Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery.

Vascular smooth muscle cells (SMCs) perform diverse functions and this functional heterogeneity could be based on differential recruitment of distinct SMC subsets. In humans, however, there is little support for such a paradigm, partly because isolation of pure human SMC subsets has proven difficult. We report the cloning of 12 SMC lines from a single fragment of human internal thoracic artery and the elucidation of 2 distinct cellular profiles. Epithelioid clones (n=9) were polygonal at confluence, 105+/-9 micrometer in length, and had a doubling time of 39+/-2 hours. Spindle-shaped clones (n=3) were larger (267+/-18 micrometer long, P<0.01) and grew slower (doubling time 65+/-4 hours, P<0.01). Both types of clones expressed smooth muscle (SM) alpha-actin, SM-myosin heavy chains, h-caldesmon, and calponin, but only spindle-shaped clones expressed metavinculin. Epithelioid clones displayed greater proliferation in response to platelet-derived growth factor-BB and fibroblast growth factor-2 and were more responsive to the migratory effect of platelet-derived growth factor-BB. Spindle-shaped clones showed more robust Ca(2+) transients in response to angiotensin II, histamine, and norepinephrine, crawled more quickly, and expressed more type I collagen. On serum withdrawal, spindle-shaped clones differentiated into a contraction-competent cell. A regional basis for diversity among SMCs was suggested by stepwise arterial digestion, which liberated small, SM alpha-actin-positive cells from the abluminal medial layers and larger SMCs from all layers. These results identify inherent SMC diversity in the media of the adult internal thoracic artery and suggest differential participation of SMC subsets in the regulation of human arterial behavior.

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.

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 Masson's 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.

Functional receptor-channel coupling compared in contractile and proliferative human vascular smooth muscle.

We have previously identified a human vascular smooth muscle clone that can reversibly convert between proliferative and contractile phenotypes. Here we compared receptor-channel coupling in these cells using fura-2 to monitor [Ca(2+)](i) and patch-clamp to record currents. Histamine elevated [Ca(2+)](i) in all cells and caused contraction of cells exhibiting the contractile phenotype. The rise of [Ca(2+)](i) persisted in Ca(2+)-free solution and was abolished by thapsigargin, indicating involvement of stores. Whole cell electrophysiological recording revealed that histamine evoked transient outward K(+) current, indicating functional receptor-channel coupling. The time-course and amplitude of the histamine-activated current were similar in cells of the proliferative and contractile phenotypes. Moreover, a large conductance K(+) channel was recorded in cell-attached patches and was activated by histamine as well as the Ca(2+) ionophore A-23187, identifying it as the large conductance Ca(2+)-dependent K(+) channel. This K(+) channel showed similar characteristics and activation in both proliferative and contractile phenotypes, indicating that expression was independent of phenotype. In contrast, histamine also elicited an inward Cl(-) current in some contractile cells, suggesting differential regulation of this current depending on phenotype. These studies demonstrate the usefulness of this human vascular cell clone for studying functional plasticity of smooth muscle, while avoiding complications arising from extended times in culture.

HSP47 expression by smooth muscle cells is increased during arterial development and lesion formation and is inhibited by fibrillar collagen.

HSP47 is a heat-shock protein that interacts with intracellular procollagen. It has been found in fibrous atherosclerotic plaque, but its involvement in acute vascular restructuring is unknown. We analyzed the expression of HSP47 and its regulation in the developing rat aorta and after balloon injury to the adult rat carotid artery. HSP47 was strongly expressed in each layer of the maturing fetal aorta (embryonic day 17 to birth). Expression declined during the first 4 postnatal days but persisted at low abundance into adulthood. HSP47 expression was substantially upregulated in the injured carotid artery, with intense immunostaining in neointimal smooth muscle cells (SMCs). HSP47 expression in SMCs was correlated with the emergence of a less mature phenotype and with expression of type I procollagen. Interestingly, a precipitous decline in HSP47 expression was evident during aortic development and after carotid artery injury, in association with the appearance of collagen fibrils in the local extracellular matrix. Furthermore, type I collagen fibrils, but not collagen monomers, inhibited expression of HSP47 by SMCs. These findings indicate that upregulation of HSP47 is a feature of vascular restructuring, including acute neointimal formation, and that the constituents of the extracellular matrix regulate the duration of expression. This feedback control may be important for self-termination of vascular development and lesion growth.

