Vascular Mural Cells in Healing Canine Myocardial Infarcts
Section of Cardiovascular Sciences, the DeBakey Heart Center, Baylor College of Medicine, and the Methodist Hospital, Houston, Texas
Correspondence to: Nikolaos Frangogiannis, MD, Section of Cardiovascular Sciences, One Baylor Plaza M/S F-602, Baylor College of Medicine, Houston, TX 77030. E-mail: ngf{at}bcm.tmc.edu
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Summary |
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(J Histochem Cytochem 52:10191029, 2004)
Key Words: myocardial infarction smooth muscle cell myofibroblast pericyte arteriole angiogenesis desmin smoothelin -smooth muscle actin non-muscle myosin heavy chain
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Introduction |
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VSMCs can express a range of phenotypes and respond to environmental cues by altering their expression of contractile proteins and by modifying their ability for protein synthesis. Vascular injury is associated with phenotypic modulation of smooth muscle cells, leading to increased expression of non-muscle myosin heavy chain (NMMHC) isoforms, such as NMMHC B/Smemb, in the vascular wall. Although structural and phenotypic alterations of VSMCs have been described in a variety of vascular lesions, the phenotype of vascular myocytes in healing wounds has not been systematically studied. Healing myocardial infarcts contain a large population of mesenchymal cells expressing -smooth muscle actin (
-SMA) (Willems et al. 1994
). The majority of these cells are phenotypically modulated myofibroblasts and do not express smooth muscle myosin (Frangogiannis et al. 2000b
). As the wound matures, many infarct neovessels become coated with vascular mural cells, leading to the formation of a scar containing predominantly coated vessels (Ren et al. 2002
). In this study we examined the progressive phenotypic changes of pericyte-coated vessels in infarcts and report significant changes in VSMC phenotype in mature scars. We describe for the first time dynamic changes in the morphological features of coated vessels that lead to a progressive increase in vessel wall thickness and the formation of a large number of vascular structures that lack an internal elastic lamina. Infarct maturation is associated with increased expression of desmin in the neovascular smooth muscle cells and with formation of vessels with large irregular deposits of matrix proteins in the forming media. In addition, we report for the first time induction of the non-muscle myosin heavy chain (NMMHC) A and B genes in the reperfused infarcted canine heart.
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Materials and Methods |
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Animals included in the study underwent 1 week (n=4), 4 weeks (n=4), or 8 weeks of reperfusion (n=4) and demonstrated evidence of myocardial infarction on light microscopic examination of hematoxylineosin-stained tissue for replacement fibrosis. Additional animals were used for RNA extraction (5-hr reperfusion, n=2; 24-hr reperfusion, n=2; 5-day reperfusion, n=2). Myocardial RNA samples were described as ischemic if they came from areas in which blood flow during coronary occlusion was less than 25% of control. Samples of control tissues were taken from the anterior septum and had normal blood flow during coronary occlusion. An animal that underwent 1-hr ischemia and 5 days of reperfusion but that showed no evidence of infarction (because of the presence of collateral circulation) was used as an additional control to demonstrate the effects of infarction on NMMHC mRNA expression.
In addition, four healthy mongrel dogs were sacrificed and the segments from the left ventricle and the following vessels were obtained: thoracic ascending aorta, thoracic descending aorta, carotid, subclavian, femoral, and coronary arteries, superior vena cava, jugular vein, and femoral vein. The samples were fixed in B*5 without formalin and embedded in paraffin.
Tissue Processing, Histology, and Immunohistochemistry
Histological samples were fixed in B*5 fixative and embedded in paraffin. Sequential 35-µ sections were cut by microtomy. Collagen and elastin fibers were identified by Verhoeff-van Gieson (VVG) staining. Immunostaining was performed using the ELITE rabbit, goat, or mouse kit (Vector Laboratories; Burlingame CA). Briefly, sections were pretreated with a solution of 3% H2O2 to inhibit endogenous peroxidase activity and incubated with 2% horse serum to block nonspecific protein binding. Then they were incubated with the primary antibody for 2 hr at room temperature. After rinsing with PBS, the slides were incubated for 30 min with the secondary antibody. The slides were rinsed with PBS and incubated for 30 min in ABC reagent (Hsu et al. 1981). Peroxidase activity was detected with diaminobenzidine (DAB; Vector). Slides were counterstained with eosin. The following primary antibodies were used for IHC: mouse anti-
-SMA (Frangogiannis et al. 2000b
), mouse anti-desmin (both from Sigma; St Louis MO), mouse anti-smoothelin (Abcam; Cambridge, MA), mouse anti-CD31 (Dako; Carpinteria, CA) (Frangogiannis et al. 2000b
), mouse anti-collagen type III (ICN; Aurora, OH) (Frangogiannis et al. 2003
), and rabbit anti-NMMHC A and B (Covance; Berkeley CA).
