Cyclic GMP–Dependent Protein Kinase Expression in Coronary Arterial Smooth Muscle in Response to Balloon Catheter Injury
Abstract—Arterial smooth muscle cells undergo phenotypic and proliferative changes in response to balloon catheter injury. Nitric oxide (NO) and cGMP have been implicated in the inhibition of vascular smooth muscle cell proliferation and phenotypic modulation in cultured-cell studies. We have examined the expression of the major cGMP receptor protein in smooth muscle, cGMP-dependent protein kinase I (PKG), in response to balloon catheter injury in the swine coronary artery. On injury, there was a transient decrease in the expression of PKG in neointimal smooth muscle cells when compared with medial smooth muscle cells. The decrease in PKG expression was observed in the population of proliferating cells expressing the extracellular matrix protein osteopontin but not in cells present in the uninjured portion of the media. Coincident with the suppression of PKG expression in neointimal cells after injury, there was a marked increase in the expression of type II NO synthase (inducible NOS [iNOS], NOS-II) in the neointimal cells. These results suggest that PKG expression is transiently reduced in response to injury in the population of coronary arterial smooth muscle cells that are actively proliferating and producing extracellular matrix proteins. The reduction in PKG expression is also correlated temporally with increases in inflammatory activity in the injured vessels as assessed by iNOS expression. Coupled with our current knowledge regarding the role of PKG in the regulation of cultured cell phenotypes, these results imply that PKG may also regulate phenotypic modulation of vascular smooth muscle cells in vivo as well.
- Received April 24, 2000.
- Accepted May 26, 2000.
Balloon angioplasty is a widely used interventional procedure to alleviate vascular stenoses. The angioplasty procedure, however, results in damage to the arterial wall, initiates a tissue response characterized by inflammation of the injured area, and promotes the proliferation and migration of vascular smooth muscle cells from the media to the intimal (see Reference 1 for a review). The vascular smooth muscle cells migrating to and proliferating in the intima of the arterial wall are phenotypically different from medial smooth muscle cells.2 3 4 Neointimal cells synthesize and secrete extracellular matrix macromolecules such as the protein osteopontin, leading to the formation and expansion of the neointima.5 6
Experiments with cultured arterial smooth muscle cells have provided a general appreciation for the role of growth factors, inflammatory cytokines, and other extracellular signals in the initiation of proliferation and migration.7 8 9 However, the intracellular mechanisms responsible for the phenotypic modulation of vascular smooth muscle cells from a contractile phenotype capable of regulating vascular tone to a synthetic phenotype responsible for the synthesis and secretion of large amounts of extracellular matrix proteins have remained obscure.
The messenger molecule nitric oxide (NO) acts as an inhibitor of cultured smooth muscle cell proliferation.10 Likewise, several in vivo studies have suggested that NO prevents intimal thickening in animal models of restenosis.11 12 13 14 On the other hand, the induction of NO synthase (NOS), particularly NOS-II, or inducible NOS (iNOS), would be predicted to restore NO to injured arterial tissue and block further proliferation and intimal growth. In fact, inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) that induce iNOS expression do not reduce cell proliferation in vivo15 16 and have been correlated with the worsening of intimal expansion.17 Therefore, there is uncertainty regarding the role of NO in vessel wall restructuring in response to injury.
NO activates the soluble form of guanylyl cyclase, leading to the formation of intracellular cGMP. cGMP activates a serine/threonine kinase, the cGMP-dependent protein kinase (PKG), leading to the phosphorylation of key regulatory proteins. Studies in our laboratory have shown that the levels of PKG decrease in adult rat aortic smooth muscle cells as they are passaged in culture and become more phenotypically synthetic.18 Restoration of PKG levels in vascular smooth muscle cells via transfection causes the synthetic cells to assume a more contractile phenotype.19 Whether a similar downregulation in PKG expression occurs in vivo in response to injury has not been examined in animal models that are relevant for human vascular disease. Such information would be important, because it would provide a link between the findings observed in vitro with cultured smooth muscle cells and those in intact arterial tissues in disease models.
