(Stroke. 1996;27:1889-1895.)
© 1996 American Heart Association, Inc.
Articles |
Protein Kinase C Has Two Different Major Roles in Lattice Compaction Enhanced by Cerebrospinal Fluid From Patients With Subarachnoid Hemorrhage
the Departments of Neurosurgery (T.S., D.H.B., A.D.P.) and Anatomy (D.H.B.), University of Mississippi Medical Center, Jackson, and Department of Neurological Surgery (K.H.), Kagawa Medical School, Takamatsu, Japan.
Correspondence to Tomomi Shiota, MD, c/o David H. Bernanke, PhD, Department of Neurosurgery, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216-4505. E-mail dhb@umsmed.edu.
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Abstract |
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Background and Purpose Compaction of extracellular matrix (ECM) lattices by cultured fibroblasts was accelerated by cerebrospinal fluid (CSF) from patients with subarachnoid hemorrhage (SAH). The rate of acceleration was significantly related to the clinical grade of vasospasm. However, the mechanism remains unclear. Evidence exists for an important role in cerebral vasospasm for protein kinase C (PKC). The purpose of this study was to help clarify whether PKC has a role in contraction of the ECM.
Methods We studied the effects of a myristoylated PKC peptide inhibitor (Myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val) (PKC peptide inhibitor), (5-isoquinolinesulfonyl)homopiperazine (HA-1077) (inhibitor of protein kinase A, myosin light-chain kinase, and protein kinase G), 7-deacetyl-6-(N-acetylglycyl)-forskolin (forskolin) (adenyl cyclase activator), and diacylglycerol-lactone (DAG-lactone) (PKC activator) on fibroblast-populated collagen lattice compaction with or without CSF from SAH patients. Four sets of fibroblasts were used: three explanted from skin and one from cerebral artery.
Results Moderate and high concentrations of PKC peptide inhibitor inhibited lattice compaction with or without acceleration by CSF. Low concentration of PKC peptide inhibitor enhanced acceleration by CSF but had no effects without CSF. HA-1077 could not inhibit lattice compaction. Forskolin inhibited compaction. DAG-lactone accelerated compaction in early phases.
Conclusions In the mechanism of acceleration of contraction of ECM under the influence of CSF, PKC seems to have two different roles. Protein kinase A and myosin light-chain kinase apparently play more minor roles than PKC in the mechanism, but no evidence was found of a role for protein kinase G activation in matrix compaction.
Key Words: extracellular matrix • protein kinase C • subarachnoid hemorrhage • vasospasm
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction |
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The mechanism of cerebral vasospasm after SAH has been the subject of many research studies, but the details of the pathophysiological mechanism remain unresolved. Spastic arteries, especially those in advanced stages of cerebral vasospasm after SAH, are known to respond poorly to the commonly used vasodilators.1 This evidence suggested that some process other than the usually expected smooth muscle contraction mechanism was involved in cerebral vasospasm. Yamamoto et al2 reported that CSF from SAH patients enhanced FPCL compaction. The degree of enhancement had a significant correlation with the grade of cerebral vasospasm after SAH. Participation of endothelin and several kinds of growth factors in cerebral vasospasm has been demonstrated.3 4 5 6 These have also shown a potential for enhancing the compaction of collagen matrices by cultured cells.7 8 9 10 11 At least two studies have reported that this kind of traction mechanism could generate significant forces in the regulation of arterial diameter.12 13 Thus, the compaction of ECMs has the potential to play a significant role in cerebral vasospasm.
Numerous reports have been published relating PKC to cerebral vasospasm after SAH.14 15 16 17 These reports showed that activation of PKC after SAH should have an important role in vasospasm. Indirect evidence also exists for a role for PKC in the mechanism of compaction of collagen matrices.18 In this study we attempted to clarify a portion of the pathway for ECM compaction enhanced by CSF from SAH patients, particularly as it relates to PKC and as contrasted with other kinases.
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Materials and Methods |
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Fibroblasts
Three sets of human dermal fibroblasts and one of fibroblasts obtained from human cerebral arteries were used in this study. All fibroblasts were obtained by explantation culture. Cultures were maintained in Dulbecco's modified Eagle's medium (Mediatech) with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL). The cultures were incubated in a water-saturated atmosphere at 37°C with 5% CO2 and 95% air.
