial blood clot or saline. All the experimental procedures performed in this study were in accordance with the institutional guidelines of Okayama University Medical School.

Angiographic Studies on the Femoral Artery
Seven days after exposure of the artery to blood, the diameter of the femoral artery was measured on x-ray films by angiography. For the angiography, the rats were anesthetized and allowed to breathe spontaneously. First, a 1F polyethylene catheter (ATOM) was inserted retrogradely into the left common carotid artery up to the aorta and fixed to the artery with 5-0 silk during observation under a surgical microscope. Contrast medium (1 mL) was injected at the speed of 1 mL/s into the inserted catheter. Films of anteroposterior projections were made. We used an Advantx/AFM (GEYMES) machine for digital subtraction angiography. The diameters of the 1-cm proximal segments of both femoral arteries in the inguinal region were measured on x-ray films. We measured the diameter within the 1-cm segment of each artery, where the blood and subsequently oligoDNAs were applied, at three points: proximal, center, and distal (the central one was 0.5 cm apart from the other two). The measurements were made by three volunteers. The average of the three figures was used for the diameter of the segment. During the procedure, blood pressure and heart rate were monitored with an automatic detector (Omniace RT3108J, NEC Corp) through the inserted catheter. Blood gases were analyzed by an automatic analyzer.

Contraction of the Femoral Artery
Under anesthesia, 0.1 mL of fresh autologous blood or saline (control) was applied directly around bilateral rat femoral arteries in the same manner as above. Two days after the blood or saline exposure, 100 µL (20 nmol/100 µL) of AD (n=13), SD (n=10), MD (n=8) dissolved in pluronic gel, or pluronic gel alone (n=15) was applied at the site of blood or saline exposure. For control experiments (n=11), the right femoral artery exposed to saline for 2 days was covered with pluronic gel, and the left artery was kept intact during the course of the experiment. The rats were allowed to recover after the treatments. Seven days after the blood or saline exposure, the diameters of the femoral arteries were measured by angiography (as described above). During this study, levels of blood pressure, heart rate, and blood gases were monitored continuously. Differences in diameters between control (saline) and gel-alone groups, and those among AD, SD, and MD groups, were assessed by Student's t test; the results are expressed as mean±SD.

RNA Assay
To determine whether ADs for {alpha}1(I) procollagen mRNA directly inhibited the transcription of the gene, we directly measured the amount of mRNA for {alpha}1(I) procollagen in the AD-treated femoral artery and compared it with that of control experiments (SD and/or gel alone). Two days after autologous blood or saline exposure, the artery was exposed again and treated with 100 µL (20 nmol/100 µL) of AD, SD, or gel alone. Only pluronic gel was applied onto the control artery. Seven days after blood or saline exposure, the arteries were removed, and mRNA was prepared from them by use of a QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech Inc). RT was performed with oligo-dT primers supplied in a T-primed First-Strand Kit (Pharmacia Biotech Inc). On the basis of our sequence data on rat {alpha}1(I) procollagen cDNAs, together with those from Genovese et al,26 we synthesized the primers for RT-PCR. The sequences of the primers were 5'-TGGAGACAGGTCAAGACCTG-3' (sense primer) and 5'-TATTCGATGACTCTCTTGCC-3' (reverse primer) for {alpha}1(I) procollagen cDNA, and the sequences for GAPDH were 5'-GCATGGAGAGCGCAGAGTTG-3' (sense primer) and 5'-CATGTAGGCCATAGGTCCACCAC-3' (reverse primer). The PCR apparatus was programmed with a thermal controller, PTC-100 (MJ Research, Inc), to perform the initial melt at 94°C for 3 minutes and 35 cycles of the following sequential steps: 94°C, 1 minute (melt); 55°C, 1.5 minutes (anneal); 72°C, 3 minutes (extend); and 72°C, 7 minutes (final extension). PCR products were electrophoresed on 1.5% agarose gels and blotted onto nylon filters (Hybond N+, Amersham). Southern blot analyses were performed by the standard method.28 The probes used for hybridization were 32P-labeled rat {alpha}1(I) procollagen and rat GAPDH cDNAs. Seven separate experiments were performed to confirm the RNA expression.