Quantitative Morphometry and Statistical Analysis
At least two ischemic and two control samples were studied from each experiment. Stained slides were examined with a Zeiss Axioskop microscope and photographed with a Zeiss digital camera. For quantitation of the arteriolar density, all coated vessels with a diameter >15 µm were counted in infarcted and control areas and the density was expressed as vessels/mm2. In addition, the density of small (diameter 1540 µm) and large arterioles (diameter 40 µm) was quantitated. Eight different fields from three control and ischemic segments from each experimental group (1 week, 4 weeks, and 8 weeks of reperfusion) were analyzed. Mean arteriolar wall thickness was quantitated for each field based on all arterioles seen in cross-section (major:minor axis <1.4). Staining for desmin, an intermediate filament protein, and smoothelin, a cytoskeletal protein expressed by differentiated smooth muscle cells, was used to assess the phenotype of vascular mural cells in healing infarcts. The percentage of desmin-and smoothelin-positive arterioles in infarcted and non-infarcted samples was quantitated by examining serial sections stained for -SMA, desmin, and smoothelin. Segments from three different experiments (control and infarcted) from each group were used for analysis. All
-SMA-positive coated vessels in the fields examined were identified, classified into one of three groups based on diameter (1525 µm, 2540 µm, >40 µm) and examined for desmin and smoothelin staining. Negative staining was defined as complete absence of desmin or smoothelin immunoreactivity in the vascular wall. Statistical analysis was performed using ANOVA followed by t-test corrected for multiple comparisons (StudentNewmanKeuls). Significance was set at p<0.05.
mRNA Extraction and Northern Hybridization
RNA isolation from myocardial tissue segments was performed using the acid guanidiniumphenolchloroform procedure. RNA (20 µg) was electrophoresed in 1% agarose gels containing formaldehyde and then transferred to a nylon membrane (Gene Screen Plus; New England Nuclear. Boston, MA) by standard procedures. The membranes were hybridized in QuikHyb (Stratagene; La Jolla, CA) at 68C for 2 hr with 1 x 106 dpm random hexamer 32P-labeled cDNA probes for NMMHC-A and NMMHC-B (Simons et al. 1991) (a generous gift from Dr. Adelstein, National Institutes of Health). Filters were washed with 2 x SSPE at 68C for 20 min, with 1 x SSPE + 1% SDS at 68C for 15 min twice, and with 1 x SSPE at 21C for 15 min with constant shaking and exposed to Hyperfilm (Amersham; Arlington Heights, IL).
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Results |
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Phenotypic Characteristics of Vascular Mural Cells in the Infarct
We hypothesized that the vascular smooth muscle cells coating the infarct neovessels may undergo a maturation process, leading to a progressive increase in expression of desmin and smoothelin in the vascular wall. In the non-infarcted myocardium, desmin expression in the arteriolar media was dependent on the size of the vessel, with larger arterioles (>40 µm) showing a significantly higher percentage of desmin-positive vessels than smaller arterioles (1540 µm) (p<0.001). After 7 days of reperfusion, desmin-expressing vessels were practically absent from the healing infarct (Figure 4). Desmin staining was found in the remaining cardiomyocytes and was particularly intense in the myocytes bordering the infarct. Infarct myofibroblasts did not express desmin or smoothelin. After 48 weeks of reperfusion a significant percentage of coated microvessels exhibited a desmin-positive vascular wall (Figures 4D4F; Table 2), suggesting a progressive maturation process. In mature scars (8 weeks of reperfusion), desmin expression by coated vessels did not depend on the size of the vessel. Smoothelin staining was found in a significant percentage of arterioles in the non-infarcted myocardium (Table 3). Smaller arterioles (1540-mm diameter) showed a higher percentage of smoothelin-positive vessels than desmin-positive vessels. In addition, larger arterioles (>40 µm diameter) were much more likely to express smoothelin than small arterioles (1525 µm) (p<0.05; Table 3). In healing infarcts, smoothelin-expressing coated vessels were noted after 7 days of reperfusion. Although the increase in density of coated vessels resulted in the presence of a large number of smoothelin-expressing vessels in mature scars (8-week reperfusion), the percentage of smoothelin-positive vessels did not change significantly over the course of infarct healing (Table 3).