FPCL
FPCL were prepared according to methods that have been described previously.2 FPCL were formed by mixing trypsin-liberated fibroblasts in Dulbecco's modified Eagle's medium with a sterile stock solution of acetic acid–extracted rat tail tendon collagen (type I) (5 mg/mL in 1 mmol/L hydrochloric acid) in sterile polystyrene tubes. The lattice mixture (0.5 mL per well) was adjusted to contain 70 000 cells per milliliter, 1.0 mg/mL of type I collagen, and 3% fetal bovine serum. The mixture was dispensed into 16-mm wells (in 24-well plates) (Corning) and incubated for 90 minutes at 37°C to form gels. The formed lattices were detached from the dish walls with a 27-gauge needle.
CSF
This laboratory has previously demonstrated the activity of CSF samples from SAH patients in the acceleration of FPCL compaction.2 Three CSF samples of high activity from SAH patients were used in this study.
Reagents
The following reagents were applied, singly or in combination, to the FPCL after the lattices were formed. Myristoylated PKC peptide inhibitor (Myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val) (Promega Corp) dissolved in water was used as a specific inhibitor of PKC-
and -ß (IC50=0.008 mmol/L). DAG-lactone (Calbiochem) dissolved in DMSO was used as a PKC activator (Ki=0.0028 mmol/L). Forskolin (7-deacetyl-6-[N-acetylglycyl]-forskolin) (Calbiochem) dissolved in DMSO was used as an adenyl cyclase activator (EC50=0.020 mmol/L). HA-1077 ([5-isoquinolinesulfonyl]homopiperazine) (Asahi Chemical Industry) dissolved in PBS was used as an inhibitor of PKA, PKG, and MLCK.
Conditions of Incubation With CSF Samples, Activators, and Inhibitors
All CSF samples and drugs were applied to FPCL immediately after detachment. The day of treatment was counted as day 0.
FPCL were treated in several combinations, as follows: PKC inhibitor: 0.0025, 0.005, 0.015, 0.025, and 0.050 mmol/L with or without CSF; PKC inhibitor: 0.005 and 0.050 mmol/L with active CSF (100 µL); PKC inhibitor: 0.005 and 0.050 mmol/L with nonactive CSF (100 µL); HA-1077: 10-4, 10-5, and 10-6 mol/L with active CSF (100 µL); forskolin: 0.001, 0.010, and 0.020 mmol/L with or without active CSF (100 µL); and DAG-lactone: 0.0003, 0.003, and 0.006 mmol/L without CSF (100 µL).
Control Treatments
The effects of the same volume of DMSO, PBS, and water used in the treatments were noted. No significant effects on lattice compaction rates were observed after administration of the treatment vehicles.
Measurement of FPCL
FPCL were placed over metric-scale graph paper, and their longer and shorter axes were measured with the aid of a dissecting microscope. The areas of FPCL were calculated and expressed as either percentage of initial area or percentage of control area. Data are presented graphically or in tabular form as mean±SD. Lattice diameters were statistically compared with ANOVA and Student's t test. Significant differences were determined at P<.01 and P<.05. We also observed the morphology of cells using Hoffmann modulation optics microscopy. Lattices incubated with water (100 µL) were used as controls.
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Results |
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PKC Inhibitors
Fig 1
and the Table show data from experiments on one dermal fibroblast set, treated with one active CSF sample and one nonactive CSF sample. Although no effect was observed in lattices treated daily with low concentration of PKC inhibitor without CSF samples, in the presence of CSF enhanced acceleration of compaction was observed. This enhancement was also observed with inactive CSF samples. Moderate concentration inhibited lattice compaction acceleration by CSF samples. High concentration inhibited lattice compaction itself. Single-day additions at all concentrations, added together with CSF samples, did not have significant effects on lattice compaction. Major morphological changes were not observed in cells in lattices after any treatment. Results obtained with other cells and CSF samples were similar to those described above, but differences in compaction rates were noted for some cells and CSF samples.
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HA-1077
There were no significant differences at concentrations of 10-5 and 10-6 mol/L compared with control. There was significant inhibition at 10-4 mol/L. Furthermore, the area was significantly larger than without CSF (Fig 2). Major morphological changes in cells at 10-4 mol/L were also observed.
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Forskolin
With all treatments of forskolin, no acceleration of lattice compaction was observed. Although the lowest concentration of forskolin did not inhibit CSF stimulation during the acute phase (day 1), forskolin treatment inhibited acceleration by CSF samples completely at the later stages (Fig 3). At a concentration of 0.020 mmol/L, forskolin stopped compaction itself by day 1, while 10 µmol/L inhibited compaction by day 3 or 4. Major morphological changes were noted at concentrations of 0.010 and 0.020 mmol/L.