Morphological Changes in the Femoral Artery
In the femoral artery vasospasm model, ie, after 2 days of autologous blood exposure, AD, SD, or MD for {alpha}1(I) procollagen mRNA was applied. Five days later, the animals were killed by perfusion-fixation with 4% paraformaldehyde. The femoral artery was then removed and immersed overnight in the same fixative at 4°C and postfixed in 1% osmium tetroxide for 1 hour at 4°C. Thin sections (3 µm) were cut transversely to the vascular axis and examined by light microscopy after aldehyde-fuchsin-Masson-Goldner staining. Three separate experiments were performed to confirm the results.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
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 down arrowIntroduction 
 down arrowReferences 
 
OligoDNA Uptake by femoral artery
To know where the labeled oligoDNAs were incorporated, we labeled ADs and MDs with fluorescence and traced the material by fluorescence microscopy. The labeled ADs or MDs mixed with pluronic gel were applied onto the femoral artery from the adventitial side; 24 hours or 5 days later, we observed the sections of the artery by fluorescence microscopy. Fluorescence-labeled material was distributed in all layers of the femoral arterial wall after 24 hours, as seen in Fig 1ADown, especially on the adventitial side. The fluorescence intensity was reduced after 5 days but clearly remained evident in all layers of the wall at that time (Fig 1BDown). These findings indicate that the oligoDNAs were directly incorporated into the arterial wall through the adventitia.




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  		Figure 1. Incorporation of oligoDNA into the femoral artery wall. To establish whether oligoDNA applied onto the adventitia of the femoral artery was really incorporated into the arterial wall, we chased the fluorescence-labeled AD or MD. Fluorescence-labeled oligoDNA (2 nmol/100 µL in pluronic gel) was applied onto the adventitia, and the wound was closed. After 24 hours (A) or 5 days (B), the femoral arteries were rapidly dissected out after perfusion-fixation with 4% paraformaldehyde. Cryostat sections were examined with fluorescence microscopy. Bar=35 µm. Note that the fluorescence-labeled material is distributed in all layers of the arterial wall from the adventitia side, indicating that the AD was directly incorporated into the inner part of the arterial wall.

Inhibition of Contraction With AD for {alpha}1(I) Collagen mRNA
In the present experiment, we used the rat femoral artery for the target tissue. Kasuya et al19 showed that TGF-ß and collagen types I and III mRNA started increasing at days 3 and 7, respectively, after the start of the blood exposure in the rat femoral artery model. Therefore, we decided to apply oligoDNAs at day 2 of the blood exposure in the experiment. After exposure to autologous blood or saline (control) for 2 days, the artery was continuously exposed to AD, SD, MD, or gel alone for 5 days. In this model, the contraction of the artery in response to fresh autologous blood was also observed after 7 days of blood exposure (note the height of the gel-alone column, which is 62.1% of the control [saline] column in Fig 2Down). Thus, the vascular contraction in response to the blood was extended up to 7 days in the present experimental system. Because we believed that the accumulation of collagen and other matrix macromolecules could be one of the factors causing the contraction, we examined the possibility that inhibition of procollagen transcription by application of antisense oligonucleotides for {alpha}1(I) procollagen mRNA might improve the pathogenesis of the vasospasm.



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  		Figure 2. Inhibition of vascular contraction by AD for {alpha}1(I) procollagen mRNA. Two days after autologous blood (0.1 mL) exposure, the femoral artery was covered with AD (n=13), SD (n=10), or MD (n=8) in pluronic gel, or gel alone (n=15), and the wound was closed. Seven days later, the diameter of the femoral artery was measured by angiography. For the control experiments, the femoral artery exposed to 0.1 mL of saline was covered with pluronic gel alone. Diameters for the groups of AD, SD, MD, and gel alone were expressed relative to the control (saline) diameter (taken as 100%). Differences in diameters between the control and gel-alone, AD, SD, or MD groups were assessed by Student's t test, and results are expressed as mean±SD. Note the significant inhibitory effect of AD on vasoconstriction, ie, 76.0%±5.6 of control diameter for AD versus 62.1%±5.8 for gel alone, 64.0%±2.4 for SD, and 63.5%±3.5 for MD. There is no obvious difference between the groups SD or MD and gel alone.