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.

The axis of interleukin 12 and gamma interferon regulates acute vascular xenogeneic rejection.

Recent advances using transgenic animals or exogenous complement inhibitors have demonstrated prevention of hyperacute rejection of vascularized organs, but not graft loss due to acute vascular rejection. Using various wild-type and cytokine-deficient mice strains, we have examined the mechanisms of acute vascular rejection. C57BL/6 mice deficient in interleukin12 or gamma interferon showed faster acute vascular rejection than did wild-type mice. Furthermore, mice defective in B-cell development showed no acute vascular rejection. These results demonstrate that the axis of interleukin 12 and gamma interferon provides a survival advantage in vascularized xenografts by delaying or preventing acute vascular rejection caused by a B cell-dependent mechanism.

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.

alpha5beta1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury.

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the alpha5beta1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

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.

Angiotensin II stimulates collagen synthesis in human vascular smooth muscle cells. Involvement of the AT(1) receptor, transforming growth factor-beta, and tyrosine phosphorylation.

Angiotensin II is an established regulator of vascular tone and smooth muscle cell (SMC) growth. However, there are little data about its effect on collagen synthesis by SMCs and none regarding the mechanism of such an effect. We studied the effect of angiotensin II on collagen production by human arterial SMCs, using uptake of [(3)H]proline into collagenase-digestible proteins, and by ribonuclease protection assay for mRNA encoding the proalpha1 chain of type I collagen, the major collagen in arteries. This revealed a dose-dependent increase in relative collagen synthesis rate and a dose-dependent increase in proalpha1(I) collagen mRNA abundance, with the half-maximal effect at 1.7 nmol/L. Angiotensin II-stimulated collagen expression was associated with a 6-fold increase in transforming growth factor-beta (TGF-beta) production and was inhibited by a neutralizing antibody to TGF-beta. Both collagen production and TGF-beta release were inhibited by the AT(1)-specific antagonist, losartan, but not by the AT(2) receptor antagonist, PD123319. To determined if tyrosine phosphorylation was functionally linked to collagen synthesis, we studied the effect of 2 mechanistically distinct inhibitors of tyrosine kinase, genistein, and tyrphostin A25. These inhibitors abrogated angiotensin II-mediated procollagen mRNA expression and angiotensin II-mediated TGF-beta production, whereas the inactive homolog tyrphostin A1 had no effect. We conclude that angiotensin II stimulates collagen production in human arterial SMCs via the AT(1) receptor and an autocrine loop of TGF-beta, induction of which requires tyrosine phosphorylation.

CD45 modulation of CXCR1 and CXCR2 in human polymorphonuclear leukocytes.

All leukocytes express the cell surface glycoprotein CD45, which has intrinsic intracellular protein tyrosine phosphatase activity. CD45 is known to play a regulatory role in activation-induced signaling in lymphocytes; however, little is known of its role in non-lymphoid leukocytes. Therefore, we examined the potential effect of CD45 on chemokine-induced signaling in human neutrophils (polymorphonuclear cells, PMN). Treating isolated PMN for 2 h with an anti-CD45RB antibody (Bra11) down-modulated expression of the chemokine receptors CXCR1 and CXCR2 to 44 +/- 10% and 47 +/- 9% of their respective controls. The tyrosine kinase inhibitors genistein and herbimycin A significantly inhibited the Bra11-induced down-modulation of CXCR1 and CXCR2. Furthermore, Bra11-treated PMN were functionally inhibited in their capacity to exhibit IL-8-induced transient intracellular Ca2+ increases. Selected targeting of CXC receptors is indicated by the fact that N-formyl-Met-Leu-Phe (fMLP) receptor expression and function were not lost following Bra11 treatment. The effect of Bra11 on IL-8-mediated function and receptor expression was paralleled by decreased tyrosine phosphorylation of a 54- to 60-kDa protein. These findings indicate that CD45 can act to modulate PMN responses to chemokines; thus agents regulating CD45 can potentially modulate leukocyte traffic and may represent a novel therapeutic approach towards the treatment of inflammatory diseases.