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Discussion |
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Although a number of studies have examined the alterations in smooth muscle cell phenotype after vascular injury, little information is available on the fate of smooth muscle cells in healing wounds. We have previously identified mature smooth muscle cells (expressing both -SMA and the smooth muscle myosin isoforms SM1 and SM2) and activated myofibroblasts (expressing
-SMA but not SM1 and SM2, markers of mature smooth muscle cells) in healing canine infarcts (Frangogiannis et al. 2000b
). Mature smooth muscle cells were found in the media of infarct arterioles, whereas myofibroblasts are predominantly located in the infarct border zone. The present study describes the morphological and phenotypical changes of the infarct arterioles, vascular structures with a potential role in scar maturation and stabilization.
Morphological Characteristics of the Normal Canine Vasculature
To understand the structure and phenotype of infarct vessels, we first systematically examined the morphological characteristics of normal canine vessels. We used staining for the cytoskeletal proteins smoothelin, a marker of contractile smooth muscle cells (van der Loop et al. 1996), and desmin, to determine the phenotypic characteristics of VSMCs in normal dog vessels. Canine muscular arteries had higher expression of desmin and smoothelin than elastic arteries. In the cardiac vasculature, large arterioles were more likely to be desmin-positive than small arterioles (Figure 1). Smoothelin and desmin expression was absent in capillaries, venular pericytes, and small veins but was often observed in large venous SMCs. These findings are similar to the previously reported pattern of localization of desmin and smoothelin in human vessels (Johansson et al. 1997
,1999
; van der Loop et al. 1997
).
The Infarct "Neoarteriole": Morphology and Phenotype
During the proliferative phase of healing, wound angiogenesis is important to provide the highly cellular and metabolically active granulation tissue with oxygen and nutrients (Frangogiannis et al. 2002b). At this stage, enlarged pericyte-poor vessels (Pettersson et al. 2000
; Ren et al. 2002
), many capillaries, and abundant myofibroblasts are noted in the healing infarct; these cells are responsible for collagen deposition in the wound (Cleutjens et al. 1995
). As the infarct matures, the highly vascular granulation tissue is replaced by a collagen-rich scar. The mature scar exhibits a relatively low capillary density but a high arteriolar density and a large number of pericyte-coated vessels (Ren et al. 2002
). We coined the term "neoarteriole" to describe these newly formed coated infarct vessels, which have certain unique morphological characteristics (Figures 2 and 3). In contrast to normal myocardial arterioles, many neoarterioles do not have a defined IEL but often exhibit irregular deposits of matrix in the media (Figure 3), which is sometimes divided into two separate layers. The lack of an IEL in infarct neoarterioles may reflect continuous vascular remodeling. In addition, because of the role of the IEL as a barrier separating the intima and the media of the vascular wall, its absence may result in increased outward diffusion of plasma macromolecules into the arteriolar media (Penn et al. 1994
). Wall thickness progressively increases as the scar matures and occasional bizarre vascular structures with multiple walls are noted. Vessels with an irregular media are also found, suggesting a continuous coating process during infarct maturation (Figure 3). Considering the low metabolic needs of the mature scar and the small number of capillaries, it is likely that coating of infarct neovessels with mural cells does not serve to create effective conduits of blood; it rather represents a mechanism necessary for inhibition of the angiogenic process. This view is supported by previous experiments (Connelly et al. 1989
) demonstrating that myocardial blood flow in reperfused rabbit infarcts 3 weeks after infarction is significantly lower than blood flow during the early post-reperfusion period. Recruitment of mural cells by the infarct neovasculature may be important for stabilization of the wound by inhibiting endothelial cell proliferation and angiogenesis and by protecting the neovessels from regression (Benjamin et al. 1998
).
VSMC Maturation in the Infarct
VSMCs demonstrate remarkable plasticity and undergo significant phenotypic changes, depending on the microenvironment. In atheroslerotic plaques, the cytoskeletal features of VSMCs are modified compared with resident medial smooth muscle cells (Kocher and Gabbiani 1986). These cells acquire a dedifferentiated phenotype as far as the cytoskeleton is concerned, showing high expression of vimentin and a relatively low expression of desmin (Gabbiani et al. 1984
). Using IHC staining for desmin and smoothelin, we investigated the phenotypic changes of vascular mural cells in dog infarcts. During the proliferative phase of healing (7 days after infarction), the majority of coated vessels contained a desmin-negative media. In contrast, in mature scars (8 weeks after infarction), coated vessels had desmin content comparable to that of non-infarcted hearts (Figure 4; Table 2). However, in contrast to non-infarcted myocardium, in which arteriolar desmin expression was found predominantly in larger arterioles (>40 µm), mature infarcts had similar desmin expression regardless of size. Although the function of desmin in vascular smooth muscle cells is not completely understood, its absence results in lower distensibility (Lacolley et al. 2001
) and microvascular dysfunction (Loufrani et al. 2002
). The increase in the number of desmin-positive microvessels during infarct maturation may reflect differentiation of the vascular mural cells and may affect the viscoelastic properties of the infarct vasculature. Surprisingly, smoothelin, a cytoskeletal protein found in mature contractile VSMCs, was similarly expressed in vessels from infarcted and non-infarcted areas (Figure 4; Table 3). Although this may suggest that infarct mural cells express certain smooth muscle cell differentiation markers at an early stage, it may also reflect the lack of sensitivity of IHC methods in detecting different levels of protein expression.
