Lawrence H. Chow

Evidence from a novel human cell clone that adult vascular smooth muscle cells can convert reversibly between noncontractile and contractile phenotypes.

Smooth muscle cells (SMCs) perform diverse functions that can be categorized as contractile and synthetic. A traditional model holds that these distinct functions are performed by the same cell, by virtue of its capacity for bidirectional modulation of phenotype. However, this model has been challenged, in part because there is no physiological evidence that an adult synthetic SMC can acquire the ability to contract. We sought evidence for this by cloning adult SMCs from human internal thoracic artery. One clone, HITB5, expressed smooth muscle alpha-actin, smooth myosin heavy chains, heavy caldesmon, and calponin and showed robust calcium transients in response to histamine and angiotensin II, which confirmed intact transmembrane signaling cascades. On serum withdrawal, these cells adopted an elongated and spindle-shaped morphology, random migration slowed, extracellular matrix protein production fell, and cell proliferation and [(3)H]thymidine incorporation fell to near 0. Cell viability was not compromised, however; in fact, apoptosis rate fell significantly. In this state, agonist-induced elevation of cytoplasmic calcium was even more pronounced and was accompanied by SMC contraction. Readdition of 10% serum completely returned HITB5 cells to a noncontractile, proliferative phenotype. Contractile protein expression increased after serum withdrawal, although modestly, which suggested that the switch to contractile function involved reorganization or sensitization of existing contractile structures. To our knowledge, the physiological properties of HITB5 SMCs provide the first direct demonstration that cultured human adult SMCs can convert between a synthetic, noncontracting state and a contracting state. HITB5 cells should be valuable for characterizing the basis of this critical transition.

Vascular Smooth Muscle Cells of Recipient Origin Mediate Intimal Expansion after Aortic Allotransplantation in Mice

Intimal expansion by vascular smooth muscle cells (SMCs) is a characteristic feature of graft vascular disease. Whether graft intimal SMCs arise from donor or recipient tissue is not well established but has important pathogenetic implications. We examined for the presence of male cells in the expanded intima of sex-mismatched mouse aortic allografts (C57BL/6-to-BALB/c) at 30 or 60 days after transplant by in situ hybridization using a Y-chromosome probe. Study groups included male-to-female allografts, female-to-male allografts, and female-to-female allografts in recipients previously engrafted with male bone marrow. Although intimal expansion developed in all allografts, male-to-female allografts lacked Y-chromosome-positive intimal cells. In contrast, such cells were abundant in female-to-male allografts and most of these cells co-labeled for smooth muscle alpha-actin by immunostain. Female-to-female allografts in recipients with male bone marrow showed a limited number of intimal Y-chromosome-positive cells. However, none of these clearly co-labeled for smooth muscle alpha-actin and their numbers declined throughout time, consistent with graft-infiltrating inflammatory cells. We conclude that intimal expansion of mouse aortic allografts is mediated by SMCs that originated from the recipient. There was little evidence of their derivation from the bone marrow, suggesting instead the adjacent host aorta as the primary source of intimal SMCs.

Coordinated Effects of Fibroblast Growth Factor-2 on Expression of Fibrillar Collagens, Matrix Metalloproteinases, and Tissue Inhibitors of Matrix Metalloproteinases by Human Vascular Smooth Muscle Cells Evidence for Repressed Collagen Production and Activated Degradative Capacity

Fibroblast growth factor-2 (FGF-2) is an established mediator of smooth muscle cell (SMC) proliferation after vascular injury. However, the influence of FGF-2 on collagen fiber remodeling, which may be a prerequisite for vascular SMC accumulation, is not well understood. We determined that FGF-2 almost completely abrogated the formation of immunodetectable type I collagen fibers in the extracellular matrix of cultured human vascular SMCs. This was associated with reduced expression of pro alpha-chains for types I and III collagen, as assessed by Western blot analysis, and a corresponding reduction in collagen synthesis. Densitometry of Northern blots indicated a potent reduction of mRNA encoding pro alpha-chains for types I and III collagen and a minor reduction in mRNA for pro alpha-chains for type V collagen. Interstitial collagenase (MMP-1), which is required for degradation of collagen types I and III, was not expressed by SMCs under basal culture conditions, but expression was induced by FGF-2, with a potent, dose-dependent increase in MMP-1 protein in conditioned medium. Metalloproteinase inhibitors TIMP-1, TIMP-2, and TIMP-3 were expressed by unstimulated SMCs and were differentially regulated by FGF-2. TIMP-1 expression increased modestly, TIMP-2 expression was repressed, and TIMP-3 was relatively unaffected. The net effect on substrate degradation, as assessed by zymography of conditioned media, was induction of MMP-1 lytic activity by FGF-2, with no effect on the activity of MMP-2, MMP-3, or MMP-9. These data indicate that stimulation of human SMCs with FGF-2 establishes a phenotype in which collagen fiber production is repressed and the capacity for fiber degradation activated. This coordinated response may be critical for SMC accumulation during vascular remodeling as well as atherosclerotic plaque destabilization.

