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(Stroke. 1997;28:799-804.)
© 1997 American Heart Association, Inc.

Articles

Gelatinase Activity and the Occurrence of Cerebral Aneurysms

Douglas Chyatte, MD Isabel Lewis, BS

From the Cerebrovascular Research Laboratory, Section of Cerebrovascular Surgery, The Cleveland Clinic Foundation (Ohio).

Correspondence to Douglas Chyatte, MD, Section of Cerebrovascular Surgery, S80, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail chyattd{at}cesmtp.ccf.org

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose Cerebral aneurysms are associated with decreased arterial collagen content; however, whether this deficiency results from impaired collagen synthesis or enhanced collagen degradation is unknown. This study tested the hypothesis that enhanced collagen degradation, not impaired collagen synthesis, is associated with the occurrence of cerebral aneurysms.

Methods Cultured skin fibroblasts and serum samples were studied in patients with angiographic evidence of aneurysm (n=31) and control subjects (n=14). Transcription of the type III collagen gene was assessed with the use of Northern blots prepared from RNA harvested from confluent cultured fibroblasts. Translation of type III collagen was assessed by Western blot analysis of proteins produced by cultured skin fibroblasts. Collagen metabolism was assessed by radioimmunoassay for type I (PICP) and type III (PIIINP) procollagen peptides in conditioned tissue culture media and serum. We assessed collagen degradation in serum and conditioned tissue culture media by evaluating gelatinase activity using quantitative zymography.

Results Type III collagen synthesis was the same in aneurysm and control patients. Neither the molecular weight nor the relative amount of type III collagen mRNA differed between aneurysm and control patient fibroblasts. Western blot analysis revealed no difference in the relative amount or molecular weight of procollagen III synthesized by aneurysm and control cells. Aneurysm patients had a threefold increase in native serum gelatinase activity compared with control subjects (P=.004). This increase occurred along with serum evidence of increased collagen metabolism. Serum levels of PICP (P=.03) and PIIINP (P=.02) were decreased in aneurysm patients. Elevated serum gelatinase activity and altered collagen metabolism could not be explained by enhanced secretion of gelatinase by cultured fibroblasts or altered net collagen synthesis by fibroblasts. High serum gelatinase activity was more common in men than in women (P=.04).

Conclusions These findings are consistent with the hypothesis that accelerated enzymatic degradation of collagens and other structural proteins compromises the mechanical integrity of the cerebral vessel wall and leads to conditions that favor aneurysm formation.

Key Words: cerebral aneurysm • collagen • gelatinases • subarachnoid hemorrhage

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Intracranial aneurysms occur in 2% to 5% of adults^{1 2 3 4 5 6} and cause catastrophic, often fatal, illness. Most commonly, patients remain asymptomatic until the aneurysm ruptures to produce a subarachnoid hemorrhage. Despite advances in diagnosis and treatment, outcome remains dismal once subarachnoid hemorrhage has occurred. One third of all patients who suffer an aneurysmal subarachnoid hemorrhage will not survive long enough to reach medical attention. Of those who do survive to hospital admission, nearly one half will die or be left neurologically devastated. Most morbidity and mortality occurs primarily as a direct result of the initial hemorrhage.^{7 8 9 10 11 12 13 14 15} Identification of patients at risk for aneurysmal subarachnoid hemorrhage and appropriate prophylactic treatment, therefore, appears to be the most practical way of avoiding the devastating effects of subarachnoid hemorrhage. However, at the present time no reliable method exists for identifying patients at risk for aneurysm formation and rupture.

Relatively little is known about the biology of cerebral aneurysms, and no hypothesis is accepted uniformly that explains the formation and subsequent rupture of intracranial aneurysms. Understanding the conditions that lead to intracranial aneurysm formation and rupture may allow new prophylactic strategies and new aneurysm treatments to be developed.