Fifteen juvenile domestic pigs (20 to 30 kg) were used for this study. All animal care and handling procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals,20 and the protocols were approved by the Institutional Animal Use and Care Committee of the University of Alabama at Birmingham.
Balloon Angioplasty Procedure for Swine Coronary Restenosis
The balloon angioplasty procedure was performed as previously described.21 After anesthesia induction and intubation, heparin (200 U/kg) and bretylium (50 mg) were given as an intravenous bolus, and 10 mg of nifedipine was given sublingually. Via a right femoral artery approach, a balloon catheter, 20 mm long with a diameter 20% larger than the arterial diameter, was used to perform the dilation injury. Three 30-second balloon inflations were performed at nominal pressure on the proximal to middle segment of either the left anterior descending coronary artery or the left circumflex artery. Each balloon inflation was separated by 1 to 2 minutes to allow for coronary perfusion. After the angioplasty procedure, an Alzet osmotic minipump (Alza Corp) containing 500 mg of 5 bromo-2′-deoxyuridine (BrdU, Sigma Chemical Co) was implanted subcutaneously. In some experiments, intracoronary artery stents (FlexStent, Cook, Inc) were deployed in pigs by using techniques similar to those described above and previously published.22
Animal Sacrifice and Tissue Preparation
Animals were killed between 4 and 14 days after angioplasty. The hearts were perfusion-fixed in 10% formalin, and segments of the coronary artery having undergone angioplasty were carefully dissected from the heart along with ≈1 to 2 cm of normal coronary artery proximal and distal to the angioplasty site, which were taken for use as controls.
The fixed vessels were sectioned at 2- to 3-mm increments perpendicular to the vessel axis so that 6 serial sections were obtained. The sections were embedded in paraffin, and tissue sections were affixed to glass microscope slides and stained with hematoxylin and eosin. In the studies described below, adjacent sections from the same vessel were used for specific histochemical analysis. All immunohistochemical analyses were performed on at least 3 separate animals at 4, 7, and 14 days.
Immunohistochemistry for PKG Expression
For examination of PKG, both swine and human tissues were stained with a goat anti-bovine PKG I antiserum.19 All immunostained tissue sections were compared with preimmune serum controls that did not stain. Tissue sections were deparaffinized by standard histological procedures and blocked for 30 minutes with 1:100 rabbit IgG (Sigma) diluted in PBS. The sections were incubated for 1 hour at room temperature with anti-PKG diluted 1:100 in PBS. After several washes, the slides were incubated with rabbit anti-goat IgG (BA-5000, Vector Labs), diluted 1:500 in PBS, for 1 hour at room temperature. The immune complexes were visualized by avidin-biotin-peroxidase staining and counterstained with Mayer’s hematoxylin.
Immunohistochemistry for Osteopontin, Calponin, and iNOS
Essentially identical procedures were used for staining osteopontin (a generous gift from Dr Charles Prince, Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, Ala), calponin (Sigma), and iNOS (Transduction Laboratories). For osteopontin, 1:200 mouse anti-human osteopontin was used; for calponin, 1:500 mouse anti–turkey gizzard calponin was used; and for iNOS, 1:200 rabbit anti-human iNOS was used. Similar controls and preimmune serum analyses were performed as described for PKG immunostaining.
Immunohistochemistry for Cell Proliferation
Two immunohistochemical methods, anti-BrdU and anti–proliferating cell nuclear antigen (PCNA), were used to quantify proliferative activity of smooth muscle cells at the balloon injury sites. Immunohistochemistry was performed on pig tissues with the peroxidase–anti-peroxidase procedure with a mouse anti-BrdU monoclonal antibody. Pig lymph nodes and duodenum were used as positive controls. Immunostaining for PCNA in pig and human autopsy specimens was performed with the Ventana ES automatic immunohistochemical stainer (Ventana Medical Systems).