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DAG-Lactone
Treatment with 0.0003, 0.003, and 0.006 mmol/L of DAG-lactone significantly accelerated lattice compaction (Fig 4) at 2 hours and day 1 compared with control lattices. Major morphological changes were observed after day 3 at concentrations of 0.003 and 0.006 mmol/L.
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Discussion |
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Studies in several laboratories have demonstrated that cells other than muscle, such as fibroblasts, have the ability to generate contractile forces.18 19 20 These reports also suggested that this is accomplished through exertion of traction force on ECM in addition to simple contractility of cells themselves.20 This ability was observed not only in fibroblasts but also in smooth muscle cells7 and mesangial cells.9 Two different groups have actually measured the isometric force produced by the action of fibroblasts and endothelial cells in their culture models. They obtained almost the same results, demonstrating that this traction force could contribute substantially to the regulation of arterial constriction. This provides the possibility of attributing at least some portion of change of tissue dimensions during vascular constriction to a reorganization, or packing, of ECM by these cells.
Guidry11 18 showed that PMA enhanced compaction activity by fibroblasts at the acute phase by approximately 20% to 50% in three-dimensional culture models. He also demonstrated that endothelin enhanced this ability two times greater than PMA and that PMA suppressed the enhancement by endothelin and serum by approximately 20%.8 11 Other reports showed that TPA causes very rapid reduction in contractility of fibroblasts cultured on a silicon rubber substratum.21 Both PMA and TPA, like other phorbol esters, are known to simulate the role of diacylglycerol in activation of PKC. These data suggest that PKC is involved, in what seems to be a complex way, in the pathway for force generation operating through the ECM.
Activation of PKC after SAH was reported in a variety of animal vasospasm models and in studies of enhancement of vascular tone.14 16 17 Other reports indicated that a variety of PKC inhibitors could reduce cerebral vasospasm in SAH models.14 15 Although these types of PKC inhibitors are known to be able to inhibit other types of kinases as well, such evidence suggests that PKC does in fact have an important role in cerebral vasospasm after SAH.
In our model, DAG-lactone treatment resulted in accelerated compaction in the early phase by approximately 10% to 20%. This also suggests that PKC activation has a role in the compaction mechanism, but DAG-lactone did not have continuous effects such as those seen with CSF samples. Moreover, the addition of higher concentrations or more frequent additions stopped the compaction and seemed to cause major morphological cell damage (not shown). The PKC activation by PMA and TPA is known to be downregulated during extended exposure to these agents.22 23 This downregulation would be consistent with the results we present here, suggesting that continuous activation of PKC is required for continuous compaction in lattices. However, this point remains unresolved.
The present study showed that PKC plays a dominant role in the pathways leading to force generation by fibroblasts, with or without enhancement by CSF from SAH patients. Some reports showed that staurosporin inhibited ECM contraction and the PKC activity related to ECM contraction in their model systems.8 18 However, staurosporin also has broader effects on PKC and other kinases. The PKC inhibitor peptide used in the present study is known to have very specific effects on membrane permeability and on PKC-, -ß, and probably -
, working as a competitive inhibitor because of the structural similarities to these isoforms. It is also known to inhibit TPA-stimulated phosphorylation activity by specific PKC isoforms to 56% at 0.001 mmol/L and to 98% inhibition at 0.100 mmol/L in fibroblast cell lines.24 Morphological changes such as those seen with staurosporin treatment18 were not observed in our cultures treated with PKC inhibitor peptide. Myristoylated epidermal growth factor receptor fragment, a different PKC inhibitor peptide, had no effect on the FPCL model with or without addition of CSF (data not shown). This suggests that a myristoylated peptide generally does not have an effect on lattice compaction. Precise and reliable measurement of the intracellular activity of each specific form (, ß, ) of PKC was technically very difficult in our models because of the presence of added inhibitor, and this measurement was not performed. However, these points also strongly suggest that the mechanism of traction force generation operating through fibroblast reorganization of ECM in our model is dependent on specific PKC pathways.