Research indicates that there is likely one or more biochemical abnormalities unique to aneurysm patients.¹ Approximately half of all aneurysm patients have an observable morphological derangement of the cerebral arteries that is related to deficiencies of structural proteins that maintain the mechanical integrity of the cerebral artery wall.^{16 17 18 19 20 21 22 23 24} Initially, it was believed that only a type III collagen deficiency was commonly associated with cerebral aneurysms.^{1 16 19 21 24} However, others have since found that mutations in the gene for type III collagen are not a common cause of aneurysms²⁵ and that total collagen, rather than only type III, is decreased in the cerebral arteries of some patients.²¹ Decreases in arterial collagen may result from impaired collagen synthesis or enhanced degradation. In this study we tested the hypothesis that enhanced collagen degradation, not impaired synthesis, is associated with the occurrence of cerebral aneurysms.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Patients and Control Subjects
Consecutive patients with cerebral aneurysms were included in the study regardless of whether or not the aneurysm had ruptured if they were treated surgically. Patients undergoing craniotomy who had no MRI or angiographic evidence of a cerebral aneurysm were selected as control subjects. After we obtained informed consent in accordance with the protocol approved by our institutional review board, skin (scalp taken from the wound edge) from aneurysm patients was collected at the time of surgery. Similarly, samples were collected from the control patients.

Experimental Design
To evaluate type III collagen synthesis, we evaluated transcription of the type III collagen gene (COL3A1) using Northern blots. Translation of both type I and type III collagen was assessed in both serum and conditioned tissue culture media by radioimmunoassay (RIA) for procollagen peptides I and III. Mature type III collagen was evaluated by Western blot analysis of trypsin-soluble proteins produced by cultured fibroblasts. Collagen degradation was measured by studying gelatinase activity in both serum and conditioned tissue culture media with the use of quantitative zymography. These biochemical data were then examined for any correspondences with clinical data.

Clinical Data and Sample Collection
Clinical data were collected by reviewing medical records. Data were collected on patient age, sex, diagnosis, presence or absence of aneurysm rupture (subarachnoid hemorrhage), aneurysm location, aneurysm size, number of aneurysms, neurological grade at admission, and family history.

Blood samples were collected immediately before surgery by venipuncture, and the serum was separated after clotting by centifugation. The samples were aliquoted and immediately frozen at -70°C for later analysis. Skin samples were immediately placed in culture (D-MEM-F12; Gibco) and transported to the laboratory where the fibroblast cultures were prepared.

Fibroblast Cultures
Fresh skin specimens were trimmed of excess fat and subcutaneous tissue and minced into 2- to 3-mm fragments. These were allowed to dry; then culture medium (D-MEM-F12; Gibco) was added, and the specimens were incubated at 37°C in a 95% O₂/5% CO₂ atmosphere. Confluent cultures were prepared by serial passage of cells. Conditioned medium was prepared with serum-free medium (Aim-V; Gibco) and a 72-hour conditioning period. Aliquots of conditioned media were stored at -70°C for later assay. The cell count (determined with a hemocytometer) and amount of deoxyribonucleic acid DNA were determined (Quigen DNA kit) and then used to normalize conditioned media protein and enzyme levels on a per cell basis. Trypsin digestion was used to solubilize proteins, including type III collagen that had been synthesized by confluent fibroblasts and deposited in the extracellular matrix. Trypsinized supernatants were aliquoted and stored at -70°C for later assay.

Northern Blots
To determine the transcription level of the type III collagen gene, Northern blots were done. Total RNA was extracted from confluent cultured skin fibroblasts (Trizol Reagent; Gibco). Each RNA sample then was dissolved in diethylpyrocarbonate-treated distilled water and denatured by heating at 65°C for 10 minutes in a standard denaturing solution. The RNA was fractioned by agarose electrophoresis and transferred to a nitrocellulose membrane. The membranes were hybridized with a cDNA probe (HF934; American Type Culture Collection) for type III collagen mRNA by means of a nonradioactive labeling technique (Photogene Nucleic Acid Detection System; Gibco). A commercially available cDNA probe for ß-actin (Oncor) was used as a standard for normalization.

Western Blots
Trypsinized samples were separated by polyacrylamide gel electrophoresis, blotted, and then probed with the use of a commercially available polyclonal antibody to type III collagen (polyclonal rabbit anti-human procollagen III antibody; Biodesign) and a peroxidase-labeled secondary antibody (Amersham).