Western Blot Analysis for PKG in Swine Neointimal and Medial Coronary Arterial Smooth Muscle Cells
To induce restenosis in swine coronary arteries, stents were implanted as described earlier.22 The animals were killed 7 days after stent implantation, and the hearts were removed as described above. The coronary arteries were dissected from the heart and placed in cold cell-isolation medium consisting of Dulbecco’s modified minimal essential medium (DMEM) containing 20 mmol/L HEPES buffer, pH 7.4, 1 mg/mL bovine serum albumin, 5 μg/mL amphotericin B, and 50 μg/mL gentamicin. The stent was removed and placed in cell-isolation medium, and the remaining vessel was opened by using a longitudinal cut. The neointimal tissue adhering to the stent and neointima from the vessel luminal surface were removed with a sterile scalpel. Isolated cells were obtained from this neointimal tissue by incubation in a digestion medium containing 1 mg/mL elastase and 200 U/mL collagenase for 1 to 2 hours (until a single-cell suspension was obtained). After being washed twice with isolation medium, the cells were resuspended in DMEM containing 5% fetal bovine serum and 5% calf serum and plated in culture flasks at a density of ≈1000 cells/cm2. Medial cells from unaffected areas were isolated from the vessels by scraping the layer into isolation medium, and single cells were obtained by digestion as described above. After 7 days in culture, the cells were photographed. Extracts were made from the cultured cells by homogenizing them in 20 mmol/L potassium phosphate buffer, pH 6.8, 1 mmol/L EDTA, 15 mmol/L 2-mercaptoethanol, 0.1 mmol/L PMSF, and 10 μg/mL leupeptin. Four micrograms of the extract protein was resolved by SDS–polyacrylamide gel electrophoresis on 8% polyacrylamide gels. After transfer to nitrocellulose, the blots were incubated in 1:1000 goat anti-bovine PKG and analyzed by the enhanced chemiluminescence (ECL, Amersham) procedure.
Human Autopsy Tissue
Human hearts from patients that had undergone angioplasty were obtained at autopsy and perfusion-fixed in 10% neutral buffered formalin. Sections of coronary vessels that had undergone angioplasty were embedded in paraffin, cut, and stained with hematoxylin, eosin, and aldehyde fuscin–Gormori’s trichrome stain. Selected regions demonstrating the angioplasty injury and neointima were selected, and serial sections were cut for immunohistochemical staining by using the same procedures as described above.
Effects of Balloon Catheter Injury on Swine Coronary Arterial PKG Expression
The time course of expression of PKG and phenotypic changes in the coronary arterial cells after balloon catheter injury are shown in Figures 1⇓, 2⇓, and 3⇓. This series of figures is a time course of changes in the arterial wall after balloon catheter injury at 4, 7, and 14 days, respectively. Panel A of each figure is the hematoxylin-eosin–stained section of the coronary artery at the site of medial laceration produced by balloon stretching of the vessel. Panels B through F of each figure are adjacent sections to those shown in A panels that have been immunostained for PKG, BrdU, osteopontin, calponin, and iNOS, respectively, after balloon catheter injury.
Panel A of Figure 1⇑ shows the site of medial laceration (arrows) from overstretch balloon injury 4 days after the angioplasty procedure. In this region, the smooth muscle cells have begun to migrate into the area of injury and have begun to form a neointima. Note that there are a few fusiform (ie, contractile) cells within the thrombotic material adjacent to the broken media and in the developing neointima. The less densely stained area at the edge of the lacerated media contains cells that appear more stellate in morphology than those in the media and are actively secreting extracellular matrix proteins, thus accounting for the pale staining in this hematoxylin-eosin–stained section. The cells in the neointima stained intensely positively for osteopontin (D) compared with the cells in the media. On the other hand, the cells in the media stained positively for calponin, whereas the neointimal cells were devoid of calponin staining (E). Panel C of Figure 1⇑ demonstrates that BrdU staining, indicative of cell proliferation, was confined to those cells appearing at the broken edge of the media and within the developing neointima. Medial smooth muscle cells, on the other hand, demonstrated little BrdU staining, indicative of very low cell proliferation and little repair activity.