Bacterial superantigens induce down-modulation of CC chemokine responsiveness in human monocytes via an alternative chemokine ligand-independent mechanism.

Staphylococcal superantigens (SAgs) are very potent T cell mitogens, but they can also activate monocytes by binding directly to MHC class II molecules in a manner independent of TCR coengagement. Induction of proinflammatory cytokines and chemokine expression in monocytes by superantigens has recently been reported. Here we report that superantigen stimulation of human peripheral blood monocytes results in a rapid, dose-dependent, and specific down-regulation of chemokine (macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-1 and MIP-1beta) binding sites (e.g., CCR1, CCR2, and CCR5), which correlates with a concomitant hyporesponsiveness of human monocytes to these CC chemokine ligands. This down-regulation occurs 15-30 min following superantigen stimulation and is specific to chemokine receptors, in that binding and responsiveness of monocytes to the chemoattractant formyl-tripeptide FMLP are not affected. We further demonstrate that SAg-induced down-modulation of chemokine binding and monocyte hyporesponsiveness to the chemokines MIP-1alpha, monocyte chemotactic protein-1, and MIP-1beta is mediated through cellular protein tyrosine kinases, and the down-modulation can be mimicked by an MHC class II-specific mAb. Additionally, our observations indicate that SAg-induced loss of chemokine binding and monocyte responsiveness is probably mediated by secreted serine proteinases. Bacterial SAg-induced down-modulation of chemokine responsiveness represents a previously unrecognized strategy by some bacteria to subvert immune responses by affecting the intricate balance between chemokine and chemokine receptor expression and function.

CXCR1 and CXCR2 are rapidly down-modulated by bacterial endotoxin through a unique agonist-independent, tyrosine kinase-dependent mechanism.

The expression of the seven-transmembrane domain chemokine receptors CXCR1 and CXCR2 modulates neutrophil responsiveness to the chemoattractant IL-8 and a number of closely related CXC chemokines. In the present study, we investigated the mechanism by which bacterial LPS induces the down-modulation of IL-8 responsiveness and CXCR1 and CXCR2 expression on human neutrophils. Treating neutrophils with LPS reduced IL-8R expression to 55 +/- 5% of the control within 30 min and to 23 +/- 2% within 1 h of stimulation. Furthermore, this down-modulation could not be attributed to increased concentrations of IL-8, TNF-alpha, or IL-1beta, since ELISA studies indicated that LPS-stimulated neutrophils did not release detectable amounts of these proteins before 2 h poststimulation. The tyrosine kinase (TK) inhibitors genistein and herbimycin A attenuated the LPS-mediated down-modulation of CXCR1 and CXCR2, indicating that the activation of a TK is required for LPS to mediate its effect. The effect of LPS on receptor expression paralleled the hyperphosphorylation of the protein TK p72syk. Although IL-8 induced a comparable down-modulation of CXCR1 and CXCR2, TK inhibitors did not attenuate this effect. These studies provide the first evidence of an agonist-independent, TK-dependent pathway of chemokine receptor regulation by endotoxin.

Evidence for a role of collagen synthesis in arterial smooth muscle cell migration.

Migration of smooth muscle cells (SMCs) and collagen synthesis by SMCs are central to the pathophysiology of vascular disease. Both processes can be induced shortly after vascular injury; however, a functional relationship between them has not been established. In this study, we determined if collagen synthesis was required for SMC migration, using ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor of prolyl-4-hydroxylase, and 3,4-DL-dehydroproline (DHP), a proline analogue, which we demonstrate inhibit collagen elaboration by porcine arterial SMCs. SMCs exposed to EDHB or DHP attached normally to collagen- and vitronectin-coated substrates; however, spreading on collagen but not vitronectin was inhibited. SMC migration speed, quantified by digital time-lapse video microscopy, was significantly and reversibly reduced by EDHB and DHP. Flow cytometry revealed that expression of beta1 integrins, through which SMCs interact with collagen, was unaffected by EDHB or DHP. However, both inhibitors prevented normal clustering of beta1 integrins on the surface of SMCs, consistent with a lack of appropriate matrix ligands for integrin engagement. Moreover, there was impaired recruitment of vinculin into focal adhesion complexes of spreading SMCs and disassembly of the smooth muscle alpha-actin-containing cytoskeleton. These findings suggest that de novo collagen synthesis plays a role in SMC migration and implicates a mechanism whereby newly synthesized collagen may be necessary to maintain the transcellular traction system required for effective locomotion.