As shown in Fig 2Up, the diameter of the femoral artery became narrower by blood exposure, ie, 62.1% of the control (saline) level (100%) as mentioned above. The relative diameter of the SD- or MD-treated artery did not change much (SD in Fig 2Up), being 64.0% or 63.5% of control, respectively. However, AD had the significant effect (AD, 76.0% of control) of increasing the diameter of the artery (P<.01 versus gel alone). This result indicated that the increase due to AD for {alpha}1(I) procollagen mRNA corresponded to 36.8% of the typical contraction observed with gel alone. Levels of blood pressure, heart rate, and blood gases did not change much during the study. Furthermore, the results suggested that AD for {alpha}1(I) procollagen mRNA was very effective in preventing the experimental vasoconstriction. The slight increase in the diameters in the SD and MD groups (64.0% and 63.5% of saline control, respectively, compared with the control [gel-alone] arteries [62.1%]) may have been due to a nonspecific reaction to oligonucleotides in general.

Inhibition of {alpha}1(I) Collagen mRNA Expression by AD
To know whether the inhibitory effect of AD described above was truly associated with blockage of transcription of the {alpha}1(I) procollagen gene, we measured this mRNA by RT-PCR as mentioned in "Materials and Methods." We compared the {alpha}1(I) collagen mRNA expression of the femoral artery treated with AD with that treated with SD. Direct application of AD by the adventitial approach resulted in a marked decrease in {alpha}1(I) collagen mRNA expression compared with that obtained with gel alone or SD (Fig 3Down). There was not much difference in mRNA expression between gel alone–treated and SD-treated groups. Densitometric analysis demonstrated that AD decreased the level of {alpha}1(I) procollagen mRNA to 10% of that obtained with SD (data not shown). The mRNA level for GAPDH did not differ significantly between AD and SD groups (Fig 3Down). On the other hand, the {alpha}1(I) procollagen mRNA expression of the gel-alone group was increased significantly compared with that of the control group. This agreed with the Northern blot analysis of Kasuya et al,19 which indicated that the rat femoral artery exposed to periarterial blood for a period of 7 days showed increased {alpha}1(I) collagen mRNA expression compared with that of the sham-operated group. Thus, the AD for {alpha}1(I) procollagen mRNA that we used in the present experiment suppressed the {alpha}1(I) procollagen mRNA expression at the transcription level and/or at the splicing step.



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  		Figure 3. Inhibition of {alpha}1(I) procollagen [Col {alpha}1(I)] mRNA expression by AD. Two days after autologous blood exposure, AD, SD, or gel alone was applied around the femoral artery and left there for 5 days (see details in "Materials and Methods"). The femoral arteries thus treated were dissected out, RNA was extracted from the tissue, RT-PCR was performed with specific primers, and {alpha}1(I) procollagen mRNA was analyzed by Southern blotting. Note suppression of {alpha}1(I) procollagen mRNA in AD-treated tissue compared with those treated with SD or gel alone. The level of GAPDH mRNA that was measured as a control did not change much in response to the various treatments.

Inhibition of Collagen Fiber Induction by AD
When the femoral artery was fixed and observed with microscopy, the relative diameters of the control, AD-, SD-, and MD-treated arteries were proportional to those from the angiographic measurements mentioned above. Collagen type I is one of the major components of the extracellular matrix in the vascular wall.29 To know whether the inhibitory effect of AD at the transcription level described above was associated with blockade at the protein level (ie, collagen type I synthesis), we performed light microscopic examination with aldehyde-fuchsin-Masson-Goldner staining, which preferentially stains collagen fibers in blue (Fig 4Down). The content of collagen fibers in the tunica media and adventitia of the gel-alone group was increased significantly compared with that of the control group. It seemed that the smooth muscle cells (stained red) in the tunica media of the gel-alone group were sparsely distributed. Furthermore, the endothelial layer of the vessel treated with gel alone was strongly deformed along the corrugated internal elastic lamina (stained violet); this deformity was associated with the contraction of smooth muscle cells.







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  		Figure 4. Morphological changes in the arterial wall observed with light microscopy. Two days after autologous blood or saline exposure, the femoral artery was covered with AD or SD in pluronic gel or with gel alone and the wound was closed. Seven days later, the femoral artery was dissected out, fixed, and stained with aldehyde-fuchsin-Masson-Goldner. Bar=35 µm. Relative to the smooth muscle cells stained in red, collagen fibers (in blue) were increased in amount in the medial layer when SD (D), MD (E), or gel alone (B) was applied onto the femoral artery. The endothelial cell lining and subendothelial matrix are deformed along the corrugated internal elastic lamina in these specimens. In contrast, neither the AD-treated artery (C) nor the control one (A) show the wrinkled lining of the endothelial cells, but the media layer in the former seems to contain more collagen fibers than that in the control (A).