Determination of Conditioned Media Procollagen Peptide Levels
To determine whether the collagen deficiency observed in some aneurysm patients results from a global depression in the synthesis rate of all collagens, the synthesis rates of type I and type III procollagen were estimated by measuring the procollagen peptide levels for type I and type III collagen in serum-free media conditioned with cultured skin fibroblasts. During the conversion of procollagen to collagen, specific proteases cleave propeptides from both the carboxyterminal and aminoterminal ends of mature procollagen. Both the carboxyterminal and aminoterminal propeptides are produced in a 1:1 stoichiometry for each procollagen molecule released. Levels of these propeptides reflect the net synthesis of collagen. Propeptide levels, however, may also be decreased by accelerated proteolytic activity.

The carboxyterminal propeptide for type I collagen (PICP) and the aminoterminal propeptide for type III collagen (PIIINP) were measured by RIA with a commercially available kit (Incstar). Propeptide levels for both collagens were measured in conditioned serum-free tissue culture media in aneurysm patients and compared with control subjects. Before the conditioning period, serum was present in the culture media. Cell counts in all conditioning flasks were similar. Normalizing peptide levels on a per cell basis did not alter results.

Determination of Serum Procollagen Peptide Levels
To determine whether the collagen deficiency observed in some patients results from a global depression in the synthesis rate of all collagens, the in vitro synthesis rates of type I and type III procollagen were estimated by measuring the procollagen peptide level for type I and type III collagen in serum.

PICP and PIIINP levels were measured in serum samples with an RIA kit (Incstar). As for procollagen produced by cultured cells, levels of these propeptides reflect net collagen synthesis unless peptide levels are decreased by accelerated proteolytic activity.

Assessment of Gelatinase Activity
Zymography was used to assess gelatinase activity in the serum samples and conditioned serum-free tissue culture media. Aliquots of conditioned media (10 µg protein) or serum (10 µL of 1:200 dilution) were loaded onto substrate gels. Gelatinase substrate gel electrophoresis^{26 27 28} was performed with the use of precast gels (10% polyacrylamide containing 0.1% gelatin; Novex). Samples were prepared for analysis by dilution into a loading buffer consisting of 0.4 mol/L Tris, pH 6.8, 5% sodium dodecyl sulfate, 20% glycerol, and 0.03% bromophenol blue. After electrophoresis at 125 V, the gels were incubated in renaturing solution (2.5% Triton X-100) for 30 minutes at room temperature and then for 72 hours at 37°C in a developing buffer containing 50 mol/L Tris, pH 7.5, 200 mmol/L NaCl, 4 mmol/L CaCl, and 0.02% Brij-35. The gels were then stained with 0.5% Coomassie blue G-250 in 30% methanol/10% acetic acid and then destained in 30% methanol/10% acetic acid. Finally, the gels were incubated for 15 minutes in 30% methanol and 5% glycerol and stored between sheets of cellophane. Gelatinase activity appears as unstained bands in the gel. The digestion was quantified with the use of a gel scanner equipped with an interface to a Macintosh computer. Gels were analyzed with the aid of NIH-Image software, version 1.41. Each gel was scanned three times, and the mean value of the integrated density for a particular unstained band was used for analysis. Gelatinase activity was quantified by normalizing sample activity to a standard composed of pooled serum or conditioned media.

Statistical Analysis
All analyses were run in triplicate; the mean of the three assays was determined and used for statistical analysis. The primary comparison made was between all aneurysm patients and control patients; a secondary comparison involved differences between patients with high or low gelatinase activity and control subjects. For biochemical measurements, the mean±SD was determined. Student's t test was used to test for differences in PICP, PIIINP, and gelatinase activity between patients and control subjects. For clinical data, differences between aneurysm and control patients were tested with the t test or Yates' correction to the ² test, as appropriate. The post hoc comparison between high gelatinase activity, low gelatinase activity, and control groups was done in a similar fashion. Bonferroni's correction, to correct for the effect of multiple comparisons, was used for these analyses. Differences were considered statistically significant at P<.05.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
A total of 31 aneurysm patients and 14 control subjects were included for study. Table 1 summarizes the clinical data for these patients. Aneurysm and control patients had similar mean ages (53±14 versus 59±17 years; P=.24) and sex (50% female versus 61% female; P=.48). Fifty-two percent of aneurysm patients had a subarachnoid hemorrhage, while the remainder had unruptured aneurysms. For the Northern and Western blots and conditioned media studies, only 21 patients and 7 control studies were studied because suitable samples for analysis were not available for all patients.