Fibroblast Growth Factor-2 Potentiates Vascular Smooth Muscle Cell Migration to Platelet-Derived Growth Factor

Fibroblast growth factor-2 (FGF-2) has been implicated in vascular smooth muscle cell (SMC) migration, a key process in vascular disease. We demonstrate here that FGF-2 promotes SMC motility by altering beta1 integrin-mediated interactions with the extracellular matrix (ECM). FGF-2 significantly increased surface expression of alpha2beta1, alpha3beta1, and alpha5beta1 integrins on human SMCs, as assessed by flow cytometry. The greatest increase was for the collagen-binding alpha2beta1 integrin. Despite this, FGF-2 did not increase SMC adhesion to type I collagen but instead promoted SMC elongation and SMC motility. The latter was evaluated by using a microchemotaxis chamber and by digital time-lapse video microscopy. Although FGF-2 was not chemotactic for human SMCs, cells preincubated with FGF-2 displayed a 3.1-fold increase in migration to the undersurface of porous type I collagen-coated membranes and a 2.1-fold increase in migration speed on collagen. Furthermore, chemotaxis to platelet-derived growth factor-BB on collagen was significantly greater in SMCs exposed to FGF-2. FGF-2-induced elongation and migration on collagen were inhibited by a blocking anti-alpha2beta1 antibody; however, SMC adhesion to collagen was unaffected. SMC migration on fibronectin was also enhanced by FGF-2, although less prominently: migration through porous membranes increased 1.8-fold, and migration speed increased 1.3-fold. Also, FGF-2 completely disassembled the smooth muscle alpha-actin-containing stress fiber network contemporaneously with the change in integrin expression and cell shape. We conclude that (1) exogenous FGF-2 promotes SMC migration and potentiates chemotaxis to PDGF-BB; (2) the promigratory effect of FGF-2 is especially prominent on type I collagen and is mediated by upregulation of alpha2beta1 integrin; and (3) FGF-2 disassembles actin stress fibers, which may promote differential utilization of alpha2beta1 integrin for motility but not adhesion. This dynamic SMC-ECM interplay may be an important mechanism by which FGF-2 facilitates SMC motility in vivo.

Heat Shock Protein 47 Is Expressed in Fibrous Regions of Human Atheroma and Is Regulated by Growth Factors and Oxidized Low-Density Lipoprotein

BACKGROUND Heat shock protein 47 (Hsp47) is a stress protein that may act as a chaperone for procollagen. Its involvement in atherosclerosis is unknown. METHODS AND RESULTS Hsp47 expression in human coronary arteries was assessed by immunostaining. Strong focal expression was evident in atherosclerotic, but not normal, arteries and was prevalent in the collagenous regions. Double immunostaining revealed that all cells expressing type I procollagen also expressed Hsp47. Moreover, parallel regulation of proalpha1(I)collagen and Hsp47 mRNA expression occurred with cultured human smooth muscle cells stimulated with transforming growth factor-beta1 or fibroblast growth factor-2. However, a proportion of Hsp47-expressing cells in plaque did not express type I procollagen, and this pattern could be reproduced in culture. Heat shock and oxidized LDL stimulated the expression of Hsp47 mRNA by smooth muscle cells, without a concomitant rise in proalpha1(I)collagen expression. CONCLUSIONS These findings identify Hsp47 as a novel constituent of human coronary atheroma. Its localization to the fibrous cap, regulation by growth factors in parallel with type I procollagen, and selective upregulation by stress raise the possibility that Hsp47 is a determinant of plaque stability.