In contrast, the amount of collagen fibers in the AD group was relatively decreased compared with that of the gel-alone, SD, or MD groups. These histological findings confirm that the collagen fiber accumulation elicited by exposure to periarterial blood was inhibited by adding AD for {alpha}1(I) procollagen mRNA. Furthermore, based on the fact that the deformity of the endothelial cell layer was inhibited by AD for {alpha}1(I) collagen mRNA, we speculate that {alpha}1(I) collagen was involved in the contraction of vascular smooth muscle cells in this model.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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 down arrowIntroduction 
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The results of the present investigation indicate that transcription of the collagen type I gene is associated with vascular contraction in a vasospasm model using the periarterial blood–treated femoral artery, and they suggest that it may be possible to introduce AD directly to inhibit vascular contraction (vasospasm) in vivo.

Our results shown in Fig 2Up indicate that 36.8% of all of the vascular contraction induced by periarterial blood was associated with the induction of collagen type I synthesis. The rest could be linked to smooth muscle cell contraction. In addition, when this induction was inhibited by AD for {alpha}1(I) procollagen mRNA, not only the decrease in vascular contraction but also the inhibition of severe corrugation of the internal elastic lamina, which indicated the vascular contraction by smooth muscle cells, was recognized. This result suggests that the increased deposition of type I collagen in the pathogenesis of vasospasm in this model introduced not only decreased compliance in the arterial wall but also the contraction by smooth muscle cells; that is, it indicated that there might be some interaction between type I collagen and smooth muscle cells in the contraction induced by periarterial blood. Several reports30 31 32 33 34 have suggested an interaction between collagen and the smooth muscle cell. Lee et al34 suggested that such interaction was mediated by integrin, which is a heterodimeric cell-surface receptor for extracellular matrix molecules that can transduce mechanical signals from the extracellular environment into the cell. In their study, collagen gel–mediated smooth muscle cell contractions were inhibited by antibody specific for the subunits of integrin (ß1 and {alpha}2). That result indicated an interaction between collagen and smooth muscle cells in the stage of smooth muscle cell contraction. Ruiz-Ortega et al30 demonstrated that endothelin-1, which is a potent vasoconstrictor, induced collagen protein production in rat mesangial cells, cells that share many properties with smooth muscle cells. Kolpakov et al32 demonstrated that the collagen synthesis by cultured rabbit aortic smooth muscle cells was upregulated by S-nitroso-N-acetylpenicillamine and sodium nitroprusside (nitric oxide–generating compounds), suggesting that nitric oxide may function as a modulator of collagen synthesis by vascular smooth muscle cells. The expression of the collagen type I gene was upregulated by cytokines (interleukin-1, interferon-{gamma}, tumor necrosis factor-{alpha} and -ß) and growth factors (platelet-derived growth factor, TGF-ß) in human vascular smooth muscle cells.31 On the other hand, the function of nitric oxide was downregulated,35 36 and those of endothelin-1,36 37 38 39 cytokines,40 and growth factors12 19 were upregulated in vascular walls subjected to periarterial blood. These results suggest that nitric oxide, endothelin-1, cytokines, and growth factors might regulate collagen synthesis in the contracted vascular wall.

This report represents the first attempt to introduce AD into the rat femoral artery directly by the adventitial approach. Furthermore, we attempted to introduce AD into the vessel covered with a clot, which might seem to be a barrier against its introduction. We found that 10 µmol/L AD was not able to inhibit the collagen deposition (data not shown); however, the much higher concentration of 200 µmol/L was effective. Further studies about how to modify AD or how to introduce AD (eg, by the intraluminal approach or by use of a delivery system such as liposome-encapsulated AD and others) are needed for more efficient introduction of AD into the clot-covered vessels. In our previous report,41 which presented the efficient introduction of low-dose (10 µmol/L) AD to the rat basilar arterial wall by the adventitial approach via the cerebrospinal fluid, AD was incorporated into all layers of the vessel but especially on the endothelial side. In the present experiment, however, AD was incorporated more into the layers on the adventitial side. This could be due to differences between the chemical components of the cerebral vessels and surrounding tissues and those of the systemic vessels. Also, cerebral vessels are suspended in the cerebrospinal fluid, whereas femoral arteries are supported by connective tissues.