View this table: [in this window] [in a new window]   Table 1. Summary of Medical History Data for All Patients With Aneurysm and Subgroups With High or Low Gelatinase Activity

Northern Blot Results
Northern blots were performed on 21 aneurysm and 7 control patients. Neither the molecular weight (5.4 kb) nor relative amount of mRNA (band densities of type III collagen mRNA) differed between aneurysm patients and control subjects. Fig 1 shows a typical Northern blot for collagen type III. Type III collagen gene expression was normal in all aneurysm patients.

View larger version (46K): [in this window] [in a new window]   Figure 1. Northern blot probed for type III collagen and prepared from RNA harvested from cultured fibroblasts. M indicates marker; C, control; and A, aneurysm. Type III collagen gene expression was normal in the aneurysm patients studied.

Western Blot Results
Western blots were performed in 21 aneurysm and 7 control patients. No difference in the relative amount or molecular weight of procollagen III synthesized by aneurysm and control cells was detected. Fig 2 shows a typical Western blot for type III collagen.

View larger version (53K): [in this window] [in a new window]   Figure 2. Western blot for type III collagen prepared from cultured fibroblasts. STD indicates standard; C, control; and A, aneurysm. No difference in the relative amount or molecular weight of procollagen III synthesized by aneurysm and control cells could be detected.

Serum and Conditioned Media Gelatinase and Procollagen Peptide
Zymograms and RIAs for PICP and PIIINP were performed on samples from 31 aneurysm and 14 control patients (Table 2). Aneurysm patients had a threefold increase in native serum gelatinase activity compared with control subjects (P=.004). This increase was accompanied by lower serum procollagen peptide levels, suggesting that enzymatic degradation of collagen was increased. Serum levels of both PICP (P=.03) and PIIINP (P=.02) were significantly lower in aneurysm patients. No significant differences were observed in media gelatinase activities (P=.23), media PICP levels (P=.08), or media PIIINP levels (P=.19) between aneurysm and control patients (Table 2). The enhanced serum gelatinase activity did not appear to be due to a gelatinase unique to aneurysm patients, as the predominant band of gelatinase activity was at 72 kD in both aneurysm and control patients (Fig 3).

View this table: [in this window] [in a new window]   Table 2. Gelatinase Activity and Collagen Metabolism Compared Between Patients Without Aneurysm and Control Patients

View larger version (25K): [in this window] [in a new window]   Figure 3. A, Serum zymogram (gelatinase). B, The 72-kD band intensity, reflecting relative activity. C indicates control; A, aneurysm.

Not all aneurysm patients had equally increased serum gelatinase activity levels (Table 3). Post hoc analysis was performed to compare aneurysm patients with obviously increased gelatinase activity to aneurysm patients not showing increased gelatinase activity. Aneurysm patients were considered "high activity" if their serum gelatinase activity was at least two times greater than mean control values and "low activity" if their serum gelatinase level was less than two times mean control values. Of the aneurysm patients, 16 had high serum gelatinase activity and differed significantly compared with aneurysm patients with low levels and with control subjects (P<.0001). Collagen metabolism was clearly altered in this high-activity subgroup. Serum levels of PICP (P=.01) and PIIINP (P=.02) were decreased compared with those of control subjects. Media gelatinase (P=.07), media PICP (P=.21), and media PIIINP (P=.26) did not differ between the high-activity group and control subjects.