PKG expression, as monitored by specific immunohistochemistry, was observed throughout the medial tissue but not in the adventitia or thrombotic material (Figure 1B⇑). Cells demonstrating fusiform morphology generally had more intense cytoplasmic staining than did cells having a less fusiform appearance. Most notably, however, the synthetic, stellate cells in the developing neointima near the leading edge of the injured area were practically devoid of PKG staining. Only the fusiform contractile cells staining positively for calponin and located adjacent to the thrombotic material stained positively for PKG. Cells devoid of PKG staining, on the other hand, stained intensely positively for osteopontin. On the basis of these staining patterns for PKG and phenotypic markers, at the earliest time point examined, PKG expression appeared to be reduced only in the synthetic cells involved in active tissue repair at the leading edge of the lacerated media and the newly formed neointima.
At 7 days after injury (Figure 2⇑), the lesion produced by medial stretching had resolved into a well-formed neointima (A). Little thrombotic material was present at day 7, and many of the smooth muscle cells composing the neointima had already assumed a fusiform appearance, aligning in a parallel fashion. At 7 days, unlike the situation at 4 days after injury, PKG staining had begun to reappear, at least in the fusiform cells of the neointima (located below the letter N in B). Much lighter staining for PKG was observed in the more disorganized neointimal area, where there were fewer fusiform cells expressing calponin (E). However, the cells in the area of disorganized neointima stained positively for osteopontin (D). The levels of cellular proliferation as indicated by BrdU staining in C were reduced at day 7 and were confined to the broken edge of the media and to the neointima. The neointimal, fusiform cells appeared capable of proliferation as assessed by BrdU staining. From these observations, it appeared that PKG staining was better correlated with the lack of osteopontin staining and increased calponin staining than with increased cellular proliferation. The results in Figure 2⇑ suggest that day 7 after catheter injury represented a transition phase in PKG expression, during which PKG expression was restored before an increased calponin expression and decreased osteopontin expression.
At day 14 after balloon angioplasty (Figure 3⇑), neointimal tissue had completely filled the space between the lacerated ends of the media (A). PKG staining was again intense in the media but was also abundant in the neointima (B). The cells in the neointima appeared to be fusiform in morphology, aligned in parallel fashion where cell density was greatest, and stained intensely positively for calponin (E). Osteopontin staining was reduced in the neointima at day 14 after balloon catheter injury, although it was still greater than that in the media (D). Sections immunostained for BrdU (C) showed much reduced cell proliferation. The results in Figure 3⇑ suggested that at day 14 after injury, PKG expression and contractile protein expression appeared together in the vascular smooth muscle cells of the neointima. This situation is in contrast to day 7 after injury, when it appeared that PKG expression preceded calponin expression.
It is well recognized that growth factors, cytokines, and NO derived from iNOS, or NOS-II, are elevated during the inflammatory response that results from balloon catheter injury.1 7 8 9 15 16 Hence, it was of interest to determine whether iNOS staining was elevated in smooth muscle cells involved in active tissue repair after balloon catheter injury to our animals. As shown in Figures 1 through 3⇑⇑⇑ (F of each figure), iNOS staining was intense in the neointimal smooth muscle cells 4 days after angioplasty but was completely absent in the medial cells. The most intense staining was observed in cells found at the edges of the medial laceration. These are the same cells that demonstrated the most intense BrdU staining and the faintest PKG staining. We have also found that the injured tissue at the edge of the medial laceration stained intensely for TNF-α, whereas the uninvolved media was devoid of staining (data not shown). By day 7 after angioplasty, iNOS staining was greatly reduced in the neointima, and by day 14 after angioplasty, there was no evidence of iNOS staining in any area of the neointima. From these results, it is evident that iNOS was transiently expressed after injury but only in the neointimal areas where active tissue repair activity was occurring.