The permeability of the cerebral arterial wall was shown to be upregulated by an increase in intracranial pressure and blood pressure.42 43 Because intracisternal injection of AD dissolved in 100 µL of Tris-EDTA buffer led to an increase in intracranial pressure, we speculate that this increase in pressure is one of the reasons why even a low dose (10 µmol/L) of AD injected into the cerebrospinal fluid penetrated into the cerebral arterial wall efficiently, resulting in greater incorporation into the adventitial side than the endothelial side. On the other hand, the clot around the femoral artery was a barrier against penetration of AD into the arterial wall, resulting in pooling of AD on the adventitial side.

We speculate that vasospasm is induced as a result of wound healing after vascular injury inflicted by periarterial blood. In general, the expression of collagen type I mRNA reaches its peak at 3 to 4 weeks in the wound-healing process.2 However, in the vasospasm model using the rat femoral artery, the expression of collagen type I mRNA reached its peak at 1 week of blood exposure.19 There might be some fundamental biological reasons to explain this temporal difference; however, the pathogenesis of both phenomena is unknown as yet. Specific factors (TGF-ß, platelet-derived growth factor, basic fibroblast growth factor, etc) associated with the pathogenesis of vasoconstriction induced by blood in the rat femoral artery may induce the expression of collagen type I mRNA earlier than it occurs during the regular wound-healing process. Furthermore, in the case of cerebral arteries, whereas the tissue-repair mechanism presumably takes place at the vessels around the tissue where SAH occurs, the process may not lead to healing or protection but rather induce vasospasm, resulting in worsening blood circulation.

In summary, AD is a powerful new tool for the inhibition of vascular contraction in the rat femoral artery model of vasospasm. AD does not contain viral sequences, does not generate immune responses, and does not integrate into host chromosomes, which are all advantageous from a safety perspective.20 On the other hand, AD is readily degraded within cells, which means that modification of its backbone structure will be required to improve its stability. High local concentration of AD and prolonged contact time with tissues may be needed to deliver AD into a sufficient number of cells to achieve gene suppression and inhibition of a biological response. We speculate that continuous development of the antisense method will facilitate the use of antisense technology to further characterize the biological role of gene products activated during vasospasm and to provide additional therapeutic agents for use in patients.


*    Selected Abbreviations and Acronyms
 
AD = antisense oligoDNA
MD = missense oligoDNA
PCR = polymerase chain reaction
RT = reverse transcription
SAH = subarachnoid hemorrhage
SD = sense oligoDNA
TGF = transforming growth factor


*    Acknowledgments
 
This work was supported in part by grants-in-aid for scientific research (B-08457367) and for exploratory research (08877189) from the Ministry of Education, Science and Culture; the Mitsui Life Social Welfare Foundation; and Kobayashi Magobe Memorial Medical Foundation. The authors would like to thank Drs H. Yoshioka and T. Oohashi for their helpful suggestions and critical discussions of the work and the members of the Department of Neurological Surgery for experimental assistance in various aspects.


*    Footnotes
 
Reprint requests to Yoshifumi Ninomiya, Department of Molecular Biology and Biochemistry, Okayama University Medical School, 2-5-1, Shikata-cho, Okayama 700, Japan. E-mail yoshinin@ccews2.cc.okayama-u.ac.jp.

Received May 20, 1996; revision received July 9, 1996; accepted July 17, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
down arrowIntroduction 
 down arrowReferences 
 