View this table: [in this window] [in a new window]   Table 3. Gelatinase Activity and Collagen Metabolism in Control Subjects and Patients With High and Low Gelatinase Activity

Of aneurysm patients, 15 showed low serum gelatinase activity. No significant differences were found in serum gelatinase activity (P=.93), serum PICP (P=.16), media gelatinase (P=.56), media PICP (P=.66), or media PIIINP (P=.20) compared with control subjects. Serum PIIINP was significantly reduced in low-activity patients (P=.003) compared with control subjects, suggesting a derangement in collagen metabolism despite normal gelatinase activity. There were nine men in the high-activity subgroup but only three in the low-activity group (P=.04). Other clinical features of the high-activity group were similar to those of the low-activity group. No difference was found in the mean age (P=.60), incidence of subarachnoid hemorrhage (P=.85), aneurysm location (P=.71), or aneurysm size (P=.71). Data regarding family history were missing in 22 patients (71%) and therefore were not analyzed.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Most morbidity and mortality after aneurysmal subarachnoid hemorrhage occurs as a direct result of the initial hemorrhage.^{7 8 9 10 11 12 13 14 15} Unfortunately, most aneurysms remain asymptomatic until they bleed, and no practical method exists for identifying individuals at risk before symptoms occur. Understanding the conditions that lead to aneurysm formation and rupture is fundamental to developing strategies for identifying and treating individuals at high risk for aneurysmal subarachnoid hemorrhage.

Currently, it is not understood why aneurysms form or rupture. Two general hypotheses regarding rupture have been investigated. In one hypothesis, exaggerated hemodynamic forces or hemodynamic imbalances are believed to lead to aneurysm formation and rupture.^{2 29 30 31 32 33 34 35 36 37 38 39} Exaggerated hemodynamic forces do appear to contribute to aneurysm formation under certain conditions in both humans and animals,^{1 40 41 42} but hemodynamic forces alone do not appear to be sufficient to cause cerebral aneurysms. For example, in animals, induced hypertension (increased hemodynamic stress) and carotid ligation (hemodynamic imbalance) must be coupled with lathyrism, which compromises the structural integrity of collagen in the vascular connective tissue, to reliably produce cerebral aneurysms.^{40 41 42} In humans, although hypertension is a risk factor for cerebral aneurysms, they rarely develop in hypertensive individuals.⁶ These observations indicate that conditions other than hypertension alone must be present to produce cerebral aneurysms.

In the second hypothesis, aneurysm formation is linked to biochemical defects that undermine the mechanical integrity of the vessel wall. As discussed below, considerable evidence exists supporting this hypothesis for a significant proportion of patients.

Cerebral artery morphology provides some insight into cerebral aneurysm formation. The extracellular scaffolding has three main components. Reticular fibers are complexes of the extracellular scaffolding composed of collagen fibrils 20 to 40 µm in diameter in close association with other structural matrix molecules^{43 44} and form a supporting framework around vascular smooth muscle cells. Aneurysm patients have a 35% reduction in histologically apparent reticular fibers in both cerebral and somatic arteries, pointing to a generalized abnormality in the arterial bed of some patients with ruptured aneurysms.^{16 19 22} Reticular fibers are irregularly distributed in aneurysm patients and often appear shorter and coarser than normal. Collagen fibers, in contrast, are cord-shaped structures 1 to 20 µm in diameter. Collagen fibers appear similar in both aneurysm and control patients.^{16 19 22} Elastin is a relatively minor component of cerebral arteries, limited almost exclusively to the internal elastic lamina. No differences have been found between elastin morphology in aneurysm patients and control subjects.^{16 19 22}

The initial biochemical investigations of cerebral aneurysms focused on type III collagen, a major structural component of the arterial wall, and examined whether cerebral aneurysm patients have a specific deficiency of type III collagen. Pope et al²⁴ and Neil-Dwyer et al¹⁹ compared tissue levels and synthetic rates of type III collagen in aneurysm patients with brain tumor patients who served as control subjects. Type III collagen levels and synthetic rates were normalized with respect to type I collagen levels. To determine type III levels in tissue, pepsin digests of skin and superficial temporal arteries were prepared, and type III and type I collagen were separated by gel electrophoresis under reducing and nonreducing conditions. Both groups concluded that approximately half of all aneurysm patients had a type III collagen deficiency relative to type I collagen. However, these differences may be merely the result of artifacts arising from unequal gel loading. Neil-Dwyer et al¹⁹ also measured collagen synthesis rates by feeding cultured skin fibroblasts with radiolabeled proline or lysine and comparing the relative amounts of type I and type III collagen as determined by carboxymethyl cellulose chromatography. Approximately half of the aneurysm patients "produced significantly less" type III collagen than control subjects (7.5% versus 10.5%). However, the -1 (III) and -2 peaks overlapped considerably such that the 3% difference may be an artifact of peak size estimation.