Evidence for Rapid Accumulation and Persistently Disordered Architecture of Fibrillar Collagen in Human Coronary Restenosis Lesions

The pattern of collagen deposition after coronary angioplasty could significantly influence recurrent lesion formation. Traditional histologic assessments of coronary restenosis lesions have not identified abundant collagen fibers in restenotic tissue; however, these methods can suffer from lack of sensitivity and are not quantitative. We analyzed collagen architecture in 40 coronary lesions retrieved from patients by directional atherectomy, by exploiting the birefringent properties of fibrillar collagen. Picrosirius red-stained sections were illuminated with circularly polarized light, and fiber content and thickness were quantified by digital image analysis. Fifteen of 19 restenosis lesions (79%) and 1 of 21 native atherosclerosis lesions (5%) displayed a pattern of reactive intimal modeling, characterized by stellateshaped smooth muscle cells variably oriented in a loose extracellular matrix. There was an apparent paucity of collagen fibers in these regions based on staining with Movats pentachrome, a traditional connective tissue stain. However, circular polarization light microscopy revealed an extensive distribution of collagen fibers in restenosis tissue, occupying 79.9% +/- 11.8% of the section area. Despite this high collagen content, the restenosis lesions were distinct from de novo atherosclerosis lesions in having a disordered collagen alignment, reduced fiber packing (p < 0.05), and thinner fibers (4.3 +/- 1.7 vs 9.2 +/- 4.3 microns, p < 0.001). Fiber diameter was greater in lesions retrieved between 3 and 17 months after angioplasty than in lesions retrieved between 1 week and 3 months (p < 0.05). However, fiber disorientation was evident in all lesions retrieved after 1 week, with little similarity to that of native plaque. Lesions retrieved within 1 week of angioplasty represented a distinct group with identical collagen features as in de novo atherosclerosis lesions, implying a different mechanism of restenosis in that population. We conclude that human coronary restenosis involves rapid accumulation of collagen fibers, which are persistently disordered. This may be critical in the development of restenosis and could significantly influence therapeutic attempts to control the process.

Cell surface-bound collagenase-1 and focal substrate degradation stimulate the rear release of motile vascular smooth muscle cells.

To migrate in the vessel wall, smooth muscle cells (SMCs) must contend with abundant type I collagen. We investigated the mechanisms used by human SMCs to efficiently migrate on type I collagen, following stimulation with fibroblast growth factor-2 (FGF-2). FGF-2-stimulated migration was inhibited by a hydroxamic acid inhibitor of matrix metalloproteinases and by a neutralizing anti-collagenase-1 antibody. Moreover, migration speed of SMCs plated on mutant collagenase-resistant type I collagen was not increased by FGF-2. Time-lapse video analysis of unstimulated SMCs migrating on collagen revealed discrete phases of leading edge membrane extension and rear retraction, the latter often after rupture of an elongated tail. FGF-2 stimulation yielded a more synchronous, gliding motion with a collagenase-1-mediated decrease in tail ripping. Surface labeling of SMCs with biotin followed by immunoprecipitation revealed that a proportion of active collagenase-1, expressed in response to FGF-2, was bound to the plasma membrane. Pericellular collagen substrate cleavage was verified by immunostaining for neoepitopes generated by collagenase-1 action and was localized to discrete zones beneath the cell tail and the leading edge. These results identify a novel mechanism by which SMC migration on collagen is enhanced, whereby rear release from the substrate is orchestrated by the localized actions of membrane-bound collagenase-1.

Enhanced detection of cardiac myocyte damage by polarized light microscopy: Use in a model of coxsackievirus B3-induced myocarditis

Although accurate detection of cardiac muscle damage is critical in the diagnosis of acute myocarditis or acute cellular rejection in both clinical and experimental settings, the histologic evaluation is frequently uncertain without specialized stains. In a study of adult male A/J mice infected with 2x10(5) plaque-forming units of myocarditic coxsackievirus B3, cardiac muscle injury causing myofibrillar disruption was detected as a loss of muscle birefringence by polarized light microscopy. The technique was corroborated by comparison with Massons trichrome stain and was helpful for histologic examination especially at the early preinflammatory stages of lesion development or in fringe territories of focal lesions. Polarized light microscopy is thus an available means to enhance the histologic determination of cardiac myocyte damage and has specific advantage in an absence of specialized stains.