  1. Rajendra R. The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB J. 1994;8:823-831.[Abstract]
  2. Glumoff V, Makela JK, Vuorio E. Cloning of cDNA for rat pro{alpha}1(III) collagen mRNA: different expression patterns of type I and type III collagen and fibronectin genes in experimental granulation tissue. Biochim Biophys Acta. 1993;1217:41-48.
  3. Fein JM, Flor WJ, Cohan SL, Parkhurst J. Sequential changes of vascular ultrastructure in experimental cerebral vasospasm: myonecrosis of subarachnoid arteries. J Neurosurg. 1974;41:49-58.[Medline] [Order article via Infotrieve]
  4. Smith RR, Clower BR, Grotendorst GM, Yabuno N, Cruse JM. Arterial wall changes in early human vasospasm. Neurosurgery. 1985;16:171-176.[Medline] [Order article via Infotrieve]
  5. Mayberg MR, Okada T, Bark DH. The significance of morphological changes in cerebral arteries after subarachnoid hemorrhage. J Neurosurg. 1990;72:626-633.[Medline] [Order article via Infotrieve]
  6. Espinosa F, Weir B, Shnitka T. Electron microscopy of simian cerebral arteries after subarachnoid hemorrhage and after the injection of horseradish peroxidase. Neurosurgery. 1986;19:935-945.[Medline] [Order article via Infotrieve]
  7. Vorkapic P, Bevan RD, Bevan JA. Longitudinal time course of reversible and irreversible components of chronic cerebrovasospasm of the rabbit basilar artery. J Neurosurg. 1991;74:951-955.[Medline] [Order article via Infotrieve]
  8. Bevan JA, Bevan RD, Frazee JG. Functional arterial changes in chronic cerebrovasospasm in monkeys: an in vitro assessment of the contribution to arterial narrowing. Stroke. 1987;18:472-481.[Abstract/Free Full Text]
  9. Yamamoto Y, Bernanke DH, Smith RR. Accelerated non-muscle contraction after subarachnoid hemorrhage: cerebrospinal fluid testing in a culture model. Neurosurgery. 1990;27:921-928.[Medline] [Order article via Infotrieve]
  10. Yamamoto Y, Smith RR, Bernanke DH. Accelerated nonmuscle contraction after subarachnoid hemorrhage: culture and characterization of myofibroblasts from human cerebral arteries in vasospasm. Neurosurgery. 1992;30:337-345.[Medline] [Order article via Infotrieve]
  11. Tekkok IH, Tekkok S, Ozcan OE, Erbengi A. Preventive effect of intracisternal heparin for proliferative angiopathy after experimental subarachnoid haemorrhage in rats. Acta Neurochir (Wien). 1994;127:112-117.[Medline] [Order article via Infotrieve]
  12. Iwasa K, Bernanke DH, Smith RR, Yamamoto Y. Nonmuscle arterial contraction after subarachnoid hemorrhage: role of growth factors derived from platelets. Neurosurgery. 1993;32:619-624.[Medline] [Order article via Infotrieve]
  13. Kim P, Sundt TM, Vanhoutte PM. Alterations of mechanical properties in canine basilar arteries after subarachnoid hemorrhage. J Neurosurg. 1989;71:430-436.[Medline] [Order article via Infotrieve]
  14. Pluta RM, Zauner A, Morgan JK, Muraszko KM, Oldfield EH. Is vasospasm related to proliferative angiopathy? J Neurosurg. 1992;77:740-748.[Medline] [Order article via Infotrieve]
  15. MacDonald RL, Weir BKA, Young JD, Grace MGA. Cytoskeletal and extracellular matrix proteins in cerebral arteries following subarachnoid hemorrhage in monkeys. J Neurosurg. 1992;76:81-90.[Medline] [Order article via Infotrieve]
  16. Nakamura S, Tsubokawa T, Yoshida K, Hirosawa T, Nakao M. Appearance of collagen fibers in the cerebral vascular wall following subarachnoid hemorrhage. Neurol Med Chir (Tokyo). 1992;32:877-882.[Medline] [Order article via Infotrieve]
  17. Wagner RW. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994;372:333-335.[Medline] [Order article via Infotrieve]
  18. Okada T, Harada T, Bark DH, Mayberg MR. A rat femoral artery model for vasospasm. Neurosurgery. 1990;27:349-356.[Medline] [Order article via Infotrieve]
  19. Kasuya H, Weir B, Shen Y, Hariton G, Vollrath B, Ghahary A. Procollagen type I and III and transforming growth factor-beta gene expression in the arterial wall after exposure to periarterial blood. Neurosurgery. 1993;33:716-722.[Medline] [Order article via Infotrieve]
  20. Nabel EG, Pompili VJ, Plautz GE, Nabel GJ. Gene transfer and vascular disease. Cardiovasc Res. 1994;28:445-455.[Free Full Text]
  21. Finkel T, Epstein SE. Gene therapy for vascular disease. FASEB J. 1995;9:843-851.[Abstract]
  22. Simons M, Edelman ER, DeKeyser J-L, Langer R, Rosenberg RD. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature. 1992;359:67-70.[Medline] [Order article via Infotrieve]