Western Blot Analysis of PKG in Medial and Neointimal Cells From the Injured Swine Coronary Artery
Neointimal and medial tissues were excised from coronary arterial tissue of the pig 7 days after angioplasty and stent deployment, and PKG levels were demonstrated by Western blot analysis. As shown in Figure 4⇓, the cells arising from the neointima appeared flattened, stellate, and fibroblastic, even after being cultured for 7 days (B). The cells from the media, on the other hand, retained their spindle-shape, fusiform morphology during culture, similar to the morphology of the contractile cells in the intact media (A). PKG expression was still detected in extracts derived from cultures of medial smooth muscle cells, whereas no PKG was detected in extracts derived from cultures of the neointimal cells (C).
PKG Expression in Human Coronary Arterial Atherosclerotic Lesions
To determine whether PKG expression varied in cells from human atherosclerotic lesions, sections of coronary artery were obtained at autopsy at various time points after clinical angioplasty procedures. From our group of autopsy patients, we were able to obtain tissue samples from 3 patients who died between 10 and 21 days after angioplasty. These coronary artery sections were stained for PKG, and 1 of these specimens obtained 14 days after angioplasty is shown in Figure 5⇓. Balloon inflation ruptured the atherosclerotic plaque (arrowheads) and stretched the media and adventitia (A). A well-formed neointima had filled in the area between the ruptured plaque and media. The cells in the neointima were generally more stellate and fibroblastic in appearance compared with the more spindle-shape cells of the media (B). Staining for PKG was intense in the media but was greatly reduced or absent in the neointima (C). Those areas of neointima that did demonstrate faint staining for PKG were confined to cells located near the endothelium. Fibrous tissue that was separated from the media and was seen adjacent to the neointima did not stain for PKG. Cell proliferation was assessed with the anti-PCNA antibody. As shown in D, there were occasional proliferating cells in the neointima but very few in the media. Cells lacking stain for PKG were among those having the most intense PCNA staining. Among the differences between PKG staining in the human compared with the pig injury model was the reappearance of PKG staining in the neointima of the pig 14 days after injury. Overall, however, the results are consistent with the concept that PKG expression is reduced in synthetic, neointimal smooth muscle cells compared with contractile, medial smooth muscle cells.
Our laboratory had found that PKG regulates events leading to the modulation of cultured vascular smooth muscle cells to the contractile phenotype in vitro.19 The purpose of the present study was to determine whether similar patterns of PKG expression exist in smooth muscle cells of different phenotypes in coronary arteries after balloon catheter injury. We discovered that there was a transient decrease in PKG expression in vascular smooth muscle cells undergoing tissue repair activity at the site of medial laceration. The evidence that these cells are synthetic in phenotype was based on their greater proliferative activity, morphology, and their ability to produce the extracellular matrix protein osteopontin. The decrease in PKG expression was most apparent within 4 days after injury and was confined to cells in the developing neointima. Cells not involved in tissue repair (eg, medial smooth muscle cells at 4 days after injury) did not appear to lose PKG expression after injury, nor did these cells appear to be synthetic in nature, since they expressed calponin but not osteopontin. Cells demonstrating losses in PKG expression were also those cells undergoing the greatest proliferation. However, the inverse relationship between PKG expression and cell proliferation is not necessarily one of cause and effect, since PKG expression was reestablished between 7 and 14 days in fusiform, contractile, neointimal smooth muscle cells that were still proliferating. The most direct correlation observed in this study was between PKG expression and the contractile phenotype.