Others evaluated this hypothesis by comparing collagen content and the biomechanical properties of the middle cerebral and brachial arteries of aneurysm patients and control patients.²¹ Collagen content was estimated by measuring the hydroxyproline content of the arteries. The relative ratio of hydroxyproline in type III to type I collagen was calculated from sodium dodecyl sulfate–polyacrylamide gel electrophoresis band sizes of the cyanogen bromide peptides of collagen. Approximately half of the aneurysm patients had a decrease in the estimated type III collagen content relative to type I in both cerebral and brachial arteries. Deficient middle cerebral arteries had normal tensile strengths but increased extensibility at stresses corresponding to physiologically relevant blood pressures. They concluded that some aneurysm patients have type III collagen deficiency and that this deficiency alters the biomechanical properties of the arterial wall. The arterial samples that were identified as type III collagen deficient, however, had a decrease in total hydroxyproline. These patients, therefore, may have had a decrease in total collagen content rather than an isolated deficiency of only type III collagen.

Kuivaniemi et al²⁵ sequenced the coding sequence of the type III collagen gene (Col3A1) in 58 aneurysm patients and found powerful evidence that mutations in the gene for type III procollagen are not a common cause of intracranial aneurysms. After cDNAs were prepared and sequenced, polymorphisms and mutations were confirmed with the use of genomic DNA. When mutations were identified, type III collagen from cultured fibroblasts was analyzed, and stability to pepsin digestion was measured. Of the 58 patients, 42 had polymorphic sites. Expression of both alleles in these 42 individuals appeared to be equal. Mutations in the type III collagen sequence were found in two individuals; both mutations resulted in an amino acid substitution (Pro 435Thr and Pro 468Leu). Neither mutation affected the relative amount of type III procollagen synthesized as determined with gel electrophoresis or stability to pepsin digestion.

Despite the evidence provided by Kuivaniemi et al,²⁵ the possibility remains that decreased type III collagen is associated with cerebral aneurysms. Majamaa et al⁴⁵ studied radiolabeled type I and type III collagen synthesized by cultured skin fibroblasts from 11 aneurysm patients. Although amounts of type I and type III collagen did not differ between patients and control subjects, a decrease in thermal stability to trypsin and -chymotrypsin digestion was observed in two of the 11 aneurysm patients (39°C versus 41°C).

Relatively little work has been done to evaluate the possibility that accelerated proteolytic activity may lead to the global decrease in arterial collagen observed in some aneurysm patients. Recently, Baker et al⁴⁶ compared serum elastase and ₁-antitrypsin levels in patients with ruptured or unruptured aneurysms with control patients. They found that the serum elastase level was significantly elevated in patients with either ruptured or unruptured aneurysms. ₁-Antitrypsin levels were normal in all aneurysm patients. Enzyme activity was not measured, and the possibility that elastase may degrade substrates other than elastin was not studied.

The present study extends the understanding of type III collagen synthesis in aneurysm patients by examining the levels of type III collagen transcription and translation. No difference in type III collagen transcription or translation was found in fibroblasts prepared from aneurysm and control patients. A primary derangement of type III collagen synthesis, therefore, does not explain the decrease in arterial structural proteins found in many aneurysm patients and is not a common cause of intracranial aneurysms. Alternatively, this decrease may be the result of accelerated collagen degradation. Such degradation would compromise the mechanical integrity of the cerebral vessel wall and lead to conditions that favor aneurysm formation. Serum gelatinase activity was increased in half the aneurysm patients studied and was accompanied by lower serum procollagen peptide levels, reflecting increased enzymatic degradation.

Conclusions
The present study links increased serum gelatinase activity to the occurrence of cerebral aneurysms. Cerebral aneurysms therefore may occur as the result of a complex remodeling process involving the degradation matrix proteins and not as the result of passive dilation alone.

	Acknowledgments

 
This study was funded by The Cleveland Clinic Foundation Research Projects Committee (grant 4564).

Received January 14, 1997; accepted January 14, 1997.

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