  23. Morishita R, Gibbons GH, Ellison KE, Nakajima M, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci U S A. 1993;90:8474-8478.[Abstract/Free Full Text]
  24. Morishita R, Gibbons GH, Ellison KE, Nakajima M, von der Leyen H, Zhang L, Kaneda Y, Ogihara T, Dzau VJ. Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J Clin Invest. 1994;93:1458-1464.
  25. Laptev AV, Lu Z, Colige A, Prockop DJ. Specific inhibition of expression of a human collagen gene (COL1A1) with modified antisense oligonucleotides: the most effective target sites are clustered in double-stranded regions of the predicted secondary structure for the mRNA. Biochemistry. 1994;33:11033-11039.[Medline] [Order article via Infotrieve]
  26. Genovese C, Rowe D, Kream B. Construction of DNA sequences complementary to rat {alpha}1 and {alpha}2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1, 25-dihydroxyvitamin D. Biochemistry. 1984;23:6210-6216.[Medline] [Order article via Infotrieve]
  27. Beltinger C, Saragovi HU, Smith RM, LeSauteur L, Shah N, DeDionisio L, Christensen L, Raible A, Jarett L, Gewirtz AM. Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides. J Clin Invest. 1995;95:1814-1823.
  28. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1992.
  29. Okada Y, Katsuda S, Matsui Y, Watanabe H, Nakanishi I. Collagen synthesis by cultured arterial smooth muscle cells during spontaneous phenotypic modulation. Acta Pathol Jpn. 1990;40:157-164.[Medline] [Order article via Infotrieve]
  30. Ruiz-Ortega M, Gomez-Garre D, Alcazar R, Palacios I, Bustos C, Gonzalez S, Plaza JJ, Gonzalez E, Egido J. Involvement of angiotensin II and endothelin in matrix protein production and renal sclerosis. J Hypertens. 1994;12(suppl 4):S51-S58.
  31. Amento EP, Ehsani N, Palmer H, Libby P. Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells. Arterioscler Thromb. 1991;11:1223-1230.[Abstract/Free Full Text]
  32. Kolpakov V, Gordon D, Kulik TJ. Nitric oxide–generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res. 1995;76:305-309.[Abstract/Free Full Text]
  33. Tatrai A, Foster S, Lakatos P, Shankar G, Stern PH. Endothelin-1 actions on resorption, collagen and noncollagen protein synthesis, and phosphatidylinositol turnover in bone organ cultures. Endocrinology. 1992;131:603-607.[Abstract]
  34. Lee RT, Berditchevski F, Cheng GC, Hemler ME. Integrin-mediated collagen matrix reorganization by cultured human vascular smooth muscle cells. Circ Res. 1995;76:209-214.[Abstract/Free Full Text]
  35. Kasuya H, Weir BKA, Nakane M, Pollock JS, Johns L, Marton LS, Stefansson K. Nitric oxide synthase and guanylate cyclase levels in canine basilar artery after subarachnoid hemorrhage. J Neurosurg. 1995;82:250-255.[Medline] [Order article via Infotrieve]
  36. Hirose H, Ide K, Sasaki T, Takahashi R, Kobayashi M, Ikemoto F, Yano M, Nishikibe M. The role of endothelin and nitric oxide in modulation of normal and spastic cerebral vascular tone in the dog. Eur J Pharmacol. 1995;277:77-87.[Medline] [Order article via Infotrieve]
  37. Yamaura I, Tani E, Maeda Y, Minami N, Shindo H. Endothelin-1 of canine basilar artery in vasospasm. J Neurosurg. 1992;76:99-105.[Medline] [Order article via Infotrieve]
  38. Foley PL, Caner HH, Kassell NF, Lee KS. Reversal of subarachnoid hemorrhage-induced vasoconstriction with an endothelin receptor antagonist. Neurosurgery. 1994;34:108-113.[Medline] [Order article via Infotrieve]
  39. Roux S, Loffler B-M, Gray GA, Sprecher U, Clozel M, Clozel J-P. The role of endothelin in experimental cerebral vasospasm. Neurosurgery. 1995;37:78-86.[Medline] [Order article via Infotrieve]
  40. Mathiesen T, Andersson B, Loftenius A, van Holst H. Increased interleukin-6 levels in cerebrospinal fluid following subarachnoid hemorrhage. J Neurosurg. 1993;78:562-567.[Medline] [Order article via Infotrieve]
  41. Onoda K, Ohmoto T, Ninomiya Y. Attempted gene therapy for experimental vasospasm: inhibition of vascular contraction with preproendothelin-1 mRNA antisense oligoDNA. Proc Spasm Symp. 1994;10:189-193.
  42. Hansson H-A, Johansson B, Blomstrand C. Ultrastructural studies on cerebrovascular permeability in acute hypertension. Acta Neuropathol (Berl). 1975;32:187-198.[Medline] [Order article via Infotrieve]
  43. Nagy Z, Mathieson G, Huttner I. Blood-brain barrier opening to horseradish peroxidase in acute arterial hypertension. Acta Neuropathol (Berl). 1979;48:45-53.[Medline] [Order article via Infotrieve]