The decrease in PKG expression was transient after injury. Seven days after injury, PKG staining reappeared in neointimal cells, particularly in those near the luminal border that demonstrated an elongated and fusiform morphology. Osteopontin staining was still evident in the neointima but appeared to be decreasing in abundance at day 7 after injury. Calponin staining was sparse in neointimal cells, but like PKG, appeared more abundant in cells adjacent to the luminal border compared with cells located near the internal elastic lamina. By day 14 after injury, both PKG and calponin staining was intense in neointimal cells. Osteopontin staining, by contrast, was decreased. Therefore, there appears to be a temporal, positive correlation between PKG expression and calponin expression on the one hand and a negative correlation between PKG and osteopontin expression on the other. It is during the first 7 days that vascular smooth muscle cell proliferation is greatest after injury, suggesting that active tissue repair is most intense during this period, when PKG staining is lowest in the neointima. Thus, both temporally and spatially, PKG expression is decreased in cells undergoing proliferation, secretory activity, and active wound healing.
The results described herein are consistent with a role for PKG in regulating cell phenotype in vivo and are also consistent with the concept that the downregulation of PKG expression leads to the development of the synthetic phenotype in vivo. This concept is supported by the observation that cells cultured from the swine neointima and media after stent implantation retained their synthetic and contractile phenotypes, respectively, and differed in PKG expression. The fusiform, contractile cells isolated from the medial tissue expressed significantly more PKG than did the synthetic cells isolated from the neointimal tissue.
The time course of events leading to the loss of PKG expression follows the pattern of the inflammatory response that occurs in the swine lesions in response to injury. It is well established that IL-1β and TNF-α are produced within the first few days after catheter injury.1 7 8 9 15 16 These cytokines induce the expression of iNOS, and iNOS elevation has been observed by others to increase in neointimal cells after balloon catheter injury.23 In the present study, we also observed a robust increase in iNOS expression in nearly all neointimal cells 4 days after injury. Medial cells, on the other hand, were devoid of iNOS staining. By day 7 after injury, iNOS staining was greatly reduced in the neointima, and by day 14 after injury, no detectable iNOS was present. These studies are important, for they provide a plausible mechanism for the reduction in arterial PKG expression after balloon catheter injury. Previous studies by our laboratory had shown that NO itself is a potent suppressor of cultured vascular smooth muscle cell PKG expression in vitro.24 We propose that the persistent and robust increases in NO generated after iNOS induction in response to injury and inflammation suppresses PKG expression in vivo as well.
It is important to distinguish between the 2 types of NO signaling that may be present in the vessel wall. On the one hand, transient increases in endothelium-derived NO stimulate soluble guanylyl cyclase, leading to the activation of PKG and the subsequent effects of PKG-dependent protein phosphorylation (ie, vessel relaxation and gene expression for the contractile phenotype). On the other hand, expression of iNOS as a result of inflammatory events leads to sustained, high levels of NO, which may be responsible for the suppression of PKG expression by mechanisms not defined at this point. This proposed pathway for PKG suppression may account for the paradoxical findings that iNOS expression in injured arterial tissues does not inhibit neointimal expansion and restenosis,1 15 16 17 25 even when lower doses of NO-donor drugs or endothelial NOS transfection does.
With respect to human arterial lesions, PKG expression was dramatically reduced in neointimal tissue when compared with medial tissue, even 2 weeks after angioplasty. In contrast, PKG levels returned to normal throughout the neointima of the swine coronary artery at this same time point after angioplasty. It is unlikely that the differences between human and swine PKG expression patterns are related to species differences, but rather the difference in PKG expression is most likely related to the presence of active disease in the human. Hence, healthy pigs having undergone balloon angioplasty demonstrate effective wound healing and lesion resolution within 14 days, whereas those factors causing human disease (eg, inflammation) are likely to still be present, leading to suppression of PKG and continual neointimal expansion and plaque formation.
This work was supported by grants from the National Institutes of Health HL34646 and HL53426 (to T.M.L.), HL58895 (to P.G.A.), and HL60186 (to D.B.M.).We wish to acknowledge the contributions of Dr Thomas Adair, a former medical student at the University of Alabama at Birmingham, for his work with human specimens.
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