Experiments in which we used the PKC inhibitor peptide provided somewhat surprising results, suggesting that the involvement of PKC in this process lies along complex pathways. The low concentrations (0.0025 and 0.005 mmol/L) of PKC peptide inhibitor accelerated lattice compaction in the presence of CSF but had no effect without CSF. The moderate concentration (0.015 mmol/L) of the inhibitor affected the more dominant PKC-dependent enhancement by CSF but had no effect without CSF. The high concentrations (0.025 or 0.050 mmol/L) of PKC inhibitor had an effect on the compaction mechanism itself. This also suggests the existence of other and/or distinct regulatory components, which might include promoters or suppressers or modifications of these, that could play either role in the mechanism. At the very least, we can say that PKC pathways have two distinguishable roles in lattice compaction under the influence of CSF from SAH patients.
Potentially, we can surmise that regulatory activity for the PKC-dependent pathways could exist in at least two entities (Fig 5). Although represented diagrammatically as single blocks, either of these units might be composed of one or more molecular species, operating as a functional unit. These two units might work either independently or in concert during the lattice compaction process, even when not stimulated by post-SAH CSF. These as yet uncharacterized units would respond differently to the PKC inhibitor, with the responses modulated by CSF. One (Fig 5A) would respond to PKC inhibitor by being downregulated and suppressed, without regard to the presence or absence of CSF. The other (Fig 5B) would have a dual role, normally in balance between enhancement (gray shading, +) and suppression (pattern shading, -) of PKC-dependent pathways for the force generation machinery. Post-SAH CSF would upregulate this unit (B), but the enhancer activity (+) would be more responsive to the CSF stimulation. The suppression activity (-), on the other hand, would be more sensitive to the PKC inhibitor peptide, and probably only in the presence of some constituent in the post-SAH CSF, not yet identified. Low concentrations of the PKC inhibitor peptide in the presence of CSF would thus downregulate the suppression activity, leaving the enhancement activity, though slightly downregulated, still operating in the pathway. Lattice compaction would thus be enhanced. Medium concentrations of inhibitor would downregulate both suppression activity (now completely halted) and enhancement activity but would still allow lattice compaction to proceed. The highest concentrations of inhibitor would downregulate the enhancement activity (and, of course, the suppression activity) to the point of halting the lattice compaction.
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PKC peptide inhibitor also accelerated lattice compaction in the presence of inactive CSF. In a single trial in which only one sample was used, inactivated CSF was applied with the lowest and highest concentrations of the PKC peptide inhibitor. The inactivation of this CSF sample occurred during long storage in the presence of air and may have been the result of oxidative changes in some components in the sample. When applied without inhibitor, this modified CSF sample did not enhance lattice compaction, and in fact some degree of inhibition of compaction was observed. Low concentrations of the PKC inhibitor peptide applied with this inactivated CSF resulted in compaction rates lower than that observed in control lattices untreated with CSF or inhibitor peptide. These data also support the concept that one of the postulated regulatory moieties with suppression activity is ineffective under normal conditions but operates under the influence of post-SAH CSF.
Several laboratories have shown cAMP activation or cAMP-dependent protein kinase regulation of ECM contraction with the use of several kinds of activators and inhibitors.11 23 25 26 27 28 29 In those reports, reagents that increased cAMP levels inhibited ECM contraction, while inhibitors of PKA either accelerated contraction or had no effect on the process. Similar to results previously reported from other laboratories, in our experimental model forskolin inhibited both ECM contraction and the acceleration by post-SAH CSF, but HA-1077 did not inhibit ECM contraction. Reports of decreased cGMP levels in cerebral arteries after SAH and of a relationship between increased cGMP levels and vessel dilation indicate that inhibition of cGMP production or decreased cGMP levels would inhibit dilation.16 Our data with HA-1077 treatment suggest that PKG activation does not play a major role in acceleration of ECM compaction by cerebral arterial fibroblasts in our model system. HA-1077 is also known to inhibit MLCK, but in other studies in which KT5926, another MLCK inhibitor, was used, ECM contraction was not only not reduced but was slightly enhanced. We found a similar lack of inhibition and slight enhancement of acceleration of lattice compaction in our own experiments at moderate concentrations. At the highest concentration, HA-1077 inhibited contraction, but the cultured cells showed major morphological damage (data not shown), indicating that the concentration was beyond an acceptable dosage level. This also suggests that the observed inhibition was not related to certain specific effects of HA-1077 on PKA or MLCK. Ehrlich et al27 showed that when calcium-calmodulin–independent MLCK is electroporated into fibroblasts, ECM contraction is enhanced, and that increased cAMP hinders these effects. Other reports showed that increased myosin light chain phosphorylation correlated with development of isometric contraction force.30 The results, which might appear controversial or contradictory, depend on differences in experimental conditions. MLCK, cAMP, and PKA could possibly exhibit different actions according to the specific environment in each model system. We can hypothesize that MLCK has an important role in force generation pathways, particularly as a modulator of the process and varying with specific conditions. In vivo these pathways probably involve more complex and modified signal transduction pathways related to MLCK.31