Editorial Comment

Inhibitory Effect of Antisense Oligonucleotide on Collagen Induction

William I. Rosenblum, MD, Guest Editor

Division of Neuropathology, Medical College of Virginia-Virginia Commonwealth University, Richmond, Va


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
This study by Onoda et al does not directly involve cerebral blood vessels. Nevertheless, the results are potentially important to all interested in cerebrovascular diseases. First, the authors use a model of vasospasm that involves the femoral artery. They show that the administration of collagen synthesis decreases the degree of vasospasm that they can produce in an in vivo model of spasm. They also cite data suggesting that collagen formation may play a role in long-term spasm of brain blood vessels after SAH. They suggest therefore that an anticollagen therapy might be a useful adjunct in the treatment of SAH.

The potential relevance of the study to cerebral vasospasm is obvious. What remains to be shown is that such treatment would ameliorate spasm in a model that uses cerebral rather than extracerebral blood vessels. In addition, it is not clear, at least to this commentator, at what stage of human vasospasm such a treatment might be useful. Are we really talking about spasm, in the sense of a reversible chemically induced narrowing, or are we talking about a later change of permanent structural change that is initiated by the earlier chemically mediated alterations?

In any case, the findings are interesting, not only because of the data but because of the method used to reduce collagen synthesis. The authors applied an antisense oligonucleotide directed against the mRNA controlling the synthesis. The use of antisense strategies offers a powerful alternative to proposed gene therapy for many diseases. Unfortunately, almost no one has tried antisense strategies to alter cerebrovascular function.1R The present authors used a non–antisense oligonucleotide control. They also demonstrated not only a decreased spasm after antisense treatment but a decline in collagen, the product of the antisense target. Finally, they demonstrate actual decline in the message. I believe that even without the evaluation of message, the data would support the conclusion that the antisense worked by reducing collagen. However, support for the hypothesis is not synonymous with absolute proof. It is still possible that the antisense diminished spasm through some other parallel and unrecognized mechanism. Indeed, even the obvious conclusion that the antisense reduced collagen by interfering with message is not really "proved" by the demonstration that message was reduced. It is still possible, although one cannot suggest how, that reduced collagen synthesis was a consequence of some other unidentified and parallel property of the antisense oligonucleotide. In fact, I believe that the hypotheses used to explain the data are appropriate and most likely to be correct. However, I also believe that the ability to design effective antisense oligonucleotides (ie, molecules that do in fact interfere with their target message) has advanced to the point where these compounds with appropriate non-antisense controls can be used "off the shelf" to test important hypotheses without the need to perform molecular analyses for message content. A positive physiological result in the predicted direction can serve as evidence that the message must have been affected. Only in the presence of a negative result would one have to perform the molecular studies to ensure that the physiologically ineffective nucleotide really did reach the putative target. I realize that this statement is controversial. Nevertheless, I hope that it encourages others to test hypotheses using antisense technology, even in the absence of molecular analysis of message. The idea that message diminution confirms the mode of antisense action is erroneous because it does not exclude other actions of the antisense. Once the authors show, as they did here, that the effect of the oligonucleotide is sequence specific, it seems to me that the hypothesis of a real antisense (ie, blocked message) action is warranted. Indeed, sequence-specific but nonantisense effects of oligonucleotides have been reported in some experimental systems, but their occurrence, even when deliberately looked for, has been vanishingly small.2R 3R


*    Selected Abbreviations and Acronyms
 
AD = antisense oligoDNA
MD = missense oligoDNA
PCR = polymerase chain reaction
RT = reverse transcription
SAH = subarachnoid hemorrhage
SD = sense oligoDNA
TGF = transforming growth factor


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. Rosenblum WI, Murata S. Antisense evidence for two functionally active forms of nitric oxide synthase in brain microvascular endothelium. Biochem Biophys Res Commun. In press.
  2. Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature.. 1990;346:818-822.[Medline] [Order article via Infotrieve]
  3. Bock LC, Griffin LC, Latham JA, Vermoss EH, Tool JJ. Selection of single stranded DNA molecules that bind and inhibit human thrombin. Nature.. 1992;355:564-566.[Medline] [Order article via Infotrieve]



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