Fibroblast growth factor-2 potentiates vascular smooth muscle cell migration to platelet-derived growth factor: upregulation of alpha2beta1 integrin and disassembly of actin filaments.

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.

Differential expression of nonmuscle myosin II isoforms in human atherosclerotic plaque.

Intimal proliferation and functional changes involving vascular smooth muscle cells are key events in the development of atherosclerosis, including restenosis after percutaneous transluminal angioplasty. Nonmuscle myosin (NMM) is required for cytokinesis and has been shown in cultures of vascular smooth muscle cells to undergo changes of isoform expression depending on the stage of proliferation and differentiation. The purpose of this study was to examine the differential expression of the two most recently identified nonmuscle myosin heavy chain isoform II (NMMHC-II) isoforms A and B in atherosclerotic plaque. Primary atherosclerotic and restenotic atherectomy specimens and non-atherosclerotic controls, were analyzed by Western Blot analysis, immunohistochemistry and in situ hybridization. Nonmuscle myosin heavy chain isoform IIA (NMMHC-IIA) was equally expressed in all types of tissue specimens both at the protein and mRNA levels. In contrast, NMMHC-IIB protein was found in restenotic specimens and normal artery but was at very low levels in primary atherosclerotic plaque. By in situ hybridization NMMHC-IIB mRNA levels were significantly greater in restenotic versus primary atherosclerotic lesions. NMMHC-IIB expression is associated with vascular restenosis but is downregulated in stable atherosclerotic lesions, whereas NMMHC-IIA is expressed in both. These results indicate that these new myosin isoforms have different functions and should be regarded separately with respect to smooth muscle proliferation and restenosis. They should prove to be useful molecular markers for the study of atherosclerosis and restenosis.

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.

Intimal thickening develops without humoral immunity in a mouse aortic allograft model of chronic vascular rejection.

BACKGROUND: The major threat to the long-term survival of cardiac allograft recipients is the development of diffuse intimal thickening in the allograft coronary arteries through mechanisms that are poorly understood. Although antidonor antibodies have been associated with the development of this condition, a causal relationship has not been established. METHODS AND RESULTS: To determine whether humoral immune responses are necessary for the development of graft vascular disease, we performed abdominal aortic allografts from normal donor mice into different immunodeficient recipient mice: those lacking all donor-specific immune responses (severe combined immunodeficient [SCID] mice and recombination activating gene-1 [RAG-1]-deficient mice) and those lacking humoral immune responses alone owing to a targeted deletion of the joining region (JH) gene segments for the immunoglobulin heavy chain. At 6 to 9 weeks after transplantation, aortic allografts in normal immunocompetent recipients showed concentric intimal thickening extending the full length of the graft (percent luminal reduction, [%LR], 31.2 +/- 9.1 [mean +/- SD] and 38.5 +/- 3.6 in different donor-recipient strain combinations). In contrast, syngeneic (histocompatible) aortic grafts showed a normal-appearing vessel wall (%LR, 1.6 +/- 0.7). In both SCID and RAG-1-deficient recipients, aortic allografts showed a virtual absence of neointimal formation (%LR, 3.7 +/- 2.1 and 3.8 +/- 1.6 in SCID and RAG-1-deficient recipients, respectively), indicating a critical etiological role for alloimmune responses in this model. Importantly, allografts in JH-deficient mice showed marked intimal thickening (%LR, 35.7 +/- 7.9), with an appearance histologically indistinguishable from that of normal immunocompetent recipients. CONCLUSIONS: Neointimal formation in graft vascular disease is critically dependent on alloimmune responses of the host. Humoral effector mechanisms, however, may not be required.