Takai et al32 reported evidence for perturbation of PKC activation with increased levels of cAMP. Their report suggests that the effects of cAMP increase could also operate through a PKC pathway in ECM contraction. On the other hand, He and Grinnell23 25 demonstrated a paradoxical transduction pathway that responded to stress relaxation and contraction. They described cAMP, phospholipase D, phospholipase A2, and phosphatidic acid relationships that were influenced by stress signals. Their studies demonstrated that stress signals could modulate these transduction pathways compared with normal culture conditions. PKC transduction pathways also seemed to interact with each of these factors. They also reported that PKC downregulation by TPA overcame the cAMP regulation as modulated by stress signals. The specific nature of the signal transduction pathway and the roles of each kinase also remain unclear in certain portions of the ECM contraction mechanism. Essentially, our experiments and other reports indicate that elevations in cAMP or PKA activation can be thought to diminish ECM contraction and correlate with stress relaxation in ECM. Metabolic failure related to SAH was reported by several researchers,33 34 who noted the possibility of prevention of relaxation of ECM contraction, which depends on cAMP elevation.
The role of PKC in the mechanism of ECM contraction, as described above, must include many aspects yet to be defined, particularly in regard to activation of PKC. However, our experiments and other reports also suggest that inhibition or disabling of PKC could reduce ECM contraction, including that enhanced by post-SAH CSF. It is likely that the true mechanism of ECM contraction, which is enhanced by post-SAH CSF, may be rather complex, involving several regulatory sites on more than one pathway. However, the results of our experiments seem to demonstrate for the first time an important role for PKC- and -ß in that mechanism. These reports, including the present study, also showed that fibroblasts and endothelial cells regulate their contractility in a manner similar to that of smooth muscle cells and that, conversely, smooth muscle cells also have the capability to regulate contractility through forces exerted on the ECM.
Many researchers have focused on collagen deposition and histological changes in spastic arteries. It is also important to focus on the functional relationship between simple structural changes and the constituent elements of the arterial wall, that is, the role of forces generated by both non–smooth muscle cells and smooth muscle cells that operate through the ECM, as well as on ECM packing or reorganization in the contractility of spastic arteries. Because the forces on the ECM could contribute substantially to vascular constriction, by reasonable extension PKC should have a role in the regulatory pathway of post-SAH cerebral vasospasm.
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Acknowledgments |
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This study was supported by the Department of Neurosurgery, University of Mississippi Medical Center. The authors thank Dr Hiroyoshi Hidaka, Nagoya University School of Medicine (Japan), for providing the HA-1077 used in this study. Samples of cerebral arteries and coronary arteries used for culture starts in this project were obtained through the Cooperative Human Tissue Network, Midwestern Division (Columbus, Ohio), Western Division (Cleveland, Ohio), and Southern Division (Birmingham, Ala). Samples used for starts of dermal fibroblasts were obtained through the Department of Obstetrics and Gynecology, University of Mississippi Medical Center. Dr Shiota is the Robert R. Smith Fellow in Cerebrovascular Research of the Department of Neurosurgery at the University of Mississippi Medical Center.
Received April 11, 1996; revision received May 28, 1996; accepted June 12, 1996.
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Editorial Comment
Department of NeurosurgeryWayne State UniversityDetroit, Mich
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Introduction |
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It becomes more and more evident that vasospasm after SAH is a multifactorial event. Smooth muscles have been investigated the longest, inflammation came next, endothelium is still a hot topic, and the accompanying article draws our attention again to the role of the ECM. The authors have shown with the use of inhibitors and activators that PKC—once again!—probably plays a central role in the ECM contraction, but the precise mechanism remains unclear and appears complicated. The long and intense search for the cause of vasospasm has thus far resulted in only one clinically proven pharmacological treatment for vasospasm: the calcium channel blocker nimodipine, which works through hitherto poorly elucidated and completely different mechanisms than originally thought. When one considers the role of calcium in PKC activation, however, this report may provide a new impetus to look again at calcium, calcium channel blockers, and the calpain-calmodulin system.
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Values are percentage of control (water-treated) lattice area at each time point.
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