Non-muscle Myosin Heavy Chain Isoforms in the Infarct
Non-muscle myosins of the myosin II superfamily are ubiquitously expressed motor proteins, potentially involved in a variety of physiological tasks, including cell migration and cytokinesis (Spudich 1989; Lofgren et al. 2003
). Cultured smooth muscle cells, while actively growing, and embryonic smooth muscle tissues predominantly express NMMHC (Kawamoto and Adelstein 1987
). In addition, atherosclerotic rabbit aortas express significant amounts of NMMHC isoforms, suggesting a dedifferentiation process towards an embryonic smooth muscle cell phenotype (Zanellato et al. 1990
). We have previously demonstrated that healing canine myocardial infarcts (Frangogiannis et al. 2000b
) and myocardial segments from patients with ischemic cardiomyopathy (Frangogiannis et al. 2002a
; Frangogiannis 2003
) contain a large number of NMMHC-B/Smemb-expressing mesenchymal cells. In healing infarcts these cells were predominantly located in the infarct border zone and were identified as myofibroblasts by their expression of
-SMA. Vascular mural cells were SM1- and SM2-positive but exhibited no staining for SMemb. In the present study we examined mRNA and protein expression of both NMMHC isoforms in healing canine infarcts. NMMHC-A expression was noted in the non-infarcted myocardium and was predominantly localized in the microvascular endothelium (Figure 6). Significant NMMHC-A mRNA upregulation was found in healing infarcts (Figure 5), and NMMHC-A protein was localized in the endothelium of infarct neovessels (Figure 6C). Myofibroblasts and vascular mural cells did not demonstrate significant NMMHC-A immunoreactivity. NMMHC-B protein was immunolocalized in the intercalated disks of cardiomyocytes in the non-infarcted heart. This is consistent with previously reported findings in human and murine tissues (Takeda et al. 2000
). In infarcted myocardial segments, a modest upregulation of NMMHC-B mRNA was noted. IHC identified
-SMA-expressing myofibroblast-like cells as the main source of NMMHC-B protein expression in the infarct, confirming our previously reported findings with a different antibody to NMMHC-B/SMemb (Frangogiannis et al. 2000b
). Vascular mural cells coating infarct neovessels did not stain for NMMHC-B. Interestingly, many border zone cardiomyocytes exhibited heavy and diffuse cytoplasmic staining for NMMHC-B, in sharp contrast to the normal cardiomyocytes, which show localized NMMHC-B expression in the intercalated disks (Figure 6D). This pattern of staining has been previously described in embryonic hearts (Takeda et al. 2000
). These cells may be resident cardiomyocytes that acquired an embryonic phenotype, or may represent a subset of stem cells expressing high levels of NMMHC-B.
Acquisition of a Muscular Coat: an Important Step for the Formation of a Mature Scar
Our study describes an important aspect of infarct healing, reporting the formation of neoarterioles in the wound. These vascular structures result from recruitment of vascular mural cells by infarct neovessels and exhibit distinct morphological features. During the proliferative phase of healing, highly vascular granulation tissue with a large number of capillaries is formed and may serve to supply the metabolically active infarct with nutrients and oxygen. However, as the scar matures, the metabolic needs of the infarct decrease and most capillaries regress as the remaining microvessels become coated with vascular mural cells. Formation of neoarterioles in the healing infarct may represent an important regulatory step in stabilizing the infarct by suppressing the angiogenic process. This investigation raises interesting questions regarding the origin of the vascular mural cells and the signals responsible for their recruitment. Blood-derived progenitors or resident myocardial mesenchymal cells may be the source of vascular myocytes coating infarct microvessels. PDGF- and endoglin-mediated pathways (Lindahl et al. 1997; Li et al. 1999
) may be important in mediating recruitment of mural cells by the infarct endothelium. Mechanistic studies, using murine models of myocardial infarction, will be required to address these intriguing questions.
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Acknowledgments |
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We wish to thank Peggy Jackson, Alida Evans, and Stephanie Butcher for their outstanding technical assistance, and Sharon Malinowski and Connie Mata for editorial assistance with the manuscript.
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Footnotes |
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